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39937855 | PMC11848293 | pmc | 2,405 | {
"abstract": "Significance Ecological interactions in nature occur between individual partners rather than species, and their outcomes determine fitness variation. By examining among-individual variation in interaction niches, we can bridge evolutionary and ecological perspectives to understand interaction biodiversity. This study investigates individual plant variation in frugivore assemblages worldwide, exploring how plant individuals “build” their interaction profiles with animal frugivores. The structure of networks composed of individuals was surprisingly similar to networks composed of species. Within populations, only a few plants played a key role in attracting a high diversity of frugivores, making them central to the overall network structure. Individuals actually interacted with a substantial diversity of partners, with individual niche “breadth” accounting for up to 70% of total interaction diversity, on average.",
"conclusion": "Concluding Remarks. We found consistent patterns of interaction assembly across biological scales using a set of biologically informative network metrics. On top of the absence of a clear hierarchy differentiation in network structure between individuals and species, we found that almost every individual-based network included a similar representation of individual interaction profiles, evidencing a common backbone in the way interactions are organized ( 8 ). Conducting future analyses on interaction types or motifs of individual-based networks may provide us with additional insights, as these approaches have proven effective in distinguishing networks between and within ecological systems ( 8 , 74 , 75 ). Intraspecific variation appears as a central ingredient in the configuration of complex networks of mutualistic interactions, driven by the widespread interaction profiles of frugivore species with plant individuals. High levels of intraspecific variation have been shown to confer greater stability to mutualistic systems ( 31 ). By zooming in on ecological interactions this study provides valuable insights into how mutualistic interactions are similarly structured at the individual level and reveal underlying, consistent, patterns of role assignment within populations and across bioregions.",
"discussion": "Discussion Our study highlights how downscaling from species to individuals uncovers consistent network structures across biological levels, mutualistic partner allocation among plants globally, and interaction profile similarities within populations, regardless of species or ecological context. This reveals aspects of ecological interactions and network assembly at the individual level. Effects of Downscaling Resolution on Network Structure. The structure of plant–animal mutualistic networks revealed fundamental heterogeneity across networks and resolution scales. We did not find major deviations in the assembly patterns of interactions as we zoomed in from species to an individual-based scale. Previous research exploring the consequences of downscaling on network architecture found significant shifts in the structure of pollination networks ( 19 , 20 ): Individual-based networks were less connected and individuals were more specialized than species. These studies examined one community using a single methodology. Our approach, however, involved diverse communities and populations from different regions with varying methodologies, capturing broader interaction patterns. Surprisingly, we found no significant differences in network connectance across scales, contrary to our initial expectations. The slightly larger connectance of individual networks after Bayesian modeling (average connectance = 0.4 ± 0.2 versus 0.3 ± 0.1 in raw networks) suggests that undersampled interaction matrices could be missing interactions. On the other hand, the slightly lower modularity in individual-based networks could stem from a decrease in the number of forbidden links as frugivores can potentially interact with virtually all the plant individuals within populations (see ref. 23 ). In contrast, species-based networks involve much more heterogeneous sets of plant species. The addition of new species or individuals with new traits provides new link possibilities in a network, yet in the case of species, potential interactions must undergo stronger trait and phenological matching filters than in individual-based networks ( 57 ). Simply put, a given frugivore species may interact with a broader range of partners within a plant population than when interacting with the full range of taxonomically diverse available plant species in a community. The former set imposes less constraints to interactions by including much more homogenous conspecific partners. In this way, downscaling from species to individuals fundamentally alters the probabilistic distribution of interactions among partners ( 58 ). Aside from minor differences in certain network metrics, the overall topology and structure of frugivory networks at different resolution scales were not sufficient to make clear distinctions. This convergence in network structure may be driven by factors like the probability distribution of interspecific encounters (PIEs), influencing network configurations consistently across scales. We argue that numerical effects are likely at the base of these emergent properties, governing interactions distribution across nodes and asymmetric interactions ( 5 , 38 , 59 , 60 ). These numerical effects can be caused by varied organism abundances in the case of species, or traits and genotypes in the case of individuals, that modulate the attractiveness of plants to frugivores, such as crop size, plant height, or phenology ( 41 ). Individual Specialization in the Interaction Niche. Individuals’ interaction niches were narrower than those of their populations, supporting that individual specialization is substantial and common in nature ( 24 ), even in mutualisms. Plant individuals’ specialization levels were similar to levels reported in other animal taxa ( 27 ). Interestingly, the degree of individual specialization varied across biogeographic regions, yet most plant species showed WIC/TNW ratios >50%, which indicates moderate generalization among plant individuals. Broader and more overlapping frugivore assemblages in Mediterranean regions versus higher specialization and variability in Southern Temperate and Tropical networks could not be attributed to differences in frugivore taxonomic diversity as all regions presented similar TNW ( SI Appendix , Fig. S5 ). Instead, plant individuals in Southern Temperate regions presented smaller relative niches. Tropical populations consisted of frugivore assemblages of variable diversity and highly variable individual specialization, yet limited area coverage of the available individual-based networks hindered comparisons. No significant niche breadth differences were found across bioregions, aligning with studies of terrestrial food webs, challenging the latitude-niche breadth hypothesis that predicts narrower niches in tropical regions ( 61 , although see ref. 62 ). The large variation in individual specialization within bioregions may be pointing to the role of fine-scale factors such as the local habitat, neighborhood effects, or the influences of individual phenological variation. Further research is needed to evaluate the ecological correlates of plant individual interaction niche utilization and its consequences. Different levels of individual specialization can have implications for population stability ( 63 ) and niche expansion ( 26 ). According to the niche variation hypothesis, populations experiencing niche expansion achieve it through increasing their interindividual variation ( 45 ). By diversifying its resources, plant individuals would be able to exploit novel and underutilized frugivores escaping competition from conspecifics. Niche shifts and expansion have become exceptionally important for adaptation to changing climate conditions ( 64 ) as well as changes in frugivore assemblages and fluctuating abundances ( 65 ). Therefore, the variation we found among populations in frugivore assemblage specialization will likely have an impact on the adaptation of plant–frugivore mutualistic interaction niche in current and future scenarios of global change. In all plant populations just a few frugivore species, even within diversified assemblages, consistently perform most of the mutualistic interactions ( SI Appendix , Fig. S6 ; 39 , 47 , 66 – 68 ). Although frugivore body mass did not prove to be a good indicator of their contribution to interactions (although see ref. 69 ), it may play a role in seed dispersal effectiveness due to its positive correlation with the number of fruits consumed per visit or the frequency of long-distance seed dispersal events ( 12 , 70 ). These highly uneven interaction patterns will result in asymmetric dependencies between plant individuals and frugivore species, where the main frugivore shows low specificity for specific plants, while most plant individuals rely mostly on the main frugivores’ service ( 16 ). Asymmetric dependency between partners also emerges at species–species interaction level ( 5 , 71 , 72 ); further downscaling into individual–individual interactions would help elucidate whether asymmetry remains consistent across scales. Finally, our analysis reveals a consistent trend for frugivory and seed dispersal service in a given plant population (estimated from the proportion of plant individuals with which a frugivore species interacts) to increase with the overall contribution to the total number of interactions by each frugivore species (i.e., link weights in the individual-based networks, Fig. 4 ). Thus, central frugivores interact with a wide range of plant individuals, most likely an emergent result of the interaction asymmetry discussed above. Consistency of Plant Individuals’ Interaction Profiles across Regions and Populations. Plant individuals’ interaction profiles were not explained by bioregion or species, pointing to fundamental architectural patterns in the assemblage of mutualistic interactions that are not strongly constrained by phylogeny or geographic location but rather by the interplay between traits and numerical effects ( 5 , 30 , 38 , 73 ). Remarkably, we found a consistent distribution of plant interaction profiles within populations, with most individuals acting in an average manner, a variable fraction standing out for their specialization or redundancy and only very few individuals having a central role, high diversity of interactions, and strong frugivores’ dependence on them (“keystone” plant individuals). Similar results are reported in food webs, where a core group of species shares ecological roles, while peripheral species have unique interaction profiles ( 8 ). It is likely that within frugivory networks these key individuals present unique phenotypic traits, such as abundant fruit crops or advantageous locations that make them reliable to many frugivores ( 39 , 41 ). Although some of the plant species considered in this study were generalists within their community, individuals in their populations showed variable interaction niche breadths ( 47 ), with populations consisting of both generalist and specialist individuals ( 31 , 45 ). This mix creates species that appear broadly interactive but actually include individuals with varied interaction patterns, from wide generalization to specific partner preferences. This highlights the complexity of species’ ecological roles within communities ( 38 ). Concluding Remarks. We found consistent patterns of interaction assembly across biological scales using a set of biologically informative network metrics. On top of the absence of a clear hierarchy differentiation in network structure between individuals and species, we found that almost every individual-based network included a similar representation of individual interaction profiles, evidencing a common backbone in the way interactions are organized ( 8 ). Conducting future analyses on interaction types or motifs of individual-based networks may provide us with additional insights, as these approaches have proven effective in distinguishing networks between and within ecological systems ( 8 , 74 , 75 ). Intraspecific variation appears as a central ingredient in the configuration of complex networks of mutualistic interactions, driven by the widespread interaction profiles of frugivore species with plant individuals. High levels of intraspecific variation have been shown to confer greater stability to mutualistic systems ( 31 ). By zooming in on ecological interactions this study provides valuable insights into how mutualistic interactions are similarly structured at the individual level and reveal underlying, consistent, patterns of role assignment within populations and across bioregions."
} | 3,232 |
32655535 | PMC7324634 | pmc | 2,406 | {
"abstract": "This study explores nitrogen removal performance, bioelectricity generation, and the response of microbial community in two novel tidal flow constructed wetland-microbial fuel cells (TFCW-MFCs) when treating synthetic wastewater under two different chemical oxygen demand/total nitrogen (COD/TN, or simplified as C/N) ratios (10:1 and 5:1). The results showed that they achieved high and stable COD, NH 4 + -N, and TN removal efficiencies. Besides, TN removal rate of TFCW-MFC was increased by 5–10% compared with that of traditional CW-MFC. Molecular biological analysis revealed that during the stabilization period, a low C/N ratio remarkably promoted diversities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the cathode layer, whereas a high one enhanced the richness of nitrite-oxidizing bacteria (NOB) in each medium; the dominant genera in AOA, AOB, and NOB were Candidatus Nitrosotenuis , Nitrosomonas , and Nitrobacter . Moreover, a high C/N ratio facilitated the growth of Nitrosomonas , while it inhibited the growth of Candidatus Nitrosotenuis . The distribution of microbial community structures in NOB was separated by space rather than time or C/N ratio, except for Nitrobacter . This is caused by the differences of pH, dissolved oxygen (DO), and nitrogen concentration. The response of microbial community characteristics to nitrogen transformations and bioelectricity generation demonstrated that TN concentration is significantly negatively correlated with AOA-shannon, AOA-chao, 16S rRNA V4−V5-shannon, and 16S rRNA V4−V5-chao, particularly due to the crucial functions of Nitrosopumilus , Planctomyces , and Aquicella . Additionally, voltage output was primarily influenced by microorganisms in the genera of Nitrosopumilus, Nitrosospira , Altererythrobacter , Gemmata , and Aquicella. This study not only presents an applicable tool to treat high nitrogen-containing wastewater, but also provides a theoretical basis for the use of TFCW-MFC and the regulation of microbial community in nitrogen removal and electricity production.",
"conclusion": "Conclusion In summary, we demonstrated that the removal of COD, NH 4 + -N, and TN from wastewater with different C/N ratios (10:1 and 5:1) using TFCW-MFC were not significantly different. Compared to the conventional CW-MFC, our TFCW-MFC achieved a high and stable TN removal efficiency (77–89%) from wastewater containing various C/N ratios. Moreover, the high-throughput sequencing analysis confirmed that a low C/N led to enhanced AOA and AOB diversities in the cathode layer during the stabilization period, causing the community structure of the dominant genera to change drastically. A low C/N ratio (5:1) significantly promoted the relative abundance of Candidatus Nitrosotenuis , Nitrosopumilus (both belong to AOA) and Nitrosomonas (belongs to AOB) in the cathode layer during the stabilization period. On the other hand, a high C/N ratio (10:1) promoted the relative abundance of Nitrosomonas (belongs to AOB) in anode and upper layers. We further observed that multiple environmental variables collaboratively contributed to change of microbial compositions; for instance, pH, DO, and nitrogen concentration could potentially change the spatial distribution of the microbial community structure. The results further revealed that effluent nitrogen concentration could be reduced through changes of certain alpha diversities indexes of microorganisms or relative abundances of the dominant genera. We also found that high voltage output in two devices can be achieved by adjusting the distribution of dominant genera in the medium. Nonetheless, to understand whether or not anammox bacteria in TFCW-MFC can be used for a different NH 4 + -N removal pathway, further experiments should be performed.",
"introduction": "Introduction Earth’s biogeochemical cycles have been largely affected by human activities ( Yu et al., 2019 ), especially by a large amount of nitrogen released from agriculture and animal husbandry, which is the root of many environmental issues such as eutrophication and drinking water pollution. To this end, various methods have been purposed to remove nitrogen. However, these traditional nitrogen removal approaches (i.e., activated sludge process, trickling filter, and oxidation pond) have some limitations, such as low removal rate or high operating cost ( Tanwar et al., 2008 ). It is acknowledged that total nitrogen (TN) in wastewater mainly consists of ammonia (NH 4 + -N), nitrite (NO 2 – -N), and nitrate (NO 3 – -N). Generally, nitrification, denitrification, and anaerobic ammonia oxidation (anammox) processes are the typical biological pathways used for nitrogen elimination ( Zhi et al., 2015 ). Also, removal of nitrogen in wastewater has been investigated by biological method, and/or its combination with natural and ecological approaches ( Wu et al., 2017 ). With two prominent challenges nowadyas, i.e., energy scarcity and non-point sourced nitrogen, finding economical and sustainable nitrogen removal technologies is particularly difficult. Constructed wetlands (CWs) have been extensively designed and studied as a sustainable technology to treat water bodies contaminated with low-concentration nitrogen ( Zhi et al., 2015 ). However, many CWs only limitedly reduce nitrogen levels with fluctuating results; thus, an improved treatment method is urgently needed ( Liu et al., 2015 ). Vertical flow constructed wetlands (VFCWs) and horizontal flow constructed wetlands (HFCWs) are the initial designs of modified constructed wetlands. Due to low TN removal efficiency of a single-stage CW, some multi-stage treatment systems, such as HF-VFCWs or VF-HFCWs, have also been developed ( Vymazal, 2007 , 2013 ). However, these systems are practically difficult to use due to complex operation conditions and a huge demand for land. Recently, an expanded system consisting of a microbial fuel cell (MFC) coupled to a CW (CW-MFC) is becoming increasingly popular because it allows simultaneous wastewater treatment and electricity generation ( Yadav et al., 2012 ; Doherty et al., 2015 ). CW and MFC share the redox gradient between an anaerobic anode and an aerobic cathode, in addition to electricity produced via MFC; thus, they have enhanced removal efficiencies ( Srivastava et al., 2015 ). Tidal flow constructed wetland (TFCW), operated through a rhythmic sequential cycle of a “feeding/flooding” phase and a “draining/resting” phase, has been proposed as a compact and efficient method that can enhance the nitrogen removal ( Zhi and Ji, 2014 ). However, the nitrogen removal pathway and the underlying mechanism that governs the nitrogen transformation process of TFCW-MFC are not clearly understood. Generally, nitrification is a biological oxidation of ammonium to nitrite and nitrate, and is the first and most crucial step in TN removal. It is conducted in two sequential steps via several microorganisms that are phylogenetically distinct, including ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB) ( Zhang et al., 2011 ). Oxidation of NH 4 + -N is considered mainly driven by chemolithoautotrophic AOB ( Guimaraes et al., 2017 ). However, a recent study has shown that AOA, which is ubiquitously distributed in aquatic environments, often has higher abundance than AOB, and its abundance is correlated with nitrification rates ( Leininger et al., 2006 ). On the other hand, oxidation of NO 2 – -N (to NO 3 – -N) depends mainly on NOB ( Zhang et al., 2019 ). The quantitative relationships between ammonia oxidation rate and nitrogen functional genes (AOA and AOB) in TFCW have been analyzed ( Li et al., 2015 ). However, the accuracy of AOA, AOB, and NOB compositions in TFCW-MFC remains unconfirmed, and the interactions between the dominant genera and environmental factors have yet to be established; these have limited our ability to optimize the treatment processes. Influent chemical oxygen demand/total nitrogen (COD/TN; generally simplified as C/N) ratio has been considered as a key factor influencing the nitrogen removal in CWs ( Meng et al., 2018 ). It is also an important parameter affecting the activity of AOB and NOB ( Li et al., 2017 ). The recommended optimal influent C/N ratio for the removal of nitrogen (from approximately 80% TN) by CW-MFC is 5.37 ( Wang et al., 2019 ). Unfortunately, to date, related research conducted using TFCW-MFC has not been reported. To fill this knowledge gap, two laboratory-scale TFCW-MFCs were developed, and their nitrogen removal performance and bioelectricity generation from synthetic wastewater under two different C/N ratios were examined. The spatiotemporal variations of the diversity and community structure of AOA, AOB, NOB, and 16S rRNA V4−V5 region at filters and electrodes were investigated using the high-throughput sequencing. The relationships between the genetic characteristics of the four types of microorganisms (i.e., diversity and relative abundance of dominant genera) and effluent indexes [i.e., pH, dissolved oxygen (DO), temperature (T), NH 4 + -N, NO 3 – -N, NO 2 – -N, and TN] were also determined. This work provides comprehensive theoretical support for improving TN removal through the optimization of main functional microbial composition and populations in TFCW-MFC.",
"discussion": "Results and Discussion Wastewater Treatment Performance Device A and Device B were stable after 30 days of operation, and they achieved COD removal efficiencies of 97.66 and 98.36%, respectively, during the stabilization period ( Figure 2A ). The obtained COD removal efficiencies were higher than previous studies of the CW-MFC to treat livestock sewage, which have typical removal efficiencies ranging from 71 to 81% ( Zhao et al., 2013 ; Doherty et al., 2015 ; Liu et al., 2020 ). The COD removal load of Device A was 40.26 g/m 2 ⋅d, which was higher than that reported previously (9.8 g/m 2 ⋅d by Albuquerque et al. (2009) , 29.20 g/m 2 ⋅d by Konnerup et al. (2009) . This enhanced COD removal efficiency is likely attributed to the intensified oxygen supply generated by the tidal flow operation ( Oon et al., 2017 ). The effluent DO in both Device A (5.31 mg/L) and Device B (4.83 mg/L) were higher than the influent DO, which may be due to the oxygen intake from the atmosphere via employing the tidal flow mode. Meanwhile, the direct reduction by heterotrophic and electroactive microorganisms in the root area and plant uptake in the system also improved COD degradation rates ( Yang et al., 2020 ). Additionally, wastewater treatment by the coupled MFC has been reported to be more effective than that by CWs. Compared with traditional CWs, the biodegradation of organic compounds is enhanced by the transfer of electrons from the anode to the cathode. Study has also shown that CW-MFC is superior to conventional CW concerning the COD removal rate ( Wang et al., 2016 ). Therefore, it is highly likely that high-efficient removal of COD by TFCW is due to the integration of MFC. FIGURE 2 Average removal efficiency and change in concentrations of COD (A) , TN (B) , NH 4 + -N (C) , NO 3 – -N (D) , NO 2 — N, (E) with various C/N ratios. As shown in Figure 2 , the removal of nitrogen other than NO 3 – -N in Device B was stable after 30 days of operation, which indicates that the microorganisms used in nitrogen removal are mature. The average removal rate of TN (82.39%) and NO 3 – -N (52.16%) in Device A were higher than those in Device B (79.25% of TN, 36.72% of NO 3 – -N). By contrast, the removal rates of NH 4 + -N in both devices were similar with values of higher than 95% (95.94% in Device A, 97.13% in Device B). The removal rates of NH 4 + -N and TN were similar, inferring that the removal of TN may be in the form of NH 4 + -N and a certain amount of the effluent NO 3 – -N may be accumulated. This indicates that the loss of carbon source may lead to incomplete denitrification, thus causing the accumulation of NO 3 – -N. The TN removal rate of TFCW-MFC was higher than that of CW-MFC previously reported (74% has been reported by Wang et al., 2017 , 69% has been reported by Wu et al., 2017 ; Table 2 ). This observation further demonstrated that the developed device has higher TN removal efficiency, which may be caused by the use of tidal flow. It could increase the abundance of nitrogen-transformation genes. The coenrichment of these genes is attributed to their environmental adaptions to the balance condition of nitrification and denitrification via oxygen transfer, as well as their related distinct ecological niches ( Zhi et al., 2015 ). Thus, the increase in gene abundance is more likely to be the reason accounting for the enhanced denitrifying activity. Furthermore, better TN removal rates (90% and 87%) in Table 2 were obtained using CW-MFC in latest reports ( Srivastava et al., 2020 ; Tao et al., 2020 ). Compared with the results in the literature, the advantage of this study was that the efficient TN removal could be achieved under relatively low C/N ratios and short retention time. However, the NO 3 – -N removal rate is low because high DO is not conducive to denitrifying microorganism growth. Although the concentration differences of NO 2 – -N in effluents between Device A and B were found, the NO 2 – -N removal rates in both systems were more than 90%. Since NO 2 – -N is in the intermediate valence state of nitrogen transformation and it has an unstable characteristic, the impact of C/N ratio on NO 2 – -N attenuation could be negligible. Nonetheless, exorbitant carbon source concentration inhibits denitrification instead of boosting it ( Liu et al., 2012 ). Thus, the C/N ratio should be further optimized to achieve higher NO 3 – -N removal rate. TABLE 2 Operating conditions and performance comparison of TFCW-MFC and other CW-MFCs. Type Liquid volume (L) Wastewater type Influent COD (mg/L) C/N Hydraulic retention time (d) COD removal rate (%) TN removal rate (%) Max. power References Vertical upflow 3.7 Swine 1058 7.25 0.9−1.25 76.5 49.7 9.4 mW/m 2 Zhao et al., 2013 Horizontal flow 96 Synthetic 250 − 3.2 80−100 − 0.15 mW/m 2 Villaseñor et al., 2013 Vertical flow 1.8 Synthetic 770−887 − 1 90.9 − 43.63 mW/m 3 Srivastava et al., 2015 Upflow−downflow 8.1 Swine 583 9.25 1 64 58 0.28 W/m 3 Doherty et al., 2015 Horizontal subsurface flow 115 Domestic wastewater 323 7.8 0.17 61 − 36 mW/m 2 Corbella et al., 2015 Vertical downflow − Synthetic 813 10 4 57.4 − 8.08 mW/m 2 Wang et al., 2016 Vertical downflow − Synthetic 207.3 3.77 2 80 74 21.53 mW/m 2 Wang et al., 2017 Vertical upflow − Synthetic 646 − 1 98 − 184.75 mW/m 3 Oon et al., 2017 Vertical upflow 3.5 Swine 647 4.72 40 69 69 112 mW/m 2 Wu et al., 2017 Vertical upflow − Synthetic − − 3 82.32 82.46 3.71 W/m 2 Xu et al., 2018 Vertical flow − Synthetic 730 6.4 − 97 − 229 mW/m 3 Oodally et al., 2019 Downflow−upflow 11.5 Swine 900 3 2 88.07 − 496.4 mW/m 3 Liu et al., 2020 Horizontalflow−vertical upflow 2.85 Synthetic 880 22 0.63 99.5 90 25 mW/m 3 Srivastava et al., 2020 Vertical upflow − Municipal wastewater treatment plants effluents − 5.4 2 64.04 88.78 6.09 mW/m 2 Tao et al., 2020 Tidal flow 40 Synthetic 523.11 10 0.77 97.66 82.39 25.78 mW/m 2 This study Tidal flow 40 Synthetic 114.05 5 0.77 98.36% 79.25 16.97 mW/m 2 This study Electricity Generation The voltage curve of TFCW-MFC under different influent C/N ratios is shown in Figure 3 . According to the curve, Device A and Device B had the average voltages of 40.25 and 26.85 mV, respectively. Moreover, the maximum power densities of the system operated with C/N ratios of 10:1 and 5:1 were 25.78 and 16.97 mW/m 2 , respectively, while the current densities were 0.18 and 0.11 A/m 2 , respectively, and the internal resistances were 151 and 174 Ω, respectively. This clearly indicated that low internal resistance causes the increase in power density. Similar and high power density values have been reported by previous studies in Table 2 : Wang et al. (2017) have reported a value of 21.53 mW/m 2 in CW-MFC planted with T. orientalis ; Corbella et al. (2015) have reported a maximum value of 36 mW/m 2 , which was harvested by implementing MFC in HFCW during the treatment of effluent from a hydrolytic up-flow sludge blanket reactor. Low power density obtained by TFCW-MFC may be attributed to the following three reasons: (1) a small ratio of electrode surface to CW-MFC system volume may obstruct the accumulation of electrochemically active microorganisms on the electrode; (2) electrons may be largely depleted due to high concentrations of electron acceptors (NO 2 – -N, NO 3 – -N, oxygen, etc.) presented in the biofilter devices ( Wang et al., 2016 ); and (3) spacing between anode and cathode electrodes (25 cm) may be too large, as has been described by Oon et al. (2017) , who observed that the voltage output in up-flow constructed wetland-MFC tends to decrease when the spacing between anode and cathode electrodes are increased from 15 to 30 cm. Hiegemann et al. (2017) have also reported that the voltage output increases with the increase of organic concentration, as the voltage output is dependent on the electrons and protons transferred from carbon sources at the anode. However, excessive organic loading is not conducive to electricity generation because the multiplication of methanogens can inhibit the power generation ( Martin et al., 2010 ), or organics may not be completely oxidized at the anode, thereby generating an anaerobic environment that causes the CW-MFC to have decreased power output or even stop working ( Villaseñor et al., 2013 ). It is worth noting that the voltage output of both devices increased after 30 days of operation, but thereafter gradually decreased until it became stable. This indicated that high electrogenic microbial activity or organic concentration can promote electricity generation in the system. After 40 days of operation, the denitrifying microorganism in the system may become more abundant than the electrogenic microorganism; consequently, the electricity-producing ability was inhibited. FIGURE 3 Voltage output of two TFCW-MFC devices operated with various C/N ratios. Diversity of Microbial Community The Chao index was used to evaluate the richness of microbial community structure. As shown in Figures 4A–D , the Chao indexes of the two systems decreased in the following order: 16S rRNA V4−V5 region > AOB > NOB > AOA. Interestingly, AOB had a higher abundance than AOA, indicating that the microenvironment of the filter is more suitable for AOB growth. While one study has reported that AOA in many marine and terrestrial ecosystems is more abundant by one to two orders of magnitude than AOB ( Mincer et al., 2007 ); by contrast, another study has indicated that AOB is more dominant ( Liu et al., 2014 ). This inconsistent result may be primarily related to the high-strength NH 4 + -N at the influent, beneficial to AOB rather than AOA reproduction. Furthermore, the spatial distribution of each type of microorganisms is different and it changes over time, due to the collaborative functions of nitrogen concentration, pH, and temperature ( Fierer et al., 2009 ; Hallin et al., 2009 ; Fan et al., 2011 ; Zhang et al., 2019 ). The microbial richness in the upper layer of V4−V5 region was higher than that in the lower layer, which may mainly be due to the aerobic microsites adjacent to plant roots and exudates from rhizomes, where the oxygen released from the Cattail ’s root into the cathode could promote the growth of microorganisms. The richness of NOB in each substrate layer in Device A was higher than that in Device B ( Figure 4C ), indicating that a higher C/N ratio is beneficial to the survival of NOB. The richness of AOA and AOB were higher in the anode layer on the 90th day than that on the 30th day, due to that the exoelectrogenic microorganism and AOA/AOB gradually adapted to coexist on the anode in a facultative oxygen environment (DO = 0.9–1.5 mg/L at the anode in Supplementary Figure S1 ). This result is consistent with the observation by Xu et al. (2018) , who have reported that the microbial richness in the anode is the highest in the CW-MFC system. FIGURE 4 Relative change of Chao indexes of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region, at different sampling points of TFCW-MFCs. The horizontal line in the box represents the median. The size of the box represents the data dispersion (Interquartile range = IQR). As shown in Supplementary Figure S2 and Supplementary Tables S3–S6 , the diversity of microorganisms in the system became unstable after 30 days, and the C/N ratio had a greater influence on the space-time distribution of all four types of microorganisms in the same layer, especially Ab1 (1.42) and Bb1 (3.1) within AOB, Aa1 (2.53), Ba1 (1.75) within NOB, and Ab1 (3.56) and Bb1 (5.58) within the V4−V5 region. It is possible that at the beginning of the operation, carbon and nitrogen concentrations may have a direct or indirect effect on the microorganism in the anode and the bottom layers of the two devices. In contrast, the microbial community structure was relatively stable after 90 days, and the lower influent C/N ratio (5:1) appeared to facilitate the growth of AOA and AOB in the cathode layer, as well as that of all types of microorganisms in the V4−V5 region. The microorganism in the cathode layer is more diverse, which is likely due to the higher DO ( Wu et al., 2011 ; Supplementary Figure S1 ) caused by the reoxygenation ability of the tidal flow, or there may be a large number of aerobic electrogenic microorganisms in AOA and AOB. The influent C/N ratio had little effect on the diversity of microorganisms in the same layer. Comparative Analysis of Microbial Community Structure The differences in microbial community structure of the two systems were evaluated based on the phylogenetic lineages by the weighted Fast UniFrac PCoA. As shown in Figure 5 , there was a certain loss of AOA in Device A and of AOB in device B, which is likely to be caused by the instability of the system at the initial operation of the tidal flow, and the oxygen concentration in the lower layer of the device may be insufficient. Additionally, the microbial community structure of AOA, AOB, NOB, and V4−V5 region were changed by 82.96%, 84.1%, 92.27%, and 65.94%, respectively. These results indicated that the microbial community structure is largely separated by space, which is consistent with the bar plot and cluster tree shown in Figure 6 . The C/N ratio also had influence on the structure of the four types of microorganisms. While that of Nitrobacter , which belongs to NOB, was not affected by the C/N ratio, the relative abundances of Candidatus Nitrosotenuis (belongs to AOA) and Nitrosomonas (belongs to AOB) in the cathode layer were significantly promoted by a low C/N ratio (5:1) during the stabilization period. This result is consistent with the influence of C/N ratio on the diversity of the microbial community, indicating that these predominant genera may cause the change of diversity. Further, as shown in Figure 6 , a low C/N ratio (5:1) significantly increased the relative abundance of Nitrosopumilus (belongs to AOA) in all layers. AOA was found to participate in ammonia removal processes; however, its functions and contributions in the processes under the influence of MFC remain unclear. These results show that at a low C/N ratio (5:1), the cathode layer is beneficial to the survival of the dominant archaea AOA. On the other hand, a high C/N ratio (10:1) promoted the relative abundance of Nitrosomonas (belongs to AOB) in the anode and the upper layer during the stabilization period. This may be caused by the increased production of carbon dioxide in the anode layer, and AOB may consume a large amount of inorganic nitrogen while immobilizing carbon dioxide ( He et al., 2016 ). These data indicate that the C/N ratio has a significant effect on the spatial distribution of the dominant bacteria AOB. The results further showed that a low C/N ratio is more suitable for the growth of Candidatus Nitrosotenuis (belongs to AOA), whereas a high C/N ratio is beneficial to the growth of Nitrosomonas (belongs to AOB). It is likely that Nitrosomonas can better adapt to a highly polluted environment than Candidatus Nitrosotenuis , as has been described by a previous study ( Fan et al., 2011 ). FIGURE 5 Difference in distribution of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region in different samples in the two devices. Data were obtained based on the phylogenetic lineages using the weighted Fast UniFrac PCoA. The red ellipse indicates that different samples with similar microbial community structure are clustered together. FIGURE 6 Bar plot and cluster tree of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region. As shown in Figure 6C , the relative abundance of Nitrobacter in each sample was the highest (from 86.95 to 99.99%). This is consistent with the previous study, which has described that Nitrobacter is the single abundant genus in NOB ( Zhang et al., 2019 ). However, it is opposite to another study using laboratory-scale devices, which has reported that Nitrospira is the predominant genus in NOB ( Sayess et al., 2013 ). The difference is likely due to that Nitrospira can better adapt to low nitrite and oxygen concentrations, and Nitrobacter can outcompete Nitrospira at high substrate concentration ( Maixner et al., 2006 ). The genera in the V4−V5 region was more abundant than those in other microorganisms, and the dominant microorganisms included Phormidium , Planctomyces , Gemmata , and Aquicella ( Figure 6D ). Trichococcus and Rhodopseudomonas preferred a high C/N ratio, while Planctomyces and Gemmata , which are anammox bacteria ( Xu et al., 2018 ), were less affected by the C/N ratio. Although the discovery of anammox genera, the role of NH 4 + -N removal via anaerobic ammonium oxidation in TFCW-MFC was negligible in the presence of relatively high DO throughout the tidal flow. Correlation Between Single or Multiple Environmental Variables and Microbial Community Structure The effects of environmental variables, including nitrogen concentration, pH, DO, temperature, and voltage, on microbial community structure were analyzed using VPA ( Supplementary Figure S3 ) and RDA ( Figure 7 ). According to the VPA diagram ( Supplementary Figure S3 ), the following results were observed; (1) AOA community structure was explained by the general chemical indexes (pH and DO, 29%), and temperature (21%); (2) AOB community structure was explained by the nitrogen concentration (12%), and the shared fraction between pH, DO, and nitrogen concentration accounted for 16%; (3) NOB community structure was explained by the shared fraction between pH, DO, and nitrogen concentration (37%), and the shared fraction between nitrogen concentration and temperature accounted for 33%; and (4) the community structure of V4−V5 region was explained by nitrogen concentration (14%), and the shared fraction between pH, DO, and nitrogen concentration accounted for 12%. These data were subjected to the RAD analysis to determine the correlation between specific environmental factors and the community structure of the four types of microorganisms under the influence of different C/N ratios. FIGURE 7 RDA analysis curves of panels AOA (A) , AOB (B) , NOB (C) , and 16S rRNA V4-V5 region (D) . Symbols indicate the samples and arrows represent environmental variables. Group A represents Device A operated with a C/N ratio = 10:1, and Group B represents Device B operated with a C/N ratio = 5:1. Environmental variables were chosen based on the significance determined from individual RDA results. The RDA results indicated that the residual concentration of NO 2 – -N is positively correlated with AOA, AOB, NOB, and microorganisms in the V4−V5 region in the lower layer of the two devices. Additionally, the residual concentration of TN and NH 4 + -N, and temperature were positively correlated with AOB, NOB, and the microorganisms in the V4−V5 region of Device A, but not with AOA. Moreover, the pH, DO, and residual concentration of NO 3 – -N were positively correlated with AOA, AOB, and NOB in the upper layer of the two devices at day 90. This is consistent with the results reported by Zhou et al. (2016) , who have observed that multiple physicochemical parameters are involved in the relative abundance of nitrifying microorganisms grown in the same environment. Combined with the VPA results, we may conclude that the ecological niche of TFCW-MFC device is mainly influenced by pH, which serves as the crucial factor that changes the diversities and compositions of all four microbial communities. It appears that each microbial species has its own optimal survival environment; for example, the optimal conditions for Nitrosomonas (belong to AOB) are 35°C and pH 8.1 and those for Nitrobacter (belongs to NOB) are 38°C and pH 7.9 ( Grunditz and Dalhammar, 2011 ). At day 90, the effluent pH and temperature in Device A were 7.47 and 23.5°C, respectively, while those in Device B were 7.34 and 23.1°C, respectively. The second most influential factor is DO, which plays an important role as an electron acceptor for nitrifying microorganisms; thus, it can determine microbial growth and reproductive rate ( Yuan and Blackall, 2002 ). DO is also a key factor affecting the positive correlation between NO 3 – -N and microorganisms in the upper layer, and NO 2 – -N and microorganisms in the lower layer. AOB population size has been reported to increase with increasing soil NH 4 + -N concentration ( Okano et al., 2004 ), and AOB and NOB growths rely on the energy provided by NH 4 + -N and NO 2 – -N ( Winkler et al., 2012 ). However, the amount of AOA is not correlated with ammonia concentration ( Di et al., 2009 ), therefore the effects of NH 4 + -N concentration on AOA, AOB and NOB are different. By considering the effects of voltage on microbial community, voltage had a greater impact on the V4−V5 region microorganisms than AOA, AOB, and NOB; it was also positively correlated with the V4−V5 region microorganisms in Device A only at day 90 and in Device B only at day 30. The V4−V5 region comprises many exoelectrogenic microorganisms ( Corbella et al., 2015 ); it is likely that electrons are largely depleted due to the relatively high concentrations of electron acceptors such as NO 2 – -N, NO 3 – -N, oxygen, etc., Wang et al. (2016) . Pearson Correlation Between Microbial Characteristic Parameters, Nitrogen Removal, and Voltage Output To further study the effect of microorganisms on nitrogen removal and bioelectricity generation, the relationship between alpha diversity index, voltage output, and effluent nitrogen concentration was analyzed by the Pearson correlation. As shown in Table 3 , TN concentration was significantly negatively correlated with V4−V5-chao and AOA-shannon with R -values of −0.646 and −0.693, respectively ( P < 0.01), as well as with AOA-chao and V4−V5-shannon with R -values of −0.608 and −0.596, respectively ( P < 0.05). This is mainly due to the dominant genera of AOA and V4−V5 region. The Pearson correlation analysis was employed to further clarify the correlation between the dominant microorganisms and nitrogen removal. The analysis was performed using the high-abundance microorganism only the genus with >10% of the total genus composition), and the effluent nitrogen concentrations from the devices using each of these microorganisms were then compared. As shown in Table 4 , TN concentration was significantly negatively correlated with Nitrosopumilus (belongs to AOA) and Aquicella (belongs to V4−V5 region) with R -values of −0.529 and −0.572, respectively ( P < 0.05), as well as with Planctomyces (V4−V5 region) with R -value of −0.666 ( P < 0.01). Nitrosopumilus is a dominant genus of AOA, and a low C/N ratio could significantly promote its relative abundance in all layers during the stabilization period. Therefore, Nitrosopumilus , which was especially rich in anode and cathode layers, could accelerate the nitrification reaction and greatly promote the conversion of nitrogen. Planctomyces (V4−V5 region) belongs to the anammox bacteria that can use NH 4 + -N as an electron donor and NO 3 – -N/NO 2 – -N as an electron acceptor to convert nitrogen compounds in water into nitrogen. The correlation between the alpha diversity of each genus and NH 4 + -N is similar to that between the alpha diversity of genus and TN. Concentration of NH 4 + -N was significantly negatively correlated with V4−V5-chao, V4−V5-shannon, AOA-chao, and AOA-shannon with R = −0.826, −0.744, −0.688, and −0.746, respectively ( P < 0.01), as well as with NOB-chao with R = −0.543 ( P < 0.05). By contrast, it was significantly positively correlated with TN concentration with R = 0.828 ( P < 0.01). The correlation between the genus and nitrogen removal ( Table 4 ) further indicated that NH 4 + -N concentration was significantly negatively correlated with Candidatus Nitrosotenuis (belongs AOA), Nitrobacter (belongs NOB), and Planctomyces (belongs to V4−V5 region microorganisms) with R -values of −0.617 ( P < 0.05), −0.648 ( P < 0.01), and −0.819 ( P < 0.01), respectively. Candidatus Nitrosotenuis was the dominant genus of AOA, and a low C/N ratio could significantly promote its relative abundance in the cathode layer during the stabilization period; thus, this genus is more conducive to the conversion of NH 4 + -N. Furthermore, as the most abundant genus of NOB, Nitrobacter is suitable for NH 4 + -N removal in wastewater, which has also been confirmed by our previous study ( Zhang et al., 2019 ). TABLE 3 Pearson correlation between alpha diversity at the genus level, voltage, and effluent nitrogen concentration ( n = 16). AOA−chao AOB−chao NOB-chao V4−V5-chao AOA-shannon AOB-shannon NOB-shannon V4−V5-shannon TN NH 4 + -N NO 3 – -N NO 2 – -N AOB-chao −0.154 a NOB-chao −0.316 0.682** V4-V5-chao 0.839** −0.371 −0.379 AOA-shannon 0.872** −0.297 −0.433 0.821** AOB-shannon −0.150 0.812** 0.522* −0.333 −0.274 NOB-shannon −0.293 0.146 0.383 −0.173 −0.264 0.117 V4-V5-shannon 0.721** −0.325 −0.341 0.927** 0.747** −0.264 −0.185 TN − 0.608* 0.477 0.460 − 0.646** − 0.693** 0.220 0.175 − 0.596* NH 4 + -N − 0.688** −0.413 − 0.543* − 0.826** − 0.746** 0.296 0.096 − 0.744** 0.828** NO 3 – -N 0.265 −0.232 0.233 0.392 0.234 −0.123 0.457 0.303 −0.169 − 0.552* NO 2 – -N −0.122 0.213 −0.188 −0.181 −0.005 0.542* −0.424 −0.212 −0.179 0.013 − 0.550* Voltage 0.740 −0.402 −0.639 0.825 0.477 −0.085 −0.244 0.758 −0.819 −0.775 0.951* 0.757 a coefficient of association ( R ); * significant correlation ( P < 0.05); ** significant correlation ( P < 0.01). Bold values mean significance correlation including p < 0.05 and p < 0.01. TABLE 4 Pearson correlation between dominant genera of each of the four types of microorganisms, voltage, and effluent nitrogen concentration ( n = 16). Candidatus_ Nitrosotenuis Nitro sopumilus Nitro sosphaera Nitro somonas Nitro sospira Alterery throbacter Nitro bacter Phor midium Planc tomyces Gem mata Aqui cella TN NH 4 + -N NO 3 – -N NO 2 – -N Nitrosopumilus 0.123 a 1 Nitrososphaera 0.367 0.032 1 Candidatus _ Nitrosopelagicus 0.791** 0.026 0.233 Nitrosomonas −0.273 −0.008 −0.017 1 Nitrosospira 0.520* −0.034 −0.185 −0.063 1 Altererythrobacter 0.008 0.938** −0.040 −0.025 −0.014 1 Nitrobacter 0.368 0.306 0.236 0.029 0.331 0.219 1 Bradyrhizobium −0.387 0.198 −0.310 −0.231 −0.262 0.212 −0.115 Phormidium 0.442 −0.158 0.423 0.122 0.559* −0.174 0.219 1 Planctomyces 0.552* 0.300 0.293 −0.308 0.233 0.218 0.399 −0.139 1 Sphingomonas 0.478 0.075 −0.066 −0.378 0.316 0.068 0.230 −0.316 0.629** Gemmata 0.370 −0.273 0.212 −0.047 0.117 −0.290 0.047 −0.176 0.737** 1 Aquicella 0.164 0.727** 0.221 0.289 0.118 0.653** 0.313 0.152 0.289 −0.100 1 TN −0.439 − 0.529* −0.177 0.186 −0.179 −0.422 −0.424 0.220 − 0.666** −0.234 − 0.572* 1 NH 4 + -N − 0.617* −0.353 −0.415 0.263 −0.401 −0.287 − 0.648** −0.109 − 0.819** −0.443 −0.446 0.828** 1 NO 3 – -N 0.527* −0.292 0.566* −0.018 0.418 −0.263 0.200 0.436 0.463 0.614* −0.040 −0.169 − 0.552* 1 NO 2 – -N −0.340 0.525* −0.101 0.215 −0.103 0.579* 0.131 0.028 −0.384 − 0.698** 0.575* −0.179 0.013 −0.450 1 Voltage 0.900 0.983* 0.464 −0.326 0.963* 0.963* 0.439 −0.064 0.854 − 0.999** 0.962* −0.819 −0.775 0.951* 0.757 a coefficient of association ( R ); * significant correlation ( P < 0.05); ** significant correlation ( P < 0.01). The selected genus accounts for >10% of the total composition of each gene. Bold values mean significance correlation including p < 0.05 and p < 0.01. The effluent concentration of NO 2 – -N was significantly positively correlated with AOB-shannon with R = 0.542 ( P < 0.05), while may be related with the dominant genus Altererythrobacter ( R = 0.579; P < 0.05). Additionally, NO 2 – -N concentration was also significantly positively correlated with Nitrosopumilus and Aquicella with R -values of 0.525 and 0.575, respectively ( P < 0.05), while was negatively correlated with Gemmata with R -value of −0.698 ( P < 0.01). NO 3 – -N concentration was significantly positively correlated with Candidatus Nitrosotenuis , Nitrososphaera , and Gemmata with R = 0.527, 0.566, and 0.614, respectively ( P < 0.05). This demonstrated that the abundance of ammonia oxidizing bacteria increases with increasing effluent NO 2 – -N and NO 3 – -N concentrations. Gemmata belongs to anammox bacteria, which can convert ammonia nitrogen using NO 2 – -N as an electron acceptor; thus a negative correlation between NO 2 – -N and Gemmata was observed. Furthermore, voltage was significantly positively correlated with the effluent NO 3 – -N concentration, Nitrosopumilus , Nitrosospira , Altererythrobacter and Aquicella with R = 0.951, 0.983, 0.963, 0.963, and 0.962 ( P < 0.05), respectively, while was significantly negatively correlated with Gemmata ( R = −0.999; P < 0.01). These data indicate the above genera and the electrogenic bacteria in the system may work synergistically so that the effect of the electrogenic bacteria is promoted, causing the voltage output to increase. On the other hand, organic matter can be oxidized by electrogens at the anode of MFC, and the produced electrons can flow to the cathode by an external circuit ( Puig et al., 2012 ). Consequently, NH 4 + -N loses its electrons and is converted to NO 2 – -N and NO 3 – -N by the action of microorganisms, as indicated by a higher abundance and diversity of the microorganism at the cathode and anode layers. Gemmata , which belongs to the phylum Planctomycetes , contains fibrillar nucleoid surrounded by electron-dense granules ( Fuerst and Richard, 1991 ) that may have an adverse effect on the electrogenic microorganism in the system. In spite of these data, the distribution characteristics of the electrogenic microorganisms and their contribution to the electricity production have not been analyzed. Thus, studies on the topics should be further conducted."
} | 9,841 |
30926658 | PMC6475390 | pmc | 2,407 | {
"abstract": "Significance Despite great success of deep learning a question remains to what extent the computational properties of deep neural networks are similar to those of the human brain. The particularly nonbiological aspect of deep learning is the supervised training process with the backpropagation algorithm, which requires massive amounts of labeled data, and a nonlocal learning rule for changing the synapse strengths. This paper describes a learning algorithm that does not suffer from these two problems. It learns the weights of the lower layer of neural networks in a completely unsupervised fashion. The entire algorithm utilizes local learning rules which have conceptual biological plausibility.",
"discussion": "Discussion and Conclusions Historically, neurobiology has inspired much research on using various plasticity rules to learn useful representations from the data. This line of research chiefly disappeared after 2014 because of the success of deep neural networks trained with backpropagation on complicated tasks like ImageNet. This has led to the opinion that neurobiology-inspired plasticity rules are computationally inferior to networks trained end-to-end and that supervision is crucial for learning useful early layer representations from the data. By consequence, the amount of attention given to exploring the diversity of possible biologically inspired learning rules, in the present era of large datasets and fast computers, has been rather limited. Our paper challenges this opinion by describing an unsupervised learning algorithm that demonstrates a very good performance on MNIST and CIFAR-10. The core of the algorithm is a local learning rule that incorporates both LTP and LTD types of plasticity and a network motif with global inhibition in the hidden layer. The SGD training of the top layer was used in this paper to assess the quality of the representations learned by the unsupervised phase of our algorithm. This does not invalidate the biological plausibility of the entire algorithm, since SGD in one layer can be written as a local synaptic plasticity rule involving only pre- and postsynaptic cell activities. Thus, it complies with the locality requirement that we took as fundamental. In the present paper all of the experiments were done on a network with one hidden layer. The proposed unsupervised algorithm, however, is iterative in nature. This means that after a one-layer representation is learned, it can be used to generate the codes for the input images. These codes can be used to train another layer of weights using exactly the same unsupervised algorithm. There are many possibilities of how one could organize those additional layers, since they do not have to be fully connected. This line of research requires further investigation. At this point it is unclear whether or not the proposed algorithm can lead to improvements on the single hidden-layer network, if applied iteratively in deeper architectures. Another limitation of the present work is that all of the experiments were done on MNIST and CIFAR-10 and only in the fully connected setting. Many biologically plausible approaches to deep learning fail on more complicated datasets, like ImageNet, even if they work well on MNIST and CIFAR-10 ( 39 ). Finally, this paper does not claim that end-to-end training is incompatible with biological plausibility. There is a large body of literature dedicated to designing biologically plausible variants of end-to-end training approximating backpropagation ( 4 – 8 , 10 – 12 ). The main difference between those approaches and the present proposal is that the algorithms of refs. 4 – 8 and 10 – 12 require the top–down propagation of information and craft the synaptic weights in the early layers of neural networks to solve some specific task (supervised or unsupervised). In our algorithm there is no top–down propagation of information, the synaptic weights are learned using only bottom–up signals, and the algorithm is agnostic about the task that the network will have to solve eventually in the top layer. Despite this lack of knowledge about the task, the algorithm finds a useful set of weights that leads to a good generalization performance on the standard classification task, at least on simple datasets like MNIST and CIFAR-10."
} | 1,073 |
29844575 | PMC5974136 | pmc | 2,408 | {
"abstract": "The β-sheet is the key structure underlying the excellent mechanical properties of spider silk. However, the comprehensive mechanism underlying β-sheet formation from soluble silk proteins during the transition into insoluble stable fibers has not been elucidated. Notably, the assembly of repetitive domains that dominate the length of the protein chains and structural features within the spun fibers has not been clarified. Here we determine the conformation and dynamics of the soluble precursor of the repetitive domain of spider silk using solution-state NMR, far-UV circular dichroism and vibrational circular dichroism. The soluble repetitive domain contains two major populations: ~65% random coil and ~24% polyproline type II helix (PPII helix). The PPII helix conformation in the glycine-rich region is proposed as a soluble prefibrillar region that subsequently undergoes intramolecular interactions. These findings unravel the mechanism underlying the initial step of β-sheet formation, which is an extremely rapid process during spider silk assembly.",
"introduction": "Introduction Spider silk has attracted great interest because of its superior mechanical properties and potential for industrial and biomedical applications 1 . Spider dragline silks consist of high-molecular-weight proteins (250–350 kDa) that contain conserved and relatively small N-terminal domains (NTDs) and C-terminal domains (CTDs) (16.5 and 10.5 kDa, respectively), as well as a long repetitive domain 2 , 3 . The molecular structure of silk fibers has been characterized using solid-state NMR and X-ray diffraction 4 – 7 . Consensus motifs identified in the repetitive domain are mainly composed of polyalanine stretches (4–12 alanine residues), which form β-sheets in a crystalline region, and the glycine-rich region (GGX), which exhibits 3 1 -helix structures in an amorphous region 4 – 6 , 8 , 9 . Prior to being spun into insoluble fibers, the precursors of dragline silk are stored as a highly concentrated protein solution in the major ampullate gland. Several studies have shown that the NTD and CTD display strong pH dependence in terms of conformation and are essential for controlling the pH-dependent assembly of silk proteins 10 – 13 . The presence of the highly conserved CTD of dragline silk is also important for directing fiber formation in vivo 14 . Although, the conserved NTD and CTD play important roles in spider silk protein self-assembly, the conformational details and role of the long repetitive domain in the soluble form are less well defined and are the focus of the present study for several important reasons. First, several studies have reported that in the absence of the CTD and NTD, a long repetitive domain of spidroin is still able to form fibers 15 – 17 , and number of the repetitive domains are proportional to the strength of the recombinant silk fibers 15 , 16 . Second, although several studies have indicated that the CTD and NTD are important for maintaining the solubility of spider silk in the major ampullate gland, the size of the CTD and NTD is much smaller than that of the repetitive domain. Therefore, the high solubility of spider silk in the ampullate gland and the fiber-forming processes likely do not depend solely on the CTD and NTD. Furthermore, the dominating role of the repetitive domain in terms of the size and fiber functions suggests that additional insights are needed in these regions of silk proteins. Based on these points, we hypothesized that the conformation of the soluble repetitive domain plays a significant role in fiber formation and determines the high solubility of spider silk. Another important point is that the conformation of the soluble precursor of spidroin (spider silk protein), particularly for the repetitive domain, remains unclear. Vibrational circular dichroism (VCD) spectroscopy data revealed the presence of PPII helix and random coil structures in soluble dragline silks 18 ; whereas, NMR data of 13 C-labeled spidroin from spider glands indicated that these proteins only consist of random coil conformations, which are highly dynamic 9 , 19 , 20 . However, the mechanism underlying the structural transition from disordered conformations to stable spider silk fibers with a β-sheet structure remains unclear because this transformation might lead to nonspecific aggregation. Therefore, we address a fundamental and specific question: is the repetitive domain fully a random coil or does it possess a certain regularity in structure in the soluble form that might explain the mechanism of transformation of soluble spider silk to insoluble silk fibers? Although, the NTD and CTD display pH-dependent conformations, the pH-gradient effect in the gland on the repetitive domain of spider silk proteins remains unknown. The importance of the pH effect on the repetitive domain was highlighted in a recent report, which demonstrated the importance of dityrosine formation in the repetitive domain of silkworm silk to promote β-sheet formation 21 . The formation of dityrosine in the repetitive domain was catalyzed by the presence of peroxidase 21 , in which the activity of peroxidase is optimal at a slightly acidic pH (pH 6.0–6.5) 22 . Thus, investigations of the pH effect on the conformation of the repetitive domain might be useful for identifying the comprehensive mechanism of β-sheet formation of spider silk, which until now has remained poorly understood. In this study, we investigated the molecular conformation and dynamics of different numbers of repetitive domains of dragline silk at neutral pH using solution-state NMR, far-UV circular dichroism (CD) and VCD spectroscopies. Our results demonstrate that the repetitive domain of dragline silk consists of two major populations: random coil (~65%) and PPII helix (~24%). We propose that the PPII helix population in the glycine-rich region might serve as the prefibrillar form of spider dragline silk and may contribute to the efficiency of the spinning process because the PPII helix can easily undergo intramolecular interactions in response to shear forces and dehydration by forming a reverse turn. Additionally, the limited flexibility of the glycine-rich region might function to maintain the solubility of spider silk in the ampullate gland. pH titration studies of the repetitive domain at acidic pH values (from 7 down to 2.6 to emulate events in the gland in vivo) show no conformational change, suggesting that the pH changes in the spinning gland does not affect this prefibrillar form. These findings provide new insights into the initiation step of rapid β-sheet formation in spider silk proteins.",
"discussion": "Discussion In this study, we investigated the conformations and dynamics of the soluble repetitive domains of N. clavipes in the absence of the terminal domains. Although, the mechanical properties of spider dragline silk are diminished in the absence of the terminal domains 16 , 41 , previous studies reported that individual domains of spider silk function independently, and no stable interactions were observed between these domains 42 , 43 . Therefore, we consider that our study on conformations and dynamics of repetitive domains in the absence of terminal domains might be highly relevant to the conformations and dynamics of the repetitive domains when considered in the presence of terminal domains. The structural propensity of the polyalanine region of the repetitive domain is similar to that of the random coil; although, the negative structural propensity score in this region suggests that a β-sheet precursor already occurred in the soluble form. A similar finding was reported for the soluble β-sheet precursor of the monomer and dimer of Aβ40 44 . Intriguingly, the soluble β-sheet precursor in the polyalanine region of spidroin is similar to the β-sheet region of spider silk fiber as demonstrated by solid-state NMR data 4 – 6 , 45 , but the conformation in this region is different from the one found in the β-sheet region (GAGAGS) in the silkworm silk of Bombyx mori , which forms a repeated β-turn type II structure prior to spinning 46 . Our finding suggest that PPII helix conformation in the glycine-rich region is retained before and after the spinning process of spider dragline silk 4 – 6 , 9 . This finding is also consistent with a previous study demonstrating that the PPII helix conformation also carried –GGX– and –GPG– motifs in Argiope trifasciata flagelliform silk 47 . The PPII helix, which is also recognized as a 3 1 helix or polyglycine II 48 , 49 , is markedly more flexible than the α-helix and β-sheets 50 , although this conformation is still less dynamic than the random coil conformation. Therefore, the presence of the PPII helix in the glycine-rich region contributes to more limited flexibility of the 15-mer than of the monomer. The presence of the PPII helix population in the glycine-rich region is reflected by more limited flexibility in this region than in the polyalanine region, even at a temperature of 15 °C. The PPII helix conformation is important because the geometry of this conformation allows the protein chain to progress directly to form a reverse turn 51 . Previously, solid-state NMR indicated that the conserved LG(G/S)QG motif in the glycine-rich region (Supplementary Note 1 ) adopts a turn conformation in MaSp1 fibers 52 . Such findings are supported by our results, which showed that the structural propensity (Supplementary Fig. 3 ) and backbone carbonyl chemical shifts in the GLGSQGTS region differ from those of the monomer and other repetitive domains (Supplementary Fig. 5 and 6 ). These findings suggest that the chemical environment of this motif in multiple repetitive domains with at least 2 repeat units (dimer, trimer, hexamer, and 15-mer), which are able to undergo intramolecular interactions, differs from that in the monomer. Our data also demonstrate that when the number of repetitive domains is increased, the helical propensity of the GLGSQGTS region also increases, suggesting that the PPII helix population is stabilized by the greater number of the repetitive domains (Supplementary Fig. 3 ). In short repeat lengths, such as the dimer and trimer, the PPII helix population is present but less stable. Therefore, the CD spectra of the dimer and trimer did not display detectable positive maxima at 215 nm, which is a clear indication of a PPII helix population. Furthermore, PPII helix stabilization with a larger number of repetitive domains enables spidroin to readily form intramolecular interactions and explains why stronger spider silk fibers can be achieved when the silk protein contains high numbers of repetitive domains 15 . The propensity of PPII helix conformation in native spider dragline silk had been reported using VCD spectroscopy 18 , which is consistent with our far-UV CD and VCD data. This previous study using VCD spectroscopy provided the overall conformation, while the conformation and dynamics of the repetitive domain as a function of amino acid sequence remained unclear. In this study, using solution state NMR spectroscopy, we demonstrated the local conformations and dynamics of the repetitive domain as the function of amino acid sequence. Our data clearly demonstrated that the PPII helix population was distributed over the glycine-rich region; whereas, the random coil population was distributed over the polyalanine region. This finding is consistent with the dynamics data, namely, the glycine-rich region has more limited flexibility compared with the polyalanine region. Despite the PPII helix population of the 15-mer represents only 25% of the total secondary structure population in the glycine-rich region, the presence of PPII helix populations in the soluble repetitive domains plays an important role in the spinning efficiency because this conformation readily undergoes intramolecular interactions. Here we propose that soluble spider silk occurs in equilibrium with two major populations (Schematic illustration in Fig. 5 ): random coils (~65%) and PPII helix (~24%). The soluble precursor of the repetitive domain containing the PPII helix serves as the prefibrillar form of the spider dragline silk. The PPII helix conformation is favorable for protein–protein interactions, such as protein complex assembly 50 . This soluble prefibrillar form of spider dragline silk might form irreversible β-sheet structures via PPII helix interactions in the glycine-rich region, which could potentially lead to the formation of silk fibers in response to a change in the biochemical environment, extensional flow, dehydration, and shear forces. The PPII helix interaction in the glycine-rich region is possibly mediated by hydrogen bonds between alpha proton and carbonyl oxygen, similar to the triple helix structure of collagen 29 . In the case of Gln in the glycine-rich region, PPII helix interactions are possibly mediated by hydrogen bonds between Gln side chain and carbonyl oxygen of the neighboring residue 53 . The PPII helix conformations in the glycine-rich regions between repeat domains enables the ultra-rapid conversion into aligned protein polymers during the silk spinning process, as well as pre-ordering of the soluble spidroins, which helps prevent premature aggregation, even at extremely high concentrations. Fig. 5 Proposed mechanism of β-sheet formation of spider dragline silk. a Spider major ampullate gland displays a strong pH gradient (reproduced with permission 70 , Copyright 2017, American Chemical Society). b Two major structural populations of spider dragline silk proteins, random coil, and PPII helix in the glycine-rich region, occur in soluble form. The presence of the PPII helix in the glycine-rich region is proposed as a soluble prefibrillar form of spider dragline silk. The polyalanine region is shown in red, and the glycine-rich region is shown in black. Schematic illustration of PPII helix interaction in the glycine-rich region is shown in the box. In response to dehydration, shearing forces, extensional flow and changes in the biochemical environment, this prefibrillar form will generate spider dragline fibers through strong β-sheet interactions Interestingly, conformation and dynamics of the recombinant repetitive domain of N. clavipes in this study are different from those of the recombinant repetitive domain of Euprosthenops australis in the presence of terminal domains 42 . These differences are possibly due to the length of the polyalanine region. The long polyalanine region (14–15 alanine residues) of the repetitive domain of E. australis shows helical structure, while the glycine-rich region has random coil conformation. As shown in the current study, a shorter polyalanine region (5 alanine residues) in N. clavipes repetitive domain leads to random coil conformation. Our results are in agreement with previous study, which revealed that the conformation of polyalanine region (6–8 alanine residues) of native Lactrodectus hesperus dragline silk was random coil 54 . In contrast, the longer polyalanine region in E. australis repetitive domain leads to the formation of helical structure 55 . The helical structure of the polyalanine in E. australis is also similar to the helical structure of a long polyalanine region (10–14 alanine residues) in Samia cynthia ricini 56 . In the case of E. australis spidroins, hydrophobicity of the repetitive domain and hydrophilicity of the C-terminal domain cause to form micelle-like structure 42 . Together, these studies suggest that the soluble form of shorter polyalanine region (4–8 alanine residues) of the repetitive domain tends to form random coil, while longer polyalanine regions (>10 alanine residues) tend to form helical structure. Furthermore, the recombinant repetitive domain of E. australis is easily aggregated even at low concentration (1 g L −1 ) 55 ; whereas, the recombinant N. clavipes repetitive domain is soluble at relatively high concentration (100 g L −1 ). The difference in solubility of the repetitive domains can be explained based on the dynamic behavior of the glycine-rich region. The limited flexibility of the glycine-rich region of the repetitive domain from N. clavipes seems to be essential for maintaining high solubility of spider silk protein by preventing premature aggregation. On the other hand, the high flexibility of the glycine-rich region of E. australis repetitive domain leads this domain to be easily aggregated 55 . Our investigation of the acidic pH effect revealed no conformational changes in the repetitive domains, suggesting that pH changes in the spinning gland do not influence the prefibrillar region of the repetitive domain. The conformational change in a protein as a function of pH is normally accompanied by protonation and deprotonation of acid and basic groups. Since no acidic residue is available in the repetitive domain, no conformational changes were observed at acidic pH. However, in vitro studies of native and recombinant spider dragline silk composed of NTD, repetitive domains and CTD demonstrated β-sheet formation at an acidic pH 54 , 57 , 58 . Thus, based on the results of the present and previous studies, we concluded that CTD and NTD might be necessary for inducing β-sheet formation in spider silk during the early stage of the spinning process. Furthermore, dehydration, shear forces, and changes in biochemical environment are also involved in the β-sheet formation of spider silk proteins. Previously, volumetric, and spectrophotometric titration studies of native spidroin ( Nephila edulis ) demonstrated two apparent p K values: 6.74 and 9.21 59 . The first p K value was hypothesized to originate from the deprotonation of different residues; whereas, the latter p K value was thought to be related to the deprotonation of Tyr side chain residues 59 . In addition, deprotonation of the Tyr side chain has been thought to be involved in the solubility of spider dragline silk at pH values >8.5 60 . In this study, since the p K a value of the Tyr side chain of the repetitive domain is ~10.3, this indicates that deprotonation of the Tyr side chain was not essential for increasing the solubility of spider dragline silk because the p K a value of the Tyr side chain at pH 10.3 is too basic for the ampullate gland. In conclusion, the soluble repetitive domains of spider dragline silk at neutral pH have two major populations: PPII helix (~24%) and random coils (~65%). The PPII helix conformation is more prevalent in the glycine-rich regions, which present less flexibility than the polyalanine region, which contains more random coils. The glycine-rich region with the PPII helix population is proposed as the soluble prefibrillar form, which readily supports the transformation into insoluble silk fiber. Our study also demonstrated that the molecular structure of the repetitive domain is not affected by pH, indicating that the prefibrillar form of the repetitive domain is not influenced by pH. This study provides a better understanding of the initial mechanism of spider silk protein self-assembly and the high solubility of spidroin in the major ampullate gland. Future designs to produce strong artificial spider silk should consider a combination of a large number of repetitive and non-repetitive domains (CTD and NTD) of spidroin as well as a combination of soft (glycine-rich region) and hard (polyalanine region) segments in the repetitive domains to form strong and desirable intramolecular and intermolecular interactions."
} | 4,897 |
31872380 | PMC6928187 | pmc | 2,409 | {
"abstract": "Hydrogen gas represents a promising alternative energy source to dwindling fossil fuel reserves, as it carries the highest energy per unit mass and its combustion results in the release of water vapour as only byproduct. The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen via the water–gas shift reaction catalyzed by a carbon monoxide dehydrogenase–hydrogenase enzyme complex. Here we have evaluated the effects of several operating parameters on hydrogen production, including different growth temperatures, pre-culture ages and inoculum sizes, as well as different pHs and concentrations of nickel and iron in the fermentation medium. All of the tested parameters were observed to have a substantive effect on both hydrogen yield and (specific) production rates. A final experiment incorporating the best scenario for each tested parameter showed a marked increase in the H 2 production rate compared to each individual parameter. The optimised parameters serve as a strong basis for improved hydrogen production with a view of commercialisation of this process.",
"introduction": "Introduction Hydrogen (H 2 ) gas is a critical component of diverse industrial applications including the synthesis of ammonia, methanol production and petroleum processing (Ramachandran and Menon 1998 ). Furthermore, H 2 is an efficient energy carrier as, compared to fossil fuel, it has higher energy per unit mass and its combustion produces zero toxic emissions (CO 2 , SO 2 and NOx). Consequently, H 2 has been projected as a formidable energy alternative to dwindling fossil fuel reserves and has become an important component of global energy dynamics (Nikolaidis and Poullikkas 2017 ). Currently, large-scale H 2 production is performed via several mechanisms, including natural gas reformation, where carbon atoms from methane separate when exposed to steam and heat, resulting in the release of H 2 and carbon monoxide (CO) (Sørensen and Spazzafumo 2011 ). Other commonly applied approaches include gasification of coal (to H 2 and CO) and electrolysis of water (to H 2 and O 2 ). However, these methods are costly, often use fossil fuels and have harmful environmental effects (Nikolaidis and Poullikkas 2017 ). As such, several biological strategies for hydrogen production have been explored including photofermentation by photosynthetic bacteria, bio-photolysis of water by algae and dark fermentation of organic substances by anaerobic microorganisms (Sokolova et al. 2009 ). Recently, there has been increased interest in microorganisms that produce H 2 via the water–gas shift reaction (WGS): CO + H 2 O → CO 2 + H 2 (Diender et al. 2015 ; Mohr et al. 2018a ). The WGS reaction couples the oxidation of CO with the splitting of a water molecule to yield CO 2 and H 2 gas (Tirado-Acevedo et al. 2010 ). This is particularly pertinent as these microorganisms can use syngas, a natural product of steam reformation of natural gas and gasification of coal and municipal waste, which primarily consists of CO, CO 2 and H 2 (Rostrup-Nielsen 1993 ). The thermophilic bacterium P. thermoglucosidasius DSM 6285 produces H 2 via the WGS reaction using a carbon monoxide dehydrogenase—NiFe group 4a hydrogenase complex (Mohr et al. 2018b ). In contrast to anaerobic organisms, Parageobacillus thermoglucosidasius is a facultative anaerobe which tolerates high concentrations of both CO and O 2 , first growing aerobically until O 2 is depleted followed by the anaerobic WGS reaction. However, a lag phase was observed between O 2 depletion and commencement of H 2 production (Mohr et al. 2018a , b ). In the current study the effects of different process parameters on H 2 production were investigated in batch experiments. The optimized parameters will form the basis for further development of up-scale biological hydrogen production with P. thermoglucosidasius .",
"discussion": "Discussion A critical aspect of microbial fermentations that involve gas as the main substrate or e − acceptor is the solubility of the gas and the threshold concentration that does not inhibit the metabolism of the microorganisms (Bertsch and Müller 2015 ). In general, high gas concentrations can have an inhibitory effect while low gas concentrations can result in a low volumetric mass transfer coefficient resulting in limited substrate availability (Daniell et al. 2012 ; Mohammadi et al. 2014 ). This was evident in the fermentations with P. thermoglucosidasius DSM 6285 as less growth (biomass) was observed with increasing CO concentrations and concomitantly lower concentration of oxygen as terminal electron acceptor during the aerobic growth phase. However, poorer growth at higher CO concentrations did not have a negative effect on the hydrogenogenic capacity of P. thermoglucosidasius DSM 6285, with the highest H 2 production rate observed with the 75:25% CO:air mixture. The higher production rate with 75% CO, which grew to the lowest optical density, suggests that hydrogen productivity is a function of the availability of CO, rather than being dependent on the amount of biomass. To investigate the influence of the amount of biomass prior the hydrogen production phase, cultivations in bottles were undertaken using different inoculum sizes. The size and age of inocula can have substantial effects on hydrogen fermentations, as has been observed in the fermentative thermophile Thermoanaerobacterium thermosaccharolyticum and the photosynthetic purple non-sulphur bacterium Rhodobacter sphaeroides (Japaar et al. 2011 ; Seengenyoung et al. 2011 ). The highest production rate was detected with the 10% inoculum size, while the lowest production rate was achieved with the highest inoculum size (20%). Similar results were obtained with the fermentative H 2 -producer Bacillus coagulans IIT-BT S1, where higher H 2 production rates were observed with a 10% inoculum volume, but decreased with larger (15% and 20%) inoculum sizes (Kotay and Das 2007 ). As such, H 2 production appears not to be directly linked to the amount of biomass but may rather be a function of the physiological state of P. thermoglucosidasius . To confirm this hypothesis, different cultivation times (4 h, 12 h, 24 h) of the 2nd pre-culture were tested. Although the maximum production rate was detected at the same time points, H 2 production with the shortest incubation time of the 2nd pre-culture (4 h) showed the highest production rate. The 4 h pre-cultures may be in the lag growth phase preceding exponential growth (12–24 h), the preparative phase where bacteria adapt optimally to new environments (i.e., the exposure of P. thermoglucosidasius to CO) (Bertrand 2019 ). This pre-adaptive physiological state may explain the highest production rate observed with the 4 h pre-culture. Similarly, the lower H 2 production rates with the 20% inoculum size may be due to the cells reaching the post-lag exponential phase more rapidly than the optimal 10% inoculum size. Parageobacillus thermoglucosidasius strains grow optimally at temperatures of 61–63 °C and an initial medium pH of 6.5–8.5 (Suzuki et al. 1984 ). The strain utilized in this study, DSM 6285, is reported to grow optimally at 55 °C, with some growth at 75 °C (Gurujeyalakshmi and Oriel 1989 ). In the current study, a growth temperature of 55 °C and a medium pH of 7.0 resulted in optimal H 2 production. Although the highest H 2 production rate was obtained with the pH = 8.5 set up, the lag phase between oxygen consumption and the commencement of hydrogen production was substantially longer (24 h later than at pH 7.0). Nickel (Ni 2+ ) and iron (Fe 2+ ) are both essential co-factors in the catalytic sites of a broad range of enzymes (Waldron and Robinson 2009 ), and both the Ni–Fe CODH and Ni–Fe group 4a hydrogenase that catalyse the WGS are reported to contain both of these co-factors (Mohr et al. 2018a ). Thus, the addition of both of these elements to the P. thermoglucosidasius growth medium might be expected to have a positive effect on hydrogenogenesis. When doubling the amount of Fe 2+ (0.08 mM FeSO 4 ·7H 2 O) normally added to mLB medium, there was an evident decrease in the lag phase between oxygen consumption and hydrogen production and the maximum H 2 production rate was 8% higher than at lower concentrations. However, the addition of NiSO 4 ·6H 2 O had a negative impact on both the growth of P. thermoglucosidasius DSM 6285, the length of the pre-hydrogenogenic lag phase, H 2 yield and maximum H 2 production rate. A study of the effects of nickel on H 2 production by anaerobic sludge bacteria showed that increasing the nickel concentration from 0.0 mM up to 0.01 mM led to an increase of hydrogen production, while higher nickel concentration had a negative effect on hydrogen production (Wang and Wan 2008 ). Furthermore, the lag phase of hydrogen production could be decreased to 6 h by using 0.01 mM nickel (Wang and Wan 2008 ). As such, further fine-tuning of the amount of nickel added may be necessary for improved P. thermoglucosidasius hydrogenogenesis. The current study highlights that WGS catalyzed hydrogenogenesis in P. thermoglucosidasius is a finely balanced process with variations in all the tested operational parameters having either a positive or negative impact on H 2 yield, maximal (specific) production rates, as well as the time frame of the lag phase preceding hydrogenogenesis and the growth. The optima for each parameter combined in a further experiment resulted in higher production rate compared to set ups in which individual parameters were tested separately. This study can serve as a basis for up-scale fermentations. However, the effects of additional parameters such as the stirrer rate and flow rate of the feed gas inherent to up-scale fermentations will also need to be evaluated. Hydrogenogenesis via the WGS in P. thermoglucosidasius is a finely balanced process, which is influenced by key operational parameters. While some parameters such as temperature and initial medium pH reflect the optimum growth conditions for P. thermoglucosidasius others such as the age of the pre-culture and inoculum volume are more complex and may rather indicate the importance of the physiological state of P. thermoglucosidasius on its hydrogenogenic capacity. Further investigations, including gene expression analysis and metabolic profiling may shed light on additional factors influencing hydrogen production which, together with additional fine-tuning of operational parameters, can be used to develop up-scale fermentations with a continuous CO feed for commercial hydrogen production using the facultatively anaerobic thermophilic carboxydotroph P. thermoglucosidasius ."
} | 2,697 |
34487902 | null | s2 | 2,410 | {
"abstract": "Soil contamination with trace metal(loid) elements (TME) is a global concern. This has focused interest on TME-tolerant plants, some of which can hyperaccumulate extraordinary amounts of TME into above-ground tissues, for potential treatment of these soils. However, intra-species variability in TME hyperaccumulation is not yet sufficiently understood to fully harness this potential. Particularly, little is known about the rhizosphere microbial communities associated with hyperaccumulating plants and whether or not they facilitate TME uptake. The aim of this study is to characterize the diversity and structure of Arabidopsis halleri rhizosphere-influenced and background (i.e., non-Arabidopsis) soil microbial communities in four plant populations with contrasting Zn and Cd hyperaccumulation traits, two each from contaminated and uncontaminated sites. Microbial community properties were assessed along with geographic location, climate, abiotic soil properties, and plant parameters to explain variation in Zn and Cd hyperaccumulation. Site type (TME-contaminated vs. uncontaminated) and location explained 44% of bacterial/archaeal and 28% of fungal community variability. A linear discriminant effect size (LEfSe) analysis identified a greater number of taxa defining rhizosphere microbial communities than associated background soils. Further, in TME-contaminated soils, the number of rhizosphere-defining taxa was 6-fold greater than in the background soils. In contrast, the corresponding ratio for uncontaminated sites, was 3 and 1.6 for bacteria/archaea and fungi, respectively. The variables analyzed explained 71% and 76% of the variance in Zn and Cd hyperaccumulation, respectively; however, each hyperaccumulation pattern was associated with different variables. A. halleri rhizosphere fungal richness and diversity associated most strongly with Zn hyperaccumulation, whereas soil Cd and Zn bioavailability had the strongest associations with Cd hyperaccumulation. Our results indicate strong associations between A. halleri TME hyperaccumulation and rhizosphere microbial community properties, a finding that needs to be further explored to optimize phytoremediation technology that is based on hyperaccumulation."
} | 558 |
26455774 | null | s2 | 2,411 | {
"abstract": "Despite recent progress, the origin of the eukaryotic cell remains enigmatic. It is now known that the last eukaryotic common ancestor was complex and that endosymbiosis played a crucial role in eukaryogenesis at least via the acquisition of the alphaproteobacterial ancestor of mitochondria. However, the nature of the mitochondrial host is controversial, although the recent discovery of an archaeal lineage phylogenetically close to eukaryotes reinforces models proposing archaea-derived hosts. We argue that, in addition to improved phylogenomic analyses with more comprehensive taxon sampling to pinpoint the closest prokaryotic relatives of eukaryotes, determining plausible mechanisms and selective forces at the origin of key eukaryotic features, such as the nucleus or the bacterial-like eukaryotic membrane system, is essential to constrain existing models."
} | 216 |
29932636 | null | s2 | 2,414 | {
"abstract": "Tensan silk, a natural fiber produced by the Japanese oak silk moth ( Antherea yamamai, abbreviated to A. yamamai), features superior characteristics, such as compressive elasticity and chemical resistance, when compared to the more common silk produced from the domesticated silkworm, Bombyx mori ( B. mori). In this study, the \"structure-property\" relationships within A. yamamai silk are disclosed from the different structural hierarchies, confirming the outstanding toughness as dominated by the distinct mesoscale fibrillar architectures. Inspired by this hierarchical construction, we fabricated A. yamamai silk-like regenerated B. mori silk fibers (RBSFs) with mechanical properties (extensibility and modulus) comparable to natural A. yamamai silk. These RBSFs were further functionalized to form conductive RBSFs that were sensitive to force and temperature stimuli for applications in smart textiles. This study provides a blueprint in exploiting rational designs from A. yamanmai, which is rare and expensive in comparison to the common and cost-effective B. mori silk to empower enhanced material properties."
} | 280 |
24481660 | PMC4241050 | pmc | 2,415 | {
"abstract": "Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C 6 to C 16 are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel-like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli , supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high-yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high-yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain-length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain-lengths and functionalities.",
"conclusion": "Conclusion and Future Challenges Classical metabolic engineering, integrated computational/experimental approach, and synthetic biology have contributed towards the improved production of FAs in E. coli , and could be extended to the development of cell factories for specific chemical production. To further dissect the regulations in FA metabolism, system metabolic engineering can be employed to pinpoint beneficial key components in the complicated genetic circuit for strain optimization. Enzymatic bottlenecks could be accurately identified with the development of detailed kinetic models that include metabolic regulatory networks constrained by system biology findings. He et al. ( 2014 ) applied a combination of system biology approaches (i.e., fluxomics and transcriptomics) to gain metabolic insights into cellular metabolism under fatty acid production. It was found the reducing equivalent NADPH and ATP as the potential bottleneck for fatty acid production, guiding the direction for future strain development and process optimization to enhance fatty acid production (He et al., 2014 ). From the industrial standpoint, fermentation using minimal medium and efficient product separation processes can lower operating costs and potentially be competitive for the production of petroleum-based chemicals. Recently, a medium optimization study showed phosphate limitation in continuous fermentation increased fatty acid yield and biomass-specific productivity compared to carbon-limited cultivation (Youngquist et al., 2013 ). It has also been noted that endogenous FA production reduced cell viability due to the loss of inner membrane integrity (Lennen et al., 2011 ). Secretion of endogenous FAs could possibly assuage the toxicity effect while reducing product extraction costs. Further investigation is warranted to address the challenges for promising commercialization.",
"introduction": "Introduction Concerns regarding crude oil depletion and climate change have encouraged the development of renewable biochemicals and biofuels using carbohydrates as the feedstock (Demirbas, 2009 ; Gabrielle, 2008 ). Microbial biosynthesis of fatty acids (FAs) for biorenewable chemicals and biofuels has recently garnered extensive attention. Free FAs can be used as precursors for the production of alkanes by catalytic decarboxylation or transesterification (Lennen et al., 2010 ; Lu et al., 2008 ; Steen et al., 2010 ). Alternatively, FAs can be converted biologically to FA ethyl esters, which have high energy density and low water solubility (Steen et al., 2010 ). Medium chain FAs with 12–18 carbon chain lengths can be effectively used for industrial applications such as detergents, soaps, lubricants, cosmetics, and pharmaceuticals. FAs can also be catalytically deoxygenated via metal catalysts to produce α-olefins, the building blocks of polymerization. The genetically suitable Escherichia coli is an excellent host for FA production, given its fully sequenced genome and well-studied FA metabolism. Type II fatty acid biosynthesis (FAB) pathway in E. coli is illustrated in Figure 2 a, which is primed with acetyl-CoA and involves reiterative condensation of malonyl acyl carrier protein (ACP) resulting in two-carbon extension of the acyl chain during each elongation cycle. Despite the intrinsic capability of synthesizing FAs for lipid and cell membrane biosynthesis, E. coli does not normally accumulate free FAs as intermediates. FA metabolism is tightly regulated at transcriptional and post-transcriptional levels by both the transcription factor and product inhibition, meaning that FA overproduction may require significant re-engineering of cellular metabolism. An excellent overview of FA biosynthesis and its regulation has been reviewed by Handke et al. ( 2011 ). The challenge then, is not only to create a microbial biocatalyst that can produce FAs at high yields, high rates, and high product titers, but also to shorten the development time in the metabolic engineering design cycle, in order to compete effectively with petroleum-based processes. The metabolic engineering design process has evolved into a Systems Metabolic Engineering design process, as shown in Figure 1 . Systems Metabolic Engineering, which encompasses systems biology, synthetic biology, and evolutionary engineering at the system level, provides powerful techniques to design new biocatalysts (Lee et al., 2011a ). The classical metabolic engineering procedures of constructing and screening strains, based on the collective wisdom of experience, are often complemented with one or more of the new tools to improve and/or fine-tune strain design. The design engineer is faced with a suite of choices in the design process, on whether to use methods in isolation or in combination, although a survey of the literature indicates that at combination of multiple approaches is still not very common to date (Lee et al., 2011a ). A plethora of engineering manipulations, although mainly classical metabolic engineering approaches, for free FA production in E. coli exist and have been reviewed in recent years (Huffer et al., 2012 ; Lennen and Pfleger, 2012 ; Liu and Khosla, 2010 ; Zhang et al., 2011a ). However, recent successes in construction of high-yielding medium chain fatty acid producing E. coli strains, rooted in different features of systems metabolic engineering, provide an emerging case study of design methods for effective strain design (Dellomonaco et al., 2011 ; San and Li, 2013 ; San et al., 2011 ; Zhang et al., 2012b ). Figure 1 Systems metabolic engineering is an integrated field of classical metabolic engineering, system biology, synthetic biology, and evolutionary engineering. The classical metabolic engineering petal exists to construct and screen strains for overproduction. The systems biology petal comprises omics technologies and computational modeling to elucidate the cellular network and generate non-intuitive insight into the biological system. Incorporation of synthetic biology petal creates novel biologically functional parts, modules, and systems using synthetic DNA tools and mathematical methodologies to expand the capacity of the production hosts. Evolution and reverse engineering improves the performance of host strain through adaptive or random evolution under a specified environment. The evolved strain can be reverse-engineered to pinpoint the beneficial mutations and further optimized by metabolic engineering cycle. Nonetheless, protein engineering, shown as a bee, acts as a catalyst to system metabolic engineering by enhancing substrate specificity and productivity of key enzymes in the production pathway. Integrations of the above discipline will increase the efficiency of metabolic engineering in strain development. In this review, we focus mainly on the recent reports regarding medium-chain FA production in E. coli using different systems metabolic engineering approaches outside the scope of traditional metabolic engineering. In particular, we describe a classical metabolic engineering technique, an integrated experimental and computational strategy, and a synthetic engineering effort for enhancing fatty acid production in E. coli ."
} | 2,212 |
28473828 | PMC5397529 | pmc | 2,416 | {
"abstract": "Arbuscular mycorrhizal fungal (AMF) community assembly during primary succession has so far received little attention. It remains therefore unclear, which of the factors, driving AMF community composition, are important during ecosystem development. We addressed this question on a large spoil heap, which provides a mosaic of sites in different successional stages under different managements. We selected 24 sites of c. 12, 20, 30, or 50 years in age, including sites with spontaneously developing vegetation and sites reclaimed by alder plantations. On each site, we sampled twice a year roots of the perennial rhizomatous grass Calamagrostis epigejos (Poaceae) to determine AMF root colonization and diversity (using 454-sequencing), determined the soil chemical properties and composition of plant communities. AMF taxa richness was unaffected by site age, but AMF composition variation increased along the chronosequences. AMF communities were unaffected by soil chemistry, but related to the composition of neighboring plant communities of the sampled C. epigejos plants. In contrast, the plant communities of the sites were more distinctively structured than the AMF communities along the four successional stages. We conclude that AMF and plant community successions respond to different factors. AMF communities seem to be influenced by biotic rather than by abiotic factors and to diverge with successional age.",
"conclusion": "Conclusion Our results support the suggestion of Zobel and Öpik (2014) that biotic host plant–fungus interactions are more important factors for AMF succession during early ecosystem development than abiotic habitat conditions. In this context; it is paradoxical that spoil bank reclamation by tree plantations had no effect on AMF communities and suggests that among host-related factors; only some are linked with AMF community assembly. Their identification in future studies will improve our understanding of AMF succession and possibly also contribute to discerning “Driver” from “Passenger” effects.",
"introduction": "Introduction Primary succession is generally characterized by soil development and changes in vegetation structure ( Odum, 1969 ). In contrast to successional dynamics of plants, primary succession of mycorrhizal fungi is still poorly understood. Despite early studies describing occurrence of mycorrhizal fungi during succession ( Allen et al., 1987 , 1992 ; Warner et al., 1987 ), the development of their communities along succession has so far received relatively little attention ( Dickie et al., 2013 ). Because of the strong connection between mycorrhizal fungi, plant, and soil, mycorrhizal fungi may play a significant role in ecosystem development. Arbuscular mycorrhiza is the most common mycorrhizal type known from c. 74% of Angiosperm species ( Brundrett, 2009 ) and plays a crucial role in terrestrial ecosystems. All arbuscular mycorrhizal fungi (AMF) belong to the phylum Glomeromycota ( Schüßler et al., 2001 ), which contains c. 250 species. Establishment and assembly of AMF communities during primary succession largely depend on AMF dispersal ability to newly exposed land. Studies focused on AMF colonization in pioneer plants show that AMF propagules are abundant at early successional stages of ecosystem development ( Püschel et al., 2008 ; Rydlová et al., 2008 ). However, it remains unclear whether these pioneer communities are formed by random filtering from local AMF species pools or by immigration of pre-adapted pioneer AMF species with a ruderal life style ( Chagnon et al., 2013 ). If the first was true, high stochasticity in AMF community assembly would be expected, resulting in higher variability (larger composition turnover, Anderson et al., 2011 ) of the AMF communities in the earliest successional stages ( Martínez-García et al., 2015 ). On the contrary, if early successional sites were predominantly colonized by AMF species with a ruderal life style, composition turnover would not be expected to change during primary succession. As root-associated mutualistic symbionts, AMF mediate nutrient flow from the soil to the host plant in exchange for assimilated carbon ( Smith and Read, 2008 ). Because of the tight coupling of the AMF life cycle with their host plants, AMF communities are strongly influenced by the identity of host plant species (e.g., Vandenkoornhuyse et al., 2002 ; Sýkorová et al., 2007 ). AMF species richness can therefore be expected to increase during primary succession due to increasing plant richness ( Walker and Del Moral, 2003 ), but evidence for that is not consistent ( Dickie et al., 2013 ). As soil dwelling organisms, AMF communities might be affected by changes in soil chemistry during the vegetation development. Particularly pH and macronutrient availability seem to be key deterministic factors governing the richness and composition of AMF communities ( Lekberg et al., 2007 ; Fitzsimons et al., 2008 ). A number of studies consistently show changes in AMF community composition during ecosystem development (e.g., Oehl et al., 2011 ; Bennett et al., 2013 ; Sikes et al., 2014 ; Krüger et al., 2015 ; Martínez-García et al., 2015 ). However, the drivers responsible for the observed differences remain unclear. In their conceptual paper, Zobel and Öpik (2014) proposed that the composition of AMF communities during ecosystem development was driven by host plant–fungus interactions (‘Passenger/Driver hypotheses’ as previously proposed by Hart et al., 2001 ), rather than abiotic habitat conditions such as soil chemistry or climate (‘Habitat filtering hypothesis’), which become more important in structuring AMF communities at late successional stages. Indeed, a recently published study showed that host plant identity was a much stronger predictor of root-colonizing AMF communities during ecosystem development than site age, i.e., the response of AMF communities to ecosystem development was mostly associated with plant community changes ( Martínez-García et al., 2015 ). Besides host plant identity, the species richness and community composition of root-colonizing AMF communities is also influenced by neighboring plants ( Mummey et al., 2005 ; Hausmann and Hawkes, 2009 ; Kohout et al., 2015 ). The neighborhood of other plant species with distinct AMF communities in roots can both increase the species richness of the root community and induce shifts in AMF community composition of a target plant species ( Mummey et al., 2005 ; Meadow and Zabinski, 2012 ), and can be as important as the identity of the host plant in structuring AMF communities ( Hausmann and Hawkes, 2009 ). This influence may be exerted by non-random assembling of AMF communities in the roots of different plant species ( Davison et al., 2012 ) as well as by allelopathy via the synthesis of antifungal secondary metabolites ( Stinson et al., 2006 ; Becklin et al., 2012a ). Thus, plant succession may influence also AMF communities associated with generalist plants species that persist in the primary successional ecosystems throughout several successional stages. Primary succession naturally occurs in environments where new substrates are deposited such as glacier forefronts or lava beds. Primary succession also occurs on spoil heaps of spoil material after mining activities ( Prach et al., 2013 ). Spoil material excavated from great depth (hundreds of meters bellow surface) has typically extremely low biological activity ( Gould et al., 1996 ; Frouz et al., 2001 ; Elhottová et al., 2006 ). Although ecosystems on man-made sites are usually of shorter history than most natural chronosequences and lack later stages of ecosystem development, understanding the factors that influence primary succession in these areas is important for their re-integration into the landscape. This is also because these man-made sites are often reclaimed with woody plants to accelerate the processes of soil and vegetation development ( Frouz et al., 2001 , 2007 ; Abakumov et al., 2013 ). Factors influencing primary succession at the reclaimed sites, including succession of AMF communities, are therefore even more complex, including the diversity and identity of the planted species and their management. The main aim of our study was to describe changes in the AMF communities of one generalist host plant during primary succession, and to determine whether they can be related to changes in soil chemistry or to changes in plant community composition, possibly also influenced by reclamation. For this purpose, we chose a unique primary successional chronosequence on a large spoil heap in the western part of Czechia, covered by a mosaic of different successional stages under two different managements (spontaneously developing and reclaimed). In this system, the AM host Calamagrostis epigejos can be found throughout the whole chronosequence spanning about 50 years at both unmanaged and reclaimed sites. We expected that: (i) The early stage AMF communities became established by random filtering and variation in AMF community composition would therefore decrease during the primary succession. (ii) The number and structure of neighboring plants rather than soil chemistry would be related to the variability in AMF community composition in C. epigejos roots. (iii) Sites reclaimed by Alnus glutinosa plantations would harbor different AMF communities than spontaneously developing sites of the same age.",
"discussion": "Discussion This study focused on soil chemistry and neighboring plants as possible factors affecting AMF community development in the roots of one constant host plant during primary succession. It has not revealed any correlation between the measured soil characteristics and AMF community composition despite changes in soil chemistry over the studied chronosequences. However, the composition of AMF community covaried with the neighboring plant community composition, which points at a more important role of biotic rather than abiotic factors in AMF community assembly during primary succession. Furthermore, our results show that AMF composition variation rather than richness is positively correlated with successional stage. It is well established that plant species richness generally increases during primary vegetation succession ( Walker and Del Moral, 2003 ), which was also the case over the studied chronosequences (12–50 years). Contrary to our expectation, however, the higher diversity of the neighboring plant community did not increase the AMF species richness in the roots of C. epigejos . Although our data are based on a single host plant only, the results confirm a weak, or even negative correlation between AMF and plant species richness during primary succession ( Johnson et al., 1991 ; Sikes et al., 2012 ; Zangaro et al., 2012 ; Martínez-García et al., 2015 ), in contrast to the situation encountered in stable ecosystems ( Hiiesalu et al., 2014 ). The contrasting patterns in the dynamics of plant and AMF richness in ecosystem development may be on one hand explained by the contrasting levels of inter and intra-specific variability between plants and AMF taxa ( Bruns and Taylor, 2016 ; Öpik et al., 2016 ). On the other hand, the explanation may lie in differential speeds of dispersal in the two groups of organisms. As previously shown by Davison et al. (2015) , AMF seem to lack dispersal limitation, at least on larger spatial and temporal scales. We therefore hypothesize that AMF dispersal to and establishment at new sites is much faster and more efficient than that of plants. While AMF taxon richness was unaffected by successional stage, AMF composition variation was positively correlated with the age of the successional stages. This relationship contradicts our expectation that the AMF communities of C. epigejos become established by random filtering of the local species pool. Most fungal communities from the earliest successional stage were dominated by the same AMF species, i.e., shared Dominika sp. 1 as the most abundant AMF taxon. With increasing site age, however, the most abundant AMF taxon became more variable. This could originate from two different mechanisms. First, early-stage AMF community assembly may have been deterministic, the early-stage conditions favoring population growth of (one) compatible species, while stochastic factors dominated in later stages with conditions favorable to a broader spectrum of species. Such a scenario is supported by previous studies in mature ecosystems that found evidence for a prevalence of stochastic processes in the structuring of AMF communities and other soil microbial communities ( Powell et al., 2009 ; Bahram et al., 2016 ). Alternatively, increasing habitat diversity with ecosystem age ( Walker and Del Moral, 2003 ) may lead to increased patchiness in the AMF communities of mature ecosystems. To further disentangle changes in the relative contribution of deterministic and stochastic processes to AMF community assembly in primary succession; more detailed sampling is need. Particularly, a spatially explicit design for each successional stage along the chronosequences would provide more detailed information. Vegetation development in primary succession is inseparably related to pedogenesis. Soil chemistry was previously described as a key factor influencing AMF community assembly ( Lekberg et al., 2007 ; Fitzsimons et al., 2008 ). However, neither a single soil chemical parameter (PERMANOVA) nor soil chemistry itself (variation partitioning) showed direct significant effect on the AMF communities in this study despite changes in soil chemistry during the ecosystem development, such as increase of soil carbon or decrease of available calcium and soil pH, which might also influence P availability in the soil. Besides, soil chemical parameters, soil physical properties, such as soil texture and structure might also change during the ecosystem development. Herrmann et al. (2016) previously described a significant effect of soil physical properties on the composition of AMF communities. Further clarification concerning the effect of soil physical properties on development of AMF communities during primary succession is needed. On the contrary, the composition of AMF communities in the roots of C. epigejos was significantly correlated with composition of neighboring plant species. Interestingly, none of these five plant species occurred at the earliest successional stage, which indicates increasing covariation between AMF and plant communities during primary succession. Covariation between plant and AMF communities has been reported repeatedly ( Fitzsimons et al., 2008 ; Hausmann and Hawkes, 2009 ; van de Voorde et al., 2010 ; García de León et al., 2016 ). However, this study for the first time shows that variation in root-colonizing AMF communities is more connected to neighboring plant communities than to soil chemistry during primary succession. This expands the conclusion of Martínez-García et al. (2015) of host plant identity as a major driver of AMF community composition during primary succession, broadening host plant identity to plant community. Altogether, both studies support the Passenger/Driver hypothesis ( Hart et al., 2001 ) rather than habitat filtering hypothesis ( Zobel and Öpik, 2014 ) of AMF community assembly during primary succession. Most AMF communities of the earliest successional stage were dominated by Dominikia sp. 1 (sister clade to Rhizophagus ), whose abundance gradually diminished along the chronosequences. Our finding is in agreement with Chagnon et al.’s (2012) trait-based framework for AMF ecology. These authors proposed that life history traits of members of Glomeraceae are consistent with a ruderal life style. Species of the Glomeraceae family were, indeed, described as early colonizers on a newly exposed artificial island ( Nielsen et al., 2016 ). However, other Glomeraceae species were found to be abundant in the later successional stages, and we still do not know the specific life history traits (e.g., dispersion efficiency) that have enabled Dominikia sp. 1 and the other pioneering AMF species to reach dominance at early successional stages. Besides the plant species ability to grow in ruderal environment, dispersal limitation is the major force structuring plant communities during primary succession ( Lichter, 2000 ). Much less is, however, known about the role of dispersal limitation in AMF communities ( Davison et al., 2015 ). In our study, we observed that roots of C. epigejos plants harbored almost all the detected AMF species already at the earliest successional stage. This observation suggests that AMF dispersal was less limited in the present study system than that of Nielsen et al. (2016) , who found that the AMF community from the earliest successional stage (also 12 years old) was a non-random subset of the communities found in the later successional stage. A potential explanation for the contradictory results may be different effectiveness of vectors of AMF propagule dispersal in the two systems. While Nielsen’s study focused on an artificial island with very restricted human access, our study was conducted on a spoil heap, which was separated from the surrounding ecosystems by more permeable barriers and exposed to relatively higher human activity (connected to ongoing depositing of spoil in other parts of the spoil heap). We therefore assume that in Nielsen’s study, the vectors of AMF dispersal were just birds and wind, which are relatively insufficient vectors of AMF dispersal ( Egan et al., 2014 ). In contrast, movement of mammals and human activity may have contributed to AMF dispersal in the present study system ( Mangan and Adler, 2002 ; Rosendahl et al., 2009 ) and by facilitating AMF dispersal, these factors also may have accelerated AMF succession. This indirect comparison therefore highlights the importance of specific site settings, especially the interconnectivity with surrounding biotopes. Interestingly, we did not find any effect of the spoil heap reclamation either on AMF species richness or community composition in the roots of C. epigejos . Previous studies focusing on AMF species compositional changes after reclamation did not make comparisons with a natural successional chronosequence (e.g., de Souza et al., 2013 ; Zhang and Chu, 2013 ; da Silva et al., 2015 ). Our expectation to find different AMF communities in the reclaimed and spontaneous succession chronosequence was therefore based merely on the intuitive assumption that reclamation fundamentally affects site conditions. However, it should be mentioned in this context that overall root colonization level of C. epigejos was higher in the reclaimed than in the spontaneous succession chronosequence. As also indicated by the model (significant negative relationship of root colonization with C tot ), this may have been due to accumulation of an organic layer on soils of the later stages of the spontaneous succession chronosequence. As previously reported ( Piotrowski et al., 2008 ; Becklin et al., 2012b ), litter of Salicaceae may inhibit the development of AMF root colonization. Thus, our study is the first comprehensive comparison of a reclaimed and a spontaneous succession chronosequence. Although reclamation significantly influenced plant community composition and root colonization by AMF, it did not lead to different AMF communities. It seems that Alnus glutinosa planting did not override the effect of site age on the understory plant communities in a way that would be affecting AMF community assembly. The situation may be different in plantations of other trees with more pronounced effect on the understory vegetation ( Mudrák et al., 2010 )."
} | 4,953 |
33806971 | PMC8004711 | pmc | 2,417 | {
"abstract": "Fire-protection coatings with a self-monitoring ability play a critical role in safety and security. An intelligent fire-protection coating can protect humans from personal and property damage. In this work, we report the fabrication of a low-cost and facile intelligent fire coating based on a composite of ammonium polyphosphate and epoxy (APP/EP). The composite was processed using laser scribing, which led to a laser-induced graphene (LIG) layer on the APP/EP surface via a photothermal effect. The C–O, C=O, P–O, and N−C bonds in the flame-retardant APP/EP composite were broken during the laser scribing, while the remaining carbon atoms recombined to generate the graphene layer. A proof-of-concept was achieved by demonstrating the use of LIG in supercapacitors, as a temperature sensor, and as a hazard detection device based on the shape memory effect of the APP/EP composite. The intelligent flame protection coating had a high flame retardancy, which increased the time to ignition (TTI) from 21 s to 57 s, and the limiting oxygen index (LOI) value increased to 37%. The total amount of heat and smoke released during combustion was effectively suppressed by ≈ 71.1% and ≈ 74.1%, respectively. The maximum mass-specific supercapacitance could reach 245.6 F·g −1 . The additional LIG layer enables applications of the device as a LIG-APP/EP temperature sensor and allows for monitoring of the deformation according to its shape memory effect. The direct laser scribing of graphene from APP/EP in an air atmosphere provides a convenient and practical approach for the fabrication of flame-retardant electronics.",
"conclusion": "4. Conclusions In this study, an intelligent fire-protection coating was fabricated on the surface of an APP/EP composite using laser scribing under ambient air conditions. Highly flame-retardant behavior and potential applications as temperature sensors in various environments were demonstrated. Direct laser scribing and multiple laser passes on the APP/EP led to a photothermal effect and a high local temperature, which resulted in the depolymerization of the APP/EP surface and the generation of graphene layers. The LIG formed on the APP/EP had a porous hierarchical graphene surface structure. The LIG was fabricated by applying a laser power of 5 W and a scribing speed of 10 mm·s −1 . It was shown that four scribing passes led to an optimal graphene structure and application performance. The prepared thin graphene coatings (LIG) increased the TTI by about 36 s, and the PHRR, THR, and TSP values all showed significant decreases of ≈ 71.1%, ≈ 75.2%, ≈ 74.1%, and ≈ 40.7%, respectively, which demonstrated a massively improved flame retardancy. Furthermore, we prepared a high electrochemical performance supercapacitor with LIG electrodes, which added further functionality to the device. Furthermore, temperature sensors were fabricated directly onto the LIG surface, displaying a metallic-type PTC effect. The temperature sensors were applied in a hot water environment, showing that they could monitor the temperature changes of electronic components in different conditions. We foresee that the laser-scribing technology that was used for forming graphene on the APP/EP surfaces may be applied to other polymers, which may lead to a quick and efficient improvement in their flame-retardant performance and electrical properties.",
"introduction": "1. Introduction In numerous technological applications and devices, unwanted ignition poses a serious risk of causing harm to human lives and damaging the respective application or device. To prevent and tackle fire hazards, considerable research has been carried out on flame protection, fire extinguishers, and fire surveying or warning technologies [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. For flame-retardant technologies, polymer materials have commonly been applied with the purpose of delaying or avoiding the occurrence of fire [ 9 , 10 ]. For example, flame-retardant epoxy resin is widely used in electronic devices, which prevents electrical appliances from burning or at least reduces the burning speed [ 11 , 12 ]. However, ideal fire-protection coatings should not only be able to protect the material itself from ignition but also have additional smart properties, such as the ability to monitor the material temperature and detect potential hazards. However, it is still a big challenge to realize the functional integration of intelligent self-monitoring fire protection in a single device with multiple functionalities, namely, fire protection, temperature detection, and hazard monitoring. This becomes even more challenging if the respective device requires reducing the size of the fire coating and integrating it into microelectronics. High-performance polymer materials, such as a flame-retardant epoxy resin (FR-EP), are an indispensable fire-protection coating due to their excellent dimensional stability and high mechanical strength [ 13 , 14 ]. For the purpose of integrating other functionalities into FR-EP materials, a viable strategy is to add additional functional materials into the FR-EPs to form composites. The traditional process consists of spraying, embedding, or filling, where such functional coatings or fillers can be carbon nanotubes, graphene, Ag, and carbon black, which can be deposited directly onto the surface of the polymer [ 15 , 16 , 17 , 18 ]. In order to integrate a functionalized temperature sensor via conventional means, multistep routes and high-cost deposition materials are required [ 19 ]. Consequently, it is still challenging to prepare smart fire-protection coatings, especially those with straightforward and facile fabrication and custom design capabilities. To circumvent such tedious multistep composite fabrication routes, graphene nanomaterials may be considered a viable alternative. Recently, graphene nanomaterials have attracted great attention in many areas of industry and academia due to their high conductivity, light weight, marked barrier effect, and excellent stability, which makes them a promising candidate material for the preparation of smart electronic coatings [ 20 , 21 ]. Traditional graphene-based electronic components are fabricated by spraying, embedding, or filling the prepared graphene onto or into the substrate [ 22 ]. Tang et al. [ 23 ] describe the creation of multifunctional coatings through assembling graphene oxide (GO)/silicone onto flammable substrate materials, which can detect a change in temperature and protect the flammable materials from combustion while the fire occurs. However, the fabrication of a GO/silicone coating needs different reagents, which may lead to a waste of reagents and waste liquid treatment, and requires a complicated preparation process. To simplify the preparation process and reduce the cost, Lin et al. [ 24 ] developed a novel, facile, and low-cost approach to generating 3D porous graphene through laser scribing on polyimide. More recently, several research groups reported the integration of graphene onto the surface of polymers, such as those of the polysulfone class and PEEK (poly-ether-ether-ketone) polymers [ 25 , 26 , 27 ]. The laser-scribing approach that is used to generate graphene is facile, efficient, and has a low cost. Due to the similar polymer structures used in this study to create FR-EP with the abovementioned precursors, we concluded that laser-scribing technology may be an interesting tool to be used with FR-EP, which may facilitate the preparation of graphene-based FR-EP materials that can be integrated into electronic devices as a fire-protection coating composite. In this work, we propose a facile strategy for preparing a fire-protection coating by integrating direct laser-induced graphene (LIG) into ammonium polyphosphate epoxy resin (APP/EP) composites. The formation of graphene in APP/EP can be attributed to the aromatic structure of the EP resin and the photothermal effect generated by the laser radiation. The resulting LIG-APP/EP device had porous graphene on the surface, which could be obtained in an ambient atmosphere in an optimized experimental process that included multiple laser-scribing process steps (laser power: 5 W, laser-scribing speed: 10 mm·s −1 ). This coating could play both a flame-retardant role and detect potential hazards through the high conductivity of the LIG and the shape memory effect of the APP/EP. The LIG-APP/EP composite exhibited high electrical conductivity (9 Ω, 1 cm × 1 cm), as required for applications in the field of electronics. The potential for application of the LIG-APP/EP composite was demonstrated in N- and P-doped supercapacitor electrodes and temperature sensors, and the hazard detection functionality was shown to be based on the shape memory effect of the APP/EP. The LIG-APP/EP may be regarded as an intelligent coating that has tremendous potential for applications in various fields."
} | 2,223 |
30739982 | PMC6358148 | pmc | 2,419 | {
"abstract": "Appropriate inoculation and maturation may be crucial for shortening the startup time and maximising power output of Microbial Fuel Cells (MFCs), whilst ensuring stable operation. In this study we explore the relationship between electrochemical parameters of MFCs matured under different external resistance (R ext ) values (50 Ω - 10 kΩ) using non-synthetic fuel (human urine). Maturing the biofilm under the lower selected R ext results in improved power performance and lowest internal resistance (R int ), whereas using higher R ext results in increased ohmic losses and inferior performance. When the optimal load is applied to the MFCs following maturity, dependence of microbial activity on original R ext values does not change, suggesting an irreversible effect on the biofilm, within the timeframe of the reported experiments. Biofilm microarchitecture is affected by R ext and plays an important role in MFC efficiency. Presence of water channels, EPS and precipitated salts is distinctive for higher R ext and open circuit MFCs. Correlation analysis reveals that the biofilm changes most dynamically in the first 5 weeks of operation and that fixed R ext lefts an electrochemical effect on biofilm performance. Therefore, the initial conditions of the biofilm development can affect its long-term structure, properties and activity.",
"conclusion": "4 Conclusions Maturing the biofilm under various R ext caused several changes in biofilm behaviour. It was shown, that growing the biofilm under higher suboptimal R ext had an adverse effect on its properties and activity. Maturing the biofilm under lower, yet suboptimal R ext , resulted in improved R int and power output. The most dynamic changes in electrochemical properties of the biofilm were observed in the first 5 weeks of operation. Implementing MPPT procedure after initial period of maturing was not sufficient to change the electrochemical profile-effect of the MFCs, i.e. dependence of the biofilm activity on initial (stage 1) R ext values. The connected R ext had a significant effect on biofilm three-dimensional structure and composition. These findings are important for developing the appropriate inoculation and maturing strategies to maximise the performance of MFCs.",
"introduction": "1 Introduction Microbial Fuel Cell (MFC) technology uses electroactive bacteria to produce electricity through oxidation of organic matter. The technology has received increased attention over past decades [ 1 ]. The bioelectrochemical reactions take place in anodic and cathodic components of the MFC have found many potential applications in the fields of wastewater treatment, electricity generation, biogas production, biosensors and bioelectrochemical synthesis [ [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] ]. Among various carbon sources that have been demonstrated as a fuel in MFCs human urine has proved to be a good substrate due to its high conductivity [ 11 ]. The ongoing development of the MFCs focuses on developing electrode materials, catalysts, membranes separating anodic and cathodic chambers, design and scale-up of the MFC-based systems [ 2 , [12] , [13] , [14] , [15] , [16] , [17] ]. Despite the broad interest in many engineering aspects of the MFCs, the most crucial role is played by the electroactive bacteria, which form the biofilm on the electrode's surface and generate electrical power from their population-level metabolism. The biofilm is a complex matrix of microorganisms and extracellular compounds which is considered to be very stable, albeit possessing physiological adaptive mechanisms [ 18 ] many of which are expressed during the initial biofilm formation period. Several research groups have previously reported on power performance and start up times when the electroactive community has been matured under different poised anode potentials. For example the anodic biofilm formed by Geobacter sulfurreducens gives highest power performance and lowest MFC start-up time, when matured under a potential range between 0 and 400 mV vs SHE (standard hydrogen electrode). This optimal potential range promoted the biofilm growth and corresponding power density of the MFCs [ 19 ]. The study reported by Aelterman et al. showed, that optimal biofilm growth and activity was obtained when the anode was poised at −200 mV vs Ag/AgCl electrode, although the original source of the bacterial inoculum was not mentioned [ 20 ]. More recently, Zhu et al. reported, that acclimating the biofilm with positive potentials may lead to the decay of the power overshoot phenomenon which leads to improved power performance [ 21 ]. Easier ways of controlling the potential of MFC electrodes is by applying an external load (R ext ), which does not require any specialist equipment that could be limiting in particular for field applications. Comprehensive investigation on the effect of R ext on biofilm formation and activity has been reported by Zhang et al. [ 22 ]. The authors investigated the ohmic range of 10–1000 Ω and indicated that optimal R ext for their MFC setup was found to be 50Ω, although biofilm matured under 10 Ω produced the highest current. The study also showed that R ext had an impact on the presence of extracellular polymeric substances (EPS) of the biofilm, and a more recent study showed that EPS plays a role in biofilm performance and in turn, power generation [ 23 ]. The influence of three different R ext values on biofilm activity (after the maturing phase) was also studied by Jung and Regan [ 24 ]. The authors focused on methane production and the inhibition of methanogenesis was found to occur in parallel with the highest power efficiency for MFCs fed with acetate and operating under lowest (150 Ω) R ext . Earlier studies also demonstrated the relationship of R ext applied during operation (after maturing phase) with performance of the MFCs in relation to the fuel supply and the best results were obtained when MFCs were operated under R ext closer to internal resistance (R int ) [ 25 ]. External resistance was also found to be a factor influencing diversity of the bacterial community [ 24 , 26 , 27 ]. Although significant work has been done to understand the interactions of R ext with the biofilm, very limited knowledge is available on dynamic evolution of biofilm subjected to various external loads. Since the biofilm forms both stable and adaptive structure, such knowledge is indispensable to develop appropriate strategies for inoculation and operation of MFCs. It is therefore important to determine, whether the conditions applied to the biofilm in the initial stage of development may leave a structural and electrochemical profile and irreversibly affect its performance thereafter. The aim of this study was to determine the temporal and long-term effects of fixed and dynamically-changed external resistance on changes of biofilm parameters and resulting MFC performance in time. The results revealed the irreversible effects that the initial R ext causes to the biofilm, which may subsequently either induce or inhibit the power performance of the MFCs in long-term perspective. This is the first of two papers in series, where we have focused on analysis of electrochemical parameters. The second part of this study will focus on biological parameters of the biofilm.",
"discussion": "3 Results and discussion All experiments were carried out using fresh human urine, collected on a daily basis as a fuel and delivered under constant flow rate. Therefore, the measured pH of fresh urine ranged from 6.15 to 6.29 and the average conductivity was equal to 11.76 ± 0.76 mS. 3.1 Electrochemical behaviour of biofilm It is known, that the shape of a polarisation and power curves and the corresponding losses are greatly dependent on the activity of electroactive bacteria [ 32 , 33 ]. Data derived from the polarisation experiments were presented in the form of power curves, as shown in Fig. 2 . The results reported in this study comprised the highest coverage of R ext within the operational range of MFCs when compared to previous studies available in the literature. Previous works used up to four R ext values below 10 kΩ, where the highest divergence of the data may be expected [ 22 , [24] , [25] , [26] , [27] ]. Fig. 2 Electrochemical behaviour of MFCs represented by matrix of power curves, obtained from polarisation experiments during the first and second stages of the experiment. Each column in the matrix represents MFCs with predefined external resistance values, which are valid for the weeks 1–4. Starting from week 8, the R ext values were continuously adjusted to the optimum (simulating MPPT). Each graph represents three individual MFC replicates. Fig. 2 Maturing the MFCs under various loads resulted in achieving similar, symmetrical power curves across all the R ext values. Nevertheless, the most uniform MFC characteristics between individual replicates were observed for 2.5 kΩ, resulting in the smallest variance of electrochemical parameters: OCV, I, P and R int . The highest dispersion of electrochemical parameters detected in polarisation experiments were reported for the two extreme R ext values: 50 Ω and OC. The most significant concentration (mass transfer) losses were observed for 10 kΩ load, while the best performing individual MFCs were reported for 500 Ω and 1 kΩ. All of the MFCs revealed only negligible overshoot phenomenon, despite this being reported for early stage of biofilm development, as well as starvation and sub-optimal running conditions, and related to its metabolic rate and activity [ 21 , 34 ]. The lack of overshoot stays in line with our previous study, where the earthenware appeared to be the most favourable ceramic membrane material to create the appropriate MFC microenvironment which induces electroactive biofilm formation and activity [ 35 ]. In the second week of operation, significant changes in the MFC characteristics were observed. All of the MFCs operating under the load within the range 2.5 kΩ - OC, and 2 out of 3 MFCs operating under 1 kΩ, underperformed. Those MFCs showed significant, rapid drop of the performance in the central part of the power curves, corresponding to the ohmic losses. In contrast, the MFCs connected to R ext of 50 and 500 Ω did not show such unfavourable changes. This trend continued until the end of the first stage of the experiment and became more significant each week, showing deterioration of the performance. Decrease of the power performance during polarisation was mainly caused by the drop of potential, while current values were similar or higher than those observed for MFCs operating under 50 and 500 Ω. The electrochemical data observed for MFCs operating under R ext ≥1.0 kΩ and OC control, showed that ohmic losses were responsible for the gradual deterioration of power performance observed with time and with increasing R ext . Since the MFCs were operating in the same hydraulic conditions and were manufactured in the same way, the observed ohmic losses were probably caused by the formation of biofilm with suboptimal electrochemical properties, which may have been the result of both the R ext and the low (but constant) flow rate. The MFCs were operating below the optimal value, which was found to be between 0.43 and 0.89 L d −1 ( Figure S2 ). Suboptimal biofilm properties (physical, chemical and biological) could have resulted in achieving a highly resistive biofilm, which could negatively affect the overall internal resistance of the MFC. As reported by Nikhil et al., biofilm conductivity was the determining factor for the performance of MFCs inoculated with Geobacter sulfurreducens [ 36 ]. Such results are supported by the findings of McLean et al. who observed that 100 Ω - matured Shewanella oneidensis biofilm had lower thickness (<5 μm), than the biofilm matured under near-OC conditions (1.0 MΩ, >50 μm). Similar results were found by Read et al. during the initial stages of biofilm formation [ 37 ]. Reaching similar values of current, within the tested resistors, suggests that even suboptimally formed biofilm, contains sufficient numbers of electroactive bacteria, capable of delivering electrons to the electrode surface. This finding can be partially explained by the work of Jung et al. [ 24 ] who showed that dominating, electroactive species belonging to Geobacteraceae are present in the quantities of the same order of magnitude in biofilms operating under various R ext values or even in OC conditions. Nevertheless, their study was constructed in a different way, i.e. the MFCs were initially matured for 3 weeks under the same R ext after which various pre-defined loads were used (150–9800 Ω). More detailed ecological studies have reported significant effects of anodic potential levels on biofilm composition and consequently, performance of these bioelectrochemical systems, which may in turn play a key role in achieving desirable electrochemical properties [ [38] , [39] , [40] ]. The differences in biofilm properties were also revealed during the flow rate experiment ( Figure S2 ). The results indicate, that only the 50 Ω-matured biofilm was capable of utilising nutrients at the highest flow/supply rate (0.888 L d −1 ). In parallel, the 50 Ω-biofilm was more susceptible to starvation (0.008 L d −1 ), which led to a drop of power output when compared to other R ext . These data perhaps suggest that the 50Ω biofilm had developed mechanisms for more efficient conversion of nutrients into electricity when the feeding rate is sufficiently high, but underdeveloped the ability to accumulate store energy reserves in such form as EPS ( Fig. 5 ) which could be used when the feeding rate is low. Flow rate is an important parameter that is bound to affect the biofilm as a whole. Previous reports [ 41 ] describe how the flow rate affects the biofilm growth rate and in turn power output. However, the biofilm structure and how this may be affected by flow rate, is an area that requires dedicated investigation. Fig. 3 Evolution of the power performance and internal resistance of MFCs and biofilm matured under different external resistance values and open circuit (here designated as 10 6 Ω) conditions. The numbers in the upper-left corner of each graph indicate the week of operation. The R ext labels reflect the real, predefined external resistance for weeks 1–5 and original external resistance of the same MFC when the external resistance was changed to follow an MPPT procedure (weeks 8–17). The colour intensity of each point (plots A and B) refers to the measured value, while colour intensity of the surface refers to the regression model. The regression model included OC (10 6 Ω) control for fitting, which was not shown in the plotting range for reasons of clarity. The points represent mean ± SD (plots 1–17) and mean values (plots A and B). Blue arrows indicate rinsing the cathodes with distilled water. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3 Fig. 4 Simultaneous effect of external and internal resistance on electrochemical parameters of MFCs in two stages of experimental period. The labels on x-axes reflect the real, predefined external resistance for weeks 1–5 and original external resistance for the same MFCs once the simulated MPPT procedure was applied (weeks 8–17). The colour intensity of each point refers to the measured value, while colour intensity of the surface refer to the regression model. The regression model was also fitted using OC (10 6 Ω) control, which was not included in the plotting range for reasons of clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 4 Fig. 5 ESEM images showing changes in biofilm three-dimensional architecture, composition and occurrence of inorganic precipitates (bright spots), when matured under different external loads. Fig. 5 After 5 weeks of operation (stage 2), the pre-defined resistor values were replaced by the optimal R ext values (derived from the polarisation data), adjusted to the optimum value at one-week intervals (simulating an MPPT approach). Changing the R ext of the MFCs to follow their optimal values (when R ext = R int [ 33 ]) resulted in changes to the shape of MFC power curves. The ohmic losses previously observed in stage 1 were no longer visible. Such an interesting phenomenon suggests, that there was perhaps a change in biofilm behaviour that adjusted to the new electrochemical conditions. This could be explained by the increased oxidation of the carbon-energy substrate due to higher metabolic rates, since the new resistors connected to the MFCs had lower resistance, when compared to the stage 1. Higher metabolic rates are proportional to higher microbial growth rates and hence biomass density of the electroactive species. In a perfusable electrode system this maintains a fixed thickness biofilm, as the outer layers, which no longer have direct contact with the electrode, are constantly washed out. This results in a thinner biofilm and improves the diffusion of nutrients into the biofilm and conductivity (as indicated by R int – Fig. 3 ). A thinner biofilm will also result in a significant reduction in extracellular polymeric substances (EPS) as previously reported by Zhang et al. [ 22 ]. The present study, suggests that R int could dynamically change over time, as also alluded to by Winfield et al. [ 34 ]. 3.2 Changes of power and internal resistance over time To further investigate the impact of R ext on biofilm maturing, the dynamic changes in R int and power over time were also monitored ( Fig. 3 ). In the first week of operation, the highest power performance was observed for 1 kΩ-matured MFCs and reached 91.6 ± 14.0 μW, whilst the R int reached a value of 471.3 ± 247.5 Ω. The lowest R int value was also observed for 1 kΩ and was equal to 205.4 Ω (derived from the polarisation experiment). Thus, in the first week, the use of 1 kΩ R ext created the most favourable conditions to obtain the lowest R int . During the first 3 weeks, the highest power performance was observed for 1 kΩ MFCs. In the following weeks the maximum power output was recorded for lower R ext values: 500 Ω MFCs in week 2 (81.3 ± 15.0 μW) and 50 Ω MFCs in week 3 (112.2 ± 23.1 μW). Similarly, the minimum R int values were observed for MFCs matured under R ext = 500 Ω in week 2 (473.5 ± 93.8 Ω) and R ext = 50 Ω in week 3 (253.8 ± 40.6 Ω). During the whole 1st stage of biofilm maturing, the lowest power performance and the highest R int values were observed for the MFCs operating under higher R ext values and under open circuit conditions, whilst the highest power values and lowest R int values were recorded for the MFCs operating under lower R ext values. The relationship between R ext , R int and power was the most notable in the 3rd week of biofilm maturing, which appears to be the critical time required for the biofilm to develop. Furthermore, the highest performance of the MFCs during the entire experiment was also observed in the 3rd week of operation and was recorded for the MFCs matured under the lowest Ohmic value, 50 Ω (112.2 ± 23.2 μW). Following the 3rd week of operation, the increase of R int with increasing R ext was observed even in the 2nd stage of operation, where optimal R ext values were applied following the simulated MPPT method. The internal resistance showed similar (but negative) trends that could be observed for power performance, which was reflected by similar patterns developed by the MFCs through time ( Fig. 3 A–B). The correlation between those two factors was later confirmed by statistical analysis. The internal resistance may be affected by various parameters such as hydraulic and environmental conditions [ 25 , 42 , 43 ]. In this study the hydraulic and other operational conditions remained constant, even though the flow rate was relatively low for this type of MFC. Therefore, such dynamic changes of internal resistance, in particular in the early period of operation (stage 1) resulted from ongoing biofilm development. Such changes may affect the conductivity of the biofilm, which is directly correlated with the current density by reducing the resistance of the electron flow and lowering the activation energy required for electron transfer between biofilm and the anode [ 36 ]. Interestingly, at the end of the 1st stage, a local power optimum was observed for 2.5 kΩ and became even more distinctive in the 5th week of operation. The 2.5 kΩ MFCs reached 93.0 ± 7.1 μW, which corresponds to 89.8% of the performance observed for 50 Ω MFCs. This state was also observed in the later stage of the biofilm growth, when the optimal R ext resistors were applied to the MFCs (stage 2). Local polynomial regression fitting ( Fig. 3 A and 3. B) revealed that this local optimum could be found between R ext of 0.5 and 2.5 kΩ (with best performing MFCs observed for 1.0 kΩ) and was maintained until 15 th -16 th week of operation. At the same time 50 Ω MFCs, which appeared to show the highest power performance in stage 1, underperformed as compared to MFCs operating under 0.5–2.5 kΩ in the initial period of stage 2 (weeks 9–11), but established a local optimum at the end of the experimental period (weeks 14–17) and outperformed the MFCs matured under higher R ext values. The overall decreasing trend of power output and simultaneous increase of R int observed in all MFCs in stage 2 resulted from the observed accumulation of inorganic salts on the cathode surfaces (Supporting information – Figure S1 ), as previously reported for this particular MFC design [ 28 ]. Therefore, at the end of stage 2, the cathodes were washed in situ with copious amounts of distilled water. As a result, the 50 Ω biofilm was brought back to show the highest power. The power profile observed across all of the MFCs in week 17 was almost identical to the one observed for the 4th week ( Fig. 3 .4 and 3.17), confirming that divergence of the data during stage 2 was partially a result of the deteriorating cathodes and indicating long-term effect of R ext on biofilm performance. The analysis of the regression surface of power and R int suggests that the deterioration rate of the cathode was increased in the 50 Ω and decreased in the 0.5–2.5 kΩ - matured MFCs. This can be explained by the occurrence of electroosmotic drag, which is related to the power output and could lead to faster accumulation of salts at the cathode observed in the best performing (50 Ω) MFCs [ 44 , 45 ]. In fact, the highest amount of salt deposits at the cathode surface were observed for 50 Ω MFCs, while the lowest for OC control and also 500–5000 Ω MFCs, indicating the interdependence between electroosmotic drag and MFC performance ( Figure S1 ). The cathode performance adds another dynamic factor to the complexity of the microbial fuel cells. 3.3 Dynamic electrochemical profile - simultaneous effect of R ext and R int resistance on MFC performance Data shown in Fig. 4 display a particular type of electrochemical profile – the pattern developed throughout time by all biofilm communities, reflecing dependence of the MFC electrochemical parameters on controlled (external) and developed (internal) resistance. The results show that the highest power efficiency of the MFCs was achieved for the biofilm which developed an internal resistance lower than 300 Ω, and such low R int was only present when the biofilm was matured under R ext between 50 and 1000 Ω. Another local optimum was found for the biofilm matured under R ext between 1000 and 2500 Ω, which developed an R int between 407.2 and 701.8 Ω. Notably, biofilms developed under lower R ext values produced the highest current and the lowest OCV, while the biofilms matured under higher values of R ext showed lower current and higher OCV. These results could be explained by the fact that the biofilm subjected to the effects of the low external resistance (higher anode potential) develops a biofilm with a different composition of electroactive species, as shown by previous studies for both biofilm [ [38] , [39] , [40] ] and planktonic communities [ 38 ]. It was shown that the strategy of maturing the biofilm under R ext values lower than the lowest observed R int may be beneficial in the long term and this is (to the best of the authors' knowledge) the first report where these phenomena have been demonstrated. Data reported in previous research focused on maturing the biofilm in R ext > R int conditions [ 22 , [24] , [25] , [26] , [27] ]. Furthermore, when comparing profiles obtained in two stages of experiment, it can be concluded that the biofilm maintained its electrochemical properties even though the environment was dynamically changing over time. The obtained profiles were similar, including the general decreasing trend in performance, current and OCV, probably due to cathodic salt accumulation, as previously described. These findings are crucial in defining the appropriate inoculation strategy and protocols for assessing and predicting the MFC performance. The internal resistance proved to be more effective parameter to determine the best performing MFCs than OCV. Nevertheless, reaching high OCV suggests that system may be better balanced and more effective in long term operation, when dynamic changes in cathode performance may occur. The data also shows, that reaching higher potential within MFC environment is not as important as acquiring the biofilm with a desirable structure and community composition as reflected by R int , which allows the MFC to reach a higher current production rate and density. This finding suggests that the electrochemical biofilm properties developed over time, are as important as the composition of the electroactive consortia, which also changes dynamically over time [ [46] , [47] , [48] ]. 3.4 Biofilm microarchitecture The biofilm structure and composition was assessed at the end of experiment (after phase 2 - switching R ext to optimal values) using environmental scanning electron microscopy, as shown on Fig. 5 . The most notable changes were in the form of inorganic precipitates. The MFCs matured under lower R ext (50, 500, 1000 Ω) showed no or negligible amounts of such precipitates. Maturing the biofilm under 2.5 kΩ and above resulted in increased amounts of graupel-shaped crystals embedded into the biofilm structure, comprising mainly Na, Cl, Mg, P and Ca, as determined by energy-dispersive X-ray spectroscopy. These salts were present in the highest amounts under open circuit conditions and in all reported cases, adjacent to neighbouring bacterial cells. Such finding may suggest a tendency from the anodic community to accumulate or induce formation of precipitates at very low or no current flow (see Fig. 5 ). This phenomenon could be one of the factors that contributed to the development of higher R int over time for MFCs that had matured under higher R ext , since the crystals are considered to be non-conductive and may have affected the resistivity of the biofilm. The differences in biofilm microarchitecture were also most notable when comparing high R ext (5 kΩ, 10 kΩ and OC) with low R ext MFCs. Images acquired for high R ext anodic biofilm and OC control, revealed development of looser structures, rich in EPS and with visibly larger water channels in comparison to the low R ext anodic biofilms. In contrast, the lowest R ext (50 Ω) MFC developed a dense and uniform biofilm structure with little EPS content. Zhang et al. [ 22 ], quantified the EPS content and reported its inverse relationship with external resistance, which is not in line with the findings of the current study. In that previous study, the results were normalised to the surface area of the carbon cloth electrode (as opposed to the biomass), in addition to using a different R ext range (10–1000 Ω). In addition to the influence of R ext on the anodic community composition, as previously reported [ 24 , 26 , 27 , 49 ], the current study suggests that the biofilm microarchitecture was affected by R ext and played an important role in the evolution of the electrochemical MFC parameters over time, reaching a desirable power efficiency. The observed differences between the lower and higher ranges of R ext , as well as the OC control, indicate that beyond a point, the biofilm was irreversibly affected by its initial maturing conditions within the timeframe of experimental period. 3.5 Quantitative analysis of dynamic changes in biofilm behaviour To investigate the dynamics of changes recorded for electrochemical parameters of the biofilm, correlation analysis has been conducted, as shown on Fig. 6 . In the first stage of biofilm maturing, when fixed R ext values were applied, positive correlation (R = 0.80) was only found for current profiles recorded in the first two weeks, which we consider as a startup period of the MFCs. The correlation coefficient gradually decreased afterwards, confirming that most dynamic changes took place during the first 5 weeks of biofilm maturing. A similar pattern was observed for the power output, while the OCV profiles reached the highest correlation coefficients (0.82–0.99) among all of the parameters, starting from the 2nd week of biofilm maturing. In contrast, following the 2nd week a positive correlation was observed for R int (0.35–0.76) and was decreasing with the increase of time; i.e. two neighbouring R int profiles (variables) were showing the highest correlation. These results suggest, that internal resistance was smoothly changing from one state to another due to the development of the biofilm growth. It can be also concluded, that current was the most dynamic biofilm parameter, while the OCV was the most stable one. Therefore, the data suggest that the power output of the MFCs was rather not affected by the redox potential of bacterial enzymes and resulting electrode overpotentials. This statement is supported by finding a positive correlation between power and current (0.51–0.93, excluding week 2) and negative correlation with R int (−0.38 to −0.91, excluding week 9), while not even a weak correlation was found between OCV and power output. Interestingly, the OCV was very well correlated with the R int during the first weeks of operation reaching R values of 0.77–0.86 following 2 weeks of biofilm maturing. Thus, R int was the main factor determining the potential of the MFCs and reaching high OCV values were not determining for the power performance. Fig. 6 Dynamic changes in correlation of electrochemical parameters reported for the whole population of MFCs in time. The numbers following each label correspond to week of operation, while colour intensity and diameter of the circle corresponds to the Pearson's correlation coefficient (r), as shown on colour key. Inverse correlation is reflected by negative R values, while positive correlation is reflected by positive r values. The following criteria were used to determine the strength of the correlation: weak (±0.1 to ±0.3), moderate (±0.3 to ±0.7), strong (±0.7 to ±0.9), very strong (±0.9 to ±1.0). Dashed lines indicate the regions of interest, which are discussed in the text. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 6 When simulated MPPT method was used to control the power performance of the MFCs in stage 2 the biofilm have readjusted its metabolism to the new conditions. Since during this period the R ext was varying to fit the R int , the dynamics of the changes in electrochemical biofilm properties have been partially inhibited. As a result, significant correlation was recorded for current throughout the second stage and reached between 0.40 and 0.89. The highest correlation coefficients were observed for the neighbouring time points and decreased with time, which suggest smooth (less dynamic) evolution of the biofilm from one state to another. In contrast, no significant correlation was observed when the changes of R int were followed in time (stage 2), while significant correlation for this parameter was observed in stage 1. This suggests that when the simulated MPPT procedure was applied, temporal and chaotic changes in R int occurred. Those changes which may have resulted from dynamic biofilm adaptation mechanisms coupled with ongoing changes in cathode performance. However, they were not reflected by the dynamic behaviour reported for current. Although strong negative correlation values were reported for R int and power in that period, power and current were also negatively correlated with original (stage 1) R ext where weak to moderate correlation was reported (R between −0.3 and −0.7). Therefore, as previously shown on Fig. 3 , Fig. 4 , the external resistance affects the properties and activity of biofilm, which thus becomes irreversibly affected by its preliminary environmental conditions. Nevertheless, the overall correlation between R ext and any other investigated parameter was the strongest in the first stage of operation. The highest (negative and positive) correlation coefficients were reported for 3rd week of operation, which we believe is the most significant period required to develop healthy and well-performing electroactive biofilm in MFC. Although MPPT method proved to be an efficient way of reducing the startup time and improving the performance of the MFCs, its application requires dedicated electronic circuit which affects the cost-efficiency of system [ 50 , 51 ]. In this study, the best performing biofilm was developed when matured under 50 Ω - an R ext approximately 5 times lower than their lowest R int , which we believe was a result of outstanding adaptive mechanisms of electroactive bacteria. Therefore, knowledge on internal resistance of the system prior to start-up is crucial for the field-applications, where the cost and available infrastructure may be the limiting factors."
} | 8,560 |
20954740 | null | s2 | 2,421 | {
"abstract": "Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces, has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species: A. trifasciata , N. clavipes , and N. cruentata . The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif, where every other amino acid is proline, and a glutamine-rich motif of 6-8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species, with particular conservation of the repetitive motifs. Northern blot analysis revealed that the mRNA is larger than 11 kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the piriform sequences form an ortholog group."
} | 340 |
31844749 | PMC6895581 | pmc | 2,422 | {
"abstract": "Coral-associated microbial communities contribute to a wide variety of useful roles regarding the their host, and therefore, the arrangement of the general microbiome network can emphatically impact coral wellbeing and survival. Various pollution sources can interfere and disrupt the microbial relationship with corals. Here, we adopted the bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP®) technique to investigate the shift of microbial communities associated with the mucus of the coral Stylophora pistillata collected from five sites (Marine Science Station, Industrial Complex, Oil Terminal, Public Beach, and Phosphate Port) along the Gulf of Aqaba (Red Sea). Our results revealed a high diversity in bacterial populations associated with coral mucus. Proteobacteria were observed to be the dominating phylum among all sampling sites. The identified bacterial taxa belong to the pathogenic bacteria from the genus Vibrio was presented in varying abundances at all sampling sites. Diversity and similarity analysis of microbial communists based on rarefaction curve and UniFrac cluster respectively demonstrated that there are variances in microbial groups associated with coral mucus along sites. The pollution sources among different locations along the Gulf of Aqaba seem to affect the coral-associated holobiont leading to changes in bacterial populations due to increasing human activities.",
"conclusion": "5 Conclusion In summary, the mucus of the coral S. pistillata from the GOA sampling sites exhibits a high diversity of associated bacterial communities. The pollution sources in different sites along the GOA affected the coral-associated holobiont, resulting in a shift of microbial communities due to increased human activity. Additionally, pyrosequencing techniques proved to be an excellent tool for the study of microbial community shifts in the marine environment. Further studies are required of coral reef-associated microbial communities in the GOA to investigate the microbial effect on coral health.",
"introduction": "1 Introduction Corals are diverse meta-organisms that provide an essential bio-habitat for many other marine species ( Mulhall, 2009 ), for instance, bacteria, Archaea, and microalgae (zooxanthellae) ( Rosenberg et al., 2007 ). Unfortunately, recent research indicates that more than 30% of coral reefs have been destroyed due to emerging diseases ( Harvell et al., 2007 ). Some of these diseases are attributed to coral-pathogenic microorganisms and other factors ( Rosenberg and Ben-Haim, 2002 ; Hughes et al., 2003 ). The microorganisms that are associated with reef–building, which is also called coral holobiont, have been widely investigated because of a real influence on coral physiology and well-being ( Carlos et al., 2013 ). Microorganisms linked to corals appear to strengthen the host's well-being by supplying a nutritional source and protecting the host from other pathogenic bacteria by the production of antimicrobial compounds ( Ritchie, 2006 ). These microbial communities exist naturally in various anatomic sites in the coral including the surface mucus layer and the coral tissues ( Bourne and Munn, 2005 ). Coral mucus contains various microorganisms that present benefits to their host by different means, including photosynthesis, nitrogen fixation, the delivery of nutrients, and inhibition of sickness ( Rosenberg et al., 2007 ). Moreover, the coral-microbial association is distinct from the surrounding habitat, containing species that are different from the free-living seawater microbes ( Rohwer et al., 2001 ; Carlos et al., 2013 ). Recently, many efforts have focused on characterizing these microbial communities and their specific metabolic role in coral health ( Lesser et al., 2004 ; Olson et al., 2009 ; Zhang et al., 2015 ; Zaneveld et al., 2016 ; Welsh et al., 2016 ). The shift of the structure and diversity of associated microbial communities could play a key role in adapting corals to rapid environmental changes ( Reshef et al., 2006 ). Recently, local and worldwide, ecological disturbances have intensely compromised the well-being of corals, which has thus influenced the entire biological community of the coral reef ( Harvell et al., 2007 ; Wilkinson, 1999 ; Green and Bruckner, 2000 ; Gardner et al., 2003 ; Pandolfi et al., 2003 ; Lesser et al., 2007 ). Recent reports show that 58–70% of coral reefs globally are endangered due to human activities ( Hughes et al., 2003 ; Weil et al., 2006 ). Different environmental stressors endanger the coral reef ecosystem in the Gulf of Aqaba (GOA). Many pollution sources threaten the Jordanian coast of GOA, including urban expansion, tourist developments, oil leakage, phosphate dust spillage during loading of phosphate minerals, and industrial and solid waste discharge ( Al-Horani et al., 2006 ). Consequently, these factors can shift the diversity of the coral-associated microbial communities which consequently affect the role of coral protection against pathogens. Coral mucus functions are essential to the coral, including providing a defense mechanism against environmental stresses (temperature and salinity in the coral surrounding water), in ciliary-mucus feeding, in sediment cleansing, a shield from UV radiation, and regulating surface bacterial growth ( Brown and Bythell, 2005 ). Different studies were conducted on the coral associated microbial communities in the GOA area using culture-based techniques for the identification and characterization of bacteria. For example, Lampert et al. (2006) characterized cultivable bacteria within the mucus of healthy Red Sea solitary coral Fungia scutaria . Jaber (2012) have also studied the mucus related bacterial communities of Stylophora pistallata and Galaxea fascicularis collected from the Jordanian coast of the GOA during different seasons. Furthermore, Telfah (2012) studied the differences in the microbial communities associated with Pocillopora damicornis and S. pistillata growing at various contaminated and uncontaminated sites in the Jordanian coast. In recent research, the bacterial communities associated with soft coral in the Red Sea were studied, and the dominant bacterial species which displayed different antimicrobial activities against various pathogens were characterized ( ElAhwany et al., 2015 ). The recent rapid advances in DNA sequencing technologies have given vital bits of knowledge into patterns of microbial diversity and function of coral reefs, improving our perception of how microorganisms impact the overall function and stability of the coral reef ecosystem ( Hussein et al., 2017 ; Meyer et al., 2014 ). Indeed, these technologies provide opportunities to explore these complex microbial associations and their response mechanisms to environmental stresses ( Kysela et al., 2005 ). In this study, we assessed the bacterial community structure associated with the mucus of the hard-coral S. pistillata inhabiting sites influenced by different sources of pollution in the GOA. Bacterial community structure was characterized using Roche GS FLX Titanium technology based on 454-pyrosequencing. Furthermore, we investigated the harmful bacterial species that affect coral health to highlight the health status of coral in the GOA.",
"discussion": "4 Discussion In this study, the microbial structure associated with coral mucus was analyzed from samples collected at five sampling sites on the Jordanian coast of the GOA using bTEFAP approach. To our knowledge, this is one of the first studies to characterize coral associated bacterial community at the GOA area using a new generation sequencing technique (NGS). Culture-independent results using the bTEFAP approach revealed a higher diversity of associated bacterial communities with corals than were previously reported using clone libraries or DGGE community profiling ( Jacob et al., 2017 ; Yakimov et al., 2006 ). The results obtained from this study were consistence with these findings of higher community diversity and abundance compared to the results obtained by Zhang et al. (2015) . The values of alpha diversity indices for our study was consistence with the study of Zhang et al. (2015) where they declared that community diversity was higher for an associated coral community using culture-free, 16S rRNA based techniques including DGGE community profiling at Luhuitou fringing reef, China (Shannon index 2.15–2.71; Simpson evenness 0.16–0.38), than what was previously reported with culture-based techniques. Most sequence readings in each sampling site were accounted for by a small number of OTUs at all sites, and this shows that there is a core bacterial community associated with mucus in all the study areas. These OTUs were categorized as alpha-, beta- and gamma- Proteobacteria, Bacilli, Flavobacteriia, and Verrucomicrobia, which were described as ubiquitous species from healthy and diseased coral using the clone-based library and pyrosequencing approaches ( Cárdenas et al., 2012 ). Changes in community composition from one site to another were probably due to changes in the presence and quantities of less abundant OTUs and changes in the abundance of dominant genera. Interestingly, 82% of identified OTUs could be assigned to species of the Proteobacteria phylum. These results were in agreement with those of Telfah (2012) ; Jaber (2012) , where they found that Proteobacteria was the predominant phyla in the mucus of S. pistillata samples in the same study area based on culture dependent methods. On the other hand, an average of 8 % of the total sequence reads was assigned for eight non-ubiquitous classes (i.e., Actinobacteria, Cyanobacteria, Bacteroidia, Cytophagia, Deinococci, Sphingobacteriia, and Clostridia). It can be postulated that these classes of bacteria belong to the 'rare biosphere', which makes them able to explain differences in community structure from one site to another, as indicated previously ( Sogin et al., 2006 ). The study of associated microbial communities over different pollution sources at the GOA revealed an influence of the anthropogenic pollutants on the coral S. pistillata mucus associated microbial communities. This is in agreement with the hypothesis that disturbances in the dynamic equilibrium of the coral microbiome may result in health deterioration ( Lesser et al., 2007 ). Previous studies revealed the fact that the structure of the microbial community is also determined by environmental factors as well as physiological conditions of the coral holobiont ( Hong et al., 2009 ). The highest number of OTUs (as indicated by the rarefaction curve, Fig. 2 ) was obtained for AQ1, which was selected as an uncontaminated site. The increased swimming and touristic activities beside the solid waste discharge at AQ4 resulted in the highest bacterial diversity among sites; this was clearly shown by the similarity obtained by UPGMA hierarchical clustering. This can be described by deterioration caused by stress of the host-microbial balance, leading to some uncontrolled appearance of irregular and harmful taxa ( Rosenberg and Kushmaro, 2011 ). For instance, a high prevalence of the pathogenic bacteria from the order Vibrionales at site AQ4 can be related to the increased disturbances and coral mucus aging as discussed by Glasl et al. (2016) . Host-species specificity decreased at the contaminated sites (AQ2, AQ3, and AQ5). However, loss of species specificity and higher similarity of coral microbial formations in corals suffering from contamination have been demonstrated previously ( Frias-Lopez et al., 2002 ; Cárdenas et al., 2011 ). Pollutants seem to negatively influence the balance between the principal microbial taxa that are linked to coral mucus. This trend has also been observed in the present study by the decrease in the bacteria belonging to the Hahellaceae family (species Endozoicomonas), Hydrogenophilaceae and Verrucomicrobiaceae. This corresponds to the microbial response due to stress in the other species and emphasizes the significance of the rare (opportunistic) bacterial biosphere, as indicated previously ( Sunagawa et al., 2010 ; Jessen et al., 2013 ). The compositional shift in the microbial communities of the stressed sites at the GAO exhibited an increased relative abundance of bacterial families such as Verrucomicrobiaceae, Oceanospirillaceae, and Flavobacteriaceae. These findings are consistent with a previous study attributing the expression of lesions in Porites astreoides colonies to the loss of Endozoicimonaceae and the proliferation of an opportunistic bacterial community ( Meyer et al., 2014 ). Furthermore, the coral mucus suffered from different pollution sources that exhibited increased relative abundances of bacterial families described as early colonizers of marine biofilms, such as Rhodobacteraceae and Oceanospirillacae, which was in agreement with the results obtained by O'Toole et al. (2000) . The diversity of microbial communities (as indicated by No. of OTUs, Table 2 ) was generally reduced at polluted sites as compared to the uncontaminated site. These findings do not agree with the observations of Jessen et al. (2013) , where exposure of Acropora hemprichii to simulated Red Sea stressors exhibited by overfeeding and excessive fishing has been shown to augment microbial diversity with time passing, notwithstanding lack of experimental control in research design. Moreover, the prevalence of diverse bacterial species also seems to be a typical characteristic of diseased coral, as shown, for instance, in White Plague stressed corals Diploria strigosa and Siderastrea siderea ( Cárdenas et al., 2011 ). This was obvious for the increased abundance of pathogenic species of the family Vibrionaceae and Pseudomonadaceae, especially at sites AQ4 and AQ5. The bacterial communities was characterized by the presence of several abundant nutrient cycling OTUs. This can be related to higher levels of nutrients in the polluted sampling areas. Hence, species of the genus Vibrio are reported to use sulfur compounds produced by coral as a cue to target stressed corals ( Garren and Azam, 2010 ). Some OTUs belonged to genera of the order Rhizobiales (Bradyrhizobium lupine), and Pseudomonadales (pseudomonas sp.) at the sites AQ4 and AQ5 subjected to sedimentation and local sewage resulting from the extensive industrial (phosphate transportation at AQ5 site) and solid waste disposal, and tourist activities (AQ4). Moreover, these OTUs include taxa that are potentially involved in cycling nitrogen ( Lema et al., 2012 ). Furthermore, some OTUs (i.e. OTU3) belonged to the human skin microbiome, mainly skin topographical and temporal forearm clones ( Kong et al., 2012 ) presented at AQ3, AQ4, and AQ5 represented by anthropogenic influences at these sites. The microbial community at site AQ1 contains OTUs of the order Oceanospirillales ( Alcanivorax sp.) and Novosphingobium (Sphingobacteriia) that have been found in environments contaminated by crude oil ( Tang et al., 2012 ; Wang et al., 2014 ) and contribute to the quality deterioration of sulfur compounds like dimethyl sulfoniopropionate ( González et al., 2003 ). These OTUs can be related to accidental oil spills from the adjacent area or result from boating activities. The shift of coral mucus associated with microbial community structure in the sampling sites is affected by pollution sources and can be interpreted as coral holobiont acclimatization methods following changes in environmental statuses that enhance the probiotic hypothesis ( Reshef et al., 2006 ). Nevertheless, the changes pose a threat to coral holobionts in related microbial structure, in which the non-stop exposure to stressors is not yet visible ( Rosenberg and Kushmaro, 2011 ). The shift of microbial communities under local pollution sources may represent a shift in favor of the disease due to loss of microbial communities specific to coral host or the increase in alpha diversity ( Ziegler et al., 2016 )."
} | 4,009 |
27788197 | PMC5082814 | pmc | 2,423 | {
"abstract": "Marine sponges are important members of coral reef ecosystems. Thus, their responses to changes in ocean chemistry and environmental conditions, particularly to higher seawater temperatures, will have potential impacts on the future of these reefs. To better understand the sponge thermal stress response, we investigated gene expression dynamics in the shallow water sponge, Haliclona tubifera (order Haplosclerida, class Demospongiae), subjected to elevated temperature. Using high-throughput transcriptome sequencing, we show that these conditions result in the activation of various processes that interact to maintain cellular homeostasis. Short-term thermal stress resulted in the induction of heat shock proteins, antioxidants, and genes involved in signal transduction and innate immunity pathways. Prolonged exposure to thermal stress affected the expression of genes involved in cellular damage repair, apoptosis, signaling and transcription. Interestingly, exposure to sublethal temperatures may improve the ability of the sponge to mitigate cellular damage under more extreme stress conditions. These insights into the potential mechanisms of adaptation and resilience of sponges contribute to a better understanding of sponge conservation status and the prediction of ecosystem trajectories under future climate conditions.",
"conclusion": "Conclusions Predicting the impact of climate change on important marine organisms, such as sponges, necessitates the elucidation of the cellular and molecular processes that contribute to their responses to elevated temperatures. This study represents the first transcriptome-wide survey of the sponge response to thermal stress. Using a high-throughput sequencing approach, we identified the potential mechanisms enabling H . tubifera to survive conditions of thermal stress and observed the different responses that are triggered at different stages of exposure. In H . tubifera , immediate stress response includes induction of heat shock proteins, antioxidants, and genes involved in signal transduction and innate immunity pathways while prolonged exposure to thermal stress affects expression patterns of genes involved in cellular damage repair, apoptosis, signaling and transcription. Differential deployment of a diverse repertoire of genes may allow the sponge to fine-tune its response to local conditions in the environment that they typically encounter. Thus, H . tubifera , which is normally located in shallow reef flats and is exposed to variable temperatures, may have a more robust response to temperature fluctuations compared to sponges found at deeper depths with colder and more stable temperatures. A similar phenomenon has been reported for several coral species, where individuals living in habitats that experience highly variable conditions exhibit greater expression of protective and metabolic genes [ 51 , 52 ]. For a more comprehensive understanding of sponge tolerance to climate change-related stressors, further comparative studies should be conducted on diverse species of marine sponges. The diversity within the sponge phylum and the relative plasticity of sponge cellular elements represent a treasure trove of gene innovations that may provide insights into the various pathways that have evolved to contribute to animal resilience [ 53 ]. It is likely that differences in ecological and physiological features of different sponges, and even their different life stages, will reflect variations in thermal tolerance and resilience that may have downstream effects on populations in areas where seawater temperatures begin to exceed tolerable temperatures. As sponges perform diverse roles in the marine ecosystem, changes in sponge distribution and abundance will have implications on reef functions. Thus, knowledge of gene expression responses in relation to organism physiology and health will support better assessment of sponge conservation status and contribute to the ability to predict reef trajectories under future climate conditions. Given the overwhelming evidence of coral decline due to climate-associated stressors, only those remarkably resistant taxa, such as sponges, are likely to survive the increasingly stressful marine environment.",
"introduction": "Introduction Global mean temperature will continue to rise over the 21 st century with the best estimates for global sea surface temperature (SST) increasing in the range of 1–3°C [ 1 ]. This increased SST may have deleterious impacts on many marine invertebrates. In fact, mass coral bleaching events triggered by elevated seawater temperatures have resulted in significantly reduced coral cover throughout the tropics [ 2 ]. This decrease in coral cover can result in changes in benthic reef communities [ 3 ], which may allow other species, such as algae and sponges, to increase in abundance. Sponges are one of the earliest multicellular animal groups [ 4 ]. Members of this phylum display remarkable ecological adaptability, having integrated into diverse marine ecosystems through the development of complex physiological and chemical properties [ 5 ]. Sponges play important roles in the functioning of these ecosystems [ 6 ]. Despite the diversity within the sponge phylum, data on sponge conservation status is still lacking [ 7 ]. Out of the thousands of known sponge species, very few are currently listed as threatened, suggesting that sponges are highly adaptable to environmental stressors, such as elevated temperatures, ocean acidification, sedimentation, and microbial pathogens [ 7 ]. In fact, compared to other reef taxa, marine sponges have the potential to be resilient to large-scale thermal stress events. Recent studies have reported that sponges are more tolerant to increased SST, with larval dynamics, ecological functions and physiological processes unaffected by increases in water temperature [ 8 – 10 ]. For example, the growth and survival of several Carribean sponges remained unaffected by exposure to thermal stress [ 11 ]. Furthermore, the sponge assemblage in Bahia, Brazil, did not change between pre- and post-El Niño Southern Oscillation (ENSO) years [ 12 ]. In contrast, some studies have described the negative effects of elevated temperature on different sponge species. For example, adult colonies of R . odorabile were found to be highly sensitive to thermal stress at 32°C [ 9 ] while severe sponge die-off due to cyanobacterial decay was triggered by elevated temperatures in the Mediterranean sea [ 13 ]. Therefore, it is important to recognize that different sponge species may have variable responses to environmental perturbations, specifically to thermal stress. The molecular mechanisms underlying sponge responses to thermal stress are poorly understood, with most studies focusing on the effect of temperature on the sponge-associated microbial community. In one example, 454 pyrosequencing of the 16S rRNA metagenome revealed that exposure to 31°C had no effect on the bacterial biosphere within the Great Barrier sponge Rhopaloeides odorabile [ 14 ]. Likewise, in the Mediterranean Sea sponge Ircinia spp ., neither thermal stress combined with food shortage nor large fluctuations in temperature and irradiance disrupted the stability of the sponge-bacteria partnership [ 15 , 16 ]. The importance of cell-cell signaling genes in the maintenance or breakdown of the sponge-bacteria interaction during thermal stress events has been explored to some extent [ 17 ]. Targeted studies on gene expression have demonstrated the upregulation of heat shock protein 70 in the Carribean sponge Xestospongia muta upon exposure to elevated temperature [ 18 ]. Expression profiling by multiplexed reverse-transcription quantitative PCR (mRT-qPCR) showed that R . odorabile larvae are remarkably able to withstand seawater temperatures up to 9°C above normal [ 9 ]. The apparent resilience of some sponge species and the sensitivity of others highlights the need to understand the genomic basis of sponge responses to environmental stressors and how they are able to adapt to rapidly changing ocean conditions. High-throughput transcriptome sequencing is a powerful tool that allows sensitive and high-resolution detection of a wider dynamic range of expression levels in contrast to other commonly used molecular approaches, such as quantitative PCR, multiplex reverse-transcription quantitative PCR, and microarrays [ 19 ]. Global transcriptome analysis can reveal how organisms respond to external stimuli and stressors by detecting changes in gene expression dynamics. This plasticity of gene expression underlies the ability of organisms to adapt to changing environmental conditions. In fact, sequencing of the genome of model demosponge, Amphimedon queenslandica , revealed the presence of a diverse genetic toolkit, or a set of conserved regulatory genes, that can integrate various signaling pathways and allow the organism to rapidly respond to its environment [ 20 ]. In this study, we performed a transcriptome-wide analysis to elucidate the changes in gene expression that occur when a sponge is subjected to different levels of thermal stress. In particular, we focused on the differential expression of genes involved in protein folding, oxidative stress response, immune response, signal transduction, transcriptional regulation, apoptosis, and tissue morphogenesis. The findings of this study unveil key processes that underlie sponge tolerance to thermal stress, which will gain importance as ocean temperatures continue to rise with the changing climate.",
"discussion": "Results and Discussion As global warming continues to raise ocean temperatures, marine ecosystems are placed at risk. However, some marine organisms, including sponges (Porifera), thrive in naturally warm environments and can tolerate high temperatures. Studies on these organisms will contribute to our understanding of the mechanisms that confer resilience to thermal stress. Thus, to elucidate the transcriptome dynamics underlying the sponge thermal stress response, we focused our study on the demosponge, Haliclona tubifera , found native to shallow reef flats in Bolinao, Pangasinan, Philippines. Based on regular monitoring by the Bolinao Marine Laboratory, sea surface temperatures in this region range from 25°C to 32°C with an annual mean temperature of 28.89±0.90°C. Sponges in this area are of great interest in exploring the determinants of ecological success, particularly in terms of tolerance to a wide range of temperatures. H . tubifera belongs to order Haplosclerida, which is the largest and most diverse group within class Demospongiae of phylum Porifera (sponges). This tubular pink sponge is found in association with branching corals in shallow water reef flats at depths of 1–2 meters ( Fig 1A ). Because of its shallow habitat, this sponge species is subjected to widely fluctuating temperatures on a daily basis. The reference transcriptome used in this study was previously assembled de novo , and contains 50,067 non-redundant transcripts, which translate into 18,000 peptides [ 21 ]. 10.1371/journal.pone.0165368.g001 Fig 1 Global transcriptome profile of adult colonies of H . tubifera exposed to thermal stress. (A) H . tubifera is a soft, pink or brownish, tubular sponge found in association with coral skeletons in shallow water reef flats. (B) Correlation of overall gene expression profiles for duplicate samples of sponges exposed to different thermal regimes (Pearson correlation coefficient, r). The correlation is based on counts per million (CPM) of reads mapping to each transcript. Only transcripts with CPM >10 in at least 2 samples were included. (C) The number of differentially expressed genes specific to or common between different treatments. Differential expression analysis was conducted on duplicate samples for each experimental treatment. Genes were considered differentially expressed if they were up or downregulated by greater than 4-fold relative to the controls with an adjusted p-value <1x10 -5 . Scatter plots of the log2 fold changes in expression relative to controls at 29°C for differentially expressed genes that are common between (D) 34°C 4hr vs 34°C 12hr, (E) 34°C 4hr vs 32°C 12hr, and (F) 34°C 12hr vs 32°C 12hr samples. Points in the upper right quadrant are upregulated transcripts while points in the lower left quadrant are downregulated transcripts. Red dots above the diagonal represent transcripts with a greater magnitude of change in the sample on the y-axis while blue dots are transcripts exhibiting a greater magnitude of change in the sample on the x-axis. Enrichment of genes with particular distributions between treatment pairs was estimated using Fisher’s exact test (p-values shown). Global expression pattern Although H . tubifera is regularly exposed to fluctuating temperatures in its shallow water habitat, different levels of thermal stress elicited observable changes in its gene expression profile. The expression profiles of sponges subjected to acute short-term thermal stress (34°C for 4 hours) showed higher correlation with controls maintained at 29°C while the transcriptome profiles of sponges subjected to longer thermal exposure (32°C for 12 hours and 34°C for 12 hours) were more similar to each other ( Fig 1B ). Although some sample replicates showed larger variability, principal component analysis was still able to differentiate the transcriptome profiles of sponges subjected to different temperature (Fig A in S1 File ). Our results show that prolonged exposure to temperatures much higher than the average encountered by the sponge triggers substantial changes in gene expression that may influence cellular characteristics or behavior. Differential expression analysis detected a total of 1,584 unique transcripts, referred to here as genes, exhibiting a significant change in expression across all treatments ( Fig 1C ). These results suggest that the sponge can rapidly deploy cellular mechanisms that support tolerance to increased temperatures. Most of the differentially expressed genes were up or downregulated by greater than four-fold relative to the controls (Table A in S1 File ). The greatest number of differentially regulated genes were observed between the control (29°C) and samples heated at 34°C for 12 hours (1,010 genes). 294 genes remained differentially regulated in all samples subjected to elevated temperature while 178 differentially expressed genes were shared between samples exposed to 34°C for 4 hours and at 32°C for 12 hours. Only 32% (505 genes) of differentially expressed genes have homology to proteins in the UniProt database (Table B in S1 File ). Of the genes with no UniProt matches, 644 exhibit similarity to sequences in the A . queenslandica reference genome (Table B in S1 File ). Further studies are needed to elucidate the functions of these unannotated genes, which may include non-coding RNAs expressed from polyadenylated transcripts [ 22 , 23 ], as well as species-specific genes that are responsive to thermal stress. By comparing the log2 fold change of genes that are commonly differentially expressed under the various treatments, we were able to determine the number that show a greater magnitude of change in expression under certain temperature regimes. Pairwise comparisons revealed that between the samples subjected to 34°C, more genes exhibited a greater magnitude of upregulation at 4 hours compared to 12 hours of exposure (142 vs. 104 genes), whereas more genes were downregulated in samples maintained at 34°C for 12 hours than for 4 hours (116 vs. 85 genes) ( Fig 1D ). Between samples exposed to different degrees of thermal stress, more genes were either up or downregulated in the samples exposed to 34°C for 4 or 12 hours relative to the samples maintained at 32°C for 12 hours ( Fig 1E and 1F ). This indicates that acute thermal stress at 34°C, regardless of duration, results in a greater change in gene expression levels compared to sublethal exposure at 32°C, which is still within the temperature range of the sponge habitat. Downregulated sequences represent genes that are normally maintained at high levels in the sponge, which may include transcripts encoding proteins that form the first line of defense or transcripts with housekeeping functions that are generally energy intensive. Upregulated sequences are mostly stress-induced genes. Interestingly, thermal stress in H . tubifera resulted in more downregulation than upregulation of genes (Table A in S1 File ). This finding is consistent with transcriptome-wide studies in other organisms, suggesting that widespread downregulation is a conserved phenomenon under stressful conditions [ 24 – 26 ]. This may be an adaptive mechanism that reflects the redirection of energy and resources towards the maintenance and repair of cellular machinery. Enriched functional groups Gene ontology (GO) enrichment analysis revealed that exposure of H . tubifera to elevated temperatures resulted in the differential expression of genes involved in the organismal stress response ( Fig 2 ). Protective mechanisms that are enriched under acute short-term stress include antioxidant activity, toll-like receptor (TLR) signaling pathway, and innate immune response activation. Signaling mechanisms that are enriched include calcium-mediated signaling, cellular ion homeostasis, messenger-mediated pathways, transporter activity, and microtubule-based movement. This suggests that the sponge responds immediately to stress exposure by inducing signaling cascades that initiate various pathways involved in the cellular stress response and by producing transcripts that encode proteins that protect the cell from damage. 10.1371/journal.pone.0165368.g002 Fig 2 Gene ontology (GO) analysis for gene groups that are up or downregulated by greater than 4-fold under various temperature regimes. Enrichment p-values for selected terms are shown. Only GO terms with a p-value <0.05 (Fisher’s exact test) were considered significantly enriched. Longer exposure to elevated temperature (34°C for 12 hours) resulted in the differential expression of genes related to the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) , protein refolding, tissue development, and proteolysis. The CSN is a protease complex with a key role in the DNA-damage response, cell-cycle control, apoptosis, and gene expression [ 27 , 28 ]. The activation of these functions indicate that sponge cells may have already sustained damage under these conditions. Genes related to tissue morphogenesis, extracellular matrix, cell cycle and cell adhesion were enriched only in samples maintained at 32°C for 12 hours ( Fig 2 ). Interestingly, stress response-related genes, such as glutathione transferase ( GST ) and the chaperones heat shock protein 90 ( Hsp90 ) and Bcl2 -Associated Athanogene 3 ( BAG3) , were also upregulated at 32°C (Fig B in S1 File ). This data suggests that exposure to the sublethal temperature of 32°C may be promoting tissue growth while, at the same time, activating the expression of protein folding chaperones that prepare the sponge for further encounters with environmental stressors. Effective acclimatization to sublethal temperature may increase the ability of the sponge to mitigate cellular damage upon exposure to a more extreme temperature. Transcriptome-wide analysis in H . tubifera reveals insights into the mechanisms that are regulated in response to variable intensity and duration of thermal stress. This analysis showed that stress triggers a series of processes that function to maintain cellular homeostasis at all stages of stress exposure. Immediate responses, which are apparent in sponges subjected to short-term stress, involve modulating the oxidative response, immune response, and intracellular signaling. These responses are designed to prevent or minimize cellular damage at the onset of thermal stress. Under prolonged exposure, where the sponge may have already sustained some tissue damage, genes that are activated include those associated with protein refolding, tissue growth, cellular repair, and apoptosis. Induction of such functions may reflect sponge health, the ability to tolerate higher temperatures, and the potential for recovery and survival under continued stress conditions. It is interesting to note that exposure to sublethal temperature activates the expression of protein folding chaperones that provide sponge cells with some protection against extended or intensified stress conditions. Network responses to stress As is evident from the results of gene ontology enrichment, the cellular stress response involves the coordination of multiple pathways that are linked through protein-protein interactions. The sponge possesses homologs of many of the genes in these pathways but it is not known whether they also function in the same manner as their homologs in other organisms. Thus, to determine if these genes potentially retain similar functions in the sponge stress response, the sponge gene expression data was overlaid upon a protein interaction network based on well-curated annotations for human genes ( Fig 3 and Table C in S1 File ). Only the samples exposed to 34°C for 4 hours and 12 hours were included in this analysis as more robust responses were observed at this temperature than at the sublethal temperature of 32°C. This analysis enables the visualization of coordinated control or co-regulation, an indicator of conservation of function for gene homologs and of co-functionality for interacting genes [ 29 – 31 ]. In general, we observed that sponge homologs of genes within the stress response-related network, including genes that function in oxidative response, protein folding, immune response, and apoptosis, exhibit coordinated expression patterns that are consistent with known functions of their human homologs and that are indicative of their cooperative function in the sponge thermal stress response. Whether the sponge stress response-related interaction network also includes sponge-specific proteins different from those in the human network remains to be determined. 10.1371/journal.pone.0165368.g003 Fig 3 Relative expression of sponge homologs of genes in the stress response-related protein network at (A) 4 hours and at (B) 12 hours of exposure to 34°C. The network shown is based on the curated human protein interaction network. Relative expression is shown as the sum of the average fragments per kilobase per million (FPKM, log2 transformed) in each treatment relative to the control at 29°C for all genes with a best blastx hit matching the human gene in the network (blue, low; red, high; gray, no match in H . tubifera ). Node size corresponds to the number of genes with the same UniProt annotation. Not surprisingly, we found that the relative expression of heat shock protein 70 ( Hsp70) and other protein folding genes was strongly upregulated in thermally stressed H . tubifera ( Fig 3 and Fig B in S1 File ). Hsp70 proteins are one of the most highly conserved groups of heat shock proteins [ 32 ]. They ensure the coordinated regulation of protein translocation processes, limit cellular damage by preventing aggregation of denatured proteins [ 33 ], and refold stress-denatured proteins [ 34 ]. We also found that Hsp90 and ubiquitin were upregulated (Fig B in S1 File ). This is in contrast to observations in adults of the Great Barrier Reef sponge, R . odorabile , where both Hsp90 and ubiquitin expression were downregulated under thermal stress [ 9 ]. This difference in response may indicate that R . odorabile has greater sensitivity to thermal stress, as the decrease in expression may be due to widespread inhibition of gene transcription accompanying extensive cellular damage. Exposure to thermal stress can induce the generation of reactive oxygen species, which can cause damage to tissues [ 35 ]. To counteract this, the organism produces genes that encode proteins with antioxidant activity. In H . tubifera , oxidative response genes such as thioredoxin ( TXN ), superoxide dismutase 1 ( SOD1 ), and peroxiredoxin ( PRDX ) exhibited an increase in relative expression at higher temperatures ( Fig 3 and Fig B in S1 File ). Oxidative stress response genes have also been identified as differentially expressed in corals subjected to thermal stress [ 36 , 37 ]. The rapid response of antioxidant mechanisms in H . tubifera suggests that the sponge is able to cope with tissue damage that may be caused by oxidative stress associated with elevated temperatures. Increased expression of heat shock proteins triggered by thermal stress can activate immune response-related genes, including toll-like receptors [ 38 ]. The toll-like receptor 1 ( TLR1 ) and the mediator of TLR signaling, myeloid differentiation primary response gene 88 (Myd88) , exhibited increased relative expression under prolonged exposure to 34°C ( Fig 3 ). TLRs initiate signal transduction pathways, which then induce the innate immune response and are important in the recognition of invading microbial pathogens [ 38 ]. These members of the innate immunity pathway, in turn, interact with genes in the intrinsic and extrinsic apoptosis pathways. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) , which serves as an inhibitor of TLR responses and apoptosis [ 39 ], was upregulated under stress. Similarly, the tumor necrosis factor receptor-associated factors (TRAFs), which act as adaptor proteins for a variety of receptors that regulate cell death and responses to stress [ 40 ], increased in expression upon exposure to 34°C. We also observed upregulation of the apoptotic protease activating factor 1 (APAF1) , which activates initiator caspases. Of the initiator caspases [ 41 ], only CASP2 was upregulated, whereas CASP8 and CASP9 were downregulated. Initiator caspases cleave and activate executioner caspases, such as CASP3 and CASP7 , both of which were upregulated in the sponge. Executioner caspases degrade cellular components [ 41 ]. Induction of caspase-like activity was similarly observed during the early stages of thermal treatment in the sea anemone, Anemonia viridis [ 42 ]. Interestingly, inhibitors of apoptosis, such as X-linked inhibitor of apoptosis protein (XIAP) and B-cell lymphoma 2 (Bcl2) , as well as the anti-apoptotic BAG3 chaperone, were upregulated upon exposure to 34°C at 12 hours. Despite the increase in expression of APAF1 and executioner caspases, the downregulation of some initiator caspases, as well as the upregulation of known apoptosis inhibitors, suggests that the induction of cell death is tightly regulated in H . tubifera under stressful conditions. Thermal stress effects on regulatory pathways Shifts in gene expression patterns can be attributed to changes in transcriptional activity. This is partly controlled by the abundance of different transcription factors that regulate the expression of specific genes in response to stimuli. We found that specific transcription factor groups exhibit distinct patterns of expression during thermal stress exposure, suggesting that these groups of factors play central roles in controlling the expression of genes commonly required to deal with the effects of increased temperatures. The most obvious trend observed for transcription factor families is an increase in expression at higher temperature with greater upregulation under prolonged exposure, as is evident for the bZIP, Tbox, ETS, bHLH, and forkhead families ( Fig 4 ). In contrast, most HMGbox and homeobox family transcription factors were not responsive to thermal stimuli. While the specific gene targets of these transcription factor families remains to be determined, the increase in their expression during stress correlates with the upregulation of stress-related genes in the sponge. This suggests that while the responses observed upon acute exposure at 4 hours may be mainly due to rapid cellular responses involving mRNA turnover and protein translation or modification, longer term stress can influence global changes in the transcriptome through shifts in regulatory factor concentrations. 10.1371/journal.pone.0165368.g004 Fig 4 Relative expression of regulatory genes in H . tubifera during thermal stress exposure. Relative expression of genes encoding transcription factors (bZIP, T-box, ETS, homeobox, and forkhead), G-protein coupled receptors (rhodopsin, glutamate, and secretin GPCRs), and scavenger receptors. Relative expression was computed as the log2-transformed average FPKM value for each gene under each treatment normalized to the average of expression across treatments (blue, low; red, high). Each column represents one treatment (A, 29°C; B, 34°C, 4hr; C, 34°C, 12hr; D, 32°C, 12hr). Enrichment of genes under particular treatments was estimated using Fisher’s exact test (p-values shown). The interaction between an organism and its environment is mediated by transmembrane receptors. G-protein coupled receptors (GPCRs) and scavenger receptors form two of the largest gene families in sponges and are thought to provide the organism with a highly sophisticated repertoire of sensors to monitor its surroundings [ 20 , 43 ]. Upregulation of a subset of rhodopsin GPCRs at 12 hours of exposure at either 34°C or 32°C ( Fig 4 ) suggests a link between thermal and light stress, which can be expected because intense solar irradiation typically correlates with increased sea surface temperatures. As such, the sponge may be able to trigger early protective responses in anticipation of seawater warming when it senses a rise in light intensity. Some specific glutamate GPCRs were upregulated upon exposure to 34°C. Glutamate GPCRs, such as the GABA-like and GRM-like receptors, may be involved in controlling the contraction of sponge canal system to regulate seawater filtration rate in response to environmental stimuli [ 44 ]. GABA receptors can either trigger canal contraction like in the demosponge, Tethya wilhelma , or they can be inhibitory, as in the freshwater sponge, Ephydatia muelleri [ 44 , 45 ]. Secretin GPCRs were downregulated at 4 hours of exposure to 34°C but increased in expression after 12 hours. These GPCRs are closely related to the adhesion GPCR family and their upregulation may be an adaptation that allows for better cell adhesion and the maintenance of sponge tissue integrity. Changes in environmental conditions, such as a rise in temperature, may lead to the proliferation of opportunistic microorganisms and pathogens whose growth and virulence is favored by warmer temperatures [ 46 , 47 ]. In general, we found that scavenger receptors, which may be important for the discrimination between symbiotic and food bacteria [ 48 ], were downregulated under acute thermal stress ( Fig 4 ). Animal lectins, which function in host defense by promoting aggregation of microbial cells for efficient phagocytosis [ 49 ], were also downregulated (Fig B in S1 File ). Similarly, strong non-self recognition challenges, such as the selection of algal and bacterial endosymbionts, has been reported to cause a drop in the expression levels of lectins in corals during post-settlement [ 50 ]. In H . tubifera , changes in the population of cell surface receptors may serve to protect the sponge during thermal stress by aiding in the recognition and clearance of potential pathogens. However, despite the downregulation of these receptors, further exposure to stress and an increase in the abundance and virulence of pathogens may override the sponge immune system, eventually resulting in pathogen invasion. It is important to note that environmental perturbations that cause shifts in the sponge microbial assemblage or loss of groups of bacteria with critical metabolic functions will have a negative impact on the overall health of the sponge holobiont [ 17 ]."
} | 7,994 |
21833344 | null | s2 | 2,425 | {
"abstract": "Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality."
} | 193 |
21833344 | null | s2 | 2,426 | {
"abstract": "Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality."
} | 193 |
25691594 | PMC4337573 | pmc | 2,428 | {
"abstract": "ABSTRACT Bacteria are extremely versatile organisms that rapidly adapt to changing environments. When bacterial cells switch from planktonic growth to biofilm, flagellum formation is turned off and the production of fimbriae and extracellular polysaccharides is switched on. BolA is present in most Gram-negative bacteria, and homologues can be found from proteobacteria to eukaryotes. Here, we show that BolA is a new bacterial transcription factor that modulates the switch from a planktonic to a sessile lifestyle. It negatively modulates flagellar biosynthesis and swimming capacity in Escherichia coli . Furthermore, BolA overexpression favors biofilm formation, involving the production of fimbria-like adhesins and curli. Our results also demonstrate that BolA is a protein with high affinity to DNA and is able to regulate many genes on a genome-wide scale. Moreover, we show that the most significant targets of this protein involve a complex network of genes encoding proteins related to biofilm development. Herein, we propose that BolA is a motile/adhesive transcriptional switch, specifically involved in the transition between the planktonic and the attachment stage of biofilm formation.",
"introduction": "INTRODUCTION Cellular stress can induce substantial physiological and molecular adaptations to ensure survival. The bolA gene is widespread in nature and was initially characterized in Escherichia coli as a stationary-phase gene that promotes round morphology when overexpressed ( 1 ). Later, it was shown that BolA can also be induced during early growth phase if cells are challenged by several forms of stress, such as heat shock, acidic stress, oxidative stress, and sudden carbon starvation ( 2 ). In addition, BolA modulates cell permeability ( 3 ) and is involved in biofilm formation not only in E. coli ( 4 , 5 ) but also in Pseudomonas fluorescens ( 6 ). Interestingly, the heterologous expression of the microalgae Chlamydomonas reinhardtii BolA in E. coli also favors the development of biofilms ( 7 ). Biofilm-associated cells display increased resistance to many toxic substances, such as antibiotics and detergents; this is also observed when bolA is overexpressed ( 3 ). The expression of bolA is tightly controlled. It is regulated both at the transcriptional and posttranscriptional levels ( 8 – 11 ). In optimal growth conditions, bolA transcription is under the control of the housekeeping sigma factor σ 70 , but in stationary phase or under stress, bolA is preferentially transcribed in the presence of sigma factor σ s ( 2 , 11 ). Direct binding of H-NS or phosphorylated OmpR to the promoter region of bolA was shown to repress its expression ( 12 , 13 ). The posttranscriptional regulation of bolA mRNA levels involves both RNase III and poly(A) polymerase (PAPI) ( 10 , 14 ). BolA-induced alterations in cell morphology are mediated by a complex network that integrates PBP5, PBP6, and MreB ( 3 , 15 – 17 ). BolA was shown to interact with the promoter region of dacA , dacC , and mreB ( 16 , 17 ). This protein is also involved in the regulation of OmpF/OmpC balance, changing bacterial permeability ( 3 ). Surprisingly, an overall picture of the specific targets and global effects of BolA is still missing. In this report, we demonstrate by chromatin immunoprecipitation sequencing (ChIP-seq) that BolA is indeed a new transcription factor that directly binds to the promoter region of a number of important genes. Complementing the ChIP-seq experiments with transcriptomic analysis gave a complete overview of the BolA regulatory network and its relevant effects on gene expression. The expression analyses were supported by in vivo and in vitro phenotypic results, showing that BolA regulates flagellar and curli biosynthesis pathways. Together, our results unravel the mechanism of action of BolA. They explain many of its pleiotropic effects and give new evidence on the impact of BolA in cell motility, significantly affecting flagellar and curli biosynthesis pathways. This involves the downregulation of the master regulator FlhDC coupled with the synthesis of curli and fimbriae and the increased production of the biofilm matrix. These results provide an important advance in the functional characterization of the BolA protein, unraveling its determinant role in the coordination of the flagellar biogenesis and biofilm formation. Therefore, this study is of utmost importance for the comprehension of genetic and molecular bases involved in the regulation of cell motility and biofilm formation and may contribute to future industrial and public health care applications.",
"discussion": "DISCUSSION BolA has been described as a protein important for survival in late stages of bacterial growth and under harsh environmental conditions ( 2 , 3 ). High levels of BolA in stationary phase and stress have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator ( 18 ). In this report, we have investigated the role of BolA as a transcription factor at a global level. We have used ChIP-seq technology to produce the first chromosome-wide direct analysis of DNA binding in vivo by E. coli BolA. Our results show that BolA directly binds to a significant number of gene sequences, from the 5′-end region to the open reading frames. Moreover, even though a blurred asymmetry in read densities between strands was observed, BolA seems to behave more as a transcription factor than a nucleoid-associated protein. The blurred effect may be related to complexes that this protein possibly form when interacting with the DNA. These results firmly support the role of BolA as a transcription factor, as suggested in previous studies ( 16 , 17 ). BolA was previously associated with positive and negative regulation of gene expression ( 15 , 17 ), an observation that prompted us to examine the overall transcription in the bacterial cells. Transcriptome analysis confirmed the dual role of BolA as an activator or a repressor of transcription. ChIP-seq and transcriptome data suggest BolA direct effects are related to the repression of flagellum-associated genes and the induction of genes related with the TCA cycle. Both flagellar and TCA cycle genes have direct consequences on bacterial motility ( 25 , 36 ). Flagella and motility have been the center of research attention of many research groups due to their roles in virulence and biofilm development ( 22 , 27 , 37 ). The remarkable effect of the presence of elevated levels of BolA in the regulation of flagellar biosynthesis and TCA cycle enzymes was observed not only at the transcriptional level but also at the phenotypic level. With the overexpression of BolA, a reduction of the swimming capacity of E. coli was observed. The gradual reduction of the bacterial swimming capacity concomitantly with the increment of BolA strongly suggests a dose effect of this protein. This observation shows cells must exercise tight control over BolA levels. The reduction of swimming can be associated with a variety of mechanisms, and the two most studied are the assembly and the rotation of flagella ( 38 ). Our results suggest BolA affects the assembly of flagella. We indeed observe a repression of genes which are flagellar transcriptional regulators and several others that encode the hook proteins and the hook filament junction proteins. Additionally, using immunofluorescence, in the strains overexpressing BolA, the flagella were practically not detected, conversely to what was observed in the wt, supporting the role of BolA in flagellar repression. Flagella are required for the initial stages of biofilm formation in E. coli ( 39 ). Even though there is a reduction in flagellar biosynthesis with the concomitant overexpression of BolA, it is plausible to hypothesize that the presence of adhesins could significantly contribute to this process, since flagellar motility was described to be dispensable for the initial adhesion in strains overexpressing adhesins ( 40 ). In this regard, the reduced motility of the strains overexpressing BolA was most likely compensated by the overexpression and production of fimbria-like adhesins. Moreover, under the conditions tested, there are evidences of differential regulation of an elevated number of genes associated with LPS biosynthesis, polysaccharide production, and membrane-associated enzymes. It was described that E. coli K-12 does not produce cellulose due to a mutation in the bcsA gene ( 32 , 33 ). However, our strains overproducing BolA showed the characteristic Calcofluor binding phenotype. This could indicate binding to other exopolysaccharides, such as colanic acid or poly-1,6-GlcNAc (PGA). Furthermore, extracellular DNA, protein, and sugar levels were all augmented in the overexpression strain compared to those in the wt condition. Noticeably, all the components mentioned above are important for the assembly and composition of the biofilm matrix, which protects bacteria in the biofilm community ( 28 , 34 ). Work previously performed in our lab has shown that in E. coli , BolA expression is correlated with the capacity to form biofilm ( 4 ). These new results have significantly contributed to understanding the mechanism by which BolA overexpression contributes to biofilm formation and to extending the role of BolA to other related pathways. For instance, our results showed BolA directly inhibits the expression of ompX , a gene whose inactivation has a positive effect on the initial step of adhesion mediated by fimbriae ( 41 ). BolA mediates the induction of the cold shock regulators cspABFGI , which were previously shown to positively regulate the first hours of biofilm formation ( 42 ). Similarly, deletions in respiratory genes such as hyaACD were shown to increase biofilm formation ( 42 ), and in fact our results indicate BolA represses the hyaABCDEF operon. The overall regulation of central carbon metabolism by BolA includes the induction of genes related to several amino acid pathways and the direct activation of the transcription of several of the TCA cycle enzymes. Together, all these regulations are connected to biofilm formation mechanisms through peptidoglycan biosynthesis (see Fig. S3 in the supplemental material). The genes upregulated at the transcriptional level 3 h after BolA induction are connected with the synthesis of peptidoglycan, a major biofilm component ( 43 ). BolA also modulates additional cellular pathways which connect to the synthesis of peptidoglycan via a number of intermediate metabolic networks, as highlighted in Fig. S3 (in red metabolic pathways upregulated in the bolA ++ strain). This interconnection is reinforced by the predicted upregulation of the TCA cycle within biofilms ( 44 , 45 ). In order to further characterize the mode of action of the BolA protein and determine a consensus DNA binding motif, an in silico analysis was performed using a pool of sequences obtained with the ChIP-seq assay. Three different motifs were obtained; however, two of them were present only in 8 and 6 of the total number of sequences tested. The third motif was in 92 of these sequences and thus considered the statistically significant consensus region for the BolA protein (TC)(TC)GCCAG(ACT). This represents a major breakthrough in the characterization of this recently discovered E. coli transcription factor, allowing to further characterize the mode of action of this protein and study the interaction with its targets. Furthermore, STRING database ( 46 ) and text-mining data indicate the possible in vivo interaction of BolA with other E. coli transcription factors, including σ S and H-NS. The question of BolA binding exclusively on its own or in synergy with another protein hence remains open. Biofilm formation is a well-studied but extremely complex process. It involves five different stages and a panoply of cellular structures and components ( 47 ). The various surface structures determinant in each step of E. coli biofilm formation have been summarized and listed ( 27 ). Briefly, initial adhesion requires flagella and motility. Type I fimbriae, curli and exopolysaccharides are involved in the second stage. In the third stage, curli, antigen Ag43, exopolysaccharides, and colanic acid have an essential role. Finally, curli and colanic acid additionally participate in the late maturation together with conjugative pili before dispersal is accomplished thanks to flagella and motility. BolA overexpression favors biofilm formation and involves production of fimbria-like adhesins and curli together with the increment of colanic acid production. In summary, BolA is an important transcription factor able to directly repress or induce gene expression and is involved in the regulation of flagellar biosynthesis, TCA cycle, and peptidoglycan synthesis. In Fig. 6 , we represent a model with the major impacts of BolA in the bacterial cell. This work constitutes a relevant step toward the comprehension of BolA protein and will have important applications for homologues in other organisms, namely, in biofilm-producing pathogenic bacteria. BolA belongs to a family of proteins that are widely conserved from prokaryotes to eukaryotes ( 48 ). Although their exact function is not fully established at the molecular level, they seem to be involved in cell proliferation and cell cycle regulation. Herein, we propose that BolA is a very important motile/adhesive transcriptional switch, specifically involved in the transition between the planktonic and the attachment stage of the biofilm formation process. Together, the molecular studies of different lifestyles coupled with the comprehension of the BolA functions may be an important breakthrough for future perspectives, with major health care and biotechnology applications. FIG 6 Model for BolA-mediated regulation of planktonic-to-sessile transition-related mechanisms in Escherichia coli . BolA is a global regulator which has a major direct effect in bacterial motility through the repression of flagellum-associated genes and the induction of tricarboxylic acid (TCA) cycle genes. It modulates the regulation of several carbon metabolism pathways connected to peptidoglycan biosynthesis. Biofilm formation is favored by BolA mainly through the differential regulation of genes involved in lipopolysaccharide (LPS) and cellulose production. The presence of elevated levels of extracellular DNA, proteins, and sugars emphasizes the role of this protein in the production of an extracellular matrix necessary for biofilm development. Concordantly, BolA induction has a positive effect in the expression of several genes encoding fimbria-like adhesins, which are possibly involved in the formation of the three-dimensional structure of biofilms."
} | 3,715 |
37888272 | PMC10608051 | pmc | 2,430 | {
"abstract": "Fungal endophytes are harboured in the leaves of every individual plant host and contribute to plant health, leaf senescence, and early decomposition. In grasslands, fungal endophytes and their hosts often coexist with large herbivores. However, the influence of grazing by large herbivores on foliar fungal endophyte communities remains largely unexplored. We conducted a long-term (18 yr) grazing experiment to explore the effects of grazing on the community composition and diversity of the foliar fungal endophytes of two perennial grassland species (i.e., Artemisia capillaris and Stipa bungeana ) across one growing season. Grazing significantly increased the mean fungal alpha diversity of A. capillaris in the early season. In contrast, grazing significantly reduced the mean fungal alpha diversity of endophytic fungi of S. bungeana in the late season. Grazing, growing season, and their interactions concurrently structured the community composition of the foliar fungal endophytes of both plant species. However, growing season consistently outperformed grazing and environmental factors in shaping the community composition and diversity of both plant species. Overall, our findings demonstrate that the foliar endophytic fungal community diversity and composition differed in response to grazing between A. capillaris and S. bungeana during one growing season. The focus on this difference will enhance our understanding of grazing’s impact on ecological systems and improve land management practices in grazing regions. This variation in the effects of leaf nutrients and plant community characteristics on foliar endophytic fungal community diversity and composition may have a pronounced impact on plant health and plant–fungal interactions.",
"introduction": "1. Introduction Leaves cover an area exceeding 6.4 × 10 8 km 2 of the Earth’s surface, making them a crucial habitat for terrestrial microbial communities [ 1 ]. Fungal endophytes have been observed in the leaves of all plant species surveyed thus far, and they are essential in the ecosystem [ 2 , 3 ]. The roles of fungal endophytes range from beneficial to detrimental, affecting the growth and reproduction of host plants and ecosystem functioning [ 4 , 5 ]. Some endophytes can enhance plant defences and increase host plant tolerance to various environmental stressors, such as drought, nutrient deficiency, temperature fluctuations, pathogen attacks, competition pressures, and herbivore damage [ 6 , 7 ]. However, endophytes may also lead to resource extraction, suppression of plant defences, disease development, and acceleration of leaf senescence or decomposition [ 8 , 9 ]. At the ecosystem level, these interactions contribute to nutrient cycling and plant community successions, driving ecosystem services [ 10 ]. Foliar fungal endophytes occur within a complex ecosystem context and are influenced by several biotic and abiotic factors across various scales, ranging from the cellular to the ecological level [ 11 , 12 , 13 ]. At the leaf scale, the infection and establishment of endophytes are affected by host resource availability [ 14 , 15 ], stoichiometry [ 16 ], host plant morphological traits [ 17 ], and the interplay between microbiomes [ 18 ]. At larger spatial scales, foliar fungal endophytes are also affected by factors such as climate (e.g., temperature, precipitation) [ 19 , 20 ], nutrient input [ 21 ], genetic or community variation in host and nonhost plants [ 22 ], spatial pattern (e.g., elevation and latitude) [ 23 ], and human activities [ 24 ]. Additionally, previous studies have identified seasonal trajectories in the diversity and composition of endophyte communities [ 25 , 26 , 27 , 28 ]. The potential mechanisms contributing to this phenomenon are multifaceted, encompassing temporal shifts in climate patterns, leaf characteristics, and fungal life cycles [ 29 , 30 , 31 ]. Livestock grazing is a globally pervasive and pivotal land use practice that significantly influences plant, animal, and microbial community dynamics and their intricate interrelationships [ 32 , 33 , 34 ]. The positive or negative associations observed between endophytes and herbivores suggest facilitation or antagonism resulting from one or more direct or indirect mechanisms [ 35 , 36 ]. Considering that plants disperse endophytes horizontally through spores or hyphal fragments, grazing potentially enhances endophyte colonization by creating wounds [ 37 , 38 ] or contributing to their dispersal between hosts [ 7 ]. However, some studies have indicated that removing plant leaves directly would also lead to a decrease in microbial abundance [ 39 ]. In addition, grazing can modify plant community diversity and composition, which indirectly alter foliar fungal endophyte communities [ 40 , 41 , 42 ]. Moreover, grazing has the potential to alter secondary metabolites such as plant leaf stoichiometry and defensive chemicals, which may impact the composition and diversity of foliar fungal endophytic communities [ 43 , 44 ]. The presence of herbivores, for instance, can influence the availability of nutrients in plants and contribute to colonization when plant tissues exhibit a higher nitrogen content [ 45 ]. Various plants employ distinct response strategies to grazing, which are associated with their growth, nutritional, or physiological traits [ 46 , 47 , 48 , 49 , 50 ]. As integral components of host plants, microorganisms inevitably exhibit diverse response strategies to grazing in accordance with their hosts. Currently, our understanding of the mechanisms and response patterns of foliar fungal endophytes under grazing during growing seasons is limited. Furthermore, there is a lack of knowledge regarding the variations in foliar fungal endophytes among different host plants. The grasslands in Northwest China occupy vast semiarid and arid areas and are sensitive to grazing disturbance [ 51 ]. Artemisia capillaris Thunberg (Asteraceae) and Stipa bungeana Trin. (Poaceae) are two dominant perennial species with important ecological and economic values in Northwest China [ 32 ]. Previous studies have revealed that A. capillaris is favoured by livestock primarily during the early season, whereas as plant tissue lignification intensifies throughout the growing season, the frequency of livestock feeding declines. In contrast, livestock preferred to feed on the upper portion of S. bungeana at all times, and it was less tolerant of grazing [ 32 , 52 ]. In the present study, we aimed to investigate (ⅰ) the variation in foliar endophytic fungal community composition and diversity in response to grazing between A. capillaris and S. bungeana across the full growing season; and (ⅱ) the factors driving the community composition and diversity of foliar fungal communities of A. capillaris and S. bungeana. To this end, we conducted a long-term (18 yr) grazing experiment and sampled the foliar fungal communities of A. capillaris and S. bungeana in the early, middle, and late growing seasons from control and grazed treatments. We expected the following outcomes: (i) grazing will decrease the mean fungal alpha diversity of A. capillaris in the early season, decrease the mean fungal alpha diversity of S. bungeana in the middle and late seasons, and change the community composition; (ii) growing season and grazing × growing season interactions will significantly affect foliar fungal community diversity and composition; and (iii) leaf nutrients, plant community characteristics, and defensive chemicals would be significantly correlated with the foliar endophytic fungal community. By performing this research, we expected to establish a theoretical foundation for understanding plant–herbivore–microbe interactions in grazed ecosystems subjected to increasing anthropogenic intervention.",
"discussion": "3. Discussion In this study, we investigated the contrasting effects of grazing in shaping the seasonal trajectory of foliar fungal endophyte communities of Artemisia capillaris and Stipa bungeana . Grazing significantly increased the community diversity of A. capillaris in the early season but significantly reduced the diversity of the foliar endophytic fungal community of S. bungeana in the late season. Both grazing and season strongly affected fungal community composition. Overall, season was more critical than grazing in shaping the composition of the foliar fungal community. Additionally, we detected a strong interactive effect of season and grazing on the community composition of A. capillaris and the community diversity and composition of S. bungeana . When partitioning the total β diversity into its components of turnover and changes in species richness, we found that the differences in the fungal community composition among the early, middle, and late seasons and between the grazed and control treatments were mainly explained by species turnover. Grazing, season, and their interactions also affected aspects of local plant community characteristics, leaf nutrients, and defensive chemicals that were expected to affect foliar endophytic fungal diversity within our two focal hosts. Although high seasonal variation dominated the diversity and composition of endophytic fungal communities, grazing and other factors also affected the endophytic fungal communities. In addition to the experimental factors, plant community characteristics had the greatest impact on fungal community diversity and were negatively correlated with ENSPIE (except for the plant Shannon index in A. capillaris ). In contrast to fungal community composition, plant community characteristics had less impact, and leaf nutrients were more nutritious. 3.1. Impact of Grazing in Shaping the Seasonal Trajectory of the Foliar Fungal Community Our observation that seasonal variation had a greater effect on the foliar fungal community is in line with several previous studies [ 19 , 53 , 54 ]. The strong seasonal changes in the foliar fungal community might be linked to physical and chemical changes in leaves and variations in environmental conditions [ 27 , 55 ]. In this study, the endophytic fungal community diversity of both A. capillaris and S. bungeana tended to decrease by the middle season before increasing by the end of the growing season. We also detected a shift in community composition, both of which were inextricably linked to the climatic conditions of the region. The region where we established the experiment is characterised by high summer temperatures, extreme drought, low rainfall, and a rainy autumn [ 32 ]. Many previous studies have shown that warming and drought decrease endophytic microbial community activity and diversity and alter community composition [ 19 , 56 ]. We also detected that the shift in community composition was mainly the result of species turnover rather than the acquisition of new species [ 19 , 57 ]. Grazing plays a significant role in shaping the seasonal trajectory of the endophytic fungal communities of the two host plants. Our experimental grazing increased the mean fungal endophytic diversity of A. capillaris in the early season but decreased that of S. bungeana in the late season, which may be related to variations in the growth–defence strategy among different plants and the feeding nature of herbivores. Previous studies have found that A. capillaris is preferred by livestock only in the early season, and as plant tissue lignification increases over the growing season, the livestock feeding frequency decreases. Thus, grazing in the early season may have promoted the entry of endophytic fungal spores into leaf tissue and increased the diversity of endophytic fungi due to the rapid growth of A. capillaris during this period, when the removal of microbial survival space and resources by livestock was not significant. In contrast to A. capillaris, we found that grazing significantly decreased the foliar endophytic fungal diversity of S. bungeana during the late growing season. In the early and middle growing seasons, the abundant nutrients of S. bungeana leaves provide resources for endophytic fungal growth; thus, the fungal diversity is high, but as the growth period progresses, S. bungeana may allocate more energy towards compensatory growth to enhance tolerance against grazing stress imposed by livestock, consequently leading to a reduction in endophytic fungal diversity. In terms of livestock feeding habits, sheep prefer to eat the upper part of S. bungeana leaves at all times of the growing season. With continuous grazing, the reduction in S. bungeana leaves leads to a reduction in photosynthetic products and reduces the survival space and resources of endophytic fungi, leading to a decrease in endophytic fungal diversity by grazing in the late growing season. In addition, a previous study on fungal diseases of S. bungeana leaves showed that grazing increased the severity of S. bungeana leaf spot disease, while pathogenic fungi in the leaves competed with endophytic fungi for resources, thus inhibiting the growth of endophytic fungi. Another study on the effect of grazing on Leymus chinensis leaf microorganisms reported a decrease in fungal α diversity under a moderate grazing treatment, and this consistency with S. bungeana may represent the pattern’s possible universality [ 53 ]. Interestingly, in a previous study regarding the response of root endophytic fungi to grazing, it was found that moderate grazing increased root fungal diversity [ 58 ]. In addition, grazing did not significantly affect endophytic fungal communities in plant stems in another study [ 53 ]. These different responses may indicate a spatial ecological niche difference in plant endophytes under grazing. We further found that grazing and its interaction with growing season influenced the fungal community composition. In line with this finding, several observational studies have reported shifts in the composition of the microbial community along a grazing intensity gradient and between different grazing methods, which are often used as an experimental design to investigate the response mechanisms of the microbiome to grazing [ 59 , 60 , 61 ]. However, most studies have focused on the effects of grazing on the soil and root microbiome. At this stage, it is unclear whether differences among studies related to plant endophytic fungi are caused by different grazing intensities, herbivores, strategies, or locations. A promising avenue for future research would be to conduct comprehensive experimental studies involving multiple perspectives (grazing intensity, herbivore, strategy, and location), thereby exploring the response mechanism of plant microbes under different grazing conditions. 3.2. Relationships between Foliar Fungal Community and Plant Community Characteristics, Leaf Nutrients and Defensive Chemicals The foliar fungal community may be affected by season and grazing, and it could also be affected by plant communities, nutrients, and defensive chemicals [ 62 , 63 , 64 ]. With the results of the model-averaged analysis, we found that the effects mediated by changes in the plant community were apparently consistent, and that plant community characteristics were the most important factors in addition to experimental factors, influencing the diversity of foliar endophytic fungal communities for both focal host plants. Notably, fungal diversity declined with plant community characteristics. This strong coupling between plant communities and microorganisms has been confirmed by previous studies [ 65 , 66 , 67 , 68 ] and confirms the need to enhance the understanding of this strong correlation between plants, microbes, and ecosystems in ecological research. The increase in fungal diversity with decreasing plant community characteristics is contrary to island biogeography theory, one of the main theories in ecology. It has been demonstrated that island biogeography theory can be applied to explain the microbial-host relationship [ 69 , 70 ], where plant hosts act as islands, and it can be predicted that an increase in host community diversity would lead to an increase in microbial diversity [ 71 , 72 ]. However, island biogeography theory assumes that dominant and inferior species have the same fitness, and that the dynamics of richness depend strictly on identical species gains or losses [ 73 ]. In contrast, we included fitness differences and competitive hierarchies; after excluding the less competitive species, the increase in dispersal can instead cause a decrease in diversity [ 14 ]. In our study, the increased plant community characteristics led to a more rapid spread of competitively dominant fungi, leading to a decline in fungal diversity within a host individual; notably, diversity and evenness were highly correlated with ENSPIE. Furthermore, host plants may devote more resources to their own growth and reproduction or to other microbes, meaning that endophytic fungi are at a competitive disadvantage [ 74 ]. It is also possible that changing plant community characteristics have altered the local microenvironment in a direction more favourable to smaller fungal taxa [ 75 ]. In the future, more research is needed to reveal the potential mechanisms. We also analysed the effects of various factors on the endophytic fungal community composition. In contrast to community diversity, plant community characteristics barely affected composition, while plant nutrients explained the most variation. Similarly, the effects of plant nitrogen, phosphorus, and carbon on the composition of endophytic fungal communities were demonstrated in previous studies by Meng, Wu, and Yang [ 76 , 77 , 78 ]. In addition, we did not find evidence for the pattern of defensive chemicals affecting the endophytic fungal community, although we detected weak effects of total flavonoids and tannins. However, a study on Pinus monticola showed that herbivores changed the leaf endophytic fungal community because herbivores induced defensive chemicals in the plant and destroyed the protective structures of the sapling (e.g., bark, leaf cuticle), independent of the nutritional status of the leaves [ 15 ]. Moreover, in our study, we found that the direction and strength of the effects of defensive chemicals on fungal endophyte diversity and composition with hosts differed greatly between A. capillaris and S. bungeana . We therefore predict that host species influence the effect of defensive chemicals on endophytic fungal communities, but at this stage, it is unclear whether these differences among studies are a result of differences in the experimental setup or the spatial scale. We note that although we detected many effects on endophytic fungal diversity and composition, direct manipulation of local plant communities, nutrients, or chemical defence substances was not included in our experimental design, which makes it difficult to provide the strongest evidence about the effects of these factors on endophytic fungal communities. In addition, controlled isolation and culture experiments of endophytic fungi would contribute to our understanding of the mechanism that governs endophyte community dynamics. Furthermore, grazing experiments can be more refined, and previous studies on grazing usually focus on grazing intensity, grazing livestock types, or split grazing effects into the removal of plant shoot tissue, dung and urine return, and trampling [ 59 , 60 , 61 ]. Finally, the enormous seasonal variation in foliar fungal endophytes is consistent with other studies, suggesting the need to follow the entire cycle of host plants from seed to litter if we are to understand the processes underlying dominant microbial community colonisation and maintenance. Although there is a wealth of data available on microbial diversity and composition, the mechanistic understanding of causal factors remains unknown. The large number of observational studies provides a solid foundation and an important starting point for subsequent studies, which should focus more on identifying underlying drivers through controlled experimentation and making predictive observations. For example, grazing experiments have revealed that grazing can affect plant symbiont communities in several ways, including direct removal, changing local plant communities, promoting microbial horizontal spread, and changing plant metabolites. However, the large span of seasonal grazing experiments is correlated with many other factors (e.g., climate, micro food network, local plant community, host species, and herbivore diet) that also change microbial communities. For this reason, controlled experiments that are replicated at the temporal scale should be integrated with existing community ecological theoretical frameworks, through which we can understand the mechanisms that determine microbial communities."
} | 5,235 |
34318611 | PMC8449669 | pmc | 2,431 | {
"abstract": "Summary The specific interaction between rhizobia and legume roots leads to the development of a highly regulated process called nodulation, by which the atmospheric nitrogen is converted into an assimilable plant nutrient. This capacity is the basis for the use of bacterial inoculants for field crop cultivation. Legume plants have acquired tools that allow the entry of compatible bacteria. Likewise, plants can impose sanctions against the maintenance of nodules occupied by rhizobia with low nitrogen‐fixing capacity. At the same time, bacteria must overcome different obstacles posed first by the environment and then by the legume. The present review describes the mechanisms involved in the regulation of the entire legume–rhizobium symbiotic process and the strategies and tools of bacteria for reaching the nitrogen‐fixing state inside the nodule. Also, we revised different approaches to improve the nodulation process for a better crop yield.",
"conclusion": "Conclusions Based on the hypothesis that the rhizobia–legume symbiotic process has evolved towards the selection of strains that best interact and induce efficient nodules for nitrogen fixation, is there a way to improve it? Despite inoculants composed of highly efficient nitrogen‐fixing rhizobia were used, the populations found in plant nodules were often diverse, with a high proportion of native strains. Native strains are certainly better adapted to the environment than those making up the inoculant. Their competitiveness for nodulation may be greater, equal, or less than that of the inoculant and may be therefore very, little or not efficient at all in nitrogen fixation. At the beginning of the nodulation process, the plant cannot discriminate rhizobia with respect to their nitrogen fixation efficiency. Once inside the nodule, some plants have the capacity to sanction non‐fixing bacteria, without preventing them from still being in the rhizosphere and competing for nodulation. In relation to the question raised, we believe that the symbiotic process between legumes and rhizobia can still be improved by seeking strains able to adapt to the environment, more competitive in the nodulation process than native strains and, at the same time, having a better capacity to fix nitrogen. This can be achieved by selecting strains with good nitrogen fixation efficiency from the most competitive ones, and then finding the appropriate inoculation conditions to increase their presence in the proximity of the root. Also, by accompanying efficient nitrogen‐fixing strains with other PGPR strains or mycorrhizas that favour proliferation in the rhizosphere and root colonization, or by modifying some functions through genetic engineering of rhizobia. The genetic modification may be targeted to improving bacterial adaptation to a particular soil condition, improving one of the multiple stages involved in the nodulation process itself or increasing the nitrogen fixation capacity. However, overexpression or mutation of a function may have pleiotropic effects that should be avoided. The resulting mechanism of action of different bacterial components may be dual and depend on the variety of the nodulated legume. As shown in various field campaigns, different varieties of the same legume species may be grown and the genetic modifications on these bacterial components may not be desirable. Accordingly, a thorough knowledge of the molecular mechanisms involved in the different stages of the nodulation process is required for the selection of strains by genetic engineering.",
"introduction": "Introduction Legumes (soybeans, clover, lotus, pea, bean and chickpea bean) are a fundamental source for human food and livestock feed. The selection of legume varieties with better growth relies on the application of traditional agronomic techniques. In the last years, transgenic legume plants with a desirable trait have been obtained using molecular biotechnology tools. Such is the case of glyphosate‐resistant transgenic soybean, an herbicide used to eliminate weed growth. In the 2018–2019 season, 364 million tons of soybean have been commercialized (FAO, 2019 ), giving an idea of the importance of this crop in the world economy. Nitrogen is one of the dominant rate‐limiting nutrients in natural systems (Ferguson et al ., 2010 ; Guignard et al ., 2017 ). Legumes have developed a particular ability to establish a symbiotic relationship with certain bacteria from the soil, whereby they can utilize the atmospheric nitrogen (Sprent, 2008 ). Thus, the application of nitrogen fertilizers can be avoided by means of this symbiosis and the consequent biological nitrogen fixation (BNF) (Ferguson et al ., 2010 ). This is auspicious because chemical fertilizers generate a negative effect on the ecosystem and involve the use of non‐renewable fossil energies (Ferguson et al ., 2010 ; Galloway et al ., 2013 ). The term rhizobia refers to bacterial species that can interact with the roots of legumes and induce the formation of structures called nodules, where gaseous di‐nitrogen is transformed into ammonium (BNF) and can thus be assimilated by the plant (Lindström and Mousavi, 2019 ). Most of the rhizobial species belong to families of the alpha‐proteobacteria class, including Rhizobiaceae ( Rhizobium, Sinorhizobium, Allorhizobium, Pararhizobium, Neorhizobium and Shinella ), Phyllobacteriaceae ( Mesorhizobium, Aminobacter, Phyllobacterium ), Brucellaceae ( Ochrobactrum ), Methylobacteriaceae ( Methylobacterium, Microvirga ), Bradyrhizobiaceae ( Bradyrhizobium ), Xanthobacteraceae ( Azorhizobium ) and Hyphomicrobiaceae ( Devosia ) (Lindström and Mousavi, 2019 ). Some species of the Burkholderiaceae family ( Paraburkholderia, Cupriavidus ) of the beta‐proteobacteria class can also induce an active nodulation process on legumes (Lindström and Mousavi, 2019 ). The nodulation process ranges from the interaction of bacteria with the root hairs to the formation of root nodules, where bacteria inside organelles called symbiosomes differentiate into nitrogen‐fixing bacteroids. While the plant benefits from the nitrogen supply, bacteria get carbon compounds provided by the plant. This is a specific process by which certain species of rhizobia induce the formation of nodules on particular legumes. However, some legumes such as Glycine max and Phaseolus vulgaris may be nodulated by more than one species of rhizobia (Ji et al ., 2017 ; Andrews and Andrews, 2017 ) and some rhizobia like Rhizobium sp. NGR234 can nodulate different legume genera (Pueppke and Broughton, 1999 ). Using in vitro culture methods and more recently by applying metagenomic approaches, it has been found that symbiotic rhizobia cohabit in the nodules with other non‐nodulating rhizobia and non‐rhizobial species (Busby et al ., 2016 ; Lu et al ., 2017 ), which may affect process performance (Peix et al ., 2015 ; Gano‐Cohen et al ., 2016 ; Lu et al ., 2017 ). The nodulation process is controlled primarily by the plant (Sachs et al ., 2018 ). Legumes benefit from and have evolved to allow this symbiosis, but such energy‐demanding process also needs to be regulated (Sachs et al ., 2018 ). The plant has developed mechanisms to enable and prevent the entry of compatible and incompatible bacteria respectively (Clúa et al ., 2018 ; Wang et al ., 2018 ). Additionally, legumes can display sanctions against the maintenance of nodules occupied by rhizobia with low nitrogen‐fixing capacity (Kiers et al ., 2003 ). Bacteria must overcome different obstacles posed first by the environment and then by the legume. In this context, rhizobia often adopt tools to survive under certain adverse soil conditions and have also developed mechanisms and strategies to facilitate the nodulation of a given legume (Soto et al ., 2006 ; Clúa et al ., 2018 ; Syska et al ., 2019 ). Below we will discuss the different pathways and mechanisms behind the limitations of the nodulation process and the tools by which bacteria could adapt to and/or overcome them."
} | 2,015 |
25989369 | PMC4817624 | pmc | 2,432 | {
"abstract": "Many cnidarians host endosymbiotic dinoflagellates from the genus Symbiodinium . It is generally assumed that the symbiosis is mutualistic, where the host benefits from symbiont photosynthesis while providing protection and photosynthetic substrates. Diverse assemblages of symbiotic gorgonian octocorals can be found in hard bottom communities throughout the Caribbean. While current research has focused on the phylo- and population genetics of gorgonian symbiont types and their photo-physiology, relatively less work has focused on biogeochemical benefits conferred to the host and how these benefits vary across host species. Here we examine this symbiosis among 11 gorgonian species collected in Bocas del Toro, Panama. By coupling light and dark bottle incubations (P/R) with 13 C-bicarbonate tracers, we quantified the link between holobiont oxygen metabolism with carbon assimilation and translocation from symbiont to host. Our data show that P/R varied among species, and was correlated with colony morphology and polyp size. Sea fans and sea plumes were net autotrophs (P/R>1.5), while nine species of sea rods were net heterotrophs with most below compensation (P/R<1.0). 13 C assimilation corroborated the P/R results, and maximum δ 13 C host values were strongly correlated with polyp size, indicating higher productivity by colonies with high polyp SA:V. A survey of gorgonian- Symbiodinium associations revealed that productive species maintain specialized, obligate symbioses and are more resistant to coral bleaching, whereas generalist and facultative associations are common among sea rods that have higher bleaching sensitivities. Overall, productivity and polyp size had strong phylogenetic signals with carbon fixation and polyp size showing evidence of trait covariance.",
"introduction": "Introduction Since the discovery of coral-hosted Symbiodinium, nutrition has been a major focus of coral symbiosis. Hand and Muscatine (1958) were among the first to demonstrate that Symbiodinium provide photosynthates to their hosts, thus settling debate over the functional significance of the intracellular algae. Since then, numerous studies have quantified the contribution of autotrophic and heterotrophic nutrition to the coral holobiont for a number of scleractinian species, even to sub-cellular scales ( Yellowlees et al. , 2008 ; Houlbreque and Ferrier-Pages, 2009 ; Pernice et al. , 2014 ). We now know that Symbiodinium translocate carbon-based photosynthates in excess of host needs and nitrogen in the form of amino acids derived from host wastes and dissolved inorganic nitrogen from the water column. By coupling the coral polyp's opportunistic heterotrophy with Symbiodinium photosynthesis, the coral holobiont can thrive despite the severe nutrient limitation typical of tropical seas ( Yellowlees et al. , 2008 ; Houlbreque and Ferrier-Pages, 2009 ). However, holobiont performance and host benefits are poorly understood across different host–symbiont interactions and changing environmental conditions. Our understanding of how coral– Symbiodinium interactions vary across diverse associations remains limited. Most studies have focused on several model coral species such as Stylophora pistillata , Pocillopora damicornis and members of the genus Acropora ( Muller-Parker et al. , 1994 ; Hoegh-Guldberg and Williamson, 1999 ; Grover et al. , 2008 ). These species of photophilic corals all possess branching colony morphologies and small polyps and are thus too similar in their morphology and nutritional physiology to reveal the full breadth of coral– Symbiodinium functional diversity ( Tremblay et al. , 2012 ; Baker et al. , 2013 ). Fewer nutritional studies have compared species within and between genera and even fewer in non-scleractinian anthozoans ( Lasker, 1981 ; Lasker et al. , 1983 ; Wilkerson and Muscatine, 1984 ; Ribes et al. , 1998 ; Swanson and Hoegh-Guldberg, 1998 ). Such critical gaps limit our understanding of the functional, ecological and evolutionary significance of these symbioses. The evolution of feeding structures occurs when species compete for limited nutritional resources ( Grant and Grant, 2006 ). For corals, niche partitioning and competition likely drive the diversity of colony and polyp morphologies. Divergence towards the utilization of novel resources at the extremes of the heterotrophic – autotrophic continuum (that is, from plankton to dissolved inorganic nutrients) can reduce competitive interactions and drive speciation. Porter (1976) argued that variation in coral colony morphology, specifically the ratio of surface area to volume (SA:V), is central to understanding the nutritional strategies of scleractinian corals. Indeed, Porter predicted that autotrophy should be maximized in corals with high SA:V, with the specific idea that branching corals would exhibit higher photosynthesis/respiration (P/R) relative to massive (non-branching) species. Moreover, such a relationship would promote the evolution of 'layered' colony morphologies, which Porter identified as a convergent character in terrestrial plants. At a smaller scale, variation in polyp size is often posited as an indicator of heterotrophic capacity as larger polyps possess larger tentacles and gastrovascular cavities for prey capture and digestion. Conversely, small polyps have higher SA:V, which maximizes the light received by Symbiodinium , inhabiting the gastroderm at densities up to 10 6 cells cm −2 , and increases the surface area for nutrient uptake. Taken together, increasing or decreasing SA:V can occur at the colony and polyp scale. Here we test Porter's hypothesis by quantifying productivity (P/R and 13 C assimilation) and utilizing morphological, phylogenetic and ecological data on a diverse collection of Caribbean octocorals (gorgonians). In the Caribbean Sea, gorgonians hosting Symbiodinium are especially diverse with at least 51 described species ( Sánchez and Wirshing, 2005 ). Moreover, there is great variation in gorgonian morphology, with many reefs dominated by species with fan, plume or rod morphologies. Perhaps due to the morphological similarity to aposymbiotic deep water species, and the paucity of symbiotic species in the Indo-Pacific, there is a perception that symbiotic gorgonians gain most of their nutrition via passive suspension feeding ( Leal et al. , 2014 ). To date, the evidence to support this contention is limited. Indeed, symbiotic gorgonians differ in their ability to capture particles from the water column ( Lasker, 1981 ). Although some species obtain sufficient carbon from particulate organic matter ( Murdock, 1978 ; Coffroth, 1984 ), they still require nitrogen supplemented via Symbiodinium photosynthesis ( Wainwright, 1967 ). Unlike Indo-Pacific soft corals ( Fabricius and Klumpp, 1995 ) previous studies of Caribbean gorgonian productivity (as determined by oxygen metabolism) support a predominantly autotrophic nutrition in many species. Here we expand on previous gorgonian productivity studies by using light and dark bottle incubations to estimate P/R in combination with inorganic stable carbon isotope tracer additions to assess autotrophic productivity among 11 species of symbiotic gorgonians. We test the following null hypotheses: (1) all gorgonian symbioses are mutualistic where the host benefits from symbiont photosynthesis, (2) the mutualisms are invariant with respect to the amount of carbon translocated to the host and (3) the host-derived benefits of mutualism are unrelated to morphology, (that is, polyp size) of a species. Further, we examined the functional significance of varied productivity through a posteriori correlations between productivity measures and eco-physiology, including symbiont specificity and thermal bleaching resistance.",
"discussion": "Discussion Gorgonian productivity varies with morphology Our data show that the sea fan G. ventalina and plume A. acerosa produced >2 times their required carbon demand with P/R values similar to many scleractinian corals. As such, we conclude that these species are net autotrophs, acquiring the majority of their nutrition from Symbiodinium photosynthesis. This contrasts with all sea rod species that had P/R<1.5 and all Indo-Pacific alcyonaceans that have negative P/R values ( Fabricius & Klumpp, 1995 ). Species with fan or plume morphologies and small polyps might maximize light exposure and optimize symbiont densities and nutrient exchange via increased SA:V. Similar observations have been made for Indo-Pacific sponges, which are more productive than their Caribbean counterparts with highly autotrophic species exhibiting flattened morphologies (encrusting, fan, dish) that are convergent traits in corals ( Wilkinson, 1983 ). In one of the first studies of gorgonian metabolism, Cary (1918) measured respiration in 12 Caribbean species and found that the highest rates of respiration were observed in species with the highest SA:V (that is, Gorgonia, Antillogorgia and Pterogorgia ). Thus, we hypothesize that some gorgonian species are more metabolically active than others, as positive NPP is maintained by high rates of photosynthesis, despite substantial respiration. Therefore, observed variations in P/R are due to overall metabolic rates rather than variation in either photosynthesis or respiration alone. Like Kanwisher and Wainwright (1967) , we found a wide range of P/R values for Caribbean gorgonians, which followed a general pattern of increasing P/R with SA:V. We stress, however, that comparisons among disparate studies should be interpreted with caution, as the method of oxygen measurement and environmental conditions can vary considerably between studies. Nevertheless, it is interesting that Caribbean gorgonians have a similar range of P/R values to some Indo-Pacific hard corals, although our data are lower than previous reports ( Roffman, 1968 ). Porter (1976) put forth several hypotheses about functional morphology of corals that are consistent with our observations on gorgonians. First, the parallels drawn between tree morphology ( sensu \n Horn (1971) ) are particularly striking as the most autotrophic species ( G. ventalina and A. acerosa ) have morphologies similar to the broad leaves and needles of trees, respectively, which are plant adaptations for enhancing photosynthesis while moderating transpiration. In sea plumes, alternating branches reduce self-shading and permit light transmission to lower portions of the colony. Sea fans have a reticulate and planar morphology with high surface area. Over time, sea fans twist their axial skeleton to a position perpendicular to water flow ( Wainwright and Dillon, 1969 ). It has been suggested that fan morphologies aid in particle capture ( Leversee, 1976 ). While this may be true for deep-water sea fan species, for G. ventalina found in high energy, shallow environments this orientation facilitates the oscillation of the colony from full illumination to shade in synchrony with the wave period. This also permits the photosynthetic centers to recover from light saturation and maximize photosynthetic efficiency over the course of the day. Colonies uprooted or twisted from this position often show heavy melanization (purpling) in response to excessive light stress to one face of the fan, whereas adjacent colonies with normal skeletal development/positioning exhibit normal coloration (pers. obs.). Alternatively, the cylindrical or bladed, digitate and branching forms common to the various genera of sea rods may be more suited to capturing plankton and particulate matter, especially those with larger polyp sizes. Thick, rounded colony branches may function to generate eddies on the side of the branch opposite an oncoming current. Such eddies can cause plankton and particulate matter to linger, affording a polyp more opportunity for capture ( Leversee, 1976 ). There is limited evidence to suggest that larger polyps facilitate prey capture. Lasker et al. (1983) found that two sea rods, Plexaura homomalla and P. nina , found on the shallow and deep forereef, respectively, differed in their prey capture abilities. With twice the polyp size, P. nina was able to capture more particles. It is interesting that the difference in polyp size and distribution of these congeners occurs over a depth gradient, which suggests that larger polyps are advantageous in deeper habitats where light is limiting. δ 13 C as an indicator of trophic position The natural abundance isotope values of gorgonians are useful indicators of both diet and localized variation in the base of the marine food web. Our data show a wide range in δ 13 C values (nearly 3.5‰) and a strong correlation between the δ 13 C host and δ 13 C symbiont , indicating either a common carbon source or tight cycling of carbon between host and symbiont ( Supplementary Figure S1 ). Interestingly, there is little support for utilization of POM in the gorgonian diet. POM from Bocas del Toro had a δ 13 C of −23‰ ( Freeman and Thacker, 2011 ), which is a reflection of a large terrestrial contribution to Almirante Bay ( Aronson et al. , 2014 ). The average δ 13 C of gorgonians (−17.4‰) greatly exceeded the 1‰ trophic enrichment expected between a consumer and its diet. Instead, these values may be enriched via photosynthetic assimilation of dissolved inorganic carbon (DIC; δ 13 C HCO3- ≈0‰). As such, gorgonian δ 13 C is a weighted average of DIC and POM contributions to the auto- and heterotrophic diet, respectively. Ultimately, we limit our interpretation of these data as different environmental sources and conditions among the three collection sites preclude a detailed view of the trophic position of gorgonians based on natural abundance δ 13 C. In this regard, the 13 C tracer study was effective in highlighting the relative utilization of DIC via photosynthesis among these species. 13 C Tracer reveals the benefits of symbiosis We have shown that Caribbean gorgonians receive important carbon resources from their associated Symbiodinium , which drives significant variation in productivity among species. The autotrophic assimilation of H 13 CO 3 _ in the light bottles was evident in δ 13 C host and δ 13 C symbiont , suggesting a tightly coupled and rapid translocation of 13 C-labeled carbohydrates and other biomolecules during the brief incubation ( Figure 2a ). The higher R 2 observed between δ 13 C symbiont and P/R ( Supplementary Figure S2 ) may indicate superior precision of direct quantification of 13 C assimilation by the holobiont versus the relatively indirect calculation of productivity based on oxygen metabolism from clonal fragments within separate bottles. The relative enrichment of δ 13 C host attained versus δ 13 C symbiont reflects the variation in photosynthate translocated from source (symbiont) to sink (host). Eunicea mammosa and E. succinea had the lowest relative δ 13 C host enrichment. Such low δ 13 C host could result from several factors including (1) higher symbiont metabolism relative to the host, (2) limited translocation of photosynthate due to lower host SA:V, (3) differential host and symbiont biomass, and similarly, (4) a relatively larger structural pool of carbon in the host fraction, which ‘dilutes' any translocated 13 C. However, if these factors were important in determining the final 13 C assimilation in the host, then similar patterns would have been observed in the Plexaurella, which have the largest polyps, lowest SA:V, and thickest host tissues among the species studied. Given that this is not the case, an alternative explanation is that Symbiodinium within E. mammosa and E. succinea are less mutualistic, retaining more of their photosynthates for their own metabolism. Such observations of cheating have been reported for a variety of microbial symbioses ( Bronstein, 2001 ; Porter and Simms, 2014 ). This contrasts with species like P. anceps that achieved higher δ 13 C host than δ 13 C symbiont , suggesting a larger carbon subsidy to the host and therefore a more mutualistic relationship. Interestingly, P. anceps had the highest natural abundance δ 13 C values reported in this study ( Supplementary Figure S1 ), suggesting a large contribution of autotrophically derived DIC. Although 13 C assimilation was not observed in any dark-incubated Symbiodinium , it was curiously evident in all host fractions ( Figure 2b ). The former is easily explained by a lack of light to drive photosynthesis. In the dark, host enrichment in the absence of symbiont photosynthesis must be proceeding by anaplerotic pathways, such as fatty-acid synthesis and oxidation ( DeNiro and Epstein, 1977 ) or by associated microbiota. Microbes other than Symbiodinium could contribute to 13 C enrichment through similar biosynthetic pathways, as their signal is inseparable from the host fraction. Quantifying the contributions of associated chemoautotrophic microbes such as cyanobacteria and archaea to holobiont productivity and biogeochemical cycling warrants further study. However, the large differences in observed δ 13 C host enrichments (>3 to 30-fold) between the light and dark treatment suggest that associated microbe 13 C assimilation was minimal relative to Symbiodinium . We found that not all hosts benefit from Symbiodinium autotrophy. Interestingly, there was no effect of light on Plexaurella or Briareum δ 13 C host and δ 13 C symbiont (except P. fusifera δ 13 C symbiont ). Given that marginal enrichments of the δ 13 C host can be attributed to light-independent assimilation ( Figure 2b ), we conclude that Symbiodinium hosted by P. fusifera, P. nutans and B. asbestinum did not translocate carbon to their hosts. Indeed, the three species were among the lowest with regards to P/R with mean values <1.0. As such, the interaction between Symbiodinium and large polyp gorgonian hosts may be more commensal than mutualistic, at least as it pertains to carbon metabolism. However, even though observed 13 C assimilation was low in many sea rod species, the role of Symbiodinium may still be critically important for obtaining growth-limiting resources such as nitrogen ( Ribes et al. , 1998 ). Symbiont identity and gorgonian productivity Caribbean gorgonians generally associate with members of Symbiodinium clade B ( Baker et al. , 2013 ), which is common on Caribbean reefs ( Pettay and Lajeunesse, 2007 ). Certain sub-clade types such as B1 are especially common, leading to the conclusion that this is a generalist symbiont associated with many hosts ( LaJeunesse, 2002 ). However, Finney et al. (2010) revealed that this clade contains substantial genetic variation with evidence of highly specialized symbionts among various host species. Such specialization could occur during divergence among host species, such has been reported for depth partitioned lineages of E. flexuosa and co-evolution of their Symbiodinium ( Prada et al. , 2014 ). Our comparison of known Symbiodinium types found evidence of symbiont specificity increasing with holobiont productivity ( Figure 5 ). Generalist hosts had lower productivities in comparison with specialists, which exhibited a higher degree of autotrophy. The mechanism underlying the enhanced metabolism afforded by specialized symbioses, including the functional significance of octocoral-associated Symbiodinium clade B warrants further investigation ( Baker et al. , 2013 ; Pernice et al. , 2014 ). Bleaching and gorgonian productivity During the 2005 mass coral bleaching in Puerto Rico only 8 out of 24 gorgonian species showed visible signs of bleaching among four monitored reefs. Remarkably, with the exception of Muricea , no species suffered subsequent mortality ( Prada et al. , 2010 ). However, the corals that readily bleached were species with larger polyps and, as we have reported here, lower rates of productivity ( Figure 6 ). Considering the observed specificity among highly productive species, we predict that such resistance to bleaching is reflective of the obligate nature of these symbioses. Essentially, bleaching may be disadvantageous to an obligate host if nutrition is derived exclusively from symbionts. This explains the difficulty in experimentally bleaching G. ventalina by elevated temperatures ( Kirk et al. , 2005 ), prolonged incubation in darkness, or with the herbicide DCMU (Baker, unpublished data). Conversely, in more facultative interactions hosts are more likely to expel symbionts when the costs (such as nutrient retention, increased respiration, or oxidative damage) outweigh the nutritional benefits, especially if the host can alternatively acquire resources easily by heterotrophy. Further work is needed to test these hypotheses among other Symbiodinium – cnidarian symbioses. Gorgonian metabolism and the evolution of symbiosis Across the diversity of gorgonians, there is a strong negative correlation between traits associated with heterotrophy and autotrophy, reflecting strong evolutionary and ecological trade-offs between their divergent life-history strategies. Indeed, we found a link between productivity and the evolutionary trajectories of host species, which may be driven by habitat specialization and niche partitioning within highly competitive coral reef environments. The strong negative phylogenetic correlation observed between polyp size and carbon translocated from symbiont to host suggests that there is an evolutionary trade-off between heterotrophic and autotrophic modes of nutrition. Species like G. ventalina and A. acerosa possess very small polyps compared with some sea rods like Eunicea and Plexaurella , whose polyps are up to an order of magnitude larger ( Thibaudeau 1983 ). As a consequence, the density and surface area of each polyp, relative to the overall colony size is much higher for the sea fans and plumes than the sea rods we studied. Thus, selection may favor the reduction of feeding structure SA:V if it increases the amount of light exposure to productive symbionts in the host polyp tissues ( Kanwisher and Wainwright, 1967 ; Wainwright and Dillon, 1969 ). The converse appears to be true for most sea rods, where larger and fewer polyps and the thick, rod shaped colony morphology would serve to limit productivity per unit of biomass but ensure that feeding structures are ready for heterotrophy during changing environmental conditions. Over deep time, gorgonian diversification in the Caribbean was less inhibited by competitive interactions with scleractinians than in the Pacific. With more niche space available, any species that acquired mutualistic Symbiodinium could boost its ability to compete for space. In doing so, selection may have favored more obligate associations with Symbiodinium with more efficient metabolisms leading to greater productivity and growth (and thus, competitive abilities) in oligotrophic environments. Such divergence is obvious within the Gorgoniidae, which display a stark contrast between clades formed by Gorgonia and Antillogorgia versus the Plexaurella ( Figure 3 ). These two host species are apparent specialists with Symbiodinium clade B1 as reported by substantial sampling efforts throughout the Caribbean Sea (Andras et al., 2011). Moreover, this specialization may have accelerated the selection for reduced polyp size and colony morphologies to enhance light capture ( Figure 3 ). A shared trajectory toward symbiont specificity and autotrophy is further supported by the observation that Gorgonia and Antillogorgia are capable of hybridization ( McFadden et al. , 2010 ). In contrast, we have shown that Plexaurella do not benefit substantially from Symbiodinium and hosts are less specific in their Symbiodinium associations ( Figures 2 and 3 ). This may reflect a divergence from the Gorgonia/Antillogorgia sister group to avoid competition for space in high light environments. While we have examined the benefits of the gorgonian- Symbiodinium symbiosis, further work must address the potential costs of these interactions. For example, with changing environmental conditions (higher SST, eutrophication, acidification), do the costs of symbiosis outweigh the benefits? We hypothesize that host energetics and related physiological mechanisms (such as immunity to pathogens) could be a key indicator of the cost of symbiosis. For example, productive and obligate hosts like G. ventalina were nearly extirpated from the Caribbean by disease ( Bruno et al. , 2011 ), whereas other gorgonian species were not as impacted. The severity of this disease has been attributed to eutrophication ( Baker et al. , 2007 ). Thus, the cost of symbiosis, including the pairing of auto- and heterotrophic metabolisms could become an Achilles' heel when the environment changes. For other species, there are benefits to being flexible in a symbiotic relationship particularly during periods of environmental change. The relatively heterotrophic sea rods (that is, Plexauridae including Eunicea ) are Caribbean endemics undergoing rapid speciation. In our study, this group displayed variable metabolic performance ( Figure 3 ). Indeed, Eunicea is the most diverse anthozoan genus with 16 described species ( Sánchez et al. , 2003 ). They are phenotypically plastic and can adapt to different habitat types along a reefscape, which contributes to their high rate of speciation ( Prada and Hellberg, 2013 ; Prada et al. , 2014 ). In the process of speciation, the association with Symbiodinium may weaken from an obligate mutualism to a commensal or parasitic relationship. In the Anthropocene, selection may favor facultative symbioses as the coastal oceans become more eutrophic and nitrogen limitation is alleviated ( Baker et al. , 2010 ). We predict that generalist and facultative hosts may be more resilient in the face of environmental stressors that specifically impact symbionts (reduced light) and capitalize on the increasing abundance of heterotrophic resources in the form of plankton and particulate organic matter to meet their nutritional needs."
} | 6,564 |
39060249 | PMC11282299 | pmc | 2,433 | {
"abstract": "Increasing demand for bio-interfaced human-machine interfaces propels the development of organic neuromorphic electronics with small form factors leveraging both ionic and electronic processes. Ion-based organic electrochemical transistors (OECTs) showing anti-ambipolarity (OFF-ON-OFF states) reduce the complexity and size of bio-realistic Hodgkin-Huxley(HH) spiking circuits and logic circuits. However, limited stable anti-ambipolar organic materials prevent the design of integrated, tunable, and multifunctional neuromorphic and logic-based systems. In this work, a general approach for tuning anti-ambipolar characteristics is presented through assembly of a p-n bilayer in a vertical OECT (vOECT) architecture. The vertical OECT design reduces device footprint, while the bilayer material tuning controls the anti-ambipolarity characteristics, allowing control of the device’s on and off threshold voltages, and peak position, while reducing size thereby enabling tunable threshold spiking neurons and logic gates. Combining these components, a mimic of the retinal pathway reproducing the wavelength and light intensity encoding of horizontal cells to spiking retinal ganglion cells is demonstrated. This work enables further incorporation of conformable and adaptive OECT electronics into biointegrated devices featuring sensory coding through parallel processing for diverse artificial intelligence and computing applications.",
"introduction": "Introduction As artificial intelligence applications continue to increase and integrate into daily life, the demand for low-cost, highly efficient, bio-interfaced computing hardware stands out as a global technological challenge 1 , 2 . Conventional silicon electronics operate following the paradigm of von Neumann architecture which depends on several circuital elements such as transistors, inverters, and their combination into logic circuits. However, these systems show poor biological compatibility on both an interfacing level, due to the inherent rigidity of conventional inorganic, CMOS transistors, and on a computing level, since they operate through serialized computational functions, thereby limiting their speed and increasing their power consumption 2 , 3 . Recent advances in neuromorphic systems specifically aim to bypass this “von Neumann bottleneck” by mimicking the massively parallel and event-driven computational functions of the human brain thus offering increased speed, efficiency, and performance over conventional computing algorithms 2 . Organic mixed ion-electron conductors (OMIECs) are emerging for bioelectronic and neuromorphic applications due to their favorable properties, including demonstrated biocompatibility, flexibility/stretchability, and ion-based tunability 4 . Specifically, organic electrochemical transistors (OECTs) require low operational voltages which serve to modulate the conductivity of their organic mixed ion-electron conductor channel resulting in high transconductance compared to field effect transistors 3 , 4 . The ion-based operation of these systems mimics neuronal ion-flux communication and neurotransmitter-receptor binding, promising future interaction with biological tissues as adaptive bio-interfaces 5 – 8 . As a result, OECTs have also been essential to design artificial neural circuits 9 – 11 . Within neuromorphic systems, OECT-based biologically-inspired synapses exhibiting both long/short term plasticity and spike timing-dependent plasticity have been developed to directly interface with living tissue 5 , 6 . Integrate and fire models have been used to design artificial spiking networks, but these circuits require multiple OECTs in a configuration comprising two complementary inverters and a switch, thereby limiting circuit miniaturization 9 . They also cannot compete with the biological realism of Hodgkin–Huxley(HH) neuron models, which closely replicates the flow of sodium and potassium ions within a biological neuron, serving as the fundamental mechanisms to generate action potentials 12 . Such biologically realistic circuit models have been demonstrated in silico for pattern classification, artificial retinas, and neurological disease models 13 , 14 . The sodium channel ion flux exhibits an inherent anti-ambipolar function which is critical for generating an action potential and must be replicated in neuromorphic systems 10 , 12 . Indeed, anti-ambipolar transistors are ideal neuromorphic devices since they naturally exhibit a characteristic positive and negative transconductance within their OFF-ON-OFF transfer curve 10 , 15 . Therefore, HH neuron circuits have been developed using anti-ambipolar OECTs based on poly(benzimidazobenzophenanthroline) (BBL), one of the few materials so far reported with stable anti-ambipolar characteristics 10 . BBL exhibits inherent anti-ambipolarity due to the overfilling of band states within the organic semiconductor that inhibits electronic conduction across the material at high gate voltages 10 , 16 . The generation of spikes in HH neurons replicates action potentials comprising an ion-dependent operation similar to in-vivo neurons 10 . However, these systems usually display fixed neuron characteristics, such as spiking threshold and frequency, and limited device footprints due to their planar structure 10 . Being able to control these parameters is essential to design bio-realistic artificial neurons and account for the high specialization of their biological counterparts in the central nervous system where these metrics vary depending on the cell’s functions 12 . For example, neurons with different action potential thresholds have been identified in the brain. These threshold variations represent a fundamental mechanism enabling neural microcircuits sharing a similar structure to perform a variety of computational tasks, as well as enabling increased robustness, synchronicity, sensitivity, and dynamic range in the whole central nervous system 17 – 20 . Another promising strategy to overcome the “von Neumann bottleneck” is represented by the development of electrically reconfigurable logic circuits, where anti-ambipolarity enables dynamic reshaping of the circuit’s connectivity and functionality. Regularly, planar OECTs have been employed to develop inverters and logic gates targeting a plethora of applications, including wearable bio-interfaced closed-loop electronics 8 , 21 – 24 . However, the footprint of these circuits are limited not only by these devices’ planar structure but also due to the device number required per logic function and the limited configurability of the circuits 21 . Thus, anti-ambipolarity allows the same transistor to be reconfigured in different logic operations depending on the use of positive and negative transconductance, enabling a bypass of the device number per logic function limitation presented by the “von Neumann bottleneck” 25 – 27 . However, the same lack of tunabilitity amongst the limited single-component anti-ambipolar OECTs hinders power consumption improvements, bio-interfacing and sensing capabilities. Despite the lack of intrinsically anti-ambipolar inorganic materials, or organic materials for field effect transistors (OFET), OFF-ON-OFF transfer characteristics can be promoted through an in-series combination of p-type and n-type materials. In these in-series field effect transistors, the high-off resistance of each material is leveraged to prevent current flow when the gate voltage exceeds the region of overlap 15 , 25 – 28 . Therefore, the selection of p-type and n-type materials of an in series-transistor alters the region of conduction overlap thereby defining the bounds of the in series devices anti-ambipolar transfer curve. This allows circuits of further reduced complexity, thus facilitating scalable Gaussian probabilistic neural networks and neuromorphic spiking systems 15 , 28 . In this work, we demonstrate a bilayer vOECT with anti-ambipolar transfer characteristics by realizing an in-series connection of p-type and n-type OMIECs with properly selected threshold characteristics, vertically arranged through stacked layers. We propose a generalizable approach for the selection of these materials realizing anti-ambipolar transistors with tailored full width at half maximum (FWHM), turn on, turn off, and peak current gate voltages. Control over these transistor metrics enables scalable, low voltage, tunable OECTs which serve to build customizable logic devices and neuromorphic spiking circuits. In the context of HH-spiking circuits, the bilayer approach serves to tune the spiking threshold of the circuit depending on the characteristics of the single anti-ambipolar transistors, in parallel with the high specialization of biological computing elements. In these cases, the vOECT architecture also enables miniaturization of circuits, while maintaining scalable fabrication methods, favoring in-sensor and point-of-care applications. Inspired by the flexibility of the human brain, neuromorphic spiking systems are designed to receive diverse stimuli such as light, pressure, temperature, and perform sensory coding 1 , 29 – 32 . Although biological encoding/computing mechanisms are far from pure Boolean logic, specific biological interactions can be described with a combination of binary algebra and multiple-state logic gates 29 – 31 . For instance, in the retina, cones and rods synapse with horizontal cells to constitute a specific preprocessing unit for wavelength and light intensity signals-encoding whose operations correspond to the boolean logic function “AND”. As such, the building blocks represented by the HH-spiking circuit and the logic gates are combined to mimic light-wavelength encoding retina-inspired functions. By exploiting the anti-ambipolar OFF-ON-OFF nature of the logic-gate units, we demonstrate preprocessing functions which activate essential firing patterns corresponding to specific light intensity and wavelength conditions 32 – 34 . This opens the possibility to seamlessly integrate anti-ambipolar logic circuits with spiking circuits to closely mimic specific architecture of the retinal circuitry and achieve complex preprocessing functions. Hence, the bilayer architecture formulates a novel organic electronics concept which enables fine control of the vOECTs anti- ambipolar characteristics, with applications that span the diverse fields of alternative-to-conventional electronics where the control of electrical characteristic in small-scale integrated circuits is of crucial importance.",
"discussion": "Discussion We have reported a general methodology for the fabrication of anti-ambipolar OECT transistors based on a vertical architecture where n-type and p-type materials are assembled as successive stacked bilayers. The bilayer approach enables control over the peak position, full width at half maximum, turn on, and turn off voltages of the vOECTs through material and thickness ratio selection. We demonstrated the versatility of the vOECT design proving its application as reconfigurable logic circuits and an HH-spiking circuit. Using such functions of bilayer devices, we replicated a retina-inspired pathway dependent on the anti-ambipolarity control of the vOECT. Our neuromorphic retinal pathway combines logic circuits which mimic the sensory preprocessing function of horizontal cells with HH neurons that act as retinal ganglion cells and encode the preprocessed signal into precise spike patterns. The unique interplay of signal preprocessing and transmission elements, herald the design of increasingly sophisticated organic neuromorphic systems comprising adaptable and reconfigurable classification and filtering functions. As such, the vertical bilayer architecture formulates a novel concept for tunable organic electronic devices, with applications that are immediate in the organic neuromorphic field but encompass the fields of bioelectronics, wearable electronics, and informatics where device footprint, stability, design flexibility, and biocompatibility parameters are key. Particularly, the material-dependent tunability of three-state logic circuits can be leveraged in the future to optimize these devices for minimal power consumption, switching speed, ion sensitivity, or bio-interfacing opening to a larger range of applications. The vertical architecture of the bilayer anti-ambipolar transistors indicates that the HH-spiking circuit can potentially be further reduced to minimal sizes. On the other hand, controlling vOECT characteristics allows for the fabrication of various neurons with differing threshold voltages, thereby mimicking the diversity of internal neuron characteristics present in the human brain and enabling a route toward the application of traditional neural microcircuits for a variety of computational tasks, such as sensory pathways and motor control 17 – 20 . For practical applications, the stability of the bilayers and integration density should be further developed and improved to enable the realization of reduced circuit sizes. In addition, synapses between photoreceptors, horizontal cells, and retinal ganglion cells are known to contribute significantly to early contrast enhancement, global and local signal processing, and key negative feedback mechanisms, and as such synaptic elements could be introduced to further develop this transduction system. This platform can serve as a building block for the future integration of the various computational components of the retina, thereby enabling bio-interfaced, closed-loop, bidirectional retinal prosthetics. These platforms rely on the combination of spike encoding processed by spiking circuits (retinal ganglion cells) and graded potentials processed by logic circuits (horizontal cells), thus they not only overcome the biocompatibility and sensing limitations of current technologies but also promote a intrinsic neuromorphic computation paradigm through advanced anti-ambipolar devices 1 , 3 , 5 , 30 , 38 – 41 ."
} | 3,497 |
25721758 | PMC4342566 | pmc | 2,434 | {
"abstract": "Theory suggests that species distributions are expanded by positive species interactions, but the importance of facilitation in expanding species distributions at physiological range limits has not been widely recognized. We investigated the effects of the nurse shrub Tamarix chinensis on the crab Helice tientsinensis on the terrestrial borders of salt marshes, a typical coastal ecotone, where Tamarix and Helice were on their lower and upper elevational distribution edges, respectively. Crab burrows were abundant under Tamarix , but were absent in open areas between Tamarix . Removing Tamarix decreased associated crab burrows with time, while simulating Tamarix in open areas by shading, excluding predators, and adding Tamarix branches as crab food, increased crab burrows. Measurements of soil and microclimate factors showed that removing Tamarix increased abiotic stress, while simulating Tamarix by shading decreased abiotic stress. Survival of tethered crabs was high only when protected from desiccation and predation. Thus, by alleviating abiotic and biotic stresses, as well as by food provision, Tamarix expanded the upper intertidal distribution of Helice . Our study provides clear evidence for the importance of facilitation in expanding species distributions at their range limits, and suggests that facilitation is a crucial biological force maintaining the ecotones between ecosystems.",
"discussion": "Discussion Our results reveal that Helice is dependent on Tamarix on the terrestrial borders of salt marshes, and that this dependence is not only because Tamarix is a food resource for Helice , but also strongly due to non-trophic facilitation. Thus, in the coastal ecotone, facilitation expands the landward distribution of marsh crabs. Our work provides an unambiguous demonstration for the critical role of facilitation in mediating species distributions in natural communities, and in maintaining ecotones. Facilitation and species range expansion at physiological range limits Our results show that thermal and desiccation stresses on the terrestrial borders of salt marshes are extreme and lethal to Helice (tethered Helice without shading all died, and Helice burrows are absent in open areas). Where Tamarix occurs, shading by its canopy retains soil moisture and decreases thermal and desiccation stress, creating microhabitats that are physically suitable to Helice . Thermal and desiccation stresses are the major abiotic factors limiting the distribution of marine animals in the upper intertidal, and alleviation of these stresses by shading has commonly been found to drive their facilitative interactions 15 16 . Although it has been argued that facilitation should collapse with extreme stress due to diminished effects of neighbors, or switch to competition for limiting resources (reviewed in Ref. 8 ), our results provide no support for these arguments (also see Ref. 27 ). The two species studied in our work are on different trophic levels and do not compete for limiting resources anyway. Although adult Tamarix persist on the terrestrial borders of salt marshes, abiotic stress such as salinity strictly limits its growth and regeneration 9 . This suggests that even in habitats where benefactor species themselves are severely limited 28 , facilitation can still function as a structuring force of communities. Our work provides a straightforward example of how facilitation expands species' realized niche at their vertical distribution limits. Facilitation by Tamarix drives the expansion of the distribution of Helice from marshes at low elevations to the upper terrestrial border. Without Tamarix , the distribution of Helice would retract to lower elevations that are more frequently flooded. Facilitation has also been shown to expand the high intertidal borders of algae and invertebrates 15 , the low intertidal limits of marsh plants 9 29 , the arid borders of plants in dry habitats 8 30 , and the landward expansion of mangroves 31 . Our work, together with these studies, support the hypothesis that including facilitation in niche theory leads to species realized niches being larger than their fundamental niches 7 8 . Biotic drivers of plant-animal facilitation in physically stressful habitats Our results also show that in addition to alleviation of abiotic stress, Tamarix 's associational defense against predation is a mechanism of facilitation of Helice . Many seabirds such as terns are abundant at our study sites and feed on Helice . It is also known that some seabirds prefer to feed in unvegetated habitats, possibly due to the ease of walking and attacking without vegetation (see Ref. 17 ). The existence of Tamarix on the largely bare terrestrial borders thus reduces Helice' s risk of predation. A previous study also suggested that the facilitation effects of vegetation on fiddler crabs in hypersaline marshes in Georgia (USA) is likely to due to association defense against avian predators 17 , but had no experimental tests. Bortolus, et al. 16 also conducted a crab tethering experiment in an Argentinean high marsh, but found no evidence for plant associational defense as a mechanism of plant facilitation on crabs. This may have been due to the fact that their tethering experiments lasted only a few hours and this may not have been long enough to detect predation. Avian predators, even those resident in an area, are often not continuously present and may move around quite a bit. Since predation is potentially high in many physically stressful habitats including the high intertidal 21 32 , our work emphasizes the overlooked importance of associational defense in plant-animal facilitations in these habitats. Highlighting the importance of facilitation in ecotones The importance of facilitation in the coastal ecotone demonstrated in our study is consistent with other studies on other types of ecotones, such as steppe-woodland 33 34 , alpine treeline 12 13 , and open water-lake shore ecotones 35 . Ecotones are often abiotically extreme to species originated from at least one of the adjacent ecosystems 12 or both (our study). These species are able to persist on ecotones where neighbors ameliorate abiotic stress to their physiological tolerance range, while beyond the ecotones they are limited by abiotic stress. Associational defenses can also be a mechanism driving facilitation on ecotones. For example, ecotones between open water and lake shores provide refugia for fish that would be at risk of predation in open waters or desiccation stress on lake shores 35 . Other mechanisms also likely occur, such as entrapment of propagules 31 . Ecotones have been long known as biodiversity hotspots, and our study together with these previous studies suggest that facilitation is likely one of the key biological forces enhancing diversity in ecotones 36 . Ecotones are often species boundaries sensitive to environmental change, and have been widely used for monitoring the effects of climate change 10 . Future research on facilitation and community organization on species distribution borders or ecotones will be critical to understanding how environmental change affects natural communities."
} | 1,810 |
35275214 | PMC9245121 | pmc | 2,435 | {
"abstract": "Abstract The complexity of lignin structure impedes efficient cell wall digestibility. Native\nlignin is composed of a mixture of three dominant monomers, coupled together through a\nvariety of linkages. Work over the past few decades has demonstrated that lignin\ncomposition can be altered through a variety of mutational and transgenic approaches such\nthat the polymer is derived almost entirely from a single monomer. In this study, we\ninvestigated changes to lignin structure and digestibility in Arabidopsis\nthaliana in near-single-monolignol transgenics and mutants and determined\nwhether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE\n(FMT) or a p -COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be\nintegrated into these novel polymers to further improve saccharification efficiency.\nMonolignol conjugates, including a new conjugate of interest, p -coumaryl\n p -coumarate, were successfully integrated into high-H, high-G and\nhigh-S lignins in A . thaliana . Regardless of lignin\ncomposition, FMT - and PMT -expressing plants produced\nmonolignol ferulates and monolignol p -coumarates, respectively, and\nincorporated them into their lignin. Through the production and incorporation of\nmonolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate\navailability, rather than monolignol transferase substrate preference, is the most\nimportant determining factor in the production of monolignol conjugates, and lignin\ncomposition helps dictate cell wall digestibility.",
"introduction": "Introduction Native lignin, although integral to the growth and development of the plant body, is the\nsource of many problems in industry due to its recalcitrant nature and the difficulties\nassociated with its degradation ( Himmel et al. 2007 ,\n Rinaldi et al. 2016 ). Lignin polymers are the\nproduct of combinatorial coupling reactions between multiple subunits through various\ncategories of covalent cross links ( Freudenberg and Neish\n1968 , Ralph et al. 2004b ). Both the number\nof subunits and the complexity of linkages between the subunits are major factors in the\nrecalcitrance of lignin toward degradation. The majority of the units found in lignin are\ncharacterized by their alkyl aryl ether (β–O–4), C–C (β–β, β–5, β–1, 5–5) or diaryl ether\n(4–O–5) bonds that are either refractory to cleavage or require strong acids or bases and\nhigh temperatures to break ( Rinaldi et al. 2016 ).\nAdding to lignin’s resistance to degradation, there is also potential for condensed and\nnon-linear structures to form during processing ( Shuai\net al. 2016 , Lan and Luterbacher 2019 , Ralph et al. 2019 ). In short, most native lignin polymers\npresent a range of traits that are undesirable for industrial processing. Recently, the\nintroduction of ester bonds into the lignin backbone by the incorporation of monolignol\nconjugates has garnered attention as a method by which to improve the digestibility of the\nlignin, and therefore the cell wall, without negatively impacting the growth or development\nof the plant ( Marita et al. 2014 , Petrik et al. 2014 , Wilkerson et al. 2014 , Smith et al. 2015 ,\n Karlen et al. 2016 , Mottiar et al. 2016 , Sibout et al. 2016 ,\n Zhou et al. 2017 , Kim et al. 2017b , Bhalla et al. 2018 ).\nNative monolignol conjugates can be found in a wide range of plants, from maize to poplar\n( Ralph 2010 , Wilkerson et al. 2014 , Lu et al. 2015 ,\n Karlen et al. 2018 ), but they may also be\nintroduced into model species such as Arabidopsis thaliana and other plants\nthat do not naturally produce them ( Wilkerson et al.\n2014 , Smith et al. 2015 ). Although such\nengineered plants represent a major milestone in improving the processing of lignin, they\nmay not yet represent the best lignins from an industrial perspective. It has been suggested that an optimal lignin for biorefinery operations is one derived from\na single type of monomer in which the resulting lignin contains primarily C–O bonds ( Li et al. 2018 ). Although native lignins do not fit this\nprinciple, there are a number of plants with mutations in lignin biosynthetic genes that\nproduce altered lignins that approximate this scenario, as exemplified by lignins from\nO-Methyltransferase (OMT)-deficient plants producing essentially homopolymers of either\ncaffeyl alcohol or 5-hydroxyconiferyl alcohol ( Ralph et al.\n2001 , Weng et al. 2010 , Tobimatsu et al. 2013 , Li\net al. 2018 ). Other lignin mutants have been well-described in the model plant\n A . thaliana . The c3ʹh null mutant\n( ref8-1 ) produces lignin that is predominantly derived from\n p -coumaryl alcohol and therefore has only\n p -hydroxyphenyl (H) units ( Franke et al.\n2002 ). These plants are dwarfed, but the growth deficiency can be overcome by\nmutating the mediator5a and mediator5b subunits in the\n c3ʹh background ( Bonawitz et al.\n2014 ). The triple mutants ( med5a/med5b/ref8-1 , hereafter denoted\n c3ʹh/med ) therefore have high H-lignin but are not substantially retarded\nin development. The flux from coniferyl alcohol (G-lignin) formation to sinapyl alcohol\n(S-lignin) formation requires the enzyme FERULATE 5-HYDROXYLASE (F5H). Null mutations in the\n F5H gene ( fah1 mutant) result in plants that deposit\nlignin primarily derived from coniferyl alcohol (G-lignin), similar to native gymnosperm\nlignins ( Meyer et al. 1996 , 1998 ). More strikingly, overexpression of the F5H gene\nunder the control of the C4H promoter ( pC4H::F5H ) results\nin the production of lignin that is primarily derived from sinapyl alcohol (S-lignin) ( Meyer et al. 1998 , Marita\net al. 1999 , Stewart et al. 2009 ). The\nlatter three mutant backgrounds all represent potential optimal lignins for lignin\nprocessing because they are dominated by one type of monomer, with the high-S lignin being\nthe best in terms of having a high β-ether content ( Stewart\net al. 2009 ). However, the processability of the lignin might be further improved\nby adding more labile bonds into the polymer as well, in a so-called ‘zip-lignin’ approach\n( Wilkerson et al. 2014 , Zhou et al. 2017 ). \n p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE ( PMT ) from\n Oryza sativa ( OsPMT = OsAT4 ) ( Mitchell et al. 2007 , Withers et al.\n2012 ) and FERULOYL-CoA MONOLIGNOL TRANSFERASE \n( FMT ) enzymes are capable of producing chemically labile ester bonds. Two\nof the best-defined monolignol transferase enzymes are from Oryza sativa \n( OsPMT = OsAT4 ) ( Mitchell et al.\n2007 , Withers et al. 2012 ) and\n Angelica sinensis ( AsFMT ) ( Wilkerson et al. 2014 ). OsPMT and AsFMT \nhave previously been successfully transformed into Arabidopsis, resulting in the production\nof monolignol p -coumarates or monolignol ferulates and their integration\ninto the lignin polymer ( Smith et al. 2013 , 2015 , 2017 ). Crucial to the determination of lignin structure and proof that monolignol conjugates are\nintroduced into the polymer is the derivatization followed by reductive cleavage (DFRC) and\ntwo-dimensional heteronuclear single quantum coherence nuclear magnetic resonance\nspectroscopy (2D-HSQC-NMR) methods. DFRC is a degradative lignin analytical technique\nreleasing the lignin-derived monomers in a reaction that specifically cleaves lignin units\nbound to the lignin polymer by β–O–4 linkages ( Lu and Ralph\n1997a , b ). Lignin end-units are cleaved to\nrelease monomer units, in addition to units bound at their β- and\n4– O -positions in β-ether units. DFRC specifically cleaves ether linkages\nbut leaves ester linkages intact ( Lu and Ralph 1999 ,\n Regner et al. 2018 ). As a result, zip-lignin\nunits/monolignol conjugates are released as intact units and create a unique chemical\nfingerprint that is distinct from that of the prototypical H-, G- or S-monomer units.\n2D-HSQC-NMR is a valuable complement to DFRC because it profiles the lignin in the intact,\nnon-degraded cell wall without requiring lignin isolation ( Kim and Ralph 2010 , Mansfield et al. 2012 ,\n Tobimatsu et al. 2019 ). NMR provides information\nregarding the total lignin units, regardless of the interunit linkage that binds them to the\nlignin polymer. When the results of the chemical analysis are taken together and if\nconjugates can be produced in the mutant lines, it is possible to ascertain the proportion\nof monolignols that are diverted to the production of monolignol conjugates. Although the integration of conjugates into mutant lignin backgrounds would seem like a\nstraightforward proposal, it is unclear whether plants are capable of efficiently\nintegrating monolignol conjugates into the lignin when there is primarily one type of\nmonomer present. The types of monolignol conjugates that would be produced, i.e. which\nmonolignol(s) and which hydroxycinnamate(s) are used for conjugate production, are also\nunknown. The aim of this study was therefore to investigate the production of monolignol\nconjugates in the different mutant backgrounds to delineate the effects of lignin-pathway\nmanipulations on the nature of the conjugating elements (hydroxycinnamates) and to determine\ntheir effects on lignin structure and, ultimately, cell wall digestibility.",
"discussion": "Results and Discussion To determine whether monolignol conjugates can be integrated into lignins dominated by\nsingle monomers and to elucidate the effect the resulting integration had on lignin\nstructure, high-H, high-G and high-S lignin Arabidopsis mutants and transgenics were\ngenerated and grown in parallel with wild-type Arabidopsis. OsPMT or\n AsFMT were overexpressed in the various mutant and wild-type backgrounds.\nThe wild-type, mutant and transgenic plants generated and analyzed in this study were as\nfollows: Col-0 wild-type Arabidopsis, Col-0 996 (empty\nvector control), Col PMT, Col FMT, c3ʹh/med, c3ʹh/med PMT, c3ʹh/med FMT, fah1, fah1\nPMT, fah1 FMT, pC4H::F5H, pC4H::F5H PMT and pC4H::F5H FMT. The 6-week-old wild-type, mutant and transgenic Arabidopsis stems were analyzed for plant\nheight and stem biomass yield. No differences in plant height were observed between\n Col-0 wild-type and Col FMT or Col PMT \nlines ( Supplementary Fig. S1 ). The mutant\nand transgenic fah1 plants also displayed wild-type plant height, with the\nexception of one fah1 FMT line that was slightly taller. The mutant and\ntransgenic pC4H::F5H and c3ʹh/med lines were somewhat\nshorter than wild type. The biomass yield of the primary inflorescence stems follows the\nsame trend as the plant height results ( Supplementary\nFig. S2 ). Both pC4H::F5H PMT lines had higher biomass\nyields than their corresponding mutant, and line 215-2 also achieved a greater plant height,\nbut the reasons for this are unclear. The lignin content of the different lines was also\nexamined to determine if differences could be observed between wild-type, mutant and\ntransgenic lines. The wild-type and mutant lines had similar cysteine-associated sulfuric\nacid (CASA)-lignin content to the transgenic lines in the same mutant or wild-type\nbackground, with the exception of the pC4H::F5H PMT lines, which had a\nslightly lower lignin content ( Supplementary Table\nS1 ). Wild-type Arabidopsis had a high percentage of G-lignin (∼80%), followed by S-lignin\n(∼20%), and a trace level of H-lignin, as has been previously reported ( Fig. 1 , Fig. 2A ) ( Mansfield et al. 2012 , Smith et al.\n2013 ). The AsFMT and OsPMT genes were\nsuccessfully transformed into the wild-type background under the control of the Arabidopsis\nC4H promoter. The Col PMT lines produced monolignol\n p -coumarate conjugates, including both coniferyl\n p -coumarate and sinapyl p -coumarate ( Fig. 1 , Fig. 2B ). The expression of\n OsPMT in these lines correlates with the amount of\n p -coumarates detected through chemical analysis ( Fig. 3A ). Prior investigation of the enzyme kinetics of the\nOsPMT enzyme determined that the enzyme had the highest substrate affinity and catalytic\nefficiency with p -coumaryl alcohol as a substrate, followed by sinapyl\nalcohol ( Withers et al. 2012 ). Coniferyl alcohol was\nthe least preferred substrate in vitro. In contrast, there is a slight preference for\nconiferyl alcohol, the most abundant monolignol, to be used over sinapyl alcohol or\n p -coumaryl alcohol in the production of monolignol\n p -coumarates in planta, suggesting that enzyme kinetic parameters are a\nless important factor than substrate availability in vivo. Similarly, the Col\nFMT lines produced monolignol ferulates and integrated them into the lignin\npolymer, and coniferyl ferulate was the primary monolignol conjugate form generated. As with\nthe Col PMT lines, there was a correlation between AsFMT \nexpression and the product detected ( Fig. 2C ,\n Fig. 3B ). The amounts of monolignol p -coumarates\ndetected by 2D-NMR ( Fig. 1 ) and released\nby DFRC ( Fig. 2C ) were substantially\nhigher than the levels of monolignol ferulates. This is most likely due to the prevalence of\nmonolignol p -coumarates as lignin end-units, facilitating their release\nfrom the polymer and subsequent detection ( Ralph et al.\n1994 , Ralph 2010 , Withers et al. 2012 , Rinaldi et al.\n2016 ). By contrast, monolignol ferulates can be integrated into the backbone of the\npolymer after which only a small percentage of the conjugates can be released by DFRC ( Wilkerson et al. 2014 ). As a result, the amount of\nmonolignol ferulates reported underrepresents the conjugates present in the lignin polymer.\nSimilarly, ferulates are difficult to detect by NMR because they can couple with each other\nand cross-couple into the lignin in a variety of ways ( Wilkerson et al. 2014 ); as such, each structure is present only at low levels,\nmaking their detectability in NMR difficult ( Ralph et al.\n1995 ). These results, together with previous reports, confirm that\n PMT and FMT can successfully be transformed into\nArabidopsis and that these transferase enzymes use whichever monolignol that is most\nprevalent regardless of enzyme kinetic preferences. Fig. 1 2D-HSQC-NMR of Arabidopsis wild-type, mutant and transgenic lines. The aromatic region\nof the spectra is shown for Col wild-type (A), and the corresponding FMT and PMT\ntransgenics (B, C), the high G-lignin mutant (fah1) (D), and the corresponding\ntransgenics (E, F), the wild-type empty vector control (G), the high-S lignin\n( pC4H::F5H ) transgenic (H), and the corresponding FMT- and\nPMT-expressing lines (I, J), and the high H-lignin (c3ʹh/med) mutant (K) and\ncorresponding FMT-expressing transgenic (L) Fig. 2 DFRC analysis of Arabidopsis wild-type, mutant and transgenic lines to determine the\nrelease of monolignols (A), monolignol p -coumarates (B) and monolignol\nferulates (C) from the lignin polymer. HA, p -coumaryl alcohol; CA,\nconiferyl alcohol; SA, sinapyl alcohol; G p CA, coniferyl\n p -coumarate; S p CA, sinapyl\n p -coumarate; GFA, coniferyl ferulate; SFA, sinapyl ferulate. The\naverage areas of p -coumaryl p -coumarates released from\nthe lignin (D) reflect the amount of p -coumaryl\n p -coumarate that was detected as pendant moieties\n( cis - or trans -Ac-HA-Ac- p CA) or\nintegrated into the lignin on both ends via β-ether linkages\n(d 3 Ac-HA-d 3 Ac- p CA) Fig. 3 Expression of the OsPMT (A) or AsFMT (B) transgene in\nprimary inflorescence stem of 4-week-old plants. Transgene expression was normalized to\nthe reference gene ACTIN2 ( At1g18780 ). Error bars\nrepresent standard deviation among biological replicates ( n = 3) One of the types of homogeneous lignin investigated was high-G lignin. High-G lignin was\nachieved in Arabidopsis through a null mutation in the F5H gene ( Humphreys et al. 1999 ). F5H catalyzes hydroxylation of\nthe 5-position of the aromatic ring of coniferaldehyde and coniferyl alcohol to yield\n5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol, respectively. Without this enzyme,\nsinapyl alcohol and syringyl lignin synthesis is blocked, and, as a result, the lignin of\nthese ferulic acid hydroxylase 1 ( fah1 ) mutants is 99.3%\nG-lignin, with only trace levels of H-lignin and S-lignin ( Fig. 1 ). The fah1 mutants were also transformed\nwith OsPMT and AsFMT to determine if high G-lignins can\nproduce monolignol conjugates and to determine what kinds of conjugates are produced. The\nplants were examined for gene expression of AsFMT or\n OsPMT , and all but one of the fah FMT lines had detectable\nlevels of the transgene ( Fig. 3 ). The\n fah1 PMT lines also had homogeneous G-lignin, with only trace amounts of\nH- and S-lignin ( Fig. 1 , Fig.\n2A ); the plants produced monolignol p -coumarates,\nspecifically coniferyl p -coumarate, and incorporated it into the lignin\npolymer ( Fig. 2B ). These results are in\nline with a previous study in which BdPMT1 was expressed in the\n fah1 genetic background ( Sibout et al.\n2016 ). Like OsPMT, BdPMT facilitated the production of coniferyl\n p -coumarate in fah1 plants, but the fah1\nBdPMT1 lines were observed to have less p -coumarates than\nwild-type BdPMT lines ( Sibout et al.\n2016 ). This was attributed to the preference of PMT enzymes for sinapyl alcohol as\nan acceptor substrate. In contrast, fah1 OsPMT lines in this study showed\nhigher levels of p -coumarates than detected in wild-type\n OsPMT plants. This may be due to differences in expression patterns of\nthe PMTs between these two studies, leading to differences in substrate availability or\ndifferences in enzyme kinetics between OsPMT and BdPMT related to coniferyl alcohol. As in\nthe fah1 PMT lines, the fah1 FMT transformants produced\nconiferyl ferulate and integrated it into the lignin polymer, as evidenced by its release\nthrough DFRC and in the whole cell wall (WCW) by 2D-NMR ( Fig. 1 , Fig. 2C ). High-S lignin is another type of homogeneous lignin available in Arabidopsis. High-S lignin\nis achieved by overexpressing the F5H gene using a strong lignin-specific\n C4H promoter ( pC4H::F5H ) ( Meyer et al. 1998 , Marita et al.\n1999 , Stewart et al. 2009 ). Overexpressing\nthe F5H gene results in a more complete conversion of coniferaldehyde to\n5-hydroxyconiferaldehyde and ultimately to sinapyl alcohol and syringyl lignin (S-lignin)\nproduction. The pC4H::F5H lines therefore have ∼97% S-lignin in contrast to\n20% S-lignin in wild-type Arabidopsis plants ( Fig.\n1 ). As with the other lignins enriched in one monolignol, the high-S lines\nwere transformed with OsPMT and AsFMT . Introducing\n PMT and, especially, FMT into high-S lines was aimed at\nproducing primarily sinapyl alcohol conjugates but was also aimed at elucidating whether\nother intermediates in the pathway, such as the various hydroxycinnamates, could be\nsatisfactorily used as substrates. Also of interest was whether the overexpression of\n F5H would result in the production of sinapate, an S-lignin intermediate\noutside the core monolignol pathway, possibly resulting in sinapyl sinapate conjugates that\nare of considerable interest in producing more readily degradable zip-lignins. Sinapyl\nsinapates would make a linear, predictable lignin structure if present in high quantities\nduring lignification. The pC4H::F5H PMT plants had similar levels of S-lignin to the\n pC4H::F5H transgenic alone, with 96% S-lignin ( Fig. 1 ). Of the total lignin in these lines, ∼6% was\n p -coumarate, the majority of it was sinapyl p -coumarate,\nwith small amounts of coniferyl p -coumarate and p -coumaryl\n p -coumarate. The pC4H::F5H FMT lines also had 97%\nS-lignin and produced significant amounts of sinapyl ferulate ( Fig. 1 , Fig. 2A, C ). There was, again, a\ncorrelation between the expression of the PMT and FMT \ngenes and the amount of monolignol conjugate detected on the lignin ( Fig. 3 ). We hypothesized that high S-lignin\n pC4H::F5H mutants expressing an FMT enzyme would accumulate higher levels\nof sinapate, as a byproduct of the S-unit branch of the pathway and therefore perhaps\nproduce sinapyl sinapate conjugates. These lines, however, produced only sinapyl ferulate\nand did not produce detectable levels of sinapyl sinapate. From these data, we can\nhypothesize that the FMT enzyme is not promiscuous enough to utilize sinapoyl-CoA as a\nsubstrate and/or that there are insufficient levels of sinapoyl-CoA to produce the\nconjugate. In Arabidopsis, only one ligase enzyme has been reported to allow sinapate to\ngenerate sinapoyl-CoA (At4CL4) ( Li et al. 2015 ). The\ngene encoding At4CL4 is expressed in the roots and cotyledon veins of Arabidopsis, neither\nof which tissues were examined for the presence of sinapyl sinapate in the pC4H::F5H\nFMT lines, and At4CL4 is not expressed in the stems, which holds\nthe majority of the lignifying tissue ( Li et al.\n2015 ). It is therefore possible that sinapyl sinapates can only be produced in\nArabidopsis or other plant species if the ligase that produces sinapoyl-CoA is co-expressed\nwith a monolignol transferase enzyme that can similarly use the sinapate substrate. High-H lignin has been produced in plants by knocking down or knocking out the\n p-COUMAROYL SHIKIMATE C3′H HYDROXYLASE \n( c3 ′ h ) gene that encodes the C3′H enzyme ( Franke et al. 2002 ). This enzyme is responsible for the\n3′-hydroxylation of p -coumaroyl shikimate to generate caffeoyl shikimate\nand, without this enzyme, plants deposit a lignin dominated by\n p -hydroxyphenyl (H) units. In Arabidopsis, null mutations in the\n c3 ′ h gene also result in severe dwarfism ( Franke et al. 2002 ). This phenotype is not the direct\neffect of the high H-lignin deposition, however, as mutations in two components of the\nmediator complex, med5a and med5b , suppress the dwarf\nphenotype of the ref8 mutant. The resulting c3ʹh/med \nplants still have lignin dominated by H-units (89%) but grow almost normally ( Fig. 1 , Supplementary Fig. S1 ) ( Bonawitz\net al. 2014 ). To determine whether these high-H lines can incorporate monolignol\nconjugates into the lignin, the triple mutant plants were transformed with\n OsPMT or AsFMT genes. No c3ʹh/med PMT \ntransformants were obtained for analysis, suggesting that the plants were seedling-lethal.\nIt is possible that the high H-lignin content results in the production of\n p -coumaryl p -coumarate units, which may be toxic to the\nplant because they cannot be efficiently integrated into the cell wall or stored in the\nvacuole. FMT expression in the c3ʹh/med background was\nhigher than in the other lines, presumably due to the upregulation of phenylpropanoid gene\nexpression previously observed in the med5a med5b background ( Bonawitz et al. 2014 ). Despite high expression of\n FMT , the c3ʹh/med FMT lines had a high H-lignin content,\nas seen in the mutant plants, but did not produce detectable levels of monolignol ferulates\n( Fig. 1 , Fig. 2C , Fig.\n3B ). The lack of ferulates in these high-H lines was expected, as a\ndisruption in the C 3ʹH gene would negatively impact the production of\ndownstream lignin biosynthetic pathway intermediates and products, including feruloyl-CoA,\nwhich is the acyl donor for AsFMT. A pattern that consistently appeared for all the FMT and PMT transgenic lines is that the\nacyltransferases were able to use multiple acceptor substrates. Both enzymes predominantly\nused whichever monolignol substrate was most prevalent. The kinetics of the OsPMT enzyme\nhave been characterized in depth ( Withers et al.\n2012 ). This study determined that OsPMT has the highest affinity and the most\nefficient activity with p -coumaryl alcohol and sinapyl alcohol as\nsubstrates with p -coumaroyl-CoA. Very little activity was observed with\nconiferyl alcohol and other alcohol or acid substrates tested. These kinetic assay results\nsuggest that, regardless of monolignol level, the plants should preferentially produce\n p -coumaryl p -coumarates and sinapyl\n p -coumarates. However, this was not the case. The most prevalent\nmonolignol, especially in homogeneous lignin lines, is almost exclusively used for the\nproduction of monolignol p -coumarates. This means that coniferyl alcohol is\nthe primary substrate for OsPMT in the high G-lignin lines, but also in the Col\nPMT lines, which have 81% G-lignin ( Fig.\n1 ). Sinapyl p -coumarates are the primary monolignol\nconjugate products in all the high S-lignin lines. p -Coumaryl\n p -coumarates were produced at very low levels in all PMT-expressing lines\n( Fig. 2D ), which reflects the low levels\nof H-lignin in all the control and transgenic lines and further supports the hypothesis that\nthe abundance of monolignols is more important than the substrate preferences observed in\nthe in vitro assays. These mutant lines presented an opportunity to test whether plants can produce certain\ntypes of monolignol conjugates and, more importantly, whether those conjugates are\nintegrated throughout the lignin polymer or exist only as pendent groups. One such conjugate\nof interest was p -coumaryl p -coumarate. As has been pointed out ( Ralph et al. 1994 ) and\nreviewed ( Ralph 2010 , Ralph and Landucci 2010 , Vanholme et al.\n2019 ), p -coumarate units in lignins are almost entirely\nfree-phenolic pendent units on the lignin side chain. p -Coumarate radicals\npreferentially undergo radical transfer to produce more stable radical species and therefore\ntend not to enter into radical coupling reactions with guaiacyl or syringyl phenolics ( Ralph et al. 2004a , Hatfield et al. 2008 , Ralph 2010 , Vanholme et al. 2019 ). In that sense, they are not\ncompatible with the radical coupling reactions occurring during lignification and do not\nintegrate into the backbone of the polymer. We have long surmised that\n p -coumarates might be compatible with H-lignin formation, however, with\nradical coupling between H-units and p -coumarates being more likely to\noccur. To assess whether p -coumaryl p -coumarates can be\nintegrated into an H-lignin polymer, p -coumaryl\n p -coumarate was synthesized and a biomimetic dehydrogenation polymer (DHP)\nwas prepared using this conjugate and the H-monolignol, p -coumaryl alcohol.\n2D-HSQC-NMR of the resulting DHP revealed cross-coupled structures from both moieties of the\nconjugate, indicating that p -coumaryl p -coumarate\nintegrated into the H-lignin polymer ( Fig.\n4 ). The most prevalent units, characterized by their diagnostic interunit\nlinkages, in the DHP were the β–O–4 A , β–5 B and β–β\n C units typical of DHP lignins. p -Coumaryl\n p -coumarates were found bound into the DHP in both\n A ʹ and B ʹ units and particularly\ndiagnostic β–β-coupled units C ʹ and C ″ \nwere evident (see Fig. 4 caption that\nexplains how the primed units are diagnostic for the incorporation of the conjugates into\nlignin). These results indicate that, in vitro, the conjugates and, importantly, the\n p -coumarate moiety can be integrated into the lignin polymer backbone, as\nrevealed by units C″ ( Fig.\n4 ). As a result, the lignin backbone contains ester units and can therefore be\nclassified as a zip-lignin, similarly to those produced using monolignol ferulates in normal\nG/S-lignins. In other words, p -coumarates can be used as zip-components\nwhen the lignification is made to be compatible with their radical coupling. It might be\nnoted that this compatibilization of the lignin is the opposite of the original zip-lignin\nstrategy of making ferulate conjugates that were compatible with the coupling of G and S\nmonomers and oligomers in ‘normal’ lignification ( Grabber\net al. 2008 , Ralph 2010 , Wilkerson et al. 2014 ). The alternative approach here to\ncreating zip-lignins in grasses, by utilizing the existing p -coumarate\nconjugates and altering the lignins to be p -coumarate-compatible H-lignins,\nmay be thwarted by the same agronomic issues noted with high-H lignin plants in general.\nIndeed, in this study, correcting for high-H lignin growth defects in Arabidopsis with the\n med5a/med5b double mutant may have resulted in seedling lethality when\nthe triple mutants were coupled with PMT expression. Fig. 4 HSQC-NMR of the synthetic lignin (DHP) prepared from p -coumaryl\n p -coumarate and p -coumaryl alcohol. Contour peaks\nare color-coded to match the assigned structures that are coded with conventional\ndescriptors [ A for β-ether, B for phenylcoumaran and\n C for resinol] for units that may be linked to other H or\n p CA units; the primed variants [ Aʹ , Bʹ ,\n Cʹ and Xʹ ] are H-units in the polymer that have\n p CA units attached and are therefore derived from the\n p -coumaryl p -coumarate conjugates in the\npolymerization; the double-primed units [( A″ ), B″ ,\n C″ ] are those from the p -coumaryl\n p -coumarate conjugates in which the p -coumarate moiety\nitself has coupled into the lignin, providing evidence for cross-coupling of those\n p -coumarate moieties to also integrate them fully into the backbone\nof the polymer. From this spectrum, we can tell little about the etherified and/or\ncross-linked nature of the β-ether units [ A , A″ (and\n A″ , not shown)], but their etherification status (to demonstrate that\nboth the H-unit and the p CA moiety are integrated into the polymer) may\nbe derived from the modified DFRC experiments (see text) The mutant lines expressing OsPMT all produced small amounts of\n p -coumaryl p -coumarates, but the DFRC and NMR analyses\ndo not indicate whether the conjugates were pendent or integrated into the lignin polymer in\nplanta. A modified DFRC approach has already been designed to directly answer this question\n( Martone et al. 2009 , Lu and Ralph 2014 ), i.e. whether the p -coumarates are\nincorporated into the polymer only as end-groups or also within the core of the polymer, as\nis the case for ferulates. The full solubilization achieved by the acetyl bromide reagent\nsuggests that complete acetylation should occur in the first step (see the ‘Materials and\nMethods’ section for further explanation). In reality, there is some potential to\nover-estimate etherification as the first acetylation step may not be able to target a\nproportion of hydroxy end-groups, depending on the access of reagents to the whole lignin\npolymer. The results from the modified DFRC provide an approximation for how much conjugate is found\nas end-groups versus within the polymer, if integration into the polymer does occur. The\n p -coumaryl p -coumarate detected was found only in two\nforms: as unlabeled conjugates or as doubly labeled conjugates. There was no evidence that\nsingly labeled conjugates were released from the lignin. The Col PMT and\n fah1 PMT lines released six times and four times as much doubly labeled\n p -coumaryl p -coumarate as unlabeled product by gas\nchromatography (GC) peak area, respectively. Even allowing for the possibility that some of\nthe doubly labeled product might be end-groups (because of incomplete acetylation in the\nfirst acetylation step of DFRC), these data indicate that p -coumaryl\n p -coumarate is being incorporated into the lignin, as we noted for the in\nvitro DHP study above. The pC4H::F5H PMT lines released as much unlabeled\nconjugate as doubly labeled conjugate, but the data still suggest that some conjugate is\nbeing integrated into the lignin polymer in these plants. These data are supported by the\nresults of a recent study in a rice c3h mutant ( Takeda et al. 2018 ). Previous studies have demonstrated that the addition of significant levels of monolignol\nferulates, and occasionally monolignol p -coumarates, into the lignin\npolymer can improve the digestibility of the plant cell wall ( Wilkerson et al. 2014 , Sibout et al.\n2016 , Zhou et al. 2017 , Kim et al. 2017b , Bhalla\net al. 2018 ). The ester linkages that are generated within the conjugates by the\nmonolignol transferase enzymes are more labile bonds than the ether (or other) linkages that\nare prevalent within the native lignin polymer. Studies in other plant species have shown\nthat engineering plants to have have high-H or high-S lignin significantly improves cell\nwall digestibility (refs) ( Huntley et al. 2003 , Bonawitz et al. 2014 ). For high-S lignin, this is due to\nthe regularity of the lignin structure and the high proportion of ether linkages, but the\nreason(s) for enhanced saccharification in H-lignin-producing plants is unclear. High-H\nlignins have elevated condensation, but may be lower molecular weight and therefore less\nrecalcitrant. Lignin with a high proportion of G-lignin, in contrast, is much more\nrecalcitrant because it has a higher propensity to form condensed linkages that are\ndifficult to cleave. In theory, therefore, the combination of high-H or high-S lignin and\nmonolignol conjugates in the lignin could lead to further improved digestibility. Partial\nsaccharification analysis was performed to determine which milled WCW plant lines have a\nhigher glucose and pentose release when pretreated with a weak base (6.25 mM NaOH) at room\ntemperature. In all lines, there was no significant difference between the control wild-type\nor mutant line and the lines also expressing AsFMT or\n OsPMT genes ( Fig. 5 ).\nThe c3ʹh/med (high H) lines had a higher glucose and pentose release than\nthe wild-type controls, as did the pC4H::F5H (high S) lines ( Fig. 5 ). The fah1 (high G)\nlines had levels of glucose and pentose release that did not significantly differ compared\nto wild-type lines ( Fig. 5 ). Although\nthese results indicate that the monomer composition of the lignin appears to be more\nimportant than the presence of monolignol conjugates with respect to the digestibility of\nthe samples, we suspect that engineering monolignol conjugates to higher than the trace\nlevels observed in this study would significantly improve the digestibility of the different\nlines. A recent study reported levels of monolignol p -coumarates in\nwild-type and pC4H::F5H poplar that rivaled those seen in grasses, in\ncontrast to the trace levels observed in this study, and the transgenic poplar lines had an\nimproved saccharification efficiency under mild alkaline treatments ( Lapierre et al. 2021 ). This study employed the gene encoding the BdPMT\nenzyme, a PMT from Brachypodium distachyon , which may reflect the\nimportance of enzyme choice and activity level when engineering monolignol conjugates into\nthe lignin backbone. Beyond the current observations, engineering plants in ways that\nintegrate conjugates more readily into the backbone rather than remaining pendent remains a\nworthwhile goal that is proving elusive. Fig. 5 Partial saccharification analysis of Arabidopsis mutant and transgenic lines. Samples\nwere pretreated with weak base (6.25 mM NaOH) at room temperature.\n* P < 0.05"
} | 8,635 |
28650444 | PMC5495174 | pmc | 2,437 | {
"abstract": "Biological interactions underpin the functioning of marine ecosystems, be it via competition, predation, mutualism, or symbiosis processes. Microbial phototroph-heterotroph interactions propel the engine that results in the biogeochemical cycling of individual elements and are critical for understanding and modelling global ocean processes. Unfortunately, studies thus far have focused on exponentially-growing cultures in nutrient-rich media, meaning knowledge of such interactions under in situ conditions is rudimentary at best. Here, we performed long-term phototroph-heterotroph co-culture experiments under nutrient-amended and natural seawater conditions which showed that it is not the concentration of nutrients but rather their circulation that maintains a stable interaction and a dynamic system. Using the Synechococcus - Roseobacter interaction as a model phototroph-heterotroph case study we show that whilst Synechococcus is highly specialised for carrying out photosynthesis and carbon-fixation it relies on the heterotroph to re-mineralise the inevitably leaked organic matter making nutrients circulate in a mutualistic system. In this sense we challenge the general belief that marine phototrophs and heterotrophs compete for the same scarce nutrients and niche space, but instead suggest these organisms more likely benefit from each other because of their different levels of specialization and complementarity within long-term stable-state systems.",
"introduction": "Introduction Marine primary production is mainly driven by microscopic phytoplankton since phototrophic picocyanobacteria and picoeukaryotes contribute to almost all photosynthesis that takes place in the vast photic zones of the oligotrophic open ocean 1 . Picocyanobacteria (i.e. Prochlorococcus and Synechococcus ) are the numerically most abundant primary producers on Earth 2 and their abundance is predicted to increase due to climate change 3 . Marine planktonic microorganisms generally show stable cell numbers, with growth and loss largely balanced 4 , 5 . Ultimately, all primary production will be converted into particulate or dissolved organic matter (DOM) which becomes the main source of carbon and energy for the complex marine food web 6 , 7 . DOM is thought to be generated by cell death, viral lysis and inefficient grazing, but also living organisms are known to be, per se , inevitably or ‘intentionally’ leaky, e.g. through the production of extracellular vesicles 8 , active efflux processes or, simply, permeable membrane leakage. In this sense, phytoplankton drive bacterial community dynamics as they are the main suppliers of organic matter 9 . Interestingly, despite reports showing how marine picocyanobacteria acquire simple organic compounds such as amino acids or glucose 10 , 11 , these organisms generally cannot utilise complex DOM as they lack the necessary pool of secreted enzymes 12 , 13 , potentially creating a dependence on remineralised nutrients released by the heterotrophic community. Here, we set out to understand the long-standing anecdotal observation that cultures of phototrophic organisms are more robust and have a much longer lifespan when indigenous heterotrophic bacterial ‘contaminants’ are present, in both natural and nutrient amended seawater. In phototroph-heterotroph systems, heterotrophs clearly benefit through the acquisition of organic matter as a source of carbon and energy 14 . From the phototroph perspective, previous studies concluded that the interaction is based on the heterotrophic scavenging of oxidative stress 15 – 18 , supply of vitamins 19 – 24 or the exchange of growth factors 25 . These are clearly important physiological dependency events that have occurred in strong mutualistic interactions during the evolution of streamlined genomes in stable environments 26 . However, these may only be species-specific co-evolution events and do not explain the general aspects that underpin phototroph-heterotroph interactions. For example, unlike Prochlorococcus , which is deficient in catalase-peroxidase mechanisms involved in oxygen radical protection and detoxification 17 , and picoeukaryotes, which are usually deficient in vitamin production 27 , marine Synechococcus are capable of both these physiological functions, but still require heterotrophic microbes for long-lasting growth as shown in this study. Whilst previous reports analysing marine picocyanobacteria-heterotroph interactions have focused on exponential phase cultures in nutrient-rich media 18 , 28 – 31 , we focus here for the first time on the long-term stable-state growth phase, both in rich media and natural oligotrophic seawater. We provide robust evidence to suggest that mutualistic phototroph-heterotroph interactions are based on nutrient cycling. The phototroph inherently produces and leaks organic matter in the form of photosynthate that allows growth of the heterotroph. In exchange, the heterotroph i) avoids the build-up of photosynthate which, for as-yet-unknown reasons, becomes toxic to the phototroph (in nutrient-rich medium), or ii) ‘unlocks’ inorganic nutrients within the photosynthate that can then be re-used by the phototroph to continue fixing CO 2 (in natural seawater).",
"discussion": "Discussion Living organisms are never alone. The general belief that microbes compete for the same resources clashes with a basic concept in ecology whereby nutrients are recycled between phototrophic and heterotrophic organisms and, hence, how evolution favours specialisation and collaborative behaviour in co-existing populations 26 , 34 . Here, we demonstrate experimentally how marine phototrophic and heterotrophic organisms represent key examples of this collaborative specialisation as highlighted by a clear functional partitioning of roles and the nutrient resources they target ( Figure 5 ). Ultimately, this complementarity of functions implies that one group of organisms cannot survive without the other which favours a mutualistic interaction based on nutrient recycling. We believe this long-term mutualistic interaction is driven passively by the availability and balance of nutrients although further research is required in order to determine if the active production of specific signalling molecules are also involved, as was recently observed in marine diatom-bacterial interactions 25 . Surprisingly, other than proteins involved in phosphate acquisition, Synechococcus appears to behave similarly in rich and poor media, dedicating a large fraction of its cellular resources to photosynthesis and CO 2 fixation (~50% of the proteome) under all culture conditions tested ( Figure 5 ). Microorganisms with streamlined genomes are known to possess minimal gene regulation capacity and marine picocyanobacteria are similar in this respect 2 . Hence, it is perhaps unsurprising to observe only small differences in their proteome under varying culture conditions 35 . Previous transcriptomic studies analysing picocyanobacteria-heterotroph interactions also report relatively low fold changes in differentially-expressed genes between axenic and co-culture conditions 28 – 31 . Overall, the proteome analysis does suggest that the presence of R . pomeroyi allows Synechococcus to allocate more resources to carbon and energy pathways as the heterotroph may help perform other functions required for growth. Synechococcus generates a considerable amount of complex photosynthate. It is unknown what fraction of this organic matter is intentionally excreted (i.e. through outer membrane vesicles 8 or protein secretion 12 ) or arises as a consequence of leakage or cell lysis 36 . Whichever is the case, these streamlined phototrophs do not encode, or produce, a large array of active membrane transporters for organic compounds nor hydrolytic enzymes for the degradation of large polymeric organic matter 12 and seem to rely on the enzymatic activities of the heterotroph, a feature concordant with previous exoproteome data from R . pomeryoi where a large number of membrane transporters and poorly-characterised hydrolytic exoenzymes were detected in the presence of Synechococcus 13 . R . pomeryoi is a highly versatile generalist organism capable of using a wide range of substrates 37 – 39 and is alone able to degrade almost all labile organic matter produced by marine phytoplankton, a facet previously suggested for this organism 13 as well as another marine generalist Alteromonas 40 . Interestingly, R. pomeroyi showed a strong transition from an energy and phosphate scavenging metabolism when alone, to the preferential use of the organic forms within the photosynthate in the presence of Synechococcus i.e. amines and vitamins. Our nutrient analyses and the proteomic response of R. pomeroyi to the presence of the phototroph suggest that Synechococcus produces N- (e.g. protein) and, probably, P-rich (e.g. glycerol-3-phosphate) DOM. The remineralisation of N from DOM or N-rich compounds by heterotrophs was previously reported 41 , 42 . The marine heterotrophic community will use DOM as a source of carbon and energy and, hence, the fraction used for the latter (i.e. respiration) will generate an excess of nutrients such as N or P. All primary-produced DOM in our long-term stable-state co-cultures or in balanced natural non-blooming euphotic marine ecosystems, where biomass is generally maintained constant 4 , 5 , will ultimately be respired and, therefore, all nutrients re-mineralised. A small fraction of non-degraded DOM may become highly recalcitrant as part of the biological pump 43 , but this recalcitrant DOM is usually poor in N and P and contains extremely high C:N and C:P ratios 44 , highlighting the preference of heterotrophs for N- and P-rich DOM and the mineralisation of these macro-nutrients. Here, we prove that the recycling of all essential nutrients (i.e. N and metals, and especially P) is essential for maintaining the viability of Synechococcus suggesting this organism cannot ‘unlock’ these nutrients from its own photosynthate and requires the presence, in this case, of a generalist heterotroph or, in nature, of the succession of a specialised microbial community 45 . Picocyanobacteria seem to have evolved into ‘photosynthetic factories’, unable to sustain the burden of its large photosynthetic machinery during extended periods without a constant supply of inorganic nutrients and light. Nutrient circulation is needed for a functional system ( Figure 5 ). Inorganic macro- and micro-nutrients are constantly being made available in marine environments despite their rapid assimilation and incorporation into organic matter by microbes via the recycling of nutrients within the microbial loop. This connects phytoplankton and heterotrophic bacterial niche space since the former is limited by inorganic nutrients (e.g. N, P and iron) whereas the latter is normally carbon and energy limited. Based on these ‘niche’ requirements, both groups of organisms reach a stable balance in marine systems where relatively constant cell numbers are maintained 5 . Interestingly, our natural seawater work shows that the simple two-strain system we present here is a good surrogate of the general heterotroph-phototroph cell ratio in the oceans 32 (10:1) where cell yields are also limited by overall nutrient availability. Nevertheless, this Synechococcus - R. pomeroyi system lacks the presence of viruses or grazers which clearly play an important role in keeping the ecosystem ‘young’ and dynamic (see 46 ) by speeding up the recirculation of nutrients. In any case, data we present here clearly demonstrate for the first time that the foundations of long-term self-sustained heterotroph-phototroph mutualistic interactions are based on nutrient recycling, which we posit is a basic concept in ecology."
} | 2,960 |
37662754 | PMC10472071 | pmc | 2,438 | {
"abstract": "Over the past half century, limited use of synthetic fertilizers, pesticides, and conservation of the environment and natural resources have become the interdependent goals of sustainable agriculture. These practices support agriculture sustainability with less environmental and climatic impacts. Therefore, there is an upsurge in the need to introduce compatible booster methods for maximizing net production. The best straightforward strategy is to explore and utilize plant-associated beneficial microorganisms and their products. Bioinoculants are bioformulations consisting of selected microbial strains on a suitable carrier used in the enhancement of crop production. Fungal endophytes used as bioinoculants confer various benefits to the host, such as protection against pathogens by eliciting immune response, mineralization of essential nutrients, and promoting plant growth. Besides, they also produce various bioactive metabolites, phytohormones, and volatile organic compounds. To design various bioformulations, transdisciplinary approaches like genomics, transcriptomics, metabolomics, proteomics, and microbiome modulation strategies like gene editing and metabolic reconstruction have been explored. These studies will refine the existing knowledge on the diversity, phylogeny and beneficial traits of the microbes. This will also help in synthesizing microbial consortia by evaluating the role of structural and functional elements of communities in a controlled manner. The present review summarizes the beneficial aspects associated with fungal endophytes for capitalizing agricultural outputs, enlists various multi-omics techniques for understanding and modulating the mechanism involved in endophytism and the generation of new bioformulations for providing novel solutions for the enhancement of crop production.",
"conclusion": "6 Conclusion The microbiome is a critical aspect of the plant ecosystem associated with vital functions like biocontrol of phytopathogens, abiotic stress tolerance, nutrient acquisition, and the production of various volatile organic compounds such as enzymes, plant hormones, and secondary metabolites essential for plant development and optimum health. Our understanding of the complexities of plant-fungi interaction has grown quickly over the last few decades. Recent advances in the development of novel biotechnological techniques such as omics technology and systems biology give an unparalleled chance to design these interactions, which can offer new alternatives to already existing pesticides and chemical fertilizers, hence taking a step towards agricultural sustainability. Along with the gene editing system, the modulation of microbial consortia for beneficial features such as phosphate solubilization, nitrogen fixation, and biocontrol has been a huge accomplishment. Custom-prepared inoculants can be employed as an excellent consortium of crop productivity. Despite the progress, the use of fungal microbiomes as bioinoculants is a study under construction. The subsequent goal should be a lab-to-land approach with the sustained translation of plant-associated microbiome, interdisciplinary research, cross-training of the next generation of scientists, and sensitizing farmers for the successful adoption of these innovative technologies.",
"introduction": "1 Introduction Booming global population, increased food security, environmental and climatic fluctuations, and a shortage of tillable land are few of the unprecedented hurdles in the envisioned sustainable agriculture goals [ 1 ]. This has resulted in the intensification of the crop yield per unit area of soil used, known as agricultural intensification which is the most efficient and prioritized method for meeting world's healthy food demands [ 2 , 3 ]. The conventional methods used for intensifying crop production are based on the excessive use of fertilizers and pesticides [ 4 ]. The uncontrolled and prolonged input of these agrochemicals adversely affects the ecosystem structure and function [ 5 ]. In view of this, microbes and microbial products have turned out to be the new frontier for fostering innovation, engineering, and the development of bioformulations for achieving ecological balance and sustainability. This has expedited the research on microbial diversity for revitalizing the green revolution by improving agricultural productivity [ 6 ]. The endophytic microbial community, in particular, has undergone millions of years of coevolution within the host plant and resulted in the generation of responsive feedback in host physiology [ 7 ]. The plant and its endobiome have been considered a mini-ecosystem in which the endobiome acts as an essential determinant for the overall growth and development of the plant. They either colonize intercellularly or intracellularly in the healthy and living tissues of host plants to complete the whole or part of their life cycle. The association with the host plant ranges from symbiotic to pathogenic, however, they are mostly in a symbiotic relationship. The host provides living space and nutrition to the endophytes, and in return, they bestow the plant with various advantages like triggering an immune response and imparting tolerance by producing secondary metabolites, proteins, and hormones [ 8 ]. The putative attributes of fungal endophytes can be suitably translated and implemented as bioinoculants, biostimulants, biofertilizers, and biopesticides. These efficacious microbial bioproducts circumvent the hazardous outcomes associated with conventional farming practices [ 9 ]. Furthermore, the spectacular progress in molecular biology, synthetic biology, high-throughput screening, enzyme discovery, protein engineering, metabolic engineering, and multiomics approaches has provided insights into the mechanistic understanding of various metabolic networks for delivering agronomic solutions [ 10 ]. Therefore, the present review provides an understanding of the beneficial attributes of plant-associated fungal endophytes, their molecular mechanisms, and modern sequencing technologies for identifying and shaping the endophytic community. Besides, various fungal bioformulations for enhancing agricultural productivity in a sustainable manner have also been discussed."
} | 1,562 |
38560681 | PMC10979216 | pmc | 2,440 | {
"abstract": "The metagenomic approach stands as a powerful technique for examining the composition of microbial communities and their involvement in various anaerobic digestion (AD) systems. Understanding the structure, function, and dynamics of microbial communities becomes pivotal for optimizing the biogas process, enhancing its stability and improving overall performance. Currently, taxonomic profiling of biogas-producing communities relies mainly on high-throughput 16S rRNA sequencing, offering insights into the bacterial and archaeal structures of AD assemblages and their correlations with fed substrates and process parameters. To delve even deeper, shotgun and genome-centric metagenomic approaches are employed to recover individual genomes from the metagenome. This provides a nuanced understanding of collective functionalities, interspecies interactions, and microbial associations with abiotic factors. The application of OMICs in AD systems holds the potential to revolutionize the field, leading to more efficient and sustainable waste management practices particularly through the implementation of precision anaerobic digestion systems. As ongoing research in this area progresses, anticipations are high for further exciting developments in the future. This review serves to explore the current landscape of metagenomic analyses, with focus on advancing our comprehension and critically evaluating biases and recommendations in the analysis of microbial communities in anaerobic digesters. Its objective is to explore how contemporary metagenomic approaches can be effectively applied to enhance our understanding and contribute to the refinement of the AD process. This marks a substantial stride towards achieving a more comprehensive understanding of anaerobic digestion systems.",
"conclusion": "8 Conclusion and future perspectives Anaerobic digestion is a widely employed process for converting organic waste into methane gas, and its efficiency is influenced by the digester microbiome, which is affected by various factors such as substrates and operational conditions. Metagenomic has emerged as a powerful tool for studying the microbial communities involved in AD processes, offering several advantages. Firstly, metagenomics enables the identification of the entire microbial community participating in the process, including non-cultivable organisms, providing a comprehensive understanding of the metabolic pathways and interactions between different microbial groups. Secondly, metagenomics can overcome limitations in the process, such as the presence of inhibitory compounds or imbalances in the microbial community, by identifying potential solutions for optimizing the AD process. Finally, metagenomics can aid in developing strategies to optimize the AD process, such as the addition of specific microbial groups or modifications to the operating conditions. Recent advances in sequencing technologies have made metagenomics more accessible and effective for studying anaerobic digestion. High-throughput sequencing allows for the rapid identification of microbial community composition and functional genes involved in the anaerobic digestion process. This information is crucial for developing more efficient and sustainable AD systems and for precisely monitoring and troubleshooting existing systems.",
"introduction": "1 Introduction Anaerobic digestion (AD) is a biochemical process with the capacity to convert a diverse range of feedstocks, primarily organic wastes including livestock manure, food wastes, sewage sludge, crop residues, agricultural byproducts, and the organic fraction of municipal solid wastes, into biogas. The growing demand for energy and the imperative to reduce greenhouse gas emissions emphasize the necessity to transition from fossil fuels to renewable energy sources, positioning biogas as a significant player in the evolving energy landscape. Biogas obtained through AD exhibits versatile applications, including combustion for heat production, electricity generation, and as a viable vehicular fuel. The conversion of wastes into biogas via AD is facilitated by a complex community of microorganisms, collectively known as the microbiota, engaging in a series of biological reactions. The intricacies of the AD process often led to the description of anaerobic digesters as “black box” and the AD microbiome as “black matter”, primarily due to the complex and heterogeneous ecosystem and the huge diversity of uncharacterized microbes. The efficiency of these processes is influenced by various environmental, microbiological and process parameters [ 1 ]. A more thorough understanding of these parameters would provide valuable insights necessary for adjusting production parameters and conditions, ultimately optimizing the process to achieve maximum product yield with minimal production costs. The increasing accessibility of high-throughput Next-Generation Sequencing (NGS) technologies and the advancement of bioinformatic algorithms have elevated metagenomic analysis to an invaluable method for understanding microbial communities within anaerobic digestion (AD) systems [ 2 ]. Thanks to sequencing methods, particularly when applied to metagenomic approaches, scientists have made several exciting discoveries about the microbial populations in anaerobic digestion processes [ 3 ]. Metagenomics involves the analysis of genetic material from environmental samples, eliminating the need for the cultivation of individual organisms [ 4 , 5 ]. Furthermore, metagenomics can be used to monitor microbial communities in anaerobic digesters and diagnose issues such as acidification or inhibition. This information can be then applied to develop strategies aimed at improving the stability and efficiency of anaerobic digestion processes [ 6 , 7 ]. Recent findings have unveiled a strong correlation between the structures of taxonomic and functional genes present in anaerobic microorganisms found in biogas-generating digesters [ 2 ]. Moreover, it has been revealed that both these gene structures are susceptible to various environmental factors including digester setup, constituents of feedstock, temperature, organic loading rate (OLR), hydraulic retention time (HRT), and levels of free ammonia [ 8 ]. In addition, the examination of functional genes using metagenomic investigations and network-based methods enables the estimation of corresponding metabolic pathways. This approach allows for the identification of new metabolic pathways and microbiological mechanisms operating within the biogas digesters [ 9 ]. The application of molecular biological methods and DNA sequencing techniques, including NGS, has significantly advanced our understanding of the microbiome in AD systems and the functional roles of different microorganisms in the AD process [ 10 , 11 ]. For example, The study of the AD microbiome has shifted to a new stage involving a genome-centric metagenomic approach [ 12 ]. This approach entails the recovery of individual genomes from metagenomic data, enabling a more detailed analysis of genome information for various novel organisms residing in AD microbiomes and facilitate insights into their potential functions and lifestyle. While it holds great promise for studying the AD microbiome, its application is still in its infancy due to the technical challenges associated with metagenomic approaches. Several works have previously discussed the application of metagenomics to study microorganisms involved in AD and biogas production (BP) [ 9 , 13 , 14 ]. These studies provide general microbiological and bioinformatic workflows and resources for examining microbial ecology in AD [ 2 , 15 ]. The present review summarizes the current and future applications of the metagenomic approaches in the AD process. It is dedicated to delving into the current landscape of metagenomic analyses, focusing on advancing our comprehension and evaluating biases and recommendations in the analysis of microbial communities in anaerobic digesters."
} | 2,001 |
26253675 | PMC4579418 | pmc | 2,441 | {
"abstract": "Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens . A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH . We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.",
"introduction": "INTRODUCTION Methods to remove, replace, and control genes are instrumental to understanding bacterial physiology. Together with DNA synthesis and high-throughput sequencing, these tools enable synthetic reconstruction of pathways and design of biological circuitry ( 1 ). Many genetic approaches were first developed in fast-growing bacteria such as Escherichia coli , while genetic techniques able to interrogate the physiology of slower-growing anaerobic organisms are often more limited ( 2 ). In some cases, heterologous expression of foreign genes can help infer function in a genetically intractable organism ( 3 ), but model hosts can lack key biochemical processes or cofactors making functional expression challenging ( 4 ). Electron transfer to metals and electrodes by Geobacter sulfurreducens is an example of a complex respiratory strategy encoded in multigene loci throughout the chromosome, requiring cytochrome maturation, protein secretion, cell surface attachment, sensing, and motility ( 5 – 8 ). The genetic study of metal reduction will ultimately require deletion and reexpression of multiple genes in the native organism, under a variety of planktonic and long-term biofilm growth conditions. A gene replacement protocol using electroporation of linear DNA fragments was first developed for G. sulfurreducens in 2001 ( 9 ), and insertion of antibiotic cassettes allowed for construction of multiple deletion mutants, such as the Δ omcB :: cat Δ omcST :: nptII Δ omcE :: aacC Δ omcZ :: aadA quintuple deletion mutant. However, due to the finite number of resistance genes available in Geobacter , it is difficult to delete additional loci or complement such complex constructs ( 10 ). Conjugal plasmid transfer from an Escherichia coli donor strain, and transposon mutagenesis via nonreplicating plasmids is also feasible in G. sulfurreducens ( 9 , 11 ). These tools accelerate mutant construction and discovery, but issues of transposon insertion polarity, growth inhibition due to use of multiple antibiotics, and unknown expression levels from promoters on plasmids limit complementation and interpretation of results ( 12 , 13 ). A cre-lox recombination gene disruption strategy used in G. metallireducens and G. sulfurreducens removes the antibiotic cassette from the chromosome ( 14 , 15 ). However, this method leaves a loxP scar sequence and creates multiple identical loxP sequences throughout the genome if additional deletions are constructed ( 16 ). In this study, we implemented a SacB/sucrose counterselection strategy to generate scarless deletions in G. sulfurreducens . We also tested the stability and the effect of commonly used broad-host-range plasmids on growth using soluble, insoluble, and poised electrode electron acceptors. New vectors based on the RK2 origin of replication with native constitutive and engineered inducible promoters were constructed for controlled gene expression in G. sulfurreducens using different electron acceptors. To accelerate the screening of constructs and promoters, we describe a real-time, high-throughput Fe(III) citrate reduction assay to measure the response of the engineered promoter system. These new tools make it possible to construct multigene deletions, promoter modifications, in-frame fusions, and inducible genetic circuitry in Geobacter sulfurreducens .",
"discussion": "DISCUSSION Precise removal and insertion of DNA into the G. sulfurreducens chromosome enabled by the SacB/sucrose counter selection strategy allows for construction of multigene deletion and insertion mutants without the effects of polarity or undesired changes in the chromosome. While weeks of anaerobic manipulation are required to generate a mutation using the two-step method, the resulting strain is not burdened by expression of antibiotic resistance cassettes, increasing the availability of markers for maintenance of expression plasmids. Researchers should take caution in the use of antibiotics, since we show the aminoglycoside kanamycin is inhibitory even when cells are expressing a resistance gene. Gentamicin, another aminoglycoside, showed similar inhibitory effects on cell growth even when respiring to a soluble electron acceptor in our preliminary studies, and therefore was not considered as we developed plasmids for this work. This underscores the need for proper empty-vector controls conducted under similar conditions in genetic studies. Ectopic expression in G. sulfurreducens previously relied on broad-host-range plasmids of unknown copy number from promoters with unknown strengths, which could partly explain the incomplete complementation of mutations in Geobacter ( 5 , 7 , 13 ). By using a more stable, low-copy-number plasmid and modifying a constitutively expressed promoter ( acpP ), we have the ability to control gene expression by the addition of a small, nontoxic, membrane permeable molecule. Since we developed these vectors using a crucial respiratory cytochrome as the benchmark for full complementation, analyses of other physiologically relevant genes involved in metal respiration should be possible. For more complex genetic circuitry or multigene expression analysis, future work will need to engineer additional inducible expression systems with other membrane permeable compounds such as benzoate or short-chain fatty acids. To accelerate the development of expression vectors and analyze the complex electron transfer pathway of Geobacter , a sensitive kinetic assay is necessary. A real-time Fe(III) citrate assay proved to be faster and use less reagent than the discontinuous assay. This nongrowth assay appears to detect residual rates of electron transfer in Δ imcH mutants that were too slow to support growth and that were not easily detectable in the traditional assay. Part of this increased sensitivity could come from the presence of the Fe(II)-trapping reagents poising the Fe(III)/Fe(II) ratio consistently high, keeping electron transfer favorable. This sensitivity and consistency will be useful in the search for secondary or overlapping electron transfer mechanisms. The genetic tools presented here will aid future genetic manipulation of G. sulfurreducens , and minimize confounding factors such as antibiotic inhibition or stability of vectors. This system offers a much needed ability to directly manipulate gene expression levels in Geobacter and provides an example of how Geobacter can be engineered to produce electrical current upon sensing an external signal ( 30 ). This combination of genetic precision and transcriptional control is a crucial part of any biosensor or biodevice based on extracellular electron transfer."
} | 2,126 |
29299059 | PMC5740764 | pmc | 2,442 | {
"abstract": "Background The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. Results A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. Conclusions A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits. Electronic supplementary material The online version of this article (10.1186/s13068-017-0991-x) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions Part of the BESC mission to produce feedstock lines for basic and applied studies in biofuel production was addressed by (a) organizing a TP to identify and select target genes that could alter cell wall structure in perennial feedstocks for better biofuel production, and (b) implementing a pipeline to produce transgenic plants altered in expression of these target genes, analyze tissue, and centralize data storage. Within this report, these activities are detailed in the context of evaluating transgenic switchgrass growth, wall traits and recalcitrance for basic and applied goals. Among constructs accepted into the TP during rounds 1–12 because of their hypothesized importance for biofuel production, some were found to be difficult to express or had no impact on measured cell wall traits in transgenic plants. Most members of the latter group were not selected for in-depth analysis. Difficulties cloning or expressing particular sequences or a lack of effect on cell wall traits in transgenic lines expressing a specific sequence are, however, useful results instructive to researchers considering future target sequences for cell wall modification. To illustrate this point, detailed analysis of the effect on lignin monomer and/or lignin content and plant growth traits for transgenic lines modified for expression of individual HCT genes was described. While no influence on lignin G and S monomer accumulation was observed after silencing two individual HCT s, simultaneous knockdown of these genes demonstrated that family silencing could result in modified lignin G and S monomer content and less total lignin in aerial tissue, but poor root growth. For a smaller number of genes, there were measurable differences in cell wall traits within the transgenic lines and these modified wall traits were correlated with reduced recalcitrance of the tissue. Findings published for some of these lines are tabulated and summarized in this report. These lines are undergoing advanced cell wall and growth trait studies. Modified expression of regulatory genes, such as transcription factors, more often yielded measurable qualitative differences in traits than when specific pathway genes were modified in expression. Also, it is clear that perturbing cell wall biosynthesis does not necessarily lead to decreased biomass and may lead to more biomass compared with control tissue. All information from these studies was captured for public dissemination through a LIMS. Thus, the target genes and transgenic plants reported here provide the research community new information when attempting to identify gene candidates for improved recalcitrance or when comparing results with those from BESC studies. Certainly, researchers could consider plant crosses based on the growth and recalcitrance phenotypes reported for these transgenic lines. Additionally, considering that these plants were heterozygous for all constructs, researchers may begin additional silencing studies by employing CRISPR/Cas9 technology to completely downregulate all homoeologous or sufficiently homologous target genes within switchgrass [ 59 ], as has been approached in another allopolyploid species, wheat [ 60 , 61 ]. The BESC switchgrass TP approach proved to be an example of a successful organizational model to study the role of target genes in cell wall biosynthesis and recalcitrance that can be used in part or in its entirety when considering methods and targets for manipulation of other traits in other species.",
"discussion": "Results and discussion Switchgrass transformation pipeline From the start of BESC in October 2007 through 2012 [submission rounds 1–12 within the TP], 128 constructs representing 88 candidate genes were accepted into the reverse genetics program for transgenesis in switchgrass. The identity of some genes accepted into the TP and the data obtained from the subsequently produced transgenic switchgrass lines are not disclosed in this report because either (a) experiments are still being performed to characterize recalcitrance traits in these lines for commercial benefit or (b) results modifying specific transgene(s) are being incorporated into single- or multi-gene-centric reports with more detailed descriptions of outcomes than can be given here. Thus, results presented here represent a subset of the total number of target genes evaluated with a bias towards genes that were not ultimately pursued for their commercial use. The findings therefore will benefit the research community not only in the identification of genes or gene fragments with potential to decrease cell wall recalcitrance, but also in indicating genes believed to be good candidates for modifying recalcitrance but which subsequently were shown to be ineffective, and in illustrating genes and gene fragments that posed cloning, expression, or plant growth hurdles. This report also will serve as a reference for the TP procedure used to produce and analyze those TP products. Membership on the TP committee, tasked with creating the TP submission form and accepting TP submissions, required experience in identifying target genes with the potential to modify recalcitrance, producing transformation constructs and transgenic plants, and/or analyzing the effects of construct expression on cell wall synthesis and biomass recalcitrance. Members of this committee [nine BESC principal investigators (PIs)] identified a set of core requirements for submissions to the pipeline. Among the requirements were: (a) name of the candidate gene; (b) cDNA or genomic sequence with accession number, if available; (c) type of expression requested (i.e. stable knockdown, stable overexpression, stable ectopic expression, or transient expression through virus-induced gene silencing (genes submitted through the latter category will be presented in a separate report); (d) evidence that the gene was expressed (e.g. through microarray, RNA-seq, northern blot, RT-PCR analyses); (e) phylogeny showing related genes within applicable gene families and highlighting potential homologs (switchgrass is a polyploid) [ 30 , 31 ]; (f) shared motifs or domains among members of the gene family; and (g) rationale for submission (i.e. the proposed mechanism for an influence of gene or gene fragment expression on cell wall constitution and how this could directly or indirectly lead to enhanced saccharification and biofuel production from switchgrass). A copy of a completed TP submission form whose construct was accepted into the TP is included (Additional file 1 ). Through TP submission round 12, a total of 615 submissions for gene overexpression or gene or gene family knockdown in switchgrass, poplar and foxtail millet ( Setaria italica ) were received from BESC member scientists. Information in submissions that were accepted into the TP made a compelling case for overexpressing or knocking down a candidate gene or gene family to decrease recalcitrance without decreasing plant growth and biomass or in providing new information about wall synthesis pathways. A small fraction of genes with unknown function, but expressed at high levels in relevant target tissue such as stems, were also accepted. The submissions that received support from the majority of the TP committee members were accepted for analysis within the TP and the completed submission form was made available to BESC team members tasked with cloning the target gene or gene fragment and transforming plants. For the 53 constructs included that were approved by the TP committee, 31 were chosen for overexpression of target genes and 22 constructs were chosen for knockdown of target genes via RNAi (Table 1 ). Multiple genes were targeted for both overexpression and knockdown and nine of these are listed. Many of the target genes were related to lignin biosynthesis (Table 1 ). Given that both altered S:G (syringyl:guaiacyl) monolignol composition and decreased total lignin in secondary cell walls can increase enzymatic sugar release [ 32 ], and therefore potentially lead to decreased recalcitrance, it is not surprising that BESC researchers nominated known lignin biosynthesis genes early in the TP. Indeed, two target genes within the BESC switchgrass TP whose modified expression resulted in significant decreases in cell wall recalcitrance were modified in lignin composition and amount [ 21 , 33 – 36 ]. Table 1 Compiled subset of constructs approved by the BESC Transformation Committee for overexpression or knockdown of target genes to modify cell wall traits Gene name BESC ID Transgene origin Expression system Gene in expression vector Expression vector Events a \n Target expression level b \n Visual phenotype Comments PI c \n Ref. \n 4 - Coumarate: coenzyme A ligase ( 4CL ) 180 Switchgrass KD Yes pVT1629 100 80% decrease Brown and red pigment in shoots d \n 2 [ 48 , 62 ] \n Cinnamic acid 4 - hydroxylase ( C4H ) 182 Switchgrass KD Yes pANIC 4B 9 50% decrease Normal growth No change in lignin or S/G monomer ratio 2 [ 48 ] \n Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase ( HCT1 ) 184-1 e \n Switchgrass KD Yes pANIC 8A 28 0–95% decrease Normal growth Revised to target HCT1. No change in lignin content or composition 7 This report \n Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase ( HCT2 ) 184-2 e \n Switchgrass KD Yes pANIC 8A 41 0–90% decrease Normal growth Revised to target HCT2. Increase in H lignin monomer in majority of transgenic lines analyzed. No change in lignin content. 7 This report \n Caffeoyl CoA 3 - O - methyl transferase ( CCoAOMT ) 186 Switchgrass KD Yes pANIC8A 17 0–90% decrease Normal growth No change in lignin content or composition 7 [ 48 ] \n Coumaroyl shikimate 3′ - hydroxylase ( C3′H ) 264 Switchgrass KD Yes pANIC 4A 48 0–80% decrease Normal growth No change in lignin content or composition 7 \n VirE2 - interactor protein ( VIP2 ) 266 Arabidopsis OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comments) Not applicable (see comment) Gene not clonable 7 \n Arabinosyl transferase 1 (Rra1 reduced residual arabinose) 274 Switchgrass KD No Not applicable (see comment) Not applicable (see comment) Not applicable (see comments) Not applicable (see comment) Gene not clonable 3 \n Endoxylanase (PttXyn10A) / Endoxyla nase 282 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comments) Not applicable (see comment) Gene not clonable 3 \n LIM transcription factor ( LIM 1 ) 292 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comments) Not applicable (see comment) Gene not clonable 2 \n R2R3 - MYB transcription factor ( MYB4 ) 294 Switchgrass OE Yes pANIC 2B 5 ninefold to 11-fold increase Varying impacts on growth, from reduced to increased yield 2 [ 33 , 34 , 36 ] \n Gibberellin 20 - oxidase ( GA20 - ox ) 318 Foxtail millet OE Yes pANIC 10A 3 No change Normal growth. No change in biomass yield No change in lignin content, S/G monomer ratio or sugar release 4 \n Gibberellin 20 - oxidase ( GA20 - ox ) 319 Switchgrass KD Yes pANIC 12A 6 No change Normal growth. No change in biomass yield No change in lignin content, S/G monomer ratio or sugar release 4 \n NAC transcription factor ( NAC 2 ) 321 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 1 \n Peroxidase - 30 \n 324 Switchgrass OE Yes pANIC 6A 52 Not done Normal growth No change in lignin content or composition 5 \n Dirigent protein \n 327 Switchgrass OE Yes pANIC 6A 23 Not done Normal growth No change in lignin content or composition 5 \n Dirigent protein \n 328 Switchgrass KD Yes pANIC 8A 30 Not done Normal growth Full length construct in antisense orientation. No change in lignin content or composition 5 \n Peroxidase - 1 \n 343 Switchgrass FKD Yes pANIC 8A 19 Not done Normal growth No change in lignin content or composition 5 \n NAC transcription factor ( NAC - AP2 ) 348 Switchgrass OE Yes pCAMBIA 1305 65 21-fold to 65-fold increase Increased biomass yield in greenhouse 6 [ 63 ] \n NAC transcription factor ( NAC - AP2 ) 349 Switchgrass FKD Yes pSTARGATE 6 No change Discontinued (regenerated plants were false positive) 6 \n NAC transcription factor ( NAC 033 ) 350 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 2 \n Dirigent protein - 2 \n 356 Switchgrass KD Yes pANIC 8A 23 Not done Normal growth 5 \n Sucrose synthase 1 ( SUS1 ) 413 Switchgrass OE Yes pANIC 10A 5 twofold to sevenfold increase Increased plant height, tiller number and biomass yield. Increased lignin 4 [ 64 ] \n Cellulose synthase - like ; subfamily D ( CslD4 ) 540 Switchgrass OE Yes pANIC 10A 10 Not done Normal growth Under further analysis 3 \n Cellulose synthase - like ; subfamily J ( CslJ ) 543 Switchgrass OE Yes pANIC 10A 11 Not done Normal growth Under further analysis 3 \n Cellulose synthase - like; subfamily F ( CslF6 ) 549 Switchgrass OE Yes pANIC 10A 23 Not done Normal growth Under further analysis 3 \n Cellulose synthase - like; subfamily F ( CslF9 ) 552 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 3 \n Cellulose synthase 8 ( CesA8 ) 558 Switchgrass OE Yes pANIC 10A 10 Not done Normal growth Under further analysis 4 \n NAC transcription factor ( NAC - AP2 ) 692 Switchgrass OE Yes modified pER8 30 No change Not determined Discontinued as plants unresponsive to estradiol treatment 6 \n Laccase 4 ( Lac 4 ) 693 Switchgrass KD Yes pANIC 8A 30 Not done Normal growth No change in lignin amount or composition 5 \n R2R3 - MYB transcription factor ( MYB63 - like2 ) 721 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 2 \n R2R3 - MYB transcription factor ( MYB63 - like3 ) 722 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 2 \n Knotted - like homeobox protein 1 ( KN1 ) 833 Switchgrass KD Yes pANIC 12A 8 No change Normal growth. No change in biomass yield Discontinued 4 \n Knotted - like homeobox protein 1 ( KN1 ) 834 Switchgrass OE Yes pANIC 10A 5 Up to sevenfold increase Normal growth. No change in biomass yield 4 [ 65 ] \n Gibberellin 2 - oxidase ( GA2 - ox ) 835-1 e \n Switchgrass OE Yes pANIC 10A 14 Up to 14-fold increase Dwarf to semi-dwarf growth 4 [ 66 ] \n Gibberellin 2 - oxidase ( GA2 - ox ) 835-2 e \n Switchgrass OE Yes pANIC 10A 7 Up to fourfold increase Dwarf growth 4 [ 66 ] \n Ethylene response factor/SHINE transcription factor ( ERF/SHN 1 ) 837 Switchgrass OE Yes pANIC 10A 6 Up to ninefold increase Normal growth. Increased biomass dry weight 4 [ 67 ] \n UTR6 \n 838 Switchgrass FKD Yes pANIC 8A 30 18–70% decrease No consistent correlation in lignin content, S/G monomer ratio or sugar release with gene expression levels 2 \n UTR6 \n 839 Switchgrass OE Yes pANIC 10A 50 Up to 38-fold increase No consistent correlation in lignin content, S/G monomer ratio or sugar release with gene expression levels 2 \n Laccase 17 - like gene A ( LAC17a ) 844 Switchgrass FKD Yes pANIC 8A 40 No change Normal growth No change in lignin content 7 \n Laccase 17 - like gene A ( LAC17a ) 845 Switchgrass OE Yes pANIC 6A 40 No change Normal growth No change in lignin content 7 \n Laccase 17 - like gene B ( LAC17b ) 846 Switchgrass OE Yes pANIC 6A 40 No change Normal growth No change in lignin content 7 \n Purple acid phosphatase 2 ( PAP2 ) 847 Switchgrass OE Yes pMDC32 40 No change Normal growth No change in lignin content 7 \n Caffeoyl CoA 3 - O - methyl transferase 2 ( CCoAOMT2 ) 848 Switchgrass KD Yes pANIC 8A 30 40–90% decrease Normal growth No change in lignin content. 7 [ 48 ] \n Coumaroyl shikimate 3′ - hydroxylase 2 ( C3′H2 ) 849 Switchgrass KD Yes pANDA 30 10–85% decrease Normal growth No change in lignin content 7 \n Basic helix - loop - helix transcription factor ( bHLH1 ) 859 Switchgrass OE Yes pANIC 10A 7 No change Normal growth. No change in biomass yield Discontinued 4 \n p - coumarate 3 - hydroxylase 2 ( C3H2 ) 861 Switchgrass KD No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 2 \n p - coumarate 3 - hydroxylase 2 ( C3H2 ) 862 Switchgrass OE No Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Not applicable (see comment) Gene not clonable 2 \n Xyloglucan endotransglucosylase/hydrolase ( XTH - like2a ) 863 Switchgrass KD Yes pANIC 8A 30 40–80% decrease Not observed No change in lignin content 2 This report \n Xyloglucan endotransglucosylase/hydrolase ( XTH - like2a ) 864 Switchgrass OE Yes pANIC 10A 40 15–70% increase Semidwarf to normal growth No change in lignin content 2 This report \n Xyloglucan endotransglucosylase/hydrolase ( XTH - like1b ) 870 Switchgrass KD Yes pANIC 8A 30 10–80% decrease Semidwarf to normal growth No change in lignin content 2 This report \n Xyloglucan endotransglucosylase/hydrolase ( XTH - like1b ) 871 Switchgrass OE Yes pANIC 10A 40 20–150% increase Semidwarf to normal growth No change in lignin content 2 This report \n Caffeic acid 3 - O - methyltransferase ( COMT ) 930 Switchgrass KD Yes pANDA 9 90% decrease Normal growth. Increased biomass yield 7 [ 21 , 35 , 47 , 48 , 68 – 70 ] \n ID identification number, OE overexpression, KD knockdown, FKD family knockdown, Ref . References \n a Number of independent events \n b Determined by RT-qPCR \n c Principal Investigator associated with construct: (1) Fang Chen; (2) Richard A. Dixon; (3) Debra Mohnen; (4) C. Neal Stewart, Jr.; (5) Yuhong Tang; (6), Michael Udvardi; (7) Zeng-Yu Wang \n d Brown coloration in parts of leaf veins; brown patches in stems; reddish-brown coloration on the inner sides of basal stems and brownish color in the mature roots \n e Homologs of HCT or GA2 - ox genes targeted after initial TP submission, designated by dashed numeral after BESC ID number \n In 11 instances of the 53 constructs targeted for cloning, the target gene sequences were not clonable (Table 1 ). Since switchgrass was the source organism for most of these genes and there was no switchgrass reference genome available in the early part of the work, we did not find the number of cloning failures to be inordinate. Most of the remaining genes or gene fragments were successfully expressed in switchgrass, indicating little or no lethality imparted due to construct expression and that the pANIC vector was effective for transformation. An average of 26 putative transgenic events per construct were produced for constructs listed in Table 1 and T0 plants were sent for analysis to the PIs who had submitted the proposals for the specific targeted genes. In a minority of cases, the transformation process was difficult in that transgenic plants representing less than 10 independent transformation events per construct were received by the submitting PI (Table 1 ). Although BESC researchers greatly improved switchgrass transformation efficiency during rounds 1–12 of the TP, transformation of this species, like that of many crops, remains a slow process (see [ 37 ] for a general discussion of crop transformation bottlenecks). In most cases, sufficient numbers of independent transgenic events (three or more per construct) were produced to gauge the potential impact of the expression of the target gene or gene fragment on recalcitrance. Where available, we have noted cell wall and recalcitrance phenotypes of these plants. Acquiring 26 independent transgenic events per construct on average for this many target genes in an experimentally challenging crop required a processing pipeline only made available to a large research center such as BESC. In order to significantly improve switchgrass as a lignocellulosic feedstock for enzymatic deconstruction [ 38 ] or consolidated bioprocessing [ 39 ], end-of-season aboveground biomass yield should be maintained or improved and cell wall recalcitrance decreased. Where available, we have noted plant growth phenotypes for the plants (Table 1 ). For 83% of the target constructs where growth and biomass characteristics were reported, growth and/or biomass yield of the individual transgenic plants was always similar to or greater than the non-transgenic parent (Table 1 ). Regarding cell wall modification, although 60% of the constructs reported here were expected to affect the lignin pathway, the manipulation of many of these did not appreciably affect lignin content. This result may be explained by the fact that cell wall biosynthesis in angiosperms marshals up to ten percent of the genome, c.a. 2500 genes [ 40 ] and many of these single gene targets reside in gene families [ 41 ]. Residence in a gene family can be indicative of redundant function between family members (e.g. [ 42 , 43 ]) and the need to silence expression of multiple family members to achieve a modified cell wall. There are also examples where silencing of a saccharide biosynthesis gene led to altered saccharide or lignin content [ 44 , 45 ]. Thus, it is possible that silencing genes involved in the synthesis of one polymer may be compensated for by those synthesizing a related or alternative wall polymer. Specific lines modified for expression of genes in different wall synthesis pathways are being crossed to evaluate any possible additive effects that become significant in decreasing wall recalcitrance [ 46 ]. Notable findings regarding altered cell walls and plant growth Although manipulated expression of multiple genes simultaneously may provide additional decreases in cell wall recalcitrance, results from multiple lines generated in the BESC TP have demonstrated that manipulated expression of single genes can be useful in molecular breeding of switchgrass for cellulosic biofuel production. For example, a gene chosen by BESC for downregulation early in the project (officially incorporated into the TP after round 12, but for which work was begun prior to round 12) was a caffeic acid 3- O -methyltransferase ( PvCOMT ; GenBank Accession No. HQ645965), which led to the selection of two transgenic switchgrass lines with lower S/G ratio, lower lignin, and higher sugar release and biofuel yield in greenhouse-grown plants [ 21 ]. Importantly, for these transgenic lines greenhouse results translated to the field [ 35 ]. After the second year of growth in the field, one transgenic event (COMT KD line 2) had increased biomass and biofuel yield, which was calculated to result in 50% more biofuel per field area over the non-transgenic parent switchgrass ([ 35 , 47 ]; also see Table 1 ). A second notable finding, involving overexpression of the gene MYB4 , encoding a transcription factor targeting the repression of a suite of lignin biosynthesis genes, was the range of growth phenotypes exhibited in individually transformed switchgrass lines [ 33 , 34 ]. Greenhouse-grown MYB4 overexpressing switchgrass lines had a wide range of transgene expression and growth phenotypes. Some greenhouse-grown transgenic lines overexpressing MYB4 had up to three times greater sugar release and 2.6-fold higher biofuel production compared with the non-transgenic parents. When transplanted into the field in Knoxville, TN, USA, the line with the highest MYB4 expression, lowest lignin, and highest biofuel yield per gram cell wall residue did not survive the first winter. Furthermore, the highest MYB4 expressers had poorly developed roots [ 36 ]. In contrast, a line with lower levels of MYB4 transcript produced 63% more aboveground biomass and 32% more biofuel than controls ([ 33 , 34 , 36 ]; also see Table 1 ). These results demonstrated that growth of the biomass in the field is required to identify lines that retain high performance characteristics under natural environmental conditions. Characterization of switchgrass transgenic lines altered for xyloglucan endotransglucosylase/hydrolase (XTH) expression Through a comprehensive microarray analysis, we found a group of xyloglucan endotransglucosylase/hydrolases (PvXTH-like1a to 1d and PvXTH-like2a to 2c) that were down-regulated during the lignification process in both stem tissue and an induced suspension cell system [ 48 ]. PvXTH - like1b and PvXTH - like2a genes were then submitted and accepted into the TP and transgenic switchgrass generated (Table 1 ). Xyloglucan endotransglucosylase/hydrolases are enzymes involved in the modification of cell wall structure through cleavage and re-joining of xyloglucan molecules in primary plant cell walls [ 49 ]. Xyloglucan binds non-covalently to cellulose, coating and cross-linking adjacent cellulose microfibrils, and the resulting extensive xyloglucan-cellulose network is thought to act as the major tension-bearing structure in the primary wall [ 50 ]. However, the function of XTHs in monocotyledon secondary cell wall formation is poorly understood. Full-length mRNA sequences of XTH1b and XTH2a were isolated from switchgrass. Sequence analysis indicated that there was 61% similarity between XTH1b and XTH2a , with 29% similarity in their GH16 XET functional domain(s). Based on these characteristics, constructs aimed toward overexpressing and silencing XTH1a and XTH1b were designed (Additional file 3 ). Notably, the fragments cloned to silence the XTH genes each would be specific for their respective targeted XTH , having no more than 16 nucleotide stretches of identity between the two XTH s (Additional file 3 ). Forty independently transformed plants expected to overexpress PvXTH - like2a (T0-Pv864) and 40 plants expected to overexpress PvXTH - like1b (T0-Pv871) were generated (Table 1 ). Clonal lines with high transgene transcript abundance compared with control plants were selected for additional analysis (Fig. 1 A). Lignin content was reduced in three PvXTH - like2a overexpression lines (#18, #27 and #34) (Fig. 1 B); however, the lignin content level did not correlate with the expression level of the transgene. In the case of PvXTH - like1b overexpression, reduced lignin content was also observed in seven out of ten plants subjected to the analysis (Fig. 1 B), although, again, transgene expression level was not correlated with lignin content. Fig. 1 Xyloglucan endotransglucosylase/hydrolase (XTH) transcript level and lignin content in transgenic switchgrass lines silenced for or overexpressing XTH - like1b or XTH - like2a . A Percent of target gene expression (target gene represents transgene for overexpressing lines and targeted plant gene for silenced lines) relative to control (mean value for three independent control plants) for plants representing listed transgenic lines at E3 stage of development. Expression of Ubq1 in these plants was analyzed and used to normalize XTH expression levels across lines. B Lignin content for plants representing listed transgenic lines and control (WT) at R1 stage. Transgenic lines: T0-Pv863-xx (silencing PvXTH - like2a ); T0-Pv864-xx (overexpressing PvXTH - like2a ); T0-Pv870-xx (silencing PvXTH - like1b ); T0-Pv871-xx (overexpressing PvXTH - like1b ) \n Thirty independently transformed plants designed to silence PvXTH - like2a (T0-863) and an identical number of plants designed to silence PvXTH - like1b (T0-870) also were generated. Through RT-qPCR, a small number of plants that expressed the transgene and were silenced were selected for further analysis (Fig. 1 A). Downregulation of PvXTH - like2a expression resulted in no changes in lignin content (Fig. 1 B). For the PvXTH - like1b silenced plants, three plants out of nine showed reduced lignin content, but as for the overexpressing lines, no correlation with measured transcript level was obtained (Fig. 1 B). The lack of correlation between transgene expression in lines over-expressing the individual XTH genes and their lignin content was perhaps unexpected based on the negative relationship between XTH expression and lignification in stems and cell cultures [ 48 ]. Furthermore, the lack of a correlation between transgene expression in the knockdown lines and lignin content for both XTH genes also was unexpected. It is possible that redundancy between XTH s compensated for reduced expression of a particular XTH . Further work is necessary to understand the influence of XTHs on lignin content in monocotyledonous plants. Clearly, although XTHs function to maintain primary cell wall extensibility during growth, they were not shown to directly impact lignification by the approach taken in the present work. Characterization of switchgrass transgenic lines altered for hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) expression Another set of genes accepted into the TP were members of the HCT family (Table 1 ). HCTs are reported to be required in two steps during the conversion of p -coumaroyl CoA to guaiacyl (G) and syringyl (S) lignin monomers, but are not involved in the biosynthesis of hydroxyphenyl (H) lignin monomers [ 32 , 51 ]. Downregulation of HCT in Nicotiana benthamiana , A. thaliana , alfalfa and Monterey pine ( Pinus radiata ) resulted in high H monomer levels, decreased recalcitrance for sugar release and/or decreased biomass [ 32 , 52 – 55 ]. Additionally, silencing HCT in Arabidopsis and alfalfa [ 55 , 56 ] showed increased flavonoid biosynthesis and improved drought tolerance and resistance to fungal infection, although plant growth was inhibited. The potential of decreasing recalcitrance in switchgrass by silencing HCT homologs was investigated through the TP. Through a BLAST search using the Switchgrass Functional Genomics Server ( https://switchgrassgenomics.noble.org/index.php ), two EST sequences, AP13ISTG44531 and AP13CTG44233, were identified as HCT genes in switchgrass and named HCT1 and HCT2 , respectively. Two full-length mRNA sequences of HCT1 and HCT2 were amplified from switchgrass genotype NFCX1. Sequence analysis indicated that there was 62% similarity between HCT1 and HCT2 , with higher similarity in their functional domain (domain PLN02663) and less toward the 5′ and 3′-ends of the ORF (Additional file 4 ). Based on these characteristics, constructs to silence HCT1 or HCT2 were designed. Additionally, a fragment within the HCT conserved domain was amplified to silence both genes (Additional file 4 ). Twenty-eight transgenic plants produced to silence HCT1 (HCT1Ri) expression were analyzed. No transgenic plant displayed a visible phenotype in the aerial tissue (Fig. 2 A). HCT1 expression levels were dramatically decreased in plants representing these T0 HCT1Ri lines with generally minimal influence on HCT2 expression (Fig. 2 D). The minimal effect on HCT2 expression in these transgenic lines demonstrated the specificity of the HCT1 silencing construct to influence HCT1 and not HCT2 expression. Based on these RT-qPCR results, six transgenic lines with more than 90% reduction in HCT1 transcript level were selected for further analysis (i.e. lines HCT1Ri-14, 19, 20, 21, 24 and 26). Lignin content and lignin monomer composition were determined for whole stem tissue (leaf and sheath removed) of T1 generation plants at the R1 developmental stage [ 22 ] for each transgenic line and a line that had lost the inserted sequence through segregation (i.e. a null segregant control). There was no significant difference in G and S monomer content between the HCT-silenced and control plants (Fig. 2 B). H monomer content also was unchanged in the HCT-silenced lines with one exception, HCT1Ri-26, which was the most silenced of the HCTRi lines studied and where H monomer content was increased (Fig. 2 B). Fig. 2 Visual and molecular phenotypes of HCT1 - RNAi transgenic plants (HCT1Ri). A T1 generation null segregant (Control; left) and HCT1Ri transgenic (HCT1Ri-24 line, right) plants at R1 stage of development. B Lignin monomer content in stems of plants from null segregant (control) and HCT1Ri lines sampled at R1 stage. Values represent mean ± S.D. of three biological (plant) replicates. For G and S monomer levels, no significant differences were seen between lines at the 0.05 significance level. For G and S monomer levels, no significant differences were observed between lines at the 0.05 significance level. Non-identical letters in red indicate significant difference in the H monomer levels between two lines at the 0.05 level determined by ANOVA and least significant difference (LSD) test. Guaiacyl (G), syringyl (S) and p -hydroxyphenyl (H) lignin monomers were measured. CWR cell wall residue. C Root architecture of 1-month-old plants. D Relative transcript levels of HCT1 and HCT2 in control and HCT1Ri plants representing 28 independent transformation events at 1 month post sub-tillering. Expression of Ubq1 in these plants was analyzed and used to normalize HCT expression levels across lines. Relative treatment level values (vertical axis) are normalized to the HCT2 control value set at 100% \n Subsequently, 41 transgenic plants targeted for HCT2 downregulation (HCT2Ri) were produced. Similar to the HCT1 downregulated plants, there was no alteration in the visible growth phenotype of HCT2Ri aerial tissue compared with the parent control (Fig. 3 A). HCT2 transcript expression was dramatically decreased in multiple lines while HCT1 expression was unaffected (Fig. 3 B). Seven transgenic lines with more than 90% reduction in HCT2 transcript level were selected for lignin monomer analysis (i.e. lines HCT2Ri-2, 14, 25, 30, 35, 38 and 39). Similar to findings with HCT1 downregulated plants, there was no difference in G and S monomer levels in HCT2 downregulated plants, but unlike results from the majority of HCT1 downregulated plants, there was an increase in H lignin monomers in most HCT2Ri lines compared with the parental control (Fig. 3 C). Fig. 3 Visual and molecular phenotypes of HCT2 - RNAi transgenic plants (HCT2Ri). A NFCX1 wild type (WT) (Control; left) and HCT2 - RNAi transgenic plants (Ri-25, -2, -39 and -38 lines, right) at R1 stage. B Relative transcript levels of HCT1 and HCT2 in control and HCT2Ri plants representing 41 independent transformation events at 1 month post sub-tillering. Expression of Ubq1 in these plants was analyzed and used to normalize HCT expression levels across lines. Relative treatment level values (horizontal axis) are normalized to the HCT2 control value set at 100%. C Lignin monomer content in stems of WT control and HCT2 - RNAi transgenic plants (HCT2Ri) at R1 stage. Values represent mean ± S.D. of three biological replicates. For G and S monomer levels, no significant differences were observed between lines at the 0.05 significance level. Non-identical letters in red indicate significant difference in the H monomer levels between two lines at the 0.05 level determined by ANOVA and least significant difference (LSD) test. Guaiacyl (G), syringyl (S) and p -hydroxyphenyl (H) lignin monomers were measured. CWR cell wall residue. D Root architecture of 5-week-old plants \n Considering that silencing HCT1 and HCT2 individually did not alter G and S lignin monomer units, a construct was created to determine if silencing both genes simultaneously would lead to an expected change in lignin monomer content. This construct was submitted to the TP after the 12th round and demonstrates the process whereby the TP committee reviewed and accepted constructs in later rounds that more extensively studied specific target gene family function based on results from plant lines expressing individual family members accepted in TP rounds 1–12. Thirty-nine plants transformed with a gene fragment having multiple regions of 100% identity for greater than 21 contiguous nucleotides between HCT1 and HCT2 (HCT1/2Ri) were analyzed. Both HCT1 and HCT2 transcript levels were decreased more than 90% in approximately half of the transgenic plants compared with the parental control (Fig. 4 ). Three lines were shown to have more than a 94% decrease in HCT1 and HCT2 expression in a repeat analysis and these lines were selected for further study (i.e. lines HCT1/2Ri-28, 34 and 37). Fig. 4 Relative HCT1 and HCT2 transcript levels in 39 independently-transformed plants targeted for simultaneous downregulation of HCT1 and 2 (HCT1/2Ri-xx) at 1 month post sub-tillering. Expression of Ubq1 in these plants was analyzed and used to normalize HCT expression levels across plants. Relative treatment level values (horizontal axis) are normalized to the HCT2 control value set at 100% \n There was little visible difference in the aerial organs between control and HCT1/2Ri plants except in plant height. All three transgenic lines were consistently slightly shorter than the parental control at the R1 stage (e.g. Fig. 5 A). HCT1/2Ri aerial (stem) tissue displayed moderate differences in lignin monomer content with both G and S monomers decreased and the H monomer increased compared with respective monomer levels in the parental control (Fig. 5 B). Compiling G, S and H lignin monomer levels, the three HCT1/2Ri lines were decreased 5–8% in total lignin content in stems. Interestingly, HCT1/2Ri plants also displayed significant differences in root architecture. HCT1/2Ri plants had increased crown root numbers and root densities, and shorter root length than the parental plant (Fig. 5 D–F). There also was a brown color correlated with the increase in root numbers and root densities (Fig. 5 D); the brown color suggesting the accumulation of flavonoids similar to findings from Arabidopsis and alfalfa where HCT activity was downregulated [ 55 , 56 ]. It will be important to determine if higher flavonoid accumulation in roots of HCT1/2Ri plants occurs and whether it is correlated with greater abiotic and biotic stress resistance in switchgrass [ 57 ]. A similar effect on visible root structure was observed when silencing HCT1 alone (Fig. 2 C), but not HCT2 alone (Fig. 3 D), suggesting that the effect on root structure was predominantly due to silencing HCT1 expression. Interestingly, HCT1 expression was observed to predominate over HCT2 expression in root tissue (Fig. 6 ), correlating well with the greater influence of HCT1 expression over HCT2 expression on root structure. As in the aerial tissue, G and S lignin monomers were decreased and H lignin monomers increased in the HCT1/2Ri root tissue compared with the parental control root tissue. Compiling G, S and H lignin monomer levels, the three HCT1/2Ri lines were decreased 7–12% in total lignin content in roots (Fig. 5 C): a difference greater than that observed in aerial tissue. Fig. 5 Visual and molecular phenotypes of HCT1/2 - RNAi transgenic plants (HCT1/2Ri-xx) simultaneously downregulated for HCT1 and 2 expression. A NFCX1 wild type (WT) (Control; left) and HCT1/2Ri transgenic (HCT1/2Ri-34 line, right) plants at R1 stage. B Lignin monomer content in stems of WT and HCT1/2Ri plants at R1 stage. Guaiacyl (G), syringyl (S) and p -hydroxyphenyl (H) lignin monomers were measured. C Lignin monomer content in roots of WT and HCT1/2Ri plants at R1 stage. D Root architecture of 1-month-old plants. E Comparison of crown root number (per plant) and length (cm) in 1-month-old WT and HCT1/2Ri plants. F Comparison of lateral root density (lateral roots number/1 cm main root) and length (cm) in 1-month-old WT and HCT1/2Ri plants. Values represent mean ± S.D. of three biological replicates. CWR cell wall residue. Non-identical letters in corresponding colors in the charts ( B , C , E , F ) indicate significant differences in analyzed trait between treatments at the 0.05 level determined by ANOVA and least significant difference (LSD) test \n Fig. 6 Expression profiles of HCT1 and HCT2 in various tissues at different stages in NFCX1 wild type (WT). E2 elongation stage 2, R1 reproductive stage 1, I1 internode 1, I2 internode 2, I4 internode 4. Values represent mean ± S.D. of three biological replicates. Expression of Ubq1 in these plants was analyzed and used to normalize HCT expression levels across tissues \n These findings indicate that, in switchgrass, silencing multiple HCTs was necessary to begin to mimic the enhanced H and minimal G and S monomer accumulation exhibited by alfalfa after silencing HCT expression [ 52 ]. The switchgrass results begin to define activities of individual HCTs in specific plant tissues. Findings here suggest redundant functions for HCT1 and HCT2 in aerial switchgrass tissue. However, redundancy between these genes may be less complete in roots where a modified growth phenotype was apparent when silencing only HCT1 , which predominates in expression over HCT2 in root tissue. The relationship between the root growth phenotype and lignin content in root cells, especially when downregulating HCT1 , requires further study. A second conclusion from these studies is that the aerial growth phenotype was only modestly affected when silencing switchgrass HCT1 or HCT2 expression alone or both HCT s simultaneously. This finding may indicate that additional HCT s with similar function in aerial tissue exist in switchgrass. Four HCT s have been identified in the grass model plant, rice [ 58 ]. Rice is a self-fertilizing diploid, while lowland switchgrass is an outcrossing tetraploid species. Taken together these findings suggest that there could be at least 4 HCT homologs in switchgrass, and further indicate the complexity associated with modifying a polyploid species to obtain a measurable influence on cell wall structure. Compilation of data from the TP Considering that hundreds of BESC researchers were producing data on thousands of feedstock and microbe samples, it was imperative to capture this information in a centralized system that provided simplified access by multiple researchers within BESC. Specific information detailing construct formation, plant transformation, plant growth and cell wall trait analysis was captured within the LIMS. Completed submission forms (e.g. Additional file 1 ) were captured as PDFs within the BESC-based Wiki website. Additionally, a committee was formed within BESC to identify the types of data to accumulate and store to provide details of construct synthesis, plant transformation, plant growth and cell wall trait analyses. Tables were added to the Oracle database to store gene names, gene models, expression type, primer sequences, gene and gene fragment sequences, recipient plant transformation vectors, plant transformation status, plant growth parameters and cell wall traits (a portion of this information is found in Table 1 ). A unique BESC identification barcode for each construct was used to link individual information (e.g. gene sequence) pertaining to each construct. BESC team members were able to share information within BESC based on the parent institute’s signature on a mutual Material Transfer Agreement (MTA). All information available in the BESC LIMS for constructs described in this manuscript can be accessed at http://bioenergycenter.org/besc/ ."
} | 11,564 |
29259591 | PMC5723320 | pmc | 2,443 | {
"abstract": "We describe experiments that follow species dynamics and gene expression patterns in synthetic bacterial communities including species that compete for the single carbon substrate supplied, methane, and species unable to consume methane, which could only succeed through cooperative interactions. We demonstrate that these communities mostly select for two functional guilds, methanotrophs of the family Methylococcaceae and non-methanotrophic methylotrophs of the family Methylophilaceae , these taxonomic guilds outcompeting all other species included in the synthetic mix. The metatranscriptomics analysis uncovered that in both Methylococcaceae and Methylophilaceae , some of the most highly transcribed genes were the ones encoding methanol dehydrogenases (MDH). Remarkably, expression of alternative MDH genes ( mxaFI versus xoxF ), previously shown to be subjects to the rare Earth element switch, was found to depend on environmental conditions such as nitrogen source and methane and O 2 partial pressures, and also to be species-specific. Along with the xoxF genes, genes encoding divergent cytochromes were highly expressed in both Methylophilaceae and Methylococcaceae , suggesting their function in methanol metabolism, likely encoding proteins serving as electron acceptors from XoxF enzymes. The research presented tested a synthetic community model that is much simplified compared to natural communities consuming methane, but more complex than the previously utilized two-species model. The performance of this model identifies prominent species for future synthetic ecology experiments and highlights both advantages of this approach and the challenges that it presents.",
"conclusion": "Conclusion We here demonstrated the utility of multispecies SCs in studying complex biogeochemical processes, such as communal metabolism of methane, in a simplified, controllable model. We demonstrate that general trends in SCs mimic the ones in natural communities, selecting for Methylococcaceae and Methylophilaceae under most conditions, thus defying the hypothesis of random distribution of “public goods.” Through metatranscriptome analysis, we uncover the unexpected complexity of transcriptional regulation of methanol oxidation, mediated through the REE switch, which, in turn, is governed by multiple environmental factors. We also demonstrate that the REE switch acts differently in different organisms and on different XoxF/XoxG enzymes. These new data will inform future development of SCs customized toward specific experimental goals, in order to target specific functions that may contribute to cooperative behavior in methane consumption.",
"introduction": "Introduction Metabolism of methane is an important part of biogeochemical cycling of carbon ( Singh et al., 2010 ). Methane is also a major contributor to climate change ( Schuur et al., 2015 ). A specialized group of microbes, the methanotrophs that consume methane, gaining both energy and carbon from this compound, represent a natural filter preventing an even faster escape of methane into the atmosphere ( Malyan et al., 2016 ). While methanotrophy has been studied for the past 100 years as a metabolic feature of individual pure cultures ( Kaserer, 1906 ; Söhngen, 1906 ; Trotsenko and Murrell, 2008 ; Chistoserdova and Lidstrom, 2013 ), a concept of communal function in methanotrophy has been gaining momentum ( Ho et al., 2016 ; Yu and Chistoserdova, 2017 ). The mechanistic details are still scarce with regard to how and why the methanotrophs share their carbon with other species, and whether and what they gain in return ( Yu and Chistoserdova, 2017 ). We have previously reported that feeding 13 C-labeled methane to natural communities of Lake Washington sediment resulted in label accumulation mainly by the Methylococcaceae and the Methylophilaceae species ( Kalyuzhnaya et al., 2008 ; Beck et al., 2013 ). Through microcosm manipulation, using methane as the sole source of carbon, followed by metagenomic analysis, we further confirmed that the Methylococcaceae and the Methylophilaceae were active in methane consumption ( Hernandez et al., 2015 ; Oshkin et al., 2015 ). A more recent study has identified methanol as one metabolic node at which community cross-talk may be taking place ( Krause et al., 2017 ), suggesting that methanol may be a major carbon source released by the methanotrophs, to support satellite communities. In most Gram-negative methylotrophs, methanol oxidation can be carried out by two alternative methanol dehydrogenases (MDH), the classic, MxaFI-type enzyme that has been studied for decades ( Anthony, 2004 ; Williams et al., 2005 ) and the recently discovered, lanthanide (Ln 3+ )-dependent, XoxF-type MDH ( Fitriyanto et al., 2011 ; Nakagawa et al., 2012 ; Pol et al., 2014 ). Moreover, Ln 3+ have been implicated in a regulatory mechanism inversely controlling expression of xoxF and mxaFI genes, this mechanism known as the rare Earth element (REE) switch ( Chu et al., 2016 ). This REE switch has been proposed to be an important factor in community function ( Krause et al., 2017 ). Methanotrophs have also been proposed to excrete multicarbon compounds such as acetate, citrate, lactate, and succinate ( Modin et al., 2007 ; Kalyuzhnaya et al., 2013 ), thus having a potential of also supporting communities of non-methylotrophic heterotrophs. A mathematical model has been recently implemented to explain carbon flow in microbial consortia consuming methane ( Modin, 2017 ). In accordance with this model, methanotrophs feed methanol to methanol utilizers, and both methanotrophs and methanol utilizers produce organics that feed non-methylotrophic heterotrophs, in conjunction with denitrification. In accordance with the model, methanol should be available to all organisms that are capable of methanol utilization, and other organics should be available to all heterotrophs, as “public goods.” However, experimental evidence is somewhat contradictory to the notion of “public goods,” as specific species cooccurrences, such as cooperative behavior of Methylococcaceae and Methylophilaceae , have been noted not only in manipulated microcosms ( Kalyuzhnaya et al., 2008 ; Beck et al., 2013 ; Hernandez et al., 2015 ; Oshkin et al., 2015 ), but also in natural populations inhabiting methane-rich environments such as permafrosts ( Martineau et al., 2010 ; Crevecoeur et al., 2015 ) or methane-fueled cave biofilms ( Karwautz et al., 2017 ). These results suggest either that many species detected in natural niches, through DNA profiling, are dormant, that some organisms may be more competitive for “public goods,” or that specific partnerships are taking place. A Black Queen Hypothesis, in accordance with which gene loss plays a role in species coevolution ( Morris et al., 2012 ), has been evoked recently to explain non-random distribution of “public goods” among species involved in a cyanobacterial consortium, and vitamin B 12 was proposed as an important metabolite ( Lee et al., 2017 ). Vitamin B 12 exchange has been previously implicated in maintaining stable cocultures of methanotrophs and non-methanotrophs ( Iguchi et al., 2011 ). In this manuscript, we describe experiments addressing the following questions: (1) whether the methanotrophs share carbon with non-methanotrophs as “public goods,” or whether they form specific partnerships, (2) whether all species capable of metabolizing methanol are equally competitive when present in physiologically active state, (3) whether non-methylotrophic heterotrophs also benefit from methane-derived carbon on “public goods” basis, and (4) whether transcription of any key methylotrophy genes changes in response to different environmental conditions. In these experiments, we employed synthetic communities (SCs) built of pure cultures of methanotrophs and non-methanotrophs, all previously isolated from the same ecological niche. A small subsample of these experiments has been previously published, to demonstrate the utility of SCs in basic research ( Yu et al., 2016 ).",
"discussion": "Discussion In this study, we aimed to make advances toward developing a synthetic model for studying syntrophy in aerobic methane-oxidizing communities. We employed defined SCs of methanotrophs, non-methanotrophic methylotrophs, and non-methylotrophic heterotrophs, to further test whether partnerships in aerobic methanotrophy are specific or non-specific, and also to potentially identify most prominent models for future SC-based experiments. Note that many methylotrophs included into SCs are capable of growth on a variety of organic compounds, and many are capable of denitrification ( Beck et al., 2015 ; McTaggart et al., 2015a , e ). These organisms have a potential to consume either single carbon- or multicarbon compounds, and some possess a potential to link carbon consumption to either oxygen respiration or denitrification. Thus, these organisms presented good models for testing the “public goods” hypothesis. The Methyloversatilis strains that require vitamin B 12 ( Smalley et al., 2015 ) also served as controls for vitamin B 12 exchange potential. All organisms were supplied into SCs in viable state, and all were presumed to be active, thus the possibility of dormancy was excluded. We assumed that members of each of the three major functional guilds would reveal competitive trends within each respective guild, and that these trends may be determined by fitness at time zero, on the one hand, and by response to specific environmental conditions, on the another hand. Thus, while we expected and indeed observed very good agreement in terms of community dynamics between the technical replicates ( Figures 1A – C ), good replication between independent experiments was not necessarily expected. In this sense, the SC approach presents some serious methodological challenges. First, as the organisms involved can survive and thrive on their own, as pure cultures, the choice of experimental conditions conducive of cooperative behavior may be important (medium composition, partial pressures of methane and O 2 , nitrogen sources, etc.). Second, the relative fitness at time zero is hard to control. For example, it is not clear on what substrate the pure cultures should be pregrown, to better reflect their diet in situ . This problem is especially profound when it comes to organisms with broad metabolic capabilities. Assuming that most organisms must be limited by some nutrient in their natural habitat (be it O 2 , carbon, nitrogen, or an essential metal), we elected to starve the premixed SCs, incubating them in a minimal medium, before supplying them with the carbon source. However, different organisms may respond differently to this mimicked “starvation,” and thus initial fitness of different organisms would depend on their stress response mechanisms and their robustness. Additionally, other factors may be important that may control species ratios in natural communities, including functional guilds not primarily involved in methane utilization or cometabolism, but sporadically influencing community structure, for example, predatory species, species harboring predatory plasmids or phages, or free-living phages. Despite some differences between the dynamics among the specific strains of Methylococcaceae and Methylophilaceae in SC1 versus SC2, general trends at the level of functional guilds remained the same. In each case, Methylococcaceae and Methylophilaceae became the dominant species, quickly outcompeting Methylocystaceae as well as other methylotrophic and non-methylotrophic heterotrophs. In most cases, the Methylomonas species became dominant. This would be predicted from their growth characteristics in pure cultures, in standard conditions, under which they outperform Methylobacter , Methylosarcina , or Methylosinus ( Auman et al., 2000 and unpublished results). However, Methylomonas species have not been detected as dominant in prior experiments that involved natural sediment samples ( Kalyuzhnaya et al., 2008 ; Beck et al., 2013 ; Hernandez et al., 2015 ; Oshkin et al., 2015 ), possibly due to existence of some specific controls (predation, antibiotic regulation, molecular signaling) that were not recreated in SCs in this study. The trend of the Methylophilaceae outcompeting other methanol utilizers, noted for natural communities ( Hernandez et al., 2015 ; Oshkin et al., 2015 ), persisted in SCs. While many species included into SCs show robust growth on methanol in laboratory (e.g., Methylobacterium , Paracoccus , Hyphomicrobium , Methylopila ; Beck et al., 2015 ), these species did not seem competitive under most regimens. As an exception, Methyloversatilis species were prominent under the “low” methane regimens in SC2. These observations suggest that the Methylophilacaea , and, under some conditions, Methyloversatilis species have some advantages over alphaproteobacterial methylotrophs in consuming methanol. Success of the latter also suggests that vitamin B 12 indeed could be shared among the community members. The heterotrophs that could potentially consume a variety of organic compounds, i.e., the Gram-positive species and the facultatively methylotrophic alphaproteobacteria, did not persist in the microcosms, in agreement with prior results with “natural” microcosms ( Hernandez et al., 2015 ; Oshkin et al., 2015 ). In contrast, Pseudomonas and Janthinobacterium were detected in some samples at over 2% abundance, suggesting that they were more competitive. However, Flavobacterium species, and also “uninvited,” contaminants belonging to Chryseobacterium (which are also Bacteroidetes) and Acidovorax (Burkholderiales, closely related to Janthinobacterium ) were measured at even higher abundances, suggesting that Bacteroidetes and Acidovorax /other Burkholderiales must have strong competitive advantages over other, methylotrophic or non-methylotrophic heterotrophs. Overall, our data continue to suggest that the carbon released by the methanotrophs is not equally accessible by all organisms present in the community, and thus some mechanisms must exist that make Methylophilaceae and, under certain conditions, Rhodocyclaceae more competitive for methanol, Acidovorax more competitive for acetate and/ other organic acids, while Bacteroidetes appear more competitive in consumption of polymeric substances that are excreted by other species. Highly covered transcriptomes were obtained for major partner species. Remarkably, among the most highly and most differentially expressed were the genes for alternative MDH enzymes, MxaFI, the classic Ca 2+ -dependent MDH and XoxF, the recently discovered MDH requiring REEs ( Chistoserdova, 2016 ; Martinez-Gomez et al., 2016 ). These enzymes have been previously found to be subjects of the so-called REE switch ( Chu et al., 2016 ), a mechanism that inversely regulates transcription from xox and the mxa gene clusters in response to the presence of REEs ( Chu and Lidstrom, 2016 ; Gu et al., 2016 ; Vu et al., 2016 ). However, the straightforward nature of the switch has already been questioned. For example, in an alphaproteobacterial methanotroph Methylosinus trichosporium OB3b, copper appears to override the switch ( Gu et al., 2016 ; Gu and Semrau, 2017 ). The switch also appears to work differently dependent on whether methanotrophs are cultivated as pure cultures or as members of communities ( Krause et al., 2017 ). Thus, further insights are required to better understand the mechanism of the REE switch. The data we present here indicate that the REE switch must be a subject to much more complex regulation than previously appreciated. (1) First, we demonstrate that the REE switch is responsive to a nitrogen source. As our experimental design did not include added REEs, high expression of the mxa genes was expected. However, this was true only for the HHNO 3 regimen, the standard laboratory condition ( Chu and Lidstrom, 2016 ). Even under this regimen, Methylophilaceae other than Methylophilus tended to overexpress xoxF over mxaF . (2) Second, we demonstrate that both O 2 and methane partial pressures also have control over the REE switch, high methane selecting for the MxaFI–MDH (under nitrate), “low” methane and “low” O 2 selecting for XoxF, and “high” methane “low” O 2 , nitrate allowing for transcription of both systems at similar levels. (3) Third, we demonstrate that, when multiple copies of xoxF genes are present in a single genome, they are not all following the same regulation pattern. (4) Finally, different organisms operate the RRE switch differently, such as in the samples where Methylophilus types express the mxa genes at a higher level, other Methylophilaceae preferentially express xoxF genes. Besides multiple xoxF genes, multiple cytochrome-encoding genes ( xoxG ) were also highly and differentially expressed, adding further complexity to the REE switch. Moreover, while Methylophilaceae tended to coexpress xoxF and xoxG genes, expression of xoxG in Methylococcaceae was not coordinated with expression of xoxF . While the meaning of such complexity for the REE switch remains enigmatic, it further points to the importance of the methanol oxidation step in communal metabolism of methane and warrants further investigations."
} | 4,360 |
39282569 | PMC11392765 | pmc | 2,444 | {
"abstract": "Thermophilic acetogens are gaining recognition as potent microbial cell factories, leveraging their unique metabolic capabilities to drive the development of sustainable biotechnological processes. These microorganisms, thriving at elevated temperatures, exhibit robust carbon fixation abilities via the linear Wood-Ljungdahl pathway to efficiently convert C 1 substrates, including syngas (CO, CO 2 and H 2 ) from industrial waste gasses, into acetate and biomass via the central metabolite acetyl-CoA. This review summarizes recent advancements in metabolic engineering and synthetic biology efforts that have expanded the range of products derived from thermophilic acetogens after briefly discussing their autotrophic metabolic diversity. These discussions highlight their potential in the sustainable bioproduction of industrially relevant compounds. We further review the remaining challenges for implementing efficient and complex strain engineering strategies in thermophilic acetogens, significantly limiting their use in an industrial context.",
"conclusion": "4 Conclusion and outlook Acetogens can fix CO 2 into acetyl-CoA with the WLP, making them promising chassis organisms for large-scale biological CO 2 fixation and compound bioproduction—a pivotal step toward mitigating climate change. Thermophilic acetogens offer additional advantages over their mesophilic counterparts by, for example, reducing gas cooling requirements and contamination risks in industrial bioprocesses. Several species with unique properties have now been isolated at temperatures above 55°C. However, most of them remain understudied, potentially due to the difficulty of cultivating and studying them under standard laboratory conditions. Consequently, significant knowledge gaps regarding their metabolism and physiology, in particular energy-conserving mechanisms remain. However, recent research efforts have started elucidating their metabolic processes, primarily in M. thermoacetica and T. kivui . Further work is, therefore, needed to fully understand their metabolism in order to design appropriate metabolic engineering strategies for industrial applications. Moreover, M. thermoacetica and T. kivui have recently been engineered for heterologous compound biosynthesis and fundamental studies, respectively, paving the way for thermophilic acetogenic microbial cell factories. However, the genetic toolkit currently available for manipulating acetogens is limited, which further prevents complex strain engineering efforts. In particular, characterized genetic parts and thermostable enzymes are missing but are key elements for metabolic engineering. Significant genetic work is therefore needed to establish thermophilic acetogens as robust microbial cell factories for simultaneous CO 2 fixation and compound biosynthesis.",
"introduction": "1 Introduction As anthropocentric industrial activities accelerate the climate change, sustainable alternatives for manufacturing essential chemical commodities are urgently needed. Microbial biotechnology processes stand out as promising solutions due to their inherent robustness, adaptability, and less energy-intensive nature as compared to traditional chemical synthesis and fossil fuel-based methods ( Ko et al., 2020 ; Cho et al., 2022 ). The development of reliable and efficient genetic tools, supporting various metabolic engineering strategies to expand and rewire microbial metabolic networks, has also allowed to further establish microbial cell factories as key production platforms. Acetogenic bacteria are becoming increasingly relevant in the current climate crisis context due to their autotrophic ability to utilize CO 2 as their sole carbon source; thus, holding great promise to mitigate global warming by abating greenhouse gas emissions. In particular, these bacteria can assimilate a combination of H 2 , CO 2, and CO (i.e., syngas) ( Liew et al., 2016 ) released by diverse industrial processes, hence offering the possibility to utilize industrial waste gas streams. Thus, acetogens can significantly contribute to industrial carbon capture and utilization efforts, alongside other non-biological strategies ( McLaughlin et al., 2023 ; Yusuf and Ibrahim, 2023 ). Although acetogens are very diverse in their metabolic capabilities, these anaerobic Gram-positive bacteria all rely on the Wood-Ljungdahl pathway (WLP) ( Drake et al., 2008 ; Ragsdale, 2008 ), also known as the reductive acetyl-CoA pathway for carbon assimilation. They use the WLP to convert CO 2 into the central metabolite acetyl-CoA, which is then channeled into both biomass and acetate formation. Operating at the thermodynamic limit of life ( Schuchmann and Müller, 2014 ), acetogens have evolved intricate energy-conserving mechanisms to thrive autotrophically with the WLP. While significant progresses in terms of genetic engineering efforts and understanding of autotrophic processes have been achieved for mesophilic acetogens, their thermophilic counterparts remain largely understudied. However, thermophilic acetogens warrant a greater attention due to their unique advantages for large-scale cultivation and industrial bioprocesses, such as high turnover rates and reduced gas cooling requirements and contamination risks in bioreactors. Despite their attractive characteristics, genetic and metabolic engineering of thermophilic acetogens for commodity bioproduction presents considerable challenges. Their metabolism is inherently constrained and the lack of efficient genetic tools complicates strain engineering. This review will discuss progresses and challenges of engineering thermophilic acetogens as microbial cell factories."
} | 1,415 |
28042198 | null | s2 | 2,447 | {
"abstract": "Social insect workers modify task performance according to age-related schedules of behavioral development, and/or changing colony labor requirements based on flexible responses that may be independent of age. Using known-age minor workers of the ant "
} | 62 |
33541405 | PMC7863362 | pmc | 2,448 | {
"abstract": "Background Mixotrophy can confer a higher growth rate than the sum of photoautotrophy and heterotrophy in many microalgal species. Thus, it has been applied to biodiesel production and wastewater utilization. However, its carbon and energy metabolic mechanism is currently poorly understood. Results To elucidate underlying carbon and energy metabolic mechanism of mixotrophy, Chromochloris zofingiensis was employed in the present study. Photosynthesis and glucose metabolism were found to operate in a dynamic balance during mixotrophic cultivation, the enhancement of one led to the lowering of the other. Furthermore, compared with photoautotrophy, non-photochemical quenching and photorespiration, considered by many as energy dissipation processes, were significantly reduced under mixotrophy. Comparative transcriptome analysis suggested that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO 2 to provide carbon sources for chloroplast organic carbon metabolism under mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped, allowing for more efficient utilization of photoreaction-derived energy. Besides, compared with heterotrophy, photoreaction-derived ATP reduced the need for TCA-derived ATP, so the glucose decomposition was reduced, which led to higher biomass yield on glucose. Based on these results, a mixotrophic metabolic mechanism was identified. Conclusions Our results demonstrate that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO 2 to provide carbon for photosynthesis in mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped in mixotrophy, which could reduce energy waste of photosynthesis while promote cell growth. This finding provides a foundation for future studies on mixotrophic biomass production and photosynthetic metabolism.",
"conclusion": "Conclusions Under mixotrophic cultivation, photosynthesis and glucose metabolism occur in a dynamic balance, such that enhancement of one results in lowering of the other. Compared with photoautotrophy, intermediates of glycolysis were supposed directly enter the chloroplast and substitute for inorganic carbon fixed by RuBisCO for organic carbon metabolism in the chloroplast. Therefore, the main rate-limiting enzyme, RuBisCO, was skipped, which resulted in decreased energy dissipation via non-photochemical quenching. Finally, compared with heterotrophy, energy provided by photosynthesis reduced the need for TCA-derived ATP, so the metabolism of glucose was reduced. Collectively, our results reveal a novel mechanism underlying mixotrophic metabolism in C. zofingiensis .",
"introduction": "Introduction Microalgae are photosynthetic organisms, which began to provide the oxygen and energy needed for life 3.5 billion years ago [ 1 , 2 ]. They are widely distributed and highly adaptable, and have adopted a variety of nutritional modes through natural selection including photoautotrophy, heterotrophy and mixotrophy [ 3 – 5 ]. Photoautotrophy and heterotrophy are two common nutrition modes which have been extensively studied. However, the underlying knowledge about mixotrophy is limited [ 6 ]. Under mixotrophic cultivation, microalgae conduct photosynthesis to fix inorganic carbon (as in photoautotrophy) while simultaneously assimilating organic carbon from the environment (as in heterotrophy). Studies have shown that, compared with photoautotrophy or heterotrophy, the growth rate, biomass accumulation, intracellular lipid content, organic carbon conversion rate and tolerance to strong light are significantly improved in certain microalgae species under mixotrophic conditions [ 7 – 9 ]. At present, mixotrophic cultivation has been utilized in microalgal biodiesel production [ 6 , 10 – 14 ], resource recycling of wastewater [ 15 ], and research on eutrophic waters [ 16 ]. Therefore, further research on mixotrophy is necessary to provide important theoretical frameworks in the fields of biology, ecology, environmental governance and the production of microalgal natural products. Chromochloris zofingiensis is a unicellular microalga, which could use a variety of organic carbon sources and has three nutritional modes including photoautotrophy, heterotrophy and mixotrophy [ 9 , 17 ]. Furthermore, it grows fast, is easy to culture, and has a well-established genetic background [ 18 ]. These characteristics make this species an excellent model for studying mechanisms underlying mixotrophic metabolism. In addition, C . zofingiensis is rich in lipids and carotenoids, which are valuable commodities in several industries including feed additives, food production and biofuels [ 3 ]. Therefore, C. zofingiensis was employed in the present study. Previous study has been proposed that there is a synergistic mechanism of photosynthesis and respiration in carbon and energy metabolism in C. zofingiensis under mixotrophic cultivation [ 9 ]. Compared with photoautotrophy, the RuBisCO activity was declined, which indicated that the CO 2 fixation rate is lower under mixotrophic conditions, thus conflicting with the “CO 2 reutilization theory” [ 7 , 9 , 11 ]. In addition, compared to heterotrophy, the citrate synthase activity was declined, which indicated that less organic carbon entered the TCA cycle. However, the detailed underlying carbon and energy metabolic mechanism is still unknown, and two key aspects are needed to resolve: Photosynthesis and glucose metabolism occur in different cellular compartments, including chloroplast, cytosol and mitochondria. How do these processes cooperate synergistically? Indeed, an enormous variety of carbon and energy metabolic processes take place in the aforementioned compartments [ 19 ]. It is interesting to note that, although chloroplasts (cyanobacteria) and mitochondria (proteobacteria) originated from different bacteria [ 20 ], each of the two organelles contains carbon and energy transporters on their membranes, which may confer them the ability to cooperate through the cytosol [ 21 , 22 ]. It is reported that several transporters localized to the inner membrane of both organelles serve to interconnect energy and carbon metabolism between the stroma (chloroplast), the matrix (mitochondria) and the surrounding cytosol [ 22 ]. Key players are thought to be triose phosphate/phosphate translocators (TPTs), glucose/phosphate transporters (GPTs), ADP/ATP carriers (AACs) and other organic carbon transporters [ 22 – 24 ]. These transporters are well studied in higher plants but have not been studied in great detail in microalgae, and considerably less is known about how they function under mixotrophic cultivation. How do photosynthesis and glucose metabolism cooperate to prevent energy loss? According to previous studies, photosynthetic light reactions can quickly absorb a large amount of light energy; however, due to the low catalytic rate and competing oxygenation activity of RuBisCO, part of the absorbed light energy cannot be used to fix CO 2 [ 25 ]. To ensure that excess absorbed energy does not damage the photosynthetic apparatus, cells can dissipate energy in three ways: non-photochemical quenching, chlorophyll fluorescence and photorespiration [ 26 – 28 ]. How these three energy dissipation pathways operate under mixotrophic cultivation merits further research. The present study was conducted to explore the detailed underlying carbon and energy metabolic mechanism of mixotrophy. The relationship between photosynthesis and glucose metabolism under mixotrophic conditions was characterized at the biochemical level at first. Then, a comparative transcriptome analysis was performed to further explore the features of carbon and energy metabolism, and a novel metabolic mechanism underlying mixotrophy was proposed. Our work established a novel mechanism for carbon and energy utilization of mixotrophy and provides a foundation for future studies on mixotrophic biomass production and photosynthetic metabolism .",
"discussion": "Discussion As the connections among different cell compartments and the ways of corporation between different metabolic pathways in eukaryotic organisms could be many and varied, their carbon and energy metabolisms are complex and fascinating [ 29 , 34 , 35 , 37 – 39 ]. Recent results showed that diatoms optimize their photosynthetic efficiency via elaborate interactions between plastids and mitochondria [ 41 ]. Besides, several studies have also noticed the synergistic photosynthesis and glucose metabolism in certain microalgae species under mixotrophic cultivation, but its carbon and energy metabolic mechanism is currently poorly understood [ 7 , 9 , 11 ]. The present study provided an interaction scenario between photosynthesis and glucose metabolism in C. zofingiensis under mixotrophic cultivation, in which the additional organic carbon source can replace the RuBisCO-fixed CO 2 for the organic carbon metabolism in the chloroplast, and provides sufficient precursors for the utilization of light energy. Thus, rather than CO 2 fixation, photosynthesis became mainly employed for light energy fixation in mixotrophy. This was similar with previous results in cyanobacteria, where the authors indicated that the photosystems are mainly employed for reducing equivalents and energy supplies with limited CO 2 fixation during mixotrophic growth [ 42 ]. As a result, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped in mixotrophy, which could reduce energy waste of photosynthesis while promote light energy utilization efficiency and cell growth. And similar results that mixotrophy can confer a higher growth rate than the sum of photoautotrophy and heterotrophy have also been reported in other microalgal species [ 7 , 43 ]. Glucose and its metabolic intermediates and ATP were presented both in photosynthesis and glucose metabolism, which could also function as regulators in many biological processes, and might coordinate photosynthesis and glucose metabolism in mixotrophy. It was reported that chloroplast-derived carbohydrates could regulate cellular metabolisms [ 34 ]. For instance, triose phosphates can trigger the expression changes of cytosolic transcription factors, and organic carbons were reported to feedback regulate photosynthesis [ 44 ]. Besides, evidence have showed that post-translational regulation may affect the Calvin cycle enzymes in microalgal species [ 45 ]. Apart from the regulations of organic carbons on photosynthesis, the regulation of photosynthesis on glucose uptake has also been reported in the present study. Besides, citrate synthase, a key enzyme of TCA cycle, was reported to be regulated by the ratio of ATP/ADP [ 46 ]. As photoreaction could provide ATP for cell metabolism, it was not hard to understand the downregulation of TCA cycle in mixotrophy. In general, the mutual regulation of photosynthesis and glucose metabolism is a complex process. The mixotrophic metabolic mechanism proposed in this study is the result of their collaboration. Previous research showed that, adg1-1/tpt-2, an Arabidopsis thaliana double mutant impaired in acclimation to high light with an 80–90% inhibition of ETR, could be rescued by exogenously supplied sugars (i.e., glucose and sucrose) [ 47 , 48 ]. A scenario was proposed that the fed sugars would be transported into chloroplast and used for anabolism. However, a recent review pointed out that this scenario would entail the assumption that CO 2 fixation by Calvin–Benson cycle would be minimized or even blocked through sugar feeding, which awaits to be tested experimentally [ 34 ]. The present study showed that the intermediates derived from exogenous glucose would directly enter the chloroplast and replace RuBisCO-fixed CO 2 to provide carbon sources for chloroplast organic carbon metabolism in mixotrophy. Therefore, CO 2 fixation was skipped, as reflected by a significant down-regulation of gene expression. And these results experimentally verified the above assumption is valid in C. zofingiensis and provide a reference for research in plants [ 47 ]. Many works have been done on directly engineering of RuBisCO to accelerate CO 2 fixation rate [ 49 , 50 ]. It was previously reported that under current atmospheric conditions, nearly 30% of the carbohydrates formed by C 3 photosynthesis are lost via photorespiration [ 33 , 51 ]. However, photorespiration is indispensable for photosynthetic organisms, since this pathway participates in photoprotection [ 32 ], amino acid biosynthesis [ 52 ] and removal of toxic intermediate metabolites [ 53 ]. Hence, reducing rather than eliminating photorespiration has become an attractive avenue for improving photosynthetic efficiency [ 26 , 33 , 51 , 54 ]. Recent work has shown that re-engineering photorespiratory pathways can significantly increase biomass production in higher plants [ 55 ]. The present study has been the first to show that skipping RubisCO could significantly reduce NPQ and photorespiration, and provided a strong evidence that increase of light energy fixation can be achieved not only by directly increasing CO 2 fixation or by modifying photorespiration [ 55 ], but also by skipping the photosynthesis rate-limiting steps. Collectively, this study not only elaborated the mixotrophic metabolic mechanisms of C. zofingiensis , but also provides a theoretical basis and new ideas for future research on photosynthesis and glucose metabolism, and provides a foundation for future industrial applications of mixotrophy."
} | 3,400 |
30116788 | PMC6081794 | pmc | 2,449 | {
"abstract": "Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change.",
"introduction": "INTRODUCTION Ruminant animals are the dominant large herbivores on Earth. Their evolutionary success is partly due to their tight symbiotic associations with commensal microorganisms that enable them to utilize otherwise indigestible plant biomass as food sources ( 1 ). Since their domestication in the Holocene, ruminants (in particular, cows) have provided humankind with various important goods. However, agricultural farming of cows is also a major source of the potent greenhouse gas (GHG) methane (CH 4 ), having a global warming potential 34 times higher than carbon dioxide ( 2 ). Cows possess a complex digestive system, including a four-compartment stomach, with the largest compartment being the rumen ( 3 ). The rumen is basically a big anaerobic fermentation chamber harboring the complex rumen microbiome (here defined as the entirety of all rumen microorganisms [i.e., the rumen microbiota] and their genetic repertoire) that catalyzes the anaerobic degradation of ingested plant biomass. During microbial hydrolysis and fermentation of plant fibers, volatile fatty acids (VFAs) are produced; the VFAs serve as the main energy source of the animal ( 4 ). A prominent end product of microbial degradation is CH 4 , produced by methanogenic archaea. Individual cows, or, more specifically, their symbiotic methanogens, produce up to 500 liters of CH 4 per day ( 5 ), making ruminant livestock one of the major anthropogenic CH 4 sources ( 6 ). Due to an increasing human world population, milk and meat demands are expected to double by 2050 ( 7 ), making the development of sustainable and productive animal farming systems a major challenge in agriculture ( 8 ). CH 4 mitigation strategies are of not only ecological but also economic importance, as ruminant CH 4 emissions represent an energy loss of 2% to 12% for the animal ( 5 , 8 ). Since the time of the pioneering work of Hungate and others ( 9 – 12 ), microbiologists have made large efforts to understand the structure-function relationships in the complex rumen microbiome, identifying the microorganisms that participate in certain steps of the anaerobic degradation pathway. More recently, the application of cultivation-independent molecular techniques has helped to uncover the high diversity of bacteria, archaea, and eukaryotes residing in the rumen and factors affecting community composition (see, e.g., reference 13 ). In addition, the usage of meta-omics techniques has paved the way for a better understanding of the rumen ecosystem and the microbial metabolic potential and activity in the rumen (reviewed in reference 14 ). These studies have revealed differences in rumen microbiome structure between animals emitting CH 4 at low levels and those emitting it at high levels (see, e.g., references 15 and 16 ) and the effects of different diets on ruminant CH 4 emissions (see, e.g., references 17 , 18 , and 19 ). New insights were also gained by identification of new members of functional groups, e.g., new fibrolytic and methanogenic community members (see, e.g., references 20 , 21 , 22 , 23 , and 24 ). Furthermore, the importance of diurnal microbiome dynamics for the understanding of VFA, H 2 , and CH 4 production in the rumen was pointed out recently ( 25 ). Despite these major advances, a holistic understanding of the rumen microbiome is still lacking, including answers to rather simple questions such as “who is doing what and when during feed degradation?” Such a fundamental understanding of the rumen ecosystem, as proposed by Hungate in the early 1960s ( 11 ), can help to specifically manipulate the rumen microbiome, to reduce CH 4 emissions, without hampering animal productivity and milk and meat quality and without being harmful to the animal ( 14 , 26 ). To obtain a more comprehensive and holistic picture of the rumen microbiome activity during plant biomass degradation in lactating cows, we performed a short-term longitudinal study using an integrated approach, combining metatranscriptomics with gas and volatile fatty acid (VFA) profiling. By applying primer- and PCR-independent metatranscriptomics, we aimed at obtaining comprehensive multidomain profiles of the active rumen microbiome members (bacteria, eukaryotes, and archaea) and their functions in the key steps of anaerobic feed degradation, i.e., polysaccharide degradation, VFA production, and CH 4 formation. We hypothesized that the microbiome exhibits a defined successional pattern, reflecting a cascade of hydrolytic, fermentative, and methanogenic steps, accompanied by distinct VFA and gas emission patterns. On the basis of a previous metatranscriptomic study from our laboratory ( 24 ) and work of others (see reference 27 and references therein), we hypothesized that the recently discovered Methanomassiliicoccales species are substantial contributors to ruminant CH 4 emissions and would therefore show high activity after ruminant feed intake. By transforming data representing the relative transcript abundances of rRNA and mRNA to abundance per gram of rumen fluid (quantitative metatranscriptomics), we were able to link rumen microorganisms and their transcription profiles to rumen processes, e.g., methane emission. Furthermore, we show extensive inter- and intradomain multifunctional redundancy among pro- and eukaryotic microbiome members at several key steps of the anaerobic degradation pathway.",
"discussion": "DISCUSSION In this study, we used an integrated approach, combining metatranscriptomics with targeted metabolomics (gas and VFA profiling) to holistically investigate temporal rumen microbiome dynamics during plant biomass degradation in lactating cows. By integrating relative transcript abundances with RNA content per gram of rumen fluid, we were able to link rumen microorganisms and their activity to processes such as gas emissions and VFA production. Relative transcript abundances, which are commonly used in (meta-)transcriptomics, were not sufficient to establish this link ( 31 , 32 ). Few studies have already applied quantitative metatranscriptomics; those that have done so predominantly examined marine ecosystems (see, e.g., references 39 and 40 ), focusing on bacteria and on nutrient cycling. Our study was the first host-associated quantitative metatranscriptomics study to link process data to microbiomes. Furthermore, our approach is different as we use total RNA concentrations instead of internal mRNA standards for “sizing up metatranscriptomics” ( 40 ). This quantitative approach allowed us to assess the contributions of major bacterial, eukaryotic, and archaeal taxa involved in the three key steps of anaerobic plant biomass degradation in the cow rumen. In fact, quantitative approaches in microbiome research have recently come to maturity ( 41 ). By taxonomic classification of the small-subunit rRNA transcripts, we investigated if the rumen process dynamics (i.e., gas emissions and VFA production) were reflected in the composition of the microbiome. Our results showed that the microbiome composition was unexpectedly stable during feed digestion. The strong increase in the level of CH 4 emissions after feeding was not related to a microbial community shift as we had hypothesized but to a fast growth response of the whole microbiome. This led to enhanced fermentation rates, reflected by the increase of CO 2 , H 2 , and VFA concentrations and an associated rise in methanogenesis rates. A similar dynamic of bacterial titers (number of SSU rRNA gene copies per milliliter of rumen fluid) as a response to feed intake was reported recently ( 25 ). While they were stable over time, the individual microbiomes differed substantially between the four cows. Despite large differences in the abundances of the core bacterial and eukaryotic community members, these microbiomes exhibited similar fermentation characteristics, evidenced by gas and VFA patterns. This points toward extensive functional redundancy among rumen microbiome members, where multiple microorganisms possess the same functional trait(s) and can replace each other ( 42 , 13 ). Interdomain functional redundancy was widespread within the fibrolytic community, where eukaryotes and bacteria contributed various amounts of CAZyme transcripts within individual cows. For instance, most cellulase transcripts stemmed from two bacterial groups ( Fibrobacter and Clostridiales ) and two eukaryotic groups ( Neocallimastigaceae and Ciliophora ), with the eukaryotes producing the largest share of cellulase transcripts in two of the four cows. Interdomain functional redundancy was also observed within hemicellulose, starch, and oligosaccharide degradation, with marked differences between individual cows. Our results add to the growing notion that the eukaryotic contribution to fiber degradation has been underestimated in the past and support recent studies suggesting an important role of ciliates and fungi in ruminant (hemi)cellulose degradation ( 21 , 43 ). Within the bacteria, Bacteroidetes , Firmicutes , and Fibrobacteres dominated the degradation of complex plant polysaccharides, with contributions of 48%, 31%, and 18% to the total CAZyme transcript pool. Similar observations were made in rumen metagenomic, metatranscriptomic, and metaproteomic studies ( 21 , 44 , 45 ). However, 10% of the CAZymes were assigned to Proteobacteria (40% Bacteroidetes , 30% Firmicutes ) in the metagenomic analysis, and Fibrobacteres seemed to play a minor role ( 44 ). On the transcript level, Comtet-Marre et al. ( 21 ) also identified Fibrobacteres , in addition to Bacteroidetes and Firmicutes , as a bacterial contributor to cellulose, hemicellulose, and pectin degradation. In a recent metaproteomic study, more than two-thirds of all identified glycoside hydrolases were assigned to Bacteroidetes ; Firmicutes and Fibrobacteres seem to play a minor role ( 45 ). However, other CAZyme categories were solely dominated by Firmicutes . Thus, the differences in the clustering of CAZymes in different categories can obstruct a direct comparison between studies. Host individuality and functional redundancy were also revealed in the fermentation of carbohydrates to VFA. Three major, well-known VFA-producing taxa ( 46 , 47 ) were identified, and their contribution to transcript pools of enzymes involved in VFA production was again found to be cow dependent. These taxa, Bacteroidetes ( Prevotellaceae ), Clostridiales , and Negativicutes ( Veillonellaceae ), produced acetate, propionate, and butyrate via different fermentative pathways; some were shared among taxa, and others were taxon specific. Although Prevotellaceae and Clostridiales in general dominated acetate/propionate and butyrate production, respectively, Negativicutes contributed substantially to butyrate production via the butyryl-CoA–acetate CoA-transferase pathway in cow 2 but not in the other three cows. Bacteroidetes and Firmicutes were the two most abundant and active bacterial community members involved in the degradation of complex plant polysaccharides and the production of VFA. Notably, only one family and only one genus (i.e., Prevotellaceae and Prevotella ) were dominant within the Bacteroidetes , while Firmicutes consisted of several families. This might explain why Firmicutes seemed to be more generalists than Bacteroidetes . Firmicutes species were involved in cellulose, hemicellulose, starch, and oligosaccharide degradation (with similar transcript abundances within these four categories), as well as in acetate, propionate, and butyrate production. In contrast, Bacteroidetes species were clearly dominant with respect to oligosaccharide hydrolysis and acetate and propionate production. We also observed functional redundancy among the methanogens. The three detected groups (i.e., Methanomassiliicoccales , Methanobrevibacter , and Methanosphaera ) differ fundamentally in their methanogenesis pathways. Methanomassiliicoccales species are H 2 -dependent methylotrophic methanogens, reducing methylamines and methanol to CH 4 with H 2 as an electron donor ( 48 , 49 ). In contrast, Methanobrevibacter species produce CH 4 mainly via the reduction of CO 2 with H 2 as an electron donor. Methanosphaera species, in turn, produce CH 4 from methanol and H 2 ( 50 ). The removal of H 2 is important for the rumen ecosystem and for the host because low concentrations of H 2 ensure high fermentation rates and efficient feed digestion ( 51 ). The longitudinal experimental setup revealed temporal dynamics in electron acceptor usage within the Methanomassiliicoccales , where the fraction of methanol-specific methyltransferase transcripts was much lower immediately after feeding, exhibiting an expression pattern opposite that seen with the methylamine-specific methyltransferases. In turn, it appeared that Methanosphaera dominated methanol reduction at these time points, showing once more the redundancy among organisms of the same functional guild. The root cause for this might be manifold, e.g., due to a higher substrate affinity of Methanomassiliicoccales for methylamines than for methanol or higher concentrations of methylamines. Alternatively, Methanosphaera could outcompete Methanomassiliicoccales for methanol under conditions of high H 2 partial pressure. Taken together, the data suggest that methyl-reducing Methanomassiliicoccales species and, to a less extent, Methanosphaera species were responsible for the increase of CH 4 emissions immediately after feed intake and not the CO 2 -reducing Methanobrevibacter . This is surprising, given that CO 2 is a much more abundant methanogenesis substrate than methylamines and methanol. The sources of methylamines, i.e., glycine betaine (from beet), choline (from plant membranes), and methanol (from the hydrolysis of methanolic side groups in plant polysaccharides), are well known ( 52 ); however, the amounts of these substrates might vary substantially with different diets. Previous, less extensively temporally resolved work suggested that Methanobrevibacter was associated with high CH 4 emissions ( 14 , 53 ). However, a comparison of sheep rumen metagenomes and metatranscriptomes indicated that Methanomassiliicoccales are highly active community members in both high-level and low-level CH 4 “emitters,” with abundances around 5 times higher in the metatranscriptomes than in the metagenomes ( 16 ). Furthermore, their transcript abundances were significantly higher in high-level CH 4 emitters. Also, it was shown that Methanomassiliicoccales can represent the predominant active methanogens in cows ( 24 ). In fact, a need for more research on methyl-reducing methanogens in the rumen was pointed out recently ( 53 ), including quantifying their contribution to rumen methane production. Further studies on Methanomassiliicoccales and Methanosphaera physiology in vitro and metabolic interactions with the substrate-providing microorganisms in situ might identify novel targets for CH 4 mitigation strategies, such as enzymes of the methyl-reducing pathway or the supply of methylated substrates. Such efforts might complement general methanogenesis inhibitors such as 3-nitrooxypropanol to achieve more-efficient methane mitigation ( 52 ). To our knowledge, our report represents the first longitudinal integrated meta-omics analysis of the rumen microbiome during plant biomass degradation. It is another step toward a comprehensive system-level understanding of the dynamic rumen ecosystem, as already envisioned by Hungate and coworkers more than 50 years ago ( 11 ). Applying a quantitative metatranscriptomics approach, our study established a time-resolved link between microbiome structure and function and rumen processes. It revealed a rather simple response to feed intake, namely, a general growth of the whole community, without distinct successional stages during degradation. The individual cow microbiomes exhibited surprisingly high functional redundancy at several steps of the anaerobic degradation pathway, which can be seen as an example of the importance of multifunctional diversity for robustness of ecosystems, similarly to what has been found in terrestrial biomes ( 54 ). Our data furthermore point toward CH 4 mitigation strategies that directly tackle the producers of CH 4 , since all other functional guilds show high organismic diversity, with individual taxa being replaceable by others."
} | 4,348 |
34626495 | PMC9298428 | pmc | 2,450 | {
"abstract": "Summary \n Many plant species simultaneously interact with multiple symbionts, which can, but do not always, generate synergistic benefits for their host. We ask if plant life history (i.e. annual vs perennial) can play an important role in the outcomes of the tripartite symbiosis of legumes, arbuscular mycorrhizal fungi (AMF), and rhizobia. We performed a meta‐analysis of 88 studies examining outcomes of legume–AMF–rhizobia interactions on plant and microbial growth. Perennial legumes associating with AMF and rhizobia grew larger than expected based on their response to either symbiont alone (i.e. their response to co‐inoculation was synergistic). By contrast, annual legume growth with co‐inoculation did not differ from additive expectations. AMF and rhizobia differentially increased phosphorus (P) and nitrogen (N) tissue concentration. Rhizobium nodulation increased with mycorrhizal fungi inoculation, but mycorrhizal fungi colonization did not increase with rhizobium inoculation. Microbial responses to co‐infection were significantly correlated with synergisms in plant growth. Our work supports a balanced plant stoichiometry mechanism for synergistic benefits. We find that synergisms are in part driven by reinvestment in complementary symbionts, and that time‐lags in realizing benefits of reinvestment may limit synergisms in annuals. Optimization of microbiome composition to maximize synergisms may be critical to productivity, particularly for perennial legumes.",
"introduction": "Introduction Microbial symbionts can play a critical role in plant fitness, and the benefits plants derive from associations with these symbionts can have consequences for plant population dynamics, community composition, and ecosystem function (Friesen et al ., 2011 ). While the effects of individual symbionts on plant performance are widely documented, in nature plants often interact with multiple symbionts simultaneously. The outcomes of multiple symbioses may vary greatly, with plant hosts showing synergistic growth responses, additive responses, or even reduced growth (Larimer et al ., 2010 ; Afkhami et al ., 2020 ). Understanding the conditions under which plants receive synergistic benefits from interacting with multiple symbionts would be a significant step toward management of the plant microbiome to optimize agronomic and environmental applications. Plants derive synergistic benefits from associating with multiple symbionts when their growth exceeds additive expectations based on their growth with individual symbionts (Fig. 1 ). This has been demonstrated in many studies of legume–arbuscular mycorrhizal fungi (AMF)–rhizobia interactions (i.e. Jin et al ., 2010 ; Abd‐alla et al ., 2014 ; Larimer et al ., 2014 ; Afkhami & Stinchcombe, 2016 ), in which AMF and rhizobia enhance plant growth by increasing availability of complementary, limiting nutrients (phosphorus (P) and nitrogen (N), respectively). Yet a meta‐analysis of studies examining the interactive effects of plant microbial symbionts found that in general, AMF and rhizobia do not act synergistically (Larimer et al ., 2010 ). Variation in outcomes of this symbiosis likely depends on several factors, including abiotic environmental conditions. For example, if soil P or N are not limiting, plants may invest fewer resources in AMF or rhizobia (Shantz et al ., 2016 ), lessening the likelihood of microbial synergism. Life history strategies of host plants may also influence microbial synergism, as plants are known to vary in their dependence on and investment in interactions with microbial symbionts (Bever et al ., 1996 ; Koziol & Bever, 2015 ; Bauer et al ., 2018 ). Notably, the previous meta‐analysis showing no overall synergism in legume–AMF–rhizobia interactions was dominated by studies of annual plants (Larimer et al ., 2010 ), while many examples of synergism come from studies of perennial plants (Larimer et al ., 2014 ; Ren et al ., 2016 ; Primieri et al ., 2021 ), suggesting that plant life‐history in particular may influence the outcome of plant–microbe interactions. Fig. 1 Graphical representation of synergism. Bars represent an average response variable (e.g. plant biomass) of uninoculated plants, plants inoculated with arbuscular mycorrhizal fungi (AMF) or rhizobia, and plants inoculated with both symbionts. Plants inoculated with both symbionts show synergistic growth responses if they perform better than expected based on the independent effects of AMF and rhizobia. Blue bars, rhizobia effects; yellow bars, AMF effects; orange bars, synergism effects. Symbionts may be more likely to act synergistically when interacting with perennial plants because of differences between perennials and annuals in terms of life cycle. Long‐lived, late‐successional plant species have been shown to be more responsive to mycorrhizal fungi than early‐successional species (Siqueira & Saggin‐Júnior, 2001 ; Pasqualini et al ., 2007 ; Koziol & Bever, 2015 ; Bauer et al ., 2018 ) and more sensitive to mycorrhizal fungal species and/or strain identity (Koziol & Bever, 2016 ; Cheeke et al ., 2019 ). Less is known about whether similar patterns exist in plant–rhizobium interactions. Differences between long‐ and short‐lived plants may arise due to the costs of associating with microbial symbionts and time lags in receiving benefits derived from them. For example, mycorrhizal fungi associations have been shown to be costly to plants in early stages of growth but provide long‐term benefits to plants in later growth stages (Johnson et al ., 1997 ), as young plants have to invest carbon (photosynthate, lipids, etc.) in symbionts to initiate interactions, but may not immediately receive benefits (nutrients) from the symbionts. If reinvestment of gain from one symbiont into the complementary symbiont is necessary for realization of synergism, then there may be an additional lag in realization of this benefit. Annual legumes can have very short life‐spans and investment in roots can be reduced with initiation of flowering (Portes & Araújo, 2012 ), when plants begin preferentially allocating carbon to flower and fruit production, potentially reducing the opportunity to realize synergism compared to perennial legumes. In addition to using legume–AMF–rhizobia interactions to examine context dependency in synergistic benefits, we can also explore potential mechanisms underlying synergism. Symbionts are predicted to act synergistically when they provide distinct functional benefits to plants (Stanton, 2003 ; Afkhami et al ., 2014 , 2020 ). In the case of the nutritional symbioses in legume–AMF–rhizobia interactions, synergism may reflect stoichiometric requirements of the legume, as has been argued for mycorrhizal fungal benefits (Johnson, 2010 ). Although AMF and rhizobia can provide more to their hosts than P and N, respectively (Qin et al ., 2011 ; Delavaux et al ., 2017 ), if we accept the widely held expectation that improved P nutrition is a primary benefit of AMF (Smith & Read, 2008 ) and improved N nutrition the primary benefit of rhizobia, associating with AMF or rhizobia alone will increase plant growth through increased access to P or N, respectively. However, growth of a legume associating with only one symbiont may become limited by the nutrient not provided by that symbiont (i.e. growth of a legume only associating with rhizobia may be limited by P, as N fixation is a P‐demanding process (Udvardi & Poole, 2013 )). Associating with AMF and rhizobia simultaneously provides the plant with both of these potentially limiting, complementary nutrients (Afkami et al ., 2020 ), which can be used for growth and further investment in each mutualism. This stoichiometric complementarity may then lead to growth greater than expected from the sum of the benefits of the two symbionts alone, because neither N nor P is limiting. Examining the N and P tissue concentration of legumes could provide insight into whether resource complementarity underlies synergistic outcomes: when grown in nutrient‐limited soil, plants associating with only rhizobia that become P limited would show increased tissue N concentration compared to uninoculated plants, and plants only associating with AMF that become N limited would show increased tissue P concentration. If synergistic growth is a result of simultaneous relaxation of N and P limitation, we may expect plants deriving synergistic growth benefits to have lower N and P tissue concentrations than plants associating with rhizobia or AMF alone, because instead of accumulating these nutrients, they can invest them in further growth. Examining plant investment in each mutualist (rhizobia nodule number/AMF colonization) will also help determine whether synergistic outcomes are linked to a greater ability of legumes to invest in their mutualists. To explore context dependence in synergistic outcomes in legume–AMF–rhizobium interactions and the potential mechanisms underlying synergism, we conducted a meta‐analysis. Specifically, we asked (1) whether annual and perennial legumes differ in the benefits they derive from AMF and/or rhizobia; (2) whether N and P tissue concentrations support a stoichiometric complementarity mechanism underlying synergism; (3) whether co‐inoculation increases investment in AMF or rhizobium, and whether this differs between annual or perennial plant species; and (4) whether plant investment in symbionts is correlated with realized synergism. We hypothesized that: (1) perennial legumes derive more benefits than annuals from rhizobia and/or AMF, and are more likely to derive synergistic benefits from dual‐inoculation; (2) plants associating with rhizobia or AMF will display higher N and P tissue concentrations, respectively, than plants associating with both symbionts; (3) co‐inoculation will increase symbiont investment more in perennial plants than in annuals; and (4) investment in symbionts is positively correlated with synergistic growth benefits.",
"discussion": "Discussion As most plants interact with multiple symbionts simultaneously, understanding the potential for, and expected patterns of, synergistic benefits of co‐inoculation represents a critical issue in microbiome–plant interactions and in the optimization of plant microbiomes for production. Our meta‐analysis reveals that plant life history can play an important role in the outcomes of legume symbioses with both rhizobia and AMF. While all plants associating with individual symbionts generally grew larger than uninoculated plants, the magnitude of this effect was greater in perennial species. Perennials also derived synergistic growth benefits from co‐inoculation, while annual species did not. Rhizobia increased in abundance in the presence of AMF regardless of the life history of host plants, while AMF colonization did not consistently increase with the presence of rhizobia. Yet for both microbial symbionts, the magnitude of the benefit to the complementary microbial partner predicted the strength of the synergistic growth benefit in all plants. Together, these results advance our understanding of the mechanisms and context dependence of synergisms. Synergism may occur in legume–rhizobia–AMF interactions because rhizobia and AMF help plants acquire complementary resources (N and P, respectively). These resources are potentially co‐limiting, as translation of acquired P into plant fitness may require additional N and vice versa. Moreover, rhizobia nodules have high P requirements (Sulieman & Tran, 2013 ), and AMF impose high demands on N, as well as plant derived lipids and carbohydrates (Johnson, 2010 ). Therefore, associating with one symbiont may enhance a plant's ability to invest in, and benefit from, the other symbiont. Our results partially support this hypothesis. When plants were inoculated with both symbionts, they produced more nodules and greater nodule biomass compared to when plants were inoculated with rhizobia alone, but AMF colonization did not consistently increase with co‐inoculation compared to inoculation with AMF alone (Fig. 4 ). In both cases, however, the responses of AMF colonization, nodule number, and nodule biomass to co‐inoculation were significantly positively correlated with synergistic plant growth – that is, as the benefits of co‐inoculation were realized by complementary symbionts, synergistic growth benefits increased (Fig. 5 ). These results suggest that while symbionts generally benefit from co‐inoculation regardless of plant life‐history, increased microbial abundance may translate to synergistic growth benefits in long‐lived legumes. Better estimates of microbial fitness, such as fungal spore production (Pringle & Taylor, 2002 ), would provide more insight into how closely plant synergistic growth and microbial fitness are correlated. Reinvestment in complementary symbionts could result in a time‐lag in the realization of synergism, which may contribute to the differences in synergism that we observed between annuals and perennial legumes. While individual studies show synergistic benefits in annuals (e.g. Abd‐Alla et al ., 2014 ; Afkhami & Stinchcombe, 2016 ), our meta‐analysis results identify that annual legumes are commonly not able to translate co‐inoculation into even the expected levels of growth promotion from the individual effect. The differences we found between perennials and annuals are consistent with a time‐lag in benefit accumulation for host plants (Johnson et al ., 1997 ). To establish associations with symbionts, plants must invest resources in symbionts that can be costly during early stages of growth. These associations can then become more beneficial to plants over time, as symbionts alleviate resource limitation by N or P to the plants, allowing plants to not only continue investing in the symbioses, but to translate increased access to N or P to enhanced biomass production. Perennial plants continue to invest in growth throughout the first growing season (and longer for long‐lived perennials), allowing them to reinvest complementary resources in the two mutualisms. However, as annuals shift resources from growth to reproduction within the first growing season (Portes & Araújo, 2012 ), they may not have as long to realize synergistic growth benefits, making this realization vulnerable to the time lag in growth benefits generated by reinvestment in complementary symbionts. As a small number of individual studies of annual legumes do show evidence of synergism (e.g. Afkhami & Stinchcombe, 2016 ), more research on the context‐dependency of synergism is needed to determine under what conditions annuals (particularly agriculturally important species) can experience synergism, which could potentially be used to enhance productivity in agricultural and natural systems. While we find clear differences between perennial and annual plants with regard to synergistic growth outcomes in our study, these results need to be interpreted with caution given other differences between our two plant groups. Namely, the annual plants in our meta‐analysis are almost all domesticated crop species, while the perennial plants are mostly nondomesticated species. Decreases in genetic diversity as a result of domestication, selection on certain traits, and the environmental conditions of agricultural systems may lead to changes in how domesticated plants interact with rhizosphere microbes comparted to nondomesticated plants (Pérez‐Jaramillo et al ., 2016 ). For example, comparisons of several domesticated species to their wild ancestors have shown that domesticated species can be less able to support AMF (Xing et al ., 2012 ), or more limited in their interactions with rhizobia (Mutch & Young, 2004 ). By contrast, a meta‐analysis showed that newer domesticated crop genotypes were more responsive to AMF compared to ancestral genotypes (Lehmann et al ., 2012 ), suggesting that the effects of domestication on plant–microbe interactions may vary. Further work examining legume–AMF–rhizobia interactions in nondomesticated annual plants is needed to tease apart the effects of life‐history from possible effects of domestication on synergistic outcomes. However, a study of nondomesticated grassland species found that annuals were relatively unresponsive to mycorrhizal fungi compared to perennials (Reinhart et al ., 2012 ), indicating that life history itself can influence the outcomes of plant–microbe interactions, and suggesting that our findings in this meta‐analysis may not only be the result of differences between domesticated and wild species. In addition to teasing apart potential effects of plant life history and domestication, further work is needed to specifically explore the role of soil nutrients in synergistic outcomes in the legume–AMF–rhizobium system. The studies in our analyses included legumes grown in a variety of substrates, which likely varied in nutrient levels. Soil N and P levels may greatly influence the benefits derived by legumes from rhizobia and AMF (Johnson et al ., 1997 ; Lau et al ., 2012 ), and thus the likelihood of synergistic outcomes. This could influence the results of our meta‐analyses, particularly if annual/domesticated legumes were grown under conditions approximating typical agricultural soil fertility levels, which are often more N and P rich than under natural conditions. While we only included control data points from studies that manipulated soil N and P, we were unable to control for natural variation in soil fertility across studies. In addition, most data points in our study came from glasshouse experiments (83%), where conditions likely differ greatly from agricultural or natural communities, and where factors such as light could be a limiting factor for plant growth, which could affect the benefits derived by legumes from rhizobia and AMF (Ballhorn et al ., 2016 ). However, we note that our analyses show that experiment type (glasshouse vs field) had limited influence on measures of synergism. More studies examining synergism in natural plant communities and/or agricultural settings, especially in the field, are needed to confirm the patterns in synergism observed in our study and to make our findings more generalizable to natural communities. In addition to exploring the effect of plant life history on synergistic growth outcomes, our analyses also find support for N and P co‐limitation and reinvestment in symbionts being a mechanism by which synergistic benefits occur. When plants were inoculated with either rhizobia or AMF, they had higher N and P tissue concentrations, respectively, than uninoculated plants, but co‐inoculation resulted in N and P tissue concentrations that were lower than the additive expectation for annuals and perennial legumes (Fig. 3a,b ). The absence of positive synergism with co‐inoculation in N and P concentration is expected because legumes are able to translate increased access to N and P into faster growth thereby diluting tissue resource concentrations, and because resources have been invested in complementary symbionts (i.e. N into increased AMF colonization and P into rhizobia nodulation). Our results then, are consistent with balanced N : P tissue stoichiometry mediating synergistic growth in legumes. We note, however, that this interpretation must be qualified, as the subset of studies of perennials that included measurements of N and P concentrations did not show strong synergism in growth promotion (Fig. S7 b,d). Further work is required to demonstrate that perennials do not have positive synergistic patterns in resource concentrations or total content, while producing synergistic benefits in total biomass. Microbial symbionts can be hugely influential on plant fitness, but the outcomes of plant–microbial interactions are notoriously context dependent. Our meta‐analysis demonstrates that plant life history can be an important factor determining when plants derive synergistic growth benefits by interacting with multiple mutualists. Moreover, we find support for a general framework that synergistic benefits emerge from microbial symbionts that provide complementary resources because of constraints on growth imposed by tissue stoichiometry. Further investigation of other context‐dependencies for synergistic growth benefits of co‐inoculation, such as dependence on the relative availability of soil N and P, would lead to a better understanding of how and when plants benefit from synergistic interactions. Our results identify synergistic benefits of multiple, complementary symbionts can be critically important for legume productivity, particularly for perennial plant species, and therefore realization of these synergisms should be a priority for management and breeding of perennial legumes."
} | 5,216 |
34641229 | PMC8512855 | pmc | 2,451 | {
"abstract": "Poly(ethyl ethylene phosphonate)-based methacrylic copolymers containing polysiloxane methacrylate (SiMA) co-units are proposed as surface-active additives as alternative solutions to the more investigated polyzwitterionic and polyethylene glycol counterparts for the fabrication of novel PDMS-based coatings for marine antifouling applications. In particular, the same hydrophobic SiMA macromonomer was copolymerized with a methacrylate carrying a poly(ethyl ethylene phosphonate) (PEtEPMA), a phosphorylcholine (MPC), and a poly(ethylene glycol) (PEGMA) side chain to obtain non-water soluble copolymers with similar mole content of the different hydrophilic units. The hydrolysis of poly(ethyl ethylene phosphonate)-based polymers was also studied in conditions similar to those of the marine environment to investigate their potential as erodible films. Copolymers of the three classes were blended into a condensation cure PDMS matrix in two different loadings (10 and 20 wt%) to prepare the top-coat of three-layer films to be subjected to wettability analysis and bioassays with marine model organisms. Water contact angle measurements showed that all of the films underwent surface reconstruction upon prolonged immersion in water, becoming much more hydrophilic. Interestingly, the extent of surface modification appeared to be affected by the type of hydrophilic units, showing a tendency to increase according to the order PEGMA < MPC < PEtEPMA. Biological tests showed that Ficopomatus enigmaticus release was maximized on the most hydrophilic film containing 10 wt% of the PEtEP-based copolymer. Moreover, coatings with a 10 wt% loading of the copolymer performed better than those containing 20 wt% for the removal of both Ficopomatus and Navicula , independent from the copolymer nature.",
"conclusion": "4. Conclusions Amphiphilic methacrylic graft copolymers, composed of the hydrophobic polysiloxane (SiMA) side chain and three different hydrophilic pendant chains—polyphosphonate (PEtEPMA), polyzwitterion (MPC), and polyethylene glycol (PEGMA)—were synthesized and used as surface-active copolymers in two different loadings (10 and 20 wt%) in a condensation cure PDMS matrix. PEtEPMA-based films derived therefrom were proven to be more susceptible to surface reconstruction after prolonged exposure to water than the corresponding MPC and PEGMA-based coatings. In particular, the extent of the decrease in the water contact angle ( θ ) increased in going from PEGMA to MPC up to PEtEPMA, for which the reduction in θ was as high as ~40°. Biological assays against two different model marine organisms revealed that the settlement of F. enigmaticus larvae was generally low (≤40%) on all of the tested films after five days of incubation. The complete removal of adults was achieved for SiMA- co -PEtEPMA20_10, which displayed the most hydrophilic surface upon immersion in water. The biological performance against N. salinicola was comparable to those of the MPC- and PEGMA-based films. However, for both of the tested organisms, a trend was observed, suggesting that removal was generally higher for films containing the lower amount of copolymer. The present study is the first to use polyphosphonate-based copolymers for marine antifouling applications. Further, we have compared the wettability and AF/FR properties of films containing polyphosphonate, polyzwitterions, and PEG. Overall, polyphosphonates were identified as a potential alternative to polyzwitterionic and PEGylated antifouling coatings. In addition, as polyphosphonates can be prepared with different hydrolysis kinetics, this can add synergistically to their amphiphilicity in producing evolving, environmentally-responsive, and erodible surfaces effective in combating marine fouling.",
"introduction": "1. Introduction Polyphosphoesters (PPEs) are an innovative class of phosphorus-based polymers that offer a platform for biodegradable and biocompatible macromolecules. In particular, main-chain PPEs are characterized by repeating phosphoester bonds in the backbone, and the pentavalent phosphorus atom can be exploited to graft different side chains, thus providing a useful tool for tuning the polymer properties [ 1 ]. Among the others, polyphosphonates are a subset of PPEs bearing an alkyl/aryl group in the side chain, linked to the phosphorus atom through a phosphonate bond [ 2 ]. The hydrolysis rate can be tuned by modifying the chemical structure of the lateral substituent, e.g., the longer and sterically bulkier the side chain is, the better it shields the main chain from nucleophilic attack [ 3 ]. Moreover, the substitution of the side-group chain changes the water solubility of polyphosphonates, e.g., the polymer becomes insoluble in water as well as partly crystalline going from isopropyl to hexyl group [ 4 , 5 , 6 ]. Tunable crystallinity, along with hydrophilicity, thermo-responsiveness, biocompatibility, potential biodegradability, possibility to introduce a functional group in the chain end, and similarity to biomacromolecules such as nucleic acids, are some of the many aspects that make polyphosphonates very appealing for biological and biomedical applications [ 7 ]. They have been studied as drug nanocarriers [ 8 ], or in protein-polymer conjugation [ 9 ]. Nonetheless, only in recent years has there been a surge in interest in this class of polymers that still represent a young field of study. Anionic ring opening polymerization (AROP) [ 10 , 11 ] and other polymerization methods [ 7 , 12 ] have proved to afford polyphosphonates with a remarkable control over molecular weight, dispersity, and architecture. This class of hydrophilic polymers could offer an attractive alternative to polyethylene glycol (PEG) and polyzwitterions, as PPEs are not plagued by uncontrolled oxidative degradation. In particular, aiming to intrinsically benign, biocide-free antifouling (AF) and fouling-release (FR) surfaces, in the last years much effort was devoted to the design of coatings with optimal performance achieved through several approaches, centered on tuning morphology, topography, surface reconstruction, segregation, and wettability of polymer films [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. Particularly promising, the FR approach relies on the combination of low surface energy, low elastic modulus, and low surface roughness typical of polydimethylsiloxanes [ 23 , 24 , 25 , 26 ], facilitating the release of the fouling biomass in presence of relatively low hydrodynamic forces, e.g., the shear stress induced on ship hulls during navigation. Nevertheless, FR siloxane coatings require improvements to overcome some disadvantages, including the higher fouling accumulation during idle time [ 26 ] and the low fouling release efficacy towards some microalgae diatoms [ 27 ]. As a result, hydrophilic fouling-resistant polymers, including PEG and polyzwitterions, were extensively investigated for the hydrophilization of hydrophobic PDMS-based FR coatings [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. PEG, especially, is considered a “gold standard” in the field of anti-biofouling coatings, even though it was proved to undergo oxidative degradation generating some toxic compounds, including 1,4-dioxane and formaldehyde [ 36 ]. Unlike PEG, polyzwitterions show significant stability to degradation even in relatively aggressive environments such as sea water, making them suitable for long-term applications in the marine industry [ 37 ]. Nevertheless, they suffer from several disadvantages, including high hygroscopicity and cost as well as poor solubility in most commonly employed solvents [ 38 ]. Thus, herein, we focused on finding a new class of water-soluble and eco-sustainable polymers that could overcome such issues [ 1 , 36 ]. Among others, we tested PPEs as an emerging and attractive class of materials for the substitution of PEG in the field of non-toxic, anti-biofouling coatings. A comparative study of protein adsorption on model monolayer surfaces composed of polyphosphates and polyphosphonates has been reported in the literature, demonstrating that protein resistance can be achieved, and antifouling properties are strongly affected by the nature of polyphosphoester side chains [ 39 ]. Except for this study, PPEs’ antifouling potential remains largely unexplored to date. To the best of our knowledge, nothing is reported in the literature about the use of polyphosphonates to combat marine fouling. For this specific application, the investigation of the hydrolysis kinetics of polyphosphonate-based polymers in seawater conditions is of main significance for their possible exploitation as hydrolysable self-polishing coatings capable of generating a dynamic, water-responsive, and evolving surface unsuitable for the settlement of marine organisms. Hydrolyzable polymers are traditionally used for the development of biocide-based erodible coatings with a self-polishing (SP) effect [ 14 ]. New strategies recently emerged in the field, which combine the advantages of biocide-free SP/FR in hybrid systems, incorporating hydrolyzable trialkylsilyl or bis(trimethylsiloxy)methylsilyl methacrylate-based polymers into a polymer matrix, either as an additive [ 40 , 41 ] or as a structural component [ 42 , 43 , 44 ]. When the polymer hydrolyzes, the surface becomes more hydrophilic and responsive to the external environment. The eventual dissolution in water of the hydrolyzed components thus generates a new fresh coating layer, according to a self-polishing mechanism. Such an evolving surface was shown to be generally beneficial in enhancing AF performance against model macrofoulers and in real seawater environments [ 15 , 45 ]. With these rationales in mind, new amphiphilic random copolymers derived from a hydrophilic poly(ethyl ethylene phosphonate) methacrylate (PEtEPMA) and a commercially available hydrophobic polydimethylsiloxane methacrylate (SiMA) were synthesized via free radical polymerization with the aim to investigate the anti-biofouling potential of polyphosphonate-based copolymers toward marine organisms. SiMA was also alternatively copolymerized with the zwitterionic phosphorus-based monomer, 2-methacryloxyethyl phosphorylcholine (MPC), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) taken as reference samples, due to the fact that their antifouling properties were well-established [ 15 ]. PEtEPMA-, MPC-, and PEGMA-based copolymers were used as surface-active additives to prepare condensation cure PDMS-based coatings for surface and biological evaluations. In particular, two different organisms, viz the serpulid Ficopomatus enigmaticus and the diatom Navicula salinicola were selected to study the antifouling and fouling release potential of the prepared amphiphilic coatings.",
"discussion": "3. Results and Discussion 3.1. PEtEPMA Macromonomer Synthesis The cyclic monomer 2-ethyl-2-oxo-1,3,2-dioxaphospholane (EtEP) was polymerized by anionic ring opening polymerization (AROP) by using 2-(benzyloxy)-ethanol as the initiator and 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) as the catalyst. The macromonomer PEtEPMA was obtained by terminating the polymerization with an excess of 2-isocyanatoethyl methacrylate ( Scheme 1 ). Successful polymerization and activation of the chain end was confirmed by 1 H and 31 P NMR spectroscopy and by GPC analysis. The average number degree of polymerization of the polyphosphonate side chain was 21, as evaluated by 1 H NMR in accordance with the previously reported procedures [ 46 , 47 ]. 3.2. SiMA-co-PEtEPMAx Copolymerization and Chemical-Physical Characterization Free radical polymerization was chosen as the polymerization technique for the synthesis of copolymers based on SiMA and PEtEPMA macromonomers ( Scheme 2 ). Furthermore, 2,2′-Azobis(2-methylpropionitrile) (AIBN) was chosen as the thermal initiator, and its concentration was kept constant at 1 wt% relative to the comonomers. All copolymerizations were carried out at 70 °C for 24 h in a mixture of ethanol and toluene, 1/2 v / v , with an initial concentration of comonomers SiMA and PEtEPMA of 0.5 M. p(PEtEPMA) and p(SiMA) homopolymers were also synthesized as a reference sample. A range of comonomer molar ratios were used in the feed in order to prepare copolymers with different molar compositions ( Table 1 ). The obtained polymers were identified as SiMA- co -PEtEPMAx, where x is the molar percentage of PEtEPMA units in the copolymer. The molar composition x was calculated from the ratio of the integrated area of the signal at 0.1 ppm and 4.6 ppm, attributed to SiCH 3 (SiMA) and O CH 2 C 6 H 5 (PEtEPMA), respectively. High conversions ( p ≥ 94%) were observed for the polymers; nonetheless, the yields were significantly lower due to the difficulty to find a selective non-solvent for the quantitative precipitation of the amphiphilic copolymers. While p(SiMA) was purified by repeated precipitations in methanol, p(PEtEPMA) purification was performed by dialysis in methanol. 3.3. SiMA-co-MPC20 and SiMA-co-PEGMA28 Copolymerizations Two copolymers, namely SiMA- co -MPC20 and SiMA- co -PEGMA28, were also prepared as reference samples for the phosphonate-based counterpart. The copolymerizations were carried out at 70 °C for 24 h in ethanol and THF (1/1 v / v ) in the case of SiMA- co -MPC20, or toluene in the case of SiMA- co -PEGMA28. AIBN was used as the thermal initiator, starting from an initial concentration of comonomers of 0.5 M. Copolymers were purified by repeated precipitations from chloroform solutions into methanol. The molar composition of copolymers ( Table 1 ) was calculated from the integrated areas of characteristic 1 H NMR peaks at 0.1 ppm attributed to SiCH 3 (SiMA), 3.4 ppm for OCH 3 (PEGMA), or 3.1 ppm for CH 2 N + ( CH 3 ) 3 (MPC). 3.4. Thermal Analysis of the Copolymers The thermal properties of polymers were investigated by differential scanning calorimetry (DSC, Figure 2 ). Polymers were studied by DSC between −140 and 120 °C, at 10 °C/min in heating and cooling scans. After the first heating, samples were kept at 120 °C for 15 min to remove most of the humidity absorbed, since water would act as a plasticizer, thus lowering the T g of the samples. This was especially true for highly hygroscopic polymers, like those carrying zwitterionic and phosphonate side groups. All of the polymers were found to be completely amorphous ( Table 2 ). Poly(alkyl ethylene phosphonate)s with short alkyl side chains are reported to have T g s around −40/−45 °C, and in particular, poly(ethyl ethylene phosphonate)s with various molecular weights exhibit T g values lower than −50 °C [ 2 , 48 ]. PEtEPMA and p(PEtEPMA) showed glass transition temperatures at ca. −31 °C. SiMA- co -PEtEP20 presented two T g s at −121 °C and −29 °C, attributed to the SiMA and PEtEPMA components, respectively. In contrast, copolymers with a higher ratio of PEtEPMA (above ≥ 53 mol%) only showed a single T g , close to the value of the polyphosphonate homopolymers. The presence of only one T g in the thermogram was likely due to the relatively low weight amount of SiMA in the copolymers. SiMA- co -PEGMA28 exhibited only the SiMA related transition at −119 °C. Since the PEGMA homopolymer with the same length of the oxyethylenic side chain is reported to have a T g of −54 °C [ 49 ], its absence in the thermogram was ascribed to the low weight fraction of PEGMA in the copolymer. Copolymer SiMA- co -MPC20 did not present any other glass transition temperatures, other than the one at −119 °C due to the SiMA units. As reported in the literature, dry polyzwitterions do not exhibit a glass transition temperature within the limits of their thermal stability, as the strong electrostatic intermolecular interactions drastically restrict the chain motion [ 50 , 51 ]. In fact, similarly, the homopolymer p(MPC) did not show any thermal transition in the investigated temperature range after the dehydration step. Overall, the results suggest that, despite the random structure of the copolymers, the chemically incompatible polyphosphonate (or polyzwitterion or poly(ethylene glycol)) and polysiloxane side chains were able to self-assemble in separated microdomains characterized by the thermal behavior of the corresponding homopolymers. No sign of mixed phases was perceived, due to the minimal shift of the T g values with respect to those of the corresponding homopolymer, even in those DSC curves where only one transition was detectable. Thus, the hydrophilic (PEtEP, MPC, PEGMA) and the hydrophobic (SiMA) side chains are supposedly immiscible in the solid state. 3.5. Hydrolisis of Poly(ethylphosphonate)-Based Polymers in Alkaline Conditions In order to evaluate the stability of the phosphonate side chains in alkaline conditions, PEtEP and p(PEtEP) were dissolved in a 50/50 v / v mixture of deuterated water and artificial seawater (pH ~ 8.2) or bicarbonate buffer (pH ~ 10). The hydrolysis reaction was followed by 31 P NMR spectroscopy. The hydrolysis of the zwitterionic homopolymer p(MPC) was also monitored at a pH ~ 10. In the p(MPC) sample, a single resonance at −0.60 ppm was observed, which did not change even after 66 days at a pH ~ 10, indicating no degradation of the polymer under these conditions. In contrast, both the macromonomer PEtEPMA and homopolymer p(PEtEPMA) showed the formation of degradation products in the 31 P NMR spectra ( Figure 3 ). The phosphorus centers in the starting material exhibited a single resonance at 38.3 ppm. Already after 15 h at a pH ~ 10, a new resonance was detected upfield at ca. 31.1 ppm that gradually increased over time, which can be attributed to the main degradation product, i.e., ethyl ethylene phosphonate (a monoester). After prolonged degradation times, a second smaller signal was detectable at a lower chemical shift (30.6 ppm), which is probably attributed to ethyl phosphonic acid, i.e., after additional cleavage of the ester bond to ethylene glycol. From the literature, it is known that monoalkyl phosphonic acid esters and alkyl phosphonic acids (or their salts) can be detected in this spectral range [ 52 , 53 ]. The P–O bonds in polyphosphoesters are hydrolyzed under basic conditions [ 3 , 6 , 54 ], possibly via a nucleophilic attack of water or hydroxyl ions at the phosphorus center ( Scheme 3 a) or via a backbiting mechanism, which had been reported to be the major degradation pathway in polyphosphates with a terminal hydroxyethyl group [ 55 ] ( Scheme 3 b). Since the herein reported polymers did not carry a free hydroxyl group at their chain end, but were functionalized with a methacrylate group, one can suppose that first a chain scission needs to occur according to Scheme 3 a, which is followed by a backbiting degradation [ 6 ]. In the further stages of the degradation, the remaining ester is also cleaved, revealing the free ethyl phosphonic acid. The degree of hydrolysis ( HD ) was evaluated according to the following equation: (1) H D % = 100 A p 1 + A p 2 A s + A p 1 + A p 2 \nwhere Ap 1 and Ap 2 are the integrated areas of the signals of the hydrolysis products at 31.1 ppm (ethyl ethylene phosphonate) and 30.6 ppm (ethyl phosphoric acid), respectively, and As is the area of the signal at 38.3 ppm of the initial polymer. The hydrolysis kinetics of polyphosphonate-based (co)polymers is reported in Figure 4 . Homopolymer p(PEtEPMA) and its monomer PEtEPMA presented similar hydrolysis kinetics at a pH ~ 10. In both cases, the degradation rate slowed down significantly after 20 days, and HD values seemed to reach a plateau at 20–30% after 35 days. Moreover, at a lower pH of ~ 8.2, typical of artificial sea water, the hydrolysis of p(PEtEP) was significantly slowed down. Hydrolysis was also observed for the copolymer SiMA- co -PEtEP20; however, as the polymer was water-insoluble, it was dissolved in deuterated THF with ~ 10 vol% of a bicarbonate buffer at a pH ~ 10. Despite the milder experimental conditions, the copolymer showed hydrolysis, even though the kinetics resulted to be significantly slower than that observed for the p(PEtEPMA) at a pH ~ 10. The lower hydrolysis rate was possibly also due to the higher hydrophobicity of the SiMA units in the copolymer. Similar findings had been reported for other non-water-soluble amphiphilic hydrolyzable copolymers, e.g., those containing silyl (meth)acrylate units [ 40 ]. Therefore, polyphosphonates were observed to undergo limited degradation (~2%) within two months at a pH ~ 8.2, while faster hydrolysis rates and more extensive degradation (20–30%) were observed at a pH ~ 10. 3.6. Preparation of Polymer Films The non-water-soluble copolymers SiMA- co -PEtEP20, SiMA- co -MPC20, and SiMA- co -PEGMA28 were selected as surface additives to prepare PDMS-based three-layer films for biological assays, in order to compare the antifouling/fouling release performance of such different hydrophilic components. The selected copolymers possessed intentionally similar and relatively low mole percentages of the hydrophilic co-units, in order to avoid or limit possible leaching of the copolymer from the matrix when in contact with water. Moreover, the predominant SiMA component was anticipated to promote the chemical compatibility with the siloxane matrix. The three-layer coatings consisted of a thin bottom layer (<5 μm thickness), a thicker middle layer (~400 μm thickness)—both composed of cross-linked PDMS—and a thin top layer of cross-linked PDMS loaded with 10 or 20 wt% surface-active copolymer ( Figure 1 ). According to this procedure, the amphiphilic copolymer was physically dispersed, i.e., not chemically linked within the PDMS matrix in a semi-interpenetrating cross-linked network. For each layer, the cross-linking reaction occurred via a condensation sol–gel process at room temperature, that was catalysed by TBAF. The final cure was carried out at 120 °C for 12 h. While the thick middle layer provides the suitable elastomeric mechanical properties to the entire coating, the thin bottom layer (<5 μm thickness) ensured firm anchorage of the film to the glass surface, thus preventing a possible delamination phenomena of the coating during underwater evaluations. PDMS-based films similar to those reported here were proven to show a low Young modulus ( E = 0.9 MPa) and high elongation at break (ε = 195%), consistent with the elastomeric nature of the PDMS-based coatings [ 20 ]. Finally, the physical dispersion of the amphiphilic copolymer in the top layer allowed for a concentration of the surface-active additive at the outer surface layers, closest to the film–water interface for more effective modulation of the surface properties. Thus, the three-layer geometry allowed the bulk/mechanical properties of the films to be independently controlled, basically due to the middle layer and its surface properties mainly depending on the nature of the amphiphilic copolymer. 3.7. Wettability and Surface Reconstruction Water contact angles ( θ ) were measured in static sessile drop conditions after 50 s from the deposition of the drop, to evaluate the wettability of the film surface. θ values of the single-layer films of the pristine (co)polymers are collected in Table 3 . Homopolymers p(MPC) and p(PEtEPMA) were completely wettable, being θ ~ 0°. For these films, a partial solubilization of the polymer film in water was also noted, as expected. All of the tested amphiphilic copolymers displayed a water contact angle in the range of 81–95°, indicating a general moderate hydrophobic nature of the film surface. In particular, SiMA- co -PEtEPMAx resulted to be the most hydrophilic copolymer class, followed by SiMA- co -PEGMA28. In any case, the θ values did not appear to be largely affected by the increasing amount of PEtEPMA co-units in the respective copolymers. One can speculate that the lower surface energy and the higher chain flexibility of the polysiloxane segments, with respect to polyphosphonate and even more to the zwitterion side chains, resulted in an efficient surface migration of such hydrophobic chains to the polymer–air interface that imparted an overall moderate hydrophobic character to the film surface. Surprisingly, despite the well-known high hydrophilicity of the zwitterion MPC, [ 50 , 56 ] SiMA- co- MPC20 films afforded the most hydrophobic surfaces with θ values of ~95°. In particular, for SiMA- co- MPC20, the migration of SiMA side chains to the surface might be predominant as a result of the ionic intra- and inter-macromolecular interactions among the zwitterion charged groups, thus limiting the migration of the hydrophilic side chains at the film surface. The contact angles of the three-layer PDMS-based films were measured before (t = 0 days) and after immersion in deionized water for different times, up to 28 days ( Table 4 ). The measurements before immersion gave similar contact angles for all of the samples, including the PDMS control, confirming that the as-coated films did not expose a noticeable amount of hydrophilic co-units to air, even for copolymer loadings of 20 wt%. All of the films, including PDMS, showed a progressive, slow decrease of θ values in the first 14 days of immersion. PDMS is known to undergo a process of surface reorganization after prolonged exposure to water to minimize the interfacial tension [ 57 ]. This is achieved by the progressive exposure of the polar Si–O–Si groups to the water–polymer interface, and reorienting the methyl groups into the bulk of the polymer film. After 21 days of immersion, all of the amphiphilic film surfaces, except SiMA- co -PEGMA28_20, showed a significant decrease in the water contact angle of 9–25° with respect to unmodified PDMS. In particular, this phenomenon was more marked for SiMA- co -PEtEP20_z-based films, which reached values as low as 65° in the case of SiMA- co -PEtEP20_10 films after immersion for 28 days. Moreover, a trend in the decrease of surface reconstruction kinetics was found in passing from PEtEP to MPC and to PEG. The continued and progressive hydrophilization of the amphiphilic PDMS-based films in general, and of SiMA- co -PEtEP20_z in particular, is attributed to the effective surface migration and major exposure of the hydrophilic co-units of the additive copolymer to the polymer–water interface. 3.8. Biological Test with Marine Organisms 3.8.1. Ficopomatus Enigmaticus Settlement and Detachment Assay The antifouling performance of three-layer films was assessed against F. enigmaticus on a laboratory scale. In the settlement (adhesion) test, competent larvae of F. enigmaticus were directly pipetted on the polymer surfaces, and the percentage of adhesion was evaluated after five days of incubation. Figure 5 shows that the percentage of adhered larvae on amphiphilic films was lower than ~40% in any case. The initial hydrophobic surface was beneficial in slowing the settlement of the larvae, which are known to better adhere to hydrophilic rather than hydrophobic substrates [ 20 ]. Overall, the different copolymer chemistries and loadings did not show clear trends and statistically significant differences, thus indicating that the PEGMA, MPC, and PEtEP hydrophilic components possess a similar antifouling performance against this specific organism. Nevertheless, it is known from the literature that a marked hydrophilic nature of the coatings promotes the release of F. enigmaticus under relatively low shear stresses [ 58 ]. Consistently, the detachment percentage of F. enigmaticus ( Figure 6 ) was found to be the highest for SiMA- co -PEtEPMA20_10, which displayed the most hydrophilic surface, wherein its water contact angle after 28 days of immersion in water was the lowest. Moreover, F. enigmaticus detachment was generally easier from the films with a lower wt% of copolymer additive, although this trend was not statistically significant. 3.8.2. Navicula Salinicola Settlement and Detachment Assay Amphiphilic films were also subjected to laboratory assays to evaluate the colonization and removal of the diatom N. salinicola . The settlement was quite similar on all of the tested surfaces, being significantly lower only on SiMA- co -MPC20_20 ( Figure 7 ). Thus, the presence of phosphorylcholine side chains in the copolymer additive improved the antifouling properties of the coatings against diatom biofilm. Figure 8 shows the percentage of diatom removal after 5 min of exposure to a low wall shear stress of 5 Pa. For all of the tested surfaces, the mean cell removal percentage was higher than ~ 75% even for the worst performing coating. In general, the chemical nature of the hydrophilic co-units in the copolymer appeared to not significantly affect the detachment of the tested diatom, with PEtEPMA performing as well as PEGMA and MPC. In agreement with what was observed for the detachment of F. enigmaticus , the removal percentage of N. salinicola was higher for films with the lower amount of copolymer loading, although the differences were not statistically significant."
} | 7,273 |
36419165 | PMC9686113 | pmc | 2,454 | {
"abstract": "Background Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO 2 /H 2 , is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae , were expressed in C. autoethanogenum . Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. Conclusions This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-022-01964-5.",
"conclusion": "Conclusions We have shown that production of ethyl acetate from the gaseous C1 substrate CO can be successfully achieved by heterologous expression of an AAT in C. autoethanogenum . The highest ethyl acetate production was achieved using the Sce Atf1, which reached 0.2 mM ethyl acetate on CO, or approximately 0.7% of the theoretical yield. C. autoethanogenum expressing Atf1 was also able to produce other acetate esters from CO, including butyl acetate and hexyl acetate, when supplied with their respective alcohols. Although there is ample room for improvement, we provide a foundation for autotrophic production of acetate esters from one-carbon substrates.",
"discussion": "Discussion We describe a successful proof-of-concept of engineering acetogens to produce ethyl acetate from CO as main carbon source. However, more efforts are needed before the envisioned autotrophic ethyl acetate production can compete with the conventional chemical catalysis or sugar-based microbial ethyl acetate production. Out of the two AATs tested, only expression of the Sce Atf1 resulted in ethyl acetate formation in C. autoethanogenum , regardless of the carbon source, background hosts, and promoters tested. The trace amounts of ethyl acetate observed for the empty plasmid control and Eat1-expression strains (but not for wild-type C. autoethanogenum ) likely originated from the chloramphenicol acetyltransferase (CatP) encoded on the plasmids as antibiotic resistance marker [ 34 ]. The higher ethyl acetate production with the promoter P Thl compared to P Pta-AckA suggests that the P Thl is stronger or more suitable for Atf1 expression on both CO and fructose. This may be specific to the microorganism and genes expressed. For instance, a study on autotrophic 2-propanol production from syngas in Clostridium ljungdahlii comparing the native C. ljungdahlii P Pta-AckA and the P Thl promoters showed that only the P Pta-AckA enabled isopropanol production [ 35 ]. On the other hand, acetone production levels were similar comparing the P Thl and the C. ljungdahlii P Pta-AckA for pathway expression in Acetobacterium woodii grown on H 2 /CO 2 [ 36 ]. Eat1 was a promising catalyst for ethyl acetate production based on previous work in E. coli [ 11 ]. However, Eat1 did not facilitate ethyl acetate production in C. autoethanogenum and was likely not functionally expressed based on lack of AAT and alcoholysis activity. Differences in AAT activity between Eat1 and Atf1 in C. autoethanogenum could be a result of differences in expression, enzyme characteristics or other factors. For instance, Atf1 was successfully expressed in C. autoethanogenum using the same promoters as for Eat1. This indicates that Eat1 might require other expression conditions than Atf1 for functional activity in C. autoethanogenum . Choice of inducible promoter and level of induction have previously been shown to strongly influence Eat1-catalyzed ethyl acetate production in E. coli , with Eat1 activity correlating positively with promoter strength [ 11 ]. Achieving functional expression of Eat1 in C. autoethanogenum might therefore benefit from a more extensive promoter library testing. Concerning enzyme kinetics, the affinity of purified Wan Eat1 for acetyl-CoA is relatively low (apparent K m = 2.43 mM) compared to that of purified Sce Atf (apparent K m = 45 μM) [ 10 , 37 ]. This might indicate that Eat1 requires higher intracellular acetyl-CoA concentrations than Atf1. However, concentrations of intracellular metabolites, including acetyl-CoA, have been described to be similar between E. coli and C. autoethanogenum [ 38 ], and Eat1 can successfully catalyze ethyl acetate formation in E. coli [ 11 ]. Metabolic pathway optimization in E. coli lysates has also been shown to be representative for C. autoethanogenum [ 39 , 40 ]. Yet there still may be physiological differences that affect activity of certain enzymes. For instance, the intracellular pH of the yeast mitochondria, where Eat1 is localized [ 41 ], is tightly regulated near pH 7.5 [ 42 ]. Atf1 appears to be located in the endoplasmic reticulum or cytosol [ 43 – 45 ], which have a pH near 7.2 [ 42 ]. Correspondingly, the intracellular pH of E. coli is near pH 7.6 [ 46 ]. However, the intracellular pH of clostridia like C. autoethanogenum and Clostridium acetobutylicum is significantly lower, near pH 6 [ 47 – 49 ]. Purified Sce Atf1 has a pH optimum near pH 7 but retains high activity at pH 6 [ 50 ]. For Eat1, the effect of pH on enzyme activity has not yet been investigated but it might be less suited for the acidic intracellular environment of clostridia. A previous study expressing the Wan Eat1 in another clostridial host, Clostridium beijerinckii, using the strong constitutive C. beijerinkcii P thl also showed low AAT activity [ 31 ]. Curiously, C. beijerinckii expressing Wan Eat1 still showed high alcoholysis activity, in contrast to C. autoethanogenum expressing Kma Eat1 in this study. Host-specific functionality is further supported by the fact that Kma Eat1 was successfully expressed in E. coli using the same expression plasmids as for C. autoethanogenum in this study. However, host-specific expression elements, such as host-specific origins of replication, may contribute to this observation. Nevertheless, this also confirms previous findings that the promoters used are also active in E. coli [ 27 , 51 ]. Overall, these results indicate that expression of Eat1 in Clostridia is troublesome and requires further investigation. For Sce Atf1, only low final ethyl acetate titers were formed, despite ethanol supplementation, in line with previous observations [ 11 , 12 ]. The inefficient ethyl acetate formation by Sce Atf1 might be due to low ethanol specificity, or other factors, such as suboptimal expression of Atf1. The first option is supported by our observations that, using the same Atf1 expression plasmid, nearly 20-fold more acetate esters could be produced with 20 mM butanol addition compared to a similar amount of ethanol addition. Therefore, this study indicates that the potential of Sce Atf1 may be limited for ethyl acetate production in acetogens under these conditions. On the other hand, these experiments indicate that Atf1 might be an effective AAT to produce other acetate esters with acetogens from C1 substrates, such as butyl acetate or hexyl acetate. Surprisingly, the amount of butyl acetate produced in this study (4.5 mM) indicated that approximately 10% of the acetyl-CoA formed out of CO could be readily directed to this non-native product without further optimization. Hexyl acetate was only produced in trace amounts, comparable to earlier findings in E. coli expressing Sce Atf1 [ 32 ]. Besides butyl acetate and hexyl acetate, Atf1 is known to be able to catalyze formation of many other acetate esters from different alcohols such as isobutyl acetate or propyl acetate [ 32 ]. Research on acetogens has already focused on autotrophic production of a variety of alcohols such as butanol, hexanol, isobutanol, and isopropanol [ 39 , 40 , 52 – 59 ]. For efficient autotrophic production of non-native products using acetogens, the limited energy availability during autotrophic growth needs to be taken into account [ 60 – 63 ]. Importantly, production of acetate esters is ATP positive in acetogens on both CO and H 2 /CO 2 assuming the bioenergetics for acetogens like C. autoethanogenum [ 49 , 63 ]. Besides several alcohols, certain acetogens like Clostridium carboxidivorans can natively also produce longer-chain acyl-CoAs, such as butyryl-CoA, which expand the possibilities of autotrophic ester biosynthesis even further."
} | 2,346 |
30718608 | PMC6361982 | pmc | 2,459 | {
"abstract": "Our understanding of diseases has been transformed by the realisation that people are holobionts, comprised of a host and its associated microbiome(s). Disease can also have devastating effects on populations of marine organisms, including dominant habitat formers such as seaweed holobionts. However, we know very little about how interactions between microorganisms within microbiomes - of humans or marine organisms – affect host health and there is no underpinning theoretical framework for exploring this. We applied ecological models of succession to bacterial communities to understand how interactions within a seaweed microbiome affect the host. We observed succession of surface microbiomes on the red seaweed Delisea pulchra in situ , following a disturbance, with communities ‘recovering’ to resemble undisturbed states after only 12 days. Further, if this recovery was perturbed, a bleaching disease previously described for this seaweed developed. Early successional strains of bacteria protected the host from colonisation by a pathogenic, later successional strain. Host chemical defences also prevented disease, such that within-microbiome interactions were most important when the host’s chemical defences were inhibited. This is the first experimental evidence that interactions within microbiomes have important implications for host health and disease in a dominant marine habitat-forming organism.",
"introduction": "Introduction Disease is emerging as a fundamentally important factor affecting the ecology and evolution of higher organisms by exerting profound effects on host performance, fitness and/or survival 1 – 3 . Disease frequency and severity are predicted to increase as environments change, due both to impacts on host reliance 4 and on pathogen abundance, behavior or virulence 5 – 7 . However, our understanding of microbial disease is undergoing a paradigm shift via the holobiont approach and both the classical concept of disease in which a single pathogen infects a single host 8 and the traditional methodology for confirming the presence of a disease via the application of Koch’s postulates 9 , are now being challenged. The holobiont approach involves assessing the health of a host organism in the context of its associated microbiome(s) 10 , 11 and studies supporting the interdependence of macro- and microbiota within holobionts have recently emerged for diverse organisms including humans 11 and demonstrate a clear association between shifts in the composition of microbiomes and disease in the host 12 – 15 . These observations imply that interactions within microbiomes are important for their hosts. However, we are only just beginning to explore interactions between microorganisms within microbiomes and we still understand very little about the mechanisms by which such interactions may lead to disease in the host (but see 16 – 18 ). One means to help understand the role of microbial interactions on hosts is to draw upon existing eukaryotic ecological theory, and its long history of theoretical frameworks for understanding interactions within and among communities. Microbiomes are communities embedded in an ecosystem (the host and the broader environment), so the application of theoretical community ecology to microbial communities can be a useful way to guide examinations into microbiomes 16 – 18 and how their interactions could influence host health. Furthermore, microbial communities are arguably ideal systems for testing ecological theory, because of their relatively short generation times (both in an absolute sense and relative to their eukaryotic hosts), which allow equilibrium states to be achieved over short periods of time (as little as 20 min 19 ); and their suitability for well-replicated, community-scale manipulations without concern for large-scale impacts on sensitive, natural environments. Here we used succession theory to investigate interactions within the microbiome of a seaweed – the dominant habitat-forming organisms on temperate reefs. Ecological succession is the process of post-disturbance community change and a fundamental concept within the field of community ecology. Since its formalization more than a century ago 20 , ecological succession has been the focus of much theoretical and experimental work e.g. 21 – 24 and can be defined as a directional, continuous and non-seasonal pattern of population colonization and extinction in a defined spatial area 25 . Various conceptual approaches to succession in ecological systems have been adopted, but one useful approach is that of Connell & Slatyer 21 , who proposed three alternative models of succession – facilitation, tolerance and inhibition, which are differentiated based on whether initial or ‘early successional species’ (ESS) have positive (facilitation), neutral (tolerance) or negative (inhibition) impacts on later successional species (LSS). Recently, some studies have investigated how microbial communities change over time, however these have mostly been limited to human 26 , agricultural 27 or controlled laboratory 28 settings with very few examples of experimental manipulations of microbiomes under natural conditions (but see 17 , 18 ). Furthermore, when the importance of priority effects on bacterial colonization 17 and diversity on dispersal 18 have been experimentally assessed, investigations have not been extended towards understanding how micro-micro interactions within microbiomes affect host health, survival or performance. Here, we apply succession theory to understand how microbiomes associated with naturally occurring macroalgae change following an experimental disturbance in situ and whether interactions between microorganisms within microbiomes can affect disease incidence or severity in the host. Delisea pulchra is a chemically defended red macroalga that produces halogenated furanones 29 , 30 , secondary metabolites that interfere with cell-cell signaling systems in many bacteria 31 , 32 . Halogenated furanones also protect D. pulchra from natural enemies, including epibiota 33 , herbivores 34 and some microbial pathogens 32 and their production can be manipulated in vitro via the removal of certain compounds from growth media 35 , 36 . When water temperatures are elevated and tissue concentrations of halogenated furanones are relatively low, this species suffers from a bacterial bleaching disease, which can affect more than 50% of individuals in populations at peak times 35 and has severe consequences for affected individuals, including dramatic reductions in fecundity and growth, and altered interactions with consumers 37 . This background understanding of host-microbe interactions in this seaweed holobiont, makes it an ideal system for investigations of within-microbiome interactions and their impacts on the host. In addition to within-microbiome interactions, D. pulchra ’s chemical defences are a potential mechanism by which this host may influence the composition of its microbiome and potentially also, interactions within it. We experimentally disturbed microbiomes associated with replicate D. pulchra thalli, then replaced them in their natural environment and followed their re-development over time to see whether they showed evidence of predictable succession. In a second group of experiments, we also manipulated succession by inoculating experimental thalli with combinations of early- and late-successional microorganisms and assessed impacts of these microbiome manipulations on the development of symptoms of bleaching disease.",
"discussion": "Discussion In one of the first studies to experimentally investigate the impacts of within-microbiome interactions on host health, we found that microbiomes associated with Delisea pulchra showed strong, directional patterns of community change following a major disturbance. After only 12 days, experimentally disturbed microbiomes returned to a pre-disturbance state, which in most cases was statistically indistinguishable from unmanipulated, ‘native’ microbiomes. In manipulative experiments designed to interfere with this succession process, we found that several early-colonizing strains of bacteria protected the algal host from putative pathogens that are more likely to colonize during later successional stages and cause disease. D. pulchra ’s chemical defences – halogenated furanones – appear to exert an important selective force on this alga’s microbiome, both with respect to recovery following a disturbance and via inhibition of potential pathogens. Thus, when the production of these chemical defences is compromised, such as during times of e.g. increased water temperatures 35 , the protective, inhibitory properties of early bacterial colonizers against later colonizing pathogens may become an important factor for the health of the host. Resistance, resilience and persistence in D. pulchra ’s microbiome The patterns of change in D. pulchra ’s microbiome following the experimental disturbance were directional (cyclic), demonstrating predictable successional processes were taking place. This cyclic pattern of recovery was consistently observed in four separate experiments conducted over two years, with some variability (e.g. in the third experiment, a clear cyclic pattern was observed but the rate of succession between sampling days was dissimilar to other experiments). The shifts observed in microbiomes on experimentally manipulated D. pulchra were greater in magnitude than those observed in seawater communities and biofilms on inanimate surfaces over the same time period. Such stability has been observed in epiphytic bacterial communities associated with another macroalga - the green alga Caulerpa taxifolia , whose microbiomes are so stable they can be used to trace the origins of the alga itself 38 . Interestingly, our results and those from C. taxifolia 38 are contrary to a systematic review conducted by Allison & Martiny 39 , who found that the vast majority of bacterial communities associated with (predominantly terrestrial) plants were not resistant to various environmental disturbances (including temperature, CO 2 , nutrient and changes in carbon sources) and often did not recover, even after years, potentially reflecting differences in holobiont dynamics between marine and terrestrial environments. One factor which may facilitate such stability is the diversity of D. pulchra ’s microbiome. Diversity has been linked to stability and resilience in soil microbial communities, which can withstand extreme perturbations (e.g. from pollution and cultivation) more effectively when bacterial diversity is high 40 . We have previously demonstrated that D. pulchra has a diverse microbiome compared to other macroalgae, using comparable methodology (>62 OTUs in the tip region alone 41 ; compared to 28 for Ulva spp. and 18 and 14 for Sargassum spp. and Porphyra spp. respectively). In the context of environmental change, a more diverse microbial community might provide insurance against perturbation, with continued stability in community structure through time 42 , 43 , although there is some debate about the generality of this for microbial communities 44 , 45 . Chemical defences and priority effects Although initial colonization following a disturbance is often a stochastic process 46 , the presence of biologically active compounds (halogenated furanones) in algal tissues may have influenced which bacterial strains were able to persist, thereby asserting a host-mediated influence on the composition of early successional communities (e.g. ESS-06 was unable to colonize chemically-defended sporelings). This suggests that succession within the microbiome of D. pulchra may be deterministic whenever ecologically relevant levels of halogenated furanones are available within the host tissues. Furthermore, prior to random selection of replicates for hierarchical analysis of DGGE gel banding patterns, we compared all replicates from within each treatment on single gels (data not shown). We observed very low levels of between-replicate variability at all time points in all experiments, indicating a high degree of uniformity among individuals at corresponding times in the succession process. Given the high diversity of epiphytic bacteria associated with D. pulchra , it is surprising that community composition remained so uniform across replicates at corresponding times and that succession patterns were indeed reproducible. Recent studies have highlighted the importance of priority effects, where the starting community has long-term implications for microbial community structure 17 , 26 , so potentially halogenated furanones, which are maintained at the algal surface boundary layer via vesicle cells 30 could strongly influence which OTUs can settle first, with long- term effects on the resulting community. Subsequently, this macroalga’s production of halogenated furanones may influence the composition of its microbiome, returning it to equilibrium following a disturbance, to the benefit of the host’s health and performance. When space becomes available after a disturbance, colonists may come from either the environment, or regrow from remnant, residual colonies that survived the disturbance 47 . In this case, the source of bacterial colonists onto D. pulchra ’s surface is not obvious because neither the communities in the surrounding seawater, nor those that developed on inanimate surfaces attached adjacent to the experimental algae had any overlap with D. pulchra ’s microbiomes, in terms of composition. Potentially, D. pulchra selects ( via the production of halogenated furanones and other chemical, physical and phenological traits), relatively rare species that may exist in the water column in low abundance (and thus remain undetectable by DNA fingerprinting methods). These may impart competitive traits suited to the specific environment of the host algal surface, where they flourish and influence the resulting composition of the developing microbiome. Our observation of complete recovery of severely disturbed microbiomes after only 12 days in situ is remarkable, given the diverse nature of the community and highlights the utility of using bacterial populations to test ecological theories. Although generation times vary greatly among bacteria (minutes to hours to days 48 ), as a group, they are dramatically shorter than most multicellular eukaryotes (weeks to years to decades). In the present study, we (conservatively) observed dozens of generations of bacterial communities. Following a similar number of generations of eukaryotes used in classical successional studies such as trees, grasses or corals during their recovery from a disturbance back to equilibrium would require decades if not centuries. Indeed, other micro-fouling (both prokaryote and eukaryote) communities provide further support for usefulness in assessing long-held ecological theories, for which empirical evidence may be lacking, varied, or inconclusive, over many generations. For example, one study of microbiomes on inanimate surfaces placed in a lake, reported rapid change in biofilm community structure in the initial 3–4 d, with stability observed after only 30 d in situ 49 . Interactions within the microbiome of D. pulchra and impacts on the host By experimentally manipulating both the production of algal chemical defences and the presence/absence of early- and late-successional bacteria, we learned that both host-mediated effects and interactions between members of the microbiome, can strongly affect succession and have major implications for host health. When algal chemical defences were left intact, colonization by the putative pathogen LSS09 was largely inhibited and algal bleaching minimal. When algal chemical defences were experimentally inhibited, LSS09 was able to colonize D. pulchra and when it did so, caused widespread bleaching. However, the presence of ESS strains significantly reduced both colonization by LSS09 and subsequent bleaching. A similar phenomenon was observed in laboratory trials using maize roots, where a simplified bacterial community prevented the growth of a pathogenic fungus 16 . Halogenated furanones inhibit bacterial cell-cell communication and the phenotypes they regulate by interfering with quorum sensing in bacteria that produce acylated homoserine lactones (AHLs 31 , 50 ). Recent related work has identified LSS09 as a dominant member of microbiomes associated with both bleached and healthy D. pulchra collected from the field 51 – 53 . Additionally, several putative virulence factors and a quorum-sensing dependent transcriptional regulator have also been identified 51 , which, in combination with our results, suggests that D. pulchra ’s chemical defences may interfere with both (i) attachment and settlement and (ii) infection and bleaching. Fernandes et al . 51 proposed that LSS09 may be a member of D. pulchra ’s native microbiome (due to its detection within the microbiomes of healthy adults from their natural environment) that can become pathogenic opportunistically, under certain conditions. Our work suggests that both a reduction of tissue concentrations of halogenated furanones and a disturbance within D. pulchra ’s microbiomes (e.g. via grazing or scouring) may facilitate LSS09 switching to a pathogenic lifestyle, leading to algal bleaching. Bleaching leads to dramatic reductions in fecundity, reduced growth and enhanced susceptibility to grazing, which all have deleterious impacts on affected individuals 37 . There is considerable spatial and temporal variability in the concentration of halogenated furanones in D. pulchra individuals 35 , 54 and under certain conditions (e.g. high water temperatures 35 ) concentrations can be very low. This is concerning given that climate conditions are predicted to become more extreme, potentially creating scenarios in which D. pulchra’s chemical defence will be compromised in the future. Under such circumstances, D. pulchra ’s microbiome may be its salvation: The protective role early-successional species appear to play may become more important for host health if algal chemical defences are reduced or depleted entirely. In our experiments, all three ESS strains prevented LSS09 colonization and significantly reduced the frequency and severity of the bleaching it caused, compared to colonization and disease that occurred in their absence. These observations provide evidence for the inhibition model of succession within D. pulchra ’s microbiome. Although compelling, an important caveat to this study is that only three ESS and one LSS strains were used – it is possible that other ESS strains may not have inhibitory effects against this or other LSS strains. However, our observations do suggest that early colonizers in D. pulchra ’s microbiome may follow the classic pattern of inhibition as observed in many other systems 55 and may be playing an important protective role for this alga when its own chemical defenses are depleted. Another limitation of our study is the use of the DNA fingerprinting methodology DGGE, which has been increasingly replaced with newer, higher throughput, next generation sequencing technology to explore the diversity and distribution of microbiomes from diverse habitats on a global scale. Whilst the use of DGGE is constraining in some respects, a major benefit of this technique is that you can obtain the full-length 500 bp sequence, providing better coverage for analysis of taxonomic patterns than shorter reads from, for example, MiSeq sequencing (discussed in ref. 56 ). Furthermore, for the D. pulchra holobiont specifically, we now know that patterns of composition of its microbiome based upon DNA fingerprinting techniques such as DGGE as described here, and sequencing from clone libraries (e.g. 35 , 51 ) are similar to patterns obtained using deep sequencing of the 16S rRNA gene 53 , so we are confident in the validity and reproducibility of our results from DGGE analysis as presented. Our results suggest that the application of ecological theory, such as succession theory, is a useful approach for investigations into interactions between microorganisms within microbiomes and their consequences for host health."
} | 5,087 |
31511505 | PMC6739400 | pmc | 2,461 | {
"abstract": "Synthetic biology and metabolic engineering have expanded the possibilities for engineered cell-based systems. The addition of non-native biosynthetic and regulatory components can, however, overburden the reprogrammed cells. In order to avoid metabolic overload, an emerging area of focus is on engineering consortia, wherein cell subpopulations work together to carry out a desired function. This strategy requires regulation of the cell populations. Here, we design a synthetic co-culture controller consisting of cell-based signal translator and growth-controller modules that, when implemented, provide for autonomous regulation of the consortia composition. The system co-opts the orthogonal autoinducer AI-1 and AI-2 cell-cell signaling mechanisms of bacterial quorum sensing (QS) to enable cross-talk between strains and a QS signal-controlled growth rate controller to modulate relative population densities. We further develop a simple mathematical model that enables cell and system design for autonomous closed-loop control of population trajectories.",
"introduction": "Introduction Advances in synthetic biology and metabolic engineering have expanded the potential for engineered cell-based systems 1 – 3 . Engineered microbes enable environmentally friendly manufacture of valuable molecular products 4 . Also, smart bacteria have appeared that sense their environments and execute desired functions such as the synthesis and delivery of therapeutics 1 , 5 – 8 . It is well recognized, however, that engineering cells to carry out multiple functions or produce products through extensive interconnected pathways leads to new challenges. These include bottlenecks, inefficient use of cell resources, and increased metabolic burden on individual cells. An emerging area of focus has been on the use of cell co-cultures or small consortia wherein individual populations work together to accomplish a desired output in cooperation with the rest of the consortia 9 – 15 . There are many potential advantages to using multi-cell systems over traditional clonal populations including the potential for division of labor and reduced metabolic burden on individual strains, ability for specialization and ease of optimization, and options for plug and play 9 – 11 . While promising, the use of co-cultures requires not only regulation of gene transcription within each population, but also regulation of each cell population within the consortia. Relatively few studies have been devoted to developing devices or systems that regulate the compositions of subpopulations within consortia. Often, studies that use multi-cell populations to carry out a coordinated task, such as producing biofuels or chemicals, rely on specific inoculation ratios or similar manual strategies to optimize the ratio of each population 16 – 18 . Alternatively, microfluidic and other devices can modulate the relative contributions of subpopulations by providing means to sequester or retain one population relative to another (e.g., using immobilization strategies) or by fluidically, but not physically, connecting populations (e.g., via porous membranes or 3D-printed microenvironments 19 ). A potentially more powerful approach that does not rely on equipment is to reengineer native cell-cell signaling systems in such a way as to enable the autonomous coordination of subpopulation densities. We and others have previously exploited quorum sensing (QS), a bacterial form of cell–cell communication, to engineer communication circuits amongst and between bacterial strains to coordinate behaviors 20 – 24 or enable density dependent activation of desired behavior 25 , 26 . QS circuits and signals have also been used to alter cell densities by, for instance, activating production of toxins or lysis genes in order to program stationary phase cell density of a monoculture 27 and to create co-cultures with defined behavior 28 , 29 . Similar strategies have been used to design co-cultures with a range of social interactions 30 . Here, we develop a platform for autonomous and targeted regulation of consortia composition based on the prevailing level of an environmental cue, autoinducer-2 (AI-2) (Fig. 1a ). The universal QS signal, AI-2, which is recognized and produced by many species of bacteria 31 , 32 , broadly indicates cell population density and is also likely to be an important signal in natural consortia or microbiomes 33 . Therefore, our synthetic system can be modulated based on an important signal often present in bacterial environments, AI-2, that is not easily measured on-line by users, either in fermentation reactions or in natural consortia. We achieve this by rewiring bacterial QS systems so that the growth rate of communicating consortia members is controlled by interspecies signaling. Thus, we present development of a signaling and control system that imparts trans-species communication and growth rate control. Our synthetic co-culture consists of an E. coli translator strain that senses AI-2 and translates this into an orthogonal QS signal (AI-1). This translator strain’s output, in turn, mediates the growth rate of the second strain. That is, a second engineered E. coli controller strain has signal-mediated tunable growth rate, regulated by the level of the second, species-specific autoinducer signal, AI-1. Thus, the translator population produces AI-1 after sensing AI-2, in turn regulating the growth rate of the AI-1 responsive controller strain and subsequently the composition of the synthetic consortia based on the prevailing AI-2 level (Fig. 1b ). Fig. 1 Design of autonomously regulated co-culture. a Engineered co-culture with AI-2 regulated composition. When the translator and controller populations are added to environments with AI-2, the resulting culture composition varies based on the AI-2 level. b Depiction of each strain in engineered co-culture. The translator strain senses AI-2 level and produces AI-1. The AI-1 growth controller strain produces HPr in response to AI-1, which alters cell growth rate and changes the culture composition There are two important and innovative aspects to our design. First, QS-mediated communication between subpopulations enables composition adjustment to occur autonomously. Importantly, the system is based on the prevailing concentration of a common naturally occurring autoinducer (AI-2) and the controller signal is based on an orthogonal species-specific autoinducer (AI-1) that has no function beyond its native host ( Pseudomonas aeruginosa ), a strain either included or not, based on system design. The second aspect of our design is signal-mediated tunable growth rate of bacteria via positive feedback. This is made possible by regulation of HPr, a phosphotransferase system (PTS) protein 34 , important for sugar (including glucose) transport in bacteria. We recently discovered that transgene expression of HPr in isogenic null mutants enables accelerated growth 35 . By controlling HPr expression via QS signaling, we enable autonomous subpopulation control. Importantly, our strategy positively modulates cell growth rate, preserving enhanced metabolic function, rather than increasing cell death (e.g., through expression of toxins or lysis genes), a strategy previously used by others 27 – 29 . Regulating expression of a critical gene for methionine synthesis has also been used to regulate cell growth, although this strategy requires use of dropout media 36 . In this paper, we develop and characterize each construct of the synthetic co-culture and then demonstrate autonomous regulation of co-culture composition based on initial AI-2 levels in batch and extended batch conditions. We create a simple mathematical model of the autonomous consortia regulator and show that the model can be used to either target a specific population composition or predict co-culture behavior given specific inputs. The model can then be used to explore parameter ranges and synthetic biology designs for future applications.",
"discussion": "Discussion Rapid advances in recombinant DNA technologies have greatly improved the ease of constructing engineered cells for applications ranging from bioprocessing (for production of valuable products) to smart bacteria capable of carrying out a multitude of functions. The bottleneck to further advancing these systems is typically not re-engineering genomes or their regulation, but optimizing cells to efficiently overproduce many proteins or carry out many functions without becoming metabolically overburdened. To work around this, many have proposed using consortia, where tasks can be divided and cells can be specialized. We and others have designed systems using QS circuits to regulate or coordinate gene expression amongst populations or between subpopulations in small consortia. However, by designing QS signal-regulated growth rate and an orthogonal translator controller, we create a tool for an additional layer of control. That is, we enable regulation of composition of the co-culture or small consortia. In our co-culture system, the composition of each subpopulation is autonomously regulated based on the level of AI-2 in the environment. The translator cells detect the level of AI-2 and produce the species specific signal molecule AI-1. The AI-1 regulated controller cells then adjust their growth rate based on the AI-1 signal level, which is a function of the initial AI-2 level. The result is an altered co-culture composition based on a native environmental cue, AI-2. We envision our system could be further engineered so that each population carries out part of a concerted effort or function that is autonomously fine-tuned when cells are placed into a particular environment. For example, we had previously reported on sensing cell networks in which the fractional level of responder cells indicates the previous environment they had surveyed 21 . Importantly, because of the partitioning of metabolic functions among subpopulations, our system reduces the potential metabolic burden on the cells—this done through use of QS to enable crosstalk between cells. Equally importantly, we have designed the system so that the AI-1 increases cell growth rate of the translator cells through increased transcription of a sugar transport protein ptsH , instead of by causing a reduced cell growth rate—the latter which may strain other engineered functions. We further believe using ptsH to regulate growth rate is likely a generalizable strategy due to its being well-conserved 37 . To estimate the behavior of a system using different strains or species, we suggest that prior to implementing the genetic circuit for growth control, one quantifies independently the growth characteristics of the individual strains. This information could be integrated into a simple model such as the one shown here, providing a range of culture dynamics that might be achieved. That is, a co-culture where the maximum growth rates are similar will be dramatically different than if they are very different, irrespective of our cell-based growth-controller module. In summary, we believe this system can easily be adjusted for further application. In this work, we described a scenario where the system is used to respond to external levels of AI-2 produced by cells in an environment of interest. As an alternative, the system could be rewired so that either population produces AI-2. In this case, both populations would grow naturally until a certain time when a threshold of AI-2 has been reached, at which time the growth rate of Population B would be signaled to change. That is, we believe the autonomous platform shown here, which functions independently and is accompanied by a simple model, could be used to design co-culture systems that allow for self-regulation of the composition of each subpopulation in multiple ways with regulation that requires no user or device intervention. Also by extension, our simple chemostat model predictions suggest we could maintain co-culture compositions at various steady states, but this would occur only with the interjection of well-defined user input (e.g., dilution rate, autoinducer addition, etc.). With such systems, or by inclusion as a subsystem within more complex environments, we expect to enable more widespread use of co-cultures—and the realization of the advantages that come with co-cultures or consortia in synthetic biology or metabolic engineering applications."
} | 3,115 |
34161255 | PMC8237665 | pmc | 2,462 | {
"abstract": "Significance Methane is a strong greenhouse gas that plays a key role in Earth’s climate. At methane seeps, large amounts of methane move upward through the seafloor, where microbial communities consume much of it. A full accounting of methane’s sources and sinks has evaded researchers—in part, perhaps, because key habitats including carbonate rock mounds have been largely neglected. We sampled seven methane seeps representing four geological settings and found that all sites had rock-hosted microbes capable of consuming methane; in lab-based incubations, some did so at the highest rates reported to date. We demonstrate several factors that help determine a sample’s methane-consuming potential and propose that carbonate rocks at methane seeps may represent a methane sink of far-reaching importance.",
"conclusion": "Conclusions It is widely accepted that ANME microbial communities play a substantial role in methane consumption in marine environments, but the contribution of rock-hosted habitats is largely absent from laboratory analyses and global estimates. Following a continental-scale assessment across a range of geological settings, we show that endolithic AOM is a common phenomenon and that rates of methane consumption by microbial communities within rocks often exceeds those associated with sediments. At the newly characterized Point Dume seep off the coast of Southern California, we report chimney-like carbonate structures that oxidized methane in laboratory-based incubations at the highest rates measured to date. We identified several factors, including cell abundance, mineral composition, kinetic parameters, and the presence of specific microbial lineages, that likely play key roles in supporting elevated endolithic AOM rates. We anticipate that future studies will better characterize how these and other aspects of the system facilitate the observed rates and will clarify the contribution of carbonate-hosted AOM to marine methane budgets. Given their frequent occurrence and elevated methane-oxidizing potential, carbonate rocks at methane seeps may constitute a major marine methane sink.",
"discussion": "Discussion Which Factors Determine Methane Oxidation Rates? Our detection of pervasive endolithic AOM potential reveals carbonate rock-hosted methane consumption as an understudied component of the methane cycle. This phenomenon was observed at all of the sites we sampled—across four geological settings and seven geochemically distinct locations—and may be ubiquitous at methane seeps across the ocean that exhibit authigenic carbonate deposits. Analysis of kinetic parameters, mineralogical constituents, electrical properties, cell abundance, microbial diversity, and the presence of particular lineages pointed to multiple factors that, taken together, help explain the range of measured rates and account for the remarkable activity observed during laboratory-based incubations of Point Dume carbonates. Cell counts indicated that changes in biomass and final cell abundances showed a stronger positive correlation with eventual long-term rates than preexisting biomass. These data suggest that environmental conditions such as in situ methane concentrations played a role in priming samples for their AOM activity, but that inherent properties of the sample—for example, mineralogy or microbial community structure—were more influential. The striking link between pyrite and higher rates is one particularly compelling avenue for further research. Framboidal pyrite, whose formation can be accelerated by sulfate reduction in the presence of reactive iron minerals ( 46 ), was also prominent in the Black Sea reefs ( 47 ), which may be the closest known analog to the poorly consolidated chimney-like structures at Point Dume ( 24 ). To further investigate pyrite prevalence in carbonates exhibiting high AOM rates, we calculated the relative abundance of putative sulfate-reducing ( 48 – 53 ) and sulfide-oxidizing lineages ( 54 – 58 ) in all samples ( Dataset S4 ). We hypothesized that more sulfate reducers and/or fewer sulfide oxidizers could allow sulfide to accumulate and ultimately precipitate as pyrite, but no significant relationship between target lineages’ relative abundance and pyrite abundance was observed. The electrical currents across all samples—up to hundreds of picoamps compared with previous cell interface measurements in the femtoamps range ( 31 )—suggest that the electrical properties of physical substrates may play a role in methanotrophic activity. Indeed, a similar phenomenon was observed in previous work, when seep sediment communities were transferred onto conductive carbon cloth and exhibited increased AOM rates ( 45 ). The presence of specific lineages directly and indirectly involved with AOM may also play an important role enhancing methane oxidation rates. ANME-2, which were more prevalent in the high-rate carbonates, possess large multiheme cytochromes and surface layer proteins that may enable them to offload reducing power through direct electron transfer between consortia partners ( 29 ). ANME-1, on the other hand, were found at higher relative abundance in lower-rate samples, and these particular genotypes may lack the physiological capacity for high-throughput extracellular electron transfer ( 59 , 60 ). In Black Sea carbonate reefs, zones dominated by ANME-2 exhibited lower δ 13 C values than those dominated by ANME-1, suggesting accelerated rates of methane oxidation and assimilation by ANME-2 ( 61 ). Atribacteria and Anaerolinaceae are not believed to be involved in the core methane-oxidizing or sulfate-reducing metabolisms, but their prevalence suggests that they may play important supporting roles. Atribacteria have been preferentially detected at marine methane seeps ( 62 , 63 ) and may enter seep habitats from below through upward-migrating fluids as was demonstrated at the Gulf of Mexico seeps ( 64 ) and mud volcanoes of the Ryukyu Trench ( 63 ). Genomic analysis suggests that they perform a fermentative metabolism capable of producing acetate, CO 2 , and H 2 —potential substrates for methanogens and/or SRB ( 65 , 66 ). Anaerolinaceae are commonly characterized as heterotrophs; in seep environments they have been posited to consume primary producer ANME biomass ( 67 ), but they may be more directly involved with hydrocarbon metabolism as in alkane-degrading enrichment cultures ( 68 , 69 ) and Gulf of Mexico seeps ( 70 ). Other lineage-specific attributes that could accelerate rates and account for the higher V max observed in Point Dume carbonate include a) increased rates of reactant uptake and product release by either syntrophic partner, b) distinct proteoforms of the enzymes performing rate-limiting reactions (methyl-coenzyme M reductase, dissimilatory sulfite reductase), c) a higher number of rate-limiting enzymes per cell, and/or d) distinct lineage-specific partnerships. Clarifying the roles of these factors will require more detailed biochemical, genomic, and proteomic studies which would likely contribute to the growing appreciation of methyl-coenzyme M reductase diversity ( 71 – 73 ). Implications of Carbonate-Hosted AOM. Existing estimates of the global extent of methane oxidation in marine settings ( 1 , 2 , 74 ) do not distinguish between sediment- and carbonate-hosted methanotrophy, despite the abundance and often dominance of carbonate rock at methane seeps ( 4 – 7 , 75 , 76 ). There are several important factors to consider when evaluating the influence of endolithic AOM on the global methane cycle, including the localized concentrations of metabolic reactants and products found within and around carbonates as well as the overall volume of carbonate at seeps. Some of these key parameters are not currently well constrained. Moreover, our experimental incubations were conducted under high-methane, sulfate-replete conditions that are not representative of all seep environments. For this reason, the absolute rates derived from our incubations reflect a sample’s methanotrophic potential that likely exceeds its in situ methane-oxidizing contribution. Nonetheless, by having maintained standardized experimental parameters for carbonates and sediments in our incubations, and by converting previously published results to equivalent conditions, our substrate-based and cross-study comparisons of potential AOM rates suggest that carbonate-hosted AOM could be an important methane sink in marine settings. Carbonate rock represents a substantial proportion of the seafloor and a majority of the methane-perfused volume at many seeps ( 4 – 6 , 8 , 9 , 77 ). While diffusion limitation may modulate the maximum rates of reaction in some carbonate edifices, many authigenic carbonates at seeps exhibit high porosities ( 4 , 28 , 78 ) and permeability values on par with those of nearby sediments ( 15 , 33 ). Accordingly, reactant transport in these two environmental regimes may be relatively similar and may even be less constrained in some carbonates. Not all seeps exhibit such pervasive carbonate structures, as cation availability could limit carbonate precipitation, and only a subset of the rock volume would be expected to experience seawater concentrations of sulfate. However, nitrogen- and metal-reducing ( 79 , 80 ) and low-sulfate AOM ( 81 ) as well as sulfate penetration of carbonate mounds caused by tidal forcing ( 33 ) or hydrologic recharge ( 4 , 34 ) could expand the endolithic AOM habitat deep into subsurface rock habitats. A final consideration when assessing the potential scope of endolithic AOM is the average rate enhancement in rocks compared with sediments. The rock-hosted to sediment-hosted scaling factor was derived from the short-term rates compiled in this study ( Table 1 ). At active sites where both endolithic and sediment-based AOM was observed, substrate-specific rate values were averaged and a rock-hosted to sediment-hosted AOM ratio was determined. These ratios were 0.88 for Gulf of Mexico samples, 8.85 for Guaymas Basin North samples, and 3.47 for Point Dume samples, indicating a wide variance but, on average, higher rates associated with carbonates than sediment per unit volume under equivalent experimental conditions. The data presented here demonstrate that carbonate samples from all seep sites examined to date, across a range of geologic settings, possess substantial anaerobic methanotrophic potential. Furthermore, some endolithic communities assessed in laboratory-based incubations exhibited the highest rates of AOM ever reported, implicating the physicochemical aspects of carbonate-hosted habitats in the promotion of enhanced methane consumption. While the full extent to which endolithic AOM influences global methane cycling remains to be established, our results demonstrate that endolithic environments may be important contributors to methane biogeochemical assessments. The consideration of methanotrophic communities in a carbonate-hosted context has implications for both mitigation of potential methane hydrate dissociation ( 82 ) and ocean acidification ( 83 , 84 ). A more detailed understanding of the structural and mineralogical attributes that enable such elevated carbonate-hosted AOM rates could also help with the rational design of methane-scrubbing systems for greenhouse gas remediation or distributed biofuel production ( 85 )."
} | 2,848 |
37953782 | PMC10638908 | pmc | 2,463 | {
"abstract": "To investigate changes in fungal community characteristics under different Cr(VI) concentration stresses and the advantages of adding arbuscular mycorrhizal fungi (AMF), we used high throughput sequencing to characterize the fungal communities. Cr(VI) stress reduced rhizosphere soil SOM (soil organic matter) content and AMF addition improved this stress phenomenon. There were significant differences in fungal community changes under different Cr(VI) concentrations. The fungal community characteristics changed through inhibition of fungal metabolic ability, as fungal abundance increased after AMF addition, and the fungal diversity increased under high Cr(VI) concentration. The dominant phyla were members of the Ascomycota, Basidiomycota, Mortierellomycota , and Rozellomycota . Dominant groups relevant to Cr resistance were Ascomycota and Basidiomycota fungi. Moreover, Fungal community characteristics were analyzed using high-throughput sequencing of the cytochrome c metabolic pathway, NADH dehydrogenase, and NADH: ubiquinone reductase and all these functions were enhanced after AMF addition. Therefore, Cr(VI) stress significantly affects fungal community structure, while AMF addition could increase its SOM content, and metabolic capacity, and improve fungal community tolerance to Cr stress. This study contributed to the understanding response of rhizosphere fungal community in AMF-assisted wetland phytoremediation under Cr stress.",
"conclusion": "Conclusion Fungal community abundance and diversity were suppressed under Cr stress, especially at high Cr(VI) concentrations (Cr(VI) = 100 mg/kg-200 mg/kg). The dominant species were Ascomycota , Basidiomycota , and Mortierellomycota . Fungi with high tolerance to Cr contamination were present in the Cysticercus phylum. After AMF addition, SOM content increased, conversion of Cr(VI) to Cr(III) increased, fungal abundance increased, and fungal diversity increased in the higher concentration groups. The fungal community has a corresponding resistance mechanism under Cr(VI) stress, as Cr(VI) can reduce the metabolic capacity of the fungal community. Cr(VI) reduces root-soil SOM, thus affecting fungal community change, however, AMF increases root-soil SOM content, which also improves the Cr(VI) resistance capacity of fungal community and its ability to enhance Cr(VI) reduction. Overall, there were significant differences in fungal community changes under different concentrations of Cr (VI), and fungal abundance increased after AMF addition. In contrast, fungal diversity increased at high Cr (VI) concentrations. AMF addition enhanced fungal community Cr(VI) reduction, but the specific fungus responsible and the principal reduction mechanism remain undetermined.",
"introduction": "Introduction Nowadays, soil Cr contamination is a global concern ( Li et al., 2019a ), as Cr is a heavy metal that is harmful to plants and humans ( Chen et al., 2018a ; Chen et al., 2018b ). It is often used in metallurgical industries, electroplating, dyeing agents and some alloy manufacturing ( Dhal et al., 2013 ; Viti & Giovannetti, 2007 ; Manikandan et al., 2016 ). Naturally Cr is often found as stable compounds Cr(VI) and Cr(III) ( Bartlett, 1991 ), with Cr(VI) mainly present in soil and water bodies as CrO 4 2− and Cr 2 O 7 2− ( Xia et al., 2019 ). Cr(VI) is more toxic and more soluble than Cr(III), reported to be more than 100 times more toxic than Cr(III), and can cause DNA damage and carcinogenesis ( Garcıa-Hernández, Villarreal-Chiu & Garza-González, 2017 ; Shi et al., 2018 ; Wang et al., 2017a ; Wang et al., 2017b ). Cr enrichment can have a severe impact on human health through the food chain. Therefore, it is crucial to remediate Cr(VI)-contaminated soils Compared to physicochemical remediation, phytoremediation is a heavy metal remediation technique that has recently been identified as cheap and with no secondary contamination ( Xue et al., 2018a ; Xue et al., 2018b ; Piyush, Singh & Anderson, 2020 ). Wetland plants, due to their well-developed root systems and vital growth capacity, play an indispensable role in the CWs (constructed wetlands) to treat wastewater containing heavy metals ( Jan, Lerika & Jaroslav, 2009 ). However, studies have shown that high levels of heavy metal pollution can be toxic to wetland plants ( Ammara & Saleh, 2020 ), and so to protect them, arbuscular mycorrhizal fungi (AMF) have been introduced (27 genera in one order, four families and 11 families in the phylum Glomeromycota , with about 300 species) ( Redecker et al., 2013 ). AMF can form symbiotic relationships with the root systems of most terrestrial, aquatic and semi-aquatic plants ( Brundrett & Tedersoo, 2018 ; Calheiros et al., 2019 ; Nataša & Gaberščik, 2010 ) which improves soil resources and responds effectively to environmental constraints ( Balestrini et al., 2018 ; Lenoir, Fontaine & Sahraoui, 2016 ; Torres, Antolín & Goicoechea, 2018 ). AMF increases plant soil nutrient uptake by colonizing and altering their root systems, while at the same time improving their tolerance to heavy metals by increasing antioxidant enzyme ( e.g. , catalase, peroxidase, and superoxide dismutase) activity and decreasing reactive oxygen species (ROS) ( Lin et al., 2017 ; Wang et al., 2018 ; Devi, Gupta & Kapoor, 2019 ). There have been numerous studies on bacterial stress to heavy metals ( Fan et al., 2017 ; Sultana et al., 2014 ), and Cr(VI) reduction by bacteria ( Wang et al., 2017a ; Wang et al., 2017b ; Xia et al., 2018 ; Viti et al., 2014 ; Xue et al., 2014 ), but few studies have addressed mechanisms of Cr(VI) reduction by fungi, especially heterotrophic fungi that are abundant in the soil/plant rhizosphere. Notably, most fungi have heavy metal chelating systems and heavy metal enrichment capabilities ( Janoušková, Pavlíková & Vosátka, 2006 ; Aly, Debbab & Proksch, 2011 ). Fungal substrates for heavy metal resistance can be divided into intra- and extracellular ( Hall, 2002 ). For example, some fungi can secrete organic acids and amino acids to chelate with heavy metals and thus reduce toxicity (intracellular; Vodnik et al., 2008 ), while others reduce heavy metal ions through electron transfer (extracellular; Xia et al., 2018 ). Cr(VI) removal by fungi studies are scarce, but some have shown that fungi also have antioxidant mechanisms to reduce Cr(VI) and thus reduce heavy metal toxic effects on themselves ( Viti et al., 2014 ; Xue et al., 2014 ; Joutey et al., 2015 ; Acevedo-Aguilar et al., 2006 ). Fungal community structure is significantly altered in response to Cr stress ( Del Val, Barea & Azcón-Aguilar, 1999 ; Nordgren, Baath & Soderstrom, 1983 ), however, few studies have investigated Cr(VI) fungal removal mechanisms and fungal community changes. In this study, the aim was to compare fungal community structure and functional predictions in an AMF group with a control group using high-throughput sequencing, to reveal fungal response mechanisms after AMF addition under Cr stress, and also provide a basis for further studies on the principle of fungal reduction of Cr(VI). This study will also contribute to our understanding of the response of AMF-assisted wetland phytoremediation to Cr stress in inter-rhizosphere fungal communities. We hypothesized that the fungal community of Acorus calamus root soil would be affected in Cr-stressed soil. However, adding AMF could increase the stress of the fungi community to Cr by increasing SOM content.",
"discussion": "Discussion Under heavy metal stress, microorganisms are more sensitive than other organisms ( Charlton et al., 2016 ; Giller, Witter & McGrath, 2009 ), and their abundance is altered in various ways. For example, Flavisolbacter and Altererythrobacter abundance is affected by Cr stress ( Zhang et al., 2020 ). Furthermore, fungal community composition was significantly altered in response to changes in heavy metal content ( Del Val, Barea & Azcón-Aguilar, 1999 ; Nordgren et al., 1983 ). In our study, fungal diversity and abundance were significantly altered by Cr(VI) stress, and overall they showed a decreasing trend with increasing Cr(VI) concentrations in both control and AMF groups. In long-term chronically Cr-contaminated soils, fungal abundance declines but fungal community diversity is not altered ( Jin et al., 2018 ). We showed significant changes in fungal community abundance and diversity at various concentrations of Cr(VI) stress. However, fungal community abundance showed an overall decreasing trend with increasing Cr(VI), especially at high concentrations. Fungal community species composition changed significantly under Cr stress, and dominant group distribution ratios also changed. The most dominant species under Cr(VI) stress were Ascomycota , followed by Basidiomycota fungi, and high Cr concentration increased Ascomycota fungi. In the correlation heat map, Ascomycota fungi were significantly correlated with Cr(VI) and Cr, so it may be that these fungi, along with some in the Basidiomycota , are sensitive, or resistant to chromium. Derxomyces ( Basidiomycota ) is very sensitive to Cr and shows a significant negative correlation with Cr and Arthrinium ( Jin et al., 2018 ). Moreover, some Ascomycota fungi can reduce Cr(VI) to Cr(III) using carbon metabolism capabilities, such as Aspergillus sp ., Penicillium sp. , and Trichoderma hamatum ( Acevedo-Aguilar et al., 2006 ). Lazarova et al. (2014) found that Trichosporon ( Ascomycota ) was able to grow under 10 mmol/LCr stress and was highly resistant to chromium because it can chelate and even reduce Cr(VI) from heavy metal ions ( Georgieva et al., 2011 ; Bajgai, Georgieva & Lazarova, 2012 ). Candida is also highly resistant to Cr and can survive in media containing 100 mmol/L Cr stress due to the presence of chromate reductase ( Ramírez-Ramírez et al., 2004 ). Aspergillus ( Ascomycota ) is also a common chromium-resistant fungus that reduces Cr(VI) to Cr(III) ( CzakóVér et al., 1999 ), and responds to Cr contamination mainly by enriching in vivo ( Mala, Nair & Puvanakrishnan, 2006 ). Coreno-Alonso et al. (2019) found that the Ed8 strain of Aspergillus tubingensis reduced Cr(VI) concentration through reduction reactions stimulated by carboxylic acids and metal chelators. Fungal reduction of Cr(VI) reduction can be in vivo as well as in vitro( Fig. 9 ). The conversion of hexavalent chromium to trivalent chromium is through an electron reduction reaction. Microorganisms generally convert hexavalent chromium to trivalent chromium through enzyme and non-enzyme reductions that perform an electron transfer role, transferring 3 electrons to hexavalent chromium. Chromate first enters the fungus through the sulfate pathway due to its similar chemical structure to sulfate, and then a Cr(VI) portion is reduced to Cr(III) through enzymatic and non-enzymatic reduction (non-enzymatic reducing substances such as GSH and cysteine) ( Thatoi et al., 2014 ). Soluble reductases are dominated by ChrR, Yief, and NfoR, and their enzymatic reductions occur under aerobic conditions ( Eswaramoorthy et al., 2012 ; Ackerley et al., 2004 ; He et al., 2011 ; Han et al., 2017 ). The CHR-1 protein, homologous to chrA, is found in a number of Cysticercus , Streptomycetes , and Seamycetes fungi. CHR-1 not only reduces Cr(VI) and immobilizes Cr in fungal vesicles, but chrA is found in many microorganisms as an efflux protein and is an additional measure of resistance to Cr(VI) ( Viti et al., 2014 ). 10.7717/peerj.15681/fig-9 Figure 9 The proposed mechanism of Cr(VI) resistance with the addition of AMF increases the root-soil SOC content and thus fungal activity. (I) Chromate enters through the sulfate channel; (II) Cr(VI) is reduced to Cr(III) through enzymatic and non-enzymatic reduction and subsequently immobilized in the vesicle via CHR-1; (III) Cr(VI) is reduced in vitro by the fungus through electron transfer; (IV) Chromate is exocytosed through chr-A; (V) The mucus adsorption on the fungal surface, as well as increased protonation levels at low pH, attract chromate ions, which are reduced and then released through electron repulsion release. In addition to the mucus produced by fungi in vitro to adsorb heavy metal ions, fungal Cr(VI) reduction in vitro is mainly associated with electron transfer. Low pH is also beneficial for Cr(VI) reduction ( Martorell et al., 2012 ). In our study, the soil was weakly acidic, which increases the fungus surface protonation level, making the positively charged surface better able to attract negatively charged chromate ions, which are thereafter reduced to Cr(III) due to electron repulsion release ( Park et al., 2005 ). Xia et al. (2018) ) found that Cr(VI) ground electron reduction was achieved by transferring them from NADH to ubiquinone in the presence of dehydrogenase CymA, MtrA, MtrB, MtrC, and OmcA, which are all cytochrome c. We found that Cr(VI) addition added to the fungal cytochrome c metabolic pathway, NADH dehydrogenase, and NADH: ubiquinone reductase, demonstrating that the fungus has in vitro reductive effects under Cr(VI) stress. Fungi are heterotrophic organisms that use soil organic matter as their primary carbon source ( Lehmann & Kleber, 2015 ; Xue et al., 2018 ), however, their ability to utilize carbon sources is limited at high heavy metal concentrations ( Georgieva et al., 2011 ). However, energy metabolism plays a vital role in the fungal response against heavy metals ( Jin et al., 2008 ). Gasch & Wemer-Washburne (2002) found that many genes involved in carbohydrate and fatty acid metabolism in Common Metal Responsive (CMR) were upregulated in response to heavy metal stress. Moreover, Acevedo-Aguilar et al. (2006) showed that Cr(VI) reduction to Cr(III) by fungi requires a carbon source that is fermented like glucose or oxidized like glycerol, and that total Cr(VI) is unaltered in the absence of a carbon source. AMF can have a beneficial effect on lowering plant exposure to heavy metals by improving water and soil nutrient uptake, increasing aboveground biomass and causing changes in root morphology, reducing oxidative stress from heavy metals ( Muhammad et al., 2020 ). Furthermore, increasing soil SOM with AMF forms fibrous roots with plants, a change that may somewhat improve Cr tolerance of fungal community. The main reason for the increase in soil SOM is that AMF can secrete glycoproteins, such as the globulin-related soil protein (GRSP), which plays a significant role in rhizosphere soil aggregation, carbon storage, and soil quality improvement ( Nichols, 2003 ; Rillig et al., 2001 ; Schüßler, Schwarzot & Walker, 2001 ) and can protect AMF mycelia from nutrient loss ( Wessels, 1996 ). AMF and GRSP play crucial roles in direct and indirect soil carbon sequestration. Directly, AMF increases soil carbon content through increased plant growth and aboveground biomass, in the context of root and root deposition input. Indirectly, GRSP improves carbon sequestration ( Miller, Reinhardt & Jastrow, 1995 ) through soil aggregation, forming stable agglomerates that protect organic compounds as well as microbial necromass from enzymatic and microbial attack ( Awad et al., 2013 ). Furthermore, GRSP can increase the active carbon pool by increasing microbial activity ( Subramanian et al., 2019 ; Wright & Upadhyaya, 1998 ). In our study, AMF addition increased SOM content ( Fig. 1C ), improved the root system soil microenvironment, improved the limitation of fungal access to carbon sources, increased the fungi metabolic level in the AMF group ( Fig. 8D ), increased fungal ability to access carbon sources, and increased their own ATP synthesis, which was also beneficial for Cr(VI) reduction."
} | 3,942 |
30310835 | PMC6176844 | pmc | 2,465 | {
"abstract": "The data included in this article provides additional supplementary information on our recent publication describing “Inducing tunable switching behavior in a single memristor” [1] . Analyses of micro/nano-structural and compositional changes induced in a resistive oxide memory during resistive switching are carried out. Chromium doped strontium titanate based resistance change memories are fabricated in a capacitor-like metal-insulator-metal structure and subjected to different biasing conditions to set memory states. Transmission electron microscope based cross-sectional analyses of the memory devices in different memory states are collected and presented."
} | 166 |
36307483 | PMC9616899 | pmc | 2,466 | {
"abstract": "Neuromorphic computing, an alternative for von Neumann architecture, requires synapse devices where the data can be stored and computed in the same place. The three-terminal synapse device is attractive for neuromorphic computing due to its high stability and controllability. However, high nonlinearity on weight update, low dynamic range, and incompatibility with conventional CMOS systems have been reported as obstacles for large-scale crossbar arrays. Here, we propose the CMOS compatible gate injection-based field-effect transistor employing thermionic emission to enhance the linear conductance update. The dependence of the linearity on the conduction mechanism is examined by inserting an interfacial layer in the gate stack. To demonstrate the conduction mechanism, the gate current measurement is conducted under varying temperatures. The device based on thermionic emission achieves superior synaptic characteristics, leading to high performance on the artificial neural network simulation as 93.17% on the MNIST dataset.",
"introduction": "Introduction In the advent of the big data era, the dramatic advance of machine learning technology and artificial intelligence have occurred, demanding the computing ability to handle the data-intensive task 1 . However, the currently exploited conventional von Neumann architecture has become the bottleneck due to its limitation to parallel computing ability and high power consumption to deal with the big data, caused by obligated data transfer through the data bus between the physically separated processing unit and memory 2 – 4 . Therefore, to perform successful big data analysis, new computing architectures have been developed. The main key idea of the new architectures is to compute the data in memory without data transfer (or small movement of data), enabling reducing power consumption and suppressing latency by parallel data processing ability 4 – 7 . Neuromorphic computing is one of the candidates for post-von Neumann architecture. By mimicking the synaptic behavior of the biological neural network, the big data can be processed by parallel computing in an energy-efficient way in real-time 5 , 8 , 9 . For accelerating the artificial neural network (ANN) with this new architecture, the neuromorphic device, which can memorize and compute the data on the same device, is required. Recently, several studies utilizing conventional memory, such as DRAM and charge trap flash memory (CTF), and emerging memory devices such as PRAM and ReRAM have been reported in neuromorphic applications 10 – 17 . In the case of conventional memories, the well-established DRAM secures fast write speed and linear conductance update 10 . Capacitor-based synaptic devices with a DRAM-like structure also have main advantages in online training for repeated updates because of their high endurance 18 . However, because of poor retention characteristics, the weight values must be transferred to nonvolatile memories very frequently during the training process, resulting in high power consumption. Moreover, these devices are difficult to create and retain analog conductance states with a single device and require a capacitor (storing charges for weight values) and several additional transistors to implement analog states 10 , 19 , 20 . This means that it has a drawback in terms of device integration density compared to a single synaptic device. On the other hand, nonvolatile memories such as CTF can distinguish between states of multi-level cells depending on how many charges are trapped in the charge trap layer, and have long retention 12 . Additionally, several studies show that the endurance characteristics of CTF can be significantly improved by structural and material engineering of the device 21 – 23 . Therefore, research using CTF devices is being actively conducted for applications in neuromorphic computing, as well as for the main memory for data storage. However, while data can be stored for long periods without data loss, CTF normally has a large operation voltage and slow speed, requiring more energy, especially for data erasing 24 – 26 . In the case of online training, using devices with low update energy is advantageous because the training demands repeated writing and erasing operations more than millions of times. The two-terminal emerging memories have been extensively studied as a promising candidate among neuromorphic devices due to their simple structure and scalability. Furthermore, they can be integrated into large-scale crossbar-array for vector-matrix multiplication, which is essential for the basic operation of neuromorphic computing 8 , 27 – 29 . However, their device variation caused by randomly formed filament during set/reset. This stochastic behavior of ion movement causes unreliable variation and it has been the significant bottleneck for the successful application as a computing device 4 , 28 . On the other hand, the three-terminal synaptic device has the advantage of enhancing synaptic weight controllability, and allows simultaneous reading and writing the data 8 , 9 , 30 – 32 . Besides suppressing the problems mentioned above, synapse device characteristics such as high linear conductance update and compatibility with the conventional Complementary-Metal-Oxide-Semiconductor (CMOS) system are commonly required to acquire the high performance of crossbar-array based neuromorphic computing as that of software-based ANN 32 – 36 . Especially, the linearity of the Long-Term Potentiation and Long-Term Depression (LTP-LTD) is regarded as one of the most important characteristics for synapse device evaluation 33 . By achieving the linear conductance update with identical consecutive pulse scheme, it is believed to enable the multi-level operation while reducing the burden on peripheral circuits to operate crossbar array 12 , 37 . The conventional three-terminal floating gate-based flash memory shows high nonlinearity in weight updates 12 , 30 , 38 , 39 due to the Fowler-Nordheim (F-N) tunnelling, a vital function of the electric field changed by electrons stored charge state 38 , 40 . Ion-conducting electrolyte-based three-terminal synapse devices show high linear conductance update for weight state 41 – 44 . However, they are vulnerable in the perspective of low on/off ratio, high programming pulse width, and incompatibility with conventional CMOS devices. In this paper, we propose a three-terminal Gate Injection-based Field-Effect Transistor (GIFET), which utilizes the CMOS compatible material and fabrication process. Through different operation mechanisms from conventional flash memory, we derive superior synapse device characteristics, such as high linearity and symmetry, high temporal and spatial uniformity (<1.64%, 9.76%), and low power consumption (50 fJ/SOP). These performances lead to a high accuracy of approximately 93.17% with the MNIST handwritten recognition dataset.",
"discussion": "Discussion In summary, we developed a three-terminal synapse device for enhancing linearity on LTP-LTD based on field-effect to control channel conductance by stored charge in CSL, which is injected from or extracted to gate metal based on thermionic emission. The effect of the conduction mechanism between CSL and gate metal through the a-Si:H blocking layer on linear conductance update was investigated by comparing the device with and without interfacial layer and observing the gate current through the blocking layer of each device under varying temperatures. The thermionic emission-based GIFET reported linear conductance update while the device with interfacial layer presented nonlinearity. Furthermore, GIFET shows superior properties such as number of conductance states, area, power consumption, on/off ratio, operating voltage, programming time, spatio-temporal variation, linearity, retention, endurance, simulation accuracy and CMOS compatibility (see Table 1 ), since the mechanism based on electron movement employs the flash memory structure. In addition, low spatio-temporal variation, reliable endurance and retention, and low power consumption of the GIFET support that the device is qualified for the large-scale crossbar array to conduct neuromorphic computing. Moreover, all the processes and materials utilized for the GIFET fabrication were CMOS compatible, which suggested low-cost and fast integration with the conventional system. Artificial neural network simulation based on MNIST dataset with the parameters extracted from GIFET measurement data shows high accuracy of 93.17%, which implies the possibility of AI acceleration with GIFET-based large-scale crossbar array. Table 1 Comparison with the various synaptic transistors for neuromorphic computing 57 44 35 32 58 41 59 This work The Number of Conductance States (LTP/LTD) 100/100 60/60 64/64 2 13 /2 13 100/50 50/50 100/100 1000/1000 Area [μm 2 ] 0.52 3 15,000 350 N/A N/A 2000 100 Power Consumption [fJ] <400 30 \\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}$$\\approx$$\\end{document} ≈ 60,000 <20 N/A \\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}$$\\approx$$\\end{document} ≈ 160 N/A <50 On/off Ratio \\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}$$\\approx$$\\end{document} ≈ 3 \\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}$$\\approx$$\\end{document} ≈ 2 >10 \\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}$$\\approx$$\\end{document} ≈ 10 3 >10 \\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}$$\\approx$$\\end{document} ≈ 2 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\approx$$\\end{document} ≈ 9 >10 Program Voltage [V] 2 1.2 2.7~4.3 4 3.5 2.5 3 1.8 Program Time [ms] 100 100 10 10 10 10 100 0.3 Temporal Variation N/A N/A 2.36% N/A N/A <6.5% N/A 1.64% Spatial Variation N/A N/A 3.93% N/A N/A <12% N/A 9.76% Linearity (ideal = 1) (LTP/LTD) 1.5/5.9 1.9/0.5 ISPP 1.51/−0.38 1.3/−0.3 N/A 0.96/−0.11 1.53/0.47 Retention N/A N/A 10 4 s 5000 s N/A 100 s N/A >1000 s Endurance (HRS/LRS switching cycles) N/A N/A >10 5 >400 N/A >40 N/A >2×10 5 Simulation Accuracy 84.6% N/A 91.1% 91.7% 86.82% 87.3% 93.26% 93.17% CMOS Compatibility No No Yes Yes Yes No No Yes ISPP (Incremental Step Pulse Programming) is not identical pulse."
} | 2,917 |
34199583 | PMC8199690 | pmc | 2,467 | {
"abstract": "Photosynthetic microorganisms are among the fundamental living organisms exploited for millennia in many industrial applications, including the food chain, thanks to their adaptable behavior and intrinsic proprieties. The great multipotency of these photoautotroph microorganisms has been described through their attitude to become biofarm for the production of value-added compounds to develop functional foods and personalized drugs. Furthermore, such biological systems demonstrated their potential for green energy production (e.g., biofuel and green nanomaterials). In particular, the exploitation of photoautotrophs represents a concrete biorefinery system toward sustainability, currently a highly sought-after concept at the industrial level and for the environmental protection. However, technical and economic issues have been highlighted in the literature, and in particular, challenges and limitations have been identified. In this context, a new perspective has been recently considered to offer solutions and advances for the biomanufacturing of photosynthetic materials: the co-culture of photoautotrophs and bacteria. The rational of this review is to describe the recently released information regarding this microbial consortium, analyzing the critical issues, the strengths and the next challenges to be faced for the intentions attainment.",
"conclusion": "4. Conclusions and Future Perspective The exploitation of photoautotrophic organisms and bacteria co-cultures showed many advantages when compared to monocultures. In fact, such co-cultures are effective farms for the production of different biomaterials with strong interest in human health, as well as source of alternative and sustainable energies. This successful combination, inspired by nature, is based on several fruitful interactions between the cells of photoautotrophs and bacteria, based on exchanges of vitamins, oxygen and carbon dioxide. In addition, algae/bacteria consortia ensure the minimization of contamination, a serious issue in axenic cultures, and it can lead to greater resistance and stability in the cell population. These are important qualities during the scale-up of the cultures [ 120 ], and, from an industrial point of view [ 121 , 122 , 123 ], these are synonyms of higher yields and economic savings, although much can be done from the engineering side (e.g., design of bioreactors) and in the management of biomasses (e.g., cells harvesting). Moreover, insights on transcriptomics, metagenomics and metabolomics are currently strongly required to reach a better understanding of microbial interactions, addressing the use of available substrates and increasing the productivity. Furthermore, a genome-editing approach could be the driving force to promote in the next future a sustainable production of molecules not conventionally synthesized by these microorganisms. The analysis of the current scenario on microbial consortia highlights another important issue to face, which is the development and consolidation of computational and mathematical assistances for co-cultures realization. In fact, this aspect, which strongly affects the total costs and the time required to obtain large volume systems, is especially essential for the achievement of the real market. Another desirable tool for achieving large-volume co-cultures, as suggested by Padmaperuma and colleagues, could be the realization of an open-access database collecting relevant metadata about tested consortia and their outcomes (both positive and negative), description of the selected strains, growth dynamics, biomolecules released and data related to the conditions in bioreactors if tested [ 24 ]. Undeniably, this would be a great support for the academic research and for the technology transfer from bench-scale to industrial applications.",
"introduction": "1. Introduction The benefits related to the photosynthetic microorganism’s cultivation (e.g., microalgae, cyanobacteria and photosynthetic bacteria) are well known owing to their ability to convert light, water and carbon dioxide into products with valuable contents in a sustainable way. The ability of these photoautotrophs to manufacture high-value compounds (e.g., vitamins, pigments and polyunsaturated fatty acids), alternative energy sources and to perform natural processes for environment safeguard (e.g., biofuel production, CO 2 mitigation and waste water treatment) founded a significant market demand from industries of food, feed, oil business, cosmetics and pharmaceutics [ 1 , 2 , 3 , 4 , 5 ]. In particular, the positive feedback related to the photoautotrophs’ use can be associated to their positive impact on the Industry 4.0 achievement. This strategic term was introduced in 2011 by the German government to indicate a technological perspective able to support sustainability challenges [ 6 ]. The expectations from the companies that embrace this new philosophy are many, including economic (as a result of a better energy use), environmental (for the reduction of scrap waste manufacturing and use of sustainable materials) and social ones (by achieving a safer and more comfortable workplace) [ 7 ]. In this context, microalgae and cyanobacteria exploitation received an increased interest for their attractive potential in the current scenario of energy savings and alternative food supplies. Many advantages derive from the use of these unicellular organisms, being capable of rapidly growing in a wide range of habitats as flexible systems. In addition, their strains can be easily edited by genetic approaches, and their cells have a shorter life cycle (1–4 days for microalgae and only 3 h for cyanobacteria) than the plants (90–180 days) [ 8 , 9 , 10 ]. This aspect is not to be underestimated, being that feedstocks with shorter life cycles are more sustainable in comparison with longer life ones [ 11 ]. However, the photoautotroph cultivations as long as they are in pilot systems and under controlled growth conditions have not shown particular worries; on the contrary, when industrial applications at large scales are reached, several concerns are brought up. In particular, two aspects heavily affect these scaled-up systems, i.e., the design of photobioreactors and the apparatus dedicated to the biomass harvesting. To this aim, evaluation in silico by mathematical simulations and dynamic modelling can be extremely useful for optimizing important parameters and for reducing the cost of expensive experiments. Process simulation is well established in chemical engineering, and its application in bio-engineering is increasing [ 12 ]. In this context, Apel and colleagues proposed a mathematical programming based on algorithms consisting of two loops: validation loop and virtual design and evaluation loop. The first one certifies that the models accurately describe reality. This step is strongly required to identify the parameters for the biology model, and it is repeated until the required accuracy is accomplished [ 12 ]. Moreover, another important issue related to the large-scale production is the culture contamination by other microorganisms (e.g., virus, bacteria, grazers and fungi) that can greatly reduce the biomass yield compromising the quality. In this regard, besides the contamination management strategies available into open ponds (e.g., use of adequate filters, selection of engineered strains, sterility conditions and antibiotics use), a new point of view is emerging on the concept contaminant organism, which is a new additional element capable of providing added value to the cultivation. In particular, the natural consortia have been a source of inspiration and offered the possibility to observe a new and complex reality able to combine productivity and high resistance to contaminations, because the ecological niche is already engaged. However, the final products of these natural consortia can be restricted due to the environment-elevated complexity. Recent efforts are focused on the design of stable, less complex and better controllable synthetic co-cultures to take advantage of microbial communities (e.g., distribution of the metabolic burden and ability to convert complex substrates) [ 13 ]. This synthetic ecological strategy is helpful for large-scale cultures, without sterile environments, and seems to be a promising approach to low-cost sustainable production of valuable compounds. Intriguing studies pointed out many strategies, such as engineering of chemical symbiosis, of quorum sensing and of ecological niches useful for this topic [ 14 , 15 , 16 , 17 ]. These parameters can be modulated purposefully to obtain a stable co-culture, in stress-free conditions and with the possibility to live together, making the most of the substrates partitioning as available resources [ 13 ]. In general, co-culture members can be identified according to the communication profile, which can be based on the analyses of metabolite content and protein assay, and/or taking inspiration from natural consortia. However, great challenges remain in the carrying out of multispecies cultures for industrial applications [ 18 , 19 ]. Several types of microalgae-based consortia have been reported in the literature, including microalgae–bacteria, algae–fungi, microalgae–protists and the multibacteria and multialgae symbiosis systems [ 20 , 21 , 22 ]. Microalgae–bacteria co-cultivation systems are extremely promising biotechnological tools, as many recent studies have revealed a positive effect of microalgae–bacteria symbiosis on microalgal growth [ 23 , 24 , 25 ]. The advantageous association of microalgae and bacteria has been attributed to various factors, e.g., (i) bacteria stimulate microalgal growth by producing growth-promoting substances as well as vitamins (cobalamin, thiamine and biotin) and cofactors and by reducing dissolved O 2 concentration [ 26 ]; (ii) microalgae produce O 2 through photosynthesis which can be utilized by bacteria, which in turn produce CO 2 photosynthetically fixed by microalgae [ 23 , 27 , 28 , 29 ]; and (iii) microalgae secrete complex compounds which serve as a source of carbon and nitrogen for bacteria. As an example, in co-culturing Chlorella vulgaris and Pseudomonas, the symbiotic bacterium showed a growth-promoting effect on the green microalga [ 30 ]. The topic of synthetic microbial consortia is still in an early infancy, and several challenges related to the intercellular communication and the design of stable/manageable systems must be still faced. In this review, the state of the art concerning the peculiar bio-materials obtainable through the photoautotrophs–bacteria co-cultures is presented, highlighting the challenges and future directions to support the development of synthetic microbial consortia."
} | 2,693 |
33983746 | PMC8280720 | pmc | 2,468 | {
"abstract": "Surfactants are often\nadded to water to increase the wetting of\nhydrophobic surfaces. We previously showed that most surfactant solutions\nbehave identically to simple liquids with the same surface tension,\nindicating that the surfactants do not change the wettability of the\nsolid surface itself. Here, we show that the superspreading surfactant\nSilwet results in a systematically higher contact angle on a hydrophobic\nsurface than other surfactant solutions of comparable liquid–vapor\nsurface tension. We also experimentally observe this “antisurfactant”\nbehavior for CTAB on hydrophilic substrates. Supported by sum-frequency\ngeneration spectroscopy results, we suggest that this effect is due\nto charge-binding of the surfactant with the substrate.",
"conclusion": "Conclusions We have experimentally demonstrated the antisurfactant\nbehavior\nof a superspreader surfactant on a variety of hydrophobic surfaces.\nThe antisurfactant behavior of the surfactant Silwet has been explained\nby considering the adsorption of the surfactant molecules on the solid–liquid\nas well as the solid–air interface. This is an important conclusion,\nsince there are many applications in which trisiloxane surfactants\nare used to improve the wetting of aqueous solutions on hydrophobic\nsurfaces, such as the deposition of aqueous pesticide solutions on\nplant leaves. A similar antisurfactant behavior is observed for the\ncharged surfactant CTAB in contact with the oppositely charged silica.\nThe adsorption of CTAB to the surface modifies the solid–air\ninterfacial free energy, thereby affecting the wetting properties\nof the solution. Our results here show that the spectacular lowering\nof the liquid–vapor surface tension by these types of surfactants\ndoes not necessarily guarantee a smaller contact angle (and hence\nbetter coverage) of the surfaces: the change in solid wetting properties\ninduced by the surfactant also has to be taken into account.",
"introduction": "Introduction Surfactants can improve the wetting of\nan aqueous solution on a\nsolid substrate. 1 As a result, they are\nwidely used in various household and industrial applications, 2 such as in detergents for cleaning clothes or\ndishes, 3 in food products, 4 and as adjuvants to help improve pesticide efficiency in\ncrops. 5 Because of their widespread use, 6 − 9 surfactant solutions, and their wetting properties have been studied\nfor almost two centuries. Nonetheless, there are still many surfactant-induced\nphenomena that remain unexplored. We have previously reported the\nwetting behavior of the surfactant solutions on hydrophobic substrates\nand showed that there is almost no difference between the behavior\nof a surfactant solution and that of a pure liquid of the same surface\ntension. 10 In contrast, in this work, we\nreport on the “antisurfactant” behavior where a surfactant\ninduces poorer wetting on hydrophobic as well as hydrophilic surfaces\nthan simple liquids of similar surface tensions. Autophobic\nand autophilic effects are two examples of intriguing\nphenomena usually attributed to the adsorption of surfactant molecules\non solid surfaces. 11 − 15 On a hydrophilic substrate, surfactants sometimes lead to an increase\nin the equilibrium contact angle. This phenomenon is known as the\n“autophobing effect”. 16 , 17 On hydrophobic\nsurfaces, however, the adsorption of other types of surfactants in\nthe thin film coexisting with the droplet leads to a decrease in the\ncontact angle. This is the so-called “autophilic effect”. 18 − 21 In many industrial applications, good wetting of the aqueous solution\non a hydrophobic substrate is crucial and hence the autophilic effect\nis used extensively to achieve (near) complete wetting. It has, however,\nalso been noticed in many cases that the autophilic effects do not\noccur on hydrophobic substrates. 22 Superspreading\nsurfactants, generically known as trisiloxanes, are widely used to\nincrease wetting on hydrophobic surfaces. They are for instance employed\nin agriculture where aqueous pesticide solutions are sprayed onto\nhydrophobic plant leaves. The specificity of the whole class of trisiloxane\nsurfactants is that they lower the surface tension of water significantly\nmore than almost all other types of surface-active molecules. 23 If the solid surface tensions (with liquid and\nvapor) are unaffected by the surfactant, this implies indeed a better\nwetting when trisiloxanes are added. In the present work, we show\nthat on different types of hydrophobic surfaces, the superspreading\nsurfactant in fact shows autophobic behavior, showing a smaller wetting\nefficiency compared to other liquids or surfactant solutions of the\nsame liquid–vapor surface tension."
} | 1,163 |
36983550 | PMC10053633 | pmc | 2,469 | {
"abstract": "Mycoremediation is one of the most attractive, eco-friendly, and sustainable methods to mitigate the toxic effects of heavy metals. This study aimed to determine the mycoremediation capacity of metallophilic fungi isolated from heavy-metal-contaminated soil containing a high Fe(III) concentration (118.40 mg/kg). Four common fungal strains were isolated, including Curvularia lunata , Fusarium equiseti , Penicillium pinophilum , and Trichoderma harzianum . These fungal strains were exposed to gradually increasing concentrations of Fe(III) of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mg/L. Sophisticated techniques and tests were employed to investigate the mycoremediation capability, including tolerance index (TI), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and adsorption isotherm. Furthermore, the impacts of initial concentration, pH, and temperature on the Fe(III) removal (%) and uptake capacity (mg/g) of the studied samples were investigated. The results were validated by statistical analysis using one-way ANOVA. It was found that the Fe(III) uptake with different ratios triggered alterations in the Fe(III) tolerance (TI) morphological (SEM), chemical (FTIR), and adsorption capacity properties. The highest Fe(III) tolerance for all studied fungal strains was achieved at 100 mg/L. Moreover, the optimum conditions of Fe(III) removal (%) for all studied fungal strains were within pH 7 and 28 °C, with similar performance at the initial Fe(III) concentration ranging from 50–200 mg/L. At the same time, the maximum Fe(III) uptake was achieved at pH 7, 20 °C, and 200 mg/L. Compared to other strains, the Fe(III) tolerance of T. harzianum was rise in the Fe(III) concentration. The Fe(III) uptake reaction was corroborated by best fitting with the Langmuir model, achieving optimum adsorption capacities of 61.34, 62.90, 63.30, and 72.46 mg/g for C.lunata , F. equiseti , P. pinophilum , T. harzianum , respectively. It can be deduced that the addressed fungi species can be applied in mycoremediation according to the utilized Fe(III) concentrations with more superiority for live T. harzianum .",
"conclusion": "4. Conclusions Based on the results mentioned above, the following deductions can be addressed as follows: (1) Four fungal species, including C.lunata , F. equiseti , P. pinophilum , and T. harzianum , were successfully isolated from contaminated soil. (2) The greatest Fe(III) tolerance was achieved for all fungal strains at 100 mg/L. Unlike P. pinophilum , the differences in Fe(III) tolerance of T. harzianum were limited at all Fe(III) concentrations, even at the highest values. (3) The morphological characterization by SEM found that there were efficient potential sites of biosorption on the mycelia surfaces responsible for Fe(III) bioaccumulation. Additionally, the Fe(III) bioaccumulation initiated changes in the morphological features of fungi, which can be observed in mycelial looping and twisting and small irregular folds. (4) Similarly, the Fe(III) removal (%) had the best performance of all experienced initial Fe(III) concentrations, including 50, 100, 150, and 200 mg/L for all studied samples. Meanwhile, the Fe(III) uptake (mg/g) gradually increased with the rising in the initial Fe(III) concentration from 50–200 mg/L to the highest at 200 mg/L. (5) For all addressed fungal strains, the pH values of 3 and 7 were found to be the optimum conditions with more superiority for the first value for Fe(III) removal (%) and uptake (mg/g). Regarding the temperature, the Fe(III) removal (%) and uptake (mg/g) had the best behavior at 28 and 20 °C, respectively. More specifically, at 28 °C, F. equiseti has the highest Fe(III) removal (89.5%), while the Fe(III) uptake (mg/g) decreases gradually from 20 to 37 °C, respectively, and was the highest at 20 °C. More specifically, T. harzianum has a maximum Fe(III) uptake of approximately 51.5 mg/g at 20 °C. (6) Unlike C. lunata , the highest values of Langmuir constants, qmax (72.46 mg/g) and K L (0.0260 L/mg), and Freundlich constants, K F (5.60 mg/g and n (2.20 L/mg), m were reported for T. harzianum , indicating the highest adsorption capacity and intensity, respectively.",
"introduction": "1. Introduction Environmental pollution is one of the most critical problems of the twenty-first century [ 1 ]. Soil contamination is one of the different forms of environmental pollution. Soil pollution is caused by the aggregation of high concentrations of toxic compounds, chemicals, salts, radioactive materials, or disease-causing agents that endanger the health of plants and animals [ 2 ]. Various chemicals or heavy metals can contaminate soil through agricultural and industrial activities. Due to soil contamination, microorganisms, aquatic life, humans, and animals are at risk. Immense industries are the most common sources of heavy metal contaminations of the soil, including cadmium, chromium, copper, iron, lead, nickel, and zinc [ 3 ]. These heavy metals can trigger serious health problems, such as skin irritation, organ damage, nervous system disorders, gastrointestinal issues, and various types of cancer [ 4 ]. Iron (Fe(III)), is one of the essential nutrients in plant nutrient cycling and primary mineral weathering [ 5 ]; however, it can be toxic in large quantities or under specific formulas. Fe(III) is one of the most common and poisonous heavy metals; it pollutes the soil and causes hazards to biodiversity, agricultural productivity, food safety, and human health when transported through the food chain [ 6 ]. However, two processes can describe the removal of such heavy metals from the soil by the live organisms: bioaccumulation and biosorption. Bioaccumulation is an active metabolic uptake process driven by energy from the living organism (e.g., fungi), in which the heavy metals accumulate inside the cell wall (i.e., intracellular binding) [ 7 , 8 ]. On the other hand, biosorption occurs when heavy metals are adsorbed on the cell wall (i.e., extracellular binding). This process can be defined as a fast and passive metabolic process independent of energy in which biological materials (e.g., fungal biomass) act as sorbents that can remove pollutants, such as heavy metals, from wastewater through metabolically mediated or physico–chemical pathways of uptake [ 8 , 9 ]. This process includes mechanisms such as redox, precipitation, electrostatic interaction, physisorption, and ion exchange, as shown in Figure 1 . Mycoremediation is a method of bioremediation in which fungi-based remediation methods are applied to neutralize or remove heavy metals and textile dyes from the environment [ 10 ]. Additionally, it is a type of bioleaching remediation that is simple in operation, costless, and biodegradable. Mycoremediation is a novel and promising remediation technology that can be applied to heavy-metal-polluted soils. Therefore, scientists are currently investigating mycoremediation methods that use microbial and associated biota (i.e., fungi) within the ecosystem to biodegrade, collect, and eliminate the contaminants [ 11 ]. Hence, the soil (as a part of the ecosystem) is privileged because it contains all main groups of microorganisms, including bacteria and fungi [ 12 ]. The microbiota of soil (i.e., bacteria, fungi, and algae) plays an essential role in the degradation and synthesis of organic compounds. Metallophilic fungi are particular fungi that can be isolated from these heavy-metal-polluted soils [ 13 ]. These fungi are characterized by their ability to adapt to heavy metals and environmental conditions. The metallophilic fungal tolerance to Fe(III) has been attributed to numerous methods, including metal capture by cell wall components, precipitation by extracellular metabolites, and intracellular complexing by metallothioneins and phytochelatins [ 14 ]. Consequently, recent studies have investigated how to isolate species of those fungal strains, which remove heavy metals from different environmental resources such as soil. To the best of our knowledge, bioremediation by these metallophilic fungi has not previously been appointed in iron remediation. Consequently, the current study aims to identify the diversity of these metallophilic fungi present in Fe(III)-contaminated soils and determine their tolerance index at different Fe(III) concentrations. This was conducted through the isolation and characterization of different metallophilic fungal types by employing macroscopic characterization, metal tolerance index (TI), scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy (FTIR). Meanwhile, Fe(III) adsorption experiments accompanied by their isotherm studies were carried out to evaluate the adsorption capacity of these fungal species.",
"discussion": "3. Results and Discussion 3.1. Soil Characterization The mean values of soil analyses are determined in association with standard deviations (SD) and listed in Table 2 . The soil pH range was 7.38, indicating that the soil is almost neutral with a slight tendency toward alkalinity. As for the electrical conductivity (EC), the soil sample has a high EC value (25.30 mS/m). This can be attributed to the high concentration of salts in the soil and heavy metal accumulation [ 27 ]. The organic matter content of the contaminated soil sample is 0.33%, with a sandy loam texture, and the moisture content is 2%. This result can be attributed to the low concentration of clay in the soil sample. Available nutrient concentrations in soil are represented by nitrogen (N), phosphorus (P), and potassium (K) with mean values of 16, 8, and 10.35 mg/kg, respectively, with SD values ranging from 0.05–0.40. Fe(III) was detected in a high concentration (118.40 mg/kg), above the ISI permissible limits for industrial effluents. This can result from long-term irrigation with industrial effluents [ 28 ]. This high concentration of Fe(III) can be a problematic issue in the efficiency of the nutrient cycle due to the reduction in the waste breakdown and nitrogen fixation [ 29 ]. 3.2. Isolation and Characterization of Metallophilic Fungi 3.2.1. Macroscopic Characterization From the contaminated soil sample, varieties of metallophilic fungi, such as Curvularia lunata, Fusarium equiseti, Penicillium pinophilum, and Trichoderma harzianum , were isolated. These species have a high resistance to heavy metals [ 30 ]. Table 3 illustrates the macroscopic characteristics of these isolated fungi. 3.2.2. Tolerance Index (TI) The TI values of all isolated fungi are < 1 with different ratios ranging from 0.35 to 0.98, which depend on the difference in the tolerance behavior of each fungus, which can be observed from the presence of filamentous fungi in contaminated sites ( Figure 2 ). These differences can be attributed to the different tolerance mechanisms of these microorganisms to metal contaminants [ 31 ]. Figure 2 shows the highest Fe(III) tolerance values in all experienced Fe(III) concentrations (100–1000 mg/L). Unlike P. pinophilum , T. harzianum species can resist and detoxify Fe(III) pollutants even with higher concentrations (i.e., 800–1000 mg/L). More specifically, the change in the Fe(III) tolerance of T. harzianum is limited at all Fe(III) concentrations, even at the highest values. 3.2.3. Colony Morphology The results of the radial growth diameters and morphological characteristics are illustrated in Figure 3 . The C. lunata colony is wrinkled in the case of the Fe(III)-supplemented media ( Figure 3 a). The pink color mycelia of F. equiseti changed to white, and the growth of the fungus was reduced in the Fe(III)-supplemented media ( Figure 3 b). A possible explanation for these changes in the colony color and growth rate can be due to the detoxification mechanisms of heavy metals [ 32 ]. P. pinophilum is accompanied by a white color around the colony in the media supplemented with Fe(III) ( Figure 3 c). This can be due to an adaptation period during which Penicillium cells produce enzymes required for Fe(III) uptake [ 33 ]. Green colonies of T. harzianum mycelia turned dark green with orange pigmentation along the edges ( Figure 3 d). This can be attributed to the pigment production and metal ion chelation on the fungal cell wall [ 34 ]. Compared to other fungal strains, T. harzianum has the highest radial growth, signifying the highest Fe(III) tolerance. 3.2.4. SEM Analysis SEM images ( Figure 4 ) illustrate alterations in the morphological features of isolated fungi after the uptake of Fe(III). Figure 4 a exhibits a regular and smooth surface of the mycelia of F. equiseti in the normal status without Fe(III) uptake. Fe(III) precipitate is observed on the mycelial surface of F. equiseti due to the Fe(III) bioaccumulation, reflecting the rough surface morphology of these mycelia ( Figure 4 b). This can be attributed to effective potential biosorption sites on the mycelia surfaces [ 35 ]. Furthermore, the bioaccumulation of Fe(III) produces changes in the morphological features of fungi represented in mycelial looping and twisting of P. pinophilum ( Figure 4 c) and small irregular folds on the T. harzianum hyphae ( Figure 4 d). All previous alterations are destructive changes with different grades that occur to the mycelia due to the penetration of Fe(III) into the cell wall. These findings are consistent with those of previous studies [ 36 ]. 3.2.5. FTIR Analysis FTIR spectra of the T. harzianum before and after Fe(III) uptake are shown in Figure 5 . Generally, negatively charged functional groups, such as hydroxyl, amino, carboxyl, phosphate, nitro, and halide groups, provide the electrostatic force required for binding positively charged Fe(III) to the cell surface [ 37 ]. The two spectra have some similarities in their profile. The broad band at 3322–3379 cm −1 is related to the −NH 2 and O−H present in carbohydrates or proteins, −NH stretching of amine (protein), and the acetamido group (chitin) [ 38 ]. Comparing the two FTIR spectra, a pronounced depletion in this band indicates that the O−H and N−H groups are bound with Fe (III). The presence of a peak at 2925 cm −1 is related to the C−H vibration group [ 39 ], which has lower intensity after Fe(III) uptake, indicating the role of C−H. The band at 2170 cm −1 is reduced in the case of Fe(III) uptake, signifying that C−H, C=O, and C=N contribute to Fe(III) adsorption. Loss of the band in the Fe(III)-laden biomass compared to the band at 1740 cm −1 in the case of raw biomass implies the role of C=O stretching vibration in carboxylates of acidic polysaccharides in the metal uptake [ 39 ]. In the raw biomass, the band at 1640 cm −1 is extinct in the case of F(III)-loaded biomass. This signifies that N−acetyl glucosamine or O−H stretching vibration (i.e., polymer of the protein–peptide bond) has significant support in Fe(III) treatment [ 40 ]. The disappearance of peaks at 1460 cm −1 at the Fe(III)-loaded sample indicates the role of methylene/alcohol groups (C−H, O−H bending) in the Fe(III) uptake [ 41 ]. The peaks at 1039 cm −1 belonged to the C–C, C=C, C–O–C, and C–O–P groups of polysaccharides [ 41 ]. This peak disappears in the Fe(III)-loaded sample, indicating the potential of these groups in Fe(III) uptake. The bands less than 1000 cm −1 concern the fingerprint zone of phosphate and sulfur groups. The decrease in the peak intensity at 617 and 705 cm −1 in the metal-loaded strain is attributed to the C−H effect [ 41 ]. Moreover, there are noticeable changes in these bands when Fe(III) uptake occurs, signifying that these bands are affected by Fe(III) adsorption. The previous Fe(III) uptake represents a type of biosorption process. 3.3. Adsorption Studies 3.3.1. Effect of Initial Fe(III) Concentration The effect of initial Fe(III) concentration on adsorption is depicted in Figure 6 . The results show that Fe(III) removal (%) increases from 81.25% to 87.50% with the increase in Fe(III) concentration from 50 to 200 mg/L ( Figure 6 a). Therefore, the Fe(III) removal (%) has an almost similar behavior with minor differences at all initial Fe(III) concentrations, including 50, 100, 150, and 200 mg/L. A possible explanation for this increase can be explained in that this initial Fe(III) concentration can provide the necessary driving force to overcome resistance to Fe(III) ions transfer between aqueous and solid phases [ 42 ]. When the initial concentration is low, there are plenty of available adsorption sites, and adsorption can reach equilibrium quickly [ 43 ]. In the same Fe(III) concentration range, the findings show that the Fe(III) uptake capacity increased gradually from 10.97 to 62.8 mg/g from 50–200 mg/L, with the highest value at 200 mg/L ( Figure 6 b). These outcomes can be attributed to the fact that high Fe(III) concentrations can enhance the interaction of Fe(III) ions with biomass, resulting in a higher Fe(III) uptake capacity [ 44 ]. 3.3.2. Effect of pH Fe(III) is a cationic metal with a surface charge of positively charged ions. The Coulombic interaction between positively charged Fe(III) and negatively charged adsorbents can explain why it is more readily adsorbed in acidic environments [ 45 ]. As shown in Figure 7 a, when the initial pH of the solution lies in the range of 3–7, all isolated fungi perform well in Fe(III) removal (%). More specifically, Fe(III) removal (%) of all fungal strains exceeded 80%. On the other hand, the Fe(III) removal (%) decreases for all isolated fungi at pH 8. As the pH rises, the fungal uptake capacity increases until reaching pH 8 ( Figure 7 b). Notably, the Fe(III) removal (%) and uptake capacity (mg/g) have almost the highest values for all fungal strains at pH values of 3 and 7, with more superiority for the second (i.e., pH 7). Although it is supposed that pH 7 is similar to pH 8 in terms of having the same deleterious impact on the Fe(III) removal (%) and uptake capacity (mg/g), this does not occur. The unfamiliar behavior at pH 7 can be confirmed by the values of Fe(III) uptake by P. pinophilum, and T. harzianum, which are equal to 64.28 and 60 mg/g, respectively. This can be attributed to the fact that the fungal strains turn the pH of the medium into acidic status as a result of their secretion of organic acids, including citric, gluconic, malic, itaconic, lactic, and fumaric acids [ 46 ]. This was validated by measuring the pH of the medium solution, which is assumed to be 7. However, it was measured and found to be closer to 3 after adsorption. These excreted organic acids not only have an effect on the pH, but also a positive effect on the Fe(III) removal (%) and uptake capacity (mg/g). Hence, there is a difference in the behavior of Fe(III) removal (%) and uptake capacity (mg/g) between the two values of pH (i.e., 3 and 7). This low pH triggers the protonation of binding sites on the microbial surface, thus imparting a negative charge and promoting Fe(III) binding on the microbial surface. On the other hand, the reduced uptake of F(III) ions at pH 8 might be attributed to the accumulation of metal ions inside the cell walls or cells by a combined sorption microprecipitation mechanism [ 47 ]. 3.3.3. Effect of Temperature It was found that the temperature significantly impacts the biosorption process. At the temperature values of 20 and 28 °C, the Fe(III) removal (%) of fungal strains increases during the temperature increase, reaching a maximum value at 28 °C ( Figure 8 a). At 37 °C, the Fe(III) removal (%) has the lowest value. At 28 °C, F. equiseti has the highest Fe(III) removal (89.5%). Regarding the Fe(III) uptake (mg/g), it decreases gradually from 20 to 37 °C, respectively, and is the highest at 20 °C. More specifically, T. harzianum has a maximum Fe(III) uptake of approximately 51.5 mg/g at 20 °C. The findings prove that various fungi have a wide range of temperature adaptability. However, the effect of extremely low- (i.e., under 20 °C) and high-temperature ranges (i.e., above 37 °C) was not investigated for the fungal growth. This can be attributed to the detrimental effect of these temperature ranges on microbial metabolic rates, metal reductase synthesis, and other active materials in the fungal cells [ 48 ]. These findings are consistent with previously reported results [ 35 ]. 3.4. Adsorption Isotherm of Fe(III) Figure 9 and Figure 10 show the Freundlich and Langmuir adsorption models applied for the adsorption of Fe(III) ions, respectively. A plot between 1/qe and 1/Ce yields a linear form of Langmuir isotherm. Similarly, the graph plotted between log qe and log Ce yields a linear form for the Freundlich isotherm. Mathematical description and isotherm constants were determined to compare the adsorption capacities of Fe(III) ions for the addressed species. The results have a high value of the correlation coefficients (R 2 ) ranging from 0.93–0.98, signifying the adsorption results are the best fit in both the Langmuir model and Freundlich model, with more superiority for the first ( Table 4 ). The high values of Langmuir constants, including q max and K L , range from 61.34–72.46 (mg/g) and 0.0390–0.0260 (L/mg), respectively. While the Freundlich constants, including K F and n, range between 4.20–5.60 mg/g and 1.80–2.20 L/mg, respectively. Unlike C. lunata , the highest values of Langmuir constants, q max (72.46 mg/g) and K L (0.0260 L/mg), and Freundlich constants, K F (5.60 mg/g and n (2.20 L/mg), are recorded for T. harzianum , indicating the highest adsorption capacity and intensity, respectively. This is validated by the highest R 2 values of 0.94 and 0.95 for Langmuir and Freundlich, respectively ( Table 4 ). Generally, the low absorption efficiency, as observed from the Fe(III) removal (%) and uptake (mg/g), of these live fungal strains is due to their active cellular metabolism, which inhibits the biosorption process."
} | 5,485 |
39863912 | PMC11762876 | pmc | 2,471 | {
"abstract": "Background Recovery of degraded coral reefs is reliant upon the recruitment of coral larvae, yet the mechanisms behind coral larval settlement are not well understood, especially for non-acroporid species. Biofilms associated with reef substrates, such as coral rubble or crustose coralline algae, can induce coral larval settlement; however, the specific biochemical cues and the microorganisms that produce them remain largely unknown. Here, we assessed larval settlement responses in five non-acroporid broadcast-spawning coral species in the families Merulinidae, Lobophyllidae and Poritidae to biofilms developed in aquaria for either one or two months under light and dark treatments. Biofilms were characterised using 16S rRNA gene sequencing to identify the taxa associated with settlement induction and/or inhibition. Results We show that light and biofilm age are critical factors in the development of settlement inducing biofilms, where different biofilm compositions impacted larval settlement behaviour. Further, we show that specific biofilm taxa were either positively or negatively correlated with coral settlement, indicating potential inducers or inhibitors. Although these taxa were generally specific to each coral species, we observed bacteria classified as Flavobacteriaceae , Rhodobacteraceae , Rhizobiaceae and Pirellulaceae to be consistently correlated with larval settlement across multiple coral species. Conclusions Our work identifies novel microbial groups that significantly influence coral larval settlement, which can be targeted for the discovery of settlement-inducing metabolites for implementation in reef restoration programs. Furthermore, our results reinforce that the biofilm community on coral reef substrates plays a crucial role in influencing coral larval recruitment, thereby impacting the recovery of coral reefs. Supplementary Information The online version contains supplementary material available at 10.1186/s40793-025-00670-0.",
"conclusion": "Conclusions Despite the importance of coral recruitment and decades of research, we still lack fundamental knowledge on the identification of taxa and mechanisms that underpin microbially induced coral larval settlement. Our research shows that biofilm development is integral to the success of larval settlement and, therefore, plays an important role in the recovery of coral reefs. Additionally, we show that certain lineages of bacteria are consistently correlated with coral settlement, such as those classified as Flavobacteriaceae and Rhodobacteraceae . Although no single universal inducer was identified, similar taxa may share functional traits leading to the production of similar biochemical cues. This knowledge offers a platform to target, isolate and begin experimentally testing these lineages for their ability to induce settlement, as well as obtaining genomic resources to address the mechanisms behind settlement induction. Resulting biochemical applications to increase coral settlement can be implemented in restoration programs worldwide.",
"discussion": "Discussion Coral larval settlement is an essential component of the coral recruitment process which contributes to sustaining healthy and resilient coral reefs [ 49 ]. While some settlement cues, or their sources, have been described [ 5 ], the majority remain uncharacterised, thereby limiting our understanding of settlement for a diverse range of coral species. Our study shows that marine biofilms developed in aquaculture can induce coral settlement, and that light exposure and development duration significantly affect biofilm composition and subsequently larval settlement. Further, we show that specific groups of taxa are consistently correlated with high or low coral settlement, suggesting these lineages have disproportionate influences on larval settlement through their associated inducing or inhibiting biochemistry. While these results have direct implications for improving coral settlement in aquaculture, importantly they also contribute to a better understanding of recruitment patterns among non-acroporid coral species, which is crucial for projecting future reef conditions under different climate scenarios [ 50 ]. Environmental factors shape settlement inducing biofilms The community composition of biofilms can impact larval settlement of marine invertebrates [ 50 , 51 ], and here we show that light exposure and biofilm maturity are two key factors that influence the development of biofilms inductive of coral larval settlement. The 2-month (2 M) light treatment developed a markedly different biofilm community compared to the 2 M dark and 1-month (1 M) light conditioned biofilms and induced the highest levels of larval settlement for all coral species tested. This aligns with previous settlement results using artificial surfaces conditioned in the field, where 2-week-old biofilms induced less than 10% metamorphosis in Acropora microphthalma, while 8-week-old biofilms induced greater than 40% metamorphosis [ 12 ]. Additionally, higher coral settlement was observed on 8-week-old biofilms that formed at a depth of 4 m compared to 8 m, which may be related to light intensity. A similar study with Acropora tenuis larvae showed that 15-day-old biofilms induced higher coral settlement than 7-day-old biofilms, and that settlement increased on biofilms that were developed further away from mariculture sites with improved water quality [ 52 ]. Hence, along with biofilm maturity, environmental parameters such as light, depth and water quality can shift the community composition of biofilms thereby affecting recruitment on shallow-water coral reefs [ 53 ]. Our results demonstrated that even small changes in the biofilm community can impact larval settlement. For instance, the pre-existing microbial communities and biofilms in the experimental tanks systems influenced the composition of biofilms that formed on the settlement substrates, which in turn affected larval settlement. Therefore, despite controlling for factors such as light intensity and water temperature, each tank functioned as a separate system, introducing variability into the conditioning process. This suggests that minor shifts in biofilm development can have broader ecological implications for coral recruitment on reefs. Biofilms are likely to vary within and between habitats [ 54 , 55 ], and this variability may influence coral recruitment patterns, ultimately shaping the community composition of reef corals. Further, since degraded reefs or those with poor water quality can have different biofilm communities compared to healthy reefs [ 53 ], this may promote a shift in coral community composition. In this study, P. lobata and L. corymbosa had less settlement on 2 M light treatment biofilms compared to other species, while E. aspera was the only species to show significant levels of settlement on dark treatment biofilms compared to controls. Hence, optimal biofilm conditions likely vary among coral taxa and this variation may correspond to the environmental conditions best suited to each species. A subset of biofilm taxa correlates with high or low settlement Community composition differences between high and low settlement biofilms within treatment were less pronounced than community differences between treatments. Therefore, it is likely that changes in the abundance of a smaller group of microbes within these communities are driving coral settlement. ASVs classified as Flavobacteriaceae were positively correlated with high settlement in three of four coral species and were more abundant in the 2 M light biofilms compared to other treatments. The Flavobacteriaceae are key components of marine biofilms with genomes that encode a diverse range of secondary metabolite biosynthesis pathways [ 56 , 57 ], and members of this family have previously been associated with settlement induction of marine invertebrate larvae. For example, biofilms that induce mussel settlement were treated with an antimicrobial agent, reducing the relative abundance of Flavobacteriaceae which correlated with a reduction in mussel settlement [ 58 ]. For coral, older biofilms that induced settlement of A. microphthalma larvae were associated with higher abundances of the Cytophaga-Flavobacterium group [ 12 ], while isolates of the Cytophaga-Flavobacterium group have shown high settlement induction of the polychaete Hydroides elegens [ 59 ]. Interestingly, high relative abundances of Flavobacteriaceae are associated with coral recruits of the species Pocillopora acuta in the first 1–2 weeks post-settlement [ 60 ]. Therefore, settlement on biofilms with specific taxa may be important for the uptake of early life stage symbionts. Similarly, ASVs classified as Pirellulaceae and Rhizobiaceae were consistently correlated with high coral settlement across three different coral species, while some Rhodobacteraceae ASVs correlated with high settlement of P. lobata and E. aspera . The Rhodobacteraceae are abundant primary colonisers of marine biofilms and thought to be important for structuring communities into high-settlement biofilms [ 13 , 61 ]. Further, an isolate of Roseivivax sp., within the Rhodobacteraceae family, was reported to induce larval settlement of the coral Porites astreoides [ 14 ], and this genus was found to correlate with high settlement for Porites lobata in this study. Hence, the Rhodobacteraceae family may be important for settlement of corals through both direct stimulation of settlement and biofilm community organisation . On the other hand, neither Pirellulaceae nor Rhizobiaceae have been implicated in the direct settlement of coral larvae and may represent new lineages for exploration. In particular, the phylum Planctomycetota (containing Pirellulaceae ) has been observed to increase in abundance in coral reef biofilms as they develop over time [ 13 , 55 ]. This suggests they are secondary colonisers and may be important members of mature biofilms that are more successful at inducing larval settlement. Although no difference was observed in Rhizobiaceae abundance between one- and two-month development times in this study, it has previously been associated with early biofilm colonisation [ 55 ]. Interestingly, many families that contained ASVs correlated with high settlement also contained other ASVs correlated with low settlement, and in some cases, this trend was observed at the genus level. For example, the genus Winogradskyella ( Flavobacteriaceae ), which was associated with low settlement for P. sinensis and high settlement for P. lobata , and the genus Limibaculum ( Rhodobacteraceae ), which was associated with both low and high settlement for P. lobata . Similar results have been observed with Acropora tenuis settlement, where some ASVs classified as the orders Rhodobacterales and Flavobacteriales had positive associations with coral settlement and others had negative associations [ 52 ]. This indicates that microbial inducers or inhibitors of coral larval settlement are species or even strain specific, and broad phylogenetic assignments are not predictive of inductive capacity. This has been observed in the genus Pseudoalteromonas , which can be a potent inducer of settlement or metamorphosis for a variety of marine invertebrate larvae including coral [ 20 , 31 , 62 ]. Yet closely related species of isolates that induce settlement have demonstrated vastly different effects on larval settlement, ranging from induction via multiple mechanisms, to no activity or even toxicity to larvae of some species [ 15 , 17 , 27 ]. Given the relatively high abundance and diversity of families such as Rhodobacteraceae and Flavobacteriaceae in marine biofilms, it is feasible they contain both inducing and inhibiting bacteria for coral settlement. Resolving strain level differences among potential inducers of larval settlement will be an important consideration when selecting prokaryotes for biochemical applications in restoration, and future studies would benefit from characterising settlement inducing biofilms beyond 16 s rRNA amplicons. Although there was consistency at the family level for which taxa correlated with high settlement across different coral species, we did not find an ASV that was correlated with high settlement across all species tested. While a lack of significant correlation does not necessarily mean no inductive capacity, the results still suggest it is unlikely that there is a universal bacterium responsible for inducing settlement across a diverse range of corals. In such a scenario, certain bacterial taxa could be cultured to induce settlement of endangered or difficult to settle coral species that are targeted for restoration. Alternatively, different bacterial lineages may be capable of similar functions, such as the production of certain metabolites, and hence it may be the metabolic capability that is important for inducing settlement. Moreover, optimal larval settlement may be reliant on a community of organisms responsible for producing a range of biochemical cues that induce settlement for a diversity of reef corals [ 17 ]. Hence, future research would benefit from investigating the genomic capability and metabolite production of settlement inducing bacteria to better understand the mechanism of bacteria induced settlement. Chemical extracts of biofilms can induce coral larval settlement Chemical extracts from 2 M light biofilms induced settlement for one of two species tested, revealing that coral larvae can respond to chemical cues within the biofilm in the absence of surface topography. Here, dichloromethane (DCM) extracts were more successful as a settlement inducer for Porites lobata compared to ethanol (EtOH) extracts, indicating hydrophobic/non-polar compounds may be more effective as settlement inducers for P. lobata than polar compounds . This result contrasts with most CCA-associated chemical inducers for the settlement of acroporid and agariciid larvae which are primarily soluble in EtOH or hot water [ 18 , 24 , 36 ]. Similarly, while both acroporid and non-acroporid corals were induced to settle by live CCA ( Porolithon onkodes ), only acroporid species settled in response to EtOH extracts [ 9 ]. This further indicates many non-acroporids may have a stronger settlement preference for hydrophobic compounds, with classes of chemical inducers differing between acroporid and non-acroporid clades. Interestingly, some bacteria may provide both soluble and hydrophobic compounds capable of settlement induction. For example, Pseudoalteromonas spp. are associated with both EtOH soluble compounds such as tetrabromopyrrole (TBP) [ 20 ] and hydrophobic compounds such as cycloprodigiosin [ 22 ]. Although the relative abundance of Pseudoalteromonas spp. was negligible within the biofilms of this study, it is possible that cycloprodigiosin or similar classes of hydrophobic inducers may be produced by other prokaryotes in the inductive biofilms. The chemical extracts of biofilms did not induce settlement for L. corymbosa , and this coral also showed the weakest response to live biofilms. L. corymbosa has previously shown a different settlement response to certain species of CCA compared to the other corals tested here [ 8 ]. For example, the CCA Porolithon sp. induced > 50% settlement for all species tested here, except L. corymbosa , which had a mean of 28% settlement. On the other hand, Sporolithon sp. induced a mean of 87% settlement in L. corymbosa and 86% settlement in the closely related E. aspera , however only induced 45–58% settlement in P. sinensis , D. favus and P. lobata [ 8 ]. These different settlement cues might arise from different ecological preferences and life-history characteristics in the diverse species tested. For example, while corals from the genus Lobophyllia and Echinophyllia (Lobophyllidae) are known to be aggressive competitors for space [ 63 ], they occupy different niches on the reef, with Echinophyllia spp. more commonly found in shaded environments compared to Lobophyllia spp. [ 64 ]. Species-specific larval responses to different settlement cues are likely to be linked to the recognition of microbial communities or CCA associated with preferred habitats, which in turn influences the spatial distribution of corals on the reef [ 65 ]. Future directions Exploring the association of biofilms with coral larval settlement offers promising opportunities for the discovery of natural biochemical inducers; however, research is still needed to understand how complex biofilms interact with coral larvae (Randall et al., 2020). Developing biofilms under dark conditions is likely to have reduced the contribution to inductive biochemistry by eukaryotes such as CCA spores or microalgae, and moderate settlement induction of E. aspera larvae was observed on these biofilms. This supports the potential role of non-phototrophic prokaryotes in the settlement of non-acroporid larvae; however, the greatest settlement was observed in response to 2 M light biofilms. Hence, it is possible that phototrophic eukaryotes may have contributed to inductive biochemistry in this treatment. Fully disentangling the role(s) of prokaryotes and eukaryotes in triggering coral larval settlement requires assessing the inductive capacity of isolates from biofilm taxa [ 16 , 17 , 20 ], and future research may benefit from targeting some of the taxa identified here. Genome sequencing paired with comparative analyses of inductive and non-inductive isolates could help reveal the molecular machinery underpinning settlement induction. However, challenges remain as most prokaryotes are not readily cultured and their biochemistry in isolation is likely to differ from that in situ [ 66 ]. Nonetheless, controlling the settlement of non-acroporids in aquaculture using biochemical inducers from cultured prokaryotes may be more practical compared to induction by CCA as it eliminates the need to continually harvest specific algal species from the reef and has potential for large-scale standardised production."
} | 4,553 |
36132304 | PMC9418881 | pmc | 2,472 | {
"abstract": "Lubricant-infused surfaces have attracted widespread attention due to their excellent liquid and organic solution repellency. On account of their high condensation heat transfer coefficient and low nucleation energy barrier, many lubricant-infused surfaces have been applied in water collection. However, they have a number of shortcomings, such as an unstable lubricating layer, poor mechanical/chemical stability and hard shedding, which severely limit the application of slippery surfaces. In this work, the silicone oil was infused into a superhydrophobic monomer (SHM) to form a flexible lubricant-infused monomer (FLIM) with outstanding sliding ability and omniphobicity for low surface energy liquids. Because the silicone oil is similar to the base molecule, there is a strong interacting force to hold the lubricant layer to the surface of the SHM. In addition, the high viscosity of the silicone oil further strengthens the lubricant layer adhesion. Therefore, the FLIM could resist hot liquid and high shear stress (up to 5000 rpm). In addition, the FLIM substrate possessed a self-similar low surface energy structure, which could endure various physical and chemical damages, such as abrasion, scratching, stretching, strong acid and alkali. Finally, pinned droplets could coalesce into large droplets to slide down its surface, resulting from the strain/release due to the high degree of deformation of the surface, which highly enhanced water/liquid coalescence and collection. The preparation of the FLIM was green and the chemicals involved were inexpensive and environmentally friendly, and thus it can be applied for large-scale water collection.",
"conclusion": "4. Conclusion Recently, lubricant-infused surfaces have attracted increasing attention due to their omniphobicity and outstanding sliding ability. 24,25 However, there are many shortcomings in the traditional lubricant infused materials that limit their development, such as poor locking lubricant ability and hot liquid repellency, and fragile structure. 9,45 PDMS with a low surface tension is used as a hydrophobic material due to its superior stability against thermal, chemical, and photo-catalytic degradation. 46,47 Herein, silicone oil was infused into an SHM to form a durable FLIM with a sliding angle of less than 4° and omniphobicity for low surface energy liquids. Herein, the lubricant was trimethylsiloxy-terminated PDMS, which is similar to the substrate prepared using PDMS. Hence, strong interactions existed between the silicone oil molecules and substrate molecules. Also, the silicone oil possessed high viscosity. Thus, the lubricant layer could be locked in the surface firmly. Due to the durable bulk substrate and stable lubricant layer, the FLIM exhibited outstanding ability to resist hot water, scratching, strong acid/alkali and high shear stress. Furthermore, the oil layer thickness was calculated based on the change in lubricant quality, and thus a reasonable lubricant thickness should be applied for water collection. Under the action of external force, the deformation of the FLIM induced by stretching reduced the pressure in the porous matrix and caused the lubricant to retreat into the pores. Therefore, the FLIM could control the sliding of the droplet by compressing and stretching. In addition, according to the high degree of deformation of the surface resulting from the strain/release, the pinned droplets could coalesce into large droplets to slide down the surface. Therefore, the water/liquid coalescence and collection were highly improved and enhanced. Accordingly, the durable FLIM has great potential applications in water collection since it can withstand extreme severe working conditions.",
"introduction": "1. Introduction Taking inspiration from lotus leaves, 1 rice leaves 2 and butterfly wings, 3 bioinspired superwettable materials have been introduced into our lives and there have been great advances in their fabrication. 4–7 Especially, superhydrophobic materials with a high contact angle and low rolling angle possess great application value in anti-icing, 8,9 self-cleaning, 10 drag reduction, 11 corrosion-resistance 12 and oil-water separation. 13 However, for their production and extensive utilization in real-world settings, their low environment friendliness and mechanical/chemical stability urgently need to be solved. 7 Although fluorinated substances and volatile organic compounds can reduce the surface energy of surfaces, they have been identified as a source of toxic pollutants, and thus their use is restricted. 14 Recently, it has been reported that some superhydrophobic materials were fabricated using nonfluorinated or waterborne materials. 15–17 However, they suffer from chemical and mechanical damage in various environments, such as strong acid and alkali, high temperature, UV irradiation, scratching, touching and abrasion, and thus easily lose their superhydrophobicity. 18 Therefore, new superhydrophobic materials need to be prepared to resist various damages. For example, an ultra-robust superhydrophobic fabric with mechanical stability, UV durability, and UV shielding property was prepared successfully. 19 Therefore, other new superhydrophobic materials still need to be fabricated to resist various damages. Recently, many researchers infused lubricants into porous substrates with nano-topography and low surface energy to form stable and inert slippery interfaces, inspired by the Nepenthes pitcher plant. 20–22 In addition, Zhu et al. prepared a lubricant-impregnated coating with long-term stable slipperiness and self-replenishment properties via a simple method. 23 Due to the existence of a lubricating liquid, slippery surfaces have the ability to realize omniphobicity and low sliding angles. However, the problems of poor locking lubricant, chemical instability and fragile structure severely limit the development of slippery surfaces. Accordingly, many articles have been reported to improve the stability of the lubricant layer. 24–26 Nevertheless, the substrate structure was easily destroyed by scratching, compression and abrasion, and thus the slippery surfaces lost their sliding and lubricant locking ability. Collecting fog by employing different strategies is an important way to solve the water shortage problem in arid regions. Accordingly, numerous biomimetic fog-harvesting materials, superhydrophobic surfaces 27,28 and hydrophobic/hydrophilic surfaces, 29,30 have been studied and fabricated to collect water based on inspiration from the Namib Desert beetle, 31 spider silk 32 and cactus. 33 The collection of water is usually divided into three parts, including water capture, water supply and water removal. 34,35 Water capture is the process of capturing tiny droplets on the surface of harvesting materials. Water supply is the process of coalescence between droplets in a fog episode and water droplets. Water removal (drainage) is the process of drainage of harvested water. Under the action of gravity, the gradually growing droplets slide down from the surface. However, compared to superhydrophobic surfaces and hydrophobic/hydrophilic surfaces, FLIM surfaces have better water collection capacity due to their excellent heat transfer efficiency and low nucleation energy, which has been proved in previous reports. 36,37 Because the thickness of the infused lubricant decreases with time, the rough base layer is exposed and the water sliding ability cannot exist for a long period. In addition, on a rigid substrate, the droplets are easily blown away by natural wind. Also, the pinned water droplets on water-harvesting surfaces limit the nucleation of new droplets, thus hindering the water collection. Thus, it is important to speed up the growth of droplets or bring together tiny droplets to slide down quickly. Recently, bulk materials with a self-similar structure, foams, sponge, and layer-by-layer coatings have attracted much attention due to their excellent mechanical properties. 7 Herein, silicone oil was infused into an SHM to form a super-durable FLIM with a sliding angle of less than 5° and omniphobicity. The lubricant in this work was silicone oil (PDMS), which was similar to the bulk substrate prepared using PDMS. Therefore, the lubricant could be locked in the SHM firmly. Due to the mechanical and chemical durability of the monomer material, the FLIM exhibited outstanding ability to resist hot water, scratching, strong acid/alkali and high shear stress. To determine the most suitable lubricant thickness for water collection, the lubricant layer thickness was calculated based on the change in lubricant quality, and a water collection experiment was carried out. Under the action of external force, the FLIM could control the sliding of the droplet by compressing and stretching. Hence, the pinned droplets could coalesce into large droplets to slide down the surface according to strain/release due to the high degree of deformation of the surface, which highly enhanced the water/liquid coalescence and collection. The FLIM greatly improved the life of the slippery material and improved the water collection efficiency under various extreme conditions.",
"discussion": "3. Results and discussion 3.1 The preparation of flexible liquid infused monomer To prepare the liquid-infused monomer, the preparation of an SHM was necessary. Due to its low surface energy ( γ SV = 20 mJ m −2 ), PDMS was modified to prepare the superhydrophobic substrate. 38 During the preparation process, water was added to PDMS dropwise to form a uniform emulsion under the conditions of no surfactant or electrolyte, as shown in Fig. 1ii(a and e) . With the constant addition of water droplets, the emulsion became thicker and the phase separation of the oil–water occurred. As shown in Fig. 1ii(i) , water droplets with a similar diameter were dispersed in the uncured PDMS. After curing at 180 °C, the water phase formed different morphologies in the uncured PDMS, which existed throughout the whole bulk material. As shown in Fig. S1b, † the microscopic pores filled the entire monomer. As displayed in Fig. 1ii(f and g) , the diameter of the cavity showed a big difference, resulting from the droplet coalescence since the emulsions were heated to initiate curing. To improve the superhydrophobicity of the monomer, a large amount of SiO 2 nanoparticles was dissolved in water and added to PDMS. Therefore, the bulk mater material was full of micro-nanostructures after heating at 180 °C. The SiO 2 nanoparticles were tightly attached to the wall of every hole (see Fig. 1ii(g and k) ). The contact angle on the SHM was 156° and the roll angle was 5° (see the Fig. 1ii(c) ). However, initially, the hydrophobicity of the SHM was blocked by the “skin” that hid the hydrophobic structure, which had a contact angle of 127° and the water droplets were pinned in the surface. Hence, the hydrophobicity of the SHM was poor, as shown in Fig. 1ii(d, h, l) and S1a. † When only water was added, the inner wall of the hole was smooth and the hydrophobicity was poor, and the contact angle was only 120° and water droplets were pinned on the surface (see Fig. 1ii(b, f, j) and S1a † ). To obtain the micro-nanostructure, SiO 2 nanoparticles were dispersed in the water/PDMS emulsion. After curing and heating, the SiO 2 nanoparticles were attached to the wall of every hole. As shown in Fig. 2a and b , the prepared sample only has three elements of C, Si, O. Also, the Si content of the SHM increased from 9.15% to 28.93% compared to the sample without the silica particles, which proved that the SiO 2 nanoparticles were embedded in the SHM. After removing the skin of the SHM, silicone oil was injected onto the surface of the sample to form a uniform oil film under high-speed centrifugation. The reason why silicone oil was chosen is that its molecule is similar to the substrate molecule and it has high viscosity, which are beneficial for its attachment to the surface to form a uniform lubricant layer. In addition, silicone oil possesses a low surface tension (less than 25 mN m −1 ) and has superior stabilities against thermal, chemical, and photo-catalytic degradation. 39,40 Accordingly, the flexible slippery monomer was successfully prepared. Fig. 2 EDS spectra of (a) flexible monomer without SiO 2 and (b) SHM with SiO 2 . 3.2 The mechanical abilities of the superhydrophobic monomer By reason of the self-similar structure and flexibility of silicone, the SHM had superior ability to resist abrasion, compression, stretching, and touching. After destruction by mechanical damage, although the surface structure was destroyed, the new superhydrophobic structure reappeared and the hydrophobicity did not change. This is because the low-surface-energy microstructures are extended to the whole volume. As shown in Fig. 3a , the sample was rubbed multiple times on sandpaper under a pressure of 1 kg. Initially, the skin of the original sample exhibited poor hydrophobicity. With an increase in the abrasion distance, no significant changes in contact angle and roll angle were observed at 156° and 5°, respectively (see Fig. 3c ). Herein, we only measured the length of 500 mm for the friction. However, the SHM could resist longer friction lengths if the sample is not depleted. Fig. 3 (a) Schematic of the abrasion under a pressure of 1 kg. (b) SEM images of the abrasion for 100 mm. (c) Water contact angle (WCA) and water roll-off angle change with abrasion distance. (d) SEM images of the abrasion for 100 mm, indicating the SiO 2 nanoparticles still existed in micron hole. (e and f) Compression and tensile test of the flexible SHM, respectively. Inset: photographs of water droplets on the tested surfaces. This is because the low surface energy microstructures were extended to the whole monomer. As displayed in Fig. 3b and d , the SHM had a new structure exposed after abrasion for 100 mm and the SiO 2 nanoparticles were still attached to the inner wall of the holes. Furthermore, the SHM could resist other mechanical damage such as finger touching, high compression for 10 MPa, tape peeling and bending, as shown in Fig. S2. † After heating at 250 °C, the SHM still maintained outstanding superhydrophobicity. Moreover, the SHM possessed superior tensile and compression resistance by reason of the flexibility of the silicone, which dispersed energy upon the deformation of the elastic material. The superhydrophobic flexible monomer had outstanding tensile and compressive properties. As shown in Fig. 3e and f , the stretching and compressing strength of the SHM was investigated by performing tensile and compression tests. Samples with a size of 10 × 10 mm 2 and 40 × 20 mm 2 were subjected to tensile and compress tests under the action of external force at a speed of 10 mm min −1 . As displayed in Fig. 3e , the compression strength and strain were about 0.5 MPa and 30%, respectively (the external force here could only reach 500 N). After the external force was unloaded, the sample returned to its original shape without hydrophobicity change. Furthermore, the stress strength and strain were about 4.5 MPa and 50% (see Fig. 3f ). Although the sample broke, the sample had good elasticity, and the hydrophobicity and microscopic morphology did not change. The reason why the SHM had excellent mechanical properties was because the SiO 2 nanoparticles were fixed throughout the sample and exhibited strong interfacial interactions with PDMS. Also, the silicone could resist external damage by dispersing energy upon the deformation of the elastic material. Therefore, the SHM had superior mechanical properties to endure various damage. 3.3 Sliding ability and stability of flexible lubricant-infused monomer After the skin of the SHM was removed, silicone oil was infused to form a uniform lubricant layer under high speed centrifugation. Due to the high viscosity and intermolecular attraction, the lubricant existed stably in the micropores, and thus could resist various harsh working conditions, such as hot water, scratching, high shear stress and compression. The surface of FLIM retained its omniphobicity for water and oleic oil. The contact angles of water and oleic oil were 96° and 26°, respectively. Also, on the surface of the FLIM, the water droplet and oleic oil could slide from it easily with sliding angles of 3.4° and 2.6°, respectively (see Fig. 4a ). The sliding angle of water was larger than oleic oil because the contact angle (CA) hysteresis of water was larger than oleic oil. As shown in Fig. 4b, the CA hysteresis of water is 2.9°, while that of oleic oil is 2.3°. Upon dropping 10 μL water and oleic oil on a 5° tilted surface, the sliding speed of water was 0.244 mm s −1 and water was 0.282 mm s −1 (see Fig. 4c ). Fig. 4 (a) Contact angle and sliding angle of water and oleic acid. (b) Receding contact angle (CA), advancing CA and CA hysteresis. (c) Sliding speed of 10 μL water and oleic acid on 5° tilt surface. Here the lubricant layer was formed at 3000 rpm. To explore the stability of the FLIM, its characteristics of resisting hot water, physical damage and high shear stress were measured. The silicone oil was locked in the SHM to form a stable lubricant layer because the intermolecular attraction of the silicone oil molecular was similar to that of the PDMS substrate. Also, its flexible self-similar structure could resist various physical damage by releasing energy after deformation. For the mechanical stability, as shown in Fig. 5b , despite the scratch on the FLIM, water droplets could still slide down from the 5° tilted surface. This is because the silicone oil had fluidity that can be filled into the scratch, reforming a uniform continuous oil layer. Fig. 5 (a) Image of hot water (10 μL) sliding on the 5° tilt surface of the FLIM. (b) Images of a water droplet (10 μL) sliding down from the scratched FLIM. (c) Change in the contact angle and sliding angle on re-oiling surface after repeated friction. (d) Changes in the contact angle and sliding angle at various pH. (e) Change in 10 μL water contact angle and sliding speed at various water temperatures. (f) Contact angle and sliding angle of water and oleic acid on a spin-coater for 40 s at various speeds. Furthermore, the FLIM could endure abrasion. Although the slide angle of the sample increased sharply after abrasion for 10 mm and the lubricant layer was reduced by 62.1% and the sliding angle of water increased to 14.5°, the slide angle returned to the original value after refilling. After abrasion several times, the large loss in lubrication caused the oil layer to disappear. However, a new similar structure was exposed after the friction (see Fig. 3b ), and the new oil layer was restored to the original sliding angle and contact angle after refilling, as shown in Fig. 5c . To explore the stability of the lubricant layer, the sample was placed on a centrifuge to undergo different shear stress. As shown in Fig. 5f , the water and oleic acid contact angle had a tendency to increase with an increase in the spin rate. Since the thickness of the lubricant layer was thinning, the contact angle of water and oleic acid became 96° and 27°, respectively. Moreover, the sliding angle of water and oleic acid increased first and then decreased. This is because initially, the lubricant layer was too thick to prevent the movement of the droplets, while the lubricant layer was too thin and the exposure of the rough substrate prevented the movement of the droplets. As displayed in Fig. 6b , the lubricant layer thinned with an increase in the spin rate. However, the thickness of the lubricant layer was still about 55 μm at 5000 rpm, which exhibited that the FLIM has excellent ability to lock the lubricant. Fig. 6 (a) Fog capture, supply and removal on FLIM during 40 s (scale bar: 200 μm). (b) Schematic illustration of the change in the lubricant layer at various spin rates. (c) H (height) and Δ M (weight of the lubricant on the SHM) of the FLIM placed on a spin-coater for 40 s at various speeds. (d) Water harvesting of FLIM placed on a spin-coater for 40 s at various speeds. In addition, the FLIM had superior ability to resist hot water and strong acid and alkali solution. The image of hot water sliding on the 5° tilt surface of the FLIM is shown in Fig. 5a . As the water temperature increased, the contact angle decreased from 94° to 83.5° because the surface tension of water decreases with an increase in temperature (see Fig. 5e ). 9 Also, the sliding speed of the hot water decreased with an increase in temperature since the condensed vapor of hot water on the lubricant layer hindered the movement of the water droplets. 20 However, the speed was not significantly reduced, which was 0.164 mm s −1 at 80 °C compared to 0.24 mm s −1 at 20 °C. The sliding state is shown in Fig. 5a . Furthermore, the FLIM exhibited outstanding ability to endure strong acid and alkali. The contact angle of water does not change under strong acid conditions. In contrast, under strong alkali conditions, the contact angle on the FLIM changed from 96.5° to 43° (see Fig. 5d ). However, the sliding angle was still less than 4°. Therefore, the FLIM had outstanding chemical and mechanical stability to endure a variety of extreme conditions, which greatly expands its range of applications. 3.4 The flexible lubricant-infused monomer for water collection Fog, which is an important water source in arid regions, is an effective way to solve water issues. Therefore, fog harvesting is a potential way to obtain freshwater in arid areas. Herein, a stable FLIM, exhibiting omniphobicity for many liquids, was fabricated to harvest water in various harsh working conditions. Usually, water collection is divided into three parts, water capture (condensation), water supply and water removal, which are mainly described in following part. For water capture, many droplets condensed on the FLIM, and the water droplets increased with increase of time (see Fig. 6a ). The reason why the FLIM was chosen to collect water was that its nucleation energy barrier is lower for infused surfaces compered to superhydrophobic surfaces, which is beneficial to collect fog. In addition, with time, the water droplets grew gradually, as shown in Fig. 6a for 5 s and 12 s. The micro-nano structures could capture more droplets and accelerate the growth rate of droplets on them. 41–43 Also, the lubricant layer has a superior heat transfer performance, which promoted the growth of the condensed droplets. As the fog continued to be captured, many droplets accumulated into larger droplets slowly. As shown in Fig. 6a by the black circles and red down arrow, the small droplets collected into larger droplets, and slid away under the force of gravity. Since the infused silicone oil replaced the air layer in the micro/nanostructures, the droplets easily grew and moved due to the higher thermal conductivity of the lubricant than gas and negligible CA hysteresis. The droplets moved continuously at high speed and removed other droplets, paving the way for the nucleation of new droplets. As displayed in the yellow box in Fig. 6a , the FLIM continued to capture fog and form new droplets in the blank area. Therefore, the FLIM could collect more water continuously. It is worth noting that the thickness of the infused silicone oil had an important effect on the water collection. Herein, the thickness of the lubricant was controlled by the spin rate and calculated based on the changes in the quality of the lubricant layer. The thickness, H (μm), can be calculated according to the following formula: 1 where m 1 is the weight of the silicone-coated substrate at different spin rates, m 0 is the weight of the original substrate, ρ is the density of the silicone oil ( ρ = 0.971 g mL −1 , 25 °C), and s is the surface area of the substrate, which is equal to 7.015 cm 2 . When the spin rate increased, the quality of the lubricant layer decreased from 0.0468 g to 0.0371 g and its thickness decreased from 68.7 μm to 54.46 μm (see Fig. 6c ). The amount of water collected varied with the thickness of the lubricant layer, as displayed in Fig. 6d . At the 3000 rpm (the thickness of the lubricant layer was 57.4 μm), the FLIM collected the most water with 0.478 g/30 min. At 1000 rpm, the thickness of the lubricating layer was thicker to form droplets with significant wrapping layers, and thus non-agglomerated droplets were formed. When the oil layer was too thin, the droplet mobility became slow and the nucleation efficiency became worse, thus the quality of water collection was 0.4103 g/30 min at 5000 rpm. Hence, a reasonable thickness of the lubricant layer is beneficial to collect water. 3.5 Enhancing water collection by the relaxation of the imposed strain In the relaxed state, droplets could slide freely on the FLIM. However, when tension was applied, the sample was deformed and changed from a flat surface into a rough and porous surface. The deformation induced by stretching reduced the pressure in the porous matrix and caused the lubricant to retreat into the pores. 44 Therefore, even at large sliding angles, the sliding of the droplets on the stretched surface slowed down or even stopped. As shown in the first image in Fig. 7a , the droplet did not slide on the surface of the vertical stretch relative to the sliding angle of 3.4° in the relaxed state. In the previous section, we explored the tensile property of the FLIM, which could be stretched by about 50% (see Fig. 3f ). Hence, the sliding state could be controlled by deforming the surface by applying an external force. When the pulling force was gradually reduced, the distance between fixed droplets was also reduced, causing them to aggregate into large droplets until the sliding threshold value was exceeded, and then removed from the FLIM. The sliding large droplets slid down together with the small droplets nearby. As displayed in Fig. 7b , as the tensile stress decreased, the small water droplets coalesced along the reverting direction until they started sliding. Similarly, when an external force was applied, the FLIM was compressed and changed into the relaxed state (see Fig. 7d ). The attached droplets coalesced in the direction of relaxation, and their size increased sharply, causing them to fall off. As shown in the green circle in Fig. 7c , the droplets moved in the direction of relaxation and pooled together to become large droplets. When the external force was released, the large droplets slid down against the resistance. By stretching and compressing the surface to be greatly deformed, a large number of adjacent tiny droplets could be brought together to become large droplets, which could be quickly removed on the surface, and this is beneficial to collect water. Thus, the movement of the droplet could be manipulated artificially. Fig. 7 (a and b) On the stretched surface, as the force was unloaded gradually, adjacent pinned water droplets slowly gathered into large water droplets and slid down from the FLIM. (c–e) In the compressed state, adjacent pinned water droplets slowly converged into large water droplets and slowly slid down as the force was unloaded. Due to its remarkably high deformation ability, the FLIM has potential to collect more water compared to undeformed surfaces. Herein, we explored the impact of different compression times (2 min per cycle, 4 min per cycle, and 6 min per cycle) on water collection. The compressed process is shown in Fig. 7d . After the measurement, more water could be collected every 2 min to reach 0.3092 g/30 min. Also, although only 0.278 g was collected at 4 min per cycle, this was slightly higher than the uncompressed FLIM, as shown in Fig. S3. † This is because in about 2 min or 6 min, many small adhered droplets formed on the slippery surface. As the force compressed, the small droplets converged into larger droplets and slid off the surface (see Fig. 7e , where the adjacent droplets were brought together into a large droplet and slid down). While in about 4 min, many large droplets slipped off, with only a small number of small droplets attached to the surface. Therefore, more water could be collected by compressing the FLIM. Furthermore, the thickness of the lubricant layer did not change after multiple compressions."
} | 7,113 |
35336324 | PMC8953929 | pmc | 2,473 | {
"abstract": "The use of biopolymers for realizing economical and eco-friendly triboelectric nanogenerators (TENGs) widens the application prospects of TENGs. Herein, an animal-sourced whey protein isolate (WPI) film, processed and prepared by a simple aqueous solution preparation and drop-casting technique, is applied to demonstrate its potential use in bio-TENGs. With the addition of formaldehyde in WPI, the films result in a free-standing and flexible film, whereas the pure WPI films are difficult to handle and lack flexibility. A TENG device based on the WPI and the laser-ablated textured polydimethylsiloxane (PDMS) for pressure-sensor application were developed. The output voltage of the TENG comprising WPI increased nearly two-fold compared to the TENG without WPI. A simple single-electrode TENG device configuration was adopted so that it could be easily integrated into a wearable electronic device. Moreover, WPI film exhibited tribo-negative-like material characteristics. This study provides new insights into the development of biocompatible and eco-friendly biopolymers for various electronic devices and sensors.",
"conclusion": "5. Conclusions In summary, an animal-sourced WPI film derived from milk was successfully applied for potential use in bio-TENG. From a simple aqueous-based solution and drop-casting method, films of WPI and formaldehyde are transparent and possess flexible film-like properties with a tensile modulus of 48 MPa; besides, the films without formaldehyde are difficult to handle. The WPI film exhibited tribo-positive material-like characteristics when subjected to mechanical loads with PDMS, a tribo-negative material. Benefitting from the relative higher triboelectric voltage among the WPI and PDMS, a TENG device comprised of WPI and laser-ablated rough PDMS for pressure-sensor application was successfully demonstrated with a maximum sensitivity of 1.61 V kPa −1 in the linear region. The TENG’s performance is nearly twice that of the TENG device without WPI film, which shows the potential use of WPI in bio-TENGs. Moreover, the ablated PDMS film by the laser-ablation technique provides great opportunities to produce films with textured surfaces without any additional use of masks and chemicals for etching. The textured PDMS film shows pressure-sensor-like characteristics owing to the increased surface-to-volume ratio of the PDMS surface. With these findings, the WPI film can be applied to other devices for wearable sensor applications.",
"introduction": "1. Introduction Biopolymers using eco-friendly, sustainable, clean, and renewable natural materials that are biocompatible and/or biodegradable are crucial for any kind of electronics or materials research field [ 1 , 2 ]. These polymers in electronic devices can substantially eliminate the global problem of electronics waste, leading to a friendly environment. Biopolymers have been successfully implemented in various electronics fields to replace conventional synthetic polymers. Recently, biopolymers have been widely applied in triboelectric nanogenerators (TENGs) [ 3 , 4 , 5 , 6 ]. TENGs are devices that convert any form of mechanical energy available in the environment into electrical energy [ 7 ]. Their uses are not limited to energy generation as they are also useful for self-powered sensing [ 8 , 9 , 10 , 11 , 12 ]. Examples of this are self-powered sensors based on modified TENG device configurations and materials such as device structure, rough surfaces, and the incorporation of nanomaterials. After the invention of TENGs by Zhong Lin Wang et al. in 2012, many synthetic polymers, including polypropylene, polyvinyl chloride, polystyrene, nylon, Teflon, and polyurethanes, typically derived from petroleum oil have been employed in TENGs. Most recently, to realize a greener environment, polysaccharide-based biopolymers such as cellulose, chitosan, starch, and lignin have been employed as dielectrics for Bio-TENGs [ 13 , 14 , 15 , 16 ]. On the other hand, protein-based biopolymers obtained from animals and plants are relatively economical because of their availability as by-products [ 17 ]. However, the extraction and purification processes of these biopolymers are complex and time-consuming [ 6 , 18 , 19 , 20 ]. Therefore, exploring new biopolymers that are straightforward and economical in terms of both processing and fabrication has become of crucial importance for successful use in TENG devices. Whey protein isolate (WPI), a by-product of cheese production, is commercially available as a powder and is economical, edible, and widely used in the food industry [ 17 ]. In addition, WPI films processed in the food industry offer flexibility, transparency, and oxygen barrier properties. Moreover, the commercially available WPI powder products are soluble in an aqueous solution, and they could possibly be used to form films with a simple drop-casting method. Although water-soluble gelatin protein has been reported, a film with freestanding ability has not been shown [ 21 ]. Aqueous solution-processed plant-based proteins have shown interesting results to enhance crop yield and quality [ 22 ]. Note that pure protein films lack film-forming properties; therefore, they require plasticizers and crosslinking agents to attain flexibility [ 20 ]. Thus, whey protein biomaterial for TENGs could certainly enable the development of low-cost devices for various sensing applications. They can even be explored based on their functional material attributes. Herein, an aqueous solution-processed WPI biopolymer film has been successfully applied in a TENG as a positive triboelectric material. WPI cross-linked with sufficient amounts of formaldehyde resulted in a freestanding, flexible, and transparent film. In many reports, the potential use of biopolymers is demonstrated by combining an opposite triboelectric material in a top-down two-electrode TENG device configuration. Besides, a single-electrode TENG configuration avoids a complex device design. Generally, opposite triboelectric materials with a relatively large difference in the ability to lose or gain charge produce high output performance. Among several triboelectric materials tested with the optimized WPI film, PDMS, a negative triboelectric material, showed better performance. PDMS is a well-known elastomer to exhibit elastic-like properties within the elastic region. Therefore, to have a related application combining WPI and the PDMS pressure sensor application was demonstrated. The WPI film attached to an adhesive aluminum (Al) tape combined with laser-treated textured polydimethylsiloxane (PDMS) elastomer exhibited enhanced triboelectric performances compared to the device without WPI film, which emphasizes the role of WPI film.",
"discussion": "4. Discussion TENG Pressure Sensor The device structure of the single-electrode TENG-based pressure sensor is shown in Figure 4 a. The device consists of a WPI film attached to an adhesive Al tape and a surface textured PDMS film. The surface of the PDMS film was textured to increase the effective surface area to enable pressure-sensitive behavior. The surface had a periodic spike-like morphology with features height and base width of 400 µm and 300 µm, respectively, distributed throughout the active area of the surface. The WPI film pressed against the surface-textured PDMS substrate exhibited an open-circuit voltage ( V oc ) and short-circuit current (I SC ) higher than those of the WPI film pressed with the planar PDMS counterparts, wherein the enlarged contact surface area between the two materials tends to induce a greater amount of surface charges on the WPI, as per the following equation: V o c = − σ A 2 C 0 \nwhere σ is the induced surface charge density, A is the effective contact area between the two materials, and C 0 is the capacitance of the TENG device. The basic principle of the Bio-TENG-based pressure sensor can be understood from previous studies [ 32 , 33 , 34 , 35 , 36 ]. Initially, under the no-pressure condition, the two different insulating materials at rest have neutral charges; upon applying pressure, a chemical bond is formed at the interface of the two materials, and the transfer of charge occurs to balance their electrochemical potential. Upon applied pressure, the electrons at the WPI surface are transferred to the PDMS surface because of their work function differences, attaining positive and negative triboelectric charges at the respective surfaces. This process of building up electrostatic charges continues significantly to a certain extent of pressure value and later saturates owing to the structural elastic limit of the pressure-sensitive PDMS film. As the transfer of charges maximizes to an upper limit of applied pressure and upon any amount of pressure release, there are a resultant definite number of electrostatic charges between the two surfaces of materials. This results in a substantially equivalent amount of potential difference generated between the electrodes because the triboelectric charges on both sides of the surfaces are separated by a gap. In return, a potential drop caused by the effective triboelectric charges induces opposite electrical charge carriers in the WPI electrode, during which current flows from the WPI electrode to the ground opposite to the electron flow. In the released condition, induced by the triboelectric charges on the WPI surface, an effective number of electrons reaches the single electrode with no further flow of current between the two electrodes. Again, further pressing the textured PDMS substrate decreases the electric field (reducing the potential difference) between the triboelectric charges, which eventually reduces the induced opposite charge carriers and, therefore, the flow of electrons back to the ground with current flowing into the single electrode. The performance of the single-electrode WPI-TENG device was examined using a series of materials, including polyvinyl chloride (PVC), polyethylene (PE), polyimide (PI), polydimethylsiloxane (PDMS), Ecoflex, copper (Cu), paper, and wool ( Figure 4 b). Depending on the type of material used for triboelectrification, the tendency of a material to lose electrons or gain electrons classifies it as a positive or negative material, respectively. When two oppositely classified materials undergo contact, the amplitude and polarization of the voltage depend largely on the ability of the materials to lose or attract electrons. The larger the number of electrostatic charges induced between the two opposite materials, the higher the output voltage, V oc . From the triboelectric series, materials such as PVC, PE, PI, PDMS, and Ecoflex are more likely to gain an electron and, therefore, become more tribo-negative in the increasing order. In contrast, positive materials such as glass, Al, paper, and wool are less likely to gain electrons, showing weak electrostatic induction with the WPI film. It was confirmed that the materials in contact with the WPI film agreed well with the triboelectric series. The output power of the Bio-TENG device was evaluated by varying the load resistance in the circuit ( Figure 4 c,d). The maximum output voltage and power density of 4 V and 2.8 mW m −2 , respectively, were obtained at a load resistance of 1 MΩ. The voltage response of the WPI-TENG under finger tapping (without a glove) at a pressure of approximately 2 kPa is shown in Figure 5 a. A slight increase in voltage with an increase in the applied frequency under the contact-separation mode is observed, which might be due to the increased pressure and reduced number of neutralized charges with the increased frequency ( Figure 5 b). The output voltage of the TENG device with and without WPI film is shown in Figure 5 c. The increase in voltage in the case of the WPI film may be due to the reduced charge neutralization compared to without the WPI film. Figure 5 d shows the rectified (positive) signal using the Wheatstone bridge connection of the diodes connected to the Bio-TENG device. With an increase in load, an increase in voltage is observed, which is due to the increased surface contact area at the interface ( Figure 5 e). The TENG-based pressure sensor is accomplished by combining a laser-ablated PDMS surface with the WPI film. Unlike photolithography and template-assisted techniques, the laser ablation technique offers additional benefits such as by being rapid, facile, mask independent, low cost, and user friendly [ 37 ]. Instead of an IR laser, a CO 2 laser was subjected to ablate the PDMS surface. The reason is that PDMS material shows high transmittance to IR wavelength of 1064 nm, which would be difficult to process. On the other hand, PDMS shows high absorption to a 10.6 µm wavelength of a CO 2 laser. The schematic representation of the laser interaction with the PDMS surface is shown in Figure 6 a and its corresponding laser ablation showing the escaped vapors of PDMS under temperatures reaching above its melting point during laser beam irradiation is shown in Figure 6 b. The PDMS surface is laser irradiated with an array of scanning lines (vertical and horizontal) of 250 µm gaps over an area of 9 cm 2 ( Figure 6 c). Real and corresponding SEM micrographs are shown in Figure 6 d. It is evidenced that the rough profile of the PDMS surface has a spike-like morphology that enables pressure-like sensitivity. The performance of the TENG-based pressure sensor device is shown in Figure 6 e. The sensor showed a linear behavior up to a pressure of 10 kPa and remained almost constant beyond that value. The increase in voltage is due to the increased surface area of the textured PDMS under pressure. This agrees with the extent of pressure where the textured PDMS undergoes elastic deformation (region I, red color-fitted line) and later saturates (structural elastic limit) with no further compression within the material (region II, orange color-fitted line). The structural elastic limit for a typical viscoelastic material depends on the physical parameters and inherent properties of the material. The physical parameters are related to the morphology of the structure, such as shape and dimensions, and inherent properties are related to the chemical composition, such as the modulus and Poisson’s ratio. Diverse materials such as PDMS, Ecoflex, polyurethane, and even biodegradable materials, in various shapes such as pyramids, domes, cones, pillars, and nature-inspired architectures, have been adopted to widen the pressure sensing range for pressure-sensor applications [ 38 , 39 , 40 , 41 ]. The single-electrode pressure sensor device exhibited pressure sensitivities of 1.61 V kPa −1 and 0.14 V kPa −1 in regions I and II, respectively. In addition to PDMS, biodegradable elastomer materials such as poly(glycerol sebacate) and poly(octamethylene maleate (anhydride) citrate) may also be applied for surface texturing by laser ablation technique. The biomaterial and laser ablation techniques proposed in this study can enable wearable TENG devices with added benefits in terms of biodegradability and conformability."
} | 3,774 |
21864340 | PMC3184077 | pmc | 2,475 | {
"abstract": "Background The ubiquity of modules in biological networks may result from an evolutionary benefit of a modular organization. For instance, modularity may increase the rate of adaptive evolution, because modules can be easily combined into new arrangements that may benefit their carrier. Conversely, modularity may emerge as a by-product of some trait. We here ask whether this last scenario may play a role in genome-scale metabolic networks that need to sustain life in one or more chemical environments. For such networks, we define a network module as a maximal set of reactions that are fully coupled, i.e ., whose fluxes can only vary in fixed proportions. This definition overcomes limitations of purely graph based analyses of metabolism by exploiting the functional links between reactions. We call a metabolic network viable in a given chemical environment if it can synthesize all of an organism's biomass compounds from nutrients in this environment. An organism's metabolism is highly versatile if it can sustain life in many different chemical environments. We here ask whether versatility affects the modularity of metabolic networks. Results Using recently developed techniques to randomly sample large numbers of viable metabolic networks from a vast space of metabolic networks, we use flux balance analysis to study in silico metabolic networks that differ in their versatility. We find that highly versatile networks are also highly modular. They contain more modules and more reactions that are organized into modules. Most or all reactions in a module are associated with the same biochemical pathways. Modules that arise in highly versatile networks generally involve reactions that process nutrients or closely related chemicals. We also observe that the metabolism of E. coli is significantly more modular than even our most versatile networks. Conclusions Our work shows that modularity in metabolic networks can be a by-product of functional constraints, e.g., the need to sustain life in multiple environments. This organizational principle is insensitive to the environments we consider and to the number of reactions in a metabolic network. Because we observe this principle not just in one or few biological networks, but in large random samples of networks, we propose that it may be a generic principle of metabolic network organization.",
"discussion": "Discussion and conclusions Our work took advantage of a new computational method [ 30 , 40 ] that uses a combination of flux balance analysis and Markov Chain Monte Carlo sampling to create large and random samples of metabolic networks with desired properties from the space of all possible metabolic networks. The property we focused on was environmental versatility, the number of chemical environments a metabolic network can sustain life in. We studied how versatility relates to a network's modularity. For our purpose, we defined modularity as the total number of reactions contained in fully coupled sets. We found that more versatile networks are more modular (they have more modules and more reactions contained in modules) than less versatile networks. We emphasize that this does not result from the fact that networks with more reactions are more versatile, because our analysis uses networks with fixed number of reactions. The reactions that form part of newly arising modules in highly versatile networks tend to be close to reactions that process nutrients. The advantage of using random samples of metabolic networks with a specific property for our analysis is that such samples have not been subject to any of the (usually unknown) selection pressures that an organism's metabolism is subject to, and that they can form a useful reference point to ask whether any one organism's metabolic network has typical or atypical properties. In such a comparison, we learned that E. coli 's network is significantly more modular than random networks of the same versatility, a feature arising mainly from the fact that it contains larger modules. Modularity in metabolic networks has been studied by several other authors [ 6 - 10 ]. Metabolic networks can be represented as graphs, allowing one to study topological (graph-based) measures of modularity; this approach has been taken for metabolic and other systems, such as protein interaction networks. Unfortunately, for any sensible definition of modularity, graph-based module identification is typically computationally very expensive, so in practice one resorts to heuristic algorithms to extract modules [ 49 , 51 , 52 ]. Additionally, in graph-based representations of metabolism, many metabolites have very high degree (number of reactions they participate in). This feature may prevent any clear modules from arising, although various heuristic tricks, such as removing high degree metabolites [ 6 - 10 ] can be used to skirt this problem. Problems like these can be avoided by using functional measures of modularity. Commonly used measures involve elementary flux mode or extreme pathways [ 25 - 27 ], but they are ill-suited for genome-scale modeling because of the complexity in computing them. The measure of modularity we used here was based on the reactions contained in fully coupled sets (FCSs) [ 28 ]. We showed that most or all reactions in a fully coupled set fall within a single metabolic pathway, which underlines the biochemical relevance of our definition of modularity. Two further technical advantages come with our definition of modularity based on FCSs: (1) the approach involves no adjustable parameters; (2) identification of FCSs is computationally efficient even for genome-scale networks. Intriguingly, the extent of modularity found in E. coli is higher than in our in silico genomes. E. coli both has more fully coupled sets and larger fully coupled sets than expected for networks with the highest versatility we consider. This high modularity may reflect the fact that E. coli is even more versatile than the most versatile networks in our samples, networks that are viable on 89 carbon sources. For example, it can also grow on sources of sulfur or nitrogen that we did not consider. The high computational cost of our analysis in multiple environments currently prohibits us from extending our study to a larger spectrum of environments. Conversely, the high modularity of E. coli might also be caused by other factors, for example, a long record of past evolutionary adaptations that may favor modularity through the high rates of adaptation it may allow and/or its high heritability, e.g. through horizontal gene transfer [ 62 , 63 ]. Indeed it has been shown that FCSs and operons in E. coli are positively associated [ 29 , 57 , 58 ]. Only future work will be able to validate which of these causes is more important in E.coli . Our network sampling approach has the advantage that it provides a rational expectation for how modular a network can be expected to be based solely on phenotypic constraints. It thus puts answering this question within reach. Given the ubiquity of modularity in biological systems, it is tempting to propose general principles that might explain its appearance. By comparing natural with man-made systems and following the original insights of Jacob [ 12 ] and others [ 1 - 6 , 8 - 11 ], it seems very plausible that modularity should emerge during adaptive evolutionary trajectories because it can increase the rate of adaptive change. This holds true in particular in artificial systems such as factories, companies and even industries, where modularity allows for lower costs and enhanced possibilities for innovation [ 64 - 66 ]. As long as a lineage of organisms is experiencing adaptive evolutionary change, modularity should remain ubiquitous, whereas in long periods of stasis modularity may become reduced. This perspective is appealing but other factors may also influence modularity, which can be seen by considering the modularity of eukaryotic cells. The organization of cells into parts with specialized tasks (organelles, ribosomes, etc.) suggests that cellular tasks are best performed in specialized modules. One may thus conjecture that modularity has not only the indirect benefit of accelerating the rate of evolutionary change, but also direct benefits such as the possibility to perform certain tasks better, and thereby allow organisms to be better adapted to the complex world around them. The question whether biological modularity may have a direct benefit can be addressed in systems where a realistically complex yet computationally tractable genotype to phenotype relationship exists. Genome-scale metabolic network models are such systems [ 32 ]. Answering the question amounts to finding out whether the best performing genotypes (according to some criterion) have a modular architecture. The criterion we used is based on the complex trait we called environmental versatility, the number of environments a metabolic network can sustain life in. The answer we found is clear: Requiring viability in additional environments requires additional pathways or modules to metabolize more nutrients and thus versatility enhances modularity. Our analysis shows that modularity can be a by-product of versatility, at least in the framework of our metabolic modeling, because our system has no selective pressure on modularity per-se; highly versatile networks that are also highly modular are simply more numerous than the less modular ones. In the language of constraint satisfaction problems [ 67 ], constraints are easier to satisfy using modular architectures, so highly modular solutions will be more numerous than the less modular ones. An analogy with the engineering of network architectures may be appropriate here. Consider the circuit layout problem where a circuit's electronic components and wires must be placed on a chip. If no constraints are imposed on the circuit's speed, many different layouts are possible. But, if one focuses on the fastest circuits, one will find that they have shorter wires and are more modular, so modularity is a by-product of circuit speed. In this example, functional constraints change the architectural characteristics in the space of possible solutions. Such a property may be expected to arise in both artificial and natural systems. Since versatility corresponds to viability in increasing numbers of environments, it can be considered as a trait associated with fitness itself. Our work suggests that modularity can emerge as a consequence of increasing functional constraints. Because our work is not just based on one or few metabolic networks from well-studied organism, but on large samples of random viable networks, we also suggest that this scenario may be generally important. Recent observations by Parter et al. [ 9 ] and Kreimer et al. [ 10 ] where generalists prokaryotes living in many different environments are more modular than specialists are fully consistent with this conclusion."
} | 2,749 |
34588437 | PMC8481357 | pmc | 2,476 | {
"abstract": "Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae , is unknown.",
"introduction": "Introduction Bacterial biofilms are consortia of surface-associated cells bound together with an EPS matrix 1 – 3 . Although individual bacteria can translocate across surfaces using flagella 4 , type-IV pili 5 , 6 , surfactants 7 or gliding motility apparati 8 , biofilms are typically stationary. In some cases, however, biofilms have been reported to move. Small Pseudomonas aeruginosa microcolonies are dynamic, using type-IV pili to merge and split before settling down to form a sessile biofilm 9 . Myxococcus xanthus displays diverse multicellular and motile behaviors. Individual cells rely on gliding motility whereas social motility for bacterial predation and the formation of fruiting bodies primarily depends upon type-IV pili 10 , 11 . Neisseria species also use type-IV pili to form large microcolonies that move across a surface as discrete units and grow via merging 12 , 13 . In these examples, type-IV pili are important contributors to the movement of microcolonies. Type-IV pili function through the extension of pilin filaments from the cell body and attachment to the substrate 14 . The subsequent force of retraction and disassembly of the pilin subunit pulls the cell forward. The same principles apply to the motility of multicellular structures using type-IV pili 13 . Here, we describe a unique collective movement that does not involve pili or flagella, in the soil-dwelling bacterium Flavobacterium johnsoniae . Surface-associated, three-dimensional (3D) biofilm-like microcolonies form, move, merge and disperse within a 24-h period (Fig. 1 ). Individual F. johnsoniae cells move by gliding motility, which is critical during early aggregation of F. johnsoniae into microcolonies. Briefly, rod-shaped cells align parallel to the surface where a rotary gliding motor moves motility adhesins, such as SprB and RemA, around the cell on a helical track to generate cell movement 8 . By contrast, in the microcolony, cells contacting the surface at the base do not arrange horizontally but interact obliquely at a cell pole. The semi-vertical base cells suggest traditional gliding, in which cells are aligned in parallel with the surface to facilitate helical movement of adhesins, is not the primary translocation mechanism. However, perturbing gliding motor function either genetically or biochemically influences microcolony development, shape, and behavior. We conclude, therefore, that the rotary gliding motor plays a crucial role in microcolony motility. Fig. 1 The microcolonies self-assembled from F. johnsoniae wild-type (WT). a Schematic of a bacterial microcolony. The rod-like bacteria (gray) self-assemble into a microcolony (diameter, D and height, h ), with extracellular polysaccharide (EPS) cores (red) and semi-vertical cells (or base cells, the dashed-line box) at the base (diameter, d ). (Callout) The typical orientation of a single base cell relative to the substrate. b z-stack slices (8 total with 6 μm between slices) of a multiphoton microscopic image confirm the spherical, three-dimensional (3D) structure of a microcolony ( D ≈ 60 μm, d ≈ 36 μm, h ≈ 45 μm). The signal indicates reduced nicotinamide adenine dinucleotide (NADH) from cell metabolism. Scale bar, 50 μm. c The under-oil open microfluidic system (UOMS) with under-oil sessile microdrops for studying the growth dynamics of F. johnsoniae . Colored water (2 μl per spot, 2 mm in diameter) was used for visualization. PDMS represents polydimethylsiloxane. Scale bar, 1 cm. (Callout) Schematic of the under-oil environment around a sessile microdrop. d Microcolonies in a microdrop with fluorescent Concanavalin A (ConA) lectin staining (12 h after initiating culture growth). Scale bar, 1 mm. e z-stack high-magnification microscopic images of microcolonies in ( d the green box). Distinct EPS cores (ConA+) enveloped by attached and actively moving cells can be detected within microcolonies that do not perfectly overlap with total cell mass. Scale bar, 100 μm. The experiments in b , d , and e were performed three times for the representative result. Source data are provided as a Source Data file.",
"discussion": "Discussion In this study, we report the discovery of a mode of collective movement of bacteria. F. johnsoniae , a non-flagellated, non-piliated bacterium, self-assembles into 3D structures with EPS cores, exhibiting translational movement across surfaces on semi-vertical, not horizontal, base cells. The closest structural relative of the F. johnsoniae collective motility appears in Neisseria species and are called microcolonies. However, due to the basis of movement and orientation of base cells, we designate this subset of microcolonies as “zorbs” to capture their dimensionality and the appearance of being moved by cells that reflect the legs on a zorb. We propose that the zorbs are biofilms, based on the cohesive multicellular structure and localization of EPS as detected by ConA binding (Fig. 1e , Supplementary Movie 2 ). In fact, localized ConA staining at the core of a biofilm has been seen previously in Pseudomonas syringae , another soil bacterium 29 , in which ConA binds levan, a branched, fructan polysaccharide. Levan is an intriguing candidate for the matrix of a motile biofilm, as it is water soluble and remains fluid in solution, perhaps contributing flexibility as well as structural integrity 30 , 31 . Comparison of annotated levansucrase genes (GO:0050053) from other species of Flavobacterium to the F. johnsoniae UW101 genome identified fjoh_2883 (65-69% identity) as a possible levansucrase-encoding homolog. A combination of polysaccharide chemical analysis, mutant analysis of fjoh_2883 and biochemical intervention in WT zorbs with polymer hydrolyzing enzymes will determine matrix components of the motile F. johnsoniae zorbs. Such studies could illuminate tradeoffs between structural stability, flexibility, merging, and dispersal of F. johnsoniae motile zorbs. Collective motility of gliding bacteria has been studied most thoroughly in Myxococcus , which coordinates rotary gliding motors and type-IV pili to form predatory swarms and fruiting bodies 6 , 32 , 33 . In addition, a relative of F. johnsoniae, Capnocytophaga gingivalis , exhibits “cargo transport,” a behavior in which C. gingaivalis transports aggregates of non-motile species within the oral microbiome across a surface via gliding 23 . Although cargo transport might explain motility of zorbs, it is not supported by the data showing that F. johnsoniae cells at the base of zorbs do not align horizontally with the surface as do C. gingivalis cells during cargo transport. By contrast, the cells within motile zorbs are dynamic and the majority seem to interact with the surface by their poles (Fig. 4b , Supplementary Movie 10 ). This collective motility is not due to Brownian motion but rather is an active process as the motility increases with size of the zorb (Fig. 2f ) and is dependent on PMF (Fig. 3 ). The direction of the motility appeared random, similar to locomotion seen in immune cells 34 – 36 , although further work will determine whether directionality can be induced through chemical signals, which could provide clues to the function of zorb movement. Collective movement and merging of microcolonies on this scale, to our knowledge, has only been reported previously in Neisseria species 37 , 38 . Although the mechanism of Neisseria colony translocation differs from F. johnsoniae as Neisseria movement depends on pili 12 , the phenotypic parallel suggests evolutionary convergence and biological relevance of social motility. While zorb development could be caused by stresses generated by the under-oil microenvironment, and thus could be an environment-dependent adaptation, the discovery of motile microcolonies generated by a gliding bacterium expands our understanding of prokaryotic collective behavior. How the activity of gliding apparatus from individual F. johnsoniae cells generates the movement of an entire zorb remains unknown. The movement of the zorbs via base cells briefly gliding when attached to the surface has not been conclusively excluded. Additionally, the many base cells may act as focal adhesions successively binding to the substrate, not unlike the pili used by Neisseria . However, the observations of pinwheeling and the dynamic nature of the cells within the zorb could signify a “power stroke” force originating from base cells tethered by their rotary gliding motors that propel the zorb forward upon contact with the surface. This power stroke could originate from cells tethered either to the surface or the zorb itself. It is our assumption that individual cell forces are equal to the drag force, which requires further clarification in the future. Agnostic to mechanism, the computational simulations overcome limitations and assumptions of the Stokes law to determine more precisely that at least the equivalent of one third of the base cells act simultaneously to propel the collective group. Future experiments with fluorescent WT cells to track behavior of single cells within the zorb, especially at the base, will begin to differentiate among the possible mechanisms. In other systems, microcolony translocation is important for virulence, specifically in N. meningiditis and N. gonorrheae 37 , 38 , providing tantalizing evidence for the connection of social motility and host health. F. johnsoniae is a common rhizosphere resident, and whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsonaie , remains to be observed. Future work to determine the chemical and physical signals regulating zorb formation and motility could reveal environmental conditions favorable for this type of social motility and possible relevance for host-microbe interactions. Understanding the mechanisms of regulation could enable manipulation of the structure, and thus behavior, of F. johnsoniae and other gliding bacteria. Manipulating bacterial collective movement may contribute to strategies that improve predictable manipulation of microbial communities. The collective forces that translocate F. johnsoniae zorbs may contribute to areas beyond microbiology including designing functional soft materials 39 , cargo transport strategies 23 , and soft robotic systems 40 with dynamic self-assembly 41 ."
} | 2,932 |
35488805 | PMC9328739 | pmc | 2,477 | {
"abstract": "Summary The growing world needs commodity amino acids such as L‐glutamate and L‐lysine for use as food and feed, and specialty amino acids for dedicated applications. To meet the supply a paradigm shift regarding their production is required. On the one hand, the use of sustainable and cheap raw materials is necessary to sustain low production cost and decrease detrimental effects of sugar‐based feedstock on soil health and food security caused by competing uses of crops in the feed and food industries. On the other hand, the biotechnological methods to produce functionalized amino acids need to be developed further, and titres enhanced to become competitive with chemical synthesis methods. In the current review, we present successful strain mutagenesis and rational metabolic engineering examples leading to the construction of recombinant bacterial strains for the production of amino acids such as L‐glutamate, L‐lysine, L‐threonine and their derivatives from methanol as sole carbon source. In addition, the fermentative routes for bioproduction of N ‐methylated amino acids are highlighted, with focus on three strategies: partial transfer of methylamine catabolism, S ‐adenosyl‐L‐methionine dependent alkylation and reductive methylamination of 2‐oxoacids.",
"conclusion": "Concluding remarks In this review, we have presented how the C1 metabolism can be harnessed for the production of amino acids or their methylated derivatives, either by use of methylotrophic cell factories or activity of specific enzymes involved in methylotrophy. Regarding biosynthesis of methylated amino acids, we focused on three strategies relying on the activity of different enzymes or enzymatic cascades (i) GMAS and NMGS derived from methylotrophic M. extorquens where they function as part of methylamine assimilation pathway, (ii) ANMT and AAMT derived from plants or (iii) DpkA derived from P. putida where it functions in D‐lysine degradation. Here, supply of precursors and co‐factors, as well as the activity of the biosynthetic enzymes seem to play major roles in the process efficiency, becoming major strain engineering targets. As an outlook, we foresee that the development of methylated amino acids may respond to market needs to a certain extent. N ‐Methylated amino acids do not only play a role as free bioactives or in peptide drugs, but they may also be co‐translationally incorporated into proteins at specific locations by codon engineering (Hoesl and Budisa, 2012 ). For example, translational amber stop codons have been re‐coded using an evolved pyrrolysyl‐tRNA synthetase‐ pylT pair (Blight et al ., 2004 ) to incorporate meta ‐nitrophenylacetate‐photocaged N ε‐L‐lysine residues. Upon photolysis in vivo , the labelled proteins were converted to proteins with monomethylated lysine residues (Wang et al ., 2010 ). The strategies used for methanol‐based production of amino acids by natural methylotrophs generally include use of classical mutagenesis and selection of best‐performing strains, or expression of genes encoding feedback inhibition alleviated enzymes or amino acid exporters. In case of non‐natural products, such as the diamine cadaverine, or the non‐proteinogenic amino acids 5AVA and GABA, expression of heterologous pathways was necessary. Considering that all these compounds are bulk chemicals, with L‐glutamate and L‐lysine serving as food and feed additives, and cadaverine, 5AVA and GABA as building blocks of polyamines of platform chemicals, it is worthwhile to investigate their methanol‐based productions. Methanol is considered a promising raw material for bioprocesses due to its stable prices, easiness of transport and storage and the fact that it can be produced sustainably from non‐food sources. We foresee that the development of new and more efficient processes for production of amino acids from methanol will be driven by a technology push. Specifically, we anticipate that the use of various CRISPR technologies will revolutionize producer strain development (Schultenkamper et al ., 2019 ; 2020 ). Adaptive laboratory evolution (Hu et al ., 2016 ; Sandberg et al ., 2019 ; Hennig et al ., 2020 ; Wang et al ., 2020 ) and enforcement of production by coupling it to growth (Haupka et al ., 2020 ) will allow for efficient selection procedures of superior strains (Prell et al ., 2021 ). Moreover, development of novel genetic tools will facilitate strain engineering of methylotrophic production hosts (Irla et al., 2016 ; Irla et al., 2021 ). In addition, synthetic consortia of different microorganisms may be developed to divide labour, for example, between conversion of a substrate such as methanol to an intermediate by one microorganism and product formation from the intermediate by another (Sgobba and Wendisch, 2020 ). In this respect it has to be noted that methanol initially is oxidized to formaldehyde and there are other sources of formaldehyde that may be used as substrates for fermentation. However, formaldehyde has to be liberated from these, for example, by degradation of formaldehyde oligomers such as trioxymethylene and hexamethylenetetramine (Kaszycki and Koloczek, 2002 ) or by demethylation of vanillin and other methylated aromatic compounds that are present in lignin (Wendisch et al ., 2018a ; Costa et al ., 2021 ). Albeit attractive, this is clearly unchartered terrain and it is questionable whether these compounds will be available at reasonable cost and quantities. Taken together, production of amino acids from methanol and production of N‐ methylated amino acids has seen substantial success. It is anticipated that future developments driven by technology push and/or market demand will shape this exiting field of microbial biotechnology.",
"introduction": "Introduction Amino acid production by fermentation is a success story that started more than six decades ago (Lee and Wendisch, 2017 ). The market demand is steadily rising, even though African Swine Fever and the COVID‐19 pandemic slowed the growth. The very efficient L‐glutamate and L‐lysine production processes that are operated at a huge scale (million tons per year) benefit from the so‐called “economy of scale” (Wendisch, 2020 ). However, since the margins are very low, two trends have emerged: a shift from commodities towards specialty amino acids (Ajinomoto, 2020 ) and a shift from traditional substrates towards alternatives carbon sources (Wendisch et al ., 2016 ). Traditional amino acid fermentation is based on sugars and molasses and costs for these feedstocks contribute notably to the operational expenditures. Considering substrate availability, costs and competing uses in the food and feed industries, a flexible feedstock concept was realized for amino acid producer strains enabling access to sustainable alternatives, for example, lignocellulosic, aqua‐ and agricultural sidestreams (Wendisch et al ., 2022 ). Specialty amino acids find applications in the pharmaceutical industry (e.g. infusions, injections, intermediates in active substance syntheses or as active pharmaceutical ingredients). Among others, Escherichia coli and Corynebacterium glutamicum strains have been engineered to produce the blood pressure‐lowering L‐arginine (Park et al ., 2014 ), the insulinotropic (2 S , 3 R , 4 S )‐4‐hydroxyisoleucine (Smirnov et al ., 2010 ; Zhang et al ., 2018 ), 5‐hydroxy‐L‐tryptophan that can be used against depression and obesity (Mora‐Villalobos and Zeng, 2018 ), and the cyclic amino acid L‐pipecolic acid used as cell protectant and precursor of, for example, the immunosuppressant rapamycin and the antitumor agent swainsonine (Pérez‐García et al ., 2016 ; 2017 ; 2019 ). In this review, we address these trends by focusing on how methanol, a feedstock without competing food and feed uses, can be harnessed for production of L‐glutamate, L‐lysine, L‐threonine and their derivatives by bacteria. In recent years, there has been substantial progress in the development of methods for methanol synthesis particularly through not only CO 2 hydrogenation but also isothermal methane conversion into methanol catalysed by copper‐containing zeolites or production of methanol from crude glycerol (Haider et al ., 2015 ; Tomkins et al ., 2016 ; Mbatha et al ., 2021 ). In this review methanol‐based production of L‐serine, an intermediate of serine cycle for formaldehyde assimilation, will not be presented as it has been thoroughly summarized elsewhere (Eggeling, 2007 ). Moreover, we cover how access to N ‐methylated amino acids, a particular class of specialty amino acids, has been gained by metabolic engineering."
} | 2,160 |
31875145 | PMC6925950 | pmc | 2,478 | {
"abstract": "Coral reef ecosystems are impacted by climate change and human activities, such as increasing coastal development, overfishing, sewage and other pollutant discharge, and consequent eutrophication, which triggers increasing incidents of diseases and deterioration of corals worldwide. In this study, bacterial communities associated with four species of corals: Acropora aspera , Acropora formosa , Cyphastrea sp., and Isopora sp. in the healthy and disease stages with different diseases were compared using tagged 16S rRNA sequencing. In total, 59 bacterial phyla, 190 orders, and 307 genera were assigned in coral metagenomes where Proteobacteria and Firmicutes were pre-dominated followed by Bacteroidetes together with Actinobacteria , Fusobacteria , and Lentisphaerae as minor taxa. Principal Coordinates Analysis (PCoA) showed separated clustering of bacterial diversity in healthy and infected groups for individual coral species. Fusibacter was found as the major bacterial genus across all corals. The lower number of Fusibacter was found in A. aspera infected with white band disease and Isopora sp. with white plaque disease, but marked increases of Vibrio and Acrobacter , respectively, were observed. This was in contrast to A. formosa infected by a black band and Cyphastrea sp. infected by yellow blotch diseases which showed an increasing abundance of Fusibacter but a decrease in WH1-8 bacteria. Overall, infection was shown to result in disturbance in the complexity and structure of the associated bacterial microbiomes which can be relevant to the pathogenicity of the microbes associated with infected corals.",
"conclusion": "Conclusion Our findings showed structural alteration of microbiomes associated with important reef-building corals in Indonesian sea revealed by a culture-independent molecular analysis approach. Coral disease pathogenesis led to disturbance of the complex microbial communities and shifts in the overall bacterial community structures as shown by well separated clustering of the heathy and infected coral microbiomes with significant changes in certain key bacterial species. This study provides insights on understanding the functions of associated symbiont bacteria in pathogenesis. Further in-depth analysis on the role of microbiomes on coral health is warrant.",
"introduction": "Introduction Coral reefs contain important ecological space harboring a diversity of marine organisms and represents the core element of the complex marine ecosystem. Southeast Asia contains the largest area of coral reefs, accounting for 34% of the world’s total ( Wilkinson, 2008 ). The reefs play crucial roles in ecology such as protecting the coastline from erosion, providing habitats for marine organisms, and supplying nutrients for the complex marine food chains. The coral reef ecosystem is estimated to provide USD 29.8 billion per year of economic benefit worldwide from various sectors, e.g., fisheries, tourisms, coastal protection, and biodiversity ( Cesar, Burke & Pet-Soede, 2003 ). From a scientific point of view, corals are also considered a rich source of unique and unexplored biosynthetic products ( Radjasa et al., 2011 ). In this ecosystem, diverse symbioses exist with complex interactive dependences between corals and associated communities of eukaryotic and prokaryotic microorganisms ( Blackall, Wilson & Van Oppen, 2015 ). For example, dinoflagellate endosymbionts, Symbiodiniaceae ( LaJeunesse et al., 2018 )utilizes light energy ( Brodersen et al., 2014 ) and secrete fixed carbon to the coral host ( Burriesci, Raab & Pringle, 2012 ). This symbiosis creates a highly complex and unique ecosystem. In the last few decades, coral reefs have been facing world-wide crisis related to coral degradation driven by complicated phenomena including anthropogenic stresses and natural factors ( Wilkinson, 2008 ). It is estimated that the world has collectively lost 19% of the original area of coral reefs while 15% are seriously endangered with expected damage within the next 10–20 years and 20% are under threat of loss in 20–40 years ( Wilkinson, 2008 ). Degradation of coral reefs ecosystem can lead to massive loss of marine biodiversity. One of the serious threats to coral reefs ecosystem comes from various coral diseases ( Harvell et al., 2007 ; Hughes et al., 2003 ). The occurrence of these diseases has been increasing dramatically during the last decade due to the rising sea-water temperature as a consequence of climate change ( Rosenberg & Ben-Haim, 2002 ). Many of these diseases are prevalent in reef-builder corals (Order Scleractinia), leading to the deterioration of entire reefs structures ( Harvell et al., 2007 ; Rosenberg et al., 2007 ). Among them, black band, white band, white plague, white pox, brown band, red band, and yellow band syndromes are the main threats on corals in the Caribbean, Indo-Pacific, and Great Barrier Reef. Coral diseases can be triggered by infection of pathogenic agents involving specific groups of bacteria, fungi, and viruses ( Bourne et al., 2009 ; Harvell et al., 2007 ). Displacement of primary symbiotic microorganisms with other members has been shown to be related to the appearance of the disease signs. For example, the yellow blotch disease is caused by infection of Vibrio spp. which targets endosymbiotic zooxanthellae rather than the coral tissue, resulting in a decrease in chlorophyll concentration ( Cervino et al., 2004a ). Bacteria are considered as the major infectious microorganisms in corals. So far, only a limited number of bacteria have been isolated and identified as causative agents of coral diseases by Koch’s postulates, including cyanobacteria for black band disease, Vibrio sp and Acrobacter sp for white band disease, Vibrio spp for yellow blotch disease and Serratia marcescens for white pox ( Sheridan et al., 2013 ). However, the symbiotic nature of bacteria to corals makes them unculturable using current isolation techniques. Therefore, identification of the causative microbes by conventional culture-dependent approaches may not be able to give the complete scenario of coral associated microbial communities upon infection. Culture-independent analysis of environmental communities through phylogenetic molecular markers allows direct study of the uncultured microorganisms, which can make up to 99% of the total diversity in many ecosystems ( Schloss & Handelsman, 2005 ). Several studies have shown that culture-independent approaches based on bacterial molecular markers have been performed to explore the microbial communities associated with healthy and corals under stresses in the Caribbean and Indo-Pacific regions and their roles in pathogenesis ( Gignoux-Wolfsohn & Vollmer, 2015 ; Meyer et al., 2017 ; Pootakham et al., 2018 ). However, variation in the taxonomic composition of the microbial consortia related to existing coral diseases can depend on different geographical, seasonal, and physical factors of the coral habitats ( Sokolow, 2009 ; Woo et al., 2017 ). In order to gain more information on the roles of uncultured bacteria on coral diseases in Southeast Asia, the bacterial communities associated with corals collected from Indonesian sea, which is considered one of the world’s most important coral habitat, in healthy and diseased stages affected by black band disease (BBD), white band disease (WBD), white plaque disease (WPD),and yellow blotch disease (YBD) were explored in this study using tagged 16S rRNA sequencing on a next-generation sequencing platform. Our work compares coral-associated bacteria in different host species and shows shifts in the bacterial community structure during the diseased stage. Our findings expand the current understanding on the microbiology of these prevalent coral diseases.",
"discussion": "Discussion Coral diseases are the result of complex interactions between host, causative agents, and environment ( Martin, Meek & Willeberg, 1987 ; Sunagawa, Woodley & Medina, 2010 ). They are characterized by a shift in microbial communities in coral mucus and tissue. However, causes and consequences of this phenomenon to pathogenesis is usually not fully understood due to the complexity and dynamics of the associated bacteria as well as effects of abiotic factors. According to our study, shifts in bacterial communities were found in all taxonomic levels upon infection of all coral species by all diseases. WBD is an important coral disease causing loss of corals in many regions of the world. It has been reported to exhibit high host specificity, particularly Acropora species, including A. cervicornis and A. palmata in the Caribbean ( Kline & Vollmer, 2011 ). Based on the findings in our work, A. aspera showed a reduced diversity index for its associated bacteria upon infection by WBD. This was related to the increasing abundance of all sub-phyla of Proteobacteria along with the decreases in Firmicutes ( Clostridia ) and Bacteroidetes ( Flavobacteria ) with the presence of Fusibacteria found only in the diseased stage. This phenomenon occurred along with a significant increase in Vibrio and WH1-8 and decreasing Fusibacter upon infection. The causative agent of WBD is currently unknown but has been shown to possibly cause by bacteria according to a study on A. cerevicornis and A. palmata using antibiotic treatment ( Sweet, Croquer & Bythell, 2014 ). Vibrio spp. were consistently found in association with both healthy and diseased corals ( Cunning et al., 2008 ; Mouchka, Hewson & Harvell, 2010 ). Comparison of bacterial diversity in healthy and WBD-infected A. palmata based on 16s rRNA showed decreasing relative abundance of Betaproteobacteria and Actinomycetes with increasing abundance of Planctomycetes and Cyanobacteria ( Pantos & Bythell, 2006 ). A putative pathogen V. charcharii has been identified and partially proven as the causative agent for WBD type II based on Henle-Koch’s postulate ( Kline & Vollmer, 2011 ). However, the causative bacteria for WBD type I has not yet been identified. BBD is a polymicrobial coral disease which is considered one of the most virulent disease of scleractinian corals caused by polymicrobial factors which result in massive destruction of framework-building corals worldwide. Although factors affecting susceptibility of corals to BBD are still not fully understood, a few works showed that BBD pathogenesis is linked to nutrient enrichment, elevated temperature and light intensity. A recent study on Caribbean corals showed that the microbial communities in heathy corals were dominated by Gammaproteobacteria , particularly Halomonas spp. while the microbiome of BBD consortia were more variable and diverse ( Meyer et al., 2017 ). Studies using a culture-independent molecular approach showed a diverse microbial community classified into four functional groups, including photoautotrophs ( Cyanobacteria ), sulfate-reducers ( Desulfovibrio ), sulfide oxidizers ( Beggiatoa ) and organo-heterotropths ( Vibrio ) ( Sere et al., 2016 ). Among them, Desulfovibrio spp. and Vibrio coralliilyticus were suspected as the primary pathogens; however, without proven by Henle Koch’s postulate ( Sere et al., 2016 ). Basically, the complex microbial consortia act to produce highly concentrated sulfide specifically by the promoted Deltaproteobacteria under anoxic conditions beneath the BBD mat that are lethal to coral tissue ( Sato et al., 2017 ). The accumulation of sulfide underneath the BBD mat was partially due to the lack of sulfur oxidizers which contributes to the lethality of the disease ( Meyer et al., 2017 ). Meta-analysis of published clonal library studies of BBD microbial communities showed that, with few exceptions, the microbial species composition of BBD communities did not correlate with the species of the host corals with the domination of OTU of Roseofilum reptotaenium in over 70% of the samples ( Miller & Richardson, 2011 ). In addition, three OTUs of Bacteroidetes and Alpha-proteobacteria were present in 13% of the samples with other OTUs found in <7% of the samples. According to our study, decreasing relative abundance of Proteobacteria , particularly the subphyla Epsilon and Gammaproteobacteria, was found with increasing Firmicutes ( Clostridia ) in the A. formosa -BBD. This was related to increasing abundance of Fusibacter and WH1-8 bacteria upon the infection. Increasing abundance of Fimicutes , Cytophaga-Flexibacter-Bacteroidetes (CFB) and Deltaproteobacteria in the infectious mat was identified in a pioneered molecular analysis work using Terminal Fragment Length Polymorphism (T-RFLP) ( Frias-Lopez et al., 2004 ). The results showed that the composition of the infectious bacterial mat was not related to the species of coral being infected. Instead, differences in the mat composition appear to be linked to the species of cyanobacteria dominant in the infection. However, the presence of cyanobacteria in A. formosa -BBD could not be detected due to the specificity of primers used for amplification ( Nübel, Garcia-Pichel & Muyzer, 1997 ). No increase in Desufovibrio was observed in the 16S rRNA sequencing analysis of A. formosa -BBD in this study. According to a previous study ( Meyer et al., 2016 ), Desulfovibrioprofundus which is thought to be responsible the production of H 2 S was detected in only 5% of the clone libraries analyzed. These discrepancies could be due to technical issues (e.g., amplification biases and low coverage of microbiome in clonal libraries), seasonal and/or regional differences in the BBD composition or function-based (rather than taxonomic-based) of the BBD community. Variations have been detected in bacterial communities associated across geographical regions and between sympatric coral species ( Sere et al., 2016 ). High variability in the BBD bacterial communities in different geographical areas and coral species suggested that this disease derives from an earlier infection, which aids subsequent infection of opportunistic microorganisms such as cyanobacteria ( Sere et al., 2016 ). Recent analysis of metageomes from Caribbean and Pacific BBD mat revealed five metagenome-assembled genomes of Roseofilum , Proteobacteria and Bacteroidetes which are proposed to play symbiotic interaction ( Meyer et al., 2017 ). However, the mechanisms of BBD development remain unclear, and no primary pathogens have yet been identified. The difference on the results observed in this study to previous works could be due to the difference of environmental factors such as climatic condition and location ( Mouchka, Hewson & Harvell, 2010 ). YBD infection in corals resulted in a decrease in microbial diversity index in Cyphastrea sp.. This was along with reduction in the relative abundance of Proteobacteria and Bacteroidetes ( Cytophaga ) and increase in Firmicutes ( Bacilli ) along the infection. Vibrio alginolyticus has been identified as a causative agent for YBD ( Cervino et al., 2004b ). Four Vibrio species ( V. rotiferianus , V. harveyi , V. alginolyticus , and V. proteolyticus ) were identified as causative agents in YBD in Caribbean corals through a series of infection and isolation experiments ( Cervino et al., 2004b ; Cunning et al., 2008 ). It is suggested that this Vibrio consortium infected the coral’s symbiotic algae and resulted in degradation of zooxanthellae leading to the pale-yellow bands observed on infected Caribbean and Indo-Pacific corals ( Cervino et al., 2008 ; Cunning et al., 2008 ). However, the pathogenesis mechanism during YBD infection is still poorly understood. A survey of Vibrio species associated with healthy corals and YBD infected corals were conducted using a culture-based approach ( Cunning et al., 2008 ). The results showed a shift from isolates taxonomically affiliated with V. fortis dominate in healthy corals to those related to V. harveyi , a known marine pathogen in diseased corals. However, this study did not find any Vibrio species that are always present in YBD lesion but not in healthy corals. Although Vibrio have been reported as pathogenic bacteria for various coral diseases, it should be noted that they are a part of common bacterial taxa found in coral microbiomes ( Bourne & Munn, 2005 ; Daniels et al., 2011 ; Gray et al., 2011 ; Nithyanand & Pandian, 2009 ). The higher overall abundance of Vibrio in the healthy corals than the infected corals as found in our study could be expected due to the taxonomic refinement of the partial rDNA sequence was analyzed to the genus level only, not specifically to the specific group of pathogenic Vibrio species. The conflicting phenomenon on the higher abundance of Vibrionales in healthy corals has been previously reported in analysis of symbioints in corals infected by WPD ( Cardenas et al., 2012 ; Kellogg et al., 2013 ). WPD has been reported to affect more than 40 coral species ( Sunagawa et al., 2009 ; Weil, Smith & Gil-Agudelo, 2006 ). However, identification of its causative agent is still problematic, suggesting a complex etiological phenomenon involving alterations in the dynamic interaction between environmental factors, and symbiotic microbiomes. According to our study, reduction in the relative abundance of all subphyla of Proteobacteria was found in Isopora sp. upon infection by WPD with increasing abundance of Firmicutes ( Clostridia ) and changes in the phylum Bacteroidetes with decreasing Cytophaga and increasing Flavobacteria along with reduction in Lentispaheria . This change in the bacterial community structure led to an increase in Acrobacter and WH1-8 bacteria along with the decrease in Clostridia and Fusibacter at the genus level. Increasing diversity and a shift in bacterial community structure in Montastraea faveolata infected by WPS Type II has been shown using high-density 16s rRNA gene microarray and clone library sequencing ( Sunagawa et al., 2009 ). Accumulation of various known bacterial families known as coral pathogens including Alteromonadaceae and Vibrionaceae has been found. However, the primary pathogen Aurantimonas corallicida ( Denner et al., 2003 ) previously proven by Koch’s postulate was not detected in this molecular study ( Sunagawa et al., 2009 ). Analysis of differentially abundant OTUs in Caribbean coral species Orbicella faceolata and O. franksi showed marked differences in bacterial communities in the heathy and diseased coral samples but not between coral species ( Sunagawa, Woodley & Medina, 2010 ). The subsequent comparison in Indo-Pacific coral species ( Pavonaduerdeni and Poriteslutea ) showed distinct bacterial community patterns associated with ocean basin, coral species and health status. Increasing bacterial richness was found in the diseased samples suggesting the role of opportunistic bacteria during pathogenesis. These studies showed microbial community patterns related to WPD that are consistent over coral species and oceans, irrespective of the putative underlying pathogens ( Sunagawa, Woodley & Medina, 2010 ). This includes various taxa involving Proteobacteria , Bacteroidetes , Cyanobacteria , and Firmicutes . A higher abundance of known coral pathogens, e.g., Alteromonadaceae , Rhodobacteraceae , and Vibrionaceae have been reported in Pavona duerdeni and Porites lutea infected by WPD using 16S rRNA gene microarray ( Roder et al., 2014 ). A significant increase in Alphaproteobacteria and a concomitant decrease in the Beta - and Gammaproteobacteria were also observed in WPD-affected reef-building corals Diploriastrigose and Siderastrea sidereal using culture-dependent methods and pyrosequencing of 16s rRNA sequences ( Cardenas et al., 2012 ). Significant shifts were also found for the orders Rhizobiales , Caulobacteriales , Burkhoderiales , Rhodobacteriales , Aleteromonadales , and Xanthomonadales , suggesting roles of these bacteria on pathogenesis."
} | 5,008 |
22473793 | null | s2 | 2,479 | {
"abstract": "The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production."
} | 500 |
28747899 | PMC5506826 | pmc | 2,480 | {
"abstract": "The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans , grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans , it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans , the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the −35 (or −24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.",
"introduction": "Introduction Among the biomining microorganisms, the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans , that obtains its energy from the oxidation of ferrous iron [Fe(II)] and inorganic sulfur compounds (ISCs), is considered as a model. The molecular mechanisms underlying the pathways involved in aerobic Fe(II) and ISCs oxidation in At. ferrooxidans have been deciphered (reviewed in Quatrini et al., 2009 ; Bonnefoy, 2010 ; Bird et al., 2011 ; Bonnefoy and Holmes, 2012 ; Nitschke and Bonnefoy, 2016 ). The expression of the genes involved in these respiratory systems is regulated according to the available electron donor in the environment i.e., the genes involved in Fe(II) oxidation are more expressed in Fe(II)- than in sulfur-grown cells and vice-versa, those involved in ISCs oxidation are preferentially transcribed in the presence of sulfur than Fe(II) (Yarzabal et al., 2004 ; Quatrini et al., 2006 , 2009 ; Bruscella et al., 2007 ; Sandoval Ponce et al., 2012 ). Furthermore, when both Fe(II) and sulfur are present in the medium, Fe(II) is immediately oxidized while sulfur oxidation takes place only after Fe(II) was completely oxidized to ferric iron [Fe(III); (Yarzabal et al., 2004 ; Sandoval Ponce et al., 2012 )]. This observation is supported by the gene expression profile since the genes involved in Fe(II) oxidation are immediately transcribed while those involved in ISCs oxidation are expressed after Fe(II) was oxidized (Yarzabal et al., 2004 ; Sandoval Ponce et al., 2012 ). The expression of the genes involved in Fe(II) and ISCs oxidation appears therefore to depend on the oxidation state of iron [Fe(II) or Fe(III)] present in the medium. We have proposed that a transcriptional regulator is activated when iron is present as Fe(II) and induces the expression of the genes involved in Fe(II) oxidation, while repressing those responsible of ISCs oxidation (Sandoval Ponce et al., 2012 ). When iron is present as Fe(III), this regulator is inactive and therefore the genes involved in Fe(II) oxidation are no longer induced, whereas those involved in ISCs oxidation are derepressed (Sandoval Ponce et al., 2012 ). This regulator is supposed to bind specifically to the regulatory region of the genes involved in Fe(II) and ISCs oxidation. The redox-responding global two-component signal transducing system RegB/RegA [functionally similar to PrrB/PrrA and RegS/RegR; reviewed in (Swem et al., 2001 ; Elsen et al., 2004 ; Wu and Bauer, 2008 )] could be involved in this regulation since in At. ferrooxidans , the redox potential increases during Fe(II) oxidation to Fe(III) but remains stable during sulfur oxidation (Sandoval Ponce et al., 2012 ). In addition, the genes encoding a redox-sensing two-component signal transducing system belonging to the RegB/RegA family was detected in the At. ferrooxidans genome sequence (Quatrini et al., 2009 ). As shown by multiple sequence alignment, the predicted RegB/RegA proteins possess all the properties of their characterized homologs (Sandoval Ponce et al., 2012 ). Indeed, RegB (AFE_3136) has a transmembrane redox-sensing domain with a quinone binding site, and a transmitter cytoplasmic domain with the conserved site of autophosphorylation (the Hbox with one histidine), the redox active box with one cysteine and the ATP binding site. The response regulator RegA (AFE_3137) has a characteristic C-terminal helix-turn-helix motif DNA binding domain, as well as a N-terminal receiver domain with an “acid box” containing the aspartate residue (D68) which accepts the phosphate from the transmitter domain of the sensor RegB and a linker region between the receiver and the activation domains (Figure S1 ). Close to the linker region is the conserved alanine residue (A102) (Figure S1 ) which, when mutated to serine, locks RegA in a stable conformation that mimics the phosphorylated state of the wild type protein. RegA-A102S allows the constitutive expression of the target genes in the absence of RegB and have a similar DNA binding affinity as the phosphorylated wild type RegA (reviewed in Elsen et al., 2004 ; Wu and Bauer, 2008 ). In At. ferrooxidans , the regBA operon is not only expressed at higher levels in the presence of Fe(II) than sulfur (Amouric et al., 2009 ; Quatrini et al., 2009 ), but is also immediately transcribed in the presence of these two electron donors (Amouric et al., 2009 ), suggesting an autoregulation as observed for this operon in other bacteria. Finally, preliminary data support that the response regulator RegA (PrrA or RegR) binds to the regulatory region of some genes/operons involved in Fe(II) or ISCs oxidation (Amouric et al., 2009 ; Sandoval Ponce et al., 2012 ). The global redox-responding two-component regulatory system RegB/RegA could therefore be involved in the hierarchy observed in the utilization of the electron donor in At. ferrooxidans and could regulate the expression of the genes involved in Fe(II) and ISCs oxidation in response to the intracellular redox state. The RegB/RegA global regulation system has been extensively studied in photosynthetic bacteria (reviewed in Swem et al., 2001 ; Elsen et al., 2004 ; Wu and Bauer, 2008 ). The RegA regulons of Rhodobacter sphaeroides (Imam et al., 2014 ), Rhodobacter capsulatus (Schindel and Bauer, 2016 ), and Bradyrhizobium japonicum (Torres et al., 2014 ) were characterized recently. In both cases, RegA was shown to regulate genes/operons involved mainly in energy-generating systems (e.g., photosynthesis, respiration, electron transport) and energy-utilizing systems (e.g., nitrogen and carbon fixation systems, iron homeostasis, motility). The sensor kinase RegB detects the redox signal in the membrane through the ubiquinone pool (Swem et al., 2006 ; Wu and Bauer, 2010 ) and in the cytoplasm through a redox active cysteine located in its cytoplasmic domain (Swem et al., 2003 ; Wu et al., 2013 ). Once activated, RegB autophosphorylates and then transfers phosphate to the global response regulator RegA that binds to its target DNA. Different effects of phosphorylation on RegA were reported (reviewed in Elsen et al., 2004 ; Wu and Bauer, 2008 ): (i) phosphorylation was shown to increase RegA DNA binding affinity/stability; (ii) both phosphorylated and unphosphorylated RegA could alter gene expression by binding to different sites (Swem et al., 2001 ; Ranson-Olson and Zeilstra-Ryalls, 2008 ; Schindel and Bauer, 2016 ); (iii) the activation of transcription of the target genes was enhanced but not the RegA binding to DNA. Such is the case for the cbbI and cbbII operons in R. sphaeroides for which RegA is required to activate transcription of the target gene by interacting with the LysR family protein CbbR, a specific transcriptional regulator of the cbb operons. This interaction enhances the affinity and the stability of CbbR for the cbb promoter DNA (Dangel and Tabita, 2009 ; Dangel et al., 2014 ). The phosphorylation of RegA leads to conformational changes in the CbbR/DNA complex that allow the recruitment of the RNA polymerase and therefore the transcriptional activation of the cbb operons. Such a synergy between RegA and an activator could also be proposed in the case of the energy generating systems encoded by cydAB and ccoNOPQ in R. capsulatus (Swem and Bauer, 2002 ), hemA (Ranson-Olson and Zeilstra-Ryalls, 2008 ) and nirK (Laratta et al., 2002 ) in R. sphaeroides and the energy-utilizing system encoded by nifA2 in R. capsulatus (Elsen et al., 2000 ). RegA could also function in synergy with a repressor as shown in R. capsulatus for the puf promoter involved in the synthesis of the photosystem (Gregor et al., 2007 ). RegA could also behave as an anti-repressor by competing with the binding of a repressor. Such is the case in the transcriptional activation of the energy-producing systems encoded by cioAB in P. aeruginosa (Comolli and Donohue, 2002 ) or puc operon in R. capsulatus (Bowman et al., 1999 ). RegA could also repress transcription by competing with the binding of an activator as shown for the energy-producing systems encoded by the hupSLC (Elsen et al., 2000 ) or dorCDA (Kappler et al., 2002 ) operons of R. capsulatus . In this work, to better understand how the RegB/RegA redox-responding global two component system controls the respiratory pathways in At. ferrooxidans , the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the promoters of the genes involved in Fe(II) and ISCs oxidation pathways have been compared by electrophoretic mobility shift assays. In addition, to determine whether RegA is a repressor or an activator of the transcription of these genes, binding of RegA relatively to the RNA polymerase binding site has been analyzed. Based on the results obtained, a model is proposed to explain the RegA mediated regulation of the Fe(II) and ISCs oxidation pathways in At. ferrooxidans .",
"discussion": "Discussion The RegB/RegA system regulates the genes involved in Fe(II) and inorganic sulfur compound oxidation in At. ferrooxidans Given that the redox potential increases during Fe(II) but not sulfur oxidation and because At. ferrooxidans oxidized Fe(II) before sulfur, we had put forward the assumption that the global redox responding RegB/RegA signal transduction system is involved in the regulation of the genes implied in Fe(II) and ISCs oxidation (Sandoval Ponce et al., 2012 ). In this paper, we have substantiated this hypothesis since the DNA binding domain of RegA (RegA-HTH; Figure 2 ) as well as the full length RegA (Figure 3 ) have been shown to bind specifically to the regulatory region of a number of genes/operons required for Fe(II) and ISCs oxidation and which are differentially expressed depending on the electron donor present in the medium (Quatrini et al., 2009 ). These include not only the operons/genes encoding the redox proteins allowing electron transfer from Fe(II) ( rus ) or ISCs ( cyo, cyd ) to oxygen or to NAD + ( petI ), but also those encoding enzymes allowing ISCs oxidation ( sqr, doxII, hdr, tet ) and those enabling the biogenesis of the hemes present in the terminal oxidases ( cta, cyo ). Furthermore, RegA-HTH and RegA specifically recognized the regulatory region of the regBA operon which expression is higher in the presence of Fe(II) than Fe(III) or sulfur (Amouric et al., 2009 ), suggesting an autoregulation as observed for the characterized regBA operons (reviewed in Elsen et al., 2004 ; Wu and Bauer, 2008 ). RegA multimerizes on its target DNA By electrophoretic mobility shift assays with RegA-HTH and RegA (Figures 2 – 4 ) and by dynamic light scattering of RegA (Table 2 ), we have shown that RegA multimerizes on its target DNA. This multimerization of RegA on its target DNA was also observed in the case of the R. capsulatus cbbI operon (Dangel and Tabita, 2009 ; Dangel et al., 2014 ). It could suggest either multimerization of RegA at a single site on its target DNA and/or multiple RegA binding sites on the DNA fragments used. Unfortunately, the search of putative RegA binding sites in the promoter regions under study failed. This is likely due to the degeneracy of the RegA DNA binding sequence. It is noteworthy that, while the helix-turn-helix domain of RegA in R. capsulatus , PrrA in R. sphaeroides and RegR in B. japonicum are 100% identical (Emmerich et al., 2000a ), the DNA binding sites of RegA (SSGNVRDNHYSNCSS; Schindel and Bauer, 2016 ), PrrA (YSCGGC(5)GWCRMA; Eraso and Kaplan, 2009 ) and RegR (GNGRCRTTNNGNCGC; Emmerich et al., 2000b ) are degenerated and dissimilar. Phosphorylation of RegA does not affect its binding to DNA Multimerization of RegA did not require phosphorylation since multimerization was also observed with the inactive form of RegA that can no longer be phosphorylated (6His-SUMO-RegA_D68A) and with the phosphorylated-like form of RegA, which conformation was proposed to mimic the phosphorylated state of the wild type protein (6His-SUMO-RegA_ A102S; Figure 3 ). In addition, the phosphorylation of RegA did not affect its binding to DNA since 6His-SUMO-RegA_D68A had a similar affinity for its target DNA than 6His-SUMO-RegA_ A102S, and the wild type RegA (6His-SUMO-RegA; Figure 3 ). Therefore, in At. ferrooxidans , like in R. capsulatus (Dangel and Tabita, 2009 ), the phosphorylation of RegA did not lead to an increase of its affinity for its specific DNA target. RegA being a global regulator, we propose that RegA acts in concert with another regulator. Like in R. capsulatus (Dangel and Tabita, 2009 ; Dangel et al., 2014 ), phosphorylation of RegA might play a role in the formation/stability of the RNA polymerase-promoter complex. RegA does not compete with RNA polymerase binding to the promoter region Having validated the hypothesis that the RegB/RegA system is controlling the genes involved in Fe(II) and ISCs oxidation, we then wondered whether RegA behaves as an activator or a repressor. Therefore, we have investigated its binding on the region corresponding to the RNA polymerase binding site. As shown in Figure 4 , in most cases RegA did not bind downstream of the −35 (or −24) box, suggesting that it is not competing with the binding of the RNA polymerase. Two exceptions have been noticed: the 140 and 251 bp fragments of the regulatory regions of the rus and tet operons, respectively. The 140 bp fragment, while overlapping the RNA polymerase binding site of PI, was just 129 bp upstream of the −35 box of the rus operon PII promoter. We could then consider that the RegA binding site on the 140 bp fragment concerned the transcription from PII rather than from PI. In agreement with this notion, it is mostly the PII, and not the PI, promoter which is regulated by Fe(II) (Yarzabal et al., 2004 ). Therefore, it can be hypothesized that RegA and the RNA polymerase do not compete for binding at PII promoter of the rus operon. Model proposed for the RegB/RegA regulation of the At. ferrooxidans T energy pathways Apart from the tet operon, we have shown that RegA was not repressing the genes/operons under study ( rus, pet, cta, reg, hdr, hdrB, sqr, doxII, cyo , and cyd ) by competing with RNA polymerase binding on their promoter(s). Therefore, RegA behaves either (i) as an anti-activator by competing with the binding of an activator (Elsen et al., 2000 ; Kappler et al., 2002 ), (ii) as an anti-repressor by competing with the binding of a repressor (Bowman et al., 1999 ; Comolli and Donohue, 2002 ), (iii) in synergy with a specific inducer (Elsen et al., 2000 ; Laratta et al., 2002 ; Swem and Bauer, 2002 ; Ranson-Olson and Zeilstra-Ryalls, 2008 ; Dangel and Tabita, 2009 ; Dangel et al., 2014 ) or (iv) in concert with a repressor (Gregor et al., 2007 ). To determine how RegA functions to control the genes/operons involved in Fe(II) and ISCs oxidation in At. ferrooxidans T , the identification of the specific regulator involved is required. In the case of petI operon, the transcription factor Fur controlling iron uptake and homeostasis is likely the co-regulator of RegA since it was shown to bind also to petI promoter (Lefimil et al., 2009 ). One Fur box was predicted at the level of the −10 site of the σ70 box (Table 3 ), indicating that Fur inhibits RNA polymerase binding at petI promoter (Lefimil et al., 2009 ). Fur is described as repressor in the presence of high intracellular levels of Fe(II). RegA could therefore impede Fur binding on the regulatory region of the petI operon when Fe(II) is present. Interestingly, a gene encoding a Rrf2 family regulator (AFE_3141) was predicted in the cta operon (Quatrini et al., 2009 ). Members of this family sense different environmental signals, in particular intracellular iron availability (Johnston et al., 2007 ; Hibbing and Fuqua, 2011 ). It is therefore tempting to propose that the Rrf2 family regulator encoded by the cta operon is iron responsive and regulates the clustered cta, reg , and rus operons and that Rrf2 binding is impeded by RegA in Fe(II) condition. The tet operon is σ54-dependent. Therefore, its initiation requires an enhancer binding protein of the AAA-class. This activator binds usually relatively far upstream of the transcriptional start site and couples the energy produced from ATP hydrolysis to remodel the initial stable inactive conformation of the σ54-RNA polymerase bound to the −24 and −12 to a transcriptionally proficient open complex (Bush and Dixon, 2012 ). In the case of the tet operon, this enhancer binding protein is likely the σ-54 dependent transcriptional response regulator encoded by the AFE_0027 gene located upstream of tetH gene. In addition, the integration host factor IHF is likely required to bend the DNA to allow interaction between the σ54-dependent transcriptional response regulator and the σ54-RNA polymerase since an IHF motif is predicted upstream of the −24 box of tet (Table 3 ). Our results suggest that RegA could block the initial closed σ54-RNA polymerase-promoter DNA complex in an inefficient conformation by preventing its interaction with the response regulator leading to the inhibition of the transcription of the tet operon in Fe(II)-growth condition. When Fe(II) is oxidized, RegA is not synthesized, the enhancer binding protein encoded by AFE_0027 could then interact with the initial closed σ54-RNA polymerase-promoter DNA complex and activate the tet operon transcription when the signal is detected by its cognate sensor histidine kinase encoded by AFE_0026. Upstream of the cyd operon a σ54-dependent transcriptional regulator (AFE_0957) was also predicted. However, while in the case of the tet operon, RegA could block the σ54-RNA polymerase in an inefficient conformation (see above and Figure 4B ), this seems not to be the case for cyd operon (Figure 4B ). We suggest that RegA could compete with the binding of either the σ54-dependent inducer (AFE_0957) or of IHF, which bends the DNA to allow interaction between the σ54-dependent transcriptional response regulator and the σ54-RNA polymerase (an IHF motif is predicted upstream of the −24 box of cyd , Table 3 ), leading to the repression of cyd operon transcription when Fe(II) is present. Concerning the other genes/operons involved in ISCs oxidation ( hdr, hdrB, sqr, doxII , and cyo ), which are σ70-dependent, no gene encoding a transcriptional regulator could be predicted inside the operon or in their immediate proximity. The identification of the other regulator(s) of these genes/operons merits further investigation. The results of this study strongly support the involvement of the RegB/RegA redox-responding global two-component regulatory system in the control of the genes/operons involved in the Fe(II) and ISCs oxidation pathways. Apart from the tet operon, RegA binding to the promoter of these genes/operons seems to not impede RNA polymerase binding. From the available data, it is tempting to propose that, in the presence of Fe(II) (i.e., low redox potential), RegA role is to block Fur or Rrf2-like repressor binding on the promoter(s) of the genes involved in Fe(II) oxidation and to prevent inducer binding on the promoter(s) of the genes involved in ISCs oxidation. RegB/RegA system in other acidithiobacilli Noteworthy, the genes encoding the global redox responding RegB/RegA signal transducing system were not detected in the available genomes of At. caldus and At. thiooxidans . In addition, it has been reported that in At. caldus , the tet operon has a σ70-, and not a σ54-type promoter and is regulated by the two-component system RsrS-RsrR belonging to the EnvZ-OmpR family (Wang et al., 2016 ). Therefore, it seems that the regulation of the genes involved in ISC oxidation is different in iron and non-iron oxidizing acidithiobacilli. Furthermore, we have noted that, in the genome of the iron-oxidizing acidithiobacilli, regBA genes are always located in the same cluster than rus and cta operons, both involved in Fe(II) oxidation. This strengthens the idea that RegB/RegA are linked to the dissimilatory Fe(II) oxidation. The fact that the genetic organization of this cluster is conserved suggests that rus, cta , and reg operons were acquired together. We put forward the hypothesis that rus, cta , and reg operons are transcribed simultaneously to allow the iron-oxidizing acidithiobacilli to coordinate optimally its energy metabolism depending on the environmental conditions."
} | 5,661 |
38280843 | PMC10821886 | pmc | 2,483 | {
"abstract": "Natural microbial ecosystems harbor substantial diversity of competing species. Explaining such diversity is challenging, because in classic theories it is extremely infeasible for a large community of competing species to stably coexist in homogeneous environments. One important aspect mostly overlooked in these theories, however, is that microbes commonly share genetic materials with their neighbors through horizontal gene transfer (HGT), which enables the dynamic change of species growth rates due to the fitness effects of the mobile genetic elements (MGEs). Here, we establish a framework of species competition by accounting for the dynamic gene flow among competing microbes. Combining theoretical derivation and numerical simulations, we show that in many conditions HGT can surprisingly overcome the biodiversity limit predicted by the classic model and allow the coexistence of many competitors, by enabling dynamic neutrality of competing species. In contrast with the static neutrality proposed by previous theories, the diversity maintained by HGT is highly stable against random perturbations of microbial fitness. Our work highlights the importance of considering gene flow when addressing fundamental ecological questions in the world of microbes and has broad implications for the design and engineering of complex microbial consortia.",
"introduction": "Introduction Natural environments harbor a substantial diversity of competing microbes 1 – 3 . Explaining such diversity is challenging: in classic ecological models, it is highly infeasible for a large number of competing species to stably coexist 4 – 6 . Several mechanisms have been proposed to resolve this apparent paradox. For instance, the niche-based mechanism assumes the resource or space partitioning among different species, which, in essence, reduces the interspecies competition 7 – 9 . This explanation, however, is challenged by the coexistence of numerous competing species in homogeneous environments with a small number of niches 10 , 11 . Alternatively, neutral theory assumes that different species have similar fitness 12 , 13 . However, due to the short doubling time and large population size of microbes, even small fitness differences can result in the fast domination of the fittest species 14 , 15 . The diversity of competing microbes is hard to explain from the static view of species fitness. However, many microbes are characterized by the fluid nature of their genomes due to the substantial interspecies or intraspecies flow of genetic materials mediated by horizontal gene transfer (HGT) 16 – 18 . For instance, ~43% of the genes in Escherichia coli’s pan-genomes are acquired by HGT 19 . In human gut microbiome, over 22,000 genes were estimated to be mobilizable 20 . The flow of mobile genes that encode growth benefits or burdens enables a dynamic change of microbial fitness within ecological timescales, which might, in turn, impact the competition outcome 21 . However, despite its conceptual importance, how HGT influences the diversity of competing species remains largely unknown. Many studies have documented the rapid evolutionary change of species fitness on ecological timescales, which leads to the emergence of eco-evolutionary dynamics 22 – 26 . For instance, in sticklebacks 22 , 23 , guppies 24 , and cichlids 25 , 26 , the eco-evolutionary feedbacks have been widely observed. A microbial species can also actively change its niche by modifying its nutrient uptake and metabolism 27 , 28 . The dynamic change of species fitness can have a significant influence on ecological outcomes, including species coexistence. HGT is a key mechanism that mediates the eco-evolutionary interplay in microbes 17 . However, a theoretical framework allowing the analysis of HGT’s effects on microbial diversity remains lacking. Understanding the interplay between HGT and microbial coexistence has implications in broad scenarios. For instance, bacterial resistance to antibiotics has become one pressing crisis facing human health. Although competing with each other in host environments, sensitive and resistant strains coexist stably, and the resistant strain often persists long even after the antibiotic selection has been removed 29 . Not knowing why sensitive and resistant strains coexist stably has become one major barrier to combat antibiotic resistance 30 . Unraveling the role of HGT in bacterial coexistence might provide insights for the design of therapeutic strategies. Indeed, antibiotic-resistance genes are often closely associated with mobile genetic elements (MGEs) like plasmids 31 . In microbiome engineering, synthetic microbial consortia have emerged as a promising tool for the production of valuable chemicals 32 . However, their applicability has been constrained by our limited ability to stably maintain the diversity of the designed communities 33 . Understanding how HGT influences microbial coexistence might provide opportunities to overcome this limitation. Here, we established a framework of species competition by accounting for dynamic gene flow among microbes. We started by studying two-species systems and generalized our analysis to random communities of multiple members. Combining theoretical derivation and numerical simulations, we demonstrated that HGT could overcome the biodiversity limit predicted by the classic model and allow the coexistence of many competitors. In contrast with the neutral theory, the diversity maintained by HGT is stable against the fluctuations of species fitness. Our results underscore the fundamental role of gene flow in shaping the ecological dynamics and evolution of microbial communities.",
"discussion": "Discussion Our work proposed an ecological mechanism of maintaining microbial diversity via gene transfer and showed the conditions where this mechanism would potentially be effective. When mobile genes only affect species growth rates, HGT allows the stable coexistence of many competing species beyond the diversity limit predicted by the classic theory by promoting dynamic neutrality of microbial fitness. In fluctuating environments, the dynamic shuffling of mobile genes across species provides a buffering mechanism against the collapse of community diversity. Our results underscore the need to consider gene flow when studying ecological dynamics and evolution of microbial communities. Recent studies have observed extensive diversity in microbial populations that seem to occupy similar niches. For instance, in Vibrio and Synechococcus communities, many strains within the same species can stably coexist in nearby spatial locations despite the fitness differences among these strains 10 , 36 . Our work provides a plausible explanation for such seemly puzzling diversity in these populations. Indeed, comparative genomics suggested that HGT within these populations is prevalent 10 , 36 . The empirically estimated gene transfer rates (denoted as \\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}$${\\eta }^{c}$$\\end{document} η c ) need to be multiplied by \\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}$${N}_{m}$$\\end{document} N m before being plugged into our model (see Supplementary Information for more details). Here \\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}$${N}_{m}$$\\end{document} N m is the maximum carrying capacity of the population and has the unit of cells per mL. Therefore, the transfer rates \\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}$$\\eta$$\\end{document} η in our model are several orders of magnitude higher than those measured in previous studies 37 , 38 . When \\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}$${N}_{m}$$\\end{document} N m is large, even slight \\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}$${\\eta }^{c}$$\\end{document} η c can significantly change the coexistence feasibility. In constrast, with small \\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}$${N}_{m}$$\\end{document} N m , the effects of \\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}$${\\eta }^{c}$$\\end{document} η c can become negligible (Supplementary Fig. 4a ). Using two empirical estimates of plasmid conjugation rates from a previous study 37 , our numerical simulations suggest that the empirical HGT rates are sufficient to promote coexistence in a wide range of natural conditions 37 , 39 (Supplementary Fig. 4b, c ). We also explored the influence of MGE fitness effects on the effective range of transfer rates. Our simulations show that when MGEs are beneficial, extremely low HGT rates can be effective in promoting diversity (Supplementary Fig. 4d ; see Supplementary Information for more details). These results suggest that the role of HGT may become prominent in many environments, especially those with high cell density and beneficial MGEs. However, for burdensome MGEs in environments with very low cell densities, the contribution of HGT can be less important than other mechanisms like growth tradeoffs or cross-feeding 40 , 41 . Natural microbial communities are often faced with constantly varying environmental conditions that affect the ecological parameters, such as species growth rates. In order for diversity to be maintained stably, the population needs to be insensitive to the perturbations of the parameters 42 – 44 . Mathematically, such robustness translates into coexistence feasibility or structural stability, which measures the volume of the parameter space that allows the positive abundances of all species 42 – 45 . We noted that structural stability, by definition, is different from dynamical stability or local asymptotic stability, which refers to the ability of a system to recover after perturbation in species relative abundances 42 , 43 . While the local asymptotic stability has been extensively studied in many systems 5 , 46 , 47 , the determinants of structural stability have been less understood. In this work, we explicitly show that HGT might promote the structural stability of microbial communities. How HGT influences the local asymptotic stability remains an open question for future studies. In the two-species model, we assumed that the metabolic burden or benefit of an MGE was independent of the host species or strains. However, in nature, the same MGE can have different fitness effects in different genetic backgrounds due to epistasis 48 – 51 . To evaluate the influence of this assumption on the conclusion, we built a model that accounted for two types of epistasis: magnitude epistasis, where the host genomic background only influences the magnitude but not the sign of the fitness effect, and sign epistasis, where the same MGE causes growth burden in one species or strain while brings fitness benefit in the other (see Supplementary Information for more details). Our numerical simulations with randomized parameters suggest that how HGT affects coexistence is dependent on the epistasis type. Magnitude epistasis does not qualitatively change the conclusion, but sign epistasis does (Supplementary Fig. 5 ). When a mobile gene causes opposite fitness effects in two different genetic backgrounds, the transfer of this gene will reduce the coexistence feasibility. These results suggest that MGE epistasis might add another layer of complexities to the interplay between HGT and the coexistence of species. Our work predicts that HGT might promote species diversity when MGEs only affect species growth rates and have no influences on inter-species competition. Certain caveats need to be considered when applying this prediction. For instance, the sharing of many mobile genes can also promote niche overlapping, leading to an increase of competition strength 52 , 53 . To understand how the transfer of these genes will influence species coexistence, we adapted the main model by considering the dynamic change of competition strength during gene transfer (see Supplementary Information for more details). The numerical simulations predict that when mobile genes promote inter-species competition, HGT can reduce the coexistence feasibility of competing species (Supplementary Fig. 6 ). These results suggest that how HGT affects species coexistence in a specific microbiota might be context-dependent. Gene transfer can promote or suppress microbial coexistence, depending on epistasis and biological traits encoded by the mobile genes. Our results are in line with the previous studies on the relationship between HGT and microbiota stability 54 , 55 . For instance, one study focused on the interaction between microbial cooperator and cheater and showed that HGT could promote the coexistence of these two genotypes 54 . Another study specifically showed that horizontal transfer of resistance genes could promote microbiome stability in response to environmental stressors 55 . While these studies only considered specific systems, our work resonates with their conclusions and generalizes the role of HGT in the broader context of microbial communities. In our extended LV model, the dynamics of species fitness during HGT arise from the changes in population structure: in each species, HGT generates subpopulations whose growth rates differ from the others due to the metabolic burden or benefits of the mobile genes. However, dynamic fitness can also emerge from the shifts of environmental factors 56 – 58 . The influence of environmental changes on species coexistence has been extensively studied 59 – 61 . Together with these previous studies, our work highlights the importance of considering the dynamic nature of species fitness in the field of ecology. The fitness effect of an MGE can be discrete. For instance, under strong antibiotic selections, only cells carrying antibiotic-resistant MGEs can survive. To examine whether our conclusion is still applicable in this scenario, we generalized our model by considering the transfer of an antibiotic-resistant MGE in a population of \\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}$$m$$\\end{document} m species (see Supplementary Information for more details). Our numerical result suggests that without HGT, only the donor species carrying the MGE can survive due to antibiotic selection. Increasing the HGT rate promotes species coexistence and diversity by allowing more species to be resistant to antibiotic killing (Supplementary Fig. 9 ). These results suggest the applicability of our conclusion to the scenario of discrete fitness effects. In our model, we assumed the number of the MGEs equaled the species number. However, in natural systems, the MGE diversity might be higher than chromosomes due to immigration or de novo mutations 62 . To understand whether our conclusion is still applicable when the diversity of MGEs changes, we established an extended model that accounts for the flow of an arbitrary number of MGEs in a community of multiple species. By numerical simulations with randomized parameters, our results show that the coexistence feasibility increases with MGE diversity (Supplementary Fig. 10 ). In addition, regardless of the MGE diversity, a faster HGT rate consistently leads to a greater possibility of coexistence. These results suggest that enhancing genetic exchange among microbes, through either increasing MGE diversity or increasing HGT rate, can promote microbial diversity. The competition between the preexisting strain and the mutants is at the core of microbial evolution 63 . The evolutionary rates are known to vary across different species, while the mechanisms shaping the evolutionary rates have been largely unknown 64 . Our work indicates that, by promoting the coexistence of different bacterial types, HGT can impede the selective sweep of the fittest strain, which might have a substantial influence on the evolutionary pace of population growth rates. A previous study suggested that HGT prevents vertical selective sweeps when migration is present 65 . Our work shows that HGT can enable the stable coexistence of strong and weak competitors, reducing the likelihood of winner-taking-all scenarios while allowing the mobile genes to spread across species, which is in line with their results. Our results also suggest the role of HGT can be amplified by greater MGE diversity. Therefore, MGE diversity through gene flow with external populations can further promote the frequency of horizontal sweeps relative to vertical sweeps. Such MGE diversity might allow the horizontal sweeps of alleles under positive selections even in the absence of immigration. Manipulation of microbial coexistence has important applications in different scenarios 66 . For instance, in waste treatments or fuel production, designing communities that maintain diversity is instrumental for overall efficiency and yield 67 . In the human gut, loss of microbial diversity is closely associated with many diseases 68 . In soil, bacterial diversity is also critical to maintaining plant productivity 69 . Our work suggests that controlling gene transfer rates can potentially be an effective strategy to engineer the diversity of complex communities. For instance, introducing efficient MGEs like broad-host plasmids can promote genetic sharing among bacteria 70 , while spatial partitioning or treatments of some small chemicals might remodel or block the gene transfer networks 9 , 71 ."
} | 4,817 |
36844751 | PMC9945171 | pmc | 2,485 | {
"abstract": "Polyesters\nare an important class of thermoplastic polymers, and\nthere is a clear demand to find high-performing, recyclable, and renewable\nalternatives. In this contribution, we describe a range of fully bio-based\npolyesters obtained upon the polycondensation of the lignin-derived\nbicyclic diol 4,4′-methylenebiscyclohexanol (MBC) with various\ncellulose-derived diesters. Interestingly, the use of MBC in combination\nwith either dimethyl terephthalate (DMTA) or dimethyl furan-2,5-dicarboxylate\n(DMFD) resulted in polymers with industrially relevant glass transition\ntemperatures in the 103–142 °C range and high decomposition\ntemperatures (261–365 °C range). Since MBC is obtained\nas a mixture of three distinct isomers, in-depth NMR-based structural\ncharacterization of the MBC isomers and thereof derived polymers is\nprovided. Moreover, a practical method for the separation of all MBC\nisomers is presented. Interestingly, clear effects on the glass transition,\nmelting, and decomposition temperatures, as well as polymer solubility,\nwere evidenced with the use of isomerically pure MBC. Importantly,\nthe polyesters can be efficiently depolymerized by methanolysis with\nan MBC diol recovery yield of up to 90%. The catalytic hydrodeoxygenation\nof the recovered MBC into two high-performance specific jet fuel additives\nwas demonstrated as an attractive end-of-life option.",
"conclusion": "Conclusions In this paper, we presented the development\nand characterization\nof a versatile set of bio-based semicrystalline polyesters, which\ndue to their high T g and T m / T d values could offer sustainable\nalternatives to atactic/syndiotactic polyacrylonitrile (PAN), polyether\nether ketone (PEEK), polyethylene terephthalate (PET), poly( tert -butyl vinyl ether) (PTBVE), and certain classes of\npolyamides (high-heat-resistant ones and nylon). Central to this invention\nis the usage of a bio-based bicyclic aliphatic diol monomer (MBC),\nwhich can impart interesting properties to the obtained thermoplastic\nproducts, and the isomerism of which is capable of tuning the polymer\nproperties. Indeed, it was found that the use of pure MBC isomers,\nvis-à-vis the original MBC mixture, allowed for fine-tuning\nof the T g and T m / T d values. The limitation of close melting\nand decomposition temperatures for the here-presented polymers is\nmitigated by polymer solubility, which opens the possibility of fiber\napplications. From a sustainability point of view, the presented polyesters\nare balanced, with MBC being directly derived from lignin and the\ndiester being cellulose-derived. Follow-up studies of mechanical properties\nwill shed further light on the further capability of the here-presented\npolymers to substitute for any of the before-mentioned polymers/polymer\nclasses in specific applications. All polymers can be efficiently\ndepolymerized using methanolysis, yielding recovered MBC in up to\n90% isolated yield. While methanolysis is a commonly applied method\nto the depolymerization of polyesters, this is nonetheless a most\ninteresting achievement, as some of the here-presented polyesters\ncould potentially rival polymer classes, which are not susceptible\nto methanolysis. Alternatively, the efficient conversion of MBC to\na competitive bio-derived aviation fuel additive was also described.\nOverall, this is an elegant example of a sustainable biorefinery concept,\none with pluridisciplinary outputs and a clear recyclability vision.",
"introduction": "Introduction Given their lightweightness\nand versatile properties, polyesters\n[e.g., poly(ethylene terephthalate) (PET)] have assumed an ever more\ndominating and beneficial role in our society for use in packaging\nmaterials, textiles, fibers, and single-use bottles, reaching an estimated\nannual production of up to 70 million tons. 1 − 3 As the main\ndownside, this has led to the accumulation of an estimated 530 million\ntons of polyester plastic waste in landfills and oceans. 1 , 4 , 5 Therefore, there is an urgent\nneed to implement circular economy approaches with regard to polymers\nthrough the development of fully bio-based and recyclable polyester\nplastics and appropriate upcycling or reconversion strategies. 6 − 13 This requires the development of novel strategies that allow us\nto source novel bio-based monomers from abundantly available renewable\nstarting materials. 14 − 17 Moreover, the polymers produced from such virgin monomers should\nreach a similar or better performance in applications compared to\ntheir synthetic analogues, but at the same time, should be readily\ndegradable. This remains a significant challenge as many fossil-resource-based\nplastics display moderate glass transition temperatures ( T g ), around or exceeding 90 °C, 18 a feature not commonly found with bio-derived polymers. 19 − 22 In this respect, notable examples, shown in Figure 1 A, are the pioneering works by Short et al.\non the design of poly(ethylene dihydroxyterephalates) with T g values up to 168 °C. 23 Llevot and co-workers reported on the copolymerization\nof a diol derived from vanillin with 2,5-furandicarboxylic acid (FDCA),\nyielding polyesters with T g values up\nto 139 °C, 24 and Curia et al. showed\nthat polyesters made from lignin-derived bisguaiacols and suitable\ndiesters could reach high T g values (up\nto 164 °C) and high thermal stabilities (>300 °C, Figure 1 A). 25 Figure 1 Overview of bio-based polyesters derived from lignocellulose: (A)\nrepresentative pioneer work on the development of fully lignocellulose-derived\npolyesters; (B) our previous work on the synthesis of MBC from product\nmixtures relating to the lignosulfonate-to-vanillin process; and (C)\noverview of the here-presented work: step 1: copolymerization of MBC\nwith the methyl esters of cellulose-derived TPA, FDCA, and AA, respectively,\nyielding poly(MBC/TPA), poly(MBC/FDCA), and poly(MBC/AA), step 2:\nmethanolysis of the obtained MBC-based polyesters to their original\nmonomers, and step 3: hydrodeoxygenation (HDO) of recovered MBC into\npromising jet fuel additives. Here, we aimed for the utilization of a new lignin-derived building\nblock MBC, in the development of novel, recyclable, and fully bio-based\nplastic alternatives. MBC is an aliphatic, bicyclic rigid diol, which\nhas been previously obtained by our group 26 from industrially relevant product mixtures relating to the lignosulfonate-to-vanillin\nprocess ( Figure 1 B). 27 − 30 Its synthesis entails the selective catalytic hydrogenation of prepurified\naromatic aldehydes into their corresponding benzyl alcohols using\nPd/Al 2 O 3 , followed by an Amberlyst-15-mediated\ncoupling of the latter compounds with phenol, yielding a mixture of\nbisphenols, and the subsequent selective Raney nickel-catalyzed demethoxylation/hydrogenation\nof these bisphenol mixtures into the single aliphatic diol MBC, the\nlatter constituting a catalytic funneling strategy. It has been\nreported that polyesters made with cyclic monomers\ntend to have rigid molecular chains and hence display higher T g values. 31 − 35 Exemplary are the FDCA/isosorbide, pimeloyl chloride/betulin, and\nFDCA/1,4-cyclohexanedimethanol (CHDM)/2,2,4,4-tetramethyl-1,3-cyclobutanediol\n(CBDO) polyesters, which hold respective T g values of 162, 32 165, 31 and 103 °C ( Figure 1 A). 35 Given the symmetric\nbicyclic nature of MBC and its inherent aliphatic alcohol functionalities,\nwe assumed that this bio-derived building block could hold great potential\nfor the synthesis of high -T g bio-derived\npolyesters. Moreover, the possibility of using different MBC isomers\ncould be valuable for the development of polymers with tunable properties.\nLess well developed, yet with relevance to biomass-derived monomers,\nis the stereochemical enhancement of polymer properties. This comprises\nmain-chain stereochemistry, stereocomplexation, and cis–trans\nisomerism. Exemplary to the latter is the general observation that\nthe higher trans content of aliphatic ring-containing monomers (e.g.,\nCHDM; 1,4-cyclohexanedicarboxylic acid; 1,4-diaminocyclohexane) in\na polymeric chain tends to give higher crystallinity, T g , and T m . 36 Based on the above, we set out for the synthesis\nof unique, fully\nbio-based polyesters composed of MBC ( Figure 1 C) 6 − 13 , 37 − 42 and cellulose-derived methyl esters of terephthalic acid (TPA), 14 , 15 FDCA, 17 or adipic acid (AA). 43 The former two polymers display industrially\ninteresting T g values in the 103–142\n°C range. Additionally, all here-presented polymers are characterized\nby high melting/decomposition temperatures in the 260–365 °C\nrange, parameters which are also markedly influenced by the type of\nMBC isomer used. Polymers displaying both a T g and a melting point T m are classified\nas semicrystalline. Finally, we subjected the prepared polyesters\nto methanolysis and\ndemonstrated efficient recovery of the individual monomers. Moreover,\na catalytic strategy for the conversion of recovered MBC to two promising\nbio-based jet fuel additives is being described. 44 − 49 Given the high GHG emissions linked to aviation, and the industry’s\ndesire to reduce these by half by 2050, this is currently an important\ntopic. 50 Overall, this work presents versatile,\nproperty-tunable, and recyclable fully bio-based polyesters, which\ndisplay excellent thermal properties and thus hold great promise for\nfuture industrial applications.",
"discussion": "Results and Discussion Analysis\nand Characterization of Lignin-Derived MBC Previously, we\nhave described catalytic strategies to obtain the\nbicyclic aliphatic diol (MBC) from the lignosulfonate-to-vanillin\nprocess via a series of efficient reaction steps involving catalytic\nfunneling, as well as the use of MBC for the construction of novel\nbio-based polybenzoxazines. Herein, we set out to explore the potential\nof this unique, semi-rigid building block for the production of bio-based\npolyesters. As MBC exists as a mixture of three geometrical isomers,\nnotably cis–cis, cis–trans, and trans–trans MBC\n( Figure 2 A), an in-depth\nNMR characterization was performed. Given the appreciable complexity\nof the 1 H NMR spectrum of MBC ( Figures 2 B and S1 ), with\nvirtually all signals showing extensive coupling and/or partial-to-full\noverlap, the unequivocal determination of the MBC isomer ratio was\nfound challenging. In this respect, the cyclohexane bridging methylene\nprotons proved most instructive, as they appeared as relatively isolated\nmultiplets in the 1–1.2 ppm range ( Figure 2 B). From these methylene signals, the relative\ncomposition of the original MBC isomer mixture could be determined\nas 10:43:47, respectively referring to cis–cis/cis–trans/trans–trans\nMBC isomers ( Figure 2 B-b). This was further confirmed using 1 H pure shift NMR,\nan NMR technique, which applies broad-band decoupling to enhance the\nresolution of proton spectra by removing all homonuclear scalar couplings,\nthe direct result being the collapse of complex multiplets into singlets\n( Figure 2 B-c). 51 , 52 The use of 1 H pure shift NMR strongly benefits correct\nintegration, especially of the low-intensity MBC cis–cis signals. Figure 2 Synthesis, purification, and analysis of MBC and its isomers. (A)\nThe production and isolation of MBC and its isomers. (B) NMR-based\nstructural characterization of MBC: (a) 2D HSQC characterization of\nthe MBC isomer mixture, (b) determination of the MBC isomer ratio\nin regular 1 H NMR by means of MBC’s bridging methylene\nprotons, and (c) determination of the MBC isomer ratio by means of 1 H pure shift NMR. 1 ′ refers to 1cis–cis\nand 1cis–trans and 1 ″ refers to 1trans–trans\nand 1cis–trans. eq stands for equatorial H and ax stands for\naxial H. More information on the NMR characterization of MBC is available\nin the Supporting Information . Interestingly, MBC trans–trans could be\neasily\nisolated in excellent purity and moderate yield (55.6%), by recrystallization\nfrom CHCl 3 . The full NMR spectroscopic characterization\nof pure MBC trans–trans is given in Figures S3–S5 . Separation of the MBC cis–cis and MBC cis–trans isomers was found possible using\ncolumn chromatography. The full NMR spectroscopic characterization\nof the pure MBC cis–cis and MBC cis–trans isomers is shown in Figures S6–S11 . Lastly, the unequivocal assignment of the trans–trans, cis–cis,\nand cis–trans connotations to the pure MBC isomers was performed\nusing nuclear overhauser effect spectroscopy (NOESY), summarized in\nthe Supporting Information . Synthesis,\nAnalysis, and Characterization of Fully Bio-Based\nPolyesters Using MBC as the Starting Material Next, a range\nof bio-based polyesters were prepared by solvent-free titanium-catalyzed\ntransesterification 53 and polycondensation\nof MBC (as a mixture of isomers) with three different cellulose-derived\ndiesters, namely dimethyl terephthalate (DMTA), dimethyl furan-2,5-dicarboxylate\n(DMFD), and dimethyl adipate (DA). These are, respectively, denoted\nas poly(MBC/TPA), poly(MBC/FDCA), and poly(MBC/AA). Figure 3 A shows the here applied polymerization\nprocedure for the specific case of poly(MBC/AA). The choice of Ti\nas the polymerization catalyst was inspired by its general high activity\nin polyester formation and the existence of ample geological reserves\nof this metal. 54 As shown in Table 1 , the latter polymers were obtained\nin good-to-excellent numerical yields, spanning the 65–94%\nrange. FTIR analysis of the final polymeric products confirmed successful\npolyesterification with the clear absence of the MBC-related diol\nmoiety (i.e., OH stretching vibration at 3200–3500 cm –1 ) 25 and the distinct presence of an ester\ncarbonyl stretching band at around 1720 cm –1 , as\nshown in Figure S54 . 25 A full structural analysis of the different synthesized\npolymers by various NMR spectroscopic methods ( 1 H NMR, 13 C NMR, 2D HSQC, and 2D COSY) is discussed in the Supporting Information and specifically shown\nin Figures S16–S35 . Most instructively,\nupon polymerization, the MBC 1 H C H –OH signals at 3.54 ppm (cis–cis; cis–trans)\nand 3.94 ppm (cis–trans; trans–trans; Figure 2 B-a) undergo a remarkable downfield\nshift beyond 4.5 ppm ( Figure 3 B-a). More specifically, the respective 1 H C H –O signals of poly(MBC/TPA),\npoly(MBC/FDCA), and poly(MBC/AA) were recorded at 4.93/5.28 ppm ( Figure S29 ), 4.90/5.22 ppm ( Figure S25 ), and 4.64/4.98 ppm ( Figure 3 B-b). Such a downfield shift of these respective 1 H NMR signals is in line with the formation of ester bonds,\ntherewith confirming effective and successful polymerization. By comparing Figures 2 B-a and 3 B-b, the relative ratio of the C H –O peaks remains the same at 1:2.3, suggesting\nnon-preferential incorporation of the MBC isomers in the polymer chain.\nConvincingly, integration of MBC’s bridging methylene protons\nin poly(MBC/AA) shows a relative ratio of (11:40:47; Figure 3 B-c), which is perfectly in\nline with the previously determined MBC isomer ratio ( Figure 2 B-b and c). Figure 3 General Synthetic procedure\nand structural characterization for\npoly(MBC/AA). (A) Exemplary depiction of the here applied synthesis\nto poly(MBC/AA). (B) NMR characterizations of poly(MBC/AA): (a) 2D\nHSQC of poly(MBC/AA), (b) integration of the 1 H C H –O NMR signals in poly(MBC/AA),\nand (c) 1 H NMR-based determination of the MBC isomer ratio\nin poly(MBC/AA) by means of the bridging methylene protons (5H). The\nNMR characterizations of poly(MBC/TPA) and poly(MBC/FDCA) are available\nfrom the Supporting Information . Table 1 Molecular-Weight Distributions and\nThermal Property Data for Synthesized Polyesters a entry products yield b [%] M w c [g·mol –1 ] M n c [g·mol –1 ] Đ T m d [°C] T d d [°C] T g e [°C] 1 poly(MBC/TPA) 93.9 8270 2890 2.9 261 272 103 2 poly(MBC/FDCA) 84.7 18 500 10 300 1.8 275 284 142 3 poly(MBC cis-cis /FDCA) 66.4 10 600 4630 2.3 263 277 101 4 poly(MBC cis-trans /FDCA) 77.8 10 200 5240 1.9 290 294 128 5 poly(MBC trans-trans /FDCA) 65.1 8230 4340 1.9 325 342 129 6 poly(MBC/AA) 76.5 25 400 9700 2.6 365 (broad) 42 a Reaction conditions: 2.5 mmol of\ndiol, 2.5 mmol of comonomers, 1 mol % titanium (IV) butoxide (TBT)\ncatalyst, 190 °C N 2 /1 h, 230 °C/1 h under vacuum\n1 mbar. b Yield (%) = weight\nof the collected\nproduct/weight of the theoretical product. c Molecular weight distribution was\ndetermined by GPC. d T m =\nmelting temperature and T d = temperature\nof decomposition—as determined by TGA/DSC characterization. e T g was\ndetermined by DSC characterization. GPC analysis revealed Mw values between 8000 and 26 000\ng mol –1 , proving effective polymerization ( Table 1 and Figures S48–S53 ). The comparison of the M w values and the obtained yields is challenging, as polycondensation\nis very sensitive to stoichiometry and hence to the purity of the\ninvolved monomers, and this determines the true reaction stoichiometry.\nIndeed, while MBC trans–trans is highly pure due\nto the crystallization process, MBC cis–trans and\nMBC cis–cis are more challenging to separate from\nthe other MBC isomers and residual solvents, hence representing a\nsomewhat lower purity. Further optimizations of the reaction conditions\nwill be carried out in the future as to obtain high Mw polyesters\nfrom which the mechanical properties can be determined. The\nDSC analysis of poly(MBC/TPA) and poly(MBC/FDCA) revealed respective T g values of 103 and 142 °C ( Table 1 , entries 1 and 2). In addition,\nthe TGA/DSC analysis of the latter two polymers showed respective\nmelting temperatures ( T m ) of 261 and 275\n°C, and respective decomposition temperatures ( T d ) of 272 and 284 °C, further underscoring their\npotential industrial relevance. The influence of the MBC pure\nisomers on the thermochemical properties\nwas investigated for the poly(MBC/FDCA) case ( Table 1 ). It was found that polymers made with a\npure MBC isomer showed a lower glass transition temperature ( T g ) compared to the original poly(MBC/FDCA) ( Figures S38–S41 ). More specifically, the T g decreased along the following series: poly(MBC/FDCA)\n[ T g = 142 °C] > poly(MBC cis–trans /FDCA) ≈ poly(MBC trans–trans /FDCA) [ T g = 128–129 °C] > poly(MBC cis–cis /FDCA) [ T g = 101\n°C], the latter T g value representing\na drop of 41 °C compared\nto that of poly(MBC/FDCA). Apart from poly(MBC cis–cis /FDCA), the use of pure MBC isomers increased the melting temperature\n( T m ) and the decomposition temperature,\nthe trend being: poly(MBC cis–cis /FDCA) [ T m = 263 °C; T d = 277 °C] < poly(MBC/FDCA) [ T m = 275 °C; T d = 284 °C] <\npoly(MBC cis–trans /FDCA) [ T m = 290 °C; T d = 294 °C]\n< poly(MBC trans–trans /FDCA) [ T m = 325 °C; T d = 342\n°C] ( Figures S42–S47 ). Thus,\nthe use of pure MBC isomers shifted both T m and T d , respectively, from 263 to 325\n°C (62 °C difference) and from 277 to 342 °C (65 °C\ndifference). Furthermore, poly(MBC trans–trans /FDCA) and poly(MBC cis–trans /FDCA) effectively\nshow higher T m values than poly(MBC/FDCA).\nThis indicates that the\nrelative stereochemistry (trans–trans, cis–trans, and\ncis–cis) in the MBC monomer plays a distinctly important role,\nalthough the molecular weight values of the respective polymers are\nalso different. Interestingly, the TGA/DSC analysis of these\npolymers generally\nreveals a dual peak around the decomposition temperature ( Figures S42–S47 ), the exception being\npoly(MBC/AA) where it concerns a broad decomposition peak ( Figure S47 ). This hints at the effective existence\nof a melting point, be it though very closely situated to the decomposition\npoint. In this respect, XRD analysis confirmed the existence of semicrystallinity\nin all analyzed polymers ( Figure S55 ). 55 It is further noteworthy that the XRD patterns\nof poly(MBC/TPA) and poly(MBC trans–trans /FDCA) show\na more pronounced fine structure, indicating a more developed crystallinity.\nAs to poly(MBC trans–trans /FDCA), this is in line\nwith the general literature statement that with aliphatic cyclic monomers,\nhigher trans contents in the polymeric chain will display higher crystallinity. 36 Of note here is also that the MBC trans–trans isomer is most prone to crystallization. Nearly all here synthesized\npolymers are soluble in THF and chloroform,\nthe exception being poly(MBC trans–trans /FDCA) which\nis only soluble in chloroform. This is tentatively explained by the\nhigher crystallinity of poly(MBC trans–trans /FDCA),\nwhich is potentially the result of additional extensive furan interactions\nbetween the polymer chains. In this respect, it is noteworthy that\nthe furan ring has a dipole moment of 0.70 Debye, which favors dipolar\ninteractions. 56 The insolubility of polymers\nengaged in extensive interchain interactions has been observed before,\nfor instance, with aromatic polyboronates. 57 Poly(MBC/AA) displayed a low T g of\n42 °C, which is attributed to the more flexible AA chain. Poly(MBC/AA)\nis also semicrystalline in nature, which is in line with other fully\naliphatic polyesters containing AA, 58 and\nit displays a high decomposition temperature ( T d ) of 365 °C ( Table 1 , entry 6). Potential for Practical Applications and\nComparisons with Other\nPolymer Classes Due to the unique steric properties of the\nlignin-derived MBC building block, many of the here-presented polyesters\ndisplay interesting properties, pointing toward very promising practical\napplications, even with respect to different classes of polymers,\nas summarized in Figure 4 . Thus, it is noteworthy that the T g and T m of poly(MBC/TPA) and poly(MBC cis–cis /FDCA) are close to the ones of polyethylene terephthalate (PET), 59 atactic polyacrylonitrile (aPAN), 60 , 61 and poly( tert -butyl vinyl ether) (PTBVE). 62 The T g and T m of poly(MBC trans–trans /FDCA)\nand poly(MBC cis–trans /FDCA) resemble those of syndiotactic\npolyacrylonitrile (sPAN) 62 but equally\nthose of the heat-resistant polyamides poly(hexamethylene teraphthalamide)\n(PA6T) 63 and poly(hexamethylene isophthalamide)\n(PA6I). 64 Moreover, a wide variety of PA6T\ncopolyamides with T g values between 90\nand 141 °C and T m values between\n235 and 325 °C have been reported, 65 spanning the same T g and T m ranges of the here-presented MBC-based polymers. Finally,\npoly(MBC/FDCA) has a similar T g as polyether\nether ketone (PEEK), 66 and poly(MBC/AA)\nresembles a range of polyamides (Nylons) such as nylon 11, 67 , 68 nylon 12, 68 , 69 and nylon 6/10. 68 Figure 4 Survey of the potential substitution of certain polymer types by\nthe here-developed MBC-based polyesters. Thus, the here-developed sustainable polymers can mimic the thermal\nproperties ( T g , T m , T d ) of certain N-containing\npolymers (polyamides, polyacrylonitrile), yet by only involving C,\nH, and O atoms in the polymeric backbone. For a classic thermoplastic\nmaterial, a melting point near the decomposition point may be a limitation.\nHowever, as the polymers are soluble, applications such as fibers\nare well within reach. In this respect, it is also noteworthy that\nthe major use of PET is as fibers (2021: 60.5 Mio t fibers 70 vs 24.3 Mio t plastics 71 ), witnessing the PET fiber trademark names Dacron (DuPont) and Terylene\n(Imperial Chemical Industries Ltd.). 72 Recycling and Upcycling Strategies With polymer recyclability\nbeing very important, we also investigated the proneness of the here-developed\npolyesters to depolymerization. It was found that all here-presented\npolymers could be fully and easily degraded into their respective\nmonomers by methanolysis, without the deliberate addition of additives.\nThat way, MBC could be recovered in 90% yield ( Figure 5 A), independent of the type of polymer (for\nrepresentative GC-FID traces, see Figure S56 ). This is an important observation as the above-mentioned PTBVE,\nPAN, and PEEK polymeric materials are not prone to facile degradation/recycling,\nsince the synthesis of PTBVE and PAN involves C–C bond formation\nthrough, respectively, cationic vinyl polymerization 73 and radical polymerization, 74 while in the case of PEEK, highly stable diphenyl ether bonds are\ncreated. Figure 5 (A) Methanolysis of poly(MBC/TPA), poly(MBC/FDCA), and poly(MBC/AA).\nReaction conditions: 200 mg of polymer, 30 mL of methanol, 190 °C,\n4 h, 1 bar N 2 , 10 mg of dodecane. (B) Influence of the\nreaction temperature on the hydrodeoxygenation of MBC over the Ni/HZSM-5\ncatalyst. Reaction conditions: 1 mmol of MBC, 30 mL of cyclohexane,\n10 mg of dodecane, 4 h. The conversion levels were determined via\na calibration curve. The selectivities were determined by the effective\ncarbon number (ECN) method. 75 Alternatively, we also investigated the catalytic transformation\nof (recovered) MBC into a valuable and high-energy dense jet fuel.\nFor that purpose, MBC was subjected to consecutive dehydration/hydrogenation\nsteps over an in-house prepared Ni/HZSM-5 catalyst. The applied Ni/HZSM-5\ncatalyst was extensively characterized using XRD ( Figure 6 A), NH 3 -TPD ( Figure 6 B), and SEM ( Figure 6 C). As shown in Figure 6 B, NH 3 -TPD reveals the presence of both weak and strong acid sites. More\nspecifically, the desorption peak in the 100–200 °C region\npoints at the weak adsorption of NH 3 on Si–OH Brönsted\nacid centers, 76 while the desorption peak\nof NH 3 in the 300–400 °C region can be attributed\nto the strong adsorption of NH 3 on mainly Al–OH–Si. 74 The XRD analysis of the Ni/HZSM-5 catalyst reveals\nthe clear presence of distinct diffraction peaks at 2θ = 44,\n53, and 77°, which points to the presence of a pure nickel metal\ncrystal phase ( Figure 6 A), 77 and SEM ( Figure 6 C) reveals the presence of highly dispersed\nNi nanoparticles, which can easily engage in the hydrogenation of\ndienes. Figure 6 General characterization of the Ni/HZSM-5 catalyst: (A) X-ray diffraction\n(XRD) analysis, (B) temperature-programmed desorption with NH 3 (NH 3 -TPD), and (C) scanning electron microscopy\n(SEM) image of the Ni/HZSM-5 catalyst. The marked white spots are\nnickel nanoparticles. It was found that at\n140 °C, MBC could be selectively (97%)\nconverted into perhydrofluorene (JF-1; IUPAC name = 2,3,4,4a,4b,5,6,7,8,8a,9,9a-dodecahydro-1H-fluorene)\nin 20% conversion ( Figures 5 B and S57a ). Additionally, the\noperation of this reaction at 180 °C yielded dicyclohexylmethane\n(JF-2) quantitatively as the sole product (>99% yield) ( Figures 5 B and S57b ). This is a most interesting observation,\nas the density\nof JF-1 (0.96 g mL –1 ) is markedly higher than that\nof JF-2 (0.88 g mL –1 ), even exceeding the density\nof the most performing JP-10 jet fuel available to date (0.94 g mL –1 ). 78 The higher density\nof JF-1 vis-à-vis JF-2 explains the higher heat value of the\nformer (40.1 MJ L –1 ) versus the latter (36.8 MJ\nL –1 ). 79 While the specific\ncatalytic formation of JF-1 and JF-2 from MBC has not been reported\nto date, the concurrent occurrence of JF-1 and JF-2 has been reported\nby Nie et al. in a study on the hydrogenation/ hydrodeoxygenation\nof 2-benzylphenol (2BP), 79 79 where it was found that 2BP was first, and invariably,\nhydrogenated over Pd/C to 2-benzylcyclohexanol (2BCH). The further\npresence of an acidic zeolite (e.g., HZSM-5) then transforms 2BCH\ninto JF-2, while the sole application of Pd/C affects 2BCH ring closure\nto JF-1. 79 Conversely, the here-observed\nNi/HZSM-5-catalyzed formation of JF-1 (from MBC) runs at a lower reaction\ntemperature and involves an HZSM-5-mediated dehydration of MBC to\na set of dienes and a Ni-mediated ring closure. 80 Separate application of sole HZSM-5 to MBC showed only\nthe formation of a range of dienes ( Figure S59 ). Application of a higher temperature predominately led to JF-2\nbecause of rapid hydrogenation of any unsaturated intermediates precluding\nany cyclization. On the potential of JF-1 and JF-2 as neat unblended\njet fuels, the currently available literature lists unfavorable freezing\npoints (JF-1 at −20 °C) and viscosities. 79 However, 50/50 blending of JF-1 with JP-10 has been shown\nto give a high-performance jet fuel with favorable properties, notably,\na density of 0.95 g mL –1 (20 °C), a viscosity\nof 17.4 mm 2 s –1 (20 °C), and a freezing\npoint below −75 °C. 79 In this\nrespect, it is also noteworthy that JP-10 is very expensive (7091\n$/ton) and only available in limited quantities, 46 making a favorable case for blending with a potentially\nless expensive fuel. All this underscores the relevance of bio-derived\nJF-1 as a high-density additive to jet fuels and hence also the practical\nvalue of this reconversion strategy in relation to establishing a\nviable circular strategy for our high-performance, bio-based polyesters."
} | 7,152 |
23525724 | PMC3695317 | pmc | 2,486 | {
"abstract": "The applicability of a newly-designed PCR primer pair in examination of methanogenic Archaea in a digester treating plant biomass was evaluated by Ribosmal Intergenic Spacer Analysis (RISA). To find a suitable approach, three variants of RISA were tested: (1) standard, polyacrylamide gel-based, (2) automated, utilized capillary electrophoresis (GA-ARISA), and (3) automated microfluidics-based (MF-ARISA). All three techniques yielded a consistent picture of archaeal community structure changes during anaerobic digestion monitored for more than 6 weeks. While automated variants were more practical for handling and rapid analysis of methanogenic Archaea , the gel-based technique was advantageous when micro-organism identification was required. A DNA-sequence analysis of dominant bands extracted from the gel revealed that the main role in methane synthesis was played by micro-organisms affiliated with Methanosarcina barkeri . The obtained results revealed that RISA is a robust method allowing for detailed analysis of archaeal community structure during organic biomass conversion into biogas. In addition, our results showed that GA-ARISA has a higher resolution and reproducibility than other variants of RISA and could be used as a technique for tracking changes in methanogenic Archaea in an anaerobic digester.",
"introduction": "Introduction Successful anaerobic treatment of organic wastes requires the stable functioning of a complex, interdependent microbial community [ 7 , 8 ]. The degradation of the organic compounds to carbon dioxide and methane occurs in four, discrete steps that are carried out by different groups of micro-organisms. At the beginning, organic molecules such as complex carbohydrates, proteins and lipids are hydrolyzed into their components [ 17 ]. The generated monomers and oligomers, such as amino acids, simple carbohydrates, and fatty acids are converted into organic alcohols, volatile fatty acids, hydrogen, and carbon dioxide. Next, the digested products are further degraded into acetate, hydrogen, and carbon dioxide. The final step is methanogenesis, which results in the production of methane and carbon dioxide from either acetate or hydrogen/formate and carbon dioxide [ 9 , 19 ]. This step is carried out by methanogens, which are especially important because methanogenesis is often the rate-limiting step in anaerobic treatment of wastes [ 7 ]. Methanogenic micro-organisms belong to Archaea , a unique prokaryotic domain of life. This group contains: (i) the acetotrophic methanogens, (ii) hydrogenotrophic methanogens, and (iii) methylotrophs which convert methyl compounds such as methanol and methylamines. Methane-producing micro-organisms are obligate anaerobes and are very sensitive to environmental changes [ 14 ]. Because methanogenesis is usually the rate-limiting step in the overall process, the appropriate control of the methanogenic phase has been a key factor in the successful operation of anaerobic processes [ 20 ]. Therefore, understanding the behavior of the archaeal community is crucial to optimize the anaerobic process for biogas production. Recently, a study of complex microbial communities has been performed by the application of culture-independent, molecular methods based on directed analysis of the 16S rRNA gene structure. Among them, Denaturing Gradient Gel Electrophoresis (DGGE) analysis is one of the most widely-used molecular techniques, enabling the identification of community members by the recovery and sequencing of amplification products. This genetic fingerprinting approach is useful for comparisons of microbial communities from different environments or in following changes in community structures over time [ 10 ]. However, DGGE has many limitations, such as limited sensitivity of detection for some rare community members and the co-migration of DNA fragments with different sequences. Thus, other DNA fingerprinting techniques have been tested for application in microbial community analysis. An alternative technique is Ribosomal Intergenic Spacer Analysis (RISA), which is based on the amplification of the intergenic region located between the 16S and 23S rRNA genes in the rRNA operon. This region is characterized by significant variability in the length and nucleotide sequence among different microbial genotypes [ 5 ]. Recently, the separation process of amplified DNA has been improved by involving a fluorescence-tagged oligonucleotide primer for PCR amplification and subsequent electrophoresis in an automated system. Due to the high resolution of the gels and the high sensitivity of the fluorescence detection, numbers of founded peaks are much higher in Automated Ribosomal Intergenic Spacer Analysis (ARISA) than in RISA profiles [ 12 ]. The main goal of this study was to develop a method facilitating the examination of changes in the methanogenic Archaea community during anaerobic digestion by RISA. The possibility of applying both traditional RISA and its automated versions (ARISA) was explored.",
"discussion": "Discussion To guarantee the correct design and application of anaerobic treatment systems, knowledge of the technological aspects, biochemistry and microbiology of anaerobic digestion is essential [ 6 ] However, the terminal phase of anaerobic treatment-methanogenesis, is still not completely understood, especially in systems where complex substrates are utilized for biogas production. A wide variety of molecular methods have been developed to assess the role of archaeal methanogens in such processes. Traditional examination of the microbial ecology in the environment is based on the microbes’ cultivation under laboratory conditions, although for many micro-organisms, especially Archaea , culture-dependent studies can be difficult [ 11 ]. However, since molecular analysis is free of limitations that are typical for culture-dependent methods, such an approach is preferred for a methodology for the examination of methane-producing micro-organisms. Unfortunately, many of these DNA-based methods often provide results which are ambiguous and difficult to interpret. In addition, they usually cannot be automated and are restricted by the limited number of samples that can be analyzed in one run. Thus, our intention was to examine the usefulness of RISA-a method that could be free of this limitation. In this study, three variants of RISA were compared to find the best solutions for controlling changes in methanogenic archaeal consortia during anaerobic treatment of silage. The standard method that relies on amplicon resolution on polyacrylamide gel was matched against the automated methods performed by an Applied Biosystems 3130 Genetic Analyzer (GA-ARISA) and microfluidics-based Agilent 2100 Bioanalyzer (MF-ARISA). All applied techniques showed similar clustering for the samples taken from the digester over time. Although, the highest values of bootstrap were estimated for the tree obtained by RISA application (Fig. 5 a), the most reliable picture of archaeal community changes was gained using GA-ARISA (Fig. 5 c), where the direction of genetic similarity changes was concordant with the time of sampling. The lower bootstrap support values are mainly due to the fact that this approach led to detect the highest number of taxons, that was supported by Shannon index values (Fig. 4 ). An additional advantage of GA-ARISA is its suitability for the routine analysis of a large number of samples (which, in the case of the Applied Biosystems 3130 Genetic Analyzer could be up to 96). For MF-ARISA, the maximum number of samples analyzed in a single run is 12 which, together with low resolution power, are the main disadvantages of this approach. The inability to load many samples on the same gel and the potential of gel-to-gel variation is a major drawback of gel-based RISA. However, the advantage of RISA is that it can be coupled with additional approaches to identify differentiating bands. Excision of selected bands and its detailed analysis by sequencing can lead to the phylogenetic affiliation of micro-organisms that are represented in the fingerprint. However, along with development of annotated 16S‐RIS‐23S region collections, the information on their source organisms could enrich ARISA by allowing micro-organism identification based on RISs lengths. The two dominant bands in polyacrylamide gel (MT-1 and MT-2) were excised and characterized by DNA sequencing. The obtained results revealed that both analyzed sequences are most similar to the sequence of Methanosarcina barkeri. Although the lengths of analyzed sequences were different than those released from the absence of tRNA Ala in the MT-2 sequence, the rest of this region was identical. Because both bands appeared at the same time (the 10th day of the process) and the intensities of these bands were similar until the end of anaerobic digestion, it is highly probable that both analyzed intergenic spacer regions originated from one micro-organism. The presence of two, different ribosomal operons was previously proven in other archaeon Haloarcula marismortui [ 3 ]. In this species, one of the operons possessed RIS lacking genes coding for tRNA Ala and tRNA Cys . This same phenomena was observed in Enterococcus hirae ATCC 9790 [ 15 ]. The presence of Methanosarcina sp. is typical for digesters with high levels of N–NH 4 \n + and VFAs [ 4 ]. According to Raskin et al. [ 13 ], Methanosarcina sp. were the most abundant methanogenic archaea in samples taken from acetate-fed laboratory chemostats. In our experiment, bands corresponding to Methanosarcina barkeri appeared on the 10th day of experiment when total biogas and methane production increased rapidly. It suggests that Methanosarcina barkeri was the main biogas producer in the performed experiment. The development of this species was promoted by high levels of N–NH 4 \n + and VFAs (Table 2 ). The Pearson correlation analysis showed that there is correlation between biogas/methane production and relative abundance of MT1, MT2 bands/peaks, that can support the finding that M. barkeri is responsible for biogas production in the studied digester. In conclusion, our results indicate that all methods are useful as fingerprinting techniques for assessing archaeal species changes and diversity in anaerobic environments. However, the results of this study indicate that ARISA performed by a genetic analyzer (GA-ARISA) is more accurate and reproducible than other tested approaches. Although, its performance requires specialized equipment, experienced staff and its execution time is quite long, the reliable results compensate for these disadvantages. Therefore, it could be concluded that the designed PCR primer pair, combined with the application of capillary electrophoresis, could be a powerful technique for methanogenic Archaea analysis."
} | 2,718 |
30507059 | null | s2 | 2,487 | {
"abstract": "Species interactions and coexistence are often dependent upon environmental conditions. When two cross-feeding bacteria exchange essential nutrients, the addition of a cross-fed nutrient to the environment can release one species from its dependence on the other. Previous studies suggest that continued coexistence depends on relative growth rates: coexistence is maintained if the slower-growing species is released from its dependence on the other, but if the faster-growing species is released, the slower-growing species will be lost (a hypothesis that we call 'feed the faster grower' or FFG). Using invasion-from-rare experiments with two reciprocally cross-feeding bacteria, genome-scale metabolic modelling and classical ecological models, we explored the potential for coexistence when one cross-feeder became independent. We found that whether nutrient addition shifted an interaction from mutualism to commensalism or parasitism depended on whether the nutrient that limited total growth was required by one or both species. Parasitism resulted when both species required the growth-limiting resource. Importantly, coexistence was only lost when the interaction became parasitism, and the obligate species had a slower growth rate. Under these restricted conditions, the FFG hypothesis applied. Our results contribute to a mechanistic understanding of how resources can be manipulated to alter interactions and coexistence in microbial communities."
} | 365 |
26630941 | PMC4668684 | pmc | 2,488 | {
"abstract": "Background Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. Results In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted other carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum -related and its potential host Ignicoccus -related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. Conclusions Our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2230-9) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions Using a metagenomic approach to acquire copious sequence data from members of SHP planktonic microbial community enabled us to not only identify community composition, but also to postulate potential interactions within the ecosystem. Specifically, we used metagenomic data to predict potential metabolite exchange, microbe-phage interaction (CRISPR analyses) and archaeal parasite-host interactions ( Nanoarchaea and Ignicoccus ) within SHP. Potential metabolite exchanges among microbes is shown (Fig. 8 ), based on existing physiological and biochemical studies of dominant microbes. Predicting potential metabolic pathways for carbon, sulfur, carbon and hydrogen with the NCBI and/or KEGG databases enabled us to elucidate metabolic ability of each dominant microbe. However, metabolic analyses cannot fully explain the Hydrogenobaculum -dominant feature. Previous studies attributed a Hydrogenobaculum -dominant feature based on carbon assimilation pathways and hydrogen utilization features of this genus. However, Hydrogenobaculum is not the only microbial genus capable of utilizing hydrogen and assimilating inorganic carbon. Thus, we proposed that Hydrogenobaculum bacteria dominate SHP due to additional abilities, e.g. temperature tolerance and ability to survive in an anaerobic environment. Together with our analytical results and literature mining, this study provided a comprehensive understanding of interactions within the microbial ecosystem in an acidic thermal environment.",
"discussion": "Results and discussion Hydrological parameters of SHP Limnological parameters of the SHP are shown (Table 1 ). Temperature and pH of the sample were 69 °C and 2.5, respectively. Concentrations of several ions (Cl − , HCO 3− , Ca 2+ , Mg 2+ , K + and Na + ) were low, as was that of dissolved organic carbon (~1 mg/L). In addition, concentrations of sulfate (378 mg/L), hydrogen sulfide (52.7 mg/L) thiosulfate (0.12 mg/L) and elemental sulfur (0.50 mg/L) in SHP water were also determined. Table 1 Geochemical and physical parameters of SHP hot spring water Parameter a \n Current study Song et al., 2005 b [ 21 ] pH 2.5 2.77 – 3.25 Temperature (°C) 69.0 49.8 – 85.1 TDS 707 177 – 1674 EC (μS/cm 2 ) 1760 266 – 1039 ORP (mv) −62 – DOC 1.0 – HCO 3 \n 2− \n <0.03 L NO 3− \n – L – 0.9 PO 4 \n 3− \n – L Cl − and other halides 9.3 L (F − ) / 4.93 – 10.3 (Cl − ) / L – 3.78 (Br − ) SO 4 \n 2− \n 378 100 – 450 S 0 \n 0.50 – H 2 S 52.7 – S 2 O 3 \n 2− \n 0.12 – Fe 2+ and Fe 3+ \n 111 1.8 – 54.9 (Fe 2+ ) Ca 2+ \n 1.32 5.8 – 977 Na + \n 12.0 L – 13.4 Mg 2+ \n 1.21 1.21 – 9.70 K + \n 2.17 1.40 – 6.00 Al 3+ \n 19.0 0.70 – 17.3 Total As 0.0012 – “L” represented concentrations below detection limits \n TDS total dissolved solids, EC electrical conductivity, ORP oxidation/reduction potential, DOC dissolved organic carbon \n a Units were mg/L for all end points except pH \n b Source: Geological survey and potential application of hot springs and geothermal energy of Yangmingshan (in Chinese); monthly report of environmental parameters in the SHP acidic hot springs in 2005 The abundant genera in SHP The top 20 abundant genera (eight bacterial and 12 archaeal genera; Fig. 1 ) were selected (from 16S rRNA gene-based diversity surveys) for phylogenetic analyses. Although there were more archaeal than bacterial genera in the top 20, bacteria clearly dominated the microbial community in SHP, based on relative abundance of 16S rRNA. In that regard, Hydrogenobaculum bacteria accounted for 86.30 % of RA 16S (relative abundance in 16S rRNA gene-based diversity survey), whereas the second most abundant genus, Nanoarchaeum (an archaeal genus) only accounted for approximately 0.99 % (Additional file 1 : Table S1). Fig. 1 Phylogenetic tree generated from top 20 most abundant genera identified with 16S rRNA gene-based diversity survey. Diameters of circles are proportional to abundances; the smallest circles represent RA \n 16S ~ 0.02 %, whereas the largest circle ( Hydrogenobaculum ) represent RA \n 16S = 86.3 %. Letters within each circle represent taxonomy levels: domain (D), phylum (P), class (C), order (O), family (F) and genus (G). Genera names are outside the circle. Genera names in bold were common genera (top 20 most abundant and information-rich genera) Relative abundances of the microbial community were also analyzed based on direct shotgun sequence (DSS) contigs. Major genera identified using this method were designated genomic information-rich genera, because contig information was used in analyses of metabolic ability. Nine genomic information-rich genera were identified, namely, Hydrogenobaculum, Vulcanisaeta, Thermoproteus, Caldisphaera, Sulfolobus, Caldivirga, Acidithiobacillus , Thiomonas, and Metallosphaera (Table 2 and Additional file 1 : Figure S1). It was noteworthy that the order of the ranking between the two lists, the information-rich genera and the 20 abundant genera from 16S rRNA gene-based method, were similar, with Hydrogenobaculum at the top of both lists and all genomic information-rich genera in the top 20 of the 16S rRNA gene-based diversity survey. Table 2 Information-rich genera and the top 20 most abundant genera derived from SNP hot springs water Rank in abundance Information-rich genera a \n Top 20 most abundant genera identified by 16S rDNA analysis 1 \n Hydrogenobaculum \n 5.73 % \n Hydrogenobaculum \n 86.31 % 2 \n Vulcanisaeta \n 3.64 % \n Nanoarchaeum \n 0.99 % 3 \n Thermoproteus \n 2.82 % \n Acidithiobacillus \n 0.85 % 4 \n Caldisphaera \n 2.66 % \n Thermoproteus \n 0.67 % 5 \n Sulfolobus \n 2.43 % \n Caldisphaera \n 0.47 % 6 \n Caldivirga \n 2.39 % \n Thiomonas \n 0.26 % 7 \n Acidithiobacillus \n 2.17 % \n Acidicaldus \n 0.23 % 8 \n Thiomonas \n 1.66 % \n Sulfurisphaera \n 0.21 % 9 \n Metallosphaera \n 1.50 % \n Acidianus \n 0.17 % 10 \n Caldivirga \n 0.16 % 11 \n Metallosphaera \n 0.16 % 12 \n Vulcanisaeta \n 0.16 % 13 \n Thiobacillus \n 0.14 % 14 \n Sulfolobus \n 0.12 % 15 \n Stygiolobus \n 0.11 % 16 \n Thermocladium \n 0.10 % 17 \n Pyrobaculum \n 0.05 % 18 \n Desulfurella \n 0.04 % 19 \n Acidimicrobium \n 0.02 % 20 \n Propionibacterium \n 0.02 % \n a Genera contained the relative abundance of contigs exceeding 1 % The name in bold represents the genus share between two lists Inconsistencies between compositional lists of 16S rRNA gene-based diversity and metagenomic information Ranking and composition of dominant microbes differed between the genomic information-rich genera list and the dominant microbe list (derived from 16S rRNA gene-based diversity surveys) identified in the present study. There were several potential reasons, including variations among microbes in genome sizes and copy numbers of 16S rRNA gene, and the threshold used. For example, although Nanoarchaeum was one of the most abundant genera in the top 20 16S rRNA gene-based list, it was absent from the list of the genomic information-rich genera (Table 2 ). This was attributed to its small genome (~490 kb; [ 27 ]), which would reduce the probability of being detected during sequencing. Advantageous characteristics of Hydrogenobaculum in SHP Hydrogenobaculum was the predominant genus in SHP, where the temperature and pH were 69 °C and 2.5, respectively. Similarly, bacteria of the same genus also predominated in other acidic hot springs with variable (albeit harsh) environmental conditions, including Dragon Spring (70 ~ 72 °C; pH 3.1; [ 25 , 26 ]), One Hundred Spring (73 °C; pH 3.5; 25, 26) and Norris Geyser (65 °C; pH 3.0; [ 26 ]). The abilities of Hydrogenobaculum bacteria to assimilate carbon and metabolize hydrogen were suggested as crucial characteristics for living in an acidic hot spring [ 28 , 29 ]. Indeed, carbon assimilation ability would be important for bacteria residing in SHP, due to the low dissolved organic carbon (DOC) concentration (1 mg/L) in spring water. However, Hydrogenobaculum was not the only microbial genus in SHP that assimilated inorganic carbon. Based on our metagenomic analysis, genes for carbon assimilation pathways were present in five of the nine genomic information-rich genera, including Acidithiobacillus , Hydrogenobaculum , Metallosphaera, Sulfolobus, and Thiomonas (Fig. 2 ). Also, physiological studies indicated that Thermoproteus tenax [ 30 ] , Sulfolobus tokodaii [ 31 ] , Acidithiobacillus [ 32 ], and Metallosphaera [ 33 ] were also capable of utilizing hydrogen as an energy source. Fig. 2 Carbon-metabolizing enzymes identified from dominant microbes using KEGG mapping. Asterisk: key enzymes in metabolic pathways. Abbreviations: hya, Hydrogenobaculum sp. Y04AAS1; hys, Hydrogenobaculum sp. SN; vdi, Vulcanisaeta distributa ; vmo, Vulcanisaeta moutnovskia ; tuz, Thermoproteus uzoniensis ; ttn, Thermoproteus tenax ; clg, Caldisphaera lagunensis ; sto, Sulfolobus tokodaii ; sso, Sulfolobus solfataricus ; sai, Sulfolobus acidocaldarius ; cma, Caldivirga maquilingensis ; acu, Acidithiobacillus caldus ; afe, Acidithiobacillus ferrooxidans ; tin, Thiomonas arsenitoxydan ; thi, Thiomonas intermedia ; mse, Metallosphaera sedula . Cell in white, not listed in the KEGG reference pathway; grey, listed in the KEGG reference pathway; black, listed in the KEGG reference pathway and identified in our metagenomic dataset. Carbon-metabolizing enzymes identified from dominant microbes using KEGG mapping Although carbon assimilation metabolism and hydrogen metabolism (Additional file 1 : Table S2) were regarded as important, they were not the only advantageous characteristics enabling the genus Hydrogenobaculum to dominate in SHP. Given substantial environmental variations among various hot springs, Hydrogenobaculum bacteria seemed to adapt to a broader temperature range compared to other detected genera; this could be another characteristic contributing to their dominance in variable acidic hot springs, with temperatures ranging from 50 to 82 °C [ 6 , 15 , 18 , 25 , 26 , 34 ]. In SHP, water temperature ranged from 50 to 85 °C in a-year-long survey [ 21 ], similar to the temperature range in other Hydrogenobaculum -dominated hot springs. On the contrary, two other relatively less well represented genera identified in SHP, e.g. Acidithiobacillus and Thiomonas , were reported to only grow under mild thermophilic conditions. For example, temperature ranges of A. caldus , A. ferrooxidans, Thiomonas arsenitoxydans, and Thiomonas intermedia , were 32 ~ 52 °C [ 35 ], 10 ~ 37 °C [ 36 ], 30 °C [ 37 ], and 30 ~ 35 °C [ 37 ], respectively (Additional file 1 : Table S3). Whether those bacterial strains have evolved additional heat tolerance mechanisms is apparently unknown. Low oxygen concentrations in SHP water could also have affected microbial dominance, as they might not have been favorable for aerobic carbon assimilators, e.g. Sulfolobus and Metallosphaera [ 31 , 38 – 40 ]. However, the oxygen requirement of Hydrogenobaculum Y04AAS1-related strain has apparently not been reported. Regardless, Y04AAS1-related strain seemed well adapted to anaerobic or microaerobic conditions, due to the presence of oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase, which catalyze carboxylation steps in the reductive citrate cycle [ 41 ]. In addition, based on previous metagenomic studies, Hydrogenobaculum bacteria dominated in two acidic hot springs with radically different dissolved oxygen concentrations (>3 and 22 μM in Dragon Spring and One Hundred Spring, respectively; [ 25 , 26 ]), suggesting substantial physiological flexibility of Hydrogenobaculum bacteria to variations in oxygen concentration. In short, dominance of Hydrogenobaculum bacteria in SHP was attributed to their inherent adaptability to withstand fluctuations in both temperature and oxygen concentration, as well as their metabolic capacity to assimilate carbon or use hydrogen as an energy source. Genomic map of Hydrogenobaculum bacteria Hydrogenobaculum was the predominant genus in SHP. Mapping DSS reads covered >90 % of the length of the Hydrogenobaculum sp. Y04SSA1 reference genome (Fig. 3 , Additional file 1 : Table S4), consistent with analysis of genomic information-rich genera, which designated Hydrogenobaculum Y04SSA1-related strain as the dominant microbe (Additional file 1 : Table S3). In addition, 16S rRNA genes and key carbon metabolic gene ccl , which encodes citryl-CoA lyase, in the genome of Y04SSA1-related strain were also on the genome map (Fig. 3 ). Fig. 3 Mapping both fosmid and DSS contigs and raw reads to Hydrogenobaculum sp. Y04AAS1 reference genome. From outer to inter circles are: DSS raw reads ( dark gray ), DSS contigs ( orange ), fosmid raw reads ( light gray ), fosmid contigs ( green ) and specific highlighted genes, respectively Comparison among acidic hot spring metagenomes Cheng et al. reported that Hydrogenobaculum was a major genus in an acidic hot spring (Huang-Shan: 82.9 °C, pH 2.2) in TVG, based on amplification and analysis of full-length 16S rRNA genes [ 24 ]. Hydrogenobaculum was also designated the major genus in acidic hot springs in Yellowstone National Park [ 25 , 26 ]. Comparative metagenomics characterize interactions between microbes and their environment. Currently, there are only two published acidic hot spring metagenome datasets [ 17 , 18 ], one from Yellowstone National Park and the other from El Coquito spring, National Natural Park Los Nevados. Functional profiles (based on KEGG or COGs) of the SHP metagenome were compared to metagenomes of Yellowstone National Park and National Natural Park Los Nevados (for the latter, see Fig. 4 and Additional file 1 : Figure S2). The SHP metagenome was closer to the metagenome from Yellowstone National Park than to National Natural Park Los Nevados. The four major pathways of the COGs category that differed between SHP/Yellowstone National Park and National Natural Park Los Nevados were: (a) amino acid transport and metabolism; (b) nucleotide transport and metabolism; (c) replication, recombination and repair; and (d) general function prediction (Additional file 1 : Fig. S2). Fig. 4 Cluster analysis of acidic hot spring metagenomes. Based on COGs clustering, YNP and SHP were more similar to each other than NNPLN. Nonetheless, the functional profiles of the three metagenomes shared a high similarity (>80 %). The clustering result based on KEGG pathways was also consistent with this clustering result (Additional file 1 : Figure S6). YNP, SHP, and NNPLN were metagenomes of Yellowstone National Park (USA), Shi-Huang-Ping (Taiwan) and National Natural Park Los Nevados (Colombia), respectively Environmental conditions shape microbial community structure, which would in turn affect functional profiles. At SHP and YNP, conditions were: temperatures >50 °C, pH approximately 2–3, concentrations of sulfur-related compounds were high, and major microbial genera were Hydrogenobaculum , Sulfolobus and Metallosphaera . That these two hot springs were on distant continents and derived by distinct geological events, we concluded that microbial communities in acidic hot springs have undergone persistent and common selection, characterized by phenotypic conservation (Additional file 1 : Fig. S2). Diverse microenvironments in SHP implied by microbial composition To further elucidate interactions between microbes and environmental factors in SHP, we critically reviewed previous reports of dominant microbes in SHP. Analyzing microbial community structures and metagenomes contribute to understanding geochemical conditions in acidic hot springs [ 42 ]. Dominant microbes in SHP microbial community had diverse oxygen preferences, including aerobic microbes (e.g., Sulfolobus and Metallosphaera ), facultative aerobic microbes (e.g., Acidithiobacillus ), microaerobic microbes (e.g., Vulcanisaeta and Caldvirga ), and anaerobic microbes (e.g., Thiomonas and Caldisphaera ). We inferred that the water environment of the hot spring had at least three distinct microhabitats, namely aerobic, microaerobic and anaerobic (Fig. 5 and Additional file 1 : Table S3). Furthermore, the lowest reported oxygen condition in SHP (2.74 mg/L; [ 43 ]) indirectly supported the presence of habitats with varying oxygen concentrations. Fig. 5 Hypothetical metabolic interactions between microbes and environments in the SHP acidic hot spring. These are potential relationships between dominant microbes and biogeochemical pathways of carbon, nitrogen and sulfur in the SHP acidic hot spring. Dominant microbial genera, species or strains are in rectangles. Dotted lines show compounds potentially derived from other sources (e.g., sediments, hot spring from underground, etc.). Thick lines are metabolic potentials detected in genera, whereas thin lines highlight alternative metabolic pathways in dominant microbial genera Although metagenomic information in the present study clearly supported the presence of microaerobic or anaerobic microenvironments in SHP water, potential sources of error could not be excluded. For example, some microaerobic or anaerobic microbes from the sediment or the soil nearby the pond might have contaminated our sample. However, that pond water was clear and calm during sampling, and sampling was carefully conducted, the probability that contamination occurred was extremely low. Carbon cycle in the SHP The carbon cycle in the acidic hot spring is highly dependent upon chemotrophic processing, as the combination of high temperature and low pH hamper photosynthesis [ 19 , 44 ]. The upper limit for photosynthesis is ~ 56 °C in an acidic (pH <4.0) environment [ 44 ]. Thus, microbes detected in the springs of Cater Hills and Horris Greyser Basin (USA) presumably used non-photosynthetic chemotrophic pathways for carbon assimilation [ 16 ]. Four chemotrophic carbon assimilation pathways were identified in our metagenomic data. Hydrogenobaculum had a reductive citrate cycle, Sulfolobus and Metallosphaera used the hydroxypropionate-hydroxybutyrate cycle [ 45 , 46 ], and T. uzoniensis and T. tenax had genes for both a reductive citrate cycle and a dicarboxylate-hydroxybutyrate cycle [ 47 , 48 ]. We identified two chemosynthesis-based carbon assimilation pathways, including a reductive citrate cycle in genus Hydrogenobaculum , and a hydroxypropionate-hydroxybutyrate cycle in genus Sulfolobus and genus Metallosphaera (Fig. 2 and Additional file 1 : Table S5). Two key genes in the reductive citrate cycle, korA (EC 1.2.7.3) and korB (EC 1.2.7.3), encoding the α and β subunits of critical enzyme 2-oxoglutarate ferredoxin oxidoreductase, were identified in the metagenome data and assigned to Hydrogenobaculum and T. uzoniensis . Furthermore, based on metagenomic data, Hydrogenobaculum bacteria had a gene encoding citryl-CoA lyase ( ccl ), an enzyme capable of catalyzing a biochemical reaction similar to another essential enzyme, ATP-citrate lyase, in the reductive citrate cycle [ 49 ]. In addition to the reductive citrate cycle, both T. uzoniensis and T. tenax had the key enzyme 4-hydroxybutyryl-CoA dehydratase (4.2.1.120 and 5.3.3.3) for dicarboxylate-hydroxybutyrate, and the key enzyme 2-oxoglutarate synthase (KorA and KorB, EC 1.7.3.2) of the reductive citrate cycle. Sulfolobus and Metallosphaera bacterial rely on the hydroxypropionate-hydroxybutyrate cycle to convert carbon dioxide into organic carbons. Genes encoding key enzymes in this pathway, including acetyl-CoA/propionyl-CoA carboxylase (EC 6.4.1.2), malonyl-CoA reductase (EC 1.2.1.75 and 1.1.1.298), methylmalonyl-CoA mutase (EC 6.4.99.2) and 4-hydroxybutyryl-CoA dehydratase (EC 4.2.1.120) [ 41 ] were identified in contigs assigned to Sulfolobus tokodaii and Metallosphaera genus. Acidithiobacillus and Thiomonas bacteria use the Calvin cycle to assimilate inorganic carbon [ 50 – 52 ]. Notably, Acidithiobacillus bacteria use electrons generated from sulfur metabolism for the Calvin cycle [ 50 , 51 ], thereby circumventing temperature limitations for photosynthesis [ 19 ]. However, it remains unclear whether Thiomonas bacteria could invoke a mechanism similar to Acidithiobacillus bacteria, enabling it to fix carbon [ 21 ] when the water temperature increased. However, the presence of cbbSL genes that encode the key enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the Calvin cycle identified in the current study and a previous report (Fig. 2 ; [ 52 ]), provided additional evidence that Thiomonas bacteria can assimilate carbon. Nitrogen cycle in SHP The only dominant SHP microbe capable of fixing nitrogen (Fig. 6 , Additional file 1 : Table S5; [ 53 ]) was A. ferrooxidans ; therefore, we inferred it played a key role in the SHP nitrogen cycle. It is noteworthy that the SHP spring is a nitrogen-limited environment (nitrate concentrations ranged from 0.9 ppm to below detection limits; Table 1 ; [ 21 ]). In addition, microbes living in SHP might have to obtain organic nitrogen from an alternative source (e.g. metabolizing existing nitrogen-containing compounds in the water). For example, several bacteria ( Hydrogenobaculum , A. ferrooxidans , and Thiomonas ) had nitronate monooxygenase (EC 1.13.12.16), the enzyme for transforming nitroalkane compounds (R-NO 2 ) to nitrite (Fig. 6 ). Fig. 6 Nitrogen-metabolizing enzymes identified from dominant microbes using KEGG mapping. For detailed descriptions for abbreviations and color codes, please see the legend for Fig. 2 \n Nitrite could be converted to ammonia (nitrogen reduction) and used to synthesize amino acids, or be converted into nitrogen (through denitrification) to generate energy. Two groups of bacteria, genus Thiomonas and A. ferrooxidans encoded several genes ( narG , narH , narI , narJ , nirB and nirD ) involved in dissimilatory nitrate reduction pathways. Nevertheless, according to the KEGG reference pathway, none of the dominant microbes had enzymes for a complete denitrification pathway (Fig. 6 ). Even though Reysenbach et al. analyzed the Hydrogenobaculum bacterial genome and suggested that str. Y04AAS1 genome harbored all genes required for this pathway, they did not detect reduced nitrate under experimental conditions [ 28 ]. However, that SHP has a low concentration of organic nitrogen compounds, microbes might prefer to use nitrate to synthesize building blocks in lieu of generating energy. Clearly, further investigations are needed to elucidate the nitrogen nutrient cycle in SHP. Sulfur metabolism Dominant microbes were dexterous in sulfur metabolism (Fig. 7 ). Vulcanisaeta archaea, Thermoproteus tenax and Caldivirga maquilingensis had the capacity to transform trithionate into sulfite with sulfite reductase (EC 1.8.99.3). Since archaea of genus Vulcanisaeta , T. tenax and C. maquilingensis had all enzymes required for dissimilatory sulfate reduction, they were capable of utilizing sulfate or sulfite for energy metabolism. Furthermore, sulfite converted from trithionate could be used for dissimilatory sulfate reduction. Thiomonas bacteria encoded genes for complete SOX complex, enabling them to convert thiosulfate into sulfate. Thiosulfate could also be converted into sulfite via thiosulfate/3-mercaptopyruvate sulfurtransferase (EC 2.8.1.1), present in most dominant microbes in SHP. Although several dominant microbes could convert thiosulfate into tetrathiosulfate, Hydrogenobaculum bacteria were the only dominant microbes capable of converting either tetrathionate or trithionate into thiosulfate. Fig. 7 Sulfur-metabolizing enzymes identified from dominant microbes using KEGG mapping. For detailed descriptions for abbreviations and color codes, please see the legend for Fig. 2 \n Searching against the KEGG database provided a basic understanding of the SHP sulfur cycle in the SHP. However, an extended literature search revealed additional or uncommon sulfur-related metabolic pathways, absent from the KEGG reference pathways, but identified in dominant microbes. For example, in addition to genus Hydrogenobaculum , based on genomic and transcriptomic analyses, we inferred that S. tokodaii [ 54 ], genus Acidithiobacillus [ 50 , 55 , 56 ] and genus Metallosphaera [ 54 ] could also convert tetrathionate to thiosulfate (Additional file 1 : Figure S3, Table S5). For bioleaching microbes like genus Acidithiobacillus and genus Metallosphaera , thiosulfate served as an oxidizer for Fe(II), which could be used to generate protons as a driving force for respiration [ 54 , 57 ]. Polysulfide mechanism is another Fe(II) oxidizing pathway [ 55 , 57 ]. Interestingly, based on the KEGG reference pathway and a literature search, almost all dominant microbes (including non-bioleaching microbes) in SHP were capable of transforming hydrogen sulfide to polysulfide (Additional file 1 : Figure S3, Table S5). Regardless, T. tenax and A. caldus were the only two dominant microbes with enzymes to recycle polysulfide [ 48 , 50 ] and thereby replenish the hydrogen sulfide pool, which would be beneficial for A. caldus during bioleaching. Hydrogen metabolism Genus Hydrogenobaculum could use hydrogen as its major energy source [ 29 ]. To explore hydrogen metabolism-related genes in our metagenomics data, we searched our DDS dataset against the NCBI database, and summarized the results (Additional file 1 : Table S2). The gene encoding Ni/Fe hydrogenase, which catalyzes the reaction: H 2 ↔ 2H + + 2e − , was identified. In addition, genes encoding Hyp, a group of proteins required during maturation of Ni/Fe hydrogenase [ 58 ], were also present in our DSS dataset. Microbial interactions in acidic hot springs In addition to several potential metabolic interactions, 16S rRNA gene-based diversity and CRISPR arrays also revealed microbe-microbe interactions. In that regard, the presence of genus Nanoarchaea and numerous viral sequences/CRISPR arrays were consistent with robust microbial interactions in the SHP. Genus Nanoarchaea (represented by Nanoarchaea -like 16S rRNA gene sequences) was a dominant genus in SHP (Fig. 1 and Table 2 ). There were apparently no previous reports of genus Nanoarchaea in an acidic thermal environment with a low NaCl concentration. The sole species of genus Nanoarchaea ( Nanoarchaeum equitans ) previously reported had a much-reduced genome and could only be grown in the presence of Ignicoccus sp. , an archaeal genus [ 59 ]. Furthermore, that an Ignicoccus -like 16S rRNA gene sequence was also detected in the present survey (highlighted in green in Additional file 1 : Table S1), suggested a potential host-parasite interaction between Nanoarchaea and Ignicoccus . It is well known that CRISPR is an antiviral defense system common in microbial genomes [ 60 , 61 ]. Furthermore, repeat sequences and spacers in CRISPR assays can be used to assign taxa, as they are strain-specific [ 62 ]. In the SHP metagenome, 1711 CRISPR-like arrays (comprising 15130 spacers) were identified, of which 123 were assigned to specific microbes (based on their unique repeat sequences; Additional file 1 : Table S6). In addition, there were several kinds of viral DNA sequences in the SHP metagenome (Additional file 1 : Table S7), providing evidence of viral infection. Spacer sequences of the CRISPR array could be used to characterize microbial evolution. Six of the CRISPR-like arrays identified from DSS dataset were assigned to Metallosphaera sedula based on their repeat sequence . The M. sedula reference genome contained four CRISPR arrays, each with a unique repeat sequence. Six CRISPR-like arrays were compared to known CRISPR arrays in the M. sedula reference genome [ 63 ]; two of the CRISPR-like arrays had identical repeat sequences with that of the longest CRISPR array (161 spacers) from the M. sedula reference genome. Furthermore, there were 65 identical spacers identified by comparing spacer sequences of those two arrays to the reference array (Fig. 8 ). More importantly, identical spacers were arranged in the same order as the reference. Since spacers are added to a CRISPR array in a chronological order [ 64 ], with 65 identical spacers in the reference genome on the 3′-end, we inferred that the two M. sedula populations, the reference strain isolated in Italy, and another identified by analyzing metagenomic data from SHP in this study, were both derived from the same ancestral population (with a common infection history). The M. sedula type strain was isolated from a hot water pond at Pisciarelli Solfatara, Italty. Multiple water samples were collected for microbial isolation, water pH was ~ 2 and temperature ranged from 25 to 52 °C [ 40 ] (cooler than SHP). Fig. 8 Alignment of CRISPR arrays with M. sedula reference arrays. Numbers of spacer are denoted. According to the CRISPR database, M. sedula has four CRISPR arrays in its genome. The repeat sequence of Array 647 and 646 (detected in the SHP metagenome by BLAST) matched repeat sequences of the longest CRISPR array (CRISPR ID: NC_009440_4, refer as “reference array” hereafter) in the M. sedula DSM 5348 reference genome. Since the order of spacers could be associated with time of virus infection, older spacers were located near the “ancient” end of the array. With a comparative analysis of the spacer sequences, all 27 spacers in Array 647 perfectly matched the array in the reference genome (with regards to sequence similarity and orientation). The 27 spacers were located at the end of the reference array. Array 646 contained 42 spacers; the last 38 spacers perfectly matched the 83 th to 121 st spacers in the reference array. Sequences of the first four spacers in Array 646 were different from all of the spacers in reference array; therefore, there was divergence of virus infection history between the M. sedula -related species in the SHP and M. sedula DSM5348. Moreover, since no spacers similar to the 1 st to 89 th and 122 nd to 124 th spacers of the reference array were detected, those spacers were designated “unknown”"
} | 8,068 |
35997428 | PMC9397054 | pmc | 2,490 | {
"abstract": "Bioinspired surfaces with special wettabilities attract increasing attention due to their extensive applications in many fields. However, the characterizations of surface wettability by contact angle (CA) and sliding angle (SA) have clear drawbacks. Here, by using an array of triangular micropillars (ATM) prepared by soft lithography, the merits of measuring the friction force of a water droplet on ATM over measurements of CA and SA in characterizing the surface wettability are demonstrated. The CA and SA measurements show ignorable differences in the wettabilities of ATM in opposite directions (1.13%) and that with different periodic parameters under the elongation ranging from 0 to 70%. In contrast, the friction measurement reveals a difference of > 10% in opposite directions. Moreover, the friction force shows a strong dependence on the periodic parameters which is regulated by mechanical stretching. Increasing the elongation from 0 to 50% increases the static and kinetic friction force up to 23.0% and 22.9%, respectively. Moreover, the stick-slip pattern during kinetic friction can reveal the periodic features of the measured surface. The friction force measurement is a sensitive technique that could find applications in the characterization of surface wettabilities.",
"conclusion": "4. Conclusions In summary, we prepared an array of triangular micropillars (ATM) by standard soft-lithography and examined the wettability by the measurements of CA, SA, and friction force. The CA and SA measurements cannot reveal the microscopic differences in the surface along the opposite directions and the change in the structural parameters upon mechanical stretching. It agrees very well with the concept proposed by Ras, that the contact angle measurement on superhydrophobic surfaces has its inherent drawbacks. The friction measurement conducted by the MPCP technique was involved, in order to examine the ATM surface. The MPCP technique revealed clear differences in the friction forces along the opposite directions and that of the ATM surface under mechanical regulation. Moreover, the kinetic friction process also revealed the surface periodicity, which cannot be achieved by the traditional SA measurement. Increasing the elongation from 0 to 50% increased both the static and kinetic friction forces over 20%, which was attributed to the increased pining effect at the receding fronts. When the elongation reached 70%, however, the friction forces slightly reduced, which was assumed to be caused by the dominating effect of the increased area of the air cushions. We therefore demonstrated the advantage of friction-force measurements in characterizing hydrophobic surfaces over the traditional CA and SA measurements.",
"introduction": "1. Introduction Adapting to the living environment, plants and animals have evolved different surface features [ 1 , 2 , 3 , 4 , 5 ]. Quite often the surface features are anisotropic, containing either anisotropic micro- and nano-scale structures or anisotropic arrangements [ 6 , 7 , 8 , 9 , 10 , 11 ]. For instance, the direction-dependent, overlapping scales on butterfly wings enable water droplets on the surface to easily roll off, maintaining dry wings [ 6 ]. The aligned hairs, together with the nanogrooves on each hair, allow the water striders to effortlessly walk on water [ 7 ]. There are many more of these types of surfaces, on which water droplets show anisotropic wetting behaviors [ 8 , 9 , 10 , 11 , 12 ]. Inspired by the anisotropic surfaces found in nature, engineered surfaces with anisotropic wetting properties could contribute significantly to the fields, such as biomedicine, ship transportation, microfluidics, smart surfaces, and so on [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. The static water contact angle (CA) and sliding angle (SA) have long been used to characterize the state of a water droplet on a solid surface [ 20 , 21 , 22 , 23 ]. The measured CA is usually an apparent CA, which is different from the inherent CA of an ideal smooth surface (CA i ), as a surface always contains a certain roughness. The apparent CA is influenced by the coordination of the surface energy and roughness. In the following text, the mentioned CAs are apparent CAs, unless otherwise mentioned. The Wenzel model [ 24 ] has been used to describe the state when water can penetrate into the roughness on the surface. In contrast, the Cassie model [ 25 ] describes the surface where air cushions are maintained at the contacting interface. In both of the models, the surface roughness has a strong influence on the CA. Therefore, the precise characterization of the CA is of critical importance to understand the surface wettabilities. However, the water-contact-angle measurement has its inherent limitations, in that a one-pixel error in the definition of the baseline and/or the outline of the water droplet will cause a large deviation in the CA, especially on the superhydrophobic surface with a CA larger than 150° [ 20 , 26 ]. Meanwhile, the measurement of the SA also has its difficulties when the water droplet keeps sticking onto the surface when the surface is turned upside down, which is known as the rose petal effect [ 27 , 28 ]. On the surfaces with a high adhesion to water droplets, the measurements of the CA and SA may have difficulties in distinguishing the surfaces with subtle differences. For instance, on the surface composed of a polydimethylsiloxane (PDMS) triangular-micropillar array, with the period ranging from 40 to 50 µm, a negligible difference in the SAs (ΔSA = 3°) in opposite directions can be found [ 16 ]. Similarly, on the microstrip surfaces, with the air fraction ranging from 0.20 to 0.50, the water droplets kept sticking to the surface when the surface was turned upside down along the direction perpendicular to the microstrips [ 17 ]. In both cases, there is no difference in the SA along certain directions, even though the surface structures are distinctly different [ 16 , 17 ]. It clearly suggests that the SA measurement cannot reveal the structural difference of these examined surfaces and what the extent of the difference could be [ 16 , 17 ]. As a complementary technique to the CA and SA measurements, we have proposed a technique termed the monitoring of the position of capillary’s projection (MPCP), to characterize the friction force (F) of a water droplet on superhydrophobic surfaces [ 17 ]. When measuring F, the displacement–time curve could also reveal the structural information of the tested surfaces [ 17 , 29 , 30 ]. For instance, when measuring a fresh lotus leaf, the F f suddenly changed when the droplet passed over the veins in the leaf. It can also measure the F f on a butterfly wing in the directions towards the body center. It is worth mentioning that the water droplets usually stick firmly to the surface in the direction towards the body center. Therefore, MPCP is capable of offering a deeper insight into the liquid–solid interactions. Here, we fabricate an array of triangular micropillars (ATM) and test the friction of a droplet on its surface in comparison with the traditional CA measurements. The CA remains the same when the period of ATM is regulated by mechanical stretching. In contrast, the MPCP reveals a clear difference in the friction forces in opposite directions on the ATM and that of the ATM with different structure parameters. Moreover, the detailed features of the kinetic friction process can also reveal the structure features of the ATM surface. Thus, the results here offer us the chance to better understand bioinspired surfaces with special wettabilities.",
"discussion": "3. Results and Discussion 3.1. Fabrication of ATM The ATM was successfully constructed by a standard soft lithography procedure. PDMS precursor was filled into the triangular microholes on a silicon mold, followed by curing at 90 °C for 1 h ( Figure 1 a). Generally, 2 mL of PDMS precursor was spread onto the whole silicon mold, which resulted in a thickness of ~1.2 mm. After peeling off from the mold, the sample was cut into a strip (~1 cm in width and ~5 cm in length) with the ATM in the center region for the convenience of the following studies ( Figure 1 b). The resultant ATM is an array of micropillars with a height of ~20 μm, period of 40 μm, and a triangular cross-section with edge length of ~23 μm, faithfully copying the structures of the silicon mold ( Figure 1 c,d). The top of the micropillars is rather smooth, with a roughness of 1.3 ± 0.2 nm. The periods of the micropillars in the ATM can be easily regulated by mechanical stress in the supporting layer. Due to the triangular shape of the micropillars, we therefore define the direction pointing to the angle of the triangular micropillar as the direction of angle (D A ), and the opposite direction towards the bottom edge of the triangle as the direction of edge (D E ) ( Figure 2 a). The stretching, in the direction of D A /D E , will thus change the period along the stretching direction (P s ) and the period perpendicular to the stretching direction (P p ) ( Figure 2 b). The as-prepared ATM has P s = P p = 40.62 ± 0.49 μm. Due to the Poisson’s ratio effect, the stretching increased P s and decreased P p ( Figure 2 c). For instance, the stretching to 30% increased P s to 54.11 ± 0.19 μm and decreased P p to 33.60 ± 0.15 μm ( Figure 2 d). The stretching to 70% further increased P s to 67.25 ± 0.26 μm and decreased P p to 30.51 ± 0.16 μm. The relaxation of the stress would recover the original structural parameters, due to the elasticity of PDMS [ 16 ]. 3.2. Characterization of Wetting Property of ATMs The wetting properties of the ATM were checked with a traditional CA measurement. The as-prepared ATM has a CA of 141.6 ± 0.3°, slightly smaller than 150° ( Figure 3 a). Moreover, the contact angle hysteresis was 8.5°, larger than 5°. Therefore, the ATM is not superhydrophobic [ 32 , 33 , 34 ]. It is reasonable that the ATM is not superhydrophobic, since a superhydrophobic surface requires micro- and nano-roughness besides the low surface energy of the material [ 35 ]. A close look at the contacting interface between the water droplet and the ATM surface revealed that the water droplet sat on top of the micropillars ( Figure 3 b). We cannot tell whether the water droplet partially penetrated into the gaps among the pillars, due to the technical limitations of our device. However, we would like to assume that the water may have partially penetrated into the gaps, due to gravity. For simplicity, the Cassie equation (2) is used to describe the measured CA: (2) cos CA = f 1 cos CA i − f 2 \nwhere f 1 and f 2 = 1 − f 1 represent the fractions of the area of micropillars on ATM contacting the water droplet and the area of air cushions at the interface, respectively. The CA i of PDMS was measured to be 117.4 ± 0.6°. While the elongation was increased from 0 to 70%, the CAs were quite close, ranging from 141.6 ± 0.3° to 143.2 ± 0.6° ( Figure 3 a). It has been argued that the diffuse edge and the uncertainty in the baseline by one pixel could introduce a substantial systematic error in the CA from about 1° to beyond 10° on superhydrophobic surfaces [ 20 ]. Here, the change in the CAs is only 1.6°, which means a difference of 1.13%, so that the difference in the CAs could be ignored. On the other hand, the air fraction changed significantly upon the mechanical stretching. Without stretching, 1118 ± 26 micropillars were involved in the contact area ( Figure 3 c), corresponding to an f 2 of 71.99% ± 0.12% ( Figure 3 d). The increase in elongation to 70% sharply decreased the number of contacted micropillars to 734 ± 30. Accordingly, the f 2 within the contact area increased to 79.01% ± 0.38%. Considering the increase in the air fraction during the sample stretching, we expected an increase of 4.5° in CA, according to the Cassie equation. The accuracy of our device for CA measurement is around 0.01°, which should be far enough to differentiate the difference of 1.6°. However, no clear difference was detected. It implies a drawback in the CA measurement [ 20 ]. It is well known that the three-phase contact line at the receding front of a water droplet on a tilted surface determines the SA. The water droplet on the ATM has different three-phase contact lines at the receding front, due to the triangular shape of the micropillars when the ATM is tilted in the directions along D A and D E . However, no significant difference between the SAs along the opposite directions was detected when P p is 40 µm (corresponding here to the stretching of 0%) [ 16 ]. Moreover, no significant difference was found when the period was increased to 51 µm. Though the SA measurement has an accuracy of 0.0001°, which is determined by the electronic tilting base unit, the SA measurement also cannot distinguish the difference between the ATM surfaces under various elongations. 3.3. Friction at Solid-Liquid Interface The MPCP technique was used to characterize the ATM surfaces. In a typical measurement, a 6 μL water droplet was dragged by the capillary sensor across the ATM surface. The deflection of the capillary sensor, which caused the resistance between the water droplet and the ATM surface, was converted into friction force (F) ( Figure 4 a). In analogs to the solid–solid friction, the typical measuring curve can be divided into three regions: static friction; kinetic friction; and the transition zone between the static and the kinetic frictions ( Figure 4 b) [ 36 ]. The peak value during the static friction period was considered as the static friction force (F S ), while the mean value during the kinetic friction was calculated to be the kinetic friction force (F K ). On the ATM, the F S of 38.7 ± 2.5 μN and 34.7 ± 1.9 μN were detected when the droplet moved in the direction of the D A and D E , respectively ( Figure 4 c). It means a difference (ΔF) of 4.0 μN, which is around 11.5% in difference along the opposite directions. The stretching to 50% increased the F S in the direction of D A to 47.6 ± 2.6 μN, while in the opposite direction the F S increased to 42.0 ± 1.1 μN. That represents a 23.0% and 21.0% increase in the direction of D A and D E , respectively. However, the further increase in the elongation to 70% slightly decreased the F S in the D A and D E directions to 43.0 ± 1.1 and 41.0 ± 1.6 μN, respectively. Accordingly, ΔF also steeply decreased to 1.9 μN, indicating a lower anisotropy of the surface. Like the solid–solid friction, the F K is smaller than the F S ( Figure 4 d). For instance, the F K s in the direction of the D A and D E were 25.3 ± 0.7 μN and 23.9 ± 1.0 μN, respectively, which are 34.6% and 28.8% smaller than the F S . Upon the increase in the elongation, the changes of the F K s in both directions are similar to that of the F S . That is, the F K s in both directions and the corresponding ΔF also reached their maximum at an elongation of 50%, and decreased upon the further stretching to 70%. Therefore, the friction force measurements (F S , F K , and ΔF) clearly revealed the microscopic anisotropy of the ATM and the structure change of the ATM upon mechanical stretching, showing obvious merits over the measurements of the CA and SA. 3.4. Mechanism for Anisotropic Friction The difference in the three-phase contact lines at the receding fronts is responsible for the anisotropic friction ( Figure 5 ). Because of the geometry of the triangular pillar, discrete contact lines (red line) and contact points (blue dots) form at the edge and the angle of the triangular pillars, respectively. When the droplet moves towards D A , the receding front is the discrete contact lines. In the opposite direction of D E , the discrete contact points locate at the receding front. Since the discrete contact lines provide a large pinning force to the water droplet than the contact points, the friction force along the D A is larger than that along the D E [ 16 ]. Due to the Poisson’s ratio, the stretching of the ATM (the increase of P s ) would decrease the space between the micropillars along P p , resulting in the increase in the total length of the discrete contact lines and the number of discrete contact points, and, therefore, the friction forces in both directions. However, when the stretching is over 50%, for example, 70%, the increase in the space between the micropillars along P s sharply increases the air fraction at the contact interface; this would dominate the change in the pinning effect of the receding front, reducing the friction forces in both directions and the corresponding ΔF. 3.5. Revealing of Surface Periodicity While the measurement of the F S discloses the moment similar to the initiation of sliding of a water droplet on a tilted surface (SA measurement), the process of the F K measurement could reveal the surface structures. On the ATM, the process of kinetic friction showed a stick-slip pattern when the water droplet moved along D A ( Figure 6 a). While the sticking should be caused by the pinning of the receding front of the droplet, the slipping is assumed to be the result of the receding front jumping from one to the next row of micropillars [ 37 ]. The period of the stick-slip (P SS ) matched the P S quite well at various elongation ratios ( Figure 6 b). For instance, the P SS revealed a period of 41.86 ± 1.73 μm of the ATM surface at an elongation of 0%, which matches the corresponding P S = 40.62 ± 0.49 μm very well. Upon the increase in the elongation to 70%, the P S and P SS were increased to 67.25 ± 0.26 μm and 63.06 ± 2.31 μm, respectively. It thus strongly suggests the possibility to study the surface geometry by measuring the stick-slip pattern during kinetic friction."
} | 4,446 |
38468949 | PMC10926130 | pmc | 2,491 | {
"abstract": "Deep sea benthic habitats are low productivity ecosystems that host an abundance of organisms within the Cnidaria phylum. The technical limitations and the high cost of deep sea surveys have made exploring deep sea environments and the biology of the organisms that inhabit them challenging. In spite of the widespread recognition of Cnidaria's environmental importance in these ecosystems, the microbial assemblage and its role in coral functioning have only been studied for a few deep water corals. Here, we explored the microbial diversity of deep sea corals by recovering nucleic acids from museum archive specimens. Firstly, we amplified and sequenced the V1–V3 regions of the 16S rRNA gene of these specimens, then we utilized the generated sequences to shed light on the microbial diversity associated with seven families of corals collected from depth in the Coral Sea (depth range 1309 to 2959 m) and Southern Ocean (depth range 1401 to 2071 m) benthic habitats. Surprisingly, Cyanobacteria sequences were consistently associated with six out of seven coral families from both sampling locations, suggesting that these bacteria are potentially ubiquitous members of the microbiome within these cold and deep sea water corals. Additionally, we show that Cnidaria might benefit from symbiotic associations with a range of chemosynthetic bacteria including nitrite, carbon monoxide and sulfur oxidizers. Consistent with previous studies, we show that sequences associated with the bacterial phyla Proteobacteria, Verrucomicrobia, Planctomycetes and Acidobacteriota dominated the microbial community of corals in the deep sea. We also explored genomes of the bacterial genus Mycoplasma, which we identified as associated with specimens of three deep sea coral families, finding evidence that these bacteria may aid the host immune system. Importantly our results show that museum specimens retain components of host microbiome that can provide new insights into the diversity of deep sea coral microbiomes (and potentially other organisms), as well as the diversity of microbes writ large in deep sea ecosystems.",
"conclusion": "4 Conclusions Our study sheds light on potential new and unique associations of corals in the deep sea with microbial lineages not previously characterized at the molecular level, which may constitute entirely new lineages. Here, we have shown that specimens held in museum collections carry meaningful information about an animal's microbiome, which is detectable through DNA barcoding sequencing. Our dataset included sequences from microbes known to be associated with deep water corals, but we also highlight the presence of microbial sequences, including chemotrophs, previously unreported in deep sea Cnidaria. In addition, and surprisingly, we found Cyanobacterial sequences associated with corals in the deep sea, as has been shown with other deep sea organisms and shallow water corals. We further suggest that Mycoplasma could aid the host immune system against viral infections. The deep sea benthic ecosystems are simultaneously the largest and most unknown habitats on Earth, which host a breadth of unexplored biodiversity, yet whose remoteness is not remote enough to protect them from the impact of anthropogenic activities. Therefore, we argue that explorations of these remote ecosystems are urgent, with important insights that can be gained through accessing resources within our reach such as specimens held in museum collections.",
"introduction": "1 Introduction The deep seafloor ecosystem (>1000 m of depth) is one of the most remote, unproductive and largest ecosystems on the planet [ 1 ], it remains mainly unexplored, covering approximately 60% of Earth's solid surface [ 2 ]. The impact of anthropogenic activities on the biota of the deep sea has been described as catastrophic [ 3 ]. Seamounts are currently being severely exploited by deep sea fishing, with global catches estimated at ∼3 million tonnes per year [ 4 ]. Similarly, it has been reported that 95% of the large sessile fauna in fished seamounts south of Tasmania is impacted by the activities of bottom trawlers [ 5 ]. Deep sea ecosystems are considered degraded environments and available data suggest that these systems are losing their biodiversity [ 2 ]. As such researches suggest that deep sea sessile organisms are increasingly endangered and given the challenges in studying these largely inaccessible habitats, any retrievable information on their biology, function, and contribution to ecosystem stability is highly valuable. The remoteness of the deep sea ecosystem hinders our understanding of many aspects of deep sea organisms' biology including their physiology, distribution and ecological interactions. Among them are cold and deep sea water corals, which encompass stony corals (Scleractinia), soft corals (Octocorallia), black corals (Antipatharia), and hydrocorals (Stylasteridae) [ 6 ]. These organisms have been reported across the oceans worldwide [ 3 , 6 , 7 ] and their biodiversity in the deep ocean is greater than that of shallow water ecosystems [ 3 , 7 ]. Corals in these ecosystems occur both as isolated colonies and larger three-dimensional reef structures that provide habitat, refuge and nursery grounds to a wide variety of organisms including commercially important species [ 6 ]. Importantly, growth rates of deep water coral species are lower than their shallow water counterparts [ 8 , 9 ] making deep sea reef structures particularly vulnerable to direct anthropogenic disturbances. Unlike most reef forming shallow water corals, cold and deep water corals lack symbiotic dinoflagellates, which provide photosynthetic byproducts that support coral growth [ 10 ]. Accordingly, microbial associates of deeper water corals may play crucial roles in the nutrient acquisition, such as recycling nutrients or degrading recalcitrant organic matter [ [11] , [12] , [13] ]. For instance, the deep sea scleractinian coral Desmophyllum pertusum (previously known as Lophelia pertusa ) relies on symbiotic bacteria to obtain fixed nitrogen [ 13 ]. A similar process has been postulated for octocorals, where the association with bacteria such as Spirochaeta , Bacillus and Propionibacterium might facilitate the nitrogen metabolism of the holobiont [ 9 , 14 ]. Several molecular surveys suggest that cold and deep water corals maintain species-specific microbial communities [ [15] , [16] , [17] ]. Indeed, anatomical compartments such as the coral mucus, tissue and when present skeleton host specific microbial communities [ [15] , [16] , [17] ], as has been observed in their shallow water relatives [ [18] , [19] , [20] ]. Currently, the number of known cold and deep water coral species exceeds 2500 [ 7 ], but despite their microbial associates' recognised importance, only the microbiomes of a few taxa have been characterized. Museums worldwide hold specimens and collections derived from decades of deep sea exploration. Many of these collections include cold and deep water coral specimens, which naturally include an abundance of microorganisms and symbionts whose biology remains unstudied. A better understanding of the role of these organisms could yield a plethora of insights into these associations and deep sea ecosystems. Given the rapid loss of biodiversity in the Anthropocene, along with the technical and logistical difficulties in studying the deep sea, museum specimens could provide new insight into its ecosystems. Here we investigated the microbial diversity of cold and deep water corals found in museum collections from tropical and temperate locations, as the different environmental conditions of these regions could affect their coral populations. We used this approach as a means to generate hypotheses on the functional roles of the microbial partners, to expand our understanding of cold and deep water coral microbiomes, and to assess the utility of using museum specimens as a tool to investigate deep water organismal and ecosystem function.",
"discussion": "3 Results and discussion 3.1 Sequencing statistics and phylogenetic inference After denoising, ASVs filtering (e.g. chloroplasts and mitochondria) and contaminant removal, the 16S rRNA gene dataset consisted of 152,262 sequences (min: 6; median: 1297; max: 51,645) of an average length of 408bp aggregated in 1916 ASVs. According to the SILVA v138 QIIME release 1235 ASVs were classified as Bacteria, 40 as Archaea and 641 were unassigned. To assess the reliability of our dataset and to clear the hypothesis that the detected microbial taxa were the result of contamination or sequencing biases, we performed further manual checks. For instance, we aligned the sequences of our dataset along with 199 archaeal partial (V1–V3 regions) 16S rRNA genes and removed 282 assigned and 352 unassigned sequences because they did not present a significant alignment distance with other sequences, or because they were chimeras. According to our phylogenetic analysis, none of the sequences assigned to the Archaea were classified within this domain but to the bacterial orders Sphingomonadales, Reyranellales, Rhizobiales, Rhodobacterales and Parvibaculales in the phylum Proteobacteria and to the order Ktedonobacterales in the phylum Chloroflexi. Misclassification of these sequences could have been determined by the high similarity (min: 70.3%; median: 85.9%; max: 100.0%; Supp. Table 3 ) of the 16S rRNA gene V1–V3 regions between these bacterial orders and the Archaea. Thus, we performed this analysis using a subset of our dataset including 41,939 sequences shared across 1282 bacterial ASVs that were curated according to the phylogenetic inference of our sequences. 3.2 The residual α-diversity By measuring the α-diversity indices observed and Shannon, we compared the residual microbial diversity of the investigated coral families, except for the Anthomastinae and Kophobelemnidae, which were represented by only one sample each ( Fig. 3 a). These measurements revealed variability in the α-diversity of the bacterial communities retrieved across specimens of the same coral family, with the Primnoidae and Umbellulidae being the least variable and the Keratosinidae and Protoptilidae showing the greatest variability across both α-diversity indices ( Fig. 3 a). The α-diversity variability measured within the families Keratosinidae and Protoptilidae can be explained by several important factors which should be highlighted in considering the data generated and any comparisons of the coral families within the study including, 1) there is ∼3277 km between the sampling locations for the two expeditions that were undertaken to compile these museum collections ( Figs. 1 ), 2) the different preservation methods of specimens (100% ethanol vs frozen); the uncertainty in taxonomic classification below family level for the specimens and 3) the prolonged preservation time for some specimens in 100% ethanol at ambient temperature [ 38 ]. Taking these factors into account, the family Umbellulidae had the most diverse and evenly distributed microbial community ( Fig. 3 a), as indicated by the Shannon index, which considers both richness and evenness [ 39 , 40 ]. In contrast, the Primnoidae had the lowest Shannon index indicating a low diverse community dominated by few taxa ( Fig. 3 a). However our data show that the specimens retained a high degree of their original biodiversity and, as found across several other marine organisms including shallow water corals [ 18 ] and reef damselfishes [ 21 ], there is a great deal of α-diversity variability across specimens belonging to the same taxon. Based on these results, we further analyzed the cold and deep sea water coral microbiome to gain insight into its structure and functions. Fig. 3 Alpha-diversity metrics (Observed and Shannon) analysed at the ASV level (a). Bacterial phyla (b) associated with each coral family. Superscript letters indicate coral families belonging to the orders Scleralcyonacea (a), Malacalcyonacea (b) and Scleractinia (c). Fig. 3 3.3 New insights into the microbial community of cold and deep sea water corals The coral families investigated here were found to have unique microbial assemblages ( Fig. 3 b). We have tested whether the microbiomes of the family Keratoisidinae (n = 20) and of the whole pool of corals from the two locations (Coral Sea and Tasman fracture) had different β-diversity, but we found no significant differences. These results suggest that the environment does not strongly influence the microbiome of cold and deep sea water corals but other processes are more likely to be key for microbiome uptake and retention. For instance, deep sea coral populations around Australia have no genetic subdivision at scales of tens to hundreds kilometres [ 41 ], and this high degree of connectivity could also be reflected in their microbiomes. At the phylum level, Proteobacteria were recorded at high relative abundance (>25.2%) in every family investigated, peaking to 89.9% in the Paragorgididae ( Fig. 3 b; Supp. Table 4 ). The phyla Verrucomicrobiota, Cyanobacteria, Acidobacteriota and Planctomycetota were present across multiple coral families but at lower relative abundance ( Fig. 3 b; Supp. Table 4 ). At lower taxonomic resolution, the Alphaproteobacteria were the most abundant class in the bacterial communities of each coral family, except in the Caryophyllidae ( Supp. Table 5 ). Within the Alphaproteobacteria, sequences associated with members of the orders Rhizobiales, Rhodobacterales and Sphingomonadales were present in every coral family and dominated the microbial communities ( Supp. Table 6 ). For instance, 43.6% of the sequences retrieved from the coral family Umbellulidae were associated with the Rhizobiales ( Supp. Table 6 ), 41.4% of the sequences retrieved from the coral family Kophobelemnidae were associated with the Rhodobacterales , and 18.2% of the sequences retrieved from the coral family Keratosidinae were associated with the Sphingomonadales. In contrast to Kellogg et al. [ 42 ], who reported a high relative abundance of sequences associated with the genus Endozoicomonas in the order Oceanospirillales in the coral genus Desmophyllum within the family Caryophiliidae [ 42 ], we detected only a low abundance of sequences associated with this bacterial genus. The specimens collected by Kellogg et al. [ 42 ] were from the West Atlantic, while ours from the Tasman and Coral Seas. Therefore it is unclear whether this discrepancy reflects biogeographical patterns affecting the microbiome composition of these corals, or is an artefact of sampling, storage, and handling. Members of the Sphingomonadales and Rhizobiales are known to play a role in the nitrogen biogeochemical cycle [ 43 ]. Evidence shows that shallow water corals rely on microbial partners for nutrient provision [ 44 , 45 ] and although there is a paucity of information regarding the metabolic interactions between cold and deep sea water corals and their microbial associates, it is likely that similar processes are also common in this group. Genomic analyses have shown that Sphingomonadales and Rhizobiales both fix nitrogen in the photic zone [ 43 ]. Whether they can perform this function in the deep dark ocean is still a mystery, but some heterotrophic Alpha- and Gammaproteobacteria do perform aphotic nitrogen fixation in abyssal waters [ 46 , 47 ]. The chemical processes by which molecular nitrogen is converted into ammonia without light are not yet fully understood. Nevertheless, it is plausible that the dark and nutrient depleted deep sea ecosystem [ 1 ] exerts selective pressure for nitrogen fixing bacteria to establish beneficial associations with corals. Our data also show that every cold and deep sea water coral family, except for the Anthomastinae, were associated with chemosynthetic bacteria (64 ASVs accounting for 4098 sequences; Supp. Table 7 ), which could aid the host metabolism through recycling important nutrients and by providing fixed carbon. For instance, we found that one specimen belonging to the family Paragorgiidae and one to the Caryophylliidae harboured sequences associated with nitrite oxidising bacteria in the genus Nitrospina [ 48 ] ( Supp. Table 7 ). We also found that one specimen in the family Protoptilidae had a high count (n = 731) of sequences associated to carbon monoxide oxidising bacteria in the family Ktedonobacteraceae ( Supp. Table 7 ; [ 49 ]). As another example, one specimen in the family Nephtheidae and one in the Paragorgiidae harboured sequences associated with the sulfur oxidising bacteria SAR324 ( Supp. Table 7 ; [ 50 ]). These data suggest that even though these Cnidaria families do not rely on chemosynthetic microbes for fixing carbon, cold and deep sea water coral might be able to establish chemosynthetic symbioses and more work is needed to unravel this aspect of their biology. 3.4 Cyanobacteria in the deep sea The generated dataset includes 41 ASVs (accounting for 2500 sequences) belonging to the orders Cyanobacteriales (n = 22), Synechococcales (n = 10), Oxyphotobacteria Incertae Sedis (n = 5) and Obscuribacterales (n = 4; Supp. Table 8 ) . Previous studies investigating cold and deep sea water corals have reported Cyanobacteria sequences associated with their samples. However, they proposed that Cyanobacteria were present in the water column and were captured with the coral samples during their retrieval from the deep sea through shallow waters [ 51 ]. A further hypothesis to explain the presence of Cyanobacteria in deep sea environments could be their association with sinking particulates that can then be retrieved at great depth [ 52 ] and/or caught from the water column at depth by corals. While we do not exclude these hypotheses for our samples, we note that sequences associated with Cyanobacteria were present in every coral family except for the Umbellulidae, ranging from 0.2% to 38.6% relative abundance ( Fig. 3 b; Supp. Table 4 ) and in corals collected in sealed containers at depth by ROV (this precludes the possibility that the corals were bathed in surface waters). Furthermore, studies investigating microbial communities associated with deep sea foraminifera [ 53 ] and subsurface rocks [ 54 ] have also found an abundance of Cyanobacteria associated in these unlit systems and proposed that they do not necessarily rely on their photoautotrophic metabolism, as their genome shows potential for a hydrogen based lithoautotrophic metabolism [ 54 ]. In fact, chemosynthesis is a widespread process in the ocean [ 55 ]. Thus, it is possible that Cyanobacteria could be a ubiquitous component of many cold and deep sea water coral microbial assemblages. The putative presence of Cyanobacteria in cold and deep sea water corals may not in fact be surprising considering that members of this phylum are consistently associated with shallow water corals [ 19 , 44 , 56 , 57 ]. Considering that Cyanobacteria are adapted to environments hostile to organisms that typically rely exclusively on photoautotrophic metabolisms such as caves [ 58 ] and the deep subsurface [ 54 ], it is worth nothing this adaptability in relation to their association with cold and deep sea water corals. The presence of Cyanobacteria in ecological niches that do not necessarily meet their typical physiological requirements suggests that they may have evolved other strategies to survive and grow in the absence of light. It is also worth noting that Cyanobacteria in partnership with cold and deep sea corals may benefit of interactions with a large and abundant microbiome. These interactions may also allow for a breadth of molecular handoffs to sustain metabolisms optimized to exploit the limited resources of the deep sea and could in part explain the success of corals in colonizing it. 3.5 Mycoplasma in the deep sea Ten unassigned sequences associated with the coral families Keratoisidinae, Kophobelemnidae and Protoptilidae clustered with the bacterial family Mycoplasmataceae in the phylum Mollicutes ( Supp. Fig. 1 ). Members of the genus Mycoplasma in the family Mycoplasmataceae have been found associated with several deep sea organisms including snails [ 59 ], chitons [ 60 ], polychaeta [ 61 ] and isopods [ 62 ]. Although their role in these organisms is unclear, it has been speculated they may aid the digestion of nutrient deficient food [ 59 ]. Using fluorescent probes, Neulinger et al. [ 11 ] identified and described Candidatus Mycoplasma corallicola, a proposed species that lives on the nematocysts of the deep sea coral Desmophyllum pertusum . Currently, the role of Mycoplasma associated with cold and deep sea water corals is unknown but given their presence in the nematocysts, Neulinger et al. [ 11 ] proposed that these bacteria could be commensal partners that benefit from leakage of hemolymph following perforation of preys by the action of the nematocysts barb. To support to this hypothesis, we screened 600 Mycoplasma genomes recovered from NCBI and found an arsenal of genes encoding transporters of micro-and macro-molecules [e.g. ABC transporters, MFS transporters and ECF transporters; [ 63 , 64 , 65 ]]. While these genes are shared across many microbial lineages, their persistence and abundance in bacteria with a reduced genome such as Mycoplasma [ 66 ] suggest their essential role in scavenging nutrients from the surroundings and maintaining homeostasis. This finding supports the hypothesis of a commensal lifestyle between families of cold and deep sea water corals and Mycoplasma [ 11 ]. Furthermore, we also found genes involved in viruses' immune response [e.g. type IV toxin-antitoxin system AbiEi family antitoxin domain-containing protein and nucleotidyl transferase AbiEii/AbiGii toxin family protein; [ 67 ]] that are activated by phage infection. Although the main aim of this defence mechanism is probably to protect Mycoplasma from viruses’ infections, it is plausible that, as a side effect, it could also aid the host immune system against infection, implying a deeper involvement of Mycoplasma in the physiology of coral holobionts. These results provide new insights into the functions of cold and deep sea water coral holobionts that can be derived from opportunistic assessments of the microbial assemblages retained in archived museum collections from these largely inaccessible habitats."
} | 5,610 |
33644523 | PMC7906494 | pmc | 2,492 | {
"abstract": "We\npresent a superhydrophobic surface capable of recovering the\nlubricious gas layer known as the “plastron” from a\nfully wetted state underwater. It is shown that full plastron recovery\nis possible without a second layer of structural hierarchy, which\nis prone to irreversible wetting transitions. This allows us to use\na cheap, fast, and potentially scalable method to fabricate the surface\nfrom silicone and carbon black in a molding process. We demonstrate\nplastron recovery from the fully wetted state and immediate plastron\nrecovery after pressure-induced wetting transitions. The wetting state\ncan be measured remotely and quickly by measuring the capacitance.\nThe slip length is measured as ∼135 μm, agreeing well\nwith the theory given the geometry of the surface. The ability of\nthe surface to conform to small radii of curvature and withstand damage\nfrom loading is also demonstrated. The work presented here could allow\nsuperhydrophobic surfaces to reduce drag on ships and in pipes where\nthe plastron would otherwise rapidly dissolve.",
"introduction": "Introduction As a large ship passes\nthrough the water, up to 70% of the fuel\nis burned to overcome skin friction drag acting on the hull 1 and in hydroelectric facilities skin frictional\ndrag can cause energy losses of up to 20%. 2 Superhydrophobic surfaces can significantly reduce skin friction\ndrag in laminar 3 and turbulent flows. 4 When submerged in water, a lubricious gas layer\nis trapped on the surface by hydrophobic surface features, which leads\nto effective slip of water over the surface. 5 Superhydrophobic surfaces may be able to minimize these losses in\nreal-world scenarios; 6 however, before\nthis technology could be adopted, there are several problems which\nmust be solved. A significant obstacle for prolonged drag reduction\nusing superhydrophobic\nsurfaces is the inherent fragility of the gas layer trapped between\nthe liquid and the solid also known as a “plastron.”\nIt can be lost through excessive pressure difference between itself\nand the water, which leads to an initial compression 7 and subsequent dissolution. 8 Once lost, the plastron is hard to recover as generally the wetted\nstate is thermodynamically more favorable. A convenient measure\nof resistance to wetting is the critical depth, d crit . This is the depth at which water pressure\novercomes capillary pressure and dissolution of the plastron starts\nto occur. To make a superhydrophobic surface more resistant to wetting,\nthe geometry should be altered to reduce the pitch, p (the center to center spacing between channel walls) and reduce\nthe gas fraction, ϕ g (the fraction of the surface\noccupied by the plastron averaged over a large area of the surface),\nas shown in eq 1 . 9 1 where θ s is sidewall\ncontact\nangle, γ is the surface tension, D is the liquid\ndensity, and g is the acceleration due to gravity. Equation 1 is valid for the\nnormal case where dissolved gases in the water are in equilibrium\nwith the atmosphere. Figure 1 a(i) shows a superhydrophobic surface made of parallel channels\nand (ii) identifies the defining parameters. Figure 1 (a(i)) Schematic of a\nsurface consisting of parallel channels.\n(a(ii)) Schematic of one channel with key parameters identified. (b)\nGraph showing the dependence of critical depth and slip length on\nthe gas fraction and pitch of a superhydrophobic surface consisting\nof parallel channels. It is not possible to have both large critical\ndepth and slip length. The isopleths of critical depth are calculated\nusing eq 1 assuming a\nsidewall contact angle of θ s of 110° and plotted\nin red. The isopleths of slip length are calculated using eq 2 and plotted in blue. However, this surface with a small pitch and gas\nfraction which\nhas good wetting resistance would be very poor at generating slip,\nthe streamwise movement of the water at the liquid/gas interface.\nA large slip length, δ, is achieved with a large pitch and large\ngas fraction as shown in eq 2 . 10 2 This\ndichotomy is shown graphically in Figure 1 , where the isopleths\nof slip length and critical depth are plotted as a function of gas\nfraction and pitch for a surface consisting of parallel channels,\nwhich has been shown to be an effective superhydrophobic surface geometry\nfor drag reduction. Although there is a benefit in choosing\na large gas fraction and\nsmall pitch to maximize both properties, even in this ideal case,\ncritical depth and slip length still oppose one another. There is\nno point on this gas fraction/pitch landscape where we can achieve\na desirable outcome of both large slip length and large critical depth;\nit will always be a compromise. While passive methods such a\nsidewall texturing, where nanosized\nstructures are added to the sides of microsized structures, have shown\nthat plastron loss from dissolution and pressure can be delayed (which\nacts to move the isopleths of critical depth upwards and rightwards\nin Figure 1 by increasing\nθ s in eq 1 ), ultimately plastron loss cannot be avoided. 11 Passive dewetting of conical pores is possible at ambient\npressures. However, this behavior requires very small pores and thus\nwould not generate a large slip length. 12 If we accept that there is no escaping the dichotomy presented\nin Figure 1 , a solution\ncould be to employ a system where the plastron is actively maintained.\nIf one had the ability to replenish the plastron, the surface could\nbe designed to prioritize large slip length over wetting resistance\nand accept that while the plastron is easily lost it could also be\nrecovered on demand. Methods to generate a new plastron when submerged\nunder water include chemical decomposition of hydrogen peroxide, 13 thermally induced supersaturation of gases in\nsurrounding water, 14 thermal vaporization, 15 pumping of pressurized air, 16 and electrolysis. 17 , 18 Very recently, the\nelectrolysis has been shown to be powered by the corrosion of magnesium\navoiding the use of an external power supply, however with the downside\nof having finite use due to consumption of magnesium. 19 To this end, Lee and Kim used electrolytic gas evolution\nas an\neffective means of plastron replenishment. 17 To achieve successful plastron recovery, they identified that the\ncapillary pressures acting on the evolved gas must be lower for lateral\nspreading along the channels than vertical growth out of them. If\nthis condition was not met, the newly generated gas was lost as bubbles\nexiting the top of the surface. A caveat to their surface design was\nthat a nanosized superhydrophobic base must be used between the microsized\nsuperhydrophobic structures and that the plastron of this nanosized\nsuperhydrophobic base must stay intact for full plastron to be recovered, Figure 2 . This meant that\nthe plastron could not be recovered when the surface was fully wetted.\nIf for some reason this became wetted, e.g., from a prolonged period\nwithout active gas generation, large pressures generated by waves\nor water with low dissolved gas concentration, then the surface would\nnot be able to recover, thus irreversibly losing its low drag properties. Figure 2 Successful\n(a) and unsuccessful (b) plastron recovery in a channel\nwith a superhydrophobic base viewed looking through the side of a\nchannel, showing that when the nanostructured base becomes fully wetted,\nthe plastron cannot be recovered. (a(i)) shows the native gas film\npresent in the channel. (a(ii)) the channel is wetted; however, the\nplastron of the nanostructured base remains intact. (a(iii)) gas is\ngenerated, and as the forces opposing lateral growth are less than\nforces opposing vertical growth, the plastron fills the channel. (b(i))\nStarting again with the native gas film present in the channel. (b(ii)),\nboth the channel and the nanostructured base are wetted. (b(iii))\nwhen gas is generated, the forces opposing lateral growth are greater\nthan forces opposing vertical growth so the plastron is lost as bubbles\nfrom the top of the channel. Another more practical problem for large-scale drag reduction is\nhow one might make a surface, which can be produced in large areas\nat reasonable cost so that it could coat something as large as a ship.\nTo give an appreciation of the areas required, a Maersk triple E has\na wetted area of 27 500 m 2 . 20 Most low drag superhydrophobic surfaces have been made using cleanroom\ntechniques on the order of centimeters squared, which could not be\nscaled to produce large areas at low cost. Furthermore, these surfaces\nare often made of silicon which would not conform to the hull and\nsurface features could be easily damaged. In this paper, we\ndemonstrate that the need for a nanostructured\nbase as used by Lee and Kim 17 can be avoided\nin some situations. This has the significant benefit that the plastron\ncan be recovered by electrolysis even when the surface has been fully\nwetted and therefore ensure that drag reduction will not be compromised,\nwhich has not previously been demonstrated. Further to this, the fact\nthat we do not need to use a nanostructured base means that the fabrication\nof our surface is easy, economical, and fast to make. We also demonstrate:\nimmediate recovery when the plastron is partially removed by an impinging\nwater jet, fast and accurate sensing of the wetting state by measuring\nsurface capacitance, measurement of the slip length and the surface’s\nmechanical flexibility, and damage resistance to loading.",
"discussion": "Results and Discussion Plastron\nRecovery without a Superhydrophobic Base To\nsuccessfully recover the plastron, the capillary pressure must be\nlower for lateral spreading than vertical spreading, otherwise the\nnewly generated gas will be lost as bubbles. This criterion can be\nexpressed in terms of the minimum channel height to pitch ratio, which\nwould lead to desired capillary pressures, as shown in eq 3 . 17 In\nthe work of Lee and Kim, h / p min was only considered as a function of gas fraction. 3 Here, in Figure 3 ,\nwe show eq 3 , the minimum\nheight/pitch ratio as a function of the\nbase contact angle for typical values of ϕ g = 0.9\nand θ s = 110°. This clearly shows that horizontal\nspreading is very much possible without the need for a superhydrophobic\nbase and is possible even using a hydrophilic base and reasonable\nh/p aspect ratios. Figure 3 Regimes of successful and unsuccessful plastron recovery\nshown\nas the minimum channel height to pitch ratio as a function of the\nbase contact angle. Plotted using eq 3 for a sidewall contact angle of 110°. Plastron\nrecovery should be possible even with a hydrophilic base at reasonable\nchannel height to pitch ratios. Fabrication of Surface Removing the requirement for\na superhydrophobic base allows us to use a molding process to generate\nthe surface, shown schematically in Figure 4 a. An acrylic (lucite) mold is used where\nchannels have been machined using a CNC router. Poly(dimethylsiloxane)\n(PDMS) is used to fill the channels in the mold by pulling the uncured\nliquid PDMS over the mold using a rubber “squigie”;\nthese regions become the walls of the channels. After curing, the\nmold is lightly polished with water and a fine polishing cloth to\nclean away any excess PDMS remaining on the top of the mold. A layer\nof conductive PDMS is applied over the top of the PDMS-filled channels\nand a wire is embedded; this is the base of the surface. After curing,\nthe surface is demolded and the exposed areas of the conductive silicone\nare covered by PDMS to be electrically insulative. Figure 4 b shows a three-dimensional\n(3D) microscopic image of the resulting surface. Figure 4 (a) Fabrication of the\nsurface using a molding process. (i) Mold\nis created. (ii) Liquid PDMS fills the channels in the mold and is\nleft to cure. (iii) Excess PDMS is cleaned from the top of the mold.\n(iv) PDMS and carbon black mixture with embedded wire covers the top\nof the mold and left to cure. (v) Surface is demolded. (b) Optical\nheight image of the surface. (c) Contact angle measurements of water\ndrops on the surface. (i) Measuring the contact angle perpendicular\nto the channel gives a contact angle of 162° and (ii) parallel\nto the channels gives a contact angle of 130°. Poly(dimethylsiloxane) (Sylgard 184 from Dow) is used for\nthe sidewall\nfor its high contact angle (110°) and electrically insulative\nproperties. For the base, we used a poly(dimethylsiloxane) and carbon\nblack composite (Vulcan XC-72R 20% by weight in Sylgard 184), which\nhad a reasonable receding contact angle of 65° and good electronic\nconductivity. We used a wall thickness of 200 μm at a pitch\nof 600 μm with a height of 850 μm. This gives a gas fraction\nof 0.66. From eq 3 , the h / p min is 0.92, the h/p of our\nsurface was 1.42, capable of recovery if the expanding plastron was\npinned to a maximum of 55°. Using eq 1 , the critical depth is 12.5 mm. Using eq 2 , the slip length is 134\nμm. Interestingly, we see that the contact angle on this\nsurface is\nheavily dependent on the orientation to the channels, Figure 4 c. We observe a contact angle\nof 162° when measuring perpendicular to the channels and 130°\nwhen measuring in the parallel direction, a similar phenomenon to\nthe crystal face-dependent spreading in earlier work. 21 Our fabrication method overcomes two key challenges\nwhen making\nsuch a surface. First, there is good selectivity over where the two\ncomponents are located; it is important that no insulative PDMS covers\nthe conductive PDMS. During fabrication, when filling the channels\nin the mold, a very thin layer of insulative PDMS also covers the\ntop of the mold. This was easily sheared off with light polishing\nwithout removing the PDMS in the channels ensuring that the conductive\nPDMS was fully exposed on the base. Second, there is excellent adhesion\nbetween the walls and the base—this was hard to achieve due\nto the necessarily low surface energy of the wall material; however,\nthis was not a problem when using a silicone for both wall and base.\nFurthermore, silicones are already established as a “foul release”\ntype antifouling coatings on ships, e.g . Hempel Silic\nOne, and therefore represent a reasonable material, choice for this\napplication. By basing the fabrication on a molding process,\nthe time-consuming\nprocess of microstructuring the surface is only required once. Replicas\nof the mold are fast to produce and do not lead to degradation of\nthe mold. Several samples were made using the method described here\nall of which behaved similarly; one of these samples was characterized\nin depth to assess full plastron recovery, immediate partial plastron\nrecovery, and sensing of the wetting state. Full Plastron Recovery The dewetting performance of\nthe surface was investigated by submerging in 10 mm of 0.06 M NaCl\nsolution and displacing the plastron. A potential difference of −10\nV vs Pt wire was applied to the surface to run the electrolysis reaction,\nand platinum gauze was used at the counter electrode. The process\nwas filmed from above using a camera mounted to a macroscope. Figure 5 shows the\ncurrent transient as the plastron is recovered with images of the\nprocess shown below. A video of this process is available in the Supporting Information S1 . We see that initially\nas the potential is applied and electrolysis begins, many small bubbles\nare generated on the conductive PDMS. As they grow, they start to\ncoalesce into larger bubbles through an Oswald ripening type process\nuntil only two gas domains occupy each channel with a small area of\nbase exposed between them. Due to reduced base area and blocking by\nthe adjacent bubbles, the gas evolution slows down throughout the\nprocess with coalescence of the last two bubbles in each channel taking\nthe longest to occur. When the channel is filled with gas, the base\nis fully blocked from the solution preventing the further passage\nof current. The average and standard deviation of time to dewet is\n42 ± 11 s, average charge required is 367 ± 56 mC cm –2 , and average energy 3.67 ± 0.56 J cm –2 , based on three full plastron recoveries. Further example full dewetting\ndata is available in the Supporting Information, Figures S1 and S2 . Figure 5 Full plastron recovery from a fully wetted state.\nThe graph shows\nthe current transient when a potential of −10 V is applied\nto the surface. Images below the graph show the growth and coalescence\nof bubbles to recover the plastron. As the circuit is closed, small\nbubbles are formed which coalesce into larger bubbles until the plastron\nis recovered which blocks the conductive base and prevents further\ngas evolution. Immediate Partial Plastron\nRecovery The ability to\nrestore a partially compromised plastron immediately after a pressure-induced\nwetting transition was also investigated. Figure 6 shows the current transient and images of\nthe process, a video is available in the Supporting Information . Initially, the plastron is intact, the potential\nof the surface is held at −10 V, but no current is drawn as\nthe plastron blocks the conductive base. At 2 s, the plastron is partially\nremoved exposing the conductive base and immediately generating gas\nby electrolysis for 11 s before the plastron is fully recovered, returning\nthe current to zero. Further example of immediate partial plastron\nrecovery is available in the Supporting Information, Figure S3 . Figure 6 Immediate recovery when the plastron is displaced by a\nwater jet\nand the surface held at a potential of −10 V. The graph shows\nthe current transient, and images show the process below. Initially\nno current passes, but when the plastron is partially removed, the\ncurrent increases, generating more gas which recovers the plastron. Sensing of Wetting State To demonstrate\nthe remote\nmeasurement of the wetted state capacitance, measurements were used\nin a similar manner to our previous work. 18 This method could be useful to quickly assess the integrity of the\nplastron without the need for a direct visual observation. To show\nthe relationship between capacitance and wetted area, the surface\nwas wetted in a stepwise fashion, removing small amounts of plastron\nover several steps and measuring the capacitance and the wetted area\nat the base of the channels from the macroscope image. Figure 7 shows a strong linear relationship\nbetween the two, where the gradient gives a capacitance density of\n0.88 μF cm –2 . The proportional scaling allows\nfast and accurate analysis of the wetting state without the need for\ndirect visual access. Figure 7 Sensing of the wetting state using capacitance measurement.\nBy\ndisplacing the plastron in a stepwise manner, we observe a proportional\nrelationship between wetted area and capacitance. Slip Length Measurement The slip length of water over\nthe surface was measured with a rheometry system using a 6 cm diameter\ncone, based on the method shown by Choi and Kim by converting the\ntorque measurement to a slip length. 22 A\nsample was fabricated with the same channel geometry as before ( p = 600 μm, ϕ = 0.66) but in a concentric arrangement\nso that the channels were always arranged parallel to the flow. Support\nstructures joining adjacent channels were introduced to channels longer\nthan 13.4 mm to improve wall stability. The slip length of water\nover a flat surface and over our superhydrophobic surface is shown\nagainst shear rate in Figure 8 a, and a 3D confocal microscope image is shown for the sample\nused for the measurements in Figure 8 b. The measured slip length shows some change with\na shear rate as also seen by Choi and Kim 22 and is ∼135 μm, which shows good agreement with eq 2 . We see that the flat\nsteel surface measured as a control holds the no-slip condition as\nexpected. There was no difference in slip length between the “native”\nplastron and recovered plastron. This slip length is large enough\nto reduce drag in macrosized laminar flows with boundary layers of\nthe order of millimeters. We would also expect a significant reduction\nto drag in turbulent flows, which also scales with pitch and gas fraction. 4 Figure 8 Measurement of slip length over the surface. (a) Good\nagreement\nbetween the experimental data, measured using a rheometry system and\ntheory, using eq 2 . Our\nsurface produces a slip length of ∼135 μm compared to\na flat surface, which holds the no-slip condition as expected. (b)\nHeight image of the test sample used for slip measurements, 6 cm in\ndiameter with concentric channels. Flexibility and Damage Resistance Due to the silicone-based\nconstruction, the surface can deform elastically without damage and\nreturn to its initial shape. Figure 9 shows a submersed sample bent to a radius of curvature\nof ∼1 cm. This property would allow the surface to conform\nto a wide range of shapes, e.g., the hull of a boat or a pipe without\nmodification to the manufacture process. To simulate a collision with\nanother body, we used an indentation test loading a 6 mm diameter\nsteel sphere on the surface. We found that the loads of up to 50 N\n(load rate of 50 N s –1 ) could be withstood without\ndamage or effect to the dewetting performance. Figure 9 Demonstration of the\nflexibility of the surface. The photo shows\na sample bent to a radius of curvature of ∼1 cm.\n\nDiscussion and Conclusions This work has shown that\nit is possible to recover the plastron\nfrom a truly fully wetted state and without the need for a superhydrophobic\nbase, which has not previously been demonstrated. This means that\nthere is no possibility of failed plastron recovery as in the case\nwhere a nanostructured superhydrophobic base is used. Our surface\nhas a significant slip length of 135 μm, large\nenough to reduce drag in laminar flows around macrosized objects.\nWe would also expect a large reduction to drag in turbulent flows. We have also demonstrated other important engineering aspects to\nthis surface. The wetting state of the surface can be quickly measured\nthrough the analysis of the capacitance. The remote sensing of the\nplastron integrity would no doubt be important in real-world scenarios.\nWe have also shown good damage resistance thanks to the silicone construction\ndeforming elastically under load. This is an important consideration\nas minor collisions with debris in the ocean is a frequent occurrence.\nHypothetically, the surface could be powered by cathodic protection\nsystems already present in ships and pipes, which are used to prevent\ncorrosion by holding the structures at cathodic potentials. Further improvements to decrease the resistance of the base material\ncould reduce the energy required to run such a system. The general\napproach presented in this work could be a viable route to bring sustained\nunderwater drag reduction to ships and pipes."
} | 5,671 |
28405373 | PMC5383830 | pmc | 2,493 | {
"abstract": "Nature has always served as an inspiration for scientists, helping them to solve a large diversity of technical problems. In our case, we are interested in the directional transport of oily liquids and as a model for this application we used the flat bug Dysodius lunatus . In this report, we present arrays of drops looking like polymer microstructures produced by the two-photon polymerization technique that mimic the micro-ornamentation from the bug's cuticle. A good directionality of oil transport was achieved, directly controlled by the direction of the pointed microstructures at the surface. If the tips of the drop-like microstructures are pointing towards the left side, the liquid front moves to the right and vice versa. Similar effects could be expected for the transport of oily lubricants. These results could, therefore, be interesting for applications in friction and wear reduction.",
"introduction": "1. Introduction The interdisciplinary field of biomimetics has been very successful in solving engineering problems by searching for solutions in nature. Through the process of evolution many living organisms developed different structural and chemical material properties that assured the continuation of a certain species. Technological challenges dealing with wetting and liquid collection and transportation also found solutions in nature. Superhydrophobic and self-cleaning surfaces were inspired by lotus leaves [ 1 ]. A highly efficient liquid retention and manipulation can be obtained by artificial open radial fibre arrays mimicking ripe dandelion seeds [ 2 ]. Fog collection and moisture harvesting of cacti [ 3 , 4 ], spider silk [ 4 ] and Stenocara beetles [ 5 ] have served as inspiration for the design and production of surfaces with special geometry and chemistry for wetting and liquid transport. In some cases (e.g. for Texas horned lizards [ 6 , 7 ]), passive liquid transport can be even unidirectional. Passive liquid transport in open triangular-shaped capillary channels is also reported in [ 8 ], here unrelated to biomimetic inspiration. Unidirectional fluid transport may be also induced by oriented nano- or microstructures. Experimental [ 9 , 10 ] and theoretical [ 9 ] studies on wetting of surfaces covered with bent nanopillars show that a water droplet spreads in a unidirectional manner, along the bent pillars' direction, while it is halted in the opposite direction. Another theoretical study regarding the water transport on a hydrophilic substrate patterned with a triangular post shows that water transport is faster in the direction of the triangles' tips [ 11 ]. The Neotropical flat bug Dysodius lunatus , a representative of the family Aradidae , is widely distributed in South and Central America [ 12 ] and lives on and under the bark of various rainforest trees, associated with and feeding on different fungi growing there [ 13 ]. Dysodius lunatus bugs have two different types of cuticle micro-ornamentation that contribute to fluid transportation. The first one is located on the dorsal surface and represents a web of capillary channels, with the function of immediately spreading water droplets that come in contact with the bug integument. This property provides camouflage, as water changes considerably the reflectance of the bug cuticle (i.e. makes it darker), protecting the bugs from visually oriented predators during rainfall [ 13 ]. Little more was known about the fluid transport mechanisms on the cuticle of these bugs as they were rarely studied, even though the bugs were first described by J.C. Fabricius in 1775 [ 12 ]. We found that D. lunatus bugs also possess caudally oriented micro-ornamentation underneath the wings, around the glands that secrete an oily defensive liquid, as an anti-predator adaptation [ 14 ]. Even though the gland system is located on the dorsal surface ( figure 1 b ), the substance is most likely evaporated at the region of the wings' base ( figure 1 a ). We suppose that specifically these microstructures contribute to the transport of the oily substance from the gland system to the wings of the bug ( figure 1 c ). These microstructures might also be used for attaching the wings to the body in their folded stage [ 15 ]. The micro-ornamentation consists of a periodical array of droplet-like structures of around 10 µm in length, with an undercut. Along the scent gland channel, the microstructures show some variation, as can be seen in the electron micrographs shown in electronic supplementary material, S1. But in all cases, the microstructures have pointed tips which are oriented caudally, i.e. opposite to the bug head. Thus, the tips are pointing to one of the three scent glands beneath. The density and size of the structures are similar over the whole channel length. The wings of D. lunatus are kept mostly in the folded state, as these bugs are bark-resting and slow-moving, and show generally a quiescent behaviour [ 13 ]. The bug cuticle containing the drop-like structures and the wing together form therefore a closed capillary channel, and the oil transport occurs in the direction from the pointed ends towards the wider ends of these microstructures.\n Figure 1. ( a ) Optical image of Dysodius lunatus (green rectangle shows the wings and blue squares show the region where the oily defensive liquid is thought to be evaporated). ( b ) SEM micrograph of D. lunatus ' cuticle under the wing, showing the scent liquid-secreting pore in the yellow square and the surface micro-ornamentation in the red square. ( c ) A higher magnification of the micro-ornamentation region, where the drop-like microstructures can be seen in detail (white arrow shows the preferential flow direction). In this study, we are using the micro-ornamentation found around the oil-secreting scent glands underneath the wings of D. lunatus as a model for surface patterning to assess oil behaviour in contact with the drop-like microstructure arrays. To produce these bioinspired microstructures at real scale, direct laser writing was used.",
"discussion": "4. Discussion In this study we have used two-photon lithography to produce bioinspired acrylic polymer microstructure on a glass substrate, and have tested wetting by transport of oil. Even though the oil front has a higher speed when the structure is not covered and when the oil moves perpendicularly towards the microstructures' orientation, we succeeded in obtaining a clear preferential direction of the oil movement (in the horizontal channel) against the tips of the micro-ornamentation when the structures are covered by a glass coverslip. In the case of a free surface (uncovered), the liquid droplet's height is larger than that of the microstructures. Thus the structure acts as surface roughness, leading to enhanced wetting, i.e. to a uniform spreading of the liquid in all directions. This can be explained by the Wenzel-model for wetting of structured surfaces [ 18 ]. In the case of capillary transport (covered), the droplet-like microstructures can be interpreted as distributed defects on a smooth surface. Joanny & de Gennes [ 19 ] derived a theory for the pinning of a liquid front, i.e. the hysteresis of the advancing and receding contact angle on a surface containing an ensemble of identical defects distributed over it. They obtained a relationship between the advancing and receding contact angles, θ A and θ R , respectively, to be\n 4.1 cos θ R − cos θ A = n W = n f m 2 2 π γ 2 ln L r , \nwhere n is the number of defects per unit area; W is the total energy dissipated by one defect; f m is the maximum force a defect can exert on the triple line before a jump occurs (maximal pinning force), which is dependent on the materials involved; γ is the surface tension; L is the average distance between adjacent defects; and r is the typical dimension (radius) of a defect. In our case, n , f m , γ as well as L are identical in both directions. However, the defect radii are different. As can be seen from figure 7 , the smaller radius r 1 of our microstructures is about 2 µm, while the larger radius r 2 is about 4.5 µm. The average distance L between the defects is approximately 13 µm. The fraction in the above formula is constant for the given material combination, but the logarithm of L/r is different for the two transport directions by a factor of approximately 2. Thus, the liquid flows and increases the contact angle at the fronts until the advancing contact angle is reached and then the liquid jumps. While at the larger radius r 2 the advancing contact angle is overcome and the liquid front jumps, the small radius r 1 pins the front better and due to the jump in the direction of r 2 , increasing the apparent wetted area, the contact angles are decreased again. This can be observed repetitively until the one side of the structure is completely filled with liquid. Then the flowing liquid can increase the contact angle at the sides of r 1 and also overcome the pinning. When the oil front moves transversally to the microstructures (i.e. down in the figures), it encounters defects with even much larger radii (almost infinite) than both r 1 and r 2 . According to the formula, this should result in less pinning making the liquid front move faster, as is observed experimentally.\n Figure 7. Different radii of the drop-like microstructures as defects on the glass slide surface. The smaller radius r 1 is about 2 µm and the larger radius r 2 is about 4.5 µm. Interestingly, our system is homogeneous in the sense that the oil contact angles are similar for the substrate and the microstructures. The same applies for oily fluids in contact with micro-patterned or rough surfaces in tribology or microfluidics. Therefore, we anticipate that this research can lead to multiple industrial applications in friction and wear reduction. From our ongoing systematic studies varying the microstructure size and geometry and applying different fluids, we already can say that the effect of directional fluid transport depends on both the viscosity and the contact angle of the fluid. The contact angle should be in the order of around 20° to 40°. Both properties have, on the other hand, to be appropriate for the envisaged technical application."
} | 2,570 |
35001476 | PMC9306861 | pmc | 2,494 | {
"abstract": "Summary Rhizosphere microbiome adapts their structural compositions to water scarcity and have the potential to mitigate drought stress of plants. To unlock this potential, it is crucial to understand community responses to drought in the interplay between soil properties, water management and exogenous microbes interference. Inoculation with dark septate endophytes (DSE) ( Acrocalymma vagum , Paraboeremia putaminum ) and Trichoderma viride on Astragalus mongholicus grown in the non‐sterile soil was exposed to drought. Rhizosphere microbiome were assessed by Illumina MiSeq sequencing of the 16S and ITS2 rRNA genes. Inoculation positively affected plant growth depending on DSE species and water regime. Ascomycota , Proteobacteria , Actinobacteria , Chloroflexi and Firmicutes were the dominant phyla. The effects of dual inoculation on bacterial community were greater than those on fungal community, and combination of P. putaminum and T. viride exerted a stronger impact on the microbiome under drought stress. The observed changes in soil factors caused by inoculation could be explained by the variations in microbiome composition. Rhizosphere microbiome mediated by inoculation exhibited distinct preferences for various growth parameters. These findings suggest that dual inoculation of DSE and T. viride enriched beneficial microbiota, altered soil nutrient status and might contribute to enhance the cultivation of medicinal plants in dryland agriculture.",
"conclusion": "Conclusions and outlook This study explored the associations between A. mongholicus roots and DSE derived from the roots and rhizosphere soil of licorice grown on farmlands of northern China. Two DSE species were effective colonizers of A. mongholicus roots. Dual inoculation with DSE and TV had a positive effect on the growth of A. mongholicus depending on the DSE species and water regime. In addition, dual inoculation and water regime also markedly affected the composition and diversity of the rhizosphere microbial communities, and such impact was greater on the bacterial community than on the fungal community. Moreover, under drought stress, combined inoculation of A. mongholicus with PP and TV exerted a stronger impact on the rhizosphere microbiome compared with combined inoculation with AV and TV. The soil factor changes caused by dual inoculation and water regime partially account for the observed variations in the rhizosphere microbiome. Furthermore, the rhizosphere microbes mediated by dual inoculation exhibited distinct preferences for various growth parameters. In this manner, dual inoculation promoted plant growth and drought tolerance, thereby facilitating survival of A. mongholicus under DS. The findings from this study may help develop efficient and eco‐friendly biofertilizers for the cultivation of medicinal plants, selecting symbiotic fungal consortia for the biofertilizers based on the soil characteristics and microbial community that such plants harbour in dryland agriculture.",
"introduction": "Introduction Drought is a major abiotic stress factor affecting plant growth and production (Bodner et al ., 2015 ). Plants respond to such adverse environments both directly and indirectly, and indirect responses through altered interactions among species have recently received increased attention (Lata et al ., 2018 ; deVries et al ., 2020 ). Utilizing beneficial endophytic fungi such as arbuscular mycorrhizal (AM) fungi, dark septate endophytes (DSE) and Trichoderma spp. is one of the most promising strategies for improving plant growth and stress tolerance through modulating morphogenesis and physiological processes of their associated plants, thereby enhancing the ability of plants to cope with environmental stresses (Mona et al ., 2017 ; He et al ., 2019 ; Xu et al ., 2020 ). Numerous studies have reported the roles of endophytic fungi in plant development under both normal conditions as well as in the presence of various abiotic stresses, such as drought, salinity and heavy metal exposure (Wani et al ., 2015 ; Li et al ., 2019a ; Hou et al ., 2020 ). Thus, the use of beneficial microbial species with the ability to promote plant growth to mitigate the adverse effects of drought on plants is an important component of sustainable agriculture (Lugtenberg et al ., 2016 ; Ku′zniar et al ., 2019 ). DSE, a major group of endophytes within plants, are characterized by melanized septate hyphae and microsclerotia (Jumpponen, 2011 ). These fungi colonize the epidermis, cortex and even the intercellular space of vascular tissue of healthy plant roots and are found in the roots of > 600 different plant species (Kauppinen et al ., 2014 ), especially plants that grow under extreme conditions, such as arid habitats (Xie et al ., 2017 ; Knapp et al ., 2018 ). Related reports have indicated that the interactions between DSE and host plants range from mutualistic to parasitic depending on the particular host‐symbiont combination (Li et al ., 2019b ). Previous studies have shown that DSE promote the growth of host plants by producing plant hormone substances, providing nutrients and decomposing complex carbohydrates to provide monosaccharides for the host plants, with the aim of protecting the host plant from various stresses (Surono and Narisawa, 2017 ; Yakti et al ., 2019 ). Trichoderma spp. colonize the roots of many plants as opportunistic, avirulent plant symbionts and these fungi have been investigated as biological control agents, biofertilizers and soil amendments for application in agricultural systems (Velmourougane et al ., 2017 ; Atieno et al ., 2020 ). Trichoderma spp. enhance plant growth predominantly by solubilizing soil nutrients (Bayoumi et al ., 2019 ), and increasing root length and secondary root number, and upregulating phytohormones such as indoleacetic acid, cytokinin, gibberellins and zeatin (Contreras‐Cornejo et al ., 2009 ; Jaiswal et al ., 2020 ). Furthermore, the interaction of both groups of microorganisms may be convenient for both plant physiology and nutrient content. For example, Metwally and Al‐Amri ( 2020 ) found that dual inoculation with AM fungi and Trichoderma viride (TV) could improve the biochemical parameters and mineral nutrient of onion plants. Liu et al . ( 2020 ) reported that the co‐inoculation with Epulorhiza repens and Umbelopsis nana increased the total dry weight of Cymbidium hybridum . Co‐inoculation with AM fungi and Trichoderma harzianum did not result in an additive effect on melon crop growth and nutritional status, but their combinations could control Fusarium wilt more effectively than each AM fungi applied alone (Martínez‐Medina et al ., 2011 ). In addition, rhizosphere soil is an intense field of microbial activity and plant stress responses (Mendes et al ., 2013 ). Rhizosphere‐associated microbes are instrumental in the decomposition of organic matter and maintenance of nutrients, and have beneficial effects on the adaptation of host plants to different environmental conditions (Bai et al ., 2015 ; Henneron et al ., 2020 ). Although the direct effects of beneficial microbial inoculants on plant growth and rhizosphere‐associated microbes have been widely reported (Santos et al ., 2017 ; He et al ., 2019 ), there is limited information available regarding the contribution of DSE, either alone or in combination with endophytic fungi, on the growth and native rhizosphere microbial community of medicinal plants under drought stress (DS). \n Astragalus mongholicus Bunge is a widely distributed herbaceous perennial medicinal plant. The roots of this plant are important medicinal materials used as a common clinical tonic in China and other parts of the world because of their pharmacological effects and biological functions such as improving immunity, promoting body metabolism and lowering blood pressure (Qin et al ., 2013 ). Therefore, considering the concept of sustainability and the need to enhance the growth status and drought resistance of medicinal plants, understanding the interaction between plants and beneficial microbes is crucial to receive benefit from the symbiotic mechanisms. In a previous study, we investigated the influences of three DSE ( Paraboeremia putaminum (PP), Scytalidium lignicola and Phoma herbarum ) from the roots of Ophiopogon japonicus and Lonicera japonica on the performance of licorice ( Glycyrrhiza uralensis ) at different TV densities under sterilized conditions in a growth chamber. The combination of DSE and TV enhanced the root morphology and biomass more effectively than either agent alone (He et al ., 2020 ). In the present study, we hypothesize that dual inoculation of DSE, either Acrocalymma vagum (AV) or PP and TV could either promote plant growth or change the rhizosphere microbiome of A. mongholicus , and that dual inoculation might have more positive effects under DS conditions than under control conditions. Therefore, we investigated the effects of dual inoculation under DS conditions on (i) plant height, leaf number, root surface area and root diameter, (ii) plant biomass, (iii) Soil physicochemical properties and (iv) soil microbial composition. Such data would display DSE and TV could withstand the drought conditions that affected host growth, and their potential for improving the stress tolerance and symbiotic performance of plants during A. mongholicus cultivation, in drought‐affected arid lands.",
"discussion": "Discussion Effects of DSE and TV on the growth of Astragalus plants As important root endophytes, DSE are reported to have negative, neutral or positive ecological roles in plant growth (He et al ., 2019 ; Li et al ., 2019a ). These organisms also increase plant resistance to a wide range of environmental stressors (Zhang et al ., 2017 ; Jin et al ., 2018 ). In the present study, plants inoculated with AV and PP exhibited higher biomass and leaf number than the control plants, indicating positive effects of these organisms on the plant growth and formed a strain‐dependent symbiosis with Astragalus plants. For example, compared with AV inoculation PP inoculation had a greater impact on biomass under well‐watered (WW), while there was no significant difference between AV and PP on biomass under DS. Furthermore, the average root diameter of PP‐inoculated Astragalus plants decreased compared with the root diameter of the control plants under DS. Roots with small diameters have been reported to exhibit faster growth and allocation of more nutrients, which is beneficial for plants under drought conditions (Comas et al ., 2013 ). TV inoculation in the present study increased plant biomass, height, leaf number, root surface area and root diameter under DS. Similarly, Guler et al . ( 2016 ) found that T. atroviride ID20G increased fresh and dry weight of maize roots and helped plants invert the adverse effects of DS. Estévez‐Gefriaud et al . ( 2020 ) reported that T. asperellum strain T34 improved the dry weight of maize plants, regardless of water regime and improved leaf relative water content, water use efficiency and photosystem II (PSII) maximum efficiency and photosynthesis under drought. TV inoculation in combination with water stress alleviated the effects of drought and these results were compatible with Khoshmanzar et al . ( 2020 ) dissecting this notion in wheat plants. There is limited information regarding the interactions of DSE and TV on host plants, especially when water availability is considered. Our previous study demonstrated that DSE associated with TV augmented plant biomass and height, shoot branching and root surface area (He et al ., 2020 ). In the present study, DSE × TV significantly affected plant biomass, height and leaf number and DSE × TV × watering regime significantly affected these three parameters and root surface area. Similarly, Parkash et al . ( 2011 ) and Commatteo et al . ( 2019 ) revealed that Dendrocalamus strictus plants and tomato plants bio‐inoculated with a consortium of AM fungi and TV showed enhancement in growth parameters. Kushwaha et al . ( 2019 ) also found that co‐inoculation of fungal endophytes and TV increased overall plant biomass and yield of Withania somnifera . The results of the present study revealed the synergistic and beneficial activity of DSE associated with TV in Astragalus growth. Relationship between soil microbe and Astragalus plant growth mediated by dual inoculation It was previously not known whether dual inoculation‐mediated changes in rhizosphere microbial communities would augment the growth and DS tolerance of Astragalus plants. The present study suggested that rhizosphere microbes associated with Astragalus were influenced by dual inoculation. For instance, soil fungi such as Stachybotrys , unclassified Ascomycota , unclassified Chaetomiaceae , Trichoderma and Chaetomium had the highest degree of enrichment in the rhizosphere of Astragalus plants exposed to different inocula under different water conditions. Certain Ascomycota , including DSE, form mycorrhizae in plant roots and enhance plant nutrient uptake and growth (Surono and Narisawa, 2018 ). The genus Stachybotrys is a marked producer of cellulose‐degrading enzymes (Fernandes et al ., 2021 ). Trichoderma is an effective biofertilizer, soil amendment and biocontrol agent (Herrera‐Parra et al ., 2017 ; Saxena et al ., 2020 ). Chaetomium is a genus of the family ‘ Chaetomiaceae ’, and application of spores and methanol extracts of C. globosum , C. lucknowense and C. cupreum to pomelo seedlings inoculated with Phytophthora nicotianae reduced the extent of root rot and increased plant weight (Hung et al ., 2015 ). Moreover, soil bacteria such as Actinobacteria , Lysinibacillus , Propionibacteriales , Paenibacillaceae , Micrococcaceae and Chloroflexia had the highest degree of enrichment in the rhizosphere of Astragalus plants treated with different inocula under different water conditions. Khan et al . ( 2019 ) reported that Actinobacteria was highly abundant in medicinal plant rhizosphere microbiomes in arid soil, and Actinobacteria can increase the amount of phosphate available to plants through P solubilization or mineralization (Soumare et al ., 2021 ). Members of the genus Lysinibacillus enhanced the growth and yield of rice ( Oryza sativa ) under greenhouse conditions (Dhondge et al ., 2021 ). Sun et al . ( 2020 ) reported that Propionibacteriales have drought, salt tolerance, alkali resistance and stress resistance, in addition to strong degradation capabilities (Ivanova et al ., 2016 ), while Paenibacillaceae are plant growth promoter and biocontrol agents (Delgado‐Ramírez et al ., 2021 ). Cui et al . ( 2018 ) found that Micrococcaceae had strong effects on microbial N metabolism, and a member of the family Micrococcaceae isolated from halophytic rangeland plants could improve wheat productivity, especially the attributes related to seed and forage quality, under salinity stress conditions (Hajiabadi et al ., 2021 ). Chloroflexia , which has been investigated in association with a variety of crop species, is considered to play a positive role in crop growth (Visioli et al ., 2018 ; Wei and Yu, 2018 ). In addition, the microbes colonizing the Astragalus rhizosphere exhibited distinct preferences for various growth parameters (Fig. S6 ), and the SEM analysis further demonstrated that soil fungi positively affected root surface area, while soil bacteria positively affected leaf number, and negatively affected plant height and biomass. Plants can recruit target microbial communities through signalling molecules, and then use the immune system to provide specific nutrients and habitat types to exert selective pressure, thereby enriching beneficial microbial communities (Foster et al ., 2017 ; Martin et al ., 2017 ; Cordovez et al ., 2019 ). Thus, the enrichment of beneficial microbial communities is essential for the survival and development of plants, allowing them to thrive in diverse environments (Gagnon et al ., 2020 ; Mengistu, 2020 ). Effects of DSE and TV on soil nutrients and microbes of Astragalus plants DSE increased organic matter, available N and available P in the soil and AV inoculation increased soil available K under WW conditions, while inoculation with PP decreased organic matter and available K in the soil under DS. Furthermore, DSE × TV significantly affected soil organic matter, available N, P and K contents and DSE × TV × watering regime significantly affected soil available N and P content. This was congruent with observations for W. somnifera after co‐inoculation with TV and native endophytic fungi (Kushwaha et al ., 2019 ). Two possible reasons can explain the interactions of DSE and TV on soil nutrient properties. First, dual inoculation of DSE and TV could improve the root system and N and P absorption by plants, which consequently led to the depletion of these common nutrients in the soil (Halifu et al ., 2019 ; Li et al ., 2019a ). Second, DSE and TV could act as decomposers, converting soil organic nutrients into available forms to promote the growth and tolerance of plants to stressful conditions (Schmoll, 2018 ; He et al ., 2019 ; Alothman et al ., 2020 ). Endophytic fungi increase the interactions between plants and soil and expand the amount of available N and P because they secrete several enzymes required for the mineralization of organic N and insoluble P in the soil into available forms. Thus, endophytic fungi provide a link between plant and soil environments that promotes the growth and tolerance of plants (Berthelot et al ., 2016 ; Saravanakumar et al ., 2016 ; Guo et al ., 2020 ). In addition, the plant rhizosphere contains a complex community of microorganisms, including bacteria, archaea and fungi. These microorganisms affect plant survival, growth and adaptability (Xiong et al ., 2021 ). In the present study, the relative abundance of microbes colonizing the rhizosphere of Astragalus differed among treatments, and Ascomycota , Proteobacteria , Actinobacteria , Chloroflexi and Firmicutes were the dominant phyla across the various treatments. The predominance of Ascomycota in arid and semi‐arid regions was previously reported (Porras‐Alfaro et al ., 2011 ). Latif et al . ( 2020 ) found that Proteobacteria , Actinobacteria , Acidobacteria , Bacteroidetes , Firmicutes , Chloroflexi and Gemmatimonadetes were the predominant bacterial phyla in the rhizosphere of Triticum aestivum , and Barraza et al . ( 2020 ) reported that the bacterial community structure of common bean roots was mainly composed of Actinobacteria , Proteobacteria , Bacteroidetes , Acidobacteria and Firmicutes . The major rhizosphere microbes may vary widely among plant species, but Actinobacteria and Proteobacteria might be the most common bacterial phyla in plant rhizospheres. Biotic and abiotic stressors alter rhizosphere microbe community structures and may augment or diminish certain microbial populations (Santoyo et al ., 2017 ; Achouak et al ., 2019 ). In the present study, water stress had a more significant impact on the distribution of bacterial communities than on fungal communities. This might be explained by the formation of stable symbiotic relationships between some soil fungi and host plants (Bouasria et al ., 2012 ; He et al ., 2021 ). Here, the number of representative fungi and bacteria between different inoculation treatments was significantly different. Among them, the number of fungi and new OTUs in Astragalus rhizosphere soil under most of the treatments was lower under DS than under normal water conditions, but the opposite result was observed for combined inoculation with PP and TV. This phenomenon was not evident when PP or TV were inoculated separately, which indicated that dual inoculation with PP and TV had a favourable synergistic effect on microbial community composition in Astragalus rhizosphere soils (Ważny et al ., 2018 ; He et al ., 2020 ). Moreover, the microbes colonizing the Astragalus rhizosphere exhibited distinct preferences for various soil factors (Fig. S7 ). These findings were consistent with those previously reported that microbial inoculation broadly influences plant rhizosphere microbial communities by altering soil chemical properties and indirectly affecting host plant growth (Raklami et al ., 2019 ; Ullah et al ., 2019 ). Conclusions and outlook This study explored the associations between A. mongholicus roots and DSE derived from the roots and rhizosphere soil of licorice grown on farmlands of northern China. Two DSE species were effective colonizers of A. mongholicus roots. Dual inoculation with DSE and TV had a positive effect on the growth of A. mongholicus depending on the DSE species and water regime. In addition, dual inoculation and water regime also markedly affected the composition and diversity of the rhizosphere microbial communities, and such impact was greater on the bacterial community than on the fungal community. Moreover, under drought stress, combined inoculation of A. mongholicus with PP and TV exerted a stronger impact on the rhizosphere microbiome compared with combined inoculation with AV and TV. The soil factor changes caused by dual inoculation and water regime partially account for the observed variations in the rhizosphere microbiome. Furthermore, the rhizosphere microbes mediated by dual inoculation exhibited distinct preferences for various growth parameters. In this manner, dual inoculation promoted plant growth and drought tolerance, thereby facilitating survival of A. mongholicus under DS. The findings from this study may help develop efficient and eco‐friendly biofertilizers for the cultivation of medicinal plants, selecting symbiotic fungal consortia for the biofertilizers based on the soil characteristics and microbial community that such plants harbour in dryland agriculture."
} | 5,539 |
28832522 | PMC5620544 | pmc | 2,496 | {
"abstract": "Artificial Neural Networks (ANNs), including Deep Neural Networks (DNNs), have become the state-of-the-art methods in machine learning and achieved amazing success in speech recognition, visual object recognition, and many other domains. There are several hardware platforms for developing accelerated implementation of ANN models. Since Field Programmable Gate Array (FPGA) architectures are flexible and can provide high performance per watt of power consumption, they have drawn a number of applications from scientists. In this paper, we propose a FPGA-based, granularity-variable neuromorphic processor (FBGVNP). The traits of FBGVNP can be summarized as granularity variability, scalability, integrated computing, and addressing ability: first, the number of neurons is variable rather than constant in one core; second, the multi-core network scale can be extended in various forms; third, the neuron addressing and computing processes are executed simultaneously. These make the processor more flexible and better suited for different applications. Moreover, a neural network-based controller is mapped to FBGVNP and applied in a multi-input, multi-output, (MIMO) real-time, temperature-sensing and control system. Experiments validate the effectiveness of the neuromorphic processor. The FBGVNP provides a new scheme for building ANNs, which is flexible, highly energy-efficient, and can be applied in many areas.",
"conclusion": "4. Conclusions In this paper, we propose a FPGA-based granularity variable neuromorphic processor FBGVNP. The traits of FBGVNP can be summarized as granularity variability, scalability, integrated computing, and addressing ability: First, the number of the neurons is variable rather than constant in one core; second, the number of internal neural computing units and the scale of the multi-core network can be extended as needed; third, the neuron addressing and computing processes are executed simultaneously. Additionally, a comparison between the FBGVNP and an existing neurosynaptic chip TrueNorth is conducted. Moreover, a neural network-based controller is mapped to FBGVNP and applied to a multi-input, multi-output (MIMO), temperature-sensing and control system. Experiments validate the effectiveness of the presented neuromorphic processor. The FBGVNP provides a new scheme for building ANNs, which is flexible and highly energy-efficient, and can be widely applied in many areas with the support of the state-of-the-art algorithms in machine learning.",
"introduction": "1. Introduction In recent years, machine learning has entered into our daily life. When we communicate with smart phones using natural language or get pictures on digital cameras using face detection, artificial intelligence plays a key role in the process [ 1 ]. Over the past decade, Artificial Neural Networks (ANNs), including Deep Neural Networks (DNNs), have become the state-of-the-art methods and achieved amazing success in machine learning, especially in visual recognition, speech recognition, and other domains [ 2 , 3 , 4 , 5 , 6 , 7 ]. With significantly higher accuracy than traditional algorithms in various tasks like face recognition and image processing [ 8 , 9 ], DNNs have attracted the enthusiastic interest of internet giants such as Google [ 10 , 11 ], Microsoft [ 12 ], Facebook [ 13 ], and Baidu [ 14 ]. There are several hardware platforms for developing accelerated implementation of DNN models, including multicore CPUs [ 15 ], General Purpose Graphics Processing Units (GPGPUs) [ 16 ], Application Specific Integrated Circuits (ASICs) [ 17 ], and Field Programmable Gate Arrays (FPGAs) [ 18 ]. CPUs and GPUs are parts of General Purpose Processors (GPPs). The classic platforms based on CPU and GPU are SpiNNaker and Carlsim, correspondingly. The SpiNNaker machine is a specifically designed computer for supporting the sorts of communication found in the brain. It is based on the connection of processing nodes, which have eighteen ARM processor cores in one node. Over hundred neurons can be modelled in each processor core and there are one thousand input synapses connected to each neuron [ 15 ]. The Carlsim is a GPU-accelerated simulator which is capable of simulating the neural model [ 19 ]. GPPs can provide a high degree of flexibility and tend to be more readily accessible. However, the hardware performs with less energy efficiency, which is of particular importance in embedded, resource-limited applications or server-based large scale deployments [ 1 ]. Recently, the development of the neuromorphic processor has received increasing attention. For GPPs, application level execution relies on the traditional von Neumann architecture. It stores instructions and data in external memory to be fetched. The von Neumann architecture is non-scalable and inefficient in executing massive neural networks, and the von Neumann bottleneck can be mitigated by colocated computation and memory [ 17 ]. As seen in Figure 1 , the centralized sequential von Neumann architecture computer is different from the brain’s distributed parallel architecture. The processor’s increasing clock frequencies and power densities are headed away from the operating point of the brain. As to implementing neural networks in a von Neumann architecture computer, a central processor has to simulate communication infrastructure and a great number of neurons. The bottleneck which serves as the communication channel between the processor and external memory causes power-hungry data movement while retrieving synapse states and updating neuron states [ 17 ]. A single processor is not suitable for simulating highly interconnected networks, which will cause interprocessor messaging explosions [ 17 ]. In comparison, the neuromorphic processor has a different architecture. The special computation structure of neural networks implies that the hardware suitable for exploiting pipeline parallelism takes advantage. When GPPs execute a parallel based on multiple cores, specially designed ASICs and FPGAs can support inherently pipelined and multithreaded applications, which are not based on the von Neumann architecture. They have the ability to exploit the large extent of pipeline parallelism and distributed on-chip memory. Similar to the brain, the neuromorphic processor has distributed and integrated computation and memory, and operate in parallel [ 1 , 20 ]. Developing the neuromorphic processor via the ASIC-based or FPGA-based approach shows their different advantages. ASICs are dedicated to a specific application. In recent years, the TrueNorth chip, which is developed by IBM, has attracted considerable attention. It is a low power, high parallel chip with 4096 neurosynaptic cores. The core is the basic block, which has a crossbar array for synaptic connections and neurons for calculation. Each core contains 256 input axons, a 64k synaptic crossbar, and 256 neurons. The TrueNorth chip is a neurosynaptic chip produced via a standard-CMOS manufacturing process [ 17 , 20 ]. Generally, the ASICs can provide high performance. At the same time, they are expensive and time consuming to produce and the architectures are relatively fixed and inflexible [ 1 ]. Traditionally, we must consider the flexibility, performance, and energy efficiency when evaluating hardware platforms. On the one hand, GPPs can be highly flexible and easy to use, but perform relatively inefficiently. On the other hand, ASICs work with high efficiency at the cost of being inflexible and difficult to produce [ 1 ]. As a compromise, the FPGA-based approach has drawn a significant number of applications from scientists and become one of the most promising alternatives, due to its low power usage, high performance, reprogrammability, and fast development round [ 21 , 22 , 23 , 24 , 25 , 26 ]. FPGAs often provide better performance per watt than GPPs and naturally fit with the neural network execution [ 1 ]. Table 1 shows a comparison of neuromorphic processors and GPPs mentioned above for implementing the neural network. Microcontroller Unit (MCU), which serves as a kind of low cost GPP, is also included for full comparison. In this paper we propose a FPGA-based granularity variable neuromorphic processor (FBGVNP) with integrated computing and addressing cells. The presented neuromorphic processor consists of neuromorphic cores structured by a router, a neural computing unit, and a data-transmission controller. The router is used to build connections and keep communications between different cores. Meanwhile, the neural computing unit is composed of neuron computing cells, which can process computing and addressing simultaneously. Moreover, the data-transmission controller is responsible for the computing result transmissions. The traits of the neuromorphic processor can be summarized as follows.\n (1) Granularity variability : The number of the cells in one neural computing unit can vary, which will enhance the flexibility of the neuromorphic core compared with fixed architectures. The neuron computing cells perform as the basic elements in this architecture. One can expand the size of the cells as required. That will make the core better suited for different applications. (2) Scalability : The scalability is achieved by connecting different cores with routers and extending inner neural computing units. The data interaction through routers links the neuromorphic cores so they become whole. Generally, routers serve as the communication nodes in the multi-core network and the network scale can be extended as needed. (3) Integrated computing and addressing ability : The neuron computing cell combines together computing and addressing abilities. The data transmission uses the broadcast mechanism. On this basis, a neuron computing cell serves as a data receiving and processing terminal and the two processes are executed simultaneously, which makes the computations perform in parallel. The paper is organized as follows. Section 2 introduces the architecture of the FBGVNP. In Section 3 , a neural network is mapped to the presented neuromorphic processor and applied in a multi-input multi-output (MIMO) temperature sensing and control test platform. Then, the experiment results and discussions are given. Finally, the conclusions are drawn in Section 4 .",
"discussion": "3. Experiments and Discussion 3.1. Experiment Setup In this section, a FCPIDNN is mapped to the neuromorphic processor and applied in a temperature sensing and control system. The control system includes a mockup, a power supply, temperature sensors distributed in the mockup, a data monitoring computer, and a data processing circuit. The presented neuromorphic processor is realized in a FPGA placed on the data processing circuit. The structure of the temperature control system is shown in Figure 7 . The configuration of the mockup contains six fans indicated as Fan1 to Fan6 and six inside modules indicated as module 1 to module 6. The modules inside the mockup have different volumes and shapes. A heater for generating heat is placed inside each module. The six heaters have different levels of power. The temperature control actuators inside the mockup are cooling fans. The local temperatures around the six modules are detected by distributed temperature sensors. The data processing circuit acquires the temperatures from the sensors through the universal asynchronous receiver-transmitter (UART) port, runs the temperature controller, and outputs the pulse width modulation (PWM) control signals to the actuators. The data monitoring computer is used to collect the temperature information via the UART port from the data processing circuit [ 28 ]. The prototypes of the data processing printed circuit board (PCB) and the temperature sensing and control mockup are shown in Figure 8 . 3.2. Experiment Results and Discussions We conduct experiments to validate the effectiveness of the FBGVNP-based temperature controller. Figure 9 shows the response process when the temperature targets for all modules are set to constant. Figure 10 shows the response process with multi-targets or the variational target. In Figure 9 , it indicates that the different modules’ temperatures approach the set target, and the differences between the actual and target temperatures decrease. The cost function value is formulated as the following Equation (1).\n (1) J ( n ) = 1 2 ∑ m = 1 6 e m 2 ( n ) \nwhere the difference between the target and actual temperature is represented by e , the number of the module is represented by m , and the sample number is represented by n . Figure 10 shows the results using the FBGVNP-based controller to deal with the temperature control with different targets, which further validates the effectiveness of the temperature control and exhibits its good robustness and adaptability to different scenarios. Figure 10 a,b shows the control results when two different targets were set. In the process, the target temperature of module 3 was set to 33 °C and others were 30 °C. It can be seen that the cost function gradually reached the minimal and the modules’ temperatures approached their specific targets. Figure 10 c,d shows the control results when the target temperature for modules 3 to 4 was set to 33 °C and the target temperature for other modules was 30 °C. The modules approached both their targets, respectively. Figure 10 e,f show the control results if the modules’ targets varied, i.e., 33 °C at the beginning and then turned to 30 °C. The FBGVNP-based controller modulated the modules’ temperature to the set targets successfully and the cost functions got to minimal at last.\n\n3.2. Experiment Results and Discussions We conduct experiments to validate the effectiveness of the FBGVNP-based temperature controller. Figure 9 shows the response process when the temperature targets for all modules are set to constant. Figure 10 shows the response process with multi-targets or the variational target. In Figure 9 , it indicates that the different modules’ temperatures approach the set target, and the differences between the actual and target temperatures decrease. The cost function value is formulated as the following Equation (1).\n (1) J ( n ) = 1 2 ∑ m = 1 6 e m 2 ( n ) \nwhere the difference between the target and actual temperature is represented by e , the number of the module is represented by m , and the sample number is represented by n . Figure 10 shows the results using the FBGVNP-based controller to deal with the temperature control with different targets, which further validates the effectiveness of the temperature control and exhibits its good robustness and adaptability to different scenarios. Figure 10 a,b shows the control results when two different targets were set. In the process, the target temperature of module 3 was set to 33 °C and others were 30 °C. It can be seen that the cost function gradually reached the minimal and the modules’ temperatures approached their specific targets. Figure 10 c,d shows the control results when the target temperature for modules 3 to 4 was set to 33 °C and the target temperature for other modules was 30 °C. The modules approached both their targets, respectively. Figure 10 e,f show the control results if the modules’ targets varied, i.e., 33 °C at the beginning and then turned to 30 °C. The FBGVNP-based controller modulated the modules’ temperature to the set targets successfully and the cost functions got to minimal at last."
} | 3,871 |
39599228 | PMC11597999 | pmc | 2,497 | {
"abstract": "In electrospinning, nanofibers are frequently produced in nonwoven web form. Their poor mechanical properties (below 100 MPa) and difficulty in tailoring the fibrous structure have restricted their applications. However, advanced materials must be highly resistant to both deformation and fracture. By combining electrospinning technology with stretching, we have overcome this disadvantage and demonstrated a polyacrylonitrile nanofiber yarn with a tensile strength of 743 ± 20 MPa. The nearly perfect uniaxial orientation of the fibrils under the stretching process is crucial for the remarkable mechanical properties of the yarn. Additionally, the nanofiber yarn was functionalized by a dip-coating process with silver nanowires (AgNWs), imparting conductive properties. This conductive, high-strength nanofiber yarn demonstrates practical applications in flexible and wearable devices. The presented strategy is versatile and can be adapted to create other high-performance nanofiber yarns, with potential uses in fields such as biomedicine and smart textiles.",
"conclusion": "4. Conclusions In summary, we have developed a novel and facile approach to fabricating PAN nanofiber yarns with high strength (743 MPa) and modulus (1.31 GPa) properties. The yarn was fabricated by using electrospinning and stretching methods. This presented process yields ultrafine electrospun yarns that are sufficiently robust for practical use, due to a combination of thousands of nanofibers with high alignment. Furthermore, benefiting from the dip-coating process, the functionalization of high-strength nanofiber yarns was confirmed to set the stage for its application in implantable flexible/wearable devices. Moreover, our fabrication strategy is versatile and can be extended to diverse materials for the preparation of functionalized nanofiber yarns.",
"introduction": "1. Introduction Nanofibers have attracted considerable attention due to their ultrafine dimensions and extensive surface area-to-volume ratio, making them invaluable in various applications, including filtration [ 1 , 2 ], biomedicine [ 3 , 4 ], and energy storage [ 5 , 6 ]. It is widely acknowledged that electrospinning is the preferred technique for fabricating nanofibers due to its straightforward and adaptable methodology, which enables the production of fibers with a diameter ranging from nanometers to micrometers [ 7 ]. Electrospun nanofibers have many applications, including tissue engineering [ 8 , 9 , 10 ], sensors [ 11 ], filtration [ 12 , 13 ] and catalysis [ 14 ]. Despite the wide applicability of electrospun nanofibers, most existing electrospun fibers are produced in the form of randomly oriented nonwoven mats [ 15 , 16 ]. This random fiber arrangement, coupled with their nonwoven structure, results in relatively low mechanical strength and limits the effective transfer of load along the fiber axes [ 17 ]. As a result, these materials often lack the structural integrity required for high-performance applications [ 18 , 19 , 20 , 21 ]. While some studies have attempted to address these issues by developing electrospun nanofiber yarns, their mechanical properties and functional capabilities remain constrained due to insufficient fiber alignment and structural cohesion [ 22 , 23 , 24 , 25 , 26 , 27 ]. Additionally, the integration of conductive elements into electrospun nanofiber yarns has proven challenging, with limited approaches to achieving reliable, uniform conductivity without sacrificing mechanical properties. To address these limitations, this study introduces a novel approach that combines electrospinning with a controlled hot drawing process to produce polyacrylonitrile (PAN) nanofiber yarns with enhanced mechanical properties. PAN, known for its semi-crystalline structure, is particularly suitable for electrospinning due to its stability and potential for high mechanical performance. The hot drawing process aligns the fibrils uniaxially, improving crystallinity and molecular orientation, which are essential for high-strength and high-modulus yarns. Moreover, we impart conductivity to these high-strength nanofiber yarns through a dip-coating process with silver nanowires (AgNWs), creating a conductive network on the nanofiber surface. This dual enhancement of mechanical strength and electrical conductivity enables the nanofiber yarns to function in advanced applications, such as wearable electronic textiles and implantable devices. The remainder of this paper is organized as follows: Section 2 details the materials and methods used, including the electrospinning, drawing, and dip-coating processes. Section 3 presents the results and discussion, focusing on the structural, mechanical, and electrical properties of the PAN nanofiber yarns. Section 4 provides the conclusions, summarizing the main findings and potential applications of this novel high-performance nanofiber yarn.",
"discussion": "3. Result and Discussion High-strength nanofiber yarns were prepared as illustrated in Figure 1 . First, a spinning solution with a concentration of 15 wt% was loaded into a syringe equipped with a metal needle to generate charged fibers under the influence of a high voltage. These fibers were then deposited into a rotating funnel, which slowly drew them into a coiled collector. During this process, nanofiber yarns were formed ( Figure 1 a). High-strength nanofiber yarns were obtained by stretching the nanofiber yarn at temperatures of 130–160 °C. The purpose of thermal drafting is to allow the fibers in the yarn to slip and align along the yarn. At the same time, stretching above the glass transition temperature can change the crystallinity of the yarn, which helps to improve the strength of the yarn. The high-strength nanofiber yarns were labeled SR x, where x represents the ratio of stretch. As the stretch ratio increases, the number of bent fibers in the yarn decreases significantly, the fibers gradually straighten ( Figure 1 b–e), and the fibers tend to be oriented along the axial direction of the yarn (in the yellow frame). The Image J software (v1.54j) as used to process the SEM of the yarn ( Figure 1 ) to obtain the diameter and twist angle of the yarns and fibers. As the stretch ratio increases, the diameter of the fibers in the yarn decreases ( Figure 2 a, red). This is due to the combined action of external tensile force and friction between fibers, whereby the curved fibers in the yarn are stretched and arrayed along the axial direction of the yarn. Yarn is defined as an aggregate composed of fibers; therefore, as the stretching ratio increases, the diameter of the nanofiber yarn also decreases ( Figure 2 a, blue). The Hermans orientation factor [ 29 ] is used to describe the degree of fiber alignment along the yarn axis.\n (1) f = 1 2 3 cos θ 2 − 1 In the formula, θ represents the angle between the fiber and yarn axes, which is equivalent to the twist angle. As the stretching ratio increases, the twist angle of the yarn gradually decreases, while the orientation degree increases significantly ( Figure 2 b, blue). As the draw ratio increases, the external force on the yarn also increases, which causes the fiber to be stretched along the axial direction of the yarn. This results in a decrease in the twist angle of the yarn and an increase in the orientation factor. To analyze the effect of stretch on the mechanical properties of the yarns, the stress, strain and modulus were analyzed for nanofiber yarns with different SRs. Such stress–strain curves can vary greatly between the same type of nanofiber yarns from different SRs ( Figure 2 c). The variability is due to differences in stretch, once again emphasizing the importance of stretching during spinning and subsequent processing to control the mechanical properties of the yarn [ 30 ]. The maximum stress ( Figure 2 d) and modulus ( Figure 2 e) increased with the SR, whereas the elongation at break decreased with the SR. SR8 yarns have a tensile strength of 743 ± 20 MPa and a modulus of 12.2 ± 0.31 GPa, which is 10 times higher than the strength (70.8 ± 30 MPa) and modulus (1.31 ± 0.5 GPa) of unstretched yarn. The performance of SR8 yarn is similar to that of dragline silk [ 31 , 32 ]. The hysteresis of the cyclic stretching curve decreases as the stretching ratio increases, which indicates that the stretched yarn has higher stability ( Figure 2 f–h). Even after 500 cycles of loading and unloading at a maximum tensile strength of 200 MPa, only a negligible change in the final tensile strength (~1%) and deformation (~1.9%) was observed at SR8 ( Figure 2 i). SEM images confirmed that the heat stretching procedure altered the orientation of the nanofibers along the yarn’s main axis ( Figure 1 ), with the percentage of aligned yarns increasing from 0.467 at no-stretch to 0.999 at SR 8 ( Figure 2 b). In addition, the diameter of the yarn decreased from 84.211 μm without stretching to 53.694 μm at SR 8. According to Peirce’s Theory [ 33 ], the smaller the yarn, the lower the probability of weak links. Consequently, the strength of the nanofiber yarn is directly proportional to its diameter. Previous research has indicated that high crystallinity resulting from stretching is also a factor affecting the mechanical properties of yarns [ 30 ]. The WAXS spectra of yarns under different stretch conditions are depicted in Figure 3 . All yarns exhibit a dominant and sharp diffraction peak around 2θ ≈ 16.8°, corresponding to the (100) plane in the hexagonal lattice of PAN. A weaker diffraction peak, attributed to the (101) plane of the hexagonal lattice, is discerned at approximately 2θ ≈ 29.0° [ 34 , 35 ] ( Figure 3 a). These observations substantiate that the PAN polymer is semi-crystalline. Notably, with an escalation in the stretch ratio, the relative intensity of the peak around 2θ ≈ 16.8° amplifies, implying an enhanced intermolecular interaction between the nitrile groups within the PAN macromolecular chains. However, the primary peak positions remain invariant, confirming that the stretching process does not disrupt the crystalline structure of PAN. Figure 3 b displays the crystallinity and crystal size of the PAN (100) plane, deduced from Figure 3 a. To determine the crystallinity and crystal size of samples, the peaks was first fitted with a Lorenz-peak function to obtain the area of separated peaks, which are depicted in Figure 3 c–f. The crystallite size of the lateral-order domains was estimated by the Scherrer equation [ 36 ] as follows: (2) L c = K λ B cos θ \nwhere λ is the wavelength of the CuK α X-ray, B is the full width at half maximum intensity (FWHM) of the peak around 2 θ = 16.8°, and K is a constant of 0.89. The crystallinity (CI) was determined by the Bell and Dumbleton method [ 36 ]: (3) CI = A c A c + A a \nwhere A c is the integral area of the crystalline zone around 2 θ = 16.8° (fit peak 1) and 2 θ = 29.1° (fit peak 3) in XRD patterns, and A a is the integral area of the amorphous zone (fit peak 2) [ 37 ]. The crystallinity of PAN increases from approximately 52.5% (in the unstretched state) to roughly 94.6% (at SR 8). Concurrently, the crystal size experiences an increment during the stretching process, evolving from approximately 2.5 nm to 10.7 nm. This underscores that stretching prominently enhances both the crystallinity and crystal size. The stretching results in the macromolecular chains in the nanofibers being more likely to be arranged parallel to the axis, with a reduction in the distance and a closer packing of the chains [ 30 , 38 ]. This allows the formation of hydrogen bonds and regular arrangement of the molecules, thereby increasing the crystallinity of the fiber. In addition, the greater the degree of crystallinity and the size of the crystal particles, the more regular the arrangement of macromolecules in the yarn, the smaller the gap holes, the stronger the bonding between macromolecules and the base protofibril [ 39 ], and the greater the fiber’s breaking strength and initial modulus, but the greater the decrease in elongation ( Figure 2 ). The TGA and DTG curves, obtained at a heating rate of 10 K/min under a nitrogen atmosphere, are presented in Figure 4 . Based on weight loss across different temperature ranges, three distinct phases can be identified in Figure 4 a. TGA parameters, such as the initial decomposition temperature and the residual weight, are listed in Table 1 . In Phase I, the temperature ascends to around 250 °C with a minimal weight reduction observed [ 40 ]. Interestingly, the weight loss for the unstretched curve initiates earlier and leaves a smaller residual weight (95.43%) compared to the stretched yarns. This phenomenon is attributed to the fluffier structure of the unstretched yarns, housing a larger number of water molecules and low-molecular-weight oligomers. Within this phase, the nitrile groups in PAN undergo cyclization reactions, forming some ladder-like structures. Phase II sees the temperature rise to about 350 °C, during which a broad and rapid weight loss occurs. This can likely be ascribed to a substantial random scission of the linear PAN chains, which do not partake in cyclization reactions during the heat treatment. Concurrently, the residual mass of the stretched yarns surpasses that of the unstretched ones (77.02). By Phase III, as the temperature approaches 500 °C and the samples exhibit a relatively rapid weight reduction pace. This is predominantly due to the dehydrogenation reaction as the ladder-like structures transition to graphite-like structures. Following this phase, all TG curves plateau, indicating the formation of a stable structure. The outcomes elucidate that the stretched nanofibrous yarns manifest enhanced thermal stability. Meanwhile, DTG curves for different stretching ratios are shown in Figure 4 b. The unstretched yarn loses weight faster than the stretched yarn. This shows that the stretched yarn can form more trapezoidal structures and has higher stability than the unstretched yarn. TGA can also be used to determine the activation energy associated with random scissions within polymer samples [ 41 ]. A higher activation energy indicates greater resistance to random scission under the same conditions. For our samples, we focused on SR 0, SR 6, and SR 8 as representatives of stretched specimens. The samples were subject to different heating rates (5, 10, and 20 K/min) to determine their respective peak temperatures. The Kissinger method can quantify the apparent activation energy without prior understanding of any reaction mechanism. It requires only a series of heating at different rates. Therefore, we employed the Kissinger method to delineate the random scission behavior in these polymers. The results of this analysis are presented in the following table. The Kissinger method [ 42 ] is described by the following equation: (4) − E a R = d ln ϕ / T 2 d 1 / T In this equation, T represents the peak temperature corresponding to a specific heating rate ϕ , R stands for the universal gas constant, quantified as 8.314 J mol −1 K −1 , and E a is the activation energy. According to the Kissinger method, the activation energies ( Table 2 ) of SR 0, SR 6 and SR 8 are 120.4, 124.8 and 239.2, respectively. The activation energy of SR8 is significantly higher than that of SR0 and SR6, indicating a higher energy barrier and a more difficult reaction for this yarn. It is noteworthy that samples with different stretching ratios show similar behavior, which is attributed to the fact that they are derived from the same type of polyacrylonitrile. As stretching progresses, the molecular chains become oriented. Such oriented chains may be susceptible to certain structural changes upon heating, such as cyclisation of the nitrile groups to form some trapezoidal structures, leading to an increase in the activation energy. At the same time, the increase in crystallinity upon stretching (as evidenced by the XRD results Figure 3 ) and the stronger intermolecular forces and tighter molecular arrangement of the more crystalline material require greater energy barriers to be overcome during the reaction process, making the stretched yarns more resistant to random breakage. Therefore, SR8 demonstrates greater stability than other yarns. This phenomenon is consistent with the observed increase in breaking strength with increasing stretch ratio ( Figure 2 ). Herein, we demonstrate a conductive composite nanofiber yarn based on our high-strength nanofiber yarn, which uses a straightforward coating method. Figure 5 a presents a schematic illustration of the preparation of the conductive composite nanofiber yarn (detailed procedures are described in the Experimental Section). To identify the morphological features of the composite yarn, we examined SEM images of the surface of the nanofiber yarn and composite yarn. From the SEM images, it is evident that the nanofibers are present on the surface of the nanofiber yarn ( Figure 5 b), whereas no such nanofibers are visible on the surface of the composite yarn ( Figure 5 c). It was found that the surfaces of the nanofiber yarn in the composite yarn were coated uniformly and randomly with interconnected AgNWs, as confirmed by circular backscatter detector–scanning electron microscopy (CBS-SEM) ( Figure 5 d). The stability of the coating was investigated using UV spectra ( Figure 5 e). The composite yarn was first stirred in deionized water for 30 min. No peaks of AgNWs were observed, indicating that the nanowires were firmly adsorbed on the surface of the yarn and that the coating exhibited excellent stability. The mechanical properties were evaluated using tensile stress versus strain measurements ( Figure 5 f). The stress of the composite yarn was found to be 343 MPa, which is less than that of the uncoated yarn ( Figure 5 g). It was postulated that the coating of AgNW on the surface of the composite yarn increased the number of weak knots on the yarn surface, which decreased the stress. The electrical performance of the composite yarn was visually demonstrated by illuminating a light-emitting diode (LED) lamp using the composite fiber as circuit wiring ( Figure 6 a). The LED lamp was observed to be lit at 3 V when AgNW/nanofiber yarn was used as an electrically conductive element ( Figure 6 b). For the uncoated nanofiber yarn, there was almost no electric current observed over the applied voltage of 3 V, while the composite yarn displayed a noticeable current ( Figure 6 c). This indicates that AgNWs are physically interconnected on the surfaces of the nanofiber yarn to form a conductive network ( Figure 5 c). The effect of bending on the electrical properties of the composite nanofiber yarn was investigated. Figure 6 d indicates that the current of the composite yarn remained constant even when bent from 0° to 180°. The results of the testing proved the potential application prospects of the composite nanofiber yarn in smart textiles as a robust electrical wiring system."
} | 4,756 |
27065954 | PMC4814501 | pmc | 2,500 | {
"abstract": "The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments into the hydrosphere. AOM is largely mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), and has mainly been investigated in deep-sea sediments. Here we studied methane seepage at four spots located at 12 m water depth in coastal, organic carbon depleted permeable sands off the Island of Elba (Italy). We combined biogeochemical measurements, sequencing-based community analyses and in situ hybridization to investigate the microbial communities of this environment. Increased alkalinity, formation of free sulfide and nearly stoichiometric methane oxidation and sulfate reduction rates up to 200 nmol g -1 day -1 indicated the predominance of sulfate-coupled AOM. With up to 40 cm thickness the zones of AOM activity were unusually large and occurred in deeper sediment horizons (20–50 cm below seafloor) as compared to diffusion-dominated deep-sea seeps, which is likely caused by advective flow of pore water due to the shallow water depth and permeability of the sands. Hydrodynamic forces also may be responsible for the substantial phylogenetic and unprecedented morphological diversity of AOM consortia inhabiting these sands, including the clades ANME-1a/b, ANME-2a/b/c, ANME-3, and their partner bacteria SEEP-SRB1a and SEEP-SRB2. High microbial dispersal, the availability of diverse energy sources and high habitat heterogeneity might explain that the emission spots shared few microbial taxa, despite their physical proximity. Although the biogeochemistry of this shallow methane seep was very different to that of deep-sea seeps, their key functional taxa were very closely related, which supports the global dispersal of key taxa and underlines strong selection by methane as the predominant energy source. Mesophilic, methane-fueled ecosystems in shallow-water permeable sediments may comprise distinct microbial habitats due to their unique biogeochemical and physical characteristics. To link AOM phylotypes with seep habitats and to enable future meta-analyses we thus propose that seep environment ontology needs to be further specified.",
"conclusion": "Conclusion Coastal sandy sediments have a higher permeability and lower porosity than the silty clays that constitute deep-sea sediments, which in turn results in a lesser interstitial volume and lesser overall particle surface. These sediment properties combined with the prevalent hydrodynamics due to wave action, currents and gas ebullition create microbial habitats in shallow methane seeps that are very different from those found at methane seeps in the deep sea. To distinguish these habitats we think that a standardized and detailed ontology of methane-fuelled ecosystems is needed. Our findings suggest that the high phylogenetic and morphological diversity of anaerobic methanotrophs, and the apparently low-efficient methane filter at the Pomonte seep site are linked to the sediment characteristics of the ecosystem. Yet, the underlying environmental processes that shape microbial diversity, abundance and function remain unclear and are promising objectives of further research. The study underlines that our understanding of shallow-water methane seeps is still incomplete, despite their widespread occurrence on active and passive continental margins and importance for the global methane budget. It is crucial to further investigate the microbial ecology and efficiency of methane removal as most of the emitted methane at shallow seeps is released directly to the atmosphere.",
"introduction": "Introduction Methane seeps are widespread features of the seafloor along continental margins, where methane ascends from subsurface reservoirs and fuels methanotrophic communities or is emitted to the hydrosphere. The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments and is mediated by anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) ( Knittel and Boetius, 2009 ). Marine AOM has mainly been investigated in deep-sea methane seeps, which are characterized by steep opposing gradients of methane and sulfate in the top centimeters of the sediment. In deep-sea cold seeps the sulfate-methane transition zones (SMTZ) have a thickness of only a few centimeters and are shaped by fluid flow and faunal activity ( Bhatnagar et al., 2008 ; Fischer et al., 2012 ; Ruff et al., 2013 ; Felden et al., 2014 ). Between 20 and 80% of the methane (around 10 6 tons of carbon per year) is consumed at the sediment water interface by methanotrophic microbial communities ( Boetius and Wenzhöfer, 2013 ). Due to the aerobic water column the top layers of seep sediments are usually oxic and sustain aerobic methanotrophic bacteria, mainly of the gammaproteobacterial order Methylococcales ( Lösekann et al., 2007 ; Tavormina et al., 2008 ; Wasmund et al., 2009 ; Ruff et al., 2013 ), whereas deeper sediment layers are depleted of oxygen and are dominated by AOM ( Knittel and Boetius, 2009 ). Here, ANME and SRB usually form dense aggregates that occur at seeps in very high abundances resulting in cell numbers of 10 10 cells per ml sediment at, e.g., Hydrate Ridge, the Black Sea ( Knittel et al., 2005 ), Hikurangi Margin ( Ruff et al., 2013 ), and Haakon Mosby mud volcano ( Lösekann et al., 2007 ). Apart from methanotrophs and their partner bacteria, seeps comprise thiotrophic Beggiatoaceae, Campylobacteraceae , and Helicobacteraceae ( Joye et al., 2004 ; Grünke et al., 2012 ; Felden et al., 2014 ) that often form thick mats on the seafloor. These organisms represent the methane seep microbiome, which is similar among deep-sea cold seeps worldwide, but very different from the surrounding seafloor ( Ruff et al., 2015 ). The anaerobic organisms (ANME and their partner bacteria) are oxygen sensitive and it is yet unclear how they disperse between these isolated ecosystems, and whether coastal, dynamic sites harbor the same microbiome that establishes at deep-sea environments. Shallow-water coastal methane seeps can be found at continental margins of all oceans, e.g., in the North Sea at 75–170 m water depth ( Wegener et al., 2008 ), the East Timor Sea at 80 m ( Wasmund et al., 2009 ; Brunskill et al., 2011 ), the Southeast Pacific at 1–5 m ( Jessen et al., 2011 ) or the Northwest Atlantic at >50 m ( Skarke et al., 2014 ). Coastal seeps at water depths of less than 100 m likely contribute large amounts of methane to the atmospheric budget as methanotrophs in the water column may oxidize only part of the emitted gas ( McGinnis et al., 2006 ; Brunskill et al., 2011 ), e.g., a single shallow seep area off the coast of Chile emitted an estimated 800 tons of the potential greenhouse gas to the atmosphere per year ( Jessen et al., 2011 ). Moreover, recent estimates indicate the presence of 1000s of coastal seeps worldwide ( Skarke et al., 2014 ). However, despite their large number, their considerable methane emission, the biogeochemistry and microbial communities of coastal seeps are poorly understood. The coastal seafloor is exposed to strong hydrodynamic forces caused by waves and tides. These high energies allow for the settlement of only larger particles of the sand fraction forming permeable sediments. Wave-driven advection furthermore greatly impacts the habitats of benthic microorganisms by the enhanced supply of electron donors, electron acceptors and nutrients ( Precht and Huettel, 2004 ; Janssen et al., 2005 ), whereas deep-sea sediments in contrast are dominated by diffusive transport ( Glud et al., 1994 ; Boetius and Wenzhöfer, 2013 ). Permeable coastal sediments harbor a high diversity of microorganisms ( Mills et al., 2008 ) that are subjected to strong seasonal and spatial dynamics ( Böer et al., 2009b ; Gobet et al., 2012 ) due to changing abiotic conditions. It is yet unclear how these dynamics and the permeability of the sediment matrix influence the distribution, community structure, and activity of seep-associated microorganisms. Here, we investigated shallow-water methane seepage off the coast of the Tuscan Island Elba (Italy). Elba is located in the Northern Tyrrhenian Sea, a relatively young (<15 Ma) back-arc basin formed by the roll-back of the Adriatic and Ionian subducting plates. The region is underlain by very thin continental crust and is tectonically very active ( Greve et al., 2014 and references therein). Since 1995 the diving team of the HYDRA Field Station on Elba observe gas flares near the coast of the little village Pomonte, the island of Pianosa and the islet Scoglio d’Africa ( Figure 1 ), which are all included in this tectonically active zone. At the Pomonte site the gas bubbles escape from permeable, organic carbon depleted sands and seagrass beds at around 12 m water depth. This unusual combination of tectonic setting, sediment characteristics and hydrodynamics turns the Pomonte seepage site into an outstanding ecosystem that differs from other known seep sites. FIGURE 1 Map of the Tuscan Island seep area (A) with the three major sites, close to the islands of Elba and Pianosa, and the islet Scoglio d’Africa. This study focused on the Pomonte seep site (B) and nearby reference sites (Ref1–3). (C) Shows the detailed location of the investigated methane emission spots (ES1a, 1b, 2–4) at the Pomonte seep site. The emission spots are characterized by gas flares (D) as well as black sulfidic sediments that are occasionally covered by white mats of sulfur-oxidizing bacteria (E) . The emission spots are situated in 12 m water depth, surrounded by seagrass and rocks and are easily accessible by scuba diving (F) . We focused this first investigation of the site on the detailed analysis of the biogeochemistry and microbial community structure. We chose four methane emission spots situated in these permeable sands and performed biogeochemical measurements, 16S rRNA gene sequencing and whole cell hybridizations. The study was based on three hypotheses: Methane seeps located in shallow, permeable sands (i) have characteristic biogeochemical profiles that are shaped by the profound hydrodynamic forces, (ii) harbor similar anaerobic methanotrophic communities than seeps found in the deep sea due to the strong selective pressure of methane as the predominant energy source, and (iii) have a higher diversity than deep-sea seeps, due to the greater number of niches available in coastal sands.",
"discussion": "Discussion Biogeochemistry of Shallow-Water Permeable Seep Sediments Most of the so far studied methane seeps are located in muddy, silty deep-sea sediments that are less affected by hydrodynamic forces and temperature changes, and are thus very stable and permanently cold. The microbial communities at these deep-sea ecosystems develop over long periods of time and are predominantly shaped by faunal activity ( Cordes et al., 2005 ; Thurber et al., 2012 ; Niemann et al., 2013 ; Ruff et al., 2013 ; Felden et al., 2014 ), or changes in the geochemistry ( De Beer et al., 2006 ; Lichtschlag et al., 2010 ; Fischer et al., 2012 ; Felden et al., 2013 ; Zhang et al., 2014 ). The Tuscan Island seepage area harbors some of the shallowest marine methane seep sites investigated to date. The Pomonte seep site is outstanding as the emission spots are situated in permeable sands that are strongly influenced by diurnally and seasonally changing hydrodynamic forces such as waves and currents and by seasonal changes in water temperature from 12 to 25°C with an average temperature of 19.4 ± 4.3°C ( Shaltout and Omstedt, 2014 ). In fine-grained or muddy seep sediments the transport of electron acceptors from the water column into the sediment is mainly diffusion regulated, or driven by small-scale advection due to gas ebullition, whereas in permeable sandy sediments this transport is largely due to advection-driven pore water circulation ( Janssen et al., 2005 ). The pore water profiles indicate that advection and lateral transport of electron acceptors are important at all studied methane emission spots, which allow AOM activity in a deeper and wider sediment horizon than what is known from the deep sea. In the upper sediment horizons recurring disturbances such as sediment relocation or advective inflow of oxic water likely prevent settlement of the oxygen-sensitive AOM consortia. In deep-sea methane seep sediments the sulfate-methane transition zone (SMTZ) and hence the zone of highest activity is usually only a few centimeters thick. Often the SMTZs are located close to the sediment surface and harbor between 10 9 and 10 10 microbial cells per milliliter sediment ( Knittel and Boetius, 2009 ). At the Pomonte shallow seep site, the zone of highest AOM activity harbored 10 8 microbial cells per milliliter sediment, was much deeper and between 20 and 40 cm thick. The sulfate concentration did not decrease as is the case in deep-sea sediments and seeps, but stayed fairly stable down to 60 cm sediment depth, indicating that sulfate replenishes from the surrounding sands. Thus, at the Pomonte site we do not find a classical SMTZ, with sharp vertical gradients of methane and sulfate, commonly found in surface sediments of methane seeps (e.g., Lichtschlag et al., 2010 ; Ruff et al., 2013 ), or in the subsurface of seeps and methane rich sediments ( Webster et al., 2011 ; Treude et al., 2014 ). We instead observed that AOM activity mainly occurred in deep sediment layers and that AOM aggregates were distributed throughout the sediment cores. This is unusual for methane seeps and indicated that the active zone, including opposing gradients of methane, and sulfate, was found laterally around the central gas conduit, rather than being confined to a thin layer close to the sediment surface. This hypothesis is further supported by studies showing that the upward flow of gas creates a downward flow of seawater in adjacent sediments ( O’Hara et al., 1995 ; Tryon et al., 1999 ), which may result in lateral advection of seawater through the permeable sediment around a central conduit ( Stein and Fisher, 2001 ). Both methane and sulfate occurred in excess throughout the sediment and were not depleted, indicating a low efficiency of the benthic filter in permeable, low-biomass sands. A large part of the methane that passes through the sand without being oxidized also passes through the shallow water column, making its way to the atmosphere, where it may act as a potent greenhouse gas ( McGinnis et al., 2006 ; Brunskill et al., 2011 ). Methane seepage was shown to cause similar biogeochemical profiles in other mesophilic, permeable sandy sediments, e.g., at Coal Oil Point in the Santa Barbara Basin ( Treude and Ziebis, 2010 ), the Skagerrak ( Knab et al., 2008 ) and the Gulfaks Oil Field in the North Sea ( Wegener et al., 2008 ). Highly permeable seep sediments also exist in hydrothermal settings, e.g., at Middle Valley on the Juan de Fuca Ridge ( Wankel et al., 2012 ), showing similar lateral advection of overlying seawater ( Stein and Fisher, 2001 ). Based on these observations it is possible that methane venting through permeable sands generally features characteristic biogeochemical profiles, vertical SMTZs and low-efficient biofilters. Many studies in recent years have tried to elucidate the niche differentiation and ecophysiology of populations that are directly or indirectly involved in the anaerobic oxidation of methane and/or hydrocarbons. Although, evidence is accumulating that microbial populations differentiate based on the availability of electron acceptors ( Green-Saxena et al., 2014 ), electron donors ( Grünke et al., 2011 ), hydrocarbons ( Stagars et al., 2016 ), temperature ( Holler et al., 2011b ), and the substratum ( Case et al., 2015 ), to name just a few, the ecological processes and niches remain unclear. To link phylotypes with habitats it is necessary to define environment ontologies ( Buttigieg et al., 2013 ; Thessen et al., 2015 ) that can be used to clearly distinguish different types of seep ecosystems and AOM habitats. The term “seep” that we used throughout this study is strictly speaking a misleading description for the investigated sites, as they have characteristic gas flares and are continuously shaped by gas ebullition. Diversity and Turnover of Microbial Communities in Shallow, Permeable Seep Sediments Diversity and turnover of the microbial communities at the Pomonte site were described and compared to those found at deep-sea methane seeps. The microbial communities of Elba shallow seeps comprised organisms that were closely related to those found at other seep ecosystems worldwide ( Figures 4 and 5 ). We detected 16S rRNA partial gene sequences that shared >97% sequence identity with 16S ribosomal genes of ANME or SRB organisms that occurred, e.g., at methane seeps in the Nankai Trough ( Miyashita et al., 2009 ), the Santa Barbara Basin ( Orphan et al., 2001 ), the Gulf of Mexico ( Lloyd et al., 2010 ) at mud volcanoes in the Mediterranean ( Pachiadaki et al., 2010 ), and the Atlantic ( Niemann et al., 2006 ), and in SMTZs of organic-rich shallow sediments of Eckernförde Bay ( Jagersma et al., 2009 ). Hence, the biogeochemical and physical constraints of the permeable microbial habitat selected for the same microbial communities that are involved in AOM worldwide. Yet, despite their proximity and their biogeochemical similarities, the studied emission spots were remarkably different concerning their richness and evenness based on amplicons of the V3–V5 region ( Table 1 ) as well as concerning their dominant microbial clades based on both partial genes ( Figure 3 ) and V3–V5 amplicons ( Figure 6 ). Permeable sands are much more heterogeneous than soft deep-sea sediments and provide a large number of niches to microorganisms ( Mills et al., 2008 ; Böer et al., 2009a ; Schöttner et al., 2011 ; Gobet et al., 2012 ). This heterogeneity may result in an increased number of niches also for anaerobic methanotrophs and sulfate-reducers. The high diversity of key players that we observed may be connected to frequent changes in the concentration of methane, sulfate and especially oxygen caused by lateral advection of seawater to the deeply buried, but permeable AOM zones. At the emission spots ES1a and ES1b we found ANME-1a, -1b, -2a, -2b, -2c and ANME-3 as well as SEEP-SRB-1a, -2, and many other sulfate reducers, among them Sva0081 and Desulfarculales . This indicated the coexistence of clades with very different habitat preferences, such as ANME-1, which predominantly occur in anoxic, deep (ANME-1b) or hot (ANME-1a) sediment layers ( Biddle et al., 2012 ; Vigneron et al., 2013 ; Ruff et al., 2015 ), seem to be oxygen sensitive ( Knittel et al., 2005 ) and tolerant to changes in temperature and sulfate availability ( Dowell et al., 2016 ) and ANME-2c, which seem to be adapted to very different conditions like shallow, bioirrigated and sulfate-rich sediment layers ( Biddle et al., 2012 ; Vigneron et al., 2013 ; Felden et al., 2014 ). Moreover, we observed associations that contained cells of both ANME-1 and ANME-2. To our knowledge this is the first time that such associations were visualized, despite the frequent co-occurrence of ANME clades in sequence-based studies of seep microbial diversity (e.g., Ruff et al., 2015 ). Monospecific occurrence of ANME archaea has been described before ( Orphan et al., 2002 ; Lösekann et al., 2007 ; Treude et al., 2007 ; Wegener et al., 2008 ; Vigneron et al., 2013 ), but the causes remain elusive. Aggregation could be a strategy to minimize stress caused by advection-driven entrainment of oxygen, which was shown for sulfate reducers ( Dolla et al., 2006 ). For sulfate-coupled AOM most physiological studies emanate from obligate syntrophy of ANME and SRB ( Girguis et al., 2005 ; Nauhaus et al., 2005 ; Orphan et al., 2009 ; Meulepas et al., 2010 ; Holler et al., 2011a ; McGlynn et al., 2015 ; Wegener et al., 2015 ) with findings that seemingly make monospecific life unfavorable. We also detected methanogens and sulfur disproportionating bacteria, which frequently occur at methane seeps and in AOM enrichment cultures. These organisms are very widespread, but rare and are involved in side reactions of AOM ( Wegener et al., 2016 ). In addition, the phototrophic enrichments suggested that sulfide-oxidizing phototrophs inhabit the sediment surface of the seeps, using the AOM-derived sulfide as an electron donor for photosynthesis. This indicated that shallow seeps are a so far overlooked habitat for anoxygenic green and purple phototrophs. Phylogenetic diversity was paralleled by an unprecedented morphological diversity, including several different forms of spherical and filamentous consortia, monospecific aggregates and even indications for AOM consortia that are comprised of two ANME clades and one SRB ( Figures 8 and 9 ). It was shown that cell aggregation decreased both cell movement through a sand column ( Vandevivere and Baveye, 1992 ) and the likelihood of being grazed by benthic predators ( deLeo and Baveye, 1997 ), and may even enhance substrate uptake per cell ( Logan and Hunt, 1987 ). In addition, grain size and permeability can influence both the abundance ( Santmire and Leff, 2007 ) and the community structure of benthic communities ( Jackson and Weeks, 2008 ; Zheng et al., 2014 ). It is possible that the high phylogenetic and morphological diversity is linked to the permeability or other parameters of the sediment. The different aggregate morphologies could be adaptations to different flow regimes, interstitial spaces, and compound concentrations. It was shown that disturbances, such as the exposition to oxygen ( Shade et al., 2011 ) or salinity ( Berga et al., 2012 ), influence community composition and function, while other disturbances may increase the microbial diversity of an ecosystem ( Flöder and Sommer, 1999 ; Buckling et al., 2000 ). Yet, the effects of sediment characteristics and disturbance largely remain understudied in particular in ecosystems that are difficult to reach and sample, such as most methane seeps. Disturbance caused by the strong hydrodynamics may also explain the high microbial turnover between the sites. Other factors may include the fluctuations in immigrating and emigrating microbial populations, which is a stochastic process that is especially important in dynamic habitats ( Böer et al., 2009b ; Gobet et al., 2012 ), or energy-diversity, as these sands are natural filters of fresh organic matter particles from both marine and terrestrial sources. It was shown that differences in available carbon sources have a significant influence on microbial community structure ( Bienhold et al., 2011 ; Sawall et al., 2012 ; Raulf et al., 2015 )."
} | 5,734 |
36772749 | PMC9920395 | pmc | 2,501 | {
"abstract": "In recent years, deep learning (DL) has been widely studied using various methods across the globe, especially with respect to training methods and network structures, proving highly effective in a wide range of tasks and applications, including image, speech, and text recognition. One important aspect of this advancement is involved in the effort of designing and upgrading neural architectures, which has been consistently attempted thus far. However, designing such architectures requires the combined knowledge and know-how of experts from each relevant discipline and a series of trial-and-error steps. In this light, automated neural architecture search (NAS) methods are increasingly at the center of attention; this paper aimed at summarizing the basic concepts of NAS while providing an overview of recent studies on the applications of NAS. It is worth noting that most previous survey studies on NAS have been focused on perspectives of hardware or search strategies. To the best knowledge of the present authors, this study is the first to look at NAS from a computer vision perspective. In the present study, computer vision areas were categorized by task, and recent trends found in each study on NAS were analyzed in detail.",
"introduction": "1. Introduction The artificial neural network (ANN) used today is the product of the combined efforts of a number of researchers, including McCulloch et al. [ 1 ], who first developed the concept in 1943. The concept of the convolutional neural network (CNN), among the most widely used neural network structures in computer vision (CV) applications, was first introduced using LeNet-5 in a 1989 study by LeCun et al. [ 2 ]. Back then, however, due to the lack of computing power of the hardware, CNN was found to be ineffective in dealing with complex and sophisticated tasks, such as object recognition and detection. After quite a while, AlexNet [ 3 ] was first introduced at the ImageNet Large Scale Visual Recognition Challenge (ILSVRC) in 2012; it made an impressive debut because the model was able to effectively overcome the limitations of deep learning (DL), making the most of GPU computing. With its unparalleled performance, AlexNet ushered in a new era of DL in earnest [ 4 ]. DL has attracted significant attention from researchers thanks to its ability to allow the automatic extraction of valid feature vectors from image data. Performing this automatic extraction requires the tuning of the hyper-parameters of DL. Thus far, extensive research for improved performance has been performed on a wide range of hyper-parameters, such as network structures, weight initialization, activation functions, operators, and loss functions [ 5 , 6 , 7 , 8 , 9 ]. However, the application of DL requires technical expertise and understanding, while incurring a significant amount of engineering time. In attempts to address these issues, automated machine learning (AutoML) has recently garnered significant attention. The AutoML process is divided into multiple steps, including data preparation, feature engineering, model generation, and model estimation [ 10 ]. The first step of AutoML, data preparation, is a process in which data are collected; factors that may negatively affect the learning process, e.g., noise, are removed to train a given model to perform the target tasks. The second step, feature engineering, extracts features from given data to be used in the model. At the same time, feature construction techniques to improve the representative ability of the model, as well as feature selection techniques used to avoid overfitting, are applied in parallel. Conventional feature engineering methods include SURF [ 11 ], SIFT [ 12 ], HOG [ 13 ], the Kalman filter [ 14 ], and the Gabor filter. Recently, CNN and recurrent neural network (RNN) applications have been used to extract high-level features. The third step, model generation, is performed to obtain optimum output based on the extracted features. Model generation can be further subdivided into search space, hyper-parameters, and architecture optimization. During this process, model optimization is performed in combination with the model estimation step. This paper summarizes the basic concepts of neural architecture search (NAS), while providing an overview of previous studies regarding the application of NAS in CV fields. NAS is a research field that encompasses feature engineering, neural networks, architecture optimization, and model estimation, which constitutes AutoML. The search space and search strategy of NAS, as well as major relevant studies, are introduced separately in Section 2.1 and Section 2.2 ., Section 2.3 introduces the NAS evaluation strategies, and Section 3 will take a close look at the major findings of previous studies on the use of NAS to address various challenges (detection, segmentation, generation, etc.) in addition to classification applications.",
"discussion": "4. Discussion This survey paper provided an extensive review of previous studies on neural architecture search (NAS) as part of AutoML. Notably, with the focus on computer vision (CV) applications, a series of field-specific algorithms based on current NAS techniques for CV, especially those that were expected to contribute to future studies, were introduced and analyzed. Thus far, fewer studies have been focused on detection, segmentation, and GAN applications of NAS for CV than on classification tasks. Furthermore, it is not simple to compare the performance of various NAS methods and approaches with those of existing algorithms reported so far because the NAS method relies on many factors other than the architecture. Most previous studies attempted to evaluate the performance of algorithms of interest using MS-COCO and CIFAR-10, datasets that were mainly used in CV tasks; however, in reality experimental evaluations also involve various other factors, such as hardware, search space, data preprocessing, calculation amount, regularization, and input size. As such, finding an accurate quantitative evaluation method for NAS algorithms for CV, rather than just simply focusing on increasing their performance with respect to each and every task, is deemed to be of great significance. When analyzing the recent keywords of Computer Vision and Pattern Recognition (CVPR) papers which are published between 2020 and 2022, the keywords of Unsupervised Learning and Self-Supervised Learning are hot topics. Thus, the research related to NAS for CV will gradually be expanded and be advanced. Furthermore, considering the latest technology, we think the search space with gradient descent might be a future direction of development in NAS for CV because it would decrease the search time and be efficient enough to handle the large amount of image data."
} | 1,697 |
35542462 | PMC9084341 | pmc | 2,502 | {
"abstract": "Resistive switching memories have been regarded as one of the most up and coming memory systems and researchers have shown great interest in them because of their simple structure, high speed and low fabrication cost. These memory systems also have great potential for scaling, however, this has been difficult to achieve without detailed understanding of underlying switching mechanisms. Meanwhile, scaling down could also raise reliability concerns in its performance. This work provides an overview of various switching mechanisms and their investigations at nanoscale levels using high resolution microscopy techniques. In this mini review, the main focus was to understand the working mechanism derived from the so-called filament model. The high resolution conductive atomic force microscope, transmission electron microscope and scanning electron microscopes are the best tools available to investigate the dynamics of filamentary switching. Several issues with the existing techniques are also highlighted.",
"introduction": "1. Introduction As a promising non-volatile memory (NVM), resistive random access memories (RRAMs or ReRAM) have quite distinct advantages of a simple structure, high speed response and small size (scaling down to few ion widths) over existing silicon-based flash memories. 1–5 Furthermore, the process of data writing in RRAMs is more or less infinitely reversible, which forms a firm basis for these memories to be more universally applicable than flash memories. A typical RRAM device can transform its inherent resistance state (either high or low) to another resistive level by means of electrical stimuli. Conventionally, there are two common resistive switching (RS) models that have been widely used to describe this transformation of resistance levels. If the RRAM device switches its resistance state at one voltage polarity and an opposite voltage polarity brings back its parent resistance state, such behaviour is called bipolar resistive switching. On the other hand, if the resistance switching process (from one to another state and vice versa ) is independent of an applied potential polarity, then this behaviour is called unipolar switching. In principle, several physical phenomena may exist that can play an equally important role in demonstrating resistive switching effects. Such phenomena may include: mechanical forces, molecular configuration, 6 electrostatic and electronic effects, domain polarization, 3,7 temperature induced effects 8 and chemical effects. 9 Among all these effects, three prominent mechanisms have been observed and microscopically studied in detail: (i) the electrochemical metallization (ECM) mechanism, (ii) the valence change mechanism, and (iii) the thermochemical mechanism. The ECM mechanism involves the presence of at least one active electrode such as silver (Ag) or copper (Cu) as a part of RRAM device. In the presence of an electrical potential, the active cations (Ag + or Cu + ) penetrate into the material to form a conducting percolation path between two electrodes, thus switching the device conductance state to an ON state (low resistive state). By reversing the potential polarity, these percolation channels can dissolve, thus resetting the device system into the OFF state. The valence change mechanism only appears in particular metal oxides and its main source is anion migration (oxygen vacancies). The applied potential may change the local stoichiometry of the system which may lead to a redox reaction by changing the valency of the cation sub-lattice and the overall electronic conductivity of the system. In the thermochemical mechanism, the change in local temperature induced by applied potential (current) could also change the local stoichiometry of the system. The RS effects in various materials/systems (even the majority of them) have been investigated using current–voltage measurements that involve a voltage source meter and a probe station. 10–14 Furthermore, various other effects such as: material thickness, electrode size, temperature dependant measurements and so on, have also been used to understand the governing mechanisms in resistive switching materials. 15–17 Although these methods provide excellent information on device performance evaluation and quantitative analysis of the underlying switching mechanisms, in order to fully understand the nature of the switching mechanism, detailed nanoscale microscopic analysis is vital. Furthermore, in situ microscopic measurements, such as atomic force microscopy (AFM) and in situ transmission electron microscopy (TEM), are very powerful tools for analysing resistive switching effects. In this paper, the state-of-the-art nanoscale microscopic investigations of the resistive switching phenomenon are reviewed. After briefly discussing conduction processes in the switching materials, how the state-of-the-art conductive AFM can be used to investigate resistive switching mechanisms will be discussed. Next, the examination of resistive switching processes using in situ and ex situ TEM will be reviewed. Thirdly, some recent work in which scanning electron microscopy (SEM) was utilized to study the resistive switching processes in various materials will be reviewed. Finally, future perspectives on microscopic investigations for resistive switching processes will be addressed."
} | 1,340 |
23565725 | PMC3630057 | pmc | 2,503 | {
"abstract": "Background Ecosystems worldwide are suffering the consequences of anthropogenic impact. The diverse ecosystem of coral reefs, for example, are globally threatened by increases in sea surface temperatures due to global warming. Studies to date have focused on determining genetic diversity, the sequence variability of genes in a species, as a proxy to estimate and predict the potential adaptive response of coral populations to environmental changes linked to climate changes. However, the examination of natural gene expression variation has received less attention. This variation has been implicated as an important factor in evolutionary processes, upon which natural selection can act. Results We acclimatized coral nubbins from six colonies of the reef-building coral Acropora millepora to a common garden in Heron Island (Great Barrier Reef, GBR) for a period of four weeks to remove any site-specific environmental effects on the physiology of the coral nubbins. By using a cDNA microarray platform, we detected a high level of gene expression variation, with 17% (488) of the unigenes differentially expressed across coral nubbins of the six colonies (jsFDR-corrected, p < 0.01). Among the main categories of biological processes found differentially expressed were transport, translation, response to stimulus, oxidation-reduction processes, and apoptosis. We found that the transcriptional profiles did not correspond to the genotype of the colony characterized using either an intron of the carbonic anhydrase gene or microsatellite loci markers. Conclusion Our results provide evidence of the high inter-colony variation in A. millepora at the transcriptomic level grown under a common garden and without a correspondence with genotypic identity. This finding brings to our attention the importance of taking into account natural variation between reef corals when assessing experimental gene expression differences. The high transcriptional variation detected in this study is interpreted and discussed within the context of adaptive potential and phenotypic plasticity of reef corals. Whether this variation will allow coral reefs to survive to current challenges remains unknown.",
"conclusion": "Conclusions In this study, we were able to genotype colonies of Acropora millepora from the reef flat surrounding Heron Island (GBR) by a high-resolution marker, microsatellites, and an additional molecular marker, intron 4–500 of a carbonic anhydrase isoform. The latter identified two different genotypes. We further explored the transcriptomic variation of six colonies by acclimatizing coral nubbins to a common garden in the same reef flat. Although no correspondence between transcriptional profiles and colony genotype was found (Figure 1 ), we revealed substantial natural gene expression variation occurring in these acclimatized coral nubbins. Some of the differentially expressed biological processes include transport and translation (Figure 2 ); these processes have previously been identified in other studies of corals examining the transcriptomic variability to various experimental factors, as well as natural variation (e.g.[ 25 , 26 , 32 , 37 , 38 , 50 , 55 ]). Genes in the category of oxidation-reduction process were also differentially expressed, most likely as a consequence of the photosynthetic activity of the dinoflagellate symbiont [ 56 ]. Genes involved in response to stimulus were also differentially expressed among colonies. This category contained several stress genes, including immune response genes. The considerable expression variation highlights the normal individual variation of coral colonies. Therefore, studies exploring gene expression either in response to stress or natural variation must consider natural variation occurring between individuals. Importantly, natural gene expression variation could be the raw material upon which natural selection can act for evolution. Furthermore, this variation at the transcriptomic level combined with epigenomic modifications may be a source of phenotypic plasticity, which could potentially allow reef corals to respond to changing environments. Whether these genetic and epigenetic responses of corals and its symbionts will allow coral reefs to cope with the rapid pace of global change remains unknown.",
"discussion": "Results and discussion Genotypic identity in A. millepora as detected by a carbonic anhydrase-intron and microsatellite loci markers We detected genotypic differences among 25 colonies tagged of Acropora millepora from the same reef flat on Heron Island (GBR) using the carbonic anhydrase 4–500 intron. We identified two different genotypes based on fingerprinting profiles (Additional file 1 ): 21 colonies as genotype 1 (displaying one single band of 550 bp) and four colonies as genotype 2 (showing two bands of 550 and 450 bp). To explore for natural gene expression variation between these two genotypes, we selected three colonies from each genotype for transcriptional profile comparison. The colonies selected for transcriptional profile comparison were also genotyped using four microsatellite loci developed for A. millepora by van Oppen et al. [ 43 ] to further assess genotypic identity in the transplanted coral nubbins from the six colonies of A. millepora in the common garden. While we initially set out to screen a total of six microsatellite loci, two sets of microsatellite loci were not successfully amplified in all colonies. A total of 13 alleles were detected within the four screened microsatellite loci (Additional file 2 ), which is within the range of alleles (2–21 alleles per locus) identified in 947 colonies of A. millepora across the GBR using these genotypic markers [ 42 ]. Additional development of microsatellite markers using EST and whole-genome shotgun sequence (WGS) databases identified 40 polymorphic loci [ 44 ]. Similarly, the number of alleles ranged from two to 16 for EST microsatellites and from five to 18 for WGS microsatellites [ 44 ]. The alleles detected here are probably common in the southern GBR, as low levels of genetic flow have been described in this area [ 42 ]. Further examination of this data in a principal component analysis (PCA) allowed the detection of differentiation exhibited among colonies at the microsatellite level (Figure 1 A). While we did not test for population structure (as this was beyond the scope of this study), the ordination of the colonies in the PCA according to microsatellites did not support the intron genotypes. Two clusters were resolved based on the axis of PC1, which explained almost 40% of the variation, but each cluster grouped colonies from the two intron genotypes. This highlights the importance of surveying genetic variation within a population of corals using different markers when carrying out molecular ecology studies (e.g. [ 45 ]). Figure 1 Principal component analysis (PCA). A ) Ordination based on microsatellite loci genotypes (13 alleles). PC1 and PC2 explain 39% and 24% of the variation, respectively. B ) Global pattern of gene expression on six colonies of A. millepora . PC1 and PC2 explain 44% and 24% of the variation, respectively. Orange = colonies genotyped as genotype 1 with the Intron 4–500. Blue = colonies genotyped as genotype 2 with the intron 4–500. Transcriptomic variation among coral nubbins within a common garden To determine the existence of natural gene expression variation and its correspondence with genotype given that some genes might respond based on the genetic background of the colony [ 29 ], transcriptional profiles of coral nubbins from six colonies (three from each of the two intron-genotypes) were determined using cDNA microarrays after bringing the coral nubbins to the same reef flat (common garden) for recovery and acclimation for four weeks. All colonies appeared healthy and coral nubbins were taken from the same tip position (see Methods). These procedures control for physiological differences between corals resulting from environmental sampling, allowing us to compare transcriptional statuses between colonies. A two-way ANOVA test in the reduced dataset found no differentially expressed genes (jsFDR-corrected; p > 0.05) in the coral colonies between the two intron genotypes. We also performed a mixed-ANOVA to detect variation in gene expression among all colonies. Unexpectedly, we detected a significant difference in gene expression, where 17% or 488 unique genes (1,021 features) from the cDNA microarray were differentially expressed between colonies ( p < 0.01, jsFDR-corrected) (Additional file 3 ). While previous studies aimed at examining natural gene expression variation detected fewer differentially expressed genes (1.31% = 114 of 8686 genes, [ 38 ], 0.046% = 4 of 8686 genes [ 37 ]) than the study at hand, the experimental design of these studies prevented the examination of naturally occurring gene expression variation uninfluenced by environmental effects. In fact, this variation probably accounts for the variation (or lack thereof) in the level of gene expression of individual genes during heat stress [ 45 , 46 ]. As such, the large variation in natural gene expression detected here after controlling for environmental effects has not been previously reported and opens new questions and avenues of research in coral adaptive evolution and phenotypic plasticity, as further discussed below. The proportion of genes differentially expressed in our study (17%) is comparable to other studies that measured gene expression in natural populations. For instance, 18% of the genes studied (161 genes) differed significantly between individuals of the same population of Fundulus heteroclitus [ 14 ]. In another study, 24% of the genes had significant expression levels among different strains of yeast [ 47 ]. Interestingly, we also found that the transcriptional difference among coral colonies was not attributed to the association with different types of Symbiodinium . Direct sequencing of the 28S nuclear rDNA from symbiotic dinoflagellates associated with the coral colonies showed that all colonies in the experiment harbored the same genetic type of Symbiodinium C3 (Acc. No. KC493130- KC493135). To identify patterns in gene expression among colonies, a multidimensional ordination based on PCA was performed using the data of the differentially expressed genes (Figure 1 B). Despite all coral nubbins acclimated to a common garden, the PCA ordination showed different transcriptional profiles among colonies, which highlights the importance of taking into account natural variation between colonies when assessing experimental gene expression differences. The first two axes explained 68% of the variation, where an important differentiation between the colonies was observed (Figure 1 B). Colonies 3, 4, and 6 grouped together under both PC1 and PC2, and were separated by PC1 from colonies 1 and 5. Additionally, colonies 1, 3, 4, and 6 were separated from colonies 2 and 5 by PC2 (Figure 1 B). The PCA ordination also allowed the comparison of transcriptional statuses of the colonies with the two approaches implemented for colony genotyping. In the case of the intron genotype, the ordination of gene expression did not correspond to the two genotypes, corroborating the lack of statistical significance examined above between genotype and gene expression profiles. In the case of the microsatellites, there was not a clear correspondence between transcriptional state and colony genetic variation. However, some colonies showed partial congruence between genotype and gene expression. Both PCA ordinations (Figure 1 A and 1 B) showed colonies 3 and 6 in close graphical proximity to each other. The lack of correspondence between genotype and gene expression profiles is not unexpected, as previous studies have shown that the correspondence is not always straightforward. Environmental factors may have effects on the patterns of gene expression (reviewed by [ 48 ]) and this is seen in corals due to their branching pattern and colonial organization [ 37 , 49 ]. General biological processes of differentially expressed genes Approximately 50% of the differentially expressed unigenes were successfully annotated in ~2,000 GO terms. The proportion of functional categories varied between colonies (Figure 2 ). The functions of transportation and translation have the highest annotation weight indicating that these GO terms had a large number of sequences and were closer to the term than other GO terms obtained. Previous studies on corals have shown various genes involved in transportation due to temperature effects (e.g. [ 25 , 26 , 32 ]), dark stress [ 50 ], the symbiotic relationship with Symbiodinium [ 51 - 53 ], metamorphosis and calcification [ 54 ], circadian clock regulation [ 55 ], and physiological plasticity [ 37 , 38 ]. Differential expression of translation has also been detected under different environmental stressors, including increase of temperature [ 26 , 30 , 32 ] and darkness [ 50 ], and associated to the life stage of corals [ 54 ] and the symbiosis with Symbiodinium [ 51 ]. We found that metabolic and cellular processes altered by the aforementioned factors are also naturally occurring in colonies of A. millepora . Figure 2 Cloud-term representation of the biological process GO terms with node score >10 in the combined graph of Blast2GO. The size of the font is proportional to the node score. GO terms with multiple colors indicated that more than one higher-level GO term was parent of the particular GO term. Oxidation-reduction processes Coral cells are subject to elevated levels of oxygen radicals during sunlight hours, which are by-products of the photosynthetic reactions carried out by Symbiodinium [ 56 ]. These reactions cause the host to activate protective mechanisms for detoxification. Genes that exhibited variation in this category included catalase , peroxiredoxin-mitochondrial- like, calcium/calmodulin-dependent kinase type II subunit delta , and ferritin , as well as the lipid metabolic gene sterol desaturase . Catalase is a common enzyme in aerobic organisms utilized for the detoxification of hydrogen peroxide [ 57 ]. The gene coding for this enzyme has been shown as differentially expressed in corals under stress [ 26 , 30 , 32 , 45 , 56 , 58 ], correlated with Symbiodinium genotype [ 51 ], diel cycle [ 55 ], and metamorphosis and calcification [ 54 ]. Peroxiredoxin also reduces hydrogen peroxide [ 57 ], but the differential expression of this gene has not been detected in previous studies on corals. Calcium/calmodulin-dependent protein kinase type II is involved in the regulation of calcium homeostasis [ 57 ]. Calmodulin genes have been detected in the response of corals to the symbiotic state, i.e. whether the cnidarian host already established a symbiosis or not [ 52 , 53 ] as well as heat-stressed corals [ 25 , 26 , 30 , 32 ], calicoblast differentiation [ 54 ], and metamorphosis [ 59 ]. Ferritin is important for iron homeostasis and also has an oxidoreduction activity [ 57 ]. This iron-storage protein-coding gene has been differentially expressed in corals [ 26 , 30 - 32 , 60 ] and sea anemones [ 61 ] under thermal stress. Interestingly, EST libraries constructed from different life stages of Acropora palmata have identified ferritin as highly expressed [ 53 ]. Examination of these EST libraries and comparison with an A. millepora EST library revealed the presence of two types of ferritins [ 53 ]. To test for positive selection, Schwarz et al. [ 53 ] compared these EST libraries with a Nematostella vectensis database and found that the dN/dS ratio of ferritin type I is particularly high, indicating potential adaptive evolution. Genes involved in response to stress Three GO terms have high node scores in the response-to-stimulus category: response to chemical stimulus, signal transduction, and response to stress. Some genes that were annotated with these GO terms are heat shock protein 70 ( hsp70 ), catalase (see above), UV excision repair protein rad 23 , ubiquitin (see below), ferritin (see above), peroxiredoxin (see above), and inhibitor nuclear factor kappa-beta ( IκΒ , see below). Hsp70 helps stabilize preexisting proteins from aggregation and acts as a molecular chaperone, mediating new protein folding under normal and high temperature conditions [ 62 ]. Hsp70 is shown to be up-regulated in heat-stressed corals [ 27 , 28 , 31 , 63 - 68 ] and is under a diel cycle [ 55 ]. However, some studies have not detected changes in gene expression of hsp70 during thermal stress [ 32 , 33 , 46 ]. Given its well-documented molecular function in heat shock, the absence of hsp70 amongst differentially expressed genes in some heat-stress studies could be due to the high level of variation that is naturally occurring as we demonstrated here, rather than due to a nonexistent response. Additionally, the timing at which the samples were collected or the use of the constitutive rather than the inducible gene could have potentially influenced this lack of detection in other studies. Other interesting genes involved in the response to stimulus category are the genes involved in immune response. Cnidarians are thought to possess only an innate immune system, lacking an adaptive immune system as the one described in jawed vertebrates (for a review of immune phylogeny see [ 69 ]). One gene found differentially expressed among the colonies is the inhibitor of the nuclear factor kappa-beta ( IκΒ ). The detection of IκΒ suggests the negative regulation of the nuclear factor kappa-beta ( NF-κΒ ) pathway, involved in different biological processes including inflammation, immunity, and apoptosis [ 70 ]. It has been shown that IκΒ is under a diel cycle [ 55 ], is a potential candidate gene for regulation of the symbiosis cnidarian- Symbiodinium [ 71 ], and may be involved in thermal tolerance [ 33 ]. Another key gene is ubiquitin , possessing a critical role in protein turnover by labeling proteins for destruction [ 57 ]. In fact, proteolysis was a GO term with strong node support (Figure 2 ). Differential expression of ubiquitin has been observed by corals under stress [ 25 - 27 , 30 , 32 , 50 , 62 , 65 ] as well as during the establishment of the symbiosis between host and Symbiodinium [ 71 ]. The myocyte-specific enhancer factor 2C ( Mef2C ) is another differentially expressed immune gene associated with apoptosis. However, Mef2C is involved components of the adaptive immune system [ 72 , 73 ], which is not found in corals. Therefore, these genes probably evolved a different, as yet unknown function and demand further exploration. Differential expression of apoptotic genes Apoptosis has received attention in coral physiology, given that it is one possible mechanism by which corals undergo bleaching (reviewed by [ 74 ]). Some genes annotated with this GO term include ras-like GTP-binding protein rho1 and CCAAT/Enhancer binding protein ( C/EBP ) gamma, which have been previously detected in stressed corals and anemones, and in response to symbiosis [ 26 , 30 , 50 , 53 , 54 , 61 ]. Interestingly, the C/ EBP was found to be stable in a study on coral thermo-tolerance [ 33 ] and expressed throughout different life stages [ 53 ]. We detected differential expression of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). However, GAPDH was not differentially expressed during natural bleaching [ 45 ] and is proposed as a potential housekeeping gene [ 75 ]. A B-cell lymphoma protein-2 like-2 ( Bcl -2) gene, which promotes cell survival by suppressing the activity of Bcl-2-associated X ( Bax ) [ 76 ], was differentially expressed. Additionally, Bax was also inhibited by another differentially expressed gene: a probable bax inhibitor 1 . This variation suggests that corals repress apoptosis under normal physiological conditions, in the absence of what is typically deemed heat stress. For example, colonies of A. millepora undergoing heat stress showed evidence of induction of apoptosis during thermal stress with a delayed up-regulation in Bcl-2 (anti-apoptosis) of surviving cells as a protective mechanism [ 77 ]. Stressed colonies of a congener species, A. palmata , showed up-regulation of an anti-apoptosis Bcl-2 family member [ 25 ], supporting the hypothesis that anti-apoptosis members protect surviving cells. This anti-apoptotic activity has also been detected during coral metamorphosis [ 59 ]. Finally, the MAPK MAK MRK overlapping kinase or MOK was another differentially expressed gene within the apoptosis GO term. MOK is a member of the mitogen-activated protein kinases (MAPKs) [ 57 ]. MAPKs are involved in signal transduction pathways integrating different biological processes, such as immune response to pathogen infection, exocytosis, and redox signaling (e.g. [ 78 , 79 ]). Genes of this class have important function in the symbiosis of reef corals and Symbiodinium . For example, it has been hypothesized that regulation of MAPK-pathway members sphingosine and sphingosine-1-phosphate ( S1P ) allows the host cell containing the algae to survive and proliferate [ 52 ]. In fact, EST libraries and microarray data for A. palmata and M. faveolata confirm the importance of MAPK signaling in host- Symbiodinium symbiosis [ 53 , 71 ]. MAPKs may also be involved during coral bleaching in the process of Symbiodinium exocytosis [ 80 ] and osmoregulation [ 81 ]. Interestingly, the expression of an MAPK member, tribble, also exhibited high inter-colony variation during a natural bleaching event [ 45 ]. Adaptive potential and phenotypic plasticity in corals Overall, there was no correspondence between transcriptional expression profiles with either intron or microsatellite genotypes in coral colonies grown under a common garden (Figure 1 ). However, the high level of gene expression variation revealed might be a natural-occurring phenomenon in wild populations of reef corals. In light of these results, two significant questions arise. Firstly, what are the sources/mechanisms driving differences in the gene expression detected in colonies of Acropora millepora acclimatized in a common garden? Secondly, what is the importance of this natural gene expression variation within an ecological and evolutionary context? Within the genome, polymorphic sites can alter transcriptional rates [ 82 - 85 ], contributing to additional variation at the mRNA level. Gene expression can also be altered through epigenetic modifications influenced by the environment (reviewed by [ 86 - 90 ]). In the case of corals, the role of epigenetics is currently unknown, but may explain some instances of acquired long-term stress tolerance (e.g. [ 91 , 92 ]). Moreover, it could provide a framework to explain the role of natural gene expression variation in corals within an ecological and evolutionary context. Most of the differentially expressed genes identified in this common garden experiment have been implicated in coral stress (e.g.[ 25 , 26 , 28 - 33 , 45 , 50 , 60 , 63 , 67 ]) and possible resilience response [ 33 ] to environmental factors, some of which are linked to global climate changes, including ocean warming. A number of these environmental drivers might be able to trigger epigenetic changes in corals generating a mosaic of transcriptional diversity within populations. This transcriptional diversity could be an important source for evolution, probably more than protein isoforms as it has been previously suggested [ 14 - 16 , 20 - 23 , 93 ]. This raises the urgent need to explore the existence of epigenetic changes in coral and its role in the physiological and adaptive response to environmental changes. It is well-known that coral reefs face a challenging future with conditions predicted to change, including an increase in temperature, a decrease in pH, and outbreaks of disease [ 5 , 7 , 9 , 94 , 95 ]. Although the response to some of these conditions might be similar across colonies (e.g.[ 36 ]), variation occurring at the transcriptomic level is vital for stress response (reviewed by [ 96 , 97 ]). Numerous studies have demonstrated that the so-called ‘core stress response’ could explain why cells can resist different stresses if they were previously treated with low levels of one stress factor [ 96 , 97 ]. Therefore, the differential gene expression generated during a specific stress is not directed towards that particular challenge, but rather form part of the generalized core stress response [ 96 ]. In fact, a recent study from our research group has showed that corals pre-exposed to sub-bleaching temperatures are able to resist bleaching by changing the magnitude of the expression levels of differentially expressed genes [ 33 ]. Fascinatingly, it has been determined that genes with a TATA box in their promoters have an increased number of binding sites for transcription factors, which increases their sensitivity when in need of being transcribed [ 85 ]. Here, we detected differentially expressed genes like hsp70 , catalase , ubiquitin , and ferritin , probably genes of the core stress response of corals. An interesting avenue of research is to determine if stress-related genes containing TATA boxes in corals also exhibit rapid regulatory evolution."
} | 6,358 |
37765664 | PMC10536798 | pmc | 2,504 | {
"abstract": "Superhydrophobic surfaces, i.e., surfaces with a water contact angle (WCA) ≥ 150°, have gained much attention as they are multifunctional surfaces with features such as self-cleaning, which can be useful in various applications such as those requiring waterproof and/or protective films. In this study, we prepared a solution from recycled polyethylene terephthalate (PET) and fabricated a superhydrophobic surface using electrospinning and electrospraying processes. We observed that the fabricated geometry varies depending on the solution conditions, and based on this, we fabricated a hierarchical structure. From the results, the optimized structure exhibited a very high WCA (>156.6°). Additionally, our investigation into the self-cleaning functionality and solar panel efficiency of the fabricated surface revealed promising prospects for the production of superhydrophobic surfaces utilizing recycled PET, with potential applications as protective films for solar panels. Consequently, this research contributes significantly to the advancement of environmentally friendly processes and the progress of recycling technology.",
"conclusion": "4. Conclusions In this study, we successfully developed an ecofriendly method to fabricate superhydrophobic surfaces using recycled PET through electrospinning and electrospraying processes without additional processing. Based on a fundamental understanding of wettability and superhydrophobicity, we aimed to produce superhydrophobic surfaces while recycling waste PET. The concentration of the solution was controlled by varying the amount of recycled PET flakes in the polymer solution. We observed and precisely analyzed how the structure produced varied depending on the solution concentration. By creating a hierarchical structure with these micro-/nanoscale structures, we minimized the contact area with water droplets and produced a surface with superhydrophobic properties. As a result, we achieved a very high WCA (>150°) in the optimized fabrication condition, which is significant in that we succeeded in producing a superhydrophobic surface using only nanostructures and without additional treatment processes such as chemical coatings. The performance of the fabricated film was verified through self-cleaning tests and solar panel application experiments, and excellent self-cleaning and efficiency protection of about 92% were confirmed, which can be improved through further research. This research enables the fabrication of environmentally friendly superhydrophobic surfaces, as an alternative to polymer waste, where the fabricated surface can be used to produce new films while recycling waste polymers such as PET.",
"introduction": "1. Introduction Wettability is an important physical property that describes the interaction between a surface and a liquid. The wettability of a solid surface refers to the degree to which a liquid flows or spreads over it, which is determined by the interaction between the liquid molecules and the solid surface molecules. A surface is said to be “hydrophilic” if it spreads and wets easily with liquids, and “hydrophobic” if it impedes the spreading of liquids. The wettability of a surface is represented by its contact angle with the water. The surface is hydrophilic if the water contact angle is <90° and hydrophobic if it is ≥90°. At a WCA > 150°, the surface is considered superhydrophobic. Manipulating the wettability of surfaces has been used to solve a variety of problems in domains such as healthcare, energy, and the environment [ 1 ]. Classic examples of superhydrophobic surfaces can be found in nature. For example, the surface of a lotus leaf or taro leaf has a very small contact area with water droplets and slides easily. The leaf surface can repel solid particles, organic liquids, and biological contaminants through the rolling action of water droplets [ 2 ]. This phenomenon is known as the “lotus effect” and is attributed to the structural and chemical properties of the lotus leaf surface [ 3 ]. Superhydrophobic surfaces have contributed to many research advances over the last 30 years [ 4 ], as they have a wide range of functions for use in a variety of fields. For example, superhydrophobic surfaces can be applied to the exterior surfaces of automobiles, aircraft, and ships to reduce fluid drag and improve energy efficiency [ 5 ]. In addition, superhydrophobic coatings can be applied to the exterior walls or glass windows of buildings to give them self-cleaning ability [ 6 ], which helps to reduce maintenance and management costs. Superhydrophobicity can also be utilized in biomedicine, where coatings can be applied to the surfaces of medical devices to prevent the adhesion of viruses or bacteria, thus contributing to the prevention of infection [ 7 ]. Other functions include anti-icing [ 8 ], anti-corrosion [ 9 ], and anti-fog [ 10 ]. Recently, various techniques and materials have reportedly achieved superhydrophobicity. Superhydrophobic surfaces can be fabricated through the processes of surface roughing, electrospinning, lithography, deposition, coating, plasma treatment, and electrochemical synthesis [ 11 ]. Studies have been reported on the fabrication and utilization of hydrophobic surfaces based on electrospinning. For example, Qi et al. fabricated superhydrophobic surfaces and oil–water separators by growing ZnO on waste PET electrospinning fiber [ 12 ]. Jun et al. made superhydrophobic filters by making waste PET electrospinning fibers porous using N-methyl-2-pyrrolidone and acetone [ 13 ]. Both studies used recycled PET as the electrospinning material, but additional processes were performed with additional materials. These additional processes are mainly chemical coating processes, which have relatively high costs, pose environmental problems, and can be difficult to apply as they use complex methods. This paper presents a simple method for fabricating superhydrophobic surfaces based on electrospinning and electrospraying, without the need for additional processes. No materials are required except for the PET and a solution to dissolve it to fabricate the surface. Electrospinning and electrospraying are electrohydrodynamic processes involving the spinning or spraying of micro-/nanoscale structures by an electric field [ 14 ]. In electrospinning, a strong electric field is applied to a polymer solution, and the resulting Taylor cone at the tip of the syringe needle forms fibers with a diameter of micro-/nanoscale order on a charged collector [ 15 ]. Electrospraying is similar in principle. The main difference between the two processes is the different physical properties, such as viscosity and concentration, and type of solution. High-viscosity polymer solutions are used for electrospinning, while low-viscosity polymer solutions are used for electrospraying [ 16 ]. Electrospinning can produce extremely thin microfibers and is characterized by a high surface-area-to-volume ratio [ 17 ]. Electrospraying allows for the fabrication of nanoscale beads. As such, electrospraying has recently been applied in various fields such as drug delivery [ 18 ] and fuel cell or electrode catalysis [ 19 , 20 ]. It is also one of the most promising superhydrophobic surface fabrication techniques as it can efficiently fabricate surfaces with a high specific surface area relative to the volume [ 21 ]. Additionally, the process is fast and simple, and a wide range of nanoscale structures can be fabricated with a small amount of polymer solution. Therefore, electrospraying and electrospinning offer new solutions for superhydrophobic surface fabrication, which has been limited by cost and complexity. Superhydrophobic surfaces can be fabricated by generating micro-/nanoscale fiber and bead structures through electrospinning and electrospraying, respectively. Various structures can be produced by controlling conditions such as the polymer solution concentration, viscosity, voltage, flow rate, tip-to-collector distance, temperature, and humidity. Depending on the concentration of the polymer solution, the thickness of the fibers and the diameter of the beads produced by electrospinning and electrospraying vary [ 22 ]. Hierarchical structures with a large surface-area-to-volume ratio can be fabricated by adjusting these parameters. Utilizing waste polyethylene terephthalate (PET) bottles to fabricate superhydrophobic surfaces is one of several promising approaches. PET, a lightweight low-cost polymer, has seen increasing use, leading to issues with its waste products [ 23 ]. Over the past 60 years, 12% of all plastic production has been incinerated, 60% has been discarded and landfilled, and only 9% has been recycled. It is estimated that 26,000 million metric tons (Mt) of PET waste will be generated in 2050, of which only 9000 Mt will be recycled [ 24 ]. PET is not biodegradable and releases toxic gases into the air during combustion [ 25 ]; thus, better management is needed to mitigate land and water pollution from PET. Our proposed approach offers a solution, as waste PET is used as the main material for our process. Recycling waste resources and transforming them into functional surfaces provides a sustainable solution while also responding to environmental issues. The fabrication of superhydrophobic surfaces by recycled PET electrospinning and electrospraying can find applications in various industrial fields, with the advantages of ecofriendliness, process simplicity, and economy. This paper presents a novel method for fabricating superhydrophobic surfaces from waste PET bottles by utilizing electrospinning and electrospray coating techniques. We present an economical and simple solution to realize superhydrophobic surfaces using only a single recycled material. The effects of process parameters such as solution concentration and jet time on the morphology and wettability of the surface are investigated. Based on the investigated parameters, hierarchical structures are formed to create a superhydrophobic surface. Furthermore, we explore the self-cleaning capability and recyclability of the fabricated surfaces and offer practical applications based on their superhydrophobic properties. Through this research, we aim to contribute to the development of environmentally friendly and functional surfaces while also responding to PET waste.",
"discussion": "3. Results and Discussion 3.1. Morphology of the Electrospun and Electrosprayed Structures Depending on the concentration of the polymer solution, different structures were observed as a result of electrospinning and electrospraying. Figure 2 shows SEM images of the electrospun and electrosprayed solutions. Figure 2 a shows a SEM image of the results from 7 wt.% solution electrospinning. Bead-on-a-string structures were observed along with nanoscale fibers. The bead-on-a-string structure showed a smaller contact area than the fiber surface, indicating a higher WCA [ 27 ]. These results were attributed to the Cassie–Baxter equation, which states that WCA increases as the fraction of contact area between liquid and solid decreases [ 28 ]. As measured by ImageJ software, fibers with a diameter of 0.229 ± 0.11 μm and beads with a diameter of 2.76 ± 0.7 μm were produced. Figure 2 b shows a SEM image of the bead morphology resulting from electrospraying with a 1 wt.% polymer solution, which formed beads with an average size of 1.46 ± 0.2 μm. Figure 2 c,d show the results obtained using 0.5 and 0.25 wt.% solution concentrations, respectively, which produced beads with average sizes of 1.04 ± 0.15 and 0.96 ± 0.27 μm, respectively. The average size of the beads produced changed with the polymer solution concentration. Fantini et al. confirmed that bead size decreases with decreasing solution concentration when electrospraying polystyrene, which is consistent with the results of this experiment with PET electrospraying [ 29 ]. Therefore, it is possible to fabricate a surface with a minimal contact area by mixing each of the fabricated structures. In the next section, a hierarchical superhydrophobic surface will be discussed. 3.2. Hydrophobicity Properties of the Hierarchical Surfaces Based on the structures described in the previous section, formation of a superhydrophobic hierarchical surface is described. The slide glass was sequentially electrospun and electrosprayed from a 7 wt.% polymer solution to a 0.25 wt.% solution. For each stage of the process, SEM images were taken and the WCA was measured. First, the 7 wt.% polymer solution was spun for 6 min ( Figure 3 a); fiber and bead-on-a-string structures were observed, and the WCA was measured to be 144.2°. This confirmed that the hydrophilic slide glass surface can be transformed into a hydrophobic surface by the electrospun fiber and bead-on-a-string structure. Figure 3 b shows the surface created by electrospraying a 1 wt.% polymer solution of waste PET onto the surface created by the 7 wt.% solution. The WCA was 150.8°, an increase of 6.6° compared to before. This is due to the reduced contact area with water due to the generation of very small beads. An even smaller contact area can be expected if smaller beads are generated on this surface. Figure 3 c shows the surface created by electrospraying a solution with a concentration of 0.5 wt.% waste PET on top of the surface in Figure 3 b. The WCA increased by 2.4°–153.2° as a result of spraying smaller-diameter beads. Figure 3 d shows the surface generated by electrospraying a 0.25 wt.% solution of waste PET over the surface shown in Figure 3 c. By spraying the smallest-diameter beads, the contact area with water was minimized and a WCA of 156.6° was achieved. Figure 3 e shows the WCA graphs of the PET surface and the fabricated surfaces in Figure 3 a–d. The values of five repeated measurements of each surface were plotted as a scatter plot, and the mean value and standard deviation for each surface are shown. As a result, conversion of a hydrophobic surface with a WCA of 92.1° to a superhydrophobic surface with a mean of 156.6° is achieved. Also, reproducibility of the fabricated surface is confirmed, as the WCA shows a close difference in five repeated measurements. Figure 3 f shows the solid surface energy of the smooth PET film and the fabricated surfaces in Figure 3 a–d. The surface energy was measured using Smartdrop Plus (Femtobiomed, Inc., Republic of Korea). This device uses Young’s equation and the equation of state to derive the surface energy value, which is shown in the equation below.\n (1) cos θ = − 1 + 2 γ s v γ l v e − β γ l v − γ s v 2 In the above equation, θ is water contact angle. β was empirically calculated with a constant value of 0.0001247 m 2 / mJ 2 , and the device measured the value of the liquid surface energy γ l v and calculated the solid surface energy γ s v [ 30 ]. DI water was used as the fluid for the measurement. In order to measure the surface energy of the same material as its morphology changed, both the surface of the PET film and the fabricated surfaces in Figure 3 a–d were measured. The surface energy on the surface of the PET film decreased sharply after the generation of 7 wt.% fibers and gradually decreased in the subsequent process, finally changing from the initial 24.48 mN/m to 0.06 mN/m. This confirmed that the same material was processed to change the surface and create a surface structure with smaller units, resulting in a shape with lower surface energy. Additionally, the roughness coefficient of the fabricated surface was calculated based on the experimental results. Since the fabricated surface was very thin, it was calculated with Wenzel’s theory, which assumes no air entrapment by water droplets on the surface. The equations used are as follows [ 31 ].\n (2) cos θ W = r f cos θ Y \nwhere θ w is the Wenzel contact angle, r f is the roughness factor, which is always greater than 1, and θ Y is the Young contact angle, which is the contact angle on a smooth surface made of the same material [ 32 ]. In the Wenzel state, for values of r f > 1, a hydrophobic surface has a larger contact angle, and a hydrophilic surface has a smaller contact angle. In other words, surface roughness always amplifies the inherent wettability of a surface [ 33 , 34 ]. The Young contact angle was characterized through thin films made by spin coating a solution of the same material as the fabricated surface. The fabricated film had a WCA of 92.1° and a cos θ Y value of −0.037, and the calculated values are shown in Table 1 . Figure 3 SEM images of the fabricated hierarchical surfaces and the water contact angle (WCA) for ( a ) 7, ( b ) 1, ( c ) 0.5, and ( d ) 0.25 wt.% waste PET on the ‘c’ surface. ( e ) Graph of the WCA (°) for each surface. ( f ) Graph of the solid surface energy (mN/m) for each surface. Because the wetting state of the surfaces in each fabrication step is the Wenzel state as mentioned above, the roughness of the surface could be derived based on Equation (2). To accomplish the superhydrophobic surface with PET, the roughness has to be over 23.4. Even though electrospun PET fiber shows good modification, it cannot reach the superhydrophobic state, but the other hierarchical surfaces with the beads achieve that state. Moreover, the more complex the structure, the greater the contact angle. It was shown that piling beads with diminishing size on the electrospun fiber is effective. 3.3. Self-Cleaning Test In this chapter, the self-cleaning ability of the fabricated superhydrophobic surfaces was tested. Self-cleaning is one of the most important properties of superhydrophobic surfaces. The fabricated surface was placed on an inclined surface and water droplets were placed on it to test its cleaning ability. The impurity was graphite powder. A large amount of graphite was placed on the fabricated surface ( Figure 4 a) and we rolled a few water droplets to check the self-cleaning ability ( Figure 4 b). After a large amount of water droplets were dropped onto the surface, the surface was completely cleaned. The experiment successfully confirmed the self-cleaning function of water droplets, which can easily roll on the surface and remove dirt. The ability of water droplets to easily remove impurities from a surface can be useful for self-cleaning coatings, waterproofing, etc. The self-cleaning capability confirmed in this experiment shows the potential for practical applications of the developed superhydrophobic surface. For example, in outdoor settings, such a surface could be utilized to prevent the accumulation of dirt, dust, and pollutants on various structures, ranging from solar panels to architectural facades [ 35 , 36 ]. It can also be used in transportation applications such as aerospace for reliability and efficiency [ 37 , 38 ]. 3.4. Surface Recyclability Test In this chapter, the recyclability of the fabricated surfaces was tested. Superhydrophobic surfaces acting as solutes were fabricated over a longer period of time to ensure the electrospinning solution volume. The fabricated superhydrophobic surface is first cleaned. After cleaning, the surface is redissolved in TFA and DCM ( Figure 5 b). The resulting polymer solution is then electrospun to produce fibers ( Figure 5 c). The recycled solution produced straight, smooth fibers ( Figure 5 d). It was also compared to a surface created with a 7 wt.% solution under the same condition ( Figure 5 d,e). Both surfaces produced ideal hydrophobic surfaces with a WCA ≥ 140°. Therefore, this reusable approach allows us to melt the fabricated superhydrophobic surface again and electrospun it, thus providing a sustainable solution for recycling plastic waste. However, for the self-cleaning test, it does not reach superhydrophobicity, showing a weak self-cleaning ability. This can be improved by the additional electrospraying process presented in this experiment. In conclusion, this method helps to reduce the consumption of additional materials for the formation of superhydrophobic surfaces, while also contributing to plastic waste management, thus expanding the application possibilities of superhydrophobic surfaces. There is also the potential to improve surface properties and impart various functionalities through the repeated electrospinning process. 3.5. Application: Solar Panel Efficiency Test In this study, experiments were conducted to explore the potential for fabricated superhydrophobic surfaces to improve the performance of solar panels. Solar panel usage continues to increase worldwide [ 39 ]. However, the efficiency and lifespan of solar panels are greatly affected by surface contaminants, with efficiency losses as high as 15% [ 40 ]. Contamination on solar panels should be cleaned immediately, as it can block the sunlight they receive or corrode the surface. The fabricated superhydrophobic surface has self-cleaning ability, which means that it can remove impurities from its surface. To verify its applicability, an Arduino-controlled solar panel voltage test device was created, and an efficiency measurement experiment was conducted ( Figure 6 a). In the experiment, the voltage generated by light in the same circumstance was measured by comparing a pure panel with nothing on top of the panel with a panel covered with a superhydrophobic surface. To measure the voltage in different situations, experiments were conducted in bright natural light (i.e., in broad daylight), dim natural light in the morning, and artificial light ( Figure 6 a,b). The surface was fabricated on the thin glass to match the size of the solar panel and the voltage was measured with all parts covered. All conditions were kept constant except for illumination and fabricated surface placement. The results showed that in broad daylight (110,000 lux), the voltage of the pure solar panel was 15.21 V, the voltage of the panel covered by the fabricated surface was 14.06 V, and the efficiency was 92%. In natural light in the morning (40,000 lux), the pure solar panel had a voltage of 14.01 V, and the panel covered by the fabricated surface has a voltage of 12.81 V and an efficiency of 91%. Under artificial light (11,000 lux), the voltage produced by the pure solar panel was 12.23 V and that of the panel covered by the fabricated surface was 11.35 V, with an efficiency of 93%. Under different illumination conditions, the efficiency remained at 91%. Plus, to test the stability of the efficiency protection of surfaces built over a period of time, the efficiency was measured after 14 days under the same conditions. For illumination control, the experiments were conducted under artificial light at 11,000 lux. From before to after 14 days, the voltage decreased from 11.35% to 11.3%, with a change rate of 0.44%. This is within the margin of error, so it is concluded that the manufactured panel shows a consistent performance for long-term use. These experimental results suggest the possibility of utilizing the fabricated superhydrophobic surfaces as solar-panel-protective films. Fabricated superhydrophobic surfaces can help maintain and protect solar panels by preventing the ingress of contaminants, dust, and other impurities from the outside through their self-cleaning capabilities. Superhydrophobic surfaces can also improve the productivity of solar panels by maintaining voltage. Furthermore, the current efficiency is expected to be further improved through optimal condition studies or surface modification studies."
} | 5,874 |
23900554 | PMC3728589 | pmc | 2,506 | {
"abstract": "The fascination for hierarchically structured hard tissues such as enamel or nacre arises from their unique structure-properties-relationship. During the last decades this numerously motivated the synthesis of composites, mimicking the brick-and-mortar structure of nacre. However, there is still a lack in synthetic engineering materials displaying a true hierarchical structure. Here, we present a novel multi-step processing route for anisotropic 2-level hierarchical composites by combining different coating techniques on different length scales. It comprises polymer-encapsulated ceramic particles as building blocks for the first level, followed by spouted bed spray granulation for a second level, and finally directional hot pressing to anisotropically consolidate the composite. The microstructure achieved reveals a brick-and-mortar hierarchical structure with distinct, however not yet optimized mechanical properties on each level. It opens up a completely new processing route for the synthesis of multi-level hierarchically structured composites, giving prospects to multi-functional structure-properties relationships.",
"discussion": "Discussion The two described processes for polymer-coating of hard building blocks at different length scales are simple, straightforward in application, and versatile in controlling the overall polymer content by adjusting the amounts introduced. The subsequent synthesis of hierarchical levels allows for individual investigation of the different levels in terms of polymer content and mechanical properties. The emulsion polymerization is a versatile process for the synthesis of a first hierarchical level, since especially at the lowest hierarchical level amounts of polymers should be kept to a minimum to get a high stiffness and resembling natural hierarchical materials 34 . Using the encapsulation approach, the polymer content can be varied through the thickness of the polymer shell 32 . Furthermore, polymer encapsulation in an emulsion polymerization can be applied to particles with sizes ranging from nano- to micrometer. However, a limitation regarding the minimum polymer content possible arises from geometrical considerations: for a given particles size distribution, there exists a maximum packing density. This can be slightly above 64%, which is the maximum packing density for random dense packing of monomodal spheres. To avoid porosity, the remaining volume has to be completely filled with polymer. As a result, the amount of polymer on the first level of hierarchy cannot be less than approximately 30 vol.-% 32 . To polymer-coat the first-level-of-hierarchy-agglomerates, the spouted bed spray granulation technique was used. This technique and the fluidization bed spray granulation in general were developed particularly to coat particles with a high homogeneity of the coating layer, which follows from the good mixing of the particles during the process and which it is widely used for in pharmaceutical industries 33 . The polymer to be applied can be solved, which requires good wetting of the polymer solution to allow spreading of the applied polymer on the agglomerate surface. Therefore, PVB was chosen as model material for the second hierarchical level, since PVB with low OH-group content adheres to hydrophobic surfaces such as PMMA. A homogeneous coating of the first-level-of-hierarchy agglomerates with PVB is envisioned in Figure 3 b . Furthermore, it is important to mention that for applying the spouted bed process, the bed particles need to have a certain minimum size (several μm) and size to density ratio, to enable fluidization and to avoid strong agglomeration during the process. For the presented studies of highly porous first-hierarchical-level-agglomerates this required particle sizes larger than 64 μm. However, by triggering the density of agglomerates, e.g. by initial pre-densification, the particles size used can be reduced. When creating another level of hierarchy, the content of polymer will increase as a matter of fact 34 . However, with the spouted bed technology even small amounts of polymer in solution can be applied to adjust the resulting polymer content. To densify the porous hierarchical agglomerates and induce an anisotropic microstructure, unidirectional pressing is applied, which requires deformable polymers (thermoplastics) or e.g. non-cured epoxy resins 23 on each hierarchical level, to allow for plastic deformation of the structural elements. Both, PMMA and PVB are thermoplastics and similar processing temperatures allow for simultaneous densification. Yet, as there is only a low miscibility for PVB and PMMA 35 blending of polymers is not supposed to occur. At temperatures applied PMMA only gets slightly viscous but does not deliquesce, allowing only just visco-plastic deformation under pressure. On the contrary, PVB is highly flowable at the processing temperature. Hence, agglomerates of the first hierarchical level are densified and also deformed anisotropically to a planar shape by movements of PMMA-coated ceramic primary units in and perpendicular to the pressure direction, with the PVB interlayer creating a barrier between the individual first-hierarchical-level particles to prevent total merging and blending. If no complete PVB-coating of first hierarchical-level agglomerates resulted from the spouted bed encapsulation, either complete merging of particles would be anticipated to occur during compression moulding, or a similar feature as observed in nacre would be created, namely solid bridges between particles 36 . The first should be visible as particles with larger dimensions in the etched microstructure. The effect of solid bridges in analogy to nacre could be of interest for further investigations. To visualize the hierarchical microstructure, the PVB-rich layers were removed by selective etching of PVB with ethanol, as PVB is soluble in ethanol, whereas PMMA only exhibits minor solubility, and hence emphasizing contours of first-level-of-hierarchy-particles ( Figure 4 a, c to g ). As reasoned from circular cross sections in parallel and elongated ones in perpendicular view ( Figure 4 a, c to e ), the first-level-particles are mostly planar and platelet-like in shape. The resulting average approximate aspect ratio (length to thickness) is 3.5, whereas maximum ratios up to 12 could be observed. Despite a high aspect ratio for the first-hierarchical-level-particles at a length scale of 100 μm, primary ceramic building units at a length scale of 200 nm still being spherical in nature. This seems to contradict Gao′s principle of a high aspect ratio being necessary for maximum load transfer 4 12 . Nevertheless, from nature there exist several examples of hard tissues which, on its smallest length scale, are comprised of spherical mineral particles, such as nacre′s platelets consisting of spherical nanograins 37 or sponge spicules 38 and hence showing the validity of our approach. As another consequence from load being transferred from one particle to another during consolidation, particles exhibit a wavy shape in cross section as obvious from Figure 4 c to f . Thus, particles of the first hierarchical level create a dense packing by shape-adjustment. This dense packing is advantageous, since the amount of polymer of the second level of hierarchy can be kept to a minimum without introducing porosity. Waviness of platelets is a feature also observed in nacre and which was found to cause interlocking effects when loaded in tension 39 . Despite the hierarchical structure obvious from etched surfaces, some encapsulated primary units must still penetrate into the PVB-layer of the 2 nd hierarchical level. This is assumed because from unetched surfaces it was hardly possible to distinguish between particles of the first hierarchical level and the separating PVB-layer, meaning that visually similar amounts of primary building units are present throughout the surface structure. In Figure 4 g this is also apparent inspecting etched regions with SEM. However, by using nanoindentation ( Figure 6 ) it is obvious, that two regions of different elastic moduli can be identified. If the two distributions are cut at 15 GPa, the one with higher elastic moduli has a mean value of 29 GPa and a standard deviation of 8 GPa, attributed to the “composite particles” of the first hierarchical level. The “soft matrix phase” results in (8 ± 3) GPa, supposedly the PVB-rich region. Yet, a modulus as low as that of pure PVB of 3.0 GPa has not been measured. There are two major reasons able to explain this discrepancy. On the one hand, with a depth of 400 nm and an area of ~4 μm 2 an influence on the measured modulus by the surrounding high-filled and stiffer material is anticipated, similar to observations for thin films 40 . On the other hand, some PMMA-TiO 2 -core shell particles are present within the PVB interlayer, as implied from Figure 4 g . These stiffen the PVB matrix and result in higher moduli. It is assumed, that an interplay of both mechanism act to yield the measured moduli. The occurrence of titania core-shell-particles within the PVB layer is supposed to be a result of processing by two means: the highly porous agglomerates exhibit only weak strength, causing abrasion of single or splits of core-shell particles upon collisions during spouted bed granulation. These will then deposit within and on the coating layer as observed from Figure 3 b . The second reason anticipated might be due to partial infiltration of PVB into the outer regions of porous first level agglomerates during hot pressing, before composite densification, and hence leading to a gradient within the boundary regions. It would be expected that by the use of dense agglomerates for the first hierarchical level both mechanisms described can be reduced. Values from mechanical bulk measurements show that elastic moduli are strongly dependent on the overall polymer content, which increased by about 65% for two levels of hierarchy. This is in accordance with predictions from hierarchical models, as organic content must increase and hence modulus inevitably decreases with increasing number of hierarchical levels 34 . As the material presented here exhibits two hierarchical levels, the analytical model of Gao and co-workers 4 12 for elastic moduli of anisotropic hierarchical materials ( Equation 1 ) can be used to compare our mechanical results with the theory for hierarchical materials. With E Particle 1 the elastic modulus ( E Particle 1 = 29 GPa from nanoindentation, see Figure 6 ) and ϕ 1 the volume fraction of hard particles of the 1 st hierarchical level ( ϕ 1 = 1 − ϕ PVB ; ϕ PVB = 0.28), G Polymer 1 the shear modulus of the polymer interlayer of the 1 st level ( G Polymer 1 ~ 3.0 GPa, calculated using elastic modulus of 8 GPa from nanoindentation, see Figure 6 , and an estimated poisson ratio of 0.35) and E 2 the measured flexural elastic modulus of the hierarchical composite with 2 levels ( E 2 = 12.5 GPa), the effective aspect ratio ρ 1 of the particle of 1 st hierarchical level yields ρ 1 = 4.7. This compares well with the optically determined value of 3.5. The bending strength does not change considerably for one or two hierarchical levels. According to the model of Gao and co-workers 4 12 , the strength of an anisotropic hierarchical material S 2 with two levels is determined either by the strength of the hard particles of the 1 st level, S Particle 1 , or the polymer strength, S Polymer 1 , on the 1 st level, magnified by the 1 st level particles' aspect ratio ρ 1 , whichever fails first ( Equation 2 ). To estimate the strength limiting constituent on the first hierarchical level, the aspect ratio of 3.5, an estimated PVB strength of S Polymer 1 = 50 MPa and a volume fraction of hard particles on the level 1 of ϕ 1 = 0.72 yield a strength of S 2 = 63 MPa for polymer failure, which corresponds well with the measured strength S 2 = 56 MPa (see Figure 5 ). However, if the strength of the hard particles on level 1 was taken to be S Particle 1 = 51 MPa, which was measured from the bulk first-hierarchical-level-composite (see Figure 5 ), the hierarchical composite with two levels would fail at only S 2 = 18 MPa. This is inconsistent with the measured strength S 2 = 56 MPa. Therefore, it can be concluded that the strength of hard particles on the 1 st level must be at least S Particle 1 ≈ 150 MPa in order to get an overall strength of 56 MPa for the two level hierarchical composite. Since this value is much higher than the measured bulk strength of the plain TiO 2 -PMMA-composite of level 1, it is anticipated that the bulk strength of non-hierarchical materials does not coincide with the strength measured on a lower length scale. As the TiO 2 -PMMA-composite fails by brittle fracture, it is assumed that this strength increase of the first hierarchical level is due to smaller defects. The woven plate-like structures of the first hierarchical level are typically 100 μm in lateral dimension and 20–30 μm thick. Therefore, cracks are restricted to dimensions smaller than that. Additionally, cracks within the first hierarchical level could be even healed to some extend by the penetrating PVB during processing. The proposed discrepancy between micro-scale and macro-scale strength behaviour could be investigated by conducting micro-beam experiments on single platelets by using focused ion beam milling. Also in situ atomic force microscopy or SEM investigations of deformation mechanisms or crack propagation could yield further insight into structure-properties relationships and are planned for future work. To increase the strength of the hierarchical composite, both particle and matrix failure have to be optimized. Matrix strength can be increased by enhancing the aspect ratio of the particles of 1 st hierarchical level by manipulating processing conditions and reducing the polymer content during the spouted bed process. Also incorporation of primary particles into the polymer layer should be prevented, as to increase the stiffness ratio between first-hierarchical-level-particles and the enchasing polymer to better activate load transfer mechanisms via shearing of the polymer matrix. The strength of the first hierarchical level particles on the other hand depends largely on the interplay between interfacial bonding between ceramic and polymer, polymer characteristics and porosity. Optimizing these is a key feature and should lead to stronger particles on a micro scale and hence can lead to improved hierarchical materials with the processing route proposed. When comparing absolute values for inorganic particle volume contents as well as elastic modulus of the two-hierarchical-level-composite with natural hard tissues it matches about the properties of dentin 34 41 . To conclude, it was shown that with this successive two-step polymer-coating process followed by directional consolidation it is possible to achieve anisotropic brick-and-mortar two-level-hierarchical-composites from two different polymers, with a method suitable for large-scale production. Polymers with appropriate glass transition temperatures and melt viscosities have been chosen to achieve good processability, and can be further tailored and enhanced with respect to mechanical properties by using other (high-performance) polymers. This work demonstrates that this processing method has the potential to synthesize multi-level hierarchical composite materials with in principle arbitrarily chosen ceramic and polymer constituents of adjustable volume contents."
} | 3,919 |
40249799 | PMC12007567 | pmc | 2,508 | {
"abstract": "Anoxygenic phototrophic sulfur bacteria flourish in contemporary and ancient euxinic environments, driving the biogeochemical cycles of carbon and sulfur. However, it is unclear how these strict anaerobes meet their high demand for iron in iron-depleted environments. Here, we report that pyrite, a widespread and highly stable iron sulfide mineral in anoxic, low-temperature environments, can support the growth and metabolic activity of anoxygenic phototrophic sulfur bacteria by serving as the sole iron source under iron-depleted conditions. Transcriptomic and proteomic analyses revealed that pyrite addition substantially up-regulated genes and protein expression involved in photosynthesis, sulfur metabolism, and biosynthesis of organics. Anoxic microbial oxidation of pyritic sulfur and consequent destabilization of the pyrite structure were postulated to facilitate microbial iron acquisition. These findings advance our understanding of the survival strategies of anaerobes in iron-depleted environments and are important for revealing the previously underappreciated bioavailability of pyritic iron in anoxic environments and anoxic weathering of pyrite.",
"introduction": "INTRODUCTION Oceanic euxinia (anoxic and sulfidic conditions) ubiquitously occurred in paleo-ocean, particularly during the Proterozoic ( 1 – 3 ). Because of the high toxicity of hydrogen sulfide (H 2 S), euxinia are believed to be an important reason for biotic crises such as the largest known Phanerozoic extinction—the end-Permian mass extinction (~252 million years ago) ( 4 – 6 ). Seasonal euxinia also frequently develop in modern environments such as the Black Sea, fjords, meromictic lakes, and the Benguela Upwelling System ( 5 , 7 – 9 ). Although euxinia are well known for containing highly toxic H 2 S, with a concentration varying from several hundreds to thousands of micromolar, such hostile environments generally accommodate unique microbial communities such as anoxygenic phototrophic purple or green sulfur bacteria (PSB or GSB) ( 5 , 6 ). These bacteria can produce long-lasting organic biomarkers, such as carotenoid isorenieratene, which can be used for robust identification of photic zone euxinia in ancient environments ( 4 , 10 , 11 ). Prosperous growth of anoxygenic phototrophic sulfur bacteria requires illumination, carbon source, and reduced sulfur compounds ( 12 ). As research progresses regarding the reaction center of anoxygenic photosynthesis, the importance of trace metals, particularly iron, in the growth of anoxygenic phototrophic sulfur bacteria has been revealed recently ( 12 , 13 ). For instance, three key Fe 4 S 4 clusters were identified within the electron transfer chain of the photosynthetic reaction center in the representative strain of GSB Chlorobaculum tepidum ( 12 ). During anoxygenic photosynthesis, light energy captured by chlorosome (i.e., the light-harvesting structure of C. tepidum ) is transferred by Fenna-Matthews-Olson proteins to its membrane-embedded photosynthetic reaction center to initiate charge separation and electron transfer reactions ( 12 , 13 ). A similarly pivotal role of iron in the growth and carbon fixation of phytoplankton has also been reported ( 14 , 15 ). In addition, iron acts as a crucial cofactor for key enzymes involved in the sulfur oxidation processes of C. tepidum ( 16 ). Therefore, fluctuations in iron concentrations over geological timescale are often closely linked to changes in ocean primary productivity and biogeochemical cycles of many elements such as carbon and sulfur ( 15 , 17 ). However, euxinic environments are iron depleted and usually characterized by a high degree of pyritization [e.g., reactive aqueous iron or iron (hydro)oxides react with free H 2 S to form fine-grained pyrite in the water column] ( 18 – 20 ). This creates an apparent paradox between iron scarcity and widespread distribution of GSB-derived biomarkers (i.e., requiring iron for GSB growth) in both the geological past and modern euxinic environments ( 8 , 10 ), prompting inquiries about the iron sources and the survival strategies of GSB in sulfidic settings. Given that pyrite is considered as the most abundant iron form in euxinic environments ( 2 , 20 ), it may be possible that pyrite-associated iron supports the growth of anoxygenic phototrophic sulfur bacteria, despite the usual belief that pyrite is stable under anoxic, low-temperature conditions ( 21 ). It is long believed that pyrite is not bioavailable in the absence of oxygen or alternative electron acceptors such as nitrate and ferric iron ( 22 – 24 ). Nevertheless, such belief is challenged by recent lines of evidence of the utilization of sulfur within pyrite by anaerobic PSB and the utilization of both iron and sulfur within pyrite by anaerobic methanogenic archaea ( 21 , 25 , 26 ). Although the underlying mechanism remains unclear, these studies suggest that anaerobic weathering of pyrite might be possible in the environment, especially under iron- or sulfur-limited conditions. Thus, we speculate that pyrite may serve as an efficient iron source to promote the growth of anoxygenic phototrophic sulfur bacteria. The primary objectives of this study, therefore, were to investigate whether anoxygenic phototrophic sulfur bacteria, using GSB C. tepidum as a representative bacterium, can use pyrite as the sole iron source under iron-depleted conditions, and if so, what the potential underlying mechanisms are. Understanding the metabolic flexibility and adaptation strategies of anoxygenic phototrophic sulfur bacteria in response to iron deficiency will shed light on microbial weathering of pyrite under anoxic condition, which is of great significance to the biogeochemical cycles of sulfur, carbon, and iron in euxinic environments ( 27 ).",
"discussion": "DISCUSSION Importance of iron in the growth of anoxygenic phototrophs Given the pivotal roles of iron in photosynthetic reaction centers of oxygenic phototrophs, it has been well recognized that iron is one of the commonly limited nutrients for the growth of marine phytoplankton because aqueous iron tends to precipitate in oxic environments. Fluctuations of iron concentration largely control the magnitude and dynamics of oceanic primary production ( 14 , 15 ) and thus are of significance to both modern and paleoclimate changes because the metabolic activities of phytoplankton regulate the carbon exchange between atmosphere and ocean ( 50 , 51 ). However, before the advent of oxygenic photosynthesis, anoxygenic phototrophs such as GSB dominantly contribute to the primary production in oceans, especially during the Proterozoic ( 17 , 52 ). In general, anaerobes, especially these early-originated species, are not believed to suffer from iron deficiency because of the presumably high solubility and concentration of reduced iron in paleo-oceans. However, widespread and recurrent euxinia in Proterozoic oceans, where aqueous iron tends to precipitate with H 2 S as solid iron sulfide minerals ( 2 , 20 ), poses a great challenge of iron deficiency to these anoxygenic phototrophs thriving in sulfidic environments ( 18 , 20 ). Because of the importance of oxygenic phototrophs in oxygenating the early Earth and in controlling carbon sequestration in the modern ocean, extensive investigations about how oxygenic phototrophs cope with iron deficiency have been conducted. Nevertheless, little is known about how iron deficiency affects the growth of anoxygenic phototrophs. Such knowledge gap largely hinders our understanding of the biogeochemical cycles of carbon, sulfur, and nitrogen in sulfidic environments. Our results here demonstrate that iron deficiency suppressed the growth of C. tepidum , as evidenced by a reduction in biomass ( Fig. 1A and figs. S1 and S2) and impairment of sulfur oxidation ( Fig. 1B and fig. S3). Furthermore, cultivation experiments also suggest that iron concentrations above 0.2 μM are required to sustain active growth and sulfur oxidation of C. tepidum ( Fig. 1 ). However, such iron concentrations are higher than those typically found in natural euxinic environments where aqueous ferrous iron is scarce, yet GSB often thrive ( 18 , 20 ). This apparent paradox suggests that alternative iron sources such as mineral-associated iron may support the growth of anoxygenic phototrophs. In euxinic environments, reactive iron may be incorporated into fine-grained pyrite, creating a spatial copresence between the suspended pyrite and the niches of anoxygenic phototrophs ( 1 ). Therefore, one possible explanation is that pyritic iron supports the growth and metabolism of GSB, considering the dominance of pyrite in the iron pool of euxinic settings. Our results demonstrated that C. tepidum can directly use pyritic iron to meet its cellular needs ( Fig. 2 ), enabling active growth and sulfur oxidation even when aqueous iron is scarce ( Fig. 1 ). However, note that a highly dynamic iron-sulfur cycle occurs in euxinic environments, particularly near or below the chemocline where other transient iron reservoirs (e.g., mackinawite, greigite, and FeS generated from the progressive sulfidation of iron oxides) may be present before complete pyritization ( 20 , 25 ). Given that GSB C. tepidum is capable of using iron in the pyrite (i.e., the thermodynamically stable phase of iron sulfide minerals), these metastable iron sulfide minerals possibly also serve as the key iron sources for the growth of anoxygenic phototrophs. Overall, our findings suggest that mineral-bound iron plays a previously underappreciated role in sustaining anoxygenic phototrophs in euxinic environments. Possible mechanisms of iron acquisition by GSB While iron is an irreplaceable element of many proteins and enzymes, previous studies have primarily focused on the iron acquisition strategies of aerobic microorganisms. In response to low iron availability, aerobic bacteria can secrete high-affinity extracellular ligands such as siderophores to bind and solubilize iron to meet their cellular iron demands ( 49 ). In addition, aerobes can also uptake more iron than immediately needed, storing the excess for later use depending on the iron concentration in environments ( 49 ). However, siderophores are generally not produced by anaerobes with a few exceptions ( 53 , 54 ). The genome of C. tepidum (i.e., the strictly anaerobic phototrophs applied in this study) does not contain any genes related to siderophore biosynthesis ( 37 ). Consistently, no siderophores or siderophore-complexed iron was detected in any treatments, implying that C. tepidum adopts different strategies to meet its cellular iron requirements under aqueous iron-depleted conditions. Our findings suggest that C. tepidum acquired iron via oxidation of sulfur on pyrite surface. Specifically, C. tepidum modified the surface of pyrite with organic compounds such as lipopolysaccharides and amino acids, as supported by the presence of organic coatings on pyrite surface ( Fig. 4 ) and up-regulation of relevant pathways, genes, and proteins ( Figs. 6 and 7 and fig. S14). Such surface modifications may facilitate oxidation of pyritic sulfur through EET mediated by cytochromes ( Fig. 4 and fig. S14). It is known that C. tepidum can oxidize sulfide to S 0 globules and sulfate ( 55 , 56 ), consistent with our results (fig. S3). However, when solid-phase S 0 purchased from reagent companies was directly added into the medium, C. tepidum could not use these exogenous S 0 globules, despite their similar composition and structure to biogenic S 0 globules ( 56 ). This biooxidizable property of biogenic S 0 globules has been attributed to their surface modifications by microbial metabolites ( 35 , 56 ) [e.g., molecular fragment with a m/z 72 (C 2 H 2 NO 2 + )]. A similar organic coating was also observed on the pyrite surface as shown by the TOF-SIMS results in this study ( Fig. 4 ), suggesting that C. tepidum may modify the pyrite surface with comparable organics. This modification could facilitate the oxidation of sulfur within pyrite ( Fig. 5 ), thereby destabilizing the pyrite structure and promoting the release of ferrous iron into the surrounding environment. Furthermore, the requirement for direct contact between C. tepidum and pyrite surface ( Fig. 3 and fig. S8) aligns with the broader understanding that microorganisms often need to establish a physical connection with a mineral surface to exchange matter and energy ( 21 , 25 , 47 ). Such mineral-microbe interaction may involve EET, where c -type cytochromes or other redox-active proteins mediate electron flow between the mineral and the microbial cell ( 47 ). A recent study demonstrated that anoxygenic phototrophic PSB can oxidize sulfur within pyrite using certain monoheme c -type cytochromes as electron carriers under sulfur-limited conditions ( 26 ). In addition, membrane-associated cytochromes play critical roles in the oxidization of extracellular solid-phase S 0 ( 30 ). Therefore, together with the results that cytochrome-related genes were among the most up-regulated genes in the presence of pyrite (fig. S14), we speculate that C. tepidum can oxidize pyritic sulfur via EET through cytochromes, as indicated by the early and direct formation of sulfate on pyrite surface (i.e., not from oxidation of sulfide and thiosulfate; Fig. 5 ), thereby destabilizing the pyrite structure and potentially facilitating its iron utilization. Although the exact mechanisms of how surface modification and EET facilitate microbial iron acquisition remain unclear, our findings offer insights into survival strategies of anaerobic microbes in iron-depleted environments. Implications for bioavailability of pyritic iron in anoxic environments Minerals and microbes have coevolved throughout much of Earth’s history ( 32 , 57 ). Various metabolic processes, including anoxygenic photosynthesis and sulfur oxidation, require enzymes that use specific transition metals such as iron for catalytic functions ( 57 , 58 ). Consequently, the origin and evolution of microbial metabolisms are tightly regulated by metal availability in the environment ( 32 , 57 ). The capability of microorganisms to use mineral-associated metals is a distinct ecological advantage that enables them to survive and function in diverse environments ( 53 , 59 , 60 ). Our study emphasizes that pyrite can potentially alleviate the iron limitation faced by anaerobic microbial communities during oceanic euxinia in paleo-ocean. It has traditionally been believed that pyrite is stable under anoxic, low-temperature conditions, and chemical weathering of pyrite typically occurs in oxic environments, resulting in a large-scale input of sulfate to oceans ( 22 – 24 ). Nevertheless, a recent study showed that anoxic photochemical weathering of pyrite induced by sunlight exposure in the presence of aqueous Fe 2+ contributes substantial amounts of sulfate to the oceans during the anoxic late Archean ( 61 ). In this reaction, aqueous Fe 2+ was photooxidized as Fe 3+ , which can lead to the chemical oxidation of sulfur within pyrite, releasing Fe 2+ and sulfate into solutions ( 61 ). Similarly, the traditional view that pyrite is only accessible to aerobic microbes or anaerobic microbes in the presence of oxidizing agents (e.g., Fe 3+ , NO 3 – , and MnO 2 ) ( 22 – 24 ) is challenged by emerging evidences ( 21 , 25 , 26 ). For example, anaerobic methanogenic archaea can use iron and sulfur from pyrite, possibly via reductive dissolution of pyrite ( 21 , 25 ). Under sulfur-limited conditions, anoxygenic phototroph PSB is capable of oxidizing pyritic sulfur, via EET, to polysulfide or elemental sulfur, but not to sulfate ( 26 ). However, under the iron-limited condition applied in this study, pyrite surface–bound sulfur was likely oxidized to sulfate by GSB, facilitating its iron acquisition. In sulfidic habitats where anoxygenic phototrophs PSB and GSB flourish, their metabolic activities should be limited by iron, not sulfur. Therefore, our observed microbial oxidation of pyritic sulfur under iron-depleted conditions may be more environmentally relevant in both ancient and contemporary euxinia, enhancing the bioavailability of pyritic iron toward anoxygenic phototrophs. These results imply that microbial weathering of pyrite may be more prevalent on early Earth than previously thought and may have contributed to the biogeochemical cycles of elements such as carbon and sulfur in the paleo-ocean."
} | 4,140 |
37386409 | PMC10311727 | pmc | 2,511 | {
"abstract": "Background Biobased 5-(hydroxymethyl)furfural (5-HMF) is an important platform that offers numerous possibilities for upgrading to a range of chemical, material and fuel products. One reaction of special interest is the carboligation of 5-HMF into C 12 compounds, including 5,5’-bis(hydroxymethyl)furoin (DHMF) and its subsequent oxidation to 5,5’-bis(hydroxymethyl)furil (BHMF), due to their potential applications as building blocks for polymers and hydrocarbon fuels. Objectives This study was aimed at evaluating the use of whole cells of Escherichia coli carrying recombinant Pseudomonas fluorescens benzaldehyde lyase as biocatalysts for 5-HMF carboligation, recovery of the C 12 derivatives DHMF and BHMF, and testing the reactivity of the carbonyl groups for hydrazone formation for potential use as cross-linking agents in surface coatings. The effects of different parameters on the reaction were investigated to find the conditions for achieving high product yield and productivity. Results The reaction with 5 g/L 5-HMF using 2 g CDW /L recombinant cells in 10% dimethyl carbonate, pH 8.0 at 30 °C resulted in DHMF yield of 81.7% (0.41 mol/mol) at 1 h, and BHMF yield of 96.7% (0.49 mol/mol) at 72 h reaction time. Fed-batch biotransformation generated a maximum DHMF concentration of 53.0 g/L (or 26.5 g DHMF/g cell catalyst) with productivity of 10.6 g/L . h, after five feeds of 20 g/L 5-HMF. Both DHMF and BHMF reacted with adipic acid dihydrazide to form hydrazone that was confirmed by Fourier-transform infrared spectroscopy and 1 H NMR. Conclusion The study demonstrates the potential application of recombinant E. coli cells for cost-effective production of commercially relevant products. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02130-1.",
"conclusion": "Conclusion and outlook This study has shown the whole microbial cells bearing benzaldehyde lyase to be an effective biocatalyst for carboligation of 5-HMF in a very short time under ambient conditions. Up to 52.7 g DHMF /L (26.5 g DHMF per g cells) was obtained during fed-batch conditions involving 20 g/L 5-HMF feeds. Performing the reaction with limited access to oxygen increased DHMF formation at higher 5-HMF initial concentrations, while also decreasing the formation of the furil BHMF. Cell recycling for repeated reactions as well as fed-batch reaction showed decreasing product yields with successive cycles/feeds due to inactivation of the enzyme activity. The potential of the keto groups in DHMF/BHMF for hydrazone formation was also demonstrated, suggesting their possible role as crosslinking agents in polymer coatings and gels. Further work should focus on increasing the kinetics of BHMF production and upscaling the process whilst maintaining high substrate conversion and good product yield and recovery. Additionally, evaluation of the product as crosslinker in coatings is planned.",
"introduction": "Introduction The climate crisis calls for substantial decrease in global dependence on fossil feedstock to avoid the release of fixed CO 2 into the atmosphere. As the demand for crude oil for making transportation fuel is expected to decline in the coming years, the petrochemical industry is still looking to use the raw material to make higher-margin chemicals [ 1 ]. In 2021, the petrochemical sector accounted for about 14.2% of total oil demand or 13.8 million barrels per day (mb/d) and is projected to increase to 17.5 mb/d in 2045 [ 2 ]. On the other hand, the production of chemicals from biomass has definite potential to lead to significant greenhouse gas mitigation compared to fossil-derived counterparts [ 3 ]. In addition, the use of biotransformation meets many aspects of more sustainable manufacturing besides CO 2 reduction. For this purpose, furan platform chemicals obtained directly from C 5 and C 6 sugars from biomass polysaccharides are promising candidates for providing an economical route for synthesis of a vast range of bio-based chemicals, materials and fuels [ 4 – 6 ]. The C 6 furan compound, 5-hydroxymethylfurfural (5-HMF), has been one of the most investigated furan compounds, both with respect to its production and its valorization to several useful building blocks for fuel, chemical, polymer and pharmaceutical industry, thanks to its reactive hydroxyl and aldehyde groups and the furan ring [ 7 , 8 ]. Production of 5-HMF from fructose at quite high yields has been achieved in our laboratory using a two-phase system of water and dimethyl carbonate (DMC) in a continuous mode in a tube reactor, followed by its recovery in a pure form [ 9 ]. Oxidation of both aldehyde and hydroxyl groups in 5-HMF to form 2,5-furan dicarboxylic acid (FDCA), has currently attracted much attention as it is expected to serve as a biobased alternative to terephthalic acid to make polyethylene furanoate (PEF), a polymer with superior barrier and thermal properties than polyethylene terephthalate (PET) [ 10 , 11 ]. Selective oxidation of the aldehyde moiety gives another interesting polymer building block, 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) [ 12 ], while its reduction results in a diol, bis(hydroxymethyl)furan [ 13 ], which can be further modified e.g. to diepoxide monomers [ 14 ]. Yet another reaction that the aldehyde group has been subjected to is aldol condensation to give a variety of value added products such as fuel precursors, polymers and biologically active compounds [ 8 , 15 ]. An interesting example is a carboligation reaction in which two 5-HMF molecules undergo self-condensation in an umpolung fashion to yield C 12 acyloin (2-hydroxyketone) product, 5,5’-bis(hydroxymethyl)furoin (DHMF) [ 16 ] (See Scheme 1 ). DHMF has three hydroxyl groups, two furan rings and one carbonyl group, all of which can be utilised for polymerisation into polyethers, polyesters, polycarbonates and polyurethanes, production of C 12 ketones, and/or production of oxygenated diesels [ 17 , 18 ]. The 2-hydroxy ketone can be further oxidised to a diketone compound, 5,5’-bis(hydroxymethyl)furil (BHMF), in which two ketone groups connect the furan rings and has potential in producing polyurethane films [ 17 ]. Scheme 1. Pathway for production of C 12 furan derivatives from sugars in the biomass via 5-HMF using recombinant E. coli cells expressing benzaldehyde lyase (BAL). The bracketed part is the focus of the present study The acyloin condensation reaction with 5-HMF has been done through organocatalysis using N-heterocyclic carbenes which resulted in > 95% yield of DHMF [ 19 ]. Mou et al. (2016) produced BHMF from DHMF through selective oxidation using either manganese dioxide (MnO 2 ) with a high isolated yield of 95% or a “greener” metal-free organocatalyst 1,8-diazabicyclo(5–4-0)undec-7-ene (DBU) with a yield of 85% [ 17 ]. Efficient oxidation of DHMF to BHMF has also been shown using molybdenum and tungsten-based catalysts with yield up to 94% [ 20 ]. However, industrial upscaling issues remain despite good DHMF and BHMF yields via organocatalysts including stability, sensitivity and synthesis of these catalysts [ 21 – 23 ]. Biotransformation through enzymatic catalysis is another option for upgrading 5-HMF. Donnelly et al. have earlier shown the carboligation of 5-HMF to be catalysed by the enzyme benzaldehyde lyase from Pseudomonas fluorescens [ 24 ]. In another study, the same enzyme was used for the carboligation of 3-furaldehyde generating good product yields and conversion and high enantiomeric excess [ 25 ]. Benzaldehyde lyase (BAL) (E.C. 4.1.2.38) is a thiamine-diphosphate dependent enzyme which catalyses reversible conversion of ( R )-benzoin to benzaldehyde, and is a valuable enzyme for the synthesis of chiral 2-hydroxy ketones. First reported in 1989, BAL was found to be responsible for giving P. fluorescens the ability to grow on benzoin as sole carbon source [ 26 ]. Due to the enzyme’s ability to catalyse C–C bond formation and high enantioselectivity, its product repertoire has since been expanded from benzoins, acetoins to hydroxybutyrophenones and aliphatic acyloins [ 27 – 29 ], and the insights into its three dimensional structure also opened access to stereocomplementary products thereof [ 30 ]. Despite the applicability of enzymes as drivers of selective catalysis, the high cost of enzyme purification is a major challenge for production at large scale. Production of recombinant whole cells is about ten times cheaper than isolated enzymes as it circumvents the need and costs of catalyst purification [ 31 , 32 ]. Moreover, due to better stability features of whole cells, the operational lifetime of the biocatalyst is higher [ 31 , 33 ]. This article investigates DHMF and BHMF production from 5-HMF in a reaction catalysed by the whole cells of recombinant Escherichia coli expressing P. fluorescens BAL. The effect of reaction parameters including co-solvent addition, varying concentrations of substrate and the biocatalyst, and other additives on the reaction performance were explored in the study. A simple purification method for purification of DHMF and BHMF was developed. Finally, reactivity of the carbonyl groups in the two products was evaluated for hydrazone formation for their potential use in crosslinking polymers and coatings.",
"discussion": "Results and discussion Screening of reaction parameters for DHMF and BHMF formation In contrast to the earlier report demonstrating the carboligation of 5-HMF using pure BAL [ 24 ], the present work used whole cells of recombinant E. coli expressing benzaldehyde lyase (activity of 5071 U/g cdw cells) for catalysing the reaction. SDS-PAGE analysis of protein in the cells showed a band density of 71.9% for the recombinant BAL enzyme (Additional file 1 : Figure S1). Initial screening of the reaction conditions was performed in 4 mL working volume at 30 °C and the reaction was allowed to continue for 72 h. The cell catalyst was applied as resting cells resuspended in phosphate buffer (pH 8.0) at concentrations in the range of 0.2–10 g cdw /L. It should be noted here that the maximum DHMF and BHMF yield is 0.5 mol (100% theoretical yield) product per mol 5-HMF. The reaction performed in the absence of any co-solvent gave low yields of both DHMF (0.23 mol/mol) and BHMF (0.20 mol/mol), at 1 h and 72 h, respectively. The reaction using pure BAL reported earlier was performed in 20% DMSO [ 24 ], which is known to have a stabilizing effect on enzymes [ 34 , 35 ] including P. fluorescens BAL [ 36 , 37 ]. As DMSO is an undesirable solvent for large scale use due to downstream processing challenges [ 36 ] and issues related to the waste handling and safety [ 38 ], we tested other co-solvents including dimethyl carbonate (DMC), 2-propanol and methanol, which are potentially bio-based [ 39 – 41 ]. Previous evaluation with respect to green chemistry metrics showed that DMC and methanol are more favourable in terms of atom economy and methylating efficiency [ 42 ], while alcohols such as methanol and 2-propanol, are generally regarded as environmentally-friendly and cost-efficient [ 40 , 43 ]. The highest DHMF yield of 0.41 mol/mol at 1 h and BHMF yield of 0.49 mol/mol at 72 h from 42.5 mmol (5.4 g/L) 5-HMF was obtained in a reaction with 10% (v/v) DMC (Fig. 1 a and b). A blank reaction performed using E. coli cells without expressed BAL did not show any consumption of 5-HMF nor the formation of any product (not shown). Fig. 1 Effect of co-solvents on the yields of ( a ) DHMF, with maximum concentration at 1 h reaction time and ( b ) BHMF, with maximum concentration at 72 h reaction time during biotransformation of 5-HMF. The reaction was performed in 4 mL volume at initial substrate concentration of 5 g/L 5-HMF, with 2 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase (5071 U/g cdw cells), 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP, at 30 °C and total reaction time of 72 h. Error bars indicate the standard deviation from duplicate experiments Adding more than 10% of DMC into the system decreased the yields of both products, which were still higher than without any solvent addition. Reactions in 10% (v/v) propanol or methanol did not have a marked influence on the DHMF yield as compared to the reaction without any solvent, but the BHMF yield was slightly increased. The higher activity of BAL in DMC containing reaction is most likely due to the relatively lower solubility of DMC in water as compared to methanol and propanol, and consequently lower impact in causing conformational changes in the enzyme. The water-miscible solvents have earlier been shown to also have a strong influence on the stereoselectivity and chemoselectivity of ThDP-dependent enzymes, the smaller the solvent molecule the higher its impact [ 44 ]. The reaction in 10% DMC was chosen for further experiments. Varying the concentration of cells between 0.2–10 g cdw /L showed maximal yields of DHMF and BHMF at 0.42 mol and 0.50 mol per mol HMF at 1 h and 72 h, respectively, at the cell concentration of 4 g cdw /L (Fig. 2 ). The yields were only marginally lower (0.41 mol and 0.49 mol DHMF and BHMF/ mol HMF, respectively) at 2 g cdw /L cells as also described above in Fig. 1 , and this cell concentration was used for further experiments. Conversely, increasing the cell concentration to 8 g cdw /L and beyond also decreased the product yields. The reason for the decreased activity at higher cell concentrations is not clear but some plausible reasons could be the limited cofactor amount or changes in properties of the reaction mixture such as viscosity, cell aggregation, etc., that may affect the enzyme–substrate interaction, and hence lower activity. A tenfold decrease in productivity of lactone production with sixfold increase in the whole cell catalyst amount has been reported earlier and was suggested to be related to limitation in oxygen supply with increase in cell density [ 45 ]. Whole-cell catalysis for production of a stable ascorbic acid derivative also showed insignificant increase in product yield even with a 50% increase in the cell catalyst amount [ 46 ]. Fig. 2 Effect of cell catalyst amount on the yields of ( a ) DHMF, with maximum concentration at 1 h reaction time and ( b ) BHMF, with maximum concentration at 72 h reaction time during biotransformation of 5-HMF. The reaction was performed in 4 mL volume using initial 5-HMF concentration of 5 g/L, 0.2–10 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase, 10% dimethylcarbonate, 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP, at 30 °C and total reaction time of 72 h. Error bars indicate the standard deviation from duplicate experiments Reactions with varying initial 5-HMF concentration ranging from 1.5 g/L to 20 g/L in 4 mL reaction volume showed that the maximum DHMF (0.41 mol/mol) and BHMF (0.49 mol/mol) yields using 2 g cdw /L cell catalyst and 10% DMC were obtained at 5.4 g/L HMF (Fig. 3 ). Fig. 3 Effect of initial 5-HMF concentration on the yields of ( a ) DHMF, with maximum concentration at 1 h reaction time and ( b ) BHMF, with maximum concentration at 72 h reaction time during biotransformation of 5-HMF. The reaction was performed in 4 mL volume using initial 5-HMF concentration of 1.5–20 g/L 5-HMF, 2 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase, 10% dimethylcarbonate, 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP, temperature of 30 °C and total reaction time of 72 h. Error bars indicate the standard deviation from duplicate experiments Increasing the initial substrate concentration to ~ 10 g/L and further to ~ 20 g/L decreased the DHMF yield to up to 27% and BHMF yield to 63%. However, in later experiments (See results in Fig. 5 , 1st cycle), when the reaction was performed in a fed-batch mode in 10 mL working volume in a 15 mL tube, providing relatively limited aeration conditions (due to low surface-to-volume ratio), DHMF yield at 20 g/L 5-HMF increased to about 0.43 mol/mol (86% yield) after 1 h. In contrast, NHC-carbene catalysed carboligation of 5-HMF (23 g/L) performed in 5 mL tetrahydrofuran at 60 °C yielded 98% DHMF in one hour [ 7 ]. Thus, the much milder conditions provided by whole-cell catalysis can generate reasonably good DHMF yields from 5-HMF that are comparable to the organocatalytic method. The reaction profile of the standard reaction with 5.4 g/L HMF in 10% DMC and 2 g cdw /L cells shown in Fig. 4 reveals that DHMF formation occurred quickly and peaked at 15 min (4.64 g/L) followed by a gradual transformation into BHMF reaching 4.92 g/L at 24 h and 5.19 g/L (0.49 mol /mol 5-HMF) at 72 h. BHMF formation occurs as a result of spontaneous oxidation of DHMF, which was confirmed using DHMF in a vial under identical standard conditions but excluding the E. coli cells. BHMF formation from DHMF with a yield of up to 0.76 mol/mol at 72 h was observed (Additional file 1 : Figure S2). To test if the spontaneous oxidation of DHMF during the biocatalytic carboligation of 5-HMF could be avoided, a parallel experiment was performed in which the reaction mixture was purged with N 2 gas in a closed reaction vessel. Under this condition, the BHMF formation was reduced to 0.24 mol BHMF per mol 5-HMF most likely due to the lower oxygen level in the system (Additional file 1 : Figure S2). Fig. 4 Reaction profiles of DHMF and BHMF production from 5- HMF. The reaction was performed in 4 mL volume using initial 5-HMF concentration of 5.4 g/L, 2 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase, 10% dimethylcarbonate, 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP, temperature of 30 °C, and total reaction time of 72 h. In the small plot embedded within the reaction profile, the time course of the first hour is shown in more detail. Error bars indicate the standard deviation from duplicate experiments On the other hand, the oxidation rate of DHMF to BHMF can be increased by increasing the aeration, as observed when the cell-catalysed 5-HMF carboligation was performed in 200 mL reaction volume in a 1 L Erlenmeyer flask, leading to a larger surface area for liquid-to-air interaction. Higher BHMF concentration was observed already at 1 h of reaction (Additional file 1 : Figure S3). For efficient selective BHMF production, the crucial parameter of aeration is to be investigated carefully. Alternatively, further transformation of DHMF can be done after removal of BAL and supplementing with an efficient oxidative catalyst like reported elsewhere [ 17 ] or even using a biocatalyst, e.g. co-expression of oxidases/oxidoreductases. 5-HMF transformation: effects of cell recycling versus fed batch reaction BAL is subject to inhibition by 5-HMF and probably also by the products, as observed with reduced yields at initial 5-HMF concentrations higher than 5 g/L (Fig. 3 ). Since the formation of DHMF occurs in a very short time, it was of interest to see if the cells retain the enzymatic activity and can be recycled for repeated transformation of 5-HMF. The biotransformation was carried out in a total volume of 10 mL with 2 g cdw /L cells. After running the reaction for 1 h, the cells were separated from the solution and added to a fresh reaction solution with the same 5-HMF concentration as in the previous batch. The cell recycling was repeated three times with initial 5-HMF concentrations of 5 and 10 g/L. As seen in Fig. 5 , the yields decreased for both concentrations of 5-HMF tested after the first cycle. During the second cycle of 5 g/L 5-HMF, a marginal decrease in DHMF yield was observed from the first reaction. In comparison, the higher 5-HMF concentration (10 g/L) saw a 67% drop of DHMF yield between first and second reactions. In the third cycle, the yield decreased by 43% for 5 g/L HMF from the initial cycle, while for 10 g/L 5-HMF the yield remained about the same as in the second cycle. Further decrease in DHMF yields was observed when the cells were recycled four to five times (results not shown). The results indicate that while it may be possible to recycle the cells more than once for the production of DHMF, replenishment of the catalyst will be needed after the third cycle. In addition, the yields for BHMF remained below 0.01 mol/mol during the cell recycling experiments. Fig. 5 Yields (mol/mol) of a DHMF and b BHMF during cell catalyst recycling. Two separate experiments with initial 5-HMF concentrations of 5 and 10 g/L were tested. The reaction was performed for 1 h using 10% dimethylcarbonate, 2 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase, 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP, at 30 °C in 10 mL working volume. The cells were separated by centrifugation after the reaction and added to a fresh 5-HMF solution. Error bars indicate the standard deviation from duplicate experiments Effect of additives for reducing the enzyme inactivation and in turn improving the operational lifetime of the whole cells was investigated. The additives included 1 wt% bovine serum albumin (BSA), 2 M glycerol and 85 mM tris(hydroxymethyl)aminomethane (Tris) (Additional file 1 : Figure S2). BSA and glycerol are often used as protein stabilizing agents, while Tris was used assuming that the aldehyde groups on 5-HMF may interact with the NH groups on Tris instead of the enzyme and minimize enzyme deactivation. However, none of the tested additives improved the production of DHMF (and also BHMF). Alternatively, the biotransformation was performed in a fed-batch mode, i.e. a fresh feed of 5-HMF (2, 5, 10 and 20 g/L, respectively) was added every hour up to 5 times. Figure 6 shows increase in DHMF concentration with every feed of 5-HMF, but with decreasing yield. The final DHMF concentration obtained was 52.97 g/L from a total of 100 g/L 5-HMF (i.e. 5 feeds of 20 g/L), while BHMF concentration was 2.13 g/L. Productivity of 10.59 g/L . h was observed after five 20 g/L HMF feeds as compared to 17.9 g/L . h after the first feed (Table 1 ). In contrast, an earlier study by Donnelly and co-workers on the 5-HMF carboligation reaction using pure benzaldehyde lyase reports a preliminary productivity value of 7 g/L.h [ 24 ]. The highest DHMF yield of 0.43 mol/mol was obtained during the first hour, decreasing by 28% to 0.31 mol/mol during the second feed, and followed by a 70% decrease to 0.12 mol/mol, and down to 0.08 mol/mol after the fifth feed. At the end of the feed, around 24% of the total 5-HMF fed into the reactor was left unreacted. Further information on yields and productivities in both cell-recycling and fed-batch experiments are presented in Table 1 . Fig. 6 Fed-batch biotransformation of 5-HMF to DHMF in 10 mL volume using 2 g cdw /L recombinant E. coli cells expressing P. fluorescens benzaldehyde lyase, 10% dimethylcarbonate, 50 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 8.0, 2.5 mM MgSO 4 , 0.1 mM ThDP and temperature of 30 °C. Four separate experiments with varying initial 5-HMF concentrations of 2, 5, 10 and 20 g/L, respectively, were tested. a DHMF yield (mol/mol) and the corresponding concentrations of 5-HMF, DHMF and BHMF in the fed-batch experiment with 5 g/L 5-HMF feeds. b DHMF and c BHMF concentrations after each substrate feed. Error bars indicate the standard deviation from duplicate experiments Table 1 Yields of DHMF and BHMF per g cell catalyst and productivities during cell-recycling and fed-batch transformations with recombinant E. coli cells Cycle/Feed Yield Yield Productivity g DHMF/g CDW g BHMF/g CDW g DHMF/Lh 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Cell recycling 5 g/L 2.05 ± 0.03 1.82 ± 0.05 0.92 ± 0.31 2.40 ± 2.05 0.25 ± 0.15 0.04 ± 0.00 0.05 ± 0.00 0.04 ± 0.01 0.06 ± 0.05 0.01 ± 0.00 4.10 ± 0.05 1.82 ± 0.05 0.61 ± 0.21 1.20 ± 1.02 0.10 ± 0.06 10 g/L 4.18 ± 0.54 1.39 ± 0.08 1.53 ± 0.11 1.60 ± 0.52 0.09 0.10 ± 0.01 0.05 ± 0.00 0.08 ± 0.01 0.05 ± 0.01 0.01 ± 0.00 8.37 ± 1.07 1.39 ± 0.08 1.02 ± 0.07 0.80 ± 0.26 0.03 Fed-batch 2 g/L 0.93 ± 0.09 1.55 ± 0.10 2.09 ± 0.25 0.00 0.00 0.02 ± 0.00 0.05 ± 0.0 0.09 ± 0.01 0.21 ± 0.00 0.20 ± 0.06 1.87 ± 0.18 1.55 ± 0.10 1.39 ± 0.17 1.30 ± 0.07 1.71 ± 0.43 5 g/L 2.46 ± 0.45 3.30 ± 0.02 3.26 ± 2.97 3.26 ± 0.28 3.97 ± 0.39 0.04 ± 0.01 0.08 ± 0.01 0.14 ± 0.13 0.22 ± 0.03 0.23 ± 0.03 4.92 ± 0.91 3.30 ± 0.02 2.17 ± 1.98 1.63 ± 0.14 1.59 ± 0.16 10 g/L 5.12 ± 0.59 7.12 ± 0.60 14.20 ± 2.89 13.99 ± 0.79 15.57 ± 0.05 0.09 ± 0.01 0.17 ± 0.03 0.52 ± 0.08 0.68 ± 0.04 0.70 ± 0.03 10.24 ± 1.17 7.12 ± 0.60 9.47 ± 1.92 7.00 ± 0.40 6.23 ± 0.02 20 g/L 8.95 ± 0.10 16.03 19.50 ± 0.00 22.95 26.48 ± 3.24 0.32 ± 0.01 0.48 ± 0.01 0.69 ± 0.00 0.91 ± 0.02 1.07 ± 0.07 17.90 ± 0.20 15.85 ± 0.24 13.00 ± 0.00 11.59 ± 0.16 10.59 ± 1.30 The initial 5-HMF concentrations in the reaction during cell recycling were 5 and 10 g/L, respectively, while the concentrations were 2, 5, 10 and 20 g/L during fed-batch experiments. Both cell recycling and feeding were done 5 times at each substrate concentration. Error values indicate margin for duplicate measurements It should be noted here that both cell-recycling and fed-batch experiments were performed without prior nitrogen sparging or tightly closed reaction vessels. However, since the 10 mL reactions were carried out in closed 15 mL Falcon tubes, except for sample withdrawal and feedings of 5-HMF, there was very limited aeration throughout the reaction. In a separate experiment where a specifically nitrogen sparged and closed vessel protocol was implemented, the product yields were similar to the reaction protocol using the 15 mL Eppendorf tubes, i.e. BHMF formation is reduced (Additional file 1 : Figure S2). However, when the working volume was increased to 200 mL and the reaction done in a 1 L Erlenmeyer flask, while maintaining identical reaction parameters (2 g cdw /L cell catalyst and 5 g/L HMF) and feeding the same substrate amount every hour, the BHMF production increased within one hour (Additional file 1 : Figure S3). Interestingly, the proportion of BHMF being produced in 200 mL working volume at 1 h of reaction was much higher compared to the 10 mL fed-batch experiments, and even more so than the initial 4 mL experiments, suggesting the conversion of DHMF to BHMF to be promoted by the larger surface area for oxygen transfer. Recovery of DHMF and BHMF, and hydrazone formation Both DHMF and BHMF were extracted from the reaction solution using ethyl acetate; most DHMF could be extracted after 1 h of the biotransformation and then recovered as a pure white substance after evaporation of the extract and dissolving the solid residue in ethyl acetate-heptane mixture. Recovery of pure BHMF was achieved using a similar procedure and separating the supernatant from the white residue of DHMF followed by evaporation. 1 H NMR analyses of the purified DHMF and BHMF showed 96.7% and 98.1% purity, respectively (Additional file 1 : Figure S4). While the potential use of DHMF and BHMF as building blocks for different polymers and also for forming deep-eutectic solvents has already been proposed [ 24 ], the utilisation of the functional keto groups in these compounds to generate crosslinks would be valuable for the enhanced performance of the products e.g. polyurethane coatings. The reactivity of the keto groups in the 2-hydroxyketone and diketone compounds was evaluated by reaction with adipic acid dihydrazide at room temperature to generate hydrazone crosslinks (Additional file 1 : Scheme S1). With a molar ratio of 2 mol DHMF/BHMF to 1 mol adipic acid dihydrazide in 50:50 methanol:water, the product formed was completely soluble in the solvent after mixing vigorously for one minute. The FTIR spectra of the product sample showed a change of signal at about 1700 cm −1 signifying C = N bond formation (Fig. 7 c, e ), in contrast to the corresponding spectrum of DHMF and BHMF with the relatively narrow peaks at about1600 cm −1 ( Fig. 7 b, d ) . The C = N bond formation characterising a hydrazone, results from a substitution reaction between the keto group in DHMF with the amide substituent of the hydrazide. Changes in the OH − group peaks (3200–3400 cm −1 ) were also apparent before and after reaction with adipic acid hydrazide to form hydrazones. The 1 H NMR spectra of the products formed further validate the formation of hydrazones as seen by a decrease in the − NH 2 peak (4.15 ppm) (Additional file 1 : Figure S5 ). The BHMF-based hydrazone sample showed nearly complete disappearance of the − NH 2 peak (4.15 ppm), whilst the N–H peak (8.93 ppm) remained visible indicating complete reaction of the dihydrazide with BHMF (Additional file 1 : Figure S5c). On the other hand, DHMF-based hydrazone showed a lower − NH 2 peak relative to the N–H peak due to an incomplete hydrazone formation (Additional file 1 : Figure S5b). Since this reaction is pH-dependent [ 47 , 48 ], and this preliminary test was done in an unbuffered aqueous methanol solution, further optimisation of the reaction is recommended. Fig. 7 FTIR absorbance spectra of hydrazone formation by reaction of DHMF and BHMF, respectively, with adipic acid dihydrazide for 2 h"
} | 7,334 |
37375932 | PMC10303550 | pmc | 2,513 | {
"abstract": "Microorganisms are an important element in modeling sustainable agriculture. Their role in soil fertility and health is crucial in maintaining plants’ growth, development, and yield. Further, microorganisms impact agriculture negatively through disease and emerging diseases. Deciphering the extensive functionality and structural diversity within the plant–soil microbiome is necessary to effectively deploy these organisms in sustainable agriculture. Although both the plant and soil microbiome have been studied over the decades, the efficiency of translating the laboratory and greenhouse findings to the field is largely dependent on the ability of the inoculants or beneficial microorganisms to colonize the soil and maintain stability in the ecosystem. Further, the plant and its environment are two variables that influence the plant and soil microbiome’s diversity and structure. Thus, in recent years, researchers have looked into microbiome engineering that would enable them to modify the microbial communities in order to increase the efficiency and effectiveness of the inoculants. The engineering of environments is believed to support resistance to biotic and abiotic stressors, plant fitness, and productivity. Population characterization is crucial in microbiome manipulation, as well as in the identification of potential biofertilizers and biocontrol agents. Next-generation sequencing approaches that identify both culturable and non-culturable microbes associated with the soil and plant microbiome have expanded our knowledge in this area. Additionally, genome editing and multidisciplinary omics methods have provided scientists with a framework to engineer dependable and sustainable microbial communities that support high yield, disease resistance, nutrient cycling, and management of stressors. In this review, we present an overview of the role of beneficial microbes in sustainable agriculture, microbiome engineering, translation of this technology to the field, and the main approaches used by laboratories worldwide to study the plant–soil microbiome. These initiatives are important to the advancement of green technologies in agriculture.",
"conclusion": "9. Conclusions In the process of moving towards sustainable agriculture, agricultural practices must reduce the extensive usage of agrochemicals. In this review, we have presented the potential of beneficial microbes to enhance growth, development, and disease suppression in the field. However, the effectiveness of beneficial microbes in field applications has been less than satisfactory. This is largely due to the fact that the newly introduced microbes must thrive in the environment and maintain a steady and stable community for the benefits to be harnessed. This has brought forth technologies such as sequencing and multiomics platforms, which have enabled us to visualize, to some extent, the diversity, communities, and structures of microorganisms in any given environment. This has created opportunities for technologies such as microbial engineering to offer designer solutions for specific environments thus achieving greater efficiency and sustainability. This technique promises answers to various gaps in knowledge, such as providing the right microbial consortia based on the plant species and soil environment to ensure proper recognition and colonization of the soil and roots by the inoculants. In this regard, the advancement of “microbiome-driven cropping systems” may herald the next agricultural revolution and a more sustainable method of plant production. Furthermore, the development of modified crops or organisms may yield the desired advancement towards zero hunger for the continuously expanding human population. This will be made possible by the application of multiomics approaches combined with genome editing techniques such as CRISPR for improving nutritional status, disease resistance, and crop yield. In the years to come, however, there is much work that needs to be conducted to comprehend the genetics and engineering of the intricacies behind the ecological and metabolic networks that govern plant-associated microbe interactions. For instance, research needs to go beyond the identification of causative or beneficial organisms into “how” this information may be used reproducibly to enhance plant growth and development and to reduce disease incidence and spread. We also need to reduce wastage and redundancies in research by producing standardized techniques and a center for the collation and annotation of meta-data. These techniques must be made such that they can be utilized by any laboratory worldwide for the translation of laboratory-raised products to the field. We also need to focus on developing new technologies that are more efficient, accurate, cost-effective, and quick to use both in the laboratory and in the field. The technologies that are developed should not be restrictive to only the well-funded research groups, but should be attainable for small laboratories. Bioinformaticians need to continuously come up with new software or upgraded versions of the existing platforms to increase the depth, speed, and quantity of information harnessed from the data. The upgrade does not stop with the equipment and the techniques; the upskilling of researchers needs to evolve along with trends in the field of plant-associated microbiome research. This field has a bountiful future, and there is room to push the boundaries of knowledge and technologies further.",
"introduction": "1. Introduction One of the major issues confronting today’s modern agriculture is optimizing sustainable crop output in order to ensure global food security. Furthermore, climate change has exacerbated the impact of environmental stresses such as drought, flooding, heat, and salinity on world food productivity [ 1 ]. In addition, the current agricultural practice of utilizing agrochemicals for optimizing yield has resulted in devastating environmental consequences to soil health and fertility [ 2 , 3 , 4 ]. Hence, to maximize crop output, it is clear that novel methods must be developed and investigated. One of these methods is the incorporation of beneficial microbes into agricultural practices [ 5 , 6 ]. While several studies have reported the role of microbes in plant fitness as well as in soil health and fertility, there is still a need to elucidate the relationship between the microbiome and plant health. Among the aspects of plant–microbe interactions that should be studied are their role in the plant immune responses, signaling pathways (both in plants and microbes), positive and negative interactions between plants and microorganisms, and microbial function in plant productivity [ 7 , 8 ] These findings will enlighten us regarding the entire process of plant–microbe interaction and the discovery of microorganisms that can be exploited to boost crop output in the near future [ 9 , 10 ]. By studying the plant microbiome, we are able to expound on the functional and structural diversities of the microbial communities linked to specific plants and ecosystems. The microbial diversity observed across regions and organs in the phyllosphere, rhizosphere, and endosphere has been well-documented by researchers [ 11 , 12 ]. Plants, in general, use a variety of tactics to favor and support microbial colonization, such as the presence of specialized structures (e.g., hairs, trichomes) or production of secondary metabolites. A comprehensive approach towards deciphering the microbial population and its relationship with plants remains a developing area of research pursued by many laboratories worldwide [ 11 , 13 ]. The diversity in the microbial population is influenced by factors such as host species, selection pressure, environment, developmental stage, and agricultural practices [ 14 ]. Several studies have focused on specialized or niche communities to link the population/community diversity with specific stresses or environmental pressures [ 15 ]. These observations clearly highlight the need for more thorough and in-depth studies to contribute towards the information and mechanisms that underly the microbiome assembly [ 16 , 17 ]. Technologies for studying microbial diversity and the composition of a specific plant microbiome have advanced significantly. These technologies have moved us from culture-dependent identification, which has its limits in terms of providing a complete picture of the microbiome, to more high-tech methods which achieve higher-resolution images of the microbiome. The development of novel high-throughput techniques and technologies has revealed multitrophic interactions in the black box of plant–microbe interactions [ 18 ]. Plant-beneficial microorganisms can now be altered thanks to the advancement of these high-throughput technologies. Microbiome engineering may be an alternate method for understanding, manipulating, and developing corresponding technology for building microbial populations which are critical to plant health and productivity in this scenario [ 19 ]. The new and emerging technologies will prove to be useful in deciphering the depth of microbiome diversity in any given ecosystem [ 20 ]. Hence, in this review we will address the microbiome in terms of its benefits; its shaping; its response to the environment; plant- and soil-associated microbiomes; and the tools that have been developed to elucidate, understand, and modify plant–microbe interactions."
} | 2,365 |
24959921 | PMC4069195 | pmc | 2,514 | {
"abstract": "Even in the absence of major disturbances (e.g., cyclones, bleaching), corals are consistently subject to high levels of background mortality, which undermines individual fitness and resilience of coral colonies. Partial mortality may impact coral response to climate change by reducing colony ability to recover between major acute stressors. This study quantified proportion of injured versus uninjured colonies (the prevalence of injuries) and instantaneous measures of areal extent of injuries across individual colonies (the severity of injuries), in four common coral species along the Great Barrier Reef in Australia: massive Porites , encrusting Montipora , Acropora hyacinthus and Pocillopora damicornis . A total of 2,276 adult colonies were surveyed three latitudinal sectors, nine reefs and 27 sites along 1000 km 2 on the Great Barrier Reef. The prevalence of injuries was very high, especially for Porites spp (91%) and Montipora encrusting (85%) and varied significantly, but most lay at small spatial scales (e.g., among colonies positioned <10-m apart). Similarly, severity of background partial mortality was surprisingly high (between 5% and 21%) but varied greatly among colonies within the same site and habitat. This study suggests that intraspecific variation in partial mortality between adjacent colonies may be more important than variation between colonies in different latitudinal sectors or reefs. Differences in the prevalence and severity of background partial mortality have significant ramifications for coral capacity to cope with increasing acute disturbances, such as climate-induced coral bleaching. These data are important for understanding coral responses to increasing stressors, and in particular for predicting their capacity to recover between subsequent disturbances.",
"introduction": "Introduction Disturbances play an important role in structuring natural communities [1] , [2] , especially in coral reef ecosystems, which are being subject to increasing frequency, severity and diversity of acute disturbances [1] , [3] . Importantly, climate-related disturbances are compounding numerous pre-existing natural and anthropogenic disturbances [1] , [4] , [5] , contributing to extensive coral loss and associated degradation of coral reef habitats [5] , [6] , [7] , [8] . Coral reefs are highly dynamic ecosystems, naturally subject to a wide range of disturbances operating at different temporal and spatial scales, ranging from widespread mass bleaching events to chronic localised removal of live coral tissue by corallivores [9] . However, increasing effects of global climate change and other more direct anthropogenic disturbances appear to be increasing rates of coral mortality beyond those which can be sustained [10] . It is also possible that the capacity of corals to recover from successive disturbance events is declining, due to sustained declines in coral growth or reproductive output [8] . Coral assemblages can recover quite quickly in the aftermath of major disturbances (e.g., [11] , [12] ), as long as there is i) sufficient time between major disturbances, ii) adequate proximity or connectivity to viable source populations, and iii) maintenance of suitable substrates for settlement and subsequent survival of coral recruits [13] . Given increasing incidence of major disturbances (e.g., cyclones, bleaching or outbreaks of crown-of-thorns starfish), the interval between these events is often less than five years [14] , exceeding the time needed for effective recovery [15] . Moreover, the increasing spatial extent of disturbances (e.g., climate-induced coral bleaching) is causing comprehensive mortality over very large areas, further undermining the capacity for recovery (but see [16] ). The capacity for coral assemblages to recover can also be negatively affected by smothering or overgrowth by macroalgae, in instances where there is insufficient grazing by herbivorous fishes [17] , and by a range of factors that potentially limit the early post-settlement growth and survivorship of corals [18] . While acute disturbances (e.g., cyclones, bleaching or outbreaks of crown-of-thorns starfish) often have very conspicuous effects on corals, causing high levels of whole colony mortality, chronic disturbances (e.g., predation, competition and disease) can have equally important effects on coral communities, further increase susceptibility to major disturbances [19] and greatly reduce recovery and resilience [20] . Wakeford and co-workers [20] attributed low coral cover recorded at Lizard Island, in northern Great Barrier Reef (GBR) to chronic disturbances and high rates of background mortality. Accordingly, coral assemblages at very isolated reefs (e.g., Scott Reef, off the north-west shelf), which are largely isolated from chronic anthropogenic disturbances, may recover quite rapidly even after very severe acute disturbances [16] . In the absence of major disturbances, corals are continually subject to a range of chronic, often small-scale disturbances that can cause relatively high rates of mortality [20] , [21] , [22] , [23] . These chronic disturbances are a normal part of the natural dynamics and turnover in coral populations and communities [24] , [25] , [26] , but may be increasing in prevalence and severity, thereby undermining the capacity for recovery (annual background mortality rates can generally vary from 1 to 30%: [20] , [23] , [26] , [27] , [28] . If for example, background mortality rates are increasing, the rate of recovery will be reduced, requiring an even longer period for complete recovery between successive major disturbances. Corals are modular organisms and can survive extensive injury (loss of polyps) or partial mortality, on a scale far beyond the regenerative capacity of most solitary organism [29] . However, partial mortality and declines in the total number of polyps that make up a colony, result in smaller colony size, which can greatly affect individual fitness [24] , . Colonies suffering from partial mortality must divert energy towards tissue repair, leading to a reduced energy expenditure towards growth, reproduction and other metabolic functions [32] . Very high prevalence and severity of partial mortality may therefore, have a stronger bearing on the fitness and fate of coral colonies and/or populations, than even colony size or other commonly used metrics of population structure. There are very few studies that have systematically quantified the prevalence or severity of partial mortality across a range of different corals or at a range of locations [33] , [34] . It is very likely however, that rates of injury will vary spatially, with greatest variation likely to occur at relatively small scales [23] . On the GBR, for example, midshelf reefs are subject to frequent and severe acute disturbances, mostly associated with outbreaks of crown-of-thorns starfish [35] , [36] , whereas offshore reefs are relatively less affected by such disturbances. However, even more apparent is the patchy nature of most disturbances, such that some reefs may be severely impacted, whereas other nearby reefs are unaffected [3] . The aim of this study was to quantify the prevalence and severity of partial mortality across four dominant coral taxa ( Acropora hyacinthus , Pocillopora damicornis , massive Porites spp and encrusting Montipora ) at a hierarchy of spatial scales (among sectors, among reefs and among sites within reefs) on midshelf reefs on the Australia's GBR. Biotic and abiotic agents of partial mortality can vary in frequency, intensity and spatial scale and can therefore have different impacts on coral colonies at different scales [3] . By measuring the prevalence and severity of partial mortality across a hierarchy of different spatial scales we hope to provide insights into local versus global causes of partial mortality. Quantifying rates of tissue loss along the entire GBR is critical for understanding spatial variation in the recovery capacity and resilience of reef-building corals. More specifically, this study tests the hypothesis that background rates of partial mortality decrease with latitude, thereby accounting for apparent discrepancies in rates of population replenishment versus overall abundance of adult corals [37] . Similar levels of adult abundance despite much higher levels of recruitment in the northern GBR imply that there must be higher levels of background mortality. The greatest variation was expected between latitudinal sectors, however, since chronic background disturbances are recurrent patchy stressors, prevalence and severity of partial mortality were also expected to vary at smaller scale (reef and site). If so, then this may have significant ramifications for the capacity of corals to cope with increasing acute disturbances associated with global climate change.",
"discussion": "Discussion Prevalence of injury was consistent across all taxa and all locations, suggesting that background mortality is common along the GBR. Similarly, Wakeford and co-workers [19] found that annual background mortality at Lizard Island on the GBR, was ca 22% for P. damicornis , ca 18% for A. hyacinthus and ca 10% for Porites massive. This is much lower than recorded in this study, but Wakeford and co-workers [20] did not account for partial mortality. Even higher rates of annual background partial mortality were recorded in the Caribbean compared to the GBR. In Curacao in 2005 corals exhibited between 14 and 48% of tissue loss mainly due to disease [42] , while in Bonairie in 2011 extent of injury ranged between 0 and 99% for Montastrea complex and was around 8% for other scleractinians [33] . Similarly, in Florida Keys, prevalence of recent partial mortality during periods of background, low-stress environmental conditions was still <5% for the 11 most abundant species [34] . High rates of background partial mortality within a population may lead to a decline in population densities through time because they can result in reduced colony growth [32] , [43] , reproductive output [44] , [45] , and reduced colony size [46] of individuals. Cumming [47] showed that recent injury can predict colony fate even more than colony size. However, even though partial mortality can negatively affect coral community dynamics [48] , it also true that it may partially enable reef recovery by providing substrate for corals to settle, thus maintaining coral dominated reefs [33] , [49] . The mean severity of background mortality varied among taxa suggesting that some species may be more resistant to routine agents and/or have better recovery potential. Massive Porites is a long-lived, slow growing coral, with generally low regenerative capabilities [50] , [51] , [52] , [53] so old injuries are likely to accumulate through time. Conversely branching corals, such as Acropora have high regeneration capacities, rapid linear growth, and short generation time (less than 30 years) [41] , [54] . The observed taxonomic differences in prevalence of injury recorded during this study broadly correspond with differences in growth rates and relative investment in repair [50] , [51] , [54] , [55] , [56] , [57] . It is possible therefore, that Acropora have equal or higher incidence of injuries compared to massive Porites , but higher rates of whole colony mortality and/or more rapid regenerative capacity, leading to lower levels of instantaneous partial mortality. Branching corals, such as Acropora spp would intuitively appear much more vulnerable to breakage and injuries than massive corals [50] . However, the agents of partial mortality are likely to vary greatly among taxa. For example, fish predation is often not visible on branching corals, while it is conspicuous on massive Porites , which shows the highest rates of grazing scars compared to other coral species [58] . It appears however, that taxonomic differences in the severity of injury are most likely due to differences in persistence of injuries, or the rate of repair. Previously published data documented marked latitudinal differences in population replenishment (highest in the northern GBR) despite similar adult abundance (measured as number of adult colonies per transect), suggesting that there are marked differences in the underlying dynamics of coral populations along the GBR [37] . More specifically, since high population replenishment did not correspond with high number of adult colonies, high rates of mortality (and/or reduced growth rates) may be responsible for differences between recruitment and adult populations. Based on these findings, it was expected background rates of mortality would be highest in the northern GBR and lowest in southern GBR. Conversely, background levels of partial mortality were found to be lower in the northern GBR and higher in the central and southern, with greatest variability apparent within sites or reefs, rather than among than sectors. Latitudinal gradients in key environmental variables (e.g., temperature and light) may in part influence rates of background mortality, modifying susceptibility or causing marked differences in recovery capacity. Temperature and light may affect background colony mortality by reducing or increasing regenerative abilities in colonies [53] , [59] , [60] . For instance reduced temperatures have been shown to cause polyps mortality [59] and declines in regeneration rates [53] and it may explain the higher rates of severity of partial mortality measured here in the southern GBR. Similarly, reduced light levels may cause a drop in regeneration rate due to reduced supply of photosynthetic products from zooxanthellae [53] , [56] , [60] . However, in the present study, latitude seemed to play a minor role in driving severity of background partial mortality, with only two of the four coral taxa showing latitudinal variation in the extent of injury. Spatial variation in background mortality can greatly affect the response (e.g., capacity for recovery) of coral populations subject to increasing acute and anthropogenic disturbances. The drivers of the observed spatial variation are still unclear as the source of mortality was often hard to determine. Many A. hyacinthus and P. damicornis colonies had injuries at the edge of the colony suggesting that partial mortality was likely due to agents that were restricted to the bottom such as competition, polychaetes, or gravity causing scouring sand and moving coral fragments [50] . The reef-to-reef and site-to-site variability within sectors observed in Porites and A. hyacinthus , were likely the result of physical and biological routine agents such as fishes, echinoids, asteroids, molluscs, polychaetes, and microorganisms [61] , [62] , acting at spatial scales smaller, equal to or larger than individual reefs. These findings suggest that both disturbance regimes, and the responses of each species to routine agents are irregular, and may vary according to small differences in environmental conditions. The observed spatial variation in tissue loss supports results from other studies [20] , [26] , [63] , [64] , [65] , [66] , [67] showing how coral populations are subject to a wide range of different levels of disturbances and trajectories of recovery. The most notable result from this study, is that variation in the prevalence and severity of partial mortality is most apparent at small (e.g., within reef) rather than larger, latitudinal scales. This shows that the disturbance history is likely to be more variable among colonies at the same site, than it is among disparate populations, suggesting that there is also likely to be marked variation in resilience to acute disturbances at this local scale [68] , [69] , [70] . Colonies with high incidence or severity of injuries are likely to have a generally lower capacity to withstand, and recover from, environmental changes or acute disturbances, leading to intraspecific differences in susceptibility to future acute disturbances. Accordingly, Pisapia et al [71] showed that adjacent colonies may vary greatly in their physiological condition due to localized differences in chronic disturbance regimes, though it is yet to be shown that this then leads to localized selectivity in the effects of major disturbances. Mortality regimes of corals are expected to be strongly size-dependent, whereby the prevalence of partial mortality is expected to increase with colony size, while the probability of whole-colony mortality decreases with colony size [24] , [46] , [72] , [73] , [74] , [75] because at least some portion of the colony is likely to persist in increasingly large colonies. In this study however, neither prevalence or severity of partial mortality showed a strong relationship with colony size; for P. damicornis there was a weak, though significant relationship between severity of injuries and colony size, but no such relationship existed for any other coral taxa. A lack of any relationship between size and severity of partial mortality was also observed in the Caribbean [33] where it was attributed to high variability in the extent of injury across all size classes. Moreover, differences in the repair and regenerative capacities of colonies of different sizes, may obscure such relationship, whereby larger colonies may experience higher incidence of injury, but also have greater capacity for tissue repair [24] , [76] , [77] . An injury of a given size is also going to require greater proportional investment in repair for smaller colonies [46] , [78] . Therefore, repeated measurements through time of observable injuries on the same colonies are needed to better investigate recovery rates and capacity of these taxa. Background levels of partial mortality are likely to have a fundamental effect on the fitness of individual colonies and the capacity of populations to withstand, and recover from, major acute disturbances [34] , [48] . This is the first large-scale study of background levels of partial mortality, testing for large (latitudinal) and small (site) scale differences in the prevalence and severity of injuries across four dominant taxa of scleractinian corals. The findings from this study provide an insight in rates of mortality along the GBR, which can be used as a guideline for global comparisons and for evaluating environmental impacts on reefs or establishing monitoring projects. Instantaneous measures of observable injuries in adult coral colonies allow quantifying mortality events that are visible, and have lasting effects on coral colonies, as well as provide baseline estimates of coral mortality [48] , [79] . However, since coral lesions regenerate at different rates, regeneration can stop before the injuries are fully healed or it can continue for over a year with lesions that do not initially regenerate healing later [80] . Future studies should combine instantaneous measures of observable injuries with repeated measurements over time to provide explicit estimates of the rate of injury. This research also needs to be combined with experimental studies to assess the effects of chronic injuries on colony physiological condition and on the capacity of colonies to withstand major acute disturbances"
} | 4,854 |
30237417 | PMC6147806 | pmc | 2,515 | {
"abstract": "A mechanical flip-flop actuator has been developed that allows for the facile re-routing and distribution of liquid marbles (LMs) in digital microfluidic devices. Shaped loosely like a triangle, the actuating switch pivots from one bistable position to another, being actuated by the very low mass and momentum of a LM rolling under gravity (~4 × 10 −6 kg ms −1 ). The actuator was laser-cut from cast acrylic, held on a PTFE coated pivot, and used a PTFE washer. Due to the rocking motion of the switch, sequential LMs are distributed along different channels, allowing for sequential LMs to traverse parallel paths. This distributing effect can be easily cascaded, for example to evenly divide sequential LMs down four different paths. This lightweight, cheap and versatile actuator has been demonstrated in the design and construction of a LM-operated mechanical multiplication device — establishing its effectiveness. The actuator can be operated solely by gravity, giving it potential use in point-of-care devices in low resource areas.",
"conclusion": "Conclusions Here we have reported on a new routing device for LMs, in the form of an actuating flip-flop switch. This flip-flop has been designed to be lightweight, easy to use, and cheap to manufacture. By optimising the design of the flip-flop and length of the arms, using PTFE washers and a PTFE-coated pivot, we are able to actuate the flip-flop switch with the very low mass and momentum of a LM. Further studies in this field could look at the miniaturisation of the flip-flop switch. Certainly LMs can be made much smaller, however our access to manufacturing techniques restricted the size we could make the flip-flop. However, other materials and manufacturing techniques could be used to make the flip-flop smaller, thereby allowing use of even smaller LMs. It is believed that LMs have a strong future in microfluidics. However for their full use to be realised, an increased toolbox for behavioural control needs to be developed. Typical tools required will include auto-generation, routing, merging and dividing of LMs. This new flip-flop design presents a new routing technique for LMs, not before seen in microfluidics. Its ability to alternate directions of sequential LMs will prove useful. In a digital microfluidic system, the swing of the flip-flop arms and its bistable positions results in sequential LMs being directed into different alternate directions. This novel distributing phenomenon can be cascaded, allowing one input to be divided between 2, 4, 8 … outputs. An example of a 1-input 4-output arrangement is shown in Fig. 3(b) where sequential LMs can be dispensed equally between four distinct channels from one input channel. The demonstration model of the LM-actuated mechanical multiplying device establishes the versatility of this flip-flop design. To the authors knowledge, this also represents the first time that LM digital microfluidics has been partnered with mechanical multiplication. We envision that this flip-flop switch could be of use in point-of-care devices in low resource environments, such as for distributing droplets into separate zones for different reaction conditions. The gravity-powered nature of the design means that no electricity is required, helping to keep running-costs down. Additionally, the systems state is not lost or modified due to a lost of power/input (either electricity or LMs). Many traditional systems will loose their current setting and/or reset when power/input is resumed, for example a counter may reset back to zero. In our mechanical set-up this is not an issue — if the stream of LMs is interrupted for any reason then the system will remain unchanged indefinitely until another LM is introduced to the device.",
"introduction": "Introduction Liquid Marbles (LMs) are small droplets of liquid that have been coated in a nano- or micro-powder 1 . Also known (less theatrically) as particle-coated droplets, the liquid is typically aqueous and the powder coating has a degree of hydrophobicity — resulting in non-wetting of said powder. Due to the hydrophobic nature of this powder, the minimal energy profile of the water droplet results in a near-spherical coated droplet, that does not wet hydrophilic surfaces. Whilst an aqueous droplet with a hydrophobic particle coating is by far the most common form of a LM, there are also examples using an organic liquid core and oleophobic coating 2 . This phenomenon results in the ability to easily transport microlitre quantities of liquid around, with zero loss due to surface adhering/wetting. The advantages to microfluidics are obvious 3 , and are increased by the high-mobility and variability of LM manipulation: controlled movement of LMs has been demonstrated using lasers 4 , magnets 5 , electrostatic-forces 6 , the Marangoni effect 7 , and gravity 1 . Another well-documented use of LMs is as miniature chemical-reactors 8 . By encapsulating one’s reaction in a LM, the reaction is conducted quickly, cheaply and with great ease of parallelisation (allowing for potential use in high-throughput screening). The practical uses of LMs are plentiful. Due to the steadily increasing widespread interest in LMs 9 – 14 , there are reports of their use in a number of fields. For example, LMs have been used as micro-incubators for the viable growth of mammalian embryonic stem cells 15 ; for rapid blood-typing assays by injection of antibodies into a “blood-marble” 16 ; and as signals in mechanical collision-based computation 17 . Previously reported computation with LMs has utilised a conservative logic collision-based approach in the construction of an interaction gate 17 . In this, the Boolean value true was portrayed by the presence of a LM and false was portrayed by the absence of a LM. By having two LMs approaching each other from different directions, computation would be performed once the LMs collide and resultantly change direction; conversely if there is only one LM, then there is no collision and no change in direction. In a pure collision-based computer the LMs can move about in free space, momentarily ‘creating wires’ as required. This, combined with the ease of tuning a LM’s core and coating, allows for an elegant system. This collision-based system, however, requires the precise synchronisation of the data signals (i.e. the LMs). Whilst this has been achieved with the use of magnetic LMs and synchronised electromagnets, it places an additional burden on the experimental computational setup. As such we have developed a sequential mechanical computing device, based on our new LM-actuated flip-flop switch, to demonstrate the new flip-flop actuator. In our design the data signals are represented by LMs, and the result of the computation is displayed in the positioning of the bi-stable flip-flop actuators. These new switches are able to be mechanically actuated by the very low mass and momentum (~4 × 10 −6 kg ms −1 ) of a LM. By having a reproducibly activated flip-flop switch, it is possible to cascade them in series, forming circuits and even computation. Such a system also has great scope as a digital microfluidic platform 18 , 19 . In digital microfluidics, discrete droplets can operate as virtual reaction chambers. In these chambers reactions are carried out on a micro-scale, minimising the use of reagents and cost. Current digital microfluidic use includes point-of-care diagnostics 20 and ad hoc synthesis of hazardous materials 21 . Our actuator allows for the facile distribution of sequential LMs into different parallel paths. There are many methods of actuating droplets in digital microfluidics, with the most common being electrowetting on dielectric (EWOD) 18 , magnetic 20 , and surface acoustic wave (SAW) 22 . These techniques all have advantages and disadvantages, which have been discussed in-depth elsewhere 23 . However, they all share two major disadvantages. Firstly, they require the surface to be pretreated to make it hydrophobic (at a minimum, there are often other surface requirements) — preparing a high-quality low-surface-energy substrate is both expensive and critical to traditional digital microfluidic devices. Secondly, they all require electricity. This second point may seem strange, but many microfluidic point-of-care devices are used in resource-poor environments, where the electrical supply can be unreliable and even batteries can be a luxury 24 . Our device manipulates discrete microlitre droplets of water, through a system of switches and pathways, while negating these two negative points of EWOD, magnetic and SAW actuation: our surface treatment is quick, cheap and easy (being applied with a commercial aerosol); and the device does not require electricity to actuate the LMs. This is achieved by encapsulating the droplets in a hydrophobic powder (i.e. forming LMs), and allowing them to roll through a series of ramps, bridges and switches under gravity. By pre-setting the switches, a series of LMs can be controlled through different pathways, thus allowing different LMs to undergo different processing, when combined with more traditional microfluidic techniques. By joining microfluidics with mechanical computing, a new dimension of processing can be accessed. A simple example of this is a system that counts the number of LMs that have passed through the device. More elaborate designs can conduct calculations, such as multiplication — discussed below and in the Electronic Supplementary Information (ESI).",
"discussion": "Discussion Used as a digital microfluidic platform, this gravity-powered mechanical flip-flop actuator provides many advantages. Firstly is the ability for the flip-flop to evenly distribute sequential LMs into alternate pathways. By arranging an additional flip-flop on each exit path (as demonstrated in Fig. 3(b) ), sequential LMs are evenly divided between four paths. An additional layer of flip-flops allows for even distribution amongst eight paths, and so on. Secondly, it can operate with zero electrical power (the main system uses no power, and the syringe pump is easily replaced by a syringe droplet), making it attractive for situations such as point-of-care diagnostics in developing countries. A potential point-of-care use of our actuator is in the loop-mediated isothermal amplification (LAMP) of DNA. In a digital microfluidic device reported by Wan et al . multiple consecutive droplets are separated into different regions using EWOD 30 . These consecutive droplets could be easily distributed into the required four separate zones using our flip-flop switches. A similar use could be implemented in the digital microfluidic polymerase chain reaction (PCR) device reported by Wulff-Burchfield et al ., with EWOD-powered droplet separation replaced with electricity-free flip-flop switches 31 . Thirdly, LMs are able to encapsulate the aqueous buffer-conditions often required for bioassays, as well as more arduous environments. And fourthly, as the LM moves through the system it is rolling, as oppose to the sliding motion often observed in microfluidics. This imparts a level of mixing to the contents of the LM, which is often highly desirable. In our view, the additional physical features are not discourteous to the ideals of microfluidics. Indeed, many existing microfluidic platforms already instigate physical barriers; such as for pathway restriction, or to physically halt a discrete droplet for dispensing or particle-removal purposes 20 , 32 , 33 . Indeed, the straightforward mechanical nature of the flip-flop means that the switch is highly stable and durable (a repetition video is available in the ESI). Being non-reliant on electricity, it is also resistant to some of the failures of other manipulation methods. The frame-by-frame video analysis revealed that there were two different, but equally effective, techniques for actuating the flip-flop switch via a LM. The first technique is visible in Fig. 4 : the LM enters the flip-flop, rolls down to the extremity of the arm, causing a rotation of the switch, and thereby release of the LM. The second technique became more prevalent if the LM entered the actuator at speed: the LM enters the flip-flop by rebounding off its top point, it then moves quickly along the arm and collides with the upwards-pointing tip of the arm, this impact causes the actuator to start rotation, meanwhile the LM squeezes between the arm and the side-wall and continues along the device before the flip-flop has completed its rotation. A high-speed video of a LM actuating a flip-flop switch was recorded using an fps1000HD-256 camera (Imagetec Ltd). The video was filmed at 1000 fps with a resolution of 1280 × 720, playback was performed at 25 fps for an effective slowdown of 40x. This video, available in the ESI, demonstrated that a combination of the two actuating techniques was sometimes used, also with great effect. Also apparent in this video is the comparatively violent nature of the LMs path. At every impact, collision or change of direction the stresses on the LM cause it to warp in shape. A common side-effect of this is the loss of particle coating from the LM. It should be noted that whilst this loss of coating is very small, it is not negligible, and therefore will eventually amount to destroy the LM by wetting of the surface. This reinforces the notion of LM lifetime 6 , 13 , 28 , 34 . The applied torque (τ app ) is the actual torque applied to the flip-flop switch by the LM. It can be calculated using Eq. ( 1 ), where r is the distance between the mass and the rotation centre (8.5 mm), F is the force acting on the flip-flop (18 mg × 9.81 ms −2 = 1.77 × 10 −4 N) and θ is the angle between the force and the lever arm. In this case, the lever arm and applied force are in line and so the only influence on θ is the tilt of the device (54.8°). This gives a value of applied torque of 1.23 × 10 −6 Nm. 1 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tau }_{{\\rm{app}}}=rF\\,\\sin \\,\\theta $$\\end{document} τ app = r F sin θ The moment of inertia of the flip-flop switch can be calculated using Eq. ( 2 ), where I is the moment of inertia, m is the mass of the object, and k is the radius of gyration. The radius of gyration was determined as 6.76 mm, using Autodesk AutoCAD 2018. This gives a moment of inertia of 7.31 × 10 −9 kg m 2 . 2 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$I=m{k}^{2}$$\\end{document} I = m k 2 The net torque (τ net ) is the resulting torque on the system, after frictional effects. Knowing the moment of inertia, it is possible to calculate the net torque using Eq. ( 3 ). Here, α is the angular acceleration of the flip-flop switch. This was determined from the high-speed video to be 152 rad s −2 . The resulting net torque is 1.11 × 10 −6 Nm. The frictional effects of the system can be quantitatively determined as the friction torque (τ F ). This value can be calculated using Eq. ( 4 ), as the ‘missing torque’ between applied and net torque values. In our system, the friction torque was 1.20 × 10 −7 Nm. 3 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tau }_{{\\rm{net}}}=I\\alpha $$\\end{document} τ net = I α 4 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tau }_{{\\rm{net}}}={\\tau }_{{\\rm{app}}}+{\\tau }_{{\\rm{F}}}$$\\end{document} τ net = τ app + τ F The total work done ( W tot ) by a LM actuating a single flip-flop can be described using Eq. ( 5 ), where θ rot is the total rotation of the flip-flop in radians. The the flipping of the switch from −23° to +23° represents a total rotation of 0.80 rad. Using the values calculated for torque, the total work performed is 9.84 × 10 −7 J. 5 \\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}$${W}_{{\\rm{tot}}}={\\tau }_{{\\rm{app}}}{\\theta }_{{\\rm{rot}}}$$\\end{document} W tot = τ app θ rot From this, it can be said that the speed of the LM entering the flip-flop is not a determining feature. As long as the LM has entered the flip-flop, it will actuate the switch using its gravitational potential energy, before exiting and rolling downhill to the next flip-flop. What is significant is the weight of the LM, as described by F in Eq. ( 1 ) — which must be large enough to have sufficient torque to rotate the flip-flop. A curious observation was made when two LMs were permitted to roll next to each other. When the two LMs make contact, they do not roll together like smooth ball-bearings. Instead, they bounce off each other. This is because the rear of the leading LM is moving upwards, whilst the front of the approaching LM is moving downwards. When the two opposite directing motions meet they clash, resulting in the chasing LM rolling backwards briefly. This is caused by the rough texture of the LM surface. An interesting feature of these flip-flops, is that they do not fully actuate if the LM is too small. Instead, the small LM moves along and around the flip-flop, leaving it in the same position. This phenomenon could be used for sorting LMs. If a string of small LMs approaches the flip-flop they will all exit along the same path, until a large LM approaches and actuates the flip-flop, after which the small LMs will exit along the other path, until a large LM approaches and actuates the switch again. By designing a system to operate on LMs, the evaporation rate of the droplets is reduced 28 . Therefore, it is not necessary to cover the droplets with oil to prevent evaporation, as is often the case with more traditional microfluidic systems; or to form a phospholipid layer around the droplets 35 . There are many advantages to this: heating the sample requires much less energy, and so can be done quicker; there is no risk of cross-contamination from reagents diffusing through the oil 20 ; and costs are kept down, by both the reduced energy use as well as the decrease in oil/solvent use. A possible future development of the multiplication device would be to investigate the use of liquid metal marbles (LMMs) 36 . Due to the powder coating inherently present on a LMM, the high surface tension of the liquid metal (e.g. Galinstan) would not be an advantage directly — however it would enable the use of a wider range of powders. The high cohesive forces within liquid metals results in a very high surface tension (534.6 mN m −1 ) 37 , and minimal adhesive forces between both the powder coating and the device surface. This means that LMMs would be less prone to premature rupture or or accidental surface wetting. Indeed, the wettability of liquid metals has already been shown to be tunable 38 . The greater density of the liquid metal core would also be advantageous in actuating the flip-flop switches. Additionally, initial investigations towards the regular formation of liquid metal droplets — important for the automatic creation of LMMs — has been reported by others already 39 ."
} | 4,986 |
28541580 | PMC5982782 | pmc | 2,516 | {
"abstract": "Abstract Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.",
"conclusion": "Conclusions The efficient conversion of lignocellulosic biomass to biofuels varies with wood composition, which itself is subject to natural genetic variation and is dependent on environmental conditions, such as water availability. Previous research on wood traits that may affect saccharification mainly focused on lignin, revealing opportunities for improving biomass quality by reducing lignin content. However, there is also growing awareness that this approach has limitations ( Voelker et al. 2010 , Tavares et al. 2015 ). By studying variation in wood traits and saccharification potential in three genotypes of P. nigra exposed to a moderate drought treatment, we show that glucose release was not impaired, but moderately improved, by gradually decreasing water availability. Interestingly, the saccharification potential was not related to lignin content or expression of genes related to lignin biosynthesis. Instead, our research identified transcriptional regulation of biosynthesis of hemicelluloses and modification of pectins as potential targets for improving wood quality for the production of biofuels. Further experiments are needed in which the expression of the identified candidate genes is modified by overexpression or suppression to test the causal relationship of the identified genes and pathways with saccharification potential.",
"introduction": "Introduction Wood is an attractive feedstock for biofuels because it is a renewable resource that can be produced in a sustainable manner ( Polle et al. 2013 , Weih and Polle 2016 ). Wood is composed mainly of cellulose (35–50%), hemicelluloses (15–35%), lignin (15–35%) and pectins (<10%) ( Plomion et al. 2001 ). Glucose can be released from cellulosic compounds in the cell wall by saccharification and subsequently used for conversion into ethanol ( Galbe and Zacchi 2002 ). Lignin and hemicelluloses and their interactions with pectin or cellulose can negatively impact the efficiency of biofuel production ( Mansfield et al. 1999 , Nookaraju et al. 2013 , Xiao and Anderson 2013 ). For example, transgenic lines of Populus trichocarpa with suppressed expression of 4-coumarate:coenzyme A ligase contained lower lignin contents and exhibited greater saccharification potential than the wild type ( Min et al. 2012 ). In Populus × canescens , in which cinnamoyl-CoA reductase, a key enzyme for lignin biosynthesis, was down-regulated, the cell walls were more easily digestible, which resulted in higher saccharification and ethanol yield ( Acker et al. 2014 ). The saccharification potential of wood is also affected by environmental conditions such as drought ( Fiasconaro et al. 2012 , van der Weijde et al. 2016 ). Drought brings about changes in cell wall composition including an increased abundance of hemicellulose, pectins and lignin, which results in strengthening of the cell wall ( Moore et al. 2008 , Le Gall et al. 2015 ). Fiasconaro et al. (2012) reported that the potential for biofuel conversion in drought-treated alfalfa plants decreased due to a higher lignin content compared with the well-watered plants. On the other hand, in a study comprising 50 different Miscanthus genotypes, drought treatment caused a reduction of cell wall cellulose content and an increase in cell wall hemicellulose content, but nevertheless resulted in a higher efficiency of the conversion of cellulose to glucose during saccharification ( van der Weijde et al. 2016 ). These contrasting findings suggest that the effect of drought on saccharification potential depends largely on the particular species studied, and on the relative changes of cell wall components under drought. In poplar, drought stress has massive consequences for the wood anatomy and cell wall metabolism ( Harvey and Van Den Driessche 1997 , Arend and Fromm 2007 , Beniwal et al. 2010 , Fichot et al. 2009 , 2010 , Schreiber et al. 2011 , Cao et al. 2014 , Guet et al. 2015 , Le Gall et al. 2015 ), but studies on the effects of drought on the saccharification potential of Populus and the underlying anatomical and molecular responses have not yet been reported, but are greatly needed ( Studer et al. 2011 ). The saccharification potential is also subject to genetic variation, as shown for Miscanthus ( Souza et al. 2015 ) and poplar ( Studer et al. 2011 ). Similarly, natural genetic variation in lignin content was reported for many taxa, including Arabidopsis thaliana ( Capron et al. 2013 ), eucalyptus ( Klash et al. 2010 , Elissetche et al. 2011 ), conifers ( Gonzalez-Martinez et al. 2007 , Gaspar et al. 2011 ) and poplars ( Wegrzyn et al. 2010 , Zhou et al. 2011 , Guerra et al. 2013 , Porth et al. 2013 , Muchero et al. 2015 ). Given the importance of the genus Populus as a second generation bioenergy crop ( Allwright and Taylor 2016 ), it is of great interest to study whether these variations translate into variation in saccharification potential. Attempts to improve the saccharification potential of bioenergy crops require insights into the molecular control of wood properties. Several studies using quantitative trait locus (QTL) mapping or association mapping approaches identified candidate genes related to cell wall composition, as well as lignin content and composition ( Ranjan et al. 2009 , Guerra et al. 2013 , Porth et al. 2013 , Fahrenkrog et al. 2016 ). However, such approaches have only rarely been applied to uncover candidate genes or QTLs related to sugar release ( Brereton et al. 2010 , Muchero et al. 2015 ). Knowledge on transcriptional networks controlling diverse aspects of wood formation including secondary cell wall formation and biosynthesis of its components has accumulated during recent years (for recent reviews: Zhong et al., 2010 , Hussey et al., 2013 , Nakano et al., 2015 , Ye and Zhong 2015 ). However, a systematic transcriptome-wide investigation of genes and gene clusters underlying genetic variation in saccharification potential in economically important bioenergy crops such as Populus is missing. The aim of this study was to identify clusters of co-expressed genes, as well as candidate genes and their putative transcriptional regulators, related to genotype- and drought-induced variation in wood anatomy, lignin content and saccharification potential. We hypothesized (i) that drought results in increased lignification and decreased saccharification, (ii) that genotypes originating from different environments differ in lignin content and saccharification potential and (iii) that these drought and genotype-effects on saccharification potential are underpinned by distinct differences in wood anatomical traits and transcript abundances of genes involved in wood formation and cell wall metabolism, especially lignin biosynthesis. To this end, we used three genotypes that were selected from large-scale common garden experiments with up to 13 different Populus nigra L. populations ( DeWoody et al. 2015 , Viger et al. 2016 ). The P. nigra populations originated from habitats with different climatic conditions across Europe and showed significant genetic differentiation as well as phenotypic variation in growth rates, plant architecture and leaf size ( DeWoody et al. 2015 , Viger et al. 2016 ). The most pronounced differences among the phenotypic traits were observed between P. nigra genotypes originating from a dry habitat in Spain and those from a wet habitat in Italy, while a French genotype exhibited intermediate properties ( Viger et al. 2016 ). Here, we exposed these contrasting genotypes to a highly controlled drought treatment, applied RNA sequencing and weighted gene correlation network analysis (WGCNA) to identify novel candidate genes related to wood properties and saccharification potential.",
"discussion": "Discussion Drought-induced variations in wood anatomical traits are independent from genotype Drought significantly reduced, but did not abolish diameter increment in all genotypes, along with a reduction in the number of cambial cell layers (Table 1 ), indicating a reduced cambial activity under these conditions. These findings agree with previous studies ( Arend and Fromm 2007 , Bogeat-Triboulot et al. 2007 ) and demonstrate that the moderate, gradually increasing drought, applied in a highly controlled manner, was effective to induce acclimation processes. Such moderate yet significant treatment effects mimicking ecologically relevant stress conditions are suited to study the molecular control of drought-induced and genotypic variation in wood traits. Along with a reduction in cambial activity, a significant increase in the fraction of cell wall area of vessels, fibers and rays under drought was noted, while vessel and fiber wall thickness were not affected by drought in any of the genotypes (Table 1 ). Interestingly, the three P. nigra genotypes studied here showed similar drought-induced changes in wood anatomy, although the wood structures clearly differed among the genotypes. Constitutive differences were also apparent among the transcriptomes because the majority, i.e., 67% of all tested genes, showed genotype-related expressional differences in the developing xylem. This observation underlines that substantial genotypic variations in transcript abundance exist not only in leaves and roots (leaves: Wilkins et al. 2009 , roots and leaves: Cohen et al. 2010 , leaves: Hamanishi et al. 2010 , DeWoody et al. 2015 ) but also in wood-forming tissues (this study). Under the current experimental conditions, which allowed gradual acclimation to drought, surprisingly no genotype × drought interaction was detected for any gene. Thus, differences in wood anatomy are the result of constitutive differences among the transcriptomes, while flexible adjustment of wood anatomy to variation in water availability is apparently mediated by similar molecular programs. Drought-induced and genotype-related variations in wood anatomical traits and lignin content are not related to variation in saccharification potential In contrast to our initial hypothesis that drought will cause an increase in lignification ( Moore et al. 2008 , Le Gall et al. 2015 ), the lignin content was not affected under drought. We had further hypothesized that along with an increased lignification, drought will cause a reduction in saccharification potential. Our data do not support these hypotheses, since we observed a significant increase in saccharification potential under drought across all genotypes (Figure 1 ) and no correlation with lignin or with wood anatomy. We are not aware of any previous studies on drought effects on saccharification potential in woody dicots, but van der Weijde et al. (2016) reported an increase in saccharification potential of Miscanthus under drought. These observations suggest that drought may cause alterations in cell wall chemistry apart from lignin that facilitate the enzymatic release of glucose. Important bioenergy crops such as Miscanthus ( Souza et al. 2015 ), Eucalyptus ( Klash et al. 2010 , Elissetche et al. 2011 ), and Populus ( Wegrzyn et al. 2010 , Studer et al. 2011 , Zhou et al. 2011 , Guerra et al. 2013 , Porth et al. 2013 , Muchero et al. 2015 , Fahrenkrog et al. 2016 ) exhibit substantial natural genetic variation in lignin content. Likewise, saccharification potential of bioenergy crops showed genetic variation in earlier studies ( Studer et al. 2011 , Souza et al. 2015 , van der Weijde et al. 2016 ). In agreement with these findings, P. nigra also showed genotypic variations in lignin content and in saccharification potential (Table 1 , Figure 1 ). Our hypothesis that adaptation of poplars to low water availability in their native habitat correlates with high lignin content and low saccharification potential has to be refuted: saccharification potential was unexpectedly highest in genotype Spain, originating from the driest habitat, whereas the lignin content of the Spain genotype was not different from that of the Italy genotype, which originated from the wettest habitat (Table 1 , Figure 1 ). Previous studies also reported inconsistent results concerning the relationship between lignin content and saccharification potential. Some studies with transgenic poplars with low lignin content reported negative correlations between lignin and saccharification potential ( Min et al. 2012 , Acker et al. 2014 ), while Voelker et al. (2010) found unaffected saccharification potential when lignin decrease was modest, and decreasing saccharification in transgenic poplar lines with strongly reduced lignin content. Studer et al. (2011) did not find a general negative relationship between natural variation in lignin content of P. trichocarpa and sugar release, but reported that the effect of lignin on saccharification depends on the lignin composition and pretreatment methods. In Miscanthus natural genetic variation in lignin content was not consistently correlated with saccharification potential ( Souza et al. 2015 ). In a study with maize, QTLs for lignin content were different from those detected for saccharification ( Penning et al. 2014 ). Altogether, these studies suggest that lignin is not a good predictor for the saccharification potential. Transcriptional regulation of genes involved in cell wall polysaccharide biosynthesis and modification is related to genotypic variation in saccharification potential To uncover the molecular basis for differences in saccharification potential we re-constructed transcriptional networks and found that co-expression modules correlated with this trait were enriched in distinct sets of GO terms (Table S2 available as Supplementary Data at Tree Physiology Online). This finding underpins the assumption that modules summarize functionally related genes. The functional categories pointed to an involvement of cell wall metabolism, especially cell wall polysaccharides (bin 10), and vesicle-associated secretory processes (bins 31.4 and 29.3.4) in the control of saccharification potential. In agreement with lacking correlation between lignin content and saccharification potential, categories related to lignin biosynthesis were not enriched, suggesting that variation in expression of genes involved in cell wall polysaccharide biosynthesis is more important for saccharification potential than moderate variations in lignin. However, among the top saccharification-correlated genes, none of the biosynthetic genes for cell wall polysaccharides was detected. Instead three putative kinases (Potri.002G019300, Potri.007G039800, Potri.010G155600) and one gene involved in brassinosteroid metabolism (Potri.011G155600) were present among the top 10 correlated genes (Figure 4 ), suggesting that signaling and hormone regulation are important check points for the molecular control of saccharification potential. Surprisingly, most GDEGs assigned to cellulose biosynthesis (bins 10.2, 10.2.1, 10.2.2) were negatively correlated with saccharification potential (Figure 5 A, Table S5 available as Supplementary Data at Tree Physiology Online). Saccharification potential depends not only on cellulose content, but also on the level of cellulose aggregation and thus accessibility for enzymatic degradation ( Nookaraju et al. 2013 ). Primary cell walls contain cellulose microfibrils with low aggregation, whereas during the synthesis of secondary cell walls, highly aggregated microfibrils are formed ( Li et al. 2016 ). Synthesis of cellulose in primary and secondary cell walls involves distinct sets of genes ( Li et al. 2016 ). It is thus possible that the GDEGs that were negatively correlated with saccharification potential (Table S5 available as Supplementary Data at Tree Physiology Online) represent poplar cellulose synthase genes involved in the synthesis of highly aggregated cellulose microfibrils. Although pectins comprise only a small fraction of secondary cell walls ( Harholt et al. 2010 ) there is accumulating evidence that they play a role in determining saccharification potential, by mediating cell adhesion, masking cellulose and hemicelluloses and thus restricting their accessibility by enzymes ( Xiao and Anderson 2013 , Tavares et al. 2015 ). The effect of pectins on saccharification is dependent on the degree of pectin acetylation and methyl-esterification ( Xiao and Anderson 2013 ). Once secreted into the apoplast, pectins can be de-methylesterified by pectin methylesterases, enabling cross-linking and possibly increasing cell wall stiffness ( Xiao and Anderson 2013 ). A reduction of de-methylesterified pectins was found to increase saccharification potential in Arabidopsis , wheat and tobacco ( Lionetti et al. 2010 ). In agreement with these findings, putative pectin methylesterases (bins 10.8.1 and 10.8.2) were mostly negatively correlated with saccharification potential (Figure 5 , Table S5 available as Supplementary Data at Tree Physiology Online). The major compound of hemicellulose and second most abundant polysaccharide in woody dicots is glucuronoxylan ( Awano et al. 1998 , Lee et al. 2009 ). Here the transcript abundances of two genes related to glucuronoxylan biosynthesis (Potri.002G132900 and Potri.016G086400, of bin 10.3.2) were negatively correlated with saccharification potential (Figure 5 A, Table S5 available as Supplementary Data at Tree Physiology Online). There is experimental evidence that the P . × canescens orthologs of Potri.002G132900 and Potri.016G086400 are functional homologs of Arabidopsis PARVUS ( Lee et al. 2009 ) and IRX9 ( Zhou et al. 2007 , Lee et al. 2011 ), respectively. PARVUS (At1g19300) and IRX9 (At2g37090) are required for the synthesis of the tetrasaccharide reducing end sequence of glucuronoxylans, and the xylosyl backbone of glucuronoxylans, respectively ( Brown et al. 2005 , 2007 , Lee et al. 2007 a , 2007 b , Peña et al. 2007 ). Lee et al. (2011) found that RNAi down-regulation of the P. × canescens ortholog of Potri.016G086400 resulted in an increased glucose release from wood. Remarkably, UDP-glucuronic acid decarboxylase (UXS) transcript abundances were negatively correlated with saccharification potential (GDEGs in bin 10.1.5, Figure 5 B, Table S5 available as Supplementary Data at Tree Physiology Online). UXS enzymes catalyze the irreversible synthesis of UDP-xylose from UDP-glucuronic acid ( Kuang et al. 2016 ), and are key enzymes for nucleotide sugar partitioning for cell wall polysaccharide biosynthesis ( Du et al. 2013 ). In transgenic tobacco, down-regulation of UXS genes led to a significant increase in saccharification efficiency ( Cook et al. 2012 ). Taken together, these findings illustrate the power of our experimental and analytical approach to identify candidate genes related to the regulation of wood traits. Putative transcriptional regulators of saccharification-related genes involved in cell wall metabolism To identify putative transcriptional regulators of saccharification-related genes involved in cell wall metabolism, we used the identified cell wall genes as baits in an in silico co-expression analysis. Among the genes with a significant correlation with saccharification, 31 potential transcription factors were identified that showed significant co-expression with cell wall biosynthesis genes in Arabidopsis and were also present among the poplar genes, significantly correlated with saccharification (Table 3 ). Among these genes, MYB domain, WRKY and homeobox domain transcription factors that belong to families known to be involved in wood formation were retrieved ( Hussey et al. 2013 , Yang and Wang 2016 ). For example, two putative pectin esterases (Potri.014G127000, Potri.011G135000) were co-expressed with WRKY, MYB and bZIP transcription factors. Interestingly, a gene encoding a putative UXS protein (Potri.010G207200), which appears to be important for the saccharification potential as discussed above, was co-expressed with a MYB family (Potri.010G193000) and TGA1 (Potri.007G085700) transcription factor. MYB transcription factors are known to play a role in the regulation of xylan biosynthesis ( Rennie and Scheller 2014 ), but to our knowledge, no published results are available for the specific MYB family members highlighted by our study. In addition, the gene set of co-expressed transcription factors contained novel candidate regulators such as TGACG SEQUENCE-SPECIFIC BINDING PROTEIN 1 (TGA1), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 (SPL4), AGAMOUS-LIKE 8 (AGL8), SCARECROW, AUXIN RESPONSE FACTOR 8 (ARF8), ANTEGUMENTA (ANT), etc. These transcription factors play roles in redox regulation (TGA) and development. Our approach suggests that developmental changes brought about by drought or genetic differentiation among the genotypes also control cell wall composition that affects saccharification. Therefore, the present correlative results provide an excellent platform for future targeted approaches to improve the saccharification potential of poplar."
} | 5,784 |
22266340 | null | s2 | 2,517 | {
"abstract": "Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30 min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme."
} | 394 |
22266340 | null | s2 | 2,518 | {
"abstract": "Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30 min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme."
} | 394 |
19000309 | PMC2614490 | pmc | 2,519 | {
"abstract": "Sequencing of the complete genome of Ignicoccus hospitalis gives insight into its association with another species of Archaea, Nanoarchaeum equitans.",
"conclusion": "Conclusion The combinations of ecophysiological and morphological features that collectively enable the I. hospitalis-N. equitans relationship are encoded within a surprisingly simple genomic blueprint. The genome of I. hospitalis is the smallest among free-living bacteria and archaea, shows evidence of gene exchange with N. equitans and encodes streamlined biochemical functions necessary for a chemoautotrophic metabolism relying on carbon dioxide, hydrogen and sulfur. Aside from selection pressure against genome expansion in a restrictive environmental niche, the two organisms have coevolved, leading to symbiotic specificity and gene exchange. In addition, I. hospitalis appears to have acquired a significant number of genes and predicted operons from Bacteria and Euryarchaeota, some of them encoding membrane-associated complexes involved in transport and energetic metabolism. This unicellular symbiotic system might resemble relationships that gave rise to eukaryotic organelles. The availability of complete genomic data for both organisms opens the possibility to study interspecific gene regulatory networks and identify proteins that might be exchanged between interacting cells.",
"discussion": "Results and discussion A minimal genome The genome of I. hospitalis consists of a single circular chromosome (Table 1 ). At 1,297,538 bp, the genome of I. hospitalis is the smallest among free-living organisms, which do not require a continuous association with another species and can replicate independently (Figure 1 ). Even the combined gene complement of I. hospitalis and N. equitans (1,434 and 556 protein-coding genes, respectively) is significantly smaller than that of average free-living bacteria (approximately 3,600 genes) or archaea (approximately 2,300 genes), based on the available completed genomes. The size distribution of 623 complete microbial genomes indicates that the 1-2 Mbp range includes both obligate symbionts/parasites as well as free living bacteria and archaea (Figure 1 ). The minimal genome for free-living organisms may therefore be on the order of 1 Mbp, several taxonomically and metabolically distant archaeal and bacterial lineages having independently reached near-minimal functional gene sets for their respective ecological niches. Table 1 General features of the I. hospitalis genome Parameter Value % Chromosome size (bp) 1,297,538 Chromosome G+C content 56.5 Total number of genes 1,494 100 Protein coding genes .1,444 96.6 RNA genes .50 3.3 Genes with function prediction .885 59.2 Genes without function prediction 559 37.4 Genes in ortholog clusters .1,149 76.9 Genes in paralog clusters .406 27.2 Fusion genes .27 1.8 Genes assigned to COGs .972 65.1 Genes assigned to arCOGs 1,155 80.5 Genes assigned to Pfam domains .875 58.6 Genes with signal peptides .213 14.3 Genes with transmembrane helices .216 14.5 Putative pseudogenes (RNA + proteins) 12 0.8 Figure 1 Relationship between the genome size and the number of protein-coding genes in 623 complete archaeal and bacterial genomes, based on data in IMG version 2.5 (March 2008). The line points to I. hospitalis having the smallest genome among independently replicating organisms. The genomes of obligate parasites/symbionts are represented by grey circles. The shaded region of genome sizes spans the transition between obligate symbionts/parasites and free-living organisms. The sizes of microbial genomes are the result of dynamic equilibria between contraction by deletions and expansion due to duplications, lateral gene transfer and insertion of mobile DNA. For free-living organisms with very large effective population sizes, genome streamlining is likely to be a selective consequence of reducing the metabolic burden to maintain DNA of little adaptive value, as illustrated by the genomes of such highly successful and widespread lineages as Prochlorococcus and Pelagibacter [ 20 , 21 ]. An alternative (but not necessarily exclusive) hypothesis links genome reduction to elevated mutation rates in large populations. Accumulation of mutations could lead to inactivation and loss of genes that make weak contribution to the fitness of the respective organisms [ 22 ]. Ignicoccus , however, inhabits heterogeneous, geographically dispersed and relatively ephemeral hydrothermal marine environments. Such organisms generally have small effective populations and experience periodic bottlenecks and limited gene flow [ 23 ]. Conceivably, in a case like this, genome contraction might have to do with the very active recombination and DNA repair that organisms inhabiting extreme environments employ for maintaining genomic integrity. Frequent recombination might not only efficiently remove deleterious mutations induced by the environmental conditions but also generate diversity and increase the fixation rate of adaptive alleles [ 24 , 25 ]. A high frequency of illegitimate, intra-chromosome recombination could also be effective in preventing genome expansion by increasing the frequency of deletions and counteracting gene duplication. This might explain the reduced genome size in many members of the Archaea and contribute to their proposed higher adaptability to chronic energy stress [ 26 ]. While we expect these general principles to be valid in the Nanoarchaeum-Ignicoccus system as well, the co-evolution of these two organisms also left unique imprints on their physiology [ 2 , 3 ]. The most striking effect of this co-evolution, however, is the massive gene loss in N. equitans , resembling that of obligate intracellular bacterial symbionts and, as an extreme case, that of eukaryotic organelles [ 12 ]. The recently published database of archaeal clusters of orthologous genes (arCOGs) provides a framework for comparing the I. hospitalis genomic data to genes from 41 previously sequenced archaeal genomes organized into sets of probable orthologs [ 27 ]. Of the 1,434 annotated I. hospitalis protein-coding genes, 1,155 (80.5%) were assigned to arCOGs, a coverage that is the lowest among the Desulfurococcales (85% on average) and overall among thermophilic Crenarchaeota. I. hospitalis lacks orthologs of 19 genes from the Crenarchaeota core (that is, genes that are represented in all 12 available genomes of thermophilic species of Crenarchaeota included in the arCOGs) [ 27 ] (Table S1 in Additional data file 1). None of these genes include components of information processing systems, indicating that these systems are largely intact in I. hospitalis despite the small genome. The missing genes encode, primarily, diverse metabolic enzymes, some of which - for example, thymidylate kinase - catalyze essential reactions. Conceivably, these enzymes are substituted for by distant homologs that so far remain undetected or by analogs. Using the assignment of I. hospitalis genes to arCOGs, we applied weighted parsimony to perform a reconstruction of gene gain and loss events in archaea [ 27 , 28 ], with an emphasis on the I. hospitalis lineage. The small genome size appears to be a result of gene loss that has vastly predominated the evolution of this lineage: it was inferred that approximately 484 arCOGs were lost, compared to the inferred gain of only 56. Approximately 946 arCOGs (1,094 genes, representing 76% of the I. hospitalis gene set) appear to have been inherited from the last common ancestor of the Desulforococcales, the order to which Ignicoccus belongs, together with Aeropyrum pernix, Hyperthermus butylicus and Staphylothermus marinus . The functional distribution of the lost genes is consistent with the fact that I. hospitalis is an obligate anaerobic autotroph. In contrast to A. pernix , numerous genes related to aerobic metabolism as well as catabolism and transport of amino acids, sugar and nucleotides were lost, along with many transcriptional regulators (Figure 2 ; Table S2 in Additional data file 1). An analysis of arCOGs that are present in N. equitans but absent in I. hospitalis does not suggest that the inferred gene loss in I. hospitalis was accompanied by transfer of potentially essential functions to the symbiont (Table S3 in Additional data file 1). Ignicoccus is far removed from the root of the tree of thermophilic Crenarchaeota (whether the tree is constructed for rRNA or various informational proteins), and the tree, including basal branches, is dominated by heterotrophs and mixotrophs (Figure S1 in Additional data file 2). Thus, the alternative scenario, namely, that Ignicoccus reflects the ancestral state for this entire group, is not supported by the phylogenetic analyses. However, this might reflect our incomplete sampling of the archaeal diversity and the bias towards isolation and characterization of heterotrophs. A better understanding of the direction of evolution in archaeal genome size and architecture will require a significant increase in the number and diversity of cultivated species and sequenced genomes, including close relatives of I. hospitalis and additional chemolithoautotrophs. Figure 2 Numbers of arCOGs in different functional categories (COG classification) lost or gained in the I. hospitalis lineage. The sets of lost and gained genes were derived on the basis of a comparison of the I. hospitalis gene compliment with the reconstructed gene set of the last common ancestor of Desulfurococcales [ 27 ] (see Additional data files). The numbers of arCOGs in each category that are present in N. equitans but are absent in I. hospitalis are also indicated. The one letter code for COG categories is the following: amino acid transport and metabolism (E); carbohydrate transport and metabolism (G); cell cycle control, cell division, chromosome partitioning (D); cell motility (N); cell wall/membrane/envelope biogenesis (M); coenzyme transport and metabolism (H); defense mechanisms (V); energy production and conversion (C); inorganic ion transport and metabolism (P); intracellular trafficking, secretion, and vesicular transport (U); lipid transport and metabolism (I); nucleotide transport and metabolism (F); posttranslational modification, protein turnover, chaperones (O); replication, recombination and repair (L); secondary metabolites biosynthesis, transport and catabolism (Q); signal transduction mechanisms (T); transcription (K); and translation, ribosomal structure and biogenesis (J). The reduced frequency of duplicated genes (paralogs) in I. hospitalis compared to all other archaea except N. equitans (Figure 3 ) and the absence of transposable elements support the hypothesis of genome streamlining. Furthermore, approximately 180 chromosomal gene clusters that are typically conserved in archaea are disrupted in the genome, including some of the ribosomal operons as well as those encoding the proteasome components, ATP synthase and DNA topoisomerase VI. As it is unlikely that so many gene clusters and operons have independently assembled in archaeal lineages not directly related, the architecture of the I. hospitalis genome suggests that recombination events have resulted in gene cluster fragmentation, deletions, and may have restricted gene family expansion. On the other hand, it is notable that several families of paralogous genes are uniquely expanded in I. hospitalis (Table S4 in Additional data file 1). The most intriguing is the presence of 10 genes that encode WD40-repeat-containing proteins. Proteins containing WD40 repeats are among the most abundant and highly conserved in eukaryotes, where they are key structural components of a variety of macromolecular complexes [ 29 ]. Proteins containing these repeats are also widely scattered among archaea and bacteria but are mostly encoded in (relatively) large genomes [ 30 ]. In particular, among archaea, we have detected comparable expansions of WD40-containing proteins only in Methanosarcinales, a group of Euryarchaeota that displays significant gene gain [ 27 ]. Conceivably, the WD40-proteins of I. hospitalis are involved in the organization of specific protein complexes and/or cellular compartments, and potentially might contribute to the interaction with N. equitans . Similarly, I. hospitalis encodes 9 proteins containing the V4R domain and 12 proteins containing the CBS domain, both small-molecule-binding domains that are likely to be involved in metabolic regulation and signaling [ 31 , 32 ] (Table S4 in Additional data file 1). Considering the homology identified between the V4R domain and a component of the eukaryotic Golgi vesicle transport machinery [ 33 ], some of the expanded V4R gene family members also might be implicated in the unique vesicle formation process that has been observed in Ignicoccus [ 9 ]. Figure 3 Paralog distribution in completely sequenced archaeal genomes. (a) The average number of paralogs in arCOGs for completely sequenced archaeal genomes. The arrows point to the vales for N. equitans and I. hospitalis genomes, respectively. (b) Paralog density in completed genomes of species from the order Desulfurococcales and in N. equitans , determined by blastclust using a variable identity threshold over at least 50% of the aligned pairs of sequences. In addition to streamlining, selection for reducing metabolic cost in I. hospitalis may have impacted its proteome composition. In hyperthermophiles, certain biases in amino acid usage have been associated with side chain physical and chemical properties that contribute to increased protein stability [ 34 , 35 ]. For example, a preference for lysine over arginine has been attributed to a greater flexibility of the lysine side chain, which entropically stabilizes the folded state of proteins [ 36 ]. While the overall amino acid usage in the N. equitans - I. hospitalis proteomes follows the distribution observed for other hyperthemophiles, there is a significant increase in lysine over arginine usage in I. hospitalis relative to the values that could be predicted from the GC content (Figure 4 ; note that the two positively charged amino acids, lysine and arginine, are often interchangeable in proteins but are encoded by contrasting codons, namely AAA/G for lysine, and CGX and AGA/G for arginine, hence the strong correlation of the abundance of these amino acids with the GC content). This discrepancy could be explained by selection at the genomic level against using the metabolically more expensive arginine. Arginine biosynthesis in Ignicoccus is predicted to proceed via carbamoyl-phosphate and would require five ATP equivalents, whereas lysine, synthesized from 2-oxoglutarate via the aminoadipate pathway, would use two ATP equivalents (Figure 4 ). Metabolic cost and nutrient availability have been proposed to play a selective role in the evolution of genome size, GC content and amino acid use in organisms that inhabit oligotrophic or energetically poor environments [ 20 , 26 , 37 ]. Since sulfur-hydrogen respiration is energetically weak [ 38 ], such genomic and proteomic adaptations allow I. hospitalis not only to be a competitive vent colonizer but also to support N. equitans . At present, in the absence of sequence data from other species of Ignicoccus , we cannot distinguish if the relationship with N. equitans has directly influenced these genomic features of I. hospitalis . Figure 4 Lysine and arginine use in archaeal proteomes, relative to genome G+C content. The dotted lines represent the linear fit to the hyperthermophile data and the goodness of fit values. The archaeal classification as hyperthermophiles, thermophiles and mesophiles follows that of the NCBI Genome Project database [ 100 ]. The proposed pathways for the biosynthesis of the two amino acids, the genes predicted to be involved and the metabolic costs of the two reactions are shown below the graphs. Lateral gene transfer The cell-cell contact between I. hospitalis and N. equitans seems to present an opportunity for extensive lateral gene transfer (LGT). LGT is considered to play a major role in microbial genome evolution and is well-documented in symbiotic systems and in environmental microbial communities [ 39 - 42 ]. Recent LGT events are readily detected with various methods based on nucleotide composition or codon usage, but methods that rely on protein sequence similarity and phylogenetic trees are more informative for ancient LGT events [ 43 ]. To analyze the I. hospitalis genome for potential LGT events, we therefore combined automatic genome-wide phylogenetic reconstruction using PyPhy [ 44 ] with similarity searches and COG distribution analysis. The LGT candidates were further analyzed using hand-curated alignments and maximum likelihood phylogenetic analyses. Identifying the LGT direction requires analysis of conflicts between the topologies of the corresponding gene trees and the adopted species tree. The position of N. equitans within the Archaea is controversial and ranges from representing a distinct and basal phylum [ 1 , 12 , 16 ] to being a derived member of order Thermococcales from the Euryarchaeota [ 17 ]. Many gene trees identify the Thermococcales as an early diverging lineage, which further complicates this distinction. Ignicoccus on the other hand has been confidently assigned to order Desulfurococcales from the Crenarchaeota based on phylogenetic and arCOG analysis. Therefore, when attempting to infer direction of LGT, we relied on the phylogenetic placing of N. equitans and I. hospitalis genes relative to other crenarchaeal homologues, especially those from the Desulfurococcales ( Aeropyrum , Hyperthermus and Staphylothermus ). A small fraction of I. hospitalis genes (approximately 6%) appear to have been transferred from lineages within Euryarchaeota, while approximately 4% seem to be of bacterial origin (Figure 5 ). Many of those genes encode subunits of protein complexes involved in energy metabolism or transporters and might have been acquired by I. hospitalis as small clusters or operons. Examples of putative 'bacterial' gene clusters include those encoding bacterial type polysulfide reductase (Igni528-530), the multisubunit putative Ech hydrogenase (Igni542-546, Igni1144-148) and a nitrate reductase-like complex (Igni1377-1379). Among the clusters of apparent origin from Euryarchaeota are genes encoding ABC-type transporters for antibiotics and molybdate (Igni146-147, Igni1340-1343) as well as a 2-oxoacid:ferredoxin oxidoreductase complex (Igni1075-1078). Other genes encoding characteristic proteins of Euryarchaeota are scattered in the genome (for example, the CrcB-like integral membrane protein Igni921, a 6Fe-6S prismane cluster-containing protein Igni960, micrococcal thermonuclease Igni1343, thermophilic glucose-6-phosphate isomerase Igni415). If N. equitans is a derived member of Thermococcales, as some gene trees and genomic analyses suggest [ 17 , 27 ], then some of the putative euryarchaeal LGTs in the I. hospitalis genome might actually represent transfers from N. equitans . Such transfers could have occurred during extensive genome degradation suffered by N. equitans associated with elimination of metabolic functions, similar to cases of nuclear transfer of symbiont genes during eukaryotic organelle formation. Additional LGTs from bacteria and/or archaea, including N. equitans , might be hidden in the large number of genes (>600 or approximately 40% of the open reading frames) that either lack detectable homologs or are placed unresolved within the Archaea due to insufficient phylogenetic signal. Figure 5 Taxonomic classification of I. hospitalis protein-coding genes based on phylogenetic and COG distribution analyses. Genes labeled in green or blue-green are of Crenarchaeota-type or are of unresolved archaeal nature, respectively. Genes that could represent horizontal gene transfers from Euryarchaeota or Bacteria are labeled in purple and yellow, respectively. Genes that have their closest ortholog in N. equitans are labeled red and are described in the table. Genes labeled in gray lack recognizable homologues in other microbial genomes or have unresolved phylogenies preventing confident affiliation to either Archaea or Bacteria. One of the possible outcomes of LGT in symbiotic associations involves orthologous gene displacement in the recipient genome and maintenance of the gene in the donor genome as well. In the N. equitans-I. hospitalis system, we identified 13 such cases, in which the orthologs in both genomes are each other's closest homologues (Figure 5 ). Several of the genes appear to have been transferred from N. equitans to I. hospitalis , including ones encoding valyl-tRNA synthetase (Igni220-Neq252), tyrosyl-tRNA synthetase (Igni347-Neq389) and a type IV endonuclease (Igni1092-Neq77a) (Figure 6a ; Figure S2 in Additional data file 2). Two genes involved in recombination and repair that form a predicted operon in N. equitans (an AP endonuclease 2 family and a DEAD/DEAH box helicase, NEQ368-369) have also been transferred to I. hospitali s, either as independent events or becoming separated later by genomic rearrangement (Igni0112 and 0145). Genes encoding aminoacyl tRNA synthetases and recombination and repair proteins are frequently exchanged in microbial communities and might increase the fitness of recipient organisms, for example, by conferring antibiotic resistance in the case of aminoacyl-tRNA synthetases [ 45 , 46 ]. Figure 6 Maximum likelihood phylogenetic trees (a) of archaeal valyl-tRNA synthetases and (b) of leucyl aminopeptidases representing the three domains of life and including all the known archaeal sequences. Numbers indicate bootstrap support based on 100 replicates. Where the value was <50, the branch was collapsed. The scale bar indicates the inferred number of substitutions per site. The sequence alignments used to generate the trees are provided in the Additional data file 4. A similar case of lateral transfer likely involved the gene encoding leucyl aminopeptidase (LAP), Igni738-Neq412 (Figure 6b ). LAPs are ubiquitous in bacteria and eukaryotes but their presence in archaea is so far strictly limited to the Desulfurococcales and the Cenarchaeales. While no specific function has been described so far for archaeal LAPs, in bacteria they are multifunctional proteins, with roles in protein turnover as well as in transcription control and recombination [ 47 ]. The absence of LAP in Euryarchaeota, in Korarchaeum cryptofilum (a potentially basal archaeal lineage with affinities with the Crenarchaeota [ 48 ]) as well as in two of the four Crenarchaeota orders for which genomic data are available may suggest that the Desulfurococcales and Cenarchaeales acquired the gene via LGT from bacteria. The phylogenetic analysis places the I. hospitalis gene close to that of N. equitans but not part of the Desulfurococcales clade. The high level of sequence similarity between the N. equitans and I. hospitalis LAP genes (40%) surpasses that between any of the other Desulfurococcales (approximately 30%). However, the direction of the transfer is uncertain. The exclusion of the I. hospitalis LAP from the clade formed by the other Crenarchaeota homologs suggests that the Ignicoccus gene may have been acquired from N. equitans followed by orthologous gene displacement. Based on this scenario, the original presence of LAP in N. equitans would be at odds with its purported affiliation with the Euryarchaeaota and specifically the Thermococcales, which are lacking leucyl aminopeptidases. The alternative hypothesis, transfer of the LAP gene from I. hospitalis to N. equitans , is challenged by the separation of the Ignicoccus-Nanoarchaeum clade from the other Desulfurococcales. Complete genome sequences of other Ignicoccus or Nanoarchaeota species may help distinguish between these competing hypotheses. Genetic information processing in I. hospitalis , as inferred from the genome sequence, is typical of the Crenarchaeota. Orthologs of two family B DNA polymerases are present in the genome (Igni62, 690); one corresponds to the aphidicolin-resistant DNA polymerase I (polA), and the other to the aphidicolin-sensitive DNA polymerase II (polB) of Aeropyrum pernix [ 49 ]. No orthologs of the third family B DNA polymerase or Euryarchaeota-type heterodimeric DNA polymerase were found. Unlike other archaeal genomes, the genes coding for replication initiation/origin recognition factor (Orc1/Cdc6) are not co-localized with the predicted origin of replication [ 50 , 51 ], a characteristic potentially related to general operon fragmentation in I. hospitalis . Unlike other archaea, including I. hospitalis , that possess DNA primases consisting of a small (catalytic) and large (structural) subunits, N. equitans seems to encode a single-subunit primase (NEQ395) in which the small subunit is fused to the carboxy-terminal domain of the large subunit [ 52 ] (EVK, unpublished observations). This may be the result of extreme genome contraction in this organism, possibly linked to its symbiotic lifestyle. Similarly, an important molecular machine absent in N. equitans but present in I. hospitalis is the RNase P complex (RNA and four separate proteins subunits, rpp14, 21, 29 and 30). It has been recently shown that tRNA processing in N. equitans is RNase P-independent, most likely because genome shrinkage led to the evolution of leaderless tRNAs that was followed by the loss of all five RNAse P complex genes [ 53 ]. Transport processes The membrane composition of hyperthermophiles is specifically adapted to reduce proton and ion permeability, which increase with temperature [ 54 ]. Cyclic tetraether-type lipids (caldarchaeol) that are present in the cytoplasmic membrane of I. hospitalis and in the cell membrane of N. equitans are especially associated with low permeability [ 13 ]. In contrast, the absence of caldarchaeol in the outer membrane of Ignicoccus and the presence of protein pores [ 11 ] indicate potentially less restrictive exchanges with the environment through the outer membrane. With only eight types of transporters, almost all predicted to be specific for inorganic ions or export of intracellular solutes (Figure 7 ), N. equitans is unlikely to import by itself all of the required metabolic precursors from its host. Consistent with its streamlined genome and autotrophic lifestyle, I. hospitalis also encodes very few transporters (<3% of its proteome), the lowest number among the sequenced species of Crenarchaeota. The types of transporters and their inferred specificities are described in Figure 7 . A number of inferred subunits of ABC transporters were found in membrane preparations of I. hospitalis cells, showing that these proteins are expressed in significant amounts [ 55 ]. An unexpected finding for an obligate autotroph was the presence of genes encoding two ABC transporters for oligopeptides and branched amino acids. Under laboratory conditions, it was indeed found that addition of peptides improved growth of I. hospitalis [ 2 ], suggesting that, in its natural environment, this organism might be opportunistic in utilizing such resources. The different lipid and protein compositions between the cytoplasmic membrane and the outer membrane of I. hospitalis [ 10 , 13 ] suggest the existence of specific partitioning mechanisms. The genome encodes a predicted gene (Igni479) from the LolE permease family, an ATP-dependent transport system involved in lipoprotein release that has been shown in Buchnera to transport lipids targeted to the outer membrane across the inner membrane [ 56 ] and that might play a role in I. hospitalis membrane synthesis. Figure 7 Predicted functional systems and metabolic pathways of the I. hospitalis-N. equitans system. The numbers refer to the corresponding genes in the I. hospitalis and N. equitans genome (green and red, respectively). Some of the biochemical pathways (carbon fixation, amino acid biosynthesis and sugar metabolism) have been experimentally validated [ 66 , 69 ]. Specific subcellular compartments and structures (periplasmic space, vesicles, tubules, pores, fibers) [ 9 , 11 , 62 ] are indicated and speculative functions are indicated with question marks. Scissors indicate proteases. Stars indicate specific regulatory proteins. Different transporter categories and their individual subunits are indicated by shape symbols and the direction of transport of specific substrates across the membrane is shown by arrows. While some proteins may spontaneously insert in the membrane, most transport into and across the membrane requires the function of specialized cellular systems [ 57 ]. All the components of the Sec pathway were identified in the I. hospitalis genome, including the 7S RNA gene component of the signal recognition particle (Figure 7 ). Even though potentially functional protein secretion complexes, including the euryarchaeal-specific SecDF are encoded in its genome, N. equitans lacks an identifiable 7S RNA gene. Since that component is critical for the assembly of a functional signal recognition particle, it might be synthesized as two separate transcripts, such as some of the tRNAs [ 58 ], or might be imported from the host. The Tat system, which transports folded protein across the membrane, is present in I. hospitalis but absent in N. equitans . For all proteins that are targeted for translocation, the signal peptide has to be removed either during or after translocation. The protease that removes some of the signal peptides in archaea, signal peptidase I, was identified in both genomes (Igni153 and Neq432). I. hospitalis also encodes a type IV prepillin peptidase (PibD, MEROPS family A24A, Igni1405), which processes membrane and secreted proteins with a class III signal peptide, including proteins involved in motility (flagellin) and pili formation [ 59 ]. Neither I. hospitalis nor N. equitans appear to have flagellins, although several genes potentially associated with archaeal flagellar or pili assemblies were identified in both genomes ( flaI, flaJ ). While the cells do not appear to be motile, certain appendages and pili-like structures have been observed in electron micrographs [ 60 - 62 ] and might play a role in the interaction between the two organisms. Central metabolism I. hospitalis is the first archaeon with sulfur-based autotrophy for which a complete genome sequence is hereby reported. Metabolic reconstruction (Figure 7 ) points to simple and efficient strategies that fit a streamlined genome. Nitrogen assimilation is predicted to rely on readily available ammonia, the most economical strategy in reduced environments [ 63 ]. Ammonia could be acquired through an AmtB transporter (Igni1293), which is apparently co-transcribed with the gene for the nitrogen regulatory protein PII ( glnK , Igni1294). These genes are widely present in bacteria and most members of the Euryarchaeota but are nearly absent from Crenarchaeota, and probably have been laterally transferred to I. hospitalis from a euryarchaeon (Figure 5 ). GlnK controls the transport of ammonium ions by interacting with AmtB and also activates a type of glutamine synthase (GS) that fixes the ammonia into glutamine. GS is present in all sequenced archaea with the exception of N. equitans and, by assimilating ammonia into the amide group of L-glutamine, makes it available to downstream glutamine-dependent amidotransferases. One such enzyme is glutamate synthase (GltS, Igni408), which is predicted to catalyze the reductive transfer of the amide group from glutamine to 2-oxoglutarate, resulting in glutamate and an amino group donor for transamination reactions. The domain architecture of GltS in I. hospitalis is unique and contains a GXGXG structural domain [ 64 ] followed by a ferredoxin (4Fe-4S) and a glutamine synthetase FMN-binding domain. The glutamate synthase domain I (GlxB domain) is expressed as a separate polypeptide (Igni407) and it is unclear if these two proteins actually assemble to form glutamine synthase or if GlxB functions independently as a type II glutamine aminotransferase. The two genes are likely cotranscribed with an aspartate/aromatic aminotransferase (Igni406), suggesting a tight coupling of the transamination processes. The GS/GltS operates very efficiently at low concentrations of ammonia and substitutes for the alternative ammonia incorporation mechanisms that use glutamate dehydrogenase (GDH) [ 63 ]. GDH catalyzes the reversible conversion of glutamate to 2-oxoglutarate and represents an alternative route for both deamination and ammonium incorporation. It has been suggested that GDH provides the major nitrogen assimilation mechanism in most hyperthermophiles [ 65 ], but this is clearly not the case with Ignicoccus . This is likely due to the absence of a steady source of exogenous amino acids; thus, the cells must rely on free ammonia, present at concentrations too low for GDH to operate at. However, in N. equitans , which lacks the ammonium transporter or a GS/GtlS enzyme pair, limited nitrogen metabolism could rely on a GDH (NEQ077), likely using glutamate imported from the host. The transfer of glutamate from I. hospitalis to N. equitans has been detected experimentally [ 3 ]. It is not clear why N. equitans has retained a GDH gene among its very few encoding metabolic enzymes. One possibility could be that GDH would contribute to the cell redox potential by oxidative deamination of glutamate. Pathways for the synthesis of almost all amino acids can be recognized in the I. hospitalis genome, with the exception of proline and homocysteine. Some of the enzymatic activities involved in I. hospitalis amino acid biosyntheses have been detected experimentally and labeling experiments have been used to reconstruct most the pathways [ 66 ]. The genome also encodes the predicted enzymes of purine, pyrimidine, NAD, riboflavin/FAD, pyridoxal and CoA biosynthesis. The mevalonate pathway for the synthesis of the characteristic archaeal membrane archaeol- and caldarchaeol-type lipids appears to be complete (Figure 7 ), although enzymes involved in some of the steps have not yet been characterized in archaea [ 67 , 68 ]. I. hospitalis utilizes a novel and so far unique autotrophic CO 2 fixation pathway, termed the dicarboxylate/4-hydroxybutyrate cycle [ 69 ]. The individual steps of the pathway have been investigated experimentally in detail and most have been confirmed biochemically [ 66 , 69 , 70 ] (Figure 7 ). Acetate in the form of acetyl-CoA is carboxylated by a pyruvate ferredoxin oxidoreductase enzyme complex (Igni1256-1259) to form pyruvate, which is then converted to phosphoenolpyruvate by pyruvate:water dikinase (Igni1113). The source of acetyl-CoA may be linked to two adjacent genes potentially encoding an acetyl-CoA synthase (Igni256, 257). Normally, that enzyme is encoded as a single polypeptide. The two genes in I. hospitalis may encode the enzyme as two subunits requiring post translational assembly or, alternatively, the two open reading frames could indicate a pseudogene. The level of acetate in the environment where I. hospitalis has been isolated from is not known, but the genome encodes a putative sodium-acetate symporter (Igni454) and uptake of acetate has been confirmed experimentally [ 66 ]. Since Ignicoccus can grow in the laboratory in the absence of acetate, the genome might also encode an acetogenesis mechanism using CO 2 . One potential route is direct reduction of CO 2 to formate using hydrogen, catalyzed by a putative membrane formate dehydrogenase complex. Genes encoding a membrane complex with similarity to both nitrate reductase and formate dehydrogenase were identified as a likely operon acquired from a bacterium (Igni 1377-1380). However, since neither nitrate respiration in I. hospitalis cultures [ 2 ] nor the biochemical activity of formate dehydrogenase in cell extracts [ 66 ] were detected, the cellular function of that complex remains unclear. The archaeal-type PEP carboxylase [ 71 ] catalyzes the second CO 2 incorporation reaction, which results in the formation of oxaloacetate, an important precursor for amino acid biosynthetic pathways (Figure 7 ). Reactions catalyzed by malate dehydrogenase, fumarase, succinate dehydrogenase and succinyl CoA-ligase lead to the synthesis of succinyl-CoA. Until recently, the fate of succinyl-CoA was unclear and, as the reactions that would close the cycle were not apparent based on experimental data or genomic information, the mechanism of acetyl-CoA regeneration remained unknown. Huber et al . [ 69 ] recently discovered that I. hospitalis uses a novel strategy to connect, through succinyl-CoA, the partial reductive citric acid cycle with the 4-hydroxybutyrate route of acetyl-CoA regeneration (Figure 7 ). Based on this finding, the proposed dicarboxylate/4-hydroxybutyrate cycle appears to be energetically less costly than other carbon fixation cycles operating in archaea [ 69 ], further supporting the notion that I. hospitalis combines a streamlined genome with efficient metabolic strategies. Phylogenetic analysis of the two I. hospitalis gene clusters encoding oxoacid:ferredoxin oxidoreductase complexes indicates that one of them (Igni1256-1259) belongs to the pyruvate:ferredoxin oxidoreductase family and, therefore, is the likely catalyst for acetyl-CoA carboxylation. The other complex (Igni1075-1078) has a close affinity to a family with oxoglutarate specificity with no close homologs in Crenarchaeota (Figure S3 in Additional data file 2), suggesting acquisition by lateral transfer. The functional inference is based on phylogenetic partitioning of archaeal oxoacid:ferredoxin oxidoreductase genes into distinct clades that correspond to enzymes specific for pyruvate, valerate/isovalerate, or 2-oxoglutarate or that have mixed specificity [ 72 - 74 ]. In addition, alignments of the I. hospitalis alpha and beta subunit sequences revealed the presence of motifs conserved in archaeal and bacterial enzymes specific for pyruvate (Igni 1258-1259) or 2-oxoglutarate (Igni 1077-1078) [ 75 - 77 ] (Figure S4 in Additional data file 2). The function of the predicted 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) complex in I. hospitalis remains unclear. 2-Oxoglutarate serves as an entry point in glutamate and lysine biosynthesis and is also linked to the biosynthesis of several other amino acids as shown by carbon tracing and inferred from genomic data [ 66 ] (Figure 7 ). In heterotrophic archaea and bacteria, oxoacid:ferredoxin oxidoreductases are involved in amino acid and sugar fermentation reactions that generate reduced ferredoxin and ATP, although OGOR has been ascribed a biosynthetic function, namely, generation of succinyl-CoA from 2-oxoglutarate [ 78 , 79 ]. By contrast, in anaerobic autotrophs, OGOR is a key enzyme in the reverse citrate cycle, where it catalyzes the fixation of CO 2 on succinyl-CoA with the formation of 2-oxoglutarate [ 70 ]. However, this reaction has not been detected in I. hospitalis cell extracts and carbon isotope tracing does not support its occurrence in laboratory cultures [ 66 ]. In fact, succinyl-CoA produced by the first half of the carbon fixation cycle is reduced by succinyl-CoA reductase to succinic semialdehyde and channeled into the hydroxybutyrate pathway [ 69 ]. The same reaction has been shown to connect the 3-hydroxypropionate with the 4-hydroxybutyrate pathways in another recently discovered novel carbon fixation cycle in the crenarchaeaote Metallosphaera sedula [ 80 ]. However, unlike the succinyl-CoA reductase from M. sedula , which uses NADPH as electron donor, the enzyme in I. hospitalis requires reduced ferredoxin [ 69 ]. A speculative role for OGOR could be to provide a ferredoxin-based electron shuttle between oxoglutarate and succinyl-CoA, at the expense of a fixed carbon (Figure 7 ). Such coupling has been shown to be important in the anaerobic metabolism based on aromatic compounds in Thauera aromatica , OGOR providing benzoyl-CoA reductase with reduced ferredoxin [ 75 ]. In Ignicoccus , under active growing conditions, such a reaction based on de novo synthesized 2-oxoglutarate does not seem advantageous as it would increase the succinyl-CoA pool at the loss of an acetyl-CoA while sufficient reduced ferredoxin may be supplied by a hydrogenase. However, under limited CO 2 and H 2 conditions, 2-oxoglutarate from the internal pool or derived from exogenous amino acids and peptides could keep the 4-hydroxybutyrate part of the cycle active and generate acetyl-CoA for maintenance functions. Experimental studies will be needed to test this hypothesis and identify the specificity of the predicted OGOR complex. Respiration and energetic metabolism Under laboratory conditions, the only energy yielding reaction that sustains the metabolism of I. hospitalis is the oxidation of molecular hydrogen coupled to the reduction of elemental sulfur. While energetically weak (-6.7 kcal/mol) [ 38 ], there are indications that this type of respiration might have been used by ancient microbes of the early Archaean [ 5 ]. Details of bioenergetic reactions and the mechanisms for generating the membrane chemiosmotic potential in anaerobic hyperthermophilic archaea are still not well understood. Minimal enzymatic components that are required include a membrane hydrogenase complex, a sulfur reductase and an electron transport chain between them. In I. hospitalis , there appear to be two clusters of genes encoding subunits of the sulfur/polysulfide reductase complex. The first such cluster (Igni801-803) contains the catalytic reductase (SreA), a 4Fe-4S ferredoxin (SreB) and the membrane anchoring component NrfD (SreC) with eight transmembrane domains. NrfD is thought to participate in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. Elsewhere in the genome, a gene cluster (Igni528-530) that appears to be of bacterial origin contains a different NrfD, a periplasmic FeS ferredoxin, as well as a membrane protein with four putative heme binding sites that may serve in the electron transfer chain through the membrane, possibly binding menaquinone. This gene cluster is also present in the related archaeon Hyperthermus butylicus [ 81 ], suggesting the possibility that it was transferred between the two archaeal lineages after one of them likely acquired it from a delta proteobacterium. Two types of reductase complexes might therefore assemble in I. hospitalis , archaeal and bacterial. In other sulfur reducers a periplasmic polysulfide-sulfur transferase (a member of the rhodanese family) facilitates the transfer of low concentrations of polysulfide to the reductase. I. hospitalis is the only crenarchaeote that is missing a rhodanese family gene. This could be a result of growing under relatively neutral pH, where polysulfide concentrations may be high enough. Therefore, access of polysulfide to the cytoplasmic membrane, where the reductase complex is likely located, could occur by diffusion across the large periplasmic space after passage though the outer membrane pores. Ignicoccus depends on molecular hydrogen as the sole electron donor. A single predicted operon contains the genes encoding the large and small subunits of a hydrogen uptake NiFe hydrogenase, including the large and small subunits (Igni1366-1369). The heterodimer is exported to the periplasm through the twin-arginine translocation (TAT) system and is assembled with a 4Fe-4S ferredoxin and a membrane protein anchor containing histidine residues that might bind a b-type heme [ 82 ]. The formation of the metal-containing active site and the assembly of the hydrogenase is a complex process requiring multiple accessory proteins [ 83 ], all of which appear to be encoded in the I. hospitalis genome (Figure 7 ). Hydrogen oxidation is coupled with electron transfer through FeS centers and a putative membrane cytochrome to the quinone pool of the respiratory chain, which contributes to the generation of a membrane potential that drives ATP synthesis. The quinone appears to be associated with the membrane component of the hydrogenase and that of polysulfide reductase, with the exchange of the electrons likely involving formation of respiratory 'supracomplexes' [ 84 ]. A separate 'energy-converting' Ni-Fe hydrogenase family complex (Ech), which is evolutionarily related to the energy-conserving NADH:quinone oxidoreductase (complex 1), appears to be encoded by genes in two clusters (Igni542-546 and Igni1144-1146). This hydrogenase is the likely catalyst in maintaining the pool of reduced ferredoxin. The I. hospitalis genome also contains a four gene putative operon with close homologues among the bacterial respiratory periplasmic nitrate reductases (Igni1377-1380). Similarity to formate dehydrogenases was also detected, so the function of the complex is not clear, as nitrate cannot serve as a sole electron acceptor in Ignicoccus [ 2 , 60 ]. In bacteria, depending on the composition of the complex, periplasmic nitrate reduction can either contribute to the generation of the proton gradient or serve as an electron sink, eliminating excess reducing equivalents from the cytoplasm [ 85 ]. A complete membrane A-type ATPase is predicted to be encoded in the genome of I. hospitalis , in contrast with only a subset of subunits in N. equitans [ 12 ]. While N. equitans might be unable to synthesize ATP, the presence of a predicted nucleoside diphosphate kinase (Neq307) suggests that regeneration of the NDP pool is feasible, which might reduce its host dependency by recycling (Figure 7 ). Since it has few ion transporters and no genes encoding membrane hydrogenases or oxidoreductases, it is unknown if N. equitans can independently maintain a membrane potential or whether it needs to acquire such capabilities from its host. As an obligate anaerobe, I. hospitalis requires a mechanism to deal with the toxicity of reactive oxygen species. A superoxide reductase is present (Igni1348) and could detoxify superoxide resulting from oxygen reduction by transition metals. According to a recently proposed mechanism [ 86 ], a ferrocyanide complex bound within the superoxide reductase active site may scavenge the superoxide by one-electron redox chemistry while the superoxide reductase iron site remains reduced. The resulting peroxide could be transferred to soluble organic compounds, resulting in the formation of alkyl peroxides that can be reduced by peroxiredoxin. A gene encoding a member of this family is encoded in the genome (Igni459) and a recent proteomic analysis of I. hospitalis in laboratory cultures has shown that its product is an abundant cytosolic protein [ 55 ]. Potential molecular and structural determinants of the I. hospitalis-N. equitans interaction Although the recognition and exchange mechanisms between I. hospitalis and N. equitans remain elusive, the available genomic and ultra-structural data suggest some possible ways of interaction between the two organisms. Since the transporters in both species are few and provide limited specificities, they are unlikely to comprise the main route of metabolite acquisition by N. equitans . Similarly, transfer of protein complexes to N. equitans from the host by secretion, especially for membrane components, would violate topological and signal sequence constraints of the translocation machinery. Potential vehicles for the transport of metabolites and proteins from I. hospitalis to N. equitans appear to be the large and variably shaped vesicles and tubes that emerge from the host's cytoplasm [ 9 , 10 ]. Such structures could provide transient or even constant contact between the two cytoplasms once the physical contact between the cells has been established, possibly fulfilling the metabolic and energetic requirements of N. equitans . This would also allow it to carry out limited respiration, transport and ATP synthesis and may explain how detached N. equitans cells or cells not in direct contact with the host can survive for some time. Electron microscopy studies have indicated that some of the I. hospitalis periplasmic vesicles fuse with the outer membrane, which likely results in their contents being released into the environment [ 9 , 10 ]. This release of small molecules and, perhaps, peptides might provide chemical cues to N. equitans for host recognition and attachment. Since neither of the two organisms appears to be motile, the actual mechanism by which they find each other and become attached in the turbulent hydrothermal vent environment remains enigmatic. Recent ultra-structural and physiological studies have shown that a physical connection can form between the two organisms [ 3 , 62 ]. Three-dimensional reconstructions point to a dynamic type of interaction, some N. equitans cells contacting the outer membrane of I. hospitalis in places where the host periplasmic space is wide and contains cytoplasmic vesicles while others are attached to regions with a very narrow periplasm and displaying fibrilar structures [ 62 ]. The steps and molecular determinants of the cell-cell recognition and interaction and the membrane and periplasm dynamics remain uncharacterized. The cytoplasmic membrane of Ignicoccus itself is highly 'corrugated', as shown in sections and three-dimensional reconstructions, thereby increasing its surface significantly; in addition, it spontaneously evaginates in the absence of N. equitans [ 2 , 9 , 10 , 62 ]. Therefore, the physiological role of the conglomerate of tubes and vesicles and the significance of the wide periplasmic space probably extends beyond their possible connection to N. equitans . As energy generation resides at the level of the cytoplasmic membrane, these structures could provide a substantially increased respiratory surface confined in the space surrounded by the outer membrane, analogous to the eukaryotic mitochondrial cristae. Vesicles might concurrently transport specific lipids and proteins to the outer porous membrane, which in this case would serve not only as a protective barrier but also for controlling gas and solute exchange. This could represent a mechanism enabling Ignicoccus species to rely exclusively on the low energetic yield of the sulfur-hydrogen respiration to sustain an elevated turnover of cellular components at high temperature. Combined with the obligate CO 2 autotrophy and efficient metabolism, such adaptations might allow Ignicoccus to outcompete heterotrophs in colonizing emerging hydrothermal vent niches that are still poor in dissolved organic compounds."
} | 12,766 |
32587290 | PMC7316772 | pmc | 2,521 | {
"abstract": "Marine biofouling can cause a biocorrosion, resulting in degradation and failure of materials and structures. In order to prevent sea creatures from attaching to the surface, in this work, a new environmentally friendly antifouling coating by incorporating antibacterial polymers and natural antifouling agents has been designed and synthesized. Surface chemical composition and changes in surface hydrophobicity were studied by FTIR spectroscopy and contact angle measurements, respectively. Measurements of mass loss of antifouling resin were also carried out and the release rate of camphor from antifouling coating was tested by using UPLC. It had been found that the changes in the content of triisopropylsilylacrylate (TIPSA) (from 4% to 12%) and isobornyl methacrylate (IBOMA) (from 50% to 16.7%) did not significantly affect the release of camphor. The content of IBOMA decreased from 50% to 16.7%, the antifouling performance of the resin system appeared slightly reduced. In addition, rosin could help regulate the release rate of the resin system to desorb camphor slowly in water in a controlled manner. Furthermore, the antifouling capability of as-prepared samples was evaluated via algae suppression experiments and marine field tests. This study highlighted the environmentally friendly antifouling coating as a potential candidate and efficient strategy to prohibit biofouling in seawater.",
"conclusion": "Conclusion A new polymer antifouling coating has been successfully synthesized by using environmentally friendly terpolymer resins and natural antifouling agent camphor. It has been demonstrated that our antifouling polymer can be released slowly in a controlled manner. Due to the hydrolysis of acrylic silicone resin and the increase of carboxylic acid radical content after immersion, the hydrophobicity of the coating surface rapidly changes to hydrophilicity. The application performance of this antifouling system also has been tested. The changes of TIPSA content have no significant effect on the release rate and antifouling performance of the resin system. Although the change of IBOMA content does not significantly affect the release rate of camphor, the decrease of IBOMA content corresponds to the antifouling performance of the resin system. Rosin may help regulate the release rate of resin system by reducing the release rate of camphor in the early stage and accelerating the release rate of camphor in the later stage. The results of marine field tests highlight the potential application of the polymer antifouling system synthesized by incorporating antibacterial polymer and natural antifouling agents to prevent fouling in the application of antifouling coatings. The results need to be further evaluated via marine field tests for longer duration.",
"introduction": "Introduction Submerged hull surface is highly susceptible to biofouling. Marine fouling organisms attach to the hull surface, grow and propagate, which will lead to the aggravation of surface corrosion, the exacerbation of surface friction resistance, the increase of fuel consumption, and a great number of economic loss 1 . In order to prevent marine organisms from attaching and adhering, a variety of methods have been adopted, such as physical method, chemical method, net changing method, new antifouling nets 2 , 3 . Among those technologies, antifouling coating is the most convenient, effective and economical method. Due to its low cost and easy implementation, antifouling coating has always been the direction of efforts to solve the problem of antifouling 4 . In recent decades, a variety of functional polymer brushes and coatings have been developed to prohibit the attachment of marine organisms 5 . However, traditional antifouling coatings rely on the release of antifouling agents to control biological adherence, which seems to show a promising antifouling effect 6 . Unfortunately, these antifouling agents are toxic molecules, because of the toxic nature of certain biocides such as tributyltin (TBT) and copper, they can cause marine pollution and bring about serious harm to the ecological environment 7 . Since 2008, the International Marine Organization (IMO) has constantly urged and continuously strengthened legislation to restrict the use of toxic antifouling agents 8 . Therefore, it is urgent to develop environment-friendly, non-toxic or low toxic antifouling compounds 9 , 10 . In the past decade, environmentally friendly antifouling coatings based on self-polishing copolymers and natural antifoulant have drawn substantial attention as a promising strategy and the most effective means of sequestering biocorrosion and biofouling 11 , 12 . Natural compounds separated from marine microbes, algae, aquatic plants, marine invertebrates, and land and other sources are a promising source of antifouling agents 13 . Compared with synthetic antifouling agents, they have the advantages of compatibility with biological systems, and are more specific to bacteria, algae and mollusks and other attached organisms than heavy metals 14 , 15 . However, despite the isolation of many potential antifouling compounds from marine natural products, their application as effective antifouling biocides has been progressed slowly 16 . Among these natural antifouling agents, 2,5,6-tribromo-methylgramine deserves consideration because of its simple structure and high antifouling capability 17 . It has been reported that the coatings with poly(m-aminophenol) (PmAP) have improved antifouling performance which can effectively inhibit the formation of biofilm 18 . There are still many challenges to be overcome in terms of cost, large-scale production, biosafety and pollution control mechanisms. In addition, the introduction of antifouling compounds into synthetic polymers and the release of natural antifouling agents from coatings are of great importance for the reliability and longevity of metals in service. And research on the synthesis and property of its analogues has attracted increasing attention 19 . Camphor is a terpene natural organic compound, which can be extracted from the trunk of Cinnamomum camphora and can also be synthesized in large quantities 20 . At room temperature, it appears white or transparent waxy solid. It is often used in the practice of daily life to repel insects and mosquitoes 21 . Theoretically, it is a potential compound with antifouling ability 22 . If one or more compounds or polymers can be designed, synthesized or blended to make them compatible with camphor, with stable controlled release of camphor and synergistic anti-fouling effect, it will be of great significance to the application of environmentally friendly antifouling coatings 23 , 24 . Borneol is a time-honored herb in traditional Chinese medicine, a crystalline cyclic alcohol that occurs in two enantiomeric forms. It can be extracted from medical herbs such as lavender, lavenrian, and chamomile 25 . It has many biological effects such as sedative, antiinflammatory, analgesic, anti-nociceptive, antithrombotic and vasorelaxant effects 26 . Natural herb-based antibacterial borneol has been reported to have an excellent broad-spectrum antibacterial capability 27 . Borneol-based polymers, isobornyl methacrylate (IBOMA), have been demonstrated a strong activity against bacterial infection and can be suitable for preparing antibacterial coating 28 . The novel approach employed in the present study is synthesis of an environmentally friendly antifouling coating based on antibacterial polymer (such as IBOMA) and natural antifouling agent (such as camphor). The as-prepared coatings can slowly degrade and release borneol in seawater, while borneol itself has antibacterial properties, and at the same time, it provides a self-renewing surface to prevent fouler from adhering. Furthermore, antifouling camphor is also released together, providing a special antibiosis and antifouling surface with sterilized polymers to play a synergistic antifouling effect. The present study aims at the investigation of the antifouling properties of as-prepared coatings and synergistic antifouling effect of polymer hydrolysis, camphor and borneol release.",
"discussion": "Results and Discussion Analysis of FTIR The chemical structure and composition of as-prepared resin samples was studied using FTIR absorption spectroscopy. Here resins were used without camphor in order to facilitate the comparison of their chemical components of samples. In Fig. 2 for anti-fouling resins characteristic bands were observed at 2960, 1730 and 1150 cm −1 . The stretching vibration absorption peaks of C = C double bond (1650 cm −1 ) did not appear in the spectra, indicating that the copolymer of binary acrylic resin had been polymerized. 2963, 2907 and 2873 cm −1 were the stretching vibration absorption peaks of saturated C-H, 1730 cm −1 was the stretching vibration absorption peak of C = O, 1450 and 1390 cm −1 were the in-plane bending vibration absorption peaks of C-H. 1240 cm −1 might be the out-of-plane bending vibration absorption peak of C-H, and medium and wide doublet peaks, 1180 and 1150 cm −1 , were the stretching vibration absorption peaks of C-O-C. Table 2 illustrated the GPC traces of the molecular weights of all prepared samples. The average molecular weight and the dispersity of all samples were only little difference. The average molecular weight of samples 1, 2 and 5 was slightly larger than that of samples 3 and 4. Figure 2 The chemical structure and composition of samples. FTIR spectra of the resin samples. Table 2 GPC of anti-fouling resins. Sample M w (GPC) M n (GPC) PDI = M w / M n 1 34790 18258 1.905 2 34332 18893 1.817 3 23802 14512 1.640 4 22112 14072 1.571 5 31999 18941 1.689 Quality loss test Measurements of the mass loss of antifouling resins were carried out and the release rate of camphor from antifouling coating was tested by using UPLC. The weight loss rate of resin and the biocide release rate of resin containing camphor were the two key factors which determined the duration and antifouling performance of an antifouling system. As shown in Fig. 3(a) that in the early stage of immersion, the mass of each sample increased to a certain extent, among which sample 3, 4 and 5 increased the most. The possible reason was that samples 3, 4, and 5 contained relatively high content of TIPSA, and the water absorbability was relatively stronger. After more than 60 days of immersion, all the samples appeared to have a certain degree of weight loss; however the results showed that the weight loss rate was stable within an error range, indicating that the change of TIPSA, IBOMA and BMA content had no significant effect on the weight loss rate of the resin. Especially compared with sample 1, the system had no obvious hydrophilic group, and sample 1 has the best water resistance in the group. Therefore, the weight loss rate of sample 1 was the lowest. Figure 3 The mass loss of resins. ( a ) The swelling rate of resin; ( b ) The weight loss rate of antifouling coatings. Fig. 3(b) illustrated the time dependence of the weight loss of as-prepared coatings on glass panels immersed in ASW. When the resin and the camphor were mixed, the changes were slightly different. In the first stage, the weight loss of each sample was basically the same. The weight loss of sample 3c with 10% rosin was slightly lower than that of other samples, but the difference was not significant. In the second stage, the weight loss of sample 4 increased rapidly. The weight loss of sample 3 was lower to that of sample 4, while the weight loss rate of other samples did not change significantly. It might be that the molecular weight of sample 3 and 4 was relatively smaller, and the content of TIPSA was higher. The smaller the molecular weight and the easier the hydrolysis of TIPSA might result in the difference of the local hydrolysis of the resin. In particular, sample 4 had the smallest molecular weight and dispersion, which might lead to the rapid increase of weight loss rate. In the second stage, compared with sample 3c, basically it continued the trend of the first stage, and the weight loss was still the lowest, which indicated that the resin might have a certain delay effect on the weight loss of coating system. The possible reason was that in the presence of seawater, resin had a better hydrophilicity than camphor. The TIPSA segment might preferentially migrate to the coating surface, reducing the exposure of camphor to seawater, thus delaying the release rate of internal camphor and reducing the weight loss rate. In the third stage, the release rate of sample 3c increased rapidly, but the variation tendency of other samples was not obvious. Sample 4 returned to the normal group of weight loss. This indicated that the proportion of TIPSA in the whole system was limited, which was not enough to change the weight loss effect of the whole system. In the fourth stage, sample 3c continued its rapid release trend in the previous stage, while other samples also had some increase, but their changes were inferior to sample 3c. This suggested that rosin might help sample 3c release camphor in the third and fourth stages. The rosin might play a certain proactive role in the release of camphor in the middle and late stage. The possible reason was that with the prolongation of immersion time, the rosin gradually degraded and released into the seawater, forming a hollow channel in the resin system, which in turn was conducive to the release of the internal camphor, improving the release rate of camphor in the later stage, thus further increasing its weight loss. This also followed the principle of the release antifouling paint that had been applied up to now 32 . Analysis of contact angle and surface roughness In order to evaluate the role of surface characteristics of the coatings, their surface properties before and after immersion were analyzed by measuring contact angle and surface roughness. As showed in Table 3 , after immersion in ASW, the contact angle of all samples decreased significantly, e.g., the contact angle of sample 3c decreased from 94 degree to 67 degree. As indicated in Table 4 , the roughness of all samples also appeared a similar trend. After immersion in ASW, the maximum value of surface roughness of sample 3c from dry surface, 12.87 μm, reduced to 6.67 μm for its wet surface. These changes could also be visualized for sample 3 and 3c as examples as illustrated in Fig. 4 . This might be due to the hydrolysis of acrylic silicone resin and the increase of carboxylic acid radical content, resulting in the increase of hydrophilic characters. The contact angle of sample 3c (with extra 10% rosin) decreased most. These might suggested that the auxiliary resin itself was hydrophilic, which further enhanced the hydrophilicity of the coatings. These hydrophilic and slippery surfaces would further reduce the chance of the initial attachment of foulers 33 . Table 3 Surface contact angles of anti-fouling resins. Sample 1 2 3 4 5 3c dry (°) 94 93 93 93 91 94 wet (°) 78 83 79 77 82 67 Table 4 Surface roughness of anti-fouling resins. Sample 1 2 3 4 5 3c dry (μm) 12.83 12.38 12.74 12.85 12.72 12.87 wet (μm) 9.34 7.75 10.36 7.61 8.13 6.67 Note: ‘dry’ represents the surface state of coatings after fully cured; ‘wet’ represents the surface state of coatings after immersion in water for 7 days and after surface water has been removed naturally. Figure 4 Surface roughness measurements. The images, sample 3 (S-3, above), and 3c (S-3c, bottom), of surface roughness of anti-fouling resins taken from an ultra-depth-of-field electron microscopy. Controlled release of camphor In order to optimize the antifouling performance of an antibiofouling system, it is necessary to study the controlled-release performance of antifoulant camphor in detail. Since the resin is not completely hydrolyzed, the release rate of antifouling agents is slower than that of more commonly used self-polishing resins 34 , 35 . Therefore, it is worth noting that it is inappropriate to use 24 hours to detect/test the release rate of the system in some other literatures. In order to resolve the problems of slow release rate and a lot of error, here we use a staged method to calculate the average value of multi-day cumulative release. In our experimental system, the total process of release rate can be divided into four stages, the first stage is 1–25 days, and the other stages are 26–41, 42–56 and 57–72 days, respectively. It can be seen from Fig. 5 that the average release rate of the first stage is the highest for all samples, and the average release rate then decreases gradually with time. In the third and fourth stages, the release rate gradually stabilized. Therefore, the first stage can be regarded as the period of high speed release, the second stage is the period of transition release, the third and the fourth stages are the periods of stationary release, which means that the release of antifouling agents in the resin system tends to be stable. Figure 5 Controlled release of camphor. Release rate of camphor in resins at different stages. During the first stage, the high speed release, as shown in Fig. 5 the release rates of sample 1, 2, 3, 4 and 5 are all around 11 μg/day·cm 2 , the release rate of sample 3c containing an extra 10% rosin is obviously lower than that of other samples. This illustrates that the rosin has a certain delay effect on the release of camphor in the period of high speed release stage during the first stage. The possible reason is that rosin has better hydrophilicity than camphor in the seawater environment, and preferentially it migrates to the top surface of the coating, so as to reduce the chance of camphor contacting with seawater and delay the release rate of internal camphor. During the second stage, the transition period, the release rates of samples 1, 2, 3, 4 and 5 are all around 7. Compared with other samples, the release rate of sample 3c is higher. In the third and fourth stages, during the period of stationary release, the release rate of each sample tends to be stable. However, the release rate of 3c is still slightly higher than that of other samples, although this advantage is not obvious. This shows that in the middle and later stages the release of the resin has a certain promoting effect on the release of camphor. The possible reasons are that with the prolongation of the soaking time, the resin is gradually released into the seawater, forming a hollow channel in the resin system, which is beneficial to the diffusion and release of the internal camphor, so as to improve the release rate of camphor at the late stage 3 . The behaviour and properties of as-prepared coatings are in accordance well with the principle of releasable antifouling coatings 32 . At the same time, Fig. 5 also shows that the changes in the content of silicon acrylate monomers (TIPSA), which increase from 4% to 12%, and IBOMA, which decreases from 50% to 16%, do not significantly affect the release amount of camphor. In addition, the antifouling performance of all samples has been further evaluated via algae suppression and marine field tests. Analysis of algae suppression data As can be seen from Fig. 6 , the antifouling test of algae cell attachment illustrates that the coatings of environmental friendly biocides have good and encouraging anti algal adhesion performance. The algae attachment intensities of sample 1, 2, 3, 4, 5 and 3c are 0.781%, 1.168%, 1.622%, 2.427%, 3.526%, 6.083% respectively, as shown in Fig. 6b . With the increase of silicon content in acrylic resin, the algae adhesion intensities of the sample increases gradually, and the algae inhibition ability of the coating decreases with the increase of silicon content in acrylic resin. Silicon surface is inert to microbial attack but fungal or algal growth occurs especially when extra carbon sources are available as nutrients, which is the case for acrylic resin 36 , 37 . On the other hand, with the decrease of isobornyl methacrylate monomer content, the inhibition ability of algae decreases, as indicated in Table 1 , sample 1, 2 and 3 with 150 g of IBOMA, and only 100 and 50 g of IBOMA in sample 4 and 5, respectively. Because IBOMA can release borneol via hydrolysis, borneol has a remarkable inhibitory effect on bacterial attachment and growth 26 . And therefore borneol based polymers can be utilized for fabricating biocompatible antibacterial coating interface 28 . Figure 6 Algae inhibition experiments. ( a ) Fluorescence images of as prepared coating samples after inhibition test of algae adhesion; ( b ) Inhibition rate of algae calculated via the attachment areas of algae. Marine field tests In order to further assess the antifouling properties of resin samples, the short-term antifouling performance of all as-prepared coating samples was evaluated by marine field test. In Fig. 7 , the typical images of panels coated with as-prepared coating samples were illustrated. When the content of IBOMA was the same for sample 1–3, with the increase of TIPSA content, the antifouling performance of the coating was not obviously improved. The possible reason was that both IBOMA and TIPSA were small functional monomers with some antifouling effects. The small change of content might not be enough to influence the antifouling performance. When the content of TIPSA was the same, the content of IBOMA decreased from 50% to 16.7%, indicating that the amount of released borneol could decrease. Therefore, the antifouling performance of the resin system could decrease slightly. This was also consistent with the above algae suppression data. The decrease of IBOMA content was accompanied by the decrease of antifouling performance of antifouling coatings, which suggested that IBOMA had better antifouling performance than TIPSA. In addition, resin could help regulate the release rate of the resin system by reducing the release rate of antifouling agents in the early stage and accelerating the release rate of antifouling agents in the later stage, which was the key process to prevent the occurrence of biofouling in the long run 31 . From the actual marine testing, the change of release rate could not significantly alter the antifouling performance of the resin system. After 4 weeks, there almost none of barnacle appeared on the surfaces of sample 1–3, there were barely a few barnacles on the surfaces of sample 4–6. However, there were many more barnacles attached on the surface of control samples. Here marine field tests did not seem to follow the same trend with those from analysis of algae suppression. In addition, all the samples seemed to have yellow brown adherents’ appearance on the surfaces, which were likely to be silts or tiny grain aggregates in the sea water. Generally, the seawater near Zhoushan emerged muddy or turbid water with sediments. How to overcome this problem might require further study. Figure 7 Marine field tests. Morphologies of the antifouling performance of coating samples at different stages, (above, 4 weeks; bottom, 9 weeks). Fig. 8 illustrated the number of barnacles attached to antifouling plates. In Fig. 8 , the number of barnacles on the control samples appeared to be the highest after 9 weeks in the marine field, especially for the control panel submerged in a depth interval 1.4–1.75 m. As discussed above, the release rate of borneol increased as the content of IBOMA increased, so that the antifouling performance of resin samples could be improved. Because of their hydrolysis of ester bonds, the antifouling performance is mainly dependent on the self-renewal of as-prepared coatings and the synergistic effect of the antimicrobial borneol released from the coatings and the desorption of camphor, a nature antifoulant. Figure 8 Evaluation of antifouling performance. The number of barnacles attached to antifouling boards."
} | 5,991 |
40064892 | PMC11894081 | pmc | 2,522 | {
"abstract": "Physical hydrogels, three-dimensional polymer networks with reversible cross-linking, have been widely used in many developments throughout the history of mankind. However, physical hydrogels face significant challenges in applications due to wound rupture and low elasticity. Some self-heal wounds with strong ionic bond throughout the network but struggle to immediately recover during cyclic operation. In light of this, a strategy that achieves both self-healing and elasticity has been developed through the construction of topological hydrogen-bonding domains. These domains are formed by entangled button-knot nanoscale colloids of polyacrylic-acid (PAA) with an ultra-high molecular weight up to 240,000, further guiding the polymerization of polyacrylamide to reinforce the hydrogel network. The key for such colloids is the self-assembly of PAA fibers, approximately 4 nm in diameter, and the interconnecting PAA colloids possess high strength, simultaneously acting as elastic scaffold and reversibly cross-linking near wounds. The hydrogel completely recovers mechanical properties within 5 h at room temperature and consistently maintains >85% toughness in cyclic loading. After swelling, the hydrogel has 96.1 wt% of water content and zero residual strain during cycling. Such physical hydrogel not only provides a model system for the microstructural engineering of hydrogels but also broadens the scope of potential applications.",
"introduction": "Introduction Hydrogel, a polymer network swollen with water, has attracted great interest in the fields of wearable devices 1 , 2 , human-machine interaction 3 , 4 and biorobotics 5 , 6 due to its biocompatibility and diverse functionality 7 . Self-healing and elastic hydrogels are crucial for such applications to restore from the unexpected mechanical damage and prolong their service life. Generally, self-healing capability in hydrogels is commonly achieved by introducing directional self-assembly to enable the dynamic bond reconstruction 7 – 15 . For instance, the polyampholytes P(NASS-co-MPTC), assembled by the ionic association of single polymer network, physically crosslinks the polymer network and enables a healing efficiency of 100% after being immersed in water for 24 h 9 . However, due to high bonding strength, the self-assembly requires considerable energy (light, thermal, etc.) and enough time to reach thermodynamically stable state 16 – 23 . Such reversible polymer network breaks upon successive stretch and hardly recovers timely, resulting in the deterioration of elasticity 22 . Double-network hydrogels introduce additional polymer network, designed to retain self-assembly from deterioration, yet the self-healing capability and elasticity are still far from satisfactory 24 , 25 . Therefore, it is necessary to design a proper reversible polymer network with recoverable mechanical dissipation to ameliorate the elasticity. Strategies for enhancing the recoverability of polymer networks include chemical crosslink and weak hydrogen-bonding (H-bonding). The presence of chemical crosslink pinches the polymer network with immobilized knots. Until these knots break, polymer chains can slip and be thoroughly recoverable within limited strain, resulting in minimal mechanical dissipation and excellent elasticity 26 – 28 . Meanwhile, weak H-bonding enables recoverable mechanical dissipation during cyclic loads. Instead of strong reversible bonds in self-assembly, weak H-bonding is noncooperative and thereupon rapidly reconstruct during cyclic loads. With the chemical crosslink as scaffold, the weak H-bonding between poly-vinyl alcohol and polyacrylamide (PAM) readily breaks and reforms, resulting in nearly 100% recoverable mechanical dissipation during 5000 cyclic loads 29 . Although both chemical crosslink and weak H-bonding endows hydrogel with high elasticity, neither constructs a reversible network for self-healing capability. Therefore, a strategy to achieve both recoverable mechanical dissipation and reversible network is urgent for designing elastic, self-healing hydrogels. The topology of a polymer network stems from the assembly of polymer chains due to intermolecular interactions and steric hinderance and plays important roles in macroscopic properties, in which multiple functionalities can be regulated 24 , 30 . For instance, hydrophobic assembly by NaCl solution reconstructed the polymer network from the random distribution into certain topology where discrete hydrophobic domains hold ionic polyelectrolyte chains, leading to 90% self-healing capability within 24 h and recovering the full mechanical property after resting for 4 min. Yet the high strength of cooperative H-bonding and ionic interaction between polyelectrolyte chains deteriorates the polymer network during successive loading 24 . Sun et al. introduced an interconnecting hydrophobic assembly in hydrogel so that the hydrophobic domains locked the non-covalent interaction, enabling elastic contraction of polymer chains and healability 31 . However, such hydrophobic domain was only healable in 333 K water due to limited mobility of hydrophobic assembly, restraining the further application. Herein, we demonstrate a topological design to simultaneously achieve full self-healing capability and high elastic recovery in physical hydrogel. We selectively utilize the topology of polyacrylic acid (PAA) colloids to assemble double-network hydrogel and locally construct the reversible elastic cooperative H-bonding domain embedded in noncooperative H-bonding matrix. PAA colloids are assembled via intermolecular cooperative H-bonding in concentrated solution, and different molecular weights lead to distinctive entangled conformation at the same concentration, as elucidated via Tyndall effect, cryo-TEM, small-angle X-ray scattering (SAXS), rheology and simulation. Cooperative H-bonding domain is composed of PAA colloids and polymerized acrylamide (AM) around PAA, contributing to the reversibility of polymer network with strong H-bonding. The excess AM polymerizes aside as noncooperative matrix connecting to the cooperative domain, facilitating the rapidly recoverable polymer network with weak H-bonding. The topology of cooperative domains critically determines the macroscopic properties of hydrogels, and only interconnecting cooperative domains assembled by high-molecular-weight PAA enable hydrogel with an autonomous self-healing capability (~100% healing in 5 h at room temperature) and excellent elasticity (<6.3% residual strain, >100% stress retention, >85% reversibility) upon successive loading. After swelling, the hydrogel exhibits 0% residual strain upon cyclic loading. We believe that the proposed methodology holds significant potential for designing with multiple functionalities for various practical applications.",
"discussion": "Results and discussion Structural evolution of PAA depending on molecular weights The hydrogel was synthesized via the free-radical polymerization of AM along the PAA colloid. PAA is a well-known water-soluble polymer with alternating carbonyl and carboxyl groups and leads to high negative charge density when the carboxyl groups dissociate. The strong negative charges on the PAA backbone induces the repulsive electrostatic screening in aqueous solution and thereupon self-assembles into colloids. Due to the intermolecular cooperative H-bonding and steric alignment, PAA of diverse chain length forms colloid with different structure (Fig. 1a ) 32 , 33 . For example, PAA chains with molecular weights of 3000 (PAA3k) and 240,000 (PAA240k) have chain lengths of approximately 10 nm and 780 nm, respectively, based on the length of monomer (~2.18 Å) and molecular weight. Due to the negligible steric hinderance, low-m.w. PAA substantially contacts with water molecules and thereupon spreads as discrete islands. Meanwhile, high-molecular-weight PAA interacts with each other due to the significant steric hinderance, self-assembling into nanostrings. Upon critical concentration, PAA nanostrings overlap and assemble highly entangled colloids with button-knot morphology (Supplementary Fig. 1 ), forming button-knot cooperative domains. Fig. 1 Molecular-weight-dependent evolution of polyacrylic acid (PAA) colloids. a Schematic of the synthesis and stretching of topological hydrogels. Low-molecular-weight PAA (orange) chains form discrete colloids and partially stabilize the surrounding polyacrylamide (PAM) (grey) chains. High-molecular-weight PAA chains form button-knot colloids and result in elastic recovery and self-healing capability. Dashed circle depicts the size of colloids. b Photos of PAA droplet dyed by Coomassie brilliant blue and Tyndall effect of PAA aqueous solution. The variation of droplet color results from the different pH values of PAA solution with molecular weight of 3000 and 240,000 (PAA3k and PAA240k). c Cryo-TEM images of 25 wt % PAA3k and PAA240k solution diluted five times. The actual concentration after blotting is much higher than the initial concentration. d Molecular dynamics of low-molecular-weight-PAA aqueous solution (repeating unit, r.u. = 30) and high-molecular-weight-PAA aqueous solution (r.u. = 256). Blue line depicts water molecules. Both purple line and blueviolet line represent the carbon backbone of separate PAA chains. e Photos of PAA3k-AM and PAA240k-AM upon deformation after self-healing for 5 h. f Photos of PAA240k-AM cycling at λ = 2 for 10 cycles. g Schematic comparison of self-healing capability and reversibility between this work and previously reported hydrogels. Before the polymerization, partial AM penetrates into PAA colloids since amide groups form Lewis-acid-base couple with carboxyl groups. Aside from the coupled AM, excess AM dissociates around the PAA-AM colloids. With the initiator ammonium persulfate (APS) activated by heat, the coupled AM in-situ polymerizes into PAM chains and keeps the supramolecular structure with PAA, forming topological cooperative domains with cooperative H-bonding. The cooperative H-bonding consists of double H-bonding between carboxylic groups and amide groups (Supplementary Fig. 2 ), contributing to high bonding strength. Meanwhile, the polymerization of dissociated AM forms noncooperative H-bonding matrix interconnecting with the topological cooperative domains. The noncooperative H-bonding matrix is instead composed of noncooperative H-bonding between amide groups and thereupon easily dissociates compared to cooperative H-bonding, making cooperative domains the scaffold upon deformation. Due to the discrete distribution, the cooperative domains built by PAA3k chains only reach surrounding PAM chains after polymerization, inducing permanent cut-off damage and continuous disentanglement of the polymer network. Instead, the button-knot cooperative domains by PAA240k colloids become reversible elastic scaffold and stabilize the entire matrix, resulting in highly recoverable mechanical dissipation. Such cooperative domains also knit the mechanical damage at interface, providing the hydrogel with fully self-healing capability. The variation of colloidal behavior leads to different macroscopic characteristics of PAA solution even at the same concentration. 25 wt % PAA240k solution exhibits typical shear-thinning effect at a shear rate of 30 s − 1 (Supplementary Fig. 3 ) whereas 25 wt % PAA3k solution is nearly Newtonian liquid, indicating that PAA240k forms colloidal suspension in water. Such PAA240k solution exhibits a more intense Tyndall effect, and PAA240k droplet is highly viscous compared to PAA3k droplet (Fig. 1b ), indicating the presence of highly entangled PAA240k colloids. Notably PAA240k and PAA3k solutions show different colors after they dye with Coomassie brilliant blue, which turns green at the pH value < 1. The color difference results from the low dissociation degree of PAA240k chains. The carboxyl group of PAA240k is associated with each other due to the steric hinderance, leading to lower H + concentration. The colloidal behavior is further evidenced by the Cryo-TEM observation (Fig. 1c ). Due to the limited size and high association with water, low-m.w. PAA solution (PAA3k, m.w. = 3000) barely exhibits discernable morphology, while high-molecular-weight PAA solution (PAA240k, m.w. = 240,000) leads to the colloids around 7 nm distributed in ice with two to four PAA nanostrings (the radius is around 2 nm). The polymer chains at high molecular weight tend to entangle at high concentration and self-assemble, thereby forming button-knot structure in water. The evolution of colloid behavior on different molecular weights of PAA is examined via molecular dynamics (Fig. 1d and Supplementary Movie 1 ). Low-m.w. PAA (repeating unit, r.u. = 30) spreads in water due to its affinity to water molecules and negligible steric hinderance, and ergo seventeen short chains barely overlap during the molecular dynamic simulation. Meanwhile, high-molecular-weight PAA (r.u. = 256) self-associates due to the significant steric hinderance so that intermolecular and intramolecular H-bonding becomes dominant. Therefore, two long PAA chains entangle with each other and extend into aqueous system, leading to button-knot cooperative domains. The resultant high-molecular-weight cooperative domains can knit the mechanical damage at the interface, which grants the hydrogel with fully self-healing capability (Fig. 1e and Supplementary Movie 2 ). Such cooperative domains also stabilize the entire hydrogel network, resulting in highly recoverable mechanical dissipation (Fig. 1f and Supplementary Movie 3 ). With the hierarchical H-bonding strength and the topological distribution of cooperative domains, hydrogel manages to completely heal from the cut-off and retain the stress upon successive loading (Fig. 1g ), making it prominent compared to chemically crosslinked hydrogel and other self-healing hydrogels with cooperative matrix. Characterization of polymerization Before the polymerization, the precursor solution was examined via small-angle X-ray scattering (SAXS). From SAXS profiles, both PAA3k and PAA240k aqueous solution at 25 wt % experience a downturn at low-q region (Fig. 2a ), which typically exists in polyanionic polymer solution where the polyanionic backbone carries negative charges and leads electrostatic screening 34 , 35 . When the separation between polymers is lower than electrostatic screening distance, polyanionic chains self-organize and form loosely ordered structure to minimize such interaction, forming colloids and resulting in a peak at low q region around q 0 = 0.04 Å − 1 in case of PAA240k solution. Compared with PAA3k, PAA240k exhibits the preferential tendency towards ordered assembly due to the narrower characteristic peak, confirming that PAA240k self-assembles due to the repulsive electrostatic screening and steric hinderance. PAA3k is more mobile and thereupon has the assembled structure only after the addition of AM. Fig. 2 The polymer network directed by PAA colloids. a SAXS profiles of PAA240k, PAA240k+AM, PAA3k, and PAA3k+AM precursor solution with Lorentzian peak approximation. The fit result is correlated with correlation length (ξ). b In-situ ATR-FTIR spectra of PAA240k-AM during polymerization. Each spectra were recorded based on the polymerization time. c SAXS profiles of PAA-AM hydrogels with correlation length approximation and Lorentzian peak approximation. d AFM images of PAA3k-AM and PAA240k-AM. e Storage modulus ( G ′) and loss modulus ( G ′′) of PAA240k-AM and PAA3k-AM hydrogels. The colloidal structure was further evaluated by Lorentzian peak fitting to obtain the correlation length (ξ), which describes the spatial extent over which the electrostatic interactions between the charged groups on the polymer chain are significant 36 , 37 . The ξ of PAA240k precursor solution is 30.3 Å (Fig. 2a and Supplementary Table 1 ), higher than that of PAA3k precursor (ξ = 18.9 Å), because the correlation length scales with the chain length in good solvent and is related to the mesh size of the polymer network in concentrated polymer solutions. Additionally, the peak position of PAA240k is significantly lower than calculated chain length of PAA240k, indicating that the peak is attributed to the structure within colloid 37 . The ξ of PAA240k is close to the diameter of PAA240k nanostrings from Cryo-TEM images, in concordance with the observed mesh size of PAA240k assembly. After the addition of AM, the resultant PAA240k+AM precursor retains the characteristic peak around 0.042 Å −1 and leads to higher ξ of 32.3 Å, suggesting enhanced intermolecular interaction and extended state of PAA240k chains upon interacting with AM. Meanwhile, PAA3k solution has the fitting peak at 0.064 Å − 1 (Supplementary Table 1 ), which is close to the calculated chain length of PAA3k, and thereupon the peak is related to inter-colloid interactions. PAA3k+AM solution results in a characteristic peak around 0.018 Å −1 and is fit to have a ξ of 17.2 Å. The decrease in correlation length suggests the further coiled state of PAA3k chains, which may result from the steric separation by AM monomers and accompanying charge compensation. The polymerization of hydrogel was characterized by in-situ attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy (Fig. 2b ). The FTIR spectra of precursor solution exhibit four characteristic peaks of acrylamide at 1666 cm −1 ,1628 cm − 1 , 1501 cm −1 , and 1460 cm −1 , corresponding to the v (C = O), v (−NH2), v (C = C) and v (C − N), respectively 38 . The v (COOH) from protonated PAA locates around 1710 cm − 1 , partially overlapping with that from AM. After the polymerization of AM, the v (C = C) vanishes and the v (C − N) shifts to higher wavenumber. The v (COOH) from carboxyl group shifts to lower wavenumber (Supplementary Fig. 4 ), suggesting the further complexation of between PAA and PAM after the polymerization. The in-situ ATR-FTIR spectrum shows that as the polymerization initiates, the AM monomer is attacked by the free-radical from initiator as the v (−CH = CH 2 ) rapidly vanishes. Notably, the v (C = O) retains at the same wavenumber until the polymerization ends, implying that the bonding environment with AM does not change along the polymerization. SAXS was further conducted to characterize the structural information of cooperative domains of hydrogels. The down-turn phenomenon retains in PAA240k-AM hydrogel after the polymerization yet is absent in PAA3k-AM hydrogel (Fig. 2c ), reflecting that only PAA240k colloids keep the interaction. The repulsive interaction between PAA3k is possibly compensated by PAM chains due to the lack of steric hinderance, leading to a minor peak around q 0 = 0.02 Å −1 . From the gaussian size fitting (SasView, 5.0.5 Version), the PAA240k-AM hydrogel shows the similar peak position at 0.0438 Å −1 with precursor solution (PAA240k: q 0 = 0.0434 Å − 1 , PAA240k+AM: q 0 = 0.0434 Å −1 ) (Supplementary Fig. 5 and Supplementary Table 2 ), again confirming that the colloidal structure is retained to form cooperative domains throughout the polymerization. The ξ of PAA240k-AM is further intensified from 32.3 Å to 40.0 Å after the polymerization (Figs. 2a and c and Supplementary Table S1 ), indicating the stronger intermolecular interaction within cooperative domain to sprawl in noncooperative matrix. Meanwhile, the ξ of PAA3k-AM decreased from 17.2 Å to 12.7 Å after the polymerization (Supplementary Table 3 ), indicating that discrete islands further contract and reach limited PAM chains 39 . PAA-AM hydrogels demonstrates different polymer network due to the directed polymerization around different PAA colloids 40 . Field-emission scanning electronic microscope (FESEM) images show that PAA240k-AM has uniform morphology at micron level, which is different from typical hydrogels with mesopores or micropores 10 , 41 , 42 , while PAA3k-AM leads to bumpy surface (Supplementary Fig. 6 ). At submicron scale, atomic force microscope (AFM) images show that both hydrogels exhibit the mixed morphology of plains and gullies where plains are polymer regions and gullies are the residual of sublimed ice (Fig. 2d ). PAA3k-AM has dense yet tiny plains at nanoscale, and PAA240k-AM leads to interconnecting and stout plains, again indicating that PAA240k-AM has continuous PAA scaffold, but PAA3k-AM has discrete domains. The viscoelastic response was examined to investigate the stability of the polymer network (Fig. 2e ) 43 . The storage modulus ( G ′) is significantly higher than loss modulus ( G″ ) at all frequencies for all hydrogels, indicating their elastic and solid-like nature. Specially, the G ′ of PAA240k-AM reaches 61 kPa at ω = 1 rad s − 1 , even higher than the value of reported chemically crosslinked PAM hydrogel 44 , 45 , and is increasing across the frequency sweep. The increase of stiffness at higher frequencies indicates the entangled and non-permanent nature of polymer network, which is observed in both polymer melts and gels. The high G ′ signifies the large stored deformation energy in hydrogel network and would act as the driving force to reform the hydrogel structure to original shape. On the other hand, the G″ of the topological hydrogel is significantly lower than that of the copolymer hydrogel (Supplementary Fig. 7 ), indicating less viscous contribution during deformation. The stable G ′ and G ′′ of the topological hydrogels stem from the rapid construction of noncooperative H-bonding and contribute to the elastic recovery upon stretching. The loss factor (tan δ), defined as the ratio of G″ to G ′, describes the proportions of elastic and viscous behavior. Due to the benefits of cooperative domains, the tan δ of PAA240k-AM is much less frequency-sensitive compared to P(AA-co-AM) and PAA3k-AM before ω = 10 Hz. It remains below 0.1 until ω = 40 Hz (Supplementary Fig. 8 ), demonstrating behavior close to ideal elasticity (tan δ = 0) 46 . The downturn of tan δ in case of P(AA-co-AM) and PAA3k-AM indicates that both hydrogel networks relax at low frequency whilst PAA240k-AM behaves like crosslinked hydrogel. The self-healing and mechanical performance based on molecular weights The topological cooperative domains, built by long-chain colloids and cooperative H-bonding, grants the PAA240k-AM hydrogel with self-healing ability. When the hydrogel is cut, the physical bonding network is separated into two parts. During the self-healing process, the nanofibers of PAA colloids diffuse through the wound interface and knit the interface with supramolecular complex. Polymers then re-entangle with each other and thereupon recover the wound. PAA240k-AM is cut and placed at room temperature in a humid atmosphere to observe the self-healing process directly under the industrial digital microscope (Fig. 3a ). Initially the gap with width of 60 μm is observed, then progressively dimmers with the increasing healing time and completely vanishes after 12 h. By attaching each other, several separate hydrogel blocks also heal together in humid condition and are able to bend and stretch without damage (Fig. 3b ). Besides, the mechanical property of hydrogel after cutting off is restored with the respect of healing time. PAA240k-AM shows humidity-dependent stretch fracture behavior and possesses the stretchability of 12 and maximum stress of 110 kPa at 60% humidity (Supplementary Fig. 9 ), while PAA3k-AM and P(AA-co-AM) only demonstrate limited stretchability and breakage stress (Supplementary Fig. 10 ). For PAA240k-AM, 50% of the break elongation and 60% of the original break strength are recovered after 3-h healing, and the mechanical property is utterly restored after 5-h healing (Fig. 3c , Supplementary Fig. 11 and Supplementary Movie 4 ). At 333 K, the self-healing efficiency of PAA240k-AM is further improved, completely restoring mechanical behavior in one hour (Supplementary Fig. 12 ). In comparison, short PAA chains in PAA3k-AM and loose cooperative H-bonding in P(AA-co-AM) fail to knit the surrounding polymer chains (Fig. 3c and Supplementary Fig. 13 ), resulting in limited self-healing capability (Fig. 3d ). The elastic behavior of PAA240k-AM is also recovered by conducting successive stretch cycles (Fig. 3e ). Fig. 3 Self-healing and mechanical performance of PAA-AM hydrogels. a Self-healing process of cut PAA240k-AM observed by a digital microscope. b Photos of PAA240k-AM blocks healing towards each other. c Stress-stretch curves of PAA240k-AM and PAA3k-AM after different healing time. The stretch rate is 2 min −1 . d Healing capabilities of PAA-AM hydrogels after 5 h. The healing capability is calculated based on the recovered extensivity. e Successive stress-stretch curves of PAA240k-AM at λ = 2 after healing for 5 h. The stretch rate is 3 min −1 . f Successive stress-stretch curves of PAA240k-AM, PAA3k-AM, and P(AA-co-AM) at stretch ( λ ) = 2 for 10 cycles. The stretch rate is 3 min −1 . g The reversibility of PAA-AM hydrogels upon 10 cycles. Error bars belong to the standard deviation. h Comparison of self-healing efficiency and toughness retention between this work and previously reported hydrogels. Solid symbols belong to non-self-healing hydrogels, and hollow symbols are subtractive self-healing hydrogels. In case of mechano-stability of PAA240k-AM, the loading curve increases with higher stretching rates due to the different amount of broken sacrificial bond and the higher interchain friction, which is a typical mechanical behavior of entangled polymer network (Supplementary Fig. 14 ) 27 . Successive stretch cycles at different humidities shows that PAA240k-AM is capable of bouncing back, dissipating energy along the loading cycle and achieving the same stress as that of the first cycle above 60% humidity (Fig. 3f and Supplementary Fig. 15 ). In contrast, PAA3k-AM and P(AA-co-AM) fail to reach the same stress during successive cycling. Notably, the second loading curves of all hydrogels overpass the previous unloading curve, reflecting that the H-bonding network reforms during successive loading. The elasticity of hydrogel is comprehensively evaluated by multiple parameters such as reversibility and residual strain. The reversibility is defined as the ratio of the toughness from the corresponding loading curve to that from the first loading curve, U n / U 1 (Supplementary Fig. 16 ) 47 . In case of cyclic loads at λ = 2, PAA240k-AM retains higher reversibility than those of PAA3k-AM and P(AA-co-AM), reflecting the robustness of button-knot PAA240k colloids in stabilizing surrounding noncooperative matrix (Fig. 3g ). With the reversibility (>85%) and self-healing efficiency (~100%), the performance of PAA240k-AM surpasses that of other reported hydrogels (Fig. 3h , Supplementary Table 6 ) 13 , 14 , 16 , 17 , 19 , 20 , 24 – 26 , 28 , 41 , 42 , 47 – 54 . The residual strain is defined as the unrecoverable deformation from cyclic loads and examined as the strain when slope of unloading curve abruptly changes (Supplementary Fig. 17 ). The initial residual strain of PAA240k-AM after the first cycle is 0.83% (Supplementary Fig. 18 ), the lowest among all PAA-AM hydrogels (PAA3k-AM: 11.4%, P(AA-co-AM): 16.7%). Notably, the molecular weight of PAA chains significantly affect the stability of resultant polymer network. The residual strains of all cyclic loading curves are tracked, and PAA240k-AM keeps the residual strain below 6.3% throughout 10 cycles while those of PAA3k-AM and P(AA-co-AM) soar to 26% and 34%, respectively, indicating that long-chain PAA as an interconnecting scaffold holds the surrounding PAM matrix from significant plastic deformation. Therefore, even with the same mass ratio, the topology of polymer network plays a significant role in determining elasticity, and the cooperative domain based on button-knot PAA colloids effectively stabilize the polymer network. The swelling behavior depending on molecular weights Since the association degree of carboxyl group is dependent on the molecular weights of PAA chains, the as-formed PAA-AM hydrogels exhibit the variation on swelling behavior. Unlike typical physical hydrogel, PAA240k-AM swelled in all dimensions and retained the structural integrity even after 48 h (Fig. 4a ), while PAA3k-AM rapidly dissolved in water and lost the initial shape after 6 h. PAA240k-AM can stabilize at the swelling ratio of 10.5 after 12 h due to high association degree and entangled polymer network (Fig. 4b ), and the water content after swelling is 96.1 wt% . In contrast, P(AA-co-AM) and PAA3k-AM continuously swell and lose integrity after 3 h and 8 h, respectively (Fig. 4b and Supplementary Fig. 19 ). Absent from entangled self-assembly, the highly hydrophilic nature of acrylic acid and arylamide leads full dissociation of polymer network. SAXS pattern shows that PAA240k-AM keeps the downturn shape near low q region even after swelling for 12 h (Fig. 4c ), reflecting that PAA240k chains maintain the self-assembly structure even though PAA is known to be highly associated with water. The Lorentzian peak approximation leads to the increase of ξ from 40.0 Å to 73.0 Å (Supplementary Table 5 ), indicating that the assembled PAA nanostrings expand upon the introduction of water and leads to highly associated yet entangled PAA nanostrings. Fig. 4 Swelling behavior of PAA-AM hydrogels. a Photos of PAA240k-AM after swelling for 48 h and PAA3k-AM after 6 h. Red lines circle out the hydrogel throughout swelling. b Swelling evolution of PAA240k-AM and PAA3k-AM across time. c SAXS profiles of PAA240k-AM before and after swelling with Lorentzian peak approximation. d Photos of swelled PAA240k-AM throughout the cycling at λ = 2. e Successive loading curves of swelled PAA240k-AM at λ = 2, λ = 3, and λ = 4. The stretch rate is 3 cm −1 . f cyclic loading curves of swelled PAA240k-AM at λ = 2 for 100 cycles. The assembled PAA fibers stabilize the polymer network of PAA240k-AM even after swelling. The swelled PAA240k-AM has a breaking stress of 6.4 kPa and a breaking elongation of λ = 6.7 (Supplementary Fig. 20 ) and exhibits no shape change upon cycling at λ = 2 for 100 loading cycles (Fig. 4d ). Upon cyclic loading, the swelled PAA240k-AM exhibits no residual strain and low dissipation even at λ = 4 (Fig. 4e ), demonstrating the fully recoverable polymer network. After swelling, the chain friction and non-cooperative H-bonding are minimized so that the mechanical dissipation in the virginal PAA240k-AM becomes negligible and stabilized by the entangled button-knot cooperative domains. One hundred cycles at λ = 2 also lead to zero residual strain and stable loading (Fig. 4f and Supplementary Movie 5 ). The evolution of microstructure during deformation The mechano-stability of PAA-AM hydrogels is first evaluated from the mechanism of bonding dissociation. The average H-bonding strength in topological polymer network is investigated via a simple molecular model (Fig. 5a ). The single H-bonding (4) between amide and carboxyl groups is −184 kJ/mol (Supplementary Fig. 21 ), higher than those other H-bondings. Considering the double H-bonding nature, the overall H-bonding strength in PAA/PAM scaffold is much stronger than the noncooperative matrix (Fig. 5b ). During stretching, the noncooperative matrix easily dissociates upon deformation, and cooperative domains immobilize PAM chains as the elastic scaffold due to the variation of H-bonding strength. Fig. 5 Structural evolution of PAA-AM hydrogels. a The molecular models of PAM matrix and PAA/PAM scaffold. b The binding energy of hydrogen bonding within PAM matrix and PAA/PAM colloids based on the theoretical simulation. c – e Time-resolved SAXS profiles of c PAA3k-AM, d PAA240k-AM and e swelled PAA240k-AM for ten successive cycles. f Schematic of stretching of topological hydrogel. High-molecular-weight PAA chains form button-knot colloids and result in elastic recovery. Time-resolved SAXS profile tracks the evolution of polymer network during cyclic loading. The characteristic peak of PAA3k-AM around 0.04 Å −1 gradually deteriorates (Fig. 5c ), and the corresponding characteristic ring weakens during deformation and almost disappears after 10 cycles (Supplementary Fig. 22 ), reflecting the inter-colloid ordering continuously decreases. Notably, the ξ of PAA3k-AM remains unchanged throughout cyclic loading (Supplementary Fig. 23 and Table S3 ), suggesting that PAA3k remains coiled state and is weakly correlated to the deformation. In contrast, PAA240k-AM shows that the colloidal peak rapidly stabilizes around 0.034 Å −1 after the second cycle (Fig. 5d ). The ξ of PAA240k-AM decreases from 40.0 Å to 30.3 Å and stabilizes after the second cycle (Supplementary Fig. 23 and Supplementary Table 4 ), conforming with the trend of toughness. Meanwhile, the ξ of PAA3k-AM continuously decreases after the second cycle, reflecting that cooperative domains within PAA3k-AM substantially contact. The corresponding 2D SAXS patterns substantially exhibit significant isotropy along 10 cycles, again indicating the excellent mechano-stability of colloidal structure under cyclic loads (Supplementary Fig. 22B ) and thereby confirming that button-knot colloids manage to stabilize the polymer network. After swelling, the characteristic peak of PAA240k-AM barely shifts throughout the cyclic loads, and the ξ of swelled PAA240k-AM increases to 77 Å and stabilizes (Fig. 5e , Supplementary Fig. 23 and Supplementary Table 5 ), indicating that the stability of polymer network is further improved when the chain friction is minimized. Therefore, due to the high bonding strength of cooperative H-bonding and entangled nature, the button-knot PAA is stable during the cyclic deformation and holds the surrounding PAM matrix (Fig. 5f ). The noncooperative PAM matrix easily dissociates upon deformation, and button-knot PAA immobilizes PAM chains as the cooperative domain, leading to elastic recovery of hydrogel. Based on the structural evolution of two PAA-AM hydrogels, molecular weight significantly impacts the packing conformation of PAA in multi-component concentrated solution and gel. Low-m.w. PAA exhibits coiled state and is separated by PAM network, thereby losing the interaction among PAA colloids. High-molecular-weight PAA instead are assembled as flexible nanostrings and form interconnecting scaffold, securing the self-healing network and structural stability. Therefore, the self-assembly conformation of PAA determines the reconstruction of polymer network and the structural integrity during deformation. In real-world applications, mechano-stability is critical for the robustness of devices, and autonomously self-healing capability prevents devices from unexpected damage. The hydrogel in this work provides strategy to surpass the trade-off between self-healing capability and elasticity, potentially solving the requirement of next-generation applications. In summary, we have developed a strategy on fabricating a self-healing and elastic physical hydrogel by topological cooperative domain. Such domains were constructed based on the topology of PAA chains and directed polymerization of AM. PAA chains formed the colloidal structure by repulsive electrostatic interaction and immobilized the PAM chains by cooperative H-bonding, retaining colloidal cooperative domains within the noncooperative PAM matrix. By tuning the chain length of PAA and thereupon the structure of cooperative domains, such topological structure resulted in a perfectly reversible and highly recoverable polymer network, achieving both ~100% self-healing efficiency and excellent elasticity (>100% stress retention, >85% toughness retention) during cyclic loads. The physical hydrogel still retains with 97.5 wt% after swelling and has 0 residual strain upon successive cycling. The cooperative domain not only provides the reversible cooperative bonding for wound-healing but also anchors the interconnecting noncooperative matrix, preventing irrecoverable mechanical dissipation. When the physical hydrogel is further swelled, the hydrogel leads 0% residual strain during cyclic loads. This topological strategy would shed light on the design of self-healing yet resilient hydrogels for the practical applications such as wearable electronics, human-machine interaction and biorobotics."
} | 9,156 |
38352797 | PMC10862693 | pmc | 2,523 | {
"abstract": "The internet of things and growing demand for smaller and more advanced devices has created the problem of high heat production in electronic equipment, which greatly reduces the work performance and life of the electronic instruments. Thermal interface material (TIM) is placed in between heat generating micro-chip and the heat dissipater to conduct all the produced heat to the heat sink. The development of suitable TIM with excellent thermal conductivity (TC) in both in-plane and through-plane directions is a very important need at present. For efficient thermal management, polymer composites are potential candidates. But in general, their thermal conductivity is low compared to that of metals. The filler integration into the polymer matrix is one of the two approaches used to increase the thermal conductivity of polymer composites and is also easy to scale up for industrial production. Another way to achieve this is to change the structure of polymer chains, which fall out of the scope of this work. In this review, considering the first approach, the authors have summarized recent developments in many types of fillers with different scenarios by providing multiple cases with successful strategies to improve through-plane thermal conductivity (TPTC) (k ⊥ ). For a better understanding of TC, a comprehensive background is presented. Several methods to improve the effective (out-plane) thermal conductivity of polymer composites and different theoretical models for the calculation of TC are also discussed. In the end, it is given a detailed conclusion that provides drawbacks of some fillers, multiple significant routes recommended by other researchers to build thermally conductive polymer composites, future aspects along with direction so that the researchers can get a guideline to design an effective polymer-based thermal interface material.",
"conclusion": "6 Conclusion Carbon-based fillers provide the best through-plane TC at reduced filler loading; yet, filler dispersion and processing remain a challenge in their implementation. They can be promising to meet all the demands but their high cost and poor electrical insulation need attention in future research. The authors believe that the h-BN is a promising candidate to meet all the demands of the high-performance TCPCs. However, the particles of BN fabricated on industrial scales exhibit a plate-like structure. Owing to the low filling density and poor processing capacity of plate-like BN, it is desirable to manufacture spherical-shaped BN nanoparticles with smaller and homogeneous sizes of particles. This might make it possible for significantly higher loading densities in polymers while maintaining the processability of the nanocomposites. The type of filler, aspect ratio, filler concentration, shape, and size of filler particle have a significant impact on the TC of polymer composites. There does not appear a distinct percolation threshold for thermally conducting composites. Future research should focus on establishing new mechanisms and models to assess the reliance of the TC of composite on the above-mentioned factors. Combining experimental and computational information may result in the understanding of trends along with quantifiable ideas for future studies. Authors believe that the hybrid fillers consisting of carbon and h-BN with proper surface treatment, high aspect ratio, less filler content, spherical shape of filler particles with large and small sizes, and the oriented arrangement of fillers make it easier to build continuous as well as efficient thermal conduction pathways in the desired direction. High TC can be produced by polymer combinations with a strongly packed framework and an abundance of hydrogen bonds. For the fabrication of strong frameworks, ice and salt-templated techniques are good, although magnetic and electrostatic techniques are the best and most reliable for creating excellent thermal interface materials that exhibit good thermal conduction in the desired direction. However, magnetic and electrostatic techniques require multiple processing steps, and a less viscous and anisotropic polymer matrix, for which BN is certainly a great option. Other fillers still need further research. Scientifically, testing the effectiveness of the developed material should not be limited to TC evaluation. The contact surface between the heat-generating chip, the TIM, and the heat sink is one of the main challenges to obtain the best performance of the TIM. Its surface must be carefully improved by modifying the design of the device and TIM. If the entire coating of the electronic equipment is composed of TIM, maximum heat can be dissipated in all directions, but much research is needed to build a reliable and mechanically strong TIM with excellent TC. This idea may be helpful in improving future thermal management developments.",
"introduction": "1 Introduction A polymer composite is a versatile material that combines various fillers with the polymer matrix to produce coordinated properties that are not possible from any one of the components alone [ 1 ]. Polymers and polymer composites are extensively used in medical [ 2 ], biological [ 3 ], energy [ 4 ], industry [ 5 ], and all fields of daily life, owing to their lightweight, stability, low cost, good processing, and excellent corrosion resistance, etc. [ 6 ]. From a technological point of view, polymers can be classified into three types: thermosets, thermoplastics, and elastomers (rubbers). Thermosets are polymers that cannot be re-formed when heated, adopt a fixed or permanent shape, and break down when heated further. Thermosets are generally amorphous types of polymers. Thermoplastics are polymeric materials that can be remolded if heated and solidified again on cooling. Thermoplastics can be amorphous or crystalline. Elastomers are those polymeric materials whose dimensions can be greatly altered by applying moderate force and when the force is released, the elastomers return to their initial dimensions. Soft thermally conductive elastomers are of interest in electronic devices because of their thermally conductive properties [ 7 ]. Elastomers illustrate a fresh category of soft and versatile composites when tiny liquid metal (LM) alloy droplets are dispersed in their matrix, these composites have the power to revolutionize wearable electronics, soft robotics, as well as biocompatible machining. However, LM alloys have to stay liquid throughout the complete range of temperatures if they are to be used in the aforementioned applications and preserve acceptable mechanical performance throughout the duty cycle [ 8 ]. As time goes by, electronics are getting more powerful and compact and have new functions. This rapid development is facing the problem of unequal temperature distribution and high heat accumulation in the device which leads to short life and unreliability of the electronic equipment. This is the main challenge for the best operation and reliability of electronics [ 9 ]. Thermal management failures cause almost 55 % of problems in electronic devices. Thermal management is defined as the heat transfer through the device to heat dissipaters, such as heat sinks and spreaders [ 10 ]. The results of earlier experiments showed that a temperature rise of just 2 °C can cause a 10 % reduction in device performance [ 11 ]. Therefore, the establishment of an effective and cost-effective procedure is crucial for the automatic cooling of electronic devices. Generally, metals like gold (Au), silver (Ag), copper (Cu), and aluminum (Al) have higher TC than polymers. But metals are heavy and come at a high price [ 12 ]. Thermally conductive polymer composites (TCPCs) have become more important due to the growing demand for smaller, lighter, and more powerful electronic devices. They represent an interesting option for efficient heat management [ 13 ]. This is undoubtedly owing to the truth that thermally conductive and electrically insulating polymers have developed considerably in the present era, mainly because of remarkable developments in material science and technology. Due to the poor transport capacity of the thermal carriers (phonons), the intrinsic TC of polymers is just 0.1–0.5 Wm −1 K −1 [ 14 , 15 ]. Because polymer composites are simple to construct, environmentally acceptable, and have affordable processability, new ways to increase the TC of TCPCs are attracting a great deal of interest [ 16 ]. These efforts can be divided into two types, depending on the approach used. The first strategy is to appropriately organize molecules and chains and their orientations in the pure polymer to improve TC, usually by creating a highly organized structure, which encourages the transport of phonons. At the same time, the rearrangement will cause a decrease in the phonon scattering at interface defects and minimize chain entanglement. These two aspects work together to encourage the intrinsic polymer to achieve a high value of TC while keeping up a low value of EC without the addition of electrically conductive materials. The second is the improvement of the TC of the polymer by including thermally conductive fillers, or a structure produced by such fillers, in the polymer matrix. By modifying the content, type, geometry, and distribution of the different fillers, the characteristics of the resulting polymer composite can be enhanced [ 17 ]. Of these two approaches, the first is challenging for large-scale production and is therefore limited to nanoscale laboratory research. Therefore, this review focuses mainly on the second approach. The TC of polymers can be effectively improved by introducing different fillers into their matrix [ [18] , [19] , [20] ]. This review gives a summary of the achievable ways to optimize the TC of polymers. There are many good reviews on the advancement of polymer TC but less research has been done on the effective TC (through-plane thermal conductivity, TPTC, k ⊥ ) and suitable types of fillers. A broad review is needed for the better development and effective design of polymer-based compounds, which would be helpful for the removal of heat particularly when insulation is required. The outline of this review article is as follows: Initially, a background section is included for a better understanding of polymeric TC and the potential of polymeric materials. Then, in the third section, the methodologies to improve the TPTC of polymer composites are presented. In the next section, the types of fillers and results of using these different types of fillers in terms of through-plane TC (k ⊥ ) of polymers are mentioned. In the last section, the authors have given a short discussion on the theoretical calculation models of effective TC. In the end, a conclusion is provided detailing the drawbacks of some fillers, challenges, multiple significant routes recommended by other authors to construct thermally conductive polymer composites, and future aspects along with some guidelines that may help other researchers in the design of new efficient polymer-based TIMs."
} | 2,769 |
35516629 | PMC9054545 | pmc | 2,524 | {
"abstract": "As a useful and renewable chemical building block from biomass, 2,5-furandicarboxylic acid (FDCA) has become an increasingly desirable platform chemical as a terephthalic acid replacement for polymerization. In this work, an efficient and highly selective biocatalytic approach for the synthesis of FDCA from 5-hydroxymethylfurfural (HMF) was successfully developed using a TEMPO/laccase system coupled with Pseudomonas putida KT2440. TEMPO/laccase afforded the selective oxidation of the hydroxymethyl group of HMF to form 5-formyl-2-furancarboxylic acid as a major product, which was subsequently oxidized to FDCA by P. putida KT2440. Manipulating the reaction conditions resulted in a good conversion of HMF (100%) and an excellent selectivity of FDCA (100%) at substrate concentrations up to 150 mM within 50 h. The cascade catalytic process established in this work offers a promising approach for the green production of FDCA.",
"conclusion": "Conclusions In this study, we developed a promising approach for the production of FDCA from HMF by a TEMPO/laccase system coupled with P. putida KT2440. This method exhibits 100% conversion of HMF and 100% selectivity of FDCA under mild conditions at substrate concentrations up to 150 mM and does not require complex gene modification, enzyme purification or expensive cofactor addition. Currently, the preparation of HMF from inexpensive and highly abundant lignocellulosic biomass has been well documented in the literature. The synthesis of FDCA from biomass resources can be attained readily by the combination approach established in this work. Additionally, the success of the coupling system will provide a similar alternative approach for further biomanufacturing of other value-added chemicals.",
"introduction": "Introduction The massive depletion of nonrenewable fossil resources and the emission of greenhouse gases have produced the widest range of changes in the world. 1 There has been growing interest in developing renewable and sustainable fuels and platform chemicals. 2–5 Lignocellulosic biomass is an important renewable carbon resource, and its derived carbohydrates can be converted into various biobased platform compounds for the production of high-value products. 6,7 Among these compounds, 5-hydroxymethylfurfural (HMF) is such a compound that has a furan ring, a hydroxymethyl group and an aldehyde group, which makes it an appealing starting material for catalytic upgrades. It can be selectively oxidized to versatile building blocks, including 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-diformylfuran (DFF), 5-formyl-2-furancarboxylic acid (FFCA), and 2,5-furandicarboxylic acid (FDCA). 8 Among them, FDCA, obtained through the full oxidation of the hydroxymethyl and aldehyde moieties in HMF, is only listed as one of the 12 key value-added chemicals from biomass by the U.S. Department of Energy. 9 It has been considered to have a huge market potential because it can be used as a ‘green’ substitute of terephthalic acid for the production of poly(ethylene terephthalate). 10 In the past few years, intensive research efforts have been conducted to synthesize FDCA by the oxidation of HMF through chemical pathways, 11 such as the use of traditional stoichiometric oxidants, 12 homogeneous catalysts, 13 and heterogeneous catalysts (various noble metal catalysts, such as supported Au, Pt, and Pd catalysts). 14 However, these chemocatalytic approaches are characterized by problems associated with harsh reaction conditions, such as high temperature, high pressure, and the presence of metal salts and organic solvents, which render the process expensive and polluting. In addition, the selectivity of some catalysts is poor in specific reactions, resulting in the formation of byproducts. 15,16 Biocatalysis is emerging as a valuable tool to address these problems in the context of green chemistry since it is typically performed under mild conditions, usually requires fewer and less toxic reagents and solvent than chemocatalysis, and exhibits excellent selectivity. 17 Currently, biocatalytic production of FDCA mainly involves microbial and enzymatic conversion approaches. Since the FDCA concentration produced by wild-type strains is too low, 18 gene modification or metabolic engineering becomes an indispensable step for the microbial conversion process. Two notable examples of microbial bioconversion are the recombinant strains Pseudomonas putida S12 ( ref. 19 ) and Raoultella ornithinolytica BF60. 20 Although both of them give relatively high FDCA titers, they still suffer from several disadvantages: complex molecular operation and low productivity or yield of FDCA. Enzymatic bioconversion is another promising alternative approach, but there are limited examples in the literature. An HMF oxidase was identified with a remarkable capability of oxidizing HMF to FDCA, which was conducted at very low concentrations (2–4 mM). 21,22 The full oxidation of HMF more often needs a combination of two enzymes, as the production of FDCA entails three consecutive oxidation steps. For example, aryl-alcohol oxidase coupled with an unspecific peroxygenase, 23 galactose oxidase coupled with lipase, 24 and galactose oxidase variant M 3–5 coupled with aldehyde oxidase 25 have been reported in the past five years. Compared with the corresponding bioconversion by microbes, the bioconversion of HMF to FDCA proceeded by enzymes needs extra expression and purification steps. Sometimes, the reaction conditions of two enzymes do not match well, which further complicates the whole process. The shortcomings of the existing methods and the potential importance of FDCA production motivate us to further explore efficient and robust oxidation method. In this study, we designed a method of coupling reactions to produce FDCA from HMF. The key feature of this approach is the oxidation of the hydroxymethyl group via a TEMPO/laccase system and the oxidation of the aldehyde moiety by P. putida KT2440 cells. Combining the advantages of two catalysts, a satisfactory HMF conversion and FDCA selectivity were realized even at high HMF concentrations (150–200 mM). This study provides a highly green and efficient biocatalytic alternative for the production of FDCA from HMF.",
"discussion": "Results and discussion Design and selection of routes for FDCA production The synthesis of FDCA from HMF requires a catalyst to act on both hydroxymethyl and aldehyde groups. Interestingly, a previous study in our group demonstrated that wild-type P. putida KT2440 could effectively convert HMF into HMFCA, and no DFF, FFCA or FDCA formation was observed, 26 which shows perfect selective oxidation toward the aldehyde group of HMF. For the oxidation of the hydroxymethyl group, TEMPO/laccase system has been employed in the literature toward HMF. 24,27 Herein, we attempted to combine the abilities of the TEMPO/laccase system and P. putida KT2440 to oxidize the hydroxymethyl group and the aldehyde group, respectively, hoping that they match perfectly and efficiently achieve FDCA production under mild reaction conditions. Based on the above assumptions, there are two possible pathways for the oxidation of HMF to FDCA ( Scheme 1 ). In the first pathway, the aldehyde group of HMF is first oxidized by P. putida KT2440 to yield HMFCA. Then, TEMPO/laccase is added to the reaction mixtures to further oxidize the hydroxymethyl group of HMFCA and thus complete full oxidation of HMF. In this pathway, it is uncertain whether TEMPO/laccase can oxidize the hydroxymethyl of HMFCA. In the second pathway, the TEMPO/laccase catalysis operates prior to P. putida KT2440. In this way, the conversion of the hydroxymethyl group of HMF is first catalyzed by the TEMPO/laccase system to generate furan mixtures, which are then converted to FDCA by P. putida KT2440. Regarding this pathway, we have not detailed and comprehensively investigated the biocatalytic ability of P. putida KT2440 toward other furanics except HMF. Scheme 1 Two pathways for oxidation of HMF to FDCA. To verify the feasibility of the first route, considering the oxidation of HMF to HMFCA by P. putida KT2440 had been proved, we set up a test reaction using HMFCA with the TEMPO/laccase system to identify whether the hydroxymethyl group of HMFCA could be oxidized. The reaction was started with addition of the commercially available fungal laccase. When HMFCA was incubated with the TEMPO/laccase system in acetate buffer, almost 64% FFCA and only 6% FDCA were detected after 24 h of incubation, and these percentages changed slightly during the subsequent incubation (approximately 19% FDCA after 60 h) ( Fig. 1A ). The TEMPO/laccase oxidation of HMFCA resulted in mixtures in which FFCA was dominant. Qin and coworkers ever reported that TEMPO/laccase could convert HMF into DFF as initial dominate intermediate and further transform into FFCA, but its oxidation efficiency of FFCA into FDCA was poor. 24 Our results further confirmed the low activity of the TEMPO/laccase system on FFCA. In general, the hydrated form ( gem -diol) of aldehydes act as the key intermediates play a central role in the aldehyde oxidation catalyzed by TEMPO/laccase system. Whereas, only a small fraction of FFCA may exist in hydrated form. 22 Thus, route 1 will not be sufficiently active to produce FDCA ( Fig. 1C ). Fig. 1 Time course of oxidation of HMFCA (A) and HMF (B) by TEMPO/laccase system and them coupled with P. putida KT2440 via route 1 (C) and route 2 (D) respectively. Reaction conditions: 30 mM HMFCA or HMF, 4 mL acetate buffer (50 mM, pH 4.5), 20 mol% TEMPO, 2.6 mg mL −1 laccase, air bubbling for 3 min each 12 h, 25 °C, 150 rpm. Next, the second route was evaluated. HMF was mixed with TEMPO/laccase to test the ability of this system to oxidize it. Most of the HMF (80% conversion) was converted into FFCA in 48 h, and very little DFF and FDCA were formed. The proportion of FFCA slowly decreased over time because of the formation of low amounts of FDCA (4.5% selectivity after 48 h) ( Fig. 1B ). Then, the ability of P. putida KT2440 to oxidize the intermediates of DFF and FFCA was tested (Fig. S1 † ). The results revealed that, given enough time, P. putida KT2440 can fully oxidize the aldehyde groups of both DFF and FFCA to yield FDCA. This means that the oxidation of HMF to FDCA via route 2 is indeed feasible ( Fig. 1D ). For this route, a critical task is determining how to improve the conversion of HMF to 100%, as the surplus HMF in the TEMPO/laccase system would be completely converted to HMFCA by P. putida KT2440, increasing the difficulty and cost of the separation and purification of FDCA. Effects of key reaction conditions on HMF oxidation To obtain higher FDCA selectivity and concomitantly eliminate HMFCA generation in the second step, a series of experiments were performed to improve the conversion of HMF. The influence of temperature on the oxidation of HMF in the TEMPO/laccase system was tested in the range of 20–30 °C for 48 h ( Fig. 2A ). The highest conversion of HMF (nearly 90%) was obtained at 20 °C. But considering that 25 °C is closer to room temperature, we selected 25 °C for the next experiment. It has been reported that the type of buffer and pH used can influence the conversion of substrates in reaction systems. 28 For this reason, the effects of buffer and pH on the oxidation of HMF were explored at 25 °C for 24 h. It was found that buffer type and pH exerted a significant effect on the catalytic performance. Compared to unbuffered aqueous solutions, all the buffered solutions tested were capable of considerably enhancing HMF conversion apart from phosphate buffer at pH 7.0. The use of sodium acetate buffer at pH 6.0 led to an almost perfect HMF conversion (100%). At the same pH of 6.0, the conversion of HMF in phosphate buffer decreased to 67.6%. For phosphate buffer, the best results were observed at pH 6.5 (HMF conversion of 83.1%) ( Fig. 2B ). In general, the results suggested that a buffered solution was necessary for the conversion of HMF because of the production of acidic intermediates during the reaction course. Thus, we selected sodium acetate buffer of pH 6.0 for subsequent work. During the aerobic oxidation of laccase/TEMPO, laccase can easily oxidise the stable oxyl-radical form of TEMPO to the oxoammonium ion. The oxoammonium ion of TEMPO as the actual oxidant could selectively oxidize the hydroxymethyl group of HMF. 27 Therefore, the dose of laccase was also crucial to HMF conversion ( Fig. 2C ). A control experiment confirmed that no substrate transformation occurred in the absence of laccase under the investigated conditions, which was in good agreement with previous reported results in the literature. 29 A maximum conversion of 95.6% was achieved at 2.6 mg mL −1 laccase. When the amount of enzyme was more than 2.6 mg mL −1 , there was no significant improvement in the conversion of HMF. Therefore, considering the cost of enzyme, a laccase dose of 2.6 mg mL −1 was used for the subsequent studies. Fig. 2 Effects of reaction temperature (A), buffer pH (B) and laccase concentration (C) on HMF conversion in TEMPO/laccase system. Reaction conditions: (A) 4 mL acetate buffer (50 mM, pH 4.5), 30 mM HMF, 20 mol% TEMPO, 2.6 mg mL −1 laccase, 150 rpm, the designated temperature, air bubbling for 3 min each 12 h. (B) 4 mL buffer (50 mM, CH 3 COOH–CH 3 COONa for pH 4.5–6.0, NaH 2 PO 4 –Na 2 HPO 4 for pH 6.0–7.0, and distilled water), 25 °C, others were the same as (A). (C) 4 mL acetate buffer (50 mM, pH 6.0), laccase of the designated concentration, others were the same as (B). Different letters represent significant differences between treatments ( p < 0.05). The oxidation of HMF in the TEMPO/laccase system at high substrate concentrations The excellent conversion at high concentrations of HMF obtained in the first step is conducive to the production of FDCA in the second step. Thus, the transformation of 10–200 mM HMF by the TEMPO/laccase system was firstly assessed under the optimum conditions obtained above. As presented in Fig. 3 , after 24 h of reaction, 100% conversion was achieved when the HMF concentration was ≤30 mM. For 50 mM and 70 mM HMF, the conversion was slightly decreased to 96% and 89%, respectively. However, the conversion was reduced markedly to 79% and 34% at HMF concentrations of 100 and 200 mM. The major reason for the unsatisfactory conversion at high concentrations might be the inappropriate amount of catalyst and poor buffer capacity in the reaction system because of TEMPO decomposition 30 and pH drop with time. Based on the above speculation, we next investigated the influence of these two factors on HMF conversion. First, the influence of TEMPO concentration on the oxidation of HMF by the TEMPO/laccase system was evaluated. As shown in Fig. 4A , when the laccase amount was constant, the conversion of HMF was promptly increased with increasing TEMPO concentration. When a TEMPO concentration of 80 mol% was used a high HMF conversion of nearly 100% was obtained after 12 h in the presence of 70 mM HMF, and further increasing the TEMPO concentration resulted in no significant changes in the conversion of HMF. This result means that a suitable TEMPO amount is crucial for the efficient conversion of HMF. A higher mediator/laccase ratio could greatly promote the conversion of HMF at high concentrations. Therefore, 80 mol% TEMPO was used for further research. Fig. 3 Effects of the initial HMF concentration on HMF conversion in TEMPO/laccase system. Reaction conditions: 4 mL acetate buffer (50 mM, pH 6.0), HMF of the designated concentration, 20 mol% TEMPO, 2.6 mg mL −1 laccase, air bubbling for 3 min each 12 h, 25 °C, 150 rpm. Different letters represent significant differences between treatments ( p < 0.05). Fig. 4 Optimization of the TEMPO dosage (A) and buffer concentrations (B) for HMF conversion. Reaction conditions: (A) 4 mL acetate buffer (50 mM, pH 6.0) containing 70 mM HMF, different concentration of TEMPO (0–150 mol%); (B) 4 mL acetate buffer (50–200 mM, pH 6.0), 100 mM HMF, 80 mol% TEMPO, 2.6 mg mL −1 laccase, air bubbling for 3 min each 12 h, 25 °C, 150 rpm. Different letters represent significant differences between treatments ( p < 0.05). Upon examining the second possibility for unsatisfactory conversion, we found that the continuous accumulation of intermediates (mainly FFCA) resulted in an appreciably decreased pH in the reaction system, which affected the progress of the reaction. For this reason, we investigated the effect of buffer concentrations (50, 100, 200 mM) on the catalytic reaction in the case of a higher HMF concentration. After a reaction period of 36 h, 100 and 200 mM sodium acetate buffer gave the best performance, and HMF conversion was close to 100% ( Fig. 4B ). Given these facts, we further increased the HMF concentration to 150 mM and 200 mM, and this TEMPO/laccase system was still capable of oxidizing HMF with conversion of 100% and 98.2%, respectively ( Table 1 ). In the literature, Qin et al. reported a conversion of 79% after 48 h starting with 30 mM HMF. 24 Apparently, the results reported in this work were better than the previous results. To our knowledge, this is the best HMF conversion at such a high substrate concentration in the TEMPO/laccase system ever reported. Two-step cascade oxidation of HMF to FDCA \n \n Entry HMF (mM) Buffer (mM) TEMPO (mol%) HMF conversion (%) \n t \n a (h) FDCA selectivity (%) Cell dosage (g L −1 ) \n t \n b (h) 1 30 50 20 100 24 100 6 1 2 70 50 80 100 12 100 10 1 3 100 100 80 100 36 100 10 1 4 150 200 80 100 48 100 16 2 5 200 200 80 98.2 60 82.4 24 5 a The reaction time of TEMPO/laccase oxidation. b The reaction time of P. putida KT2440 biotransformation. Synthesis of FDCA via the TEMPO/laccase system and P. putida KT2440 in tandem With improved HMF conversion, we turned our attention to the second step. Considering that the intermediate FFCA and the acetate buffer in the first step might affect the biocatalytic performance of P. putida KT2440 in the last step, the effects of these factors on P. putida KT2440 were tested by employing the commercially available FFCA as a substrate. As shown in Fig. S2, † when the pH values varied from 4.5 to 6.0, P. putida KT2440 cells exhibited good catalytic performances, and a conversion of 100% was achieved after the short reaction time of 0.5 h. In addition, all FFCA was oxidized to FDCA with an excellent selectivity of 100%. Better yet, P. putida KT2440 maintained its outstanding capability under all tested FFCA and acetate buffer concentrations (Table S1 † ), which further demonstrated its great potential for oxidizing various furan aldehydes. Based on the above experimental results, the TEMPO/laccase system and P. putida KT2440 cells were coupled to test the conversion of HMF to FDCA in a sequential manner at different concentrations of HMF. When HMF was completely converted by the TEMPO/laccase system, the pH of the reaction mixtures was adjusted to 6.0 with NaOH, and P. putida KT2440 cells were added for further oxidation. The results of the two-step cascade oxidation of HMF to FDCA are summarized in Table 1 . In most cases, almost 100% conversion of HMF was acquired in the TEMPO/laccase system. Increasing the initial concentration of TEMPO and buffer was beneficial for HMF conversion at high concentrations. The complete oxidation of HMF by TEMPO/laccase resulted in the predominant formation of FFCA and minor amounts of FDCA. HMF to FDCA conversion was faltered at the aldehyde acid stage (FFCA) because of the low activity of the TEMPO/laccase system on FFCA. FFCA was not efficiently oxidized by TEMPO/laccase system, which correlates with the low degree of hydration. After P. putida KT2440 cells, which can directly oxidize the aldehyde group of FFCA, were added, FDCA was synthesized quickly, and FFCA decreased sharply in a short reaction time. A full conversion of intermediates from the previous step was realized even at HMF concentrations up to 150 mM. Moreover, excellent selectivity (100%) was retained during the reaction. Yang and coworkers exploited Comamonas testosteroni SC1588 cells for synthesis of FDCA from FFCA in TEMPO/laccase system. 31 However, it took about 15 h in the last step and achieved around 90% of conversion. Because a relatively high concentration of TEMPO in the catalytic system significantly inhibited the activity of the cells. However, P. putida KT2440 in this study can completely convert FFCA to FDCA within few hours in the second step. These results suggested that P. putida KT2440 is an excellent biocatalyst for the oxidation of FFCA. Besides, P. putida KT2440 has good compatibility with TEMPO/laccase system, which would likely be a potential and promising workhorse in biological conversion. Meanwhile, it was observed that improvement of the biocatalyst concentration was pivotal to obtaining a full conversion of FFCA with good product selectivity. When the concentration of starting HMF was increased to 200 mM, only 82.4% selectivity of FDCA was observed. Unsatisfactory FDCA production may be attributed to insufficient amount of biocatalyst at higher substrate loadings. The results obtained in this work were compared with those of previous studies. As shown in Table 2 , the electrochemical oxidation of HMF to FDCA was achieved using a catalytic anode under strongly acidic conditions. 32 Although the method appeared to be clean, the initial HMF concentration and FDCA yield reported in the literature were relatively low and accompanied by the formation of byproducts. Homogeneous catalysts have some problems in the synthesis of FDCA, such as high FDCA yield usually being obtained at the expense of harsh reaction conditions. 13 Compared with homogeneous catalysts, heterogeneous noble metal catalysts for the conversion of HMF to FDCA have been extensively studied due to their recoverability and reusability. 14 However, noble metal materials are expensive, and slashing reaction conditions was required. The biosynthesis of FDCA from HMF is of considerable interest because it offers many advantages: mild conditions, excellent selectivity and relatively little environmental pollution. Although some biocatalytic oxidation methods have been reported for the synthesis of FDCA, they still suffer from some weaknesses, such as long reaction time, low HMF loading, and unsatisfactory FDCA yield. For example, the sequential oxidation of HMF to FDCA by fungal aryl alcohol oxidase (AAO) and unspecific peroxygenase (UPO) has been described, 23 but its transformation efficacies were very poor (a reaction time of 120 h was required to achieve maximum yield). The TEMPO/laccase system coupled with P. putida KT2440 proved to be a good biocatalytic approach for the oxidation of HMF to FDCA because of its good conversion and excellent selectivity even at HMF concentrations up to 150 mM and 200 mM. HMF oxidation catalyzed by various catalysts Catalysts HMF (mM) Reaction conditions \n t [h] HMF C a [%] FDCA S/Y b [%] Ref. Manganese oxide (MnO x ) anode 20 pH 1H 2 SO 4 solution, 60 °C, 1400 rpm, E = 2.0 V vs. RHE, 250C charge passed n.a. c >99.9 53.8 (Y) \n 32 \n Co/Mn/Br ∼377 Co/Mn/Br = 1/0.015/0.5, T = 180 °C, P = 30 bar (molar CO 2 /O 2 = 1), H 2 O/HOAc = 7/93 (v/v), n = 1200 rpm 0.5 n.a. 90 (Y) \n 13 \n Pt/C 150 690 kPa O 2 , 2 equiv. of NaOH, 22 °C 6 100 79 (S) \n 14 \n Recombinant Raoultella ornithinolytica BF60 100 45 g per L cells, 30 °C, 50 mM phosphate buffer (pH 8) 170 n.a. 89 (Y) \n 20 \n HMF oxidase (HMFO) 4 20 μM HMFO, 20 μM FAD, 25 °C, potassium phosphate buffer of pH 7 24 100 95 (Y) \n 22 \n AAO + UPO successively 3 AAO (5 μM), UPO (0.65 μM), sodium phosphate (pH 7) 120 100 91 (Y) \n 23 \n TEMPO/laccase + C. testosterone SC1588 100 20 mol% TEMPO, 8.1 mg mL −1 laccase, 140 g per L cells, phosphate buffer (200 mM, pH 7) 36 100 87 (Y) \n 31 \n TEMPO/laccase + P. putida KT2440 in tandem 150 80 mol% TEMPO, 2.6 mg mL −1 laccase, 16 g per L cells, acetate buffer of pH 6 50 100 100 (S) This work a C: conversion. b Y: yield, S: selectivity. c n.a.: not available."
} | 6,057 |
28861250 | PMC5574787 | pmc | 2,525 | {
"abstract": "Abstract Differences in the direction and degree to which invasive alien and native plants are influenced by mycorrhizal associations could indicate a general mechanism of plant invasion, but whether or not such differences exist is unclear. Here, we tested whether mycorrhizal responsiveness varies by plant invasive status while controlling for phylogenetic relatedness among plants with two large grassland datasets. Mycorrhizal responsiveness was measured for 68 taxa from the Northern Plains, and data for 95 taxa from the Central Plains were included. Nineteen percent of taxa from the Northern Plains had greater total biomass with mycorrhizas while 61% of taxa from the Central Plains responded positively. For the Northern Plains taxa, measurable effects often depended on the response variable (i.e., total biomass, shoot biomass, and root mass ratio) suggesting varied resource allocation strategies when roots are colonized by arbuscular mycorrhizal fungi. In both datasets, invasive status was nonrandomly distributed on the phylogeny. Invasive taxa were mainly from two clades, that is, Poaceae and Asteraceae families. In contrast, mycorrhizal responsiveness was randomly distributed over the phylogeny for taxa from the Northern Plains, but nonrandomly distributed for taxa from the Central Plains. After controlling for phylogenetic similarity, we found no evidence that invasive taxa responded differently to mycorrhizas than other taxa. Although it is possible that mycorrhizal responsiveness contributes to invasiveness in particular species, we find no evidence that invasiveness in general is associated with the degree of mycorrhizal responsiveness. However, mycorrhizal responsiveness among species grown under common conditions was highly variable, and more work is needed to determine the causes of this variation.",
"introduction": "1 INTRODUCTION Many natural plant communities are either invaded or at risk of invasion by non‐native plant species (Pimentel, Lach, Zuniga, & Morrison, 2000 ). The negative consequences of such invasions have stimulated extensive research aimed at identifying the responsible mechanisms, so as to identify efficacious control/management strategies (Mack et al., 2000 ). Although several mechanisms are likely in play (Gurevitch, Fox, Wardle, Inderjit, & Taub, 2011 ), exotic species are generally thought to be less reliant on specialist mutualists than native taxa (Richardson, Allsopp, D'Antonio, Milton, & Rejmanek, 2000 ; Vogelsang & Bever, 2009 ) but whether this also extends to generalist mutualists is less known. Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that colonize about 75% of all plant species (Brundrett, 2009 ) with apparent low specificity (Lekberg & Waller, 2016 ). These ubiquitous fungi can provide increased nutrient uptake, drought tolerance, and pathogen protection to plants in return for carbon (Smith & Read, 2008 ). AMF could contribute to invasiveness if exotic plants benefitted more from these services than native plants, or if exotic plants were less dependent on AMF and/or could disrupt this mutualism in native plants. To determine whether exotic plants are less reliant on the mutualism with AMF, an important first step is to compare the mycorrhizal status and responsiveness (measured as the biomass difference between inoculated plants and non‐inoculated controls; Janos, 2007 ) of native and exotic plants. Some have suggested that a weak mycorrhizal responsiveness may be a general mechanism of plant invasion (van der Putten, Klironomos, & Wardle, 2007 ; Vogelsang & Bever, 2009 ) because invasions often occur in disturbed habitats (Mooney & Hobbs, 2000 ) that tend to harbor lower AMF abundance (Abbott & Robson, 1991 ). Consistent with this hypothesis is the fact that some well‐studied noxious invaders in North America are either nonmycorrhizal or have a low mycorrhizal responsiveness (e.g., Busby, Gebhart, Stromberger, Meiman, & Paschke, 2011 ; Stinson et al., 2006 ) and that exotic plants in California are disproportionally from nonmycorrhizal families (Pringle et al., 2009 ). Also, careful comparisons of native and exotic populations of both St. John's Wort (Hypericum perforatum; Seifert, Bever, & Maron, 2009 ) and yellow star thistle (Centaurea solstilialis; Waller, Callaway, Klironomos, Ortega, & Maron, 2016 ) have documented more ruderal traits and a reduced AMF responsiveness in the exotic populations. On the other hand, AMF can increase growth and competitiveness of spotted knapweed (Centaurea stoebe; Marler, Zabinski, & Callaway, 1999 ), which is one of the most invasive weeds in the intermountain west of the USA. This and another invasive forb ( Euphorbia esula ) can also increase AMF abundance and diversity compared to remnant native communities (Lekberg, Gibbons, Rosendahl, & Ramsey, 2013 ). And, contrary to California grasslands, exotic plants in Great Britain and Germany were more likely to be from families that were more dependent on AMF (i.e., greater proportion that were obligatory mycorrhizal and lower proportion that were facultatively mycorrhizal) than native plants (Hempel et al., 2013 ; Pringle et al., 2009 ). Thus, it is clear that interactions with AMF differ drastically among invasive plants and possibly across regions being invaded. A recent systematic literature search and summary (Bunn, Ramsey, & Lekberg, 2015 ) reported no appreciable difference between invasive and native plants in their growth responses to AMF, suggesting that invasions do not select for directional shifts in AMF associations. While this meta‐analysis represents the most comprehensive summary of published studies to date, there are some important limitations to the inferences drawn from such analyses. Specifically, meta‐analyses cannot always account for a high level of experimental heterogeneity among the included studies. The Bunn et al. ( 2015 ) analysis, for example, included data for 55 invasive and 70 native species from 67 studies that varied in experimental conditions (e.g., soil chemistry, identity of AMF taxa, growth conditions), which are known to affect plant–AMF interactions (Johnson, Wilson, Bowker, Wilson, & Miller, 2010 ; Klironomos, 2003 ; Stahl & Smith, 1984 ). These experimental nuances are not easily incorporated into meta‐analysis as factors or covariates (Koricheva, Gurevitch, & Mengersen, 2013 ) and may have prevented detection of actual differences between invasive non‐native vs. native taxa that are apparent only with a dataset obtained from a common set of experimental conditions. Also, plant responses to AMF are often phylogenetically conserved (Anacker, Klironomos, Maherali, Reinhart, & Strauss, 2014 ; Reinhart, Wilson, & Rinella, 2012 ), and phylogenetic nonindependence can bias results if the pool of species in comparison groups is disproportionately from clades that are highly responsive (or unresponsive) to AMF. To circumvent these limitations, we analyzed two relatively large datasets that approximated the total sample size of the Bunn et al. ( 2015 ) meta‐analysis where plants were grown under the same environmental conditions and where the effects of shared ancestry were controlled. We generated new mycorrhizal responsiveness data for 68 plant species from the Northern Plains (14 invasive: 54 noninvasive) and performed separate tests for a published dataset with 95 taxa (11 invasive: 84 noninvasive) from the Central Plains (Wilson & Hartnett, 1998 ). A previous analysis of the Central Plains dataset which did not control for phylogeny found that native grassland plants were more responsive to AMF than taxa from outside the region (fig. 3 in Pringle et al., 2009 ), and we tested whether the same result would be found if the effects of shared ancestry were included.",
"discussion": "4 DISCUSSION Overall, using phylogenetically controlled statistical tests, we found no evidence that invasive grassland plant species responded differently to inoculation with AMF relative to other plant taxa in either the Northern or Central Plains dataset. Plant invasive status in both datasets was nonrandomly distributed across the phylogeny; invasive plants were disproportionally from two plant families (Asteraceae and Poaceae; Figures 1 , 2 , 3 ). Although there was a significant phylogenetic signal for mycorrhizal responsiveness with the Central Plains dataset (Figure 3 ), the overall pattern ( K < 1) indicated that closely related taxa were more likely to differ than expected from a Brownian random walk model of evolution (Ackerly, 2009 ). Others have also reported that invasive plant taxa occur predominantly in a few plant clades (Lim, Crawley, de Vere, Rich, & Savolainen, 2014 ) and that mycorrhizal responsiveness exhibited greater divergence than convergence ( K < 1) for grassland taxa (Anacker et al., 2014 ). The relatively weak effects of phylogeny, and the finding that mycorrhizal responsiveness is more likely to be divergent than conserved suggest that predicting mycorrhizal responsiveness in different ecosystems may be enhanced by considering key functional traits associated with root function or key genes related to mycorrhizal interactions, rather than phylogenies constructed with neutral molecular markers (e.g., matK , rbcL ) used to identify plants. Our findings are consistent with a recent meta‐analysis that compared invasive non‐natives to natives, albeit without the inclusion of phylogenetic information (Bunn et al., 2015 ), but contrary to a prior analysis of the Central Plains dataset (Fig. 3 in Pringle et al., 2009 ). In the latter case, inclusion of phylogenetic information helped to partition variance due to invasive status and phylogenetic relatedness which was not possible by Pringle et al. ( 2009 ). When phylogenetic relatedness was not controlled (i.e., standard ANOVA), results for the Central Plains dataset continued to provide marginally significant evidence that invasive taxa were less responsive to AMF (ANOVA, F \n 1,93 = 3.745, p = .056) similar to related comparisons by Pringle et al. ( 2009 ). Phylogenetic analyses, however, indicated a nonrandom phylogenetic signal for AMF responsiveness with the Central Plains dataset and enabled us to differentiate whether AMF responsiveness varied by invasive status and/or phylogeny. Nevertheless, phylogeny was a weak overall predictor of mycorrhizal responsiveness for the Central Plains dataset (Reinhart et al., 2012 ) partly because related taxa tended to have greater trait divergence ( K < 1) than convergence ( K > 1). Trait divergence may be a result of related (and neighboring) plants associating with divergent AMF communities (Reinhart & Anacker, 2014 ; Veresoglou & Rillig, 2014 ) and/or other factors that contribute to niche partitioning among related plant species. The Northern Plains region is known to support some of North America's most invasive plant taxa, including Bromus tectorum , Centaurea stoebe , and Euphorbia esula . Among the 14 invasive species in the Northern Plains dataset, three ( Agropyron cristatum , C. stoebe , and E. esula ) responded positively to AMF and three ( A. cristatum [Fairway], B. japonicus , and B. tectorum ) responded negatively to AMF. These divergent responses likely contributed to the lack of differences between invasive and other taxa. These findings support the hypothesis that a multitude of factors (e.g., anthropogenic disturbance, plant–microbe interactions, traits) contribute to invasiveness (Hierro et al., 2016 ; e.g., Maron et al., 2013 ; Müller, Horstmeyer, Rönneburg, van Kleunen, & Dawson, 2016 ). It is not too surprising that on average invasive taxa are not less responsive to AMF as invasiveness may be facilitated by either decreased or increased mycorrhizal responsiveness. For example, the invasive grass B. tectorum is known to reduce AMF abundance (Busby, Stromberger, Rodriguez, Gebhart, & Paschke, 2013 ; Lekberg et al., 2013 ) and responded either negatively or neutrally to AMF (Figures 1 and 3 ). Other invasive grasses ( A. cristatum , B . inermis ) also appear capable of reducing AMF diversity (Jordan, Aldrich‐Wolfe, Huerd, Larson, & Muehlbauer, 2012 ) and had varying responses to AMF (Figures 1 and 3 ). Some invasive plants are thought to degrade AMF communities (Hale & Kalisz, 2012 ; Pakpour & Klironomos, 2015 ; Stinson et al., 2006 ; Vogelsang & Bever, 2009 ; Zhang et al., 2010 ) which may contribute to soil legacy effects that negatively impact the resilience of resident plant communities (Jordan, Larson, & Huerd, 2008 ; Perkins & Nowak, 2012 , 2013 ). In contrast, some invasive forbs (i.e., C. stoebe and E. esula ) increased the abundance and diversity of AMF communities (Lekberg et al., 2013 ), responded positively to AMF (Figure 1 ), and AMF enhanced the invasiveness of C. stoebe (Callaway, Mahall, Wicks, Pankey, & Zabinski, 2003 ; Marler et al., 1999 ). Therefore, mycorrhizal responsiveness facilitates the invasion of particular plant species, but weak mycorrhizal responsiveness is seemingly not a general mechanism of plant invasion. Dominant native plants in the Northern Plains tended to be unresponsive to AMF. Specifically, four of the most prominent plants in the Northern Plains ( Artemisia tridentata , Bouteloua gracilis , Hesperostipa comata , and Pascopyrum smithii ) routinely associate with AMF (Bethlenfalvay & Dakessian, 1984 ; Carter, Smith, White, & Serpe, 2014 ; Reinhart & Anacker, 2014 ), but we found that only H. comata increased shoot biomass with AMF (Figure 1 b). Artemisia tridentata actually had less shoot biomass with AMF than without. Other studies reported no effect of AMF on A. tridentata during mine reclamation (Biondini, Bonham, & Redente, 1985 ; Stahl, Williams, & Christensen, 1988 ), restoration postfire (review by Dettweiler‐Robinson et al., 2013 ), and a controlled mycorrhizal responsiveness test (Busby et al., 2011 ). Yet, other experiments indicated that A . tridentata seedlings had increased drought tolerance (Stahl, Schuman, Frost, & Williams, 1998 ), biomass (Stahl et al., 1988 ), and survival [fall but not spring transplants] with AMF (Davidson, 2015 ). Prominent congeners responded both positively ( A. dracunculus ) and negatively ( A. frigida ) to AMF. Bouteloua gracilis and P. smithii were also used in the Central Plains dataset, and B. gracilis plants were appreciably larger with AMF (Figure 3 ). Although not a study aim, we are compelled to mention the apparent differences in AMF responsiveness for the two Plains ecoregions (Figure 1 vs. 3). Specifically, 61% of plants in the Central Plains were larger with AMF than without. Yet, appreciable positive effects of AMF on total plant biomass and shoot biomass were detected for only 28% of grassland plants in the Northern Plains. This apparent difference between Plains regions is intriguing, but we cannot reconcile whether such a difference is due to important methodological differences between studies or important ecological differences between Plains regions. There is a small body of literature suggesting ecological differences are possible—agronomic plants in calcareous soils were often less responsive to AMF (Li, Zhu, Marschner, Smith, & Smith, 2005 ; Zhu, Smith, & Smith, 2003 ). Calcium carbonates (which are prevalent in dryland soils like the Northern Plains) are known to reduce the bioavailability of phosphorus (e.g., Belnap, 2011 ) and may impact mycorrhizal mutualisms. Further testing is necessary to determine whether these regions truly have divergent interactions with AMF and the factors contributing to such differences. In conclusion, despite substantial differences in functional characteristics, taxa sampled, and growth conditions for the experiments, we found no evidence to support the hypothesis that invasive taxa differ from native taxa in the degree of mycorrhizal responsiveness. Although we considered interspecific differences in the present analysis (i.e., a single genotype represented most plant species), others have noted that plant–AMF interactions vary by cultivars of invasive taxa like A. cristatum (Jun & Allen, 1991 ; Figure 1 ). Genotypic and geographical variation in plant–AMF interactions likely complicates formulation of robust generalizations for taxa with broad distributions. For instance, other plant–AMF interactions are conceivable with other pairings of AMF communities (species and genotypes), plant genotypes (e.g., Klironomos, 2003 ; Stahl & Smith, 1984 ), and growth conditions. Additional experiments that incorporate population and genotypic variability are necessary to quantify general effects of soil microbes across a plant's range (Callaway, Bedmar, Reinhart, Silvan, & Klironomos, 2011 ; Reinhart, Royo, van der Putten, & Clay, 2005 ; Wagner, Antunes, Ristow, Lechner, & Hensen, 2011 )."
} | 4,234 |
38932022 | PMC11207373 | pmc | 2,526 | {
"abstract": "In this study, a series of amine-modified mesoporous silica (AMS)-based epoxy composites with superhydrophobic biomimetic structure surface of Xanthosoma sagittifolium leaves (XSLs) were prepared and applied as anti-corrosion and anti-biofilm coatings. Initially, the AMS was synthesized by the base-catalyzed sol–gel reaction of tetraethoxysilane (TEOS) and triethoxysilane (APTES) through a non-surfactant templating route. Subsequently, a series of AMS-based epoxy composites were prepared by performing the ring-opening polymerization of DGEBA with T-403 in the presence of AMS spheres, followed by characterization through FTIR, TEM, and CA. Furthermore, a nano-casting technique with polydimethylsiloxane (PDMS) as the soft template was utilized to transfer the surface pattern of natural XSLs to AMS-based epoxy composites, leading to the formation of AMS-based epoxy composites with biomimetic structure. From a hydrophilic CA of 69°, the surface of non-biomimetic epoxy significantly increased to 152° upon introducing XSL surface structure to the AMS-based epoxy composites. Based on the standard electrochemical anti-corrosion and anti-biofilm measurements, the superhydrophobic BEAMS3 composite was found to exhibit a remarkable anti-corrosion efficiency of ~99% and antimicrobial efficacy of 82% as compared to that of hydrophilic epoxy coatings.",
"conclusion": "4. Conclusions This study successfully prepared a series of amine-modified mesoporous silica (AMS)-based epoxy composites with superhydrophobic biomimetic structures mimicking the surface of Xanthosoma sagittifolium leaves (XSLs). By integrating the concepts of “gas barrier properties” and “hydrophobicity”, the materials were endowed with dual-functional properties of anti-corrosion and antibacterial. A biomimetic negative mold of PDMS replicating the micro-nanostructure of XSL surfaces was prepared through soft lithography, synthesizing BEMS3 materials with biomimetic structures and adding 3 wt% AMS. The contact angle of water droplets on the material increased from 69° to 152°, significantly improving by 83°, and exhibiting superhydrophobic performance. Electrochemical corrosion tests demonstrated that the addition of inorganic filler (AMS) could increase the gas permeation path, obstructing the diffusion of corrosive factors (water, oxygen) and further enhancing anti-corrosion capability, with the polarization resistance (Rp) increasing from 349 KΩcm 2 to 20,292 KΩcm 2 , achieving a protection efficiency of up to 99.95%. In terms of antibacterial applications, short-term bacterial anti-adhesion effects were observed with SEM, revealing that the BEMS3 material, with its biomimetic structure and the addition of 3 wt% AMS, exhibited the best anti-adhesion effect against both S. aureus and E. coli . The long-term anti-biofilm growth effect was quantitatively monitored using microscopy and an Elsa Reader. After a 7-day test period against E. coli , the BEMS3 composite material achieved an antibacterial efficiency of up to 82%, maintaining the best long-term anti-biofilm growth effect. In both antibacterial tests, the effect was better against E. coli , as the shape and size of bacteria could affect their adhesion to the membrane, with S. aureus being closer in size to the gaps on the BEAMS3, making it easier to adhere compared to rod-shaped bacteria [ 12 ]. The geometric shape, size, and height of the surface biomimetic structures influenced the contact area and the number of bacteria adhering to the membrane surface, limiting the motility of flagellated E. coli and delaying direct contact between bacteria, thus inhibiting biofilm formation. The papillary nanostructures of XSLs and the air layer formed an antibacterial mechanism on the superhydrophobic surface to reduce the bacterial adhesion area [ 13 ]. The presence of air, moisture, and salts in the atmospheric environment promotes the corrosion of metal materials and provides suitable conditions for bacterial growth. Based on this, the study developed a dual-functional coating with anti-corrosion and antibacterial properties, addressing the need for metal corrosion prevention and antibacterial action in daily life and preventing biofilm formation. It is also hoped that this coating can be used in food processing facilities to inhibit microbial growth and prevent the corrosion of processing equipment, thereby avoiding food contamination. Moreover, the coating can be applied to marine environments to resist microbial and seawater corrosion, thus reducing damage to ships, offshore platforms, and other metal structures, and ensuring the safety of personnel and property. The development of this innovative coating not only addresses the dual challenges of metal corrosion and microbial growth but also opens new possibilities for future application areas.",
"introduction": "1. Introduction The impact of anti-corrosion and anti-biofilm coatings has extensively attracted a significant number of industrial and research activities in the past decades. Among these coatings, superhydrophobic surfaces usually exhibit remarkable anti-corrosion and anti-biofilm performances. The development of these superhydrophobic surfaces requires distinctive techniques [ 1 ], including chemical etching [ 2 ], hydrothermal treatment [ 3 ], anodization [ 4 ], electrodeposition [ 5 ], sol–gel processes [ 6 ], and templating [ 7 ], as well as electrospinning [ 8 ]. A study by Yeh et al. replicated the surface structure of a fresh Xanthosoma sagittifolium leaf (XSL) to fabricate biomimetic materials containing micron-sized papillae decorated with lots of nano-scaled creases, which exhibited remarkable superhydrophobicity and high surface area [ 9 ]. The superhydrophobic property of biomimetic surfaces replicated from XSLs envisioned two applications of corrosion protection [ 10 , 11 ] and anti-biofilm [ 12 ]. On the other hand, the high surface area property of biomimetic surfaces envisioned another four distinctive applications of supercapacitors [ 13 , 14 , 15 ], biological scaffolds [ 16 ], photocatalysis [ 17 , 18 ], and gas sensing [ 19 ]. Conversely, distinctive hydrophobic metal oxide particles such as SiO 2 , TiO 2 , etc., functioned as inorganic fillers and had been reported to promote the hydrophobicity of organic polymer coatings to give the organic–inorganic hybrid materials. The sol–gel process is a convenient method for preparing different coatings on the surface of galvanized steel substrates to prepare the metal oxide particles. Silica-based protective coatings are very efficient as corrosion protectors of metals under different circumstances [ 20 , 21 , 22 ]. Mesoporous sol–gel coatings and nanoparticles are suitable as inhibitor carriers for self-healing corrosion-resistant systems [ 23 , 24 , 25 ]. Recently, novel results and trends were reviewed on this topic thoroughly by Montemor [ 25 ]. Silica-based superhydrophobic sol–gel coatings are also promising for preventing metal surfaces against corrosion [ 26 , 27 , 28 ]. Mesoporous silica with worm-like porosity prepared from a non-surfactant template route was first reported by Wei et al. [ 29 ]. Subsequently, Yeh et al. explored the comparative study on the physical properties of PMMA–silica mesocomposite (PSM) and nanocomposite (PSN) membranes [ 30 ]. They reported that the PSM was found to show effectively enhanced mechanical strength, thermal stability, thermal insulation, optical clarity, surface hydrophobicity, and surface roughness properties compared to that of the corresponding PSN based on a series of systematic studies. Additionally, fillers treated with a silane coupling agent and subjected to ultrasonic treatment can significantly enhance the physicomechanical properties of epoxy composites, improving their impact toughness, heat resistance, and thermal conductivity [ 31 ]. After introducing aminopropyl trimethoxysilane as an interfacial modifier to the surface of graphene nanoplatelets, different loadings of silane-modified graphene significantly improved the tensile, compressive, interlaminar shear strength (ILSS), and tribological properties of the epoxy nanocomposites [ 32 ]. Hameed et al. investigated the effects of functionalization and different weight fractions of multi-walled carbon nanotubes (CNTs) on the mechanical properties of CNT/epoxy composites. The results showed that increasing the weight fraction of CNTs and applying functionalization treatments significantly enhanced Young’s modulus, tensile strength, and thermal stability of the materials [ 33 ]. In this current study, we prepared a superhydrophobic surface of organic–inorganic hybrid materials by integrating the hydrophobic inorganic mesoporous silica particles and hydrophobic biomimetic organic epoxy coating simultaneously for the applications of anti-corrosion and anti-biofilm. Initially, the AMS was synthesized by the base-catalyzed sol–gel reactions of TEOS and APTES through a non-surfactant templating route. Subsequently, a series of AMS-based epoxy composites were prepared by performing the ring-opening polymerization of DGEBA with T-403 in the presence of AMS spheres. Furthermore, a nano-casting technique with PDMS as a soft template was utilized to transfer the surface pattern of natural XSLs to AMS-based epoxy composites, forming AMS-based epoxy composites with biomimetic structure. The anti-corrosion and anti-biofilm of as-prepared materials were intensively characterized by electrochemical corrosion measurements and contact angle measurements.",
"discussion": "3. Results and Discussion 3.1. Characterization and Property Analysis of Mesoporous Materials Prepared via the Non-Surfactant Templating Method Mesoporous materials are renowned for their high surface area, thereby serving as potential catalyst supports with vast applications in chemical adsorption and catalytic reactions. However, their applicability often becomes constrained when mesoporous materials comprise inert amorphous silica. Enhancing their utility and boosting their stability through surface modification has emerged as a primary research focus in recent studies [ 2 ]. This paper explored the concurrent introduction of amino functional groups (amino groups) onto the surface of mesoporous materials using the non-surfactant templating method, followed by a detailed characterization and examination of their morphology and pore structure. 3.1.1. Microscopic Morphology of AMS Utilizing electron microscopy, the morphology of the synthesized material was observed. SEM images ( Figure 1 a,b) demonstrate that the average particle diameter of the amino-functionalized silica spheres prepared via the non-surfactant templating method is roughly between 220 and 250 nm post-template removal, with all samples presenting a spherical appearance. The transmission electron microscopy (TEM) images of the mesoporous material of amino-modified AMS produced using the non-surfactant template approach are depicted in Figure 1 c. Furthermore, AMS exhibits a notably brighter porous pattern. This suggests that the amino-functionalized mesoporous silica spheres (AMS) are indeed replete with channels, and the distribution of these pores is remarkably uniform. 3.1.2. Chemical Structure Identification and Analysis of Mesoporous Material (AMS) To ascertain whether the mesoporous material successfully incorporated amino functional groups onto the silica surface, we initially utilized the 13 C-Solid State Nuclear Magnetic Resonance Spectrometer ( 13 C-Solid State NMR) for identification. Figure 2 A represents the 13 C-NMR spectrum of the sample derived from the copolymerization of the precursor TEOS and the modifying agent APTES. Carbon positions C(a), C(b), C(c), and C(d) indicated within the APTES structure exhibit chemical shifts of approximately 59 ppm, 43 ppm, 21 ppm, and 10 ppm, respectively. Meanwhile, the carbon positions C(a) and C(e) within the TEOS structure possess chemical shifts of 59 ppm and 17 ppm, respectively. When TEOS and APTES undergo hydrolysis followed by sol–gel condensation reactions, most TEOS and APTES form an O-Si-O crosslinked network structure. However, a few silicon atoms would retain either the methyl or methylene functional groups. Moreover, chemical shift signals displayed in the 29 Si Solid State MAS-NMR spectrum facilitate the determination of the bonding situations between silicon and oxygen atoms within the mesoporous material. Organic silicon (T) and inorganic silicon (Q) chemical structures are predefined in silicon atom structures. In cases where Si-O-Si bonding condensation becomes more complete, electron shielding effects will be reduced, leading to a shift in the absorption peak toward a higher magnetic field. The identification results of the sample’s 29 Si SS-NMR are depicted in Figure 2 B. Upon the sol–gel condensation of TEOS and APTES resulting in AMS, chemical shifts at 102 ppm and 110 ppm are the signals associated with Q 3 and Q 4 belonging to the inorganic silicon series. The silica precursor, TEOS, mainly contributes to these. After modification by APTES, signals for T 2 and T 3 from the organic silicon series were observed at chemical shifts of −63 ppm and −66 ppm, respectively. By consolidating the evidence from 13 C SS-NMR and 29 Si SS-NMR, it can be affirmed that the -NH 2 active functional group has been successfully bonded within the SiO 2 mesoporous material [ 34 ]. 3.1.3. BET Analysis of Mesoporous Properties Figure 3 A,B represent the nitrogen adsorption/desorption isotherms and pore size distribution of the aminosilane-functionalized silica spheres before (ARS) and after (AMS) template removal, respectively. Before the sample testing, they were heated under a vacuum at 120 °C to remove water vapor and other gases. Subsequently, they were cooled to cryogenic temperatures with liquid nitrogen, followed by nitrogen gas introduction to achieve equilibrium before measurements were taken. From the pore distribution in Figure 3 A, the curve is characteristic of a type V isotherm, indicative of a mesoporous material structure. At high P/P 0 , capillary condensation is observed. Using the non-surfactant template D-(-) Fructose, mesoporous materials can be fabricated with an average pore size distribution of approximately 6.4 nm. The specific surface area for ARS is 16.3 m 2 /g, whereas for AMS, it increases to 260 m 2 /g. Likewise, the pore volume for ARS is 0.04 cm 3 /g, and for AMS, it escalates to 0.42 cm 3 /g, marking an approximately tenfold increase. This confirms the successful preparation of mesoporous materials from aminosilane-functionalized silica spheres [ 34 ]. 3.2. The Identification and Application of the Epoxy Resin/Silica Mesoporous Composite with a Structure Biomimetic of the XSF Surface Fourier Transform Infrared Spectra Analysis of Epoxy Resin Epoxy resins exhibit high mechanical strength, exceptional electrical insulating properties, and commendable adhesive capabilities. As such, they frequently serve as adhesives, waterproof coatings, IC encapsulants, and functional polymeric materials in fiberglass composites. This study primarily synthesized epoxy resins by uniformly blending the diglycidyl ether of Bisphenol-A (DGEBA) and polyoxypropylene triamine (T-403) in specific ratios. As evidenced by the FTIR spectrum in Figure 4 , the hardener with terminal amine groups (T-403) exhibits characteristic peaks of primary amines at absorption positions between 3400 and 3200 cm −1 . In contrast, DGEBA contains a bis-epoxide resin characterized by epoxy peaks at an absorption position of 910 cm −1 . As the reaction gradually heated to 150 °C, the ring-opening reaction approached completion. From the epoxy infrared spectrum in Figure 4 c, it is apparent that the epoxy absorption peak at 910 cm −1 disappeared, as did the characteristic peak of primary amines at 3400–3200 cm −1 . These changes in the infrared spectra suggest that DGEBA underwent ring-opening, subsequently crosslinking with the primary amine of T-403. Under these reaction conditions, epoxy resins with a high degree of crosslinking can be prepared. Additionally, the epoxy with 3 wt% AMS (EAMS) displays the characteristic absorption peak of Si-O-Si at 1100 cm −1 ( Figure 4 d). This reveals that the mesoporous silica spheres have been successfully mixed into the epoxy resin. 3.3. Observation of Surface Morphology of Biomimetic Epoxy/Silica Mesoporous Composites In this study, Bisphenol A epoxy was employed to transfer the micro-nanostructure of the XSF surface, with subsequent observation and verification of successful replication of the micro-nanostructure using SEM, as presented in Figure 5 . Figure 5 a,b depict the SEM images of the natural XSF surface morphology. At the same time, Figure 5 c,d illustrate the SEM images of the negative mold of the micro-nanostructured XSF leaf back surface, replicated via PDMS imprinting. These images highlight the presence of microscale depressions and nanoscale wrinkles, mirroring the structure found on natural leaves. On the other hand, Figure 5 e,f present the SEM images of the surface microstructure of the biomimetic BEP film. The observed microstructure on the BEP coating appears highly reminiscent of the natural XSF morphology, exhibiting numerous microscale papillary structures and nanoscale folding. By incorporating 3 wt% AMS into the epoxy and utilizing the PDMS negative mold for biomimetic imprinting of the XSF surface, the SEM images of the resultant BEAMS3 are depicted in Figure 5 g,h. The microstructure on the BEAMS3 coating can be observed, effectively replicating the XSF surface morphology, and featuring microscale papillary formations and nanoscale folding patterns. We subsequently examined the surface morphologies of the biomimetic microstructures on the BEP material, as illustrated in Figure 6 . Figure 6 a,b show that the average distance between the papillae is approximately 13.6 μm, with a papilla diameter of about 15.5 μm. Additionally, from Figure 6 c, the papillary columns’ height is around 12 μm. Through SEM analysis, it is ascertained that the PDMS template can successfully transfer the micro-nano composite structure of the XSF surface. As a result, coatings of BEP and BEAMS3 with micro-nano composite structures are obtained. Further investigations will focus on the influence of these microstructures on the material’s surface properties and related physicochemical analyses. 3.4. Static and Dynamic Contact Angle Measurements and Analysis Using the CA measurement, the hydrophilic and hydrophobic behavior of the epoxy/mesoporous silica composite film was evaluated ( Table 2 ). The contact angle of water droplets on the natural XSF leaf backside is 130°. The non-biomimetic EP coating exhibited a contact angle of 69.2° ± 0.2°, while the biomimetic BEP has an angle of 134.9° ± 0.4°. After introducing the XSF biomimetic template, the originally hydrophilic surface was transformed into a hydrophobic one, increasing the water droplet contact angle by 65°. This validates that the micro-nano structure of XSF indeed enhances the surface hydrophobicity of the material. TEM images from Section 3.1.1 indicate that the AMS is filled with pores internally. The introduction of air blocks water molecules, and the modified silica surface, covered with amino functional groups, enhances the compatibility between inorganic material (AMS) and organic material (epoxy), ensuring a more uniform dispersion of AMS in the epoxy matrix. When AMS is added up to 3 wt%, the static contact angle reaches 91.7° ± 0.5°. Using biomimetic template transfer, by increasing the surface roughness, the contact angle increased to 152.9° ± 0.3°, achieving a superhydrophobic effect ( Figure 7 ). The incorporation of mesoporous silica and biomimetic structure in this study significantly increased the static contact angle by 83°, substantially enhancing the material’s hydrophobicity. Figure 7 i–l present the SEM-EDX mapping analysis for the Si element in the epoxy/mesoporous silica composite. It is evident that when epoxy incorporates AMS, the EDX images display a high distribution of red dots ( Figure 7 k,l). The uniform distribution of these red dots signifies that the AMS is evenly dispersed within the epoxy. Both static and dynamic contact angles play pivotal roles in exploring practical applications for superhydrophobic surfaces. This is attributed to the fact that for materials exhibiting surface roughness or chemical heterogeneities, the contact angle of a droplet does not remain constant but rather fluctuates within a specific range. The maximum and minimum of this range are termed the advancing and receding contact angles, respectively. The difference between these angles is called the contact angle hysteresis ( Figure 8 .) Typically, the advancing contact angle represents the hydrophobic nature of the surface (corresponding to regions of low surface energy on the solid). In contrast, the receding contact angle reflects its hydrophilic tendencies (corresponding to areas of high surface energy). Notably, a more pronounced roughness on the material’s surface generally increases hysteresis, signifying enhanced hydrophobicity. This study prepared a series of epoxy/mesoporous silica composites (EAMS1, EAMS2, EAMS3) exhibiting relatively small hysteresis angles ranging from 12° to 22° ( Table 2 ). This suggests a relatively uniform surface, stable surface energy, fewer surface defects, and consistent chemical and physical properties. This further underscores the uniform integration of mesoporous silica with the epoxy matrix. In contrast, biomimetic structures such as BEP and BEAMS3 exhibited a significantly increased hysteresis of 27° and 32°, respectively. Due to the biomimetic XSL surface structure, these materials manifest superhydrophobic properties akin to the lotus leaf effect. Their heightened surface roughness, combined with chemical and physical heterogeneities, alters the interplay between the liquid and solid, leading to distinct advancing and receding angles, thereby resulting in a more significant contact angle hysteresis. 3.5. Corrosion Protection Application Testing 3.5.1. Electrochemical Potentiodynamic Testing Epoxy/mesoporous silica composites were coated onto cold-rolled steel strips to fabricate working electrodes for metal corrosion resistance tests. Parameters such as corrosion potential (E corr ), corrosion current (I corr ), and polarization resistance (R p ) were measured to evaluate the anti-corrosive performance of the coated materials. The open circuit potential (OCP) was monitored until it reached a stable equilibrium state, after which cyclic potential measurements commenced. Scanning from −500 mV to +500 mV, the resulting data was plotted on a Log I vs. E graph, known as the Tafel plot. This yielded individual anodic and cathodic polarization curves. The intersection of these curves determines the corrosion potential (E corr ), with the corresponding current being the corrosion current (I corr ). Polarization resistance was ascertained by scanning at a rate of 100 mV/s until surpassing the corrosion current. A potential–current graph was plotted, with the resulting slope indicating the polarization resistance (R p ). In general, a higher corrosion potential (E corr ), a more considerable polarization resistance (R p ), and a smaller corrosion current (I corr ) suggest an enhanced corrosion resistance of the material. The corrosion rate (R corr ) decreases correspondingly. The material’s protective efficiency (P EF ) can be calculated using the following formula: Protection Efficiency P E F % = I corr C R S − I corr c o a t e d I corr C R S × 100 % From the Tafel curves shown in Figure 9 , one can observe the composite curves formed by both anodic and cathodic polarization curves. The consolidated data are presented in Table 3 . A shift of the Tafel curve towards the bottom right indicates an elevated corrosion potential (E corr ) and a decreasing trend in corrosion current. It is hypothesized that including mesoporous silica enhances gas permeation pathways, hindering the diffusion of corrosion agents (water, oxygen), thus achieving corrosion protection. By incorporating 3 wt% AMS into the epoxy resin, its corrosion potential can be improved to −583.9 mV. By further employing a biomimetic surface structure transfer onto this material and conducting corrosion protection tests, its corrosion potential can be further elevated to −556.5 mV. Moreover, its polarization resistance experiences a significant increase, rising from 349 KΩcm 2 to 15,469 KΩcm 2 . This suggests that the drainage performance inherent to the biomimetic superhydrophobic structure can bolster its anti-corrosion capabilities. 3.5.2. Electrochemical Impedance Testing Under stable conditions, impedance describes the relationship between current and potential. It is referred to as the Z value when determined using alternating current impedance methods. Electrodes comprise resistance and capacitance, where R S is the electrolyte resistance, R ct is the charge transfer resistance, and C denotes capacitance or the double-layer capacitance. The following equation can express these relationships: Z = Z′ + j Z″ = R S + R ct /(1 + ω j R ct C) The Nyquist plot derived from this equation is semicircular. A larger radius or area of the semicircle from a low to high frequency indicates higher impedance values, signifying superior barrier properties and corrosion resistance. A more significant real impedance part, Z’, suggests the excellent anti-corrosion performance of the material. Figure 10 shows that the epoxy resin/mesoporous silica composite superhydrophobic coating with a biomimetic taro leaf surface structure, labeled BEAMS3, possesses the highest surface impedance. As the content of mesoporous silica gradually increases in the epoxy resin, the impedance value also escalates ( Table 3 ). A primary factor for this is the drainage mechanism of the superhydrophobic surface, which effectively impedes water vapor. Moreover, the uniform dispersion of mesoporous silica within the epoxy resin interferes with oxygen penetration. It prolongs the oxygen pathway, decelerating the onset of rust formation on the metal, thereby substantially enhancing the anti-corrosion effect. In Figure 11 , the electrochemical impedance Bode plot reveals trends in the impedance analysis of the epoxy resin/mesoporous silica composite material. These trends align perfectly with the tendencies derived from the Nyquist plot in the electrochemical impedance analysis. Notably, BEAMS3 exhibited the highest impedance value, indicating its superior anti-corrosion effect. Subsequently, the BEAMS3 coating, showcasing the optimal anti-corrosion performance, will be subjected to the next phase of antimicrobial experiments, with EP, BEP, and EAMS3 materials serving as the control group. 3.6. Inhibition of Biofilm Formation In assessing short-term bacterial attachment inhibition, SEM enabled the observation of bacterial adhesion to the material at 2 and 24 h post-exposure. The images presented in Figure 12 depict the material’s surface morphology amplified 3000-fold under SEM. The findings of S. aureus attachment to the materials after 2 and 24 h incubation are shown in Figure 12 , in which a large number of clustered cocci on the EP are spread all throughout the surface of EP ( Figure 12 (Aa,e)). However, only a few sporadic cocci appeared on the surface of EAMS3, BEP, and BEAMS3 ( Figure 12 (Ab,f)). After soaking the materials for 24 h, a cocci-covered EP surface was found ( Figure 12 (Ae)). Meanwhile, only a few cocci grew within the space between the papillae in EAMS3, BEP, and BEAMS3 ( Figure 12 (Ab–d,f–h)), possibly due to the limited attachment and proliferation of cocci to the papillae. Figure 12 B illustrates the adherence of E. coli to the materials following 2 and 24 h of incubation. Notably, only a few E. coli cells were observed adhering to both EP and EAMS3 surfaces with the bacteria uniformly distributed across these surfaces. On the BEP and BEAMS3 films, E. coli attachment was markedly less frequent, with only isolated bacteria noted after a 2 h incubation ( Figure 12 (Ba,c)). The panels from Figure 12 (Be–h) detail the extent of bacterial colonization on the materials after a prolonged 24 h soaking period. The EP and EAMS3 surfaces were covered with E. coli . Contrastingly, E. coli was only sporadically observed on the BEP and BEAMS3 surfaces. These materials exhibited superior efficacy in inhibiting bacterial attachment compared to EP and EAMS3. The aforementioned findings underscore that the biomimetic surfaces of BEP and BEAMS3 effectively restrict the proliferation of both bacterial strains. In evaluating the long-term anti-biofilm efficacy of the materials, crystal violet (CV) staining was employed for quantitative analysis, complemented by a microscopic examination to visualize biofilm development. The CV staining distinctly marked the bacterial presence, as indicated by the purple regions on the material. In this study, a biofilm inhibition assay was performed using S. aureus and E. coli over periods of 1 and 7 days. Figure 13 A depicts the biofilm formation of S. aureus on the material at these time intervals. After the initial day of bacterial exposure, a more pronounced white area was noticeable on the EP, suggesting a lower extent of biofilm formation with limited bacterial adhesion (45.2% coverage) ( Figure 13 (Aa)). By the 7th day, the white area had noticeably diminished, indicating a substantial increase in biofilm presence ( Figure 13 (Ae)). The purple spots distributed across the image reflect a significant escalation in bacterial accumulation on the material (78.5% coverage). For EAMS3, a smaller number of purple spots were formed throughout the image, indicating a remarkable decrease in bacteria on the material with a reduced coverage of 24.2% and 34.6% for days 1 and 7 ( Figure 13 (Ac,g)). It indicates that EAMS3 has better anti-biofilm properties than EP. For BEP and BEAMS3 with a superhydrophobic surface, there are only a few purple spots throughout the image indicating a remarkable decrease in bacteria on the material with a reduced coverage of 10.9% and 5.8% for day 1 ( Figure 13 (Ab,d)) and 27.7% and 12.1% for day 7 ( Figure 13 (Af,h)), respectively. It indicates that BEP and BEAMS3 have better anti-biofilm properties for their biomimetic surface structure. The proliferation of E. coli on the material was also monitored and compared over 1 and 7 days ( Figure 13 B). Initially, after 1 day of bacterial growth, scattered purple spots indicating limited bacterial presence (16.2% coverage) were observed on the EP, suggesting that biofilm formation was still in its early stages ( Figure 13 (Ba)). By contrast, on day 7, the density of purple spots markedly increased on the material, denoting a substantial escalation in E. coli growth (41.6% coverage), indicative of significant biofilm development ( Figure 13 (Be)). For EAMS3, a smaller number of purple spots were formed throughout the image, demonstrating a remarkable decrease in bacteria on the material with a reduced coverage of 19.7% and 26.6% for days 1 and 7 ( Figure 13 (Bc,g)). This indicates that EAMS3 has better anti-biofilm properties than EP. However, the superhydrophobic BEP significantly inhibited the growth of E. coli with a reduced coverage of 5.9% and 15.3% for day 1 and day 7 ( Figure 13 (Bb,f)). These findings indicate that BEP’s surface is relatively inhospitable to E. coli , with only minimal bacterial growth observed. This suggests that BEP possesses enhanced anti-biofilm capabilities compared to EP. Furthermore, the superhydrophobic BEAMS3 exhibited almost complete inhibition of E. coli proliferation, with bacterial coverage limited to just 4.2% and 9.6% on day 1 and day 7, respectively ( Figure 13 (Bd,h)). This evidence strongly suggests that the growth of E. coli on BEAMS3 is significantly impeded, underscoring its exceptional anti-biofilm properties. OD 590 values showed the quantitative CV stain data in Figure 14 . For S. aureus , significant differences ( p -value < 0.05) were observed in BEP and BEAMS3 membranes on day 1, and EAMS3, BEP, and BEAMS3 membranes on day 7, when compared with the EP membrane. In addition, significant differences were also observed in BEAMS3 when compared with EAMS3 or BEP, which proved that BEAMS3 has the best anti-biofilm formation property ( Figure 14 A). For E. coli , the OD 590 values significantly decreased in EAMS3, BEP, and BEAMS3 membranes both on day 1 and day 7 when compared with the EP membrane. Moreover, significant decreases were also observed in BEAMS3 compared to EAMS3 or BEP on day 1 and day 7 ( Figure 14 B). All these results proved that BEAMS3 has the best anti-biofilm formation property. The reduction in bacterial adhesion/growth was calculated using the following formula for each AMS3-contained or micro-structured EP membrane: (1) Efficiency of biofilm inhibition ( % ) = OD590 value of EP membrane − OD590 value of Sample membrane OD590 value of EP membrane × 100 % Sample membranes include EAMS3, BEP, or BEAMS3. Table 4 shows that after a day of S. aureus cultivation, the efficiencies of biofilm inhibition in EAMS3, BEP, and BEAMS3 were 11.2%, 22.2%, and 25.8%, respectively. On day 7, the number of attached/grown bacteria did not continue to increase with higher antibacterial properties, 32.6%, 62.3%, and 68.6%, respectively. For E. coli , the bacterial growth in the membrane had a similar trend and better results compared to that of S. aureus ( Table 4 ). The efficiencies of biofilm inhibition in EAMS3, BEP, and BEAMS3 were 35.1%, 56.0%, and 70.5%, respectively, after one day of cultivation. With the increase in time (day 7), the antibacterial properties increased to 55.9%, 71.7%, and 82.0%, respectively. These results prove that adding AMS3 and biomimetic feather micro-structures could specifically inhibit biofilm formation."
} | 8,542 |
25763004 | PMC4340175 | pmc | 2,528 | {
"abstract": "Cadmium (Cd) is a toxic, biologically non-essential and highly mobile metal that has become an increasingly important environmental hazard to both wildlife and humans. In contrast to conventional remediation technologies, phytoremediation based on legume–rhizobia symbiosis has emerged as an inexpensive decontamination alternative which also revitalize contaminated soils due to the role of legumes in nitrogen cycling. In recent years, there is a growing interest in understanding symbiotic legume–rhizobia relationship and its interactions with Cd. The aim of the present review is to provide a comprehensive picture of the main effects of Cd in N 2 -fixing leguminous plants and the benefits of exploiting this symbiosis together with plant growth promoting rhizobacteria to boost an efficient reclamation of Cd-contaminated soils.",
"conclusion": "CONCLUSION AND PERSPECTIVES Extensive research on the valuable cooperation of PGPRs and N 2 -fixing legumes for phytoremediation purposes has been performed and it is ongoing due to its enormous potential to renew Cd-contaminated soils. However, there are several knowledge barriers which need to be addressed. Prominent among them are optimization of SNF under stressful conditions and a greater understanding of the ecology and dynamics of PGPRs under field conditions. In this respect, before inoculating soils with PGPRs, it must be considered that some strains might be pathogenic to some plant species and even allergenic for humans. Moreover, if the strains inoculated have been genetically modified the potential of horizontal gene transfer should be born in mind. It is also especially important the use and safe disposal of legume edible parts after phytoremediation process (i.e., roots, shoots, and seeds), since they could constitute an important route of Cd introduction in the food chain. For this reason, legumes used as phytoremediation tools should not be considered as products for animal feed or human consumption. Finally, to boost the use of PGPRs–rhizobia–legume partnership the use of metagenomic approaches are essential to identify new bacterial strains with PGPR traits. Moreover, research should be focused in understanding the molecular mechanisms underlying the benefits of PGPRs on nitrogen fixation. In this sense, genetic engineering, a powerful tool that has still been poorly exploited in this area, should lead to the generation of strains better adapted to field conditions and with enhanced abilities to help legume–rhizobia symbiosis for effective Cd phytoremediation."
} | 634 |
39546572 | PMC11588079 | pmc | 2,530 | {
"abstract": "Significance How do nonmotile organisms protect themselves from intense light? While motile single-celled algae can swim toward or away from light (phototaxis), many photosynthetic algae are nonmotile. Studying the nonmotile dinoflagellate Pyrocystis lunula , we reveal the dynamics of a complex chloroplast network that undergoes rapid intracellular morphological reorganization in response to changing light. We found that the topologically complex network structure facilitates strong intracellular deformation under cell wall confinement. The cell’s response resembles a low-pass filter for light-fluctuations, distinguishing between irrelevant and relevant light-stimuli. These results illustrate an elegant adaptation process at a single-cell level toward environmental fluctuations. Besides the physiological and ecological relevance, the light-regulated morphodynamics of the chloroplast network represents a well-controlled active matter system, manifesting a biological metamaterial.",
"discussion": "Discussion In our experiments, we show that the chloroplasts of P. lunula retract toward the cell’s center under strong white or blue light conditions and expand under weak red light conditions. This bidirectional movement of chloroplasts toward and away from light, mirrors the chloroplast photorelocation motion seen in leaves of green plants ( 2 , 6 , 9 , 50 ), nonetheless, using a fundamentally different mechanism. The significant light-induced retraction of chloroplasts leads to increased light transmission through the cell ( Fig. 1 C ), suggesting that, similar to green plants, P. lunula employs this mechanism as a means of light avoidance ( 2 , 6 , 7 , 9 ). Notably, under low white light conditions, we observe a transient response in the chloroplasts—initial fast contraction followed by slow expansion—suggesting a dynamic competition between these processes. This shows similarities with the observed counteraction of phototropin 1 and 2-mediated chloroplast motion ( 50 , 68 ). In fact, the transcriptome of P. lunula bears various phototropin 1 and phototropin 2-like sequences and Light-oxygen-voltage-sensing (LOV) domains ( 67 ), pointing toward potential similarities in light sensation. The similarity of this organism’s light response to the one of green plants is surprising, as the origin of chloroplasts in dinoflagellates is very distinct: While green plants obtained their chloroplasts from primary endosymbiosis of cyanobacteria, dinoflagellates underwent tertiary endosymbiosis, including the incorporation of a red-algae ( 33 , 34 , 47 ). At light intensities exceeding the natural physiological conditions of dinoflagellates, chloroplasts contract fully toward the cell center. Under such extreme conditions, the crowding of the chloroplast strands poses a mechanical limit for contraction, and consequently for this photoavoidance mechanism. However, within their native physiological light conditions, P. lunula responds via light-dependent chloroplast compaction, indicating the evolution of a gradual adaptation response to various ecologically relevant light conditions. Furthermore, we find that the relaxation phase following strong light-induced contraction occurs over a longer time scale, suggesting a different driving mechanism for the expansion of the chloroplasts. The large-scale transport of organelles observed likely depends on the coordinated action of the actin and microtubule networks, together with molecular motors such as myosin, as has been demonstrated in the context of the diurnal intracellular reorganization between the photosynthetic phase during the day and bioluminescent phase at night ( 45 ), in line with pharmacological perturbation experiments for light-adaptation ( SI Appendix , Text IV and Fig. S5 ) in which we confirm that actin is necessary for bidirectional chloroplast motion. However, the exact driving mechanism of the chloroplast relocation in P. lunula remains unidentified and may differ from that of green plants, where chloroplast movement is primarily driven by the assembly of short actin (cp-actin) filaments and the transmission of polymerization forces toward the plasma membrane ( 9 , 15 , 68 ). We elucidate the details of the reticulated morphology of chloroplasts and show that such a structural “design” offers mechanical advantages. Structured metamaterials, like this chloroplast morphology, facilitate buckling and other complex deformations ( 58 , 59 , 69 ), enabling efficient chloroplast contraction under the confinement of the cell wall. However, to strengthen this analogy, a more in-depth study of the organelles’ architecture and contractile apparatus is required. Interestingly, P. noctiluca , another species within the Pyrocystis genus, was found to have a reticulated cytoplasm, assisting with the vertical migration ( 56 ), showing another intricate link between the morphology of cytoplasmic space and its function in dinoflagellates. We also showed that the dynamics of chloroplast contraction can be effectively modeled using a “viscoelastic” framework with chemically controlled stress applications. Although our coarse-grained model does not pinpoint the precise origins of observed elasticity and viscosity—whether from passive or active cellular components—it importantly enabled us to identify adaptation time scales and compare them with the ecologically relevant fluctuations. These time scales suggest that chloroplast motion serves as a feasible light adaptation strategy for environmental light variations persisting longer than 3 to 5 min . Indeed, such fluctuations notably exceed the duration of second-long light changes induced by waves but are in line with the motion of clouds obscuring the sun ( 53 ) and might complement nonphotochemical quenching. Our experiments have also shown that chloroplast contraction is driven by local sensing, with directed relocation observed when chloroplasts are locally stimulated ( Fig. 4 ). However, even though locally stimulated, the chloroplast network on the opposite side of the cell contract within 20 to 40 s , indicating a long-ranged signal transfer via fast diffusive signals ( D ≈ 160 to 500 μ m 2 / s ), such as calcium, recognized for its important roles in photosensory downstream signaling ( 66 ) and in P. lunula bioluminescence ( 70 ). Interestingly, stimulation in the cytoplasmic core prompts chloroplasts to move toward rather than away from the center. This counterintuitive behavior might result from the network’s topologically conserved structure and a photo movement that is inherently biased toward the cell center. This hypothesis, however, needs further investigation. Moreover, the intricate relationship between the chloroplast and nucleus, with the latter hosting a significant portion of the chloroplast genome ( 2 , 71 ), suggests that chloroplast movement toward the nucleus in the cytoplasmic core ( 39 ) could also serve a photoprotective function, shielding genetic material from intense light damage. Our study provides evidence for such a mechanism in dinoflagellates, however, a comprehensive examination of the chloroplast-nucleus relationship is necessary to fully understand these dynamics. Overall, the complex relationship between the geometry and topology of chloroplast structure and its dynamics provides a fertile ground for exploring intriguing physical dynamics with significant physiological implications, in the context of light–life interactions."
} | 1,873 |
24915802 | PMC4065386 | pmc | 2,531 | {
"abstract": "Background Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense . Results Deletion of Mgfnr not only resulted in decreased N 2 production due to reduced N 2 O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. Conclusions We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O 2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.",
"conclusion": "Conclusions We demonstrated for the first time that MgFnr is a genuine oxygen regulator in a magnetotactic bacterium and mediates anaerobic respiration. The expression of MgFnr is required to be precisely controlled, which is regulated by oxygen. In addition, MgFnr is also involved in regulation of magnetite biomineralization during denitrification, likely by controlling proper expression of denitrification genes. This allows the transcription to be adapted to changes in oxygen availability, and thus maintaining proper redox conditions for magnetite synthesis. Despite of general similarities with Fnr proteins from other bacteria, MgFnr is more insensitive to O 2 and further displays additional functions for aerobic conditions, which might result from some non-conserved amino acids. Although oxygen is known to be a major factor affecting magnetite biomineralization for decades, the mechanism of this effect in MTB is still unknown. The common observation that magnetosomes are only synthesized under oxygen-limited conditions raised the possibility of protein-mediated regulation of the biomineralization process. However, although MgFnr mediates oxygen-dependent regulation, its relatively subtle and indirect effects on magnetite biomineralization cannot account for the observed complete inhibition of magnetite biosynthesis under aerobic conditions. In addition to a possible effect caused by directly perturbing the redox balance of iron ions required for magnetite synthesis, another level of genetic regulation may exist in MSR-1. Since MgFnr only affects expression of denitrification genes but not genes encoding O 2 respiration enzymes, magnetite biomineralization is also probably regulated by other unknown O 2 sensors. Therefore, further research on respiratory pathways in MTB is likely to gain more insights into the mechanism of oxygen-dependent regulation of biomineralization.",
"discussion": "Discussion Our previous findings have implicated denitrification to be involved in redox control of anaerobic and microaerobic magnetite biomineralization [ 5 , 6 ]. In E. coli and other bacteria the switch between aerobic and microaerobic respiration such as nitrate reduction is primarily controlled by the Fnr regulator [ 16 , 17 ]. In this study, we have characterized the effect of the MgFnr protein on growth and magnetite biomineralization in MSR-1. Deletion of Mgfnr did not affect the growth yield, but impaired magnetosome formation under microaerobic conditions only in the presence of nitrate (i.e., when denitrification was active) but not in its absence. This implies that MgFnr might be involved in magnetite synthesis by regulation of denitrification genes, whereas expression of terminal oxidases for O 2 respiration is likely not under the control of MgFnr, similar to Fnr from Shewanella oneidensis [ 33 ]. In fact, we found that neither the rates of oxygen consumption nor transcription of terminal oxidase genes [ 34 ] displayed any difference between the WT and Δ Mgfnr mutant. The presence of putative Fnr binding sites in the promoter regions of all operons of denitrification further indicates that MgFnr is involved in controlling the transcription of denitrification genes in response to different oxygen concentrations. Consistent with this, transcription patterns of denitrification genes in Δ Mgfnr mutant were different from WT. For example, in the Δ Mgfnr strain the expression of nap was no longer upregulated by oxygen, expression of nirS was much higher under aerobic conditions than WT, and aerobic expression of nor and nosZ was no longer repressed but upregulated by oxygen. Furthermore, we failed to identify a putative Fnr protein encoded in the genome of the nondenitrifying magnetotactic bacteria Magnetococcus marinus or Desulfovibrio magneticus strain RS-1, which also suggests that Fnr of MTB is likely only responsible to regulate genes encoding for denitrification, but not required for aerobic respiration. In addition, we also observed significantly decreased N 2 evolution in deep slush agar tubes in Δ Mgfnr mutant. This raised the question at which step(s) of denitrification is affected by the loss of MgFnr. We propose that this is not likely caused by the reduction steps from NO 3 - to N 2 O based on the following observations: (i) The consumption rate of NO 3 - and NO 2 - did not decrease in Δ Mgfnr mutant; (ii) NO is lethal to the cells while no defective growth was found in Δ Mgfnr mutant, and no NO emission was observed during mass spectrometry experiments which also implies that the activity of NO reductase is not decreased; (iii) The N 2 O emission rate after addition of nitrate was similar for Δ Mgfnr mutant and WT. Therefore, we conclude that loss of MgFnr affects the last step of denitrification, the reduction of N 2 O to N 2 . In agreement, the emission rate of N 2 was lower for Δ Mgfnr mutant than for the WT. However, we cannot exclude the possibility that loss of MgFnr has an impact on further pathways involved in biomineralization other than denitrification. For instance, we have previously shown that (i) besides acting as nitrate reductase, Nap also plays a role in redox control for magnetosome formation, (ii) nitrite reductase NirS is capable to oxidize ferrous iron to ferric iron for magnetite synthesis, and (iii) NO reductase Nor also participates in magnetosome formation by yet unknown functions [ 5 , 6 ]. On the other hand, in the magnetotactic Magnetovibrio blakemorei strain MV-1 which is capable of anaerobic respiration with N 2 O as electron acceptor, a putative periplasmic Fe (II) oxidase was identified and proposed as N 2 O reductase NosZ [ 35 ], which suggests that N 2 O reductase might be also involved in magnetite biomineralization by unknown functions. In addition, in Δ Mgfnr mutant the different phenotypes observed under anaerobic and microaerobic conditions in the presence of nitrate indicate that MgFnr plays a more important role in magnetite biomineralization when O 2 respiration and denitrification occur simultaneously. Our recent findings showed that maintaining a balance between aerobic respiration and denitrification is crucial for WT-like magnetite biomineralization [ 34 ]. In this case, MgFnr might provide the main contribution to mediate the expression of denitrification genes and therefore, poise the redox state for magnetosome formation. Since deletion of Mgfnr altered oxygen-dependent regulation of denitrification genes under aerobic conditions, we hypothesized that MgFnr protein is active under aerobic conditions. Consistent with this, the expression of Mgfnr was upregulated by oxygen, which, however, was never reported for any Fnr protein from other bacteria. Studies on EcFnr mutants in E. coli have established the important role of a [4Fe-4S] 2+ cluster in regulating EcFnr activity, and some single amino acid substitutions at positions not conserved in the Fnr family led to increased stability of Fnr to oxygen and activated transcription of nitrate reductase genes under aerobic growing conditions [ 24 , 25 , 30 , 32 , 36 ]. None of these reported amino acids of EcFnr are conserved in MgFnr, which might cause a more active MgFnr under aerobic conditions. Among them, Asn-27 and Ile-34 of MgFnr are located very closely to Cys-28 and Cys-37, two of the four cysteine residues that bind the [4Fe-4S] 2+ cluster [ 37 , 38 ]. An E. coli EcFnr mutant protein containing amino acid substitution at either of these two positions showed increased expression of an EcFnr-dependent lac promoter under aerobic conditions [ 30 , 32 , 36 ]. In agreement with these observations, MgFnr mutants including N27D and I34L showed increased aerobic expression of nosZ promoter, suggesting that Asn-27 and Ile-34 of MgFnr are required for a functional MgFnr and likely play a role in maintaining the stability of [4Fe-4S] 2+ cluster. However, MgFnr was able to complement Δ Ecfnr mutant back to WT-like growth, which indicates that MgFnr also has the universal properties of EcFnr. Nonetheless, Δ Ecfnr mutant and Δ Mgfnr mutant displayed significantly different phenotypes during anaerobic growth, such as a largely decreased growth yield in Δ Ecfnr mutant, but no defective growth in Δ Mgfnr mutant. These differences might be explained by different media used for cultivation because in E. coli deletion of Ecfnr only resulted in growth defect in some minimal media [ 11 ] while there is no minimal medium available, which provides reliable growth for MSR-1. In addition, not only deletion of Mgfnr but also overexpression of Mgfnr in WT affected anaerobic and microaerobic magnetite biomineralization in the presence of nitrate and caused the synthesis of smaller magnetosome particles, which indicates that the balanced expression of MgFnr is crucial for WT-like magnetosome synthesis and the expression level is under precise control, be regulated by oxygen. Therefore, MgFnr might play an important role in maintaining redox balance for magnetite synthesis by controlling the expression of denitrification genes, and thus the expression of MgFnr is required to be strictly regulated. On the other hand, since MgFnr serves as an activator for expression of denitrification genes ( nor and nosZ ) under microaerobic conditions while as a repressor on the same genes under aerobic conditions, it is proposed that other oxygen sensors involved in expression of nor and nosZ are regulated by MgFnr. For example, a NosR protein has been shown to be required to activate the transcription of nos gene in Pseudomonas stutzeri [ 39 ]. However, our data cannot rule out the possibility that MgFnr is also regulated by other yet unknown proteins and that other genes involved in magnetosome formation is controlled by MgFnr."
} | 2,941 |
24885133 | PMC4019354 | pmc | 2,532 | {
"abstract": "Background 3-hydroxypropionic acid (3HP) is an important chemical precursor for the production of bioplastics. Microbial production of 3HP from glycerol has previously been developed through the optimization of culture conditions and the 3HP biosynthesis pathway. In this study, a novel strategy for improving 3HP production in Escherichia coli was investigated by the modification of central metabolism based on a genome-scale metabolic model and experimental validation. Results Metabolic simulation identified the double knockout of tpiA and zwf as a candidate for improving 3HP production. A 3HP-producing strain was constructed by the expression of glycerol dehydratase and aldehyde dehydrogenase. The double knockout of tpiA and zwf increased the percentage carbon-molar yield (C-mol%) of 3HP on consumed glycerol 4.4-fold (20.1 ± 9.2 C-mol%), compared to the parental strain. Increased extracellular methylglyoxal concentrations in the Δ tpiA Δ zwf strain indicated that glycerol catabolism was occurring through the methylglyoxal pathway, which converts dihydroxyacetone phosphate to pyruvate, as predicted by the metabolic model. Since the Δ tpiA Δ zwf strain produced abundant 1,3-propanediol as a major byproduct (37.7 ± 13.2 C-mol%), yqhD , which encodes an enzyme involved in the production of 1,3-propanediol, was disrupted in the Δ tpiA Δ zwf strain. The 3HP yield of the Δ tpiA Δ zwf Δ yqhD strain (33.9 ± 1.2 C-mol%) was increased 1.7-fold further compared to the Δ tpiA Δ zwf strain and by 7.4-fold compared to the parental strain. Conclusion This study successfully increased 3HP production by 7.4-fold in the Δ tpiA Δ zwf Δ yqhD E. coli strain by the modification of the central metabolism, based on metabolic simulation and experimental validation of engineered strains.",
"conclusion": "Conclusions In conclusion, the production of 3HP from glycerol in E. coli was improved by the modification of central metabolism, based on metabolic simulation and experimental validation of the engineered strains. Gene knockout simulations using the genome-scale metabolic model identified tpiA and zwf as candidates to be modified and in this double knockout strain, TK52tz, 3HP yield was increased 4.4-fold relative to the TK52 parent strain, as predicted. Increased extracellular methylglyoxal in TK52tz suggested that glycerol catabolism through the methylglyoxal pathway was consistent with metabolic simulation. Since TK52tz produced 1,3-PDO as an abundant byproduct, yqhD , which encodes an enzyme involved in 1,3-PDO production, was deleted in TK52tz. The resulting strain, TK52tzy, exhibited a 1.7-fold increase in 3HP yield relative to TK52tz and a 7.4-fold increase (33.9 ± 1.2 C-mol%) relative to TK52. The double knockout of tpiA and zwf contributed to the reduced production of byproducts such as succinate and lactate that are associated with NADH oxidation, likely due to reduced NADH production by the inhibition of glycolysis. The successful increase in 3HP production, based on metabolic simulation, demonstrated the effectiveness of metabolic modeling in designing a metabolic engineering strategy. Moreover, experimental validation of the engineered strains and comparison with the simulation results provided additional modifications to the engineering strategy to increase target production.",
"discussion": "Results and discussion Construction of a 3HP-producing strain in E. coli The 3HP biosynthetic pathway from glycerol consists of two reactions: the dehydration of glycerol to 3-hydroxypropionaldehyde (3HPA), catalyzed by glycerol dehydratase, and the oxidation of 3HPA to 3HP, catalyzed by aldehyde dehydrogenase [ 16 ] . Since E. coli does not possess the 3HP biosynthetic pathway, the 3HP-producing strain (TK52) was constructed by the overexpression of dhaB and gdrAB , which encode for glycerol dehydratase and glycerol dehydratase reactivase (from K. pneumoniae ), respectively, and aldH , which encodes for aldehyde dehydrogenase (from E. coli ), as described in a previous study [ 17 ]. TK52 was cultivated in M9 medium in a Sakaguchi flask and 3HP was produced at 4.6 ± 0.8% carbon-molar yield of 3HP on consumed glycerol (C-mol%) (Figure 1 A–B and Table 1 ). Acetate was produced as a major byproduct (9.9 ± 1.8 C-mol%) and small yields of 1,3-PDO (0.8 ± 0.4 C-mol%) and succinate (0.9 ± 0.1 C-mol%) were also produced. Ethanol and formate were not detected in this strain and other strains that were constructed. Although E. coli does not possess 1,3-PDO biosynthetic pathways, the introduction of dhaB for 3HP production enabled production of 1,3-PDO as follows: glycerol dehydratase converts glycerol to 3HPA, and an endogenous alcohol dehydrogenase (encoded for by yqhD ) further converts 3HPA to 1,3-PDO [ 13 , 32 ]. Figure 1 Culture results of the 3HP-producing strains. The culture results of the strains TK52 (A, B) , TK52z (C, D) , TK52t (E, F) , TK52tz (G, H) , and TK52tzy (I, J) are shown. Open diamond, 3HP; closed square, glycerol; open triangle, 1,3-PDO; closed circle, OD 600 ; open circle, acetate; open square, succinate; closed triangle, lactate; closed diamond, methylglyoxal. Error bars represent standard deviation of triplicate experiments in TK52 and TK52z strains, and of nine replicate experiments in other strains. Some of the error bars are smaller than the symbols. Table 1 Summary of the experimental results TK52 TK52z TK52t TK52tz TK52tzy Simulation \n *4 \n iAF1260-3HP Simulation \n *4 \n \n ΔtpiA Δzwf \n Specific growth rate (1/h) *1 0.73 ± 0.00 0.71 ± 0.00 1st: 0.55 ± 0.01 1st: 0.54 ± 0.01 1st: 0.56 ± 0.01 2nd: 0.03 ± 0.01 2nd: 0.03 ± 0.00 2nd: 0.04 ± 0.01 Maximum 3HP production rate (mmol/(g DC●h)) *2 0.08 ± 0.02 0.09 ± 0.01 0.22 ± 0.14 0.27 ± 0.11 0.94 ± 0.05 Consumed glycerol (mM) *3 192.7 ± 5.0 193.2 ± 2.4 168.2 ± 24.4 119.0 ± 44.6 117.6 ± 3.9 Biomass (C-mol%) *3 37.0 ± 3.5 36.1 ± 1.4 19.6 ± 3.2 21.7 ± 11.0 20.8 ± 1.2 47.7 22.8 (5.2 ± 0.5) (5.1 ± 0.2) (2.4 ± 0.6) (1.6 ± 0.3) (1.8 ± 0.1) 3HP (C-mol%) *3 4.6 ± 0.8 5.7 ± 0.6 14.7 ± 7.0 20.1 ± 9.2 33.9 ± 1.2 0 70.5 (8.9 ± 1.3) (11.1 ± 1.0) (24.3 ± 11.7) (21.2 ± 7.7) (39.9 ± 2.4) 1,3-PDO (C-mol%) *3 0.8 ± 0.4 0.7 ± 0.2 22.9 ± 5.2 37.7 ± 13.2 5.9 ± 0.5 0 0 (1.5 ± 0.8) (1.3 ± 0.3) (38.1 ± 9.4) (40.5 ± 14.0) (7.0 ± 0.7) Succinate (C-mol%) *3 0.9 ± 0.1 1.3 ± 0.2 0.3 ± 0.3 0 0 0 0 (1.3 ± 0.2) (1.9 ± 0.2) (0.4 ± 0.3) (0) (0) Acetate (C-mol%) *3 9.9 ± 1.8 12.9 ± 0.2 8.2 ± 9.9 2.2 ± 4.1 6.6 ± 1.9 27.4 0 (28.7 ± 5.4) (37.3 ± 1.1) (20.5 ± 24.3) (5.1 ± 9.6) (11.6 ± 3.0) Maximum methylglyoxal concentration (mM) 0.03 ± 0.02 0.03 ± 0.02 0.29 ± 0.02 0.22 ± 0.02 0.60 ± 0.02 *1 Specific growth rates were calculated using OD 600 at 0–6 h for the TK52 and TK52z strains. For the TK52t, TK52tz and TK52tzy strains, specific growth rates during the 1st and 2nd growth phases were calculated using the OD 600 at 0–6 h and 48–72 h, respectively. *2 Maximum 3HP production rates were calculated from the data at 24–48 h for the TK52 and TK52z strains, and at 48–72 h for other strains. *3 C-mol% was calculated from the carbon-mol of the product per carbon-mol of the consumed glycerol. The values in the parentheses indicate the final concentrations of biomass (g/L) and products (mM). Standard deviations were obtained from triplicate experiments in the TK52 and TK52z strains, and from 9 replicate experiments in other strains. For calculation of biomass yield, OD 600 was converted into dry cell weight using the conversion factor 0.37 g DC/L, and carbon-mol in the biomass was calculated based on the biomass composition described in the iAF1260 model [ 21 ]. *4 Metabolic simulation was performed with following parameters: GUR was 15 mmol/(g DC●h) and OUR was 10 mmol/(g DC●h). Gene knockout simulation for 3HP production Metabolic simulation was carried out to improve 3HP production by considering the whole metabolic network. A genome-scale metabolic model of E. coli , iAF1260, which includes 2,077 metabolic and transport reactions and 1,038 unique metabolites [ 21 ], was employed with FBA [ 24 , 25 ] to simulate 3HP production in E. coli . Since the 3HP biosynthesis pathway does not exist in E. coli , seven reactions involved in the 3HP biosynthetic pathway were added to the iAF1260 model (Table 2 ), which was subsequently referred to as the iAF1260-3HP model. Using the iAF1260-3HP model, multiple gene knockout simulations were performed to identify candidate genes that could enhance 3HP production when deleted. For simulation parameters, glycerol was used as the sole carbon source, and its uptake rate (GUR) was set to 15 mmol/(g dry cell (DC)●h). The oxygen uptake rate (OUR) was set to 10 mmol/(g DC●h), which corresponds to a microaerobic condition. Table 2 The metabolic reactions added to the iAF1260 model Reaction name Metabolic reaction EC number Glycerol dehydratase Glycerol → 3HPA + H 2 O 4.2.1.30 3HPA dehydrogenase 3HPA + NAD + + H 2 O → 3HP + NADH + 2H + 1.2.1.3 3HP transporter 3HP + H + + → 3HP[e] + H + [e] – 3HP exchange 3HP[e] → – 1,3-PDO oxidoreductase 3HPA + NADPH + H + → 1,3PDO + NADP + 1.1.1.202 1,3-PDO transporter 1,3-PDO → 1,3-PDO[e] – 1,3-PDO exchange 1,3-PDO[e] → – Metabolites with “[e]” indicate extracellular metabolites. In single-gene knockout simulations, solutions in which the metabolic flux of 3HP production was higher than zero were not obtained. Double-gene knockout simulations identified some candidate genes that when deleted together could enhance 3HP production (Table 3 ). Among these, Δ tpiA Δ pgi , Δ tpiA Δ zwf , and Δ tpiA Δ edd models displayed the highest carbon-mol yield of 3HP on glycerol (70.5 C-mol%). In this study, we focused on the double knockout of tpiA and zwf for further analysis. tpiA encodes for triosephosphate isomerase, which converts dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP). zwf codes for glucose-6-phosphate-1-dehydrogenase, which converts glucose-6-phosphate to 6-phospho-glucono-1,5-lactone. Table 3 Knockout candidate genes for enhancing 3HP production obtained by gene knockout simulation Knockout genes 3HP yield (C-mol%) Specific growth rate relative to wild-type iAF1260-3HP 0 100% Δ tpiA Δ zwf 70.5 48% Δ tpiA Δ pgi 70.5 48% Δ tpiA Δ edd 70.5 48% Δ tpiA Δ gldA 69.0 47% Δ tpiA Δ fsaB 69.0 47% The simulated flux distributions of the iAF1260-3HP, Δ tpiA , Δ zwf , and Δ tpiA Δ zwf models are shown in Figure 2 . In the iAF1260-3HP model, a high flux of glycolysis was predicted and ATP required for cell growth was mainly produced by glycolysis and the respiratory chain (Figure 2 A). The Δ zwf model exhibited the same flux distribution as the iAF1260-3HP model (Figure 2 B) because the flux of the glucose-6-phosphate-1-dehydrogenase reaction was zero in the iAF1260-3HP model (Figure 2 A). In the Δ tpiA model, glycerol was mainly catabolized through the Entner-Doudoroff pathway via dihydroxyacetone (DHA) and entered into gluconeogenesis and the TCA cycle. The flux into glycolysis was blocked by the inability to generate GAP from DHAP because the amount of GAP consumed by the fructose 6-phosphate aldolase reaction, which converts DHA and GAP to fructose 6-phosphate, was equal to the amount of GAP produced by the Entner-Doudoroff pathway (Figure 2 C). Figure 2 Metabolic flux distributions based on the genome-scale metabolic model. Metabolic flux distribution for the iAF1260-3HP (A) , Δ zwf (B) , Δ tpiA (C) , and Δ tpiA Δ zwf (D) models are shown. Flux values are normalized by the glycerol uptake rate to 100%. Width of the black arrow corresponds to the relative flux value of glycerol uptake rate. Gray arrow indicates the flux of the corresponding reaction was 0. Abbreviations: 3HP, 3-hydroxypropionic acid; 3HPA, 3-hydroxypropionaldehyde; 1,3-PDO, 1,3-propanediol; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; G6P, glucose 6-phosphate; 6PG, 6-phospho-gluconate; 6PGL, 6-phospho-glucono-1,5-lactone; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; Ru5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7-phosphate; GAP, glyceraldehyde 3-phosphate; X5P, xylulose 5-phosphate; E4P, erythrose4-phosphate; MGO, methylglyoxal; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; AcetP, acetyl phosphate; EtOH, ethanol; Oxa, oxaloacetate; Cit, citrate; IsoCit, isocitrate; α-KG, α-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Glyox, glyoxylate; Q, ubiquinone; QH 2 , ubiquinol. In the Δ tpiA Δ zwf model, the zwf knockout blocked flux into the Entner-Doudoroff pathway, which was active in the Δ tpiA model. This resulted in glycerol catabolism through the glycerol kinase reaction, which converts glycerol to glycerol-3-phosphate, and the methylglyoxal pathway, in which DHAP is converted to pyruvate via methylglyoxal. 3HP was produced instead of acetate in the Δ tpiA Δ zwf model. In the Δ tpiA model, acetate production was preferred since ATP was also generated. However, in the Δ tpiA Δ zwf model, ATP was consumed by the glycerol kinase reaction, which is why total ATP was not generated by acetate production from glycerol. When the OUR was limited, 3HP production was increased, instead of acetate production, to balance the reduced capacity of the respiratory chain due to the reduction in NADH generation by 3HP production from glycerol (Figure 2 D). 3HP production based on the metabolic simulation Based on the results from the metabolic simulation, both tpiA and zwf were disrupted in the TK52 strain, generating strain TK52tz. The 3HP yield of TK52tz was successfully increased 4.4-fold relative to TK52 (20.1 ± 9.2 C-mol%) (Figure 1 G-H and Table 1 ). TK52tz exhibited a two-step growth phase that was not observed in TK52, with specific growth rates of 0.54 1/h in the first growth phase (0–6 h) and 0.03 1/h in the second growth phase (48–72 h). 3HP was mainly produced in the second growth phase with the consumption of acetate and lactate that was produced prior to the second growth phase. The growth rate of TK52tz was decreased compared to TK52 (0.73 1/h), and glycerol was not completely consumed in TK52tz. Acetate and 1,3-PDO were produced as byproducts. Although 1,3-PDO production was not predicted by metabolic simulation, the experimental results of TK52tz were consistent with the simulation results, including the improvement in 3HP yield, the decrease in growth rate, and the lack of succinate production. This indicated that metabolic modeling is an effective strategy for the improvement of target production. Based on the metabolic simulation, glycerol was predicted to be catabolized in TK52tz through the methylglyoxal pathway, which converts DHAP to pyruvate. This pathway is not usually active in E. coli due to allosteric inhibition by inorganic pyrophosphate and the low activity of enzymes involved in this pathway [ 37 , 38 ]. The extracellular concentration of methylglyoxal, an intermediate of the pathway, was increased significantly in TK52tz (0.22 mM at maximum) compared with TK52 (0.03 mM at maximum). This suggested that flux into the methylglyoxal pathway was increased as predicted, and this pathway might be a bottleneck for glycerol catabolism. This could result in decreased growth and incomplete glycerol consumption because methylglyoxal is a toxic cellular electrophile that reacts with the nucleophilic centers of macromolecules such as DNA, RNA, and protein [ 39 ]. Abundant production of 1,3-PDO was observed as a primary byproduct (37.7 ± 13.2 C-mol%) in TK52tz. Increased 3HP production might lead to the accumulation of intracellular 3HPA, a precursor of 3HP as well as 1,3-PDO, and result in the overflow of flux toward 1,3-PDO production. We speculated that changing the aldehyde dehydrogenase to the superior enzyme [ 17 ], or disruption of yqhD , whose product converts 3HPA to 1,3-PDO, would increase 3HP production and decrease 1,3-PDO production. The single knockout strains of tpiA and zwf , TK52t and TK52z, respectively, were constructed to analyze the effects of the knockout of each gene. Metabolic simulation predicted that the zwf knockout would not affect metabolism, but the tpiA knockout would alter metabolic flux into the Entner-Doudoroff pathway and decrease the growth rate. Similar culture results between TK52z and TK52 (Figure 1 A– 1 D, Table 1 ) suggest that the flux into the oxidative pentose phosphate pathway was small, and the deletion of zwf had a small impact on metabolism in this condition, as predicted by the simulation. The culture results of TK52t and TK52tz were similar (Figure 1 E- 1 H, Table 1 ). The 3HP yield of TK52t was increased by 3.2-fold relative to TK52 (14.7 ± 7.0 C-mol%) and slightly reduced when compared to TK52tz. TK52t produced 1,3-PDO (22.9 ± 5.2 C-mol%) as a byproduct, as predicted by metabolic simulation. Acetate production by the tpiA knockout was also predicted, however measurements of acetate production (8.2 ± 9.9 C-mol%) in TK52t contained significant variation, thus it was difficult to compare the experimental results with the results from the metabolic simulation. The increased 3HP yield in the tpiA knockout, which was not predicted by metabolic simulation, might be due to the conversion of glycerol to glycerol 3-phosphate by glycerol kinase, which may have had a higher activity than the glycerol dehydrogenase that converts glycerol to DHA [ 40 ]. This would lead to increased flux into the methylglyoxal pathway, as indicated by elevated methylglyoxal levels (Figure 1 F), resulting in similar culture results for TK52tz (Figure 1 H), such as 3HP production. Further increase in 3HP production by yqhD deletion, based on measurement of 1,3-PDO production 3HP production was successfully increased in TK52tz, however a high yield of 1,3-PDO was also produced (37.7 ± 13.2 C-mol%). yqhD , which is involved in 1,3-PDO biosynthesis, was deleted in TK52tz, generating the strain TK52tzy, in order to decrease 1,3-PDO production and further increase 3HP production. As a result, the 3HP yield of TK52tzy was increased 1.7-fold (33.9 ± 1.2 C-mol%) and the 1,3-PDO yield was drastically reduced (5.9 ± 0.5 C-mol%) relative to TK52tz (Figure 1 I-J, Table 1 ). Compared to the parental strain, a 7.4-fold increase in 3HP yield was achieved in TK52tzy. Despite the deletion of yqhD , 1,3-PDO was still produced in TK52tzy (5.9 ± 0.5 C-mol%), likely due to the presence of other alcohol dehydrogenases that might convert 3HPA to 1,3-PDO. The deletion of yqhD increased the maximum concentration of extracellular methylglyoxal in TK52tzy (0.60 mM at maximum) relative to TK52tz (0.22 mM at maximum), since YqhD also utilizes methylglyoxal as a substrate [ 41 ]. The other culture results of TK52tzy, such as consumed glycerol and biomass yield, were similar to those of TK52tz (Table 1 ). The culture results of TK52t and TK52tz revealed large variations in the production of 3HP, 1,3-PDO, and acetate and the consumption of glycerol (Figure 1 E-H). On the other hand, the results from TK52tzy displayed smaller variations in these measurements (Figure 1 I-J). Furthermore, TK52t and TK52tz produced a large amount of 1,3-PDO, which accompanies NADPH oxidation. The deletion of yqhD in TK52tz decreased the magnitude of the error, suggesting that the high production of 1,3-PDO in TK52t and TK52tz might cause redox imbalance, resulting in the large variations in the measurements. The 3HP yield of TK52tzy (33.9 ± 1.2 C-mol%) was comparable to previous studies producing 3HP from glycerol via flask cultivation. Mohan et al. achieved 3HP yield of 39.0 ± 0.01 C-mol% by the optimization of culture conditions such as the initial culture medium pH [ 18 ]. Rathnasingh et al. achieved a yield of 40 C-mol% by the expression of α-ketoglutaric semialdehyde dehydrogenase instead of aldehyde dehydrogenase, and periodic supplementation with vitamin B 12 , a coenzyme for glycerol dehydratase [ 17 ]. Jung et al. constructed an engineered E. coli strain by knocking out ackA, pta , and yqhD to reduce byproduct generation and knocking out glpR and overexpressing glpF to enhance glycerol metabolism [ 42 ]. They achieved high 3HP production (42 g/L) by fed-batch cultivation using a jar fermenter, but the 3HP yield was lower (26.2 C-mol%) than that achieved in this study. In previous studies [ 17 , 18 ], acetate was a major byproduct, as it was in this study, and higher yields of succinate, lactate, and ethanol were also produced. Succinate, lactate, and ethanol production might serve to oxidize the excess NADH that accompanies 3HP production and glycerol catabolism via glycolysis, since production of these metabolites requires NADH as a reducing agent. Conversely, the yields of these metabolites were small in TK52tzy. This might be because the deletion of tpiA and zwf prevented flux into glycolysis, reducing excess NADH production, as predicted by metabolic simulation. In this study, metabolic simulation was performed under the conditions of OUR/GUR = 0.67 (OUR = 10 mmol/(g DC●h) and GUR = 15 mmol/(g DC●h)), and additional simulations were also performed under other various OUR/GUR conditions (data not shown). Given the 3HP yield of TK52tzy (33.9 ± 1.2 C-mol%), the metabolic state of the cell in this study was estimated under the condition of OUR/GUR ≈ 1, by the metabolic simulation. Although the adjustment of oxygen supply to the simulation result was difficult using flask cultivation (oxygen transfer coefficient, k L a, of a shaking flask is 10–100 1/h), the metabolic simulation was used effectively for improving 3HP production. Further increases in 3HP production can be expected by the optimization of aeration conditions using a bioreactor. Metabolic simulation is a powerful tool for the design of metabolic engineering strategies to improve target production and has been used successfully in many studies. Metabolic simulation using FBA is simply based on the assumption of steady state metabolism without considering the complex cellular mechanisms such as enzyme activity and regulation. This can cause discrepancies between the simulation and experimental results, i.e., the unpredicted production of 1,3-PDO in the present study. Thus, examination of the differences between experimental and simulation results, and conformation of the constructed strains to the simulated metabolic state, is important for further improvement of target production."
} | 5,638 |
23610641 | PMC3631411 | pmc | 2,534 | {
"abstract": "Ocean acidification and warming are considered two of the greatest threats to marine biodiversity, yet the combined effect of these stressors on marine organisms remains largely unclear. Using a meta-analytical approach, we assessed the biological responses of marine organisms to the effects of ocean acidification and warming in isolation and combination. As expected biological responses varied across taxonomic groups, life-history stages, and trophic levels, but importantly, combining stressors generally exhibited a stronger biological (either positive or negative) effect. Using a subset of orthogonal studies, we show that four of five of the biological responses measured (calcification, photosynthesis, reproduction, and survival, but not growth) interacted synergistically when warming and acidification were combined. The observed synergisms between interacting stressors suggest that care must be made in making inferences from single-stressor studies. Our findings clearly have implications for the development of adaptive management strategies particularly given that the frequency of stressors interacting in marine systems will be likely to intensify in the future. There is now an urgent need to move toward more robust, holistic, and ecologically realistic climate change experiments that incorporate interactions. Without them accurate predictions about the likely deleterious impacts to marine biodiversity and ecosystem functioning over the next century will not be possible.",
"conclusion": "Conclusions Quantitative syntheses of the published literature can provide powerful inferences, however, like all analyses they are subject to caveats. We identified and incorporated the available literature that met our selection criteria, however, this also outlines the current gaps in knowledge and highlights opportunities for further study. Moreover, species-specific sources of heterogeneity are always likely to make some results from the literature greatly context dependent (e.g., Fabry 2008 ; Kurihara 2008 ; Dupont et al. 2010a ; Hendriks et al. 2010 ; Kroeker et al. 2010 ). Hence, our findings highlight the complexity of marine organism responses to ocean warming, acidification, and their interaction. The magnitude, direction, and interaction of the effects varies between response types, likely a result of the pathways driving the biological responses. Responses also differ between taxonomic groups, trophic levels, and life-history stages. Most importantly, we observed synergistic interactions between ocean acidification and warming in four of the five biological responses measured (calcification, photosynthesis, reproduction, and survival), highlighting the difficulties in making inferences from single-stressor studies. However, single-factor studies in junction with those that manipulate multiple stressors can play a vital role in understanding the pathways through which particular stressors operate and will enable a more accurate assessment of the likely outcomes of interactions between warming and acidification. Importantly, we must also consider further abiotic and biotic stressors in the marine environment that are likely to also interact with warming and acidification (Halpern et al. 2007 ) as well as scaling up studies from individuals and populations to communities and ecosystems (Harley et al. 2006 ). Such large-scale multifactorial experiments would not only increase our knowledge of the functioning and resilience of marine ecosystems, but provide explicit evidence to policymakers on the effectiveness of conservation and management strategies in response to future environmental change.",
"introduction": "Introduction The concentration of atmospheric carbon dioxide (CO 2 ) has increased from 280 ppm in preindustrial times to a present day level of 391 ppm (Le Quéré et al. 2012 ). Over the last 100 years, this has led to changes in global sea surface temperatures (+0.74°C) and ocean carbonate chemistry (Orr et al. 2005 ), which have included ocean acidification by 0.1 pH units (Caldeira and Wickett 2003 ; Kleypas et al. 2006 ). By the year 2100, sea surface temperatures are expected to rise by a further 1–4°C while increased CO 2 (aq) will result in the decreased availability of carbonate ions and a further reduction in pH by 0.3–0.5 units (Caldeira and Wickett 2005 ; IPCC 2007 ; Gooding et al. 2009 ). These changes in temperature and ocean carbonate chemistry are considered two of the greatest threats to marine biodiversity (Kleypas et al. 1999 ; Doney et al. 2009 ), leading to changes in the physiological performance of individual organisms, which will in turn alter biotic interactions, community structure, and ecosystem functioning. A range of marine biological responses have already been observed in response to ocean warming including hypoxia (Pörtner and Knust 2007 ), coral bleaching (Hoegh-Guldberg et al. 2007 ), species range shifts (Parmesan and Yohe 2003 ; Root et al. 2003 ), changes to phenology (Walther et al. 2002 ), and reduced organism body size (Daufresne et al. 2009 ). Experimental manipulations simulating predicted future ocean temperatures have suggested that warming will also lead to increased metabolic costs for plants and animals (O'Connor et al. 2009 ), increased consumption rates (Sanford 1999 ), and changed food-web structures (Petchey et al. 1999 ). Observed responses of marine organisms to recent ocean acidification are limited (but see Iglesias-Rodriguez et al. 2008b ; Moy et al. 2009 ), but are expected to become increasingly apparent in the next 50–100 years (Doney et al. 2009 ; Feely et al. 2009 ). Experimental evidence, however, suggests that responses are likely to be varied (Hendriks et al. 2010 ; Kroeker et al. 2010 ) and will include hypercapnic suppression of metabolism (Christensen et al. 2011 ), acid–base balance disturbances (Miles et al. 2007 ), plus both positive and negative effects on skeleton formation (related to a decrease in carbonate saturation; Doney et al. 2009 ; Ries et al. 2009 ). The vulnerability of marine species and ecosystems to temperature, in particular, is well established (for reviews; Hoegh-Guldberg and Bruno 2010 ; Richardson et al. 2012 ; Wernberg et al. 2012 ). Conversely, the resilience of marine organisms to ocean acidification still remains a reasonably challenged concept (Dupont et al. 2010a ; Hendriks and Duarte 2010 ). Recent meta-analyses assessing the biological effects of ocean acidification (Dupont et al. 2010a ; Hendriks et al. 2010 ; Kroeker et al. 2010 ) concurred that it is unlikely to act in a uniform manner as variation exists in marine organism responses and resilience. Hence, if any meaningful comparisons are to be made on the response of marine organisms, they need to be hypothesis driven based on a priori assigned groupings, such as taxonomic groups or life stages (Dupont et al. 2010a ). Studies of the biological effects of elevated temperature and acidification on marine organisms in isolation have provided some insight into the potential tolerance of species to these changing conditions (Gattuso and Hansson 2009 ). However, given that these stressors are unlikely to operate independently (Halpern et al. 2007 ), there is now a need to gain a more ecologically realistic understanding of how the combined effects of temperature and acidification will affect marine biota (Sala et al. 2000 ; Fabry et al. 2008 ). This is vital in order to inform future adaptive management strategies. Other recent meta-analyses, across ecological systems, have also shown that multiple stressors can lead to nonadditive interactions with responses dependent on the type of stressor as well as the level of ecological organization investigated (e.g., population vs. community, autotroph vs. heterotroph) (Crain et al. 2008 ; Darling and Côté 2008 ; Tylianakis et al. 2008 ). Moreover, the mechanism through which the stressor acts upon the organism will affect the response. Multiple stressors acting through a similar pathway may have an additive effect (Crain et al. 2008 ). In contrast, any stress-induced tolerances could lead to antagonisms (Blanck 2002 ), while those stressors that act on different, but dependent mechanisms may act synergistically (Kneitel and Chase 2004 ). These reviews did, however, contain few, if any, studies that investigated both warming and acidification. Therefore, the concurrent effect of temperature and ocean acidification via elevated CO 2 remains unclear, but is likely to lead to complex biological outcomes. Organisms vary widely in their individual responses to ocean warming and acidification as a result of differences in their physiological and ecological characteristics (Dupont et al. 2008 ; Fabry 2008 ). For example, many marine organisms possessing a calcium carbonate (CaCO 3 ) structure would be considered more susceptible to ocean acidification as this process will impair their capacity to produce calcified skeletons (Doney et al. 2009 ). Conversely, some species, including some calcified species, will have the capacity to buffer against the deleterious effects of acidification by utilizing acid–base compensation (Claiborne and Evans 1992 ; Larsen et al. 1997 ), active mobility and metabolism (Widdicombe and Spicer 2008 ; Whiteley 2011 ) or energy reallocation (Wood et al. 2008 ; McDonald et al. 2009 ). Warmer temperature, up to a limit, stimulates metabolism in ectotherms, resulting in faster growth and development (Byrne 2011 ). Moreover, it has been speculated that warming could even ameliorate the negative impacts of acidification (McNeil et al. 2004 ; Kleypas and Yates 2009 ). Species responses to ocean warming and acidification are also likely to vary among life-history stages (Byrne 2011 ). Early life-history stages are considered most susceptible to changes in both temperature and ocean acidification (Byrne 2011 ). These stressors may, however, have positive and/or negative effects for the successful recruitment of juveniles to the adult population. Trophic level is also likely to determine how species respond due to differences in environmental sensitivity (Petchey et al. 2004 ; Raffaelli 2004 ). Previous study has suggested that the effects of multiple stressors are likely to act antagonistically in autotrophs, but synergistically in heterotrophs (Crain et al. 2008 ). Furthermore, as higher trophic levels contain less “biological insurance” (sensu Yachi and Loreau 1999 ), that is, less taxonomic, physiological, and genetic diversity, they are predicted to be more susceptible to multiple environmental perturbations (Christensen et al. 2006 ) which could act upon them synergistically (Crain et al. 2008 ). Using a meta-analytical approach of the peer-reviewed literature, we assessed the impacts and interactions of ocean acidification and warming on marine biological responses. Given that variability in the strength and direction of responses was expected, we classified data according to taxonomic groups, calcifiers and noncalcifiers, life-history stage and level of trophic organization (autotroph and heterotroph) in terms of changes in rates of calcification, growth, photosynthesis, reproduction, and survival. Specifically, we aimed to address three questions: (i) How do marine organisms respond to warming and acidification in isolation? (ii) How do marine organisms respond to the combined effects of warming and acidification? (iii) How do warming and acidification impacts interact?",
"discussion": "Discussion This study used a meta-analytical approach to assess the impacts and interactions of ocean acidification and warming on marine biological responses. Responses were classified according to taxonomic groups, calcifiers and noncalcifiers, level of trophic organization (autotroph and heterotroph), and life-history stage in terms of changes in rates of calcification, growth, photosynthesis, reproduction, and survival. We assessed marine organisms responses to warming and acidification in isolation and in combination and assessed the biological implications of their interaction. Effects of ocean acidification Ocean acidification generally had an adverse effect on a large range of marine biota with more specific differences between life-history characteristics. Calcifying organisms were generally negatively affected by ocean acidification, with significant biological variability in responses, such as molluscs being negatively affected, which may be due to their poor ion regulation and inability to buffer their internal compartments (Fabry et al. 2008 ; Widdicombe and Spicer 2008 ; Melzner et al. 2009 ; Dupont et al. 2010a ). Conversely, crustaceans were generally unaffected by acidification perhaps due to their active mobility, higher metabolism, and capacity to control intracellular pH (Gaillard and Malan 1983 ; Widdicombe and Spicer 2008 ; Whiteley 2011 ). Overall, less is known about how noncalcifying organisms are likely to respond to acidification (Connell and Russell 2010 ), particularly marine fishes (Ishmatsu et al. 2008 ; Munday et al. 2009 ), however, our results show that noncalcifiers were generally unaffected by acidification and for growth were positively affected when analyzed together as a group. Fishes are thought to have more efficient acid–base regulation compared to invertebrates (Widdicombe and Spicer 2008 ), which, when coupled with an increased food intake or reduced energy expenditure (Munday et al. 2009 ), could explain the positive growth response observed. Similarly, noncalcifying marine autotrophs demonstrated an increased growth to ocean acidification. This is possibly via their capacity to derive dissolved inorganic carbon from the increased CO 2 (aq) (Beer and Koch 1996 ). Autotrophs may be capable of increasing their inorganic carbon assimilation (Rost and Riebesell 2004 ) and buffering the negative effects on calcification (Ries et al. 2009 ). However, negative effects on growth were observed in calcifying autotrophs. Our results suggest that the negative effects on calcifying autotrophs may outweigh positive effects associated with the increased availability of CO 2 (aq) as a substrate for photosynthesis. We also show some evidence that calcification and survival in early life-history stages were more negatively affected by ocean acidification, highlighting not only the susceptibility of early life stages, but also the subtle nature of life-history responses when compared to species-specific effects of heterogeneity (Kurihara 2008 ; Kroeker et al. 2010 ). Importantly, our results agree with the findings from previous meta-analyses (Dupont et al. 2010a ; Kroeker et al. 2010 ) while introducing a further 48 key studies (168 additional data points). Effects of ocean warming Moderate elevations in temperatures will increase metabolic rates (Hochachka and Somero 2002 ), which influences key biological processes that regulate life-history characteristics (O'Connor et al. 2007 ). While marine organisms are capable of acclimation to a range of temperatures, once their thermotolerance limits are exceeded, organism fitness is reduced and the risk of mortality increases (Hofmann and Todgham 2010 ; Tomanek 2010 ). We found that both noncalcifying organisms and autotrophs demonstrated increased growth under warming conditions, likely due to this increase in metabolic rate (Hochachka and Somero 2002 ). Warming, however, had no effect on the growth of heterotrophs and even negatively affected their calcification. Possibly because the metabolism complexes of autotrophs (photosynthesis-limited) are less sensitive to ocean warming than the respiration-limited metabolism of heterotrophs (Lopez-Urrutia et al. 2006 ). Moreover, our results also support the hypothesis that the threshold for deleterious warming may vary between developmental stages (Byrne et al. 2009 , 2010 ) with survival significantly lower in larvae than in juveniles, and significantly lower in juveniles than in adults. Simultaneous acidification and warming Meta-analysis of the full dataset revealed that the combined stressors caused significant negative effects on calcification, reproduction, and survival, and a significant positive effect on photosynthesis, but no effect on growth. Importantly, we also found that four of the five responses (calcification, photosynthesis, reproduction, and survival) showed a synergistic interaction between acidification and warming. Although, such synergistic interactions between stressors (i.e., where the outcome was greater than the sum of the individual stressors; Folt et al. 1999 ) are relatively common (e.g., Sala et al. 2000 ; Harley et al. 2006 ), they are concerning because they are also unpredictable. Hence, such synergies limit our capacity to predict potential future impacts from single-stressors studies. Ecological synergies are important to marine systems (Paine et al. 1998 ; Harley et al. 2006 ; Sutherland et al. 2006 ) because they can further exacerbate adverse effects and reduce ecosystem resilience (Folke et al. 2004 ). They can also introduce indirect effects via biotic interactions (Darling and Côté 2008 ; Tylianakis et al. 2008 ). For example, climate-driven changes in plankton communities can regulate top predators through bottom-up control (Beaugrand et al. 2003 ). Moreover, as marine systems are subject to multiple interacting stressors (Halpern et al. 2007 ), it is possible that the addition of further stressors would introduce additional adverse consequences (e.g., Przeslawski et al. 2005 ). Therefore, our results highlight the need to move away from single-stressor studies and toward more ecologically realistic research incorporating multiple stressors, in order to more fully understand how near-future anthropogenic change will affect marine biodiversity. There was, as expected, variation in our analyses among the biological responses to combined warming and acidification between different taxonomic groups, calcifiers and noncalcifiers, trophic levels, and life-history stages. The combination of warming and acidification generally exhibited a stronger effect (either positive or negative) than when exposed to the stressors in isolation. For instance, echinoderms are highly vulnerable to ocean acidification (Dupont et al. 2010b ), likely due to their skeletons being formed from highly soluble magnesium calcite (Politi et al. 2004 ). However, the addition of moderate increases in temperature (+4°C), as predicted for the end of the century, resulted in further adverse effects (e.g., Byrne et al. 2011 ), such as the highly negative calcification responses shown here. In some instances, the combined effects of warming and acidification were reduced compared with the individual effects. Corals had both calcification and photosynthesis negatively affected by ocean acidification in isolation, while they were unaffected by the combined effects, suggesting that the addition of warming may ameliorate the adverse effects of acidification (McNeil et al. 2004 ; Kleypas and Yates 2009 ). These differences in the resilience of marine organisms will have important implications for ecosystem level responses. Interestingly, the combined effect of warming and acidification positively affected growth in echinoderms, which could be explained by energy allocation, where the cost of homeostatic regulation can be influenced by changes in somatic and reproductive growth performance (Melzner et al. 2009 ). Hence, if more energy is utilized to maintain growth, then calcification responses could be more adversely affected (e.g., Arnold et al. 2009 ; McDonald et al. 2009 ). Other studies have shown an alternative strategy for energy allocation where growth was negatively affected, while calcification was maintained (e.g., Wood et al. 2008 ; or crustaceans, this study), highlighting the species-specific nature of biological responses. Heterotroph responses to ocean acidification and warming individually differed, with acidification reducing growth, but not affecting calcification or survival; while warming alternatively did not affect growth, reducing both calcification and survival responses. However, the energetic demands of the combined effects of acidification and warming on heterotrophs resulted in calcification, growth, and survival all being reduced. Conversely, the combined effects of warming and acidification positively affected growth in autotrophs, likely due to the effect of temperature on metabolic rate (Hochachka and Somero 2002 ), while CO 2 , acted as a substrate for photosynthesis and possibly indirectly promoted growth (e.g., phytoplankton; Loehle 1995 ). Moreover, as multiple stressors affecting autotrophs are likely to act antagonistically (Crain et al. 2008 ), and the dissolved inorganic carbon sources utilized by marine autotrophs is set to increase (Raven 2005 ), photosynthesizing organisms will likely be more resilient to conditions predicted for the end of the century; as long as they do not exceed their thermotolerance (Hofmann and Todgham 2010 ; Tomanek 2010 ) and are not limited by other factors, such as inorganic nutrient availability (Langdon and Atkinson 2005 ; Cohen and Holcomb 2009 ; Ries et al. 2009 ). Our results also show that the combined effects of warming and acidification had significant negative effects on the survival of early life-history stages. Although there was insufficient data to compare across all life-history stages, both larvae and juveniles were highly susceptible to changes in temperature and ocean acidification, supported by previous research (Gosselin and Qian 1997 ; Hunt and Scheibling 1997 ; Byrne 2011 ). Indirectly, ocean warming can reduce the mortality of larvae by shortening the planktonic duration (Lamare and Barker 1999 ), when they are most vulnerable to predation (O'Connor et al. 2007 ). Our results, however, identified that the combined effects of ocean acidification and warming increased mortality, indicating that multiple stressors will have important implications for population persistence, potentially acting as a bottleneck for some species (Dupont et al. 2010b ; Byrne 2011 )."
} | 5,565 |
25323862 | null | s2 | 2,535 | {
"abstract": "Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula-Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula-Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology."
} | 416 |
34977392 | PMC8671096 | pmc | 2,536 | {
"abstract": "Currently, the establishment of synthetic microbial consortia with rational strategies has gained extensive attention, becoming one of the important frontiers of synthetic biology. Systems biology can offer insights into the design and construction of synthetic microbial consortia. Taking the high-efficiency production of 2-keto- l -gulonic acid (2-KLG) as an example, we constructed a synthetic microbial consortium “ Saccharomyces cerevisiae-Ketogulonigenium vulgare” based on systems biology analysis. In the consortium, K. vulgare was the 2-KLG producing strain, and S. cerevisiae acted as the helper strain. Comparative transcriptomic analysis was performed on an engineered S. cerevisiae (VTC2) and a wild-type S. cerevisiae BY4741. The results showed that the up-regulated genes in VTC2, compared with BY4741, were mainly involved in glycolysis, TCA cycle, purine metabolism, and biosynthesis of amino acids, B vitamins, and antioxidant proteases, all of which play important roles in promoting the growth of K. vulgare . Furthermore, Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG. Therefore, VTC2 would be of great advantage in working with K. vulgare . Thus, the synthetic microbial consortium \"VTC2- K. vulgare \" was constructed based on transcriptomics analyses, and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K. vulgare , reaching 13.2 ± 0.52 g/L. In addition, the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes ( sod , cat and gpd ) in K. vulgare. The results indicated that the oxidative stress in the synthetic microbial consortium was efficiently reduced. Thus, systems analysis confirmed a favorable symbiotic relationship between microorganisms, providing guidance for further engineering synthetic consortia.",
"conclusion": "5 Conclusions In this study, systems biology analysis was applied to construct a meaningful synthetic microbial consortium. The yield of 2-KLG was significantly improved in the co-cultured system compared with that of the mono-cultured system. To the best of our knowledge, this is the first report on increasing 2-KLG production via taking engineered S. cerevisiae as helper strain. This study provided a reference for subsequent rational design and the construction of synthetic microbial consortia.",
"introduction": "1 Introduction Oxidative stress could lead to macromolecular damage and disruption of redox signaling [ 1 ]. The accumulation of reactive oxygen species (ROS) is one of the main causes of oxidative stress [ 2 ]. The production of ROS can be scavenged by the cellular antioxidant system, including enzymatic components (superoxide dismutase, catalase, peroxidase, etc.) and non-enzymatic components (glutathione, NADPH, thioredoxin, glutaredoxin, vitamin C, etc.) [ 3 ]. Vitamin C is a water-soluble vitamin with important physiological functions, which has been proven to inhibit the generation of free radicals and reduce the oxidative damage caused by ROS [ 4 , 5 ]. Currently, 2-keto- l -gulonic acid (2-KLG) is mainly produced through a two-step fermentation process developed in the 1980s [ 6 ]. The second step is to convert l -sorbose into 2-KLG by microbial co-cultivation, which is also the focus of microbial transformation research. 2-KLG-producing strain ( Ketogulonigenium vulgare ) and the helper strain ( Bacillus sp.) are usually used as industrial strains for the synthesis of 2-KLG. Exploring the interaction between the two strains during co-culture has always been one of the hotspots in the field of 2-KLG fermentation. K. vulgare shows poor growth and low yield when cultured alone. Based on the research results of genomics, transcriptomics, proteomics, and metabolomics, scholars have done a lot of work in exploring the mechanism of interaction between the two strains from the perspective of amino acids, vitamins, and environmental stress [ [7] , [8] , [9] , [10] ]. K. vulgare lacks key enzymes for the synthesis of amino acids such as histidine, threonine, leucine, and isoleucine [ 11 ], while helper strains can synthesize amino acids lacking in K. vulgare . Our lab reconstructed the threonine biosynthetic pathway in K. vulgare to improve the biomass and 2-KLG production [ 12 ]. The expressions of genes related to amino acids transport and metabolism were significantly up-regulated in K. vulgare [ 13 ]. Studies have shown that K. vulgare needs the helper strain to provide B vitamins and purines to maintain normal metabolism [ 14 , 15 ]. In addition, the helper strain could secrete proteases (glutaredoxin, catalase, etc.) and up-regulate the expression of the antioxidant enzymes (superoxide dismutase, thioredoxin, etc.) in K. vulgare to alleviate oxidative stress caused by ROS metabolism disorder [ 16 ]. The construction of the synthetic microbial consortium is of great significance for the production of 2-KLG by K. vulgare . The helper strains for the industrial production of 2-KLG mainly include Bacillus megaterium [ 17 ], Bacillus thuringiensis [ 18 ], and Bacillus cereus [ 19 ], all of which are prokaryotic microorganisms. However, taking eukaryotic microorganisms as helper strains were rarely reported. S. cerevisiae is a conventional and well-studied model organism for which advanced genetic tools for metabolic pathway manipulation are available [ 20 ]. The co-cultured relationships between cells in synthetic microbial consortia are dynamically balanced, leading to greater adaptability and stability to variable environments, thus providing an important new field for synthetic biology. There are mainly two strategies for designing and constructing synthetic microbial consortia [ 21 ]. The first one is to construct microbial consortia based on genetic elements, modules, and metabolic pathways. The other one is to de novo design microbial consortia based on multiple omics analyses. Transcriptome analyses can discover the dynamic expression and regulation mechanism of microbial genes, laying a foundation for exploring microbial community structure and ecological functions [ 22 ]. In this study, the synthetic microbial consortium was rationally designed based on system biology. Transcriptomic analyses were performed on the wild-type S. cerevisiae (BY4741) and the engineered S. cerevisiae (VTC2), which was a vitamin C producing strain constructed by our lab [ 4 ]. Taking advantage of VTC2, we constructed a synthetic microbial consortium system containing the K. vulgare and VTC2, which significantly increased the production of 2-KLG. This research could provide an effective reference for rational design of the synthetic microbial consortium to efficiently produce 2-KLG or other high value-added products.",
"discussion": "4 Discussion Synthetic microbial consortia could perform complicated tasks and endure the changeable environment, thus providing an important new frontier for synthetic biology [ 38 ]. Recent advances in multi-omics and automation technology have enabled researchers to focus on engineering the microbiome's metabolic network and microbial interactions. The design principles for synthesizing microbial consortia are mainly based on the patterns of microbial interactions, including cell-cell communications, and exchange of metabolites and energy, etc. To achieve predictions of microbiome function at a systems level, measurements of the microbiome's in situ metabolic network structure and activity are essential [ 39 ]. Systems biology approaches enable the objective description of genetic and metabolic pathways in microbial consortia, which facilitates the design and optimization of synthetic consortia [ 40 ]. Systems biology analysis [ 21 ], particularly meta-omics techniques (e.g. meta-genomics [ 41 , 42 ], meta-proteomics [ 16 ], meta-transcriptomics [ 43 ], and meta-metabolomics [ 8 ]), can be combined with bioinformatic tools to enable the analysis of individual species in the microbiome and global measurements of proteins and metabolites. This provides insights into the design and construction of functional genes, functional modules, and entire synthetic consortia. In this study, comparative transcriptome analyses of VTC2 and BY4741 were performed to objectively characterize the genetic and metabolic pathways of VTC2, which facilitated the design and optimization of the synthetic consortium. As described in previous studies, the glycolysis pathway and the TCA cycle were central metabolic pathways of glucose metabolism in S. cerevisiae [ 44 ]. The genes involved in glycolysis were significantly up-regulated in VTC2 compared with those of BY4741. Thus, VTC2 had higher glucose utilization efficiency compared to BY4741 and could provide more precursors for the synthesis of vitamin C. The transcription levels of most genes were significantly down-regulated in VTC2 throughout the TCA cycle compared with that of BY4741, indicating a decrease in the production of reducing substances and ATP, i.e. the strains were in a stable growth phase at 72 h. And 2-ketoglutarate decarboxylase (encoded by kgd1 ) catalyzed the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A, a key reaction of the TCA cycle [ 45 ]. The down-regulation of kgd1 transcription might lead carbon flux into the biosynthetic of glutamate, the source of 80% of cellular nitrogen [ 46 ]. The PPP is also a fundamental component of central carbon metabolism, maintaining carbon homeostasis. The PPP provided precursors for nucleotide and amino acid synthesis, and also provided reducing substances to eliminate oxidative stress [ 47 , 48 ]. The up-regulation of the transcription of key metabolic genes in the PPP pathway could lead to an increase in the synthesis of Ru5P and PRPP. Ru5P is a precursor for the synthesis of nucleotides. PRPP is an important precursor of de novo synthesis of purine nucleotide and pyrimidine nucleotide [ 49 , 50 ]. The establishment of symbiosis between strains via metabolites exchanges is a common mechanism in synthetic microbiota [ 51 ]. Modularity is introduced into the pathway design of microbial metabolite production, in which metabolic intermediates provided by one microorganism were transferred to the other one. This unidirectional interaction can stabilize the Escherichia coli - S. cerevisiae co-culture for high production of oxygenated taxanes [ 52 ]. In addition, the mutualism is enhanced by the exchange of metabolites between strains. It is considered that cross-feeding of metabolites in synthetic consortia would facilitate the heterologous production of flavonoids [ 53 ]. The expression of vitamin C heterologous biosynthetic pathway in VTC2 resulted in the transcriptional up-regulation of genes involved in the biosynthesis of amino acids, B vitamins, purines and antioxidant proteases compared with BY4741. Amino acids play an important role in cell growth, metabolism, and cell survival in severe environments. Proline is an essential stress protector in yeast cells and also participates in numerous important intracellular physiological processes, such as maintaining protein and membrane stability and scavenging ROS [ 54 ]. Valine, leucine, glycine, and serine are derived from the glycolysis pathway. Higher intracellular levels of amino acids derived from the glycolysis pathway reflect the higher glycolytic capacity of the cell [ 55 ]. Cysteine can be used to synthesize glutathione, which is the most important intracellular antioxidant. And cysteine oxidation can be used to control protein function and cell signaling pathways [ 56 ]. Previous studies have shown that K. vulgare itself could not synthesize B vitamins [ 15 , 57 ], whereas S. cerevisiae contained the B vitamins synthesis pathway and supplied the synthesized B vitamins to K. vulgare to maintain its normal growth metabolism in our constructed consortium. The B vitamins comprised a group of water-soluble vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, folic acid, biotin, etc.) that acted as cofactors, precursors, and substrates for biological processes [ 58 ]. Pyridoxine is a cofactor for more than 140 biochemical reactions in cells [ 59 ], most of which are related to amino acids biosynthesis. In addition, pyridoxine can also quench ROS [ 60 ]. Folic acid and its derivatives are also important factors for the growth of K. vulgare [ 61 ]. However, K. vulgare is unable to utilize folic acid for deficiency of folate reductase, while S. cerevisiae can metabolize folic acid to dihydrofolate and tetrahydrofolate that can be utilized by K. vulgare . In addition, adenine, guanine, xanthine, and hypoxanthine produced by helper strains could contribute to the resistance of the microflora to ROS [ 14 ]. Cellular redox systems included glutathione, thioredoxin, pyridine nucleotides, and NADPH [ 62 ]. Among them, antioxidant proteases mainly include superoxide dismutase, catalase, glutathione, and glutathione. In summary, the metabolites released by VTC2 could compensate for the defective growth of K. vulgare . In addition, the results that exogenous addition of vitamin C raised 2-KLG production also further confirmed that VTC2, producing high vitamin C, had better concomitant effects. The consumption of the same carbon source by two strains can lead to competitive interactions and unstable symbiosis in microbial communities. Differences in nutrient metabolism in symbiotic systems can reduce or eliminate competitive exclusion, and make symbiosis possible. Therefore, avoiding carbon source competition is one of the strategies to achieve effective symbiosis. For example, a bacterial consortium consisting of a glucose-selective strain and a xylose-selective strain was constructed to achieve high yields of n-butanol [ 63 ]. Here, we used a dual carbon source of glucose and l -sorbose for fermentation. The results showed that S. cerevisiae did not compete with K. vulgare for l -sorbose, thus achieving optimal growth of K. vulgare . S. cerevisiae , as a model organism, is widely used in large-scale industrial production due to intensive research on its genetic and growth characteristics. The focus of this study was to explore the advantages of applying engineered S. cerevisiae to the microbial consortia. K. vulgare suffered from weak growth and low production due to oxidative stress and lack of nutrients required for growth. However, the results of the transcriptomic analysis indicated that VTC2 had the potential to promote the growth of K. vulgare . Therefore, this study took VTC2 as the helper strain with K. vulgare to form the synthetic microbial consortium, and successfully achieved a significant increase in the production of 2-KLG. In addition, ROS are harmful byproducts of aerobic metabolism. When ROS levels increase, oxidative stress will occur, destroying cellular biomolecules (DNA, proteins, and lipids) [ 2 ], limiting cellular growth and 2-KLG production. In order to explore the antioxidant capacity of different 2-KLG fermentation systems, the SOD and CAT activities were evaluated, respectively. The results showed that the synthetic microbial consortium had a stronger antioxidant capacity compared to the mono-cultured system, which was associated with the high vitamin C production of VTC2. It was also demonstrated that VTC2, as the helper strain, induced a significant up-regulation of 2-KLG producing and antioxidant-related genes in K. vulgare . These results further validated the advantages of the VTC2 as the helper strain in the synthetic microbial consortium. This microbial consortium also has implications for continued research and improvement. For example, engineered K. vulgare could work with VTC2 to form a more efficiently microbial consortium for 2-KLG production. And transcriptomic analyses of the microbial consortium are indeed an important direction to obtain more information of the microbial consortium. In addition, the constructed consortium could be expanded in controlled fermenters to achieve a higher yield of 2-KLG."
} | 4,060 |
36050493 | null | s2 | 2,541 | {
"abstract": "The functions of many microbial communities exhibit remarkable stability despite fluctuations in the compositions of these communities. To date, a mechanistic understanding of this function-composition decoupling is lacking. Statistical mechanisms have been commonly hypothesized to explain such decoupling. Here, we proposed that dynamic mechanisms, mediated by horizontal gene transfer (HGT), also enable the independence of functions from the compositions of microbial communities. We combined theoretical analysis with numerical simulations to illustrate that HGT rates can determine the stability of gene abundance in microbial communities. We further validated these predictions using engineered microbial consortia of different complexities transferring one or more than a dozen clinically isolated plasmids, as well as through the reanalysis of data from the literature. Our results demonstrate a generalizable strategy to program the gene stability of microbial communities."
} | 245 |
36188008 | PMC9522894 | pmc | 2,542 | {
"abstract": "The current economic and environmental context requests an accelerating development of sustainable alternatives for the production of various target compounds. Biological processes offer viable solutions and have gained renewed interest in the recent years. For example, photosynthetic chassis organisms are particularly promising for bioprocesses, as they do not require biomass-derived carbon sources and contribute to atmospheric CO 2 fixation, therefore supporting climate change mitigation. Marine cyanobacteria are of particular interest for biotechnology applications, thanks to their rich diversity, their robustness to environmental changes, and their metabolic capabilities with potential for therapeutics and chemicals production without requiring freshwater. The additional cyanobacterial properties, such as efficient photosynthesis, are also highly beneficial for biotechnological processes. Due to their capabilities, research efforts have developed several genetic tools for direct metabolic engineering applications. While progress toward a robust genetic toolkit is continuously achieved, further work is still needed to routinely modify these species and unlock their full potential for industrial applications. In contrast to the understudied marine cyanobacteria, genetic engineering and synthetic biology in freshwater cyanobacteria are currently more advanced with a variety of tools already optimized. This mini-review will explore the opportunities provided by marine cyanobacteria for a greener future. A short discussion will cover the advances and challenges regarding genetic engineering and synthetic biology in marine cyanobacteria, followed by a parallel with freshwater cyanobacteria and their current genetic availability to guide the prospect for marine species.",
"introduction": "Introduction Modern society faces various challenges that must be addressed promptly to preserve most living organisms. The current environmental crisis is particularly pressing and threatening and demands that the present industrial and economical behaviors are entirely revised ( Levi and Cullen, 2018 ). As such, many research, political and industrial efforts have focused on developing sustainable solutions to mitigate the climate emergency. In this context, bioprocesses, relying on living organisms for biological catalysis, stand as a key solution due to their flexibility, robustness and sustainability. Moreover, the current omics data and synthetic biology techniques available to strengthen and diversify bioprocesses further support their wider use for various industrial applications, including fuels, chemicals or pharmaceuticals production. Of particular interest for sustainable industrial bioprocesses, cyanobacteria perform oxygenic photosynthesis, using solar energy for their metabolism while fixing atmospheric CO 2 . These prokaryotes are significant contributors to global carbon fixation in many environments, participating in ecosystem maintenance. The tremendous diversity and strain-specific variation of pigments and photosystems ( Stephens et al., 2021 ), essential elements for photosynthesis, provide great opportunities for tailored biotechnological applications, with strains metabolically adapted to specific environmental conditions, such as high salinity, temperature and fluctuating light. To date, hundreds of cyanobacterial species have been isolated in marine and freshwater environments with new species continuously being identified and further characterized. Historically, a few model freshwater species have previously led researchers to understand cyanobacterial metabolism and photosynthetic capabilities and develop synthetic biology tools for strain engineering ( Gale et al., 2019 ). However, marine cyanobacteria display several advantages over their freshwater counterparts, including their capacity to grow in seawater, their robustness to environmental changes and specific metabolic abilities, including biosynthesis of complex and unique biomolecules. Currently, more work is still needed to adapt many synthetic biology techniques to marine cyanobacteria and fully unlock their biotechnological and industrial potential ( Khalifa et al., 2021 ). This short review will briefly discuss the impressive diversity of marine cyanobacteria and the associated opportunities for unique bio-applications. The recent development of genetic tools and the advances of synthetic biology in marine cyanobacteria will be further reviewed, highlighting some of the challenges that must be addressed to move forward with these species and expand their applications. Finally, some of the more advanced synthetic biology tools developed for freshwater cyanobacteria will be summarized to draw a parallel between marine and freshwater species. While the focus of this review will be synthetic biology for industrial applications, it is worth mentioning that synthetic biology methods are also relevant for fundamental studies, such as phenotypic screening or regulation characterization, but will not be discussed here.",
"discussion": "Discussion and outlook The potential of marine cyanobacteria for various biotechnological applications and, more importantly, sustainable compound bioproduction is remarkable. The diversity of these species and their unique metabolic properties, evolved to survive in changing environments, offer tailored possibilities for industrial bioprocesses, such as large-scale photobioreactors for target bioproduction. To fully unlock their potential, marine cyanobacteria must be genetically accessible to further engineer strains for industrial purposes. While some progress has already been made, the current genetic toolkit remains insufficient. In fact, a clear discrepancy persists between species where some Synechococcus species have been and are still leading the field. On the other hand, despite the current knowledge and techniques available on genetic systems, some species continue to resist genetic modifications, as exemplified by the studied Prochlorococcus . While some biological challenges must participate in this discrepancy, additional aspects, such as historical attention and devoted resources, further contribute to the uneven genetic accessibility of marine cyanobacteria. Moreover, the genetic toolkit for freshwater cyanobacteria is undeniably more extensive, with complex tools developed for several genera. Considering how phylogenetically close some marine and freshwater species are, the transferability of methods is highly probable. In fact, a proteomic analysis of Synechocystis sp. PCC 6803 and PCC 7338 showed how close species can be, although protein regulation reflected their respective natural habitat ( Kwon et al., 2020 ). Similarly, genetic elements from freshwater species have shown to be functional in marine cyanobacteria ( Markley et al., 2015 ), suggesting possible transferability. As such, adaption of genetic techniques similar to freshwater species is hopeful, especially in a growing context for the search for bioprocesses. In fact, the available research on marine cyanobacteria does not suggest that challenges specific to these species prevent their genetic engineering. Instead, the current lack of tools reflects that these species have been historically less explored than their freshwater counterparts. In addition, their extreme diversity prevents from addressing these obstacles globally as it would be inappropriate to assume one strategy would succeed in all species. Instead, strain-specific methods will be needed for efficient genetic manipulation. To achieve this, optimized growth conditions and robust methods for foreign DNA introduction are key first steps to build on for more complex genetic tools. This, of course, might prove difficult for interesting chassis organisms, such as Prochlorococcus , for which engineering obstacles remain unclear ( Laurenceau et al., 2020 ). However, it is worth pointing out that, despite the discussed advances in freshwater species, cyanobacteria in general remain behind in terms of complex genetic tools compared to model prokaryotes, such as Escherichia coli , for which, for example, sophisticated CRISPR tools, such as prime-editing ( Tong et al., 2021 ), have been developed. Although adapting genetic tools to any organism is difficult, the polyploidy of cyanobacteria is particularly challenging to address and modulate and significantly contributes to limiting tool development. In particular, the lack of understanding of chromosomal copy fluctuations and their related environmental clues prevent to overcome these issues systematically. As such, further research efforts are needed to complete the genetic toolset for both marine and freshwater cyanobacteria for their unlimited industrial use."
} | 2,193 |
34073438 | PMC8161448 | pmc | 2,545 | {
"abstract": "Computers nowadays have different components for data storage and data processing, making data transfer between these units a bottleneck for computing speed. Therefore, so-called cognitive (or neuromorphic) computing approaches try combining both these tasks, as is done in the human brain, to make computing faster and less energy-consuming. One possible method to prepare new hardware solutions for neuromorphic computing is given by nanofiber networks as they can be prepared by diverse methods, from lithography to electrospinning. Here, we show results of micromagnetic simulations of three coupled semicircle fibers in which domain walls are excited by rotating magnetic fields (inputs), leading to different output signals that can be used for stochastic data processing, mimicking biological synaptic activity and thus being suitable as artificial synapses in artificial neural networks.",
"conclusion": "4. Conclusions In a recent study, neurons were defined as three coupled semicircle fibers in which domain walls are excited by rotating magnetic fields. These inputs, defined by pairs or rotational orientations (clockwise/counterclockwise), result in different outputs, which were investigated in terms of “learning” and “forgetting”. Depending on the number of added signals per time, defining “learning”, and the leaking rate, defining “forgetting”, these artificial neurons are found to reach a defined stimulus value with a certain probability. Such simple systems, which can be prepared by lithographic processes, can thus be used as parts of neuromorphic hardware.",
"introduction": "1. Introduction Neuro-inspired signal processing presently relates to the most intensively studied topics of technical sciences in a wide range of completely different realizations. It can be implemented from an electronic perspective, using, for example, field-programmable gate array (FPGA) circuits [ 1 , 2 , 3 , 4 ] or employing the recent achievements of spintronics [ 5 , 6 , 7 ], phase change materials [ 8 , 9 ] or other components. Different computational schemes have been developed, such as artificial neural networks (ANNs), support vector machines or reservoir computing as a special type of recurrent ANN [ 10 , 11 , 12 ]. Simulating the human brain with recurrent neural networks necessitates calibration of weights and connections of the nodes in the network; this time-consuming task can be avoided by using a reservoir in which weights between input values and reservoir neurons are either set arbitrarily or optimized [ 13 , 14 ], while the weights between reservoir neurons and output layer are linearly trained, thus speeding up the training process [ 15 , 16 ]. For all these approaches, it is necessary to produce artificial neurons and synapses that are able to transfer and modulate data. Often, such artificial neurons and synapses are produced by electronic devices with a variable resistance which represents the synaptic weight [ 17 , 18 , 19 ]. Several approaches to neuro-inspired signal processing are based on magnetic materials. There is a rich tradition of such research, firstly introduced by works of Allwood, Cowburn et al. [ 20 , 21 , 22 ] and more recently by Grollier et al. [ 23 , 24 ], to name just a few. Generally, signal processing necessitates a deterministic or stochastic correlation between input and output values. This means that not only input and output need to be defined, but also data transport and processing between them. In several approaches, nanofibers or nanofiber networks are used for these tasks, in which data can be transported and manipulated in the form of domain walls [ 25 , 26 , 27 ]. The static and dynamic magnetic properties of such nanofibers depend strongly on their geometry, material and orientation with respect to the external magnetic field [ 28 , 29 , 30 , 31 ]. From the perspective of neuroscience and neuronal spikes, the effect of neuron excitation if a given threshold level is exceeded is fundamental [ 32 , 33 , 34 ]. In the language of bioelectronics, this can be interpreted in the following way: If the excitations from several inputs overlap constructively, a defined energy barrier is overcome, and the output action potential is different from zero. Hence, since biological signals have different amplitudes, it can be imagined that the implementation of a small network of ferromagnetic fibers might be a good choice to use magnetization dynamics to provide a wide enough range of vector values, mimicking neuronal and synaptic activity. Here we investigate three-semicircle systems with two inputs and three outputs with dynamic micromagnetic simulations. Our results show that, depending on the chosen threshold values, different data processing operations can be defined in this simple system. It must be mentioned that the nanofiber network presented here forms a single artificial neuronal cell body, receiving signals and performing data processing, which can be used as a part of a forward-propagating neural network [ 35 , 36 , 37 ]. It does not form a full artificial neural network with axons and synapses, which has to be implemented after establishing the functionality of these devices. Generally, the functionalities of the elements found in the human brain are not 100% reflected by such a neuromorphic approach.",
"discussion": "3. Results and Discussion As an example, the calculation\n (1) M x ( t o t ) = w A M x ( A ) + w B M x ( B ) + w C M x ( C ) , \nimitates similar combinations found in neural networks, with w i defining the weights of the respective outputs. The total signal M x ( t o t ) is then normalized by its maximum value that occurs during simulations, related usually to 150 ns or 200 ns. In this way, the M x ( t o t − n o r m ) component values fall in the range of < − 1 ; + 1 > . Next, for the assumed threshold value M t h , a digitization operation is performed; i.e. the transformation from M x ( t o t − n o r m ) into M x ( t o t − d i g ) , namely\n (2) M x ( t o t − d i g ) = { 1 i f M x ( t o t − d i g ) ≥ M t h 0 i f M x ( t o t − d i g ) < M t h . Since M x ( t o t − n o r m ) ∈ < − 1 ; + 1 > , we tested M t h ∈ < − 1 ; + 1 > with a resolution of 0.1 . The steps of data preparation are shown in Figure 2 for w A = w C = 0.45 , w B = 0.1 , M t h = 0.7 , and RL combination of rotating fields. Firstly, Figure 2 a–c shows the single outputs. It is visible here that outputs A and C show very fast oscillations between maximum and minimum x components, i.e., fast-moving and oscillating domain walls (cf. domain walls near output A in Figure 1 ). Output B behaves differently, as also visible in Figure 1 . Here, the interaction between the left and the right semi-circle results in a more stable situation, with some “spikes” visible when domain walls move through this output. It should be mentioned that these spikes are not directly suitable to be used as logic results, as in spin-torque oscillators used to prepare neural circuits [ 47 ]. Instead, weighted sums of the three outputs are applied here to allow for differentiation between the input combinations LL, LR, RL and RR. In all three outputs, it is visible that the signal starts only at approx. 20–25 ns. This time gap between the onset of the rotating input fields and the onset of receiving an output value is correlated with the velocity of the domain walls moving through the nanostrips (cf. Figure 1 ). After this time gap, the initial starting configuration is overwritten with the introduced signals. Due to the spatial symmetry of our fiber-based neuron, for all simulated cases we assume w A = w c ≠ w B , while w A + w B + w C = 1 . As an example of calculation results, we present in Table 1 the case RL along with w A = w c = 0.45 , w B = 0.1 ; w A = w c = 0.40 , w B = 0.2 ; or w A = w c = 0.35 , w B = 0.3 , for several representative threshold values of M t h . Here, the influence of the threshold values is obvious. Smaller values of M t h are easier overcome by M x ( t o t − d i g ) , so that smaller threshold values will lead to more positions being 1 than 0 and vice versa. In this way, it is possible to define the output with the desired probability. This, on the other hand, is the basis for the common process of adding up signals. For this, it is necessary that not only “learning” is realized, but also “forgetting”; i.e., if a certain stimulus value (here named in this way to avoid confusion with the threshold values defined before) is not reached after a certain time, the sum of the signals is set back to its original value (here zero) and summing up starts again [ 48 ]. This process can be realized, e.g., by a mono-domain magnetic tunnel junction in which the input stimuli frequency must be high enough to allow for crossing the energy barrier that separates two stable states [ 49 ]. Quite similarly, here it is possible to define a certain time (i.e., number of simulation steps) after which a stimulus value must be crossed; else the state reached is “forgotten” and the process starts again from zero. Figure 3 depicts the corresponding summing mechanism of two examples using the weights w A = w c = 0.35 , w B = 0.3 and thresholds of 0.4 or 0.2, respectively. The first 25 ns in which the input signals do not fully influence the output signals are neglected. While Figure 3 a,b show the original signals derived for these thresholds, Figure 3 c,d show the corresponding “learning and forgetting” simulation. Here, each “1” in Figure 3 a,b adds a defined value in Figure 3 c,d (“learning”), while for each “0” in Figure 3 a,b, a value > 0 in Figure 3 c,d is reduced by a defined value (“forgetting”). The leaking rate, defining the “forgetting”, is set to values from 0.1 to 0.3. A value of 0.1, e.g., means that one “learning” step” is “forgotten” after 10 “forgetting” steps, etc. Comparing these exemplary threshold values (cf. Table 1 ), it becomes clear that they should be correlated with different stimulus values, here chosen as 0.5 ( Figure 3 c) or 2.5 ( Figure 3 d), respectively, as marked by the horizontal lines. Comparing “learning” (i.e., ranges of increasing values) and “forgetting” (i.e., ranges of decreasing values), it can be noted that the first shows a certain stochastic behavior, as mentioned before, while “forgetting” is here realized by a simple linear function, as explained in the caption of Figure 3 . This can be modified in a next step to mimic the human brain more adequately; however, this was not within the scope of this project. While until now we concentrated on the input case RL to describe the system and its basic functionality, a discussion of the influence of the input on the weighted output is still necessary. Table 2 , Table 3 and Table 4 depict the values of the digital signals, averaged over the time in which a signal can be measured at the outputs (i.e., after 25 ns, cf. Figure 2 ), derived for the different input cases LL, LR, RL and RR in case of the aforementioned combinations of weights and threshold values. It must be mentioned that these weights are just a few from a broad range of possible combinations. The column RL corresponds to the values depicted in Figure 2 ; Table 2 , Table 3 and Table 4 correspond to the weight combinations in columns 2–4 of Table 1 . In particular, a threshold value of 0 (marked in grey) seems to be suitable to differentiate between different input combinations. It must be mentioned that the differences in the averaged values found for this threshold value (and others) strongly depend on the chosen weights. While for a combination of w A = w c = 0.45 , w B = 0.1 , i.e., the smallest value of w B chosen here, LL and RR give quite similar results for M th = 0, LR and RL differ clearly from each other and from the symmetric cases ( Table 2 ). This is different for the cases in which larger values for w B were chosen ( Table 3 and Table 4 ), where LL and RR give quite different results, while LL and RL ( Table 3 ) or LL and LR ( Table 4 ) give nearly identical averaged values. As this short overview shows, the system suggested here cannot be used to build up a classical binary logic, as it is available in a transistor, etc. Instead, a more complicated logic is enabled with a broad range of possible correlations between inputs and outputs, defined by the combinations of weights of the single outputs. Similar to artificial neural networks, setting these weights will define the results of the performed logic operations, i.e., the correlation between inputs and output. In a full neural network, the averaged values can also be used to define new weights in the next layer. As these examples show, domain wall motion in small nanowire networks can serve to simulate neuronal behavior, including “learning” and “forgetting”."
} | 3,205 |
37405163 | PMC10315665 | pmc | 2,546 | {
"abstract": "Introduction The diversity, nitrogen-fixing capacity and heavy metal tolerance of culturable rhizobia in symbiotic relationship with Pongamia pinnata surviving in vanadium (V) - titanium (Ti) magnetite (VTM) tailings is still unknown, and the rhizobia isolates from the extreme barren VTM tailings contaminated with a variety of metals would provide available rhizobia resources for bioremediation. Methods P. pinnata plants were cultivated in pots containing the VTM tailings until root nodules formed, and then culturable rhizobia were isolated from root nodules. The diversity, nitrogen-fixing capacity and heavy metal tolerance of rhizobia were performed. Results Among 57 rhizobia isolated from these nodules, only twenty strains showed different levels of tolerance to copper (Cu), nickel (Ni), manganese (Mn) and zinc (Zn), especially strains PP1 and PP76 showing high tolerance against these four heavy metals. Based on the phylogenetic analysis of 16S rRNA and four house-keeping genes ( atpD , recA , rpoB , glnII ), twelve isolates were identified as Bradyrhizobium pachyrhizi , four as Ochrobactrum anthropic , three as Rhizobium selenitireducens and one as Rhizobium pisi . Some rhizobia isolates showed a high nitrogen-fixing capacity and promoted P. pinnata growth by increasing nitrogen content by 10%-145% in aboveground plant part and 13%-79% in the root. R. pachyrhizi PP1 showed the strongest capacity of nitrogen fixation, plant growth promotion and resistance to heavy metals, which provided effective rhizobia strains for bioremediation of VTM tailings or other contaminated soils. This study demonstrated that there are at least three genera of culturable rhizobia in symbiosis with P. pinnata in VTM tailings. Discussion Abundant culturable rhizobia with the capacity of nitrogen fixation, plant growth promotion and resistance to heavy metals survived in VTM tailings, indicating more valuable functional microbes could be isolated from extreme soil environments such as VTM tailings.",
"conclusion": "Conclusion The application of the symbiotic remediation systems of rhizobia and leguminous plants is a major research area with a focus on bioremediation of the multiple heavy metal-polluted environments. There are at least three genera of culturable rhizobia in symbiosis with P. pinnata in VTM tailings, namely, Bradyrhizobium , Ochrobactrum , and Rhizobium . Some rhizobia have high N-fixing efficiency, plant growth-promoting capacity, and resistance to heavy metals, indicating there are abundant functional microbial resources in extreme soil environment. Interestingly, the phenotype of strong N-fixing capacity seemed to coincide with the resistance to multiple metal ions, which could explain why Bradyrhizobium was the dominant genus of rhizobia around the P. pinnata rhizosphere in the soil contaminated with heavy metals. Because of resistance to several heavy metals, these isolates were competent candidates for the bioremediation of soils contaminated with multifarious metals. This study did not only reveal the genetic diversity and phylogeny of P. pinnata rhizobia in VTM tailings, but also provided important resources for the development of soil remediation techniques using rhizobium-legume symbiotic systems.",
"introduction": "Introduction Tailings are the part of the product of separation operation in mineral processing that has low content of useful target components and cannot be used for production. Vanadium (V) – titanium (Ti) magnetite (VTM) is a widely distributed mineral ore containing oxides of V, Ti, and Fe, which become large amounts of heavy metal-containing slag after being subjected to the iron and steel smelting process. Because of its physicochemical characteristics, soil often becomes the most direct acceptor of pollutants from the processing of minerals such as titanium and magnetite ( Wang et al., 2018 ; Demková et al., 2019 ). The soil around a mining or smelting area will continuously accumulate the byproducts of the mining processes, resulting in serious heavy metal pollution, e.g., Cd was determined to be the main heavy metal pollutant in the Dahuangshan mining area ( Zeng et al., 2022 ). Heavy metals are not easily degraded and can persist for years in the soil ( He et al., 2019 ). Plants can absorb metal ions through their roots and invertebrates can ingest metal-containing particles so that they enter the food chain where they may be ingested by larger animals or even humans, harming the environment and endangering human health ( Jin et al., 2014 ). Meanwhile, plant extract remediating metal in contaminated environmental has been considered as sustainable and environmentally friendly way ( Upadhyay et al., 2023 ). Therefore, in order to achieve sustainability in the mining industry, one of the most urgent tasks is to concentrate on the reclamation of land contaminated with mine tailings and soil remediation in mining areas. There are some sustainable measures to deal with unavoidable heavy metal and fly-ash pollution, e. g. arsenic contamination in rice agro-ecosystems is migitated by using biochar, organic fertilizers, nanomaterials ( Upadhyay and Edrisi, 2021 ; Upadhyay et al., 2023 ). Some emerging methods such as CRISPR and nanotechnological approaches along with PGPR also can manage degraded soil effectively ( Upadhyay et al., 2022 ). Pongamia pinnata is a deep-rooted Asian tree in the Fabaceae family, which has strong tolerance to salt, drought and heat, and can withstand submersion in fresh water ( Marriboina and Attipalli, 2020 ). The root system of P. pinnata is extensive, and the root nodules are large and numerous with strong nitrogen fixation ability. Kumar et al. (2017) found that P. pinnata increased antioxidant and nutrient accumulation to protect plants under heavy metal stress. P. pinnata can grow well in the soil polluted with heavy metals and already shows good remediation potential ( Yu et al., 2019 ). These characteristics make P. pinnata an excellent pioneer plant for removing heavy metal contaminants from soils ( Marriboina and Attipalli, 2020 ). As an important nutrition of plant growth, nitrogen is supplied through the biological nitrogen fixation by some endophytic diazotrophs of crops or soil microorganism ( Singh et al., 2022 ). Legume-rhizobium symbiotic system shows strong nitrogen fixation capacity and strong resistance to heavy metal through the mutually beneficial relationship between rhizobia and the host plant ( Zhang P. et al., 2019 ). Rhizobia can increase the heavy metal tolerance of a leguminous plant such as alfalfa by sequestering the metals or changing their forms in the soil ( Teng et al., 2011 ). The fixation of nitrogen by rhizobia also improves the plant’s resistance to metal stress by increasing soil fertility ( Fagorzi et al., 2018 ). This is a unique advantage of the joint symbiosis between leguminous plants and rhizobia to mitigate heavy metal pollution in soil. Recent research on different types of legume-rhizobia symbiosis systems has mainly focused on: (1) isolation of heavy metal-tolerant rhizobia and screening for plant growth-promoting traits ( Wani and Khan, 2013 ; Fan et al., 2018 ), (2) mechanisms of resistance of rhizobia to heavy metals ( Adediran et al., 2015 ; Nocelli et al., 2016 ), (3) screening for legumes that are tolerant to heavy metals ( Abdelkrim et al., 2018 ), and (4) evaluation of the ecological remediation effect of legume-rhizobia symbiosis systems on heavy metal pollution ( Ju et al., 2015 ; Shen et al., 2019 ). Because the number of symbiotic remediation systems that have been studied and tested is very limited, the diversity of rhizobial populations offers great opportunities for discovering high quality strains that can be used for bioremediation of heavy metal-contaminated soils. However, resources for rhizobia-legume nitrogen fixation systems with high efficiency are still lacking, especially those from some extreme environments. Previous studies have found that there was abundant growth-promoting bacteria, such as rhizobia, in the VTM tailings ( Yu et al., 2014 ). Culturable Bradurhizobium genus aymbiotic with P. pinnata was also isolated from the VTM tailings, and then a aymbiotic bioremedition system of P. pinnata and rhizobia was established for ecological remediation of the VTM tailings ( Yu et al., 2017a ). High-throughput sequencing technology found some other genus of rhizobia in the VTM tailing ( Yu et al., 2019 ). It was hypothesis that there are more genus of rhizobia symbiotic with P. pinnata , and these rhizobia could show high nitrogen-fixing capacity and strong heavy metal tolerance, which would provide more high quality rhizobia resources for bioremediation of the VTM tailings or other heavy metal-contaminated soil. So, this study more comprehensively understand diversity, nitrogen-fixing capacity and heavy metal tolerance of culturable P. pinnata rhizobia in the VTM tailings, providing a basis for the development and utilization of rhizobia.",
"discussion": "Discussion Physicochemical properties and metal contaminations in the VTM tailings The quality of the soil depends on its physicochemical properties. Soil pH affects soil microbial diversity and function ( Blum et al., 2018 ), and the pH of VTM tailings in Sichuan Province, China, was found to be quite acidic, which is similar to the soil near a zinc blende mine north of Spain ( Pérez-Esteban et al., 2014 ). Thus, the microbial communities in the VTM tailings must be more adaptable to an acidic environment. The available N, P, K and organic matter were as low as in other mine tailings (e.g., in Mexico), which indicated that the VTM tailings was not very fertile ( Armienta et al., 2019 ). The Ti concentration was up to 38 times higher than that in the similar soil near a Ti mining site in Kenya, and the V concentration was up to 7.5 times higher than that in Cuban soils on average ( Alfaro et al., 2014 ; Maina et al., 2016 ). The concentration of Fe was approximately 3,000 mg/kg, which is higher than the soil near a steel plant in India ( Kaur et al., 2019 ). Compared to the soil near a coal mine in China with severe Cu, Zn, and Cr pollution, the concentration of these elements in Sichuan VTM tailings was 6.70, 3.69, and 1.54 times higher, respectively; but Pb was lower at 1.45 mg/kg ( Liu et al., 2020 ). The concentration of Mn and Ni in VTM tailings was approximately 5 and 3 times higher, respectively, than in the magnetite tailings after growth of Imperata cylindrica ( Yuan et al., 2018 ). The Cd concentration already exceeded the minimum inhibitory concentration for plant growth ( Zhang F. et al., 2019 ). Consequently, the reason why plants grown in the VTM soil were infertile may be due to the high heavy metal contents and low available N, P, K, and organic matter. Therefore, when carrying out ecological restoration, attention must be paid to reducing the concentration of heavy metals in the soil and increasing the content of nutrients. Diversity and phylogeny of Pongamia pinnata rhizobia in the VTM tailings As an biofules resource, P. pinnata is a fast-growing leguminous tree with the potential for high oil seed production and can grow on marginal land ( Scott et al., 2008 ). Only two genera of Rhizobium genera including R. pongamiae, R. miluonense , and Bradyrhizobium genera including B. liaoningense, B. elkanii , B. yuanmingense were found to be symbiotic nitrogen fixation with P. pinnata in India and Australia ( Rasul et al., 2012 ; Arpiwi et al., 2013 ; Kesari et al., 2013 ). However, three genera rhizobia of Rhizobium , Bradyrhizobium and Ochrobactrum symbiotic with P. pinnata were isolated from the VTM tailings, which revealed there were abundant rhizobia in the VTM tailings. These rhizobia included B. pachyrhizi , R. nepotum , R. nepotum , and O. lupini , indicating P. pinnata rhizobia isolated from the VTM tailings were different form previous reported others. So, the VTM tailings was a resource pool including abundant functional microbiology. Although it was the first time that Ochrobactrum was found to have symbiotic nodulation with P. pinnata , their symbiotic nitrogen fixation efficiency were not high ( Table 2 ). The distribution of the Rhizobium population can easily be changed by the influence of different environmental factors ( Stefan et al., 2018 ). Because of multiple heavy metal pollution and barren environmental factors, rhizobia symbiotic with P. pinnata for the VTM tailings were different from others. The proportion of Bradyrhizobium strains was highest among three rhizobia genera in the VTM tailings, probably because of stronger resistance of Bradyrhizobium to heavy metals. Compared with R. pongamiae VKLR-01 isolated from root nodules of P. pinnata, their genetic similarity is not high ( Kesari et al., 2013 ). Some of the strains had specific genetic traits which helped to enhance their adaptability to the toxic environment of heavy metal ions. These special rhizobia from the VTM tailings also proved that microbial composition in a rhizobial system has host-specific and bio-geographical distribution characteristics ( Zhang et al., 2011 ). Heavy metal tolerance of Pongamia pinnata rhizobia in the VTM tailings Although there are multiple heavy metal pollutants and very poor nutrition in there VTM tailings, abundant heavy metal resistant and plant growth promoting bacteria survival in the extreme environment ( Yu et al., 2014 ). Because of the extreme heavy metal environment, P. innata rhizobia from the VTM tailings also showed heavy metal resistance. Environmental factors following the order: soil pH > heavy metals > nitrogen > soil texture had distinct impacts on microbial community ( Deng et al., 2018 ), indicating that heavy metals were very important affection factor for soil microbe. Only 40% rhizobia from the VTM tailings showed tolerance against Cu, Ni, Mn, and Zn. The tolerance concentrations of these metals (except for Mn) for these strains were higher than those in the VTM tailings, and different isolates had different level of tolerance to heavy metals, indicating soil heavy metals are not the only factor affecting strain resistance. Some microbes metabolize and transform heavy metal into a less hazardous form for surviving in such harsh environments, resulting in the formation of heavy-metal-resistant microbes ( Prabhakaran et al., 2016 ), so these microbes had their own unique resistance characteristics. From BOXA1R-PCR fingerprints and Phylogenetic characteristic, rhizobia form the VTM tailings had different genotype, so their tolerance to heavy metals was different, and even some isolates did not showed tolerance to the four tested heavy metals. In other research on rhizobial systems, most bacteria were only tolerant to a single heavy metal and only a few were resistant to multiple types of heavy metals ( Stan et al., 2011 ; Fan et al., 2018 ), which was consistent with the tolerance to heavy metals of rhizobia from the VTM tailings. Bradyrhizobium sp. PP76 was resistant to Ni, Cd, Mn of the four metal ions and Rhizobium sp. PP1 was resistant to Cd and Mn, which makes them the best choices for the establishment of symbiotic systems of leguminous plants and rhizobia in heavy metal-contaminated soils. Nitrogen fixation capacity of Pongamia pinnata rhizobia in the VTM tailings As a typical function of rhizobia, symbiotic nitrogen fixation is relation to nod , nif and fix genes, such as nifH named as dinitrogenase reductase ( Shamseldin, 2013 ). The nifH gene is the biomarker most widely used to study the ecology and evolution of nitrogen-fixing bacteria ( Gaby and Buckley, 2014 ), so the amplified nif H gene was an initial evidence of the nitrogen-fixing capability of the rhizobia isolates from the VTM tailing. From the phylogenetic tree, the nifH genes of three genera rhizobia were also consistent with the 16S rRNA and house-keeping genes with high similarity among the same genus rhizobia. Because symbiotic nitrogen fixation was decided by series of nif genes, e.g., S. meliloti and R. leguminosarum bv. viciae have a restricted set of 9 and 8 nif genes, respectively ( Masson-Boivin et al., 2009 ). Therefore, although the nifH genes of same genus with high genotype similarity was on the same branch, these rhizbia showed different nitrogen fixation and plant growth capacity. These rhizobia had symbiotic N-fixation ability with the leguminous host plant of P. pinnata and were facilitative in promoting plant growth. Almost of rhizobia showed consistence between symbiotic nitrogen fixation and plant biomass, except for Ochrobactrum sp. PP7 and PP14. Although Ochrobactrum sp. PP7 and PP14 showed lowest symbiotic nitrogen fixation efficiency among the eleven isolates, their plant-growth promoting activity was stronger than some Bradyrhizboium sp. isolates, indicating that Ochrobactrum sp. PP7 and PP14 might had some of other plant-growth promoting capacity ( Yu et al., 2017b ). The excessive metal concentrations cause undeniable damage to rhizobia, legumes and their symbiosis to affect efficiency of symbiotic nitrogen fixation ( Zahran, 1999 ; Ahmad et al., 2012 ), which does not hinder that rhizobia increase phytoremediation by nitrogen fixation and production of plant growth-promoting factors and phytohormones ( Pajuelo et al., 2011 ). So, legume–rhizobium symbioses has been considered as a tool for bioremediation of heavy metal polluted soils ( Pajuelo et al., 2011 ). However, rhizobium should have heavy-metal resistance to improve legume–rhizobium symbiosis in bioremediation of heavy metal polluted soil ( El-Tahlawy and Ali, 2021 ). P. pinnata rhizobia from the VTM tailings did not only show nitrogen fixation capacity but also heavy metal tolerances, so these rhizobia can be used to build symbiosis bioremediation system for heavy metals. P. pinnata inoculated with B. liaoningense PZHK1 was proved to show huge potential for phytoremediation of mine tailings, which had applied for soil and ecological remediation at the VTM tailings ( Yu et al., 2017a , 2019 ). These rhizobium isolates with nitrogen fixation capacity and heavy metal resistance provided excellent microbial resources for bioremediation of the VTM tailings to other heavy metal polluted soil."
} | 4,600 |
27222209 | PMC4894961 | pmc | 2,549 | {
"abstract": "Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l −1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l −1 ) and fatty alcohols (1.5 g l −1 ), to our knowledge the highest titres reported in S. cerevisiae . This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.",
"discussion": "Discussion The budding yeast S. cerevisiae is an attractive host for biosynthesis of specific products because of its robustness in industrial harsh conditions and easily transfer to existing bioethanol production plants. In this study, we undertook a major metabolic engineering effort to engineer S. cerevisiae for high-level production of FFAs and then their further transformation into alkanes and fatty alcohols. We demonstrated for the first time the significant conversion of FFAs to alkanes and fatty alcohols in yeast, and we also showed that this FFA dependent pathway is far more efficient than the earlier reported route from fatty acyl-CoA ( Fig. 3b and Supplementary Fig. 8 ). The production of alkanes and fatty alcohols benefited from our effort to streamline the fatty acid overproduction by taking the advantage of high cellular FFA levels (>200-fold higher than fatty acyl-CoA). Oleaginous yeasts have been engineered for high-level production of neutral lipids such as triacylglycerol 36 37 , an ideal feedstock for biodiesel production through transesterification. However, the intracellular accumulation requires very high cell density fermentation and also makes it challenging to recover the products 38 . FFAs are another ideal feedstocks for deoxygenated production of renewable hydrocarbon-based biofuels that are entirely fungible with fossil fuels 39 . More importantly, FFAs can be secreted ( Supplementary Fig. 4c ), which is beneficial for high-level production by decoupling it from the cell growth ( Fig. 2b ). Aiming to overproduce FFAs, several researchers disrupted FFA activation and enhanced FFA biosynthesis, for example, through expression of different thioesterases, which enabled FFA production at 0.1–0.5 g l −1 in minimal media in shake flask cultures ( Table 1 ) 14 40 41 . More recently, disruption of FFA activation and neutral lipid recycle enabled production of 2.2 g l −1 in complex (YPD) medium 21 . However, due to its high costs, complex makeup and variable composition, YPD medium would not be suitable for industrial production. Furthermore, the final engineered strain had a 20% lower biomass level in YPD medium, which indicated that the combination of disrupting FFA activation and neutral lipid recycle was harmful to the cell, and might retard growth further in minimal media with lower and less diverse nutrient availability. In this study, we systematically optimized the primary metabolism by disrupting FFA activation, constructing a more efficient fatty acid synthesis system and a chimeric citrate lyase cycle for enhanced precursor supply. More importantly, we are the first to construct a plasmid-free FFA overproducing strain by integration of all pathway components into the genome, which is important for application in industrial processes. These strategies enabled high-level FFA production in yeast under shake flask with minimal media ( Fig. 2a ) without a decrease in the biomass yield ( Supplementary Fig. 4a ). Fed-batch cultivation not only led to accumulation of a high FFA titre (10.4 g l −1 ), but also a high biomass titre of 48 g l −1 , which is at the same level as a wild-type CEN.PK strain in fed-batch cultivation 42 . Before our study, the highest FFA titre (8.6 g l −1 ) was reached by an engineered E. coli in fed-batch culture 29 . This is the first time that S. cerevisiae surpassed E. coli in regards to oleochemical production. It is worthy to mention that the FFA titre is also higher than oleaginous yeast Yarrowia lipolytica of 0.5 g l −1 ( Table 1 ) 43 , which shows the potential of S. cerevisiae for FFA production. Though lower in titre, the alkane production was much higher by using the FFA-based pathway compared with the fatty acyl-CoA-based pathway ( Fig. 3b ). By-product accumulation can hamper metabolic engineering endeavours. Because of the low ADO activity 44 , the alkane titre remained low and fatty alcohols were being produced as major by-products ( Fig. 3c ). To overcome this problem, we first identified Adh5 as a key enzyme for conversion of fatty aldehydes to fatty alcohols by screening a series of ALR/ADH deletion strains. By deleting Adh5, we could significantly improve alkane production. However, their indispensable role in the biosynthesis of essential metabolites makes it impossible to delete all these enzymes. Increased expression of enzymes involved in conversion of fatty aldehydes to alkanes further increased alkane production, pointing to this step as having major flux control. In contrast to alkane production, fatty alcohol biosynthesis relies on efficient reduction of fatty aldehyde ( Fig. 5a ). We therefore took advantage of our screening of different ALR/ADH deletion strains and found that overexpression of ADH5 and deletion of ADH6 could significantly improve fatty alcohol production ( Supplementary Fig. 7 ). Combined with enhanced precursor supply, our final strain produced 1.5 g l −1 fatty alcohols in fed-batch culture, which to our knowledge is the highest reported titre by S. cerevisiae . Current heterologous fatty alcohol biosynthesis pathways in yeast are designed to utilize fatty acyl-CoA as precursor, which enabled producing ∼90 mg l −1 fatty alcohols in shake flasks 14 45 . Recently, increasing acetyl-CoA supply and relieving the inhibition on fatty acyl-CoA biosynthesis, resulted in production of fatty alcohols at 330 mg l −1 in shake flask and 1.1 g l −1 in fed-batch cultivation with high concentrated cells 35 . In that study, concentrated cells were used in fed-batch cultivation, which might result in an overestimated titre since concentrated cells should carry high-level initial cellular fatty alcohols. Moreover, the higher titre compared with our study for shake flask cultures might be attributed to the use of a dodecane overlay, which has been shown to be beneficial for fatty alcohol production 46 . However, a dodecane overlay will result in higher costs for product recovery due the similar boiling points of fatty alcohols and dodecane. Here, our strain produced much more fatty alcohols in fed-batch culture without a dodecane overlay. In the future, identification of fatty alcohol transporters might realize in situ product separation and recovery. In conclusion, we have developed yeast cell factories for the production of FFAs and fatty alcohols, as well as demonstrated the significant production of alkanes in yeast. These strains represent a starting point for establishing yeast-based commercial bioprocesses for the production of oleochemicals and advanced biofuels from renewable resources. Our metabolic engineering strategies of pathway balancing at the fatty aldehyde node not only facilitated the production of fatty aldehyde-derived products but also provide valuable insights for construction of yeast cell factories for production of other valuable aldehyde chemicals, for example, vanillin 47 , because of the similarity of the competition from ALR/ADHs."
} | 1,999 |
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