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30792432 | PMC6385266 | pmc | 7,344 | {
"abstract": "Structural complexity underpins the ecological functioning of coral reefs. However, rising ocean temperatures and associated coral bleaching threaten the structural integrity of these important ecosystems. Despite the increased frequency of coral bleaching events, few studies to date have examined changes in three-dimensional (3D) reef structural complexity following severe bleaching. The influence of local stressors on reef complexity also remains poorly understood. In the wake of the 2015-2016 El Niño-induced mass coral bleaching event, we quantified the effects of severe heat stress on 3D reef structural complexity across a gradient of local human disturbance. Using Structure-from-Motion photogrammetry we created 3D reconstructions of permanent reef plots and observed substantial declines in reef structural complexity, measured as surface rugosity and terrain ruggedness, and a detectable loss of habitat volume one year after the bleaching event. 3D reef complexity also declined with increasing levels of human disturbance, and with decreasing densities of branching and massive corals. These findings improve our understanding of the effects of local and global stressors on the structural foundation of coral reef ecosystems. In the face of accelerating climate change, mitigating local stressors may increase reef structural complexity, thereby heightening reef resilience to future bleaching events.",
"introduction": "Introduction Habitat complexity has long been known to play an important role in structuring natural communities 1 . This is particularly true in highly-complex aquatic habitats such as coral reefs, given the unique physical challenges of the aquatic environment 2 . The physical structure provided by living corals, and the underlying topographic complexity of the substrate itself, play a critical role in the maintenance of biodiversity in coral reef ecosystems 3 , 4 . Complex structure supports high reef fish abundance and diversity 5 – 7 , mediates the effects of competition and predation on coral reefs 8 , facilitates the settlement of reef fish 9 , and may even make reefs more resilient to severe disturbances 10 . However, the increasing frequency and intensity of severe heat stress and associated coral bleaching events 11 threatens the structural integrity of these vital ecosystems 12 , 13 . Coral bleaching occurs when environmental stressors, such as elevated water temperatures 14 , 15 , disrupt the relationship between corals and their endosymbiotic algae (Symbiodiniaceae), resulting in expulsion of the algae from the coral tissue 16 and heightening the chances of coral mortality 17 . In 2015-2016, heat stress associated with an extreme El Niño triggered the third major global coral bleaching event 18 , resulting in severe coral bleaching and mortality throughout all three tropical ocean basins 12 . Beyond impacts on live coral cover, extreme warming events such as this may also affect the three-dimensional (3D) structure of coral reef ecosystems. Mass coral bleaching can severely reduce reef carbonate budgets 19 , shifting reefs to a state of net erosion and limiting their ability to recover lost structure. This loss is compounded by increases in the abundance of common bioeroding organisms 20 following mass bleaching, and the proliferation of dead coral substrate, which is more easily eroded 21 . Declines in reef structural complexity were reported following the 1998 mass coral bleaching event 3 , 22 , 23 , however these descriptions were primarily qualitative. To date, relatively few studies have quantified changes in 3D reef structural complexity following a severe heat stress event. Quantitative measures of structural complexity on coral reefs have traditionally been made using the ‘chain-and-tape’ method for assessing linear rugosity 24 , however this technique is time-intensive, measures a small spatial area, and suffers from high variation due to the particular placement of the chain on the reef 25 . Recent advances in the quantification of coral reef structural complexity, most notably Structure-from-Motion (SfM) photogrammetry 26 – 28 , enable more precise and robust measures of fine-scale complexity, including a wider array of biologically-relevant structural complexity metrics 25 , 29 . However, the application of SfM photogrammetry to answering questions about reef structural change has so far been relatively limited (but see 30 – 34 ). The few studies that have used SfM to quantify changes in reef complexity following coral bleaching have employed these techniques within only one 31 , 32 to three 30 reef plots, limiting our ability to understand the effects of bleaching on reef structure at a wider scale. Gaining a comprehensive understanding of the effects of bleaching-associated coral mortality on the physical structure of reefs and associated ecological processes will require quantification of these changes across environmental gradients and levels of human impact 35 (e.g. from fishing 36 or nutrient enrichment 37 , 38 , which can influence coral reef resilience and recovery from bleaching), using modern techniques capable of capturing fine-scale structural changes. This study capitalized on a severe pulse heat stress event to examine the effects of bleaching-associated mass coral mortality on 3D reef structural complexity and the influence of underlying local human disturbance on these changes. During the 2015-2016 El Niño, coral reefs around Kiritimati (Republic of Kiribati) in the central equatorial Pacific Ocean were subjected to globally unprecedented levels of heat stress and suffered over 80% loss of coral cover by the end of the bleaching event (Baum, unpublished data). Kiritimati also presents the opportunity to examine the effects of local stressors on coral reef structural complexity: the atoll is characterized by a gradient of local human disturbance with the majority of its population concentrated on the northwest side of the island 39 (Fig. 1 ), resulting in a diverse spectrum of reef states ranging from highly-degraded sites near the villages to near-pristine ones on the eastern side of the island (Fig. 2 ). Within the context of this ecosystem-scale natural experiment, we used photogrammetric techniques to quantify fine-scale reef structural complexity (surface rugosity, terrain ruggedness, curvature, and habitat volume) across the atoll’s disturbance gradient over the course of the bleaching event. Specifically, we aimed to (1) quantify the change in 3D reef structural complexity on Kiritimati resulting from the 2015-2016 El Niño, (2) determine the effect of local anthropogenic stressors on levels of structural complexity and the degree of structural change, and (3) examine the relationship between shifts in benthic composition and changes in coral reef structural complexity. Figure 1 Map of forereef study sites and villages on Kiritimati, Republic of Kiribati. Sites are divided into three levels of local human disturbance, and villages (red circles) are scaled to human population size. Inset shows Kiritimati’s location in the equatorial central Pacific Ocean. Figure 2 Photos of three permanent photoquadrats (PPQs) on Kiritimati, one from each of the low ( a , b ), medium ( c , d ), and high ( e , f ) human disturbance levels. Photos show the reef before (left) and after (right) the 2015-2016 El Niño and mass coral mortality event, and represent approximately 2 m × 2 m sections of each PPQ. In each row, the exact same area of the same PPQ is shown, with ellipses highlighting examples of visible changes in reef structure over time.",
"discussion": "Discussion Given the importance of 3D structural complexity in mediating the responses of reefs to disturbances such as coral bleaching 10 , understanding the drivers of reef complexity will be critical for the preservation of reefs under future scenarios of climate change. Here, we examined three different metrics of coral reef structural complexity (surface rugosity, terrain ruggedness, and absolute curvature) and found that declines in both rugosity and terrain ruggedness were detectable only a year after the 2015-2016 El Niño and mass coral bleaching event, with some PPQs losing close to 1 m 3 of reef substrate (Figs 3 – 5 ). The declines in surface rugosity that we observed corroborate the findings of Burns et al . 31 and Couch et al . 32 , who used SfM techniques to document changes in reef complexity on a smaller number of plots within one year of severe bleaching events in the Hawaiian Islands. Conversely, our results suggest that curvature was not substantially affected by the heat stress event, which is also consistent with the findings of Burns et al . 31 . Additionally, we provide what is, to the best of our knowledge, the first evidence that terrain ruggedness is negatively impacted by heat stress in coral reef ecosystems. Although we did not find strong evidence for an interaction between heat stress and local human disturbance, we did observe a decrease in variance around the mean values of surface rugosity and terrain ruggedness from before to after the heat stress event. This suggests that the declines in reef structure measured here are leading to a homogenization of structural complexity values, with sites across the human disturbance gradient becoming increasingly similar in their levels of structural complexity. Despite the influence of baseline levels of structural complexity on reef resilience and recovery capacity 10 , few studies to date have examined the effects of local anthropogenic stressors on 3D reef structure. Our results suggest that 3D reef complexity declines with increasing levels of local human disturbance. Reefs exposed to intermediate to high levels of local disturbance due to stressors such as fishing, pollution, and coastal infrastructure had substantially lower levels of structural complexity compared to reefs with very little local human disturbance. However, because our human disturbance metric is based on a combination of fishing pressure and proximity to local villages (which may serve as a proxy for stressors such as pollution and sedimentation), we are unable to pinpoint the precise mechanism behind this pattern. Possible drivers include high levels of sedimentation associated with coastal infrastructure that limit coral recruitment 53 , overfishing of important functional groups such as herbivores that help to maintain the reef in a coral-dominated state 54 , and direct physical damage to coral structure by fishing gear 55 or boat anchors. However, more research will be needed in the future to tease apart and test the impacts of each of these factors on reef structural complexity. The abundance of particular coral growth forms (e.g. complex branching corals) is also thought to be an important factor driving levels of 3D structural complexity on coral reefs. Here, we found evidence that levels of reef structural complexity are heavily influenced by the densities of both branching and massive corals (Fig. 4 , Table 1 ). While some previous studies have found negative associations between the abundance of branching corals and structural complexity 56 , we found that branching coral density had a positive impact on all three structural complexity metrics, with this effect being particularly strong for terrain ruggedness. Massive coral density was also positively related to all three metrics, with the strongest effects for terrain ruggedness and curvature. This strong influence of massive coral density on reef structural complexity may seem counterintuitive, given the domed structure of many massive coral genera. However, the massive coral assemblage on Kiritimati includes corals with a variety of finer-scale morphologies that may contribute to increased structural complexity. For example, certain submassive coral species such as Favites pentagona and Goniastrea stelligera can take on columnar forms, while Pavona duerdeni may produce ridge-like structures. The contribution of massive corals to measures of reef complexity may also depend both on the scale at which complexity is measured and the particular metrics that are quantified. For example, massive corals, with their rounded surfaces, may achieve higher values for metrics based on vector dispersion (e.g. terrain ruggedness) or changes in slope (e.g. curvature) compared to plating corals, which have large planar surfaces. These predictions reflect the findings of previous research on reef structure suggesting that complexity is higher for larger massive and branching coral colonies than for plating colonies 40 , 57 , 58 . The lack of a relationship between plating coral density and reef structural complexity is nevertheless surprising, given the recognized role of tabular corals as “keystone structures” on coral reefs 59 and the high surface-area-to-volume ratio of these species, which would be expected to contribute to high levels of surface rugosity. However, our results may be an artifact of the type of 3D model used in our analysis. While digital elevation models (DEMs) are commonly used in photogrammetry to represent 3D surfaces, including in other coral reef studies 26 , 31 , 32 , 57 , DEMs are projected from one planar angle and thus are not truly three-dimensional, since they cannot model multiple z points at a single ( x,y ) coordinate 60 . Given that the structural function of plating corals relies largely on the existence of sheltered spaces beneath the coral plates 61 , analyzing reef structure from 2.5D DEM projections may underestimate the complexity of foliose and tabulate coral morphologies, compared to other coral growth forms 40 . These effects may be especially severe in areas where plating corals form complex, multi-tiered structures. As such, we suggest that future studies work towards developing methods to extract complexity data from true 3D digital surface models. The strong dependence of 3D reef structural complexity on the presence of branching corals has implications for the maintenance of reef structure under future climate change. Branching corals, such as acroporids and pocilloporids, are highly susceptible to heat stress and bleaching-associated mortality 62 as well as physical damage from storms 63 . Based on our PPQ benthic data, the density of live branching corals on Kiritimati declined by 95% following the mass coral bleaching event. Although many dead branching coral skeletons remain and continue to provide structure to the reef, it is likely that these structures will soon erode, resulting in further declines in reef complexity. While our results, and those of previous studies 64 , suggest that massive corals may play an important structural role on coral reefs, it remains to be seen how the increased dominance of this coral growth form will affect the ecological functioning of the reefs around Kiritimati. Shifts in coral assemblages from complex reef-building species to smaller, weedy coral species have previously been shown to result in substantial declines in coral reef calcification rates and linear rugosity values 65 . Continued monitoring of the reef system will be necessary to determine the magnitude of the shift in benthic composition following the mass coral mortality event, and how this change impacts both the physical structure and ecological function of the reef over the long term. Given the many important ecological processes and ecosystem services facilitated by coral reef structural complexity, the loss of reef structure has negative implications for both marine organisms and human coastal communities. Declines in reef complexity are expected to compromise coral reef fisheries, potentially leading to up to a three-fold decline in fisheries productivity in severe cases 66 . This problem is of particular concern for local communities on small, isolated islands such as Kiritimati, where the majority of people depend on reef fisheries for income and subsistence and opportunities for alternative livelihoods are scarce 39 , 67 . A key question for future research will be to determine how levels of structural decline similar to those observed here, as well as those occurring in subsequent years with further degradation of the reef substrate, impact the structure of reef fish assemblages and populations of other ecologically and socioeconomically-important reef organisms. Prior to the advent of SfM photogrammetry techniques, studies documenting bleaching-induced changes in reef complexity using conventional methods usually took place several years after the end of a severe disturbance event, and documented the extreme and highly-visible collapse of reef structure 3 , 22 , 23 . While it is vital to understand the effects of severe levels of reef degradation on coral reef communities, studying multiple time points along the trajectory of reef degradation will allow us to gain a better understanding of the rates and impacts of fine-scale structural change, and detect threshold levels of structural complexity below which the ecological functioning of the reef is impaired. As such, it is vital that coral reef monitoring programs incorporate modern methods for quantifying structural complexity into their standard reef monitoring protocols. Photogrammetry techniques provide a time- and cost-effective approach that can be used to this end to objectively quantify multiple measures of fine-scale structural complexity on coral reefs. Coral reefs are currently being impacted at multiple scales by a suite of natural and anthropogenic disturbances. In this study we have shown that levels of 3D structural complexity, a vital component of healthy coral reef ecosystems, are impacted by both local and global stressors. Our finding that local human disturbance is a strong predictor of structural complexity suggests that chronic local stressors may have indirect impacts on reef recovery potential through their influence on reef structure. As such, management should focus on mitigating local stressors in order to maintain reefs at ecologically functional levels of structural complexity. However, action at the local level will also need to be accompanied by policy changes at the global scale. Recent research suggests that limiting global warming to 2 °C, the upper limit of the recent Paris Agreement, will still result in annual severe bleaching on the majority of coral reefs within the next few decades 68 . Our results provide further evidence for the negative effects of ocean warming on coral reef ecosystems, demonstrating the need for drastic reductions in greenhouse gas emissions and support for the development of sustainable low-carbon infrastructure."
} | 4,680 |
34956738 | null | s2 | 7,345 | {
"abstract": "The dynamics of small particles, both living such as swimming bacteria and inanimate, such as colloidal spheres, has fascinated scientists for centuries. If one could learn how to control and streamline their chaotic motion, that would open technological opportunities in the transformation of stored or environmental energy into systematic motion, with applications in micro-robotics, transport of matter, guided morphogenesis. This review presents an approach to command microscale dynamics by replacing an isotropic medium with a liquid crystal. Orientational order and associated properties, such as elasticity, surface anchoring, and bulk anisotropy, enable new dynamic effects, ranging from the appearance and propagation of particle-like solitary waves to self-locomotion of an active droplet. By using photoalignment, the liquid crystal can be patterned into predesigned structures. In the presence of the electric field, these patterns enable the transport of solid and fluid particles through nonlinear electrokinetics rooted in anisotropy of conductivity and permittivity. Director patterns command the dynamics of swimming bacteria, guiding their trajectories, polarity of swimming, and distribution in space. This guidance is of a higher level of complexity than a simple following of the director by rod-like microorganisms. Namely, the director gradients mediate hydrodynamic interactions of bacteria to produce an active force and collective polar modes of swimming. The patterned director could also be engraved in a liquid crystal elastomer. When an elastomer coating is activated by heat or light, these patterns produce a deterministic surface topography. The director gradients define an activation force that shapes the elastomer in a manner similar to the active stresses triggering flows in active nematics. The patterned elastomer substrates could be used to define the orientation of cells in living tissues. The liquid-crystal guidance holds a major promise in achieving the goal of commanding microscale active flows."
} | 511 |
34135383 | PMC8209060 | pmc | 7,346 | {
"abstract": "Current use of mineral nitrogen (N) fertilizers is unsustainable because of its high fossil energy requirements and a considerable enrichment of the biosphere with reactive N. Biological nitrogen fixation (BNF) from leguminous crops is the most important renewable primary N source, especially in organic farming. However, it remains unclear to which degree BNF can sustainably replace mineral N, overcome the organic to conventional (O:C) yield gap and contribute to food security. Using an agronomic modelling approach, we show that in high-yielding areas farming systems exclusively based on BNF are unlikely to sustainably reach yield levels of mineral-N based systems. For a high reference wheat yield (7.5 t ha −1 ) and a realistic proportion of fodder legumes in the rotation (33%) even optimistic levels of BNF (282 kg N ha −1 ), resulted in an O:C ratio far below parity (0.62). Various constraints limit the agricultural use of BNF, such as arable land available for legumes and highly variable performance under on-farm conditions. Reducing the O:C yield gap through legumes will require BNF performance to be increased and N losses to be minimised, yet our results show that limits to the productivity of legume-based farming systems will still remain inevitable.",
"conclusion": "Conclusions In many cases, sustainable OF systems will have lower attainable yields than current conventional systems, even with good management practices. According to our modelling, average O:C values for cereals cited in the literature are likely to overestimate the sustainably maintainable productivity of OF. In return, OF produces a higher process quality for environmental impact categories such as biodiversity 52 – 54 , animal welfare 55 and water nitrate contamination 56 especially in regions with intensive farming. Permanent and intensive use of key inputs such as mineral nitrogen fertilizers and herbicides in simplified cropping systems is neither sustainable nor ecologically sound 7 , 57 . Consequently, a more ecological management of agroecosystems is globally required 58 , 59 . Many routine practices in OF could help to increase the resource efficiency and resilience of conventional production systems, including crop diversification using legumes and regular application of farmyard manure. More research is needed to increase the productivity of OF systems and the adoption of BNF. Promising approaches include plant breeding, improved management of biotic and abiotic constraints, reducing N-losses from the systems and recycling nutrients from human waste. However, squeezed between the general limitations to sustainably maintainable organic yields on the one hand, and the environmental unsustainability of mineral N input on the other, approaches to meet the current and future demand for calories and protein are likely to depend on adjusting consuming behaviour with respect to dietary patterns 5 , 60 and food waste as well as reduction of nutrient losses, especially through higher nitrogen use efficiency and improved nutrient recycling.",
"introduction": "Introduction The question whether the global conversion of agriculture to Organic Farming (OF) systems would be able to supply the current and future demand for food and feed is the subject of scientific inquiry, public discussion, and political debate 1 – 3 . A rigorous analysis needs to distinguish between political, socio-economic and agronomic aspects of world food supply, before they are integrated into complex strategies. Although there is some agreement that the current food production level is principally sufficient to feed the approximately 7.8 10 9 people living today, the number of people suffering undernourishment is unacceptably high 4 , 5 . However, the causes for food insecurity are to a large degree independent of farming systems and are mainly due to political and economic reasons 6 , 7 . In contrast, the production of calories and proteins needed for human nutrition as an agronomic issue mainly depends on the available agricultural area and its productivity, i.e. the yield level, but also on the stability of a cropping system. Any scenario on effects of global conversion to OF on food production needs to be based on a realistic estimation of attainable yields. In this context, the productivity of cereals is of primary importance due to their dominant role for world food supply 8 .\n A recent meta-analysis compared conventional and organic cereal yields of 156 data sets. Cereal yields in organic systems were on average 78% of those obtained in conventional systems 9 . Yield differences showed a wide range, e.g. 40–130% for wheat (average = 73%). Two further meta-analysis confirmed these estimations (Fig. 1 ). Across 161 comparisons, organic cereal yields were 26% lower (40% for wheat) than conventional ones 10 . In another meta-analysis (n = 559) organic cereal yields were 22% lower than under conventional management (37% for wheat) 11 . O:C ratios generally get closer to parity with increasing nitrogen input in organic systems (Fig. 2 ), i.e. when the N-input ratio in both systems is close to 1. From a methodological point of view, however, the O:C ratios cited here are questionable and therefore at least partly inconclusive. Figure 1 The wheat yield gap. Ratio of organic to conventional wheat yields (O:C ratio) from various data sources. Quartiles (box), mean (filled circle), 2%- and 98%-percentile (whiskers); absolute maximum (1.76) and minimum (0.13) not shown; number of observation pairs given at top. The second box shows data compiled by Ponisio et al. 11 that was not included in the meta-analysis by Seufert et al. 10 . Further data not included in either study is shown in the third box. Figure 2 Dependence of the O:C yield ratio on nitrogen input. Based on data compilation by Ponisio et al. 11 ; cases restricted to those where N input was 120–150 kg N ha −1 in conventional system. The two main problems related to the validity of yield comparisons are the determination of an adequate reference system, and the proper selection of representative production techniques. Paired comparisons of individual crop yields are generally only of limited validity if yield only refers to single crops and ignores the crop rotation 12 . Organic cereals grown after a two-year legume ley with additional farmyard manure (FYM) application, for example, may have a similar N supply as a conventional crop 13 . Subsequent organic crops in contrast will have a lower N-supply resulting in decreasing yield levels over the rotation. While organic farmers may then choose less N-demanding crop species later in the rotation, a direct comparison with a much simpler conventional rotation is difficult. Many studies comparing yields from organic and conventional systems, however, have ignored rotational constraints, in particular, in terms of N supply from legumes to cereals. The validity of a study is also limited if the empirical data has been generated using non-representative amounts of nutrient inputs. Unfortunately, the approach of applying high amounts of FYM or other organic inputs to organic crops not generated on the farm (substitution method) is commonly practiced in research 14 , 15 , and this hampers the generalisation of yield comparisons, as organic practice is much more limited in N supply. There is also some evidence that O:C yield gaps calculated on the basis of field experiments are lower than those found under on-farm conditions 16 . For meaningful comparisons, it is also important to distinguish between actual and attainable yields. Comparisons of actual yields, which are determined by biotic stress and limited nutrient and water supply in farmer fields, may sometimes result in low or no organic to conventional yield gaps. They are mainly a function of the specific degree of suboptimal management in the systems under comparison. Under conditions of extensive non-organic agriculture with missing access to agricultural inputs, conversion to OF may therefore lead to equal or even higher actual yields compared with conventional production 3 . From an agronomic perspective, however, comparisons also need to include the attainable yield, where water and or nutrients are non-limiting and biotic stress is controlled 17 . Because of these various shortcomings, a realistic estimate of the potential of organic and other legume-supported cropping systems is currently lacking. We therefore aimed to include aspects of rotation and N balance into an assessment of legume-supported crop productivity. Here we propose a novel deductive method for calculating an upper limit of sustainably achievable O:C ratios for wheat for a typical cropping system in a temperate climate, assuming that nitrogen is the decisive yield limiting factor in organic cereal production. To be sustainable, rotational nitrogen budgets in OF must be balanced, i.e. nitrogen supply from within the system needs to match nitrogen demand of all crops. Thus, our aim is to determine an upper limit of wheat yields that can be maintained sustainably by legume-derived nitrogen. By this it is possible to quantify O:C ratios for a crop species paradigmatic for world food supply and hence suitable for world nutrition scenarios. Because we aim at an upper yield limit we follow the basic principle to assume optimal conditions for the legume-supported organic system. The complex dynamics of nitrogen flows through agroecosystems are therefore simplified in the model. An important component of our approach was to choose some key conditions to ensure that the yield of the legume-supported cereal in the model will always be greater than an actual realistic value. The spatial level of our scenarios is an aggregate of individual farms at the regional level. By taking the availability of arable land into account, other than with many previously published pairwise O:C comparisons, the essential aspect of land use competition is included in our model.",
"discussion": "Discussion To our knowledge, in the literature on organic to conventional (O:C) yield gaps, approaches assessing the yield potential based on nitrogen balances have not been published so far. The approach for calculating O:C ratios presented here is based on the sustainability concept of OF and the assumption that the productivity of cereals in most organic cropping systems is limited by nitrogen availability 13 , 18 , 19 . To ensure short-term productivity and long-term sustainability of OF it is important to maintain soil fertility by achieving a balance between nutrient input and output. Field level nutrient budgets, which consider the differences between total inputs and removals from a field are used to determine nutrient requirements of a single crop. For sustainable yields, however, nutrient management and budgeting need to be understood and planned over rotation cycles 20 . System budgets—with data on nutrient losses and internal flows within a rotation cycle—are more suitable to evaluate the sustainability and productivity of a cropping system. Sustainable yields can only be attained if the nutrient budget, in particular for N, of the system is balanced 21 . Long-term yields of organic non-legume crops are determined by the net nitrogen input across the crop rotation. The only relevant net gain of nitrogen for organic cropping systems is symbiotic BNF provided by legumes. Leaving legume cultivation out of organic crop rotations inevitably leads to soil N-depletion and in the medium-term to decreasing yields. Other nitrogen inputs such as deposition and non-symbiotic BNF only play a minor role and were estimated for our model with 20 and 5 kg N ha −1 a −1 respectively, although some authors consider the latter to be higher 8 . Non-legume crop residues as well as FYM application delineate the temporary field balance’s nutrient turnover, but not the long-term system N-balance. External nitrogen sources, such as a purchased manure only play a role for individual farm nutrient budgets 21 , but are not relevant on a larger scale. In a simplified model, organic yields are therefore determined by the productivity of legume based cropping systems. The performance of BNF is mainly a function of the proportion of legumes in the rotation, legume total dry matter production and the percentage of nitrogen derived from the atmosphere 22 . Net contribution to the soil nitrogen balance further depends on legume use either as green manure, fodder for livestock or cash crop. The net soil nitrogen input of grain legumes used as cash crops is generally low unless the nitrogen harvest index is low 23 . To delineate the upper boundary of O:C it is therefore reasonable to refer to forage legumes known to have the highest BNF performance of legumes 24 . Our model compared a high and medium conventional reference cereal yield level with three scenarios of BNF productivity (282, 183 and 132 kg N ha −1 a −1 ) based on forage legumes. For the assumption of high reference yield levels (7.53 t ha −1 86% dm) we demonstrated that even with high BNF and a P L of 33% the upper boundary of O:C en is 0.62. Higher O:C ratios can only be achieved under conditions that do not seem feasible on a large scale. A recently published yield comparison between conventional and organic systems in the Kenyan highlands, for example, showed comparable corn yields for high input systems 14 . Grain yield of sole cropped organic maize with an N-input of 96 kg ha −1 was on average of two cycles 5.14 t ha −1 versus 4.89 t ha −1 (n.s.) under conventional management. The total annual N input over two seasons and crops (corn and cabbage) amounted 251 kg N ha −1 . Organic inputs were based on composted farm yard manure and both Tithonia mulch and tea taken from hedges and wild collection. Such an approach is feasible for individual organic farms but might reach a limit on larger scale due to resource unavailability. In this context it is interesting to compare the model-derived yield ratio (O:C dm ) (Fig. 4 d), with the empirically derived O:C-ratio for wheat yields in the meta-analyses (Fig. 1 ). When the proportion of legumes was restricted to practically sustainable levels of P L = 0.33, the model showed, under realistic and favourable conditions maximal O:C-ratios of 0.44 and 0.68, respectively. We can also calculate corresponding ‘unprotected’ O:C-ratios that do not consider the availability of land for legumes vs. cereals. These values are 1.22 and 1.52, respectively, and are, as expected, much higher than the overall median of empirical yield O:C-ratios, of 0.59. While many factors may contribute to discrepancies between the different approaches, it is likely that organic wheat in the empirical studies experienced additional yield losses via pests, weeds and diseases which were not included in our model. Simultaneously, it is likely, and confirmed by Fig. 2 , that N inputs in the organic relative to the conventional systems will have played a major role in determining the OC-ratio in the empirical studies. Our results show that the O:C ratio strongly depends of the conventional reference yield level. Lower reference yields mathematically result in narrowing the O:C ratio. However, independent of the cause for low conventional yield levels, the attainable yield O:C gap may remain relatively wide. If the actual conventional reference yield is limited by nitrogen, higher conventional yields could be attained just by N-fertilizer application. If site specific factors, e.g. soil or climatic conditions, especially water availability limit cereal yield, it is likely that correspondingly, legume production and BNF will be low as well, compensating the effect of low conventional reference yields and thereby resulting in relatively low O:C values. Optimistic estimations about the potential of legume supported cropping systems to feed the world 2 assuming maximum O:C ratios of 0.75 appear somewhat doubtful against this background. The O:C ratios suggested here refer to wheat, one of the three cereals predominant for world nutrition. Theoretically, higher maximum O:C ratios could be attained for crop types with a lower N demand (also see supplementary material), and this is supported by yield comparisons 25 ; for rice, which is less N demanding than wheat, using green manure crops or Azolla water fern is known to have a potentially considerable nitrogen fertilizer equivalent 26 , 27 . These approaches have been neglected during the past decades because of the large scale adoption of mineral nitrogen fertilizers. Azolla water fern, for example is estimated to only play a minor role in global paddy rice production 24 . The basic interdependence of those systems with the requirement of balanced N-budgets and the permanent competition for arable land use however remain unaltered, also for crop categories other than cereals such as oil seeds and vegetables. The O:C en ratio of 0.62 calculated for high BNF of 282 kg N ha −1 a −1 , 33% P L and a high reference wheat yield level typical for Western Europe delineates the nitrogen limited theoretical maximum value assuming no losses during the internal nutrient flow. Under on-farm conditions, however, the amount of BNF-N available for non-legumes will often be lower than this level for two main reasons. First, organic forages are generally mixed swards of legumes and grasses for reasons of yield stability and fodder quality which may, depending on the legume proportion in the mixture, result in a reduction of BNF-N input into the soils 22 . Second, and more importantly, legume-based cropping systems are linked with unavoidable N-losses from the system. Recycling of legume nitrogen harvested on the field via animal feeding, subsequent manure production in the stable and final broadcasting on the fields results in N-losses which can range between 17 to 46 kg N cow yr −1 \n 28 . Further nitrogen losses relevant for the system budget may also occur from leaching 29 , 30 . Moreover, the actual performance of BNF is often restricted by abiotic factors such as temperature, water and nutrient supply 31 , in particular phosphorous 32 , 33 , sulphur 34 and molybdenum 35 . Biotic constraints such as abundance and persistence of specific Rhizobium strains in the soil and soil borne diseases such as Sclerotinia trifoliorum may additionally limit rotational BNF performance 36 . While many of these issues may be solved through fertilization, irrigation or microbial inoculation, it is likely that even under ideal legume management, BNF will often remain below the high level assumed in our optimistic scenario. Organic cereal yields not only depend of the rotational BNF input. The actual yield in each field further depends of the temporal N-availability over the season. The primary determinants for N-availability are the net rate of N release from SOM, the contributions from organic and inorganic N-sources such as FYM and losses from the plant available N pool 37 . Favourable environmental conditions and adequate management may easily result in high nitrogen supply and a positive N-balance of an organic field, when combining pre-crop effects of ley with manure application. However, unfavourable environmental conditions such as drought and cold often result in low soil nitrogen mineralization and subsequent lower productivity of organic cereals. Due to the trend of delayed N release from legume residues, cereal N demand in the early season is often not adequately matched 38 . The missing synchrony between crop demand and N supply from the soil tends to result in a lower crop N recovery of organic fertilizers 18 and partly explains the high variation of actual yields in OF. For the ultimately attainable O:C ratio biotic factors reducing cereal yields need to be considered as well. Global estimates for wheat carried out for 19 world regions over a period of three years 2001 to 2003 showed considerable actual yield losses of 28.2% 39 . Pathogens (10.2%) were the main cause for actual yield losses in wheat. The few comparative studies on disease incidence suggest that there is a lower disease pressure in cereals in low-input compared with intensive conventional systems with fungicide use 40 . Site-specific climatic conditions however may also lead to fungal epidemics in organic cereals, e.g. rust diseases, if resistance levels are insufficient, as no curative fungicides are available. In contrast, several effective control options are available for organic pest and weed management. In addition to preventive measures, pests in organic crops can often be controlled by using specific approved pesticides 41 . Weeds, often considered to be a major challenge in organic crop production can be managed with a wide range of indirect and direct control methods, though some are not always economically viable 42 . Apart from the single farm-oriented view on O:C as discussed above, the global dimension of a full conversion scenario needs to be considered as well. Since the end of the Second World War the global use of mineral nitrogen fertilizers has steadily increased. Together with animal manure they constitute the major input of nitrogen in conventional arable land 43 . The total global nitrogen input in agricultural soils managed as both cropland and grassland was estimated to be ~ 249 Tg in the year 2000. At that time, mineral nitrogen already covered one third of the input ~ 83 Tg, and in 2013 already 107 Tg 44 , while the contribution of BNF was only ~ 30 Tg 43 . Only 93 Tg were removed with the harvested crops resulting in a considerable enrichment of the biosphere with reactive N. When assuming that (i) the major part of mineral N fertilizer is applied to arable land, (ii) the current production level of staple crops needs to be at least maintained and (iii) the N use efficiency (43%) remains unaltered, (iv) the average global BNF amounts to 165 kg N ha a −1 \n 24 , the replacement of ~ 83 Tg N would at least require some ~ 500∙10 6 ha of fodder legumes, which corresponds to approximately one third of the current total global arable land area of ~ 1.5 10 9 ha. In countries with a high population density and low availability of arable land, e.g. Bangladesh or Indonesia, this scenario currently seems improbable. Intensive, often unsustainable rice production systems for example in Indonesia with up to three seasons per year produce high amounts of caloric energy in a short period and cannot be based on legumes only, unless accepting a significant reduction of the production, or a global shift in human diets. For this reason mineral N has been considered to be indispensable for middle and low income countries such as India, China and Indonesia, which have achieved staple food self-sufficiency 45 . Some authors claim that human population now exceeds the carrying capacity of agricultural systems that exclusively depend on legumes for N input and that a sizable percentage of the world population depends on mineral N-fertilizers 46 . At the same time, however, caution is due when applying the results from our study across the globe. The data feeding our models was mostly collected from temperate climates and transferability to tropical regions is limited, not only for ecological and agronomic but also for socio-economic reasons. Exclusively legume based cropping systems require time and space for growing fertility building crops and may therefore be best suited for areas where cropland availability per capita is high. Growing more legumes has been repeatedly claimed for crop management schemes that aim at enhancing sustainability and buffering against the dependence of mineral N-fertilizer 46 – 48 . The current political will to significantly reduce greenhouse gas emissions is expected to feed a greater interest to reduce mineral N-fertilizer input by using legumes. Experiments in the USA have shown that including alfalfa in a cereal rotation can help to reduce the N-fertilizer need by 25% 49 . In the tropics BNF can significantly contribute to reduce dependence of mineral N fertilizers, if there is no competition for arable land 50 and soil P-supply not limiting, which, however, is not the case for some parts of the world 51 ."
} | 6,063 |
37258699 | PMC10232518 | pmc | 7,347 | {
"abstract": "We experimentally investigate the role of illumination on the collective dynamics of a large school (ca. 50 individuals) of Hemigrammus rhodostomus . The structure of the group, defined using two order parameters, is quantified while progressively altering the visual range of the fish through controlled cycles of ambient light intensity. We show that, at low light levels, the individuals within the group are unable to form a cohesive group, while at higher illuminance the degree of alignment of the school correlates with the light intensity. When increasing the illuminance, the school structure is successively characterized by a polarized state followed by a highly regular and stable rotational configuration (milling). Our study shows that vision is necessary to achieve cohesive collective motion for free swimming fish schools, while the short-range lateral line sensing is insufficient in this situation. The present experiment therefore provides new insights into the interaction mechanisms that govern the emergence and intensity of collective motion in biological systems.",
"introduction": "Introduction Collective behavior during locomotion is a fascinating phenomenon observed in many living systems, ranging from bacterial colonies 1 , 2 to human crowds 3 , 4 and starling murmurations 5 , 6 . These motions are characterized by synchronized movements on large scales of time and space 7 , emerging from local, short-range interactions between nearest neighbors 8 . Fish provide a typical example of such self-organization, with a natural tendency to form ordered groups, known as swarms or schools 9 . Over 50% of fish species exhibit schooling behavior 10 , which confers benefits such as protection against predators 11 , improved foraging 12 , and reduced cost of locomotion to the group 13 , 14 . From a practical point of view, schooling involves, for each individual in the group, a knowledge of both position in space and kinematics of close neighbors 15 , 16 . In order to get this information, fish rely on vision, sensing of hydrodynamic disturbances and chemo-olfactory cues 17 , 18 . The role of each of these senses is not clearly elucidated today 19 , but it is generally accepted that vision and hydrodynamic sensing are the most predominant 20 , 21 . To sense hydrodynamic disturbances, fish use their lateral line system 22 . This ability has been suggested to be a factor in the formation of fish schools 23 . It is possible to impair the functioning of the lateral line of fish, resulting in a modified schooling behavior 23 – 25 . However, this kind of invasive procedure may alter the behavior of the fish in an unexpected manner. Another way of quantifying the main sensory mechanisms for swimming interaction is to evaluate the role of vision. For instance, the ambient light level can modify the collective response of schooling fish in different situations 26 , 27 . Recently, McKee et al. 28 compared the role of the lateral line and vision in schooling fish. They suggested, based on experiments with 5 fish, that although both lateral line and vision are involved in the interaction between individuals, vision should be sufficient for schooling. Previous studies 20 , 29 have also addressed the problem of vision with larger schools (20–30 fish), and showed that fish wearing opaque eye covers were able to maintain collective motion, using their lateral line system only. However, in these experiments, only one fish was blinded and placed back in a normal school, which limits the conclusions in terms of collective motion. It has been found that fish reduce or completely suppress schooling behavior below a certain light threshold, that can vary across species 30 , 31 . However, these experiments were conducted on 4 to 6 fish and therefore do not provide evidence for specific behaviors that may occur when increasing the number of individuals in the school. Furthermore, the question was tackled in terms of an abrupt limit between a cohesive and a non-cohesive state, without considering the effect of an increase in light level over a wide range once these thresholds are exceeded. In this work, we go further in addressing the role of vision in the formation of large groups of fish, by altering the vision of all individuals at once. For that purpose, we chose to work with a species of highly cohesive fish, Hemigrammus rhodostomus , freely swimming in a large and shallow water tank. The available visual information is gradually altered by modifying the illumination over time, with two cycles of increasing then decreasing ramps. In addition to quantifying the role of vision, our study allows us to evaluate the role of the lateral line in a non-invasive way (typically, the response of fish in an experiment without light informs on their hydrodynamic sensing capabilities). Moreover, the progressive nature of the light variation enables use to fully resolve the transition from non-cohesive to cohesive motion. In contrast with similar previous studies, we worked on schools composed of a large number of individuals (around 50), allowing for a robust statistical analysis of the collective behavior parameters. The evolution of the parameters characterizing the group cohesion clearly shows that the fish group is unable to organize collectively until the light intensity is sufficient for the fish to see each other. This conclusion is supported by a complete description of the transition from disordered to ordered group dynamics as a function of individual visual capacities.",
"discussion": "Discussion The reading of both Figs. 3 and 4 is here straightforward. In the absence of light, or with insufficient lighting, fish are unable to give rise to coherent and cohesive group dynamics. We also observed that above a certain threshold, the properties characterizing the collective dynamics do not statistically change with the degree of light intensity and tend to saturate to a constant value. This remark of course holds for the range of illuminance used for this work ( E ∈ [0, 900] lx) and the global behavior of the group might change with higher values of E . However, the range used in this work corresponds to lighting values in natural habitats for this kind of animals 34 . This sheds light on the recent discussion on the respective roles of vision and lateral line sensing in the appearance of cohesive behaviors. Our observation in the absence of light suggests that lateral line sensing is not sufficient for the group to form a school in free swimming. Moreover, the quality of the visual cue seems to be paired with the capacity of the individuals to achieve collective swimming. It is worth noting again that the conclusions brought with this work are based on a large number of individuals constituting the group. This contrasts with most of past studies 35 – 37 that characterized cohesion and collective dynamics under a changing illuminance using a reduced group of fish (<10 individuals), then mainly focusing on local interactions. Thus, this study constitutes the first experimental work examining vision-based global behavior of a large scale group of fish. In addition, the stable milling motion observed here with sufficient lighting may in fact be induced by the interaction with walls 38 . Figure 4 a shows that the group polarization starts decreasing after exceeding the visual threshold. This decrease is coupled with the amplification of the milling parameter characterizing the group rotation around its center of mass. Thus, considering that fish tend to align with each other as their ability to see other individuals in the group is enhanced by a brighter environment, the milling behavior could be the simple consequence of being aligned in a confined space. Indeed, the alignment can be either quantified by the polarization or milling parameter, both having the same role in that particular geometry: while the polarization quantifies the alignment along lines, the milling parameters can be understood as a measure of an alignment along circles around the center of the group. The effect of confinement on this transition from polarization to milling is the subject of further investigations."
} | 2,045 |
36866038 | PMC9971824 | pmc | 7,348 | {
"abstract": "Summary Animal search movements are typically assumed to be mostly random walks, although non-random elements may be widespread. We tracked ants ( Temnothorax rugatulus ) in a large empty arena, resulting in almost 5 km of trajectories. We tested for meandering by comparing the turn autocorrelations for empirical ant tracks and simulated, realistic Correlated Random Walks. We found that 78% of ants show significant negative autocorrelation around 10 mm (3 body lengths). This means that turns in one direction are likely followed by turns in the opposite direction after this distance. This meandering likely makes the search more efficient, as it allows ants to avoid crossing their own paths while staying close to the nest, avoiding return-travel time. Combining systematic search with stochastic elements may make the strategy less vulnerable to directional inaccuracies. This study is the first to find evidence for efficient search by regular meandering in a freely searching animal.",
"conclusion": "Conclusion and limitations of the study We found that ants meander regularly, cross their own paths less, and disperse from the nest more than expected at random. We assume that ants in the arena are searching for targets, but cannot infer what they are searching for, if at all. Different targets or ‘states’ could explain the relatively large variation in meandering behavior. Nevertheless, the combination of systematic and random search by these ants is an argument against the widely used assumption of animals moving in random-only walks and indicates that the search strategies of many animals are more sophisticated than currently assumed. Our findings show that theoretical work and empirical analyses of animal movement behavior should pay more attention to non-random elements in ants and other species. With more such data, it will be possible to create and test hypotheses about the behavior's efficiency in real-life resource scenarios, its evolution, and generating processes. Incorporating non-random elements in movement models could lead to more accurate models and a richer understanding of these processes.",
"introduction": "Introduction Mobile animals spend much of their life searching for resources, be it for food, water, mates, shelter, or group members. Typically, they need to stay in a particular area (habitat), and often even stay close to a ‘central place’, like a nest, to return quickly to it. If resources are unpredictably distributed, the animal must adopt a search strategy to maximize encounters with such resources. Systematic search patterns like spirals or meanders, where right and left turns of similar sizes alternate, are mathematically optimal in ideal scenarios, because they cover 100% of the area exactly once. However, their efficiency drops below that of random movements in the presence of navigational and locomotory inaccuracies 1 or unknown obstructions, 2 as small local deviations from the pattern can lead to long periods of overlapping search paths and unsearched areas close to the central place. Random movements are robust to these factors but less efficient than non-random strategies in low-noise scenarios. Here, we ask whether and how animals combine systematic and random elements in their search for resources. Most studies of animal movement classically assume that animals move according to a ‘Correlated Random Walk' (CRW). 3 In this framework, the movement path is typically represented by discrete steps between turns (but see 4 ), where two consecutive steps point in similar directions (hence ‘correlated’). 5 Another often used type of random walk is the Lévy Walk, where step lengths are drawn from a power-law distribution and turn angles are uniformly distributed. 6 , 7 A Correlated Random Walk, and by extension animal movements, are typically described by the distribution of turn angles (usually a Wrapped Cauchy Distribution) at a defined step length (or with a defined step length distribution). Similarly, most of the work on the efficiency of search strategies analyzes the properties of different random walks, like turn angle distributions and combinations of different distributions in various resource environments, 8 , 9 rather than how systematic behaviors would change the resource finding efficiency. Only recently have more studies focused on the non-random aspects of animal movement patterns beyond responses to external stimuli like gradients, 10 environmental structures, 11 or encounters with resources or conspecifics. 12 Although theoretical work suggests systematic search behaviors arising from interactions with resources patches, 13 , 14 we still have a limited understanding of systematic movements might be intrinsically generated and combined with random factors to create the search strategy before reacting to extrinsic cues. On one extreme end, some animals like leaf-grazing moths or ancient seafloor dwellers move(d) in completely or mostly systematic, space-filling meanders (see Figure 1 A). Such movement is theoretically one of the most efficient ways of completely covering a given area, 14 but it is rarely performed perfectly in nature. However, elements of systematic movement patterns are present in many other trace fossils, 15 as well as extant species like hungry collembola 16 and roundworms, 17 or different species of amoeba. 18 Spirals as search strategies are often used by animals, but only when their target is known, as in homing isopods 19 and ants 20 , 21 or when returning to the location of a previous food find. 22 We would also expect larger animals to adopt a regular left-right pattern when searching for food, because of its high efficiency. 18 Smooth meandering, as observed in rivers, has been anecdotally observed in different ants, but never confirmed or quantified, 23 , 24 maybe due to the difficulty of accounting for the random elements and the continuous character of the movement. Figure 1 Ant meanders and fossil meanders are detectable through autocorrelation Left column: Cosmorhaphe tremens (fossilized trace, from Sims et al. 25 ), right column: Temnothorax rugatulus ants. Simulations are created by shuffling turn angles (see results : random walk simulations for details). (A and B) Example tracks in black and one simulation of it in red. (C and D) Correlograms of turn angle autocorrelations of the example tracks. An autocorrelation value toward 1 means that angles are always followed by very similar angles in the same direction, a value near −1 means turns in opposite directions, and a value of 0 means equal likelihood of turns in the same or the opposite direction. The x-axis represents the time lags between the two angles being correlated. In (D), there is a negative autocorrelation between 4 and 22 mm, indicating that successive turns into the opposite direction are approx. 4 to 22 mm apart. Phases of significant positive and negative autocorrelation should appear alternatingly at multiples of this timeframe. That this is not the case here is because of sufficient noise in length and amplitude of meanders. (E and F) Turn angle distributions of the tracks. Distributions of empirical and simulated tracks overlap perfectly because turn angles of the empirical tracks are used to create the simulated tracks. Here, we use a Correlated Random Walk derived from empirical data as a null-model to identify meandering as a non-random, systematic movement in an ant likely to be generated without external cues. Specifically, we compare the turn autocorrelations between ant tracks and a realistic Correlated Random Walk. Turn autocorrelations are positive for a track when two turn angles some distance δ away from each other are similar, negative in the case of zig-zagging or meandering, and random movement contains no autocorrelation among turn directions ( Figure 1 ). Turn autocorrelation is not to be confused with the heading angle correlation, also called ‘persistence’, namesake of the Correlated Random Walk. In previous studies, turn autocorrelations were also used to explain anomalous diffusion by explicitly incorporating them into random walk models. 4 , 16 , 17 , 26 , 27 , 28 We use the ant species Temnothorax rugatulus , which has been the subject of studies on collective decision making and recruitment. 29 , 30 , 31 Their colonies are of typical size for ants 31 (50–300 workers) and do not employ group- or mass recruitment, leading to most resources being collected by individually searching foragers. Anecdotal evidence suggests they mostly forage on small living or dead arthropods and opportunistically lick up sugary liquids. 32 , 33 , 34 Individuals can discriminate their own pheromone from that of other ants 35 , 36 , 37 and are not attracted to or follow the trails of nestmates toward food. 35 , 38 Their traits of foraging individually, having a relatively small range compared to other ant species, 32 and small colony size make these ants a good study species to investigate search efficiency of Central Place Foragers. Research on ant search is typically done in contexts where the goal location is already known, such as food baits or the nest entrance. Generally, ant search becomes more dispersed the less ‘confident’ ants are in their knowledge about the location of the goal. 36 , 37 , 38 , 39 , 40 A rare example of a study on search of unknown resource locations shows that Cataglyphis bicolor search is consistent with a correlated random walk with no systematic elements. 41",
"discussion": "Discussion We found evidence that Temnothorax ants systematically meander left and right on a scale of around 5.4 mm (a half ‘wavelength’ of ∼1.5 body lengths). This is the first time such meandering behavior has been shown in ants or in any central place forager. We also find that ant tracks cross themselves less often than random walk tracks, while being as or more dispersive than them. This lends credence to the idea that the meandering seen here is an element of systematic search which allows ants to cover an area more efficiently than pure random walking. How this changes our view on systematic/random search This study adds to the body of literature showing that even relatively ‘simple’ organisms employ more sophisticated movement patterns than simple correlated (or biased) random walks or purely reacting to the environment. 18 , 50 , 51 Instead, the ant species studied here employs a combination of random and systematic elements which are combined in a way to substantially decrease path self-crosses. This finding has implications for both the methods used in trajectory analysis and our conceptual understanding of animal search. Models of animal movement generally assume independence of successive turn angles and process empirical tracks in a way to account for this assumption. However, this processing discards a lot of important and interesting information, which may lead to drawing false conclusions. For example, our results indicate that the non-random component of the movement behavior is important for high search efficiency. Systematic behaviors are probably widespread in animals and including them in process models of animal movement might increase not only the fit of the generated behavior, but also the simplicity of the algorithm. In the example of meandering ants, this could be a formulation of a turn direction oscillator, removing the need to explicitly specify angle distributions. The field of search and area surveying using drones and robots currently mostly use regular patterns. 52 In particular, swarms of such agents could greatly benefit from combining random with systematic elements, solving some of the problems of robustness and adaptability. 53 Turn autocorrelation as a metric Our main finding corroborates the anecdotal reports of a meandering behavior in other ant species 23 , 24 and a desert isopod. 19 Our simple but powerful approach could also be applied to these datasets and give evidence for a meandering behavior in other moving organisms. We analyzed the autocorrelation of turning angles, which is long known to be an important part of movement. However, it requires including data over a large range of scales, 54 which is why turn autocorrelation analysis is still comparatively rare. Here, we collect such a dataset, and use turn autocorrelations to quantify systematic elements and to specifically discover regular meandering during search. Turn autocorrelation analysis can be a useful descriptor of any high frequency sampled movement paths, similar to its use in Gurarie et al., 2017. 4 This measure can add information to other trajectory metrics describing, for example, sinuosity, space use, and behavior changes. 55 Our approach is in stark contrast to most movement analysis, where the turn angle autocorrelations are seen as a nuisance and avoided by track resampling, because these methods assume a pure Correlated RandomWalk 56 , 57 and might give spurious results if this assumption is not met. 58 Other species ‘Zig-zagging’ is a common behavior, observed mostly in insects. It is present, e.g., in flying insects approaching objects, 59 , 60 probably to gain spatial information about them, moths following a pheromone plume, 61 ants following a pheromone trail, 62 , 63 or ants visually navigating toward a goal location. 64 , 65 However, these examples are rather different from searching for resources of unknown locations in a gradient-free environment. In such a scenario, zig-zagging has been suspected in harvester ants but could not be confirmed. 24 Highly regular and tight meandering without a goal is observed, for example, in some worms in sediment (see introduction ), leaf-mining or leaf-grazing insect larvae, 66 and bark beetles. 67 These cases have in common that the foraging space is severely restricted, and the visited surface or substrate is permanently altered. The combination of these factors may increase the value of highly regular patterns for the mover. 14 However, regular movement patterns may be underreported in species which do not leave such conspicuous trails. In fact, meandering elements in search might be widespread, as it can be found across a large portion of the Tree of Life, from slime molds, 18 over worms 25 to ants, despite these organisms having different foraging environments, life histories, and substrate of behavior. This suggests that this behavior either arose multiple times or is very ancient, but in either case does not require a brain. Area coverage efficiency We also showed that ant tracks 1) cross themselves less than random walks, and 2) sometimes disperse more than random walks. The first property indicates that the area coverage efficiency of ant movements is higher than that of a random walk because ants waste less time searching the same places multiple times. Although the higher dispersivity leads to longer return trips once a resource is found, it leads ants more quickly away from areas near the nest, which are probably well-searched on a colony level. However, modeling studies are necessary to show in which resource and cost scenarios this behavior is indeed significantly more efficient. Random search, for example, might be just as efficient when resources replenish or change position quickly or when the probability of detecting resources is low, 68 or when navigational noise is high. 1 Likewise, more dispersive movements tend to improve search in resource sparse or patchy environments, where they are ideally combined with more tortuous local search within patches. 8 , 69 , 70 Systematically testing resource scenarios and specific meander properties, such as amplitude and frequency, will be useful to create a framework of when which pattern is theoretically optimal and generate hypotheses about ant navigation and foraging ecology. A meandering-like pattern can also be found in certain space-filling curves, which are mathematically ideal ways of filling spaces without curve-intersections. 71 This property is important for applications like land surveillance drones, which need to gather information about a given area in a time- and energy efficient manner, similarly to ants. 52 However, the currently employed rigid, a priori path planning is difficult in complex bounding shapes and impossible for unknown environments. A more ant-like area coverage strategy incorporating stochasticity is a possible solution to this problem. Coordination with nestmates Because ants need to coordinate with their nestmates to maximize colony efficiency, it is likely that even solitary ant searchers are influenced by the movements of other ants of their colony. 23 , 72 , 73 , 74 We find that ants in larger colonies and those entering the arena later in the experiment meander less strongly, cross their paths less, and move faster and farther away from the nest. These movement characteristics likely reduce the repeated coverage of the same area by different ants of the same colony and seem to be upregulated with increasing number of ant steps in that area. This could be facilitated by the perception of the concentration of chemical marks on the ground. It is also plausible that properties of the meandering behavior, like turn length and amplitude are modulated by physical interactions with nestmates or perceiving their pheromone traces. Although we are not able to test this hypothesis here, it could be done systematically in a future study by controlling interactions and pheromone deposition. Conclusion and limitations of the study We found that ants meander regularly, cross their own paths less, and disperse from the nest more than expected at random. We assume that ants in the arena are searching for targets, but cannot infer what they are searching for, if at all. Different targets or ‘states’ could explain the relatively large variation in meandering behavior. Nevertheless, the combination of systematic and random search by these ants is an argument against the widely used assumption of animals moving in random-only walks and indicates that the search strategies of many animals are more sophisticated than currently assumed. Our findings show that theoretical work and empirical analyses of animal movement behavior should pay more attention to non-random elements in ants and other species. With more such data, it will be possible to create and test hypotheses about the behavior's efficiency in real-life resource scenarios, its evolution, and generating processes. Incorporating non-random elements in movement models could lead to more accurate models and a richer understanding of these processes."
} | 4,664 |
36417558 | PMC10100090 | pmc | 7,350 | {
"abstract": "Abstract Waste plastics represent major environmental and economic burdens due to their ubiquity, slow breakdown rates, and inadequacy of current recycling routes. Polyethylenes are particularly problematic, because they lack robust recycling approaches despite being the most abundant plastics in use today. We report a novel chemical and biological approach for the rapid conversion of polyethylenes into structurally complex and pharmacologically active compounds. We present conditions for aerobic, catalytic digestion of polyethylenes collected from post‐consumer and oceanic waste streams, creating carboxylic diacids that can then be used as a carbon source by the fungus Aspergillus nidulans . As a proof of principle, we have engineered strains of A. nidulans to synthesize the fungal secondary metabolites asperbenzaldehyde, citreoviridin, and mutilin when grown on these digestion products. This hybrid approach considerably expands the range of products to which polyethylenes can be upcycled.",
"conclusion": "Conclusion We have presented a method to rapidly upcycle post‐consumer polyethylenes into structurally diverse and medically useful SMs. We degrade these polyethylenes using oxidative catalysis to generate a distribution of diacids. These diacids are rapidly isolated and upgraded by engineered strains of A. nidulans to synthesize bioactive SMs. Taken together, this two‐step process dramatically expands the catalogue of products to which polyethylenes can be upcycled to thousands of SMs.",
"introduction": "Introduction Plastic production is currently accelerating at a rate faster than any other material on the planet,[ \n 1 \n , \n 2 \n ] and is estimated to reach a global production volume of 1.1 billion tons annually by 2040. Only 9 % of plastics were recycled as of 2015. \n [3] \n Millions of tons of plastics, in the form of trillions of plastic particles, \n [4] \n leak from waste management systems into the environment, posing increasing threats to our food supply and ecosystems. \n [5] \n Generally, polyesters are frequently recycled (ca. 30 % of polyethylene terephthalate (PET)), unlike polyolefins (ca. 6 % of low‐density polyethylene (LDPE)).[ \n 2 \n , \n 3 \n ] Due to their robust microstructures and excellent physicochemical properties, polyethylenes have been utilized to deliver countless improvements to quality of life and health. Polyethylenes are, thus, likely to remain ubiquitous in society. To minimize the environmental hazards they present, we must reclaim value embedded in these materials by developing viable upcycling approaches for them. The same physicochemical properties that make polyethylenes useful also hinder their degradation and recycling. Further exacerbating this problem are additives that necessarily accompany any post‐consumer waste stream, e.g. colorings and plasticizers. Unlike polyesters and nylons, the chemical methods known to recycle or remanufacture polyethylenes are limited. Some forcing methods (e.g. refluxing nitric acid, deep UV radiation) are known to cleave polyethylenes, the former to give carboxylic acids.[ \n 6 \n , \n 7 \n , \n 8 \n ] This type of strategy was exemplified in an oxidative process in which O 2 and nitric oxide (NO) were shown to cleave polyethylenes to carboxylic acids, nitrates, and other oxygenates at 170 °C and 40 atm with 65 % total yield. \n [9] \n \n Separately, oxidant‐free, catalytic approaches are emerging for polyethylene upcycling, including alkane metathesis, hydrogenolysis, and related pathways to convert polyethylenes to light alkanes.[ \n 10 \n , \n 11 \n ] While these have modest yields and require energy‐intensive conditions, they avoid the potential uncontrolled reactions that can result from heating organics with O 2 . Still, oxidative conditions have the important advantage of tolerance to impurities associated with post‐consumer polymer waste. These impurities, especially salts, are a particular concern in samples recovered from the oceans or recycling centers. Chemical approaches to polyethylene degradation generate a diverse distribution of products because there are no functional handles in their pure hydrocarbon structures to direct a catalyst to where the polymer should be cleaved. We've shown, by contrast, that thermoset epoxies and fiber‐reinforced polymers (FRPs) can be upcycled selectively, owing to the special chemistry of their linking nitrogen atoms.[ \n 12 \n , \n 13 \n , \n 14 \n ] In polyethylenes, the diversity of products arising from cleavage either limits the value of these products or creates a challenge of separating them. Thus, there is growing interest in employing biological systems to break down plastics. Over the past two decades, several groups have made tremendous progress toward the biological upcycling of plastic degradation products. The discovery of enzymes capable of depolymerizing PET has inspired extensive interest in both PET degradation and upcycling.[ \n 15 \n , \n 16 \n , \n 17 \n , \n 18 \n ] Separately, whole‐cell biocatalysts have also been employed to reclaim value embedded in PET. Several groups have demonstrated the microbe‐facilitated generation of polyhydroxyalkanoates (PHAs) or related products from plastic‐derived substrates.[ \n 19 \n , \n 20 \n , \n 21 \n ] Others have shown that PET‐derived PHAs can be converted to both alkenoic acids and hydrocarbon fuels. \n [22] \n One group utilized E. coli to convert PET‐derived terephthalic acid into diverse aromatics, including gallic acid and catechol. \n [23] \n Others have engineered E. coli to upcycle terephthalic acid into vanillin. \n [24] \n \n In contrast to PET, however, fewer biological upcycling approaches have been developed for polyolefins such as LDPE and HDPE. Recently, one group utilized Pseudomonas putida to biologically upcycle HDPE, in addition to other plastics, to PHAs and β‐ketoadipate. \n [25] \n While several groups have investigated the chemical[ \n 26 \n , \n 27 \n , \n 28 \n ] and biological degradation of these polymers,[ \n 29 \n , \n 30 \n , \n 31 \n , \n 32 \n , \n 33 \n ] approaches to biologically valorize these polymers are limited. We aimed to exploit fungi, which produce products worth billions of dollars each year \n [34] \n to biologically upcycle polyethylenes. Their biosynthetic products include medically valuable secondary metabolites (SMs) including antibiotics, the cholesterol‐lowering statins, immunosuppressants, and antifungals. \n [35] \n Because they have been reported to use diacids as carbon sources,[ \n 36 \n , \n 37 \n ] we sought to generate structurally diverse and pharmacologically active SMs directly from polyethylene‐derived substrates. We show here that post‐consumer polyethylenes can be rapidly degraded to generate substrates that are suitable for upgrading by fungal metabolism. As a proof of principle, we demonstrate that these plastic‐derived substrates can be used to produce the diverse SMs asperbenzaldehyde, citreoviridin, and mutilin in useful yields (Scheme 1 ). We also demonstrate robust genetic engineering strategies that permit the expression of biosynthetic gene clusters (BGCs) from many different organisms. Thus, in principle, this method expands the catalogue of products to which polyethylenes can be upcycled to thousands of SMs. Scheme 1 The upcycling of polyethylenes to SMs. Polyethylenes are chemically degraded using metal catalysts and pressurized oxygen to generate a distribution of diacids, which are metabolized by fungi to rapidly produce structurally diverse SMs.",
"discussion": "Results and Discussion Optimization of Polyethylene Digestion By adapting conditions for the conversion of cyclohexane to adipic acid, \n [38] \n we were able to optimize an initial system for polymer cleavage. Using O 2 consumption and 1 H NMR integration for indicative signals as our characterization handles (Figure S1), we eventually found conditions based on cobalt and manganese salts and a phthalamide‐based NO source \n [9] \n that give useful oxidative cleavage results (Table S1). The distribution of α,ω‐diacid products that are produced by the oxidative chemistry was further quantified by GCMS (see Supporting Information, Table S2 and Figures S2–4). We observed that re‐charging our reactor with additional O 2 did not restart the polymer cleavage reaction and hypothesized that N ‐hydroxyphthalimide (NHPI) serves as a source of NO, which is vented from the reactor headspace upon O 2 recharge. We see rapid hydrolysis of NHPI to phthalic acid upon reaction initiation. We further observed that our metallic catalysts lost reactivity in the recharge process (Table S3, entries 4–6), and that a better result was obtained when metal salts were added portion‐wise along with O 2 and NHPI at recharge (compare entries 6–7). Under conditions optimized for full polymer conversion to relatively small diacid products (Figure S5), we observed 86 wt % mass recovery (entry 8) from a 5 g sample of clean polymer. Note that branched products were not tabulated in this yield, because they could not be unambiguously identified by identity to an authentic sample. Further, addition of oxygen to the polymer adds weight, so the molar yield of carbon atoms was 52 % to the named products. Its highly tolerant O 2 ‐based conditions give this method the critical and distinguishing feature of tolerance of post‐consumer wastes. We demonstrate that feature here with four examples (Figure 1 & Table S4). Note that plasticizers and branched fragments from this LDPE film were omitted from the product accounting, although a large portion of these products are likely suitable for fungal digestion. A plastic grocery bag was homogenized into diacids of length C4–C12 with 34 wt % mass recovery, with an additional 2 % of longer diacids (Table S4, entry 1). The balance of material comprises branched diacids derived from polymer branches, which are likely suitable for fungal metabolism. The bag must also contain colorings and plasticizers, which we account as non‐products. A plastic milk jug and laboratory squeeze bottle (entries 2–3) were homogenized, respectively in 63 and 54 wt % mass recovery. These gave a distribution of products generally of higher mass than the grocery bag as shown in Figure 1 B. The higher and lower recoveries are explained by the difference of HDPE versus LDPE: the HDPE milk jug does not have polymer branches that are omitted from the recovery calculation.\n Figure 1 Post‐consumer plastics degraded in this study. A) From left to right: LDPE plastic grocery bag, HDPE milk jug, LDPE laboratory squeeze bottle, Pacific gyre waste collected from Santa Catalina Island, CA. B) The distribution of diacid products after post‐consumer polyethylene waste degradation using our optimized reaction. Fungal Upgrading of Polyethylene Digestion Products Fungi represent attractive candidates for diacid upgrading due to their robust growth capabilities, inexpensive cultivation requirements, engineerable metabolic pathways, and potential to synthesize metabolites with potent and diverse bioactivities. Short chain diacids, however, have been reported to inhibit fungal growth. \n [39] \n We confirmed that C4–C8 (studied individually, Figure S6) were toxic to the model filamentous fungus A. nidulans (strain FGSC A4) even when glucose was present as a carbon source. We found, however, that A. nidulans utilizes C10 and C12 diacids as sole carbon sources (Figure S7) without signs of toxicity. We thus devised a system to separate polyethylene digestion products of ≥10 carbons from those <10 carbons. A series of pH‐controlled liquid‐liquid extractions permitted the rapid separation of C10+ diacids from light diacids and metal salts (Figures S8–9). In a representative example (vide supra), 27 wt % of ocean‐sourced polyethylenes were converted to diacids that were discretely identifiable using authentic standards. It should be noted that light diacids are not waste products. They may be used in large‐market applications such as in the synthesis of PBCx, a biodegradable plastic emerging in agricultural applications. \n [40] \n Our data also suggest that these light diacids possess antifungal properties (Figure S6) that may be exploited. For attempts to produce SMs from polyethylene‐derived diacids, the heavy diacid extract was added to liquid minimal media at a concentration of 10 g L −1 . Liquid cultures were inoculated with fungal strains and incubated for several days (see Supporting Information for a full extraction protocol, culture conditions, and media recipes). SMs were analyzed and quantified from culture extracts via HPLC‐DAD and HPLC‐DAD‐MS. Initial attempts to elicit SM production from various wild‐type fungal strains resulted in only small amounts of SMs as detected via HPLC‐DAD‐MS. We consequently genetically engineered A. nidulans to overexpress SM biosynthetic genes or biosynthetic gene clusters (BGCs) and this proved effective, allowing robust and efficient SM production. In order to determine the versatility of this system, we attempted to engineer fungal strains to produce various SMs using several BGC activation/expression approaches (Table S5). The SM used as a readout for the first of these systems was asperbenzaldehyde, a major polyketide intermediate in asperfuranone biosynthesis. \n [41] \n Asperbenzaldehyde and its derivatives disassemble tau filaments, inhibit lipoxygenases, and inhibit the interactions of the oncogenic RNA‐binding proteins HuR and Musashi‐1 with their target mRNAs.[ \n 42 \n , \n 43 \n , \n 44 \n ] We chose to target a biosynthetic intermediate because it can serve as a discovery platform that can easily be synthetically modified. We developed three strains with different systems for driving asperbenzaldehyde production: LO2955, LO8355, and LO10050. All molecular genetic modifications were executed using previously described fusion PCR‐based construct generation and transformation protocols. \n [45] \n In strain LO2955, the afoD gene was deleted, blocking asperfuranone biosynthesis such that asperbenzaldehyde, its biosynthetic precursor, accumulates. Further, the promoter of the afoA gene that encodes the transcription factor (AfoA) that drives expression of the asperfuranone BGC was replaced with the alcA promoter ( alcA (p)), which is highly inducible with a variety of alcohols and ketones, including methyl ethyl ketone. \n [46] \n To increase expression of AfoA, we next replaced the promoter of the alcR gene with the strong, constitutive gpdA promoter \n [47] \n in LO2955, creating strain LO8355. The alcR sequence encodes a transcription factor that drives expression of alcA . \n [48] \n \n In addition, we developed a new, strong constitutive promoter system that employs a positive feedback loop (Figure 2 ) and incorporated it in strain LO10050. This system requires no induction and should drive strong expression on any carbon source, whereas the AlcA system is repressed by a number of sugars including glucose. The positive feedback system is designed to drive very high levels of transcription. In addition to the new promoter system and the deletion of afoD , LO10050 also carries deletions of the entire sterigmatocystin BGC (genes AN7804‐AN7825) and the emericellamide BGC (genes AN2545‐AN2549). Deletion of these highly expressed BGCs increases the pool of SM precursors, which are then free to feed into asperbenzaldehyde biosynthesis.\n Figure 2 Overview of the novel promoter system driving production of asperbenzaldehyde in strain LO10050. The constitutive promoter gpdA (p) drives expression of afoA , encoding the AfoA transcription factor. AfoA binds to the promoter regions of genes afoG , afoE , and afoC within the asperbenzaldehyde BGC, leading to their expression and subsequent asperbenzaldehyde production. AfoA also binds to the afoE promoter ( afoE (p)) controlling a second copy of afoA inserted elsewhere in the genome, driving additional AfoA production. This results in a positive feedback loop that generates high levels of both AfoA and asperbenzaldehyde. Note that afoD is deleted, halting conversion of asperbenzaldehyde to downstream metabolites. Other genes responsible for conversion of further downstream products to asperfuranone, the final product of the pathway, are not shown. Yields of each strain grown in liquid lactose minimal media (LMM) were quantified via HPLC‐DAD (Figure S10). Each strain gave substantial yields but yields from LO10050 were the highest (4.3 g L −1 from 15 g L −1 lactose, or 29 % mass conversion of lactose to asperbenzaldehyde). We therefore selected this strain to assay for asperbenzaldehyde production on polyethylene digest. To determine the general utility of the system, we also attempted to express the diterpene antibiotic platform mutilin from the basidiomycete Clitopilus passeckerianus and the F1‐ATPase β‐subunit inhibitor citreoviridin from A. terreus var. aureus . \n [49] \n Mutilin is an intermediate in the biosynthetic pathway for pleuromutilin, which binds to the peptidyl transferase center of the bacterial ribosome, thus halting protein synthesis. \n [50] \n Mutilin is therefore an attractive platform for medicinal discovery efforts toward overcoming bacterial antibiotic resistance. Further, basidiomycetes are phylogenetically distant from ascomycetes such as A. nidulans and the ability to produce mutilin would indicate that this system works for BGCs from very diverse fungi. Citreoviridin is a potent mycotoxin that uncompetitively and noncompetitively inhibits ATP hydrolysis and ATP synthesis, respectively, by binding to the β‐subunit of F1‐ATPase. \n [51] \n Compounds in this class of mycotoxins have been investigated for the treatment of cancer. \n [52] \n In total, four genes from A. terreus var. aureus and five genes from C. passeckerianus were transferred into an A. nidulans recipient strain and placed under control of alcA (p) to generate robust producers of citreoviridin and mutilin, respectively. Engineered fungal strains were incubated in liquid minimal media supplemented with 10 g L −1 polyethylene digest extracts (PMM, polyethylene minimal media). Culture media and/or mycelia were extracted with appropriate organic solvents (see Materials and Methods), which were then analyzed via HPLC‐DAD or HPLC‐DAD‐MS (Figure 3 ). Standard curves were generated (Figures S11–13) using purified standards to quantify SM yields in PMM relative to glucose minimal media (GMM) or minimal media controls. SMs were purified from polyethylene digest cultures and confirmed via 1 H NMR (Figures S15–17) and tandem MS (Figures S18–20).\n Figure 3 A) Paired extracted ion chromatograms generated via HPLC‐DAD‐MS. Asperbenzaldehyde production in (I) GMM and (II) PMM; citreoviridin production in (III) GMM and (IV) PMM; mutilin production in (V) GMM and (VI) PMM. Intensities are normalized for metabolites in each condition. B) SM yields produced by engineered fungal strains when grown in PMM and GMM liquid media. Bars represent means±SD ( n =3). *P≤0.05; **P≤0.01; ***P≤0.005. Our results indicate that engineered fungal strains can efficiently produce useful quantities of each target SM in under one week. Interestingly, microscopic examination of LO10050 when cultured in liquid PMM revealed initial stunted growth relative to GMM controls (Figure S21). However, we observed ample hyphal growth after 48 hours and abundant asperbenzaldehyde crystals after 72 hours of incubation in PMM, which is consistent with our findings regarding asperbenzaldehyde titers. These yields are in contrast to other metabolic engineering efforts; while high‐yielding strains have been reported following extensive engineering, \n [53] \n ample SM production typically requires much larger quantities of carbon source(s) to achieve comparable yields.[ \n 54 \n , \n 55 \n , \n 56 \n ] It is also noteworthy that our yields were obtained from shake flasks with minimal optimization. Alteration of other culture parameters known to influence fermentation titers (e.g. culture length, media components, etc.) should permit significantly higher yields. Use of the strong constitutive promoter system may increase production of citreoviridin and mutilin and codon optimization may further increase mutilin production. We further note that it was not necessary to employ metabolic engineering strategies to confer the ability to metabolize polymer‐derived diacids to the fungi; rather, simple extraction protocols selectively isolated diacids suitable for fungal metabolism. Finally, it is quite likely that polyethylene degradation products can be used as a carbon source in the production of other SMs. The BGCs that we have expressed are from diverse fungi and the approaches we have developed should permit the expression of BGCs from many sources. Furthermore, it should be possible to produce other fungal fermentation products such as organic acids and proteins using the same approach. The combination of the catalytic degradation of polyethylenes with genetic engineering of filamentous fungi represents a promising strategy to plastic upcycling."
} | 5,273 |
37799596 | PMC10548237 | pmc | 7,351 | {
"abstract": "Background The ruminant gastrointestinal contains numerous microbiomes that serve a crucial role in sustaining the host’s productivity and health. In recent times, numerous studies have revealed that variations in influencing factors, including the environment, diet, and host, contribute to the shaping of gastrointestinal microbial adaptation to specific states. Therefore, understanding how host and environmental factors affect gastrointestinal microbes will help to improve the sustainability of ruminant production systems. Results Based on a graphical analysis perspective, this study elucidates the microbial topology and robustness of the gastrointestinal of different ruminant species, showing that the microbial network is more resistant to random attacks. The risk of transmission of high-risk metagenome-assembled genome (MAG) was also demonstrated based on a large-scale survey of the distribution of antibiotic resistance genes (ARG) in the microbiota of most types of ecosystems. In addition, an interpretable machine learning framework was developed to study the complex, high-dimensional data of the gastrointestinal microbial genome. The evolution of gastrointestinal microbial adaptations to the environment in ruminants were analyzed and the adaptability changes of microorganisms to different altitudes were identified, including microbial transcriptional repair. Conclusion Our findings indicate that the environment has an impact on the functional features of microbiomes in ruminant. The findings provide a new insight for the future development of microbial resources for the sustainable development in agriculture.",
"conclusion": "5. Conclusion In-depth exploration of ruminant gastrointestinal microbes is necessary to understand the function of the microbiome and its interactions with the host animal. This study enhances our comprehension of both the structure and function of the ruminant gastrointestinal microbiota, a critical aspect for investigating microbial-host symbiotic functional dynamics. Furthermore, it advances our understanding of the gastrointestinal microbiota adaptations necessary for herbivores. In addition, it informs strategies to decrease contamination and increase the robustness and efficiency of ruminants.",
"introduction": "1. Introduction Ruminants, as ancient animals, exhibit a wide range of morphological and ecological diversity ( Mennecart et al., 2021 ). They have adapted to diverse habitats, from tropical jungles ( Díaz-Céspedes et al., 2021 ) to the plateau ( Guo et al., 2020 ); range in size from 2 kg ( Pickford, 2001 ) to 1.5 tons ( Sauer et al., 2016 ); show great variations in diet, feeding on objects ranging from moss ( Ihl and Barboza, 2007 ) to ordinary standard feed ( Li et al., 2019 ); and have adapted to almost all ecosystems on Earth. Ruminants are distinguished by their plant digestion patterns and have evolved the rumen. As one of the most vital organs, the rumen allows partial microbial digestion of feed before it enters the true stomach ( van Lingen et al., 2017 ). The rumen is a crucial factor underlying the domestication of ruminants. The productivity of ruminant livestock depends on their gastrointestinal microbiota, which can transform plant material that humans cannot digest into easily accessible animal products ( Shabat et al., 2016 ). The gastrointestinal microbiota of ruminants is characterized by its diversity and dynamic nature, making it prone to alterations due to changes in diet ( Malmuthuge and Guan, 2016 ), environmental factors ( Cholewinska et al., 2021 ), and the presence of enteric pathogens ( Cortés et al., 2020 ). These perturbations play an integral role in host nutritional intake, behavior, metabolism, immunological function, and development. Natural selection has allowed hosts and symbiotic microbes to evolve as integrated systems. In both the ecological and social realms, ruminants are immensely valuable. Due to rising consumer demand for animal products resulting from population growth ( Seshadri et al., 2018 ), ruminants play an increasingly vital role in ensuring agricultural security. They generate a significant amount of the meat and milk that are the primary sources of protein in the human diet ( Stewart et al., 2019 ). Nonetheless, sustainable manufacturing confronts significant obstacles due to the depletion of natural resources and the resulting rise in production costs. The ability of ruminants to utilize microorganisms is one of their key traits. Microorganisms bring significant benefits to ruminant animals. However, due to the diverse functionalities and species diversity of microorganisms, they exhibit intricate physiological and biochemical characteristics, making their in-depth analysis quite challenging ( Ban and Guan, 2021 ). We have uncovered inconsistencies in predicting microbial community structures, primarily stemming from a limited grasp of the mechanisms governing microbial community assembly. In order to mitigate this unpredictability, it is imperative to comprehensively understand the microbiome as a cohesive entity. With the ongoing increase in the depth of metagenomic sequencing, the range of sequencing is progressively expanding ( Vestergaard et al., 2017 ), while the cost of the technology is decreasing. Thus, large amounts of data can be generated for analysis. Consequently, substantial volumes of data can be generated for analysis. However, despite genomics being inherently data-driven, the resultant datasets are becoming both exceedingly large and complex, thereby giving rise to technological challenges. Recent publication of the most recent collection of gut microbial genomes includes a ruminant whole gastrointestinal tract microbial gene set and the reconstruction of over 10,000 nonredundant ruminant gastrointestinal microbial genomes ( Xie et al., 2021 ). This represents a significant change in the ability to understand the ruminant microbiome. This study used public database collections to characterize the microbiomes and functional groups and applied a metagenomics approach to achieve the following objectives: (1) building microbial cooccurrence networks for exploring linkages in microbial communities, (2) the influence of the network’s special microstructure on the survivability of gastrointestinal networks in ruminant was explored, (3) detecting antibiotic resistance in different ruminants on a large scale, and (4) exploring the adaptation of ruminant microbes to their environment.",
"discussion": "4. Discussion Ruminants are among the most successful herbivorous mammals ( Decker et al., 2009 ). In this study, we took full advantage of the largest and most comprehensive database of ruminant gastrointestinal microbes available. We employed a range of approaches, encompassing complex networks and interpretable machine learning, to characterize the state of environmental microbial populations. In this study, we employ network science theory to examine the properties of gastrointestinal networks and evaluate the robustness of these networks by examining these properties in more detail. Diverse profiles of topological features in diverse environmental networks reveal the unique co-occurrence patterns of microorganisms in ruminant species. The prevalence of cross-feeding relationships in the network may be indicated by the high clustering coefficient, which suggests that these settings have abundant degradation pathways, niche filtering, or environmental unpredictability. Different models built to test the resilience of networks reveal that they are more resilient to random faults and more vulnerable to deliberate attacks. Understanding the resilience of networks and the various approaches to averting catastrophic failures of these networks is essential. Only a tiny number of significant species have been eliminated, which may have an effect on the general structure of the network of microbes in a healthy microbiome. This finding highlights the importance of using antibiotics sparingly once more. By investigating antibiotic resistance genes, we gain insights into how microbial communities respond to antibiotics. This understanding is pivotal, as it unravels the intricate interplay among microorganisms and is fundamental to comprehending the intricate web of interactions that shape ecosystems ( Sharland et al., 2015 ). The presence of antibiotic-resistant microbiomes (ARBs) and ARGs in supermarket meat and dairy products suggests that ARBs/ARGs from ruminants can penetrate the food system. It would be helpful to make an effort to compile a list of significant ARG-carrying species for monitoring and control based on the assessment of the total antibiotic resistance risk at the species level. It is alarming that the farming environment contains high-risk MAGs. Additionally, microbiomes communities frequently experience an increase or decrease in ARGs as a result of genetic changes or HGT. Human health would be seriously endangered by these high-risk MAGs. The Qinghai–Tibet Plateau, sometimes known as the “Third Pole,” is a huge, high-altitude region with a unique and fragile ecological environment ( Yao et al., 2012 ). The region is characterized by a harsh climate of extreme cold, drought, high ultraviolet radiation and a lack of oxygen, making it a challenging living environment for humans and other mammals ( Zhu et al., 2018 ; Pan et al., 2021 ; Shen et al., 2021 ). It is essential to determine precisely which genetic features give ruminants their exceptional digestive capacity and ability to live in harsh conditions ( Zhu et al., 2020 ). To answer this question, we developed interpretable machine learning methods to deeply mine complex, high-dimensional metagenomic data. We found a significant increase in the transcription repair coupling factor (K03723) in the yak gastrointestinal microbiota. K03723 regulates transcriptional processes and recognizes DNA damage. In addition, such phenomena were also found in samples from plateau-based animals for Rhodobacter sp. ( Pérez et al., 2018 ) and nitrogen-fixing microbiomes ( Suyal et al., 2018 ). We hypothesize that this may be a common measure adopted by microorganisms facing extreme environments. In addition, we found that the K03498 trk/ktr system potassium uptake protein contributes significantly to plateau acclimatization. We speculate that the numerous high-salinity sites in the plateau region have resulted in a microbial response to salt stress ( Li et al., 2021 ). Similar to previous studies ( Guo et al., 2021 ),the results indicate a preference for an amino acid metabolism gene (K00817) and polysaccharide and lipid metabolism genes (K05989 and K01181) in the yak gastrointestinal microbiota. These pathways provide additional adaptive responses to the lack of energy intake in yaks. In conclusion, our study provides important insights into ruminant plateau adaptation and highlights the key role of the microbial genome as a “second genome” for adaptation, contributing to a more comprehensive understanding of mammals living in extreme environments."
} | 2,763 |
39704780 | PMC11662053 | pmc | 7,352 | {
"abstract": "Abstract Syngas fermentation to ethanol has reached industrial production. Further improvement of this process would be aided by quantitative understanding of the influence of imposed reaction conditions on the fermentation performance. That requires a reliable model of the microbial kinetics. Data were collected from 37 steady states in chemostats and from many batch experiments that use Clostridium authoethanogenum . Biomass-specific rates from CO conversion experiments were related to each other according to simple reaction stoichiometries and the Pirt equation, with only the ratio of ethanol to acetate production remaining as degree of freedom. No clear dependency of this ratio on dissolved concentrations, such as CO or acetic acid concentration, was found. This is largely caused by the lack of knowledge about the dependency of the CO uptake rate (and hence all other rates) on the CO concentration. This knowledge gap is caused by a lack of dissolved CO measurements. For dissolved H 2 , a similar gap applies. Modelling H 2 consumption adds more degrees of freedom to the system, so that more structured experiments with H 2 is needed. The inhibition of gas consumption by acetate and ethanol is partly known but needs further study. Key points \n • Set of Clostridium autoethanogenum syngas fermentation data from chemostats. \n \n • Unstructured kinetic models can relate most biomass-specific rates to dilution rates. \n \n • Lack of dissolved gas measurements limits deeper understanding. \n Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13364-3.",
"conclusion": "Conclusions A considerable set of experimental C. autoethanogenum fermentation data is available. The chemostat data have been reconciled to improve their consistency. Most published chemostat experiments use CO without H 2 . Biomass-specific rates from these CO experiments are related to each other according to simple reaction stoichiometries and the Pirt equation, with only the ratio of ethanol to acetate production remaining as degree of freedom. No straightforward dependency of this ratio on dissolved concentrations, such as CO or acetic acid concentration, was found. This is largely caused by the lack of knowledge about the dependency of the CO uptake rate (and hence all other rates) on the CO concentration. This knowledge gap is caused by a lack of dissolved CO measurements. For dissolved H 2 , such a lack also applies. H 2 use adds unknown kinetic parameters, so much more experimental data with H 2 will be required. Moreover, more experiments with added products such as acetate and ethanol are needed to determine the influence of these products on the fermentation and its inhibition. Overall, using basic kinetic equations has demonstrated knowledge gaps that cannot be filled by introducing additional equations that describe intracellular kinetics. When sufficient useful experimental data are available, more complex equations than treated here will be needed to describe the experiments. Although the focus of this review has been on C. autoethanogenum , most of the reasoning will be also applicable to gas fermentation by other microbes.",
"introduction": "Introduction Mixtures of CO, H 2 , and CO 2 are collectively called syngas. Traditionally, they are produced from fossil resources or waste-streams. Novel production methods, which are potentially more sustainable, use gasification of biomass or municipal waste and electroreduction of CO 2 and water (Bachmann et al. 2023 ; Detz et al. 2024 ). Conversion of syngas to useful products could contribute to a circular economy. Acetogenic bacteria can anaerobically grow on syngas, leading to interesting metabolic product such as acetate (acetogenesis) and ethanol (solventogenesis). Especially, the gas fermentation model organism Clostridium autoethanogenum stands out as a robust and versatile platform for gas fermentation, being already used in industrial processes for producing ethanol (Liew et al. 2016 ) and being developed for many other products. Minor natural products of C. autoethanogenum are 2,3-butanediol and lactate (Köpke et al. 2014 ), and after metabolic engineering, it has produced other useful compounds such as acetone and isopropanol (Liew et al. 2022 ), ethylene glycol (Dang et al. 2021 ), ethyl acetate (Dykstra et al. 2022 ), and poly-3-hydroxybutyrate (de Souza Pinto Lemgruber et al. 2019 ). Because of increasing commercial and scientific interest, the literature on anaerobic syngas fermentation by C. autoethanogenum and many other acetogenic bacteria is expanding rapidly. Many recent reviews have treated the bacteria, strain engineering, bioenergetics, growth nutrients and inhibitors, fermentation operation, process strategies, and commercialization (Bae et al. 2022 ; Elisiário et al. 2022 ; Fackler et al. 2021 ; Fernández-Blanco et al. 2023 ; Katsyv and Müller 2020 ; Khalid et al. 2024 ; Kim et al. 2023 ; Köpke and Simpson 2020 ; Owoade et al. 2023 ; Stoll et al. 2020 ; Yang et al. 2021 ). However, no systematic attention has been given to the status of knowledge on the microbial kinetics, even though such knowledge is a central aspect in this field. For rational optimization of the design and operation of syngas bioreactors, predictive kinetic models are crucial. These need to be obtained from lab-scale experiments, since industrial experimentation is too expensive. It is known how to combine lab-scale microbial kinetics with computational fluid dynamics to predict industrial-scale performance (Puiman et al. 2024 ), but the models used for lab-scale microbial kinetics still have large degrees of uncertainty, as will be shown in this review. The microbial kinetics depend on factors that have been varied widely, namely all fermentation operation conditions including the used microbial strain. Not all these conditions will be elaborated here. The focus will be on the influence of concentrations of extracellular substrates and products on their rate of consumption and production by wild-type C. autoethanogenum . Deeper knowledge of these kinetics will facilitate the design of syngas fermentation processes for producing acetate and ethanol. To minimize the number of unknown parameters, established kinetic models will be used, which has been useful in many cases (Heijnen and Kleerebezem 2010 ). As we will focus on basic models, intracellular kinetics will not be addressed. These add another layer of complexity and require additional degrees of freedom, but that is especially useful for finding targets for metabolic engineering (van Rosmalen et al. 2022 )."
} | 1,664 |
22927158 | null | s2 | 7,354 | {
"abstract": "Mechanical forces are among important factors that drive cellular function and organization. We present a microfabricated device with on-chip actuation for mechanical testing of single cells. An integrated immersible electrostatic actuator system is demonstrated that applies calibrated forces to cells. We conduct stretching experiments by directly applying forces to epithelial cells adhered to device surfaces functionalized with collagen. We measure mechanical properties including stiffness, hysteresis and visco-elasticity of adherent cells."
} | 136 |
38303509 | PMC11009365 | pmc | 7,357 | {
"abstract": "Symbiotic nitrogen fixation (SNF) facilitated by the interaction between legumes and rhizobia is a well-documented and eco-friendly alternative to chemical nitrogen fertilizers. Host plants obtain fixed nitrogen from rhizobia by providing carbon and mineral nutrients. These mineral nutrients, which are mostly in the form of metal ions, are implicated in various stages of the SNF process. This review describes the functional roles played by metal ions in nodule formation and nitrogen fixation and specifically addresses their transport mechanisms and associated transporters within root nodules. Future research directions and potential strategies for enhancing SNF efficiency are also discussed.",
"conclusion": "Concluding remarks and prospects In a symbiotic relationship, legumes provide rhizobia with photosynthetically derived carbohydrates and all essential minerals in exchange for fixed N. However, excessive fixed N can deplete the plant’s photosynthetic products and mineral nutrients. One model predicted that the N fixation cost was 4.13 g C g −1 N, resulting in a grain yield reduction of 27% compared with non-nodulating plants that obtained N from the soil ( Holland et al., 2023 ). Thus, legumes adjust nodule quantity and SNF efficiency based on soil N levels to maintain nutrient balance ( Udvardi and Day, 1997 ; Kiers et al., 2003 ). For instance, they possess an autoregulation of nodulation system composed of long-distance signals from both shoot and root, which enables the systematic regulation of nodule numbers based on external N availability. During the SNF period, N availability can regulate SNF efficiency by influencing the expression level of leghemoglobin and nitrogenase enzymes. Under N-rich conditions, legumes prioritize soil N uptake, and nodulation and N fixation are suppressed ( Ferguson et al., 2019 ). However, research on how N regulates metal-nutrient balance and transport is limited. It is believed that high N levels can lead to premature nodule senescence, which limits nutrient import into nodules and accelerates nutrient recycling ( Matamoros et al., 1999 ; Van de Velde et al., 2006 ). Nonetheless, no specific signaling pathways that mediate these processes have been identified. The metabolic processes of nodule growth and nutrient utilization are highly coordinated. Optimal C–N exchange efficiency can be achieved only when other mineral nutrients meet the nodules’ ideal conditions. Unlike in non-symbiotic organs, these mineral elements play specialized roles in nodule development. For instance, iron and molybdenum are crucial for nitrogenase synthesis in the N fixation zone ( Rubio and Ludden, 2008 ; Brear et al., 2013 ). Magnesium not only contributes to energy metabolism during SNF but also regulates cell permeability within the nodule’s inner cortex ( Cao et al., 2022 ). However, most of our understanding of metal ions in root nodules still relies on knowledge gleaned from non-legumes like rice and Arabidopsis . Future research should focus more on understanding the synergistic effects of these metals’ unique contributions to SNF. Metal transporters in root nodules are important for maintenance of nutrient balance. These proteins, located on the membranes of vascular cells, infected cells, and symbiosomes, mediate transport across three distinct spatial dimensions ( Table 1 ). The SM has received more attention because of its distinctive localization. One proteomic study identified 197 proteins located on the SM that may be involved in nutrient exchange in soybean nodules ( Clarke et al., 2015 ). A recent study using liquid chromatography–tandem mass spectrometry combined with label-free quantitative proteomic approaches identified 1759 proteins from soybean and 204 proteins from rhizobia on the SM ( Luo et al., 2023 ), suggesting that some of the protein components in the SM may derive from rhizobia. Despite growing interest in the SM, few proteins identified on it have been confirmed to mediate metal transport ( Figure 2 ). Furthermore, several reported transporters on the SM are also located on the plasma membranes of infected cells ( Kryvoruchko et al., 2016 , 2018 ; Ram et al., 2021 ; Wu et al., 2022 ). Typically, the transport of metals into the cytoplasm, either from the apoplast or from the symbiosome, would involve transporters with unidirectional activity localized on both membranes. This is logical for citrate efflux transporters, as citrate is presumably needed in both the apoplast and the symbiosome space to maintain the availability of iron prior to its uptake by rhizobia. However, for metal-uptake transporters, it seems implausible that the same transporter would facilitate metal import into both the cytoplasm and the symbiosome. Future research should therefore focus on understanding the direction and function of SM-localized transporters. Table 1 Plant transporters involved in metal transport in legume nodules. Gene Species Subcellular localization Ion Physiological function References LjKUP L. japonicus Plasma membrane Potassium Plays roles in cell expansion during nodule development and in ion homeostasis during SNF Desbrosses et al. (2004) GmHAK5 G. max Plasma membrane Potassium Plays a vital role in maintaining potassium balance within nodules Liu et al. (2022) MtDMI1 M. truncatula Nuclear envelope Calcium A cation channel necessary for symbiotic calcium oscillations Ané et al. (2004) ; Capoen et al. (2011) MtCNGC15a/b/c M. truncatula Nuclear envelope Calcium Responsible for nuclear calcium ion oscillations in the symbiotic signaling pathway Charpentier et al. (2016) MtMCA8 M. truncatula Nuclear envelope/endoplasmic reticulum Calcium Required for nuclear calcium signaling during symbiotic interactions Capoen et al. (2011) ; Del Cerro et al. (2022) GmMGT4/5 G. max Plasma membrane Magnesium Facilitates magnesium uptake and accumulation within the inner cortical region of the soybean nodule Cao et al. (2022) MtNRAMP1 M. truncatula Plasma membrane Iron Responsible for apoplastic iron uptake from the apoplast into infected cells Tejada-Jiménez et al. (2015) LjMATE1 L. japonicus Plasma membrane Iron Mediates citrate efflux transport, assists in translocation of iron to the apoplast surrounding infected cells, and facilitates iron uptake Takanashi et al. (2013) MtMATE67 M. truncatula Plasma membrane/symbiosome membrane Iron Mediates transport of citrate into the symbiont space, increasing the solubility and availability of ferric iron for rhizobia bacteroids Kryvoruchko et al. (2018) GmYSL7 G. max Symbiosome membrane Iron An oligopeptide transporter and possibly an iron–nicotianamine transporter at the symbiosome membrane that contributes to N fixation and symbiosome development Wu et al. (2022) ; Gavrin et al. (2021) GmVTL1a G. max Symbiosome membrane Iron Mediates transfer of iron from the cytoplasm to the symbiosome Liu et al. (2020) ; Brear et al. (2020) MtVTL4/8 M. truncatula Symbiosome membrane Iron Mediates transfer of iron from the cytoplasm to the symbiosome Walton et al. (2020) LjSEN1 L. japonicus Symbiosome membrane Iron/molybdenum? Belongs to the VIT-like protein family, possibly mediates the transfer of iron and molybdenum from the cytoplasm to the symbiosome Suganuma et al. (2003) ; Hakoyama et al. (2012) ; Chu et al. (2022) MtFPN2 M. truncatula Symbiosome membrane Iron Facilitates iron transfer to N-fixing bacteroids Escudero et al. (2020a) GmDMT1 G. max Symbiosome membrane Iron Transports ferrous iron and maintains iron homeostasis in the soybean nodule Kaiser et al. (2003) MtCOPT1 M. truncatula Plasma membrane Copper Responsible for shuttling copper from the apoplast into nodule cells Senovilla et al. (2018) MtYSL3 M. truncatula Plasma membrane Zinc/iron Transfers iron and zinc from host root tissues to nodule cells Castro-Rodríguez et al. (2020) MtZIP6 M. truncatula Plasma membrane Zinc Responsible for mobilizing zinc from the apoplast to infected cells Abreu et al. (2017) GmZIP1 G. max Symbiosome membrane Zinc Facilitates translocation of zinc to the symbiosome Moreau et al. (2002) MtMTP2 M. truncatula Intracellular compartment Zinc A zinc efflux protein involved in nodule development and SNF León-Mediavilla et al. (2018) MtMOT1.3 M. truncatula Plasma membrane Molybdenum Mediates transport of molybdate into infected cells Tejada-Jiménez et al. (2017) MtMOT1.2 M. truncatula Plasma membrane Molybdenum Mediates molybdate delivery by the vasculature into the nodules Gil-Díez et al. (2019) In addition, attention should be given to metal transporters in uninfected cells within the N-fixation zone, as they make a significant contribution to SNF in the nodule, even if they are not directly involved in this process. Recently developed single-cell transcriptomics approaches may help to identify metal transporters in different cell types, such as infected and uninfected cells ( Wang et al., 2022 ; Liu et al., 2023 ). Transport of metal ions from the soil to the rhizobium is a complex process that involves the coordinated action of multiple transporters. Although our understanding of the roles of these metal elements has been developing, the specific mechanisms of their transport, regulatory networks, and interactions within root nodules remain largely unexplored, revealing a significant gap in our understanding of metal-ion dynamics in the legume–rhizobium symbiosis. This gap represents an opportunity for future research, which could potentially lead to improved SNF efficiency and legume crop yields. Notably, these metal transporters often serve as downstream output elements in signaling regulatory pathways, making them excellent candidates for the manipulation of SNF efficiency in legume root nodules. Indeed, overexpression of certain transporter genes has been shown to enhance nodulation or SNF ( Chen et al., 2019 ; Lu et al., 2020 ; Deng et al., 2022 ). However, the regulatory system within root nodules is a highly intricate and finely tuned mechanism; potential side effects of gene overexpression are yet to be fully understood. Because excessive N fixation is not an optimal growth strategy for legumes, future research should not merely focus on increments in nodule number and SNF capacity but rather aim to find a balanced approach that harmonizes nodule N fixation with soil N fertilization and stress conditions (e.g., darkness, drought, herbicide, and wounding). Such an approach could minimize agricultural input costs and environmental risks.",
"introduction": "Introduction Although nitrogen (N) constitutes 78% of the Earth’s atmosphere, it is predominantly in a gaseous state, rendering it biologically unavailable to most plant species. This biological impasse is overcome in legumes through symbiotic relationships with rhizobial bacteria. These bacteria colonize root nodules, specialized organs formed on legumes, and execute the critical conversion of atmospheric N into bioavailable ammonia ( Sprent and Platzmann, 2001 ; Udvardi and Poole, 2013 ). This mutualistic interaction brings many advantages: it provides host plants with essential nutrients while enhancing the N content of the soil, thus also benefitting the growth of non-N-fixing plants. Moreover, symbiotic N fixation (SNF) of legumes can serve as an eco-friendly substitute for chemical N fertilizers, contributing to lower production costs and diminished environmental risks ( Kebede, 2021 ). This is of heightened importance in developing countries, where cost-effective and environmentally sustainable N sources are pivotal for both sustainable agriculture and food security. Upon infection by rhizobia, root cortical cells of host plants begin to differentiate, leading to formation of a symbiotic organ known as the root nodule ( Roth et al., 1988 ; Vessey, 2003 ; Oldroyd and Dowine, 2008 ). These bacteroids catalyze the reduction of atmospheric N (N 2 ) to ammonia (NH 3 ), supplying host plants with vital N nutrition. In return, the host plants provide rhizobia with carbon (C) sources and other essential mineral nutrients to sustain their growth and metabolic functions ( Udvardi and Poole, 2013 ; Roy et al., 2020 ). Effective nutrient exchange between legumes and rhizobia is crucial for establishing this mutualistic symbiosis. The symbiotic relationship terminates under conditions of high inorganic N concentration or when bacteroids fail to supply N to the host ( Goh et al., 2019 ; Lin et al., 2021 ). Conversely, if the host plant cannot meet the C and mineral nutrient requirements of the bacteroids, SNF is also interrupted ( Krusell et al., 2005 ; Liu et al., 2020 ). These mineral nutrients, which are mostly in the form of metal ions provided by legumes, are implicated in various stages of the SNF process, including signal recognition, rhizobial infection, and nodule formation and development, as well as N 2 reduction. Therefore, legumes strategically modulate the expression of genes related to metal-ion-transport pathways to finely tune the uptake and transport of essential metal ions involved in the SNF process ( Slatni et al., 2012 ; González-Guerrero et al., 2014 ; Chou et al., 2019 ). Uptake of metal elements within root nodules is mediated by two distinct approaches ( Figure 1 C). One is a direct uptake process whereby metal elements are absorbed into the nodule cells through the nodule epidermal and cortical cells ( Slatni et al., 2009 ). Alternatively, the host plant can take up these elements via the root system, subsequently transporting them into the nodule cells through the vascular tissue ( Rodríguez-Haas et al., 2013 ). Once inside the nodule, these metal elements can be transported directly to infected cells through a symplastic pathway or reach the periphery of infected cells via an apoplastic pathway, subsequently entering infected cells through metal transporters located on the plasma membrane ( Figure 1 D). Regardless of the pathway employed, metal ions must ultimately traverse the symbiosome membrane (SM) to enter the symbiosome ( Figure 1 E), the basic N-fixing unit that bacteroids colonize ( Brear et al., 2013 ; Rodríguez-Haas et al., 2013 ; González-Guerrero et al., 2014 ; Roy et al., 2020 ). The SM is initiated from the plasma membrane but may then develop via a vesicular membrane system ( Oldroyd et al., 2011 ; Ivanov et al., 2012 ; Roy et al., 2020 ). It serves as both a physical barrier for nutrient exchange between legumes and rhizobia and a protective shield against immunological attacks from the host plant, thereby playing an essential role in SNF efficiency ( Udvardi and Day, 1997 ; Gage, 2004 ; Udvardi and Poole, 2013 ). Figure 1 Hierarchical structure diagram of the soybean nodule. (A) Soybean seedling. (B) Soybean nodule. (C) Cross section of the soybean nodule. Metal ions can be absorbed into nodule cells through the nodule epidermal and cortical cells (1). Alternatively, the host plant can take up these elements via the root system, subsequently transporting them into nodule cells through the vascular tissue (2). V, vasculature; FZ, fixation zone; C, cortex. The red color indicates the RFP-tagged rhizobia in the fixation zone. (D) Ultrastructure of infected cells. PM, plasma membrane; IC, infected cell. The yellow dotted line indicates the PM of the infected cell. (E) Ultrastructure of the symbiosome. SM, symbiosome membrane; B, bacteroid. The yellow dotted line indicates the SM at the periphery of the bacteroid. Images in (D) and (E) are modified from Liu et al. (2020) . Although a significant amount of research has been directed at understanding the mechanisms behind nutrient exchange in nodules, the processes required for transport of C, N, and mineral nutrients are still not well studied. Several existing reviews have summarized the functions and transport of metal ions in SNF ( Brear et al., 2013 ; Roy et al., 2020 ; González-Guerrero et al., 2023 ). Building on this foundation, the present review provides an update on research progress on various metal nutrients in terms of their functions and transport pathways. Moreover, this review broadens its coverage by providing an in-depth exploration of macronutrients, including potassium, calcium, and magnesium, as well as less-explored micronutrients like nickel and cobalt."
} | 4,071 |
26636956 | null | s2 | 7,358 | {
"abstract": "No abstract available"
} | 5 |
38792135 | PMC11123716 | pmc | 7,359 | {
"abstract": "The hydrolysis and biotransformation of lignocellulose, i.e., biorefinery, can provide human beings with biofuels, bio-based chemicals, and materials, and is an important technology to solve the fossil energy crisis and promote global sustainable development. Biorefinery involves steps such as pretreatment, saccharification, and fermentation, and researchers have developed a variety of biorefinery strategies to optimize the process and reduce process costs in recent years. Lignocellulosic hydrolysates are platforms that connect the saccharification process and downstream fermentation. The hydrolysate composition is closely related to biomass raw materials, the pretreatment process, and the choice of biorefining strategies, and provides not only nutrients but also possible inhibitors for downstream fermentation. In this review, we summarized the effects of each stage of lignocellulosic biorefinery on nutrients and possible inhibitors, analyzed the huge differences in nutrient retention and inhibitor generation among various biorefinery strategies, and emphasized that all steps in lignocellulose biorefinery need to be considered comprehensively to achieve maximum nutrient retention and optimal control of inhibitors at low cost, to provide a reference for the development of biomass energy and chemicals.",
"conclusion": "6. Conclusions and Perspective This article provides an in-depth analysis of how different steps in lignocellulose biotransformation affect the nutrients and inhibitors in lignocellulosic hydrolysates, highlighting the differences in raw material selection, pretreatment methods, and biorefinery strategies in terms of nutrient production and inhibitor control. We found that, although each strategy has its unique advantages, it also faces various challenges such as cost, efficiency, inhibitory product generation, and difficulties in strain development. A vast amount of research focuses on nutrient retention (increasing fermentable sugar yields or conversion rate), the control or removal of inhibitors, and strain development for specific strategies, but there are only limited studies that considered the overall compatibility of steps in the entire lignocellulosic bioconversion process. Our analysis shows that both nutrients and inhibitors can be produced throughout all biorefinery stages, and, at the same time, each subsequent step is closely related to the nutrient retention and inhibitor generation in the previous step. To achieve the techno-economic viability of lignocellulosic biorefining, it is necessary to consider all steps comprehensively, design the most suitable biorefining strategy based on the characteristics of the raw materials and final target products, and optimize the whole process to maximize nutrient retention and conversion and minimize inhibitor generation. Therefore, we propose that future research on lignocellulosic biorefinery should focus more on matching the overall biorefinery strategy and process, especially the compatibility of the pretreatment, enzyme production, and fermentation strain development, to maximize nutrient retention and control inhibitors at a lower cost. For example, pretreatment technologies should be developed based on the requirements of downstream saccharification and fermentation processes. It is necessary to analyze the role of residual chemicals or byproducts during pretreatment more meticulously and thoroughly because they can be inhibitors for subsequent saccharification and fermentation, act as activators for enzyme activity, or provide nutrients for fermentation. For instance, nitrogen sources required for the growth of downstream fermentation microorganisms often need to be added additionally. The ammonium salts, such as ammonia or ammonium sulfite, from nitrogen-containing pretreatment processes, may move into the microbial conversion stage with residual lignin, but their stimulative or inhibitory effects on fermentation still require further studies. Most previous research on nutrient retention, inhibitor control, or removal focused on off-site strategies (SHF and SSF), while very limited studies target more recent on-site strategies (CBP and CBS). Clostridium thermocellum is currently used in on-site strategies, whose core of the saccharification process is their secreted cellulosomes. However, research about the effects of various sugars, residues, and byproducts from pretreatment on the activity and stability of Clostridium thermocellum with their cellulosomes is still very few and should be focused on in the future. For the CBS strategy first developed in our lab, we are committed to the investigation of the overall compatibility of steps in the entire lignocellulosic bioconversion process. For example, the composition of the produced saccharification liquid is complex, and it should be developed in collaboration with downstream processes. The medium used in the CBS process for enzyme production and saccharification contains various nutrients required by Clostridium thermocellum growing, and most of them can remain in the final saccharification liquid in various forms, along with some products generated from Clostridium thermocellum metabolism. More detailed studies need to be carried out about the suitability between these components and downstream fermentation strains, which are ongoing in our lab for the CBS strategy. Moreover, the salts in the CBS hydrolysates come from not only the medium but also ash in the substrate dissolved during pretreatment and saccharification. These salts have potentially significant effects on downstream fermentation equipment and processes. The choice of medium in an on-site saccharification strategy not only determines the production status of the enzyme-producing microorganisms but also affects crucial carbon source supply. Moreover, the inhibitory effects of metabolites and the changes in nutrient components during the enzyme production process of the on-site saccharification strategy also require an overall assessment of their impacts on the performance of production strains. More TEA and LCA studies for different biorefinery approaches, particularly the more recent on-site strategies (CBP and CBS), with improved nutrient retention and inhibitor control are needed to validate their feasibility and sustainability.",
"introduction": "1. Introduction Lignocellulosic biomass (LCB), as one of the most abundant renewable resources in the world, plays an increasingly important role in the circular economy and sustainable development, thus attracting great attention in various research areas. These studies are dedicated to developing techniques in the bioconversion process known as biorefinery which converts lignocellulosic substrates into products such as biofuels, bioplastics, bio-based chemicals, and bio-gases, to substitute non-renewable fossil-based fuels partially or completely [ 1 , 2 , 3 , 4 ]. The biorefinery processes of lignocellulosic biomass can be classified into two groups based on conversion approaches and intermediate products (platform molecules). One is the thermochemical conversion process involving pyrolysis which converts biomass into hydrogen and carbon monoxide (the syngas platform) and downstream chemical conversion or biological fermentation which synthesizes various downstream products [ 5 ]. The other is the biochemical or biological process that converts lignocellulosic biomass into sugars (the sugar platform) for subsequent conversion. Many studies and recent advances in thermochemical conversion processes have been reported and summarized [ 6 , 7 , 8 , 9 ]. In this review article, we will mainly focus on the strategies related to lignocellulosic biorefinery via the sugar platform. Cellulose and hemicellulose are major polysaccharides in lignocellulose. In the process via the sugar platform, these carbohydrate polymers are enzymatically hydrolyzed and release fermentable sugars such as monosaccharides or oligosaccharides, generating lignocellulosic hydrolysate (sugar solution), which can be further utilized by microbes [ 10 ]. Lignocellulosic biomass is a tightly interwoven matrix, and pretreatment is a required step for breaking this highly complex structure with a recalcitrant nature by various chemical, physical, physicochemical, and biological methods [ 11 ]. Then, the saccharification of cellulose and hemicellulose polymers is achieved by enzymatic hydrolysis with cellulases and hemicellulases [ 12 , 13 ]. In addition to cellulose and hemicellulose, lignin is also one major component of lignocellulose, as a network phenolic polymer providing structural reinforcement and resilience [ 14 ]. Moreover, other substances such as small amounts of pectins, cutins, waxes, lipids, tannins, terpenes, alkaloids, and resins are also found in lignocellulosic biomass, and they vary significantly with the species [ 15 , 16 ]. These structural constitutes can be retained in the lignocellulosic hydrolysate with varying degrees in different biorefinery strategies and processes. Most pretreatment processes are accompanied by complex physical and chemical changes, and all lignocellulosic compositions may undergo chemical reactions and produce new compounds that are potentially present in the lignocellulosic hydrolysate [ 17 , 18 , 19 ]. Therefore, the chemical composition of lignocellulosic hydrolysate is often complex and heterogeneous ( Figure 1 ). Lignocellulosic hydrolysate is typically used for further microbial fermentation to produce the final biofuels and bio-based chemicals, but the relatively low price of the end products makes it economically impractical to separate and purify fermentable sugars from lignocellulosic hydrolysate. Therefore, it is crucial that we understand the various chemical compositions (including the nutrients and inhibitors) of the lignocellulosic hydrolysate and their impact on the subsequent fermentation step in depth for the development of economically feasible biorefinery strategies. In the downstream fermentation process of biorefinery, the nutritional requirements for fermenting microbes include carbon sources, nitrogen sources, trace elements, and other essential nutrients. In most studies, lignocellulosic hydrolysates are used as carbon sources, and whether hexoses (such as glucose) or pentoses (such as xylose) in the hydrolysates can be fully utilized by microbes or not will be taken into account [ 20 ] and determine the sugar conversion rate. In fact, due to the complex structural components of lignocellulosic materials themselves and the complexity of the pretreatment and saccharification processes, not only carbon sources but also many other complex components are contained in lignocellulosic hydrolysates. For example, nitrogen sources are necessary for microbial growth and often present in the lignocellulosic hydrolysate. The raw materials themselves, especially those agricultural wastes, naturally contain certain amounts of nitrogen, such as proteins, amino acids, and other nitrogen compounds [ 15 , 16 , 21 ]. These nitrogen compounds may be retained during certain pretreatment and saccharification processes and, ultimately, remain in lignocellulosic hydrolysates. Furthermore, if ammonia or nitrates are added during the pretreatment or saccharification in some process designs, they may also remain in the lignocellulosic hydrolysates with higher concentrations than the amount of nitrogen possibly required for downstream fermentation [ 22 , 23 ]. Other minor but essential nutrients such as phosphorus, calcium, magnesium, and iron also have significant impacts on growth and production, and all their contents in the lignocellulosic hydrolysates depend on the raw materials, pretreatment methods, and saccharification strategies [ 24 ]. Their amounts might directly meet the needs of downstream fermentation production [ 25 , 26 ], be insufficient and require supplementation, or be excessive and have inhibiting effects [ 27 ]. Additionally, in some pretreatment and saccharification strategies, especially those whole-cell-based saccharification processes, amino acids, organic acids, vitamins, and other biostimulants might be produced. Although the content of these substances is low, they may have significant impacts on the downstream fermentation process [ 28 ]. Nutrients and inhibitory compounds in lignocellulosic hydrolysates are highly dependent on the composition of the raw material, and the methods, strategies, and technologies used in specific pretreatment and saccharification processes [ 19 , 29 , 30 , 31 , 32 , 33 , 34 ]. The pretreatment process aims to open the recalcitrant structure of lignocellulose and separate different components as much as possible for subsequent performance [ 35 ]. There are many pretreatment methods, including physical (grinding, microwave, ultrasonic, and pyrolysis), chemical (acid, alkali, ozonolysis, organic solvents, and ionic liquids), physicochemical (hot water, steam explosion, ammonia fiber explosion, wet oxidation, and carbon dioxide blasting), biological, and their combinations [ 36 ]. While the dense structure of lignocellulose is broken, different pretreatment processes may involve some additional chemical reactions because of the chemical reagents added, high temperature, and high pressure, and some byproducts may be inhibitors for the subsequent saccharification or fermentation [ 37 , 38 ]. For example, some lignin-derived phenolics may be released or converted into more toxic forms during the pretreatment and saccharification, disrupting the integrity of the microbial cell membranes, interacting with or changing the structure of the enzyme’s active sites, thereby inhibiting the enzyme activities [ 39 ]. In addition, sugar degradation products (such as furfural) produced during the pretreatment process may directly inhibit enzyme functions and affect the efficiency of the saccharification process [ 32 ]. After pretreatment, biomass materials often undergo the washing step for detoxification, but this will significantly increase the cost of pretreatment and the burden of wastewater treatment, so strategies for biomass pretreatment need to be considered comprehensively by combining subsequent saccharification and fermentation processes. The saccharification process is a key step in biorefinery for producing fermentable sugars. Various saccharification strategies have been developed based on the techno-economic feasibility and difficulty level in implementation. Based on the source of the enzymes used, current saccharification strategies can be divided into off-site and on-site approaches [ 40 ]. In the off-site approach, separately produced cellulases are used to convert pretreated lignocellulose into fermentable sugars by enzymatic hydrolysis, such as simultaneous saccharification and fermentation (SSF) which is currently used in the majority of pilot-scale demonstrations and industrial plants. Only enzymes with buffers are usually added in the off-site saccharification approach, and monosaccharides and oligosaccharides are produced, along with other nutrients and inhibitors generated mainly from raw materials and pretreatment processes [ 41 , 42 ]. In the on-site approach, the enzyme production is integrated with the saccharification process in one system, which can significantly reduce the cost of enzyme production and separation, such as consolidated bioprocessing (CBP) and consolidated bio-saccharification (CBS) [ 40 ]. Since enzymes are produced directly by microorganisms for saccharification in the on-site approach, metabolic products and partial cell lysates from the enzyme-producing microorganisms are often contained in the resulting lignocellulosic hydrolysate. They can serve as nutrients or inhibitors for downstream fermentation. For example, organic acids may be accumulated by metabolic activities of microorganisms during enzyme production, lead to changes in the pH of the medium, or have toxic effects on the microorganisms, thus affecting the growth of microorganisms and the efficiency of the downstream biorefinery process [ 39 , 43 , 44 , 45 , 46 ]. In summary, lignocellulosic hydrolysates contain carbon sources and other nutrients, as well as various inhibitors. The composition and concentration are closely dependent on the lignocellulosic raw material types, pretreatment processes, and saccharification strategies and processes. This article aims to explore the key stages in the biorefinery process of lignocellulose, summarize how various factors in the biomass, pretreatment, saccharification, and fermentation steps affect nutrient supply and inhibitor formation, and offer new insights for designing lignocellulosic biorefinery processes and improving the efficiency and yield in biorefinery."
} | 4,198 |
36641904 | null | s2 | 7,362 | {
"abstract": "Stress response mechanisms can allow bacteria to survive a myriad of challenges, including nutrient changes, antibiotic encounters, and antagonistic interactions with other microbes. Expression of these stress response pathways, in addition to other cell features such as growth rate and metabolic state, can be heterogeneous across cells and over time. Collectively, these single-cell-level phenotypes contribute to an overall population-level response to stress. These include diversifying actions, which can be used to enable bet-hedging, and coordinated actions, such as biofilm production, horizontal gene transfer, and cross-feeding. Here, we highlight recent results and emerging technologies focused on both single-cell and population-level responses to stressors, and we draw connections about the combined impact of these effects on survival of bacterial communities."
} | 219 |
38913407 | PMC11261841 | pmc | 7,365 | {
"abstract": "Abstract Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the Golden Gate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least five members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the uncharacterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.",
"conclusion": "Conclusion In this study, we demonstrate that the synthetic biology tools developed for the yeast Y. lipolytica can be effectively repurposed for genetic engineering of other members of the clades, in particular for Y. oslonensis , Y. deformans , Y. galli , and Y. hollandica for which we were able to express a multi-gene heterologous pathway. Y. phangngensis and C. hispaniensis exhibit a distinct GC content compared to other members of the clades and significant reduction in size for some protein families [ 7 ], indicating a greater sequence divergence. Furthermore, both strains are highly resistant to typical antibiotics, which may suggest lower compatibility, if any, with Y. lipolytica ’s dedicated tools. On the other hand, Y. alimentaria is much closer to Y. lipolytica in terms of GC content, and its protein expression failure could help to determine the homology threshold for compatible expression, especially for promoters, although further investigation is required. While it is widely recognized that the choice of host is crucial in metabolic engineering applications, there is still limited understanding of the strain diversity among non-type strains, particularly for non-conventional microorganisms. Therefore, exploring this aspect of diversity is a critical route toward achieving success in microbial cell factories. By evaluating other relevant traits, we anticipate that the results presented in this study will facilitate new biotechnological applications involving strains within the Yarrowia clade that were previously neglected and not thoroughly characterized.",
"introduction": "Introduction Bio-based production through the microbial fermentation process to replace petroleum-based chemical production is extensively studied as an essential way to develop sustainable and environmentally friendly bioproduction. A key point for microbial cell factory is the choice of the chassis microorganism for its metabolite production and its metabolic rewiring capacity as well as the availability of synthetic biology tools. Yeasts are particularly attractive eukaryotic production hosts due to their capacity to grow on a broad variety of renewable carbon sources, to produce complex target molecules, and their well-established scale-up process. Apart from Saccharomyces cerevisiae , non-conventional yeasts have been investigated due to their particular industrial traits. Among them, Yarrowia lipolytica has been extensively studied as a chassis for many applications from fuels and chemicals to foods. This yeast is genetically amenable, can grow on a large range of substrates, has the GRAS (Generally Regarded as Safe) status, and can produce high yields of various biomolecules, in particular lipids and organic acids [ 1 ]. Target molecules produced by this yeast have been further expanded to plant-based derivative products like terpenes [ 2 ]. This continuously growing interest in using this yeast as an industrial chassis has led to an exponential advancements in the development of synthetic biology tools including fast and modular cloning systems and CRISPR/Cas9 methods [ 3 ]. Yi et al. recently reviewed the importance of considering strain diversity for establishing microbial cell factories by highlighting the fact that a wide range of strain variations already exists even within a specific yeast genus and species [ 4 ]. An illustrative example has been provided by a primary screening of the potential producer of triacetic acid lactone by expressing a 2-pyrone synthase in 13 industrial S. cerevisiae strains with different genetic backgrounds [ 5 ]. The production levels varied up to 63-fold among different strains, highlighting the importance of considering the strain variation for optimal microbial cell factories. Moreover, basal production does not consistently signal the potential for rewiring, and there are variations in phenotypes and physiology when it comes to non-native metabolites [ 4 ]. Therefore, heterologous expression must be screened in various backgrounds to evaluate the best strains/species for further genetic engineering. This emphasizes the growing importance of considering strain-level characteristics when selecting host microorganism. However, this requires extensive study of variants or screening with the requirements of genetic tools adaptations to each strain. Most studies focus on a limited set of industrial or type strains and rarely extend to other strains or related species with limited knowledge on the physiological and metabolic state of such species or strains. Nevertheless, for some interesting traits like lipid production, physiological data of clade or set of strains have been explored. The diversity of closely related species in the Yarrowia clade has been investigated through comparative studies of their oleaginous properties [ 6 ]. These species including Y. lipolytica , Yarrowia alimentaria , Yarrowia deformans , Yarrowia galli , Candida hispaniensis , Yarrowia hollandica , Yarrowia oslonensis , Yarrowia phangngensis and Yarrowia yakushimensis which have exhibited different levels of tolerance to salt, different optimal temperatures for growth [ 7 ], maximum lipid content, and cell dry weights ranging from 30 % in C. oslonensis to 67 % in C. hispaniensis [ 8 ]. In the past decade, the clade has grown up to 15 potential species including Yarrowia keelungensis , Yarrowia divulgata , Yarrowia porcina , Yarrowia bubula , Yarrowia brassicae and Yarrowia parophonii [ 9 11 ]. Quarterman et al. investigated the biomass and lipid production of 13 species of the Yarrowia clade by using a non-detoxified acid-pretreated switchgrass hydrolysate as a feedstock [ 9 ] with further characterization of a subset of them for inhibitor tolerance. This study highlighted the diversity in terms of behaviour, production and resistance, for example, Y. hollandica and Y. phangngensis reached higher cell biomass and lipid titre compared to Y. lipolytica W29 in these conditions. In other studies, up to thirteen of these yeast species from the Yarrowia clade were screened for erythritol, arabitol, mannitol, citric acid, lipids, and protein production [ 12 13 ]. In particular, Y. divulgata and Y. oslonensis were identified as robust producers of polyol compared to Y. lipolytica when grown on glycerol [ 13 ]. Protease production among the Yarrowia clade has been confirmed the potential of certain Yarrowia species to secrete significantly high amounts of proteases and revealed the influence of culture media when benchmarking species for a particular trait [ 14 ]. Therefore, the physiological aspect and diversity of the Yarrowia clade are now well-documented, highlighting the variation in industrially relevant traits among different species, which can outperform Y. lipolytica . Following this, genetic engineering of the other species in the clade starts to be considered. A first transformation method for the most distant species C. hispaniensis allows using sucrose as a carbon source by expressing the S. cerevisiae invertase as a marker and a heterologous enzyme [ 15 ]. C. hispaniensis is resistant to antibiotics like gentamycin, nourseothricin, and hygromycin, which are typically employed as selective markers in yeast genetic engineering. This limits the development of genetic tools in this species [ 15 ]. Moreover, this yeast is refractory to the standard transformation method and requires the use of biolistic approach for transformation [ 15 ]. Similarly, Y. phangngensis has been transformed for the first time by using genetic tools and the standard LiAc/PEG transformation method neutralizing the cell membrane by lithium cations developed for Y. lipolytica with hygromycin and zeocin as markers for selections [ 16 ]. The recycling of Y. lipolytica tools in this species shows possibility for more complex genetic engineering. However, it is worth noting that the hygromycin concentration required for selection in Y. phangngensis is three to five times higher compared to Y. lipolytica transformants selection. It opens the way for reusing the existed tools or developing the standardized tools in non-conventional and non-type organisms, allowing rapid screening of numerous potential hosts for diverse applications. Here we evaluated the functionality of a Golden Gate modular synthetic biology tool developed for Y. lipolytica [ 17 ] in six species from the Yarrowia clade by using two different selection markers and the expression of a fluorescent protein as a validation system with a fast transformation method. As a proof of concept, the genes in the carotenoid pathway were expressed in five of the six species."
} | 2,561 |
26109501 | null | s2 | 7,366 | {
"abstract": "Biohybrid microcylinders are fabricated using electrohydrodynamic cojetting followed by a surface chemistry approach to maximize cell-adhesive characteristics. As proper cell alignment and mechanical stiffness are important components of bioactuator design, spatial cell selectivity and stress/strain properties of microcylinders are characterized to demonstrate their capability of response to rat cardio-myocyte contraction. These microcylinders can find applications in a host of micromechanical systems."
} | 126 |
30913219 | PMC6435185 | pmc | 7,367 | {
"abstract": "Biodiversity loss is driven by interacting factors operating at different spatial scales. Yet, there remains uncertainty as to how fine-scale environmental conditions mediate biological responses to broad-scale stressors. We surveyed intertidal rocky shore kelp beds situated across a local gradient of wave action and evaluated changes in kelp diversity and abundance after more than two decades of broad scale stressors, most notably the 2013–2016 heat wave. Across all sites, species were less abundant on average in 2017 and 2018 than during 1993–1995 but changes in kelp diversity were dependent on wave exposure, with wave exposed habitats remaining stable and wave sheltered habitats experiencing near complete losses of kelp diversity. In this way, wave exposed sites have acted as refugia, maintaining regional kelp diversity despite widespread local declines. Fucoids, seagrasses and two stress-tolerant kelp species ( Saccharina sessilis , Egregia menziesii ) did not decline as observed in other kelps, and the invasive species Sargassum muticum increased significantly at wave sheltered sites. Long-term monitoring data from a centrally-located moderate site suggest that kelp communities were negatively impacted by the recent heatwave which may have driven observed losses throughout the region. Wave-sheltered shores, which saw the largest declines, are a very common habitat type in the Northeast Pacific and may be especially sensitive to losses in kelp diversity and abundance, with potential consequences for coastal productivity. Our findings highlight the importance of fine-scale environmental heterogeneity in mediating biological responses and demonstrate how incorporating differences between habitat patches can be essential to capturing scale-dependent biodiversity loss across the landscape.",
"introduction": "Introduction Ongoing biodiversity loss is expected to reduce ecosystem functioning and services [ 1 ] but uncertainty remains about the spatial scale at which to investigate the environmental drivers of such loss [ 2 – 4 ]. Global stressors can interact with local factors to exacerbate or ameliorate community responses to ongoing global change [ 5 – 7 ]. Yet, fine-scale microclimatic differences between sites are often ignored by both climate envelop models–which predict systematic shifts in the latitudinal ranges of species [ 8 ]–, and in meta-analyses of local diversity change–which group plots only by habitat-type (e.g. forest, marsh, grassland) or by region [ 9 – 11 ]. These common approaches, although insightful, may miss functionally important trends in community diversity change or local abundance loss if the stresses associated with a habitat patch depend more on local conditions than on regional patterns [ 12 ], or if even the most consistent declines occur at only a subset of sites within each habitat type. Understanding how to detect and predict functionally-relevant biodiversity changes will therefore depend on determining the relative importance of both broad-scale and fine-scale stressors in driving community shifts through time. While much work has focussed on how broad-scale stressors are driving the biological responses of communities [ 6 , 8 , 10 , 13 ], fewer studies have examined the role that local, fine-scale conditions play in magnifying or ameliorating them [ 6 ]. The rocky intertidal zone is predicted to be particularly sensitive to ongoing changes in climate [ 14 ] because intertidal organisms live near their physiological limits [ 5 ] and are sensitive to air temperatures, which tend to be more variable and extreme than seawater temperatures [ 5 , 15 ]. Local environmental gradients also have profound effects on intertidal systems. Environmental heterogeneity in the form of wave action plays a significant role in structuring intertidal communities [ 16 , 17 ]. Water movement from waves can eliminate nutrient-depleted or oxygen-rich boundary layers that are associated with low-flow environments, thereby increasing productivity [ 18 ]. Furthermore, wave splash can ameliorate the stressors associated with aerial exposure, such as desiccation and thermal stress [ 5 , 15 ]. Thermal profiles have suggested that wave exposed intertidal sites experience reduced thermal stress and emersion times compared to sheltered sites [ 15 , 19 ], suggesting that perhaps they are more resilient to rising air temperatures. Given the importance of wave action to the physiology and ecology of organisms that live along rocky shorelines, exposure to waves is likely to mediate the biological responses of intertidal organisms to ongoing changes in environmental stressors. However, the scarcity of appropriate baseline community data has made this hypothesis difficult to test in the field. Here we investigate the influence of a local wave exposure gradient on temporal changes in intertidal kelp bed habitats in Barkley Sound, British Columbia, Canada following 22 years of broad-scale stressors and extreme temperature events ( Fig 1 ) [ 20 , 21 ]. Rocky shore kelp beds are composed of a wide variety of marine flora (e.g. seaweeds and seagrasses) and fauna (e.g. mussels, barnacles, echinoderms) many of which compete for space on the shore [ 22 – 24 ]. In these systems, kelps (hereafter referring only to Laminariales) act as foundation species in both intertidal and subtidal regions [ 22 ], driving ecosystem productivity through rapid growth and formation of habitat for many ecologically important species [ 23 ]. Kelps are sensitive to high temperatures [ 25 , 26 ], however, and as such are expected to respond negatively to climate change and climate change-amplified heat wave events [ 27 – 29 ]. This sensitivity to high temperatures can be made worse by the tendency for heat waves to be associated with nitrogen poor waters [ 30 – 32 ] that can magnify the impacts of increased temperatures [ 33 ]. Increases in marine heat wave prevalence and intensity have begun to cause negative impacts on kelp forests near their geographical range limits [ 13 , 27 ]. However, interactions between global, regional and local processes have led to complex responses of kelp communities, with large variability in the magnitude and direction of change [ 11 , 28 , 34 , 35 ]. Studies of local-scale temporal change in the abundance of kelps and other large brown algae are increasingly common [ 11 , 36 – 38 ] and have collectively demonstrated that local conditions can interact with global stressors to drive variation in ecosystem responses [ 11 , 35 – 37 ]. However, these studies have focused on a small number of species, generally in the subtidal zone, and have not examined temporal changes in the diversity or composition of entire kelp assemblages. Moreover, there is still broad uncertainty as to how natural variation in site-level environmental conditions will influence the responses of kelp-dominated ecosystems to increases in the prevalence of broad-scale stressors. 10.1371/journal.pone.0213191.g001 Fig 1 Temperature anomalies between 1990 and 2018. Data are presented for (A) air temperature and (B) sea surface temperature (SST) in relation to the timing of surveys. Data were taken from lighthouses near the opening of Barkley Sound and are calculated with respect to 33-year historical averages (dating to 1985). Also shown (as black lines) are one-year moving averages of temperature anomaly. Note that between 2014 and 2018, air temperature anomalies were consistently positive and reached an unprecedented extreme of more than 3°C in 2015. SST anomalies were also consistently positive between late 2014 and 2018. To assess temporal changes in the diversity and abundance of kelps on intertidal rocky shores, we resurveyed sites (n = 49) in 2017 and 2018 that had previously been surveyed by Druehl & Elliot between 1993 and 1995 [ 39 ]. Sites occurred broadly throughout the region, and were situated across a range of wave exposures, slopes, aspects, and types of rocky substrates. We also analysed other long-term monitoring data for one centrally located site (Wizard Islet) to better assess the timing of any widespread changes in kelp bed composition, diversity or abundance. We found substantial changes in the diversity of Barkley Sound kelp communities and widespread declines in the abundance of many kelp species. We discuss potential drivers and consequences of changes in kelp communities as they relate to gradual warming [ 40 ], the recent marine heatwave [ 20 , 21 , 41 ] and possible changes in trophic dynamics [ 42 , 43 ].",
"discussion": "Discussion Timing and causes of declines Between 1993–1995 and 2017–2018 kelp beds in Barkley Sound have changed substantially with losses in kelp diversity at wave-sheltered and moderate sites. Most kelp species were found at fewer sites in 2017 and 2018 than during 1993–1995 and kelp communities in 2017–2018 tended to consist of fewer species that were less abundant on average. The spatial extent and magnitude of species loss, as well as the multiannual life cycle of many investigated kelp species suggest that these declines are a result of widespread responses to broad-scale stressors that are occurring or have occurred throughout Barkley Sound. While temperatures in Barkley Sound have gradually increased over the past century [ 40 ], this gradual change is not detectable over the 22 year period between 1995 and 2017 ( Fig 1 ). Instead, temperature data bear a clear signal of the 2013–2016 marine heatwave, with anomalously warm temperatures detected consistently between 2013 and 2018. Thermally tolerant fucoids ( Sargassum muticum ) and seagrasses ( Phyllospadix spp.) showed greater persistence than most kelp species, and have not declined significantly between the 1990s and 2017. Climate-mediated shifts from kelp-dominated to Sargassum -dominated communities have been documented elsewhere [ 28 , 29 , 58 – 61 ]. Therefore, these data are consistent with the hypothesis that changes in kelp communities have resulted from increases in climate stress. Data from Wizard Islet also support this hypothesis, demonstrating a substantial drop in kelp cover between 2009 and 2017, consistent with the timing of temperature anomalies. Splashing of cool water at exposed sites could alleviate air temperature stress during low tide, leading to the patterns that we show here [ 62 ] or local mixing at sites with increased water motion could mediate these stresses by preventing pockets of warm water from forming at small scales. Out of the nine common kelp species that we investigated using proportional odds models, only two species have not declined since 1993–1995: Saccharina sessilis and Egregia menziesii . Both of these species are found higher in the intertidal zone than most other kelps, suggesting resistance to desiccation and thermal stress at low tide [ 39 , 63 – 65 ]. Egregia menziesii has been described as the kelp with the highest upper limit [ 65 ], although Postelsia palmaeformis , Saccharina sessilis and a wave exposed ecotype of Alaria marginata (i.e. A . nana ) may be found as high or higher at wave exposed sites [ 64 ]. Both S . sessilis and E . menziesii possess complex three-dimensional morphologies that could promote the retention of water and may therefore improve survival during particularly stressful low tides. Although S . sessilis specializes in the high intertidal zone, it is less resistant to warm water temperatures than some species that experienced significant declines at moderate and sheltered shores (e.g. Alaria marginata and Costaria costata ) [ 25 ]. This suggests that air temperature is likely a stronger driver of the observed patterns of kelp loss than SST. During the recent 2013–2014 “Blob” and the 2015–2016 El Niño, nitrogen levels were also abnormally low [ 38 , 56 ]. Nutrient availability may limit productivity [ 66 ] and influence thermal tolerance of kelp species [ 33 ]. So, multiple stressors could have interacted to result in the declines that we observed [ 33 , 67 ]. Given the multiple stressors associated with the heatwave, it is possible that different species have declined in abundance as a result of distinctive broad-scale drivers. An alternative hypothesis, separate from the direct effects of recent temperature anomalies, is that kelp declines were caused by changes in the trophic dynamics of intertidal kelp beds. Sea stars have declined in abundance along the coast of British Columbia as a result of sea star wasting disease [ 43 , 68 ], an epidemic that was possibly amplified by the 2013–2016 heatwave [ 68 , 69 ]. This loss of sea stars has led to increases in sea urchin biomass and declines in kelp abundance in some areas [ 42 , 43 ]. While it is well established that herbivory by urchins can cause declines in kelp abundance, urchins are generally absent in the intertidal zone in our system, with the exception of tidepools, and therefore are not likely to be responsible for observed kelp losses. Herbivory by intertidal grazers however, especially Katharina tunicata , has been shown to influence kelp bed diversity and species composition in some areas [ 70 , 71 ]. It is unknown whether intertidal grazers are more abundant following sea star wasting disease outbreaks of 2013–2014, and it is possible that changes in trophic dynamics could have contributed to kelp losses. However, K . tunicata is predominantly found at wave exposed sites, rather than at sheltered sites [ 72 – 74 ], and therefore cannot have driven the ubiquity of kelp declines at moderate and sheltered sites. Declines at sheltered and moderately exposed sites occurred regardless of substratum (boulder versus bedrock) or slope (steep versus shallow), factors known to influence the distribution of invertebrates [ 75 – 77 ]. Therefore, the observed declines are likely too widespread to have resulted from increases in abundance of a single grazer species. Moreover, increases in grazers would have been expected to influence fucoids, such as Sargassum , along with kelps [ 78 ], a result which did not occur. Although we cannot rule out a role of herbivory in driving some declines, it is unlikely to be the most important driver. Local stressors caused by human activity such as run-off or pollution are also unlikely to be drivers of the declines that we document. Barkley Sound has very low population densities, limiting human disturbance [ 79 ] and many of our sites occurred within Pacific Rim National Park, a region that is largely uninhabited and protected from human disturbance. In sum, while changes in kelp bed composition, diversity and abundance may have resulted from multiple interacting factors, evidence is consistent with the hypothesis that temperature anomalies during the 2013–2016 heatwave drove widespread declines in kelp bed diversity and species abundance. Regardless of the timescale over which these declines occurred or the exact combination of factors that have driven them, our results suggest that wave-sheltered habitats are more sensitive to regional stressors than wave exposed habitats. Implications of kelp loss Given the important ecological role of kelp [ 22 , 23 , 80 ], the substantial declines that we document are likely to have cascading effects on the diversity of other organisms and on ecosystem functioning and productivity of intertidal communities [ 22 ]. While the affected kelp communities may yet recover following the 2013–2016 heatwave, our results offer a novel prediction for how communities will be affected by increasing climatic stressors. In particular, these results suggest that kelp communities at wave-sheltered sites may be particularly sensitive to the increasing prevalence of broad-scale stressors, such as more frequent and intense marine heat waves [ 81 – 84 ]. It could be hypothesized that declines on wave-sheltered shores may not affect regional productivity or habitat availability as much as would declines on wave-exposed shorelines, which are more diverse and more productive [ 17 ]. Yet, positive interactions generated by kelp canopies may be especially important on wave-sheltered shores because these shores are more physiologically stressful [ 85 ]. Furthermore, the lower diversity and productivity of sheltered shorelines is far outweighed by their sheer abundance in the Northeast Pacific ( Fig 8 ). 10.1371/journal.pone.0213191.g008 Fig 8 Northeast Pacific intertidal habitat classified by wave exposure. Wave-sheltered habitat makes up the majority of Northeast Pacific shorelines (A) and is abundant in British Columbia (C) and Alaska (B) but rare along the outer coast of Washington and Oregon (D). Bar plots show the proportions of rocky shoreline of different wave-exposures and are accompanied by the length of predominantly rocky shoreline in each region (A) or in each inset (B-D). Mosaic plot inset in A shows the relative proportions of rocky shoreline of different wave-exposures scaled by the length of coastline for each region. Regions from South to North: (1) Oregon coast, (2) Washington outer coast, (3) Salish Sea, Puget Sound, and Strait of Georgia, (4) western Vancouver Island, (5) northern British Columbia, (6) southern Alaska, (7) central Alaska. Approximately 57,000 km of wave-sheltered rocky shoreline exists from Oregon to central Alaska, virtually all of which (99.8%) occurs north (and east) of Washington’s outer coast ( Fig 8A ). Therefore, even small changes in kelp diversity on more sensitive wave-sheltered shores could have large effects on intertidal productivity if magnified across the landscape. While some of this shoreline may not be suitable kelp habitat due to limitations from salinity and other factors, it is clear from our analyses that wave sheltered shorelines are common and extensive in northern Washington, British Columbia and Alaska. Given that these types of habitats are uncommon further south, it is likely that some northern shorelines will experience losses in kelp abundance and diversity before southern ones. The sensitivity of wave-sheltered sites in our system is contrasted by the apparent resilience of wave exposed kelp beds, a novel finding that has important implications for conservation and management. Wave exposed sites are highly productive and often represent hotspots of diversity in our system [ 39 ]. Our results demonstrate that these sites may also be especially resilient against broad-scale stressors. Wave exposed sites may act as refugia during times of stress, potentially buffering kelp ecosystems against regional extinctions and playing a key role in maintaining regional species diversity. The role of climatic refuges in maintaining species diversity through geological time has been widely discussed in the paleoecological literature [ 86 , 87 ]. However, few studies illustrate this phenomenon under ongoing global change. Recent efforts to understand biodiversity change in ecologically important biogenic habitats have identified areas where ecosystems are performing substantially better (“bright spots”) or worse (“dark spots”) than average [ 88 ]. Our results demonstrate that a fine-scale environmental gradient–one that can vary over tens of metres [ 44 ]–has mediated the formation of bright spots and dark spots in kelp-dominated ecosystems. Importantly, given the distribution of wave exposure in the Northeast Pacific ( Fig 8 ), such dark spots are likely to be much more common than bright spots across the landscape. As a result, although wave exposed sites might maintain regional diversity, abundant dark spots could have profound effects on ecosystem functioning and coastal productivity [ 22 , 24 , 89 ]. In addition to reductions in diversity, we document widespread declines in the abundance of intertidal kelps in Barkley Sound. While the magnitude of decline was dependent on wave exposure ( Fig 3B ) and varied between species (Figs 6 and 7 ), sites from all wave exposure categories declined significantly in average kelp abundance and 7 of the 9 species most common species declined in abundance. Losses of kelp cover are common worldwide [ 29 ] and a recent global meta-analysis found that more than one third of published subtidal kelp bed surveys showed declines over the past 50 years–significantly more than had increased [ 11 ]. While many negatively affected kelp forest ecosystems are found near the warm-edge of kelps’ latitudinal range [ 13 , 28 , 34 , 90 ], our data suggest that similar declines have occurred in the intertidal zones of British Columbia, reasonably far from the warmer latitudinal limit of northeast Pacific kelp ecosystems [ 91 ]. This supports previous work suggesting that central-and not just edge- populations of brown algae may be susceptible to broad-scale stressors brought on by heat waves [ 37 , 92 ]. This may be especially true for intertidal communities that show limited correlations between latitude and thermal stress [ 5 ]. Although declines may be attributable to stressors occurring over short timescales [ 56 ] ( Fig 1 ), rather than a response to gradual warming, the recovery from ecosystem-wide declines may not occur rapidly in either case. Four of our sites lost all kelp species and thirteen others were reduced to a single, sometimes rare (< 5% cover) species. Thus, many of our sites have experienced complete or near-complete collapses of kelp-dominated communities. For the 17 sites that had the fewest kelps in 2017, similar results were found in 2018: four sites with no kelps and 13 sites with only one kelp species (data in S1 and S2 Tables). Thus, even if declines did occur recently, they have persisted for two years, indicating that recovery has not occurred immediately following the heatwave. Kelp bed collapses have been documented previously in various regions worldwide and many have yet to recover following initial kelp bed collapse [ 28 , 93 , 94 ]. Scale-dependence of diversity loss and the importance of local gradients A broader implication of our results, one that extends beyond rocky shores, is that important biodiversity loss could easily remain hidden from studies not specifically designed with environmental heterogeneity in mind. We found that the total diversity of kelps in Barkley Sound has not changed throughout the region, yet a majority of sites experienced large losses in local diversity. This clearly demonstrates how declines in diversity can be concentrated in only some habitats that may be stressful and lower diversity to begin with. Studies that focus on regional patterns or only investigate certain types of sites could miss losses mediated by local gradients. Thus, differences between local conditions in distinct habitat patches may directly contribute to the disconnect between diversity measurements taken at different spatial scales [ 1 , 89 ]. In our study, species accumulation curves demonstrate how we could have missed the widespread biotic homogenization that has occurred only at wave-sheltered sites were we to assess all sites together ( Fig 5 ). Capturing these losses in between-site diversity can be essential to monitoring and conservation efforts because ecosystem functionality can depend on having many species combinations across the landscape [ 1 , 95 ]. Yet, while our results support growing evidence that local environmental heterogeneity explains important variation in diversity loss [ 7 , 14 ], few studies that examine responses to ongoing global change incorporate these gradients into their analyses. Our results point to the need for a framework that better incorporates the interacting effects of stressors at different scales. Such an approach would hold much promise for identifying and predicting diversity loss and changes in abundance not only at species range edges but also along local gradients throughout the range of each species. Heterogeneity in environmental variables, like wave exposure, is ubiquitous in the natural world, but its importance in determining the responses of communities to broad scale stressors is often underappreciated [ 6 ]. As global change continues to drive shifts in ecosystem structure, heterogeneity of habitats will lead to variation in microclimates [ 5 , 19 ] and could strongly affect the biological responses of organisms. Rather than assessing the average responses across all communities in a region or across the globe [ 9 , 10 ], we should work to identify the habitats that are most vulnerable to declines and determine whether they are abundant enough to influence ecosystem functioning across the landscape. Consistent declines across all habitat patches or at the most diverse, high quality habitats may not be reasonable predictions for how communities will respond to global change [ 96 ]. Instead, some sites may act as refugia, while diversity is lost from marginal habitats; if these sensitive habitats are common, as they are in our system, then the consequences to ecosystem functioning could be profound."
} | 6,246 |
35423531 | PMC8695597 | pmc | 7,369 | {
"abstract": "Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD + . Determination of the structure of the cofactor–enzyme complex (PDB ID 6PX2 ) revealed variable NAD + occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.",
"introduction": "Introduction Temperature has a profound influence on enzyme activity, not least because protein stability depends on temperature. 1–3 Maintaining metabolic throughput at a range of temperatures is a challenge faced by the vast majority of the world's organisms. 4,5 In the case of mutualism or parasitism, both the host and the symbiont must be able to tolerate the same temperature ranges. Coral reefs have been seen to be particularly susceptible to temperature increases in ocean water that lead to a breakdown in symbiosis between coral and the photosynthetic algae that they host. 6–8 Under normal physiological conditions, a healthy coral enters into a symbiotic relationship with one of a variety of species of photosynthetic algae. 9–11 These algae are hosted intracellularly and provide the coral with glucose, the product of photosynthesis. Increased water temperatures cause a breakdown of symbiosis and concomitant bleaching of the coral. 12–15 The precise signal that prompts this symbiosis to break down is unknown, but some research has focused on the thermal stability of both coral and algal metabolic enzymes, since metabolic throughput is important to the calculus of mutual benefit. 16–19 Glyceraldehyde-3-phosphate dehydrogenase is a cytosolic enzyme central to sugar metabolism. During glycolysis, GAPDH oxidatively phosphorylates glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, concomitantly reducing NAD + to NADH, and under aerobic conditions these reducing equivalents are passed to the electron transport chain. Because of its importance in cellular ATP generation, regulation of GAPDH is an important regulator of metabolic rate. 20,21 It should be noted that GAPDH is well-known for its ‘moonlighting’ role in other non-metabolic biochemical pathways, so it is possible that the enzyme has a more expansive role in coral. 22,23 GAPDH is generally thought to be active as a tetramer, although dimeric forms have been identified. 24 Each monomer has an NAD + binding domain related to the one first identified by Rossmann in lactate dehydrogenase. 25 In addition, there is a C-terminal catalytic domain that provides the environment to facilitate hydride transfer and phosphorylation among its three substrates, NAD + , glyceraldehyde-3-phosphate, and inorganic phosphate. In the reef-building coral Acropora millepora , expression of the metabolic enzyme GAPDH has been observed to be temperature-dependent. 26–28 Thermal stability of the enzyme itself has also long been the subject of study, as GAPDH was one of the earliest enzymes to be examined in order to understand the structural basis of the extreme heat stability of enzymes from hyperthermophilic organisms. 29–33 GAPDH is a complex enzyme, with three substrates, important quaternary structure, and sensitivity to oxidation. The present structure does not provide a complete picture of its thermostability. The goal of the present study is to begin to characterize the structural basis of thermostability in metabolic enzymes of reef-building corals and their endosymbionts. We report here the melting temperature for Acropora millepora GAPDH, along with observation of a significant increase in the melting temperature of the enzyme upon binding its NAD + cofactor. We determined the crystal structure of this complex to a resolution of 2.4 Å (PDB ID 6PX2 ). This is among the first structures of any protein from this genus of coral.",
"discussion": "Discussion The GAPDH enzyme from the reef-building stony coral Acropora millepora is a rather typical eukaryotic GAPDH, comprising an N-terminal NAD + -binding Rossman fold and a catalytic domain that provides important active site residues. Its nearest structurally characterized homologs are mammalian (human, porcine and bovine), along with the nematode Brugia malayi , all of which share 75–76% amino acid identity with the coral sequence. 36,37 The thermal stability of GAPDH from both corals and their algal symbionts could be important, as anthropogenic temperature increases in ocean water are causing the symbiosis to break down ever more frequently. 6 \n A. millepora GAPDH is a relatively thermostable enzyme, retaining its folded structure until 65 °C, as assessed by a Thermofluor assay. In this assay, a fluorescent dye that is highly quenched by water is incubated with the purified protein. 38 The temperature is slowly raised, with continuous measurement of the fluorescence. When the protein denatures, the dye gains access to the hydrophobic residues of the protein's formerly folded core, and the fluorescence increases dramatically. The resulting melting curve gives a measure of the thermostability of the protein. Like many proteins, AmGAPDH appears to be greatly stabilized in the presence of one of its cofactors, NAD + ( Fig. 1 ). 39 We used a simple two-point experimental design here, because the goal was not to determine the binding constant of NAD + , but rather to confirm that we had created the experimental conditions to produce a form of the protein with higher thermal stability. In order to assess the structural basis for this increase in thermostability, we sought to crystallize the protein in the presence of NAD + . In principle, the tetrameric GAPDH protein could bind four NAD + cofactors. In practice, a wide range of occupancy has been reported, depending on the precise details of protein preparation. In addition to these experimental factors, there is likely cooperativity in binding NAD + , though we have not collected data on the cooperativity of this enzyme. Some eukaryotic GAPDH structures contain two NAD + molecules, while others crystallize with three or four. 36,41 Under the experimental conditions here (exposure to NAD + during protein expression in E. coli , absence of NAD + during purification, incubating the crystal in 50 mM NAD + during cryoprotection), we see evidence of NAD + in all four binding sites of the tetramer, although the electron density is particularly weak in two of them (chain C and chain H). In two of the chains (D and G), every atom is clearly delineated by the electron density contoured at 1.5 sigma ( Fig. 2 ). In cases where specific atoms or molecules in the crystal are disordered and cannot clearly be modeled into electron density, numerous representational strategies are available. 42 The two most common options are to model the atoms as present with full occupancy, allowing increased temperature factors to indicate to the reader whether or not the atoms are within the observed electron density. The other strategy is to decrease the occupancy, to represent the fact that not every protein within the crystal has clearly identifiable atoms at this position. 43 We have taken the latter approach, with occupancy ranging from 0.65 (chain C) to 1.0 (chains D and G). Another observation is that phosphate is bound in all eight active sites ( Fig. 3 ). The mechanism of catalysis requires two phosphate binding sites, one for the 3-phosphate of glyceraldehyde-3-phosphate (the substrate, or Ps, site) and the other for the inorganic phosphate that adds to the activated substrate to generate the 1,3-bisphosphate (the inorganic phosphate, or Pi, site). The actual picture of the active site that emerges from the consideration of GAPDH structures from many different organisms is more complex. It has been observed that there is more than one anion site in the active site. The Pi binding site identified original observations made on GAPDH from Bacillus stearothermophilus was not seen in subsequent structures of Thermotoga maritima GAPDH (reviewed in Cook 44 ). By the consensus terminology of GAPDH anion-binding sites, the site occupied here in the Acropora millepora GAPDH structure is the new Pi site. The mechanistic consequences of these different inorganic phosphate binding sites are not entirely clear, nor is the significance of having one or the other site occupied in different protein preparations. 45 In the case of A. millepora , we didn't seek to experimentally enforce a particular type of phosphate occupancy, or even phosphate occupancy at all, but it is true that the protein was initially harvested from the E. coli host bacteria in a phosphate-containing buffer (50 mm sodium phosphate, pH 8.0). The observation that the melting temperature of the protein increases in the presence of NAD + suggests some structural change upon binding. 39 A goal of these experiments was to compare atomic-resolution structures of the protein at 0 and 1 mM NAD + concentrations. Though the protein crystallized under both conditions, the diffraction was poor for the 0 mM NAD + condition. In addition, despite the fact that no NAD + was added, electron density consistent with NAD + occupation was observed in this position, apparently from endogenous NAD + captured by the enzyme during expression in E. coli . Thus, we were not able to cleanly make the comparison between the apo and NAD + -bound structures. The fact that this structure has eight chains with differing degrees of NAD + occupancy allows us to consider the idea that we might be able to detect conformational differences associated with NAD + binding from the single structure obtained at high NAD + concentration. The occupancy was determined during structural refinement. After the apo enzyme had been refined, the NAD + cofactors were placed into the electron density visible in the omit map. This was done via a combination of hand-fitting the cofactor into the electron density and by analogy to the structural homolog 4K9D ( Brugia malayi GAPDH). It was observed during this process that a sphere of positive difference density correlated well with bound phosphates that have been observed in other GAPDH structures. These phosphates were observed in every chain, and were placed in the electron density by the same combination of fitting by eye and by analogy with the structure 4K9D . With both phosphate and NAD + roughly placed, further refinement was carried out in Phenix using both individual B factors and occupancy, with automatic weighting of the geometrical restraints and the observed electron density. A comparison was also made to the maps generated by using the same strategy without refinement of occupancy. In the case where occupancy was set to 1.0, marked negative and positive difference density were observed in all chains except D and G. Allowing the occupancy to be refined did not completely resolve this problem, but this model, as expected, was fit much better by the 2 F o − F c map. Having arrived at a model with NAD + occupancy varying by chain, we then used the occupancy to roughly rank the extent of binding. In order to examine chain differences, we used chain D as a reference and aligned each chain to it in turn ( Fig. 4 ). Areas of lower spatial agreement were colored red, while regions where the chains aligned well were colored white and blue. For clarity, the RMSD was plotted using only the alpha carbons and not the side chains. The plot reveals that regions of difference are fairly localized. The consensus difference is in the catalytic domain, from residues 187–199. This helix-loop structure is part of the S-loop region (residues 179–201), which has been seen to vary in GAPDH structures with and without bound NAD + . 46,47 Indeed, a recent study has shown that this loop's flexibility is such that it may be perturbed by the presence of cryoprotectants. 48 In addition, one of the helix–helix interactions of the Rossman fold is seen to vary among the eight chains of the AmGAPDH structure. This region comprises residues 75–90 and 102–113 and appears at the top of the structures in Fig. 4 . For chain C, specifically, which is in the same tetramer as chain D but which has the poorest NAD + occupancy, the turn at Phe33–Pro34 is acutely red, signifying the highest level of structural difference from the reference (D) subunit. This Pro is fairly common in GAPDH sequences but is not universally conserved. A recent structure of mammalian (bovine) GAPDH also shows these two residues to give the largest difference between NAD + -bound and unbound chains. 36 Globally, the alignment of all eight chains does make clear that the overall RMSD correlates well with the occupancy of NAD + ( Table 2 ). The present study illuminates the regions of the protein that change under conditions of high NAD + occupancy. High NAD + occupancy correlates to a state of increased thermal stability for the protein overall ( Fig. 1 ). In order to tease out which conformational changes are associated with functional aspects of NAD + binding (positioning of the cofactor for catalysis, for example) and which are most associated with thermal stability, a more complete study of stability across temperature would need to be undertaken, ideally with crystal structures providing snapshots of AmGAPDH mutants of varying thermal stability under conditions of varying NAD + occupancy. The question of whether or not the melting behavior at elevated NAD + concentration represents either a physiologically or functionally relevant observation depends on a certain degree of extrapolation from the Thermofluor assay. Acropora is a widely distributed coral that inhabits waters of varying depth, across the world's oceans. Shallow-water coral may experience significant day–night temperature variation, but certainly the observed melting temperatures are outside the range of physiological exposure. Though it is reasonable to suppose that proteins with melting temperatures that are 10 °C apart might have different thermally allowable motions at lower (more physiological) temperatures, the Thermofluor methodology provides no direct insight into this question. The extent of these motions or packing of the structure are presumably the mechanisms whereby different melting behavior at high temperature might affect either enzymatic activity or structure at physiological temperature. We cannot definitively conclude that the effect we see has relevance to GAPDH activity. Nonetheless, the residues identified in this study probably represent a good starting place for further investigation into temperature–activity relationships in this enzyme. In conclusion, we are interested in the temperature-sensitivity of metabolic enzymes from coral, because rising ocean temperatures have resulted in widespread coral bleaching. The GAPDH from the stony coral Acropora millepora was observed to melt at a significantly increased temperature in the presence of NAD + . The crystal structure of this co-complex reveals both that the eight active sites in the asymmetric unit have very different NAD + occupancy and that the extent of cofactor occupancy correlates with conformational changes in the polypeptide backbone."
} | 3,924 |
39597525 | PMC11596547 | pmc | 7,371 | {
"abstract": "Natural vegetation restoration has emerged as an effective and rapid approach for ecological restoration in fragile areas. However, the response of soil microorganisms to natural succession remains unclear. To address this, we utilized high-throughput sequencing methods to assess the dynamics of soil bacterial and fungal communities during forest succession (shrubland, secondary forest, and primary forest) in a karst region of Southwest China. Our study revealed that bacterial α-diversity was significantly higher in secondary forest compared to both shrubland and primary forest. Intriguingly, the soil bacterial community in primary forest exhibited a closer resemblance to that in shrubland yet diverged from the community in secondary forest. Conversely, the soil fungal community underwent notable variations across the different forest stages. Furthermore, analysis of the microbial co-occurrence network revealed that, within these karst forests, the relationships among soil fungi were characterized by fewer but stronger interactions compared to those among bacteria. Additionally, soil properties (including pH, soil organic carbon, total nitrogen, moisture, and available potassium), soil microbial biomass (specifically phosphorus and nitrogen), and plant diversity were the drivers of soil bacterial community dynamics. Notably, soil pH accounted for the majority of the variations observed in the soil fungal community during karst forest succession. Our findings provide valuable insights that can inform the formulation of strategies for ecological restoration and biodiversity conservation in karst regions, particularly from a microbial perspective.",
"conclusion": "5. Conclusions Forest succession exerts diverse influence on soil bacterial and fungal diversity, community composition, and co-occurrence patterns. Specifically, bacterial diversity in secondary forest significantly differs from that in shrubland and primary forest, whereas fungal diversity is distinctly differentiated across the three stages of karst forest succession. The co-occurrence patterns suggest that soil fungi exhibit fewer but more intense relationships compared to bacteria in these karst forests, indicating that bacteria and fungi adopt distinct strategies during forest succession. Furthermore, soil properties, including pH, SOC, Total N, AK, MBP, and MBN, along with woody plant diversity, collectively influence the bacterial community. Among these, soil properties, particularly pH, are the most dominant factor controlling the fungal community. Variations in soil nutrient status can effectively predict the changes in soil bacterial and fungal community composition and diversity throughout forest succession. In summary, intermediate pH levels and nutrient-rich soil conditions favor both bacterial and fungal communities and their interactions in karst forests. Therefore, understanding the intricate interactions between soil properties, plant diversity, and microbial communities is crucial for comprehending the ecological processes that shape forest ecosystems.",
"introduction": "1. Introduction Belowground biodiversity, notably microbial diversity, plays a crucial role in regulating aboveground biodiversity and ecosystem functioning [ 1 ]. Furthermore, microbial diversity is a key driver of multifunctionality in terrestrial ecosystems, encompassing critical aspects such as climate regulation, soil fertility, material production, and provision [ 2 , 3 ]. These contributions ultimately enhance human well-being [ 4 ]. As a vital component of terrestrial ecosystems, microbial biodiversity in forests has garnered attention comparable to that of macro-organisms such as plants [ 5 , 6 ]. Furthermore, it has been suggested to apply existing macro-ecological theories to the field of soil microbial ecology, as proposed in [ 7 ]. Various environmental factors play regulatory roles in shaping soil microbial diversity. For instance, temperature and soil carbon content influence soil archaea, while aridity, vegetation characteristics, and soil pH regulate bacterial communities, as reported in [ 8 ]. During secondary succession, the soil bacterial community is affected by multiple factors, including plant diversity and composition, as well as soil nutrients such as total organic carbon and total nitrogen, as detailed in [ 9 ]. Additionally, soil fungal diversity and functionality are influenced by plant species during afforestation processes [ 10 ]. When compared to arable land, forest ecosystems exhibit more stable and complex microbial networks [ 11 ]. The karst landscape in the southwest of China spans over 0.54 million km 2 , constituting one of the three largest continuous distribution areas in the world [ 12 ]. This landscape is particularly susceptible to disturbances, exhibiting unstable characteristics, and posing challenges for self-adjustment [ 13 , 14 , 15 ], partly due to slow species turnover and infertile soils [ 16 ]. Despite these challenges, substantial increases in vegetation growth and carbon stocks have positioned the karst region as a hot spot of global greening [ 17 ], with arbuscular mycorrhizal fungi playing a potentially crucial role in the maintenance of multiple ecosystem functions [ 18 ]. Recent research [ 7 ] has demonstrated that microbial communities in forest ecosystems undergo distinct changes across successional stages. Specifically, early successional stages tend to be dominated by bacteria (r-strategists), whereas late successional stages are prone to be dominated by fungi (K-strategists). This shift may be attributed to the differing responses of bacteria and fungi to succession [ 19 ], influenced by factors such as size, growth, and turnover rates [ 20 ]. Additionally, some studies [ 21 ] have indicated that karst forests exhibit greater connectivity among bacterial and fungal communities compared to non-karst forests, suggesting that increased microbial diversity strengthen the complexity of co-occurrence networks. Despite the growing interest in understanding the dynamics of soil microbial communities during forest succession, our knowledge of their response magnitude and direction in karst forest succession, characterized by specific plant communities and soil properties [ 22 ], remains limited. In particular, the specific drivers of microbial community changes in different succession stages in karst regions in southwest China are still poorly understood. Gaining these insights into their drivers is vital to increase ecosystem stability and function, particularly in the context of achieving international carbon sequestration and carbon neutrality goals. To bridge this knowledge gap [ 9 , 18 , 19 , 21 ], we conducted a study in the karst region of Southwest China. In this study, we employed the sequencing of 16S rRNA gene and ITS gene amplicons to gain insights into the community composition and diversity of soil bacteria and fungi across various stages of forest restoration, including shrubland, secondary forest, and primary forest. Subsequently, we quantified the soil microbial community, its diversity, and the co-occurrence network in relation to karst forest succession. Our ultimate goal was to identify the key factors influencing soil microbial dynamics during the process. This research contributes to a deeper understanding of the critical role that soil microorganisms play in maintaining ecosystem functions and services in a typical karst environment.",
"discussion": "4. Discussion 4.1. Dynamics in Soil Microbes and Environmental Variables Along Karst Forest Succession The implementation of ecological engineering, including natural restoration, has been widespread adopted worldwide [ 40 ]. For instance, in a karst region of southwest China [ 14 , 16 ], natural restoration has undoubtedly enhanced carbon sequestration through accumulation in biomass and soil organic carbon [ 41 ]. Additionally, without human or animal disturbance, plant natural succession alters vegetation growth, community composition, and productivity [ 21 ]. These changes, in turn, impact the belowground status and functions [ 42 , 43 ]. In our study, Proteobacteria , Actinobacteria , and Acidobactria dominate the bacterial community at the phylum level ( Figure 1 a), while Ascomycota and Basidiomycota dominate the fungal communities ( Figure 1 b), regardless of the forest succession stage in the karst region. This finding aligns with previous studies conducted in karst regions [ 19 , 44 ], indicating that forest succession had no significant effects on the dominant soil bacterial and fungal community structure. It is worth noting that the Proteobacteria communities are higher in the secondary forest compared to the shrubland ( Figure 1 a). This suggests that soil conditions improved during early plant succession by favoring Proteobacteria in a copiotrophic environment with available labile substrates [ 45 ]. Similar observations have been made in other studies. For example, Protebacteria communities increased with secondary succession after abandonment in the Loess Plateau in China [ 8 ] and with vegetation succession along the Franz Josef chronosequence in New Zealand [ 46 ]. We find that the bacterial α-diversity is lowest in the primary forest ( Figure A2 a,c), despite the high diversity of woody plants listed in Table 1 . This finding contrasts with the commonly held belief that plant diversity is positively related to soil bacterial diversity [ 47 , 48 ]. This contradiction might be explained by the differences in the substrates availability between our study’s forest ecosystems and the grassland ecosystems previous examined [ 47 , 48 ]. Furthermore, fungal α-diversity followed an initial downward and then upward trend during karst forest succession ( Figure A2 b,d). These findings suggest that the fungal and bacterial communities respond differently to forest succession [ 19 ]. The distinct responses may be attributed to how these communities respond differently to changing soil properties during forest succession [ 49 ]. 4.2. Divergent Patterns of Bacteria and Fungi Along Karst Forest Succession The NMDS results show that the compositions of soil bacterial and fungal communities in primary forest exhibit greater similarity to those in shrubland, in contrast to secondary forest ( Figure 2 ). The phenomenon may be caused by two factors. Firstly, the soil physical and chemical properties under shrubland and primary forest are comparable ( Table 1 ), fostering a similar environment for microbial survival and proliferation. Secondly, evergreen tree species prevalent in primary forests possess higher C:N ratios than deciduous tree species, stemming from their unique woody plant composition [ 20 , 50 , 51 ]. Although microorganisms may prefer the litter of evergreen trees, their litter production is relatively scant compared to deciduous tree species, resulting in a microbial community composition that mirrors that of shrublands with a higher proportion of deciduous trees [ 50 ]. However, in subtropical non-karst regions, soil bacterial communities vary with forest succession, potentially due to the increased production and accumulation of bacterial residues as succession progressed [ 52 ]. The divergence suggests that karst forests may undergo a more intricate succession process compared to non-karst forest. Generally, soil fungal communities undergo changes during forest succession ( Figure 2 b), indicating that specific fungal species or taxa fluctuate with successional stage. For instances, arbuscular mycorrhizal (AM) fungi tend to be earlier colonizers of successional habitats [ 53 ]. The results from the microbial co-occurrence network ( Figure 3 and Table A1 ) indicate that, despite their lower abundance, the correlations among soil fungi in forests are notably stronger than those observed among bacteria. This may be due to the more pronounced and enduring interactions between various fungal species [ 19 ], such as the symbiotic relationship between diazotrophs and arbuscular mycorrhizal fungi [ 54 ]. Our findings further imply that the diversity of bacteria and fungi across forest succession stages in karst regions may surpass that observed in non-karst regions, such as the Loess Plateau [ 49 ]. Researchers have emphasized that the prevalence of highly interconnected taxa, such as kinless hubs, within soil microbial networks correlate with elevated functional potential in terrestrial ecosystems [ 55 ]. Our study’s co-occurrence network reveals that JG30-KF-CM66, Subgroup_17, Subgroup_9, Anaerolineae , Dehalococcoidia , Gitt-GS-136, Gemmatimonadetes , and Chloroflexia are the most prominent bacterial nodes. Meanwhile, Kickxellomycetes stands out as the most significant fungal node ( Figure 3 b). These nodes exhibit robust connection with other taxa within the network, indicating a strong link to functional potential. Notably, JG30-KF-CM66 has been documented to play a role in global cobalamin production via the cobinamide to cobalamin salvage pathway [ 56 ]. Additionally, Sordariomycetes , composed of typical saprotrophic fungi, excel at decomposing labile C [ 57 ], whereas Mortierllomycetes show a robust response to readily degradable, N-rich substrates [ 58 ]. Therefore, the observed co-occurrence patterns indicate that species interactions play a more pivotal role in soil nutrient processes or functions compared to microbial diversity [ 19 ]. 4.3. Drivers of Soil Microbial Dynamics Along Karst Forest Succession Bacterial community structure along vegetation succession is predominately influenced by variations in soil nutrients, plant diversity, and composition [ 8 , 21 ]. Our analysis, utilizing db-RDA and variance partition analysis, has revealed that soil properties such as pH, SOC, Total N, moisture, and AK, along with soil microbial biomass P and N, and woody plant diversity are the key factors driving soil bacterial community dynamics during karst forest succession ( Figure 4 a and Figure 5 a, Table A2 ). Our findings further indicate that the diversity and biomass of woody plants, which are highly positively related with DBH [ 59 ], can negatively impact soil bacterial diversity during forest succession ( Table A2 ). This may explain why secondary forests, despite having lower SOC and soil N content compared to other forests ( Table 1 ), harbor the most abundant bacteria ( Figure A2 ), and why primary forests, with the highest available K ( Table 1 ), exhibit lower bacteria richness ( Figure A2 ). Our observation contrasts with studies conducted in undisturbed grasslands [ 47 ] and during plant secondary succession following abandoned farmland [ 8 , 48 ], where plant diversity is positively associated with soil bacterial diversity. This finding indicates that the relationship between plant diversity and bacterial diversity evolves across different succession stages. One plausible explanation for this shift is that in undisturbed grasslands or during the early succession stages, plant diversity provides a diverse range of niches for bacteria colonization and growth. However, in the late succession stage, such as forest, woody plant biomass, indicated by DBH [ 59 ], exhibits a significant negative correlation with soil bacterial diversity ( Table A3 ). This implies that higher woody plant diversity and biomass may promote plant–soil feedback that enhances the stability of soil bacteria rather than the diversity [ 42 ]. Such feedback may include nutrient cycling and modulation of the soil microenvironment, which can indirectly influence bacterial community structure and function [ 42 ]. As forest succession advances, plant regeneration occurs and exerts a notable influence on soil properties, including pH, organic inputs, and available nutrients ( Table 1 ). Notably, there are significant correlations between SOC, total N, AN, NO 3 − -N, and MBN with soil bacterial alpha diversity ( Table A3 ). This underscores the pivotal role of soil carbon and nitrogen in shaping bacterial diversity [ 60 ]. Intriguingly, while NH 4 + -N does not exhibit a significant correlation with the relative abundances of dominant bacterial phyla, NO 3 − -N does ( Figure 4 a, Table A3 ). This finding aligns with previous research suggesting that variations in bacterial communities during forest succession can be attributed to different N fractions [ 61 ]. The distinct relationship between NH 4 + -N and NO 3 − -N with bacterial abundances highlights the intricate interplay between nitrogen availability and bacterial dynamics during forest succession. It has been established that plant diversity exerts a global influence on soil fungal communities [ 61 ], a finding that is corroborated by research examining plant secondary succession on the Loess Plateau in China [ 21 ]. This influence primarily arises from the diverse range of food resources that plants provide to fungi, encompassing root exudates and litter [ 62 , 63 ]. In particular, fungal groups such as Ascomycota and Basidiomycota play crucial roles in the decomposition and rhizodeposition of organic substrates [ 62 , 64 ]. In our study, we observed that soil pH is the primary factor explaining the response of the soil fungal community to forest succession ( Figure 4 b and Figure 5 b, Table A2 ). This finding contrasts with the results showing that shrubland and primary forest have similar pH values ( Table 1 ), yet there is considerable variation in fungal richness ( Figure A2 ). This discrepancy may be attributed to the higher tree species richness in primary forest ( Table 1 ), as many ectomycorrhizal fungi belong to the phyla of Ascomycota and Basidiomycota identified in our study. Furthermore, Ascomycota and Basidiomycota show significant correlations with most soil properties, but not with woody plant diversity ( Table A3 ). This indicates that fungal community compositions, particularly the dominant phyla Ascomycota and Basidiomycota , respond significantly to forest succession, which is dependent on soil pH, C, and N dynamics. This phenomenon can be attributed to the dynamics of soil properties during forest succession in the subtropical climate and unique karst habitat. The karst terrain, characterized by its distinct geology and hydrology [ 16 ], combined with the subtropical climate, creates a unique environment that shapes soil properties and nutrient cycling. These conditions, in turn, directly affect fungal communities, which heavily rely on soil resources and conditions for their growth and reproduction. The significant role of soil pH, C, and N in determining fungal community structure and diversity underscores the importance of considering soil properties when studying fungal ecology in forest ecosystems."
} | 4,696 |
40213443 | PMC11935023 | pmc | 7,372 | {
"abstract": "As the field of wearable electronics continues to expand, the integration of inorganic thermoelectric (TE) materials into fabrics has emerged as a promising development due to their excellent TE properties. However, conventional thermal methods for fabricating TE fabrics are unsuitable for wearable applications because of their high temperatures, resulting in rigid TE materials. Herein, a nonthermally fabricated silver selenide (Ag 2 Se) TE fabric is developed that can be effectively integrated into wearable applications. Ag 2 Se nanoparticles are densely formed within the fabric through a simple in situ chemical reduction process, resulting in remarkable electrical stability even after 10 000 cycles of mechanical deformation, such as stretching and compression. Notably, the fabricated Ag 2 Se TE fabric exhibits superior stretchability, stretching ≈1.36 times more than the thermally treated Ag 2 Se TE fabrics, while retaining its excellent electrical conductivity. Moreover, the TE unit exhibits 9.80 μW m −1 K −2 power factor, 134.45 S cm −1 electrical conductivity, and −26.98 μV K −1 Seebeck coefficient at 370 K. A haptic sensing glove based on the Ag 2 Se TE fabric as a sensor for detecting potential hazards is demonstrated. The glove effectively distinguishes between simple touch, physical pain, and high‐temperature hazards, ensuring user safety and prompt response.",
"conclusion": "3 Conclusion In this study, we successfully fabricated a stretchable Ag 2 Se TE fabric through a simple in situ reduction process without thermal treatment. This nonthermal fabrication method resulted in the formation of durable Ag 2 Se TE NP networks within the fabric, providing high stretchability up to 350% tensile strain. The Ag 2 Se TE fabric exhibited remarkable electrical reliability under 10 000 cycles of mechanical deformation such as 20% tensile strain and 16 kPa pressure, making it suitable for use as a wearable sensor. Moreover, the Ag 2 Se TE fabric achieved a PF of 15.58 μW m −1 K −2 , electrical conductivity of 250 S cm −1 , and a Seebeck coefficient of −24.97 μV K −1 at 370 K. The Ag 2 Se TE fabric demonstrated the capacity to generate different output voltages in response to various temperature differences based on the Seebeck effect. To demonstrate its practical application, we integrated the Ag 2 Se TE fabric into a knitted glove, resulting in the development of a haptic sensing glove. This glove was designed to detect various stimuli, such as touch, physical pain, and high‐temperature hazards, achieved by utilizing analog input signals derived from the resistance and output voltage of the Ag 2 Se TE fabric, influenced by pressure, strain, and temperature differences. This facile and nonthermally created Ag 2 Se TE fabric‐based electronic device can pave the way for the application of inorganic TE materials in a stretchable form in wearable devices.",
"introduction": "1 Introduction With rapidly advancing textile‐based electronics, smart fibers have emerged as a critical component of functional textiles, distinguished by their remarkable responsiveness to various external stimuli. [ \n \n 1 \n , \n 2 \n , \n 3 \n , \n 4 \n , \n 5 \n \n ] These smart fibers boast an extensive range of capabilities, including sensory functions [ \n \n 6 \n , \n 7 \n , \n 8 \n , \n 9 \n \n ] and energy‐harvesting abilities. [ \n \n 10 \n , \n 11 \n , \n 12 \n \n ] This innovation is currently driving the emergence of novel fiber‐based wearable electronics, encompassing applications such as electronic skin, [ \n \n 13 \n , \n 14 \n \n ] health sensors, [ \n \n 15 \n , \n 16 \n \n ] and interfaces for human–machine interaction. [ \n \n 17 \n , \n 18 \n \n ] As the field of fiber‐based wearable electronics continues to evolve, the integration of thermoelectric (TE) materials into fibers has become a significant development. [ \n \n 19 \n , \n 20 \n , \n 21 \n , \n 22 \n , \n 23 \n , \n 24 \n \n ] TE materials efficiently convert temperature gradients into electrical output voltage and vice versa. [ \n \n 25 \n \n ] This integration introduces a groundbreaking ability to harvest electrical energy from the natural heat generated by the human body. [ \n \n 26 \n , \n 27 \n \n ] Furthermore, TE fibers enable the detection of temperature variations and changes in posture by measuring output voltage and resistance. [ \n \n 28 \n , \n 29 \n , \n 30 \n \n ] \n The pursuit of using inorganic TE materials for wearable applications has garnered significant attention in recent research. [ \n \n 22 \n , \n 23 \n , \n 24 \n , \n 31 \n , \n 32 \n , \n 33 \n \n ] Among these efforts, silver selenide (Ag 2 Se)‐based materials have emerged as promising candidate because of their unique TE properties. [ \n \n 34 \n , \n 35 \n , \n 36 \n \n ] The incorporation of Ag 2 Se into fibers has been a cautious endeavor in several studies, primarily due to the inherent rigidity associated with inorganic TE materials. [ \n \n 31 \n , \n 32 \n , \n 33 \n \n ] Yang et al. suggested an approach for creating a three‐dimensional (3D) TE generator with biaxial stretchability. [ \n \n 31 \n \n ] By seamlessly integrating inorganic Ag 2 Se filmstrips into a knit fabric, aligned with the vertical heat flux direction, a stable temperature difference of 5.2 °C was realized when the fabric was in contact with the wrist at room temperature. Research is currently underway to explore TE units in fiber form, rather than in film form, to improve their suitability for wearable electronics applications. Vinodhini et al. reported a one‐step solvothermal method for integrating Ag 2 Se and Ag 2 S with conductive carbon fabric (CF). [ \n \n 32 \n \n ] Ag 2 Se CF and Ag 2 S CF exhibited power factors (PFs) of 6.7 and 24 μW mK −2 . However, during the material synthesis process, applying heat at 180 °C for 24 h frequently resulted in the formation of rigid structures. The only research on nonthermal Ag 2 Se fabric has been recently reported. Liu et al. introduced a two‐step impregnation method for fabricating a 3D TE network. [ \n \n 33 \n \n ] The TE network demonstrated an elongation of over 100%. The resulting network‐based TE generator achieved an output power of 4 μW cm −2 . Nevertheless, the process can be complicated and time‐consuming due to multiple steps, including solution preparation and impregnation. For the effective integration of Ag 2 Se into wearable electronics, a simple and nonthermal approach is essential. Here, we fabricated a stretchable Ag 2 Se TE fabric through a simple and nonthermal process. Ag 2 Se nanoparticles (NPs) were densely and rapidly formed within the cotton fabric using a chemical reduction method without the need for thermal treatment. Due to the durable NP networks, the stretchable Ag 2 Se TE fabric exhibited remarkable electrical stability under repeated conditions of 20% lateral strain and 16 kPa normal pressure. Remarkably, it maintained its electrical path even under the strain of 200%, surpassing the thermally treated Ag 2 Se TE fabric in terms of electrical and mechanical performance. Moreover, the TE unit exhibited a PF of 9.80μW m −1 K −2 , electrical conductivity of 134.45 S cm −1 , and a Seebeck coefficient of −26.98 μV K −1 at 370 K. To demonstrate the practical utility of the fabricated fabric, a haptic sensing glove, capable of detecting changes in strain, pressure, and temperature, was developed, which effectively alerted users to potential hazards related to elevated temperatures and exhibited accurate sensing capabilities for pressure and tensile forces, thereby enhancing its real‐world applicability.",
"discussion": "2 Results and Discussion The stretchable Ag 2 Se TE fabric is fabricated by incorporating Ag 2 Se NPs into a cotton fabric, as illustrated in Figure \n \n 1 a . This process was achieved using a simple and nonthermal chemical reduction method. To ensure the TE fabric stretchability, commercially available cotton fabric was used as the foundational structure. The nonthermal chemical reduction method comprises two distinct steps. Initially, the cotton fabric was immersed in the Ag 2 Se precursor solution for 20 min, allowing it to swell and absorb the solution. The Ag 2 Se precursor solution was formulated by blending two distinct precursor powders with an ethylene glycol (C 2 H 4 (OH) 2 ) solvent. Specifically, silver trifluoroacetate (CF 3 COOAg) and sodium selenite pentahydrate (Na 2 SeO 3 ) were selected as Ag and Se precursor. Subsequently, hydrazine hydrate (N 2 H 4 ) was applied to the fabric to reduce the absorbed Ag 2 Se precursor solution, resulting in the rapid formation of dense Ag 2 Se NPs within the fabric. The reduction reaction between the precursor solution and the reductant occurred instantly without any thermal treatment. Following the reduction process, the fabric is washed with deionized (DI) water to remove any residual hydrazine hydrate. Figure 1b shows the X‐ray diffraction (XRD) patterns of the Ag 2 Se TE powder synthesized using the nonthermal chemical reduction method. These patterns revealed the crystallinity of Ag 2 Se and were aligned with the diffraction lines of (0 0 2), (1 1 1), (1 0 2), (1 2 0), (1 1 2), (1 2 1), (0 1 3), (1 0 3), (1 1 3), (0 3 2), (0 0 4), (1 2 3), (2 2 1), (1 1 4), and (0 4 2). [ \n \n 34 \n , \n 37 \n \n ] Figure S1, Supporting Information, shows an optical image of the Ag 2 Se TE fabric with a deep brown color. Figure 1c shows the top‐view images acquired through scanning electron microscopy (SEM) and energy‐dispersive X‐ray spectroscopy (EDS) for the Ag 2 Se TE fabric. The SEM image provided a detailed depiction of the structure of the TE fabric with a bundle of fine fibers. The stretchability of the TE fabric can be attributed to its interwoven fiber‐based structure. In EDS images, the formation of NPs was depicted in color, with Ag and Se NPs appearing as red and green dots. Figure 1d shows SEM images of the Ag 2 Se TE fabric with and without thermal treatment. Figure 1d ‐(i) shows the surface of the Ag 2 Se TE fabric treated at 200 °C for 2 h. The surface became film‐like, making it susceptible to mechanical deformation caused by routine human activities, potentially resulting in the formation of cracks on the surface and potential loss of electrical conductivity. The film‐like Ag 2 Se layer can be easily peeled off after cyclic mechanical deformation (Figure S2, Supporting Information). Conversely, the surface of the nonthermally treated Ag 2 Se TE fabric maintained an NP morphology (Figure 1d ‐(ii)). This NP morphology allowed external forces to disperse between NPs, making the fabric resistant to mechanical deformation and enhancing its suitability for wearable applications. The Ag 2 Se NPs remained attached to the surface of the fiber even after cyclic mechanical deformation (Figure S3, Supporting Information). Figure 1e shows the resistance of the Ag 2 Se TE fabric with and without thermal treatment under tensile strain. The Ag 2 Se TE fabric without thermal treatment maintained electrical conductivity over a broader range of tensile strain, stretching ≈1.36 times more than the thermally treated Ag 2 Se TE fabric. Notably, the Ag 2 Se TE fabric exhibited high electrical conductivity even without thermal treatment. Figure 1 a) Schematic of the fabrication process of the Ag 2 Se TE fabric using the nonthermal chemical reduction method. b) XRD patterns of the Ag 2 Se TE fabric. c) Top view of SEM and EDS images of the Ag 2 Se TE fabric. d) (i) SEM image of Ag 2 Se TE fabric with thermal treatment. (ii) SEM image of Ag 2 Se TE fabric without thermal treatment. e) Resistance of the Ag 2 Se fabric with and without thermal treatment under different tensile strain levels. © 2024 WILEY‐VCH GmbH \n Figure \n \n 2 a shows the relative changes in resistance according to the tensile strain of the Ag 2 Se TE fabric. As the applied tensile strain increased, the relative changes in resistance increased due to the intrinsic piezoresistance of Ag 2 Se NPs. This increase was reflected in the change in the gauge factor of the Ag 2 Se TE fabric, increasing from 1.9 to 16.5. The electrical reliability of the Ag 2 Se TE fabric was evaluated under repeated mechanical deformations. As shown in Figure 2b, a stretching test with 20% tensile strain was conducted. Remarkably, the Ag 2 Se TE fabric maintained high electrical reliability throughout 10 000 stretching cycles. Figure 2c shows the relative changes in the resistance of the Ag 2 Se TE fabric under stretching and releasing deformations at 20%, 40%, 60%, 80%, and 100% tensile strains. The increase in tensile strain corresponded to a proportional increase in the relative changes in resistance, demonstrating the ability to distinguish between different levels of tensile strain. Figure 2d shows the relative changes in resistance according to the pressure of the Ag 2 Se TE fabric. The relative changes in resistance decreased, reaching ≈−90% at the maximum compression point of 32 kPa due to the increased contact area between Ag 2 Se TE fibers under pressure, thereby improving electrical conductivity. The electrical stability of the Ag 2 Se TE fabric was confirmed under 16 kPa pressure. Figure 2e shows that even after 10 000 repeated compression cycles, the Ag 2 Se TE fabric maintained its electrical reliability. Figure 2f shows the relative changes in resistance observed in the Ag 2 Se TE fabric during cyclic compression and release deformations at 30 kPa pressure over five cycles. These cycles consistently exhibited a substantial reduction in relative changes in resistance, reaching ≈−90% when the Ag 2 Se TE fabric was touched. Under tensile strain, the Ag 2 Se TE fabric exhibited an increase in resistance, while pressure decreased the resistance. This distinct electrical behavior of the fabric depicted a clear differentiation between tensile strain and pressure. Figure 2g shows the strain–stress curve of the Ag 2 Se TE fabric with and without thermal treatment up to mechanical fracture. The Ag 2 Se TE fabric without thermal treatment fractured at 325% tensile strain, exhibiting Young's modulus of 870 Pa. However, after thermal treatment, the fabric exhibited a lower fracture point at 260% tensile strain, along with an increased Young's modulus of 2100 Pa, indicating that the nonthermally treated fabric was more flexible and stretchable than the thermally treated fabric. Figure 2h shows the force of the Ag 2 Se TE fabric as a function of tensile strain, 50–200% values. Mechanical hysteresis is in all loading and unloading cycles; however, its impact is negligible, emphasizing the mechanical stability of the fabric. Figure 2i shows the results of the thermogravimetric analysis (TGA) conducted on the Ag 2 Se TE fabric, revealing information about the weight percentages of Ag 2 Se NPs. The TGA graph indicated that Ag 2 Se NPs were densely formed within the TE fabric, constituting ≈45 wt%. Figure 2 a) Relative changes in the resistance of the Ag 2 Se TE fabric under tensile strain levels up to 200%. b) Resistance changes of the Ag 2 Se TE fabric during 10 000 stretching cycles from 0% to 20% tensile strain. c) Relative changes in the resistance of the Ag 2 Se TE fabric during cyclic stretching and releasing deformation at a 20%, 40%, 60%, 80%, and 100% tensile strain. d) Relative changes in the resistance of the Ag 2 Se TE fabric under pressure up to 30 kPa. e) Resistance change of the Ag 2 Se TE fabric during 10 000 compressing cycles from 0 to 16 kPa. f) Relative changes in the resistance of the Ag 2 Se TE fabric during cyclic compression and release deformation at a pressure of 30 kPa for five cycles. g) Strain–stress curve of the Ag 2 Se TE fabric with and without thermal treatment up to mechanical fracture. h) Force of the Ag 2 Se TE fabric against tensile strain every 40% up to 200%. i) Weight (%) of Ag 2 Se NPs in the TE fabric determined by TGA. © 2024 WILEY‐VCH GmbH \n Figure \n \n 3 a–c shows the thermoelectrical performance of the Ag 2 Se TE fabric (5 × 1 × 10 mm 3 ). Figure 3a shows the output performance with the current of the fabric at various temperature differences (Δ T ). The maximum power output was determined by achieving a balance between external and internal resistances. [ \n \n 38 \n , \n 39 \n \n ] The maximum power values were ≈0.01656, ≈0.05376, ≈0.19392, ≈0.5250, and ≈0.082236 μW at various Δ T values of 30, 45, 60, 70, and 80 K, respectively. Figure 3b shows the experimentally measured output voltage of the Ag 2 Se TE fabric at several Δ T values, showing a linear increase as Δ T increases. Figure 3c shows the temperature‐dependent TE properties of the Ag 2 Se TE fabric, including the Seebeck coefficient ( S ), electrical conductivity ( σ ), and PF. Electrical conductivity, represented by the black line, increased but then sharply decreased from 250 to 140.36 S cm −1 as the temperature increased from 370 to 420 K. The blue line indicates the Seebeck coefficient with a negative sign, indicating that electrons predominate as the majority carriers in this n‐type material. The absolute Seebeck coefficient gradually decreased and then rapidly decreased from 24.97 to 13.24 μV K −1 within 370–420 K. Consequently, the PF increased from 9.79 μW m −1 K −2 at 300 K to a maximum of 15.58 μW m −1 K −2 at 370 K, followed by a sharp decrease when the temperature exceeded 370 K. Above 300 K, intrinsic conduction prevailed, resulting in enhanced thermal excitation and subsequent growth in the carrier concentration, and contributing to enhanced electrical conductivity. The abrupt change in TE performance from 370 to 420 K can be attributed to the phase transition of Ag 2 Se, transitioning from semiconducting orthorhombic to superionic cubic, occurring at ≈407 K. [ \n \n 40 \n , \n 41 \n \n ] Figure 3d shows the consecutive electrical responses of the Ag 2 Se TE fabric across various temperature gradients (10–95 K). The Ag 2 Se TE fabric efficiently transformed the thermal energy into electrical energy, yielding an output voltage. The peak output voltage, observed under the largest temperature difference of 95 K, reached ≈2.7 mV. Figure 3e shows the Seebeck coefficients of the Ag 2 Se TE fabric under various applied tensile strains. Notably, the Seebeck coefficients remain constant. As the Seebeck coefficient directly correlated with the output voltage, a parameter crucial for Δ T sensing, it indicated that the Δ T sensing ability of the fabric remained consistent under different tensile strain levels. Figure 3f shows that the output voltage of the Ag 2 Se TE fabric remained consistently stable across varying pressures (0–20 kPa) for each Δ T value, indicating that Δ T can be reliably detected using the fabric, even when it is subjected to applied pressure. Figure 3g shows current–voltage ( I – V ) curves under different applied pressures and tensile strains, showing the pressure and tensile strain sensing mechanism of the Ag 2 Se TE fabric. The curve slopes increased around the origin with increasing pressure levels due to the decreasing resistance of the fabric under applied pressure. Specifically, the resistance of the fabric decreases from 1.43 kΩ under 0 kPa to 1.11, 0.83, 0.56, and 0.33 kΩ under 0.5, 1, 2.5, and 5 kPa. Conversely, slopes exhibited a decrease around the origin with increasing tensile strain levels, as shown in Figure 3g,h . This decrease in slope was due to the increasing resistance of the fabric under the applied tensile strain. Figure 3h provides a closer look at the I – V curves from Figure 3g under different tensile strain levels. The resistance of the Ag 2 Se TE fabric was 1.43 kΩ under strain of 0% and increased to 2, 3.33, 5, 10, 20, 22.2, 27.03, 40, 62.5, and 100 kΩ under strain of 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, and 200%, respectively. Due to these distinctive resistance behaviors under pressure and tensile strain, these two mechanical stresses were distinguished using the Ag 2 Se TE fabric. Consequently, the Ag 2 Se TE fabric enabled the ability to distinguish touch that occurred during repeated 5% and 10% tensile strains as shown in Figure 3i . Notably, when the fabric was touched, the relative changes in resistance decreased to ≈−40%. This contrasts starkly with the resistance changes of ≈150% and 200% observed during cyclic 5% and 10% tensile strain. Figure 3 a) Output voltage and power of the Ag 2 Se TE fabric with current at various temperature differences (Δ T ). b) Experimental output voltages of the Ag 2 Se TE fabric at various Δ T values. c) Temperature dependence of Seebeck coefficient ( S ), electrical conductivity ( σ ), and PF of the Ag 2 Se TE fabric. d) Sequential electrical responses of the Ag 2 Se TE fabric at various Δ T , ranging from 10 K to 95 K. e) S of the Ag 2 Se TE fabric under varying tensile strain up to 100%. f) Output voltages of the Ag 2 Se TE fabric under different pressures (0–20 kPa) at each Δ T , ranging from 10 K to 60 K. g) I – V curves of the Ag 2 Se TE fabric taken at different pressures (0–5 kPa) and tensile strain (0–100%). h) I–V curves of the Ag 2 Se TE fabric taken at different tensile strains, ranging from 0% to 200%. i) Relative changes in the resistance of the Ag 2 Se TE fabric under finger touch and repeated 5% and 10% tensile strain. © 2024 WILEY‐VCH GmbH The Ag 2 Se TE fabric exhibited electrical responsiveness to pressure, strain, and temperature differences through changes in resistance and output voltage. We used these features to detect potential hazards such as physical pain and high temperatures using a glove integrated with Ag 2 Se TE fabrics as a haptic sensor. The haptic sensing glove was fabricated by simply weaving Ag 2 Se TE fabrics onto the fingertips and palms of a knitted glove. Figure \n \n 4 a shows a schematic of the hazard detection system using the fabricated haptic sensing glove. The haptic sensing glove transmits analog signals such as resistance and output voltage in response to pressure, strain, and heat generated during contact with Arduino. These signals are then transmitted to a mobile phone via a Bluetooth module. The application analyzed the received information about pressure, strain, and heat to determine whether it was a simple touch, physical pain, or high‐temperature hazard. The results were then displayed within the application accordingly. Figure 4b shows optical images of the haptic sensing glove, showing the Ag 2 Se TE fabrics embedded into the fingertips (1 × 0.7 cm 2 ) and palm (2 × 1.2 cm 2 ) of the knitted glove (Figure 4b ‐(ii)). Figure 4b ‐(i) shows a magnified view of the fabric structure at the fingertip of the haptic sensing glove. By integrating Ag 2 Se TE fabrics as sensing components, the haptic sensing glove exhibited great potential as a wearable electronic device, offering exceptional conformability, flexibility, and stretchability. Figure 4c–e shows the practical implementation of the haptic sensing glove for detecting simple touch, physical pain, and high‐temperature hazards. Figure 4c shows a demonstration of touch sensing detection, where glove touch interactions lead to a noticeable decrease in the resistance of the Ag 2 Se TE fabric, enabling the detection of simple touch inputs. Figure 4d shows pain detection, where the application of stimuli such as pinching induces tensile strain in the Ag 2 Se TE fabric, increasing its resistance and allowing the detection of physical pain through changes in fabric resistance. Figure 4e shows heat detection, where the temperature difference caused by holding hot objects triggered the generation of an output voltage in the Ag 2 Se TE fabric. This distinctive characteristic has enabled the detection of potential high‐temperature hazards. The applications of the haptic sensing glove, including the detection of simple touch, physical pain, and high‐temperature hazards, are presented in Movie S1 (Supporting Information). Additionally, the Ag 2 Se TE fabric demonstrates superior stretchability and a straightforward, nonthermal fabrication process compared to previously reported TE fibers and fabrics, as detailed in Table S1 (Supporting Information). Figure 4 a) Schematic illustration of the haptic sensing glove‐based potential hazard detection system. b) Optical image of the haptic sensing glove. c) (i) Demonstration of touch sensing using the haptic sensing glove. (ii) Pressure sensing‐based touch sensing mechanism. d) (i) Demonstration of pain detection using the haptic sensing glove. (ii) Strain sensing‐based pain detection mechanism. e) (i) Demonstration of heat detection using the haptic sensing glove. (ii) Output voltage sensing‐based heat detection mechanism. © 2024 WILEY‐VCH GmbH"
} | 6,165 |
39347544 | PMC11542596 | pmc | 7,373 | {
"abstract": "ABSTRACT The ruminant-microorganism symbiosis is unique by providing high-quality food\nfrom fibrous materials but also contributes to the production of one of the\nmost potent greenhouse gases—methane. Mitigating methanogenesis in\nruminants has been a focus of interest in the past decades. One of the\npromising strategies to combat methane production is the use of feed\nsupplements, such as seaweeds, that might mitigate methanogenesis via\nmicrobiome modulation and direct chemical inhibition. We conducted\n in vitro investigations of the effect of three seaweeds\n( Ascophyllum nodosum , Asparagopsis\ntaxiformis , and Fucus vesiculosus ) harvested\nat different locations (Iceland, Scotland, and Portugal) on methane\nproduction. We applied metataxonomics (16S rRNA gene amplicons) and\nmetagenomics (shotgun) methods to uncover the interplay between the\nmicrobiome’s taxonomical and functional states, methanogenesis rates,\nand seaweed supplementations. Methane concentration was reduced by\n A. nodosum and F. vesiculosus , both\nharvested in Scotland and A. taxiformis , with the greatest\neffect of the latter. A. taxiformis acted through the\nreduction of archaea-to-bacteria ratios but not eukaryotes-to-bacteria.\nMoreover, A. taxiformis application was accompanied by\nshifts in both taxonomic and functional profiles of the microbial\ncommunities, decreasing not only archaeal ratios but also abundances of\nmethanogenesis-associated functions. Methanobrevibacter \n“SGMT” ( M. smithii, M. gottschalkii, M.\nmillerae or M. thaueri ; high methane yield) to\n“RO” ( M. ruminantium and M.\nolleyae ; low methane yield) clades ratios were also decreased,\nindicating that A. taxiformis application favored\n Methanobrevibacter species that produce less methane.\nMost of the functions directly involved in methanogenesis were less\nabundant, while the abundances of the small subset of functions that\nparticipate in methane assimilation were increased. IMPORTANCE The application of A. taxiformis significantly reduced\nmethane production in vitro . We showed that this reduction\nwas linked to changes in microbial function profiles, the decline in the\noverall archaeal community counts, and shifts in ratios of\n Methanobrevibacter “SGMT” and\n“RO” clades. A. nodosum and F.\nvesiculosus , obtained from Scotland, also decreased methane\nconcentration in the total gas, while the same seaweed species from Iceland\ndid not.",
"conclusion": "Conclusions The in vitro application of A. taxiformis as a\nfeed supplement resulted in a drastic reduction of CH 4 concentration\nproduced with minor effects on nutrient degradation. The reduction was closer to\n100% for three of the four replicates, indicating a significant potential for\nCH 4 reduction. The present study suggests the AT mitigation of\nCH 4 concentration is caused not only by the competitive\ninhibition of F 430 coenzyme ( 18 ) but also through a decreased portion of the archaeal domain in\nthe microbiome, as well as lower ratios of Methanobrevibacter \n“SGMT” to “RO” clades, and changes in the abundances\nof methane-associated microbial functions. Abundances of most of the KEGGs that\nare directly involved in methanogenesis were decreased, as well as KEGGs that\nare associated with it indirectly through the synthesis of\nmethanogenesis-related compounds, such as F 420 cofactor, coenzyme M,\nand methanofuran biosynthesis and extension of 2-Oxocarboxylic chain.\nAdditionally, a small group of KEGGs that participate in CH 4 \nassimilation via the serine pathway were more prevalent. A.\nnodosum and F. vesiculosus also decreased methane\nconcentration in the total gas (2%–19%) at the 2.5% inclusion level;\nhowever, only the seaweed samples from Scotland decreased it significantly.",
"introduction": "INTRODUCTION Ruminants are an important source of meat and dairy products. They also produce\nmethane (CH 4 ) ( 1 , 2 ) through microbial fermentation ( 3 ) mainly occurring in the reticulorumen.\nCH 4 is known as one of the greatest contributors to greenhouse gas\nemissions ( 4 ). Moreover, CH 4 \nproduction contributes to feed energy loss by the host ( 5 ). Numerous efforts have been dedicated to investigating energy\nloss through methanogenesis in ruminants in the last decades ( 6 , 7 ). One of the most\npromising approaches to mitigate CH 4 production by livestock is the\napplication of specific feedstuffs and feed supplements, including seaweeds ( 8 – 10 ). Such studies were\nimplemented using both in vivo ( 11 ) and in vitro ( 12 – 15 ) experiments. The red seaweed\n Asparagopsis taxiformis is particularly effective in\nmethanogenesis inhibition ( 13 ) due to its\nhigh bromoform content ( 16 ). Bromoform acts\nas a competitive inhibitor of methanogenesis in virtue of its high chemical\nsimilarity to the F 430 coenzyme ( 17 , 18 ). However, it has been\nreported that A. taxiformis mitigation of CH 4 production\ncannot be explained by only direct competition of bromoform with F 430 \ncoenzyme and considerably surpasses it ( 19 ).\nThe CH 4 reduction effect of brown seaweeds like Ascophyllum\nnodosum ( 15 , 20 ) or Fucus vesiculosus \n( 21 ) is proposed to be caused by\nphlorotannins. However, the effect of these seaweeds on CH 4 production is\nnot as clear as that of A. taxiformis . In addition, tannins have\nbeen described to affect the protein metabolism in the rumen. Seaweeds containing\ntannins may also exert effects on the microbial degradation of dietary proteins\n( 22 ). Enteric CH 4 in ruminants is mostly produced by archaeal methanogens in\nsymbiosis with fiber-degrading bacteria and hydrogen (H 2 ) producing\nprotozoa ( 23 , 24 ). Therefore, CH 4 reduction may be associated with reduced\nfiber degradation, an undesirable outcome because the degradation of fiber is a big\nadvantage of ruminants compared to other animals. Although numerous studies have\nbeen performed on the effects of seaweeds on rumen microbiome, to the best of our\nknowledge, there is no study investigating the effect of seaweed additives\n(particularly, A. taxiformis ) on microbial functions. The rumen is an important part of the ruminants’ digestive tract with a very\ncomplex microbial community, and therefore, it is difficult to create strictly\ncontrolled conditions for in vivo studies. Moreover, increased\nawareness of animals’ welfare stimulates researchers to develop and use\n in vitro alternatives to in vivo studies, such\nas the rumen simulation technique (Rusitec) or Hohenheim gas test (HGT). Rusitec is\na semi-continuous cultivation system and allows constant inflow and outflow of the\nsubstrates and artificial saliva and, therefore, is well-regulated and balanced\n( 25 ) The HGT is a widely accepted method\nfor gas production (GP) measurements used for the estimation of digestibility or\nscreening of feed additive effects on methane production ( 26 , 27 ). Our objective was to study the effect of five seaweeds on total gas and\nCH 4 production, nutrient degradation, and microbial composition and\nfunctions in in vitro systems. We hypothesized that seaweeds affect\nmethanogenesis not only through biochemical inhibition but also by alteration of\nmicrobial (specifically methanogens) composition and functions. Additionally,\nseaweeds were compared by species and sampling places as two species were harvested\nat different locations.",
"discussion": "DISCUSSION In vitro fermentation characteristics Among all seaweed supplements tested, AT resulted in the largest decrease in\nCH 4 concentration of total gas production in both HGT and\nRusitec. These findings are in agreement with previous results ( 11 – 13 , 28 ). Negative effects of AT supplementation\non nutrient degradation ( Table 1 ) were\nhardly observed, making this seaweed a desirable candidate as a feed supplement\nto mitigate methanogenesis. Only CP degradation was significantly reduced by AT\nsupplementation. This indicates that a higher amount of CP was not degraded by\nmicrobes in the rumen and can therefore be used at the duodenum by the animal\ndirectly ( 29 ). Despite the great\npotential of AT in reducing CH 4 production by ruminants, there are\nincreasing concerns regarding the safety of such applications since the high\ncontent of its main anti-methanogenic compound—bromoform—has been\nreported to be toxic ( 30 ) and able to\naccumulate in milk ( 31 ). In addition, AT\nis inherently high in iodine concentration, and there are limits on how much\niodine can be fed to animals producing meat and milk for human consumption in\nsome countries ( 32 ). Therefore, further\nstudies are required to investigate if AT inclusion can be reduced to levels\nthat would avoid negative effects on animals and limit bromoform and iodine\nlevels in the end products while minimizing methanogenesis. In the Rusitec experiment, there was a high variation in fermentation traits and\nmicrobial data among the fermenters with AT supplementation, indicated by high\nvariability in CH 4 concentration compared to other seaweeds and\ndistribution of FL samples in metataxonomics. This variability was also observed\nin the HGT experiment for the AT treatment, suggesting that either A.\ntaxiformis itself or the heterogeneity of the applied stock\nmaterial led to these changes. CH 4 production was almost non-existent\nin three out of four fermenters, likely due to fermenter instability or\ninconsistent anti-methanogenic compounds in A. taxiformis. The seaweeds sampled in Iceland (AN1 and FV1) had a non-significant reduction on\nCH 4 concentration produced in the Rusitec (2.4% and 4.8%,\nrespectively). However, the same seaweed species harvested at a similar time in\nScotland (AN2 and FV2) did significantly decrease it (7.7% and 19%,\nrespectively), especially FV2. Previous research on Icelandic AN and FV did show\na reduction in CH 4 concentration in total gas produced (reduction of\n17% and 11%, respectively, at 5% seaweed inclusion) ( 15 ). The differences in whether or not these species reduce\nCH 4 production are likely due to their bioactive content, such as\nconcentrations of phlorotannins or total phenolic content. This extent of\nreduction in CH 4 production for the AT harvested in the North\nAtlantic Ocean is consistent with previous findings with AT harvested in the\nPacific Ocean which also generally showed a substantial reduction in\nCH 4 production ( 12 , 13 , 28 ). Microbiome composition and domain ratios Among all seaweeds applied, the most prominent effect on methane concentration\nwas observed for AT supplementation ( Fig.\n1 ). The reducing effect of AT supplementation on CH 4 \nproduction in ruminants was already reported in in vitro ( 12 , 13 , 33 ) and in\nvivo ( 11 , 28 ) experiments. In our data, decreased\nmethane production for AT-supplemented TMRs was accompanied by changes in\narchaeal counts, overall microbiome composition, and functional profiles. Based on the “shotgun” metagenomic analysis, A/B ratios were lower\nin AT compared to all other supplementations and TMR alone in FL and FR samples\n( Fig. 7 ). This finding indicates that\nAT caused growth suppression of archaea and therefore the main rumen\nmethanogens. Previous studies indicated that methanogen abundances are affected\nby various compounds, such as carbohydrates, lipids, peptides, phlorotannins,\nbromoform, and others, leading to a decline in methanogenesis ( 34 , 35 ). Similar to our metagenome analysis, it was shown that AT\nsupplementation resulted in drastically lower counts of archaeal methanogens in\nthe Rusitec compared to the control, based on metataxonomics data ( 36 ). When A/B ratios were assessed based on\nour metataxonomics data, they were also lower in AT-treated samples compared to\nthe TMR, AN1, and AN2 treatments in FL and compared to TMR and FV2 in FR (Fig.\nS2). Moreover, five samples from the 16S rRNA gene library with the lower counts\nof archaeal reads were attributed to the AT treatment (data not shown). It is well known that archaeal methanogens are closely associated with protozoa\n( 37 , 38 ), and it was shown that abundances of protozoa are declining with\ntime in the Rusitec ( 13 ). To test if the\ndecline of the A/B ratio in AT-supplemented samples is associated with Eukaryota\nabundances, we also tested E/B and A/E ratios ( Fig. 7 ). In the FL sample type, both ratios were significantly\naffected by seaweed supplementation. However, the E/B ratio demonstrated no\ndifferences between treatments when tested pairwise, while the A/E ratio was\nlower in AT-supplemented samples compared to the TMR alone and to all other\ntreatments, suggesting that lower archaeal counts in the AT were not provoked by\nthe decline in the total eukaryotic community. However, even if the total amount\nof eukaryotic microorganisms was not affected by the AT treatment, the abundance\nof some protozoa changed ( Fig. 6 ). Thus, AT\nsupplementation resulted in the decline of S. strix , T.\nvaginalis , T. foetus , and unclassified\n Entamoeba . Though it was not yet directly shown that\n S. strix produces hydrogen, several hydrogenases were\nidentified in its single-cell metagenomics study ( 39 ). Both T. vaginalis and T.\nfoetus are parasitic protists ( 40 ) and possess the ability to produce hydrogen due to the presence\nof special organelles—hydrogenosomes ( 41 ). Entamoeba species were shown to host similar\nto hydrogenosome organelles in their ability to produce hydrogen-mitosomes\n( 42 , 43 ). An important implication of these findings is that the decrease\nof the archaeal community representation under AT supplementation is associated\nwith the decline of specific protozoa that are producing hydrogen, rather than\nwith the overall decrease of Eukaryota. These results are consistent with a\nrecent study that stated the decline in the methanogens activity was not solely\ndependent on the Rusitec-specific shifts in the microbiome composition but was\ndue to AT supplementation, as it was the only treatment tested that caused it\n( 36 ). It has also been suggested that the composition of archaeal methanogens from the\n Methanobrevibacter genus, rather than their joined relative\nabundances, is responsible for methanogenesis inhibition ( 44 ). It was proposed that greater abundances of the\n Methanobrevibacter “SGMT” clade, which\nincludes M. smithii , M. gottschalkii ,\n M. millerae , and M. thaueri , and its ratio\nto another Mbb. clade “RO” ( M. ruminatium and\n M. olleyae ) are associated with higher production of\nCH 4 ( 45 – 47 ). Our results are consistent with that hypothesis and\ndemonstrated that “SGMT” to “RO” ratios were lower\nin AT-treated samples ( Fig. 7 ) when\ncompared to the TMR alone in FL. Though 16S rRNA gene amplicon sequencing\napproaches do not provide reliable species-level annotations, our results\ndemonstrate that at the ASV level, numerous sequences assigned to the same\ngenus, Methanobrevibacter (A), were separated into two clusters\nbased on the positive or negative effect of the AT treatment at their abundances\n( Fig. 5 ). Both metataxonomics and\nmetagenomics revealed the negative effects of AT treatment on\n Methanomicrobium abundances. In addition, based on the\nmetagenomic data, the abundance of M. millerae , one of the\n“SGMT” clade members, decreased when AT was supplemented ( Fig. 6 ). Of note, both FV1 and FV2 treatments\nincreased abundances of Methanobrevibacter A and decreased\n Methanobrevibacter B. This indicated that the modulation of\nboth methanogen abundances and their composition are important aspects in\ndeveloping CH 4 mitigation strategies. Regarding bacterial genera, our data demonstrated that\n Prevotella abundances were positively affected by AT\nsupplementation. It is likely that excessive availability of H 2 ,\naccumulated due to suppressed overall methanogenesis, resulted in greater\nrelative abundances of that genus members ( Fig.\n5 ), which are competing with methanogens for hydrogen utilization.\n Prevotella abundances were previously shown to be reversely\nassociated with methane production ( 15 ,\n 35 , 48 ). It was also recently shown that AT treatment caused an increase\nin Prevotella abundance in a Rusitec study ( 36 ). However, we should not completely\nexclude the possibility that hydrogen-consuming bacteria are somehow favored by\nAT treatment and decrease relative abundances of archaeal methanogens by direct\ncompetition. Some other bacteria that increased their abundances in AT-treated\nsamples belonged to the Streptococcus ,\n Limosilactobacillus , Ruminobacter , and\n Limimorpha genera. One of them,\n Streptococcus , is known as an anti-methanogenic bovicin\ncomponent producer ( 49 ) .\nLimosilactobacillus member Lactobacillus reuteri \ninhibits methanogenesis ( 50 ).\n Ruminobacter is a genus of bacteria that produces formate,\nacetate, and succinate ( 51 ). Like\n Methanobrevibacter A methanogen,\n Limimorpha sequences were clustered into two groups of\nASVs, positively or negatively affected by AT supplementation, though the exact\nreason for it is yet not known to us. Microbiome functional profiles Among all seaweeds tested, A. taxiformis affected the most\nmicrobial functional profiles, especially functions associated with\nmethanogenesis. The abundances of more than 60% of such functions were decreased\nafter AT supplementation, while around 10% increased ( Fig. 8 ). Such drastic effects corresponded with changes in\n Methanobrevibacter clades ratios and indicate that AT\nmodulates methanogenesis not only through suppression of the total methanogens\npopulation but also via modulation of their taxonomical and functional profiles.\nFor instance, among KEGGs that are directly involved in CH 4 \nproduction, two from the acetoclastic pathway were augmented by AT, while the\nremaining DAFs, included in the hydrogenotrophic, methylotrophic, and\nacetoclastic methanogenesis pathways, decreased their abundances ( Fig. 9 , “Methanogenesis”). The\nreduction of methanogenesis can be accomplished not only by decreasing\nCH 4 production but also by enhancing its utilization. In our\nstudy, we observed that most of the KEGGs, which were positively affected by the\nAT supplementation, were involved in the CH 4 utilization through\nformaldehyde assimilation ( Fig. 9 ,\n“Formaldehyde assim.”), especially involved in the serine pathway.\nOther important pathways that were affected by AT supplementation and that are\nrelated to the methanogenesis are 2-Oxocarboxylic chain extension, Acetyl-CoA\npathway, and biosynthesis of such components as cofactor F 420 ,\ncoenzyme M (MCR), and methanofuran. It was shown that 2-oxocarboxylic acid is a\nprecursor for coenzyme B and methanofuran biosynthesis, both of which\nparticipate in methanogenesis ( 52 – 54 ). In our study, KEGGs associated with 2-Oxocarboxylic chain\nextension ( Fig. 9 , “Methanogenesis\nrelated”) significantly decreased their abundances when AT was supplied.\nAcetyl-CoA is one of the intermediate products of acetoclastic methanogenesis\n( 55 ) and one KEGG (cdhC) from its\npathway was augmented in FL. Both cofactor F 420 and coenzyme M (MCR)\nare crucial for hydrogenotrophic methanogenesis ( 56 – 58 ). In our analysis, KEGGs participating\nin the cofactor F 420 and MCR biosynthesis were significantly reduced\nin AT-treated samples compared to TMR. Finally, methanofuran, as already\nmentioned, is an important component of methanogenesis ( 55 ). In our data, the abundance of one KEGG from its\nbiosynthesis (mfnC) was increased by AT treatment, while the rest of the\nassociated DAFs decreased their counts. Conclusions The in vitro application of A. taxiformis as a\nfeed supplement resulted in a drastic reduction of CH 4 concentration\nproduced with minor effects on nutrient degradation. The reduction was closer to\n100% for three of the four replicates, indicating a significant potential for\nCH 4 reduction. The present study suggests the AT mitigation of\nCH 4 concentration is caused not only by the competitive\ninhibition of F 430 coenzyme ( 18 ) but also through a decreased portion of the archaeal domain in\nthe microbiome, as well as lower ratios of Methanobrevibacter \n“SGMT” to “RO” clades, and changes in the abundances\nof methane-associated microbial functions. Abundances of most of the KEGGs that\nare directly involved in methanogenesis were decreased, as well as KEGGs that\nare associated with it indirectly through the synthesis of\nmethanogenesis-related compounds, such as F 420 cofactor, coenzyme M,\nand methanofuran biosynthesis and extension of 2-Oxocarboxylic chain.\nAdditionally, a small group of KEGGs that participate in CH 4 \nassimilation via the serine pathway were more prevalent. A.\nnodosum and F. vesiculosus also decreased methane\nconcentration in the total gas (2%–19%) at the 2.5% inclusion level;\nhowever, only the seaweed samples from Scotland decreased it significantly."
} | 5,190 |
27446060 | PMC4923064 | pmc | 7,374 | {
"abstract": "Bacteria adopt alternative cell fates during development. In Bacillus subtilis , the transition from planktonic growth to biofilm formation and sporulation is controlled by a complex regulatory circuit, in which the most important event is activation of Spo0A, a transcription factor and a master regulator for genes involved in both biofilm formation and sporulation. In B. cereus , the regulatory pathway controlling biofilm formation and cell differentiation is much less clear. In this study, we show that a novel gene, comER , plays a significant role in biofilm formation as well as sporulation in both B. subtilis and B. cereus . Mutations in the comER gene result in defects in biofilm formation and a delay in spore formation in the two Bacillus species. Our evidence supports the idea that comER may be part of the regulatory circuit that controls Spo0A activation. comER likely acts upstream of sda , a gene encoding a small checkpoint protein for both sporulation and biofilm formation, by blocking the phosphor-relay and thereby Spo0A activation. In summary, our studies outlined a conserved, positive role for comER , a gene whose function was previously uncharacterized, in the regulation of biofilm formation and sporulation in the two Bacillus species.",
"introduction": "Introduction Bacillus subtilis and B. cereus are closely related, soil-dwelling spore-forming bacteria. In the environment, both species are found in the rhizosphere and both are considered as biological control agents that help plants fend off infections caused by plant pathogens and sometimes even fungi and parasites ( Emmert and Handelsman, 1999 ; Berg et al., 2005 ; Aliye et al., 2008 ). Therefore they have drawn great interest in the agricultural field. In both B. subtilis and B. cereus , it is proposed that the biological control activities in part have to do with their ability to form multicellular communities, or biofilms, on the root surface of the plants ( Bais et al., 2004 ; Chen et al., 2012 , 2013 ; Beauregard et al., 2013 ). Studies show that wild-type (WT) strains of B. subtilis capable of forming robust biofilms have a much higher efficacy in the biological control activity than the mutants deficient in biofilm formation ( Chen et al., 2013 ). For B. cereus , aside from being a biological control agent, some strains are also known to cause foodborne illness or even more severe diseases such as endophthalmitis and meningitis ( Kotiranta et al., 2000 ). The pathogenesis of B. cereus is related to several enterotoxins and hemolysins produced by some B. cereus strains, such as hemolysin BL (Hbl), non-hemolytic enterotoxin (Nhe), and cytotoxin K (CytK; Gohar et al., 2008 ). In B. subtilis , the genetic circuitry that controls biofilm formation has been well characterized ( Aguilar et al., 2007 ; Shank and Kolter, 2011 ; Vlamakis et al., 2013 ). Multiple histidine kinases (KinA, KinB, KinC, KinD, and KinE) sense various environmental and physiological signals and collectively act, either directly on the master regulator Spo0A through protein phosphorylation, or indirectly via a phosphor-relay (mediated by the phospho-transfer proteins Spo0F and Spo0B; Figure 1 ; Burbulys et al., 1991 ; Jiang et al., 2000 ; McLoon et al., 2011b ). Spo0A functions as a master regulator for endospore formation by controlling hundreds of genes involved in the sporulation process in B. subtilis ( Molle et al., 2003 ; Fujita et al., 2005 ). Spo0A also regulates biofilm formation by activating a small gene sinI , which encodes an anti-repressor for the biofilm master repressor SinR ( Figure 1 ) ( Bai et al., 1993 ; Kearns et al., 2005 ; Chai et al., 2011 ; Newman et al., 2013 ). SinR directly represses two operons, tapA-sipW-tasA and epsA-O , that are responsible for making the protein fibers (TasA) and exopolysaccharides (EPS) of the biofilm matrix, respectively ( Figure 1 ) ( Kearns et al., 2005 ; Chu et al., 2006 ). Recent studies suggest that the biofilm matrix of B. subtilis also consists of a small hydrophobin BslA ( Hobley et al., 2013 ). The gene for BslA was shown to be under the control of the response regulator DegU and the transcription repressors, SinR and AbrB, either directly or indirectly ( Verhamme et al., 2009 ). The biofilm repressor SinR also represses the gene for an additional regulatory protein SlrR ( Chu et al., 2008 ; Kobayashi, 2008 ), which shares strong amino acid sequence similarity with SinR ( Chu et al., 2008 ). Evidence indicates that SinR and SlrR constitute a self-reinforcing double-negative loop that locks cells in the matrix-producing state ( Figure 1 ) ( Chai et al., 2010 ). A third small antagonist of SinR, SlrA, was also shown to directly interact with SinR and relieve SinR-mediated repression ( Figure 1 ) ( Chai et al., 2009 ; Newman and Lewis, 2013 ). Molecular details of how SinR interacts SinI, SlrR, and SlrA were further characterized by recent studies using structural and biochemical approaches ( Newman and Lewis, 2013 ; Newman et al., 2013 ). FIGURE 1 A schematic presentation of the regulatory circuit for the control of alternative cell fates in B. subtilis . Spo0A is positioned at the center of the regulatory circuit, controlling genes involved in both sporulation and biofilm formation. Spo0A is activated by protein phosphorylation (Spo0A~P), often through a phosphor-relay (initiated from multiple Kin kinases and mediated by Spo0F and Spo0B). Sda is a checkpoint protein that blocks the phosphor-relay from KinA to Spo0F and thus Spo0A activation during cell exponential growth. sda is activated by DnaA during exponential growth. SinR is the biofilm master repressor for the matrix genes tapA-sipW-tasA , espA-O , and bslA . SinR is counteracted by two parallel anti-repressors (SinI and SlrA) during biofilm induction ( Kearns et al., 2005 ; Kobayashi, 2008 ; Chai et al., 2009 ). SlrR is another counteracting protein of SinR and shares strong amino acid sequence similarity with SinR ( Chu et al., 2008 ). These two proteins constitute a self-reinforcing double-negative loop for the mutually exclusive control of matrix genes and free-living genes ( Chu et al., 2008 ). Red, gene regulation; blue, protein–protein interaction. In the genetic network for the control of alternative cell fates in B. subtilis (planktonic growth, biofilm formation, sporulation, etc.), Spo0A is positioned at the center of the network ( Figure 1 ). A spo0A null mutant is severely defective in both sporulation and biofilm formation ( Branda et al., 2001 , 2004 ; Hamon and Lazazzera, 2001 ). Activation of Spo0A does not simply rely on protein phosphorylation, but is under the control of complex regulations ( Ireton et al., 1993 ; Perego et al., 1994 ; Jiang et al., 2000 ). For instance, the activity of Spo0A is counter-regulated by protein dephosphorylation by multiple phosphatases ( Perego et al., 1994 ). Spo0A activation is also reinforced by a positive feedback mechanism, in which the expression of several genes involved in the phospho-relay (such as spo0F and spo0B ) is further activated by Spo0A ( Fujita and Losick, 2005 ; Chastanet et al., 2010 ). Lastly, Spo0A activity is also controlled by Sda, a small checkpoint protein for sporulation by blocking the phospho-transfer from the sensory histidine kinase A (KinA) to the intermediate phosphor carrier Spo0F, thereby blocking or delaying Spo0A activation ( Figure 1 ) ( Burkholder et al., 2001 ; Whitten et al., 2007 ). Bacillus cereus has also been reported to be capable of forming submerged or surface-attached biofilms under laboratory conditions as well as on the surface of plant roots ( Emmert and Handelsman, 1999 ; Chandramohan et al., 2009 ; Shemesh and Chai, 2013 ; Gao et al., 2015 ). In contrast to B. subtilis , few genes involved in biofilm formation have been characterized in B. cereus and the regulatory mechanisms that control biofilm formation are poorly understood ( Lindbäck et al., 2012 ; Caro-Astorga et al., 2015 ; Gao et al., 2015 ). One recent study suggested that the homologous gene to spo0A of B. subtilis is important for biofilm formation in B. cereus ( Gao et al., 2015 ). Another study showed that genes homologous to sipW and tasA of B. subtilis also seem to be important for production of adhesion-like fibers for the biofilm matrix in B. cereus ( Caro-Astorga et al., 2015 ). A global regulator CodY for cell stationary phase growth was also shown to be important for biofilm formation in B. cereus ( Lindbäck et al., 2012 ). However, even with the recent progresses, current knowledge about B. cereus biofilm formation is still largely lacking. We aimed to identify genes that are important for biofilm formation in B. cereus and further characterize the function of those genes. In our study, we used an environmental isolate of B. cereus (AR156; Niu et al., 2011 ). AR156 is capable of forming thick floating pellicle biofilms under laboratory conditions (presented in this study) and shows strong biological control activities toward various plant pathogens ( Niu et al., 2011 ). In a parallel study, we conducted a genome-wide random insertion mutagenesis in AR156 by using the mini-Tn10 based transposon system. A total of ~10,000 transposon insertion mutants were screened for alteration of the biofilm phenotype. About 23 such mutants were subsequently obtained (see section “Materials and Methods”). In this study, we focused on one such mutant that has a transposon insertion in the gene annotated as comER ( Figure 2A ). comER encodes a protein that resembles Δ 1 -pyrroline 5-carboxylate reductase, an enzyme involved in the last step of proline biosynthesis ( Belitsky et al., 2001 ). However, previous evidence suggests that comER does not have any significant role in proline biosynthesis in B. subtilis ( Inamine and Dubnau, 1995 ; Belitsky et al., 2001 ). Therefore, the exact function of comER remains unclear. In this work, we show that the comER gene plays an important role in biofilm formation and sporulation in both B. cereus and B. subtilis . Based on our evidence, we propose that comER may be part of the regulatory pathway involved in activation of Spo0A, the master regulator for biofilm formation and sporulation in the two Bacillus species. FIGURE 2 comER is important for biofilm formation in both B. subtilis and B. cereus. (A) A schematic drawing of the chromosomal region in B. subtilis containing divergently transcribed comER and the comEA-EB-EC operon (indicated by arrows). \n comEA and comEC encode structural proteins involved in DNA uptake during genetic competence ( Hahn et al., 1993 ). The role of comEB is unclear and the gene is dispensable for genetic competence ( Hahn et al., 1993 ). The position of the mini-Tn10 transposon insertion in the comER gene on the chromosome of B. cereus AR156 is indicated by the triangle. (B) Pellicle biofilm formation by the wild type (WT) (AR156) and the comER mutant (B168), and the comER complementation strain (YY298) of B. cereus . Scale bars, 4 mm. (C) Quantitative analysis of the biomass of pellicle biofilms from the WT (AR156), the comER transposon insertion mutant (B168), and the comER complementation strain (YY298) of B. cereus . Values in y-axis represent average dry weight (mg) per pellicle. Assays were done in triplicate. (D) Pellicle and colony biofilm formation by the WT (3610), the comER mutant (B165), and the comER complementation strain (YL46) in B. subtilis . Scale bars in the upper panels (pellicles) represent 4 mm in length and those in the lower panels (colonies) represent 3 mm in length. Arrows point to putative suppressors of B. subtilis Δ comER emerged during both pellicle and colony biofilm development.",
"discussion": "Discussion The role of the comER gene in the Bacillus species was not identified in previous studies ( Inamine and Dubnau, 1995 ; Belitsky et al., 2001 ). In those previous studies, highly domesticated laboratory strains of B. subtilis were used. Those domesticated strains are now known to be poor in the ability of forming robust biofilms ( Branda et al., 2001 ; McLoon et al., 2011a ). Our investigations carried out in the undomesticated strains of B. subtilis (NCIB3610) and B. cereus (AR156) show that the comER gene plays an important role in the regulation of biofilm formation and sporulation in both B. subtilis and B. cereus . Results from our study further suggest that comER may be part of the regulatory pathway that controls activation of Spo0A, the master regulator essential for both biofilm formation and sporulation. We propose that ComER may regulate Spo0A activities through its effect on the small checkpoint protein Sda ( Figure 1 ). Sda is known to down-regulate Spo0A activities by blocking the phospho-transfer from the histidine kinase A to Spo0F ( Whitten et al., 2007 ). In B. subtilis , the important role of Sda in sporulation as a checkpoint mechanism was already shown previously ( Hoover et al., 2010 ). It may seem obvious that sda is likely involved in biofilm formation as well due to its strong regulation on Spo0A, but nevertheless it was not shown. In this study, we demonstrated that this checkpoint protein also plays an important role in the transition from free-living motile cells to sessile, biofilm-forming cells. Taken together, our results suggest a broader role of the Sda protein during decision-making for alternative cell fates (planktonic growth, biofilm, sporulation, etc.) in B. subtilis. The regulation of Sda activities has been investigated previously and was shown to occur at different levels ( Veening et al., 2009 ; Hoover et al., 2010 ). At the transcriptional level, sda is primarily regulated by the replication initiation protein DnaA, in response to cellular physiological conditions ( Figure 1 ) ( Veening et al., 2009 ; Hoover et al., 2010 ). When cells are in rapid growing mode, levels of the DnaA proteins are relatively high, which activate expression of sda . Sda in turn effectively blocks Spo0A activation and entry of spore development. Thus, Sda acts as a checkpoint protein to prevent cells from entering sporulation prematurely. This can be reversed when cellular physiological conditions and DnaA activities change ( Veening et al., 2009 ; Hoover et al., 2010 ). Sda proteins are also regulated at the post-translational level by proteolysis ( Ruvolo et al., 2006 ). During the initiation of sporulation in B. subtilis , a proteolysis mechanism triggers degradation of Sda by ClpXP and subsequently allows Spo0A activation ( Ruvolo et al., 2006 ). In this study, we postulate that Sda may be regulated by another mechanism at the post-translational level, even though the details are still unclear. In particular, we speculate that ComER may regulate the activities of Sda, instead of the gene expression of sda or Sda protein production since our results did not support that idea that the comER mutation may cause either altered expression of sda or altered production of the Sda proteins. Based on structural predictions (HHPred 1 ), ComER most strongly resembles Δ 1 -pyrroline-5-carboxylate reductases (100% probability) and prephenate dehydrogenases (99.6% probability) from various sources (FY, personal observations), indicating that ComER is possibly an oxidoreductase for a small metabolite. In future studies, it will be interesting to further understand how ComER regulates Sda activities. In this study, we also observed that the protein levels of Spo0F, an important phospho-transfer protein for mediating activation of Spo0A by Sda, were reduced in the comER mutant. Apparently, altered activities of Sda (presumably caused by Δ comER ) alone are not sufficient to explain this result since the primary activity of Sda is to block phospho-transfer from Kin histidine kinases to Spo0F. However, it is known that genes for the intermediate phospho-relay proteins (Spo0F and Spo0B) and Spo0A are under the control of a feedback regulation ( Chastanet et al., 2010 ). Lowered levels of Spo0A should further decrease the expression of spo0F indirectly through the effect of Spo0A on the sigma factor H, which is required for expression of spo0F as well as other genes whose products are involved in phospho-relay ( Predich et al., 1992 ). Therefore, lowered Spo0F levels could be due to lowered activities of Spo0A and the feedback mechanism. In summary, our studies suggest that the small checkpoint protein Sda may have a broader role in the cell development processes in the Bacillus species."
} | 4,177 |
34932877 | PMC8719810 | pmc | 7,375 | {
"abstract": "Summary Our epoch is largely characterized by the growing realization and concern about the reality of climate change and environmental deterioration, the surge of global pandemics, the unacceptable inequalities between developed and underdeveloped countries and their unavoidable translation into messy immigration, overpopulation and food crises. While all of these issues have a fundamentally political core, they are not altogether removed from the fact that Earth is primarily a microbial planet and microorganisms are the key agents that make the biosphere (including ourselves) function as it does. It thus makes sense that we bring the microbial world—that is the environmental microbiome—to the necessary multi‐tiered conversation (hopefully followed by action) on how to avoid future threats and how to make our globe a habitable common house. Beyond discussion on governance, such a dialogue has technical and scientific aspects that only frontline microbial biotechnology can help to tackle. Fortunately, the field has witnessed the onset of new conceptual and material tools that were missing when the journal started."
} | 282 |
26759604 | PMC4709877 | pmc | 7,379 | {
"abstract": "Background Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions. Results Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 % for Caldicellulosiruptor bescii to 65 % for Clostridium thermocellum , with intermediate values for a thermophilic horse manure enrichment, Clostridium clariflavum, Clostridium cellulolyticum , and simultaneous saccharification and fermentation (SSF) featuring a fungal cellulase cocktail and yeast. Under a variety of conditions, solubilization yields were about twice as high for C. thermocellum compared to fungal cellulase. Solubilization of mid-season harvested switchgrass was about twice that of senescent switchgrass. Lower yields and greater dependence on particle size were observed for Populus as compared to switchgrass. Trends observed from data drawn from six conversion systems and three substrates, including both time course and end-point data, were (1) equal fractional solubilization of glucan and xylan, (2) no biological solubilization of the non-carbohydrate fraction of biomass, and (3) higher solubilization for three of the four bacterial cultures tested as compared to the fungal cellulase system. Brief (5 min) ball milling of solids remaining after fermentation of senescent switchgrass by C. thermocellum nearly doubled carbohydrate solubilization upon reinnoculation as compared to a control without milling. Greater particle size reduction and solubilization were observed for milling of partially fermented solids than for unfermented solids. Physical disruption of cellulosic feedstocks after initiation of fermentation, termed cotreatment, warrants further study. Conclusions While the ability to achieve significant solubilization of minimally pretreated switchgrass is widespread, a fivefold difference between the most and least effective biocatalyst—feedstock combinations was observed. Starting with nature’s best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substitution of cotreatment for pretreatment. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0412-y) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions Our results provide directional guidance for development of advanced processes that look beyond the fungal cellulase/thermal pretreatment paradigm. Key process-relevant lessons we take from these results are as follows: Some biocatalysts, some feedstocks, and some biocatalyst-feedstock combinations are much more effective than others at achieving high solubilization with minimal pretreatment, with the extents of solubilization achieved by several bacterial systems substantially higher than for fungal cellulase and a fivefold difference between the most effective and least effective combinations; Starting with nature’s best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substituting cotreatment for pretreatment. Although promising, further work is required to translate these results into industrial practice. In particular, the biocatalysts we found to be most effective at solubilizing biomass are non-model microorganisms for which limited molecular tools are available and extensive development and testing under industrial conditions are required, e.g. with respect to solids loading. In addition, optimization, innovation, and evaluation pursuant to a diversity of cotreatment strategies in conjunction with these biocatalysts have yet to be undertaken.",
"discussion": "Discussion The extent of solubilization of lignocellulosic substrates by various biocatalysts and with no pretreatment other than autoclaving was documented under controlled, but not industrial conditions, in the most comprehensive such comparative study to date. Glucan and xylan solubilization yields obtained by three bacterial cultures ( C. thermocellum , C. clariflavum, and C. cellulolyticum ) were substantially higher (Fig. 1 ) than the yields obtained by SSF. At an initial substrate concentration of 5 g glucan/L, the extreme thermophile C. bescii exhibited the lowest solubilization of the systems tested. However, at an initial glucan concentration of 1 g/L, C. bescii exhibited higher solubilization than the SSF, suggesting that this organism has strong intrinsic biomass solubilization capability but that growth and/or enzymes are more susceptible to inhibition. A strong correlation between the rate of microbial growth on model cellulosic substrates with increasing temperature has previously been observed [ 31 ], although conditions were not controlled. Although the highest solubilization yields observed herein were at 60° ( C. thermocellum and the horse manure enrichment), the range of yields observed for thermophilic systems overlapped that for mesophilic systems. The observation here of roughly twofold higher lignocellulose solubilization yields for C. thermocellum as compared to SSF on microporous lignocellulosic substrates is remarkable—and as yet not understood—in light of the much larger size of the C. thermocellum cellulosome complex as compared to the components of the non-complexed T. reesei cellulase system. We saw little additional solubilization in response to increased enzyme loading above 5 mg/g mid-season switchgrass for either fungal or C. thermocellum cellulases. Increasing hydrolysis yields from similar enzyme loadings have been seen for fungal cellulases acting on pretreated substrates [ 7 , 8 , 22 ], as might be expected due to increased substrate accessibility following pretreatment. Our results differ from those of Resch et al., who reported similar solubilization of minimally pretreated senescent switchgrass by equal loadings of either fungal or C. thermocellum cellulases [ 37 ]. This discrepancy remains to be explained. Enhanced rates of cellulose solubilization have been observed in the presence of metabolically active microorganisms as compared to cell-free enzyme systems [ 29 , 30 ]. We looked for, but did not find, increased extents of solubilization in experiments involving actively fermenting cells as compared to cell-free cellulase preparations. Adding high levels of C. thermocellum cellulase (Fig. 2 ) does not result in higher 5-day solubilization as compared to microbial cultures (Fig. 1 ). The results reported here do not preclude such enzyme-microbe synergy with respect to the rate of biomass solubilization. Data drawn from three feedstocks, six biocatalysts, and including both time-course and end-point measurements exhibit a strong correlation between glucan and xylan solubilization, which are equal on a fractional basis. This is consistent with coordinated and mutually dependent solubilization of these two components, although different enzyme systems are known to be involved [ 31 , 38 ]. This extensive data set is consistent with the non-carbohydrate fraction of cellulosic feedstocks being inert and is not consistent with proportional biological solubilization of carbohydrate and non-carbohydrate components. A prior report of proportional solubilization [ 28 ] was based on characterization of residual solids that would not pass through a glass filter with a pore size of 40–60 µm and included non-biological as well as biological solubilization. Although the data in Fig. 4 b are for biological solubilization with subtraction of solubilization observed in uninoculated controls, the same pattern of declining residual carbohydrate fraction with increasing fractional conversion was observed whether non-biological solubilization was subtracted or not, which is inconsistent with proportional solubilization of all biomass components. We hypothesized that glycome profiling might reveal large differences in the composition and linkages present in unsolubilized substrates in light of the different mechanisms employed by C. thermocellum and fungal cellulase as well as the substantially different extents of solubilization observed for these two biocatalysts. However, only small differences were in fact observed. Notable disruption of feedstock particles was observed microscopically following cultivation with C. thermocellum and was significantly enhanced by ball milling. Since even the most effective systems examined here achieve less than 40 % solubilization of senescent switchgrass under favorable conditions, it is logical to look to non-biological strategies to increase solubilization. Disruption of the lignocellulose matrix prior to biological processing (pretreatment) has been investigated extensively. Considerably less attention has been given to non-biological disruption after biological attack has begun, or “cotreatment”. As noted by Weimer et al. [ 39 ], alternating mechanical and biological disruptions are important factors underlying the high solubilization of grass realized by ruminants, and this approach has promise in the context of industrial processes. Brief ball milling of residual solids from fermentation of senescent switchgrass by C. thermocellum followed by a second fermentation nearly doubled overall solubilization yields as compared to that achieved after a single fermentation without ball milling. It is notable that the fractional solubilization of carbohydrate present at the start of the respective fermentation was higher for the second fermentation (51 ± 2 %) than for the first fermentation (35 ± 2 %). We saw little impact of brief milling on crystallinity, consistent with prior reports [ 20 , 40 , 41 ]. Our results highlight the importance of accessibility in determining hydrolysis yields. We observed that milling after partial biological solubilization was more effective at enhancing solubilization than milling prior to fermentation, as have others [ 14 , 15 ], and also found that milling after partial solubilization was more effective at reducing particle size. The substantial impact of brief milling observed here supports the possibility of mechanical disruption in a vessel much smaller than the fermenter. By contrast, prior investigations of cotreatment, under various names, have generally employed continuous milling [ 14 – 16 ] requiring that milling occur in the hydrolysis reactor or a comparably sized vessel. Whereas we studied the impact of milling during fermentation, prior reports using milling to enhance solubilization of lignocellulose have focused on enzymatic hydrolysis in the absence of cells. Energy requirements for milling as a pretreatment for solubilization using fungal cellulase are known to prohibitively high [ 42 , 43 ]. We are optimistic that energy requirements for cotreatment-enhanced thermophilic fermentation can be much lower, but leave this important topic to a future report."
} | 2,862 |
36133208 | PMC9473203 | pmc | 7,380 | {
"abstract": "The anti-soiling (AS) performance of highly reflective, superhydrophilic (SPH, 0° water contact angle) coated mirrors was characterized and compared with that of superhydrophobic (SP, >165° water contact angle) coated mirrors. A simple one-step nanotextured silica nanoparticle coating on a mirror exhibited SPH properties associated with hydrophilic rough surfaces. Another mirror surface post-functionalized with low-surface-energy ligand molecules displayed SP behavior. Both coated mirrors, with no solar reflectance loss, demonstrated excellent AS performance because the engineered surface roughness reduced the adhesive force of dust particles. The daily degradation in solar reflectance induced by dust accumulation under outdoor field testing demonstrated that the SPH- and SP-coated mirrors, compared with an uncoated mirror, maintained higher solar reflectance, which was associated with the designed self-cleaning behavior and natural cleaning. However, over the long term, dust-moisture cementation—evidenced by organic hard water stains on the mirror—initiated unrecoverable reflectance loss on the SP-coated mirror after 3 months, whereas the SPH-coated mirror maintained higher reflectance for 7.5 months. Considering fabrication costs and maintenance, SPH-coated nanotextured mirrors offer potential benefits for application in solar energy harvesting.",
"conclusion": "4. Conclusions Facile and environmentally friendly silica oxide NP-textured coatings revealed excellent AS and unique self-cleaning performance associated with superhydrophilicity. The engineered surface roughness associated with superwetting significantly decreased the adhesive force of dust particles on a mirror surface, resulting in an intrinsic repellence of inorganic soil and dust particles and enhancement of self-cleaning behaviour due to facile water layer sliding. Compared with the AS performance of SP coatings, the SPH coating exhibited an ∼2.5 × enhancement of AS performance in outdoor field testing. Superhydrophilicity was more effective in reducing organic dust-cementation soiling on the mirror surface. Considering the fabrication process, cost, and extra energy-intensive cleaning cycles, SPH NP-textured coatings are expected to result in highly efficient solar energy harvesting.",
"introduction": "1. Introduction Surface engineering of an anti-soiling (AS) feature on the reflective surfaces of solar energy devices is important for stable solar energy harvesting, because accumulating dust layers induce significant scattering and absorption of incident solar irradiation. 1–9 For example, concentrated solar power (CSP) technologies generate power by using mirrors to reflect and concentrate a large area of sunlight onto a small area of a receiver. 1,7,10–14 Maintaining clean mirror surfaces is important because CSP plants, mainly built in arid desert areas, experience severe dust accumulation. In efforts to mitigate soiling and maintain clean surfaces, many studies have explored natural soiling of reflective surfaces and environmentally friendly cleaning methods in the field. 1–3,5,8–14 Recent progress in nanotechnology ( e.g., transparent superhydrophobic [SP] coatings 6,15–22 ) suggests a potential breakthrough in a unique surface self-cleaning technology that could significantly enhance the reliability and efficiency of solar panel glass and solar mirrors while drastically reducing cleaning and maintenance costs. A reduction in maintenance costs as a result of self-cleaning surfaces could have a significant impact on energy harvesting. Self-cleaning coatings can be divided into two categories: hydrophobic and hydrophilic. Both types of coatings clean themselves through the action of water, the former by rolling water droplets off and the latter by forming water into a thin sliding layer that carries away dirt. 6,16,20 Recently, we reported that highly transparent SP nanoparticle (NP) -textured coatings exhibited excellent AS performance, which was associated with surface roughness at the scale of <100 nm and with a non-wetting property. 21,22 The rough surface structure of the SP NP coatings provided an intrinsic capability to repel small dust particles by reducing the adhesive force between dust particles and the coated surface. An SP-coated mirror maintained high reflectance by resisting dust accumulation during testing in an outdoor environment. 21 Also, the results indicated that the NP-textured coating before post-fluorination for SP performance was highly hydrophilic, with a measured water contact angle (WCA) of 0–5°. Based on the accepted definition of superhydrophilicity ( i.e., textured materials having a surface roughness factor of [ r > 1], on which water spreads completely), NP-textured surfaces can be called superhydrophilic (SPH) materials. 23 Inspired by the performance of the one-step SPH NP-textured coating with a significant reduction in adhesive force, in this study, we systematically compared the AS behaviour of an SPH coating with that of an SP coating to evaluate their feasibility for use on solar energy-harvesting mirrors under outdoor environments. Many studies predict that hydrophilic and hydrophobic surfaces have potential to benefit AS performance. 21,22,24 However, to our best knowledge, a rigorous comparative study of SP and SPH self-cleaning surfaces has not been reported. We found that the adhesive force of simulated dust particles on SPH surfaces was similar to the force on SP surfaces, because both have NP-textured surfaces that afford excellent AS performance against inorganic dust particles. Outdoor field tests demonstrated a significant reduction in dust accumulation and outstanding self-cleaning performance for an SPH-coated mirror compared with an SP-coated mirror and an uncoated mirror. Note that without the toxic and expensive post-fluorination step, the simple, environmentally friendly SPH textured coating outperformed the SP coating, according to the field testing.",
"discussion": "3. Results and discussion 3.1. Transparent SPH-coated mirrors A transparent SiO 2 NP self-assembled thin layer was deposited by rod drawdown coating. The transparent SPH coatings on a solar mirror were fabricated as two drawdown coating layers with SiO 2 NPs and silicate sol–gel binder [ Fig. 1a ]. SEM images of the two-layer NP coatings show a uniformly textured thin layer with a 200–250 nm thickness [ Fig. 1b and c ]. The coating exhibited strong hydrophilicity, with complete spreading of water (0° WCA) due to hydroxyl groups on the rough surface. After thermal fluorosilane vapor deposition, the textured coating exhibited SP characteristics, having a >165° WCA compared with the ∼45° WCA of an uncoated mirror. The surface morphology and thickness of the coating showed no distinguishable difference after fluorination. The surface chemical component of the XPS analysis showed high silicon and oxygen contents at the surface of the SPH coating compared with other elements, which was explained by the presence of the intrinsic SiO 2 NP layers [ Table 1 ]. The SP-coated surface showed the existence of fluorine due to the fluorination process. Further XPS analysis is available in the ESI. ‡ Fig. 1 (a) Highly reflective, anti-soiling nanoparticle-texture solar mirrors (7.6 × 7.6 cm 2 ) with no coating, superhydrophilic (SPH) coating, and superhydrophobic (SP) coating. Insets are water contact angle measurements on the coated mirrors (the measured water droplet volume = 5–10 μl). (b) SEM images of coated mirror surface, showing characteristics of SPH nanotexturing. (c) SEM cross-sectional view of SPH coating. Chemical composition of SP and SPH coating determined by XPS O–Si O Si–O Si C C-Fx F O–Si/Si–O C-Fx/F Uncoated 53.7 6 27.1 0 7.2 0 0 2.0 — SPH 53.1 \n 10.0 \n 27.1 \n 3.0 \n 6.3 0 0 2.0 — SP 45.8 0.4 22.5 0.0 7.7 \n 7.0 \n \n 15.5 \n 2.0 0.45 The NP-coated mirror exhibited good durability against the abrasive effect of falling sand ( e.g., simulation of 15 years of sand falling on the coating) and the aging effect of an accelerated ultraviolet light. 21 The added sol–gel ( i.e., acidic catalysed hydrolytic condensation of TEOS) acted as a strong binder, resulting in good mechanical stability that could be attributed to the noncovalent bonding ( e.g., hydrogen bonding with hydroxyl groups via hydrolytic condensation) formed between the NP, binder, and glass surface. 25,26 The SPH textured coating was studied for characterization and AS performance compared with the SP coating. The SiO 2 -NP-based drawdown coating exhibited excellent uniform optical properties on a large scale. 21 Fig. 2 shows the average specular reflectances at seven wavelength bands extending from 335 to 2500 nm on the SPH-coated and uncoated mirrors at a single measurement location for each mirror. The spectrum bands of the SPH coating had a slightly higher reflectance than those of the uncoated mirror from 700 to 2500 nm, resulting in similar or higher overall average solar specular reflectance measured on the uncoated mirror. The difference in the reflectance distribution between the SPH coating (0.937 ± 0.002, n = 6) and the uncoated mirror (0.940 ± 0.002, n = 6) was negligible [ Fig. 2c ]. Fig. 2 Solar specular reflectance measured in wavelength bands across the solar spectrum for SPH-coated and uncoated mirrors. Inset is average specular reflectance of an SPH surface and an uncoated mirror surface (7.6 × 7.6 cm 2 , n = 6). Error bars are the standard deviations in the mean values. 3.2. Adhesive force reduction of the SPH coating The NP-textured coating significantly reduced the adhesive force of dust particles on the surface. The adhesion force between a particle and a substrate can be expressed in terms of van der Waals (vdW), electrostatic, and capillary forces. 27–31 The contribution of the capillary force can be significant when water is present between the interacting surfaces as a result of water condensation on the surface of the coating. The SP and SPH coatings were tested in a dry environment. Therefore, water condensation and capillary forces were not considered in this study. The electrostatic force between the particle and the surface is also dependent on the environmental conditions and was not considered. In the present study, we focused on fine particles ( i.e. , airborne dust) with diameters less than 70 μm. 13 When the particle size is less than 50 μm, the primary adhesion force of the particle is the vdW force. 25 Excluding the electrostatic interactions, the adhesion force between dry sand particles and a glass surface is predominantly vdW force. Several models have been developed to predict and understand the adhesion force between a spherical particle and a nanostructured surface. 21,31,33–36 We developed the adhesion force prediction based on the vdW force ( F vdW ) between a particle and a surface asperity and expressed the F vdW as a function of the root mean square (RMS) surface roughness value and the distance between the surface asperities. 21,33 1 In eqn (1) , A is the Hamaker constant, D is the particle diameter (15 μm), a is the distance between the particle and the surface (∼0.3 nm, when the particle is in contact with the surface), rms is the RMS surface roughness, k 1 is a constant (1.817), and λ is the distance between the asperities. The Hamaker constant was calculated according to the mixing rule for dissimilar surfaces , where A 1 is the Hamaker constant for the fused silica (6.5 × 10 −20 J) and A 2 is the Hamaker constant for the substrate ( e.g. , polytetrafluoroethylene [3.8 × 10 −20 J] for the SP surface, silica for the SPH surface). 27 \n Fig. 3a shows that the vdW attraction ( i.e., adhesion force) between the SP/SPH surfaces and a model particle (15 μm diameter) decreases when the surface roughness increases. The calculated adhesion force on the SPH coating is ∼30% higher than the force on the SP coating. Interestingly, the experimentally measured adhesive force on the SPH coating was ∼4.1 times smaller than the adhesive force on the uncoated substrate and slightly lower than on the SP coating. Note that the good agreement between the measured and predicted values indicates the adhesion force between the dust particles and the coated substrates is dependent on the surface roughness, regardless of the surface functionalities. This behaviour agreed well with the modelling results. 21 Fig. 3b–d show the AFM surface morphology analysis of the coated substrates. The bare SiO 2 NP layer of the SPH coating had a uniform surface texture with 25.1 nm of RMS roughness at 2.5 × 2.5 μm 2 , whereas the flurosilane molecule coating on the SP surface slightly smoothened the surface texture, giving it 21.6 nm of RMS roughness. The uncoated surface ( i.e. , bare silicon wafer) showed 3.0 nm of RMS roughness. Fig. 3 (a) Measured and calculated adhesion force between a silica sphere (15 μm diameter) and SP and SPH surfaces. AFM surface morphology analysis of (b) uncoated surface (c) SPH coating, and (d) SP coating. \n Fig. 4 shows the adhesive force of a model particle (6 μm) on SPH-coated, SP-coated, and uncoated mirror surfaces as a function of RH. The adhesive forces on the SPH- and SP-coated mirrors were >2.5 times lower than on the uncoated surface at 2–80% of RH. The adhesive force of the particles on the uncoated mirror surface increased with increasing RH. The behaviours of the SP coating as a function of humidity were well matched with the previous results. 21 The observation that the SPH-coated mirror retained similar adhesive forces as the humidity increased was counterintuitive. Because of strong capillary condensation, the adhesion force between particle and substrate generally increases with an increase in humidity. 29,31 However, the humidity effect is more complicated because of the interaction between electrostatic force and capillary force associated with the surface chemistries of particles and substrates. 29 For example, for hydrophilic surfaces, excessive water adsorption attenuates the surface charge by providing a path for leakage, which might cancel out the electrostatic force, leading to a reduction in the adhesive force. 29 Fig. 4 Adhesive force of spherical dust particles with 6 μm diameters on SPH-coated, SP-coated, and uncoated mirrors as a function of relative humidity. 3.3. Anti-soiling and self-cleaning performance of SPH-coated mirror The SPH-coated mirrors demonstrated excellent soil repellence and a unique self-cleaning capability with facile water layer sliding. Fig. 5 shows a photographic observation of soiling when 1 g of dust (=220 g m −2 ) was applied to a mirror, inclined at 45°, with half its area SPH coated and other half uncoated. Mostly large dust particle agglomerates (a few millimetres in size) were sparsely scattered on the SPH-coated surface, and large agglomerates and small particles were densely spread on the uncoated surface [ Fig. 5b ]. To simulate natural winds and the surface cleaning effect, 13 airbrushing with a bulb dust blower (air volume ≅ 40 ml) was applied to remove the loose dust. The small amount of blowing air effectively removed most of the dust particles on the SPH-coated surface and restored the clean surface. An optical microscopy image shows a few sparse fine particles (<5 μm) remaining on the surface [ Fig. 5c ]. The particle size distribution on the SPH-coated surface was narrow and centred at 1.32 ± 0.62 μm ( n = 30 from 8977 μm 2 of image analysis area), which was similar to the soiled-particle size distribution on the SP-coated surface. 21 The standard test soil for the dust soiling has a very broad size range of 0.9–352 μm. Only 12.5 vol% of the test soil had fine particles of <5 μm. The image shows that a very small portion of the fine particles were adhered to the NP-textured surfaces because of the reduction in the adhesion force. However, as reported in the previous work, the small dust particles (∼10 μm) were still densely and strongly adsorbed onto the uncoated mirror surfaces as a result of the vdW force, a charge double layer, surface energy, capillary forces, and electrostatic force on the mirror surfaces. 13,14 The particle size distribution on the uncoated surface was broad and centred at 2.03 ± 1.49 μm ( n = 375 from 8977 μm 2 of image analysis area). Typically, most soiled dust particles on reflective substrates are <40 μm in dry and desert areas. 7,9 After a few water droplets were deposited on the SPH-coated mirror, unidirectional water layers were observed sliding to the bottom of the mirror edge along the 45° slope of the mirror [ Fig. 5d ]. The uncoated mirror experienced nonuniform water sliding, which stopped two-thirds of the way from the top edge of the mirror. The facile water sliding results indicated that the SPH-coated mirror demonstrated effective self-cleaning. Fig. 5e shows water-dispersed soil suspensions dropped on clean mirror surfaces. The soil suspended in water easily spread out on the SPH-coated surface, and soil residues were left on the surface after drying. For the uncoated mirror, soiled water droplets formed on the surface and left dense cake-like residues. The soil residues on the SPH-coated surface were not simply rinsed out, because the wet condition added a very large capillary force to the adhesion force between the particles and the surface. 31,32 In humid conditions, the capillary force could be 1 order of magnitude larger than the vdW force. 31 The soil residues were removed by gentle brushing with water, and the coated surface exhibited superhydrophilicity after the brushing. Fig. 5 Observation of soiling on a mirror (15.2 × 7.6 cm 2 ) with half its area coated with nanoparticles to give it SPH properties. (a) A half-coated mirror at 45° elevation, (b) initial soiling (1 g) on the mirror, (c) dust accumulation on the mirror surface after airbrushing. Insets are optical microscope (5000× magnification) images of soiling on an SPH-coated mirror and an uncoated mirror after airbrushing. (d) Water dripping on the mirror surfaces. The water was coloured with a green dye. (e) Water disperses soil dripping on the mirror surfaces. Inset is the suspended soil that was collected from the suspension in 0.1 g ml −1 of soil and water mixture (ISO 12103-1 A4 Coarse Sand). The AS performance of the SPH-coated mirror was compared with that of the SP-coated and the uncoated mirrors [ Fig. 6 ]. Solar reflectance measurements were carried out with three mirrors inclined at 45° elevations after gravimetric soiling and following airbrushing. The applied soiling amount was increased to the simulated 1 year accumulation expected in the Arizona desert area ( i.e. , ∼909 g m −2 ). 21,36 Our previous work reported that an SP-coated mirror (called the AS-coated mirror) exhibited excellent AS performance, resulting in no reflectance loss after soiling in an indoor soiling experiment. 21 The uncoated mirror had a reduction in reflectance associated with the soiling rate and elevation. As we reported, the reflectance of the uncoated mirror dramatically decreased with an increasing soiling rate. Note that the solar reflectance of SPH-coated mirrors showed no decrease as soiling rates increased. Both the SP and SPH coatings showed similar outstanding AS performance due to the engineered surface roughness, which was associated with an adhesion force reduction. This finding suggests that the engineered surface roughness is a key factor in AS performance, regardless of the surface functionality. Fig. 6 Solar specular reflectance analysis of dusting effects on SPH-coated, SP-coated, and uncoated mirrors as an increasing amount of dust was applied to the mirrors at elevations of 45° after airbrushing. The number of measurements per data point was ≥5, and the error bars are the standard deviations in mean values. Lines are drawn for illustration. 3.4. Field evaluation of comparative SPH- and SP-coated mirrors A field examination of environmental soiling on SPH-coated, SP-coated, and uncoated mirrors (15.0 × 20.0 cm 2 in size) was carried out for 234 days on a sloping roof (45° elevation) at Oak Ridge National Laboratory during the late fall, winter, spring, and summer seasons. The soiling conditions in Oak Ridge, Tennessee, USA ( e.g. , high humidity, high concentrations of pollen and organic aerosols produced by the surrounding forest, frequent rain) are much different from those in the dry or semi-arid desert environments where CSP plants are sited. Some reduction in daily soiling was alleviated by natural cleaning provided by rain, heavy morning dew, snow, and frost formation during the field test period. Specular reflectance measurements across the full solar spectrum showed that the daily degradation of reflectance (DDR) on the SPH-coated mirror was quantitatively lower than the DDR of the uncoated mirror [ Fig. 7 ]. Both SPH- and SP-coated mirrors showed a similar higher retention of reflectance, compared with the uncoated mirror, over 79 days. The SP- and SPH-coated mirrors exhibited their unique self-cleaning capability after rain events. (Photographs of mirror statuses 1, 2, and 3 in Fig. 7a are available in the ESI. ‡ ) Our previous work showed the SP-coated mirror maintained a lower DDR than the uncoated mirror for 61 days during the late summer and the early fall seasons. 21 However, over long-term exposure, the SP-coated mirror experienced a distinguishable decrease in reflectance, with a significant loss of dewetting performance, after 99 days. (Photographs of mirrors at status 4 in Fig. 7a are available in the ESI. ‡ ). The entire SP-coated mirror surface showed hard water marks, and rainwater droplets formed on the entire mirror surface after light rain events. It appears that the distinguishable DDR of the SP-coated mirror, induced by hard water marking, was associated with the beginning of the pollen season ( e.g. , late February). After that time, the DDR of the SP-coated mirror was similar to the DDR of the uncoated mirror. Fig. 7 (a) Solar specular reflectances of SPH-coated, SP-coated, and uncoated mirrors measured during 234 days of outdoor exposure. Photographic images of each mirror status, designated by number, are available in the ESI. ‡ The number of measurements per data point was ≥6, and the error bars are the standard deviations in mean values. Lines are drawn for illustration. (b) Corresponding weather conditions in Oak Ridge, Tennessee, USA. Blue bars and orange lines indicate the precipitation levels and airborne pollen counts (from www.weather.com ) during the field test period. The maximum pollen count was 5 grains per m 3 during the test period. Some data points for air pollen counts in the figure were not reported. Note that the reflectance of the SPH-coated mirror was 2 to 7% greater than that of the SP-coated and the uncoated mirrors over a period of 220 days. Also, the standard deviation in the average value of the reflectance of the SPH-coated mirror was significantly smaller than the deviation in the average reflectance of the other mirrors. After 160 days, all mirrors experienced a distinguishable reduction in reflectance ( i.e. , ∼4% for SPH-coated and ∼8% for SP-coated and uncoated mirrors) because of the high concentration of organic foulants ( e.g., airborne pollen particles) and low precipitation amounts in the late spring. (Photographs of the mirrors at status 6 in Fig. 7a are available in the ESI. ‡ ) After 160 days, the reflectance of the SP-coated mirror was not restored by natural cleaning, whereas the reflectance of the uncoated mirror recovered slightly after frequent rains. It appears the hydrophilic surface (which induced fast water layer sliding) had better self-cleaning performance associated with weathering than the hydrophobic surface (which induced water droplet rolling). Our previous study reported that a possible dust fouling mechanism manifested as a loss in reflectance could be induced by a dust-moisture cementation process. 21 Atmospheric dust contains a distribution of inorganic and organic particulates ( e.g. , pollen) that contain some water-soluble and insoluble salts. At high humidity, water-soluble dust particles on the surface form microscopic droplets of salt solutions that also retain any insoluble particles. When dried, the precipitated salt acts as a cement to anchor insoluble particles to the surface. Another possible dust-cementation mechanism of the SP-coated mirror in this field area could be airborne dust absorption on microscopic water droplets on mirror surfaces under humid and wet conditions. Generally, an SP-coated surface has bulk water repellence, but microscopic water droplets can form between NPs, on possible defects due to the SP-coated surface, and on direct fouling from the field area ( e.g., bird droppings, fouling from the portable reflectometer measurement). Airborne organics ( i.e. , pollen and its fibres) were adsorbed on the microscopic droplets, and microorganic fouling accumulated over time [see ESI ‡ ]. It appears that the hydrophilic self-cleaning mechanism may be more effective in mitigating dust cementation and airborne organic adsorption than the hydrophobic self-cleaning mechanism. The cemented organic dust aggregations and water marks were not rinsed away until mechanical brushing was used. \n Fig. 8 shows the characteristics of the three weathered mirrors after 234 days of outdoor exposure. The samples were cut to 7.6 × 7.6 cm 2 for further characterization. The SPH-coated mirror still exhibited an average WCA of 7.2 ± 2.3° ( n = 6), slightly increased from the average WCA of 4.8 ± 3.5° ( n = 10) at 0 day. The SP-coated mirror exhibited an average WCA of 111.0 ± 3.9° ( n = 6), significantly decreased from an average WCA of 164.9 ± 0.9° ( n = 10) at 0 day. The change in WCA was associated with organic dust accumulation on the mirror surfaces [ Fig. 9 and ESI ‡ ]. Fig. 9 shows an SEM image of the weathered SPH-coated and SP-coated mirrors. The NP coatings on the weathered SPH-coated and SP-coated mirrors were present, providing the surface roughness for hydrophilicity and hydrophobicity, respectively. The uncoated mirror had an average WCA of 42.7 ± 1.9° ( n = 6), a decrease from the average WCA of 52.6 ± 19.0° ( n = 30). Then 1 g soiling followed by airbrushing was carried out on the weathered mirrors to evaluate the AS performance. Note that the weathered SPH- and SP-coated mirrors still showed no adhesion of soiling dust particles, corresponding to no decrease in solar reflectance, whereas the weathered uncoated mirror exhibited a large reflectance reduction with soiling [ Fig. 10 ]. This is a significant result for the design of AS-coated mirrors for CSP mirror applications in arid areas. Fig. 8 Surface characteristics and optical microscopic images (500× magnification) of SPH-coated, SP-coated, and uncoated mirrors after 234 days of outdoor exposure. Insets are the water contact angle measurements on the mirrors (the measured water droplet volume = 5–10 μl). Fig. 9 SEM image of (a) SPH-coated and (b) SP-coated mirror after a 234 day outdoor field test. Fig. 10 Optical microscope images of soiling (1 g) after 234 days of weathering of SP-coated, SPH-coated, and uncoated mirrors after airbrushing. (a) Uncoated mirror surface at 5000× magnification, (b) SPH-coated mirror surface at 5000× magnification, (c) SP-coated mirror surface at 5000× magnification. Another outdoor weathering test was performed on mirrors with various WCAs during a 1 day interval in the field area. The topography and surface functionality of the coatings were controlled by varying the sol–gel/NP weight ratio from 4 to 64. First, drawdown coating was conducted on a 20 × 30 cm 2 second-surface solar mirror. Half of the coated mirror was cut to fabricate a mirror with a hydrophobic coating. For the as-is mirrors ( i.e. , hydrophilic, H-1, 2, 3, and 4), as the sol–gel/NP ratio increased, the WCA increased from 4.8° to 57.5°. For the post-functionalized mirrors (hydrophobic, P-1, 2, 3, and 4), as the sol–gel/NP ratio increased, the WCA decreased from 164.9° to 111.2°. The H-series and P-series mirrors had different surface functionalities based on the same surface morphology structure. Fig. 11 shows that the mirrors with hydrophilic behaviour outperformed the mirrors with hydrophobic behaviour in maintaining clean surfaces. For mirrors with hydrophobic surfaces, the reflection reduction of the P-1 mirror, assigned to the SP-coated mirror, was less than that of the other hydrophobic mirrors; whereas the H-1 mirror, assigned to the SPH-coated mirror, exhibited the lowest reflectance reduction among the hydrophilic mirrors. All SPH-coated mirrors maintained their unique surface morphology after the field test [ESI ‡ ]. Therefore, we carefully suggest that the SPH coating had better AS performance than the SP-coated mirror in the humid valley area around Oak Ridge. Fig. 11 Solar specular reflectance analysis of outdoor dusting effects on various mirrors from 5 to 165° WCA. Mirrors were exposed for 234 days in Oak Ridge. The one bar result is the average reflectance over the period. The number of measurements per data point was ≥30. Error bars are standard deviations in mean values of reflectance. The red dotted line indicates 0.9 of reflectance."
} | 7,348 |
21798749 | null | s2 | 7,381 | {
"abstract": "Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (∼12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified."
} | 296 |
38465733 | PMC10926176 | pmc | 7,383 | {
"abstract": "Abstract Microbes are powerful upgraders, able to convert simple substrates to nutritional metabolites at rates and yields surpassing those of higher organisms by a factor of 2 to 10. A summary table highlights the superior efficiencies of a whole array of microbes compared to conventionally farmed animals and insects, converting nitrogen and organics to food and feed. Aiming at the most resource‐efficient class of microbial proteins, deploying the power of open microbial communities, coined here as ‘symbiotic microbiomes’ is promising. For instance, a production train of interest is to develop rumen‐inspired technologies to upgrade fibre‐rich substrates, increasingly available as residues from emerging bioeconomy initiatives. Such advancements offer promising perspectives, as currently only 5%–25% of the available cellulose is recovered by ruminant livestock systems. While safely producing food and feed with open cultures has a long‐standing tradition, novel symbiotic fermentation routes are currently facing much higher market entrance barriers compared to axenic fermentation. Our global society is at a pivotal juncture, requiring a shift towards food production systems that not only embrace the environmental and economic sustainability but also uphold ethical standards. In this context, we propose to re‐examine the place of spontaneous or natural microbial consortia for safe future food and feed biotech developments, and advocate for intelligent regulatory practices. We stress that reconsidering symbiotic microbiomes is key to achieve sustainable development goals and defend the need for microbial biotechnology literacy education.",
"conclusion": "CONCLUSION: EDUCATION, COMMUNICATION AND PROGRESS BY MEANS OF EXPERIMENTAL TESTBEDS With as much as 800 kilograms of food ingested per capita per year on average, issues related to food supply, safety, risks and innovations are sensitive and often spark passionate debates. So far, the dominance of conservative discourses and adverse effects of the cautionary approaches of regulatory agencies have limited to tails the promises of microbial biotech towards food and feed production. Yet, to achieve a circular bioeconomy and an eco‐friendly food system, it is vital to be able to make use of the power of microbiomes, including refurnishing soil microbiomes (i.e. soil health) and mimicking their synergistic resource recovery strategies (Shayanthan et al., 2022 ). Microbiomes can already adequately be managed and provide quality assurance. These facts should reach the public, its lawmakers and regulatory agencies through open science, interactive citizen‐science demonstrations, biotechnology literacy education and communication campaigns. Such actions could lead to a more open vision in society towards microorganisms in general and their beneficial roles in particular. Several decades were needed before it became generally accepted that climate change was due to human activities. The concept that we need to use at large‐scale microbiomes to reach the Sustainable Development Goals is still in its infancy. Experimental testbeds, in which open‐minded innovators, entrepreneurs, regulators and consumers cooperate over extended periods of time to explore the potentials of new feeds and foods based on fermentations, both axenic and natural symbiotic types, are warranted, particularly if we want to progress towards a much more sustainable feed and food supply for the future."
} | 862 |
36846501 | PMC9947057 | pmc | 7,385 | {
"abstract": "Abstract Materials science is about understanding the relationship between a material’s structure and its properties—in the sphere of mechanical behavior, this includes elastic modulus, yield strength, and other bulk properties. We show in this issue that, analogously, a material’s surface structure governs its surface properties—such as adhesion, friction, and surface stiffness. For bulk materials, microstructure is a critical component of structure; for surfaces, the structure is governed largely by surface topography. The articles in this issue cover the latest understanding of these structure–property connections for surfaces. This includes both the theoretical basis for how properties depend on topography, as well as the latest understanding of how surface topography emerges, how to measure and understand topography-dependent properties, and how to engineer surfaces to improve performance. The present article frames the importance of surface topography and its effect on properties; it also outlines some of the critical knowledge gaps that impede progress toward optimally performing surfaces. Graphical abstract",
"conclusion": "Conclusion In conclusion, we urge the materials-science community to view surface topography as a material property. Using the concept of the surface tetrahedron, the performance of a material’s surface can be understood and controlled analogously to how we routinely modify bulk material parameters. The ultimate goal for the field is to be able to systematically design, impart, and measure surface topography for the rational control of surface properties from adhesion to friction to biocompatibility to electrical and thermal transport across a contacting interface. The recent advances described in the present issue, and ongoing fundamental investigations in the field, are moving the community toward achieving this goal.",
"introduction": "Introduction: Surface topography as a multiscale material parameter stretching from the size of a component down to the atomic scale A surface is a planar defect, the outermost layer of a material. Depending on context, it can refer to the outermost atoms, or it can include some depth of near-surface material. The geometry of this surface is a two-dimensional contour, the surface topography. The series of articles in this issue describe how, besides the chemical composition of the material itself, this arbitrarily complex shape of the boundary between a material and the outside world plays a key role in determining the surface properties of that material. Surface topography can control whether paints and coatings flake off of a consumer product, how much energy is wasted in automobile and airplane engines, how quickly a cutting tool wears out, the biocompatibilty of a medical device, and whether a flooring tile will cause slip-and-fall injuries. We contend that the conventional ways of measuring and describing surface topography impede advancement in this area. Common reference standards (e.g., ISO 4287 or ASTM B46) specify that a surface topography measurement should be separated into three components: large-scale waviness, medium-to-small-scale roughness, and small-scale noise. For example, for the commonly used stylus profilometer, “noise” is typically defined as topography below 2.5 μm of lateral size scale (or wavelength), whereas “waviness” is commonly defined as topography with wavelength larger than 80, 250, or 800 μm (depending on topography and measurement conditions), with everything in between designated as “roughness.” These distinctions serve a practical purpose in a machine shop, where large-scale machining defects can be considered “waviness,” and polishing or other finishing techniques can be used to control “roughness.” However, we contend that these distinctions impede the scientific understanding of topography-dependent properties because the final performance of the part often depends on all scales of topography and does not acknowledge these arbitrary distinctions. We advocate instead to think of the surface topography as a multiscale material parameter, which stretches from the size of a component down to the atomic scale. In an extreme case, the size of the component could be on geologic scales: In this issue, Aghababaei et al. 1 discuss earthquake faults ( Figure 1 a), where surface topography has been measured for the same fault on scales stretching from 100 km down to the scale of micrometers with common scale-independent features across all scales. Describing the multiscale nature of surface topography requires spectral analysis, rather than simple scalar metrics. One such example is the power spectral density (PSD), which is a mathematical tool for decomposing a surface into contributions from different spatial frequencies (wave vectors). 2 An example of the multiscale roughness of an engineering material is given in Figure 1 b, where a wear-resistant diamond coating has been characterized from centimeters down to the atomic scale. Taken together, the examples of Figure 1 illustrate that roughness features can exist, and can be statistically characterized, over more than 16 decades in length. All of this multi-scale surface structure can contribute to surface performance. Figure 1 Surface topography can range from kilometers to the atomic scale. (a) The Corona Heights Fault in San Francisco, Calif., provides an example of the multiscale nature of roughness, where roughness features exist over length scales from tens of kilometers down to microns. The surface topography shows self-affine fractal-like scaling, here manifested as a power-law \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$C\\left(q\\right)\\propto {q}^{-1-2H}$$\\end{document} C q ∝ q - 1 - 2 H in the PSD, over this whole range. (b) The topography of ultrananocrystalline diamond, a common wear-resistant coating, deviates from self-affine behavior, but still shows roughness across a wide range of scales from centimeters to Ångströms. TEM, transmission electron microscopy; LIDAR, light detection and ranging. (a) Adapted with permission from Reference 3 . © 2012 American Geophysical Union. (b) Adapted with permission from Reference 4 . © 2018 American Chemical Society."
} | 1,606 |
25884952 | PMC4401573 | pmc | 7,386 | {
"abstract": "Microbially enhanced coalbed methane technology must be used to increase the methane content in mining and generate secondary biogenic gas. In this technology, the metabolic processes of methanogenic consortia are the basis for the production of biomethane from some of the organic compounds in coal. Thus, culture nutrition plays an important role in remediating the nutritional deficiency of a coal seam. To enhance the methane production rates for microorganism consortia, different types of nutrition solutions were examined in this study. Emulsion nutrition solutions containing a novel nutritional supplement, called dystrophy optional modification latex, increased the methane yield for methanogenic consortia. This new nutritional supplement can help methanogenic consortia form an enhanced anaerobic environment, optimize the microbial balance in the consortia, and improve the methane biosynthesis rate.",
"introduction": "Introduction Methane is an important fuel for energy production worldwide, and due to growing markets, demand is increasing. Many methods have been used to extract methane from oil deposits and coal fields. If certain nutrients are injected into a coalbed, methanogenic consortia can metabolically convert some organic compounds into methane. Microbially enhanced coalbed methane (MECoM) technology is a procedure for producing methane and secondary biogenic gas to increase the methane content in mining. Nearly all previous studies regarding coal biogasification have focused on using a microbial community to convert coal into methane, carbon dioxide, or other organic molecules in surface bioreactors [ 1 – 6 ]. Research on methane reservoirs has shown that microbial gas is typically located at shallow depths in thermally immature coals (at a vitrinite reflectance R 0 <0.6%), where formation waters exhibit relatively low salinity (<2 mol/L Cl - ) and low S O 4 2 - concentrations (<10 mmol/L). The infiltration of dilute meteoric waters into these systems plays an important role in the transport of nutrients, the dilution of coalbed formation waters, and the stimulation of methongen growth. Most organic compounds in coal cannot be directly used as nutrition for methanogens. Three functionally different trophic groups of bacteria ( Fig 1 ) are required to convert organic material to methane [ 7 – 9 ]: (1) hydrolytic fermentative bacteria, (2) syntrophic acetogenic bacteria, and (3) methanogenic bacteria. 10.1371/journal.pone.0124386.g001 Fig 1 Generalized flow diagram for anaerobic decomposition of organic matter and generation of methane. Hydrolytic, hydrogen-reducing, acetogenic, and hydrogen-utilizing bacteria provide the organic compounds metabolized by methanogens. To achieve a greater yield of secondary biogenic gas in an Illinois basin coalbed, the nutritional components have been studied for the growth of, and stimulation of biomethane production in, methanogenic consortia [ 10 , 11 ]. The biomethane productivity of a methanogenic community can be improved if the culture conditions within the coal seam environments of the coalbed are improved. Dystrophy optional modification latex (DOL) contains sulfanilic acid, fatty acid oil, amide fat polymers, and other inorganic constituents. However, these emulsions are unstable. A DOL emulsion can improve the organic compound makeup of coal slurries and change the surface tension of the coal cleat system. The objective of this paper is to study microbial methane yields in the presence of DOL emulsions.",
"discussion": "Discussion and Conclusions This research examined the efficacy of DOL emulsion nutrition for improving methanogenic consortia biomethane production. The yields of methane and carbon dioxide were monitored in real time. The effects of pH on methanogenic consortia were also examined. Studies on the influence of DOL emulsions showed that the methane yield is variable and dependent on the stability of the methanogenic consortia. The results of this study revealed the following: (1) The DOL emulsion improved the biomethane yield compared with the control experiment. The highest methane production rate achieved was 40.32 mL/g•day -1 . (2) Real-time monitoring of the methane yield indicated that the coalbed methanogenic microbial fermentation process was not stable but could be continuously maintained when the microbial cultures were kept in balance. (3) Carbon dioxide was an important intermediate in the coal fermentation process and the main carbon source of methanogens. It is important to measure the concentration of carbon dioxide in mixed gas as it affects the stability of methanogenic consortia. The carbon dioxide concentration could be kept low when the methanogenic and hydrolytic bacteria consortia were in a balanced condition. (4) Methanogenic consortia have the ability and tendency to alter the culture environment. pH 6.10 was the optimum pH in this experiment. These experiments were simple but very important for the feature research of MECoM. In some cases, methanogenic bacteria are inhibited because of nutritional deficiency. The modification of the coalbed microbial environment is an important method for maintaining the microbial consortia in a good condition. Controlling the available nutrients is necessary for improving the effective carbon source availability and decreasing the carbon dioxide concentration in mixed gas. DOL emulsion nutrition is an effective method for improving the nutritional capacity of coal. To fully understand the effect of DOL emulsions in enhancing the methane yield of methanogenic consortia, further study is required. There are three mechanisms that could be responsible for the effects of DOL. (1) DOL could adsorb at the coal surface ( Fig 7 ). This would assist anaerobic fermentation in the cleat system in coal. (2) DOL could improve the dissolution of the organic compounds of coal, and it could improve the nutrition availability of coal for microbials. (3) Some DOL compounds might provide nutrients for certain bacteria and improve the microbial balance between methanogenic and hydrolytic bacterial consortia. Determining which of these mechanisms is ultimately responsible requires more research. Real-time monitoring was used in this study. The experimental data revealed that the methane biosynthesis rate and carbon dioxide yield rate need further study. Methane biosynthesis was not a stable process. The data demonstrated that coalbed methanogenic bacterial cooperation may have a periodic nature. Exploring the mechanism of this periodicity could improve research into coalbed methanogenic consortia ecology. Carbon dioxide is an important factor that governs microbial group balance. Further study on this correlation is needed."
} | 1,679 |
21689458 | PMC3145561 | pmc | 7,387 | {
"abstract": "Background The robustness of Saccharomyces cerevisiae in facilitating industrial-scale production of ethanol extends its utilization as a platform to synthesize other metabolites. Metabolic engineering strategies, typically via pathway overexpression and deletion, continue to play a key role for optimizing the conversion efficiency of substrates into the desired products. However, chemical production titer or yield remains difficult to predict based on reaction stoichiometry and mass balance. We sampled a large space of data of chemical production from S. cerevisiae , and developed a statistics-based model to calculate production yield using input variables that represent the number of enzymatic steps in the key biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability. Results Based on the production data of about 40 chemicals produced from S. cerevisiae , metabolic engineering methods, nutrient supplementation, and fermentation conditions described therein, we generated mathematical models with numerical and categorical variables to predict production yield. Statistically, the models showed that: 1. Chemical production from central metabolic precursors decreased exponentially with increasing number of enzymatic steps for biosynthesis (>30% loss of yield per enzymatic step, P-value = 0); 2. Categorical variables of gene overexpression and knockout improved product yield by 2~4 folds (P-value < 0.1); 3. Addition of notable amount of intermediate precursors or nutrients improved product yield by over five folds (P-value < 0.05); 4. Performing the cultivation in a well-controlled bioreactor enhanced the yield of product by three folds (P-value < 0.05); 5. Contribution of oxygen to product yield was not statistically significant. Yield calculations for various chemicals using the linear model were in fairly good agreement with the experimental values. The model generally underestimated the ethanol production as compared to other chemicals, which supported the notion that the metabolism of Saccharomyces cerevisiae has historically evolved for robust alcohol fermentation. Conclusions We generated simple mathematical models for first-order approximation of chemical production yield from S. cerevisiae . These linear models provide empirical insights to the effects of strain engineering and cultivation conditions toward biosynthetic efficiency. These models may not only provide guidelines for metabolic engineers to synthesize desired products, but also be useful to compare the biosynthesis performance among different research papers.",
"conclusion": "Conclusions Although S. cerevisiae has been widely used as a robust industrial organism for metabolic engineering applications, many metabolic features of this organism for biosynthesis under various conditions remain unknown. In this study, the statistic model for yeast biosynthesis permits a priori calculation of the final product yield achievable by current biotechnology. Unlike other in silico models based on mass balance or thermodynamics (such as FBA model) [ 36 , 37 ], our model is based on a statistical analysis of published data using numerical and ordinal variables (categorized experimental conditions). The model has three applications. 1. The yield prediction takes into account the genetic design of the microbial host system and the \"suboptimal\" conditions under which the fermentation process occurs. 2. The model may identify effective metabolic strategies and at the same time, quantitatively provide the degree of uncertainty (i.e., possibility for failure). For example, statistical analysis shows that, for S. cerevisiae , metabolic bottlenecks may be more likely to be in the secondary metabolic pathways rather than primary pathways, and thus it can narrow down the genetic targets and avoid futile work. 3. This model may be used to qualitatively benchmark yields of different engineered production platforms.",
"discussion": "Result and Discussion We constructed simple models which linked several numerical and ordinal variables that affected the yield of chemical production from S. cerevisiae . These ordinal variables consisted of the number of modified genes or pathways (OVE), the number of gene knockouts in known competitive pathways (KNO), nutrient source (NUT), intermediate (INT), cultivation mode (CUL), and oxygen availability (OXY). We described the yield of chemical production as the summation of these independent variables in Equation 2. We fitted Equation 2 and determined the coefficients of the variables using linear regression analysis of ~40 compounds. Although multiple data of production yields were often reported in each literature, the model only considered the best yield under a denoted experimental condition. Then, all experimental conditions were categorized by numerical and ordinal variables. The linear regression coefficients obtained for Equation 2 were given in Equation 4, such that: (4) The accuracy of obtained coefficients in Equation 4 was evaluated based on R 2 and the P-value. Here, we used a P-value of 0.1 as the limit below which the result was considered significant [ 14 ]. Out of the eight variables specified in our model, SEC, OVE, KNO, NUT, INT and CUL had P-value of less than 0.1. The summary of the P-value of each variable was listed in Table 3 . Figure 2A showed a plot of the production yields obtained experimentally and those obtained from model prediction for the corresponding conditions. The correlation of this model to the dataset had an R 2 value of 0.55, which reflected the moderate discrepancy between reported yields and the model-predicted yields. Figure 2B plotted the residuals of model fitting. The residuals appeared to scatter around zero randomly, so the linear model was proper to describe the experimental data. Table 3 Regression coefficients and P-values for S. Cerevisiae Model Model 1 Model 2 Model 3 With primary steps Without primary steps Ethanol as a primary metabolite Variable Coefficient P-value Std . Error Coefficient P-value Std . Error Coefficient P-value Std . Error Intercept -1.53 0 0.42 -1.60 0 0.34 -1.73 0 0.41 Primary step -0.01 0.76 0.04 - - - 0.003 0.93 0.03 Secondary step -0.19 0 0.02 -0.19 0 0.02 -0.19 0 0.02 OVE C2 0.007 0.98 0.26 0.0003 0.99 0.25 0.05 0.84 0.24 OVE C3 0.52 0.07 0.29 0.50 0.079 0.28 0.56 0.05 0.28 KNO C2 0.31 0.08 0.18 0.31 0.078 0.18 0.37 0.03 0.17 NUT C2 0.73 0 0.18 0.73 0 0.18 0.71 0 0.17 INT C2 0.77 0.02 0.31 0.82 0.001 0.25 0.86 0.004 0.29 CUL C2 0.51 0.02 0.22 0.51 0.02 0.21 0.51 0.02 0.21 OXY C2 0.27 0.32 0.27 0.28 0.31 0.27 0.12 0.65 0.27 Multiple R 2 0.55 0.55 0.58 Figure 2 Model results . A) Plot of the actual logarithmic yields against the logarithmic yields generated by the regression model. The line drawn as diagonal to the plot is one-to-one and passes through the origin. The data points have an R 2 value of 0.55. B) Plot of residuals against fitted values. C) Model validation using newly published data (2010~2011) 1 - β-amyrin[ 22 ]; 2 - ascorbic acid [ 23 ]; 3 - monoterpene [ 24 ]; 4 - vanillin [ 25 ]; 5 - succinic acid [ 26 ]. Interestingly, the number of enzymes in the primary pathway (PRI) did not significantly affect production yield (P-value = 0.76) (Table 3 ). This suggested that rate-limiting steps to increase chemical production flux often lay in the downstream pathway of central metabolism. The coefficient of SEC was negative. This suggested that the length of a pathway downstream of central metabolism negatively affected production yield. Specifically, addition of a new enzymatic step in a secondary metabolic pathway reduced product yield by 36% (for numerical variable SEC: ). A good demonstration of the effect of pathway length on product yield was found in the case of naringenin production [ 15 ]. With the following inputs of variables PRI = 10 (Galactose to PEP), SEC = 14 (i.e., 10 steps from PEP to phenylalanine; 4 steps from phenylalanine to flavanone), KNO = INT = CUL = OXY = category 1, NUT = Category 2; OVE = Category 3; the model calculated: Yield = 10 -1.53- (0.01 × 10) + (-0.19 × 14) + 0.52+0.73 = 0.0009 (The reported experimental production yield was 0.00058). In most cases, our model-predicted yields were within the range of one order of magnitude compared to the experimental values. Since the number of steps in central metabolism (PRI) did not significantly affect production yield, we computed another set of regression coefficients for Equation 2 without the variable PRI, to yield a simplified form Equation 5. (5) As shown in Table 3 , regression using Equation 2 with the exclusion of the variable PRI did not change the R 2 value. This result indicated that the number of enzymatic steps in primary metabolism did not significantly affect product yield. Presumably, fluxes in central metabolic pathways were typically high and robust [ 16 ], when compared to those downstream secondary pathways. It has been demonstrated recently that production of chemicals was significantly improved, only when the capacity of a downstream pathway was increased [ 17 ]. Metabolic engineering typically involves pathway modification [ 16 - 22 ] to shift metabolic fluxes into a desired product or to permit the use of an alternative carbon source. We defined the variable OVE, and KNO in Equation 2 to capture the effect of pathway overexpression, and deletion, respectively. The regression of experimental data using Equation 2 showed that the coefficients of OVE C2 and OVE C3 had positive values (Table 3 ). The model successfully captured the contribution of both pathway overexpression and gene deletions to increase product yield in S. cerevisiae . The high P-value of OVE C2 (0.98) indicated that statistically, the overexpression of a small number of genes (1-2) was uncertain to improve production yield. However, the coefficient of OVE C3 (= 0.52; P-value = 0.07) indicated the effectiveness of multiple gene modification to resolve the bottleneck steps. This observation is consistent to the fact that metabolic fluxes generally do not sensitively respond to changes of single enzyme activity, but are controlled by all key enzymes along the biosynthesis pathway. On the other hand, the regression coefficients of KNO C2 had positive value (= 0.31, P-value = 0.08), and thus the removal of competitive pathways could be effective to increase production yield. It is a general knowledge that bioprocess conditions affect cellular viability and product yield. Our model suggested fermentation using a well-controlled bioreactor improved production yield by 3.2 times . The model further suggested that fermentation under anaerobic or microaerobic condition could enhance yield compared to aerobic fermentation. However, such enhancement was not statistically significant (P-value = 0.32). This observation could be explained by the fact that S. cerevisiae produced fermentative products (ethanol and glycerol) (Crabtree effect) [ 18 , 19 ] under aerobic and glucose-sufficient medium. Therefore, aerobic metabolism in S. cerevisiae could operate similarly to metabolism under oxygen-limited condition. The coefficient for the variable INT was 0.77, which represented that the supplementation of a precursor metabolite translated to an approximately six fold increase of the product yield (P-value = 0.02). Similarly, the addition of nutrients (such as yeast extract) also significantly increased production yield (the coefficient of NUT C2 was 0.73). The contributions of INT and NUT to product formation indicated that intermediates/nutrients provided building blocks or energy sources that reduced the rate-limiting steps in biosynthetic pathways. We used Equation 2 to compute the production yield of chemicals according to the specifications listed in Table 2 . We observed that, for ethanol production, the experimental values were generally higher than the empirical model predictions. In reality, the reported maximum ethanol yield could reach 0.5 mol C-ethanol/mol C-glucose [ 20 ], which could be several folds higher than model predictions. To mitigate this discrepancy, we re-categorized the ethanol synthesis pathway as the primary pathway to generate Equation 6. (6) Regression of the data using Equation 6 improved the R 2 value from 0.55 to 0.58, demonstrating that ethanol could be better assumed as a central metabolite for S. cerevisiae . Using Equation 6, we predicted ethanol production based on a recent reference [ 21 ] by specifying PRI = 11, SEC = 1 (cellulose degradation step), OVE = C3, KNO = C1; NUT = C2, INT = C1, CUL = C1, and OXY = C2. The ethanol production yield calculated by Equation 6 was 0.31. This value was in good agreement with the reported values of ~0.4 [ 21 ]. Model Applications and Limitations The main application of the model is to predict the biosynthesis yield from S. cerevisiae . The model were validated by \"unseen data\" (Figure 2C ) from some randomly selected new publications (2010~2011). The model predicted the yields based on the reported experimental conditions described by these papers [ 22 - 26 ]. Most yield data were close to model predictions. The predictive power of the model was consistent with the model quality described in Table 3 . Furthermore, the model can reveal the metabolic features of S. cerevisiae . For example, the modified model Equation 6 showed that it was better to treat ethanol pathway as the primary routes in cell metabolism, because of the strong ability for ethanol fermentation by yeast, possibly due to long-term process for selecting yeast as alcohol producer through human history. The model can also be useful for comparing the productivity among other yeast species (Figure 3 ). For example, riboflavin producer, Candida famata , exhibits a high riboflavin productivity (2~3 order of magnitude higher than model prediction) [ 27 ]. Pichia pastoris , a common species for protein expression, shows high S-adenosyl-L-methionine productivity if a large amount of the intermediate methionine was repeatedly added in the medium [ 28 ]. Besides, Pichia stipitis also has high yields of L-lactic acid and ethanol from glucose and xylose [ 29 ]. Figure 3 demonstrated that some yeast species were able to explore their native pathways for biosynthesis of certain products with extraordinary efficiency (better than S. cerevisiae ), therefore, these yeast species may be alternative hosts for certain biotechnology applications. Figure 3 S. cerevisiae model prediction of biosynthesis yields for other industrial yeast species [ 27 - 29 , 38 - 40 ]. Ethanol: ■ or ◆. L-lactic acid: ▲. Lycopene: ●. Riboflavin: + or × . S-adenosyl - L - methionine: ─. The accuracy of the model predictions for some products could be poor due to several limitations during model development. First, the category was a rough estimation of experimental conditions especially for variables related to gene modifications (OVE and KNO), and the yields could be very different even in the same category. Second, some products, despite large synthesis rates, were either not very stable or difficult to accumulate in a large quantity due to consumptions by downstream pathways or product degradations (e.g., Glycerol 3-phosphate [ 30 ]). Their yields could be significantly lower than model predictions even though the actual flux to the product was high. Third, the coefficient β SEC from model regression could not account for the big variances of biosynthesis efficiency or potentially feedback inhibitions in secondary pathways. For example, butanol synthesis is significantly improved via non-fermentative amino acid pathways compared to traditional acetyl-CoA routes [ 31 ], because amino acid synthesis pathways in microorganisms are more effective than other heterogeneous pathways. Fourth, because of limited information from the references, the yield calculation could not precisely include the CO 2 fixation (e.g., overexpression of the native carboxylase pathway: pyruvate + CO 2 → oxaloacetate) [ 32 ] or the nutrients utilization in the rich medium. Fifth, the model neglected enzyme steps related to energy metabolism (such as ATP and NADPH synthesis), while cofactor imbalance can also affect the product yields. Comparison to the previously published E. coli model [ 33 ] Recently, we have constructed the E. coli model using same modeling approach. Compared to the E. coli model, S. cerevisiae shows several differences: 1. Oxygen conditions made a more significant impact on biosynthesis yield in E. coli than that in S. cerevisiae ; 2. The genetic modification in E. coli had higher uncertainty for metabolic outcomes; 3. For metabolic pathways from precursors to final products, loss of yield per biosynthesis step (~30%) in S. cerevisiae is higher than that in E. coli (10~20%). Interestingly, E. coli model states that primary metabolism influences product yield (a relatively small P-value of 0.06) which indicates the balance of precursor production from central metabolism is also an important consideration for metabolic engineering of E. coli . For example, it has been demonstrated that lycopene production with E. coli was enhanced by redirecting the carbon flux from pyruvate to G3P [ 34 ], but feeding other central metabolite precursors (such as pyruvate) could not improve lycopene production. On the other hand, the S. cerevisiae model indicates that it is less likely that the number of steps in central metabolism play a bottleneck role in the production of metabolites derived from it, while the bottlenecks are more likely in the secondary pathways (from central precursors to the final product). Therefore, the metabolic strategies should focus on the secondary pathways to have a better chance for increasing final yield. Although modification of central metabolism may affect microbial physiologies, a few studies indicate the robustness of the central metabolism in S. cerevisiae because of its importance to cell vitality. For example, S. cerevisiae may maintain central metabolic fluxes via gene duplication and alternative pathways under different environmental and physiological conditions [ 16 , 35 ]. Therefore, the inflexibility of central pathways in S. cerevisiae is likely to render metabolic engineering strategies ineffective when targeting enzymes in central metabolism. In general, the unique metabolic features of yeast and bacteria can be of important consideration when choosing a production host."
} | 4,643 |
38009763 | PMC10832539 | pmc | 7,388 | {
"abstract": "Abstract Single engineered microbial species cannot always conduct complex transformations, while complex, incompletely defined microbial consortia have heretofore been suited to a limited range of tasks. As biodesigners bridge this gap with intentionally designed microbial communities, they will, intentionally or otherwise, build communities that embody particular ideas about what microbial communities can and should be. Here, we suggest that metaphors—ideas about what microbial communities are like —are therefore important tools for designing synthetic consortia‐based bioreactors. We identify a range of metaphors currently employed in peer‐reviewed microbiome research articles, characterizing each through its potential structural implications and distinctive imagery. We present this metaphor catalogue in the interest of, first, making metaphors visible as design choices, second, enabling deliberate experimentation with them towards expanding the potential design space of the field, and third, encouraging reflection on the goals and values they embed.",
"introduction": "INTRODUCTION Two traditions in microbial engineering traditions are merging. One has relied on individual microbial strains to drive processes, whereby control is exerted through selecting a suitable strain, maintaining stable culture conditions, and preventing contamination. The other has relied on complex co‐cultures—potentially including microbes that are neither individually selected nor individually identified—whereby control is exerted through cultivating a stable community with predictable properties, as in wastewater treatment. In light of increasingly sophisticated biosynthetic goals, however, microbial consortia are being deliberately assembled to carry out complex metabolic transformations, requiring new ways of thinking about engineering microbial relationships: among microbes, among microbes and environments, and maybe among microbes and scientists who work with them, too. This movement to engineer stable, productive microbial consortia begs a question: what kinds of communities do and can microbes form? Communities in the more easily observable macrobial world can be structured in many different ways. Identifying which of those ways applies in the microbial world—or whether new concepts of community are required—is not obvious because complex microbial communities can only ever be studied indirectly. Researchers conducting observational or experimental studies must infer microbial relationships from metagenomic or other meta‐omics data obtained in a way that destroys much if not all of the internal structure that a microbial consortium might have had. Assembling that picture requires having some idea of what they are trying to piece (back) together. Better‐characterized communities of other kinds—ecologies, economies, towns, and so on—serve as a conceptual repository for doing so. Conceptual tools for working with microbial consortia shape what kind of consortia can be built just as physical or computational tools do, constraining what can be imagined and what goals or targets seem reasonable (Thibodeau & Boroditsky, 2013 ). Expectations about what microbial consortia are like shape how parts and wholes are understood, and therefore how protocols are described and enacted (Keller, 2017 , 2020 ). Metaphors—expectations about microbial consortia built into the everyday language of science—are therefore important construction tools for building consortia‐based biotechnologies (Döring & Zunino, 2014 ; Martínez & Carrillo, 2021 ). As science and technology studies researchers interested in how metaphors and other features of language shape emerging biotechnologies, we have addressed the question “what kind of communities are microbial communities?” by surveying metaphors applied to microbiomes (a term we employ interchangeably with microbial communities and microbial consortia in keeping with the literature we analyse) in recent peer‐reviewed articles. This article reports the results of that study as a catalogue of metaphors that represents the current range of images through which microbial community structure is being imagined and understood. This catalogue indicates how microbes are being configured in contemporary research now that they are understood as simultaneously social, engineerable, and tools for addressing global challenges. We hope that it serves as a resource to enable explicitly understanding and deliberately employing metaphors as part of the toolkit for investigating varied aspects of microbial lifestyle and behaviour, and for engineering microbial consortia. We also hope that juxtaposing the variety of extant empirically supported metaphors works against the tendency that sometimes appears in the literature to argue that microbial consortia fundamentally are any one of these possible ways of imagining them to the exclusion of others.",
"discussion": "DISCUSSION AND CONCLUSIONS We present this metaphor catalogue as a snapshot of the microbiome field and an invitation to researchers to explicitly consider and experiment with the conceptual tools employed to make sense of what microbial consortia may be. Our overarching observations are, first, that a profusion of metaphors are being employed to imagine, understand, and construct microbial consortia, and second, that most of these are fundamentally ecological. Different metaphors are not mutually exclusive, and are routinely mixed. Because each emphasizes different facets of what microbial communities can be, any one metaphor will be insufficient to make sense of microbial behaviour; in other words, metaphor targets invariably exceed their sources. Mixed metaphors are a common feature of everyday language and, as a general rule, present no challenges for communication even when they present some conflicting expectations (Kimmel, 2010 ). Many of these metaphors are clearly more useful for some purposes than others, and it is reasonable to assume that designing custom microbial consortia will require multiple metaphors in combination. Designing stable microbial consortia is notoriously difficult. In microeconomic terms, they tend to suffer from the “winner‐takes‐all” problem: even a small advantage in resource consumption and growth efficiency tends to lead to one member of the would‐be consortium dominating the others (e.g. Wang et al., 2022 ). In contrast, finding stable microbial consortia is easy. Through at least one lens, this discrepancy is unsurprising. Designed consortia must be constructed through conceptual models of what they are expected to be like—through metaphor—and those conceptual models are invariably partial and imperfect; they account for some but not all possible parameters. Found microbial consortia, for example, may not obey principles derived from human economics, e.g., by not prioritizing growth as the ultimate good or resource competition as the primary driver of community structure. Constructing stable de novo consortia may thus require either novel approaches to making sense of community structure or a combination of extant approaches that have yet to be synthesized. In other words, in addition to in silico and wet‐lab experimentation to identify essential parameters, researchers may need to experiment with the metaphors employed to conceptualize the structure of what they are trying to build. Experimenting with metaphors is easier when metaphors are recognizable as such, when a range of alternatives can be imagined, and when one's current choice does not seem like the only possible choice. While present challenges in constructing functional consortia might appear discouraging in some lights, this moment can also be seen as opportune and even hopeful. Microbial biodesigners have an opportunity (no longer available in many neighbouring fields) to actively consider and experiment with the metaphors that best serve their goals, both in a given project and in what the field aims to accomplish in a global sense. We hope that this moment is not cut off too soon, and that researchers are inspired to think broadly and creatively, for example, about metaphors for microbial consortia that are not fundamentally economic or even fundamentally ecological (active matter and companion species being only two examples) not only to build biotechnologies but also to preserve space to consciously reflect on what they are trying to achieve."
} | 2,105 |
25240674 | PMC4191913 | pmc | 7,391 | {
"abstract": "Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly, and structure-function relationship of those natural amyloid fibers remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibers. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibers have an underwater adhesion energy approaching 20.9 mJ/m 2 , which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibers taken on their own at all pHs and exhibit better tolerance to auto-oxidation than Mfps at pH ≥7.0. This work establishes a platform for engineering multi-component self-assembling materials inspired by nature."
} | 336 |
34115922 | PMC8313247 | pmc | 7,392 | {
"abstract": "Summary There are a need for novel, economical and efficient metal processing technologies to improve critical metal sustainability, particularly for cobalt and nickel which have extensive applications in low‐carbon energy technologies. Fungal metal biorecovery processes show potential in this regard and the products of recovery are also industrially significant. Here we present a basis for selective biorecovery of Co and Ni oxalates and phosphates using reactive spent Aspergillus \n niger culture filtrate containing mycogenic oxalate and phosphate solubilized from struvite. Selective precipitation of oxalates was achieved by adjusting phosphate‐laden filtrates to pH 2.5 prior to precipitation. Co recovery at pH 2.5 was high with a maximum of ~96% achieved, while ~60% Ni recovery was achieved, yielding microscale polyhedral biominerals. Co and Ni phosphates were precipitated at pH 7.5, following prior oxalate removal, resulting in near‐total Co recovery (>99%), while Ni phosphate yields were also high with a recovery maximum of 83.0%.",
"introduction": "Introduction In recent years, much attention has focussed on improving the security of supply and sustainability of critical metal resources through the development of cheaper and environmentally friendly systems for metal processing and extraction from low‐grade ores, sludges and recycled materials (Watling, 2015 ; Werner et al ., 2018 ). Co is one such critical metal resource that faces steep projected increases in demand because of strategically important industrial applications in alloys, electrochemical materials and catalysts (Petavratzi et al ., 2019 ). Low‐cost metal processing technologies may improve the operational viability of Co extraction from under‐utilized resources, such as low‐grade ores and tailings, or those with additional costs associated with mining, e.g. deep‐sea manganese nodules and ferromanganese crusts. Microbial alternatives or adjuncts to traditional hydrometallurgical technologies are now considered to be important options with regard to sustainable metal and mineral processing, with proven industrial applications over many years in the field of metal bioleaching (‘biomining’) (Watling, 2015 ; Johnson, 2018 ). Several other microbial processes for efficient biorecovery of metals from leachates and other metal‐laden solutions also show applied potential (Gallegos‐Garcia et al ., 2008 ; Yang et al ., 2020 ) and these rely on biomineralization and precipitation of metals from solution by direct or indirect processes involving microorganisms or their metabolites (Gadd and Pan, 2016 ; Yang et al ., 2020 ). In this work, we present a new system for selective, high‐yield biorecovery of cobalt (Co) and nickel (Ni), as their corresponding oxalates and phosphates. Ni is a valuable element that is frequently geochemically and industrially associated with Co, and therefore relevant to Co bioprocessing options. To achieve biorecovery of these two metals, we employed a reactive spent Aspergillus \n niger culture filtrate containing excreted oxalate and solubilized inorganic phosphate (P \n i \n ) from the P‐containing mineral, struvite, which was incorporated in the medium. Since metal phosphates are soluble at low pH while simple divalent metal oxalates readily precipitate, we hypothesised that selective recovery of oxalates and phosphates could be achieved by manipulation of solution pH, thereby avoiding the simultaneous precipitation of oxalates and phosphates. Oxalic acid secretion is central to a range of environmental processes mediated by fungi, including lignin degradation, plant pathogenesis, element cycling, and mineral colonization and bioweathering (Cessna et al ., 2000 ; Fomina et al ., 2010 ; Guggiari et al ., 2011 ; Gadd et al ., 2014 ; Ferrier et al ., 2019 ). Furthermore, the ability of oxalate‐producing fungi, such as A . niger , to solubilize phosphate from insoluble inorganic sources is well known, including release from rock phosphate and other fertilizers as well as natural and synthetic struvite (Schneider et al ., 2010 ; Mendes et al ., 2015 , 2013 ; Ceci et al ., 2018 ; Suyamud et al ., 2020 ). Such released mobile phosphate can subsequently precipitate with available metal species forming insoluble secondary metal phosphates, such as chloropyromorphite (Pb 5 (PO 4 ) 3 Cl) with lead, and uranyl phosphates with uranium following depleted uranium or uranium oxide solubilisation by oxalate‐producing fungi (Fomina et al ., 2007 , 2008 ; Rhee et al ., 2012 . Struvite (NH 4 MgPO 4 ·6H 2 O) precipitation has received considerable attention in the water industry for the recovery of phosphate from wastewaters and is produced commercially in several countries (Le Corre et al ., 2007 ; Manning, 2008 ; Parsons and Smith, 2008 ; Peng et al ., 2018 . In water treatment plants, struvite can also extensively crystallize and accumulate in wastewater pipes leading to speculation as to the use of this waste product as a fertilizer (Le Corre et al ., 2009 ). For these reasons, struvite was considered to be an appropriate source of inorganic phosphate for metal phosphate bioprecipitation, specifically the monohydrated form, dittmarite (NH 4 MgPO 4 ·H 2 O). The objective of our work was to provide a proof‐of‐concept demonstration of a versatile and sustainable selective Co and Ni biorecovery system based on fungal oxalate excretion and phosphate solubilisation from struvite which provided a reactive culture filtrate capable of precipitating supplied Co or Ni as insoluble oxalates or phosphates.",
"discussion": "Discussion A shift towards new sustainable concepts in metal processing industries has led to the investigation of bioprecipitation approaches for the recovery of soluble metal species from solution, notwithstanding the long‐standing appeal of such systems for the bioremediation of metal‐contaminated wastewaters (Cessna et al ., 2000 ; Gadd and Pan, 2016 ; Liang and Gadd, 2017 ). In this work, XRD and EDXA analysis confirmed that oxalates were selectively precipitated from solution at pH 2.5. Particularly high Co oxalate precipitation was achieved for both single and mixed‐metal solutions, which contrasted with Ni showing consistently lower recovery efficiencies relative to Co. This can be explained by the lower solubility product of CoC 2 O 4 ∙2H 2 O ( K \n sp = 2.7 × 10 −9 ) compared with NiC 2 O 4 ∙2H 2 O ( K \n sp = 1.2 × 10 −3 ) (Li et al ., 2014 ). With the exception of Ni alone, the highest recoveries were obtained at a 20 mM initial metal concentration, and while Ni recovery at 20 mM was less than at a 10 mM concentration in terms of efficiency, in absolute terms a greater amount of Ni oxalate was precipitated at a 20 mM concentration. While oxalates of divalent metals are sparingly soluble or insoluble and readily precipitate from solutions, soluble metal complexes can also form, comprised of oxalate anions linked to a central metal cation via coordinate bonds (Verma et al ., 2019 ). The frequency of oxalate complex formation increases with excess oxalate and this may contribute to the reduced Co and Ni recovery efficiency at lower initial concentrations due to the lower metal ion to oxalate ratios since both Co 2+ and Ni 2+ can form M(C 2 O 4 ) 2 \n 2− and M(C 2 O 4 ) 3 \n 4− complexes (Krishnamurty and Harris, 1960 ; Tang et al ., 2016 ). Following oxalate removal by Ca‐oxalate precipitation, phosphate precipitation at pH 7.5 was carried out. Phosphate precipitation showed very high recovery efficiencies, with near‐total Co being recovered at each concentration tested in both single and mixed‐metal solutions, while Ni recovery reached 86.1% in the single‐metal precipitations and over 85% in the mixed‐metal solutions at each concentration tested. As before, the dominance of Co recovery from the mixed‐metal solutions may be explained by the differing solubility products of Co 3 (PO 4 ) 2 ( K \n sp = 2.05 × 10 ‐35 ) and Ni 3 (PO 4 ) 2 ( K \n sp = 4.74 × 10 ‐32 ), although both phosphates are highly insoluble and Co recovery was only ~10–15% more efficient than that of Ni. XRD analysis showed clear patterns for Co phosphate in the single‐metal Co and mixed‐metal precipitates. Clear XRD patterns were not detected for Ni phosphate, and the XRD patterns obtained for the Ni precipitates only showed two broad peaks which broadly corresponded with the highest‐intensity reference peaks at 26° and 36°. The absence of clear Ni phosphate patterns in the mixed‐metal precipitates was primarily due to their amorphous nanoscale morphology. However, the clear peaks for P determined using EDXA provided supporting evidence that these precipitates were phosphates. Similar XRD patterns have been reported for Co, Co+Ni and amorphous Ni phosphates in other studies (Tang et al ., 2016 ). Selective metal phosphate precipitation was attempted at pH 7.5 without oxalate removal, but this resulted in simultaneous precipitation of both oxalates and phosphates (data not shown). The data presented here show clearly that phosphates may be selectively precipitated from the solution following an oxalate removal step. Future investigations could seek to optimise a sequential approach, involving total oxalate removal as Co/Ni oxalates, prior to phosphate precipitation. A simplified process summary is shown in Fig. 6 . In this work, oxalate was removed by the addition of an excess of CaCl 2 resulting in calcium oxalate precipitation, which clearly facilitates the efficient biorecovery of pure metal phosphates. Our proposed metal biorecovery system may be envisioned as a modular process that could be applied in combination with any metal processing technologies which produce an aqueous metal‐containing solution, such as wastewaters and streams, hydrometallurgical processing streams and leachates, and recycling processes. While this study used struvite due to its established use in phosphate recovery from wastewaters, and also availability as waste material from water treatment facilities, both aspects implicit with resource sustainability, there is scope for the use of other inorganic phosphate‐containing materials that can be solubilized, such as tricalcium phosphate (TCP), bone char, and rock phosphate (Mendes et al ., 2015 , 2013 ; Ceci et al ., 2018 ; Suyamud et al ., 2020 ). Co and Ni oxalates and phosphates are industrially significant materials and receive wide‐ranging applications as catalysts, pigments and, in the case of Co/Ni oxalates, can be used as precursor materials for the production of oxides which are components of lithium‐ion batteries and other energy storage devices (Wang et al ., 2011 ; Che et al ., 2013 ; Kim et al ., 2015 ; Sun et al ., 2020 ). Co and Ni oxalates and phosphates are also receiving attention for direct application as energy storage materials and appear particularly suited for application in batteries and supercapacitors (Zhang et al ., 2015 ; Li and Gadd, 2017 ; Theerthagiri et al ., 2017 ; Li et al ., 2018 ; Yeoh et al ., 2018 ). There is, therefore, a clear opportunity to explore the suitability of fungal biorecovery products for useful future applications in this area. In summary, a fungal‐struvite biorecovery system showed potential as a low‐cost high‐yield Co and Ni biorecovery system through the production of micro‐ and nanoscale biomineral products. I n principle, such a process could be applied to any metals which form insoluble metal oxalates and phosphates. Fig. 6 Overview of the A . niger struvite reactive spent culture filtrate, metal oxalate and phosphate biorecovery system. The diagram demonstrates the stages required to prepare the reactive oxalate‐ and phosphate‐laden supernatant, following appropriate pH adjustment required for final product precipitation. Protocol stages are displayed in dashed boxes with text in italics, inputs and products are shown in solid boxes with standard text."
} | 2,992 |
23770768 | PMC3709513 | pmc | 7,393 | {
"abstract": "The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum . The 8,355 predicted genes in P. purpureum , hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life.",
"discussion": "Discussion Analysis of the first genome sequence from a mesophilic, unicellular red alga turned up several surprises. First, the genome is tightly packed with coding regions and is intron poor, reminiscent of a bacterial genome. There are very few large gene families, suggesting that unicellular red algal extremophiles and mesophiles may have undergone a phase of genome reduction, perhaps in an extremophilic common ancestor of all Rhodophyta. It is also interesting (if not surprising) that hundreds (5.4–9.3%) of the 8,355 P. purpureum genes show evidence of a reticulated evolutionary history and are likely to be implicated in E/HGT, with many more gene with phylogenies that cannot be readily interpreted using existing data. These data shed light on recent debates about the role of HGT in microbial eukaryote genome evolution, in particular whether phagotrophic and parasitic lineages are more likely to capture foreign genes than strict photoautotrophs 46 47 . In contrast to expectations, we demonstrate that anciently diverged relatives of the free-living, photosynthetic P. purpureum were mediators of HGT between prokaryotes and photosynthetic eukaryotes, vis-à-vis endosymbiosis. We have, however, no way of knowing for certain whether the red algal (or Plantae) ancestor was phagotrophic and therefore more prone to HGT, because evidence of HGT has also been described in non-phagotrophic lineages 48 . Regardless of the mechanism of ancient or more recent HGT, these data underline the fundamental importance of red algae to the evolution of eukaryotic plankton. Red algal plastids and red algal nuclear genes are now widespread in chlorophyll c -containing lineages such as diatoms, haptophytes and cryptophytes. Other highlights of this genome include the finding of a nuclear-encoded PBP that apparently has a plastid function (that is, supported by the presence of a putative plastid-targeting signal), an unexpected diversity of CYP genes compared with green plants, and a simpler pathway for starch biosynthesis (see Supplementary Data 3 for a list of the plastid encoded genes). As red algae have the longest fossil record known among eukaryotes (1.2 billion years 41 ) and the lineage contains up to 14,000 species 1 , P. purpureum provides promise that a wealth of novel information awaits to be unearthed as additional genomes are completed from Rhodophyta."
} | 852 |
25699214 | PMC4330909 | pmc | 7,395 | {
"abstract": "Empirical studies in salt marshes, arid, and alpine systems support the hypothesis that facilitation between plants is an important ecological process in severe or ‘stressful’ environments. Coastal dunes are both abiotically stressful and frequently disturbed systems. Facilitation has been documented, but the evidence to date has not been synthesized. We did a systematic review with meta-analysis to highlight general research gaps in the study of plant interactions in coastal dunes and examine if regional and local factors influence the magnitude of facilitation in these systems. The 32 studies included in the systematic review were done in coastal dunes located in 13 countries around the world but the majority was in the temperate zone (63%). Most of the studies adopt only an observational approach to make inferences about facilitative interactions, whereas only 28% of the studies used both observational and experimental approaches. Among the factors we tested, only geographic region mediates the occurrence of facilitation more broadly in coastal dune systems. The presence of a neighbor positively influenced growth and survival in the tropics, whereas in temperate and subartic regions the effect was neutral for both response variables. We found no evidence that climatic and local factors, such as life-form and life stage of interacting plants, affect the magnitude of facilitation in coastal dunes. Overall, conclusions about plant facilitation in coastal dunes depend on the response variable measured and, more broadly, on the geographic region examined. However, the high variability and the limited number of studies, especially in tropical region, indicate we need to be cautious in the generalization of the conclusions. Anyway, coastal dunes provide an important means to explore topical issues in facilitation research including context dependency, local versus regional drivers of community structure, and the importance of gradients in shaping the outcome of net interactions.",
"introduction": "Introduction The role of positive interactions, or facilitation, between plants as key drivers of plant community dynamics and structure is widely recognized and reviewed ( Brooker et al., 2008 ; McIntire & Fajardo, 2014 ). Most empirical studies show that facilitative effects are more important in severe environments because neighbors frequently buffer other individuals from abiotic stressors ( He, Bertness & Altieri, 2013 ). The classical systems that generated this research topic are deserts ( Franco & Nobel, 1989 ; Flores & Jurado, 2003 ), salt marshes ( Bertness & Hacker, 1994 ; Bertness & Leonard, 1997 ), and more recently, alpine systems ( Badano et al., 2006 ; Cavieres et al., 2014 ). However, positive interactions may also be important in many other ecosystems, and there are similar gradients that likely shift the relative frequency of positive interactions. Recent research in coastal dune vegetation has increasingly focused on facilitation between plants. Coastal dune vegetation, here defined as a mosaic of plant communities in the coast that occupy sandy plains formed by marine deposits (modified from Scarano, 2002 ), is both stressful and highly disturbed, with soil moisture and nutrient limitations, wind exposure, sand burial, salt spray and soil salinity, potentially negatively impacting plants ( Wilson & Sykes, 1999 ). Similar to desert systems, the presence of some plants, such as shrubs, ameliorate some of these limiting factors and can provide an opportunity for association by other species ( Martínez & García-Franco, 2004 ). Several studies in coastal dunes have shown that the performance of plants established in the neighborhood of other plants are higher than in open areas ( Shumway, 2000 ; Martínez, 2003 ; Forey, Lortie & Michalet, 2009 ; Castanho & Prado, 2014 ). However, the occurrence and intensity of facilitation in coastal dunes is also highly variable within and between the studies ( Forey, Lortie & Michalet, 2009 ; Castanho, Oliveira & Prado, 2012 ) thereby suggesting that facilitation is dependent on the local environmental conditions or the gradients ( He, Bertness & Altieri, 2013 ) and also on the traits of interacting plants ( Soliveres et al., 2014 ). As demonstrated in dunes and other systems, the magnitude of facilitation depends on plant life-stage ( Miriti, 2006 ; Armas & Pugnaire, 2009 ) and plant life-form ( Gómez-Aparicio, 2009 ; Castanho, Oliveira & Prado, 2012 ) with higher intensities associated with adult woody benefactors and woody beneficiary species at relatively earlier life-stages. Furthermore, the enviromental severity also shapes the outcome of interactions, with more intense facilitation commonly detected under increasingly harsh conditions ( He, Bertness & Altieri, 2013 ). Consequently, coastal dunes may also be an ideal system to explore net interactions in plants communities. However, without synthesis, the context dependency of these positive interactions is not broadly accessible ( Gómez-Aparicio, 2009 ) and research gaps are not easily identified. Therefore, a formal quantitative analysis of the literature in these systems is required. Such analysis can provide an estimate of the general influence of facilitation on the organization and dynamics of the coastal dunes, further the scope of hypothesis testing in this ecological subdiscipline, and contrast the relative importance of local versus regional drivers of plant community structure ( Thebault et al., 2014 ). We note that the scale of drivers of community structure is an important contemporary issue in ecology ( Powers et al., 2009 ; O’Halloran et al., 2013 ) that most likely needs to be resolved on an ecosystem-by-ecosystem basis. Moreover, the restoration of degraded coastal dunes is a pressing issue in many regions of the world ( Lithgow et al., 2013 ), and facilitation by dominant coastal plant species is an obvious potential management solution. To meet these research needs, we present a systematic review and meta-analysis of facilitation in coastal dune plant communities. The systematic review synthesizes current literature and highlights research gaps, while the meta-analysis tests if factors at distinct scales (local versus regional) such as environmental severity, life-form, or life-stage of the interacting plants significantly explained the variation in the intensity of plant facilitation in coastal dunes.",
"discussion": "Discussion Empirical studies support the hypothesis that facilitation between plants is an important ecological process in severe environments ( Brooker et al., 2008 ) including as demonstrated herein coastal dunes. The first goal of the systematic review was to highlight general research gaps in the plant facilitation research in coastal dunes. We found a clear concentration of dune facilitation studies in temperate dunes, indicating that to assess the impact of climate differences and gradients on facilitation between plants, future studies need to be done in other dune systems such as the tropics. The systematic review also identified a predominance of observational studies over experimental studies, and this is unfortunate in many respects, given that the former is a weaker form of inductive inference. The second goal of this study was to examine the importance of factors quantitatively and contrast different scales of drivers on interaction strengths. The response variables measured in facilitation studies were an important determinant of the factors determining the strength of interactions. For the three response variables considered (density, growth, and survival), we found significant evidence for the importance of geographic region in determining the magnitude of facilitation, but no evidence for the effects of climatic and local factors within a region such as life form and life stage of the interacting species. Collectively, this indicates that facilitation is important in coastal dunes and that its relative intensity is best described by the regional context. As highlighted previously, the clear concentration of dune facilitation studies in temperate dunes suggests that we need to expand the scope of coastal dune interaction studies to other geographic regions. This is important for a number of reasons. Macroecological synthesis is an important, novel, and dominant source of theory validation in community ecology ( Keith et al., 2012 ). Alpine and semi-arid syntheses ( Maestre, Valladares & Reynolds, 2005 ; Arredondo-Núnez, Badano & Bustamante, 2009 ) and large-scale integrated experiments ( Fraser et al., 2013 ) are a powerful means to test global issues including the importance of small-scale processes ( Paine, 2010 ) such as interactions. Considering that coastal dunes are subject to significant global change effects ( Van der Meulen, Witter & Ritchie, 1991 ), understanding how plant interactions vary between geographic regions increases predictive ecology on important issues such as climate change effects on plant community structure globally ( Michalet et al., 2014 ). As highlighted by Hesp (2004) , even though few comparative studies have been carried out, differences in factors such as species, adaptative strategies and rates of plants growth indicate that ecological processes may be distinct between tropical and temperate dunes in many systems. Another major limitation identified by the systematic review was the predominance of observational studies over experimental studies. Observational studies included spatial association analyses among species ( McIntire & Fajardo, 2009 ; Cushman, Waller & Hoak, 2010 ; Castanho, Oliveira & Prado, 2012 ). Although positive associations provide evidence of facilitation, this associational pattern does not exclude alternative explanations such as shared physical microhabitats requirements and the tendency of some plants to act as foci for seed deposition ( Callaway, 1995 ). Alternatively, experimental manipulations provide a causal form of verification for plant facilitation because the mechanistic pathways can be identified ( Callaway, 2007 ). Consequently, we also recommend that coastal dunes be studied more comprehensively using manipulative approaches or a combination of observational and experimental methodologies to decouple direct from indirect effects ( Kunstler et al., 2006 ), identify mechanisms ( Shumway, 2000 ; Maestre, Bautista & Cortina, 2003 ; Cushman, Lortie & Christian, 2011 ), and examine the importance of local variation ( Lu et al., 2011 ; McIntire & Fajardo, 2014 ). The extent that facilitation or plant interactions in general can be used to manage or restore highly impacted/stressed systems such as coastal dunes is generally best examined through manipulation. The quantitative examination of plant facilitation magnitude across studies, i.e., the meta-analyses, showed that the factors influencing the occurrence and magnitude of facilitation in coastal dunes depended on the response variable measured. Whilst geographical region influenced the magnitude of facilitation for plant growth and survival, no effect of region was observed on interactions regarding plant density ( Table 2 ). This result supports the general findings of another meta-analysis on facilitation for arid and semi-arid environments that concluded that the effect of abiotic stress on the outcome of interactions depended on the plant response ( Maestre, Valladares & Reynolds, 2005 ). In order to explain this difference between response variables, we need to better understand how the neighbor presence changes the conditions and resources in its neighborhood and how it affects the distinct species-specific responses ( Michalet et al., 2014 ). Therefore, to better understand the factors affecting the magnitude of positive interactions, we must investigate the mechanisms behind facilitative interactions in coastal dunes and, importantly, also record multiple target responses to neighbors ( Hastwell & Facelli, 2003 ; Brooker et al., 2008 ). This is rarely done in a single study (but see for instance Rudgers & Maron, 2003 ; Cushman, Lortie & Christian, 2011 ) but is nonetheless an important avenue of research that will benefit assessment of restoration efforts. Growth and survival trends suggest that geographic region mediates the presence of facilitation more broadly in coastal dune systems. This is a very novel finding ( Thebault et al., 2014 ). Altogether, these results showed that the presence of a neighbor was positive for plant survival and growth in the tropical region, whereas in the temperate and subarctic regions, the effects were neutral for both plant response variables. The environmental severity is relative to the stress tolerance and resource use adaptations of the species within a system ( Lortie, 2010 ). The species composition and predominant life-forms differ between tropical and temperate dunes ( Hesp, 2004 ). Therefore, the observed result can be the product of different sets of traits associated with the species in each region respectively, and consequently, distinct sensitivities to the changes in conditions and resources generated by neighbor presence in the tropical and temperate dunes. However, the limited number of tropical studies indicates that we need to be cautious in the generalization of this alternative hypothesis at this junction. In the present synthesis, we focused on only facilitation studies to test hypothesis related to the magnitude of this interaction, although competition and facilitation are of course both subsets of plant–plant interactions. To explore and contrast the relative importance of competition and facilitation, primary studies in coastal dunes must now test them directly and concurrently, and the scope of a synthesis must be expanded to also include plant competition studies. The capacity for regional drivers of change to mediate positive, local interactions is a novel and important challenge to traditional community ecology and suggests that studies must also now consider the regional context in studying plant–plant interactions even at relatively fine scales in these systems. From a restoration and management perspective, this also suggests that best practices in using facilitation to reduce potential anthropogenic or disturbance effects may need to be tested and/or applied via different mechanistic pathways depending on the importance of regional drivers on productivity gradients and specific local limitations to key target plant species. The use of remote sensing data together with meta-analytical techniques could be a powerful tool to explore the importance of climatic and environmental covariates (usually not provided by primary studies) driving ecological processes. In the present study we did not find an effect of NDVI and MAP on the variability of plant interaction in coastal dunes. One possible explanation for the failure to detect a significant effect of the remote sensing covariates and the other local covariates also tested (plant life-form and life-stage) is the highly variable nature of ecological data together with the small number of primary studies available to construct a big picture. However, as we accumulate primary data testing plant facilitation in coastal dunes, we should be able to drawn synthesis with more definitive conclusions about the factors driving facilitation intensity."
} | 3,850 |
37312147 | PMC10262425 | pmc | 7,396 | {
"abstract": "Background Characterization of microbial activity is essential to the understanding of the basic biology of microbial communities, as the function of a microbiome is defined by its biochemically active (“viable”) community members. Current sequence-based technologies can rarely differentiate microbial activity, due to their inability to distinguish live and dead sourced DNA. As a result, our understanding of microbial community structures and the potential mechanisms of transmission between humans and our surrounding environments remains incomplete. As a potential solution, 16S rRNA transcript-based amplicon sequencing (16S-RNA-seq) has been proposed as a reliable methodology to characterize the active components of a microbiome, but its efficacy has not been evaluated systematically. Here, we present our work to benchmark RNA-based amplicon sequencing for activity assessment in synthetic and environmentally sourced microbial communities. Results In synthetic mixtures of living and heat-killed Escherichia coli and Streptococcus sanguinis , 16S-RNA-seq successfully reconstructed the active compositions of the communities. However, in the realistic environmental samples, no significant compositional differences were observed in RNA (“actively transcribed — active”) vs. DNA (“whole” communities) spiked with E. coli controls, suggesting that this methodology is not appropriate for activity assessment in complex communities. The results were slightly different when validated in environmental samples of similar origins (i.e., from Boston subway systems), where samples were differentiated both by environment type as well as by library type, though compositional dissimilarities between DNA and RNA samples remained low (Bray–Curtis distance median: 0.34–0.49). To improve the interpretation of 16S-RNA-seq results, we compared our results with previous studies and found that 16S-RNA-seq suggests taxon-wise viability trends (i.e., specific taxa are universally more or less likely to be viable compared to others) in samples of similar origins. Conclusions This study provides a comprehensive evaluation of 16S-RNA-seq for viability assessment in synthetic and complex microbial communities. The results found that while 16S-RNA-seq was able to semi-quantify microbial viability in relatively simple communities, it only suggests a taxon-dependent “relative” viability in realistic communities. \n Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-022-01449-y.",
"conclusion": "Conclusions In conclusion, our results found that while 16S-RNA-seq alone may never fully quantify viability in microbial communities, it can provide a qualitative profile of which community members are generally viable across similar environments and remain to be coupled with additional molecular approaches to understand the mechanisms of persistence, metabolism, and potential health consequences in the BE, environmental, and human microbiomes [ 62 , 63 ].",
"discussion": "Discussion The role of rRNA as an indicator of microbial activity has long been scrutinized, with the concern that the correlation between real-time microbial activity and rRNA quantities in environmental samples is inherently inconsistent due to differences across microbes biologically and across environments [ 12 , 44 , 45 ]. Before this limitation could be addressed, 16S-RNA-seq became an established method for microbial community activity assessment and has been applied in various environments [ 11 , 16 – 18 ]. The reliability of this technique remains to be tested across different applications and is especially needed in complex microbial communities. This study performed a systematic evaluation of 16S-RNA-seq using synthetic, spiked realistic communities, and environmental microbial communities and for the first time explored its quantitative potential. 16S-RNA-seq was qualitatively appropriate and provided at least semiquantitative evaluation on active taxa in simple synthetic communities. However, this approach was unable to distinguish active microbes from relic DNA in complex realistic situations, with the differentiation most likely affected by the sustainability of rRNA molecules of each taxon and underlying growth rates of the microbes rather than viability per se (e.g., higher transcription/translation rate during exponential growth period, more ribosomes and more lingering rRNA in actively reproducing microbes). On the other hand, while 16S-RNA-seq presented minimal differentiations in spiked realistic samples, it did provide some insight into the discriminative ability between DNA and RNA libraries in samples of similar origin in our study (Fig. 3 ) as well as in a previous study with low biomass built environment samples [ 40 ]. These findings highlight the need for alternative approaches that accurately assess community activity in a way that allows us to further understand the basic biology of microbial communities and human-microbiome interactions. The performance of 16S-RNA-seq in real microbial communities varied by environment, likely as a result of both biological and technical factors, including the biochemical characteristics of each sample, the biology of the microbes within communities (e.g., gene copies in the bacterial genomes, transcription rate, stability difference from microbe to microbe), and community composition (microbial diversity, viability, etc.). Previous reports have indicated that many biochemical factors can influence 16S-RNA-seq readouts [ 12 ]. Especially in non-host-associated contexts, moisture level, pH, light (UV) exposure, and the presence of nucleases can all vary dramatically among microenvironments, affecting the longevity of rRNA molecules as well as their absorption to their matrices (dust and other small particles) [ 46 – 48 ]. This biochemical phenomenon may help explain the non-differentiable results in soil and some of the BE samples (Figs. 2 and 4 a), where highly moisturized (soil, bathroom), particle-rich (soil, outdoor/MBTA samples), and light (UV)-protected (bathroom, kitchen, and other indoor surfaces) environments tended to retain rRNA molecules longer, thus diminishing the differences between active vs. whole communities. The greater biochemical diversity and instability in such environments are usually also accompanied by greater microbial diversity and by more extensive differences in microbial activity, further affecting 16S-RNA-seq results. It should be noted that the physical and biochemical characteristics of BEs also bring additional technical challenges to accurate interpretation of 16S-RNA-seq readouts, inasmuch as individual protocol steps can also be differentially affected by different environmental conditions. Increasing this potential complexity, different microbial members of these communities may also be differentially affected — DNA/RNA yields may not be comparable directly after extraction, the efficiency of reverse transcription of RNA molecules can differ, and library construction may be variable among different environments. These technical factors also contribute to making 16S-RNA-seq somewhat more consistent in lower diversity, less biochemically complex environments, as also observed in this study [ 12 , 38 , 49 ]. Despite these potential limitations, ribosomal RNA has been widely used to investigate active microorganisms in human and environmental samples on the basis that they are relatively stable and accessible both inter- and intracellularly [ 12 ]. However, this raises the question of whether rRNA in microbial communities accurately represents which microorganisms are active in “real time.” The half-life of recently produced rRNA in soil bacterial communities was reported to have strong temperature dependency, which increased from days to over a year as the temperature decreased [ 50 ]. This suggests that rRNA may remain long after the microbe is dead and thus may not accurately indicate activity, especially when the samples are from chemically and geographically diverse backgrounds. Aside from the persistence of rRNA molecules, the 16S rRNA gene is not linearly correlated with actual bacterial count by nature [ 12 , 51 ]. Copy number of the 16S rRNA gene varies greatly across microbial species. This does not even consider the gene’s transcription rate, which varies with factors such as growth rate, life stages, and exposure to stressors [ 12 , 52 ]. Thus, 16S RNA/DNA abundance ratios vary between and within microbial communities. Particularly, some active, highly transcribable taxa may look “dormant” within a mixed community, as dormant cells may accumulate high numbers of ribosomes [ 1 ]. On the other hand, it is also possible that dead microorganisms or those with low metabolic output would appear as active, given the large amount and persistence of rRNA molecules [ 44 ]. This is worth considering especially in BE samples, where desiccation, regular disinfection, and lack of nutrients contributed to a relatively harsh environment and thus more microbes being dead or dormant [ 6 ]. The higher RNA/DNA ratios of the Lachnospiraceae and Tissierellaceae families in stool samples therefore do not necessarily indicate their being “more active” compared to BE or oil facilities (Fig. 4 c) but a reiteration of the biological differences (i.e., life stages, microbial compositions) in the underlying microbial communities. Quantitation of active microbes using ribosomal RNA transcripts would thus be affected by the community structure as well as environments biochemical background — e.g., the readouts from 16S-RNA-seq would not linearly reflect the active composition outside very simple communities that contain mono- or closely related species and that are growing at a similar rate, which closely resembles our results (Fig. 1 ). 16S-RNA-seq was qualitatively and semiquantitatively informative in simple synthetic communities, but it is not always effective in complex communities and only indicates relative activity trends in BE samples. It is thus important to set proper criteria for better interpretation of 16S-RNA-seq results. This may be achieved by filtering the taxa based on appropriate prevalence or absolute abundance (read count) so that some systematic interference, such as contamination or taxa ubiquitous in those environments, may be minimized. This, however, risks losing information on rare taxa, which were reported being more differentially abundant in RNA vs. DNA libraries in BE samples [ 18 , 40 ]. As in shotgun metagenomic sequencing, microbial community structure represented by 16S-RNA-seq is dependent on sampling effects and sequencing depth [ 7 , 44 , 53 ]. This is particularly true in activity assays in low biomass BE samples, where many rare taxa can only be detected in larger read libraries, and the differences between RNA and DNA libraries can be misrepresented due to the sampling stochasticity [ 12 ]. This explained some of the “phantom taxa” (taxa detected in RNA libraries by not the DNA libraries) [ 20 ] across various families (Fig. 4 c) — the disproportionately high activity of those rare taxa is more likely a technical flaw than a real sign of activity. Therefore, it is important to consider the ceiling of sequencing technologies (depth, sampling statistics) when interpreting 16S-RNA-seq readouts in complex communities. Another potentially helpful consideration is to include a threshold 16S abundance ratio to determine active taxa [ 54 ]. A ratio threshold of 0.1 to 10 might simply provide a conservative view of activity in microbial communities (Supplementary Fig. 6 ). Additionally, ensuring that the average abundances of a taxon in both RNA and DNA libraries are sufficient for confident detection can help ameliorate the effects of sequencing depth. For example, we can be fairly confident that the Comamonadaceae family is likely to be less active in indoor air samples given its high DNA/RNA abundance, which makes it less possible to be misrepresented by technical variance (sampling effect, sequencing depth, etc.). Similarly, the Tissierellaceae family, less active in oil facilities, is more transcriptionally active in stool samples and well-detected as such (Fig. 4 c). However, for the reasons introduced above, it is not possible from sequencing alone to determine that these consistently enriched/depleted families simply have more/less persistent rRNA molecules within these particular environments. Overall, the results of 16S-RNA-seq may be better interpreted with the consideration of proper filtration, sequencing parameters, and overall abundance of the taxa. A comprehensive evaluation of 16S-RNA-seq in microbial communities would require extensive effort, and there are limitations of the current study as a result. Our synthetically co-cultured and spiked “communities” use only a very small number of representative microbes, if anything leading us to underestimate the variability of 16S-RNA-seq between protocols and settings. While it may be impossible to directly interpret 16S-RNA-seq results for accurate viability quantifications, the thoughts here are promising in terms of qualitative assessment: determining which microbes are generally more active in a (or similar) environmental source. Other potential improvements might include the combination of other microbiological experiments, such as metabolic capacity measurements that directly capture the metabolism activities [ 55 – 57 ], biochemical colorimetry that based in the membrane integrity of viable cells [ 58 ], or to explore alternative activity markers from mRNA transcribed from protein-coding genes. A small number of protein coding genes have also been proposed for microbial community viability profiling in previous work, such rpoB , gyrB , and cpn60 [ 59 – 61 ]. These housekeeping genes are potentially conserved enough to be detected and amplified using universal primers as with the 16S rRNA gene while similarly retaining variable regions used to discriminate microbes at high resolution. mRNAs from such functional genes have shorter half-life compared to ribosomal RNA and may thus better represent currently active members of a community. Additionally, they present in one copy (or conserved copies) in bacterial genomes and are transcribed stably but exclusively in the active growth phase of the cellular life cycle, so that their copies directly correlate with the active bacterial amounts. If their transcripts could be targeted as universally and reliably as those of the 16S rRNA gene, this combination of properties would make these genes of potential interest for viability assessment as well. Last but not least, activity assessment in microbial communities will benefit substantially from multiomic integration, e.g., combining 16S-RNA-seq with functional indicators such as metatranscriptomic or metaproteomic profiles. These somewhat circumvent the drawbacks of using 16S rRNA gene as activity markers, providing a complementary definition of viability, and directly observing activities such as virulence, pathogenicity, or antimicrobial resistance that are not captured by amplicon sequencing."
} | 3,809 |
33369139 | PMC8085927 | pmc | 7,398 | {
"abstract": "A 6‐step cascade is distributed among P. taiwanensis and E. coli to synthesize the Nylon‐6 monomer 6‐aminohexanoic acid from cyclohexane under environmentally friendly conditions. The appropriate shuttle compound, avoidance of substrate toxicity, and mass transfer considerations finally enabled 86% 6‐aminohexanoic acid yield in a mixed‐species biotransformation.",
"conclusion": "Conclusion and future perspectives This is the first study that demonstrates a one‐pot biocatalytic synthesis of 6AHA and 7AHA from cycloalkanes with 100% conversion and high selectivities (86% and 49% respectively), which outcompete the one‐pot chemical process with yields below 10% (You et al ., 2008 ). Due to coexpression issues for the 8 genes required, the cascade was apportioned among P. taiwanensis and E. coli . As a shuttle compound, є‐CL turned out feasible as it readily diffuses through cell membranes enabling high 6AHA production rates. In shake flasks, the interplay between the cyclohexane and O 2 mass transfer and respective reaction rates was found to be critical for attaining a high 6AHA yield. Respective optimization of reaction conditions finally enabled the complete conversion of 10 mM cyclohexane with 86% 6AHA yield. For the technical scale, sophisticated feeding regimes and reaction engineering are prone to facilitate the balancing of oxygen and cyclohexane mass transfer and conversion rates. Suitable concepts include two‐liquid phase systems (Kuhn et al ., 2012 ) or a feed of cyclohexane‐saturated air. Furthermore, the mixed‐species phototrophic biofilm concept enabling continuous photosynthetic O 2 supply and operation at high cell densities (Hoschek et al ., 2019 ) may be adapted for 6AHA production from cyclohexane. Besides the in situ oxygen supply, the utilization of light as the energy source and water as the electron source enabling redox cofactor regeneration constitutes another possible advantage of this approach, when cascade parts are implemented in phototrophic microbes.",
"introduction": "Introduction Synthetic fibres, especially Nylon 6 and Nylon 66, account for 95% of the total polyamide production (Bellussi and Perego, 2000 ), with applications in the automotive, textile, electronics and packaging industries (Moody and Needles, 2004 ; BASF, 2020 ). The industrial synthesis of Nylon 6 from cyclohexane, with a scale of 4.2 million tons year ‐1 , involves multiple reaction steps and thus is highly resource‐intensive (Ritz et al ., 1986 ). With an industrial history of 75 years, it suffers from a low cyclohexane per pass conversion of 10–15%, multiple unit operations at high temperature and pressure variations, huge efforts necessary for intermediate product isolation, extensive waste generation, and formation of explosive intermediates (Fischer et al ., 2010 ; Wittcoff et al ., 2012 ). Thus, there is a growing demand to develop sustainable synthetic routes that allow high substrate conversion and product yield, with reduced waste generation and energy consumption (Bellussi and Perego, 2000 ). In this context, the one‐pot (bio)synthesis of 6‐aminohexanoic acid (6AHA as precursor of Nylon 6) from cyclohexane would offer a greener and more sustainable process route. Sattler and coworkers demonstrated an in vitro approach combining 6 isolated enzymes to synthesize 6AHA from cyclohexanol (Sattler et al ., 2014 ). Although balancing the enzyme ratio in the in vitro approach seems to be more straightforward than in vivo , finding the right enzymes with matching kinetics and catalytic efficiencies to synthesize 6AHA without accumulation of intermediate products turned out to be challenging (Sattler et al ., 2014 ). However, the tedious purification of all enzymes, the provision of necessary cofactors and their respective regeneration systems, and enzyme instability under process conditions favour the use of whole‐cell biocatalysts (Duetz et al ., 2001 ; Walton and Stewart, 2004 ; Leak et al ., 2009 , Schrewe et al ., 2013 , Ladkau et al ., 2014 ). In past decades, advances in the metabolic engineering toolbox enabled the rational design and expression of complex biosynthetic pathways in a single host for the production of high‐value chemicals (Ladkau et al ., 2014 ). Although cascades involving more than 5 steps have been rationally engineered (Jeschek et al ., 2017 ), several challenges including redox imbalance, excess metabolic burden, poor expression levels, and toxicity issues often resulted in low product yields (Muschiol et al ., 2015 ; Rudroff, 2019 ). In this context, significant progress has been achieved by segregating complex pathways via the use of more than one engineered microbial strain and thus distributing the metabolic burden (Zhang and Wang, 2016 ; Jones and Wang, 2018 ). For example, the coupling of limonene synthesis and oxyfunctionalization in one Escherichia coli strain resulted in low perillyl acetate yield, which could be improved by distributing this complex reaction cascade among two recombinant E. coli strains (Willrodt et al ., 2015 ). Recently, the Nylon 66 monomer adipic acid (AA) has been synthesized from cyclohexane employing a consortium of three E. coli strains achieving a conversion of 42% and > 99% AA yield (Wang et al ., 2020 ). In this study, we aimed at 6AHA production from cyclohexane in a one‐pot approach with high conversion and yield. For this purpose, previously established Pseudomonas taiwanensis VLB120 strains containing cascades for the conversion of cyclohexane to є‐caprolactone (є‐CL) and/or 6‐hydroxyhexanoic acid (6HA) (Schäfer et al ., 2020 ) served as the basis. The respective biosynthetic pathways involve a cytochrome P450 monooxygenase (Cyp), a cyclohexanol dehydrogenase (CDH), a cyclohexanone monooxygenase (CHMO), and, facultatively, a lactonase (Lact). In this study, we set out to amend this cascade by the alcohol dehydrogenase AlkJ from Pseudomonas putida GPo1 (van Beilen et al ., 2001 ) and the ω‐transaminase CV2025 from Chromobacterium violaceum (Ladkau et al ., 2016 ) to finally synthesize 6AHA from cyclohexane (Fig. 1A ). In order to enable modularity and a high overall pathway flux, we distributed the pathway genes among two vectors and also considered a mixed‐species approach, which requires efficient shuttling of appropriate pathway intermediates among the strains (Jones and Wang, 2018 ). Finally, the operation of resulting systems was investigated by varying biomass amounts, gas–liquid phase ratios, and cyclohexane amounts in the aqueous phase to maximize substrate conversion and 6AHA yield. Fig. 1 Mixed‐species strategies to produce 6‐aminohexanoic acid (6AHA). A. Reaction scheme for the conversion of cyclohexane to 6AHA employing the enzymes Cytochrome P450 monooxygenase (Cyp), cyclohexanol dehydrogenase (CDH), cyclohexanone monooxygenase (CHMO), lactonase (Lact), alcohol dehydrogenase (AlkJ) and ω‐transaminase (TA), (B) combination of P. taiwanensis VLB120 (pSEVA_CL_2) ( P. taiwanensis _CL) and E.coli JM101 (pJ10Lact, pAlaDTA) ( E. coli _CL) with ε‐caprolactone (ε‐CL) as shuttling compound, and (C) combination of P. taiwanensis VLB120 (pSEVA_6HA_2) ( P. taiwanensis _6HA) and E.coli JM101 (pJ10, pAlaDTA) ( E. coli _6HA) with 6‐hydroxyhexanoic acid (6HA) as shuttling compound.",
"discussion": "Discussion In recent years, co‐cultures have been applied to synthesize various compounds such as antibiotic building blocks (Zhang and Stephanopoulos, 2016 ), biofuels (Shin et al ., 2010 ), monoterpenoids (Willrodt et al ., 2015 ) or intermediates for polymer production (Zhang et al ., 2015 ; Wang et al ., 2020 ). In comparison with a single‐species culture, the co‐culture approaches are rationally designed to distribute the metabolic burden of long and complex biocatalytic pathways to different strains (Jones and Wang, 2018 ). However, the use of defined co‐cultures requires the selection of compatible strains, easy access to shuttling compounds, and optimization of reaction conditions to achieve good biocatalytic performance. Selection of biocatalysts to establish a mixed‐species approach The ability of AlkJ to oxidize ω‐hydroxyacids and its tolerance towards the carboxylic acid moiety in 6HA were key findings in this work enabling the establishment of a more direct route for the transformation of cyclohexane to 6AHA compared previous studies (Sattler et al ., 2014 ). Although AlkJ originated from the Pseudomonas genus (van Beilen et al ., 2001 ) and can efficiently be synthesized in E. coli (Schrewe et al ., 2014 ; Ladkau et al ., 2016 ), recombinant expression of alkJ in P. taiwanensis VLB120 was not successful. On the other hand, employing E. coli for cyclohexane conversion was also not feasible, probably due to the high metabolic burden caused by the cytochrome P450 and Baeyer–Villiger monooxygenases. Even leaky expression of respective genes may have prevented successful transformation. For these enzymes, E. coli often does not constitute an optimal host organism (Biggs et al ., 2016 ; Jones and Wang, 2018 ). Reasons include cofactor demand (e.g. heme), uncoupling involving the formation of reactive oxygen species, and drainage of cellular resources (redox equivalents, building blocks). More fine‐tuned gene expression appears to be required to solve issues hampering upper and lower pathway expression in E. coli and P. taiwanensis , respectively, and finally may enable the establishment of a single strain catalysing 6AHA production from cyclohexane. However, the approach to distribute this complex biocatalytic pathway among P. taiwanensis and E. coli proved to be suitable for efficient 6AHA synthesis from cyclohexane and allowed to overcome the issues encountered in the single‐species approaches. A similar approach was successful for the production of oxygenated taxanes (Zhou et al ., 2015 ) and of ethanol from xylan (Shin et al ., 2010 ). Besides alleviation of metabolic burden, mixed‐species/strain approaches can be employed to circumvent inhibitory effects of pathway intermediates (Martínez et al ., 2016 ), to meet cofactor and precursor requirements (Jones et al ., 2016 ), or simply to establish physical compartmentalization for the prevention of sideproduct formation (Chen et al ., 2017 ). Consequently, mixed‐species cultures constitute a solid approach for the efficient operation of otherwise inefficient compex pathways/cascades by exploiting the capacities of two or more strains. Selection of appropriate shuttle compounds in a mixed‐species approach The export and subsequent import of a shuttle compound that connects the pathway between the different species is a prerequisite for the efficient operation of a mixed‐species set‐up. The transport of organic acids, alcohols, simple sugars, amino acids, certain flavonoids and alkaloids can be realized by the selection of appropriate microbes (Zhang and Wang, 2016 ). The inner and outer membranes of Gram‐negative bacteria, such as E. coli and P. taiwanensis, represent barriers for the diffusion of hydrophilic and large hydrophobic compounds, respectively (Leive, 1974 ). In this study, ε‐CL and 6HA were investigated as possible shuttling compounds. Their small size (< 600 Da) allows them to pass the outer membrane by non‐specific transmembrane pores (Denyer and Maillard, 2002 ; Nikaido, 2003 ). The inner membrane allows for passive diffusion of hydrophobic molecules but demands specific transport proteins for more hydrophilic compounds (Chen, 2007 ). 6HA is more polar than ε‐CL and charged at the applied pH, leading to hampered transport of 6HA, as only its protonated form diffuses at a reasonable rate through the cytoplasmic membrane. This slower diffusion as compared to the ε‐CL is reflected by the higher substrate concentration required for half‐maximal activity (Fig. 2 A and B). In certain cases, transporters such as AlkL, a hydrophobic outer membrane pore facilitating the passage of large hydrophobic molecules (Julsing et al ., 2012 ), ShiA as a transporter for 3‐dehydroshikimic acid (Zhang et al ., 2015 ), or glycoside transporters (Shin et al ., 2010 ) are required to enhance the transfer over the cellular membrane. The membrane transfer rate of pathway intermediates thus can constitute a major limitation for the performance of a mixed‐species approach. A careful selection of the intermediate or strain can enhance conversion. Optimization of reaction conditions to maximize product yield Co‐cultivation of different species in growth mode is challenging due to possibly fluctuating community structures caused by differing induction times and growth rates (Jones and Wang, 2018 ). Avoidance of growth by the omission of a growth substrate other than the energy source, for example the N‐source, constitutes a more straightforward approach. The different species can be grown separately according to their specific requirements (e.g. induction time, temperature) and then combined in defined ratios in a resting cell format (Willrodt et al ., 2015 ; Martínez et al ., 2016 ). In this study, a closed reaction system was required to avoid the loss of the volatile substrate cyclohexane. Figure 7 gives a schematic overview on substrate mass transfer and reaction rates. Gas–liquid mass transfer rates for cyclohexane (m 1 ) and O 2 (m 2 ) have a direct influence on r 1 , the lumped rate of the three upper cascade reactions involving two oxygenases (Schäfer et al ., 2020 ), and r 3 , the rate of AlkJ‐catalysed 6HA oxidation, which is linked to the respiratory chain (Kirmair and Skerra, 2014 ). Furthermore, intrinsic dehydrogenases in both strains can convert 6‐oxohexanoic acid into the by‐product AA (r 5, r 6 ), thus minimizing 6AHA yield (Karande et al ., 2017 ; Schäfer et al ., 2020 ). Thereby, r 5 in P. taiwanensis also involves dehydrogenase‐catalysed 6HA oxidation. The challenge was to minimize 6HA accumulation and AA formation. Fig. 7 Schematic representation of substrate mass transfer rates and cascade reaction rates in the mixed‐species set‐up designed in this study. The scheme refers to the consortium composed of P. taiwanensis _CL ( Ps ) and E. coli _CL ( E ). m 1 , gas–liquid cyclohexane mass transfer; m 2 , gas–liquid O 2 mass transfer; r 1 , upper cascade reaction rate lumping the three reactions catalysed by P. taiwanensis _CL; r 2 , rate of lactonase‐catalysed ε‐CL hydrolysis; r 3 , rate of AlkJ‐catalysed 6HA oxidation; r 4 , rate of transaminase‐catalysed 6AHA formation; r 5 , rate of dehydrogenase‐catalysed AA formation in E. coli _CL; r 6 , rate of dehydrogenase‐catalysed AA formation in P. taiwanensis _CL. If m 1 < r 1,max , the cascade performance is governed by cyclohexane mass transfer (substrate limitation). If m 2 < 2r 1,max + r 3,max , O 2 mass transfer becomes limiting. The high lactonase activity available led to r 1 = r 2 . r 2 > r 3 leads to 6HA accumulation and possibly AA formation via r 6 . r 3 > r 4 can lead to AA formation via r 5 . r 1 = r 2 = r 3 = r 4 enables high 6AHA yield. Optimization of reaction conditions led us to utilize a gas–liquid phase ratio of 23 with biomass and cyclohexane concentrations of 2 g CDW l ‐1 and 5 mM, respectively, as well as additional O 2 supply to avoid limitations of r 1 and r 3 (Fig. 6 A and C). Under these conditions, m 1 governed overall cascade performance with the upstream cascade (r 1 = 1.02 mM h ‐1 (all products considered, first 4 h of reaction)) operated under first‐order kinetics (Table 1 , Fig. 6 A and C). The effect of m 2 was minimized combining an appropriate biomass concentration with an O 2 feed. Due to the very high activity of the lactonase, the 6HA formation rate always was equal to the rate of ε‐CL formation, r 1 = r 2 . In set‐ups only containg E. coli _CL (Fig. 2C ) or P. taiwanensis _CL (Fig. S4 ), AA was formed at r 5 = 0.019 or r 6 = 0.041 mM h ‐1 , respectively (see Supporting Information, Section 3 for calculation details). However, within the first 4 h in the optimized mixed‐species set‐up (Fig. 6A ), AA production rates (r 5 + r 6 = 0.03 mM h ‐1 ) were well below the sum of these rates. Thereby, r 6 may have been limited by 6HA availability/uptake. The volumetric 6AHA formation rate r 4 reached 0.92 mM h ‐1 (Table 1 ), which almost equalled r 3 = 0.92 – 0.95 mM h ‐1 , and thus enabled 100% cyclohexane conversion and 82% selectivity for 6AHA formation within 18 h. The step‐wise assessment of crucial reaction parameters and their optimization allowed full cyclohexane conversion, which was not achieved previously (Wang et al ., 2020 ). In comparision with the biological process, a one‐step chemical process using Mn/AlVPO resulted in 9.8% of cyclohexane conversion and 72.9% of selectivity to є‐caprolactam (You et al ., 2008 ). Substrate limitation was selected in this study to minimize inhibition/toxification by cyclohexane and to avoid extensive 6HA accumulation. In the experiment shown in Fig. 5A , the lower cascade did not reach its maximum rate and could not keep up with the upper cascade, although O 2 was not limiting and the necessary cyclohexane limitation was in place (Fig. 5C ). This may be due to the different cyclohexane : biomass ratio applied (1.67 mmol g CDW \n ‐1 ) compared to the experiments shown in Fig. 6 (2.5 mmol g CDW \n ‐1 , applied at two different concentration levels). This effect of the cyclohexane : biomass ratio and a possible interconnection with TA instability as indicated by AA accumulation (Fig. 5A ) remain to be further investigated. The low aqueous cyclohexane concentrations in the optimized set‐up also can be considered beneficial regarding biocatalyst stability. With a logP of 3.44, cyclohexane is expected to cause membrane disintegration at a concentration above 0.6 mM (Sikkema et al ., 1994 ) and, already at lower concentrations, can have effects on membrane fluidity, electron transduction, and, consequently, reactions connected to metabolism (Sikkema et al ., 1994 ). Thus, cyclohexane can affect r 1 (via redox metabolism), r 3 (via electron transport chain), r 4 (via alanine uptake and regeneration) and r 5 /r 6 (via redox metabolism). The establishement of an appropriate feeding regime will be crucial for future reaction engineering and scale up. Further optimization may include strain ratio variation to equilibrate the rates of upstream and downstream cascades as demonstrated by Wang et al . ( 2020 ). The system presented here has been optimized regarding cyclohexane conversion and 6AHA yield and not specific activity. A higher E. coli _CL share can be considered promising to limit 6HA accumulation but has been tested without success (Fig. S2 ). However, O 2 depletion kinetics (Figs 4 and 5 ) indicate that cells were O 2 ‐limited at the high cell concentration and low gas : liquid phase ratio applied in the respective experiment. Thus, strain ratio variation should be combined with a strategy to overcome O 2 limitation. Further, suppression of AA formation can be targeted via the knockout of 6‐oxohexanoic acid oxidation catalysing dehydrogenases in both strains. 7AHA could be synthesized from cycloheptane by the same enzymes and mixed‐species set‐up, although the titre achieved was 2‐fold lower than that obtained for 6AHA, going along with stronger overoxidation to PA. It is known that AlkJ also converts longer‐chain substrates such as 12‐oxododecanoic acid methyl ester with high activity (Schrewe et al ., 2014 ). Thus, competition among TA and host dehydrogenases for 7‐oxoheptanoic acid may be critical. In a previous study, it has been shown that the C. violaceum TA prefers small aliphatic and aromatic molecules (e.g. glyoxylate, butanal, benzaldehyde, phenylacetaldehyde) (Kaulmann et al ., 2007 ). Hence, TA affinity for 7‐oxoheptanoic acid constitutes a promising target to optimize the 7AHA yield, with enzyme engineering and screening of alternative TAs as possible approaches (Guo and Berglund, 2017 )."
} | 5,027 |
26547794 | null | s2 | 7,400 | {
"abstract": "The importance of microbial root inhabitants for plant growth and health was recognized as early as 100 years ago. Recent insights reveal a close symbiotic relationship between plants and their associated microorganisms, and high structural and functional diversity within plant microbiomes. Plants provide microbial communities with specific habitats, which can be broadly categorized as the rhizosphere, phyllosphere, and endosphere. Plant-associated microbes interact with their host in essential functional contexts. They can stimulate germination and growth, help plants fend off disease, promote stress resistance, and influence plant fitness. Therefore, plants have to be considered as metaorganisms within which the associated microbes usually outnumber the cells belonging to the plant host. The structure of the plant microbiome is determined by biotic and abiotic factors but follows ecological rules. Metaorganisms are co-evolved species assemblages. The metabolism and morphology of plants and their microbiota are intensively connected with each other, and the interplay of both maintains the functioning and fitness of the holobiont. Our study of the current literature shows that analysis of plant microbiome data has brought about a paradigm shift in our understanding of the diverse structure and functioning of the plant microbiome with respect to the following: (i) the high interplay of bacteria, archaea, fungi, and protists; (ii) the high specificity even at cultivar level; (iii) the vertical transmission of core microbiomes; (iv) the extraordinary function of endophytes; and (v) several unexpected functions and metabolic interactions. The plant microbiome should be recognized as an additional factor in experimental botany and breeding strategies."
} | 444 |
32011873 | null | s2 | 7,401 | {
"abstract": "Polymer topology dictates dynamic and mechanical properties of materials. For most polymers, topology is a static characteristic. In this article, we present a strategy to chemically trigger dynamic topology changes in polymers in response to a specific chemical stimulus. Starting with a dimerized PEG and hydrophobic linear materials, a lightly cross-linked polymer, and a cross-linked hydrogel, transformations into an amphiphilic linear polymer, lightly cross-linked and linear random copolymers, a cross-linked polymer, and three different hydrogel matrices were achieved via two controllable cross-linking reactions: reversible conjugate additions and thiol-disulfide exchange. Significantly, all the polymers, before or after topological changes, can be triggered to degrade into thiol- or amine-terminated small molecules. The controllable transformations of polymeric morphologies and their degradation herald a new generation of smart materials."
} | 238 |
34380615 | PMC8357239 | pmc | 7,402 | {
"abstract": "Kinking induced local deformation and rotation can lead to autonomous crack-healing in atomically layered ceramic materials.",
"introduction": "INTRODUCTION The lack of materials capable of withstanding extreme environments, more often than not, imposes the greatest technological barrier to the development and deployment of a host of next-generation technologies, such as efficient jet engines, hypersonic flights, and safer nuclear reactors ( 1 – 4 ). While ceramic materials provide outstanding chemical and structural stability at high temperatures and in hostile environments, their insufficient plastic deformability and damage tolerance compared to metallic materials severely limit their applicability ( 5 , 6 ). In particular, at room temperature, ceramic materials readily undergo catastrophic fracture by cohesive bond breaking at the crack tip. Traditional approaches to partially overcome this issue in ceramic materials rely on microstructure-specific toughening mechanisms such as crack-deflection, crack-bridging, microcracking, and stress-induced phase transformations ( 5 , 6 ). The quest to enhance damage tolerance also includes the development of nature-inspired ceramic-based (hybrid) materials with hierarchical structures that can trigger a combination of toughening mechanisms ( 7 – 12 ). Although activating toughening mechanisms during crack growth can postpone catastrophic fracture, a more potent toughening mechanism is to heal the cracks as they form. Crack-healing requires local flow of material to close and heal the cracks by physical or chemical interactions ( 13 ). In principle, this can be realized intrinsically (without healing agents) or extrinsically (with healing agents) and autonomously (without external triggers) or nonautonomously (with external triggers) and is not limited to a single class of materials ( 13 ). Nonetheless, polymers and polymer composites ( 14 – 16 ), as well as cementitious materials ( 17 , 18 ), are by far the biggest beneficiaries of crack-healing mechanisms, while only limited successes have been reported for ceramic ( 19 – 23 ) and metallic ( 24 ) materials. In ceramic materials, except for a few, crack-healing has been mainly realized extrinsically by incorporating healing agents and/or nonautonomously (postmortem) by applying high pressure at high temperatures. In very few cases, autonomous crack-healing in ceramic materials is achieved by high-temperature oxidation of the cracks that form during loading ( 20 – 23 ). Crack-healing by high-temperature oxidation, however, has several limitations. For example, it at least requires the presence of an oxidative phase, accessibility of the crack surfaces to the oxidizing environment, and good adhesion of the oxide scale to the crack surfaces. Also, selective oxidation of some microstructural features (e.g., grain boundaries) can lead to stress concentration sites, while thermal expansion mismatch between the material and the crack-healing oxide can lead to crack initiation during thermal cycling. Here, we demonstrate that intrinsic and autonomous crack-healing is possible even at room temperature in crystalline atomically layered ceramic materials such as MAX phases ( 25 – 27 ) by kinking-induced crystallographic rotation and plastic deformation. MAX phases are a family of ternary carbides/nitrides with general formula M n +1 AX n , where M is an early transitional metal, A is mostly an element from groups 13 to 16, X is carbon or nitrogen, and n varies from 1 to 3, and comprise alternating M n +1 X n and A atomic layers within a hexagonal crystal structure ( 25 – 27 ). Numerous studies have shown that several individual grains in polycrystalline MAX phases can readily kink under a variety of external loading conditions, such as tensile, compressive, creep, fatigue, and impact ( 28 – 33 ). Kinking leads to localized plastic deformation in bands referred to as “kink-bands,” wherein the material is microstructurally or crystallographically misoriented from the unkinked region. Kinking is not unique to MAX phases and has been observed in a variety of natural ( 34 – 37 ) and engineered ( 38 – 46 ) materials. While in engineering composites kinking results in failure ( 40 – 42 ), in polycrystalline MAX phases it has been postulated that kinking restricts the rapid crack growth along the weakly bonded planes by crack-bridging ( 28 , 29 ) and is solely responsible for their unconventional damage tolerance ( 25 , 26 , 30 ). However, our discovery that kinking in MAX phases heals the cracks as they form reveals that it is an even more potent toughening mechanism than previously envisaged. Specifically, we report on the evolution of kinking and its role in crack-healing during room temperature in situ indentation of carefully grown single crystals of the Cr 2 AlC MAX phase (fig. S1) ( 47 ) in a scanning electron microscope (SEM). The in situ indentation is carried out using an in-house design and build fixture that allows us to load single-crystal specimens with a punch along predetermined directions, with and without lateral deformation constraint. The lateral deformation constraint considered here mimics the presence of neighboring grains that affect the deformation and damage evolution in individual grains of a polycrystalline aggregate. Until we used this fixture, it was extremely difficult to study the effect of local deformation constraint on kinking and the implications of kinking on toughening in MAX phases from mechanical testing of their polycrystalline aggregates ( 25 , 26 , 28 – 32 ) or micropillars milled from individual grains ( 48 – 50 ).",
"discussion": "DISCUSSION In summary, it is evident from our results that catastrophic fracture of atomically layered ceramic materials, such as Cr 2 AlC, can be effectively overcome by not only suppressing the crack growth along the weakly bonded planes but also healing them as they form. For instance, by indenting unconstrained specimens, we showed that even a small Poisson’s expansion of the crystal normal to the weakly bonded basal planes is enough to cause initiation and rapid growth of cracks along these planes. However, indentation of these materials while under slight deformation constraint normal to the weakly bonded basal planes enables buckling and kinking of the ligaments between multiple parallel cracks, and formation of kink-bands. Once a kink-band forms, the faces of the portion of the crack that lie within the kink-band undergo permanent tilting, twisting, and closure, leading to physical healing. Furthermore, the growth and widening of multiple adjacent kink-bands lead to physical healing of the entire crack with length greater than hundreds of micrometers. The importance of this finding cannot be overstated as this explains the puzzling observation that the MAX phases, instead of undergoing catastrophic fracture, demonstrate unconventional damage tolerance ( 25 , 26 ). The unconventional damage tolerance of polycrystalline MAX phases not only is limited to monotonic compressive loading but also has been observed under a variety of loading conditions ( 28 – 32 ). From our results, it can be concluded that, in polycrystalline MAX phase aggregates, even the grains that are oriented for easy cleavage along weakly bonded planes can undergo kinking due to the deformation constraint imposed by the neighboring grains and consequently heal the cracks. Thus, the kinking-induced crack-healing mechanism endows these otherwise brittle materials with exceptional resistance to catastrophic fracture. This also implies that the toughness of this class of atomically layered ceramic materials can be further enhanced by carefully designing microstructures to promote extensive kinking and crack-healing. In ceramic materials, limited autonomous crack-healing, i.e., healing of the cracks as they form during loading, has been previously realized by high-temperature oxidation of the cracks ( 21 – 23 ). However, we are not aware of any realization of intrinsic and autonomous crack-healing at room temperature in a monolithic ceramic material. Although we have demonstrated room temperature kinking-induced crack-healing in a particular class of atomically layered ceramic material, i.e., MAX phases, it should be noted that MAX phases are an extremely diverse class of material with approximately 155 MAX phase compositions found to date ( 27 ). Moreover, kinking is not unique to MAX phases and has also been observed in a variety of other atomically layered ceramic materials, and thus, it is reasonable to assume that kinking-induced crack-healing can be achieved in these materials as well. These other atomically layered ceramic materials include graphite ( 37 ), micas ( 34 ), hexagonal carbides ( 43 ), boron nitride ( 44 ), and layered borides ( 46 ), among others, whose broader applications have been limited by their poor damage tolerance and propensity to cleave along weakly bonded planes."
} | 2,237 |
28449106 | PMC5570177 | pmc | 7,403 | {
"abstract": "Abstract The widespread application of next-generation sequencing technologies has revolutionized microbiome research by enabling high-throughput profiling of the genetic contents of microbial communities. How to analyze the resulting large complex datasets remains a key challenge in current microbiome studies. Over the past decade, powerful computational pipelines and robust protocols have been established to enable efficient raw data processing and annotation. The focus has shifted toward downstream statistical analysis and functional interpretation. Here, we introduce MicrobiomeAnalyst, a user-friendly tool that integrates recent progress in statistics and visualization techniques, coupled with novel knowledge bases, to enable comprehensive analysis of common data outputs produced from microbiome studies. MicrobiomeAnalyst contains four modules - the Marker Data Profiling module offers various options for community profiling, comparative analysis and functional prediction based on 16S rRNA marker gene data; the Shotgun Data Profiling module supports exploratory data analysis, functional profiling and metabolic network visualization of shotgun metagenomics or metatranscriptomics data; the Taxon Set Enrichment Analysis module helps interpret taxonomic signatures via enrichment analysis against >300 taxon sets manually curated from literature and public databases; finally, the Projection with Public Data module allows users to visually explore their data with a public reference data for pattern discovery and biological insights. MicrobiomeAnalyst is freely available at http://www.microbiomeanalyst.ca .",
"conclusion": "CONCLUSIONS As a new frontier in biomedical research, current microbiome studies and data analyses are mainly exploratory in nature. Despite the development of many new statistical algorithms in recent years, there is no single statistical method that performs universally well, as clearly shown by a recent large-scale benchmarking test ( 33 ). It is therefore critical to enable researchers in the microbiome field to easily explore their own datasets using a variety of algorithms, in real-time and through interactive visualization, to facilitate data understanding and hypothesis generation. MicrobiomeAnalyst fulfills these requirements by offering comprehensive support for diversity profiling, comparative analysis and metabolic network visual exploration. It also provides novel functions that allow users to interpret their findings with regard to curated taxonomic signatures or to compare their own data with public datasets. We believe MicrobiomeAnalyst fills a critical gap in current microbiome research. The microbiota is complex and dynamic, and to fully understand its behavior as a system and its interactions with the host, more than one type of omics data needs to be collected, analyzed and integrated. Indeed, multi-omics approaches are increasingly adopted for many microbiome studies ( 47 ). The future development of MicrobiomeAnalyst will focus on supporting these expanding trends, particularly in the integration of metabolomics data and systems biology ( 48 – 51 ).",
"introduction": "INTRODUCTION The past decade has seen an immense growth in the number of studies that aim to characterize the structures, functions and dynamics of host-associated microbial communities (microbiota) within the context of host development, pathophysiology, diet and environment perturbations ( 1 , 2 ). These studies have revealed a wide array of important roles that the microbiota play in human and animal health. Due to drastic reduction in costs and its high-throughput capacity, next-generation sequencing has become the preferred method to study the collective genetic contents of microbial communities (microbiome). Currently, microbiome datasets are mainly generated using one of the three common sequencing strategies including marker gene (i.e. 16S rRNA) survey to characterize microbial community compositions, shotgun metagenomics to study their functional potentials, and shotgun metatranscriptomics to identify those actively expressed genes. These studies usually generate datasets that are both large (with regard to data size) and complex (with regard to data structure), posing substantial ‘big data’ challenges in downstream data analysis. The initial computational effort in microbiome data analysis focused on raw sequence processing, clustering and annotation. This led to the development of several powerful tool suites such as MEGAN, MG-RAST, mothur and QIIME ( 3 – 6 ), which together helped to establish the essential pipelines and procedures for processing raw reads generated from microbiome studies. Given the ever-increasing data sizes and computational costs, raw data processing is now typically handled at the same sequencing center following standardized protocols. These procedures produce a key summary table containing feature (Operational Taxonomic Units (OTUs), taxa or genes) abundance information across samples, along with various annotations and sample metadata. The Biological Observation Matrix (BIOM) file was recently developed to store all these types of information to facilitate the interoperability of existing bioinformatics tools and future meta-analyses ( 7 ). For most researchers, their primary challenge in data analysis is how to make sense of the abundance tables or BIOM files within the context of different experimental factors or study conditions. Microbiome data analysis can be placed into four general categories: (i) taxonomic profiling - to characterize community compositions based on methods developed in ecology such as alpha-diversity (within-sample diversity) or beta-diversity (between-sample diversity); (ii) functional profiling - to assign genes into different functional groups (i.e. metabolic pathways or biological processes) to understand their collective functional capacities; (iii) comparative analysis - to identify features that are significantly different among conditions under study and (iv) meta-analysis - to integrate user data with public data or knowledge accumulated for improved statistical power or biological understanding. The first two categories are now relatively straightforward to perform, while the last two categories still remain very challenging and become the focus of intense research efforts. Microbiome abundance data presents several unique challenges including sparsity (containing many zeros), vast differences in sequencing depth, and large variance in distributions (over-dispersion) ( 8 ). These unique characteristics have made it inappropriate to directly apply methods developed in other omics fields to perform comparative analysis on microbiome data. As a result, non-parametric permutation-based methods are often employed for identification of significant features in microbiome data ( 9 , 10 ). Although robust, the main limitations of such approaches are the lack of statistical power and the inability to model confounding factors to accommodate complex experimental designs. To deal with uneven sequencing depth, researchers often resort to two common approaches: rescaling the total reads in each sample to a constant sum (using proportions), or resampling the reads in each sample to an equal amount (rarefying). The former will lead to typical issues associated with compositional data ( 11 ), and the latter may lead to the loss of important information. In general, it is statistically more appropriate to develop suitable statistical models for sparse count data to accommodate differences in sequence depth, or to develop strategies to transform data to have distributions that fit the models assumed by other well-established algorithms. There has been significant progress towards these directions in recent years. For instance, the metagenomeSeq algorithm integrates cumulative-sum scaling (CSS) method and a statistical model based on the zero-inflated Gaussian (ZIG) distribution to improve the power for differential abundance analysis of microbiome data ( 12 ). It has also been shown that, following proper data normalization, the methods developed for RNAseq such as edgeR and DESeq2 perform similarly to or better than many other algorithms developed specifically for microbiome data ( 13 – 15 ). To account for compositional data, different data transformation approaches have been proposed such as the centered log-ratio (CLR) transformation ( 16 ). The majority of these recent methods have been implemented as R packages. In particular, the phyloseq package has been developed to provide a unified framework to allow R users to explore different statistical algorithms for microbiome data analysis ( 17 ). Although powerful and flexible, learning R programming and the underlying statistics can be demanding for most clinicians and bench researchers. There is an urgent demand for user-friendly tools that support these recent approaches for comprehensive statistical analysis of microbiome data. In addition, with the increasing number of public datasets and our growing knowledge about microbiome, it is now possible to perform meta-analyses to reveal larger pictures or novel insights beyond a single study, such as using compatible public datasets for contextualizing new experiments ( 18 ), pooling new data with existing cohorts for increased power ( 19 ), or comparing microbial signatures with those reported from other studies ( 20 ). To address these gaps as well as to meet new requests arising from current microbiome data analysis, we have developed MicrobiomeAnalyst, a web-based program to allow clinical and basic scientists to easily perform exploratory analysis on common abundance profiles and taxonomic signatures generated from microbiome studies. The key features of MicrobiomeAnalyst include:\n Support for a wide array of common as well as advanced methods for taxonomic diversity analysis, functional profiling, visualization and significance testing; Comprehensive support for various data filtering and transformation methods coupled with well-established as well as more recent algorithms for differential abundance analysis; A powerful, fully-featured metabolic network visualization framework for intuitive exploration of results from functional profiling; Support for meta-analysis with compatible public datasets for context reference and pattern discovery using 3D visual analytics; Enrichment analysis based on >300 taxon sets manually collected from literature and public databases. MicrobiomeAnalyst also contains a comprehensive list of frequently asked questions (FAQs) and tutorials to help researchers easily navigate different analysis tasks. Collectively, these features consist of comprehensive tool suites for microbiome data analysis. MicrobiomeAnalyst is freely available at http://www.microbiomeanalyst.ca ."
} | 2,705 |
35056571 | PMC8781833 | pmc | 7,405 | {
"abstract": "Eukaryotic organelles supposedly evolved from their bacterial ancestors because of their benefits to host cells. However, organelles are quite often retained, even when the beneficial metabolic pathway is lost, due to something other than the original beneficial function. The organellar function essential for cell survival is, in the end, the result of organellar evolution, particularly losses of redundant metabolic pathways present in both the host and endosymbiont, followed by a gradual distribution of metabolic functions between the organelle and host. Such biological division of metabolic labor leads to mutual dependence of the endosymbiont and host. Changing environmental conditions, such as the gradual shift of an organism from aerobic to anaerobic conditions or light to dark, can make the original benefit useless. Therefore, it can be challenging to deduce the original beneficial function, if there is any, underlying organellar acquisition. However, it is also possible that the organelle is retained because it simply resists being eliminated or digested untill it becomes indispensable.",
"conclusion": "7. Conclusions It is believed that the evolution of endosymbiotic organelles is driven by a benefit to the endosymbiotic partners. While the benefit of photosynthesis seems to be obvious in the case of plastids, the original beneficial function of mitochondrion is the subject of discussion. The mosaic evolutionary origin of mitochondrial proteome opens a possibility of serial endosymbiotic events behind the current mitochondria, analogous to that of complex plastids. Plastids and mitochondria can represent two different types of endosymbiotic events: invasion of the alphaproteobacterial intracellular parasite into the pre-eukaryotic host and predation on the cyanobacterial ancestor of plastids. In the mitochondrial evolution, the host has adapted to the presence of the parasite, with a gradual transition of the parasitic symbiosis to a mutualism. Plastids evolved from cyanobacterial prey resistant to digestion by the host cell. In both these models, a benefit for the host was not a driving force for organellogenesis.",
"introduction": "1. Introduction A eukaryotic cell is typical by hosting semiautonomous organelles, such as mitochondria and plastids. These organelles are deeply integrated into the host cell; however, they usually keep some level of independence by encoding a fraction of the organellar proteome and RNAs in their genomes [ 1 , 2 , 3 , 4 ], living to some extent like endosymbiotic bacteria [ 5 , 6 ]. Mitochondria and plastids are, with few exceptions, essential for the host cell survival; once the cell has captured an organelle, it can hardly get rid of it [ 1 , 2 , 3 , 4 , 6 , 7 ]. It is believed that mitochondria and plastids evolved in endosymbiotic events, involving an engulfment or invasion of a free-living organellar ancestor, followed by the endosymbiotic transfer of genes from the captured entity to the nucleus of the host cell, with a consequent import of nuclear-encoded proteins into the organelle [ 3 , 8 , 9 ]. Symbiosis is an intimate, long-time relationship of two dissimilar organisms living together [ 10 ]. Although it is often understood as mutualism, the state beneficial for both partners, symbiosis, in fact, involves a continuum of relationships ranging from mutualism to parasitism [ 11 ]. The evolutionary history of plastids by domesticating a cyanobacterium is apparent because they are evolutionarily younger, and a cyanobacterial ancestor was likely acquired by the regular eukaryotic cell capable of phagocytosis [ 3 , 8 , 9 ]. On the other hand, the origin of the evolutionary older mitochondrion is more elusive. It is not straightfoward as to what kind of cell engulfed the mitochondrial ancestor, what ancestor it was, what the original mitochondrial beneficial function was, if it had any, and what kind of a symbiotic relationship the endosymbiotic partners had [ 6 , 12 , 13 , 14 ]. The veil of time successfully obscures the evolutionary history of the mitochondrion."
} | 1,010 |
37687393 | PMC10490184 | pmc | 7,406 | {
"abstract": "Soil contamination with cadmium (Cd) is a severe concern for the developing world due to its non-biodegradability and significant potential to damage the ecosystem and associated services. Industries such as mining, manufacturing, building, etc., rapidly produce a substantial amount of Cd, posing environmental risks. Cd toxicity in crop plants decreases nutrient and water uptake and translocation, increases oxidative damage, interferes with plant metabolism and inhibits plant morphology and physiology. However, various conventional physicochemical approaches are available to remove Cd from the soil, including chemical reduction, immobilization, stabilization and electro-remediation. Nevertheless, these processes are costly and unfriendly to the environment because they require much energy, skilled labor and hazardous chemicals. In contrasting, contaminated soils can be restored by using bioremediation techniques, which use plants alone and in association with different beneficial microbes as cutting-edge approaches. This review covers the bioremediation of soils contaminated with Cd in various new ways. The bioremediation capability of bacteria and fungi alone and in combination with plants are studied and analyzed. Microbes, including bacteria, fungi and algae, are reported to have a high tolerance for metals, having a 98% bioremediation capability. The internal structure of microorganisms, their cell surface characteristics and the surrounding environmental circumstances are all discussed concerning how microbes detoxify metals. Moreover, issues affecting the effectiveness of bioremediation are explored, along with potential difficulties, solutions and prospects.",
"conclusion": "10. Conclusions High levels of Cd are being released into the environment due to human activity, directly and indirectly affecting all living things. Reports have indicated that contaminated soil contains numerous HMs simultaneously, and conventional detoxification techniques are less effective than the bioremediation procedure. It has been established that bioremediation procedures are significantly cheaper than other physicochemical remediation methods. Numerous bacterial and fungal strains have recently been isolated and characterized from metal-contaminated and mining-abandoned soils. Many strains of Bacillus spp., Pseudomonas spp., Aspergillus spp. and Penicillium spp. are present and exhibit excellent metal resistance in soil, especially against Cd. A variety of contaminated areas throughout the world are currently using bioremediation, with variable degrees of effectiveness. The main worry for the considerable yield of bioremediation is the inclusion of appropriate supplements and improving environmental conditions. To address the issue, adding organic matter and a group of microorganisms can increase microbial metabolic activity and enhance the potential for bioremediation. Moreover, more research is still needed to identify the best microbes and hyperaccumulator plants with a high tolerance for multi-metal-contaminated and multi-stress environmental locations and to accumulate several metals simultaneously. More emphasis will be paid to plant–microbe-based bioremediation strategies to find novel plant–microbe pairs with high metal removal effectiveness and to create an environment that is conducive to other microbial strains. This will indirectly improve the health of the soil. Additionally, more research is required on combining nanomaterials, biochar and microorganisms with bioremediation.",
"introduction": "1. Introduction In the last two decades, the quality of human life has improved significantly. However, developmental activities have occurred at the expense of the environment’s quality [ 1 ]. Soil and the environment are contaminated due to higher concentrations of metalloids and heavy metals (HMs) resulting from rapidly expanding industrial wastes, excessive use of automobiles, resource extraction, petrochemical spillage, metallurgy and anthropogenic activities [ 2 ]. A heavy metal is any metallic substance with a relatively higher density and is toxic even at low concentrations [ 3 ]. Heavy metals include elements such as aluminum (Al), arsenic (As), antimony (Sb), beryllium (Be), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), mercury (Hg) and nickel (Ni) [ 4 ]. The persistent nature of these toxic HMs causes harm to humans, plants and animals at higher levels [ 5 ]. Cadmium is one of the most dangerous HMs to living organisms [ 6 ], mainly due to its higher toxicity and severe extent of bioaccumulation [ 7 ]. It adversely impacts human health by accumulating in the kidney and causing renal tubular damage and emphysema [ 8 ]. Cd has persisted in soil for decades, depending on multiple factors, including soil type, redox potential, pH, clay contents, organic matter, plant uptake and leaching [ 9 ]. Cd presents a unique concern due to its notable mobility in soil environments. Unlike some heavy metals, Cd exhibits a relatively high degree of mobility within soils, facilitated by factors such as soil pH, organic matter content and redox potential. This mobility renders Cd more hazardous even at relatively low soil concentrations, as it can readily leach into groundwater and accumulate in crops, posing environmental and human health risks. Cd toxicity negatively affects plant functioning by inhibiting carbon fixation, reducing chlorophyll synthesis and minimizing photosynthetic activity [ 10 ]. Cd-induced phytotoxicity leads to plant morphological alterations, such as chlorosis and the suppression of lateral root formation [ 11 ]. Additionally, Cd exposure induces osmotic stress in plants by reducing relative leaf water content, stomatal conductance and transpiration, ultimately leading to tissue damage [ 12 ]. Furthermore, the toxicity of Cd results in the overproduction of reactive oxygen species (ROS), damaging plant membranes and destroying cell organelles [ 13 ]. Cd toxicity also reduces the uptake and transportation of mineral elements, leading to stunted growth with ultimate yield penalties on field crops [ 14 ]. The increased mobility of Cd underscores the urgency of effective remediation strategies to mitigate its potential widespread contamination and its subsequent adverse effects on ecosystems and agriculture. Cd remediation mitigates or eliminates Cd contamination from environmental systems, particularly soil. Cadmium, a highly toxic heavy metal, poses significant health and ecological risks even at relatively low concentrations due to its mobility within soils [ 12 , 15 ]. This process involves various strategies to reduce Cd’s presence, minimizing its potential harm to human health, ecosystems and agricultural productivity. Remediation methods can be broadly categorized into physicochemical approaches, which involve chemical treatments and physical processes, and bioremediation, which employs living organisms such as microorganisms and plants to transform or remove Cd from the soil matrix. The most commonly employed approaches include chemical oxidation and reduction, precipitation, electrochemical treatment, solvent extraction, ion exchange, filtration, reverse osmosis, recovery by evaporation and soil washing with chelating chemicals [ 16 , 17 ]. However, one major drawback of these traditional processes is the creation of toxic heaps, sludge and secondary pollutants [ 18 ]. Therefore, it is necessary to continuously monitor the stability of immobilized HMs such as Cd [ 19 ]. Moreover, conventional remediation techniques can only remove Cd to a certain degree. In addition, traditional remediation requires expensive chemicals, significant energy and investment [ 20 ]. In contrast to physicochemical procedures, bioremediation is an environment-friendly technique that utilizes plants and microorganisms (such as fungi, bacteria and algae) to aid in the restoration of contaminated soil to its original state [ 21 ]. Bioremediation harnesses the natural metabolic capabilities of these organisms to convert Cd into less harmful forms, offering a sustainable and eco-friendly solution to Cd contamination. Biological techniques such as biosorption and bioaccumulation offer an advantage in removing HMs from polluted resources [ 18 , 22 ]. In a natural ecosystem, microbes are widely distributed and thrive in HM-polluted environments [ 12 ]. However, the ability of microbes to remediate contaminants can halt when they run out of food [ 23 ]. An enrichment method for the isolation of microbes that combines the properties of (1) the degradation of a chosen pollutant and (2) excellent root colonization has been developed [ 24 , 25 ] to ensure that these microbes can access the best available food source in soil, namely root exudates [ 26 ]. Plant root exudates, including organic acids, alcohols and sugars, serve as energy sources for soil microflora and promote microbial activity and growth [ 27 ]. According to Sabae et al. [ 28 ], some root exudates may also function as chemotactic signals for microbes. Furthermore, plant roots enhance water movement and loosen the rhizosphere, which improves microbial colonization [ 29 , 30 ]. As a result, these microbes transform hazardous HMs into non-toxic forms. Throughout bioremediation, these microbes transform organic pollutants into end products, including H 2 O, CO 2 and metabolites, which are the primary substrates for cell growth [ 31 ]. Microbes maintain a defense system against HM contamination in the rhizosphere via two mechanisms: (i) the biosynthesis of enzymes that break down specific contaminants and (ii) persistence that can withstand associated HMs [ 18 ]. Despite numerous individual efforts to evaluate the potential of various microbes for remediating Cd, such as bacteria, fungi and algae, no comprehensive review covers the multivariate features of plant growth-promoting microbes and their strategies and mechanisms for decontaminating Cd-contaminated soil. This review covers some new aspects and dimensions of the bioremediation of Cd-contaminated soils. Here, we mainly review the recently published literature from 2010 to 2022. The main objective of this review is to highlight the bioremediation potential of various microorganisms, especially bacteria, fungi and algae, individually and in combination with plants. Different mechanisms, i.e., indirect and direct mechanisms, adapted by microorganisms to detoxify Cd, are also discussed. Furthermore, factors, i.e., water content, temperature, pH, nutrient availability, moisture content and pollutant bioavailability, which can influence the bioremediation of Cd in contaminated soil, are also explored. Finally, the present review explores field application knowledge through case studies, challenges and prospects."
} | 2,695 |
24047255 | null | s2 | 7,407 | {
"abstract": "Quorum sensing (QS) is a cell-to-cell communication system responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, autoinducers (AIs), including AI-2 that is secreted by both Gram-positive and -negative microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via autoinducer-binding remains poorly understood. Here, we provide detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phospho-isobutyl-AI-2, D8P). Interestingly, the bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. However, the same movement of F124 by the D8P'-binding did not cause the unfolding of the α7 segment. Although the LsrR-binding affinity of R5P (Kd, ∼1 mM) is much lower than that of D5P and D8P (∼2.0 and ∼0.5 μM), the α-anomeric R5P molecule fits into the binding pocket without any structural perturbation, and thus stabilizes the LsrR tetramer. The binding of D5P, not D8P and R5P, disrupted the tetrameric structure and thus is able to activate LsrR. The detailed structural and mechanistic insights from this study could be useful for facilitating design of new antivirulence and antibiofilm agents based on LsrR."
} | 408 |
39910851 | PMC11799749 | pmc | 7,409 | {
"abstract": "SUMMARY There is a need for ground‐breaking technologies to boost crop yield, both grains and biomass, and their processing into economically competitive materials. Novel cereals with enhanced photosynthesis and assimilation of greenhouse gasses, such as carbon dioxide and ozone, and tailored straw suitable for industrial manufacturing, open a new perspective for the circular economy. Here we describe the vision, strategies, and objectives of BEST‐CROP, a Horizon‐Europe and United Kingdom Research and Innovation (UKRI) funded project that relies on an alliance of academic plant scientists teaming up with plant breeding companies and straw processing companies to use the major advances in photosynthetic knowledge to improve barley biomass and to exploit the variability of barley straw quality and composition. We adopt the most promising strategies to improve the photosynthetic properties and ozone assimilation capacity of barley: (i) tuning leaf chlorophyll content and modifying canopy architecture; (ii) increasing the kinetics of photosynthetic responses to changes in irradiance; (iii) introducing photorespiration bypasses; (iv) modulating stomatal opening, thus increasing the rate of carbon dioxide fixation and ozone assimilation. We expect that by improving our targeted traits we will achieve increases in aboveground total biomass production without modification of the harvest index, with added benefits in sustainability via better resource‐use efficiency of water and nitrogen. In parallel, the resulting barley straw is tailored to: (i) increase straw protein content to make it suitable for the development of alternative biolubricants and feed sources; (ii) control cellulose/lignin contents and lignin properties to develop straw‐based construction panels and polymer composites. Overall, by exploiting natural‐ and induced‐genetic variability as well as gene editing and transgenic engineering, BEST‐CROP will lead to multi‐purpose next generation barley cultivars supporting sustainable agriculture and capable of straw‐based applications.",
"conclusion": "CONCLUDING REMARKS Current environmental and societal issues, together with existing and future policies, are driving industries to develop eco‐efficient, bio‐based compounds, and materials in various sectors, such as chemical industry, building & construction, transportation, and sports & leisure. Besides the market size and growth expectations, the current availability of sustainable raw materials offers great opportunities. To this end, a variety of lignocellulosic sources and agricultural production processes are being considered, including traditional plant fibers and agricultural and forestry residues. The deployment of crop‐straw and plant‐based products will promote the creation of new industrial facilities, offering new growth opportunities, particularly for farm‐based rural areas, where the diversification of economic activities and new industrial outlets are crucial for generating added value in agriculture‐related fields, while contributing to the decarbonization of corresponding industrial sectors. The multi‐purpose barley cultivars delivered by BEST‐CROP will support straw‐based applications and innovative technologies that can be arranged, even on a small scale, in any rural area. This is a huge advantage for the supply chain, allowing the entire production process to be covered in a single compact plant—from raw material to final product. Finally, BEST‐CROP knowledge and innovations in terms of genetic improvement of photosynthesis and straw quality, together with straw transformation technologies could also be transferred to other major crops, first of all wheat, making the optimization of canopy photosynthesis and valorization of straw in different industrial sectors a powerful lever for the circular bioeconomy and climate change mitigation.",
"introduction": "INTRODUCTION The continuous growth of the world population is driving an increase in the demand for food and feed up to 50% by 2050 (van Dijk et al., 2021 ). This, in turn, will require a boost of agricultural productivity, while simultaneously reducing the negative environmental impacts (Haughey et al., 2023 ), like freshwater consumption and greenhouse gas (GHG) emissions, and without increasing cultivated land area. BEST‐CROP ( https://www.bestcrop.eu )—Boosting photosynthESis To deliver novel CROPs for the circular bioeconomy—is a research and innovation action aiming to contribute to these challenges in the frame of the circular and bioeconomy transition, supporting the European Green Deal. Specifically, BEST‐CROP targets barley ( Hordeum vulgare L.), a major crop worldwide, with 153.6 million tons of grain produced in the 2022/23 season and an almost equivalent amount of straw covering around 60 million ha of world arable land (Taner et al., 2004 ). The ambition is to develop next generation multi‐purpose barley plants with: (i) increased uptake of carbon dioxide (CO 2 ) and ozone (O 3 ); (ii) enhanced total biomass production without modification of the harvest index; (iii) straw composition tailored for transformation into high‐value bio‐based industrial products for the feed, green chemistry (biolubricants), construction, and composites sectors (see Figure 1 ). Figure 1 Overview of the main objectives and products of BEST‐CROP. Next generation barley plants with optimized canopy photosynthesis will provide high‐quality straw to produce biolubricants, construction panels, and feed. The experimental plan builds on evidence that genetic variability impacting photosynthesis (Croce et al., 2024 ; Flood et al., 2011 ), canopy architecture (Mantilla‐Perez & Salas Fernandez, 2017 ; Sakamoto et al., 2006 ; Shaaf et al., 2019 ), and barley straw composition, in terms of lignin (Daly et al., 2019 ; Grove et al., 2003 ; Halpin, 2019 ; Li et al., 2003 ; Zhang et al., 2010 ) and protein content (Bellucci et al., 2017 ; Przulj & Momcilovic, 2001 ; White et al., 1981 ), exists within barley germplasm and mutant collections and can be exploited to design a next generation barley plant. Research conducted in different laboratories worldwide has demonstrated that genetic manipulation of model organisms has the potential to improve photosynthesis efficiency (for a review see Croce et al., 2024 ), O 3 uptake (Brosché et al., 2010 ; Morales et al., 2021 ; Sierla et al., 2018 ; Yamauchi et al., 2016 ), and straw quality (Araus et al., 2016 ; Guo et al., 2020 ; Halpin, 2019 ; Karunarathne et al., 2022 ; Wang, Nian, et al., 2018 ). The performance of advanced breeding lines, carrying the selected traits, will be evaluated under field conditions in different European agroclimatic scenarios by monitoring, among other things, total biomass production, grain yield, and O 3 uptake. Finally, BEST‐CROP tailors the straw composition to different industrial transformation processes by increasing protein content for feeding insects capable of transforming straw into animal feed and biolubricants, as well as by modulating (up and down) the lignin content to make barley straw suitable to produce mycelium‐based construction panels (Alaneme et al., 2023 ), structural straw board (Amziane & Collet, 2017 ; Li et al., 2023 ; Tlaiji et al., 2022 ), and sandwich panels, as well as polymer composites (Bourmaud et al., 2018 ; Mohanty et al., 2018 ). During more than one century of barley/wheat ( Triticum ssp.) breeding, straw traits have been selected mainly to maximize yield and minimize lodging (semidwarf plants), while no attention has been given to straw composition. Although some straw transformation processes have been proposed, straw composition has never got attention from breeders and the current straw composition might not be the most suitable for every possible transformation. By tailoring straw composition to specific industrial needs, the project innovations will contribute to replacing non‐renewable with renewable resources. The production of chemical lubricants has a high environmental impact that could be reduced by substituting oil‐derived lubricants with biodegradable alternatives. Moreover, transforming straw into high‐nutrition feed using insects could reduce the need for dedicated protein crops. The increase in root biomass and the use of straw as raw material in the construction and composite sector promote carbon sequestration contributing to mitigate the effects of climate change. By shifting to utilizing straw‐based materials which are locally and widely available, BEST‐CROP will aid in decarbonizing relevant industries and will contribute to reducing their environmental impact. These objectives will be driven forward based on highly innovative biotechnology approaches that exploit natural‐ and induced‐genetic variation, gene editing and genetic engineering techniques for improving the photosynthetic efficiency, and building a next generation barley plant that could be exploited directly in breeding programs, while also serving as proof‐of‐concept of gene function."
} | 2,261 |
40309222 | PMC12041475 | pmc | 7,411 | {
"abstract": "Abstract Aerobic ammonia oxidation is crucial to the nitrogen cycle and is only known to be performed by a small number of bacterial lineages [ammonia-oxidizing bacteria (AOB)] and a single lineage of archaea belonging to the Nitrososphaeria class of Thermoproteota [ammonia-oxidizing Archaea (AOA)]. Most cultivated AOA originate from marine or soil environments, but this may capture only a limited subset of the full diversity of this clade. Here, we describe several genomes of AOA from metagenomic sequencing of a hot spring microbial mat, representing several poorly characterized basal lineages that may be important for understanding the early evolution of archaeal ammonia oxidation. These genomes include a novel genus most closely related to Nitrososphaera as well as novel species belonging to the genera Nitrosotenuis , Nitrososphaera and Nitrosotalea . Furthermore, the distributions and phylogenetic relationships of key metabolic genes support a history of vertical inheritance of ammonia oxidation and carbon fixation from the last common ancestor of crown group AOA.",
"introduction": "Introduction The biogeochemical nitrogen cycle is essential both to convert relatively inert atmospheric N 2 into bioavailable forms and to return fixed nitrogen to the atmosphere to maintain long-term steady state (e.g. [ 2 ]). Ammonia oxidation is only known to occur in four extant clades, which include a single lineage of archaea [ammonia-oxidizing archaea (AOA)] and three known lineages of ammonia-oxidizing bacteria (AOB). In the most current version of the Genome Taxonomy Database (GTDB; the taxonomic system that we use throughout this work), AOA are restricted to a clade within the Nitrososphaerales order of phylum Thaumarchaeota [ 3 ]. AOB include two distinct lineages of ammonia-oxidizing Pseudomonadota (formerly Proteobacteria ) and one clade of commamox (complete ammonia oxidation to nitrate) bacteria in the Nitrospira class of Nitrospirota [ 4 ]. Though AOB and AOA both perform the first step of ammonia oxidation using the ammonia monooxygenase (AMO) enzyme, divergences in AMO structure and in the downstream biochemical pathways employed by these groups affect the environmental signals and distributions of different ammonia oxidizers. In particular, variations in oxygen tolerance, carbon fixation efficiency and substrate affinity [ 5 10 ] carry strong significance for the ecological niches [ 11 12 ] and primary productivity of AOB and AOA [ 4 13 14 ]. Of particular note to this study, the discoveries of oligotrophic and thermophilic members of the AOA within the last two decades have expanded the potential range of ammonia oxidation into habitats where AOB have not been detected, including high-temperature environments like hot springs (e.g. [ 15 18 ]). Although the presence and significance of AOA in terrestrial hot springs are now widely recognized (e.g. [ 11 19 ]), only a few thermophilic AOA species have been previously characterized in detail, potentially leaving large gaps in our understanding of the clade’s taxonomic and metabolic diversity [ 15 20 ]. A thorough genome-resolved metagenomic sampling of geographically and geochemically diverse hot springs is essential for understanding the diversity, phylogenetic distribution and evolutionary history of microbial metabolic pathways (e.g. [ 21 ]). This issue is further exacerbated by biases in hot spring research in general, which has disproportionately drawn from well-studied springs in Yellowstone National Park and their associated geochemical conditions (e.g. [ 22 23 ]) leading to a relative dearth of knowledge about hot springs elsewhere in the world. Recent years have seen an increase in sampling in some regions (e.g. [ 24 ]), yet the southern hemisphere remains underrepresented in available datasets. Here, we describe several metagenome-assembled genomes (MAGs) of novel AOA from hot spring microbial mats in Aotearoa New Zealand, substantially improving genomic sampling across basal AOA clades. Organismic and metabolic protein phylogenies incorporating these MAGs provide further support to the hypothesis that ammonia oxidation and carbon fixation via the 3HP/4HB pathway have been vertically inherited from the last common ancestor of extant AOA.",
"discussion": "Discussion and conclusion The evolutionary history of AOA has been confounded by a limited understanding of Thermoproteota diversity, with uneven sampling and cultivation leading to discrepancies between which AOA are most well-characterized and which are most ecologically significant [ 47 ]. Most of the diversity of previously characterized AOA is from the so-called ‘Shallow-water group’, (which includes Nitrosopumilus ), with relatively few isolates or genomes representing the ‘Terrestrial group’, which appears to be a paraphyletic grade at the base of the AOA clade [ 10 ]; the phylogenetic placement of the ‘Terrestrial group’ suggests that they may be more representative of the most evolutionarily ancient AOA, with marine groups representing a much younger, evolutionarily derived radiation (e.g. [ 48 ]). Recent phylogenetic analyses also tend to infer a thermophilic ancestor for all AOA due to the basal placement of AOA from specifically geothermal environments (especially the lineage including Nitrosocaldus [ 10 49 ]). The added diversity of the genomes described here improves our sampling across the AOA evolutionary tree, both supporting the basal placement of Nitrosocaldus and adding new information about early-diverging ‘Terrestrial group’ lineages more generally. Our protein phylogenies further target specific biochemical mechanisms and demonstrate that the evolutionary relationships among proteins involved in respiration, ammonia oxidation and carbon fixation are broadly congruent with organismal relationships ( Figs1 2 , Data S2–S4 and Figs S2–S4), which suggests that these traits were vertically inherited rather than acquired via horizontal gene transfer. Thus, expanded hot spring sampling of Nitrososphaerales supports vertical inheritance from an aerobic, ammonia-oxidizing last common ancestor capable of carbon fixation. These early divergences are key to our understanding of nitrogen cycling, which only recently expanded to include the AOA. In extant ammonia oxidizers, bacterial and archaeal AMOs differ significantly, especially through structural disparities in transmembrane helices. Notably, recent work on putative additional archaeal subunits also suggests that although bacterial AMO complexes contain only three subunits (AmoABC), AOA may utilize six subunits (AmoABC and AmoXYZ [ 50 ]). The relationship between these structural distinctions and any functional differentiation is not yet understood, but commamox, other AOB and AOA do show wide-ranging substrate affinities for ammonia [ 9 ]. Within the AOA, the distribution of substrate affinities may be tied to ecological niche and phylogeny [ 7 ]. Diverging downstream biochemical pathways further compound the biochemical and ecological contrasts between AOA and AOB. Paired with processes like respiration and carbon fixation, which are carried out via different mechanisms in different groups, the relative contribution of AOA versus AOB affects models for nitrogen cycling and net primary productivity over time [ 4 ]. Whilst several steps in the nitrogen cycle (e.g. nitrogen fixation and denitrification) are performed by diverse organisms and are generally thought to have evolved early in Earth’s history (e.g. [ 51 ]), the limited set of lineages that perform ammonia oxidation may not have arisen until more recent time [ 4 48 ]. The phylogenetic divergence of AOA from closely related clades can help to untangle the relative timings of various evolutionary events. For example, of the traits highlighted in this paper, only aerobic respiration is present in the nearest relatives of extant AOA (so-far unnamed basal families of Nitrososphaerales known only from MAGs [ 33 ]), (Figs S2–S4). This suggests that the evolution of autotrophic ammonia oxidation in stem group AOA occurred relatively rapidly following the divergence of these groups. Whether aerobic respiration was independently acquired along this AOA stem branch several times or vertically inherited from the last common ancestor of all Nitrososphaerales has yet to be determined. Further investigation of this trait’s inheritance may be crucial for understanding the timing of the evolution of archaeal ammonia oxidation relative to the Great Oxygenation Event ~2.3 Gya when biologically meaningful concentrations of O 2 first accumulated. In either case, the full diversity of characterized AOA appears to be adapted to relatively high O 2 environments (given the utilization of a low-affinity A-family heme-copper oxidase [ 52 ] and the O 2 -tolerant 3HP/4HB carbon fixation pathway, e.g. [ 53 ])."
} | 2,220 |
38250293 | PMC10797516 | pmc | 7,412 | {
"abstract": "Plant specialized metabolites (PSMs) are considerably diverse compounds with multifaceted roles in the adaptation of plants to various abiotic and biotic stresses. PSMs are frequently secreted into the rhizosphere, a small region around the roots, where they facilitate interactions between plants and soil microorganisms. PSMs shape the host-specific rhizosphere microbial communities that potentially influence plant growth and tolerance to adverse conditions. Plant mutants defective in PSM biosynthesis contribute to reveal the roles of each PSM in plant–microbiota interactions in the rhizosphere. Recently, various approaches have been used to directly supply PSMs to soil by in vitro methods or through addition in pots with plants. This review focuses on the feasibility of the direct PSM application methods to reveal rhizospheric plant–microbiota interactions and discusses the possibility of applying the knowledge gained to future engineering of rhizospheric traits.",
"conclusion": "Conclusion and future perspectives The rhizosphere microbiome plays a key role in plant growth and tolerance to various stresses ( Carrion et al. 2019 ; Kwak et al. 2018 ); therefore, engineering the rhizosphere microbiome has high potential to achieve sustainable agriculture to support the growing demand of foods and alleviate negative effects on environment ( Ke et al. 2021 ). The inoculation of plant growth-promoting bacteria and fungi into soil rhizosphere has been conducted in both laboratories and fields for decades. Although microbial inoculants have been commercialized to improve plant health and crop yield, impediments for their long-term success in agriculture lay in root colonization, persistence in the rhizosphere, and consistent responses under different soil and climatic conditions ( Rilling et al. 2019 ). Engineering the rhizosphere metabolites is a propitious solution to overcome the limited root colonization by microorganisms. This review summarized recent findings on PSM-mediated alterations in soil microbiome with a special focus on the direct application of metabolites ( Table 1 ). Transgenic Arabidopsis expressing the gene to synthesize octopine, an opine released from crown gall tumors, secretes octopine into the rhizosphere and favors the growth of Ensifer that can utilize octopine as a carbon source ( Mondy et al. 2014 ). Furthermore, transgenic Medicago truncatula and barley expressing genes for producing rhizopine, a scylloinosamine, harbor Rhizobia that can utilize rhizopine ( Geddes et al. 2019 ). In addition to the modification of host plant trait to secrete PSMs, the direct application of PSM in the soil can alter the rhizosphere metabolome, thereby favoring the growth and colonization of microorganisms attracted to and/or capable of utilizing PSMs. The enriched microorganisms often possess metabolic pathways that allow them to tolerate the inhibitory and adverse effects of the PSMs ( Nakayasu et al. 2021a ; Shimasaki et al. 2021 ); however, it is not yet clear whether enriched microorganisms exert a beneficial or harmful influence on host plants. It is crucial to test the effects of exogenously applied PSMs on root colonization by microorganisms and their influence on plant growth. When using PSMs for field-grown plants, possibility of toxicity to soil microorganisms and environment should also be considered, similar to the risks associated with agrochemicals such as pesticides ( Karpouzas et al. 2022 ). Additionally, the manipulation of microbial genomes to improve the capability of the strain to utilize PSMs as a signaling or carbon source could enhance the ability of transgenic microorganisms to colonize host roots efficiently while competing with indigenous soil microorganisms. Table 1. Chemical structures of plant specialized metabolites and analysis of their effects on soil microbiota through the addition of pure chemicals. Group Compound Chemical structure Treatment Effects on microbiota References Flavonoids Apigenin \n \n Maize grown in pot •Altered soil bacterial microbiome •Growth promotion under nitrogen deficiency \n Yu et al. (2021) \n Daidzein \n \n Field soil in a vial •Altered soil microbiome \n Guo et al. (2011) \n Field soil in a test tube •Altered soil bacterial microbiome closer to that of soybean rhizosphere than that of bulk soil •Enrichment of Comamonadaceae \n Okutani et al. (2020) \n 7,4′-Dihydroxyflavone \n \n Field soil in a conical tube •Altered soil bacterial microbiome •Enrichment of Acidobacteria \n Szoboszlay et al. (2016) \n Flavonoid mixture Soybean grown in a pot •Altered soil bacterial microbiome •Enrichment of Burkholderiaceae, Methylobacteriaceae, and Sphingobacteriaceae \n Liu et al. (2021) \n Genistein \n \n Field soil in a vial •Altered soil microbiome \n Guo et al. (2011) \n Luteolin \n \n Peanuts grown in a pot •Altered soil microbiome •Reduced nodulation \n Wang et al. (2018) \n Quercetin \n \n Field soil in a pot •Altered soil bacterial microbiome •Enrichment of ASVs belonging to Proteobacteria \n Schütz et al. (2021) \n Triterpens Cucurbitacin B \n \n Melon grown in a pot •Altered soil bacterial microbiome •Suppression of melon wilt disease caused by Fusarium oxysporum \n Zhong et al. (2022) \n Steroidal saponins Dioscin \n \n Field soil in a test tube •Altered soil bacterial microbiome •Enrichment of Sphingobium \n Nakayasu et al. (2021b) \n α-Solanine \n \n Field soil in a test tube •Altered soil bacterial microbiome •Enrichment of Sphingobium \n Nakayasu et al. (2021b) \n α-Tomatine \n \n Field soil in a test tube •Altered soil bacterial microbiome closer to that of tomato rhizosphere than to that of bulk soil •Enrichment of Sphingobium \n Nakayasu et al. (2021a) \n Triterpenoid saponins Ginsenoside Rg1 \n \n Hillside soil in a bottle •Altered soil fungal microbiome •Increase in Fusarium oxysporum \n Li et al. (2020) \n Natural and conditioned soil in a pot •Altered soil bacterial and fungal microbiome \n Xu et al. (2021) \n Glycyrrhizin \n \n Field soil in a test tube •Altered soil bacterial microbiome •Enrichment of Novosphingobium \n Nakayasu et al. (2021b) \n Soyasaponin Bb \n \n Field soil in a test tube •Altered soil bacterial microbiome •Enrichment of Novosphingobium \n Fujimatsu et al. (2020) \n Alkaloids Gramine \n \n Barley grown in a pot •Altered soil bacterial microbiome •Enrichment of Nitrosotaleales \n Maver et al. (2021) \n Field soil in a pot •Altered soil bacterial microbiome •Enrichment of ASVs belonging to Proteobacteria \n Schütz et al. (2021) \n Nicotine \n \n Field soil in a test tube •Altered soil bacterial microbiome closer to that of tobacco endosphere than to that of bulk soil •Enrichment of Arthrobacter \n Shimasaki et al. (2021) \n Benzoxazinoids BOA \n \n Field soil in a pot •Altered soil bacterial microbiome •Enrichment of ASVs belonging to Actinobacteriota \n Schütz et al. (2021) \n DIMBOA \n \n Field soil in a vial •Altered soil microbiome \n Chen et al. (2010) \n Glucosinolates Sinigrin \n \n Soil from tree nurseries in a glass vial •Altered soil bacterial microbiome \n Hanschen et al. (2015) \n Isothiocyanates 2-Phenylethyl isothiocyanate \n \n Luvisol soil •Altered soil bacterial microbiome \n Rumberger and Marschner (2003) \n 2-Propenyl isothiocyanate \n \n Field soil in a container •Altered soil bacterial and fungal microbiome \n Hu et al. (2015) \n Rapeseed extract treated with myrosinase (goitrin) \n \n Field soil in a pot •Altered soil bacterial and fungal microbiome •Enrichment of Trichosporon \n Siebers et al. (2018) \n Others Santhopine \n \n Field soil in a test tube •Altered soil microbiome closer to that of tobacco endosphere than to that of bulk soil •Enrichment of Arthrobacter \n Shimasaki et al. (2021) \n PSMs have been used as a biostimulant to alleviate the unfavorable effects of abiotic and biotic stresses such as drought, high temperature, and salinity, although their mechanisms of action remains largely unclear ( Ben Mrid et al. 2021 ). Less focus was given to the microbiome in PSM-oriented research before the recent discoveries in PSM-mediated modification of microbiota. Because PSM-treatment to soil can modify the rhizosphere microbiota and improve plant growth under unfavorable conditions ( Yu et al. 2021 ; Zhong et al. 2022 ), comprehensive understanding of PSM–microbiome–host plant communication is critical to develop approaches for PSM-mediated rhizosphere engineering for crop production. Host plant and its microbiota are regarded as a unique biological entity called holobiont, in which the host and microbiota interact with each other to affect the development and physiology within the holobiont ( Hassani et al. 2018 ; Rosenberg and Zilber-Rosenberg 2016 ). PSMs could be key mediators in the holobiont. Practically, the combined application of PSMs with potentially plant growth-promoting cocktail of microorganisms to establish a holobiont would be a propitious solution to overcome the limited root colonization of soil microorganisms and improve the effectiveness of inoculants in the agricultural fields; this approach together with appropriate fertilizer and pesticide application could eventually enhance crop production ( Figure 3 ). Figure 3. Integration of metabolites and microorganisms to engineer the rhizosphere for promoting plant growth and mitigating stress. The combination of plant growth-promoting microorganisms and plant specialized metabolites can promote the root colonization of microorganisms, leading to the establishment of “good” microbiome.",
"introduction": "Introduction Plant specialized metabolites (PSMs) play important roles in the adaptation of plants to both biotic and abiotic stresses. Plants store PSMs with antimicrobial activities in vacuoles and use them as chemical defense agents following infection with pathogenic microorganisms and attach by herbivores. Plants also use PSMs to establish beneficial relationships with other organisms. Symbiotic interactions with rhizobia and arbuscular mycorrhizal fungi are well-known examples, where PSMs such as flavonoids and strigolactones function as signaling compounds to initiate symbiosis ( Zhang et al. 2015 ). The rhizosphere, which is defined as the soil region adjacent to plant roots ( Hartmann et al. 2008 ), harbors a microbiome that exerts multiple effects on plant growth, fitness, and potential for crop production ( Canto et al. 2020 ; Chialva et al. 2022 ). Recent studies have revealed the involvement of PSMs in shaping the rhizosphere microbiome by analyzing the root/rhizosphere microbiome of plant mutants with disruption in a particular biosynthetic pathway for PSMs ( Jacoby et al. 2021 ; Pang et al. 2021 ; Pascale et al. 2020 ) ( Figure 1 ). The loss-of-function approach is robust, although it lacks the ability to determine the effects of other metabolites in the rhizosphere, and both direct and indirect influence from plant mutant roots cannot be differentiated with this approach. Figure 1. Model for plant specialized metabolite-mediated root microbiota formation and its effects on host plant. The application of pure chemicals to the soil is a promising method to directly assess the effect of PSMs on soil microbiomes. By repeated additions of PSMs to the soil, PSM concentration can be maintained at levels observed in the plant rhizosphere. Our group used the “pseudo-rhizosphere system” to reveal the effects of PSMs on shaping rhizospheric microbiota. In the present review, we summarize the functions of PSMs revealed by the methods for adding metabolites to the soil with or without plant growth and discuss the potential of the soil application of PSMs to engineer and regulate the rhizospheric environment to improve crop growth."
} | 2,907 |
38326309 | PMC10850122 | pmc | 7,413 | {
"abstract": "Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed G ene E xpression M odification b y L oxPsym-Cr e R ecombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.",
"introduction": "Introduction Microbial cell factories are increasingly used for the sustainable production of biofuels, bioplastics, food substitutes, medicines and other high-value compounds 1 , 2 . However, the successful implementation of heterologous pathways and perturbation of native microbial metabolism often requires extensive optimization to ensure economically viable production titers. Metabolic engineering allows rewiring of cellular metabolism to increase product titers by enhancing precursor supply 3 , 4 , tackling co-factor limitations 5 – 7 , interrupting competitive pathways 8 , 9 , preventing buildup of toxic intermediates 10 and improving heterologous pathway flux 11 . Over the past years, several tools and strategies have been developed to facilitate yield optimization, including chassis strain improvement 12 – 14 , directed mutagenesis 15 – 17 , synthetic compartmentalization 18 , 19 and pathway gene expression optimization 20 . One of the most important yet challenging strategies for optimizing production yields is fine-tuning the expression levels of individual genes in a biosynthetic pathway. Tuning gene expression levels allows to balance reaction flux, thereby maximizing product yields, while minimizing detriment to cell fitness, for example by maintaining native metabolic flux and reducing the burden of excessive protein synthesis 21 – 25 . Given the importance of tuning gene expression levels, it is not surprising that many studies choose to focus on the characterization of expression modulators, including promoters, terminators, ribosomal binding sites, transcription factors and untranslated regions (UTRs) 26 – 29 . In addition to natural elements, sets of artificial expression modulators have been developed 30 – 37 . Together, these provide a diverse molecular toolbox for precise modulation of gene expression. While modification of expression and regulation of genes has become relatively easy, optimizing expression levels of a full metabolic pathway remains challenging. Various computational approaches for metabolic flux modeling have recently been exploited 38 , 39 , but the development of these models relies on large, well-defined datasets, which are laborious and expensive to obtain 40 . Moreover, construction of kinetic models is hampered by the difficulty of acquiring exact kinetic reaction parameters due to uncertainties intrinsic to biological systems. These uncertainties need to be hypothesized or modeled based on approximates, which requires intense computational power and often renders researchers to oversimplification and unreliable predictions 41 . Therefore, the use of such in silico tools for targeted pathway optimization is currently still limited. To experimentally improve pathway flux by generating large libraries of gene variants with different expression levels, several tools have been developed. For example, several papers describe the (ex vivo) parallel assembly of genes and expression modulators, such as (artificial) promoters 42 , 43 , transcription factors 44 , intergenic regions 45 , and ribosomal binding sites 46 , which can then be integrated in the microbial host. Other strategies often rely on CRISPR-based gene (in)activation systems that exploit gRNA oligo pools to target Cas proteins, either as functional editors 47 , deactivated road blocks 48 or transcription factor fusion proteins 49 , to specific loci for gene expression regulation. However, these strategies often require high technical skill and large, expensive oligonucleotide libraries, which strongly limits their applicability. Therefore, despite the large number of tools and techniques for gene expression diversification, none of these technologies provide cheap, fast, in vivo, multiplexed, large-range expression modification of multiple genes in parallel. In this study, we therefore aimed at developing a technique that would allow to quickly and easily generate a vast library of variants of a given starting strain, with each variant showing different expression levels and regulation of a set of selected genes. Our strategy is based on the use of the site-specific Cre-LoxP recombinase system. Site-specific recombinases, that invert or excise DNA sequences flanked by specific target sites, are often used in synthetic biology circuits to control gene (in)activation and cellular behavior, e.g., by deleting a promoter upstream of a target gene 50 – 52 . Among these recombinases, Cre recombinase is widely used due to its efficiency in many organisms and its independence from any accessory proteins 53 – 55 . Cre recombinase recognizes a short 34 bp DNA sequence (LoxP), binds it as a dimer and forms a tetrameric complex with another LoxP-bound dimer to recombine the two target sequences and enforce DNA cutting and pasting 56 . Depending on the orientation of the LoxP sequence, the flanking DNA will either be inverted or deleted. To bypass this directionality, a symmetrical LoxPsym site was developed, thereby expanding the types of structural variation that can be achieved upon recombination 57 . LoxPsym is a key element in the Saccharomyces cerevisiae 2.0 project, a synthetic biology project that aims to synthesize the first eukaryotic genome 58 , 59 . In Sc 2.0, LoxPsym sites are inserted across the genome after the ORF of each non-essential gene to enable massive genome shuffling and rapidly generate phenotypic diversity, a technology referred to as SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution). Several applications of SCRaMbLE for the improvement of industrial production traits have been reported in recent years, demonstrating the potential and importance of structural variation for strain optimization 60 – 63 . In this study, we develop an approach called GEMbLeR ( G ene E xpression M odification b y L oxPsym-Cr e R ecombination) that exploits site-specific recombination 63 for rapid and in vivo gene expression diversification in yeast. We first characterize a pool of hybrid promoters and terminators flanked by LoxPsym sites and use a minimal yet diverse selection to design a hyper-evolvable Gene Expression Modulator (GEM) construct. To demonstrate the capabilities of GEMbLeR, we use a fluorescent reporter system to generate variants with protein expression spanning over two orders of magnitude. Finally, we apply GEMbLeR for multiplexed, combinatorial expression optimization of six heterologous genes of the astaxanthin biosynthesis pathway and show an improvement in production titers of more than two-fold. Together, this shows that GEMbLeR is an efficient, fast and inexpensive technology for gene expression optimization of metabolic pathways.",
"discussion": "Discussion By enabling the production of various metabolites, microbial cell factories can contribute to reducing our fossil fuel consumption and help pave the way towards a more sustainable economy. These metabolites are typically synthesized by complex metabolic pathways involving multiple enzymes that work together in an assembly line fashion to convert substrates into desired products. Fine-tuning the expression of individual pathway genes has proven important for optimizing production yields and limiting byproduct formation 21 , 24 . However, rational tuning of expression levels has proven challenging, often relying on trial-and-error approaches that are labor- and time-intensive. Here, we developed a strategy called GEMbLeR that overcomes these challenges. GEMbLeR allows for the generation of large libraries of strain variants with diversified expression levels of multiple genes without requiring prior knowledge. By creating this diverse pool of variants, GEMbLeR enables the identification of variants that show optimal production characteristics. Current approaches for targeting and altering gene expression profiles often rely on combinatorial assembly techniques, such as COMPASS 44 and VEGAS 24 , or high-throughput CRISPR-based approaches, such as CRISPR-AID 49 and BETTER 47 , to generate a pool of strains with altered expression profiles. While these strategies have proven to be effective, their implementation is hampered by the design and cost of the required oligonucleotide libraries, as well as technical complexities. GEMbLeR avoids these drawbacks and simply requires a one-step pathway assembly reaction followed by a short Cre recombinase induction period to induce structural variation and generate a library of cells with highly diversified expression profiles for sets of target genes. Importantly, GEMbLeR does not rely on highly technical or costly equipment or reagents, making it easy to implement in a standard laboratory setting. Moreover, GEMbLeR can be used iteratively to further improve production traits of superior variants. Our study showed that GEMbLeR allows to diversify expression over a wide range, spanning from complete shutdown of gene expression to levels higher than those obtained from the TDH3 promoter, which is generally considered one of the strongest yeast promoters 71 . Covering expression diversification in the low levels is crucial for avoiding potential toxicity associated with certain metabolic steps 18 , 85 . Importantly, the range was covered in an almost continuous manner, allowing to obtain variants that only show subtle, but potentially important differences in gene expression. This continuity is achieved by combining 5’ and 3’ GEMs, as well as by using symmetrical LoxPsym variants, which ensure that the recombination outcome is independent of the LoxPsym orientation and allows to generate a much larger pool of diversified GEM-layouts compared to the canonical non-symmetrical LoxP site. We showed that LoxPsym-mediated inversions were crucial for obtaining this dense coverage, as we observed that inverted elements contributed to small expression modifications. In fact, our findings are consistent with observations from RNAseq experiments showing that eukaryotic promoters are often bidirectional 86 , 87 , explaining why inverted elements still play a role. To demonstrate the potential of GEMbLeR for strain optimization, we set out to improve astaxanthin production titers in S. cerevisiae . After only one round of GEMbLeR and testing a relatively limited number of variants, we identified several clones with a two-fold increase in production titers. Notably, our heterologous pathway included a bifunctional phytoene synthase/lycopene cyclase ( CrtYB ), that is needed for two non-consecutive pathway steps. To further improve pathway flux, it would probably be better to use two separate enzymes for these steps. This would allow their expression levels to be balanced individually with respect to up- and downstream enzymes. GEMbLeR is a black-box approach that relies on chance rather than rationale to improve pathway expression profiles. One might argue that this strategy, along with most existing approaches, is therefore only applicable for optimizing phenotypes or production of molecules that can be easily measured. However, diverse fast high-throughput screening methods have recently been developed, such as biosensors 88 , 89 , microfluidic based approaches 90 , 91 and high-throughput mass spectrometry 92 , 93 , which enable rapid identification of optimal GEMbLeR variants, even when large variant pools need to be screened. One important consideration when using GEMbLeR is the repetitive usage of the GEM construct for multiple heterologous genes. While we have shown that this does not cause genomic instability when targeting six heterologous genes simultaneously, it does limit the applicability of GEM, which relies on the available pool of sixteen orthogonal LoxPsym variants and can therefore target a maximum of eight genes simultaneously 63 . Although this is sufficient for many heterologous pathways, expanding GEMbLeR to target more genes can be achieved through the design of an array of heterologous genes with alternating directions. This would enable dual use of GEM for two genes simultaneously, which would allow to target twice as many genes with the same amount of GEM constructs. Moreover, if there is a need to reduce the number of GEM repeats, some of the building blocks could be replaced by other UPEs or terminators of similar strength, either from this study or from other high-throughput screens on hybrid promoters and terminators 27 , 94 . This flexibility provides opportunities for customization based on specific pathway requirements. Importantly, while we only tested GEMbLeR in S. cerevisiae , the technique should be easily expandable to other organisms since we have previously demonstrated functionality of LoxPsym variants in various host species, including prokaryotes and plants 63 . Applying GEMbLeR in other hosts would simply require characterization of host-specific cis- regulatory elements and LoxPsym site activity. Another interesting and easy expansion of GEMbLeR would be to incorporate an array of LoxPsym-flanked alleles for each pathway gene. This would enable the generation of variants that express different alleles of specific genes. It has been shown that alleles derived from different organisms can drastically improve microbial production titers 11 , 39 , 95 , 96 and combining allele and GEM shuffling would allow to tackle this extra layer of metabolic engineering. In conclusion, GEMbLeR offers an efficient strategy for multiplexed gene expression diversification in vivo in yeast. By leveraging site-specific recombination and orthogonal LoxPsym sites, GEMbLeR diversifies the layout of a GEM thereby randomizing gene expression in a simple yet functional and effective way. A key advantage of our approach is that it is fast, inexpensive and low-tech, making it highly applicable for routine implementation in metabolic engineering approaches to improve the performance of microbial cell factories."
} | 3,792 |
22253602 | PMC3257284 | pmc | 7,415 | {
"abstract": "Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli . We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.",
"introduction": "Introduction Changes in gene regulation are an important and common cause of adaptation. Support for this comes from bioinformatic evidence that changes in regulatory elements are associated with presumably adaptive phenotypic changes (reviewed in [1] , [2] ), comparative experimental studies [3] and from experimental evolution studies, which often find regulatory changes occurring during adaptation to novel environments [4] – [12] . Indeed, in some of these cases direct links have been established between regulatory changes and adaptation [6] – [8] , [10] . These experiments directly demonstrate that small and local regulatory changes can significantly contribute to adaptation. In most cases, however, the physiological basis for selection of regulatory changes is unknown. Previously, we described the evolution of populations of Escherichia coli in defined environments that differed only in the number and presentation of the limiting resource [13] . Populations were evolved in environments supplemented with a single limiting resource or combinations of two limiting resources either presented together or fluctuating daily. These populations adapted to the environments in which they were evolved and this adaptation was, at least to some extent, environment-specific [13] . Here, we focus on a subset of 24 populations that evolved in environments supplemented with glucose and/or lactose and examine changes in the regulation of the lac operon in these populations. Several attributes make the lac operon a good candidate in which to observe regulatory changes and relate them to their physiological and fitness effects. First, the costs and benefits of lac operon expression are environmentally dependent. Expression of the lac operon is necessary for utilization of lactose for growth, but expression in the absence of lactose can impose a significant cost [14] – [16] . Second, the molecular components of the lactose utilization network are well characterized and their activity can be measured in vivo . The ability to assay changes in the lac regulatory network ‘output’ provides a means to identify and test activity of evolved regulatory changes. Third, the lac operon has been the subject of much theoretical work, leading to the development of mathematical models to explain important features of lac operon physiology [14] , [17] – [22] and evolution [16] , [23] , [24] . Fourth, the utility of the lac operon for examining evolution of regulatory changes has been demonstrated experimentally. For example, lac operon constitutive [25] , [26] , loss of function [14] and duplication [11] mutants have been recovered following growth in different selective environments, demonstrating that lac operon regulation is evolutionarily flexible and can be a target of selection. It has even been possible to predict the evolution of regulatory changes on the basis of their expected fitness effects. Dekel and Alon (2005) used a cost-benefit analysis to predict the optimum expression level of the lac operon in different inducer concentration environments. They found that populations selected in environments containing a high level of gratuitous inducer, but various concentrations of lactose, generally evolved to regulate expression of the lac operon to the predicted level. In this study, we examine changes in lac operon regulation associated with selection in environments differing in the presentation of its natural substrate and inducer, lactose, and repressor, glucose. In addition to quantitatively characterizing the changes that have occurred, we examine the genetic and demographic basis for selection of different modes of lac regulation. We find that regulatory changes in the lac operon evolved in many replicate populations selected in environments containing lactose. Much, but not all, of these changes were due to mutations in the LacI repressor or its major operator binding site within the lac promoter. By themselves, these mutations conferred significant fitness benefits in all of the evolution environments that contained lactose. We also present evidence for interactions between lac mutations and mutations in genes outside of the canonical lac utilization network and show that these interactions impact lac operon regulation and fitness. Finally, operator and repressor mutations fixed at different frequencies in different selective environments, although the selective basis of this is currently not known.",
"discussion": "Discussion We sought to test whether evolution in environments that differed only in the availability and presentation of lactose selected for changes in the regulation of the lac utilization network. Our analysis of inducer response profiles found three broad classes of lac regulation change among evolved clones. Two of these classes, constitutive expression and a lower threshold/graded inducer response, represent substantial changes from ancestral regulation and were observed only in populations evolved in environments containing lactose. Sequencing of lac regulatory regions in evolved clones uncovered mutations in the lac repressor ( lacI ) and the primary lac operator ( lacO1 ) that correlated with the constitutive and the lower threshold/graded inducer response, respectively. Addition of these mutations to the ancestor demonstrated that they explained many, but not all, of the broad scale changes in regulation we observed and that, by themselves, they can confer fitness benefits in environments containing lactose. These fitness benefits were relatively large, representing 20%, 27% and 28% of the total mean fitness improvement in the Lac ( lacI mutation), G+L ( lacO1 mutation) and G/L ( lacI mutation) evolved populations, respectively [13] . Together these results indicate that regulatory changes were common, complex — occurring both within and outside of the recognized lac regulatory elements — and adaptive. Extensive previous study of lac operon regulation offers the opportunity to connect the genetic and phenotypic changes we observed. Twenty-one of the 22 lacI mutants we identified mapped to a mutational hotspot within lacI \n [30] , [31] . These mutations generate a frameshift in the coding sequence that results in expression of a nonfunctional repressor, which provides a good explanation for the complete loss of negative regulation we observed in lacI mutants. Similarly, the three lacO1 mutations we identified in the G+L evolved populations have been reported as reducing the binding affinity of LacI for this binding site [33] – [35] . Consistent with the mutations reducing, but not completely preventing, LacI binding, strains containing lacO1 mutations are able to repress the lac operon, but are induced at much lower TMG concentrations than the ancestor. lacO1 mutations also conferred a graded response to increasing inducer concentration, which contrasted with the canonical bimodal response of the ancestor. The same regulatory outcome was demonstrated by Ozbudak et al. (2004), who found that decreasing the effective concentration of LacI by providing extra copies of lacO1 binding sites resulted in a graded unimodal induction of the lac operon [19] . The similarity in regulatory changes suggests that the graded induction we observe is a consequence of decreased LacI- lacO1 affinity, reducing the effective concentration of LacI repressor. More generally, our results support the concept that small changes in the activity of cis-regulators have the potential to transform the output of a regulatory network between binary and graded responses [38] . Both lacI and lacO1 mutations were shown to confer significant fitness benefits in the three lactose containing evolution environments (Lac, G+L and G/L). Analysis of the growth dynamics of strains containing only these mutations indicated that a large part of this benefit is due to a reduction in lag phase when switching from glucose to lactose utilization. Interestingly, when added to the ancestor, both lacO1 and lacI mutations abolished the diauxic lag that separates glucose and lactose growth phases during growth in the G+L environment. This phenomenon is well documented for lacI mutants [39] , but to the best of our knowledge has not been demonstrated for lacO1 mutants. In the case of lacI mutants, constitutive expression of the lac operon primes the cell for utilization of lactose as soon as glucose resources are exhausted. In contrast to lacI mutants, lacO1 mutants are still capable of repressing lac expression in the absence of inducer. However, when grown in media containing both glucose and lactose the lacO1 mutation essentially phenocopies a lacI mutant, causing constitutive lac expression. Evidently glucose-mediated blockage of lactose import through LacY (inducer exclusion) is insufficient to prevent lactose accumulating in cells to a concentration sufficient to allow lac operon induction in lacO1 mutants [39] , [40] . The loss of lac repression in lactose (3/6), but not glucose (0/6), evolved populations is consistent with the ‘use it or lose it’ hypothesis [23] , [24] . This hypothesis proposes that negative regulation will be maintained during evolution in environments in which gene products, in this case the LacI repressor, are used because mutants that lose the repressor will needlessly express the lac operon and be selected against. If a repressor is seldom used, as in the lactose evolution environment, loss of function mutations will not be effectively selected against and can fix through genetic drift. However, in its simplest form, this mutation accumulation mechanism does not capture the dynamics of the regulatory changes we see. First, loss of the lacI repressor actually confers a benefit during growth on lactose, so that underlying mutations will increase in frequency faster than expected if they were neutral. Second, repressor mutations also occurred in environments where glucose was just as common as lactose. Analysis of growth curves suggest a mechanism for this; lac repressor mutants were able to quickly begin growth following a switch from glucose to lactose. Fitness measurements indicated that this advantage outweighed the cost of unnecessary lac expression during growth in glucose. Given the large benefit conferred by lacI mutations in the Lac environment, it is interesting that only three of the six Lac populations were enriched for lacI mutations. We identify two possible explanations for this observation. First, clonal interference may have resulted in lacI mutations being outcompeted by higher effect beneficial mutations. Second, populations that did not enrich lacI mutations may have fixed alternative mutations that genetically interact with lacI mutations to reduce their fitness benefit. To distinguish between these possibilities, we are continuing the evolution experiment and tracking the frequency of lacI mutations in the Lac populations. In addition, we are examining the fitness benefit conferred by lacI mutations when introduced into clones from Lac evolved populations that did not fix lacI . We can explain why lac mutations occurred only in lactose containing selective environments. A second layer of environment specificity is less clear; why do lacO1 mutations only reach high frequency in the G+L environment? The lacI and lacO1 mutations had no differential effect on fitness in either the ancestor or an evolved background and conferred indistinguishable growth dynamics in all evolution environments. Without a selective advantage over lacI mutations it is difficult to understand how lacO1 mutants were fixed in 4 of 6 G+L populations, especially considering that the rate of lacI mutations is likely on the order of 1000-fold greater than for lacO1 mutations. In the absence of a plausible mechanism to explain enrichment of lacO1 mutations in the G+L environment, we investigated whether environment-specific selection of lacO1 mutants was reproducible. This was not the case, with all 12 of the independent replay populations selected in G+L eventually fixing (>98%) lacI mutations. It remains formally possible that subtle differences in media or experimental conditions during competition assays or the replay evolution experiments could affect the fitness advantage experienced by lacO1 mutants in focal G+L evolved populations. However, taken at face value, the different outcome between replay and primary populations suggests that, notwithstanding mutation rate differences and the strong statistical association between environment and mutation type, the enrichment of lacO1 mutations over lacI mutations in the G+L environment might have occurred by chance. Models incorporating interactions between key regulatory elements can successfully predict aspects of lac operon regulation [18] , [19] . Nevertheless, recent studies demonstrate that regulation of the lac operon is evolutionarily plastic, such that interactions can arise or be altered to fine tune regulation and better fit E. coli to its environment [11] , [14] , [41] . By characterizing changes in regulation without a priori assumptions as to the nature of regulatory changes or the mutations causing them, we were able to identify evolved clones with changes in lac regulation that are likely due to novel interactions with mutations in genes outside of the canonical lac operon. Two results support this conclusion. First, we identified numerous clones with maximal steady state expression levels of the lac operon that were higher than the ancestor. Further examination of evolved clone G+L3-1 indicated that the increase in maximal lac expression level could not be explained by the lacO1 mutation present in this clone. Second, the fitness benefit conferred by the lacO1 mutation in this same evolved clone was significantly greater than in the ancestor, indicating that one or more evolved mutations interact with the lacO1 mutation to determine its effect on fitness. Whole genome sequencing of G+L3-1 identified six additional mutations, however, none of these mutations mapped to the lac operon or genes known to directly impact CRP-cAMP activity. It seems likely, therefore, that one or more mutations in the G+L3-1 clone have directly or indirectly led to new regulatory control of the lac operon. Is there an optimal level of lac expression in each of the three lactose environments? Dekkel and Alon (2005) found that, in the presence of a gratuitous inducer, lac operon expression evolved to a level predicted on the basis of a cost-benefit analysis, dependent on the concentration of lactose in the selection environment [14] . Our results support the idea that maximal expression level is a plastic feature of the lac operon and can be tuned to best fit the environment. At this time, however, we do not know the genetic or molecular basis for the widespread increase in maximum lac expression observed in many evolved clones. Possible ‘local’ explanations include: increases in the level of cAMP, mutations in the lacZYA genes that affect mRNA stability, or changes in DNA supercoiling that increase lac operon transcription. It is also possible that changes in lac maximum expression reflect an alteration in some global process. For example, changes in the function or concentration of ribosomes could affect expression of all genes. In future work we aim to identify the evolved mutations that are responsible for changes in maximum lac expression and then construct strains that will allow us to test the adaptive value of different expression levels as well as probe the underlying molecular mechanisms. An additional widespread change in lac regulation was that evolved clones displaying bimodal inducer responses tended to also have higher induction thresholds (TMG ½Max ) than the ancestor. This trend was not environment specific, occurring in clones isolated from Glu, Lac and G/L evolved populations. However, the parallel and large-scale increases in induction threshold observed for Glu-evolved clones suggests that this change in lac regulation is a direct or correlated response to adaptation. The mechanistic bases of increases in induction threshold are currently not understood, but could be the result of both direct and indirect mechanisms. For example, reduction in the activity and/or concentration of the permease LacY could increase the concentration of extracellular inducer required to achieve intracellular levels of inducer sufficient to inactivate LacI. In glucose evolved populations, changes in LacY activity may result from mutations in the PTS system that optimize glucose transport but lead to elevated levels of unphosphorylated EIIA glc , which is a known inhibitor of LacY activity [40] . Alternatively, higher growth rates of evolved strains will also tend to decrease the steady state intracellular concentration of inducer thereby increasing the external concentration required for induction of the lac operon. Further study will be required to discern between these and other hypotheses. In summary, we have identified and characterized widespread changes in lac operon regulation that occurred during selection of replicate populations in different lactose containing environments. In our view, the most important aspects of our findings are how common these changes were and that they likely involve mutations both within and outside of the set of genes that are recognized as regulating the lac operon. Identification of these changes will provide a rare insight into how regulatory networks can be rewired in response to an environmental change."
} | 4,853 |
34855411 | null | s2 | 7,416 | {
"abstract": "Ocean microbial communities are important contributors to the global biogeochemical reactions that sustain life on Earth. The factors controlling these communities are being increasingly explored using metatranscriptomic and metaproteomic environmental biomarkers. Using published proteomes and transcriptomes from the abundant colony-forming cyanobacterium "
} | 89 |
25247208 | PMC4163452 | pmc | 7,417 | {
"abstract": "Lignocellulosic biomass is a complex biopolymer that is primary composed of cellulose, hemicellulose, and lignin. The presence of cellulose in biomass is able to depolymerise into nanodimension biomaterial, with exceptional mechanical properties for biocomposites, pharmaceutical carriers, and electronic substrate's application. However, the entangled biomass ultrastructure consists of inherent properties, such as strong lignin layers, low cellulose accessibility to chemicals, and high cellulose crystallinity, which inhibit the digestibility of the biomass for cellulose extraction. This situation offers both challenges and promises for the biomass biorefinery development to utilize the cellulose from lignocellulosic biomass. Thus, multistep biorefinery processes are necessary to ensure the deconstruction of noncellulosic content in lignocellulosic biomass, while maintaining cellulose product for further hydrolysis into nanocellulose material. In this review, we discuss the molecular structure basis for biomass recalcitrance, reengineering process of lignocellulosic biomass into nanocellulose via chemical, and novel catalytic approaches. Furthermore, review on catalyst design to overcome key barriers regarding the natural resistance of biomass will be presented herein.",
"conclusion": "7. Conclusion Lignocellulosic biomass is the most abundant and biorenewable polymer on earth with great potential for sustainable nanocellulose production. The efficient and controlled breakdown of natural cellulose would produce nanocellulose, which is a mother compound for the synthesis of a large number of chemicals for food, energy, advance material, health, and environmental applications. Nanocellulose can be used as an immobilization support for chemical, microbial, and enzyme-catalysts. Synthesis of synthetic rubbers, bioplastic, pharmaceuticals materials, methyltetrahydrofurans, butanediol, and lactones from nanocellulose would be a quantum leap in nanocellulose industry. Nanocellulose and its derivatives could be further used to synthesize conducting polymers which could be used in biosensor appliances as well as molecular sieves. The complex hierarchy structure of lignocellulose is the main obstacle for major components separation. Overcoming the recalcitrance of lignocellulosic biomass is a key step in separating the biopolymer. Existence of lignin and the stability induced by inter- and intramolecular hydrogen bonding of cellulosic materials makes it a challenge for catalyst design. The current methods to convert cellulose into nanocellulose are based on acid, alkali, supercritical water, and thermal hydrolysis which often destroy the hierarchical structure of cellulose microfibrils and subsequently introduce impurities to the final products and produces unwanted by-products. The current methods also consume a lot of energy during and after the process and thus are deemed to be unprofitable and nonenvironmentally friendly. The typically used liquid-based catalysts are mineral acids and alkali. Introduction of novel catalysts such as iron-based catalyst, heteropoly acids, and ion exchange resin offer an integrated approach combining physical-chemical catalysts for the controlled structured degradation of natural cellulose into nanocellulose which promote more reliable methods in degradation of cellulose into nanocellulose. This shall further encourage more researches towards nanocellulosic field as more nanocellulose is readily available for utilization as a result of more efficient cellulose degradation process.",
"introduction": "1. Introduction Owing to the overconsuming of petroleum resources and increasing demand of fossil-based fuels and chemical, it is necessary to develop renewable resources to produce biofuels and biochemical for economical and sustainable development. Lignocellulosic biomass industry has become green, possible alternative of fossil resources in order to compensate the increasing trend of world's demand for petroleum usage [ 1 ]. This type of biomass is the most abundantly available biopolymer in nature. It is estimated that the worldwide production of lignocellulosic biomass is about 1.3 × 10 10 metric tons per annum [ 2 , 3 ]. The lignocellulosic resources included (i) agricultural residues (palm trunk and empty fruit bunch, corncobs, wheat straw, sugarcane bagasse, corn stover, coconut husks, wheat rice, and empty fruit bunches); (ii) forest residues (hardwood and softwood); (iii) energy crops (switch grass); (iv) food wastes; and (v) municipal and industrial wastes (waste paper and demolition wood) [ 4 , 5 ]. The high availability of biomass has appeared to be one of the most potential resources of transportation fuels and chemicals platform. Transformation of cheaper biomass into value-added product by the mean of converting “carbon source” into “carbon sink” indicates that carbon can be fully utilized before it would be released into the atmosphere [ 6 – 8 ]. Reconstruction of low cost lignocellulosic materials to products with superior functions presents a feasible option for improvement of energy security and greenhouse emissions reduction. With the availability of biomass, it is believed that this technology is capable of turning negative cost of biomass (plant waste) into positive-earning materials. Lignocellulose is a complex carbohydrate polymer, containing polysaccharides built from sugar monomers (xylose and glucose) and lignin, a highly aromatic material. Lignocellulosic biomass fractionation into reactive intermediates, such as glucose, cellulose, hemicellulose, and lignin, is a critical process prior to further development into liquid fuels, chemicals, and other end products. The lignocellulosic biomass consists of defensive inner structure which has contributed to the hydrolytic stability and structural robustness of the plant cell walls and its resistance to microbial degradation. On the other hand, the presence of cross-link between cellulose and hemicellulose with lignin via ester and ether linkages [ 9 – 11 ] leads to the biomass recalcitrance. Thus, it is important to understand the chemistry of biomass in order to deconstruct the material into component that can be chemically or catalytically converted into biomass-derived fuels, chemicals, or reactive intermediate. This paper provides an overview of lignocellulosic biomass reengineering into nanocellulose reactive intermediate by discussing (i) biomass recalcitrance, (ii) chemical approaches for lignocellulosic biomass fractionation, (iii) nanocellulose synthesis via chemical route, and (iv) new prospects of solid catalyst for nanocellulose synthesis. Finally, conclusions on the catalyst development for targeted cellulosic nanomaterial products from lignocellulosic biomass deconstruction will be drawn based on some literature study."
} | 1,702 |
29793752 | null | s2 | 7,418 | {
"abstract": "Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities."
} | 323 |
24957641 | PMC3901219 | pmc | 7,420 | {
"abstract": "Formation and adaptation of metabolic networks has been a long-standing question in biology. With recent developments in biotechnology and bioinformatics, the understanding of metabolism is progressively becoming clearer from a network perspective. This review introduces the comprehensive metabolic world that has been revealed by a wide range of data analyses and theoretical studies; in particular, it illustrates the role of evolutionary events, such as gene duplication and horizontal gene transfer, and environmental factors, such as nutrient availability and growth conditions, in evolution of the metabolic network. Furthermore, the mathematical models for the formation and adaptation of metabolic networks have also been described, according to the current understanding from a perspective of metabolic networks. These recent findings are helpful in not only understanding the formation of metabolic networks and their adaptation, but also metabolic engineering.",
"conclusion": "11. Conclusions This review has described the current understanding of metabolic network formation and adaptation revealed through data analysis and theoretical studies. Specifically, the classical view of metabolic evolution such as retrograde evolution and patchwork evolution has been largely confirmed, and several novel findings regarding formation and adaption, such as the role of chemical properties of metabolites and chaperonin in metabolic networks and the relationship between metabolic network structure and environmental factors, have also been discussed. Moreover, recent mathematical models for metabolic network formation have also described. The current understanding and mathematical models are still limited because they remain hypothetical; thus, further evaluations and biological evidences are required in order to complete the understanding of metabolic network formation and adaptation. For this, we need to consider the construction of more accurate databases and further development of effective analytical tools of metabolic networks in the future. In addition to, the large-scale mutational analyses and laboratory evolution experiments and are also useful for hypothesis testing and deeper understating of evolution and adaptation of metabolic systems. Nevertheless, these findings and mathematical models indicate global mechanisms for metabolic evolution and adaptation, and they are helpful in the field of not only general biology but also biotechnology, e.g., metabolic engineering. Furthermore, knowledge of evolutionary selection and environmental adaptation of enzymes in metabolic networks is directly linked to the modification and design of metabolic systems, and it is expected to establish a basis for identifying enzymes and metabolites with a great deal of potential in industry. Moreover, the mathematical models may be used to devise simple methods for determining important metabolic reactions and predicting interactions between biomolecules ( i.e. , link prediction, described in [ 110 ]). For example, the TFB or impact degree is useful for easily identifying key metabolic reactions. Enzyme promiscuity [ 111 ], which implies that enzymes can catalyze multiple reactions, act on more than 1 substrate, or exert a range of suppressions [ 112 ], in which an enzymatic function is suppressed by overexpressing enzymes showing originally different functions, suggests the existence of many hidden metabolic reactions. These biological features in metabolism are important for designing metabolic pathways and understanding metabolic evolution [ 113 , 114 ]; the models may be helpful in identifying such hidden metabolic reactions. In addition to this, the analysis and modeling of metabolic networks in ecosystem will become more exciting. Especially, they may be useful for understanding and controlling enteric, soil, and marine environments thorough microbes by combining metagenomic data [ 115 , 116 , 117 ]; thus, studies on metabolic networks in ecosystem will be more important in terms of medial ecological sciences in addition to evolutionary biology and metabolic engineering. Considering that metabolic networks have not been fully understood, network biology presents a research field with high future growth potential.",
"introduction": "1. Introduction Because metabolism is responsible for physiological functions and for maintaining life, it is an important topic not only in general biology but also in applied biological research fields such as biotechnology and medical science. In recent years, several new technologies and high-throughput methods have generated considerable genomic and metabolic data. The data regarding metabolic information are collected in several databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) [ 1 ] and the Encyclopedia of Metabolic Pathways (MetaCyc) [ 2 ]; these databases are widely used and contain the metabolic pathways of many living organisms. Thus, the overall picture of the metabolic world has gradually become clearer. How should we capture this metabolic world? When addressing such a question, many works have considered metabolic network modeling based on differential equations [ 3 ] and flux balance analysis (FBA) [ 4 ]. There have also been attempts to understand biological systems, including metabolism, from a network viewpoint—generally called Network Biology [ 5 , 6 , 7 ]. Metabolism can be defined as a series of chemical reactions, and is often presented as a network or graph, which consists of a set of nodes and edges (called metabolic network ). Although simplification through such a network representation results in several gaps in biological information, these networks provide new insights into metabolism. Thus far, many data analysis studies have discussed, in particular, the mechanisms involved in the formation (or evolution) of metabolic networks [ 8 , 9 , 10 , 11 , 12 , 13 ] and environmental adaptation from the viewpoint of metabolic networks [ 14 , 15 ]. It is believed that most positively selected mutations cause changes in metabolism, resulting in a better-adapted phenotypes based on natural history, phylogenetics, genetics, etc [ 16 ]; thus, metabolic networks are expected to be characterized by a long evolutionary history of adaptive shape-shifting with changing environments. For example, thermophiles possess metabolic enzymes with higher thermal stability. These thermo-stable proteins might be acquired through the selection of specific amino acid residues (e.g., charged residues) helping to increase protein structure stability. Due to changing nutrient availability and growth environments (e.g., pH and salt concentration), moreover, transporters and pumps might be newly obtained through horizontal gene transfer and/or its substrate specificity might be modified. As a result, the structure of metabolic network might be changed through the addition, deletion, and rewiring of metabolic pathways (see Section 3 and later for details). Moreover, mathematical models for the formation and adaptation of metabolic networks have been constructed, based on current knowledge regarding metabolic networks. Despite the simplicity of these models, they have shown excellent agreement with empirical metabolic networks, and have helped in understanding the evolution and design principles of metabolic networks. In recent years, in addition, these data analysis and theoretical approaches have been integrated with large-scale mutational analyses and laboratory evolution experiments, and this attempt provides deeper and more realistic understanding of evolution and environmental adaptation of metabolic systems at the molecular level (e.g., see [ 17 , 18 ]). This review focuses on a wide range of data analysis and theoretical studies, including our previous studies, on metabolic systems from the network perspective. By reconsidering the findings obtained in these studies, we provide a bird’s-eye view of the formation and adaptation of metabolic networks."
} | 1,998 |
35701391 | PMC9198039 | pmc | 7,422 | {
"abstract": "There is a growing trend to design hybrid neural networks (HNNs) by combining spiking neural networks and artificial neural networks to leverage the strengths of both. Here, we propose a framework for general design and computation of HNNs by introducing hybrid units (HUs) as a linkage interface. The framework not only integrates key features of these computing paradigms but also decouples them to improve flexibility and efficiency. HUs are designable and learnable to promote transmission and modulation of hybrid information flows in HNNs. Through three cases, we demonstrate that the framework can facilitate hybrid model design. The hybrid sensing network implements multi-pathway sensing, achieving high tracking accuracy and energy efficiency. The hybrid modulation network implements hierarchical information abstraction, enabling meta-continual learning of multiple tasks. The hybrid reasoning network performs multimodal reasoning in an interpretable, robust and parallel manner. This study advances cross-paradigm modeling for a broad range of intelligent tasks.",
"introduction": "Introduction Different from task-specific narrow artificial intelligence, artificial general intelligence (AGI) with the characteristics of human intelligence is expected to excel in scenarios that lack the following conditions: sufficient data, clearly defined problems, complete knowledge, static states, and a single system. The incorporation of computer-science-oriented and neuroscience-oriented computing approaches is widely regarded as a promising direction in the development of AGI 1 – 5 . Spiking neural networks (SNNs) and artificial neural networks (ANNs) are the representative models of these two approaches, and each has unique advantages. Hence, there is a growing trend of merging these models to leverage their advantages 6 – 13 . However, the radical differences between SNNs and ANNs 8 , 10 , 11 , 14 – 16 , such as coding schemes, synchronization methods, and neuronal dynamics, pose great challenges for merging. Recently, a cross-paradigm hybrid neuromorphic computing hardware platform was developed to support a wide range of ANN and SNN models 6 . A growing number of research teams are adopting various features to better support different networks in their neuromorphic designs. For example, Intel, IBM, and the University of Manchester recently undertook hybrid designs in their Loihi 17 , In-memory computing 11 , and Spinnaker 18 , respectively. Meanwhile, a unified system hierarchy with neuromorphic completeness 7 for brain-inspired computing has also been developed, providing general support for executing different types of programs and network models on various typical types of hardware 7 . Collectively, these provide powerful hardware platforms and software deployment tools for the development of hybrid neural networks (HNNs). On the other hand, there are also some attempts to combine SNNs and ANNs to build hybrid models from different perspectives, such as information processing 8 , 9 , computational efficiency 10 , or establishing models that incorporate more biological attributes 11 , 12 . However, they narrowly focus on using certain features of ANNs and SNNs to solve specific tasks. A general framework for versatile tasks that can take full advantages of both models is essential but still lacking. In this study, we propose a framework to support the general design and computation of HNNs at multiple scales and multiple domains by decoupling ANNs and SNNs models and using hybrid units (HUs) as their linkage interfaces. In particular, we consider that, unlike the homogenous information in pure SNNs or ANNs, the hybrid information flows in HNNs have heterogeneity at different spatial and temporal scales. If SNNs and ANNs were directly coupled neuron-to-neuron, HNN models would be inefficient and unmanageable when their architecture becomes complex. With the support of HUs, our decoupled approach not only inherits the key characteristics of SNNs and ANNs, but also provides greater flexibility in the design of hybrid models, thereby making their strengths and techniques more self-contained. An HU is an information transformation model with intermediate representations so that it can bridge the gap between SNNs and ANNs, whose parameter configurations can be designed according to domain knowledge or learned to adapt. Under mild conditions, HUs would enable the development of HNNs with general-purpose computation. This framework provides a high degree of freedom for building interwoven hybrid network models, and an intrinsic capability to process rich spatiotemporal information in a hierarchical and multi-domain manner 6 .",
"discussion": "Discussion Biological neuronal systems embrace multi-scale and multimodal signal communication and information integration. Various coding strategies have been proposed, which suggests that hybrid information transformation is likely to be a requirement for normal cerebral function. Considering these features, we argue that it might be feasible to combine ANNs and SNNs using a general transformation scheme to model “hybrid” properties in neural systems. For instance, mean-field activities, such as fMRI recordings or MEG recordings, can be efficiently modeled using ANNs 3 . Transient activities, such as spike timing synchrony, can be modeled using SNNs. Furthermore, combining different neurons and HUs can enable HNNs to implement various artificial and spiking neuron models, ranging from the classic leaky integrate-and-fire to the complex Hodgkin-Huxley model, which facilitates implementation on neuromorphic computing chips (see Methods). Thus, HNN models can not only be inspired by biological functions but also serve as powerful prototypes to promote future studies on functional neuroscience and practical brain simulations, which can be used to reveal the relationship between a certain human behavior or disease and a certain brain network mechanism 41 – 43 . Our proposed framework can greatly exert the strengths of ANNs and SNNs, and facilitate the formation of complementary multi-network models. The abilities of this framework in facilitating HNNs are multifaceted, which we demonstrated experimentally. The HSN appropriately combines the high-precision of ANNs and the high efficiency of SNNs to achieve a record-breaking performance in tracking tasks. The HMN explores the advantages of hybrid modulation in MCL problems, demonstrating the diversity of hybrid information flows for multi-network collaboration. The HRN develops a hybrid architecture to integrate multimodal information and support interpretable logical reasoning in a dynamic environment, demonstrating robustness, high parallelism, and scalability for solving large-scale and complex problems. When encountering complex environments with large uncertainties, learnable HUs can facilitate more adaptive transformation for hybrid representations, leading to superior performance (see Supplementary Material). In summary, the HNN framework can provide flexible and versatile strategies to coordinate heterogeneous networks and exploit their complementary advantages. This paves the way for the development of cross-paradigm network systems for real-world applications and potentially contributes to the development of AGI."
} | 1,822 |
32345736 | PMC7190382 | pmc | 7,424 | {
"abstract": "As the major facilitators of the turnover of organic matter in the marine environment, the ability of heterotrophic bacteria to acquire specific compounds within the diverse range of dissolved organic matter will affect the regeneration of essential nutrients such as iron and carbon. TonB-dependent transporters are a prevalent cellular tool in Gram-negative bacteria that allow a relatively high-molecular-weight fraction of organic matter to be directly accessed. However, these transporters are not well characterized in marine bacteria, limiting our understanding of the flow of specific substrates through the marine microbial loop. Here, we characterize the TonB-dependent transporters responsible for iron and carbon acquisition in a representative marine copiotroph and examine their distribution across the genus Alteromonas . We provide evidence that substrate-specific bioavailability is niche specific, particularly for iron complexes, indicating that transport capacity may serve as a significant control on microbial community dynamics and the resultant cycling of organic matter.",
"introduction": "INTRODUCTION Iron (Fe) is an essential cofactor in many enzymes facilitating fundamental life processes, such as photosynthesis, respiration, and nitrogen fixation. As such, dissolved iron is a necessary micronutrient for all microbial growth in the marine environment and is tightly linked to the cycling of carbon (C) and other macronutrients. In oxygenated seawater, iron is most thermodynamically stable in the form of Fe(III) oxyhydroxides, which are characterized by low solubility and the tendency to be further scavenged by sinking particulate matter ( 1 ). This results in extremely low dissolved iron concentrations in most regions of the world’s oceans and exerts significant control on marine primary production ( 2 ). Of the dissolved iron present in seawater, over 99% is associated with a heterogeneous pool of organic matter referred to as ligands ( 3 ). While much remains to be learned about the chemical composition of these ligands, they are thought to include humic substances, polysaccharides, metalloproteins with associated cofactors, and siderophores ( 4 – 8 ). Moreover, these ligands comprise a fraction of the total pool of marine dissolved organic matter (DOM). Marine DOM is one of the largest pools of carbon on Earth, and it is now recognized that marine heterotopic bacteria are the key determinant in the fate of this carbon and its associated macro- and micronutrients ( 9 – 11 ). Given that marine DOM consists of a highly diverse matrix of organic compounds ( 12 , 13 ), marine bacteria must use an assortment of cellular tools in order to access it. Many of the molecular mechanisms underlying these acquisition processes have yet to be explored. In particular, little is understood regarding these mechanisms at a molecular level for micronutrients such as iron. Measurements indicate that marine heterotrophic bacteria have iron quotas similar to or possibly greater than those of marine phytoplankton ( 14 ), and most of this iron resides within the respiratory chain, indicating a significant linkage between iron availability and carbon metabolism. Indeed, iron and carbon colimitation of marine heterotrophic communities has been observed ( 15 ). The specific uptake systems and enzymatic pathways that bacteria use to metabolize available substrates for both iron and carbon will influence rates of nutrient regeneration, the chemical composition of the remaining DOM, and, ultimately, the fate of fixed carbon within the ocean ( 12 , 13 , 16 ). One identified pathway for the acquisition of organic complexes by prokaryotes is the use of TonB-dependent transporters (TBDTs). TBDTs transport larger compounds (generally greater than 600 Da) across the outer cell membrane in Gram-negative bacteria. The TBDT is coupled to the energizing proton motive force of the inner membrane through the TonB complex (TonB, ExbB, and ExbD). Subsequent transport across the inner membrane is often accomplished via an associated ATP-binding cassette transporter (ABCT). ABCTs also function independently of TBDTs for the transport of small substrates, such as inorganic Fe(III) or monomeric carbon substrates, that can diffuse across the outer membrane. Known compounds transported via TBDTs include those important to iron metabolism, such as Fe-siderophore complexes and heme, as well as solutes critical to carbon metabolism and cell growth, including amino acids, vitamins, and polysaccharides ( 17 ). Bioinformatic analysis of marine prokaryotic genomes and metagenomes reveals that TBDTs are fairly widespread and especially enriched in Gammaproteobacteria ( 18 – 20 ). However, sequence analysis alone cannot predict the substrate specificity of TBDTs, and very little is known about their regulation and use in marine heterotrophic bacteria. Members of the genus Alteromonas are widespread marine copiotrophs of the class Gammaproteobacteria . The genus is globally distributed in oceanic waters, with several strains present in up to 80% of published samples from the Tara Oceans expedition ( 21 ). In addition, Alteromonas spp. have been found to become highly abundant in environments enriched in organic matter and nutrients ( 22 – 26 ). Observations of Southern Ocean bacterial communities have also found Alteromonadaceae to contribute significantly to the pool of iron uptake transcripts in this system ( 27 ). As “first responders” to bloom events and other episodes of particle enrichment, Alteromonas spp. play an important role in the biogeochemical cycling of organic matter ( 28 ). Due to their ability to disproportionately affect the processing of organic matter, we hypothesize that specific taxa will play a significant role in both carbon and iron remineralization processes. We used Alteromonas macleodii ATCC 27126 ( 29 ) as an ecologically significant model bacterium in order to study the transport mechanisms underlying substrate-specific bioavailability of carbon and iron in the marine environment. We present iron- and carbon-regulated transcriptomes of A. macleodii ATCC 27126, with a focus on the expression of TBDTs and the regulation of central metabolic processes. This is the first study to directly compare the cellular response of a heterotrophic marine bacterium to both iron and carbon limitation under controlled conditions and reveals an unexpected contrast in the stress response to these two nutrient limitations. Additionally, we identified two distinct sets of TBDTs utilized for the transport of carbon and iron compounds and allowed for the putative identification of a wide range of specific substrates. These results give new insight into our understanding of substrate specific bioavailability of iron and carbon to the marine heterotrophic community and the potential effects this has on the biogeochemical cycling of organic matter in the marine environment.",
"discussion": "DISCUSSION Metabolic responses to iron and carbon limitation are distinct. Limitations of iron and carbon in heterotrophic bacteria are typically studied separately. Remarkably, despite the shared aspect of growth limitation, iron and carbon limitation resulted in distinct reorganizations of cellular metabolism and nutrient acquisition in A. macleodii ATCC 27126. Iron limitation was characterized by a decreased dependence on iron-containing proteins, while cellular resources were directed toward iron acquisition. Fe-sparing mechanisms included a downregulation of the major Fe-containing enzymes of the CAC, ED glycolysis, and electron transport chain with a corresponding upregulation of Fe-lacking metabolic replacements (nickel superoxide dismutase [Ni-SOD] and fumarase C). A shift to the glyoxylate cycle as an alternative to the CAC was also observed in this work and has been observed under iron limitation for two additional strains of Alteromonas ( 52 ). An increase in the expression of isocitrate lyase has also been observed in the gammaproteobacterium Photobacterium angustum under iron limitation ( 53 ). Here, the authors were able to demonstrate decreased growth rates and respiration under iron limitation in a knockout mutant lacking isocitrate lyase compared to the wild type, suggesting that this pathway is an effective mechanism for coping with iron limitation ( 53 ). However, this transition to the glyoxylate cycle has also been observed in a range of taxa as a response to multiple different nutrient limitations as well as oxidative stress and is unlikely to be an iron-specific response ( 54 – 56 ). Similar cell-wide responses to iron limitation have been observed for additional Proteobacteria strains ( 57 , 58 ). This work demonstrates that these responses extend to the marine environment and reinforces the control that iron availability may have on carbon metabolism in heterotrophic bacteria. Under carbon limitation, there was no observed switch to the glyoxylate pathway. However, components of the CAC along with upstream pathways for glucose processing were similarly depleted, likely driven by the lack of glucose as a starting substrate. In striking contrast to iron limitation, carbon scavenging was seemingly enhanced by an increase in motility and particle formation as a result of a dramatic upregulation of chemotactic response systems as well as flagellar biosynthesis and agglutinin production. An increase in motility has been observed under changing environmental conditions in additional strains of Alteromonas ( 59 , 60 ). This has been observed under growth in minimal medium ( 59 ) as well as in transition to copiotrophic growth upon phytoplankton decay and the input of organic matter ( 60 ). An increase in motility under both copiotrophic and carbon-limited growth may suggest differing regulatory pathways governing these responses. Under copiotrophic growth, this was attributed to a multifaceted response involving the small RNA-binding protein CsrA and its activation of the FliA sigma factor ( 60 ). In the current study, we did not observe differential expression of either csrA or fliA , highlighting the complexity of motility regulation in Alteromonas spp. Furthermore, the lack of a motility response under iron limitation is intriguing and may suggest that this strategy quickly became too energetically expensive under these conditions. Transporters inform our view of the chemical matrix of the marine environment. When considering the specific response to iron and carbon limitation of the 66 putative TBDTs found in ATCC 27126, we find that ATCC 27126 uses a distinct set of TBDTs to acquire a wide range of iron and carbon complexes ( Table 1 ). These results emphasize the importance of experimental evidence for validating TBDT annotations. Each of these sets responds strongly and exclusively to the corresponding nutrient limitation. This indicates that there is a high degree of regulation tuning the expression of these transporters to specific environmental conditions. For the Fe-responsive TBDTs, this is likely primarily controlled by Fur regulation; however, we have also found preliminary evidence of regulatory systems that may respond to the presence of specific iron-ligand complexes. In contrast, carbon acquisition appears to be primarily controlled by a substrate-specific response. While the TBDT components of these systems were broadly sensitive to carbon limitation, the expression of hydrolytic enzymes and supporting proteins may depend on the presence of a given substrate, much like the canonical lac operon. This is supported by the identification of transcriptional regulatory systems in a majority of these gene neighborhoods. These results also highlight that the transport of substrates across the inner membrane of ATCC 27126 is likely accomplished by a diverse set of mechanisms. We have detected 6 putative inner membrane permeases with a PepSY domain that show significant structural similarity to characterized permeases for the transport of siderophores in well-studied pathogenic bacteria ( 36 – 38 ). To our knowledge, this is the first description of such permeases in a marine bacterium and provides a new, highly specific target for understanding the role of siderophore utilization in the marine environment. One of the most notable aspects of this data set is not only the total number of putative TBDTs found in ATCC 27126, but also the similarity in magnitude between the number of iron-responsive and carbon-responsive TBDTs. This implies that the diversity of organic carbon and iron complexes encountered and utilized by ATCC 27126 in its environment must be of a similar scale. Particularly regarding iron-ligand complexes, relatively few compounds have been isolated and structurally characterized directly from seawater. Even for the case of siderophores, where there has been a diverse range of structures isolated from cultured marine strains ( 61 ), only a small subset have been detected in natural seawater ( 8 , 62 , 63 ). Given these analytical challenges, the ability to test the bioavailability of relevant complexes in the marine environment has been limited. By focusing on cellular transport systems, this work has suggested an unexpected diversity of complex iron and carbon substrates that are bioavailable in the marine environment. Further characterization of these transport systems, both within Alteromonas spp. and additional taxa, will continue to inform our understanding of the bioavailable iron and carbon pools in the marine environment. Niche specialization drives differentiation in the distribution of TBDTs. Across sequenced representatives of the Alteromonas genus, there is a high capacity for transport via TBDTs, with strains possessing between 38 and 76 putative TBDTs ( Fig. S6 ). However, across the genus, the distribution of specific TBDTs is not uniform, particularly with regard to predicted iron transporters ( Fig. 5 ). While a majority of the C-responsive TBDTs in ATCC 27126 are widely distributed across Alteromonas spp., many of the Fe-responsive TBDTs, particularly those for the predicted transport of exogenous siderophores, were constrained to fewer taxa and notably absent from A. mediterranea . This may indicate that exogenous siderophore utilization (i.e., a “cheater” strategy [ 64 ]) is niche specific, or perhaps that distinct sets of siderophores prevail in different niches. Phylogenetic randomness in the distribution of trace metal transporters within two lineages of marine Alphaproteobacteria has been described, which the authors interpret as trace metal niche differentiation ( 65 ). Additionally, in an evaluation of the pangenome of Alteromonas spp., López-Pérez and Rodriguez-Valera ( 66 ) find that even between strains of a single Alteromonas species, genes encoding transporters, including TBDTs, exhibited some of the highest dN / dS ratios. High dN / dS ratios indicate positive selective pressure on these genes and support the idea of niche specialization in substrate utilization. Specifically, trace metal availability in a given environment may impart a significant control on genomic content, differentiating phylogenetically similar strains. Compared to other well-studied marine copiotrophic bacteria, the results presented here fit along an emerging spectrum of strategies for substrate utilization that ranges from the dedicated transport of monomeric substrates through ABCTs to the use of TBDTs for the acquisition of large, complex compounds. It has been suggested that substrate availability, as dictated by the use of either TBDTs or ABCTs by a given taxonomic group, creates distinct ecological niches and can be a primary control on the succession of heterotrophs during a phytoplankton bloom ( 67 – 69 ). For example, flavobacteria, whose genomes are enriched in TBDTs and hydrolytic enzymes, are known for their ability to break down and acquire high-molecular-weight DOM ( 70 , 71 ) and therefore dominate during the peak and decay stages of a bloom ( 72 , 73 ). In contrast, well-studied roseobacters, with large numbers of ABCTs, specialize in the transport of low-molecular-weight DOM ( 65 , 74 ). This can be released following the breakdown of high-molecular-weight DOM or directly from phytoplankton during early bloom stages. Previous studies have highlighted the potential utilization of complex substrates by Alteromonas spp., particularly with regard to carbon acquisition ( 23 , 26 , 28 , 75 ). With the identification of hydrolytic enzymes and associated C-responsive TBDTs in ATCC 27126, this work further supports the conclusions that members of the Alteromonas genus contribute significantly to the degradation of complex marine DOM. Furthermore, we provide the first evidence suggesting that Alteromonas spp. are likewise enriched in cellular machinery for the transport of larger, organically complexed iron substrates. Given the significant role that iron plays in carbon metabolism in heterotrophic bacteria, understanding the molecular transport mechanisms and resulting bioavailability of both of these nutrients will important steps in understanding the turnover of organic matter in the marine environment. While a majority of previous work has focused on the acquisition of carbon by heterotrophic bacteria, the results presented here show that for Alteromonas spp., the transport of large, complex substrates holds true for both carbon and iron acquisition. However, the relationship between the substrate-specific bioavailability of iron and carbon remains to be explicitly tested for many copiotrophic taxa. It is likely that the interplay of the acquisition of these two nutrients by different taxa of heterotrophic bacteria will underlie dynamics such as ecological succession and ultimately affect the balance between export and recycling in a given marine environment."
} | 4,484 |
32849330 | PMC7418181 | pmc | 7,425 | {
"abstract": "The rhizosphere hosts a complex web of prokaryotes interacting with one another that may modulate crucial functions related to plant growth and health. Identifying the key factors structuring the prokaryotic community of the plant rhizosphere is a necessary step toward the enhancement of plant production and crop yield with beneficial associative microorganisms. We used a long-term field experiment conducted at three locations in the Canadian prairies to verify that: (1) the level of cropping system diversity influences the α- and β-diversity of the prokaryotic community of canola ( Brassica napus ) rhizosphere; (2) the canola rhizosphere community has a stable prokaryotic core; and (3) some highly connected taxa of this community fit the description of hub-taxa. We sampled the rhizosphere of canola grown in monoculture, in a 2-phase rotation (canola-wheat), in a 3-phase rotation (pea-barley-canola), and in a highly diversified 6-phase rotation, five and eight years after cropping system establishment. We detected only one core bacterial Amplicon Sequence Variant (ASV) in the prokaryotic component of the microbiota of canola rhizosphere, a hub taxon identified as cf. Pseudarthrobacter sp. This ASV was also the only hub taxon found in the networks of interactions present in both years and at all three sites. We highlight a cohort of bacteria and archaea that were always connected with the core taxon in the network analyses.",
"conclusion": "Conclusion In this work, we have shown that the bacterial component of the core microbiota of canola rhizosphere is stable across years despite dissimilarity in precipitations. We identified the single core bacterial ASV in the microbiota of canola rhizosphere as cf. Pseudarthrobacter sp. In both years of the study, this single bacterial core microbiota member was a hub taxon in stable association with a cohort of bacteria. Chloroflexi were somewhat typical of canola monoculture, but the influence of crop diversification level on bacterial community structure, was only marginal, showing that the bacterial component of the microbiota of canola rhizosphere is more stable than its fungal component. This study provides information about bacterial and archaeal species in canola rhizosphere that could be important for future enhancement of canola production through microbiota manipulation or development of new cohorts for bio-inoculants.",
"introduction": "Introduction A plant in its natural environment coexists with myriads of archaea, bacteria, fungi, as well as with other unicellular eukaryotic microorganisms that constitute its microbiota. The rhizosphere is a hotspot of microbial interactions between species that have various ecological functions. These microbial communities are particularly important for plant health as they influence its development and its productivity ( Barriuso et al., 2008 ; Bulgarelli et al., 2013 ; Bakker et al., 2014 ). Throughout their life, plant roots exude compounds creating the rhizosphere environment ( Bais et al., 2006 ). Spatial and temporal variation in rhizodeposition allows plants to shape their rhizosphere microbial communities to their benefit ( Tkacz et al., 2015 ; Pii et al., 2016 ; Eisenhauer et al., 2017 ). Plant rhizosphere can host mutualistic microbes such as mycorrhiza or plant growth promoting bacteria (PGPB) that facilitate nutrient uptake, mitigate abiotic stress, and prevent root infection by pathogens ( Barriuso et al., 2008 ; Farina et al., 2012 ; Fincheira and Quiroz, 2018 ). Plant-microbe and microbe-microbe interactions are diverse. Plants live in symbiotic and commensal relationships with numerous organisms, but they must also face pathogenic attacks ( Hajishengallis et al., 2012 ). Rhizosphere organisms may influence each other, thus forming a complex web of interactions. For example, we know that mycorrhizal fungi have their own bacterial microbiota ( Bianciotto et al., 2003 ; Iffis et al., 2014 , 2017 ). These bacteria can be endophytic or form biofilm at the surface of the hyphae and can facilitate symbiosis formation in plants ( Fitter and Garbaye, 1994 ; Iffis et al., 2014 ; Taktek et al., 2017 ). Since the last decade, new generation sequencing (NGS) improved our access to microbial genetic information leading to significant advances in microbial ecology. This technological improvement lead to new ways of analyzing plant microbial communities ( Duffy et al., 2007 ; Bulgarelli et al., 2013 ; Mendes et al., 2015 ). Now, we can identify with confidence the factors shaping the microbial communities of the rhizosphere ( Kuramae et al., 2011 ; Agler et al., 2016 ). The microbiome of the rhizosphere is extremely large and diverse. To summarize this complexity, we can divide it into pools of microbes based on their functions or occurrence ( Ridout and Newcombe, 2016 ). In a given community, microbial taxa are likely to be favored by their host plant throughout its existence ( Rout, 2014 ). These taxa are expected to be always part of the plant microbiota at a defined time t , regardless of environmental conditions. According to Vandenkoornhuyse et al. (2015) , the taxa always present in association with the plant forms the core microbiome and have preferential interaction with their host. The definition of a pool of microorganisms always present at t time in the plant microbiota is appropriate for most ecological studies concerning the plant microbiota as they mostly rely on a single sampling time. However, it was necessary to consider temporal variation in our definition of the core microbiota, and this is what we did in this study. The interactions between microbes in the plant rhizosphere remains largely obscure. Next Generation Sequencing techniques can provide information on the abundance of the taxa interacting in a microbiome, but cannot reveal the biochemistry of interacting microbes in the ecosystem. That is why computational approaches aiming at identifying the nature of the links between the variations in the abundance of microbial taxa were developed as a complement to NGS ( Ings et al., 2009 ; Deng et al., 2012 ; van der Heijden and Hartmann, 2016 ). Network analysis allows us to identify microbial taxa that are functionally linked to others within the microbiome. Highly connected microorganisms may have a greater impact on plants and ecosystem functioning than others, because they theoretically interact with many partners and antagonists; these highly interacting species are named hub taxa ( Agler et al., 2016 ). Interactions occurring in microbial communities are known to be complex and difficult to retrieve with usual statistical methods ( Kurtz et al., 2015 ). However, the information provided by NGS can be processed through network analysis to identify cohorts represented by hub taxa. Simplifying the study of complex microbiome, Taktek et al. (2017) showed taxa that recruit organisms beneficial to the host plant, but hub taxa could also be pathogens. Some hub taxa in the human microbiome can articulate infection by consortia of pathogens ( Hajishengallis et al., 2012 ). As pathogens can affect the plant microbiome, pathogenic hub taxa may occur in the rhizosphere. The hub taxa are a useful concept and help to understand the ecology of the root and rhizosphere ecosystems, which could lead to the development of applications in crop plant root systems. Canola was shown to possess a specific bacterial component of the core microbiota conserved across the Canadian prairie ( Lay et al., 2018 ). Floc’h et al. (2020) reported the temporal stability of the fungal component of the core microbiota in canola rhizosphere, despite considerable changes in the plant rhizosphere microbiota across years. In the present study, we aimed to test if the bacterial component of the canola microbiota has a similar pattern of temporal variation. We investigated the temporal stability of the bacterial component of the core canola rhizosphere microbiota in order to ascertain whether a persistent bacterial component exists. Another aim was to determine if the canola rhizosphere harbors bacterial hub taxa, and to visualize the variation between years in the structure of interactions among the bacteria living in the canola rhizosphere microbiota. We sought to identify a universal bacterial component of the core microbiota in the rhizosphere of a plant species, specifically canola grown over the years under a range of climatic conditions and biological environments. We thus used a gradient of crop diversification levels to create variation in the biological environment of rhizosphere soil and examine over two years what in the bacterial component of the canola microbiota is invariable: the core microbiota. Canola is a crop of economical importance for Canada. It is also a good model plant to study the rhizosphere microbiome as canola produce antimicrobial isocyanates ( Zheng et al., 2014 ) leading to simpler microbial communities in its rhizosphere ( Rumberger and Marschner, 2003 ).",
"discussion": "Discussion We validated that a core bacterial component of the canola rhizosphere microbiota cannot only be stable across pedoclimatic zones but also through years. This core bacterial component was formed of only one taxon, ASV1 identified as cf. Pseudarthrobacter sp., which was also identified as a hub taxon and had a cohort of seven bacterial taxa with stable relationships across the two years of the study. ASV1, cf. Pseudarthrobacter sp. ASV1 was the only bacterial member that fit the definition of a core microbiota member that was detected in the canola rhizosphere and it was the most abundant ASV in both years of sampling. With our current sequencing technology (Illumina MIseq), it is likely that prokaryotes can go unseen if their abundance is low in a sample. ASV1 was the only bacterial core member identified, but it is probable that other less abundant prokaryotic members of this core microbiota were undetected. Furthermore, 16S rRNA gene sequences obtained with Illumina MiSeq technology do not have enough taxonomic resolution to distinguish between closely related species and uncertainty exists: ASV1 matches with 100% identity with at least 100 Arthrobacter and Pseudarthrobacter sequences in NCBI database. Arthrobacter is a genus of gram-positive bacteria from the Micrococcaceae family that was subdivided in several other genera like Pseudarthrobacter ( Busse, 2016 ). This genus includes mainly soil bacterial species ( Busse, 2016 ). Arthrobacter is also a genus with many species known as PGPB ( Chan and Katznelson, 1961 ; Manzanera et al., 2015 ; Ullah and Bano, 2015 ; Aviles-Garcia et al., 2016 ; Fincheira and Quiroz, 2018 ) colonizing the roots and rhizosphere of a large spectrum of agricultural crops, such as rice or tomato. Lay et al. (2018) reported a member of canola rhizosphere core microbiota identified as Arthrobacter that shared 100% identity with ASV1 in similar sites of the Canadian Prairies in 2014. They also reported that their Arthrobacter was positively correlated with canola yield as it was the case here with ASV1 in 2016. Furthermore, an Arthrobacter sp. was previously shown to increase canola yield and acts as PGPB ( Kloepper, 1988 ). This genus was reported as a highly competitive and fast growing bacteria in canola rhizosphere ( Tkacz et al., 2015 ). Lay et al. (2018) also reported the presence of Arthrobacter sp. in wheat and pea rhizospheres in rotation with canola, but in smaller proportions than in canola rhizosphere. That omnipresence and abundance of ASV1 (cf. Pseudarthrobacter sp.) in all our plots suggest a selection by canola and highlight this taxon as a good PGPB candidate. Variations in Bacterial Microbiota Bacterial communities are known to be sensitive to changes in abiotic factors such as pH and humidity, or nutrient availability ( Norman and Barrett, 2016 ; Wan et al., 2020 ). As plants actively control their rhizosphere microbiota through root exudates ( Bais et al., 2006 ; Eisenhauer et al., 2017 ), we expected important differences in the bacterial communities of our crop diversification treatments. This was not the case. In 2013, no effect of crop rotation on bacterial community structure was detected and in 2016, the only significant difference was between the two extreme treatments, i.e., canola monoculture and the highest level of crop diversification, and the difference was marginally significant ( P = 0.047). Indicator species analysis showed those two crop diversification treatments as the ones that had the highest number of indicator species. It is possible that the number of indicator species (26) of the monoculture in 2016 with a dominance of Chloroflexi ( Table 4 ) could be the source of the difference in community structure, with the highest level of crop diversification with the BMRPP, even if no significant differences was found in 2013 between those two crop diversification treatments. Long lasting effect of agricultural management such as crop rotation were reported in the literature ( Buckley and Schmidt, 2001 ). In the Brazilian Amazon for example, crop management seems to have a significant impact on microbial community structure ( Jesus et al., 2009 ). For temperate environments, our results are consistent with Jesus et al. (2016) who did not find any influence of crop rotation on soil microbial communities in Michigan. In our study, we examined the bacterial community in the canola rhizosphere, a component of the microbiota that is principally influenced by canola root exudates ( Rumberger and Marschner, 2003 ), mitigating the effects of other crops in the rotation systems. We do not know if the crop diversification levels influenced the bulk soil bacterial communities. However, our results showed that canola recruited similar bacterial communities between all crop diversification levels in 2013. Even if most of the microbes in the rhizosphere are probably selected by the plant from its surrounding soil, it is also possible that a part of the canola rhizosphere microbiota can be inherited maternally with the seed microbiome as it is known to be the case for a wide range of plants ( Shade et al., 2017 ). That could explain the similarities of canola rhizosphere community structure in systems with different levels of diversification. It is also possible that the bacterial communities in our diversified system were not host-specific, but colonize the roots of all crop species used in rotation, as it was reported by Lay et al. (2018) . They found that the bacterial microbiota of canola rhizosphere was more similar to the one found in pea than the one found in wheat rhizosphere. But here, we did not find significant difference in community structure between the low, medium and high crop diversification in 2013 and only a slightly significant difference in 2016, suggesting that rotation crops have a limited influence on the bacterial communities of canola rhizosphere. Thus, we can consider the influence of abiotic variation on bacterial community in our study. A previous study showed that soil type and the frequency of rainfall have stronger effects on the microbial community of canola rhizosphere than crop rotations ( Schlatter et al., 2019 ). Floc’h et al. (2020) also found a large variation in fungal rhizosphere community structure that was linked with difference in water availability in canola rhizosphere. In the present study, the experimental plots and sampling times were the same as those used in Floc’h et al. (2020) . But the difference in precipitation between years ( Supplementary Figure S2 ) did not affect the stability of the bacterial community structure observed in 2013 and 2016, contrarily to what was found for the fungal community in Floc’h et al. (2020) . This stability is noteworthy. Bacterial interactions in canola rhizosphere microbiota also showed stability through years, here. Interactions in the Bacterial Component of the Microbiota Using the same rhizosphere soil samples, Floc’h et al. (2020) reported drastic changes between years in the dynamics of fungal interactions in the microbiota of canola rhizosphere. In the present work, if the complexity of the interaction network changed between the two years of sampling, the pool of bacteria forming its nucleus remained the same. The hotspot of interaction was always articulated around ASV1 ( Pseudarthrobacter sp.). ASV1 was the only core bacterial member of the microbiota of canola rhizosphere and the only hub taxa detected with network analysis for both years of the present study. The fungal hub taxa in canola rhizosphere were subject to change between the years of the study, but it was not the case for bacterial hub taxa. For both year of sampling, ASV1 was interacting with seven other taxa: ASV2 (cf. Yersinia sp.), ASV3 (cf. Nitrososphaeraceae sp.), ASV4 (cf. Stenotrophomonas sp.), ASV6 (cf. Chloroflexi KD4-96), ASV11 (cf. Stenotrophomonas sp.), ASV25 (cf. Candidatus Nitrosocosmicus sp.) and ASV71 (cf. Paenarthrobacter sp). The persistence of these interactions across time suggests a close interaction of ASV1 with these other members of the community. The fact that ASV6 was negatively linked with ASV1 and negatively correlated with canola yield raises interest. This phylum is associated with several agricultural plants like potato ( Ýnceoğlu et al., 2011 ), lettuce ( Cardinale et al., 2015 ) or maize ( Peiffer et al., 2013 ) and was found in a large spectrum of soil ecosystems including forest, grassland, and tundra ecosystems ( Fierer et al., 2012 ). Chloroflexi appears as characteristic of the rhizosphere of canola monoculture: 3 of 9 ASVs in 2013 and 9 of 26 ASVs were identified as indicator species in 2016 ( Table 4 ). Monoculture of canola was found to have lower yield values across time and favour accumulation of microbial pathogenic taxa in soil ( Hummel et al., 2009 ; Harker et al., 2015 ). Chloroflexi have been reported in the canola rhizosphere previously, but there was no mention of Chloroflexi species being pathogenic to canola ( Gkarmiri et al., 2017 ). Correlations do no indicate that there is a causal relationship between the abundance of the different bacterial ASVs and canola yield. Correlations may point to bacteria that benefit from higher canola growth, or to a condition favorable to both canola and these bacteria, rather than an effect of the bacteria on plant productivity. However, the correlation values can be used as an index for identifying potential bacterial ASV of interest for the enhancement of canola production, since the bacteria directly beneficial to canola would be among those showing positive correlation with yield. It is possible that ASV6 could be commensal of canola fungal pathogens or of other microbes that are favored by monoculture ( Floc’h et al., 2020 ), or pathogenic itself. Tests of pathogenicity should be made, or cross-kingdom network interactions studies conducted to verify the occurrence of ASV6 with pathogenic microbes. In the cohort of taxa associated with ASV1, two other taxa were positively correlated with canola yield in 2016: ASV3 and ASV71. ASV71 was identified as Arthrobacter , so it is phylogenetically closely related to ASV1, and could be a potential PGPB with ASV1 ( Manzanera et al., 2015 ; Ullah and Bano, 2015 ; Pereira et al., 2019 ). ASV3 is an archaea identified as a member of the Nitrososphaeraceae family that was poorly correlated with canola yield. Little information about this family is available. The presence of Nitrososphaeraceae was previously reported by Gkarmiri et al. (2017) , and Lay et al. (2018) found core microbiota members of canola rhizosphere that were genetically close to Nitrocosmicus spp. Another study mentioned Nitrososphaeraceae as a microbial taxa retrieved from spacecraft surfaces ( La Duc et al., 2012 ). This family appears to be widely distributed in the environment. As hub taxa can have very strong influence on the whole microbiota and on plant performance, ASV1 and its cohort members could be important. These bacteria should be isolated and tested under controlled conditions in structured experiments to examine their potential PGPB activity or pathogenic behavior on canola."
} | 5,078 |
35875522 | PMC9301000 | pmc | 7,426 | {
"abstract": "The recalcitrance of biofilms to antimicrobials is a multi-factorial phenomenon, including genetic, physical, and physiological changes. Individually, they often cannot account for biofilm recalcitrance. However, their combination can increase the minimal inhibitory concentration of antibiotics needed to kill bacterial cells by three orders of magnitude, explaining bacterial survival under otherwise lethal drug treatment. The relative contributions of these factors depend on the specific antibiotics, bacterial strain, as well as environmental and growth conditions. An emerging population genetic property—increased biofilm genetic diversity—further enhances biofilm recalcitrance. Here, we develop a polygenic model of biofilm recalcitrance accounting for multiple phenotypic mechanisms proposed to explain biofilm recalcitrance. The model can be used to generate predictions about the emergence of resistance—its timing and population genetic consequences. We use the model to simulate various treatments and experimental setups. Our simulations predict that the evolution of resistance is impaired in biofilms at low antimicrobial concentrations while it is facilitated at higher concentrations. In scenarios that allow bacteria exchange between planktonic and biofilm compartments, the evolution of resistance is further facilitated compared to scenarios without exchange. We compare these predictions to published experimental observations.",
"conclusion": "5. Conclusion Many mechanisms contribute to the recalcitrance of biofilms against antimicrobials. Non-genetic mechanisms can be crucial for driving resistance evolution, as they alter the expression of the genotype and interfere with evolutionary processes. However, the individual and combined effects of these mechanisms are difficult to study experimentally. Given the multiple, possibly conflicting effects of biofilm lifestyle on the evolution of antibiotic resistance, population genetics models are a useful tool for clarifying the population genetic effects of each mechanism on their own and in combination. For instance, Roberts and Stewart ( 2004 ) modeled antibiotic tolerance (recalcitrance) by accounting for nutrient limitation. Adaptive responses to antimicrobial agents in biofilms were modeled by Szomolay et al. ( 2005 ). However, more comprehensive models accounting for the polygenic nature of antibiotic resistance and the complex phenotypic aspects of biofilm recalcitrance are still largely missing. The model proposed here offers new quantitative insights into the evolution of resistance, population dynamics, and consequences of phenotypic recalcitrance mechanisms on antibiotic resistance evolution. By exploring the combinations of mechanisms that contribute to biofilm recalcitrance using computer simulations, we add quantitative support for two previous verbal predictions (Trubenová et al., 2022 ). First, we hypothesized that biofilms could slow down resistance evolution. In our simulations, this holds true under low antibiotic concentrations that are sufficient to select for antibiotic resistance in planktonic cultures (Gullberg et al., 2011 ; Santos-Lopez et al., 2019 ). Second, we showed that biofilms could promote resistance evolution under drug concentrations that are high enough to suppress most resistant planktonic mutants. Finally, when we allowed for a continuous exchange between planktonic cells and biofilms, the evolution of resistance was further accelerated. Our simulations hence show that evolutionary outcomes of populations experiencing selection pressure from the presence of antibiotics are expected to depend heavily on many parameters, most notably the concentrations of antibiotics and the treatment regimen. The timing of treatments and frequency of dosing will influence the population dynamics of sensitive and resistant strains and determine the probability of resistance evolution. Our modeling study makes many assumptions, in part due to uncertainty about the parameters that characterize the processes involved and, in another part, for conceptual and computational simplicity. Below, we briefly mention and discuss these assumptions and the limitations to which they lead and briefly mention how we could approach a more comprehensive quantitative description of the pharmacodynamics and population genetics of bacterial populations that grow as biofilms. The values of parameters used in the simulations were taken from real-world scenarios whenever possible (see Supplementary Table 1 ). For instance, the degradation rate used in our simulations (rate 6 × 10 −4 MIC/min, which translates into the drug half time of approximately 19 h) is consistent with in vitro measures (Lallemand et al., 2016 ): in soil it has been shown that the typical halftime of antibiotics is 2 − 80 days, depending on the soil composition and other conditions (Pan and Chu, 2016 ). In our simulations, the effect of the degradation rate is therefore only visible in the first set of illustrative simulations, while in other simulations (repeated treatment and evolutionary experiment), it does not have any noticeable effect. However, in living organisms treated for infections, drugs are actively degraded and excreted, and drug concentrations decrease faster. Therefore, a significantly higher degradation rate would be more suitable for designing and analyzing treatment strategies. When the parameter values were not known, and not even their magnitude could be estimated (e.g., dispersal and attachment rates), we investigated a range of these values across several orders of magnitude. While we could gain qualitative insights into the impact of exchange between biofilms and planktonic populations on resistance evolution despite these uncertainties in the dispersal and attachment rates, better estimates of these rates would allow us to gain quantitative insights into the impact of exchange. In most of our simulations, we assumed a starting population without pre-existing resistant strains. This allowed us to investigate the emergence and the subsequent selection of resistant strains. Had we assumed pre-existing resistance in the simulations, we would have been able to obtain insights into the selection phase only. Furthermore, starting with a population consisting purely of wild-type strains allowed us to identify whether the mutants are more likely to appear in plankton or biofilm, a topic we discuss in Section 4.3. To investigate the effects of various treatment strategies, initiating the model with a bacterial population that already contains resistant mutants would be an important addition to the analysis presented here because, in many infections, resistant mutants are either co-transmitted with the wild type or arise de-novo before treatment in the infected host. Our modeling approach was designed to investigate the effects of recalcitrance mechanisms known in biofilms on the evolution of resistance without explicitly describing the intricate spatial characteristics of biofilms. Instead, we based our investigations on the population- and pharmaco-dynamic differences between the two lifestyles, some of which are direct consequences of the spatial aspects. As a result, in our simulation, planktonic bacterial populations and biofilms differ only in terms of their growth rates and pharmacodynamic parameters. By defining homogeneous planktonic populations and biofilms this way, we ignore the physicochemical and physiological heterogeneity of biofilms and the heterogeneous physiological states between these two extreme lifestyles (e.g., persister cells in the plankton). Formally, these aspects could be incorporated into the modeling framework, stopping short of a full-blown spatial simulation of bacterial populations by introducing multiple subpopulations. In particular, multiple populations of the same strain but with different “biofilm related” properties could be added, simulating multiple layers of a biofilm. Similarly, heterogeneity could be introduced into planktonic populations as was done in, for example, (Balaban, 2004 ; Wiuff et al., 2005 ; Levin-Reisman et al., 2017 ; Rodriguez-Rojas et al., 2021 ). A promising avenue of future research will be to investigate if the bacterial lifestyles and their intrinsic heterogeneities facilitate or hinder resistance evolution.",
"introduction": "1. Introduction Biofilms are heterogeneous communities of bacteria attached to a substrate or each other, forming aggregates that can be visible by the naked eye. Biofilms are very difficult to remove and cause significant problems in many aspects of human lives: from industry and households, where their large colonies block water pipes, to human health, affecting all body systems: they grow on teeth, tongues, eyes and skin, contact lenses, catheters, and medical implants (Donlan, 2002 ; Ciofu et al., 2017 ). Mature biofilms can survive in antibiotic concentrations thousands of times higher than those killing planktonic cells (Nickel et al., 1985 ; Sharma et al., 2019 ). This ability, denoted recalcitrance , allows biofilms to serve as reservoirs of bacterial cells that survive antibiotic treatment, further releasing bacterial cells. Biofilms cause chronic infections in wounds, tooth decay, and can cause tissue damage by eliciting persistent immune responses or even cancer (Ciofu et al., 2022 ). It has been shown that some genetic , heritable mutations conferring antibiotic resistance arise in biofilms (Sharma et al., 2019 ). These represent a significant problem for health care, as the resistance is retained by bacterial cells upon dispersal, leading to difficulties in subsequent infection treatments and the spread of antibiotic resistance (Jorge et al., 2019 ). However, bacterial cells often lose their recalcitrance when they disperse from the biofilm. Therefore, the recalcitrance cannot be attributed solely to mutations and genetic changes in biofilm cells. Other, phenotypic adaptations of individual cells, as well as the presence of an extracellular matrix, must contribute to biofilm recalcitrance. Numerous explanations for the observed recalcitrance of biofilms have been proposed and thoroughly reviewed (Stewart, 2002 ; Venkatesan et al., 2015 ; Ciofu et al., 2017 ; Hall and Mah, 2017 ; Hathroubi et al., 2017 ; Valquier-Flynn et al., 2017 ; Roy et al., 2018 ; Crabbé et al., 2019 ; Gebreyohannes et al., 2019 ; Sharma et al., 2019 ; Yan and Bassler, 2019 ; Bottery et al., 2021 ). For instance, biofilms can resist penetration by antimicrobials, degrade them by enzymes present in the extracellular matrix, or interact with other extracellular polymeric substances (EPS), such as enzymes, lipids, or extracellular DNA (eDNA); these have been recently shown to provide cooperative fitness to biofilm populations (Belcher et al., 2022 ). Cells with low metabolic activity offer fewer targets for antimicrobials, rendering many of them ineffective. It is now widely accepted that multiple genes of various effects determine antibiotic resistance in planktonic bacteria (Petchiappan and Chatterji, 2017 ; Apjok et al., 2019 ; Igler et al., 2021 ). Similarly, multiple mutations were recently implicated in biofilm recalcitrance (Santos-Lopez et al., 2019 ; Santos-Lopez and Cooper, 2021 ). Evolutionary experiments have shown that when biofilm and planktonic bacteria are exposed to increasing concentrations of antibiotics, the biofilm populations harbor even higher genetic diversity than planktonic populations that experienced the same treatment (Ahmed et al., 2018 , 2020 ; Santos-Lopez et al., 2019 ; Santos-Lopez and Cooper, 2021 ). Understanding interactions between the phenotypic and genotypic factors influencing biofilm recalcitrance is crucial for maximizing the probability of successful treatment and minimizing the risk of antibiotic resistance evolution. These interactions and their consequences have recently been discussed in Trubenová et al. ( 2022 ). We reasoned that the effects of individual recalcitrance mechanisms combine in non-intuitive ways and can either hinder or promote resistance evolution, depending mainly on the concentration of the antibiotics. Building on these verbal arguments and hypotheses, we here provide a quantitative modeling approach, investigating the role of the different drivers of biofilm recalcitrance. Despite the fact that the biofilm is a predominant bacterial lifestyle, most experiments are performed with planktonic bacteria. Similarly, models of antibiotic resistance evolution are abundant. However, mathematical models and simulations of biofilm growth often focus on the formation of the spatial biofilm structure, modeling physical and chemical processes such as cell and nutrient transport, metabolic reaction, hydrodynamics, biomass growth, and detachment (Picioreanu et al., 2007 ; Kragh et al., 2016 ; Ali and Wahl, 2017 ; Brockmann et al., 2020 ). For instance, Stewart ( 2003 ), Stewart et al. ( 2016 ) modeled diffusion in biofilms, while Picioreanu et al. ( 2007 ) modeled 3D structure of Pseudomonas aeruginosa biofilms, that form mushroom-like colonies. These models and predictions are useful for industry, where biofilm mass, shape, and other physical properties are important. They typically do not deal with multiple strains and the possibility of resistance mutations. Only a minority of models focuses on the population dynamic and genetic aspects that are central to the evolution and spread of resistance in biofilms (Torella et al., 2010 ; Eastman et al., 2011 ; Raynes et al., 2018 ). Here, we develop a polygenic model of biofilm recalcitrance that allows us to simultaneously study the main phenotypic recalcitrance mechanisms proposed in the literature: (a) those relying on extracellular polymeric substances (EPS, such as extracellular DNA, enzymes, or lipids) acting as a physical barrier, reducing the antibiotic concentration that bacteria experience inside the biofilm; and (b) those relying on physiological alterations, such as slow replication or metabolism, or high fraction of persisters. We will use a pharmacodynamic modeling framework to capture both the genetic and phenotypic mechanisms acting in biofilms, as discussed in Trubenová et al. ( 2022 ), and derive population replication, death, and mutation rates in the presence of antibiotics. The main question we address is under which conditions biofilms accelerate or delay antibiotic resistance evolution. Based on arguments discussed in the literature (Trubenová et al., 2022 , and references therein), the biofilm lifestyle is expected to reduce selection pressure for mutations at low concentrations, thus slowing down the evolution of resistance. By contrast, it is expected to enable the evolution of resistance under higher concentrations that kill the free-living bacteria. Here, we will show under which conditions these arguments hold and what other factors influence biofilm survival and resistance evolution."
} | 3,751 |
36534468 | PMC9788563 | pmc | 7,430 | {
"abstract": "Purple phototrophic bacteria use a ‘photosystem’ consisting of light harvesting complex 1 (LH1) surrounding the reaction centre (RC) that absorbs far-red–near-infrared light and converts it to chemical energy. Blastochloris species, which harvest light >1000 nm, use bacteriochlorophyll b rather than the more common bacteriochlorophyll a as their major photopigment, and assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple genes. To assign a role to γ, we deleted the four encoding genes in the model Blastochloris viridis . Interestingly, growth under halogen bulbs routinely used for cultivation yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC only, but growth under white light yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose gradient fractionation demonstrate that the white light-grown mutant assembles RC–LH1, albeit with an absorption maximum blue-shifted by 46 nm. Wavelengths between 900–1000 nm transmit poorly through the atmosphere due to absorption by water, so our results provide an evolutionary rationale for incorporation of γ; this polypeptide red-shifts absorption of RC–LH1 to a spectral range in which photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids encoding natural LH1γ variants and demonstrate that the polypeptide found in the wild type complex red-shifts absorption back to 1018 nm, but incorporation of a distantly related variant results in only a moderate shift. This result suggests that tuning the absorption of RC–LH1 is possible and may permit photosynthesis past its current low-energy limit.",
"introduction": "Introduction Chlorophototrophic organisms use chlorophyll (Chl) and/or bacteriochlorophyll (BChl) pigments to capture solar radiation and convert it to chemical energy to power cellular metabolism [ 1 ]. Oxygenic chlorophototrophs, such as plants, algae and cyanobacteria, use Chls to primarily absorb photons of visible wavelengths, which have sufficient energy to drive the thermodynamically challenging extraction of electrons from water, liberating molecular oxygen as a by-product [ 2 ]. Anoxygenic phototrophic bacteria are generally found below oxygenic organisms in water columns and microbial mats, and use BChls to capture lower-energy wavelengths in the far-red and near-infrared region of the spectrum that have not been utilised by the Chl-containing organisms above [ 3 ]. These bacteria use alternative sources of electrons than water, and thus do not generate O 2 . The vast majority of anoxygenic phototrophs use BChl a to harvest in the 780–900 nm range, while a few phototrophs found within the Proteobacteria use BChl b , and harvest wavelengths greater than 1000 nm [ 4 ]. BChl b was discovered in 1963 as the sole BChl extracted from an organism tentatively identified as a species of Rhodopseudomonas ; the extracted pigment displayed a Q y maximum red-shifted by 23 nm compared with that of BChl a [ 5 ]. An additional ‘ Rhodopseudomonas ’ isolate containing this pigment was found to have a whole-cell absorption maximum at ∼1020 nm, 142 nm further into the near-infrared than the BChl a -containing proteobacterium Rhodospirillum rubrum to which it was compared [ 6 ]. This strain was subsequently named Rhodopseudomonas viridis due to its intense green colour [ 7 ]. Phylogenetic analysis of this strain, and closely related species synthesising BChl b , led to transferral to the novel Blastochloris genus; the isolate described above being designated Blastochloris ( Blc .) viridis [ 8 ]. The reaction centre (RC), the site of charge separation that initiates photosynthetic electron transfer, is encircled by an antenna known as light-harvesting complex 1 (LH1) to form the ‘core’ RC–LH1 supercomplex, the key functional unit for phototrophy in Proteobacteria. RC–LH1 complexes contain five universal components: the L, M, and H subunits of the RC, and the α and β polypeptides of LH1. Most proteobacterial RCs, including that of Blc. viridis , have a bound cytochrome subunit, C, containing four haem cofactors. The RC from Blc. viridis was the first membrane protein to have its structure solved, earning the 1988 Nobel Prize in Chemistry [ 9 , 10 ]. Some LH1 antennas also contain additional subunits; the Rhodopseudomonas palustris LH1 ring contains a single transmembrane helix known as protein W [ 11 , 12 ], LH1 from Rhodobacter spp. contains a PufX polypeptide [ 13–15 ], and a further subgroup of these organisms additionally contain PufY [ 16–18 ]. These polypeptides create a channel in the LH1 ring allowing quinone/quinol exchange between the RC and the cytochrome bc 1 complex [ 19 , 20 ]. An additional LH1 subunit was also identified in Blc. viridis [ 21 ]. However, unlike those mentioned above, this LH1γ polypeptide was found to be in apparent equimolar ratio with the α and β polypeptides [ 22 ]. A recent cryo-electron microscopy structure of the RC–LH1 complex from Blc. viridis revealed the location of γ, packing between β polypeptides on the outside of the ring ( Figure 1 ) [ 16 ]. The α : β : γ ratio was found to be 17 : 17 : 16; in this case the ‘missing’ γ subunit creates the channel for quinone diffusion. We proposed that the role of the γ subunit in this complex is to tighten packing of the BChls in LH1, increasing excitonic coupling of these pigments, which results in the extreme ‘red-shift’ of the complex past 1000 nm to the current low-energy limit for photosynthesis on Earth. Figure 1. Cryo-EM structure of the Blc. viridis RC–LH1 complex, displaying γ subunit locations. ( A ) Side-on view of the RC–LH1 complex, with the periplasmic side facing up. The individual components are indicated in text of the respective colour. The gap created by the ‘missing’ LH1γ polypeptide is at the anterior of the complex. ( B ) View of the RC–LH1 complex from above the periplasmic surface of the membrane, with the gap in LH1 at the bottom of the ring. ( C ) View as in previous with only LH1γ subunits displayed around the LH1 BChls (green). The route for diffusion of quinone/quinol (tan) is indicated by the orange arrow. Peptide analysis of γ isolated from Blc. viridis indicated that the polypeptide is 36 amino acids in length, and variance at position 34 was detected with threonine and valine residues identified in a 2 : 1 ratio, respectively [ 22 ]. This suggested that multiple copies of LH1γ-encoding genes were present in the Blc. viridis genome. Subsequent genome sequencing has revealed that Blc. viridis has four genes encoding putative γ polypeptides, three of which are clustered and share high sequence identity (LH1γ 1–3 ), and the fourth being more divergent (LH1γ 4 ) [ 23 , 24 ] ( Figure 2 ). Figure 2. Amino acid sequence alignment of translated LH1γ-encoding genes from Blc. viridis . Genetic loci for each ORF are listed in parentheses. Full-length sequence comparisons between clustered LH1γ polypeptides 1–3 (blue box), and with the additional, divergent LH1γ 4 (red box); identical, highly similar and similar amino acids are indicated by asterisks, colons, and periods, respectively. The sequences of the polypeptides found in the purified complex, identified by Edman degradation [ 22 ], and those resolved in the cryo-EM structure [ 16 ], are indicated by light and heavy underlining, respectively. In the present study, we constructed mutants of Blc. viridis that do not produce LH1γ, permitting the confirmation of the role of this unusual core complex component. Our results indicate that the loss of γ results in large blue-shifts in the absorption maxima of whole cells and of the isolated RC–LH1 complex to a region of the solar spectrum within which photons poorly transmit through the atmosphere, providing an evolutionary rationale for the recruitment of this additional subunit into the bacterial photosystem.",
"discussion": "Discussion The vast majority of anoxygenic phototrophs in diverse bacterial phyla discovered to date use BChl a as the RC pigment for phototrophy [ 37 ]. Only a few purple bacteria use BChl b [ 4 ]. Of these, only Blastochloris spp. have orthologs of LH1γ. In each case, the common components of the photosystem are encoded by single ORFs within the photosynthesis gene cluster (PGC), which also contains genes encoding pigment biosynthesis enzymes and photosystem assembly factors [ 38 ]. However, each sequenced strain contains multiple LH1γ genes that are located distant from the PGC [ 24 , 25 , 39 , 40 ]. This suggests that these genes evolved separately from the other PGC genes, and that assembly of RC–LH1 without LH1γ is safeguarded against by the presence of multiple paralogs in the genome. The evolutionary rationale for this is provided by our results; our mutant strains assembling RC–LH1 lacking LH1γ absorb maximally at 972 nm, which sits in a range of the solar spectrum that poorly transmits through the atmosphere due to absorption by water vapour ( Figure 8 ). In this range, few photons reach the surface of Earth, and those that do also poorly transmit through liquid water at the surface [ 41 ]. Therefore, it is likely that Blastochloris spp. evolved or recruited an additional LH1 subunit to influence absorption of the complex, shifting to a region of the spectrum where photons are of lower energy but are more abundant. Figure 8. Absorption spectra of RC–LH1 complexes relative to available solar energy. The purified complex from Rsp. rubrum containing BChl a (blue trace) is blue-shifted in comparison the BChl b -containing complexes from WT Blc. viridis (green trace) and the ΔLH1γ 1–4 mutant (black trace). The overlayed red trace displays the spectrum of solar radiation that reaches the surface of the Earth [ 42 ]. Absorption bands from O 2 and H 2 O in the atmosphere are labelled, and the corresponding spectral regions from which photons poorly transmit are highlighted in grey. LH1γ polypeptides 1 and 2 are identical, and LH1γ 3 differs by a single amino acid (see Figure 2 ); their encoding genes are clustered in the genome. The original proteomic analysis of Blc. viridis demonstrated that highly similar polypeptides containing threonine or valine at position 34 could be isolated from its photosystem and are present at a 2 : 1 ratio, while a polypeptide equivalent to LH1γ 4 was not detected [ 22 ]. This indicates that the genes encoding LH1γ 1–3 are transcribed together, and that LH1γ 4 is not produced when the cells are grown under full-spectrum light from incandescent bulbs. Similarly, we could not detect any phenotypic change in the cells of the ΔLH1γ 1–3 mutant when compared with the mutant lacking all γ-encoding genes under any condition tested that we could ascribe to the production of LH1γ 4 . Our finding that expression of the LH1γ 4 gene from a plasmid in the ΔLH1γ 1–4 mutant leads to the cells having an absorption maximum of 1003 nm indicates that incorporation of γ 4 into RC–LH1 induces a moderate red-shift, which may provide more favourable absorption under certain environmental conditions (e.g. when in competition with other BChl b phototrophs such as Halorodospira spp. displaying similar absorption at 1018 nm, see below). It may be that incorporation of LH1γ 4 into the WT RC–LH1 can be induced by growth under specific LED regimes in the laboratory, which we intend to explore further. We found that the ΔLH1γ 1–3 and ΔLH1γ 1–4 mutants grown under our standard laboratory conditions, with illumination provided by halogen bulbs, resulted in the assembly of RCs, but no intact RC–LH1 complexes. However, growth under white-light fluorescent tubes that would be routinely used for cultivation of oxygenic cyanobacteria permitted assembly of RC–LH1, with an absorption maximum far outside the range of emission from the light source. This appears counterintuitive, but it is possible that the emission from the halogen bulbs, which reduces sharply at ∼850 nm (see Supplementary Figure S1 ), provides wavelengths that efficiently excite the BChls contributing the major absorption feature of the RC at 831 nm (see RC band spectrum in Figure 5 ). This RC contains only a single 15- cis -1,2-dihydroneurosporene carotenoid, providing limited absorption in the visible range of the spectrum. The observation that RC–LH1 lacking γ only assembles when the mutants are grown under white light might be explained by the carotenoid content of LH1; the 17 all- trans pigments bound in the ring provide strong absorption in the 400–550 nm range, which overlaps with the narrow, intense emission band of the fluorescent tubes in this range (see Supplementary Figure S1 ). Taking this observation together with the very slow growth rate of WT and ΔLH1γ 1–4 under white light (see Table 1 ), an alternative explanation might be that Blc. viridis senses this regime as very low light and so may compensate by increasing expression of the LH1 α and β genes. A complete elucidation of this phenomenon will form the basis of a future study. Our results will be noteworthy to those studying anoxygenic phototrophy; although it is possible to purchase LED panels that mimic ‘photosynthetically active radiation’ for oxygenic phototrophs, panels emitting wavelengths to at least 1200 nm that more accurately mimic full-spectrum natural sunlight do not yet exist. As it becomes more difficult to source incandescent and halogen bulbs there may be a need for researchers to assemble broad-spectrum LED regimes themselves, to accurately study phototrophic bacteria under conditions similar to those experienced in nature. The mutants lacking LH1γ assembling RC–LH1 in white light also accumulate RCs, as can be seen in the sucrose density gradients, while the WT does not ( Figure 5 ). This suggests that the presence of the 16 LH1γ subunits provide a stabilising role; the interaction with the adjacent β polypeptides may fix the complex in place in addition to imparting the red-shift in absorption, and the presence of γ may orient the RC in such a position that efficient quinone/quinol shuttling can occur at the ‘missing’ 17th γ position [ 16 ]. This stabilising effect may be analogous to the role that PufX plays in the primarily dimeric Rhodobacter sphaeroides RC–LH1 [ 43 ]. Deletion of the pufX gene leads to formation of solely monomeric RC–LH1, and the recent cryo-EM structure of this complex was solved at much lower resolution than the PufX-containing dimer or monomer, attributed to PufX's role in orienting the RC within LH1 [ 43 , 18 ]. It is interesting that we do not observe assembly of RCs without LH1 in the WT under either white light or halogen light conditions. This could be due to the stability imparted by γ; it is likely that the formation of RC–LH1 is a highly co-ordinated process and that γ is incorporated into LH1 at the same time as the α and β polypeptides by the assembly machinery, and this may not be easily reversed once γ has stabilised the complex. Turnover and recycling of light-harvesting and RC complexes is a well-studied process; photosystem II of oxygenic phototrophs is damaged by the reaction it catalyses so is constantly reassembled [ 44 ], and the metabolically diverse purple bacteria using BChl a can assemble and disassemble RC–LH1 depending on whether conditions suit aerobic respiration [ 45 ]. However, Blastochloris spp. are unable to grow in the presence of O 2 and they generally inhabit anoxic ecological niches such as the bottom of microbial mats, where rapid turnover of the photosystem is not required [ 46 ]. Here, extremely red-shifted absorption provided by the γ-stabilised complex affords a competitive advantage over the abundant BChl a -containing phototrophs in the mat where wavelengths are highly filtered, and thus a route to survival and growth. Halorhodospira ( Hrs .) halochloris and Hrs . abdelmalekii (formerly Ectothiorhodospira halochloris and Ectothiorhodospira abdelmalekii ) are additional BChl b -containing purple bacteria isolated from hypersaline and alkaline soda lakes in Wadi El Naturn, Egypt, that also display whole-cell absorption maxima at 1018 nm [ 47 , 48 ]. The isolated RC–LH1 complexes from these organisms were found to contain three low molecular weight components, and the LH1 absorption at 1018 nm could be reversibly blue-shifted to 964 nm when titrated with acid in the pH range of 7.5–5.7 [ 49 ]. The small polypeptide of a similar size to Blc. viridis LH1γ was isolated from the RC–LH1 complex of Hrs. halochloris and was assigned to be analogous, although its sequence was not presented in the publication [ 50 ]. The recent complete genome sequence of Hrs. halochloris has revealed than an ortholog of a Blastochloris spp . gene encoding LH1γ is absent [ 51 ]. The inability to find LH1γ orthologs in the genome of Hrs. halochloris — as well as in other non- Blastochloris BChl b -containing organisms — via BLAST search is unsurprising since the polypeptide is a small, single transmembrane helix (e.g. the sequences of the single transmembrane PufX polypeptides from Rhodobacter spp. display low sequence identity across the genus [ 52 ]). Hrs. halochloris was found to have an unusual complement of genes encoding RC–LH1 subunits [ 51 ]. This organism has 2 pufBALMC operons, and an additional two pairs of pufBA genes found elsewhere in the genome, one pair of which ( pufB 3 A 3 ) is more divergent, sharing only 41% and 42% identity with LH1β 1 and LH1α 1 , respectively, which may lead to assembly of LH1 containing multiple forms of α- and/or β-polypeptides, as was found in the RC–LH1 of BChl a -containing Thiorhodovibrio strain 970 [ 53 ]. A recent study demonstrates that desalting the purified Hrs. halochloris RC–LH1 complex results in the same blue-shift described previously with a decrease in pH, which can be reversed by supplementation with salts [ 54 ]. Taking all of these results together, it is likely that Halorhodospira spp. have evolved an alternative approach to accessing photons of wavelengths >1000 nm to that taken by members of the Blastochloris genus; the red-shift displayed by these organisms appears to be strongly influenced by electrostatic charge. This study elucidates the role of the γ polypeptide in the RC–LH1 of Blc. viridis . The presence of the highly conserved γ isoform stabilises the complex and shifts its absorption 46 nm further into the near-infrared. This extreme red-shift allows Blastochloris spp. to access the more abundant photons >1000 nm that are not absorbed by water in the atmosphere. The discovery that plasmid-borne expression of a divergent isoform of LH1γ not found in the WT complex results in only a moderate red-shift of the complex upon assembly provides a route to explore the extent to which polypeptide sequence influences the absorption shift, and whether the low energy limit of natural photosynthesis can be surpassed by rational design of the LH1γ subunit."
} | 4,764 |
40216764 | PMC11992226 | pmc | 7,431 | {
"abstract": "Benzamides constitute an important class of bulk and fine chemicals as well as essential parts of many life science molecules. Currently, all these compounds are majorly produced from petrochemical-based feedstocks. Notably the selective aerobic oxidative conversion of lignin and lignin-derived compounds to primary, secondary, and tertiary amides and phenols offers the potential for a more sustainable synthesis of valuable building blocks for fine chemicals, monomers for polymers, biologically active molecules, and diverse consumer products. Here we present the concept of “lignin to amides” which is demonstrated by a one-pot, multi-step oxidation process utilizing molecular oxygen and a 3d-metal catalyst with highly dispersed and stable cobalt species (Co-SACs) supported on nitrogen-doped carbon in water as solvent. Moreover, our cobalt-based methodology allows for the cost-efficient transformation of a lignin and its variety of derivates simply using O 2 and organic amines. Mechanistic investigations and control experiments suggest that the process involves an initial dehydrogenation of C α -OH, cleavage of the C β -O as well as C(O)-C bond and condensation of the resulting carboxylic acids with amines. Spectroscopic studies indicate that the formation of superoxide species (O 2 ●− ) and specific Co-nitrogen sites anchored on mesoporous carbon sheets are key for the success of this transformation.",
"introduction": "Introduction Over the past two centuries, the growing world population and the excessive consumption of fossil resources have caused significant environmental problems. Among other problematic issues, billions of tonnes of CO 2 have been released into the atmosphere, far exceeding the capacity of the Earth’s ecological cycle and leading to the global climate problems of today 1 – 4 . In this context, “carbon peaking” 4 and “achieving carbon neutrality” 5 have become important topics of concern for the global scientific community including researchers from social, economic, natural, and other sciences. In addition to the development of climate-neutral and carbon dioxide-free energy technologies that are in line with the Paris Agreement, circular chemistry can make a significant contribution to achieve sustainability 6 – 9 . The United Nations Intergovernmental Panel on Climate Change (IPCC) found that the chemical and petrochemical industry, which manufactures important products from fossil raw materials, is responsible for over seven percent of greenhouse gas emissions 10 . According to a report from the International Energy Agency (IEA) on the future of petrochemicals, this feedstock accounts for 12% of global oil demand and continues to grow. Explicitly, it was stated: “Our economies are heavily dependent on petrochemicals, but the sector receives far less attention than it deserves” 11 . To move away from the use of oil and gas as feedstocks in the chemical industry, it is necessary to develop a toolbox of technologies which allows the production of all kinds of valuable chemicals without concomitant formation of carbon dioxide. Specifically, the increased use of waste as well as abundant, non-eatable biomass as feedstocks can be part of the solution to meet the ever-growing demand and to reduce the dependence on fossil resources in this area 12 – 28 . One of the most abundant biomass resources is lignocellulose, which is available from agricultural residues, forestry wastes, and energy crops 4 , 20 – 28 . It has an annual production of around 170 billion tons via plant photosynthesis and consists of three main components: cellulose (40–50 wt%), hemicellulose (20–30 wt%), and lignin (10–35 wt%). While cellulose and hemicellulose are polymers of C5 and C6 sugars, lignin is a heterogeneous, highly crosslinked polymer akin to phenol-formaldehyde resins, which is made up of various building blocks with certain bond linkages, e.g., β-O-4, β-5, α-O-4, β-β, 4-O-5, β-1 and others (see Fig. 1a ) 4 , 20 – 28 . Due to the large content of aromatic structures, it is regarded as a potential alternative resource to replace fossil-based aromatic chemicals. Hence, research on lignin depolymerization and its downstream products have attracted much attention from both academic and industrial researchers in the past years. The majority of the investigated lignin transformations include hydrogenolysis 29 – 33 , hydrolysis 34 – 36 , and nucleus exchange processes 18 , 37 – 44 , which provide a variety of interesting building blocks. Compared to these works, selective oxidations, especially using air or molecular oxygen, to provide carboxylic acid derivatives are scarce 12 , 40 – 42 , 44 – 47 . Among these derivatives, amides are highly valuable compounds, which are widely used in organic synthesis. Meanwhile, these products are used in numerous agrochemicals and pharmaceuticals 48 – 51 . In fact, 73 of the top 200 selling drugs of the year 2022 were based on amides and its derivatives, mainly aromatic one (Fig. 1b ) 52 . Fig. 1 State-of-the-art of synthesis amides. a Representative lignin structure and typical linkages, ( b ) selected benzamide containing drugs, ( c ) classical and current methods for the preparation of amides, ( d ) this work on the renewable synthesis of amides with Co-SACs. Conventional strategies for amide bond formation typically involve the condensation of a carboxylic acid or its derivatives with an amine, leading to the elimination of one equivalent of water (Fig. 1c ) 53 – 55 . Functionalized amides are generally synthesized through the reaction of activated carboxylic acid derivatives, such as acyl chlorides and anhydrides, with amines. Alternatively, direct amidation of carboxylic acids with amines can be achieved using stoichiometric amounts of coupling reagents. However, these methods often generate considerable waste, raising environmental and sustainability concerns. Additionally, other methods, such as nitrile hydration 56 , Ritter reaction 57 , Passerini reaction 58 , Schmidt reaction 59 , and Beckmann rearrangement 60 are applied for amide synthesis (Fig. 1c ). With respect to lignin model compounds, Loh and Chiba reported the oxidative synthesis of α-ketoamides by C β -H oxidation and C β -O bond cleavage of β-O-4 models in the presence of CuI and secondary amines 40 . More recently, the groups of Wang and Liu reported the synthesis of amides by oxidation of 2-phenoxyacetophenones applying homogeneous copper complexes 41 , 42 . In 2022, Xu and co-workers reported the preparation of benzanilides by oxidation of 2-phenoxyacetophenones with H 2 O 2 43 . In the same year, Dai and co-workers reported the heterogeneous manganese oxide-catalyzed cleavage of C-C bonds in alcohols using oxygen as the environmentally benign oxidant and ammonia as the nitrogen source 44 . Despite these advancements, all reported methods make only use of a limited number of lignin model compounds and have not been used for “real” lignin. With the aim of developing a general methodology for the synthesis of various aromatic amides from renewable raw materials, we became interested in the oxidative amidation of lignin, its derivatives, and model compounds. Due to stability, ease of separation, and potential recycling, heterogeneous materials are generally preferred over homogeneous catalysts, and they might be readily used in flow reactors, facilitating production using continuous processes. Unfortunately, to the best of our knowledge, there are rare examples of heterogeneous materials for amides synthesis starting from lignin derivates. To ensure sufficient reactivity, we aimed to use highly dispersed redox-active 3d-metal centers merging the advantages (superior reactivity, high selectivity, and stability) of both homogeneous and heterogeneous catalysts 61 – 66 . Herein, we describe a generally applicable protocol which allows for highly selective oxidative amidation of lignin derivates in the presence of molecular O 2 and H 2 O. The identified single cobalt atom catalyst (Co-L1@NC-800) shows high stability and broad functional group tolerance."
} | 2,022 |
35222085 | PMC8867213 | pmc | 7,432 | {
"abstract": "The cnidarian-dinoflagellate symbiosis is a mutualistic intracellular association based on the photosynthetic activity of the endosymbiont. This relationship involves significant constraints and requires co-evolution processes, such as an extensive capacity of the holobiont to counteract pro-oxidative conditions induced by hyperoxia generated during photosynthesis. In this study, we analyzed the capacity of Anemonia viridis cells to deal with pro-oxidative conditions by in vivo and in vitro approaches. Whole specimens and animal primary cell cultures were submitted to 200 and 500 μM of H 2 O 2 during 7 days. Then, we monitored global health parameters (symbiotic state, viability, and cell growth) and stress biomarkers (global antioxidant capacity, oxidative protein damages, and protein ubiquitination). In animal primary cell cultures, the intracellular reactive oxygen species (ROS) levels were also evaluated under H 2 O 2 treatments. At the whole organism scale, both H 2 O 2 concentrations didn’t affect the survival and animal tissues exhibited a high resistance to H 2 O 2 treatments. Moreover, no bleaching has been observed, even at high H 2 O 2 concentration and after long exposure (7 days). Although, the community has suggested the role of ROS as the cause of bleaching, our results indicating the absence of bleaching under high H 2 O 2 concentration may exculpate this specific ROS from being involved in the molecular processes inducing bleaching. However, counterintuitively, the symbiont compartment appeared sensitive to an H 2 O 2 burst as it displayed oxidative protein damages, despite an enhancement of antioxidant capacity. The in vitro assays allowed highlighting an intrinsic high capacity of isolated animal cells to deal with pro-oxidative conditions, although we observed differences on tolerance between H 2 O 2 treatments. The 200 μM H 2 O 2 concentration appeared to correspond to the tolerance threshold of animal cells. Indeed, no disequilibrium on redox state was observed and only a cell growth decrease was measured. Contrarily, the 500 μM H 2 O 2 concentration induced a stress state, characterized by a cell viability decrease from 1 day and a drastic cell growth arrest after 7 days leading to an uncomplete recovery after treatment. In conclusion, this study highlights the overall high capacity of cnidarian cells to cope with H 2 O 2 and opens new perspective to investigate the molecular mechanisms involved in this peculiar resistance.",
"introduction": "Introduction The evolutionary success of symbiotic cnidarians is based on a mutualism with dinoflagellates of the family Symbiodiniaceae. The symbionts, living inside the gastrodermal host cells, find a protected and stable environment and benefit from inorganic compounds provided by the animal cells (e.g., nitrogen, phosphorus, and sulfate) for their photosynthetic activity. Conversely, the animal host benefits from the organic compounds produced by algal photosynthesis (e.g., glucose and subsequently amino-acids, lipids) and largely transferred from the alga to the animal cell ( Davy et al., 2012 ). This partner cooperation allows autotrophy to the animal host, leading to the colonization of oligotrophic waters by the symbiotic holobiont. Concomitantly with those advantages, some constraints appear, especially the photosynthetic-dependent production of oxygen in the animal tissue. Such oxygen production causes diurnal hyperoxia condition in a symbiotic cnidarian, leading to a pro-oxidant state with reactive oxygen species (ROS) overproduction ( Dykens et al., 1992 ; Richier et al., 2003 ; Saragosti et al., 2010 ; Shaked and Armoza-Zvuloni, 2013 ). Both partners have the pathways for cross-regulating the intracellular redox state, especially by ROS detoxication through a full suite of antioxidant enzymes to avoid cellular damages ( Shick and Dykens, 1985 ; Richier et al., 2003 , 2005 ; Plantivaux et al., 2004 ; Furla et al., 2005 ; Merle et al., 2007 ; Pey et al., 2017 ). The study of ROS sensitivity in these organisms is also of environmental interest. Environmental perturbations (especially variations in temperature and UV radiation) induce oxidative stress that may lead in extreme cases to symbiosis breakdown, a process commonly called bleaching. Thus, under stressful oxidative conditions, Symbiodiniaceae can be eliminated from or exit the host through different cellular processes, like exocytosis, cell detachment, necrosis or apoptosis (see for review Suggett and Smith, 2020 ). Oxidative stress is known to induce specific cellular damages such as DNA modification (DNA adducts), lipid peroxidation and protein oxidation. In symbiotic cnidarians, several biochemical biomarkers (e.g., protein carbonylation, lipid peroxidation, and protein ubiquitination) were validated in studies following imbalances between ROS overproduction and antioxidant defenses during environmental stress, resulting in the disruption of the symbiotic association (e.g., Lesser and Farrell, 2004 ; Richier et al., 2006 ; Pey et al., 2011 ). Among ROS, hydrogen peroxide (H 2 O 2 ) is a relatively stable chemical formed from O 2 and is naturally present in the aquatic systems ( Ndungu et al., 2019 ) with concentrations ranging from 0.3 μM in the water column to 4 μM in intertidal areas ( Abele-Oeschger et al., 1997 ). It originates from marine biota ( Hansel and Diaz, 2021 ) or is carried by the rain ( Cooper et al., 1987 ). In all the organisms, intracellular H 2 O 2 levels can reach tens of micromolar and is generated during normal cellular metabolism (i.e., photosynthesis and respiration) playing crucial roles in the intracellular signaling such as hypoxic signal transduction, cell differentiation and proliferation as well as for immune responses ( Halliwell et al., 2000 ; Apel and Hirt, 2004 ; Giorgio et al., 2007 ). At high production levels, the H 2 O 2 effect can be mitigated by several antioxidant defenses including peroxidases, catalases, thioredoxin reductase, peroxiredoxins, and glutathione S-transferases family enzymes which can lead to rapidly decreasing intracellular H 2 O 2 concentrations. However, if cellular redox homeostasis cannot be maintained, H 2 O 2 leads to reversible and irreversible oxidative modifications of proteins (e.g., carbonylation), enhancing protein ubiquitination and subsequent proteasome activation. In addition, cell cycle arrest or apoptosis could also be observed (see for review Fulda et al., 2010 ). Although the impact of H 2 O 2 has been more widely investigated in mammalian cells and particularly in tumor cells (see for review Lennicke et al., 2015 ), studies have shown a similar impact in marine invertebrates, such as bivalves or polychaetes ( Abele-Oeschger et al., 1994 ; McDonagh and Sheehan, 2006 , 2007 ; Da Rosa et al., 2008 ; Friedman et al., 2018 ; Nguyen, 2020 ). In the coral symbiont, H 2 O 2 has been shown to be a by-product of photosynthesis processes ( Suggett et al., 2008 ; Armoza-Zvuloni and Shaked, 2014 ). Thanks to its cell-permeable properties, H 2 O 2 may diffuse from algal to animal host cells. Interestingly, some studies reported a release of H 2 O 2 from non-stressed corals ( Armoza-Zvuloni and Shaked, 2014 ) and an extracellular production by the dynamics of the superoxide anion ( Saragosti et al., 2010 ). Therefore, due to their symbiosis lifestyle, animal host cells are daily exposed to H 2 O 2 , raising the question of their intrinsic potential to resist a massive influx of H 2 O 2 . Nevertheless, in excess, ROS (including H 2 O 2 ) cause negative impact (mostly on protein and lipids) on the symbiont, leading to photosynthesis impairment, even if no bleaching phenomenon is induced ( Higuchi et al., 2009 ; Roberty et al., 2015 , 2016 ). Anemonia viridis is a temperate sea anemone deeply studied as biological model of the cnidarian-dinoflagellate symbiosis. Its enzymatic antioxidant properties, tissue distribution and regulation have been intensively investigated ( Hawkridge et al., 2000 ; Richier et al., 2003 , 2005 ; Plantivaux et al., 2004 ; Merle et al., 2007 ; Ganot et al., 2011 ; Pey et al., 2017 ). In addition, the sensitivity of A. viridis to thermal and UV stresses has been well described and some mechanisms of bleaching have been decrypted, including oxidative stress and apoptosis ( Richier et al., 2006 ; Moya et al., 2012 ). Recently, we succeeded in the establishment of primary cell cultures from A. viridis exhibiting a gastrodermal signature ( Barnay-Verdier et al., 2013 ; Ventura et al., 2018 ; Fricano et al., 2020 ). Thus, to test the hypothesis of an intrinsic resistance of animal cells to H 2 O 2 , we exposed A. viridis specimens and primary cell cultures at the same H 2 O 2 concentrations (200 and 500 μM) during the same periods of time (24 h and 7 days), and compared the respective responses. For each treatment, we monitored global health parameters (symbiotic state, viability and cell growth) and stress biomarkers (global antioxidant capacity, oxidative protein damages, and protein ubiquitination). This allowed us to assess the cnidarian cell susceptibility to H 2 O 2 exposure, highlighting the putative influence of the tissue organization or/and of the presence of symbionts.",
"discussion": "Discussion Extended Tolerance of Symbiotic Cnidarians to Hydrogen Peroxide In this study, we used H 2 O 2 to induce pro-oxidative condition and to investigate the stress response in the symbiotic sea anemone A. viridis at both whole animal and cellular scales. The H 2 O 2 concentrations used in the present study correspond to extremely high levels never measured in the seawater. However, rainwater can temporally induced increase of H 2 O 2 to tens of micromolar ( Ndungu et al., 2019 ; Jones and Lee, 2020 ). Benthic marine organisms from coastal areas are therefore regularly facing H 2 O 2 , that could lead to oxidative stress ( Abele-Oeschger et al., 1997 ). Indeed, among ROS, H 2 O 2 is the most abundant and long-lived in sea water and contrary to other ROS, H 2 O 2 could rapidly diffuse across membranes (see for review Halliwell and Gutteridge, 2015 ). In our study model, at both whole animal and cellular scales, 200 μM of H 2 O 2 did not create a condition of stress, since very weak impact was observed in global health: no sign of mortality or bleaching on specimens ( Figure 1 ), no intracellular ROS accumulation ( Figure 4 ) and only very slight effect on viability after 7 days exposure in cell cultures ( Figure 5 ). In addition, no effect was observed on any stress biomarkers we tested ( Figures 2 , 7 ). In cell cultures, we nevertheless measured a significant cellular growth arrest ( Figure 5 ), reflecting a common feature of stress response and could be interpreted as an usual resistance mechanism (see for review Davies, 1999 , 2000 ). H 2 O 2 usually represents a threat for most organisms. Indeed, in diverse mammalian cell lines, a cytotoxic effect of H 2 O 2 could be observed from 60 μM ( Coyle et al., 2006 ) with drastic and irreversible impacts (i.e., apoptosis and necrosis) induced at 400 μM H 2 O 2 ( Xiang et al., 2016 ). Some studies performed on marine invertebrates showed that micromolar concentrations of H 2 O 2 , ranging from 0.5 to 20 μM, could impact the metabolism of the whole animal. For example, a 40% drop in O 2 consumption was observed in the Polychaete Nereis diversicolor under 5 μM of H 2 O 2 ( Abele-Oeschger et al., 1994 ), while higher level of H 2 O 2 (50 μM) can cause oxidative damages (i.e., lipid peroxidation), as observed in another Polychaete species, Laenoereis acuta ( Da Rosa et al., 2008 ). Contrasting with those results, our study highlighted an extended tolerance of symbiotic cnidarian facing even greater H 2 O 2 concentrations. This resistance to H 2 O 2 is, however, not a general cnidarian feature as it has been shown that concentrations exceeding 163 μM H 2 O 2 caused mortality of the non-symbiotic sea anemone Nematostella vectensis ( Friedman et al., 2018 ). Therefore, these results reinforced the hypothesis of the adaptation of symbiotic cnidarians to pro-oxidative conditions, due to their lifestyle with a photosynthetic symbiont. Indeed, it has been already highlighted that symbiotic cnidarians exhibited a wide diversity of biochemical antioxidant actors, compared to non-symbiotic species ( Furla et al., 2005 ). For instance, higher number of superoxide dismutase (SOD) isoforms was identified in the symbiotic cnidarian A. viridis compared to the non-symbiotic one Actinia schmidti . In complement, another comparative analysis showed that the glutathione peroxidase (GPx) isoforms were less numerous in the non-symbiotic sea anemone N. vectensis than in the symbiotic sea anemone A. viridis ( Pey et al., 2017 ). To confirm the hypothesis of the adaptative process, it will be required to extend the comparison of the antioxidant battalion between symbiotic and non-symbiotic cnidarians at multiple scales, even by including the non-enzymatic actors. Importance of Host Cells to Hydrogen Peroxide Defense In the present study, H 2 O 2 exposure on whole organism affected mainly the endosymbiont, Philozoon sp. genus ( LaJeunesse et al., 2021 ), rather than the animal host tissues. Although antioxidant defenses were stimulated after 7 days, an increase of protein carbonylation was measured in the symbiont fraction, whereas no increase was observed in the animal compartments ( Figure 2 ). The susceptibility of free-living algae to H 2 O 2 is well documented and highlighted an important heterogeneity in H 2 O 2 response. For example, although the cyanobacterium Synechococcus aeruginosus tolerated until 2 mM H 2 O 2 , another cyanobacterium species, Microcystis aeruginosa , was affected by around 20 μM (EC50) and the diatoms Navicula seminulum by 200 μM ( Drábková et al., 2007 ). Few studies have been performed on Symbiodiniaceae sensitivity to ROS and again they highlighted species-specific impacts on photosynthesis ( Wietheger et al., 2015 ; Roberty et al., 2016 ). For example, cultured Symbiodinium microadriaticum strain showed high resistance to 1 mM H 2 O 2 , compared to Fugacium kawagutii showing drastic damage to photosystem function at the same H 2 O 2 concentration ( Wietheger et al., 2015 ). In addition, light exposure increased the photosynthesis impairment of cultured Symbiodiniaceae from 30 min of >1 mM H 2 O 2 exposure ( Wietheger et al., 2015 ). Protected inside the gastrodermal host cell, the endosymbiont would not have shown any signs of oxidative stress under H 2 O 2 exposure, but the sensitivity assessed in our study did not confirm this assumption. This sensitivity to H 2 O 2 could, however, be correlated to previous studies demonstrating that Symbiodiniaceae living in hospite present a reduction of the antioxidant enzymatic defenses (i.e., SOD, catalases or peroxidases) compared to the free-living condition ( Lesser and Shick, 1989 ; Richier et al., 2005 ; Pey et al., 2017 ). Nevertheless, it has been shown that in hospite , Symbiodiniaceae harbor a higher surface volume of thylakoid lamellae ( Lesser and Shick, 1990 ), increasing definitely the photosystem density and consequently the source of ROS associated with electron chain transports ( Saragosti et al., 2010 ). These data, in addition with measurements of H 2 O 2 diffusion from the symbiont ( Suggett et al., 2008 ; Armoza-Zvuloni and Shaked, 2014 ), support the conclusion that, in hospite , the redox homeostasis of the symbiont is bolstered by the antioxidant defenses of the animal host cells. For example, in A. viridis it has been frequently observed that the animal compartment constitutes the major contributor to the holobiont antioxidant potential, with higher amount of antioxidant defenses compared to the symbiont fraction ( Richier et al., 2003 , 2005 ; Plantivaux et al., 2004 ; Merle et al., 2007 ; Pey et al., 2017 ). This agrees with studies performed on other symbiotic cnidarian species ( Yakovleva et al., 2004 ; Levy et al., 2006 ; Krueger et al., 2015 ). By consequence, an experimental burst of H 2 O 2 leads to more deleterious effects in the endosymbiont than in animal cells, illustrating their capacity to cope with H 2 O 2 . No Bleaching Induction by Hydrogen Peroxide Even at highest H 2 O 2 exposure, no bleaching was observed neither during, nor after the exposure period in the treated A. viridis specimens ( Figure 1 ). Interestingly, despite the protein damages observed in the symbiont, the equilibrium of the symbiosis was maintained. It has been largely documented in symbiotic cnidarians that stress-induced bleaching (e.g., thermal stress) is linked with the over-production of ROS by the endosymbiont, leading to significant oxidative damages in the host cells (see for review Suggett and Smith, 2020 ). Due to its permeable properties and its overproduction in several strains of Symbiodiniaceae exposed to thermal stress ( Lesser, 1996 ; Suggett et al., 2008 ; Roberty et al., 2015 ), this ROS has then been suggested to be responsible of oxidative stress occurring in host cells during bleaching events. However, in this study, the absence of bleaching and of oxidative damages in host cells under H 2 O 2 exposures can support the conclusion that H 2 O 2 may not be the most important ROS associated with coral bleaching. Limits of the Resistance At cellular level, pro-oxidative condition can elicit a broad spectrum of responses from proliferation to growth arrest, or senescence and cell death, depending on the cell capacity to overcome the stress by repairing or removing damaged molecules. The observed effect reflects the balance between intracellular pathways activated in response to the oxidative injury and can vary significantly with the concentration of the oxidant agent and the treatment exposure. In our in vitro study model, 500 μM of H 2 O 2 induced a decrease of cell viability, particularly pronounced after 7 days of treatment and associated with a drastic growth arrest ( Figure 5 ). This response could be explained by a strategy of the “sacrifice” signaling pathway set up by the cells to eliminate the most damaged cell population ( Davies, 2000 ). Indeed, a change in the cell population pattern was highlighted after 7 days at 500 μM H 2 O 2 ( Figure 6 ) and no protein damages were observed in these surviving cells ( Figure 7 ). Nevertheless, the increase of autofluorescence measured in surviving cells after 7 days at 500 μM H 2 O 2 ( Figure 6 ) might be correlated with changes in metabolic activity of mitochondria that cells undergo during apoptosis, as it has been previously observed in mammalian cells ( Levitt et al., 2006 ). In addition, the partial recovery of cell growth after the resilience period ( Figure 5 ) suggested a non-reversible impact of 500 μM H 2 O 2 injury on surviving cells, whose mechanisms should be deeply addressed in the future. Finally, comparing the in vitro and the in vivo approaches, we highlighted that the sensitivity of A. viridis gastrodermal cells at 500 μM of H 2 O 2 exposure is less pronounced in the gastrodermal tissue than in the isolated cultivated cells ( Figures 2 , 7 ). This is likely due to the contribution of the tissue organization thanks to host cell/cell communication, more efficient turnover of damaged cells and/or by a signaling pathway linked to the presence of the symbiont. Moving forward thanks to the in vitro cnidarian cell culture, an ambitious perspective of this study will be to disentangle the mechanisms of H 2 O 2 resistance of cnidarian cells and more specifically to assess the impact of other ROS (as the superoxide anion and the hydroxyl radical), thus contributing to decipher the adaptative tools that have evolved for a successful symbiosis stability and conversely to better understand the bleaching processes."
} | 5,000 |
24031461 | PMC3768596 | pmc | 7,433 | {
"abstract": "Influence of iron-depletion on twitching motility and quorum sensing (QS) system in P. aeruginosa was evaluated. The results demonstrated iron-depletion can retard biofilm formation and increase the twitching motility and expression of QS-related genes, suggesting a potential interaction between twitching motility and QS system in P. aeruginosa biofilm formation."
} | 92 |
34650235 | PMC8879416 | pmc | 7,434 | {
"abstract": "Synthetic biology enables microbial hosts to produce complex molecules that are otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by chemical reactions catalyzed by natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically by microorganisms. We report an engineered microbial cell expressing a heterologous biosynthetic pathway, which contains both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. To create this hybrid biosynthetic organism, we engineered Escherichia coli (E. coli) with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, together with natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.",
"conclusion": "Conclusions Production of an unnatural product using a microorganism engineered with a heterologous natural product biosynthetic pathway, an ArM, and a heterologous cofactor transport system demonstrates the feasibility of creating organic molecules by artificial biosynthesis comprising synthetic biology and synthetic chemistry. One can envision this combination of biosynthesis and artificial metalloenzymes being applied to engineered pathways that produce unusual core structures, with enzymes that catalyze functionalization of C-H bonds to install abiotic groups at positions where P450s might operate naturally, or with the artificial metalloenzyme operating in the middle of a pathway followed by diversification of the abiotic product with evolved downstream enzymes. One can even envision creating reagents, such as diazoesters, biosynthetically based on known pathways to these species 34 . Such goals could be achieved in E. coli or in a range of organisms that produce diverse classes of natural products, such as polyketides or various alkaloids, thus opening a realm of biosynthesis beyond that limited by the scope of natural reactions.",
"introduction": "Introduction Molecules produced by natural biosynthetic pathways are the inspiration or the actual matter in many products, ranging from medicines and agrochemicals to commodity chemicals and biofuels. Although many molecules with structures that are the envy of chemists are produced by these natural or engineered biosynthetic pathways, the structures remain limited by the available chemical reactions catalyzed by natural enzymes. If artificial enzymes could be incorporated into such biosynthetic pathways, then unnatural products could be produced from a combination of nature’s reactions and chemists’ imaginations. Recently, artificial metalloenzymes (ArMs) have been created that catalyze a series of unnatural reactions 2 – 13 . However, most of these reactions do not occur on the types of molecules that could be produced by biosynthesis. In contrast, an iridium-containing analog of a heme enzyme catalyzes the cyclopropanation of unconjugated, hindered, terminal alkenes that are present in natural products, such as terpenes 4 . This reactivity provides the potential to create an artificial biosynthetic pathway that combines natural pathways manipulated by synthetic biology with the catalysis created by ArMs. The assembly of artificial metalloenzymes has advanced from in vitro reconstitution of purified proteins with organometallic complexes to assembly of ArMs in the periplasm 13 , 14 or on the outer-membrane 15 , 16 of living cells. However, methods to assemble ArMs in more complex intracellular environments are lacking 17 , 18 . We hypothesized that artificial cytochrome P450s containing synthetic heme derivatives could be generated in the cytoplasm by employing the natural heme transport machinery 19 and the affinity of hemoproteins to heme derivatives. Thus, artificial cytochrome P450s are potential ArMs for conducting unnatural biotransformations in vivo in a fashion that enables the assembly of artificial biosynthetic pathways. Terpenes are the largest and most structurally diverse family of natural products 20 . Their core skeletons are assembled in one step and modified by a suite of tailoring enzymes including cytochrome P450s 21 . While native P450s typically catalyze oxidations of terpenes, extensively engineered P450s, such as P450 BM3 22 , and artificial P450s substituted with abiotic metalloporphyrins, could catalyze unnatural transformations 3 – 5 , 23 , 24 that would introduce an artificial dimension to the diversification of terpene structures. Here, we report the combination of limonene biosynthesis by a series of heterologously expressed natural enzymes and the catalysis by an artificial metalloenzyme Ir-CYP119 (CYP119 containing Ir(Me)MPIX as cofactor; CYP119, a P450 enzyme from Sulfolobus solfataricus; MPIX, mesoporphyrin IX) to produce a terpenoid that was previously inaccessible in a microbial host. This unnatural combination of synthetic biology and synthetic chemistry mimics the production of a terpene and subsequent conversion of this hydrocarbon to a terpenoid by P450 enzymes during natural biosynthesis. Our overall strategy for production of the unnatural terpenoid is depicted in Fig. 1 . The artificial metalloenzyme is assembled in E. coli with the aid of a cofactor transport system and overexpression of the apo-enzyme. In concert, the same E. coli is engineered with the pathway for production of a natural product that serves as the substrate for the artificial metalloenzyme. This combination of natural and unnatural reactions in the same cell would produce an unnatural derivative of a classic natural product.",
"discussion": "Results And Discussion Assembling the ArM in vivo The first major challenge facing this integration of an artificial metalloenzyme with a cellular biosynthetic process is to assemble such enzymes in the cytoplasm 25 . Previously, we reported the in vitro construction of Ir-CYP119 by incorporation of Ir(Me)MPIX into the heme binding site of the purified apo-form of CYP119. To construct this artificial enzyme in a cell, the cofactor must enter into the cytoplasm from the medium through the cell membranes. Metal-substituted heme derivatives have been reported to be transported across cell membranes when the outer-membrane receptor ChuA is overexpressed 26 – 28 . Thus, we first considered whether the co-expression of such a heme transporter and CYP119 would enable the artificial cofactor Ir(Me)MPIX to be transported into E. coli and incorporated into the apo-CYP119 mutant in the cytoplasm. To test the feasibility of assembling Ir-CYP119 and catalyzing a reaction in E. coli, we studied the cyclopropanation of the monoterpenoid (−)-carvone. We used the diastereoselectivity of the reaction as a parameter to distinguish any background reaction or reaction catalyzed by free cofactor from the reaction catalyzed by the assembled artificial metalloenzyme. During our previous study, Ir-CYP119 generated from in vitro reconstitution of Ir(Me)MPIX and purified apo-CYP119 mutants catalyzed the cyclopropanation of (−)-carvone with EDA (ethyl diazoacetate) to give an 8 : 1 : 1 : 1 ratio of diastereomeric products. The major diastereomer of this reaction is a minor diastereomer of the 1 : 1 : 3 : 3 ratio of diastereomers formed from the analogous reaction catalyzed by the free iridium porphyrin complex 4 . Thus, a reaction that forms a mixture of diastereomers resembling the ratio produced by the pure ArM in vitro would denote reaction by the assembled ArM in the cell. To this end, E. coli BL21(DE3) cells co-expressing the heme transporter ChuA and the CYP119 mutant were supplemented with 10 µM Ir(Me)MPIX, and the cell pellets containing in vivo -assembled Ir-CYP119 were directly employed for catalyzing the reaction of (−)-carvone with EDA after re-suspension in reaction buffer. However, the diastereomeric ratio (dr) of the whole-cell reaction was 1.7 : 1.0 : 2.7 : 3.3 ( Supplementary Fig. 1a ), indicating that the majority of the product of the whole-cell system was formed from a reaction catalyzed by the free iridium porphyrin complex, rather than by an assembled ArM. In addition, no product was detected when E. coli cultures were incubated in the absence of Ir(Me)MPIX ( Supplementary Fig. 1a ). To reduce interference from the background reactivity of the free iridium porphyrin, we evaluated the reaction of (−)-carvone with EDA catalyzed by cells grown in the presence of Ir(Me)MPIX in concentrations ranging from 10 µM to 0.02 µM. These experiments showed that the dr of the cyclopropane product was much higher (23 : 3.5 : 1.0 : 1.2) when the concentration of Ir(Me)MPIX was 0.02 µM ( Fig. 2a ). We surmised that the lower concentrations of Ir(Me)MPIX reduced the amount of product formed by a background reaction catalyzed by the cofactor unbound to CYP119. Control experiments showed that the dr was low for products formed in E. coli expressing ChuA without co-expression of CYP119 ( Supplementary Fig. 1b ). Under the conditions of this control experiment, the cyclopropanation would be catalyzed by the free iridium cofactor. Next, we sought to combine the Ir-CYP119 with a natural biosynthetic pathway to produce an unnatural terpenoid. The analysis of Ir(Me)MPIX uptake for cyclopropanation in whole cells described above was conducted with carvone because we had shown previously that cyclopropanation of this terpenoid occurred in high yield and high diastereoselectivity 4 . However, the biosynthesis of (−)-limonene, which has the same carbon skeleton as (−)-carvone, occurs with much higher titer 29 , 30 , so we sought to assess the potential to combine heterologous biosynthesis with artificial metalloenzymes by generating unnatural derivatives of limonene. Initial attempts to express a heterologous biosynthetic pathway to form limonene in concert with expression of CYP119 regulated by a T7 promoter caused the titer of limonene to decrease drastically from 188 mg/L without co-expression of CYP119 to 28 mg/L with co-expression ( Supplementary Fig. 2a ). To restore the production of limonene, we expressed CYP119 with a weaker promoter. However, when the reaction of exogenously added (−)-carvone with EDA was conducted with cells co-expressing ChuA and CYP119 with the weaker lac UV5 promoter, the cyclopropane was formed with low diastereoselectivity, again implying that assembly of the artificial metalloenzyme was inefficient ( Supplementary Fig. 3 ). Thus, we sought conditions that would assemble the artificial metalloenzyme more efficiently at low expression levels of CYP119 to generate product with high diastereoselectivity. To increase the efficiency of the cellular uptake of Ir(Me)MPIX, we replaced the ChuA transporter with a heme transport system encoded by the hug operon from Plesiomonas shigelloides, which has been used less frequently than ChuA, but has been shown previously to increase expression yields of hemoglobin to unusually high levels with heme supplementation 31 , 32 . Reactions of (−)-carvone with EDA catalyzed by E. coli cells expressing the HUG transport system, in concert with CYP119 (expressed from the lacUV5 promoter), formed the cyclopropane product with a high diastereoselectivity of 82% (the % diastereoselectivity in this case is the percentage of the major diastereomer in the 24.0 : 3.1 : 1.0 : 1.2 ratio of diastereomeric products) under conditions that are identical to those of the reactions with cells expressing the ChuA transporter ( Fig. 2b ). To elucidate the origin of the higher efficiency of the assembly of ArMs in cells expressing the HUG transport system, the distribution of iridium was analyzed by ICP-MS (inductively coupled plasma mass spectrometry) ( Fig. 2c ). In cells harboring CYP119 in the presence of 0.1 µM Ir(Me)MPIX, 65% of the iridium was located in the cytoplasm of the cells expressing the HUG transport system, while only 10% of the iridium was located in the cytoplasm of the cells expressing no transporter. Further, the cytoplasmic fraction isolated from the cells co-expressing the HUG transport system catalyzed the cyclopropanation of (−)-carvone with a diastereoselectivity of 80%, and the yield of cyclopropane was more than 13 times higher than that of the reaction catalyzed by the cytoplasmic fraction from cells expressing no transporter ( Supplementary Fig. 4 ). These results indicate that the HUG system significantly enhances the uptake of Ir(Me)MPIX into the cytoplasm. Cyclopropyl Limonene Biosynthesis Having achieved the diastereoselective cyclopropanation of (−)- carvone with EDA in cells expressing CYP119, even at a relatively low level, we sought to integrate such reactivity with the biosynthesis of limonene from a simple sugar ( Fig. 3a ). Again, we assessed whether the expression of the artificial metalloenzyme would affect the production of limonene. The production of limonene was unaffected by the expression level of CYP119 achieved with the pJHA135 plasmid ( Fig. 3b ). Moreover, the limonene titer was unaffected by the presence of 0.1 µM Ir(Me)MPIX ( Supplementary Fig. 2b ). Finally, both Ir(Me)MPIX and cyclopropyl limonene did not inhibit cell growth within the concentration range relevant for our study ( Supplementary Fig. 5 ). The cyclopropanation of limonene added exogenously to the whole-cell system catalyzed by E. coli expressing the hug operon and CYP119 in the M9-rich medium was then evaluated. These cells produced the cyclopropyl limonene with a diastereoselectivity of 52% (1.0 : 3.3 : 1.1 : 1.0 dr) ( Fig. 3c ). This ratio of diastereomers is distinct from that of the reaction catalyzed by free Ir(Me)MPIX (1.0 : 1.2 : 3.5 : 4.2 dr) and the diastereoselectivity was fortuitously higher than that of the reaction catalyzed by the same Ir-CYP119 mutant in vitro (1.0 : 2.4 : 2.3 : 2.9 dr) ( Fig. 3c ). We suggest that the higher diastereoselectivity in the whole-cell system results from a greater stability of the enzyme in vivo than in vitro . To fully combine terpene production and artificial catalysis within the same cell, gene expression for limonene biosynthesis and CYP119 was induced concurrently with addition of Ir(Me)MPIX ( Fig. 3d ). After overnight incubation, the synthesis of cyclopropyl limonene was initiated by addition of EDA. Under these conditions, 250 µg/L of cyclopropyl limonene was produced after 4.5 h with diastereoselectivity (43%, 1.0 : 3.2 : 1.8 : 1.5 dr) that was within 10% of that of the whole-cell reaction with exogenously added limonene. This similarity in diastereoselectivity indicates that the cyclopropyl limonene is formed from the combination of the heterologous biosynthesis of limonene and cyclopropanation catalyzed by the ArM. To assess the possibility that formation of the cyclopropane was catalyzed by the Ir-CYP119 released from dead cells, the cells were separated from the culture by centrifugation. The catalytic activity of the supernatant and of resuspended cells was evaluated by the reaction of (−)-carvone and EDA. The formation of cyclopropyl carvone with resuspended cells occurred with high diastereoselectivity and a yield more than 10 times that of the reaction conducted with the supernatant ( Supplementary Fig. 6 ). These data show that the product was produced primarily by intact cells. Increasing diastereoselectivity and titer To increase the diastereoselectivity and titer of the cyclopropyl limonene from the artificial pathway, we evolved the CYP119 mutant. We constructed a small library of CYP119 mutants containing additional changes to the amino acids around the binding site of the cofactor and tested the diastereoselectivity of these mutants for the cyclopropanation of limonene in whole cells ( Supplementary Fig. 7 ). After three rounds of screening, mutant P/R256W/V254A (P designates the parent) catalyzed the cyclopropanation of limonene with a higher diastereoselectivity of 72% (1.0 : 9.6 : 1.7 : 1.1 dr) and a higher yield ( Fig. 4a , b ). This evolved mutant was then introduced into the E. coli strain containing the limonene pathway and hug operon to perform artificial production of cyclopropyl limonene. With the procedures previously developed ( Fig. 3d ), the titer and diastereoselectivity of the products formed from this new pathway were 265 µg/L and 59% (1.0 : 7.0 : 2.3 : 1.5 dr) respectively ( Supplementary Fig. 8 ). The conversion efficiency of limonene by natural P450s in heterologous hosts is known to be low 30 , 33 , and the titer of cyclopropyl limonene is limited, in part, by the volatility of limonene. The titer of limonene is just 2 mg/L when the cells are grown without an organic overlay, but is 145 mg/L when the cells are grown with a dodecane overlay ( Supplementary Fig. 9 ). However, an overlay is used only if limonene is the final product; it would be incompatible with our system in which we must preserve limonene intracellularly as a substrate for the ArM. Thus, we sought to optimize the titer of cyclopropyl limonene by alternate methods. To do so, we investigated the potential toxicity of EDA to E. coli in the process of producing cyclopropyl limonene and investigated the effect of adding EDA portion-wise ( Supplementary Fig. 10 ). We found that addition of EDA at an initial concentration of 2 mM and refeeding EDA three times over 10 h led to a titer of cyclopropyl limonene that was almost 3 times its initial value (from 258 ug/L to 760 µg/L) with little change in diastereoselectivity (54%, 1.0 : 6.4 : 2.6 : 1.9 dr) ( Fig. 4c ). Additional increases in product titer would likely be gained by further optimization of the protocol for feeding EDA, investigation of additional enzyme mutants, and manipulating the expression levels of proteins in the pathway."
} | 4,546 |
34616952 | PMC8489177 | pmc | 7,435 | {
"abstract": "Abstract Ants show collective and individual behavioral flexibility in their response to immediate context, choosing for example between different foraging strategies. In Pachycondyla striata , workers can forage solitarily or recruit and guide nestmates to larger food sources through tandem running. Although considered more ancestral and less efficient than pheromone trail-laying, this strategy is common especially in species with small colony size. What is not known is how the decision to recruit or follow varies according to the immediate context. That is, how fine adjustments in information transfer affect immediate foraging decisions at the colony level. Here, we studied individually marked workers and evaluated their foraging decisions when food items varied in nature (protein versus carbohydrate), size, and distance from the nest at different temperatures and humidity levels. Our results show that tandem run leaders and potential followers adjust their behavior according to a combination of external factors. While 84.2% of trips were solitary, most ants (81%) performed at least 1 tandem run. However, tandem runs were more frequent for nearby resources and at higher relative humidity. Interestingly, when food items were located far away, tandem runs were more successful when heading to protein sources (75%) compared with carbohydrate sources (42%). Our results suggest that the social information transfer between leaders and followers conveys more information than previously thought, and also relies on their experience and motivation.",
"discussion": "Discussion In this study, we tested hypotheses about the factors influencing recruitment decisions in tandem running. This behavior has previously been studied only in very restrained laboratory conditions ( Richardson et al. 2007 ; Franklin 2014 ; Glaser and Grüter 2018 ; Richardson et al. 2021 ; but see Kaur et al. 2017 ), and in most instances in a nest emigration rather than in a foraging context ( Healey and Pratt 2008 ; Franklin 2014 ; O’Shea-Wheller et al. 2016 ; but see Kaur et al. 2017 ). Our results provide novel insights about the mechanisms associated with information transfer and decision-making of foragers, both leaders and potential followers ( Grüter and Czaczkes 2019 ). Most ants in our sample used tandem running ( Table 1 ), which shows that, although less frequent than solitary return trips, this strategy is regularly used for foraging in P. striata . The structure of the foraging bouts varied according to food type and quantity, as well as distance from the nest and external factors such as RH and temperature. For solitary foraging, we observed a larger number of trips to the protein food source near the nest (see Figure 1 ). Besides, more ants performed tandem runs in far distances toward protein food of very large size. Workers therefore seem to tune their behavior as a function of the encountered food type and size, similar to collective-foraging trail-laying species for which this allows regulating the colony-level nutrient intake ( Feldhaar 2014 ; Csata and Dussutour 2019 ). Similarly, another ant from the same genus, P. harpax , also seems to perform faster trips when foraging for protein, despite also consuming both protein and carbohydrate ( Grüter et al. 2018 ). In tandem runs at far distances, workers were probably able to identify not only the food type but also its size. This unexpected result suggests that ants can evaluate quantitatively the size of food items without direct comparison ( d’Ettorre et al. 2021 ) and increase the motivation to recruit fellow workers in specific conditions. This can be related to the possible intense competition at these resources over time ( Glaser et al. 2021 ). In the case of P. striata , a generalist species with a preference for insect prey, brood demand probably explains this higher motivation to exploit protein (and possibly lipid, from the tuna bait) sources. This difference is not present for solitary foraging at a far distance ( Figure 1 ). This suggests that travel distance may not be the important factor for the continued exploitation of a particular food source (near and far), but rather a differential satiation effect according to the food type ( Grüter and Czaczkes 2019 ) and also of a proportionally shorter time window for exploration since trips at far distances are longer. Optimal foraging and cognitive theory predict that more tandem runs should be observed if far sources were more at risk of being exploited by other species, and a fast exploration would allow limiting competition ( Glaser and Grüter 2018 ; Grüter et al. 2018 ). In our experiment, more tandem runs were observed near the nest ( Table 1 ). In this population, competition is more frequent at the food sources near the nest, which is often in a shaded and more protected area ( Silva et al. 2017 ). Grüter et al. (2018) suggested that more distant food resources are more at risk to be exploited by competing species. In our study site, the nests of P. striata coexist with nests of different species, Gnamptogenys striatula being the most common and most frequent competitor ( Lanhoso and Châline 2017 ; Silva et al. 2017 ). Our results suggest that competition may be intense even in the proximity of the nest. It must be noted, however, that as hypothesized by Glaser et al. (2021) for P. harpax , P. striata is often successful in excluding supposedly more dominant or aggressive species ( Lanhoso and Châline 2017 ) and shows a diversity of responses such as guarding the resource, robbing from other species, or tandem runs to exploit a source efficiently despite the presence of aggressive species such as Wasmannia auropunctata or Solenopsis saevissima ( Silva et al. 2017 ). We found that RH and temperature influenced probability and duration of solitary foraging bouts and tandem runs. Higher RH increased the likelihood of tandem runs in the far food source condition. Solitary trips were faster with higher RH and at near distance also when temperature was low; tandem runs were faster near the nest and at higher air temperature. We do not know how workers perceive external humidity and temperature. However, if potential recruits stay in chambers close to the nest exit, as other studies have shown ( Pinter-Wollman et al. 2013 ), workers could experience the conditions they will face if going out to forage. Since ants are ectotherms, temperature and humidity can directly interfere with their foraging ( Traniello 1989 ; Gordon 2013 ). Thus, foragers prevent excessive dehydration which would occur during the long trips when following or recruiting when the humidity is high ( Levings and Windsor 1984 ). In another ponerine ant, Dinoponera quadriceps , also from the Atlantic forest, humidity is positively correlated with foraging activity ( Medeiros et al. 2014 ). It is expected that ants are faster at higher temperatures, which occurred in near tandem runs but not other situations. The fact that solitary trips are faster with higher RH and low temperature is puzzling, but we can hypothesize that ants in these conditions stop less to assess potential risks associated with desiccation. Indeed, P. striata workers are slow foragers which often stop for long periods en route to the food source under leaves in the typical cluttered environment and die within minutes if exposed to high and dry temperature (N. Châline, personal observation). We observed that there was a decrease in duration in both solitary foraging and tandem runs on consecutive trips for the 2 distances ( Figure 3 ). This suggests that the route learning process allows ants to become familiar with their environment, making increasingly linear paths and foraging more efficiently ( Wystrach et al. 2011 ). Route learning allows to decrease exposure time as well as the probability for ants to be lost during foraging ( Azevedo et al. 2014 ). Since P. striata does not use chemical trails, its orientation and route learning probably rely on visual cues or path integration (reviewed by Wehner and Srinivasan 2003 ). Accordingly, in a study where the eyes of Temnothorax albipennis workers were experimentally covered with paint, the use of visual landmarks seemed important to assume the role of leader, while followers mostly rely on olfaction and path integration ( Franklin et al. 2011 ). Importantly, we conducted this study in natural conditions, where leaf litter and lower vegetation hamper foragers’ movement. Learning processes also improve trip efficiency in conditions more complex than those created in the laboratory and in ants living in desert environments ( Wystrach et al. 2011 ). Although consecutive tandem runs were faster, we did not find any improvement in tandem run success with time. That is, experience did not make workers better at leading followers. Despite the cluttered environment, tandem running success was high (80% excluding the far carbohydrate treatment) and stable over time and across treatments. Contrary to what is found in Temnothorax spp., where 3 quarters of tandem runs are unsuccessful ( Pratt 2005 ; Pratt et al. 2005 ), high success rates in ponerine ants such as P. harpax and Diacamma indicum ( Kaur et al. 2017 ; Grüter et al. 2018 ) suggest that, in natural environments, communication about food location and the subsequent route learning are very efficient. It also indicates that the motivation of leader/follower pairs is high, or that leaders and/or followers both have previous knowledge of the environment, which may help route learning and limit delays during the tandem ( Schultheiss et al. 2015 ; Stroeymeyt et al. 2017 ). In the far carbohydrate treatment, success was lower than 50% ( Table 1 ) and a lower number of tandem runs were registered ( Figure 4 ). This also suggests that followers can receive information about the nature of the food source (the only modified variable), and/or that the probability of tandem running recruitment and giving up probability en route to the food source in cases of break-ups by leaders and/or followers depends on this complex interacting information ( Schultheiss et al. 2015 ). We cannot exclude the hypothesis that part of our data can be explained by the existence of experienced leaders preferring certain food types and having higher success ( Richardson et al. 2021 ). However, a complementary hypothesis that needs to be tested would be that followers prefer following experienced leaders. Figure 4. Percentage of successful tandem recruitments separated by food type and distance. Success percentage was significantly lower for carbohydrates located far away (see Table 1 ). *GLM, z = −6.073, P < 0.001. As we already saw with the number of trips for solitary foraging in the near condition and the percentage of ant leading successful tandem runs in the far condition, workers seem less motivated to forage for carbohydrate sources. This probably happens because foraging costs become higher as ants move away from their nests, due to energy expenditure and exposure to predators and adverse environmental conditions ( Fewell 1988 ). Therefore, our results support the novel hypothesis that communication during tandem run initiation is more complex than previously envisaged. All through the tandem running, from recruitment to completion, information about the food source affects the motivation of both actors. These complex interactions can finely modulate colony-level foraging efforts, since potential tandem run followers are probably weighing out decisions according to repeated interaction with potential scouts or leaders. One clear missing element in our study, and in foraging behavior studies in general, is how the recruitment process occurs through interactions between the informed leader and the available potential follower. In nest emigration, such interactions may not be so important since choices of a new nest are limited so both leaders and followers maintain high motivation. Studies on ants relying on chemical trails, such as Pogonomyrmex , suggest that nest entrances are the theatre of complex interaction regulating colony foraging, mostly mediated by specific hydrocarbons present on the cuticle of forager scouts ( Gordon 2013 ; Pinter-Wollman et al. 2013 ). In ponerine ants, tactile interactions are common between nest mates ( Denis et al. 2008 ; Yagound et al. 2014 ; Kaur et al. 2017 ). Although we did not elucidate the mechanisms, our results suggest that differences in the decision, success, and duration of tandem runs are not stochastic events, but are probably influenced by how the follower perceives the recruiting motivation of the leader, as occurs in dancing honey bees ( Núñez and Giurfa 1996 ; Hrncir et al. 2011 ; George et al. 2020 ). In 3 occasions, we observed leaders being led in subsequent trials. This is an indication that followers are not necessarily always naive ( Schultheiss et al. 2015 ) but evaluate from public and private information treatment whether to exploit a discovered food source ( Grüter and Leadbeater 2014 ). Thus, tandem running can be influenced not only by spatial learning cognitive abilities, but also by the internal and motivational states of both leader and follower, depending on the evaluation of distance, food type, RH, and temperature. Perhaps in our study the motivation was changed by offering preferred and non-preferred food types. An example of follower’s decision-making was previously observed in D. indicum where the leader performs a stereotyped invitation call and the start of tandem run depends on follower’s acceptance ( Kaur et al. 2017 ). In a situation of lesser immediate risk than emergency nest emigration poses, followers could play an important role in the recruiting process, based on their prior experience and immediate evaluation of the present context. This is suggested by the fact that tactile and/or olfactory signals are indeed exchanged in early phases of recruitment, as both our results and research in other species suggest ( Crawford and Rissing 1983 ; Greene and Gordon 2003 ; Pinter-Wollman et al. 2013 ). Future studies can elucidate the influence of these stimuli in tandem running and other foraging strategies. Ponerine ants constitute a great monophyletic group to study such diversity, since they also occur in ample sympatry ( Schmidt and Shattuck 2014 ). An important inference in this study is that the use of tandem runs is not an all-or-nothing process. This is because we did not observe a unique ideal situation where recruitment or solitary foraging was prioritized. A possible explanation is that ants may recruit as long as there are available foragers or until a certain number are recruited. When foragers become rarer, the increased time windows required to find new recruits, as well as the crowding at food sources, would prevent a linear increase in the benefits associated with more foragers ( Grüter and Czaczkes 2019 ). Contrarily, the tandem runs in our study were not concentrated in the first trips after food discovery. This means that neither forager disponibility nor the number of foragers already recruited explain the combined use of solitary trips and tandem runs. Rather, we propose that decision-making is the result of how both leaders and the potential followers evaluate the current parameters according to their own experience (as foragers and of their social environment) together with more simple mechanisms (e.g., colony nutritional need) and environmental factors. We are left with the question of how P. striata and other individual characteristics of Ponerinae lead to interindividual differences in foraging behavior, and how the interaction between leaders and followers with different experience lead to the initiation and completion of the tandem run ( Jeanson and Weidenmüller 2014 ; Lihoreau et al. 2021 ; Richardson et al. 2021 ). In conclusion, our results suggest that foraging decision-making is also complex in species that do not use foraging trails and are considered ancestral regarding their social organization and division of labor ( Châline et al. 2015 ). Communication between leaders and followers seems to be modulated in many species by both internal, external, and social factors, as well as immediate and prior experience and knowledge. This flexibility begins to be described in species with larger colonies that rely on pheromone trails ( Czaczkes et al. 2019 ; Oberhauser et al. 2019 ). To determine the dynamics underlying this diversity and flexibility, it is crucial to produce comparative datasets using species with diverse social organization and foraging strategies, and occurring in different environments. Further research should explore possible complex information transfers between leaders and followers, and how these may be integrated with the followers’ physiology and experience to determine decisions in constant feedback loops ( Lihoreau et al. 2021 )."
} | 4,257 |
35727978 | PMC9245662 | pmc | 7,436 | {
"abstract": "Significance Natural selection enriches a population with the best-adapted phenotypes. But collective behaviors can also shape a population’s phenotype composition, even without selection. We study this in the context of collective migration of bacteria, in which the spatial arrangement of individuals by their chemotaxis abilities determines which individuals keep up with the migrating group. Since this spatial organization is environment-dependent, we find that a slow loss of low-performing phenotypes enables an isogenic population to nongenetically adapt its phenotype composition to migrate in changing environments. An important part of this adaptation strategy is the time scale on which individuals with new phenotypes are produced. Nongenetic inheritance provides a way to tune this time scale and may be widespread among microbes.",
"discussion": "Discussion Here, we investigated how growth in a nongenetically diverse population balances with loss of phenotypes during collective behaviors and how this balance impacts population performance. We used collective migration of chemotactic bacteria as a model system, where the demands of migration change in environments of different porosity. We realized that collective migration causes differential loss of phenotypes in an environment-dependent manner: Migration excludes whichever individuals perform poorly in the current environment. Our central finding is that, with growth and phenotypic inheritance, this differential loss dynamically enriches the population phenotype composition with high performers for the current environment, thus enhancing group migration speed in multiple environments. Growth of diverse phenotypes requires specifying how much daughters “remember” the phenotypes of their mothers, and we found that this nongenetic inheritance controls a general trade-off between larger composition shifts relative to the batch culture and slower responsiveness to new environments. In varying environments, this trade-off resulted in an optimal level of inheritance that maximized the average migration speed and enabled a diverse population to outrun a nondiverse one composed of the generalist phenotype. Differential loss of phenotypes during group migration resembles the effects of natural selection: Individuals that are “better adapted” to the current environment become enriched in the traveling group over time. However, these composition changes were not due to differences in growth or death, by assumption. Instead, differential loss of migrating individuals emerged from their spatial arrangement by chemotaxis performance. This distinguishes our results from past work on the role of nongenetic diversity in well-mixed, growing populations ( 41 – 60 ) and populations undergoing undirected range expansion ( 61 – 63 ). Spatial structure in past work has affected population growth or evolutionary success by governing which individuals access limited nutrients in dilute conditions ( 64 , 65 ), which populations occupy finite habitats ( 22 , 23 ), or the ability of populations to exchange metabolites ( 66 ). Our results point to another way that spatial structure can shape the composition of a population: by dictating which individuals participate in a collective behavior. Since this spatial structure is environment-dependent, it enables migrating populations to dynamically shape their compositions to migrate effectively in multiple environments. The dynamic nature of these nongenetic changes in phenotype composition also differentiate our results from past work by us ( 65 ) and others ( 47 , 53 , 54 , 57 , 67 ). In Frankel et al. ( 65 ), diversity enabled a population to avoid trade-offs among multiple tasks by having phenotypes in the population that performed well at each task. These tasks were performed independently by each individual cell, without collective behavior. While mutations allowed the population to evolve its standing distribution of phenotypes, this distribution was fixed for a given genotype during performance of a task. Our results here imply that, over cell divisions, diverse populations can do better: Growth and selection-like effects (either natural selection or the kind described here) dynamically shift the population’s neutral distribution of phenotypes toward those that are high performing at each task, without mutations, as the task is being performed. Likewise, approaches to studying the fitness effects of diversity that geometrically analyze Pareto fronts or optimize phenotype distributions without considering how the phenotype distribution dynamically responds to selection or selection-like effects will miss this ( 47 , 53 , 54 , 57 , 67 , 68 ). The problem is that the population can have a different phenotype composition in each environment, giving it more flexibility than these procedures allow. As a result, the population is not necessarily limited to the convex hull of nondiverse populations’ performance across environments. This, in turn, affects the conditions in which a diverse population performs better than a nondiverse, generalist population. Predicting changes in phenotype composition requires knowing how new phenotypes are produced. We modeled this as resulting from imperfect inheritance of swimming phenotypes at cell division ( 40 , 42 , 48 , 69 – 73 ), consistent with recent measurements ( 8 ). Stochastic switching is another known mechanism for producing new phenotypes ( 41 , 43 , 49 – 52 , 74 , 75 ), such as in the case of persisters ( 50 , 51 , 55 , 76 ). Since these mechanisms produce phenotypes independently of the cells’ environment, we expect that they are both subject to the trade-off that increasing the “susceptibility” of the population’s phenotype composition to selection-like pressures comes at the cost of slower responses to new pressures. This trade-off underlies past results showing that an optimal level of inheritance or an optimal switching frequency maximizes population growth in changing environments ( 41 , 42 , 56 ). Despite the very different context, we found that migrating groups of bacteria exhibit the same qualitative behavior. The existence of an optimum suggests that, on longer time scales, the population can evolve the level of nongenetic inheritance to match the time scales of environmental changes. Emergent adaptation of phenotype composition during collective behaviors could have important effects on a range of biological processes and could interact with natural selection. For example, the adaptation studied here could play a role during competition among multiple comigrating species of bacteria ( 22 , 23 , 28 ). More generally, group migration with spatial ordering of phenotypes is not limited to bacterial chemotaxis: It can arise whenever individuals with diverse motility phenotypes consume or deplete a signal in the environment that they then chase. Self-generated gradients drive collective migration of eukaryotic cells during development, wound healing, and cancer metastasis ( 77 – 82 ), in which a leader–follower structure emerges ( 83 – 85 ), and, thus, adaptation mediated by spatial organization of motility phenotypes could affect these processes. A similar kind of nongenetic adaptation could potentially occur during biofilm formation, in which the collective behavior confers a selective advantage to phenotypes that produce costly biofilm-associated materials ( 86 ). Collective behaviors may generally adapt the phenotype compositions of diverse, growing populations to perform a variety of collective tasks."
} | 1,895 |
23239051 | null | s2 | 7,439 | {
"abstract": "No abstract available"
} | 5 |
33951861 | null | s2 | 7,440 | {
"abstract": "Acid mine drainage-affected environments are interesting microbial niches for the isolation of metal-resistant microorganisms. In this sense, the aim of the present work is to isolate and characterize metal-resistant microorganisms from sediments of an abandoned gold mine located in San Luis (Argentina). For these purposes, the metal removal capacity and the microelemental composition of the biomass exposed to metals were evaluated. Likewise, proteomic techniques were applied to understand the removal and resistance mechanisms. Fusarium tricinctum M6 was isolated and identified as tolerant to Cu(II), Fe(II) and Cr(VI). When faced with 40 µg mL"
} | 162 |
39825415 | PMC11748598 | pmc | 7,441 | {
"abstract": "Biochips are widely applied to manipulate the geometrical morphology of stem cells in recent years. Patterned antenna-like pseudopodia are also probed to explore the influence of pseudopodia formation on gene delivery and expression on biochips. However, how the antenna-like pseudopodia affect gene transfection is unsettled and the underlying trafficking mechanism of exogenous genes in engineered single cells is not announced. Therefore, the engineered microarray biochips were conceptualized and prepared by the synthesized photointelligent biopolymer to precisely manage geometric topological structures (cell size and antenna-like protrusion) of stem cells on biochips. The cytoskeleton could be regulated in engineered cells and large cells with more antennas assembled well-organized actin filaments to affect cell tension distribution. The stiffness and adhesion force were measured by atomic force microscope to reveal cell nanomechanics on microarray biochips. Cytoskeleton-mediated nanomechanics could be adjusted by actin filaments. Gene transfection efficiency was enhanced with increasing cell nanomechanics, which was also confirmed by the evaluation of cell internalization capacity of nanoparticles and DNA synthesis ability. This work will provide a new strategy to study functional biomaterials, microarray chips and internal mechanism of gene transfection in patterned stem cells on biochips. Graphical abstract \n Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03101-x.",
"conclusion": "Conclusion The engineered microarray biochips were successfully designed and created on fibronectin-coated CCP substrates to manipulate cell size and antenna-like protrusions of the engineered hMSCs. The various cell morphologies could induce different cytoskeleton structures and distribution on these biochips. AFM was applied to measure cell nanomechanics by cantilever nanoindentation. Cell stiffness and adhesion force were regulated in the engineered cells. Young’s modulus was enhanced in cells with well-organized actin filaments, while cell adhesion force was reduced in these cells. The cytoskeleton-mediated nanomechanics could also affect gene delivery and transfection in the patterned hMSCs, and antenna-like protrusions could improve gene transfection in larger cells (60 μm in diameter), independently of small cells (30 μm in diameter). The transfection results showed the similar results with cell nanomechanics and were also regulated by cell internalization and nuclear DNA synthesis ability in mechanotransduction.",
"introduction": "Introduction The biochip generally refers to biological information molecules on biofunctional microarrays, such as gene or DNA fragments, polypeptides, proteins, sugars and tissues [ 1 – 3 ]. Based on the interaction of specific biomacromolecules, the biochip can integrate the bioelectronic information for biochemical analysis to achieve high-throughput rapid detection and diagnosis of RNAs, DNAs, proteins and various biological components [ 4 – 7 ]. Meanwhile, the biochips are also applied to monitor cell morphogenesis and functions [ 8 , 9 ]. Microarray chip, microfluidic chip and liquid biochip are considered as the new biochip technologies for biomolecular recognition and cell behavior evaluation [ 10 – 13 ]. An angiogenesis microfluidic chip is designed as a human blood–brain barrier (BBB)-like microvasculature to regulate the reparative cell therapeutics for ischemic stroke in vitro [ 14 ]. Human bone marrow mesenchymal stem cells (hBM-MSCs) can serve as perivascular cells in tight BBB reconstruction, and have better vasoconstriction ability than human pericytes (hPCs), indicating that engineered stem cells can repair BBB rather than paracrine on a chip. Tumor metastasis in circulating tumor cells (CTCs) is also diagnosed to realize simultaneous on-chip isolation for subtype verification [ 15 ]. Reprogramming and pluripotency of somatic cells are also explored in a predictable manner on the superhydrophobic microwell array chip (SMAR-chip) and the results show that microarray-controlled biophysical stimuli can enhance the pluripotent reprogramming on SMAR-chips [ 16 ]. Biofunctional microarray chip is regarded as the crucial tactic in vitro to simulate extracellular microenvironment and probe cell functions [ 17 – 19 ]. Pseudopodium, a kind of cell protruding structure related with cytoskeleton formation, can determine some important physiological functions, such as cell adhesion and spreading, cell tension sensing and nanomechanics, extracellular matrix degradation and cell internalization of exogenous particles [ 20 – 22 ]. Macrophages in vitro can gradually form dense pseudopodocyte clustering superstructures to play a crucial role in matrix degradation [ 23 ]. The microtubule skeleton can penetrate the actin core of pseudopodocyte at the myosin ring level to support the cluster superstructure of pseudopodocyte. Pseudopodocytes are disintegrated by the treatment of microtubule depolymerization drug and associated with the activation of Rho/ROCK/Myosin signaling pathway [ 24 ]. The formation of podocyte is the key factor for osteoclast adhesion and bone absorption. The assembly of osteoclast foot is a multimolecular co-regulation process. Src kinase and Rho GTP enzyme pathways are involved in the regulation of osteoclast foot assembly and ring formation [ 25 ]. As a subfamily of non-receptor tyrosine kinases, Src kinase family participates in the assembly and disassembly of podiums, and affects the assembly of podiums by modifying key protein with phosphorylated tyrosine residues. Rho GTP enzyme family is a component of small GTP binding proteins, and it is a key molecular switch to mediate intracellular signal transduction and cytoskeleton remodeling in many cell types. Further, filamentous pseudopodia can also rotate and coil by actively generating distortions in its actin axis to adjust cell functions [ 26 ]. Therefore, pseudopoda is a very important module to understand the underlying mechanism of cell migration and internalization on biochips. Gene delivery and expression are regarded as a great potential technique for genotype-related disease treatment and cancer progression [ 27 – 30 ]. Cells can perceive force-mediated stimuli from microenvironment or extracellular matrixes (ECMs) to transfer the mechanical signals into cells by adhesion proteins (fibronectin, integrin, RGD, vinculin and focal adhesions) and cytoskeleton structures (actin, myosin and actinin) to affect cell internalization and gene transfection [ 31 – 35 ]. However, the influence of cell size and pseudopoda on gene delivery and expression is vague and the underlying mechanism of gene delivery and trafficking also needs to be explained in stem cells. In this work, we used a functional microarray biochip to command the geometrical morphology of human mesenchymal stem cells (hMSCs), including cell size and antenna-like protrusions. The patterned biochips were designed and fabricated by the synthesized photoreactive biopolymer. The biochips were further functionalized by adhesion proteins and applied to pattern the hMSCs on cell culture plates. The cytoskeleton structures (actin filaments) were investigated to explore the cytoskeleton-induced tension and nanomechanics by atomic force microscope nanoindentation. Gene delivery and expression were associated with cytoskeletal nanomechanics by the regulation of the internalization of exogenous nanoparticles and nuclear DNA synthesis on microarray biochips. The results will be helpful for the design of functional array biochips and the understanding of gene trafficking mechanism in stem cells with pseudopodia.",
"discussion": "Results and discussion Conceptualization and performance of engineered microarray biochips The biofunctional photoreactive PVA was chemically synthesized according to previous report [ 30 ]. The photoreactive azidophenyl group in 4-azidobenzoic acid was grafted into PVA chains by the polymerization of carboxyl radical in 4-azidobenzoic acid and hydroxyl radical in PVA (Fig. S1A). The synthesized reaction of biofunctional PrPVA was confirmed by the characterization of UV–Vis analysis and 1 H-NMR evaluation. The absorbance peak at 275 nm of AzPhPVA showed that the benzenoid group was successfully introduced in PrPVA (Fig. S1B). In addition, we also measured PVA, azidobenzoic acid, and the mixture of PVA and azidobenzoic acid by UV–Vis analysis. Absorbance peak of benzenoid group appeared at about 270 nm in non-treated azidobenzoic acid. Configuration alternation in azidobenzoic acid caused the bathochromic shift in benzenoid absorbance peak of PrPVA. Moreover, 1 H-NMR spectrum was applied to further analyze the percentage of azidophenyl group in PVA chains (Fig. S1C). The typical peaks at 7 and 8 ppm were the proton peaks of benzenoid structures. The peaks of methylene and methylidyne protons were observed at about 1.4 and 3.8 ppm in synthesized PrPVA chemicals, respectively. The grafting degree was measured to be 3.7% through the integration of each peak in 1 H-NMR spectrum of PrPVA. The chemical results indicated that the PrPVA biomolecules were synthesized by the introduction of azidophenyl group of 4-azidobenzoic acid in PVA. Biofunctionalized patterned platform was considered as microarray biochip to supervise delivery and expression of pDNA nanolipoplexes in stem cells via mechanotransduction (Fig. 1 ). Cell protrusion could regulate cell adhesion and heterogeneity on biochips to control cytoskeletal nanomechanics of patterned hMSCs. The various cell force and tension determined gene delivery and expression of nanolipoplexes for gene therapy. Photoresist PVA is regarded as an anti-protein-adhesion biopolymer during cell culture [ 36 ]. To construct the engineered microarray biochips, bioreactive PVA solution was synthesized and further applied to contrive the biofunctional chip system for cell culture (Fig. 2 A). The PVA solution was dropped on CCP substrates and dried at room temperature in the dark to develop a nanoscale PVA-coated sheet. A conceptualized mask with different microcircle arrays was firmly enshrouded onto the PVA sheets and irradiated under UV light for photoreactive PVA crosslinking, while the PVA regions under dark microcircles were not crosslinked. After UV exposure, the designed microcircles were established in CCP substrates. Then, the CCP substrates were washed in water to remove the uncrosslinking PVA regions to expose the patterned cell culture surface for cell adhesion in sectional view (Fig. 2 B). The mask was designed to possess two-type microcircles of 30 μm and 60 μm in diameter (Fig. 2 C). The microcircles included 0, 1, 2, 3, 4 and 6 antenna-like protrusions, respectively. Fig. 1 Illustration of biofunctionalized patterned platform as microarray biochip to supervise delivery and expression of pDNA nanolipoplexes in stem cells via mechanotransduction. Cell protrusion could regulate cell adhesion and heterogeneity on biochips to control cytoskeleton-mediated nanomechanics of the patterned hMSCs. The various cell force and tension determined gene delivery and expression of nanolipoplexes for gene therapy on biochips Fig. 2 Fabrication and characterization of the engineered microarray biochips. A Schematic flowchart of the engineered microarray biochips by intelligentized PrPVA on CCP plates. B Sectional view of the microarray biochips. C Representative images of the conceptualized masks. The diameters were designed as 30 and 60 μm, and the number of antenna-like protrusions was defined to be 0, 1, 2, 4 and 6, thus defined as P0, P1, P2, P3, P4 and P6. Scale bar: 200 μm. D Representative images of 3D view of the engineered microarrays. Scale bar: 200 μm. E Diameter of the engineered microarrays. (F) Length of antenna-like protrusions in the engineered microarrays. G Width of antenna-like protrusions in the engineered microarrays. The data present the mean and SD, n = 5, n.s. presents no significance, *** p < 0.001 The prepared microcircles were analyzed by AFM scanning to confirm the integrity of microcircles (Fig. S2). The AFM results showed that engineered circle microarrays were successfully built on CCP substrates. 3D view of microcircles was further created to observe the excellent maneuverability of microcircles on biochips (Fig. 2 D). The diameter of microcircles was measured and close to the designed sizes of 30 μm and 60 μm (Fig. 2 E). In addition, the length (Fig. 2 F) and width (Fig. 2 G) of protrusions were also calculated to investigate the formation of protrusions. The length of protrusions in 30-μm-in-diameter microcircles was measured to be 15.7 ± 1.1, 15.3 ± 1.4, 14.9 ± 0.9, 15.9 ± 1.9 and 14.9 ± 1.7 μm, and the width was measured to be 2.5 ± 0.1, 2.5 ± 0.2, 2.6 ± 0.3, 2.4 ± 0.2 and 2.5 ± 0.1 μm for P1, P2, P3, P4 and P6, respectively. For 60-μm-in-diameter microcircles, the length of protrusions was measured to be 14.9 ± 1.4, 15.7 ± 1.8, 15.5 ± 0.9, 15.6 ± 1.6 and 15.7 ± 0.8 μm, and the width was measured to be 2.5 ± 0.2, 2.6 ± 0.3, 2.6 ± 0.2, 2.5 ± 0.3 and 2.6 ± 0.3 μm for P1, P2, P3, P4 and P6, respectively. There was little significant difference each other. The length was about 15 μm and the width was 2.5 μm, respectively, similar to designed ones. Therefore, the AFM scanning results revealed that the microcircle arrays were triumphantly equipped on PVA-coated CCP biochips. Fibronectin decoration and cell microarray pattern on biochips The engineered microarray biochips were decorated by fibronectin to improve cell adhesion ability and the hMSCs were seeded on CCP substrates to form the uniform cell array chips (Fig. 3 A). The immunofluorescent images were observed to substantiate the prosperous modification of fibronectin (Fig. 3 B). Specially, antenna-like protrusions were also packed in slender microgrooves. After fibronectin coating, hMSC suspension was dropped to the microcircle arrays for cell culture to construct different cell geometry on biochips. Cell morphology was captured by the optical microscope to observe cell adhesion and spreading on microarrays (Fig. 3 C). The cell culture results showed that the hMSCs could accommodate to microcircle sizes (30 μm and 60 μm in diameter) and spread their area to cram into each available space of microarrays on biochips, including cell diameter (Fig. 3 D) and cell spreading area (Fig. 3 E). Interestingly, the cells also assembled the antenna-like protrusions along with designed ones on microarrays and the length of cell protrusion was same results with designed size (Fig. 3 F). The cell results demonstrated that cell microarray pattern was well manipulated by fibronectin decoration on microarray biochips. Fig. 3 Fibronectin (Fn) modification and cell engineering on the engineered microarray biochips. A Illustration of Fn coating and cell patterning. B Representative fluorescence images of Fn staining on the microarray biochips. Scale bar: 200 μm. C Representative images of cell morphology. Scale bar: 50 μm. D Diameter of the engineered cells on biochips. E Spreading area of the engineered cells on biochips. F Length of antenna-like protrusions in the engineered cells. The data present the mean and SD, n = 50, n.s. presents no significance, *** p < 0.001 Strong compliance of protrudent focal adhesion on biochips Cell adhesion and protrusion formation is related with the development of focal adhesion (FA) between typical adhesive proteins integrin and extracellular matrix (ECM) to regulate cell functions. The patterned cells were seeded on microarray biochips to induce mature integrin and focal adhesion for cell attachment, further promoted integrin activation and interaction with vinculin, talin and paxillin (Fig. 4 A). Therefore, Integrin was measured by WB analysis (Fig. 4 B). The WB results showed that integrin expression level was enhanced with increasing the number of cell protrusion in 60-µm cells, while there was little difference in 30-µm cells even though small cells had 6 protrusions (Fig. 4 C). Fig. 4 Protrudent compliance of focal adhesion on the microarray biochips. A Illustration of focal adhesion (FA), integrin and ECM to regulate integrin activation and interaction with vinculin, talin and paxillin. B WB analysis of integrin. C Quantitative integrin expression level by WB analysis (n = 3). The data present mean and SD, n.s. presents no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. D Heatmap images of vinculin Vinculin, key component of FA, was studied to investigate the formation and compliance of FA on microarray biochips. The patterned cells could weave the FA fibers along with the protrusion and induce mature FA formation on microarray biochips (Fig. S3A). The islands of FA serve as the decisive role in the tension and stability of cell adhesion, and positive feedback pathway is found between the mechanical signals and FA islands [ 27 ]. The mechanical force may regulate the aggregation of adhesive molecules, FA island enlargement and mechanical signal stimulation to control cell activity [ 37 , 38 ]. Further, the total area of FA and average size of FA were also analyzed to reveal the relationship of cell protrusion and FA formation. Total area of FA was increased with protrusion increasing in large cells, regardless of small cell size and protrusion (Fig. S3B). Average vinculin size in each cell was analyzed to explore the maturity of FA on microarray biochips (Fig. S3C). The average FA size increased from about 2 µm 2 to 4 µm 2 with increasing the protrusion number in 60-µm cells. Nevertheless, average FA size wasn’t affected by protrusion number in 30-µm cells, similar results with total FA area. In addition, the heatmap of vinculin showed that mature vinculin was accumulated in the periphery of cells, especially in cell protrusion regions (Fig. 4 D). The results showed that the formation and maturity of FA presented the distinguishing diversification on different biochips. The FA maturation may adjust the contractive property of cytoskeletal distribution and regulate the cell adhesion ability between talin and integrin to affect the cell mechanics of patterned cells on biochips. Cytoskeleton-induced cell nanomechanics on microarray biochips Extracellular stimuli from microenvironments can induce cells to form more focal adhesions to mechanosense biophysical cues and transfer these signals into cells and nuclei for mechanical regulation [ 39 , 40 ]. Cytoskeleton structures are the most important composition in cytoplasm to perceive the stimulation on the cell membrane and respond to it based on cell identification and diagnosis [ 41 ]. The cytoskeletons, actin filaments, were stained green to investigate the regulation of cell microarray chips on cytoskeleton structures and assembly (Fig. 5 A). The actin results indicated that all cells could develop cytoskeleton structures, including cortex actin and actin filaments. Nevertheless, when the size of cells was small (30 μm in diameter), the cells just assembled cortex actin at the periphery of microcircles. Even though the cells presented more antenna-like protrusions, the cells still developed the incomplete actin structures, similar results with 30-μm microcircle arrays. Notably, when cell size was increased to 60 μm and even 80 μm in diameter (Fig. S4), actin filaments showed totally different results with 30-μm cells. Interconnected actin filaments were assembled along with microcircle periphery. Especially, the cells would gather their actin filaments in protrusion regions with increasing antenna number. Further, the thickness of actin fibers in microcircle periphery was measured to reveal the underlying mechanism of cell protrusion on actin fiber formation (Fig. S5A). The thickness was enhanced with increasing cell protrusion number in 60-μm cells, independently of protrusion number in 30-μm cells (Fig. S5B). These differences in cytoskeleton assembly could transform the chip information of microcircle arrays into cells to administer cell mechanical behaviors. Fig. 5 Cytoskeleton-mediated cell nanomechanics on the micropatterned biochips. A Representative fluorescence images of actin filaments (green) and nuclei (blue) staining. Scale bar: 50 μm. B Schematic diagram of cytoskeletal structures to regulate cell nanomechanics by AFM cantilever nanoindentation. Extracellular biophysical cues are transferred into cells or nuclei by mechanical biosensors (focal adhesion, integrin and RGD) to administer actin organization. The reorganized actin filaments will manage cell nanomechanics by the contractile movement. C AFM-nanoindentation force/distance curves of 30 μm and 60 μm. D Cell stiffness (Young’s modulus) of engineered hMSCs (n = 20). E Cell adhesion force of engineered hMSCs. The data present mean and SD, n.s. presents no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 Based on above actin results, the engineered hMSCs adhered on fibronectin-coated microarray biochips to sense extracellular force by the regulation of cytoskeleton structures, focal adhesion, arginine-glycine-aspartate (RGD), and integrin (Fig. 5 B). To bear out the hypothesis of cytoskeleton structures and mechanical behaviors, AFM nanoindentation was applied to analyze cell nanomechanics. Representative force-distance curves of 30-μm and 60-μm cells in diameter were obtained by AFM cantilever measurement (Fig. 5 C). Young’s modulus was calculated to evaluate cell stiffness (Fig. 5 D). The Young’s modulus was about 1.0 kPa in 30-μm cells and there were little significant difference, even increasing the number of antenna-like protrusions. Interestingly, 60-μm cells presented higher Young’s modulus than 30-μm cells due to the well-organized cytoskeleton structures. When the number of protrusions was increased on microarray chips, Young’s modulus had a significantly upward trend and the cells with 6 protrusions showed the highest modulus (1.7 kPa). The stiffness of the pseudopodia of thin-body was also analyzed to reveal the spatial distribution of cell stiffness in a topology-dependent manner. The results showed that the stiffness of pseudopodia in the cells with a diameter of 60 μm was stronger than that of the cells with a diameter of 30 μm, independently of the number of pseudopodia (Fig. S5C). Nevertheless, after cytochalasin D treatment, cell stiffness was further measured and the results showed that all type cells presented similar Young’s modulus (less than 1.0 kPa) due to actin deploymerisation (Fig. S5D). On the other hand, cell adhesion force usually decreased with increasing cell stiffness (Fig. 5 E). Adhesion force was the important element to evaluate cell nanomechanics. When cell size was defined to be 30 μm, adhesion force was about 440 pN, independently of protrusion number. Nevertheless, adhesion force was reduced to less than 400 pN when cell size was 60 μm in diameter. With the increasing of protrusion number, the adhesion force was further decreased to 330 pN for the 60-μm cells with 6 protrusions. It may be due to the increasing spreading area to form network cytoskeleton for more antenna-like protrusions in 60-μm cells. The non-specific adhesion of bioengineered hMSCs was adjusted by cytoskeleton distribution and cell stiffness. Therefore, the cytoskeleton and AFM results showed that the engineered hMSCs on microcircle arrays could adjust their nanomechanics to affect cell functions. Regulation of engineered microarray biochips on gene delivery and transfection by nanomechanics Extracellular cues, such as biological, chemical and physical stimulations, are applied to regulate the delivery and transfection of exogenous genes in engineered or patterned cells [ 42 – 44 ]. To administer the compliance of cell nanomechanics and gene transfection on the engineered microarray biochips, the gene delivery and transfection were investigated by pDNA and cationic lipids in the engineered hMSCs. The pDNA and cationic lipids were measured by dynamic light scattering (DLS) to disclose the uniform nanostructures of about 90–100 nm in diameter (Fig. 6 A). After transfection, the transfected cells were captured to express GFP proteins by a fluorescence microscope (Fig. 6 B). Green GFP proteins were lighted in the engineered cells to disclose gene delivery ability. Meanwhile, the nuclei and actin were stained blue and red to determine cell contour. Further, the efficiency of gene transfection was also supervised to reveal the influence of engineered microarray biochips on gene delivery and expression by cytoskeleton-induced nanomechanics (Fig. 6 C). Gene expression was enhanced with increasing the number of antenna-like protrusions in 60-μm cells, while transfection was not affected in 30-μm cells and independent of protrusion number. The transfection results showed the similar tendency with cytoskeleton structures and cell nanomechanics. The well-organized actin filaments and large cell nanomechanics could provide stronger phagocytosis stress for gene delivery and expression in the engineered hMSCs (Fig. 6 D). Fig. 6 Regulation of cytoskeleton structures on gene delivery and expression (transfection) on the engineered microarray biochips. A Diameter and PI of Lipo2000 and Lipo2000-pDNA nanoparticles by dynamic light scattering (DLS). B Fluorescent images of successfully transfected hMSCs to express green GFP proteins on the engineered microarray biochips. Actin: red; nuclei: blue. Scale bar: 50 μm. C Gene transfection efficiency by calculating the number of green cells (GFP). The data present mean and SD, n = 5, n.s. presents no significance, * p < 0.05. D Illustration of gene delivery and expression in the engineered hMSCs. The hMSCs would sense extracellular tension from microenvironment to regulate the distribution of actin cytoskeleton. Gene delivery was controlled by the bioengineered cytoskeleton structures, which was related with clathrin-mediated endocytosis, and further mitochondria could provide necessary energy (ATP) for the internalization of exogenous genes to promote transcription and translation The hMSCs could sense extracellular tension from microenvironment to regulate the distribution of actin cytoskeleton. Gene delivery was driven by force-sensing cytoskeleton structures and mitochondria could provide ATP energy for the internalization of exogenous genes. After cationic uptake and lysosomal escape, the DNA was transferred into nuclei for transcription. The mRNA was carried out from nuclei to cytoplasm and ribosome for GFP translation. To monitor the delivery level of exogenous genes in the engineered hMSCs, the delivery ability of pDNA/L2000 lipoplexes was studied by pDNA labeling (Fig. S6A). The pDNA (YOYO-1) was marked green by fluorescence staining. The nucleus was marked blue. The location of pDNA was captured to evaluate the delivery level in the engineered cells. When the nanolipoplexes were added for 5-min transfection, pDNA and endosome/lysosome were observed in cytoplasm of stem cells, rather than in nuclear regions. After the cells were transfected for 15 and 30 min, pDNA would gradually appear in nuclear regions. At 1 or 2 h, more pDNA content could locate and accumulate in nuclear regions of engineered cells. After 4 and 6 h transfection, pDNA would almost localize in nuclear regions. In this process, more pDNA content would localize in nuclear regions in larger spreading cells. The pDNA fluorescent intensity was decreased with increasing time and pDNA content in 60-μm cells was almost twice than that in 30 μm cells (Fig. S6B). The gene delivery results indicated that the engineered cells with large spreading area showed high delivery level of pDNA and strong lysosomal escape ability under clathrin-mediated endocytosis on microarray biochips. Gene transfection is enhanced in larger spreading and adhesion area cells, and confirmed by previous reports [ 45 ]. Furthermore, the patterned hMSCs having large diameter and elongation can improve internalization level of SiO 2 and Au NPs [ 41 ]. Fibroblasts are cultured on micropitted substrates (under 10-μm diameter) to reveal the relationship between engineered cell morphology and transfection mechanism [ 46 ]. Nanopatterns are also applied to adjust cell morphology for gene delivery and expression [ 47 ]. For instance, nanogrooves are prepared to regulate gene delivery and transfection in fibroblasts [ 48 ]. The size and depth of nanogrooves can administer gene expression ability in the engineered cells. Nanopillars are modified by adhesive proteins to guide gene delivery and transfection based on hollow nanotubes for high gene expression level [ 49 ]. These studies have demonstrated that gene delivery and expression would be affected by engineered cell morphology through cytoskeleton-induced nanomechanics. Cell uptake and internalization in the engineered hMSCs on biochips To verify gene uptake ability in the engineered hMSCs, transmembrane delivery capacity of exogenous NPs was applied to reveal the influence of the engineered hMSCs on cellular internalization ability. As the first process of gene transfection, cell phagocytosis plays an important role in gene delivery, especially for efficient and selective transportation of semipermeable cell membrane [ 50 ]. Therefore, the cell internalization ability of exogenous NPs was evaluated by fluorescence-marked SiO 2 NPs on the microarray biochips. When spreading area of the engineered cells was small (30 μm in diameter), the fluorescent images of SiO 2 NPs showed that all-type cells could achieve the internalization of NPs, while the amount of fluorescent NPs didn’t seemingly have obvious significance (Fig. 7 A). The fluorescence intensity of NPs was also analyzed to further explore cell uptake and internalization ability, and the results indicated that all cells presented the similar fluorescence intensity, independently of the number of antenna-like protrusions (Fig. 7 B). On the other hand, 60-μm cells in diameter were also investigated by the fluorescence uptake of NPs and showed the increasing fluorescence amount with protrusion number (Fig. 7 C). The quantitative result indicated that fluorescence intensity was enhanced with increasing the number of antenna-like protrusions (Fig. 7 D). The internalization data were closely consistent with the transfection results. Fig. 7 Influence of cytoskeleton-mediated nanomechanics on cell uptake and internalization on the microarray biochips. A Fluorescent images of cell uptake of SiO 2 NPs (green) in 30-μm cells. Actin: red; nuclei: blue. Scale bar: 50 μm. B Fluorescence intensity of NPs in 30-μm cells. C Fluorescent images of cell uptake of SiO 2 NPs (green) in 60-μm cells. Actin: red; nuclei: blue. Scale bar: 50 μm. D Fluorescence intensity of NPs in 60-μm cells. The data present mean and SD, n = 5, n.s. presents no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 Evaluation of nuclear DNA activity and YAP mechanotransduction by cell nanomechanics Cytoskeleton-mediated nanomechanics and mechanotransduction on the engineered biochips can regulate cell functions and fate, including morphogenesis, kinematic dynamics, and cell development [ 39 , 51 , 52 ]. Based on the important interplay of nanomechanics and mechanotransduction on transfection, DNA activity and division ability of the engineered hMSCs could improve gene transfection efficiency, especially for transcription and translation. Herein, nuclear DNA activity was investigated by BrdU staining to disclose the regulation of cell nanomechanics on cell mitogenesis (Fig. 8 A and Fig. S7). Further, the DNA synthesis activity was also inspected to probe the influence of cytoskeleton-mediated mechanics on nuclear proliferation (Fig. 8 B). The DNA synthesis in large cells (60 μm) was promoted with increasing protrusion number, while the small cells (30 μm) were not affected by antenna-like protrusions. DNA synthesis could reflect nuclear activity of the microarrayed cells, which was beneficial for gene transfection. The DNA results were used to indicate the close relationship between microarray-induced DNA synthesis and nuclear activity to improve gene transfection ability. Further, YAP nuclear translocation was investigated by WB analysis (Fig. 8 C). The percentage of YAP nuclear translocation was enhanced with increasing the number of antenna-like protrusions in 60-μm-in-diameter cells, independently on small cell size (30 μm in diameter) (Fig. 8 D). In addition, the immunostaining results of YAP also presented an enhanced tendency of YAP nuclear translocation with increasing cell size (Fig. S8A). The difference of YAP expression may originate from the force-sensing mechanotransduction of nuclear skeleton structures. LaminA/C, a nuclear skeleton structure, can affect cell functions, including nuclear structure stability, cell mobility, mechanical sensing, cell differentiation, DNA damage repair and telomere protection. Furthermore, nuclear skeletal structures (LaminA/C) were also explored by WB analysis and showed the similar results with YAP expression (Fig. S8B). These results displayed that the hMSCs were cultured on microarray biochips and sensed extracellular tension from microenvironment to assemble ECMs by integrin, RGD and vinculin (Fig. 8 E). These force-sensing proteins on cell membrane could regulate cytoskeleton formation (such as talin, paxillin, myosin and actin) under RhoA signaling pathway on the engineered microarray biochips. Synchronously, the cytoskeleton-mediated nanomechanics could be induced into nuclei to adjust DNA synthesis. Therefore, gene delivery and transfection on microarray biochips could be modulated by cytoskeleton-mediated nanomechanics through the regulation of cell internalization of NPs and DNA synthesis in mechanotransduction. Fig. 8 Nuclear DNA synthesis evaluated by BrdU staining and YAP mechanotransduction. A Representative fluorescence images of BrdU (green) staining on biochips. Nuclei: blue. The arrows point out the BrdU-positive cells. Scale bar: 200 μm. B Percentage of BrdU-positive cells (DNA synthesis ability) evaluated by BrdU staining. The data present mean and SD, n = 5, n.s. presents no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. C WB analysis of nuclear YAP in the patterned cells. (D) Quantitative analysis of nuclear YAP. The data present mean and SD, n = 3, n.s. presents no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. E Illustration of cell nanomechanics to affect nuclear DNA activity. The hMSCs were cultured on the microarray biochips and sensed extracellular tension from microenvironment to assemble ECMs by integrin, RGD and vinculin. These force-sensing proteins on cell membrane could regulate cytoskeleton formation (such as talin, paxillin, myosin and actin) by RhoA signaling pathway on the engineered microarray biochips. Synchronously, the cytoskeleton-mediated nanomechanics could be induced into nuclei to adjust DNA synthesis ability"
} | 8,792 |
34584038 | PMC9628787 | pmc | 7,443 | {
"abstract": "Scenedesmus obliquus ABC-009 is a microalgal strain that accumulates large amounts of lutein, particularly when subjected to growth-limiting conditions. Here, the performance of this strain was evaluated for the simultaneous production of lutein and biofuels under three different modes of cultivation – photoautotrophic mode using BG-11 medium with air or 2% CO 2 and heterotrophic mode using YM medium. While it was found that the highest fatty acid methyl ester (FAME) level and lutein content per biomass (%) were achieved in BG-11 medium with CO 2 and air, respectively, heterotrophic cultivation resulted in much higher biomass productivity. While the cell concentrations of the cultures grown under BG-11 and CO 2 were largely similar to those grown in YM medium, the disparity in the biomass yield was largely attributed to the larger cell volume in heterotrophically cultivated cells. Post-cultivation light treatment was found to further enhance the biomass productivity in all three cases and lutein content in heterotrophic conditions. Consequently, the maximum biomass (757.14 ± 20.20 mg/l/d), FAME (92.78 ± 0.08 mg/l/d), and lutein (1.006 ± 0.23 mg/l/d) productivities were obtained under heterotrophic cultivation. Next, large-scale lutein production using microalgae was demonstrated using a 1-ton open raceway pond cultivation system and a low-cost fertilizer (Eco-Sol). The overall biomass yields were similar in both media, while slightly higher lutein content was obtained using the fertilizer owing to the higher nitrogen content.",
"introduction": "Introduction Microalgae are considered a good source of biomass for the production of renewable energy that can replace fossil fuels over plant-derived biomass. The biofuels produced from microalgae are carbon neutral as microalgae consume the same amount of CO 2 during cultivation and they can be directly used in existing facilities. Compared to the 1 st and 2 nd generation biofuels from woods and crops, microalgae-derived biofuels are advantageous in terms of land usage, cultivation periods, and ethical issues [ 1 , 2 ]. However, algae-derived biofuels are not considered economically feasible, as the current prices of microalgae products remain higher than those of conventional sources [ 3 ]. Hence, efforts to reduce production costs have been made in each process, including strain development, cultivation, harvesting, and conversion. In recent years, the utilization of algae-derived materials to produce high-value products such as bio-jet fuel, cosmetics, and nutraceuticals has been studied worldwide to gain economic feasibility. Scenedesmus is one of the most common algal species in freshwater and marine systems worldwide [ 4 ]. Several species have been investigated for the production of biofuels [ 5 , 6 ], and some have been examined as potential candidates for production of lutein, which is one of the main photosynthetic pigments in nature [ 7 - 10 ]. Lutein is well characterized for its important role in maintaining eye health and strong antioxidant properties [ 11 ]. Since humans lack essential enzymes to synthesize lutein, sufficient amounts of lutein must be ingested from species such as plants, algae, bacteria, and certain fungi. In particular, as the use of computers and smartphones has increased, the importance of lutein for the protection of the retina has also increased. Currently, marigold petals are the main source of lutein production, which requires a lot of labor, land, water, and time for cultivation and harvesting [ 12 , 13 ]. To overcome these disadvantages, microalgae are emerging as a potential candidate to replace marigold petals for lutein production. The lutein content in microalgae is 3–6 times higher than that in marigold petals and microalgae can be grown in any region year-round [ 12 ]. Various microalgal species such as Chlamydomonas , Chlorella , Muriellopsis , and Scenedesmus have been examined for their lutein production rate, and the highest lutein yield of 7.62 mg/l/day was reported in Chlorella sorokiniana using two-stage mixotrophic cultivation [ 14 ]. However, most studies were restricted to lab-scale cultivation, and there is a lack of actual investigation on large-scale cultivation for industrial applications. In this study, we isolated a novel strain of Scenedesmus obliquus ABC-009, and investigated the potential of lutein production by modulating cultivation mode. Our finding provides important insights for lutein production that meet the increasing demand in industrial field.",
"discussion": "Results and Discussion Phylogenetic and Phenotypic Analysis of Novel Strain S. obliquus ABC-009 A total of seven algal isolates were obtained from nature (Namwon, Jeon-ra-do, South Korea) as explained in the Materials and Methods section, and one of the strains that turned into orange-like color after cultivation for 10 days in TAP media, was temporarily designated as ABC-009 ( Fig. S1 ). As the color of algal cells often provides information on the pigment composition [ 16 ], we assumed that the strain possesses some types of carotenoids. HPLC analysis verified that the dominant pigment was lutein. Considering that lutein is a high-valued product with the potential to be used in diverse industrial areas, including healthcare products and cosmetics, we further analyzed the unknown ABC-009 strain. The morphology of the ABC-009 strain was first investigated via light microscopy of cells cultivated at 25°C in TAP media. Most mature vegetative cells had spherical or elliptical shapes, with a chloroplast occupying approximately two-thirds of the whole cell ( Fig. 1C ). The average size of mature vegetative cells was 4.5 × 5 μm. A few vacuoles were observed in the cytoplasm. In general, the shape of Scenedesmus sp. is widely known for its unique feature consisting of 4, 8, 16, or 32 cells arranged in a row, with spines or bristles [ 4 ]. However, a few Scenedesmus species, such as S. obliquus or S. rubescens , also exhibit spherical shapes similar to the ABC-009 strain, and some are known to change their form upon cultivation conditions [ 17 ]. Accordingly, we could not determine the exact algal species of the ABC-009 strain because these features are often found in various species, including Chlorella , Scenedesmus , and Ettlia [ 18 , 19 ]. The phylogenetic analysis based on the sequence of 18S rDNA, however, could provide more clues on the genus to which it belongs ( Fig. 1A ). The phylogenetic tree showed clear similarity of the 18S rDNA with most Scenedesmus species, especially with Scenedesmus obliquus . In addition, as the novel strain contained lutein and neoxanthin as the main xanthophyll pigments, similar to Scenedesmus sp. ( Fig. 1B ), we concluded that the isolated strain was S. obliquus ABC-009 after considering overall aspects of the morphology, pigments, and 18s rDNA sequence. Cultivation under Different Conditions Although most microalgae are considered photoautotrophs, heterotrophic cultivation is also adopted for the production of high-value products in several species, including Scenedesmus sp. [ 14 , 20 ]. Often, heterotrophic cultivation has the advantage of better cell growth compared to phototrophic cultivation, allowing higher final cell densities [ 20 ]. In contrast, heterotrophic cultivation often results in lower contents of light-harvesting pigments and lipids, which is relevant to the lack of sufficient light. To determine the optimal conditions for producing lutein and lipids from S. obliquus ABC-009, we cultivated the microalgae under three different conditions: photoautotrophic cultivation with ambient air, photoautotrophic cultivation with 2% CO 2 , and heterotrophic cultivation with 10 g/l glucose. Through a preliminary study in 96-well plates, the optimal temperature for cultivation was found to be 29°C, where YM media showed the best performance over BG11 or TSB (Tryptic Soy Broth) media ( Fig. S2 ). The growth rate according to cell numbers showed similar results in CO 2 supplemented photoautotrophic cultivation and heterotrophic cultivation, while cultivation with ambient air was insufficient to provide sufficient CO 2 ( Fig. 2A ). However, the dry cell weight of heterotrophically cultivated cells was3-fold higher than that of autotrophically cultivated cells ( Fig. 2B ). As an increase in dry cell weight without any changes in cell density may imply variations in cell morphology or contents, we first analyzed the cells with a microscope and observed significantly increased cell size in heterotrophically cultivated cells ( Fig. 2C ). The average size of cells was the largest in the order of heterotrophically cultivated cells (6.71 μm), CO 2 supplemented cells (4.37 μm) and cells cultivated with ambient air (3.72 μm). A similar phenomenon of increased cell size under heterotrophic cultivation was reported in Chlorella sp. without clarification of the exact underlying mechanism [ 21 ]. Since large cells require less energy for harvesting [ 22 ], the cultivation of S. obliquus ABC-009 under heterotrophic conditions may be a good option for industrial-scale cultivation. Furthermore, as microalgae with increased cell sizes are reported to exhibit higher transformation efficiency [ 23 ], this effect of cell enlargement can be applied for genetic engineering of S. obliquus ABC-009 in the future. FAME and Lutein Content In order to compare the lutein and lipid productivity of cells cultivated under different conditions, pigments and FAME were analyzed using HPLC and GC, respectively ( Fig. 3 ). The highest FAME content was observed in cells cultivated photoautotrophically with CO 2 supplementation ( Fig. 3A ). Due to the existence of an organic carbon source, the lipid accumulation was rather limited under heterotrophic conditions, showing even lower FAME contents than the photoautotrophically cultivated cells with ambient air on day 6 (8.2% and 7.1%, respectively). The maximum FAME content in CO 2 supplemented cells (15.8%) and heterotrophically grown cells (10.5%) was achieved on day 9. However, due to high biomass, the FAME productivity was 1.8 folds higher in heterotrophically cultivated cells (66.38 mg/l/day) than in photoautotrophically cultivated cells with CO 2 supplementation (36.85 mg/l/day) ( Fig. 3B ). The lutein content of cultivated cells was between 0.1% and 0.45% of its biomass, and the highest content was observed on day 3 in the aerobically cultivated cells (0.45%) ( Fig. 3C ). However, the actual quantity of lutein in aerobically cultivated cells on day 3 was merely 0.03 μg/cell, which was much lower than the cases of cells cultivated with CO 2 (0.08 μg/cell) or YM media (0.11 μg/cell) ( Fig. 3D ). Accordingly, the high lutein content in aerobically cultivated cells was only due to the low cell biomass, and as a consequence, the lutein content actually decreased over time under aerobic conditions. In contrast, lutein content increased from 0.25% DCW to 0.35%DCW in CO 2 supplemented cells, whereas no significant changes were detected in heterotrophically cultivated cells. As photosynthesis does not take place under dark conditions, it was difficult for the lutein, a kind of photosynthetic pigment, to increase above 0.2% of DCW in heterotrophic condition. Post-Cultivation Light Stress for FAME and Lutein Accumulation Compared to the high biomass productivity under heterotrophic conditions, the productivity of FAME and lutein was not as high as expected because of their low contents. To increase the lutein and FAME contents, fully cultivated cells were exposed to 150 μmol photons/m 2 /s of LED light for 5 days without shaking ( Fig. S3 ). Induction of post-cultivation stress by light, salinity, or nutrient starvation has been studied in a few microalgae to increase the amounts of metabolites [ 24 , 25 ]. As our target products were lipid and lutein, we decided to increase only light intensity to minimize the extra energy input for post-cultivation stressing. Five days of light irradiation on cells cultivated in each condition resulted in a color change from green to yellow, which may be the result of increased carotenoid content and degradation of chlorophylls. After post-cultivation light stress period, the overall biomass was increased in all three conditions, and the highest increase observed was 4.8 g/l in the heterotrophically cultivated cells ( Fig. 4 ). However, the FAME content of heterotrophically cultivated cells increased by only 17%, while increases of 336% and 86% were measured in photoautotrophically cultivated cells with air or CO 2 , respectively. These results suggest that nutrients were not completely depleted in YM media, and cells continued to grow in a semi-mixotrophic condition, resulting in a large increase in biomass and small changes in lipid content. On the other hand, photoautotrophically cultivated cells face nutritional starvation, causing them to accumulate lipids instead of biomass. Likewise, lutein content was decreased by 39–51% under photoautotrophic conditions, while a 34% increase was observed in heterotrophically grown cells. As heterotrophic cultivation was carried out in a completely dark condition, exposure to light triggered photosynthetic metabolism, including biosynthesis of light-harvesting pigments. In conclusion, we have successfully increased the FAME and lutein productivity by post-cultivation light irradiation, achieving the highest FAME and lutein productivity of 92.78 mg/l/day and 1.006 mg/l/day from heterotrophically cultivated cells. Among the diverse Scenedesmus sp. studied for lutein production, the productivity of S. obliquus ABC-009 was around the average value ( Table 1 ). However, it is difficult to directly compare the lutein productivity of each strain from different studies as growth conditions (scale, media composition, temperature, light source, extraction method, osmotic stress, etc.) are not the same. Previous studies have revealed many favorable conditions for lutein production in selected Scenedesmus species. For example, white LEDs resulted in better production efficiency than other LEDs, with the best performance at 300 μmol photons/m 2 /s [ 7 ]. In addition, Chen et al . found that mixotrophic condition with a 12 h light period followed by a 12 h dark period can increase lutein productivity [ 8 ], and Sanchez et al . reported that pH 8 and low salt stress conditions work well for Scenedesmus almeriensis [ 10 ]. Many variables that may affect lutein productivity remain, and the optimal conditions can vary depending on the specific strain. Hence, further studies on S. obliquus ABC-009 are necessary to determine the optimal conditions for maximizing lutein productivity. Large-Scale Cultivation of S. obliquus ABC-009 In particular, S. obliquus is a robust microalga that has stable biomass productivity in moderate climates [ 28 , 29 ]. Moreover, the stability of scale-up outdoor cultivation of microalgae over long periods is important for the successful commercialization of microalgae-based products [ 30 ]. Several previous studies have reported the enhancement of lutein productivity in Scenedesmus sp. These include the modulation of cultivation modes, such as two-stage cultivation, mixotrophic and heterotrophic cultivation, and light induction strategies [ 7 - 9 , 31 ]. However, studies on large-scale photoautotrophic cultivation using low-cost media for lutein production have rarely been reported. To evaluate the potential of large-scale outdoor cultivation using low-cost media, 1-ton scale outdoor cultivation was carried out for 2 months, and biomass and lutein content, and productivity were analyzed. The results showed that the lutein content of cells grown in Eco-Sol, the low-cost media, was generally much higher (111 ~ 382%) than that in cells grown in BG-11, while biomass production was generally higher in culture using BG-11 (50 ~ 216%) ( Fig. 5A ). From the comparison of elemental compositions in each medium ( Table 2 ), we can see that Eco-Sol media contained a much higher amount of nitrogen (20x), phosphorous (252x), and potassium (196x) compared to BG-11, while the abundance of other trace elements was similar or richer in BG-11. It has been reported that a sufficient amount of nitrogen is essential for lutein accumulation, and lutein content may depend on residual nitrogen concentration in green algae [ 32 , 33 ]. Hence, the high lutein content in cells cultivated with Eco-Sol may be due to the higher nitrogen concentration, which was approximately 20 folds of that in BG-11. As a result, overall lutein productivity considering biomass and lutein content over the long-term cultivation was approximately 16% higher in the culture grown using Eco-Sol than that in culture grown using BG-11, indicating that low-cost media employed for stable biomass and lutein production through the large-scale outdoor cultivation of S. obliquus ABC-009 ( Fig. 5B )."
} | 4,250 |
25520953 | PMC4253960 | pmc | 7,445 | {
"abstract": "The primary focus in the network-centric analysis of cellular metabolism by systems biology approaches is to identify the active metabolic network for the condition of interest. Two major approaches are available for the discovery of the condition-specific metabolic networks. One approach starts from genome-scale metabolic networks, which cover all possible reactions known to occur in the related organism in a condition-independent manner, and applies methods such as the optimization-based Flux-Balance Analysis to elucidate the active network. The other approach starts from the condition-specific metabolome data, and processes the data with statistical or optimization-based methods to extract information content of the data such that the active network is inferred. These approaches, termed bottom-up and top-down, respectively, are currently employed independently. However, considering that both approaches have the same goal, they can both benefit from each other paving the way for the novel integrative analysis methods of metabolome data- and flux-analysis approaches in the post-genomic era. This study reviews the strengths of constraint-based analysis and network inference methods reported in the metabolic systems biology field; then elaborates on the potential paths to reconcile the two approaches to shed better light on how the metabolism functions.",
"introduction": "Introduction Metabolic network is the outmost layer of cellular activity from the genome. The genome of a cell is a comprehensive and condensed information base, defining a boundary for the biochemical capacity of the cell. The processing of genetic information passes through several layers of fabrication and regulation before reaching their end products. This is from information to the function, from genotype to phenotype. Metabolic enzymes count for a significant percentage of the end products of genes, and their activity sets the physiology of the cell. Since metabolic network activity is the major representative of cell functionality, it is of great importance to gain as much knowledge as possible on the active metabolic network at a specific cellular state. Systems-based approach to molecular biology has contributed to an increased knowledge of metabolic pathways for an increasing number of organisms, and led to almost complete metabolic networks for a number of major organisms, from yeast to human. Such static networks are available in a condition-independent manner through web-based databases such as KEGG or MetaCyc (Altman et al., 2013 ), or reconstructed in a format suitable for simulation by several researchers at genome scale (Oberhardt et al., 2009 ; Kim et al., 2012 ). There are several mathematical approaches to process such networks to come up with condition-specific networks, the most common one being the Flux-Balance Analysis (FBA) framework (Orth et al., 2010 ). This is a bottom-up direction toward the active network since already-known “parts,” interactions, are used as inputs (Bruggeman and Westerhoff, 2007 ; Petranovic and Nielsen, 2008 ). In parallel to the developments on the knowledge of metabolic networks, techniques to measure metabolite levels at high throughput, termed metabolomics, have arisen (Kell, 2004 ; Dunn et al., 2005 ). Quantitative or semi-quantitative metabolome data, although one of the most challenging compared to other omic sciences, have come a long way in a decade, from the detection and quantification of about 50 metabolites (Devantier et al., 2005 ) to more than 1000 metabolites (Psychogios et al., 2011 ). Metabolome data are a snapshot of the condition-specific status of the investigated organisms. Reverse-engineering metabolome data to discover the underlying network structure is the goal behind metabolic network inference approaches (Srividhya et al., 2007 ; Çakır et al., 2009 ). The information content of metabolome data is revealed by processing it with correlation or optimization-based methods (Weckwerth et al., 2004 ; Hendrickx et al., 2011 ; Öksüz et al., 2013 ). Such an approach to discover metabolic network structure is termed top-down approach since the parts, interactions, are not known a priori , and predicted from the whole set of available biomolecules (Bruggeman and Westerhoff, 2007 ; Petranovic and Nielsen, 2008 ). In this review, we will cover the basic developments in bottom-up and top-down approaches to discover active metabolic network, and then ponder over the possible ways of reconciling these two approaches for a better prediction of active network structure. Figure 1 illustrates the two alternative network discovery approaches. Figure 1 Comparative demonstration of bottom-up and top-down approaches to discover active metabolic network . The white box in the figure defines different levels of network structure information."
} | 1,211 |
35530221 | PMC9072136 | pmc | 7,446 | {
"abstract": "The aim of this study was to investigate hydrogen production enhanced by methanogenesis inhibition in the single-chamber microbial electrolysis cell (MEC) under alkaline conditions. With 50 mM bicarbonate buffer and 1 g L −1 acetate, the MEC was tested at pH = 8.5, 9.5, 10.5, and 11.2, respectively, within 124 d operation. Effective methanogenesis inhibition in the MEC increased with pH from 8.5 to 11.2. At pH 11.2, Methanobacteriaceae reached the lowest absolute quantity ( i.e. , biomass and mcrA gene copy number of methanogens) within the microbial community in the cathodic biofilm among the pH values. Under the alkaline conditions, a hydrogen percentage of 85–90% and a methane percentage < 15% were achieved within 25 cycles (50 d) of operation. The maximum current density in the MEC reached 83.7 ± 1.5 A m −3 with the average electrical recovery of 171 ± 18% and overall energy recovery of 72 ± 3%. The excellent performance of the MEC at pH = 11.2 was attributed to the low abundance of methanogens within the cathodic biofilm (2.23 ± 0.46 copy per cm 2 ), low cathodic biomass (0.12 ± 0.01 mg protein per g), and low anode potential (−0.228 mV vs. saturated calomel electrode). Results from this study should be valuable to expand applications of the MEC with methanogenesis inhibition in alkaline wastewater treatment.",
"conclusion": "5. Conclusions It was the first time to report the excellent performance for H 2 production of single-chamber MEC at pH 11.2 with effective methanogenesis inhibition within 50 d operation. The maximum current density reached 83.7 ± 1.5 A m −3 with the electrical recovery of 171 ± 18% and overall energy recovery of 44–81%. At pH 11.2, H 2 production was kept at 85–90% and CH 4 production was <15% within 25 cycles (50 d). The good performance of the MEC at pH 11.2 was attributable to low abundance of methanogens within the cathodic biofilm, low cathodic biomass, and low anode potential.",
"introduction": "1. Introduction With high energy density and clean combustion product, hydrogen (H 2 ) has been considered as an ideal alternative energy source to fossil fuel. 1,2 Compared with the conventional H 2 production processes, such as chemical refinery, water electrolysis, and dark-fermentation, the microbial electrolysis cell (MEC) emerges as a promising and attractive technology with advantages of mild operation conditions, efficient biomass conversion to biohydrogen gas, and high coulombic efficiency (CE). 2,3 Under an applied voltage of 0.30 V ( vs. standard H 2 electrode), electrons and protons released by electrochemically active bacteria (EAB) in the anodic biofilm can transfer to the cathode to form H 2 with the cathodic catalyst. 4,5 The H 2 energy harvested in the MEC can be 2–4 times the input electrical energy. 1,3 However, a notable challenge in the single-chamber MEC is the H 2 sink during long term operation, mainly attributable to the H 2 scavenging by undesirable methanogenesis process. 6–8 For example, the hydrogenotrophic methanogenesis can consume 4 mol H 2 (combustion heat of 285 kJ mol −1 ) to produce 1 mol CH 4 (combustion heat of 890 kJ mol −1 ), resulting in significant decrease of H 2 and energy recovery. 6,9 To inhibit the methanogenesis process in the single-chamber MEC, different strategies have been developed in recent years. 6,9,10 Chemical agents, such as 2-bromomethane sulfonate (2-BES), 4 chloroform, 11 and acetylene, 5 have been tested for the methanogen inhibitors in the MEC. With addition of 286 μM 2-bromoethanesulfonate in the MEC, methanogenic electron loss decreased from 36% to 2.5% and the overall H 2 recovery increased from 56% to 80%. 4 With the chloroform dosage of 5‰, the methane (CH 4 ) production was efficiently inhibited within 11 batch cycles. 11 However, the chemical inhibitors above were toxic and not sustainable during long-term operation in the MEC. 2,6 The negative pressure (40.5 kPa) control in the single-chamber MEC improved H 2 production rate and electrical energy recovery due to the inhibition of methanogen growth. However, CH 4 production became dominant in the biogas production once the negative pressure was removed. 10 It was shown that continuous H 2 extraction via a gas-permeable hydrophobic membrane in the MEC ( i.e. , dual-chamber MEC) could eliminate the CH 4 production. 12 Some conditions, such as ultraviolet irradiation and low temperature, can be applied to effectively inhibit the methanogenesis in the single-chamber MEC during long-term operation. Nevertheless, applications of such conditions require high operation costs. 4,9,13 Therefore, it is necessary to develop efficient methods for methanogenesis inhibition in the single-chamber MEC. Since most of methanogens prefer neutral pH conditions, pH control in the MEC has been tested to enhance H 2 production in recent years. At pH = 5.8, the CH 4 production was suppressed in the single-chamber MEC within the first two batches but recovered in the following batches, indicating that acidic pH control was not sustainable to the methanogenesis inhibition. 14,15 At pH = 9.3, H 2 production rate in the single-chamber MEC increased by 117% compared with that under the neutral condition. 16,17 The dominant species of bacterial community in anodic biofilm of the MEC are Geoalkalibacter sp. and Geobacter sp. under the alkaline and neutral conditions, respectively. 16,18,19 However, the development of methanogens in the cathodic biofilm of MEC under alkaline conditions has not been studied. The objective of this study was to investigate H 2 production with methanogenesis inhibition in the single-chamber MEC under alkaline conditions during long-term operation. Performance of the MEC was examined at different pH values for 124 d of operation. Bacterial communities in the anodic and cathodic biofilms were analyzed and the mechanism of methanogenesis inhibition in the MEC was discussed related to H 2 production.",
"discussion": "4. Discussion The performance for H 2 production of our MEC at pH 11.2 was comparable to that of other MECs with different strategies of methanogenesis inhibition. The H 2 production in our MEC at pH 11.2 was much higher than that with addition of 1% acetylene and 5 mM 2-bromoethanesulfonate inhibitors, respectively (85–90% vs. 60–70%). 5 The H 2 yield in our MEC was comparable to that in the MEC with UV irradiation (2.64–3.36 vs. 2.87–3.70 mol H 2 per mol acetate). 9 The current density in our MEC at pH 11.2 was higher than that at pH = 9.3 (83.7 vs. ∼2 A m −3 ). 16 Moreover, many strategies ( e.g. , UV irradiation, chemical agents, and negative pressure control) could not inhibit CH 4 production effectively once methanogenesis was well established in the MEC. 6,9,10 Our alkaline adjustment with pH 11.2 could significantly improve H 2 production even after produced CH 4 dominated the whole biogas in the MEC. Therefore, the alkaline adjustment should be an effective method for methanogenesis inhibition in the single-chamber MEC. The better performance of our MEC at pH 11.2 was attributed to low cathodic biomass (0.02 ± 0.00 mg protein per g), low abundance of methanogens in the cathodic biofilm (2.23 ± 0.46 × 10 8 copy per cm 2 ), and low anode potential (−0.228 mV vs. SCE). The low abundance of methanogens in the cathodic biofilm resulted in less CH 4 production in the single-chamber MEC. 9,28 The low cathodic biomass resulted in less acetate consumption in the cathode, and enhanced acetate utilization by EABs on the anode. 9 The low anode potential was beneficial to improve the activity of EABs on the anodic biofilm. 29 Many bacteria and Archaea identified in our single-chamber MEC have been reported in various bioelectrochemical systems (BESs). In the cathodic biofilm of BESs, Methanobacteriaceae has been widely identified as a dominated hydrogenotrophic methanogen. 22 The relative abundance of Methanobacteriaceae in the microbial community reached 77.2% in the thickness of 45–60 μm within the cathodic biofilm in the single chamber MEC. 22 The copy number of mcrA gene within the cathodic biofilm in the MEC greatly varied because of different substrates, inoculums, and operation conditions. 30–33 The mcrA gene copy number in the cathodic biofilm of our MEC was similar to that of the mini-MEC with the inoculums of anaerobic digestion sludge at pH 7.0 (∼10 8 copy per cm 2 ). 30 The relative abundance of Methanobacteriaceae was not equivalent to the absolute quantity of Methanobacteriaceae in the microbial community. At pH 11.2, Methanobacteriaceae reached high relative abundance of 73%, but low absolute quantity ( i.e. , mcrA gene copy number). The growth of Methanobacteriaceae was greatly inhibited with significant decrease of the mcrA gene copy number and cathodic biomass. The low amount of Methanobacteriaceae resulted in low CH 4 production and high H 2 production. The change of Methanobacteriaceae was consistent with the performance of our MEC under the alkaline condition. Although Geoalkalibacter and Corynebacterium have seldom been found in the cathodic biofilm of MEC under alkaline conditions so far, other EABs ( e.g. , Geobacter and Desulfobacteraceae) have been identified in the cathodic biofilm under the neutral condition. 23,32,34 The relative abundance of Geobacter could reach 30–45% within the bacterial community in the cathodic biofilm. 32 The synergistic effect among Geoalkalibacter , Corynebacterium , and Archaea within the cathodic biofilm on H 2 production is unclear, which needs to be further explored. Homoacetogens have been frequently identified in the MEC fed with acetate, which can utilize H 2 and produce acetate to form an internal H 2 cycle in the MEC. 35 High presence of homoacetogens may greatly decrease the efficiency of H 2 production and result in CE as high as 1242%. 35 However, homoacetogens ( e.g. , Acetobacterium ) were not identified in the microbial communities in this study. Efficient inhibition of homoacetogen growth should be helpful for increasing H 2 production, 35 attributable to better performance of our MEC compared to other MECs with different strategies of methanogenesis inhibition. In the anodic biofilm of BESs, Geoalkalibacter has been identified as an EAB with high electricity generation. 16,18 The relative abundance of Geoalkalibacter reached 43% and 9.8% in the bacterial community of the anodic biofilm in the MEC fed with acetate and glycerol at pH 9.3, respectively. 16,17 The relative abundance of Geoalkalibacter greatly changed at pH = 8.5–11.2 in this study, indicating that the growth of EABs in the anode biofilm was sensitive to pH in the solution. The total biomass in the anode significantly decreased with pH from 8.5 to 11.2. Therefore, it is necessary to determine the optimal pH for Geoalkalibacter growth using pure Geoalkalibacter strain in the future. In addition, Corynebacterium ( e.g. , Corynebacterium sp. strain MFC03) was capable of generating electricity in a pH range of 8.0–10.0. 36 The optimal pH for Corynebacterium sp. strain MFC03 to produce electricity was 9.0. 37 Corynebacterium was detected with the relative abundance of 32.8% in the bacterial community in the anodic biofilm of MFC fed with glucose and p -nitrophenol at pH = 7.0. 31 As a strictly anaerobic and an alkaliphilic bacterium, Alkalibacter has been identified in the alkaline MFC and MEC. 16 Therefore, Geoalkalibacter and Corynebacterium identified in this study might have high activity under alkaline condition. Moreover, the protons released by exoelectrogens ( e.g. Geoalkalibacter and Corynebacterium ) can significantly decrease the pH in solution close to the anode biofilm, resulting in high alkaline endurance of EABs. The carbonate buffer used in this study is also useful for accelerating the proton transfer from anode to cathode and improving the reaction activity of the anode and cathode ( e.g. , potential) in the MEC. To the anode, acetate can be degraded by EABs with to produce electrons and protons as follows: 38,39 1 In the electrolyte with carbonate buffer solution, excess H + can be neutralized by OH − from the CO 3 2− hydrolysis as follows: 2 H + + OH − → H 2 O 3 CO 3 2− + H 2 O → HCO 3 − + OH − To the cathode, HCO 3 − can release H + on the cathode to produce H 2 : 38 4 HCO 3 − → CO 3 2− + H + 5 Therefore, bicarbonate (HCO 3 − ) plays an important role as pH buffer and proton carrier under an alkaline condition. 38 With indirect transportation of H + , our MEC could produce hydrogen efficiently. Moreover, high electron transfer ability under high pH was helpful for high H 2 production. According to the Nernst equation, the anode and cathode equilibrium potentials can be calculated as follows: 40 6 7 Here E an and E cat are the equilibrium anode and cathode potentials, respectively; E an 0 and E cat 0 are the standard anode and cathode potentials, respectively; [CH 3 COO − ] and [HCO 3 − ] are the concentrations of acetate and bicarbonate in the solution, respectively; [H an + ] and [H cat + ] are the concentrations of protons in the anode and cathode, respectively; pH 2 represents the H 2 concentration; 40 R and F are constants; and T is the temperature (K). With pH increase from 8.5 to 11.2, the concentrations of HCO 3 − decreased in the solution, resulting in increase of the value. Thus the equilibrium anode potential ( E an ) decreased with pH from 8.5 to 11.2. Due to the H 2 production improvement with the pH increase from 8.5 to 11.2, the values of increased, resulted in the decrease of the equilibrium cathode potential ( E cat ). The results of the theoretical potential analysis were consistent with the measurements on the anode and cathode potentials in this study. And the activity of EABs on the anodic biofilm can be improved by the low anode potential. Our results demonstrate for the first time the operation of MEC at pH 11.2. Various EABs such as Geoalkalibacter and Corynebacterium in the anodic biofilm at pH 11.2 suggest that the extracellular electron transfer to anode may be independent of the optimal pH for the growth of EABs. High current density in the MEC at pH 11.2 indicated that the extracellular electron transfer rate under alkaline condition may be faster than that under neutral condition. It should be interesting to explore the extracellular electron transfer mechanism among mixed EABs under alkaline condition. Moreover, the configuration and position of the anode and cathode in the scale-up MEC should be optimized to enhance the bubble formation and accelerate the release dynamics of H 2 . Alkaline wastewater discharged from various industries may contain many organics. 41,42 For example, the pH value in yogurt wastewater can be > 11.0. 41 Such alkaline industrial wastewater may be used for H 2 production in the MEC."
} | 3,735 |
35425567 | PMC8982057 | pmc | 7,448 | {
"abstract": "Rich iridescent structural colors in nature, such as peacock feathers, butterfly wings, beetle scales, and mollusc nacre, have attracted extensive attention for a long time and they generally result from the interaction between light and periodic structures. However, non-iridescent structural colors, such as silvery structural colors, have received relatively little attention, and they usually result from non-periodic structures. Here, using optical microscopy, fiber-optic spectrometry, field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and laser Raman spectroscopy, we investigate the origin of a novel structural color occurring at the edge of a bivalve shell ( i.e. , an otter shell). We find that: (1) the structural colors are observed to be uniform metallic silvery when viewed with the naked eye; (2) they are surprisingly multicolored with various colorful pixels juxtaposed together when viewed with an optical microscope; (3) each individual pixel shows a single color originating from a periodic, multilayered organic film with definite spacing ( d ); and (4) different pixels vary significantly in size, shape, and color with different d values (202–387 nm). Finally, we confirm that the macroscopic silvery color results from the pointillistic mixing of nano-to microscale iridescent pixels. We also discuss the special photonic structure responsible for the silvery color. We hope that this work can not only accelerate our comprehension of photonic materials, but also provide new inspiration for the synthesis of silvery white materials.",
"conclusion": "5. Conclusions The shell edge of Lutraria lutraria shows a bright silvery color, which is caused by the pointillistic mixing of iridescent pixels. The causes of these pixels were studied via examining the structure of the surface and a cross-section of the porous layer of the shell using SEM, XRD, FTIR, and laser Raman spectroscopy. Through Fourier analysis, we have demonstrated that the silvery color originates from the organic film, which acts as a broadband reflector. Moreover, we also confirm that this reflector consists of different photonic crystallites, which lead to different pixels with different colors. Finally, we hope that this organic–inorganic composite structure can provide new inspiration for the design and synthesis of photonic materials.",
"introduction": "1. Introduction Color is a visual effect that is produced when light reflected from objects stimulates our eyes and brain. 1–5 It can be simply divided into chemical and structural colors, 3,6 which are mainly related to the material's chemical composition or structure, respectively. 3,6 To date, structural color is extremely attractive in a variety of fields such as materials science, since it does not require toxic dyes to prevent it from fading, and it can find a wide range of applications such as in displays, decorations, cosmetics, and textile dyeing. 7–9 The structures that can produce colors usually exhibit nanoscale variations in their refractive index in 1-, 2-, or 3-dimension (D), and are, therefore, called 1D, 2D, or 3D photonic structures (reflectors), respectively. 10 Among these, 1D photonic structures, also called multilayer reflectors, are widespread both in the artificial and natural world and have been heavily studied. 11 Essentially, multilayer reflectors are made of alternating films of high and low refractive index materials, and can usually be divided into two types: periodic and amorphous. 1,11 The former reflect light with specific wavelengths, which results in defined structural colors and so are called narrow-band reflectors, while the latter reflect most or all wavelengths of light, which results in silvery (or golden) structural colors and so are called broadband reflectors. 12 So far, four types of broadband reflectors have been found in nature ( Fig. 1 ): 1,13–17 (a) chirped, where the film thickness ( t ) changes systematically; (b) chaotic, where t and the center-to-center distance of two adjacent films ( d ) change randomly; (c) triple composite, where three narrow-band reflectors are superimposed together, each tuned to a different wavelength or color; and (d) curved, where both t and d are constant but the film direction ( N ) changes globally. Fig. 1 A schematic diagram of types of natural broadband reflectors (adapted from ref. 12 , 13 , 15 and 16 ): (a) chirped, (b) chaotic, (c) triple composite, and (d) curved. t : film thickness; d : center-to-center distance of two adjacent films. The latter two types of reflectors have the following characteristics: (a) locally, films are orderly arranged with uniform t and d , which act as small narrow-band reflectors (named “photonic crystallites” in this work) and result in specific pure colors (named “color units” or “pixels” in this work); (b) globally, t , d and N may vary in different crystallites; (c) these composite or curved reflectors often lead to an unusual mixing of colors ( i.e. pointillistic mixing). To date, silvery structural colors have been found in numerous species of organisms such as butterflies, fish, and spiders. 17–19 They invariably originate from the four broadband reflectors as described above and are depicted in Fig. 1 . Moreover, little has been reported for mollusc shells. Here, we aim to investigate (1) the silvery structural colors occurring in the shell layers of bivalve animals named Lutraria lutraria ( Fig. 2b ); (2) the corresponding nanostructure and composition of the shell layers responsible for the silvery color. We first report that this color is caused by pointillistic mixing of iridescent pixels, resulting from a novel composite broadband reflector. We hope that this natural broadband reflector offers a potential strategy in the future for the design of ideal reflectance-based materials, such as the back reflector used in the field of solar cells. 20,21 Fig. 2 Photographs of Lutraria lutraria : (a) a general view of the shell in the wet state, and (b) the brilliant silvery white color at the surface of the shell.",
"discussion": "4. Discussion The class Bivalvia contains about 10 000 species. 28 However, the iridescent color of the bivalve shell is uncommon. Studies on the iridescent color of the shell are relatively limited and focus on regular multilayers, such as of mother of pearl (nacre). 29,30 To the best of our knowledge, there are few studies that report a silvery color of the bivalve shell. Interestingly, we found a distinctive silvery color at the shell edge of Lutraria lutraria . We found that the organic film act as a broadband reflector, which consists of different photonic crystallites. Accordingly, the various pixels juxtapose together, and then form a pixelated surface (the pixelated surface consists of many ordered or disordered color units, which are beyond the resolution of the naked eye 21,31,32 ). Finally, this surface gives rise to a silvery color due to pointillistic mixing. In contrast with the four types of broadband reflectors that have been discovered (as detailed in the Introduction, Fig. 1 ), the broadband reflector in this work has the following characteristics: (1) as compared with type (a), t changes horizontally rather than vertically; (2) as compared with type (b), t and d change gradually rather than randomly; (3) as compared with type (c), the number of crystallites are far more than three (there are only three crystallites in the single scale 33 ); (4) as compared with type (d), t and d change gradually but N is constant. These characteristics indicate that the broadband reflector in this research is unusual. Accordingly, we hereby term this reflector as a “hybrid broadband reflector”. For the whole structure in the porous layer, the aragonite fiber is rigid while the organic film is flexible. We believe that the whole structure formed a tensegrity system to self-balance. In addition, this structure may play a key role for buffering from the viewpoint of the Lutraria lutraria live in soft substrates such as sand."
} | 2,027 |
35744293 | PMC9229385 | pmc | 7,451 | {
"abstract": "In this study, ball mill pretreated iron ore tailings were modified with tetraethoxysilane (TEOS) and hexadecyltrimethoxysilane (HDTMS) to obtain iron ore tailings/polysiloxane (IOT/POS) superhydrophobic powders, which were subsequently mixed with chloroprene rubber solution (CRS) to prepare durable superhydrophobic composite coatings. The effect of HDTMS amount and reaction time on the wettability of the superhydrophobic powder was investigated. The influence of the superhydrophobic powders concentration on the wettability of the composite coatings as well as the degree of damage of the superhydrophobicity of the composite coating was analyzed by using the sandpaper abrasion and tape peeling tests. Further, SEM and FTIR were used to analyze the formation mechanism of the IOT/POS superhydrophobic powders and coatings. The results showed for an HDTMS amount of 2.5 mmol and reaction time of 4 h, the contact angle of the IOT/POS powder was 157.3 ± 0.6°, whereas the slide angle was determined to be 5.9 ± 0.8°. For an IOT/POS powder content of 0.06 g/mL in CRS, the contact angle value of the superhydrophobic composite coating was 159.2 ± 0.5°, whereas the slide angle value was 5.5 ± 0.8°. The superhydrophobic composite coating still maintained the superhydrophobicity after the sandpaper abrasion and tape peeling tests, which indicated the iron ore tailings solid waste has the potential to prepare superhydrophobic coatings.",
"conclusion": "4. Conclusions In conclusion, a low-cost method was developed in this study to prepare durable superhydrophobic composite coatings, which were facilely designed by spraying a mixed suspension of iron ore tailings/polysiloxane (IOT/POS) and chloroprene rubber solution (CRS). The amount of HDTMS and reaction time exhibited a significant influence on the wettability of IOT/POS powders. Further, the concentration of IOT/POS superhydrophobic powders also had a marked effect on the wettability of the composite coating. Owing to the irregular stacking of the SiO 2 nanoparticles on the surface of IOT with a micrometer scale, a micro/nano composite structure was generated. Grafting of long-chain alkylsilane of HDTMS endowed IOT/POS and composite coating with a low surface energy. CRS not only contributed toward fixing the IOT/POS particles, but also formed a strong adhesion between the coating and substrate. Therefore, the developed superhydrophobic composite coatings displayed a high water CA, low SA, and excellent durability and self-cleaning property. Further, this study also provides the basis for the preparation of superhydrophobic coatings from other solid wastes.",
"introduction": "1. Introduction The superhydrophobic coatings have attracted an increasing attention due to their promising applications in various fields, such as self-cleaning [ 1 , 2 , 3 ], anti-icing [ 4 , 5 , 6 ], corrosion resistance [ 7 , 8 ], and drag reduction [ 9 , 10 ]. The cooperation of appropriate micro/nano scale composite structure and the low-surface-energy modifier is the basic strategy for fabricating superhydrophobic coatings [ 11 ]. Based on this strategy, researchers used SiO 2 , TiO 2 , and carbon nanotubes as skeleton materials to produce the superhydrophobic powders or suspensions through hydrothermal synthesis, sol-gel synthesis, graft coating and other processes, followed by spraying or dipping to prepare superhydrophobic coatings [ 12 , 13 , 14 , 15 ]. Alternatively, aluminum, copper, and other bulk metal surfaces are employed as raw materials, followed by the corrosive dissolution, mechanical abrasion, and other processes to form a rough surface. Subsequently, spraying the nanoparticles modified with low surface energy modifier leads to the preparation of the superhydrophobic coatings [ 16 , 17 , 18 , 19 ]. However, the complicated and expensive preparation methods and the low stability of most of the reported superhydrophobic coatings limit their practical application. Thus, a few research studies have employed inexpensive nanostructured clay minerals as skeleton materials, along with their modification with silane coupling agents and introduced an adhesive into the coating to prepare superhydrophobic coatings. For instance, Li et al. [ 20 ] prepared a superhydrophobic coating with excellent anti-corrosion properties by spraying the water-based polyurethane and octadecyltrimethoxysilane-modified diatomite powders. Zhang et al. [ 21 ] prepared a superhydrophobic coating with excellent stability and transparency by spraying the organosilane/attapulgite nanocomposites. Chen et al. [ 22 ] used tetraethylorthosilicate and hexadecyltrimethoxysilane to modify sepiolite and incorporated it in an epoxy resin suspension to prepare superhydrophobic, mechanically strong and chemically stable coatings. However, high quality clay minerals are still scarce, and the price of beneficiation and purification is still high. In this respect, the use of industrial waste, which is similar in composition as the clay minerals, to prepare the superhydrophobic coating materials represents a functional strategy. Therefore, it is necessary to find lower-cost skeleton materials to prepare high-performance superhydrophobic coatings. As a kind of bulk solid waste, coarse-grained iron ore tailings were widely used as sand and stone material for construction and produced glass ceramics [ 23 , 24 ], porous ceramsite [ 25 ], cement clinker [ 26 ]. However, the high value-added industrial utilization of microfine iron ore tailings has become an urgent problem that needs to be solved [ 27 ]. Because of the similar composition between iron ore tailings and clay, microfine iron ore tailings may be an ideal skeleton material for developing superhydrophobic coatings to further improve its application value. Therefore, this study focuses on the feasibility of iron ore tailings as superhydrophobic coating materials. In addition, rubber materials have excellent high elasticity and wear resistance. Compared with epoxy and polyurethane adhesive, rubber adhesive can not only effectively improve the durability of superhydrophobic coatings but also provides enhanced elasticity to the coating, allowing the coating to better dissipate wear. Milionis et al. [ 28 ] first sprayed a liquid rubber adhesive (Plasti Dip™) on the aluminum substrate as a primer, then sprayed a polymer-MWCNTs mixed solution to enhance the thermal stability of the coating, and finally sprayed acrylonitrile buta-diene benzene. The mixed solution of ethylene rubber and hydrophobic fumed silica was used as the hydrophobic layer. After heat curing, a superhydrophobic coating was obtained. The strong adhesion from the binder made the coating resistant to 1700 linear wear cycles at 20.5 kPa, and the presence of multi-walled carbon nanotubes made it resistant to high temperatures of 42 °C. Wimalasiri et al. [ 29 ] first mixed polychloroprene adhesive and ethyl acetate to prepare adhesive solution, then the solution was sprayed on the surface of vulcanized natural rubber, and then superhydrophobic coating solution made of organosilicon sealant, organically modified silica aerogel and turpentine oil was sprayed. After thermal curing, superhydrophobic coating with good durability was prepared. Therefore, using rubber as adhesive is an important measure to improve the durability of superhydrophobic coatings. Based on the above discussion, in this study, the industrial waste iron ore tailings after ball milling are used as a skeleton material, followed by modifying with TEOS and HDTMS to obtain superhydrophobic powders, and using chloroprene rubber solution as adhesive, to prepare the durable superhydrophobic coatings.",
"discussion": "3. Results and Discussion 3.1. Effect of the HDTMS Amount on the Wettability of IOT/POS The effect of the HDTMS amount on the wettability of IOT/POS is shown in Figure 2 (TEOS amount was 6.5 mmol and reaction time was set to 4 h). In the absence of HDTMS, the CA value of the modified IOT powder was 60.9 ± 1.4°. On increasing the HDTMS amount, the CA values of the powder presented an initial increasing trend, followed by stabilization, and the SA values gradually decreased and eventually became stable. On adding 2.5 mmol HDTMS, the CA value increased to 157.3 ± 0.6°, whereas the SA decreased to 5.9 ± 0.8°. On further increasing the amount of HDTMS, the change in the CA and SA values of the powder tended to be slow. Therefore, an amount of 2.5 mmol HDTMS was selected for the subsequent studies. 3.2. Effect of the Reaction Time on Wettability of IOT/POS The influence of reaction time on the wettability of the IOT/POS powders is shown in Figure 3 (TEOS amount was 6.5 mmol and HDTMS amount was 2.5 mmol). On increasing the reaction time, the CA value of the powder first increased and subsequently stabilized, whereas the SA value first declined and gradually tended to be stable. The CA and SA values reached 157.3 ± 0.6° and 5.9 ± 0.8° after 4 h. The wettability of the powder tended to be stable with the reaction time. As the reaction time increased, the silane amount was also enhanced through hydrolysis and condensation, thus, resulting in a high degree of IOT surface coating modification and enhanced hydrophobic characteristic of the generated powder. After 4 h, the coating modification of IOT was basically complete, and further extending the reaction time had a little effect on the wettability of the powder. Considering the wettability trend and time, 4 h was chosen as the optimal reaction time for subsequent experiments. 3.3. Effect of IOT/POS Powder Concentration on Wettability of Composite Coatings Figure 4 shows the relationship between the concentration of IOT/POS powders and wettability of the superhydrophobic coatings. The CA value of pure CRS coating was 74 ± 1.3°. The value was observed to be significantly increased, whereas the SA value decreased, with the increase of IOT/POS powders concentration. For a concentration of 0.06 g/mL, the CA value of the coating was 159.2 ± 0.5°, whereas the SA value was determined to be 5.5 ± 0.8°. However, on further increasing the concentration of IOT/POS powders, the CA value of the coatings was reduced, whereas the SA value was increased. Therefore, the composite coatings prepared with 0.06 g/mL IOT/POS powders were selected for subsequent studies. 3.4. Durability Testing of Composite Coatings One of the challenges for practical application is the inferior durability of the superhydrophobic surfaces. Most artificially fabricated superhydrophobic surfaces swiftly lose their superhydrophobicity when exposed to the harsh mechanical conditions [ 32 , 33 ]. Thus, sandpaper abrasion and tape peeling tests were carried out to analyze the durability of the superhydrophobic composite coatings. The superhydrophobic behavior of the composite coatings as the number of abrasion cycle is shown in Figure 5 a. Owing to the sandpaper abrasion, the roughness of the coating increased in the beginning, leading to a slight increment in the CA value. As the abrasion cycles reached 50, the CA value of the composite coating reduced to 151.1 ± 1.3°, whereas the SA value increased to 9.4 ± 0.7°. Figure 5 b shows the variation of the superhydrophobicity of the composite coating during the tape peeling test. After 15 cycles, the CA value of the composite coating decreased to 151.1 ± 1.3°, whereas the SA value increased to 11.0 ± 0.5°. These results indicated that the superhydrophobic composite coatings exhibited an excellent durability. 3.5. Analysis of Surface Energy and Surface Topography Changes The micro/nano scale rough structure and low surface energy are the two key factors for obtaining a superhydrophobic surface [ 34 , 35 ]. In order to explore the changes in the surface energy of IOT before and after modification as well as after adding CRS to the coating, the surface energy of IOT, IOT/POS, and composite coating was calculated according to Owens’ theory, as shown in Equations (1)–(3): (1) γ s = γ s p + γ s d \n (2) γ l = γ l p + γ l d \n (3) ( cos θ + 1 ) γ l = 2 ( γ s d γ l d ) 1 / 2 + 2 ( γ s p γ l p ) 1 / 2 \nwhere, γ s is the surface energy of the solid;\n γ s p \nand γ s d \nare polar and nonpolar sections of γ s , respectively; γ l is the surface energy of the liquid, γ l p and γ l d are polar and nonpolar sections of γ l , respectively; and θ is the CA value of the test liquids. Choosing diiodomethane and water as the typical liquids ( Table 1 ), the surface energy of IOT, IOT/POS and composite coating was calculated as 80.1 mN/m, 35.4 mN/m, and 30.7 mN/m, respectively. Thus, the surface energy of the modified IOT was obviously reduced, and the addition of CRS further reduced the surface energy. It can be observed from the previous experiments that changing the HDTMS amount affects the wetting performance of the IOT/POS powders, attributed to the changes in the surface microstructure. Figure 6 a is the SEM image of IOT prior to modification. IOT after ball milling was observed to be broken into multi-shaped particles. Figure 6 b,c presents the SEM images of modified iron tailings after the addition of HDTMS (1.5 mmol and 2.5 mmol, respectively). As observed, the surface of IOT became rough after modification. This was attributed to the hydrolysis and condensation reaction of TEOS under alkaline conditions, leading to the generation of SiO 2 nanoparticles. These nanoparticles were irregularly stacked on the surface of IOT at a micrometer scale to form the micro/nano composite structure. For a HDTMS amount of 2.5 mmol, the SiO 2 nanoparticles were more uniformly coated on the surface of IOT. This indicated that a high amount of HDTMS could induce the SiO 2 nanoparticles to aggregate on the surface of IOT, imparting the IOT/POS powders a high surface roughness. The observed phenomenon could be attributed to the superior wettability. The previous experiments also prove that the concentration of IOT/POS superhydrophobic powders has a significant effect on the wettability of the composite coatings. This is also related to the changes in the surface microscopic topography. Figure 7 presents the SEM images of the composite coatings prepared using different addition concentration of IOT/POS powders. As shown in Figure 6 a, the surface of the CRS coating was smooth with no rough structure, thus, indicating that the rough surface required for the superhydrophobic coating could not be provided by spraying the CRS reagent solely. However, as can be seen from Figure 7 b–e, the addition of IOT/POS powder directly led to the formation of the micro-nanoscale composite structure on the surface. The micro-nanoscale composite structure was led by the cross-linking of CRS and IOT/POS powders during the curing process. At a high concentration of the IOT/POS powder, as shown in Figure 7 d,e, the IOT/POS particles were observed to be prone to agglomeration, thus, producing holes of different sizes and depths on the coating surface. The unique micro-nano structure and holes can capture a large amount of air and reduce the solid–liquid contact area, thus, improving the wetting performance of the coating. At a low concentration of the IOT/POS powders, as shown in Figure 6 b,c, a majority of the IOT/POS particles were completely wrapped by CRS, and the coating surface had only a few holes. Further, the ability to capture air lessened, thereby resulting in a smaller contact angle of the coating. However, the difference between Figure 7 d,e was not obvious. In order to further explore the reasons for the change in wettability, the surface roughness of the composite coatings with IOT/POS concentration of 0.06 g/mL and 0.08 g/mL were measured by optical profilometer. The result is shown in Figure 8 . Results showed that when the IOT/POS concentration is 0.06 g/mL, the average roughness value of the coating is 3.471 μm. The average roughness value of the composite coating with an IOT/POS concentration of 0.08 g/mL is 2.619 μm. This indicates that the increased concentration of IOT/POS powders leads to a decrease in the average surface roughness of the coating, thereby resulting in a decline in wettability of the coating. To further explore the reasons for the excellent durability of the coating, the morphology of the coating surface after abrasion was observed with SEM. Figure 9 a,b presents the low and high magnification SEM images of the composite coating surface after 1000 cm wear distance, respectively. It can be seen that the macrostructure was partially destroyed from the low magnification SEM image. The high magnification SEM image of the coating surface demonstrated no obvious change in the micro/nano structure, which was the key reason explaining the retention of superhydrophobicity of the composite coatings after abrasion. It indicated that the cured CRS contributed toward fixing the IOT/POS particles, thus, preventing the microstructure of the coating surface from being damaged during the friction process. In addition, strong adhesion between the coating and substrate was also generated, thus, the coating did not fall off during the abrasion processes easily. 3.6. FT-IR Analysis The surface wetting behavior is not only affected by the surface microstructure, but also by the surface functional groups. The FTIR spectra, as shown in Figure 10 , were used to analyze the changes in the functional groups. For the raw IOT, characteristic peaks were observed at nearly 3308 cm − 1 and 1604 cm − 1 attributed to the O-H bond. As for the IOT/POS powders, new peaks at 2923 cm −1 and 2850 cm −1 were attributed to the -CH 3 and -CH 2 groups, respectively, which originated from the HDTMS modifier. In addition, the peaks at 1089 cm − 1 and 797 cm − 1 were attributed to the Si-O-Si bond. The appearance of the new peaks indicated that IOT was successfully modified with POS. Moreover, it was noted from the FTIR spectra of the superhydrophobic coating that the C=C stretching vibration peak from chloroprene rubber appeared at 1658 cm − 1 . Further, the characteristic peaks of the C=O group appeared at 1791 cm − 1 and 1724 cm − 1 , which was due to the reason that the (CH=C)-Cl structure of CRS combined with oxygen to produce oxygen-containing groups during the curing process. These results indicated that the IOT/POS powders could be effectively combined with CRS. In addition, grafting of CH 3 and CH 2 from the long-chain alkyl groups in HDTMS endowed the IOT/POS powders and superhydrophobic composite coatings with low surface energy. 3.7. Self-Cleaning Test Self-cleaning is a unique feature for superhydrophobic surfaces that automatically cleans surface dirt with water. In order to investigate the self-cleaning properties of superhydrophobic composite coating that we obtained, we deposited red attapulgite as a contaminant on the coating surface, with a slight inclination angle ( Figure 11 a). When water droplets dyed by methyl blue rolled down from the coating surface, it was found that the water droplets retained the round shape and washed away the contaminants ( Figure 11 b). This is related to the low surface energy of the coating. After rinsing with water, the surface was clean ( Figure 11 c). The above results demonstrate that the composite coatings we obtained have good self-cleaning property."
} | 4,824 |
31749767 | PMC6843077 | pmc | 7,453 | {
"abstract": "A constitutive, host-specific symbiosis exists between the aboveground fungal endophyte Epichloë coenophiala (Morgan-Jones & W. Gams) and the cool-season grass tall fescue ( Lolium arundinaceum (Schreb.) Darbysh.), which is a common forage grass in the United States, Australia, New Zealand, and temperate European grasslands. New cultivars of tall fescue are continually developed to improve pasture productivity and animal health by manipulating both grass and E. coenophiala genetics, yet how these selected grass-endophyte combinations impact other microbial symbionts such as mycorrhizal and dark septate fungi remains unclear. Without better characterizing how genetically distinct grass-endophyte combinations interact with belowground microorganisms, we cannot determine how adoption of new E. coenophiala -symbiotic cultivars in pasture systems will influence long-term soil characteristics and ecosystem function. Here, we examined how E. coenophiala presence and host × endophyte genetic combinations control root colonization by belowground symbiotic fungi and associated plant nutrient concentrations and soil properties in a 2-year manipulative field experiment. We used four vegetative clone pairs of tall fescue that consisted of one endophyte-free (E−) and one E. coenophiala -symbiotic (E+) clone each, where E+ clones within each pair contained one of four endophyte genotypes: CTE14, CTE45, NTE16, or NTE19. After 2 years of growth in field plots, we measured root colonization of arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE), extraradical AMF hyphae in soil, total C, N, and P in root and shoot samples, as well as C and N in associated soils. Although we observed no effects of E. coenophiala presence or symbiotic genotype on total AMF or DSE colonization rates in roots, different grass-endophyte combinations altered AMF arbuscule presence and extraradical hyphal length in soil. The CTE45 genotype hosted the fewest AMF arbuscules regardless of endophyte presence, and E+ clones within NTE19 supported significantly greater soil extraradical hyphae compared to E− clones. Because AMF are often associated with improved soil physical characteristics and C sequestration, our results suggest that development and use of unique grass-endophyte combinations may cause divergent effects on long-term ecosystem properties.",
"conclusion": "Conclusion In this study, we found that although tall fescue symbiotic genotype and aboveground E. coenophiala symbiosis did not significantly alter total colonization by belowground AMF or DSE, they did affect the abundance of specific AMF structures such as arbuscules in roots and extraradical hyphae in soils. Tall fescue genotypes differed in their inclination to form nutritional symbioses with AMF, while E. coenophiala presence appeared to indirectly alleviate AMF stress, indicated by decreased vesicle production, potentially through stimulatory effects on tall fescue biomass or resource availability. Endophyte symbiosis significantly decreased the length of ERH in mesic conditions, perhaps through reduction in plant C allocated to AMF, but only in one tall fescue genotype. Here, we have demonstrated that both host-symbiont genetic variation and E. coenophiala symbiosis in tall fescue result in different plant and endophyte interactions with AMF, resulting in different AMF structures and potentially altering the functional role of these belowground symbionts. In this mesic environment, development of new commercial grass cultivars based on relatively small genetic differences in plants and constitutive aboveground fungal symbionts may have long-term effects on soil properties and ecosystem functioning.",
"introduction": "Introduction Cool season grasses of the family Poaceae form the basis of many agricultural grasslands worldwide that are used to support grazing livestock, and substantially contribute to the over 178 million ha of pastureland in the US alone ( United States Department of Agriculture National Agricultural Statistics service [USDA-NASS], n.d. ). Many of these grasses naturally form host-specific symbioses with endophytic Epichloë spp. fungi ( White, 1987 ; Leuchtmann, 1993 ; Clay and Schardl, 2002 ; Schardl et al., 2004 ). In the United States, researchers estimate that over 75% of the widespread perennial forage grass tall fescue ( Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort = Festuca arundinacea Schreb.) naturally contains the asexual symbiotic endophyte Epichloë coenophiala (Morgan-Jones & W. Gams) [ = Neotyphodium coenophialum (Morgan-Jones & W. Gams) = Acremonium coenophialum Morgan-Jones & W. Gams] ( Shelby and Dalrymple, 1987 ; Leuchtmann et al., 2014 ; Banfi et al., 2017 ). The interaction between tall fescue and E. coenophiala is a constitutive mutualistic symbiosis in which the asexual fungus is only transmitted vertically within seeds ( Schardl et al., 2004 ). E. coenophiala can benefit its grass host through increased forage production, seed dispersal, drought tolerance, and protection from pathogen damage ( Clay, 1988 ). One of the primary contributions to this symbiosis is that E. coenophiala can produce bioprotective compounds including ergot, loline, peramine, and indole diterpene alkaloids to deter mammalian and insect herbivory ( Bush et al., 1997 ; Clay and Schardl, 2002 ). Yet, herbivore-deterring ergot alkaloids produced by the most common E. coenophiala strain found in the United States also causes toxicity symptoms such as reduced reproductive success and increased susceptibility to heat stress in grazing livestock ( Schmidt and Osborn, 1993 ). Many populations of tall fescue and related grasses in their native habitats across Europe and North Africa ( Gibson and Newman, 2001 ) harbor asexual Epichloë strains that do not specifically produce these livestock-toxic ergot alkaloids, yet still defend hosts against insect herbivores and pathogens using similar alkaloid compounds such as lolines and peramine ( Johnson et al., 2013 ). Researchers have isolated many of these Epichloë strains, now deemed non-toxic endophytes or novel endophytes (NTE), and inserted them into improved tall fescue cultivars for agricultural use to circumvent livestock stress and loss of cattle productivity caused by common toxic endophyte (CTE) strains ( Bouton et al., 2002 ; Johnson et al., 2013 ). Although frequently classified as either CTE or NTE tall fescue, breeding efforts and natural selection have resulted in endophyte strains that differ in alkaloid production patterns, even within these CTE and NTE categories ( Takach and Young, 2014 ; Young et al., 2014 ). Forage breeding programs have produced myriad genetically distinct grass-endophyte combinations, some of which eventually result in commercially available fescue seed. Examples include the combination of Jesup and GA-5 tall fescue cultivars containing the selected AR542 endophyte strain, commercially available as Jesup and GA-5 MaxQ tall fescue ( Bouton et al., 2002 ), or insertion of the non-livestock toxic endophyte strain AR584 into Texoma tall fescue which created Texoma MaxQ II ( Hopkins et al., 2011 ). Multiple additional cultivars of tall fescue containing selected NTE strains are commercially available and increasingly planted and evaluated for their use in forage livestock production (e.g., Kenyon et al., 2019 ), and researchers are continually developing new methodology to increase the efficiency of selecting and manipulating E. coenophiala strains to develop new grass-endophyte combinations (e.g., Ekanayake et al., 2017 ; Hettiarachchige et al., 2019 ). Considering the large acreage of natural and agricultural land occupied by both NTE and CTE-symbiotic tall fescue (NTE+ and CTE+, respectively), these associations can have potentially large impacts on ecosystem properties and functions such as plant community dynamics, above- and belowground herbivore activities and soil food webs, and nutrient cycling ( Rudgers and Clay, 2007 ; Omacini et al., 2012 ). For example, CTE+ tall fescue can steadily dominate plant communities or forage mixtures over time due to herbivore deterrence and enhanced competitive ability ( Clay, 1996 ; Clay and Holah, 1999 ; Iqbal et al., 2013 ). Beyond its effects on aboveground plant communities, CTE+ tall fescue can reduce decomposition rates, increase C accumulation over time and alter soil microbial communities ( Franzluebbers et al., 1999 ; Siegrist et al., 2010 ; Iqbal et al., 2012 ). Less is known about NTE+ tall fescue effects on plant communities and belowground dynamics. NTE+ tall fescue has demonstrated fewer negative effects on surrounding plant diversity than CTE+ tall fescue ( Rudgers et al., 2010 ), but these effects are not easily generalizable between specific NTE+ strains and tall fescue cultivars ( Yurkonis et al., 2014 ). Specific NTE genotypes also differ in their contributions to soil greenhouse gas emissions and C-cycling dynamics, as well as root exudate composition ( Iqbal et al., 2013 ; Guo et al., 2015 , 2016 ). These genetically distinct effects of different grass-endophyte combinations on both above- and belowground properties will likely have divergent long-term impacts on nutrient cycling and ecosystem productivity. The underlying mechanisms that regulate how various CTE+ and NTE+ tall fescues impact belowground ecosystem properties have not been fully characterized. Potential drivers of these effects may include differences in root architecture and nutrient uptake dynamics ( Malinowski and Belesky, 1999 ; Malinowski et al., 2000 ; Ding et al., 2016 ), and altered rhizosphere chemical profile differences between E. coenophiala -tall fescue associations referenced above ( Guo et al., 2015 , 2016 ). Another potential driver for belowground effects of E. coenophiala symbiosis in tall fescue are context-dependent interactions with other microorganisms within or on plant tissues. The myriad assemblages of bacteria and fungi in plants may or may not be host-specific and behave on a continuum from beneficial to negative outcomes for the host plant ( Carroll, 1988 ; Johnson et al., 1997 ). For example, most land plants including tall fescue can form non-specific root symbioses with soil-borne fungi such as arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) ( Mandyam and Jumpponen, 2005 ; Smith and Read, 2008 ). The exact role and function of DSE is still unclear, although they can improve plant performance ( Newsham, 2011 ). The role of AMF in plants is typically that of a nutritional mutualist, although the relative benefits of this association are context-dependent and vary based on plant and soil resource availability ( Johnson et al., 1997 ). For example, AMF colonization is most beneficial to plant hosts under P-limitation despite some involvement in N-uptake, and varies based on plant functional or taxonomic group ( Hoeksema et al., 2010 ). Commonly examined in agricultural ecosystems for their contributions to improved water and nutrient uptake in hosts ( Augé, 2001 ) and enhancing long-term soil aggregation and C sequestration ( Wilson et al., 2009 ), AMF may also play important roles in stress tolerance and pathogen defense ( Barto et al., 2010 ; Abhiniti et al., 2013 ; Tao et al., 2016 ). Prior work has shown that CTE+ tall fescue can reduce the presence of AMF propagules and spores ( Chu-Chou et al., 1992 ), and lower abundance of the AMF-associated microbial lipid biomarker 16:1ω5cis in soil ( Buyer et al., 2011 ). Colonization and subsequent sporulation of inoculated AMF can also be reduced in CTE+ tall fescue compared to E. coenophiala -free (E−) plants ( Guo et al., 1992 ; Mack and Rudgers, 2008 ). Even in other grasses, addition of CTE+ tall fescue litter can inhibit AMF colonization compared to E− or NTE+ tall fescue ( Antunes et al., 2008 ). Yet, the effects of both CTE+ and NTE+ tall fescue on AMF are inconsistent. Climate change factors such as warming and added precipitation can moderate the effects of distinct CTE and NTE strains in tall fescue on root symbionts such as AMF and DSE ( Slaughter et al., 2018 ). In addition, some studies have found that neither CTE+ nor two genotypes of NTE+ tall fescue (AR542 and AR584) significantly affected root AMF colonization or extraradical hyphae in soil ( Slaughter and McCulley, 2016 ; Kalosa-Kenyon et al., 2018 ), or the abundance of AMF lipid biomarkers in rhizosphere soil samples ( Ding et al., 2016 ). In contrast, Rojas et al. (2016) found that E+ tall fescue regardless of CTE or NTE status increased relative abundance of the AMF phylum Glomeromycota via ITS1 rRNA sequencing but not the AMF lipid biomarker 16:1ω5cis in rhizosphere soil samples. Evaluation of other asexual Epichloë symbionts in cool-season grasses have similarly revealed negative ( Müller, 2003 ; Liu et al., 2018 ), positive ( Novas et al., 2005 , 2009 , 2011 ; Kazenel et al., 2015 ; Vignale et al., 2016 ), or mixed ( Omacini et al., 2006 ; Liu et al., 2011 ; Larimer et al., 2012 ; Kalosa-Kenyon et al., 2018 ) effects on belowground AMF symbionts or microbial communities, with positive effects most prevalent in native or non-agronomic grasses. The inconsistencies described above highlight the need to better characterize the interactions between unique grass-endophyte combinations and belowground root symbionts, especially considering how new selected combinations are continually being developed and planted in agricultural grasslands. Because both aboveground and belowground microbial associations are supported by photosynthetically produced plant C, tradeoffs likely exist in the capacity for a single host plant such as tall fescue to optimally benefit from, and meet the resource needs of, multiple mutualists. For example, previous studies have suggested that differing access to, or availability of, photosynthetically produced C ( Mack and Rudgers, 2008 ) or competition between symbionts for plant C ( Liu et al., 2011 ) may play an important role in mediating interactions between Epichloë endophytes and root fungi such as AMF or DSE. Because of its location in aerial tissues where plant C is initially fixed, as well as its initial presence in seeds rather than colonization from soil, Mack and Rudgers (2008) suggest that E. coenophiala may benefit from both spatial and temporal priority and thus exhibit greater competitive ability for plant resources compared to belowground symbionts. Both plant-fungal genetics and environmental conditions such as nutrient and moisture availability that regulate photosynthetic rates and C allocation between above- and belowground plant tissues are important context-dependent biotic and abiotic factors. These may, therefore, alter interactions between multiple fungal symbionts of grasses such as competition for plant resources, where E. coenophiala may have an advantage in accessing newly fixed plant C. All three of these symbionts are also typically regarded as having mutualistic interactions with host plants, a relationship that is likely to fluctuate between positive or negative outcomes across ecological contexts ( Chamberlain et al., 2014 ). Therefore, we suggest considering this theoretical framework of biotic and abiotic context-dependency to delineate how evolutionarily coupled aboveground symbioses, such as between tall fescue and E. coenophiala , impact concurrent belowground associations with root-dwelling symbiotic fungi across different grass-endophyte combinations is necessary to determine how these agronomically selected plant-microbe interactions will impact future ecosystem functioning. In this study, we examined how E. coenophiala presence and host × endophyte genetic combinations affect root colonization by belowground symbiotic fungi and associated plant nutrient concentrations and soil properties in a 2-year field experiment. We hypothesized that distinct E. coenophiala and tall fescue genotypic combinations would uniquely alter belowground colonization by AMF and DSE, potentially due to varying spatial and temporal priority effects or altered resource competition by different E. coenophiala strains. For example, limited plant C resources under drier conditions that reduce photosynthesis may promote antagonistic relationships between E. coenophiala and root symbionts, where E. coenophiala is better positioned to access C, compared to more synergistic relationships developing in resource-rich conditions where less symbiont competition is experienced. We expected the greatest reduction of AMF and DSE in CTE+ tall fescue due to greater competitive ability of CTE E. coenophiala genotypes for plant resources, and that this effect would be greatest under more stressful environmental conditions when symbiont antagonism might be most likely to occur.",
"discussion": "Discussion Because E. coenophiala and tall fescue genotype interactions may differentially impact signaling and carbon allocation to belowground symbionts, we hypothesized that unique E. coenophiala and tall fescue genotypic combinations would elicit different belowground colonization by AMF and DSE, and that modifying plant photosynthate due to added water would alter these interactions. However, this was only partially supported by our study results. No effects of tall fescue and E. coenophiala genotype were observed on DSE colonization in tall fescue roots ( Table 1 ). This contrasts with prior analysis in Slaughter et al. (2018) that considered only two of the four genotypes used in this study (CTE45 and NTE19) across four climate change treatments and found both significant endophyte symbiosis and warming effects, where DSE colonization was decreased in E+ samples but stimulated by warming. Such discrepancies indicate that the greater variety in CTE and NTE genotypic combinations used in this study created a more variable, less clear effect of E. coenophiala presence on DSE colonization, and demonstrates the importance of examining multi-symbiont interactions within multiple host genotypes. The occurrence of AMF arbuscules was strongly controlled by tall fescue genotype regardless of E. coenophiala presence. Genotype CTE45 exhibited 4–10% less arbuscule colonization than the other three genotypes ( Figure 1 ). Endophyte symbiosis significantly reduced the length of ERH in soils associated with NTE19, but had inconsistent effects in the other three genotypes ( Figure 2 ). Yet, tall fescue genotype had little influence on AMF vesicles, although endophyte presence significantly reduced the occurrence of AMF vesicles. Surprisingly, none of these interactions were modified significantly by reduced shoot biomass and potentially less abundant photosynthate in E− clones due to added moisture ( Figure 4A ). Despite the large difference in biomass between E+ and E− clones under the+ Precip treatment, combined with previous findings that+ Precip reduced photosynthesis rates in E− clones (significant in NTE16; Bourguignon et al., 2015 ), this effect of added precipitation, and presumably reduced C availability, had no impact on belowground fungi or plant nutrients ( Table 1 and Supplementary Tables S1 – S3 ). This directly contradicts our hypothesis regarding increased symbiont antagonism under more C-limited conditions. Similarly, greater root biomass in E+ clones than in E− clones regardless of plant genotype ( Table 3 ) may also have resulted in greater root C availability to belowground symbionts, yet belowground fungal colonization was not altered. Further, the lack of a clear relationship between belowground fungal measurements and plant nutrient or biomass parameters, despite significant endophyte- and tall fescue genotype effects on plant N and P characteristics ( Tables 2 , 3 ), suggests that plant and E. coenophiala genetics more heavily control plant nutrition at this site than belowground nutritional symbionts such as AMF. Previous studies have shown that Epichloë endophyte symbiosis and grass genotype alters P uptake, potentially due to root architecture or metabolic differences ( Cheplick et al., 1989 ; Malinowski and Belesky, 1999 ). We also found increased shoot P in E+ plants, as well as increased shoot N:P in NTE genotypes compared to CTE regardless of endophyte status ( Tables 2 , 3 ), yet none of these effects were related to measured root fungal parameters. Arbuscules are the primary nutrient-transfer interface between host and AMF symbiont, where exchange of photosynthetic plant C for nutrients acquired by AMF, such as N and P, occurs ( Smith and Smith, 1989 ), and are considered a sign of vitality and active nutrient exchange between host and symbiont. As such, arbuscule presence can vary with time according to when nutrient uptake and transfer is demanded by the host plant ( Mullen and Schmidt, 1993 ). Given the linkages between AMF colonization and plant nutrient status (e.g., Nouri et al., 2014 ), we expected to find a relationship specifically between arbuscules and plant nutrients such as N and P. Although we observed tall fescue genotypic differences in occurrence of arbuscules, there were no tall fescue genotype-specific effects on plant nutrients or biomass that might be related to the significant arbuscule reduction in CTE45. There is some evidence that both host and AMF genotypes may interact to determine presence, abundance, and morphology of different AMF structures such as arbuscules ( Demuth et al., 1991 ; Smith and Smith, 1997 ). Our results suggest that some tall fescue genotypes, such as CTE45, are less inclined than others to form nutrient-transfer symbioses with AMF. This contradicts results in Slaughter et al. (2018) , where analysis of only two of the four genotypes used in this study (CTE45 and NTE19) across four climate change treatments revealed significant effects of endophyte presence and added moisture, but no genotype effect on arbuscules. Our results in this study therefore highlight the importance of examining multi-symbiont relationships within a variety of host genotypes, as interactions may become more or less apparent depending on plant genetics and abiotic conditions. AMF vesicles are thought to function as energy storage organs or as resting spores within or between root cortex cells ( Smith and Read, 2008 ), yet little is known about what host or environmental characteristics specifically control vesicle production ( Smith and Smith, 1997 ). Because endophyte presence reduced vesicle presence regardless of symbiotic genotype in this study, the mechanisms producing this effect must be related to characteristics shared by both CTE and NTE strains. Reidinger et al. (2012) found that total concentration of pyrrolizidine alkaloids (senecionine, seneciophylline, jacobine, jacozine and jacoline) was negatively related to AMF vesicle colonization in Senecio jacobaea . Because both CTE and NTE endophytes can usually produce loline alkaloids, it is tempting to suggest that presence of loline alkaloids such as N-formylloline, the dominant alkaloid produced by E. coenophiala ( Bush et al., 1997 ), may have played a role in the endophyte-related reduction in AMF vesicle abundance observed in this study. However, of the four tall fescue genotypes examined, NTE16 does not produce loline alkaloids while the remaining three genotypes vary significantly in the total concentration of lolines produced [NTE19 > CTE14 > CTE45 ( Bourguignon et al., 2015 )]. Because the endophyte effect on vesicles was consistent across genotypes, loline alkaloid production was likely not the dominant causal factor of the response. In Antunes et al. (2008) , presence of CTE+ tall fescue thatch stimulated vesicle production but decreased arbuscule colonization in Bromus inermis compared to E− thatch, suggesting that inoculated AMF were stressed by characteristics unique to CTE+ tall fescue, such as the presence of ergot alkaloids. These results contrast with our study, where vesicles decreased in response to endophyte presence regardless of strain. It is possible that differences in other non-loline alkaloids, or even other non-alkaloid metabolites, shared by both CTE and NTE strains were responsible for decreasing vesicle occurrence, or endophyte-related alteration of plant or mycorrhizal stress in plant tissues. Some research suggests that AMF vesicles may be formed in response to stressful environmental conditions ( Cooke et al., 1993 ; Smith and Read, 2008 ). If endophyte presence improved overall plant vigor and reduced plant or microbial stress, then a reduction in vesicle occurrence might be expected. Differences in plant shoot biomass due to endophyte status were only observed with increased moisture, which significantly reduced shoot biomass in E− plants ( Figure 4A ). This result is counter-intuitive because tall fescue typically prefers moist conditions, and the +Precip treatments increased growing season moisture as intended ( Bourguignon et al., 2015 ; Slaughter et al., 2018 ). Alternative explanations for the reduction in E− shoot biomass with added moisture such as increased pest or disease pressure are possible, but we did not observe symptoms in the field or lab that might account for this effect. Our results are supported by a similar observation in Bourguignon et al. (2015) , where lower photosynthesis rates in E− tall fescue clones were significantly exacerbated by+Precip treatments in NTE16 ( Figure 2B in Bourguignon et al. (2015) . Although endophyte effects were also observed on root and shoot N:P, shoot P, and root weight ( Table 3 ), it is difficult to assess whether these effects indicate improved plant vigor subsequently reducing vesicles, especially given that we observed no strong correlations between vesicle colonization and plant nutrient or biomass measurements (Pearson r < 0.3 in all cases, not shown). Consistent with our hypothesis, both tall fescue genotype and endophyte status influenced the length of ERH in mesic conditions. NTE16 had less ERH than CTE45 and NTE19, but only when endophyte-free. When E+, all symbiotic genotypes had similar ERH levels, which differed from our expectation that NTE symbioses would express intermediate effects compared to CTE and E−. These results contrast with prior studies that have reported a negative effect of CTE+ tall fescue on soil AMF either in terms of lipid biomarker abundance ( Buyer et al., 2011 ), or through interfering with AMF colonization of neighboring plants, potentially through effects on soil hyphae ( Antunes et al., 2008 ). We found significant differences between E+ and E− clones only in NTE19 ( Figure 2 ). Inconsistencies in E. coenophiala effects on soil AMF also conflict with prior reports that E+ plants increased the relative abundance of Glomeromycota in soil regardless of endophyte genetics ( Rojas et al., 2016 ), yet our study did not measure other AMF structures such as multinucleated spores in soil that could contribute to greater detection through sequencing methods ( Viera and Glenn, 1990 ; Bécard and Pfeffer, 1993 ; Marleau et al., 2011 ). The fact that we only detected endophyte-symbiosis effects on ERH in NTE19 also suggests that production of ergot alkaloids, the predominantly recognized difference between CTE+ and NTE+ tall fescue, was not a causal factor in these results, as NTE19 does not produce ergot alkaloids ( Bourguignon et al., 2015 ). Mummey and Rillig (2006) suggest that plants influence extraradical AMF in soils through allocation of C resources to AMF, changes in the rate of hyphal decomposition, or changes in active plant biomass present to support extraradical hyphae. However, we did not find consistent tall fescue genotype or endophyte status-mediated effects on plant nutrients or biomass that would help explain the mechanisms underlying these ERH results, nor were there any strong correlations between ERH and plant nutrient or biomass characteristics (Pearson r < 0.3 in all cases, not shown). One reason that tall fescue genotype and endophyte presence significantly affected occurrence of specific AMF structures and abundance of ERH may have been differences in AMF species colonizing these plants, as different species exhibit different developmental and functional dynamics of AMF structures such as arbuscules and vesicles ( Dodd et al., 2000 ). In addition, although AMF are often morphologically and functionally distinguished by their production of arbuscules and vesicles, these structures are not necessarily found in all AMF species ( Smith and Smith, 1997 ). We were unable to distinguish between AMF species based on structures or to test whether AMF species differences were driving the trends in these data, but future studies should focus on characterizing how AMF community composition and morphological traits are altered due to E. coenophiala symbiosis and host-symbiont genetic variability in tall fescue. Overall, our results suggest that both endophyte symbiosis and tall fescue genotype influence AMF investment in different structures, such as arbuscules, vesicles, and extraradical hyphae. These may lead to divergent long-term responses in ecosystem processes such as nutrient cycling through alterations in presence and functioning of AMF structures such as arbuscules used for nutrient transfer. Although we did not observe immediate effects on soil C or N concentrations in this study, altered abundance of extraradical hyphae in soils could also lead to long term changes in C sequestration ( Miller et al., 1995 ; Wilson et al., 2009 ; Duchicela et al., 2013 ). The results of this study combined with other investigations of aboveground asexual Epichloë symbiosis in grasses on belowground fungal symbioses highlight the complexity of genetic and environmental controls on plant-microbe-soil interactions. Further, our measurements in this study were taken at one time point after 2 years of field growth even though these tripartite relationships are the result of dynamic processes that fluctuate with time and environmental conditions, which means we likely observed only a portion of these complex interactions. We recommend that future studies monitor interactions between these symbionts across multiple timepoints, and further suggest that more consideration be given to potential changes in community composition of root AMF and DSE communities, which may have been altered rather than total colonization but was not measured in this study. Still, our results suggest that differences in both plant and fungal genetics within constitutive symbioses, such as that between asexual Epichloë endophytes and cool-season grasses, may induce subtle shifts in belowground root symbiont associations that subsequently affect long-term ecosystem outcomes. These results potentially indicate that development of new grass-endophyte combinations and use in forage settings may have long-term impacts on soil properties and management. Forage breeders frequently manipulate plant-microbe symbioses to produce myriad genetically distinct grass-endophyte combinations from similar source materials, many of which have been successfully developed into commercial forage cultivars ( Gundel et al., 2013 ; Johnson et al., 2013 ), yet the ecosystem consequences of these selected lines remain unknown. Our results and that of others clearly indicates that there is substantial diversity in the response of belowground fungal symbionts to grass host genetics and aboveground fungal endophyte strains. Given the prevalence of this symbiosis in nature and in human-constructed ecosystems (e.g., lawns), and the importance of belowground symbioses to ecosystem characteristics, a better understanding of the physiological mechanisms driving these interactions and ecosystem consequences is required."
} | 7,990 |
32845442 | PMC7450020 | pmc | 7,454 | {
"abstract": "A polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [P(3HB- co -3HV)], was biosynthesized from biphenyl as the sole carbon source using Alcaligenes (currently Achromobacter ) denitrificans A41. This strain is capable of degrading polychlorinated biphenyls (PCBs) and biphenyl. This proof-of-concept of the conversion of aromatic chemicals such as the environmental pollutant PCBs/biphenyl to eco-friendly products such as biodegradable polyester PHA was inspired by the uncovering of two genes encoding PHA synthases in the A. denitrificans A41 genome. When the carbon/nitrogen (C/N) ratio was set at 21, the cellular P(3HB- co -3HV) content in strain A41 reached its highest value of 10.1% of the cell dry weight (CDW). A two-step cultivation protocol improved the accumulation of P(3HB- co -3HV) by up to 26.2% of the CDW, consisting of 13.0 mol % 3HV when grown on minimum salt medium without nitrogen sources. The highest cellular content of P(3HB- co -3HV) (47.6% of the CDW) was obtained through the two-step cultivation of strain A41 on biphenyl as the sole carbon source. The purified copolymer had ultra-high molecular weight (weight-average molecular weight of 3.5 × 10 6 ), as revealed through gel-permeation chromatography. Based on the genomic information related to both polymer synthesis and biphenyl degradation, we finally proposed a metabolic pathway for the production of P(3HB- co -3HV) associated with the degradation of biphenyl by strain A41.",
"introduction": "Introduction There are currently two biotechnological approaches for managing environmental problems: the bioremediation of target pollutants and the synthesis of eco-friendly products. As representative players of bioremediation approaches, Achromobacter and Alcaligenes spp. contain chromosomally encoded genes for the catabolism of phenol and catechol via the meta -cleavage pathway (Hinteregger and Streichsbier 2001 ; Collard et al. 1994 ). Some species have also acquired additional genes that extend their metabolic capabilities to include the degradation of polychlorinated biphenyls (PCBs) and other halogenated aromatic compounds (Springael et al. 1993 ; Springael et al. 1994 ). The degradation of these cytotoxic molecules typically proceeds through a series of dehalogenations and aromatic ring oxidations that convert the molecule into catechol, after which it enters into the meta -cleavage pathway and undergoes further metabolic conversion to acetate and pyruvate (Hughes and Bayly 1983 ). A PCBs degrader , Alcaligenes denitrificans strain A41 is a Gram-negative, organic solvent tolerant, alkalitrophic, and denitrifying bacterium (Ohta et al. 1996 ; Maeda et al. 1998 ). The taxon of Alcaligenes denitrificans was reclassified as Achromobacter denitrificans (Coenye 2003). In our previous studies (Maeda et al. 1997 ; Tomizawa et al. 2015 ), a set of genes responsible for the metabolism of PCBs and biphenyl was found in the genome of Alcaligenes denitrificans strain A41. While sequencing the whole genome of strain A41, we also found genes related to the synthesis of biodegradable polymers known as polyhydroxyalkanoates (PHAs) at different loci. This finding is in accordance with the fact that strain A41 is related to Alcaligenes eutrophus (currently Ralstonia eutropha ), a known PHA producer (Schlegel et al. 1961 ; Raberg et al. 2018 ). Considering these findings, we aimed to establish a promising platform based on strain A41 for the conversion of PCBs and biphenyl to PHAs through a one-pot fermentation process by combining both degradation and synthetic pathways. In the present study, this proof-of-concept (Fig. 1 ) conversion of xenobiotic compounds into value-added products such as PHAs was studied using nitrogen limitation conditions, as routinely performed for PHA production with R. eutropha . As a result, we achieved the biosynthesis of a PHA copolymer, poly(3-hydroxybutyarate- co -3-hydroxyvalerate) [P(3HB- co -3HV)] from biphenyl as the sole carbon source. The copolymer production was further improved by conducting a two-step fermentation with growth and polymer production phases. Moreover, we evaluated the molecular weight and monomeric composition of the biosynthesized polymer. Finally, we proposed a pathway for P(3HB- co -3HV) production from biphenyl based on our experimental results and on the available genome sequence information. Fig. 1 A conceptual scheme of the conversion of environmental pollutants, such as polychlorinated biphenyls/biphenyl, to eco-friendly products, such as polyhydroxyalkanoates (PHAs)",
"discussion": "Discussion To date, many studies on the utilization of renewable carbon sources have been reported for the production of value-added products such as PHAs (Matsumoto and Taguchi 2013 ; Kourmentza et al. 2017 ). The use of xenobiotic compounds as carbon sources for future bioprocesses would be preferable as a solution for environmental problems. The following aromatic compounds have been reported as environmental pollutants: polycyclic aromatic hydrocarbon (Sangkharaket al. 2020 ) and lignin (Salvachüa et al. 2020 ). In this study, PCBs/biphenyl were chosen as promising targets and PHA production was achieved from biphenyl as the sole carbon source using A. denitrificans A41, which is capable of degrading PCBs/biphenyl. In this study, PHA production by strain A41 was first demonstrated under varying C/N ratio conditions using a one-step cultivation process. The amount of cell mass obtained through this process was low and PHA production was not expressive. Thus, a two-step cultivation process was used in order to improve PHA productivity. In this case, it was important to address the consumption of biphenyl considering two fermentative events: cell growth and PHA production. It is well known that the precise quantification of biphenyl is not easy due to the high volatility of this compound. As shown in Fig. 4 a, biphenyl was constantly consumed for cell growth until 48 h of culture, which corresponded to the beginning of the stationary phase. The consumption rates remarkably decreased after 72 h. Approximately 96% of the added biphenyl was consumed after 108 h. This kinetic transition of biphenyl consumption (from 48 h to 72 h) suggests that cell growth was probably decreased because of the generation of a cytotoxic benzoic acid as an intermediate metabolite. The next aspect is the feasibility of the use of biphenyl for PHA production. The cellular PHA content was 18.3% after 12 h of cultivation under nitrogen limitation conditions (Fig. 4 b). Furthermore, the highest cellular content (47.6%) was obtained by two-step cultivation with biphenyl as the sole carbon source (Fig. 5 ). By controlling culture conditions, the conversion from biphenyl to P(3HB- co -3HV) was achieved by the strain A41. This successful result may indicate that initial cell growth should first be achieved before PHA production, preferably reducing the cytotoxicity of biphenyl. Regarding the molecular weight of the polymer product, much higher values were obtained than those obtained in the other cases such as the conversion of aromatic hydrocarbon styrene to PHA (Ward et al. 2005 ; Tan et al. 2015 ; Arshad et al. 2017 ). It is of interest to address the reason behind this in our microbial conversion system. The biosynthesis of copolymer P(3HB- co -3HV) can also be supported by the amino acid sequence deduced from genes encoding PHA synthase PhaC1, which are possibly categorized into the class I PHA synthase family (Fig. 6 b). Class I PHA synthases could exhibit substrate specificity toward short-chain-length monomers, 3HB and 3HV (Rehm 2003 ). PhaC1 could at least be involved in polymer biosynthesis, as demonstrated by our gene disruption experiment. The precursor CoA forms 3HB-CoA and 3HV-CoA are generated via two condensation reactions: one with two molecules of acetyl-CoA and the other with one molecule of acetyl-CoA and one molecule of propionyl-CoA, respectively (Anderson and Dawes 1990 ). A possible regulator protein (PhaR), encoded downstream to the phaC1 gene, could be a promising target for future PHA biosynthesis strategies. Considering these findings, we propose a hypothetic pathway for PHA production associated with a previously described biphenyl degradation pathway (Brenner et al. 1994 ), as illustrated in Fig. 7 . Biphenyl is converted to 2-hydroxy-6-oxo-phenylhexa-2,4-dienoic acid (HOPDA) by BphA, BphB, and BphC; benzoic acid and 2-hydroxypenta-2,4-dienoic acid (HPDA) by BphD. Among these compounds, HPDA is metabolized to pyruvate and acetyl-CoA, and this pyruvate is further converted to acetyl-CoA by intracellular enzymes. These acetyl-CoA molecules are used to produce polyhydroxybutyrate (PHB) by β-ketothiolase (encoded by phaA ), NADPH-dependent reductase (encoded by phaB ) and PHA synthase (encoded by phaC1 ). Considering the generation of 3HV-CoA, it can be speculated that a possible sequential conversion from succinyl-CoA to methylmalonyl-CoA, and consequently to propionyl-CoA, would be carried out through intracellular metabolism. Finally, 3HV-CoA would be generated by condensation of propionyl-CoA with acetyl-CoA. Accordingly, copolymer P(3HB- co -3HV) could be biosynthesized by the copolymerization of two molecules, 3HB-CoA and 3HV-CoA. Fig. 7 Proposed pathway for PHA production associated with biphenyl degradation. The biodegradation pathway for biphenyl (shown to the left) has already been described (Brenner et al. 1994 ). HOPDA, 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate, is degraded to HPDA, 2-hydroxypenta-2,4-dienoate, and benzoate. HPDA is metabolized to pyruvate and acetyl-CoA, and the pyruvate is further converted to acetyl-CoA through common cellular functions such as that by pyruvate dehydrogenase. Acetyl-CoAs generated here would be used to produce polyhydroxybutyrate (PHB) via β-ketothiolase (encoded by phaA ), NADPH-dependent reductase (encoded by phaB ), and PHA synthase (encoded by phaC1 ). As for the generation of 3HV-CoA, it can be speculated that a possible sequential conversion from succinyl-CoA to methylmalonyl-CoA, and consequently to propionyl-CoA, would be carried out by the intracellular metabolism. Finally, 3HV-CoA would be generated by the condensation of propionyl-CoA with acetyl-CoA. Accordingly, copolymer P(3HB- co -3HV) can be biosynthesized by the copolymerization of two molecules, 3HB-CoA and 3HV-CoA In conclusion, the native strain A41 provided here the prototype of a microbial platform for the production of copolymer P(3HB- co -3HV) by supplying a cytotoxic biphenyl. In the near future, a recombinant technology (Zhou et al. 2020 ) will be applied for the strain A41 platform, in order to improve PHA productivity and alter properties related to molecular weight and monomeric composition."
} | 2,715 |
36185017 | PMC9608157 | pmc | 7,455 | {
"abstract": "Abstract Nutrient stimulation is considered effective for improving biogenic coalbed methane production potential. However, our knowledge of the microbial assembly process for profuse and rare microbial communities in coals under nutrient stimulation is still limited. This study collected 16S rRNA gene data from 59 microbial communities in coals for a meta-analysis. Among these communities, 116 genera were identified as profuse taxa, and the remaining 1,637 genera were identified as rare taxa. Nutrient stimulation increased the Chao1 richness of profuse and rare genera and changed the compositions of profuse and rare genera based on nonmetric multidimensional scaling with Bray-Curtis dissimilarities. In addition, many profuse and rare genera belonging to Proteobacteria and Acidobacteria were reduced, whereas those belonging to Euryarchaeota and Firmicutes were increased under nutrient stimulation. Concomitantly, the microbial co-occurrence relationship network was also altered by nutrient addition, and many rare genera mainly belonging to Firmicutes, Bacteroides , and Euryarchaeota also comprised the key microorganisms. In addition, the compositions of most of the profuse and rare genera in communities were driven by stochastic processes, and nutrient stimulation increased the relative contribution of dispersal limitation for both profuse and rare microbial community assemblages and that of variable selection for rare microbial community assemblages. In summary, this study strengthened our knowledge regarding the mechanistic responses of coal microbial diversity and community composition to nutrient stimulation, which are of great importance for understanding the microbial ecology of coals and the sustainability of methane production stimulated by nutrients.",
"introduction": "Introduction Coal is the most vital fossil fuel on earth ( Sekhohola et al. 2013 ; Iram et al. 2017 ), the value of which is far greater than that of petroleum and natural gas. The formation of coal is driven by geological events ( Emery et al. 2020 ), geologic settings ( Li et al. 2018 ), and microorganisms ( Liu et al. 2019 ). Microbes are the dominant form of life in subsurface ecosystems, including coals, and play vital roles in biogeochemical cycles such as the carbon cycle ( Iram et al. 2017 ). In addition, the synergistic interaction of microbial complexes in coal seams drives the production of a large proportion (20–40%) of global methane reserves ( Thielemann et al. 2004 ; Faiz and Hendry 2006 ; Rathi et al. 2019 ). Therefore, the development of biogenic coalbed methane has gradually attracted more attention, and scholars expect to stimulate the production potential of biogenic methane in coalbeds through various methods, particularly nutrient addition, to improve coalbed methane (CBM) production ( Jones et al. 2010 ; in ’t Zandt et al. 2018). Researchers have mainly focused on the abundance and activity of methanogenic archaea in addition to microbial diversity under nutrient stimulation (in ’t Zandt et al. 2018; Wang et al. 2019b ; Bucha et al. 2020 ; Pytlak et al. 2020 ). These methanogenic groups are the drivers of the final step in degrading organic matter into methane in coal seams ( Vick et al. 2019 ). However, little attention has been given to the process of microbial assembly (including profuse and rare taxa) under nutrient stimulation. This knowledge is of great importance for understanding the microbial ecology of coal seams and judging the sustainability of methane production stimulated by nutrients. Generally, the dominant taxa in microbial community changes have received more attention ( Wu et al. 2017 ). However, microbial taxa with low abundance are often identified as the “rare biosphere”; these taxa represent most of the biodiversity on Earth ( Ji et al. 2020 ), undertake essential ecological functions ( Ji et al. 2020 ), and play vital roles in community function and stability in ecosystems ( Jousset et al. 2017 ). For example, the majority of turnover in community composition was observed in rare taxa in sandy soils ( Gobet et al. 2012 ). These rare microbes also drive anaerobic respiration, such as sulfate reduction ( Pester et al. 2010 ) and respiratory denitrification ( Philippot et al. 2013 ) in anaerobic environments. Thus, understanding the assembly process for profuse and rare microbial taxa is vital for knowledge on microbe-driven biogenic methane production processes in coals. It is generally believed that deterministic and stochastic processes co-occur and control the aggregation of microbial communities ( Chase 2010 ; Chase and Myers 2011 ). Traditional niche theory assumes the dominant role of deterministic processes and holds that deterministic factors, including species characteristics, interspecific interactions, and environmental conditions, determine community structure ( Chesson 2000 ; Fargione et al. 2003 ). In contrast, neutral theory considers that stochastic processes control the aggregation of microbial communities, including birth, death, colonization, extinction, and speciation, which are independent of species characteristics ( Chesson 2000 ; Fargione et al. 2003 ). The importance of stochastic processes in controlling microbial diversity has received little attention until recently ( Zhou et al. 2014 ; Stegen et al. 2015 ). Many studies have found that changes in microbial communities can be driven by stochastic processes such as historical contingency, ecological drift, and dispersal limitations ( Chase 2010 ; Ofiţeru et al. 2010 ; Zhou et al. 2014 ; Evans et al. 2017 ). However, our knowledge of microbial assembly processes in underground environments, particularly in coals and profuse and rare microbial communities in coals under nutrient stimulation. It limits our understanding of the mechanistic responses of coal microbial diversity and community composition to nutrient stimulation. This study extracted 16S rRNA data on coal sample microbial composition under different treatments from the NCBI database and reanalyzed the assembly process of profuse and rare taxa under nutrient stimulation. This knowledge is of great importance for understanding the microbial ecology of coals and the sustainability of methane production stimulated by nutrients.",
"discussion": "Discussion This study assessed the assembly processes of profuse and rare microbial communities in coals under nutrient (such as organic carbon, and nitrogen) stimulation. The analyzed taxonomic unit was used at the genus level to avoid OTU sequence differences caused by various amplified primers. In microbial research on coal seams, including these referenced studies, the most significant attention has been given to groups related to the formation of biogenic coalbed methane ( Szafranek-Nakonieczna et al. 2018 ; Plyatsuk et al. 2020 ). These studies are critical hubs for applying microbial knowledge to practical production. Coal seams are important habitats for the co-existence of underground microbial communities, and improving the activity of functional microorganisms also requires consideration of the relationships between multiple microbial groups. Coal seams possess many bacterial taxa, including Firmicutes, Spirochetes, Bacteroidetes, and Proteobacteria ( Dawson et al. 2012 ; Chen et al. 2018 ). This study found that nutrients have a selective stimulating effect on profuse and rare groups. In addition, these investigated studies highlighted that nutrient addition can effectively accelerate CBM production and that biomethane production is closely related to coal decomposition (in ’t Zandt et al. 2018; Pytlak et al. 2020 ). Therefore, the focus of this study was to identify the core profuse and rare genera stimulated by nutrients, which have potential value in the study of biological CBM. The main components of nutrients added in the surveyed studies were similar ( Davis et al. 2018 ; Detman et al. 2018 ; in ’t Zandt et al. 2018; Su et al. 2018 ; Wang et al. 2019b ; Bucha et al. 2020 ; Pytlak et al. 2020 ), mainly including organic carbon such as tryptone and yeast, ammonia salts, and potassium and sodium salts. The changes in profuse and rare taxa caused by adding nutrients are different for different coal ranks. Most studies also support the finding that the microbial community structure of coals differs among various ranks ( Su et al. 2018 ; Liu et al. 2019 ). In this study, the shifts in the composition of profuse and rare microbial communities caused by nutrients mainly occurred in bituminous and anthracite coals. This result may have been overlooked in previous studies ( Davis et al. 2018 ; Detman et al. 2018 ; in ’t Zandt et al. 2018; Su et al. 2018 ; Wang et al. 2019b ; Bucha et al. 2020 ; Pytlak et al. 2020 ). In addition, the Chao1 richness for lignites was higher when nutrients were added to both profuse and rare genera. This may be related to the selective stimulation of nutrients ( Bucha et al. 2020 ). For example, this study found that Euryarchaeota phylum in profuse and rare taxa may prefer increased NH 4 + , and Firmicutes phylum in profuse and rare taxa may prefer increased organic carbon. Increased organic carbon stimulated the development of the profuse and rare genera associated with Firmicutes in bituminous coals, including the profuse bacterial genera Tissierella_Soehngenia , Bacillus , Clostridium , and the rare bacterial genera Lysinibacillus and Proteiniclasticum . Firmicutes were often detected in coal seams with high microbial abundance ( Midgley et al. 2010 ; Wang et al. 2019b ; Bucha et al. 2020 ), which played a vital role in coal decomposition. These groups were the main active heterotrophic and syntrophic bacterial consortia and dominated kerogen degradation, and the abundance of these fermentation bacteria can even restrict the generation of coal biomethane ( Meslé et al. 2013 ). In addition, it was key that nutrients increased the methanogenic archaea, particularly the profuse archaea genera Methanosaeta and Methanobcterium , in a study covering multiple research areas. The activation of these methanogens directly affects the yield increase of biogenic coalbed methane ( Lupton et al. 2020 ; Pytlak et al. 2020 ). Coal quality properties can directly restrict microbial community structure ( Meyer et al. 2018 ; Plyatsuk et al. 2020 ), which was also evident in this study ( Fig. 5a and 5b) . However, not all coal quality properties can influence profuse and rare microbial communities under nutrient stimulation ( Fig. 5c and 5d) , and the main components of nutrients added in the surveyed studies restricted the assembly process of profuse and rare microbial communities ( Fig. 5e and 5f) . Little difference in microbial coal structures of the ck group was found among multiple regions, indicating that the microbial coal structures in different areas have high similarity at the genus level. Nutrients stimulated the profuse and rare microbial Chao1 richness and affected community structure, and the average Bray-Curtis dissimilarity of community composition in the nutrient group was significantly greater than that in the ck group. This result indicated that nutrient deficiency, particularly in available organic carbon, NH 4 + , and Na + , may be an important factor limiting the development of microorganisms in coal seams. Once the nutrients in coal seams are supplemented, the change in the microbial community may have undergone spatial niche partitioning ( Vick et al. 2019 ). The positive interaction in a co-occurrence network was mainly regarded as cooperation ( Ju et al. 2014 ). In this study, there were more positive connections (profuse-profuse, rare-rare, and profuse-rare) than negative connections, and the number of negative connections in the treatment group was lower than that in the ck group. In coals, the interactions between microorganisms might be an important factor in maintaining the stability of underground communities ( Abreu and Taga 2016 ). Frequent cooperation within profuse and/ or rare taxa may contribute to community resilience in changing environments because of the buffering function of the interaction network among microbes against environmental disturbances ( Konopka et al. 2015 ). In addition, nutrients can enhance the interaction between rare taxa, including archaea (particularly methanogenic archaea) and bacteria with profuse taxa, which may be another potential factor influencing yield enhancement of biogenic coalbed methane. The process of biological methane production in coals requires the collective action of microorganisms involving at least three major metabolic groups, including hydrolyzing and fermenting bacteria, hydrogen- and acetogen-producing bacteria, and methanogenic archaea ( Wang et al. 2018 ; Vick et al. 2019 ). To our knowledge, bacteria attach to the surface of the coal seams ( Vick et al. 2016 ; McLeish et al. 2021 ) and drive the anaerobic fermentation of these organic materials in coal seams ( Strąpoć et al. 2008 ; Penner et al. 2010 ). Methanogens also require bacterial partners to depolymerize and oxidize complex organic molecules into simple fermentation products (CO 2 , H 2 , acetate, formate, or other compounds). For methanogenic archaea in coal seams, symbiosis and aggregation with bacteria may be the main factor impacting their survival and sustainable methane production in coal seams ( He et al. 2020 ). Stochastic processes drive the most rich and rare communities in coals. Similarly, in many cases, microbial community changes may occur due to stochastic processes in communities via historical contingency (such as priority effects), ecological drift, and/or dispersal limitation ( Chase 2010 ; Ofiţeru et al. 2010 ; Zhou et al. 2014 ; Evans et al. 2017 ). In previous experiments adding nutrients directly affected the carbon and nitrogen in the coal environments and caused changes in the microbial community. Thus, intuitively, the microbial community structure governed by environmental conditions such as the nutrients in this study should be referred to as deterministic processes ( Fargione et al. 2003 ). It is despite the nutrient group increasing dispersal limitation (a stochastic process) for profuse and rare microbial community assembly and only increasing the variable selection (a deterministic process) for rare microbial community assembly. A previous study considered that stochastic processes could play more important roles than the functional differences of species in community pattern generation ( Zhou and Ning 2017 ). The samples selected for this study came from coal seams in different regions, and dispersal limitation is the most important factor shaping large-scale biogeographic patterns ( Hanson et al. 2012 ; Meyer et al. 2018 ). In addition, the increased contribution of variable selection by nutrient stimulation in the rare community suggested that heterogeneous abiotic and biotic factors, particularly chemical properties, can impose selective solid pressure by filtering rare species ( Li et al. 2021 ) and drive changes in rare community compositions ( Bottos et al. 2018 ). Nutrients have been demonstrated to drive a highly deterministic process for rare groups in various ecosystems and influence the diversity of rare microbial communities ( He et al. 2018 ; Guo et al. 2020 ; Cao et al. 2021 ; San Roman and Wagner 2021 ). In conclusion, this study is the first to focus on the assembly processes of profuse and rare microbial communities in coals under nutrient stimulation and showed that dispersal limitation played an important role in changing the profuse and rare microbial communities in coals. Nutrient stimulation intensified the relative contribution of dispersal limitation for both profuse and rare microbial community assemblages. It is the most crucial reason for shifts in microbial community diversity. In addition, nutrients increased the variable selection for rare microbial community assembly and enhanced the role of rare groups in the microbial co-occurrence network. Overall, this study strengthened our knowledge of the mechanistic response of coal microbial diversity and community composition to nutrient stimulation."
} | 4,061 |
26295944 | PMC4546601 | pmc | 7,456 | {
"abstract": "Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.",
"introduction": "Introduction As yeasts have been used for ethanol production for thousands of years they are also considered to be the obvious candidate for lignocellulosic ethanol production. The high ethanol tolerance of yeast and high yields of ethanol obtained in starch or sucrose derived fermentations are unsurpassed by any other microorganism and the combination of a low pH during fermentation and high ethanol titers assist in preventing contamination. Lignocellulosic hydrolysates, however, create a completely different fermentation environment as they contain organic inhibitors, including acetic acid and phenolic lignin degradation products, and significant amounts of sugars that are not naturally fermented by industrial ethanol-producing yeasts. Today, 20 years after the first patent application claiming a xylose-utilizing Saccharomyces cerevisiae was filed [ 1 ], development of yeast strains able to ferment all hexose and pentose sugars in non-detoxified lignocellulosic hydrolysates still remains a major challenge [ 2 ]. For example some sugars are metabolized sequentially due to metabolic regulation and competition for transporters; xylose is still metabolized at a slower rate than glucose and high xylose/glucose ratios can affect the tolerance of yeast to inhibitors. To our knowledge, the combination of an ethanol titre of above 45 g/L and an overall yield of more than 90% of theoretical (0.46 g ethanol per g available sugar, not including arabinose) has not been described using a genetically modified yeast growing on non-detoxified lignocellulosic hydrolysates with no additional sugar additions [ 2 ]. It has been demonstrated that many thermophilic bacteria are highly efficient ethanol producers, with the natural ability to metabolize both pentoses and hexoses found in lignocellulosic hydrolysates. The advantages of using these thermophilic bacteria include the prevention of contamination from mesophilic bacteria and fungi due to high temperature fermentation, energy savings as cooling after enzymatic hydrolysis and during fermentation is avoided and a broad sugar metabolism spectrum that enables the use of almost all sugars in the biomasses [ 3 – 7 ]. It has been shown that the Thermoanaerobacter BG1 can grow and produce ethanol from hemicellulose hydrolysate of wheat straw and corn stover with the same ethanol yield as with synthetic medium [ 4 , 5 ]. To increase the ethanol yield of this organism, lactic acid as well as acetic acid production has been eliminated by knocking out the genes encoding Lactate dehydrogenase, Phosphotransacetylase and Acetate kinase, resulting in the strain Thermoanaerobacter italicus —Pentocrobe 411 (PC 411) [ 8 ]. In this paper, data from continuous fermentation studies on a range of different biomasses using Pentocrobe 411 are presented showing the organism’s ability to grow on non-detoxified complex biomasses with high dry matter concentrations and produce ethanol with high titer, yield and productivity.",
"discussion": "Discussion Lignocellulosic material is potentially the world’s largest source of fermentable sugars for biofuels and chemicals, but to become a significant contributor to the world fuel production, ethanol derived from lignocellulosic material must be produced efficiently and with high yields. Fermentation performance is one of the pieces of this puzzle that must be optimized, not only separately, but also in relation to pretreatment, enzymatic hydrolysis and downstream processing. Over the last 30 years, hundreds of different bacteria and yeasts have been proposed as candidates for fermentation of lignocellulosic biomasses [ 11 – 13 ]. The data presented here demonstrate that Thermoanaerobacter Pentocrobe 411 is capable of achieving high ethanol yields and productivities on a broad range of biomasses including wheat straw, birch, corn cob, sugar cane bagasse, oil palm empty fruit bunches and frond, mixed waste from food production and cardboard in high temperature continuous fermentation systems. In addition, cane syrup and cane molasses were efficiently fermented. Ethanol yields were between 0.47–0.49 g/g (92%-96% of theoretical maximum) when based on glucose, xylose and arabinose and between 0.47 and 0.51 g/g (92%-100% of theoretical) when based on glucose and xylose. As most genetically modified yeasts do not consume arabinose, the most common basis for comparison is glucose and xylose only. The volumetric ethanol productivity varied from 1.2 g/L/h for biowaste to 2.7 g/L/h for cane molasses and the final ethanol concentration achieved varied from 23 g/L (cardboard) to 104 g/L (cane molasses). The difference in the maximal productivity is likely to be caused by the difference in the concentration of inhibitory compounds relative to the sugar concentration. Inhibitors such as acetic acid cause the cells to generate more ATP in order to maintain intracellular pH thereby forcing the cell to produce ethanol at the expense of cell growth [ 14 ]. The reduced cell growth sets a lower limit to the hydraulic retention time allowed if wash-out is to be avoided and, as the reactor is controlled on the basis on sugar conversion, the result is therefore an upper limit to the ethanol productivity. The maximal growth rate of Pentocrobe 411 is approximately 0.3 h -1 in minimal medium with no inhibitors present [ 9 ] and a minimal hydraulic retention time is therefore expected to be in the range of 4–5 hours. The lowest hydraulic retention time achieved in the current fermentations was 12 hours in the relatively low sugar WS80 fermentation, while the highest ethanol productivity was achieved on sugar cane molasses, containing a low concentration of inhibitors and a high concentration of sugar. None of the biomass hydrolysates used in this study were detoxified using e.g. overliming and significant amounts of inhibitors were therefore present. As can be seen from Table 3 , the maximal levels of acetic acid, lactic acid and furfural applied to the fermentations were 6.2 g/L, and 12.3 g/L, and 0.88 g/L respectively ( Table 3 ). 10.1371/journal.pone.0136060.t003 Table 3 Concentration of acetic acid, lactic acid and furfural in the final and most concentrated influents applied to the fermentations (g/L). WS80 WS97-N2 WS10 BI13B CC02-N2 SB10B CA20-1 BW20-2 EFB16-1-N2 FR16-2-N2 CS02B-N2 CM02B-N2 Acetic acid 4.89 6.18 2.65 4.19 4.39 0.39 0.44 1.95 5.02 3.62 0.20 0.74 Lactic acid 0.00 0.00 0.00 0.00 0.37 9.82 12.3 6.81 0.33 0.00 1.76 3.21 Furfural 0.41 0.35 0.88 0.14 0.29 0.69 0.09 0.00 0.67 0.72 0.00 0.00 The names of the biomass influents (WS80, WS97-N2 etc.) relate to the compositions shown in Table 1 . \n Thermoanarobacter italicus Pentocrobe 411 is tolerant to up to 38 g/L (0.46 M) sodium acetate and up to around 0.5 g/L of furfural when this is added to defined medium in batch culture ( S2 Fig , Fig 5 ). However, furan derivatives such as furfural are known to act synergistically with other inhibitory compounds including phenolic compounds, and acetic, formic and levulinic acids [ 14 ] all of which are present in the lignocellulosic biomasses, and the medium may therefore be inhibitory even if acetic acid and furfural are not at a critical level (e.g. Table 3 , WS80). Comparing Table 3 to Fig 5 , the maximum limit to the concentration of pretreated biomass can be explained by the combined inhibitory effect of furfural and acetic acid for the media containing wheat straw, sugar cane bagasse, empty fruit bunch, and oil palm frond. For birch wood, corn cob, cardboard, biowaste, cane syrup, and cane molasses, other inhibitors such as lignin degradation products, lactic acid, waxes, and inorganic salts may be contributing to the inhibitory effect since acetic acid and furfural are both relatively low in the final fermentation influents. Furfural has been shown to be a potent inhibitor of both bacteria and yeast fermentation and the high hydrophobicity of furfural has been shown to lead to cell membrane disruption, causing a reduction in cell replication rate, ATP production, and inhibition of glycolytic and fermentative enzymes in central metabolic pathways [ 14 – 15 ]. Thermoanaerobacter is at least five-fold more tolerant to biomass hydrolysates when grown in continuous fermentation as opposed to batch fermentation ( S3 Fig compared to Table 3 , WS80). This had previously been attributed to immobilization of the organisms in continuous culture [ 4 ], but as the current study has demonstrated high tolerance in non-immobilized reactor systems, this view will have to be revised. HPLC analysis shows non-detectable furfural levels in the reactor effluents indicating that furfural is converted during the continuous fermentation. Thermoanaerobacter pseudoethanolicus 39E has been shown to possess an NADH dependent alcohol dehydrogenase that reduces furfural to the less toxic furfuryl alcohol leading to reduced toxicity, a pathway that is also known to be present in yeast and other bacteria [ 16 ]. In batch culture, the microorganism will be presented with the full concentration of furfural from the beginning of the fermentation whilst in the continuous fermentation the biomass medium is gradually introduced allowing the cell to increase the expression of the relevant alcohol dehydrogenase and thereby to prevent the full effect of the furfural. Increasing reactor ethanol concentration beyond 25–30 g/L inhibits cell growth and nitrogen sparging was therefore introduced when fermenting on medium with sugar concentrations above 70–75 g/L to increase the natural evaporation of ethanol in the high temperature fermentations. In industrial fermentations, nitrogen sparging may be economically prohibitive and alternative solutions may be used instead. Because of the high temperature of the fermentation, only a modest vacuum would be necessary to remove significant amounts of ethanol from the broth for instance in a recirculation loop connected to the reactor. Pentocrobe 411 has been shown to be tolerant to both vacuum and to increased pressure as long as anaerobic conditions are maintained. The high temperature of the fermentations (66°C) is not only beneficial with respect to removal of surplus ethanol but is also important for the prevention of contamination. As shown in the WS97-N2 wheat straw fermentation, influents can be contaminated with for instance lactic acid bacteria leading to decreased yields and productivities. However, the data also show that as soon as the influent was changed, the lactic acid level decreased in the reactor even though no attempts were made to decontaminate the main reactor. Particularly for continuous fermentations it is an important advantage that decontamination of the main fermentor can be reduced to a minimum. This study shows that Thermoanaerobacter Pentocrobe 411 can efficiently convert glucose, xylose and arabinose from a broad range of different biomasses with yields close to the theoretical maximum. The ability of strains of the Thermoanaerobacter genus to co-ferment hexoses and pentoses is well known from literature [ 17 – 19 ]. Being a strictly anaerobic microorganism, Thermoanaerobacter lacks an oxidative pentose phosphate pathway (PPP) for converting hexoses to pentoses. Consequently, any preference for hexose would have compromised the many crucial metabolic processes where pentoses play essential roles [ 18 ]. Here, the co-utilization of glucose, xylose, and arabinose is demonstrated in continuous culture using pretreated lignocellulosic substrates. In media where the glucose concentration is low such as with the x wheat straw pentose fraction (WS80), the conversion of xylose is even observed to exceed that of glucose. The ability to use very different biomasses under similar conditions makes it possible to base a bioethanol plant on a variety of local biomasses which will reduce the plant transport radius and will decrease the risk of local increase in feedstock prices due to limited biomass availability. \n Thermoanaerobacter Pentocrobe 411 is deposited in an open strain collection (DSMZ, Germany) under accession number DSM 29083, to allow a wider research into the possibilities of thermophilic bacteria for ethanol production. As the complicated process steps of lignocellulosic ethanol production are highly intertwined and the current economy of the overall process is just bordering on profitability, testing and collaboration of stains across fields is a necessity for a successful outcome. In contrast to yeast, the major challenge to be overcome for thermophilic bacteria as an industrial fermentation organism is not related to changes to sugar metabolism or to inhibitor tolerance but rather to the demonstration of performance in pilot and demonstration scale of the more complicated continuous fermentation systems."
} | 3,496 |
22457751 | PMC3310061 | pmc | 7,458 | {
"abstract": "Animal groups can show consistent behaviors or personalities just like solitary animals. We studied the collective behavior of Temnothorax nylanderi ant colonies, including consistency in behavior and correlations between different behavioral traits. We focused on four collective behaviors (aggression against intruders, nest relocation, removal of infected corpses and nest reconstruction) and also tested for links to the immune defense level of a colony and a fitness component (per-capita productivity). Behaviors leading to an increased exposure of ants to micro-parasites were expected to be positively associated with immune defense measures and indeed colonies that often relocated to other nest sites showed increased immune defense levels. Besides, colonies that responded with low aggression to intruders or failed to remove infected corpses, showed a higher likelihood to move to a new nest site. This resembles the trade-off between aggression and relocation often observed in solitary animals. Finally, one of the behaviors, nest reconstruction, was positively linked to per-capita productivity, whereas other colony-level behaviors, such as aggression against intruders, showed no association, albeit all behaviors were expected to be important for fitness under field conditions. In summary, our study shows that ant societies exhibit complex personalities that can be associated to the physiology and fitness of the colony. Some of these behaviors are linked in suites of correlated behaviors, similar to personalities of solitary animals.",
"introduction": "Introduction Variation in heritable traits such as morphology or behavior is expected to be constantly removed from natural populations by drift and natural or sexual selection and it is therefore interesting to study the factors that maintain variation [1] , [2] . Behavioral syndromes, defined as the consistency in behavior across different situations and contexts, can explain why behavioral variation is kept. The same behavior may be beneficial in certain contexts but may be maladaptive in other situations (e.g., [3] ). This could result in non-directional selection on distinct behavioral types. For example, aggression can be useful against prey or competitors, but it can discourage or even lead to the premature death of potential mates, as it has been shown in a fishing spider [4] . In addition, being active in the absence of predators may be beneficial, but high activity levels in their presence are often risky [5] . Therefore, detecting correlations between behavioral traits is valuable for understanding how intra-population variation is maintained. Behavioral syndromes have been described in various animal systems, with recurrent correlations between certain behaviors. A common behavioral syndrome is the aggressiveness-boldness syndrome. Aggressive individuals often tend to be more active and they take more risks (e.g., [5] , [6] ). For instance, aggressive male field crickets went faster out of refuge in a novel environment, that is, they were also bolder [7] . Another common behavioral syndrome is related to activity in general. Many behaviors tend to be positively associated with the activity level of an individual, because they are simultaneously affected by the metabolic rate or time constraints [6] , [8] . This should lead to a triplet positive association among aggressiveness, boldness and general activity. Behavioral syndromes in solitary animals are often linked to physiological or life-history traits of the organism, which can help understanding the proximate correlates of behavior. For example, metabolic rate differences are a possible physiological explanation for consistent inter-individual differences in activity and aggression, explaining in part the aggressiveness-boldness-activity syndrome [6] , [8] . While behavioral correlations are well documented for some solitary animals, there is still little evidence for behavioral correlations in animal societies (but see three recent papers: [9] , [10] , [11] ). Using behavioral methodologies typical for solitary animals, these studies deal with research questions relevant to social insects, such as behavioral differences between castes or cooperative behavior of the whole colony. For example, Chapman et al. [9] showed that the patroller caste in Myrmica ants exhibited the common aggressiveness-boldness syndrome, while the brood-carer caste did not. In addition, the behavior of these two castes is correlated on the colony level (i.e., the whole colony is sometimes more aggressive and bold). Interestingly, social groups, as a whole, may differ in behavior, which can affect their success. Wray et al. [11] showed that the defensive response of honey bee colonies was correlated with fitness components (the colony weight). In social insects, selection predominantly acts on the colony level and collective behaviors such as communal defense, networking in foraging and nest construction are expected to be strongly linked to colony productivity. Hence, colony behavior can be shaped by natural selection similar to the behavior of multicellular organisms. We used colonies of the European cavity-dwelling ant, Temnothorax nylanderi , to study consistency, variation and the relationship of four important behaviors: (1) aggression towards an intruder, (2) nest relocation, (3) removal of an infected corpse, and (4) nest reconstruction after partial destruction. These behaviors represent important activities of animal societies in general. Collective defense is typical for many groups, and is evident in many bird species living in groups, as mobbing of predators gets more efficient with colony size [12] . In social insects, aggression is vital in defending the nest and has fitness consequences (e.g., [11] ). Collective movement is an important trait of animal groups expressed by fish schools, bird flocks, locust swarms and social insects (reviewed in [13] ). Nest relocation is a common behavior in many social insect species, which is exhibited when the present nest becomes unsuitable for some reason, such as decomposition of the nesting material in cavity-dwelling ants [14] , [15] or local food depletion in army ants (e.g., [16] ). Other reasons for nest relocation are reproduction – a large colony splits into two parts and one of them leaves, i.e., reproduction by budding [15] – and simply moving into a better-larger nest [17] . Living in groups increases the parasite burden and the risk of infection by contact-transmitted parasites [12] , [18] . Therefore, removal of waste and corpses of dead group members is crucial for colony health [19] . Indeed, waste management has been found in various group-living animals, which live in the same nest sites for longer time periods (e.g., aphids, mites and ants [20] , [21] , [22] ). Nest sites provide a safe environment to raise offspring, but they have to be constructed and maintained, and failure to repair may lead to exposure to external risks, such as predators or parasites [23] . In order to increase defensiveness against intruders, colonies often block or reduce the nest entrance by using soil, sand or wooden pieces (e.g., [14] , [24] , [25] ). Moreover, colonies of the ant genus Temnothorax prefer nest sites with very small entrances so that a single ant could control colony entry [14] . Even before testing behavior under different conditions, testing for repeatability of behavior under the same conditions is a necessary step in characterizing behavioral syndromes and personality [26] . Second, searching for collective colony personality, we looked for positive and/or negative correlations among the four behavioral traits. We predict that similar to the aggressiveness-boldness-activity syndrome in solitary animals, aggressive colonies should be bolder and more active. Therefore, they should show a better performance in the other collective behaviors such as nest reconstruction. However, as ant colonies are also energy limited we expect some behavioral trade-offs. In addition, it is intriguing to relate behavior and personality to fitness components [26] , [27] , and to understand whether different colony personalities result in the same final fitness. We therefore tested how the four behaviors correlate with per-capita productivity as a measure of colony efficiency (e.g., [28] , [29] ). The four documented behaviors are important for colony survival, representing ways to overcome stress and threat. We expect in general a positive effect of the measured behaviors on per-capita productivity. However, we do not expect a perfect match, because the same goal can be achieved in parallel ways, and specializing in one behavior may lead to another becoming superfluous. Immune defense is an important physiological trait in social insects, because frequent interactions of genetically similar individuals lead to a great risk of contagious infections [18] , [19] . The level of immune defense may correlate with inter-colony differences in some behaviors, such as corpse removal and nest relocation. Encounters with infected corpses pose a direct threat to the colony, and the ants should react by increasing their immune defense [22] . However, ant colonies that recognize and remove infectious material from the nest faster and thus show a high social immunity, might be able to invest less in the physiological immune defense. Nest relocation may be triggered by exposure to parasites, but during the move ants and their brood are also vulnerable to infection and predation [19] . Similarly, intruders might increase micro-parasite exposure [18] . We therefore expected that colonies which show a high tendency to expose their members to parasites either during nest migration, nest defense or by failing to reconstruct their nest site should invest highly in their immune function. In contrast, ant colonies that remove infected corpses fast from the nest are expected to show low immune functions. Nest relocation should show the best positive correlation with the immune defense level, because during emigrations all colony members are exposed to the surrounding environment.",
"discussion": "Discussion Behavioral syndromes and personalities/temperament have often been demonstrated for solitary animals, but evidence for syndromes in insect societies or characterization of collective personality are still rare (but see [9] , [10] , [11] ). Our study is one of the first to show collective personality on the colony level. The most important result is the evidence for a collective personality: colonies that defend their nest, either by fighting against intruders more aggressively or by removing infected corpses more efficiently, are less likely to relocate after a disturbance. It fits a common trade-off between competitiveness and emigration tendencies (e.g., [39] , [40] ). The behavioral consistency was the highest for nest reconstruction and relocation, less strong for removal of corpses and non-significant for aggression. This difference is probably related to the level of specialization each activity requires. Interestingly, the immune defense level was correlated with the nest relocation tendency, but with no other behavior, possibly because nest emigration is the only action exposing the whole colony to the surrounding environment. Finally, there is a positive correlation between per-capita productivity and nest reconstruction, suggesting a link between behavior and a fitness component. The trade-off between territory defense, either by defending against intruders (elevated aggression) or parasites (efficient corpse removal), and the tendency to relocate is often shown by solitary animals. We suggest several explanations for this trade-off in ant societies. First, some nests might be considered to be of better quality than others and were therefore fiercer defended and less easily abandoned. Temnothorax colonies easily distinguish between nest types and often move if a better nest is available [14] , [17] . Second, defending the nest implies that ants invested effort and energy, and therefore are reluctant to move out. Third, after colonies fail for some reason to fight back against intruders or to remove potential source of infection, they tend to relocate more readily to another nest, which might be more defensible. This trade-off between defense against intruders and relocation tendency has parallels in solitary animals owning a territory. For example, Cichlid fish males tended less to abandon their territory in the presence of predators if they were defending it before the encounter with predators [41] , and less aggressive Cichlid females were more likely to emigrate than more aggressive ones [42] . Yet, the aggressiveness-relocation trade-off shown here also fits a more general ecological pattern: more competitive animals usually stay while less competitive ones emigrate, e.g., flour beetles [39] , [40] . In related T. longispinosus colonies, aggressive colonies were more often found in dense areas [43] . This suggests that aggressive colonies remain in these dense areas and do not relocate despite frequent disturbances by intruders while less aggressive ones may move away. The consistency in behavior was higher for nest reconstruction and relocation than aggression and removal of corpses. The two latter behaviors are performed by specialist ants, while the two former are a true collective behavior of the whole colony. Therefore, the repeatability of behaviors based on only a few specialist ants is possibly weaker, because specialist ants could have died between trials or on the other hand can also improve their efficiency with trials. Similarly, corpse removal in other social insects is done by few specialists, to minimize the exposure to contagious elements. This behavior may vary according to response thresholds to corpse/waste removal [19] , [22] , [44] . Similarly, aggression and kin discrimination are presumably carried out by a small group of specialist workers [45] , leading to the same pattern of low consistency in behavior. In comparison, nest relocation requires a more coordinated effort: In a related Temnothorax species, one third of the colony recruits nestmates, actively participating in nest relocation [46] . We believe that the link between worker specialization and the consistency in colony behavior is important for further understanding behavioral syndromes in social insects. An interesting future direction would be to increase environmental heterogeneity and look for behavioral consistencies, expecting that consistency would be negatively correlated with environmental heterogeneity in space or time [47] . The immune defense level was positively correlated with the tendency of nest relocation, but not with any other behavior. Further research is required to establish more firmly the relation between immune defense level and relocation tendency, also in interaction with other environmental factors. We suggest that nest relocation is the only tested behavior exposing all colony members to the external environment, while the three other behaviors are carried out by a small fraction of the colony workers. Specialized workers can prevent the exposure of the whole colony to external risks in such cases, but exposure is inevitable during emigration. We suggest that colonies try to behaviorally adjust to the risk, but when not possible, react physiologically. The relationship between different behaviors and fitness components is often taken for granted but is a fundamental issue in behavioral ecology (i.e., behavior is assumed to optimize fitness). Specifically in the field of behavioral syndromes and animal personality, there is a need for a better link with fitness [5] , [26] . Smith and Blumstein [27] reviewed fitness consequences of animal personality, and showed that exploration was positively correlated with animal survival, and aggression increased with reproductive success. In social insects, Wray et al. showed a link between foraging and defense behaviors with productivity and survival [11] , and Modlmeier and Foitzik demonstrated a positive relationship between the within-colony variance in behavior and productivity in the field [43] and in the laboratory [48] . We showed here a possible link between the nest reconstruction behavior and productivity. Social insect colonies often prefer small entrances to their nests, as small entrances are more easily defendable (e.g., [14] ). Nest usurpation of Temnothorax colonies is a common phenomenon, often by other ant species of larger colonies (e.g., [49] ). Presumably to avoid invasions of different natures, Temnothorax species often reduce the entrance further more by accumulating dirt particles [25] . Other social insect species close their entrance in various ways in order to protect the colony against invasions [24] , [50] . But to the best of our knowledge, this is the first study showing a positive correlation of nest reconstruction with some fitness component. Such a link can result from the positive contribution of this behavior to fitness or alternatively may indicate that colonies having more brood relative to workers block their entrance more intensively, because they have more to lose from invasions to their nests than colonies with less brood. The other observed behaviors did not correlate with per-capita productivity. A possible reason may be that after a while in lab conditions, behaviors which enhance survival and productivity under field conditions become less relevant. In general, measuring fitness of insect societies is challenging, because of their complex life cycle and long life span. It is also plausible that colonies would employ different short- and long-term strategies, resulting in various effects on fitness components [29] . Finally, a more experimental approach would be to investigate the suggested trade-off of aggression with nest relocation. Manipulating the nest condition/quality or alternatively inducing different levels of aggression can be a promising approach."
} | 4,545 |
36894632 | PMC10066037 | pmc | 7,460 | {
"abstract": "Members of the bacterial genus Sulfurimonas (phylum Campylobacterota) dominate microbial communities in marine redoxclines and are important for sulfur and nitrogen cycling. Here we used metagenomics and metabolic analyses to characterize a Sulfurimonas from the Gakkel Ridge in the Central Arctic Ocean and Southwest Indian Ridge, showing that this species is ubiquitous in non-buoyant hydrothermal plumes at Mid Ocean Ridges across the global ocean. One Sulfurimonas species, U Sulfurimonas pluma , was found to be globally abundant and active in cold (<0−4 °C), oxygen-saturated and hydrogen-rich hydrothermal plumes. Compared with other Sulfurimonas species, U S. pluma has a reduced genome (>17%) and genomic signatures of an aerobic chemolithotrophic metabolism using hydrogen as an energy source, including acquisition of A2-type oxidase and loss of nitrate and nitrite reductases. The dominance and unique niche of U S. pluma in hydrothermal plumes suggest an unappreciated biogeochemical role for Sulfurimonas in the deep ocean.",
"discussion": "Discussion In this comparative study of Arctic Ocean and global deep-sea microbiota, we identified an uncharacterized aerobic Sulfurimonas species inhabiting non-buoyant hydrothermal plumes and fluids, and subsurface aquifers. The genome of U S. pluma shows substantial genome reduction (17−40%), including the loss of genus-specific functional genes (that is, for denitrification) and acquisition of genes allowing growth in pelagic oxygen-saturated environments. Previous studies described SUP05 as a widespread and dominant chemolithoautotroph in hydrothermal plumes, using both sulfur compounds and hydrogen as energy source 25 , 55 . The ability of U S. pluma and SUP05 to rely on the same substrates for energy to grow and their variable co-occurrence in hydrothermal plumes (Table 2 ) suggest that they could be competitors, as described for marine pelagic redox gradients 19 . Due to the low amounts of substrates (that is, tens to hundreds of nM) and the lack of a redoxcline in non-buoyant hydrothermal plumes, the mechanisms controlling niche partitioning between Sulfurimonas and SUP05 in this environment might be different from those proposed for pelagic redoxclines of the Baltic Sea 19 . Our results suggest hydrogen as an essential energy substrate for the growth of U S. pluma in the studied hydrothermal plumes. This agrees with its dominance in other hydrogen- and metal-rich non-buoyant hydrothermal plumes (Table 2 ). Of note, ‘ Candidatus Sulfurimonas marisnigri’ could grow at atmospheric oxygen concentrations, but only when MnO 2 was supplied as a terminal electron acceptor 10 . It remains to be clarified whether metals and other hydrothermal compounds favour U S. pluma growth and contribute to niche differentiation, and how U S. pluma adapts to growth at temperatures close to the freezing point. Table 2 Comparison of hydrothermal systems and vent fluid types for non-buoyant hydrothermal plumes hosting chemolithotrophic U S. pluma and SUP05 cluster (family Thioglobaceae) Ridge system Vent field Vent site Rock hosting system Fluid type U S. pluma SUP05 Reference % % Mid Atlantic Ridge Logatchev Irina I Ultramafic Hydrogen-metal-rich up to 70 <10 22 , 135 Rainbow − Ultramafic Hydrogen-metal-rich up to 60 <10 23 , 136 , 137 SouthWest Indian Ridge Longqi DFF1, DFF3, DFF5, DFF6 Basalt Sulfide-metal-rich up to 21 <5 24 , 138 Guaymas Basin − − Basalt and organic-rich sediments Methane-ammonia-rich <2 30 34 , 55 Gakkel Ridge Aurora − Basaltic/ultramafic Hydrogen-metal-rich up to 79 a <10 a this study 29 , 58 Polaris − Basaltic Hydrogen-rich <18 a up to 40 a this study 59 Kermadec Arc Macauley, Brothers − Submarine volcano Sulfide-metal-rich <4 up to 80 56 a Based on 16S rRNA sequences from metatranscriptomes (Extended Data Fig. 2 ). Percentages refer to the proportion of 16S rRNA gene sequences in sample from non-buoyant hydrothermal plumes. The global presence of a hydrogenotrophic Sulfurimonas species in transient environments such as non-buoyant hydrothermal plumes opens new paradigms in the microbial ecology of this and other aquatic habitats. So far, it has been postulated that microbes growing in the plume, such as the sulfur-oxidizing Gammaproteobacteria of the SUP05 clade and mixotrophic SAR324 deltaproteobacteria, are derived primarily from ambient seawater 26 , 56 . These microorganisms are also abundant and active in other marine pelagic environments (for example, surface and deep ocean, oxygen minimum zones), suggesting that their habitat is not exclusively hydrothermal plume but is widespread in the oceans 25 . Our results showed that non-buoyant hydrothermal plumes are a suitable environment for the growth of microorganisms typically inhabiting hydrothermal vents such as Sulfurimonas . We suggest that the hydrothermal plume does not act exclusively as a vector for dispersing microorganisms from benthic hydrothermal environments, but it might also support ecological connections between pelagic and seafloor/subsurface habitats. The phylogenetic analysis suggests that the U S. pluma lineage could have been derived from a hydrothermal vent-associated ancestor (probably by sympatric speciation), which acquired higher oxygen tolerance and then spread across the oceans. However, it remains to be further investigated whether U S. pluma originates from vent-associated environments or from background seawater. On one hand, the presence of a very similar ribotype (>99.5% 16S rRNA gene sequence similarity) in hydrothermal plumes across the globe (Fig. 1 ) suggests that the Sulfurimonas cluster, including U S. pluma , is part of the ocean microbial seed bank, and therefore that background seawater might be the source of U S. pluma . On the other hand, it may be that U S. pluma enters into the hydrothermal plumes from populations living on seafloor vent-associated environments, which due to oxygen tolerance have a higher dispersal potential than benthic Sulfurimonas species, resulting in higher global connectivity 17 . Future studies on uncultivated Sulfurimonas species described here will be needed to verify these hypotheses, and to shed light on environmental and ecological forces that shape the connections and composition of microbial communities between different environments such as subsurface aquifers, diffusive flow and hydrothermal plumes."
} | 1,607 |
26656113 | PMC4676022 | pmc | 7,461 | {
"abstract": "Inspired by the dendritic integration and spiking operation of a biological neuron, flexible oxide-based neuromorphic transistors with multiple input gates are fabricated on flexible plastic substrates for pH sensor applications. When such device is operated in a quasi-static dual-gate synergic sensing mode, it shows a high pH sensitivity of ~105 mV/pH. Our results also demonstrate that single-spike dynamic mode can remarkably improve pH sensitivity and reduce response/recover time and power consumption. Moreover, we find that an appropriate negative bias applied on the sensing gate electrode can further enhance the pH sensitivity and reduce the power consumption. Our flexible neuromorphic transistors provide a new-concept sensory platform for biochemical detection with high sensitivity, rapid response and ultralow power consumption."
} | 211 |
38137995 | PMC10745984 | pmc | 7,462 | {
"abstract": "As one of the environmental factors that seriously affect plant growth and crop production, drought requires an efficient but environmentally neutral approach to mitigate its harm to plants. Soil microbiomes can interact with plants and soil to improve the adverse effects of drought. Medicago ruthenica (L.) is an excellent legume forage with strong drought tolerance, but the key role of microbes in fighting drought stress remains unclear. What kind of flora plays a key role? Is the recruitment of such flora related to its genotype? Therefore, we selected three varieties of M. ruthenica (L.) for drought treatment, analyzed their growth and development as well as their physiological and biochemical characteristics, and performed 16S rRNA high-throughput sequencing analysis on their rhizosphere soils to clarify the variety-mediated response of rhizosphere bacteria to drought stress. It was found that among the three varieties of M. ruthenica (L.), Mengnong No.2, Mengnong No.1 and Zhilixing were subjected to drought stress and showed a reduction in plant height increment of 24.86%, 34.37%, and 31.97% and in fresh weight of 39.19%, 50.22%, and 41.12%, respectively, whereas dry weight was reduced by 23.26%, 26.10%, and 24.49%, respectively. At the same time, we found that the rhizosphere microbial community of Mengnong No. 2 was also less affected by drought, and it was able to maintain the diversity of rhizosphere soil microflora stable after drought stress, while Mennong No. 1 and Zhilixing were affected by drought stress, resulting in a decrease in rhizosphere soil bacterial community diversity indices to 92.92% and 82.27%, respectively. Moreover, the rhizosphere of Mengnon No. 2 was enriched with more nitrogen-fixing bacteria Rhizobium than the other two varieties of M. ruthenica (L.), which made it still have a good ability to accumulate aboveground biomass after drought stress. In conclusion, this study proves that the enrichment process of bacteria is closely related to plant genotype, and different varieties enrich different types of bacteria in the rhizosphere to help them adapt to drought stress, and the respective effects are quite different. Our results provide new evidence for the study of bacteria to improve the tolerance of plants to drought stress and lay a foundation for the screening and study mechanism of drought-tolerant bacteria in the future.",
"conclusion": "5. Conclusions We investigated the changes of rhizosphere bacteria in three varieties of M. ruthenica (L.) under drought stress and combined them with the phenotype and 16S high-throughput sequencing results. We screened a variety of M. ruthenica (L.) with outstanding drought resistance, Mengnong No. 2. The results showed that among the three varieties of M. ruthenica (L.), Mengnong No. 2, Mengnong No. 1, and Zhilixing, the increase in plant height decreased by 24.86%, 34.37%, and 31.97%, the fresh weight decreased by 39.19%, 50.22% and 41.12%, and the dry weight decreased by 23.26%, 26.10%, and 24.49%, respectively, after being affected by drought stress. The state of Mengnong No.2 was also optimal in physiological tests. We found that there were some differences in the population structure of soil bacteria in the rhizosphere of different varieties of M. ruthenica (L.). At the phylum level, Proteobacteria was the most abundant. At the genus level, Novosphingobium belonged to the dominant group. We also selected the biomarkers of Mengnong No.2, Mengnong No. 1, and Zhilixing under drought stress, Xanthomonas, Chloroplast, and Caulobacter, respectively. Finally, our experiments showed that drought stress had a greater impact on the composition of the rhizosphere microbial community of M. ruthenica (L.) and that its physiological and biochemical reactions and bacterial enrichment processes were clearly driven by plant genotype. The drought-tolerant cultivar Mengnong No. 2 already had a high abundance of drought-resistant bacteria in the rhizosphere under the condition of sufficient water, while the other two varieties with poor drought tolerance could accumulate a certain abundance of drought-resistant bacteria in the rhizosphere after drought stress. Therefore, drought-tolerant varieties can respond effectively and quickly to drought stress, while varieties with poor drought-stress tolerance need to spend more time adapting to the drought environment, which affects their growth and yield. In this way, beneficial strains can be isolated to improve the drought resistance of M. ruthenica (L.). From the aspect of the interaction between soil microbiome and plants, we have laid an important foundation for exploring the mechanism of plant resistance to drought stress, the screening of drought-resistant bacteria, and the construction of microbiota.",
"introduction": "1. Introduction Water scarcity affects between 100 and 200 million people globally, especially those in developing regions, which are home to about 70% of the world’s drylands, making their lives very difficult [ 1 ]. According to the projected climate change, droughts will become more frequent and severe [ 2 ]. Whereas drought is one of the most important factors affecting crop yields, increasingly drastic climate change and growing water scarcity are challenges to global crop production [ 3 ]. Medicago ruthenica (L.) is widely distributed in northern China and plays an important role in windbreak, sand fixation, soil and water conservation, and natural grassland improvement [ 4 ]. Meanwhile, it is characterized by barrenness tolerance, trampling resistance, and outstanding cold and drought resistance, which can provide excellent genetic resources for pasture grass [ 5 ]. Its high nutritional value and good palatability make it an important forage legume [ 6 ]. In summary, M. ruthenica (L.) has high ecological value, scientific research value, and economic value. Previous studies on drought tolerance in M. ruthenica (L.) have mostly focused on morphology, physiology, and genomics [ 7 , 8 , 9 , 10 ]. However, there are many problems in improving drought tolerance in lentils: traditional breeding methods are time-consuming and labor-intensive, and mutations brought about by mutation breeding are full of uncertainties. The unstable expression of drought-resistant transgenic crops and the public acceptance of transgenic crops have all affected the process of drought-resistant M. ruthenica (L.) breeding and application. In contrast, positive effects from plant–microbe interactions are gradually becoming a viable pathway for drought tolerance [ 11 , 12 ], which is a more effective approach than plant breeding and genetic improvement techniques [ 13 ]. Currently, there is a gap in exploring the effect of rhizosphere bacteria on drought tolerance in M. ruthenica (L.) based on 16S RNA amplicon sequencing technology. Rhizosphere microbiome maintains a symbiotic relationship with plant growth and development. Soil microbiomes that inhabit and symbiose in the rhizosphere of many plants exert a variety of beneficial effects on host plants through different mechanisms, such as nitrogen fixation and nodulation [ 14 ]. At the same time, they can also improve plant growth through siderophore production, the dissolution of insoluble phosphates, and the release of hormones [ 15 ]. Aspergillus flavus CHS1 could significantly increase the chlorophyll content, root–shoot length, and biomass yield of soybean plants under NaCl stress by regulating endogenous plant hormone levels and antioxidant enzyme activities up to 400 mM [ 16 ]. They have also shown that the soil microbiome can effectively increase the nutrient content of crops and improve the quality and quantity of crops by improving soil properties and secreting enzymes [ 17 ]. Yu [ 18 ] et al. significantly increased plant biomass, stem diameter, and plant height by inoculating plants with plant growth-promoting rhizobacteria (PGPR), while the presence of AMF promoted root length and the number of root shoots and reduced root volume, mean diameter, and the root–crown ratio. Singh [ 19 ] et al. studied the structural and functional changes in the root microbiome of 10 plant species of indica rice varieties and found that the α and β diversity indices of the rhizosphere microbiome with host genotypes revealed the changes in the structure of the root microbiome as well as the strong correlation with the host genotypes. In this study, we analyzed the differences in the rhizosphere soil microbial diversity of different M. ruthenica (L.) varieties and their changing rules by examining the growth, physiology, and rhizosphere microbial changes of three varieties of M. ruthenica (L.) under drought stress, revealing the relationship between plant response to drought and the role of key bacteria and providing a theoretical basis for screening and constructing drought-resistant flora of M. ruthenica (L.) and its dry cultivation.",
"discussion": "4. Discussion 4.1. Differences in Growth Response of Materials of Different Genotypes of the Same Plant following Drought Stress In this study, growth indices, physiological and biochemical indices, and the composition of rhizosphere soil bacteria were determined in three varieties of M. ruthenica (L.) under normal watering and drought stress ( Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 ). Numerous studies have shown that changes in habitat will inevitably lead to changes in plant phenotypic values, and the characteristics of the changes can well reflect the effects of environmental factors or the adaptability of this plant to habitat changes [ 27 , 28 , 29 ]. In this experiment, three varieties of M. ruthenica (L.) showed a decrease in plant height and a decrease in fresh and dry weight after drought stress ( Figure 1 and Figure 2 a,b), which was similar to the results of a large number of previous studies. In other words, a certain degree of drought can have a negative impact on the phenotypic values of plants, and the impact of water scarcity is very large, which can reduce the ability of plants to accumulate organic matter and reduce production [ 30 , 31 ]. At the same time, we found that M2 had the least amount of plant height increment and fresh weight and dry weight reduction after experiencing drought stress, indicating that it has the strongest ability to adapt to drought stress aggression. The root–crown ratio in this experiment also showed an increasing trend with drought stress ( Figure 2 d), which may be because drought stress caused the aboveground part of the plant to lose weight due to lack of water and, at the same time, promoted the growth of the belowground part of the plant to better search for water sources, increasing the root–crown ratio. These results indicated that drought stress would cause plants to preferentially allocate photosynthetic products to roots, which had a great effect on promoting the growth of roots. In previous studies, such morphological characteristics have been suggested to improve the drought resistance of plants, which is conducive to their timely response to drought stress [ 32 ]. At the same time, the three varieties of the M. ruthenica (L.) species showed different magnitudes of changes in the physiological indicators ( Figure 3 ) when they were simultaneously increased or decreased, which reflects that drought stress triggers different plant responses depending on the plant genotype [ 33 ]. 4.2. Differences in the Physiological Response of Different Genotypic Materials of the Same Plant Species after Drought Stress When experiencing drought stress, plants adapt to drought not only by altering their epimorphology but also their osmoregulation, as well as increasing their activity of antioxidant enzymes. These are important ways for plants to cope with drought [ 34 ]. The results of this study showed that both Pro and CAT, excepting MDA, showed an increasing trend under drought stress ( Figure 3 ), which indicated that M. ruthenica (L.) can resist damage by increasing their osmoregulatory substance content and antioxidant enzyme activities under drought stress [ 35 , 36 , 37 ]. At the same time, we can find that before being subjected to drought stress, there was no large Pro and CAT content in the three varieties of M. ruthenica (L.) difference, but after drought stress, M2 had the highest Pro and CAT content. Pro has the ability to scavenge reactive oxygen species; CAT can effectively scavenge hydrogen peroxide in plants and catalyze the decomposition of H 2 O 2 , which is a key functioning enzyme for plants to protect themselves from the toxicity of H 2 O 2 , indicating that its ability to resist drought stress was stronger. The more MDA accumulates in the plant, the more it can reflect the degree of damage brought by environmental stress to the plant [ 38 ]; this experiment also showed that M2 had the least amount of MDA accumulation and had a highly significant difference with M1 and ZL. It can also be seen that M2 has an excellent antioxidant capacity, which may evince the better drought resistance ability of M2. 4.3. Differences in Rhizosphere Bacteria of Different Genotypic Materials of the Same Plant Species after Drought Stress The diversity of the bacterial community in the non-rhizosphere soil was significantly different from the diversity of the bacteria in the inter-rhizosphere soil of each variety of M. ruthenica (L.) ( Figure 4 , Figure 5 c,d and Figure 6 a,b). At the same time, the experimental results showed that the inter-rhizosphere soil recruited bacteria from the non-rhizosphere soil and that this recruitment was affected by both drought and plant genotypes. Indeed, the non-rhizosphere soil determines the microbial reservoir in the rhizosphere soil. The rhizosphere selects its microbiome from the non-rhizosphere soil, after which the plant selectively recruits certain bacteria to colonize its rhizosphere [ 39 ]. Aira M. [ 40 ] et al. found significant differences in the abundance and diversity of bacteria in the rhizosphere of maize of type su1 and type sh2. In the present experiment, it can be seen that the rhizosphere soil bacterial community composition analysis of M2, M1, and ZL M. ruthenica (L.) had significant differences in their rhizosphere microbial abundance and diversity ( Figure 4 , Figure 5 and Figure 6 ), which suggests that even for the same plant species, the different genotypes create significant differences in the microbial community. This suggests that species-driven bacteria are selectively recruited to the rhizosphere surface to interact with plants [ 41 ]. At the same time, it can be seen that at the phylum level ( Figure 6 a), drought can negatively affect the abundance of Proteobacteria in plant rhizosphere soils, resulting in a downward trend in the abundance of Proteobacteria. However, it is worth noting that the first type of bacteria gathered in the rhizosphere soil of all varieties of M. ruthenica (L.) was Proteobacteria, regardless of normal watering or drought stress. These results indicated that Proteobacteria was the dominant flora in the rhizosphere bacteria of M. ruthenica (L.), which was contrary to Wang K. et al. [ 42 ]. It may be due to the different core bacteria recruited by different plants. In non-rhizosphere soils, it was found that this phylum was not affected by drought stress and did not change much. For the Actinobacteriota phylum, drought brings positive effects, which led to an increase in the abundance of Actinobacteriota phylum in the rhizosphere soil of plants after drought stress, which suggests that drought stress environment is more favorable for the survival of Actinobacteriota phylum, and Actinobacteria have better drought resistance. Also, they can reduce the harmful effects of drought on plants [ 43 ]. At the genus level ( Figure 6 b), we can see that the abundance of Novosphingobium decreases in M2 and M1 but increases in ZL, reflecting the different genotypes of the same plant to recruit the bacterium under drought stress. At the same time, the abundance of Rhizobium in the rhizosphere soil of each variety of M. ruthenica (L.) showed an increasing trend after drought stress, indicating that Rhizobium was more conducive to colonization in an arid environment and helped plants adapt to arid conditions. Rhizobium has been found to increase the growth rate of alfalfa [ 44 ], and it is worth noting that the nitrogen-fixing bacterium Rhizobium was present in large quantities in the M2 rhizosphere soil under normal watering, which may be one of the reasons why the M2 was able to better accumulate aboveground biomass under drought conditions. It has been shown that Novosphingobium is a genus that is enriched every time alfalfa is subjected to drought stress, and it has been speculated to be the core bacteria of alfalfa [ 45 ], whereas in this study, we found that Rhizobium was enriched in large quantities in all varieties of M. ruthenica (L.) after they were subjected to drought stress, and it might be considered as the core bacteria associated with M. ruthenica (L.). Combined with the results of this experiment, we can clearly understand ( Figure 7 ) that the core bacteria recruited by different plants vary greatly, and even the rhizosphere-specific bacteria driven to be recruited by different varieties of the same plant are different. 4.4. Rhizosphere Bacteria’s Growth and Drought Tolerance It is worth noting ( Figure 4 b–d and Figure 5 c,d) that the bacterial diversity of Rhizosphere soil in M1 and ZL decreased after drought stress, and only the value of M2 remained stable. However, the diversity index of the soil bacterial community in the rhizosphere decreased to 92.92% and 82.27%, respectively, due to drought stress in M2 and ZL. We can guess that the rhizosphere soil of M2 has more bacteria suitable for drought conditions. Moreover, after drought stress, the differences between the soil bacterial communities of the three kinds of M. ruthenica (L.) narrowed ( Figure 5 a,b and Figure 6 ) and tended to be similar. This may be because the drought stress eliminated some soil bacteria that were not suitable for drought conditions and left those that were adapted to drought conditions, which resulted in the similarity of the soil bacterial structure among the three varieties of M. ruthenica (L.) rhizosphere. At present, it seems that M2 has a more stable ability to recruit specific bacteria, or it recruits mostly drought-resistant bacteria itself, which may be one of the reasons for the more outstanding growth and drought-resistant ability of the M2. The rhizosphere is an important site where plants connect to the outside world in response to drought stress. Understanding the recruitment of the rhizosphere soil microbiome under drought conditions and identifying core taxa in rhizosphere soils provides an outstanding contribution to the exploration of potential mechanisms by which the rhizosphere enhances plant drought tolerance. Our study provides evidence that rhizosphere soils recruit bacteria from non-rhizosphere soils, that their microbial composition and abundance are regulated by drought stress and the plant host, and that the process of bacterial enrichment is indeed driven by the genotype of the plant."
} | 4,838 |
37050296 | PMC10097333 | pmc | 7,464 | {
"abstract": "Superhydrophobic substances were favored in wood protection. Superhydrophobic treatment of wood is of great significance for improving the service life of wood and expanding its application fields, such as improving dimensional stability, durability, UV stability, and reducing wetting. The superhydrophobic phenomenon is attributed to the interaction of micro/nano hierarchical structure and low surface energy substances of the wood surface. This is the common method for obtaining superhydrophobic wood. The article introduces the common preparation methods of superhydrophobic wood material coatings and their mechanisms. These techniques include lithography, sol–gel methods, graft copolymerization, chemical vapor deposition, etc. The latest research progress of superhydrophobic wood material coatings application at domestic and overseas is reviewed, and the current status of superhydrophobic coating application in wood materials and construction is summarized. Finally, superhydrophobic on wood in the field of applied research is presented, and the development trend in the field of functional improvement of wood is foreseen.",
"conclusion": "4. Conclusions Under the global trend of carbon peaking and carbon neutrality, as a typical natural material with carbon sequestration properties during its life cycle, wood has been widely used in construction, furniture, and energy production as well as other functional materials due to its layered and porous structure. In order to further improve the physical and mechanical properties of natural wood, hydrophobic modifications are applied to wood to extend its service life and broaden its application areas. Superhydrophobic coatings have great potential for application in the field of wood. Although research on superhydrophobic wood has been going on for many years, there are still problems with the durability of superhydrophobic coating surfaces, mechanical resistance, etc. (1) It is necessary to develop simple and feasible preparation processes that are economical and environmentally friendly. In the preparation of superhydrophobic coatings on wood, the graft copolymerization method takes longer preparation time. The demanding harsh reaction conditions of the CVD method have limited the large-scale application in the preparation of superhydrophobic wood. The etching method has a higher cost and is less economical. In addition, there are modification reagents that are not friendly to the environment. Therefore, a preparation method of superhydrophobic wood with a simple preparation process, low cost, and suitable for large-area preparation is also needed. (2) Improving the mechanical resistance. The mechanical strength of superhydrophobic wood includes two aspects. One is the interfacial bond between the superhydrophobic coating and the substrate. Another one is the mechanical strength of the micro/nanostructure on the surface of the superhydrophobic wood. During processing and use, poor bonding between superhydrophobic coatings and substrates can lead to their peeling off, and low surface energy substances on the surface of superhydrophobic wood can easily decompose under the stimulation of temperature, light, and strong oxidants. The micro/nanostructure is damaged by physical action. This all affects the hydrophobic properties of superhydrophobic woods. It is a challenge to find superhydrophobic coatings that are easy to manufacture and robust in practical applications. (3) A comprehensive standard system is imperative for the evaluation and comparison the of the durability of wood superhydrophobic coatings. Under the rapid development in the field of superhydrophobic wood materials, the definition of coating parameters and testing procedures has not yet been standardized. So far, there is no unified test method including test conditions and evaluation criteria to compare artificial superhydrophobic coatings.",
"introduction": "1. Introduction As a typical natural material with the characteristic of carbon fixation during its life cycle, renewable and environmentally friendly, wood has been widely applied in areas of building, furniture, and energy generation as well as other functional materials due to its hierarchical and porous structure. However, due to the hydrophilic nature of wood, it has a tendency towards hygroexpansion, which affects its dimensional stability and durability and limits its practical application range. Therefore, it is necessary for wood to be modified against water to prolong its service life and broaden its application area. In recent years, with further research on superhydrophobic coatings, the multifunctional coatings achieved via numerous methods have become a trend. Multifunctional coatings achieved by many methods have become more visible in various fields [ 1 , 2 ]. The application of superhydrophobic coatings in the field of wood materials has been widely studied. Wood superhydrophobic surfaces are constructed mainly by different methods of forming rough structures and functionalized with low surface energy substances, such as sol–gel, lithography, and so on. Functionalization of wood surfaces improves and enhances the properties of wood, including flame retardancy, dimensional stability, and abrasion resistance, and endows wood with new biomimetic functions, such as self-cleaning, superhydrophobicity, phase change energy storage, and so on [ 3 , 4 , 5 , 6 ]. Researchers have developed a sustainable method to fabricate a highly compressible wood sponge-based sensor with good water resistance and fast electrical response for human motion detection [ 7 ]. The wood sponge-based sensor exhibits superhydrophobicity with a WCA of 152°, maintaining its superhydrophobic state even after 100 compression cycles. In addition, the sensor had high sensitivity, fast response time, and strong fatigue resistance, capable of undergoing 1000 compression cycles at 60% strain with no change in resistance response. The sensor has been successfully applied to detect a variety of physical activities including joint flexion, walking, running, and squatting, but also subtle movements such as voice recognition and wrist pulses. The wood sensor also holds great promise for applications in wearable electronics, artificial intelligence, and electronic skin. It has a broad application prospect. It is necessary to build superhydrophobic surfaces for the effective development and utilization of wood resources and to further broaden the application of wood in environmental protection, intelligent construction, chemical, and other fields. Wood products with superhydrophobic properties will be greatly appreciated as high-value-added products by a more discerning and demanding consumer market. Superhydrophobic wood coatings with environmental friendliness are becoming a mainstream direction for popular research [ 8 ]. Wax-based materials have been widely used as environmentally friendly materials for the preparation of superhydrophobic wood coatings [ 9 ]. Ideally, wood finishes should provide efficient protection from water while retaining the overall bio-based character of the material. In order to meet this challenge, superhydrophobic coatings on the wood are achieved by using materials that are environmentally friendly. It is in line with today’s social theme of “green and sustainable development” to use green, renewable, and biodegradable resources to promote human social development. This paper mainly reviews the latest research progress of domestic and foreign superhydrophobic wood materials coatings. Based on the basic principle of superhydrophobic coating constructed on the surface of wood, this paper summarizes the latest research status, the common methods, and application prospects of superhydrophobic coatings on wood, which will make an important contribution to the potential multifunctional application development of superhydrophobic coatings in the field of wood and intelligent buildings in the future."
} | 1,992 |
31163164 | null | s2 | 7,466 | {
"abstract": "Mitochondria, a nearly ubiquitous feature of eukaryotes, are derived from an ancient symbiosis. Despite billions of years of cooperative coevolution - in what is arguably the most important mutualism in the history of life - the persistence of mitochondrial genomes also creates conditions for genetic conflict with the nucleus. Because mitochondrial genomes are present in numerous copies per cell, they are subject to both within- and among-organism levels of selection. Accordingly, 'selfish' genotypes that increase their own proliferation can rise to high frequencies even if they decrease organismal fitness. It has been argued that uniparental (often maternal) inheritance of cytoplasmic genomes evolved to curtail such selfish replication by minimizing within-individual variation and, hence, within-individual selection. However, uniparental inheritance creates conditions for cytonuclear conflict over sex determination and sex ratio, as well as conditions for sexual antagonism when mitochondrial variants increase transmission by enhancing maternal fitness but have the side-effect of being harmful to males (i.e., 'mother's curse'). Here, we review recent advances in understanding selfish replication and sexual antagonism in the evolution of mitochondrial genomes and the mechanisms that suppress selfish interactions, drawing parallels and contrasts with other organelles (plastids) and bacterial endosymbionts that arose more recently. Although cytonuclear conflict is widespread across eukaryotes, it can be cryptic due to nuclear suppression, highly variable, and lineage-specific, reflecting the diverse biology of eukaryotes and the varying architectures of their cytoplasmic genomes."
} | 426 |
17264114 | PMC1865044 | pmc | 7,468 | {
"abstract": "Macroscopically realizable applications of DNA-based molecular devices require individual molecules to cooperate with each other. However, molecular crowding usually introduces disorder to the system, thus jeopardizing the molecular cooperation and slowing down their functional performance dramatically. A challenge remaining in this field is to obtain both smarter response and better cooperation simultaneously. Here, we report a swift-switching DNA nanodevice that is enhanced by an alternating electric field. The device, self-assembled from folded four-stranded DNA motifs, can robustly switch between closed and open states in smart response to pH stimulus, of which the closed state forms a nanometer-height container that is impermeable to small molecules. This character was used to directly and non-specifically catch and release small molecules emulating mechanical hand in a controllable way. The alternating electric field was used to accelerate molecular cooperative motion during the device switching, which in turn shortened the closing time remarkably to thirty seconds.",
"introduction": "INTRODUCTION Surface-based molecular devices have exhibited many advantages owing to their chemical designability and widespread applications ( 1–11 ). Combination of DNA materials ( 2–8 ) with surface-based ‘bottom-up’ approach has led to several important technological innovations, such as DNA microarray ( 9 ), DNA computation ( 10 ) and DNA-based nanolithography ( 11 ). Macroscopically realizable applications of DNA-based molecular devices require individual molecules to cooperate with each other. However, molecular crowding usually introduces disorder to the system, thus jeopardizing the molecular cooperation and dramatically slowing down their performance ( 12 , 13 ). One challenge in this field is to obtain both smarter response and better cooperation simultaneously. For instance, we recently demonstrated that single-stranded DNA (ssDNA) capped with a short piece of double-stranded DNA (dsDNA) can form a switching compartment nanostructure on surface by a compact array of DNA molecules ( 13 ). However, the switching of this nanostructure highly relies on the cooperative hybridization between complementary DNA molecules, which is usually very slow, cumbersome for operation and contaminated for cycling. In principle, the DNA-formed compartment nanostructure is still far away from an applicable DNA nanomechanical device. Here we report a swiftly switching DNA-nanocontainer device which can non-specifically catch and release small molecules emulating mechanical hand in a controllable way. In contrast to the previous studies, our new approach greatly simplifies working principle of the DNA nanodevice, in which the switching can be fulfilled by itself without assistance of complementary strands, driven by clean energy sources (pH changes), and accelerated by alternating electric fields. The device was implemented with the surface-supported self-assembly monolayer (SAM) of thiol-terminated DNA molecular motifs on gold surface. As shown in Figure 1 , each DNA motif contains two functional domains: a 21-mer i-motif domain which can fold into a four-stranded structure via protonated cytosine–cytosine (C · C + ) base-pair formation (inset of Figure 1 ) ( 14–22 ); a single-stranded poly-(dA) n spacer, where n is an integer in the range of 10–35. To create a monolayer with sufficient spacing between the single-stranded poly-(dA) n domain, the i-motif domain was prefolded into its four-stranded i-motif form at pH 4.5 before being applied to the gold surface, which results in a SAM that is densely packed with respect to the space-filling i-motif structures but shows low-density packing with respect to the poly-(dA) n domain. Previous studies suggest that the repulsion force between DNA molecules is too weak to prevent the dsDNA from aggregating into densely packed states ( 23–31 ). The molecular repulsion interaction between dsDNA helices was measured to be consistently exponential with 2.5–3.5 Å ( 24 ), allowing the energetically favorable separation of 25–35 Å between duplex DNA in densely packed arrays ( 24 , 25 ), which was further verified by theoretical calculations ( 26 , 27 ). It was also well established that small particles, such as ferricyanide anions, methylene blue, and so on, are impermeable through a dsDNA SAM with complete coverage ( 13 , 28–30 ). While the dsDNA molecules were observed approximately perpendicular to gold surface in a densely packed SAM ( 31 ), our previous studies further established that in a DNA SAM possessing the nanocompartment structure, the ssDNA spacer is sufficiently stretched and approximately perpendicular to gold surface ( 13 ), providing functioning rationality of the DNA nanocompartment. In this study, we will show that although the i-motif structure is quite different from the dsDNA helices, similar functionality can also be achieved by the use of i-motif structure instead of dsDNA. At pH 4.5, a closed nanocompartment structure is expected to form between the densely packed i-motif membrane and gold surface with an effective height (basically commensurate with the contour length of the poly-(dA) n spacer). At pH 8.0, DNA motifs are denatured into single-stranded form, causing the SAM to become much loose, and deforming the nanocompartment structure.\n Figure 1. Working principle of the switching DNA nanocontainer. At pH 4.5, the i-motif domain folds into four-stranded structure and packs into a membrane impermeable for small molecules on gold surfaces. At pH 8.0, the i-motif structures are transformed into single strands, making the packing density of the DNA SAM relatively loose to allow small molecules to diffuse freely.",
"discussion": "RESULTS AND DISCUSSION AFM Characterization The quality of the SAM and its conformational transition of the fundamental nanostructure described above were characterized in situ by atomic force microscopy (AFM) in buffer solutions ( 32 , 33 ). High-resolution AFM imaging of soft matters in aqueous solution up to tens of nanometers is difficult to achieve due to disruption of sample surface by the AFM probing tips. Recent technical improvement by using magnetic field to drive oscillating cantilevers presents much reduced perturbation of AFM probing tips to the liquids near the imaging site as well as the soft sample itself, where less noise and smaller amplitude of the cantilevers can be achieved ( 33 ). This advanced technique helped us to image stably and reproducibly, the SAM topography in both folded and unfolded states without obviously perturbing or damaging the samples (see materials and methods section for technical details). As shown in Figure 2 , the surface roughness of the SAM is 1–2 nm at pH 4.5 ( Figure 2 A and D) and 5–7 nm at pH 8.0 ( Figure 2 B and E), indicating that a compact SAM at the closed state transformed into low-density packing SAM at pH 8.0. The surface roughness of 1–2 nm could be recovered after switching the SAM to the closed state ( Figure 2 C and F), verifying the reversibility of the device switching. The AFM image at pH 8.0 ( Figure 2 B) contains pore-like lower regions and mountain-like higher regions, both with diameters on the order of tens of nanometers. The cross-sectional height analysis ( Figure 2 E) shows that the lower regions and the higher regions are connected smoothly with height difference within 5–7 nm. We have estimated the surface coverage of the folded i-motif SAM to be about 1.2 × 10 13 molecules/cm 2 (see Supplementary Data), which indicates the average separation of ∼3 nm between nearest neighboring folded i-motif DNA molecules. At pH 8.0, the folded four-stranded i-motif domain was denatured into single-stranded form with cross-sectional diameter of ∼0.6 nm. Comparing with the diameter of i-motif structure, the packing of ssDNA is relatively loose and consequently leads to irregular pileup on the gold surface. These ssDNA pileups have an inhomogeneous nature, leading to a random distribution of the dense pileup regions and loose pileup regions, corresponding to the mountain-like higher regions and pore-like lower regions, respectively as observed in Figure 2 B. In contrast, when the i-motif domains are in the well-folded state at pH 4.5, the same surface coverage makes the folded i-motif densely packed, as the cross-sectional diameter of i-motif structure is 1.9 nm ( 17 ), in which case the irregular pileup effect for the unfolded ssDNA is not allowed and the topography of the SAM is expected to reflect the flatness of the gold surface on which they are immobilized ( Figure 2 D and F). In the large area topography of AFM observations, the SAM shows good homogeneity although there are inevitable defects, mainly pinholes on gold substrates, demonstrating that an essentially complete coverage of the DNA SAM was achievable ( 13 , 28–31 ). These characterizations make sure that an expected conformational transition between closed and open states was well defined in our devices, which was further confirmed by fluorescence quenching experiments (see Supplementary Data, Figure S1) and electrochemical analysis described below. Switching the device Since the diameter of i-motif structure is 1.9 nm ( 17 ), giving a 0.5–1 nm separation between the surfaces of the nearest neighboring i-motif structures at pH 4.5, the nanocompartment formed at the closed state may be impermeable for small molecules with about 0.5–3 nm sizes due to intermolecular repulsion interactions ( 13 , 28–30 ). This character enables the device to catch and release small molecules emulating mechanical hand on molecular level. To illustrate this process, ferricyanide anions [Fe(CN) 6 ] 3− , whose size is ∼0.8 nm, were employed as object molecules to translate the closed and open states of the device into electrochemical signals by cyclic voltammetry (CV) method ( 34 ). As shown in Figure 3 A, there was obvious peak currents in CV curves when Fe[(CN) 6 ] 3− anions were confined in the closed SAM and no peak current was observed in CV curves after the device was turned open. The obvious peak currents in Figure 3 A can only be attributed to the Fe[(CN) 6 ] 3− anions encaged inside the closed SAM, which refers to molecular encaging effect ( 13 ), for the following reasons. First, circular dichroism spectroscopy analysis ( 35 ) show that [Fe(CN) 6 ] 3− has no obvious intercalation with i-motif structure (see Supplementary Data, Figure S2). Second, a control experiment ( Figure 3 B) was performed in the presence of 10 mM [Fe(CN) 6 ] 3− in the electrolyte, without encaging any [Fe(CN) 6 ] 3− in the closed SAM in advance, showing that the closed SAM can prevent Fe[(CN) 6 ] 3− anions in surrounding buffers from diffusing to bare electrodes surface because of the electrostatic repulsive interactions between [Fe(CN) 6 ] 3− and DNA ( 28–30 ). Third, the negative charges on the sugar–phosphate backbones have higher spatial density in the i-motif structure than in the ssDNA spacer. The spatial density of the negative charges in i-motif structure versus ssDNA spacer is estimated to be roughly 8:1. Thus, the [Fe(CN) 6 ] 3− anions have much weaker electrostatic repulsion with the ssDNA spacer inside the nanocompartment than with the i-motif structure. Fourth, DNA functionalized surface without compartment nanostructure cannot confine [Fe(CN) 6 ] 3− ( 13 , 28–30 ). In our experiments, no obvious peak current was observed when spacer length was set to zero ( Figure 3 C) at both pH 4.5 and 8.0, indicating that no obvious [Fe(CN) 6 ] 3− intercalated in i-motif SAM. Finally, even though [Fe(CN) 6 ] 3− can intercalate into the i-motif membrane, the exponential decay of electron transfer across thickness ≥ 5 nm (corresponding to n ≥ 10 in poly-(dA) n domain) can result in that the electrochemical signal contributed from the intercalated reporters is not detectable ( 13 , 36–38 ).\n Figure 3. Using the switchable DNA nanocontainer to catch and release small molecules. ( A ) Cyclic voltammogram for the closed and open states of the device with different [Fe(CN) 6 ] 3− concentration. Scan rate is 0.3 Vs −1 . No [Fe(CN) 6 ] 3− presented in the electrolyte of CV analysis. The linear relationship between peak currents and scan rates shown in inset confirms that the redox species were confined to the electrode surface. Note that the peak current is defined as the difference between maximum current and baseline current. ( B ) Control experiments show that the [Fe(CN) 6 ] 3− can not diffuse into the nanocontainer at the closed state, and can quickly diffuse to the electrode surface at the open state. ( C ) Isotherms for the molecular encaging effect when applied to capture [Fe(CN) 6 ] 3− . Solid line fit to Langmuir isotherm model: y = σ m x /( x + K −1 ), where x is the concentration of the redox reporters and σ m is the saturation value of the concentration and K is the association constant. K = 13.7 M −1 in the simulation. ( D ) Cyclic switching of the device indicated by the peak current in the cyclic voltammograms. It was found that a well-prepared device can be cycled more than twenty times without obvious performance decay. Capacity and performance The capacity of the device has been investigated by CV method. As shown in Figure 3 C, the concentration dependence of peak current fits to a single-site occupation isotherm, suggesting that the closed SAM could be considered as a nanocontainer ( 13 ). The number of Fe[(CN) 6 ] 3− anions trapped in the closed nanocontainer has been estimated from experimental results ( Figure 3 ) as about 100 per i-motif DNA in the case of 10-bp poly-dA spacer length (for detailed discussion please see Supplementary Data). Several other electroactive small molecules, including phenothiazinium dye (methylene blue), metal derivative ([Co(CN) 6 ] 3− ) and organic reagent ( p -benzoquinone), were used as object molecules to further examine the molecular encaging effect (see Supplementary Data, Figure S3). Results similar to [Fe(CN) 6 ] 3− anions have been obtained, which demonstrate that the nanocontainer is non-specific with respect to the object molecules, i.e. the molecular encaging effect is a generic property of the device. We also investigated the effect of the length and the base content of the ssDNA spacer on the performance of the encaging effect. First, the base content of the ssDNA spacer is found to be irrelevant to the performance of the device. Second, the device capability is positively correlated to the spacer length in the range of 10–30 bp, which indicates that the performance is maintained in this range; when the spacer length is longer than 35 bp, the performance decreases dramatically, as the device shows incapability of encaging object molecules (see Supplementary Data, Figure S4). We propose that the DNA nanocompartment structure breaks down when very long ssDNA spacer is applied (with the length of i-motif fixed), where the conformational entropy of very long ssDNA spacers ( 39 , 40 ) may overwhelm the lateral interaction energy between neighboring i-motifs ( 24–27 ), preventing the i-motifs from packing into a regular membrane. Accelerating with alternating electric field The kinetics of the device switching has been studied by tracking the stoichiometry of the encaged reporters in the compartment during the device switching. As shown in Figure 4 , typical relaxation time constants of the device opening and closing were measured to be ∼300 and ∼330 s respectively, which are much slower than folding and unfolding of i-motif structure in solution phase ( 14 , 41 ). In this study, alternating electric field was used to modulate molecular cooperative motion during the device switching, which provides a scalable means to accelerate the device response ( 12 , 42–45 ). As shown in Figure 4 C, appropriate alternating electric field (frequency ∼1 kHz, amplitude 0.2 V, initial potential 0.3 V, also see Supplementary Data) can remarkably shorten the closing time from 326 to 30 s. However, the same electric field did not prominently accelerate the opening kinetics ( Figure 4 D). In contrast, direct electric fields cannot result in any acceleration effect (data not shown).\n Figure 4. Accelerating device switching by alternating electric field. ( A ) Closing of the device when switching pH from 8 to 4.5 at t = 0. ( B ) Opening of the device when switching pH from 4.5 to 8 at t = 0. ( C ) Closing of the device under alternating (a.c.) electric field. ( D ) Opening of the device under a.c. electric field. Control in (C) and (D) is based on the same experimental setup only in the absence of a.c. electric field. Redox reporter [Fe(CN) 6 ] 3− used in the measurement is 4 mM in (A) and (B), and 40 mM in (C) and (D). Solid lines fitting the data are simulated from model y = A (1 − exp(− t /τ)) in (A) and (C), model y = A exp(− t /τ) in (B) and (D), where τ is the relaxation time, and A is a constant parameter. In principle, in all possible i-motif structures (intramolecular, dimeric and tetrameric), the intercalation of C · C + base pairs between two parallel-stranded duplexes takes place in an antiparallel fashion, which means that each two neighboring strands within i-motif structure are in opposite directions ( 15–20 ). In our design, all DNA chains are immobilized on gold surface via thio-gold bonds at 5′ end; this design has ruled out the possibility of i-motif formed by four parallel ssDNA strands. Even though intermolecular i-motif structures are topologically possible by appropriate looping, the steric limitation made by the immobilization of each DNA chain may make the intermolecular i-motif structures imperfect, which contain mismatches and hanging single strands. The conformational entropy contributed by these imperfects will make them less stable than intramolecular i-motif structures ( 39 , 40 ). Based on these discussions, we may propose that relatively high surface coverage of DNA motifs can cause entanglement between neighboring molecules via intermolecular C · C + base-pair formation, making the device to be trapped in the metastable states during the process of refolding, and dramatically delaying the switching process ( 41 , 46 ). Our recent experimental study on the detailed i-motif folding pathway and dynamics in the dense SAM suggest that the intermolecular metastable i-motif structures take place in the middle stage of the folding process and are on-pathway intermediates that disappear at the completeness of the folding procedure (unpublished data), thermodynamically consistent with the above arguments. The external alternating electric field applied may promote intramolecular diffusion ( 12 , 42–45 ), which could mitigate the entanglements by driving the device out of the metastable states, thus speeding up the intramolecular C · C + base-pair matching during DNA folding. However, the opening of the device is a process of breaking the hydrogen bonds among intramolecular C · C + base pairs, where no entanglement status is involved, so that the same electric field has nearly no effect on the opening kinetics. Detailed underlying mechanism for this phenomenon is under further theoretical investigation and will appear elsewhere. To validate that the device is based on the conformational transition of i-motif structures, pH and temperature dependence of this device has been studied. The half transition of this device at pH 6.2 was in close agreement with the reported transition of i-motif structure in bulk phase at pH 6.3 (see Supplementary Data, Figure S5) ( 41 , 46 ). The melting profile of the device at pH 4.5 was also found to be well consistent to the i-motif structure in bulk solution at pH 4.5 (see Supplementary Data, Figure S5). These results indicate that enough spacing between immobilized DNA molecules was established and possible entanglements of neighboring molecules during their collective folding did not seriously demolish the performance of the device ( 21 , 22 ). Therefore, good cooperation between individual DNA molecules has been achieved in this system, providing an understanding to the feasibility of high closing rates of this device under alternating electric field. In summary, we demonstrate how DNA nanocontainer devices emulating mechanical hands, which require high degree of molecular cooperation, obtain rapid switching without suffering ‘molecular crowding consequence’ by the use of alternating electric field. In contrast to the previous systems whose switching relies upon intermolecular hybridization, the DNA nanocontainers can switch without the aid of complementary strands, which substantially simplifies the system architecture. According to its functional characters, we believe that this DNA nanocontainer could be reconstituted on any substrate that is chemically available to DNA immobilization with appropriate packing density ( 10 , 13 , 44 ). It is promising to enable applications like controlled drug release via DNA molecular machinery. Moreover, it is possible to further modify the ssDNA spacer's backbone with appropriate chemical groups or design its sequences which may selectively and reversibly bind object molecules to introduce selectivity on targets. In addition, when redesigning the motif sequence to include DNA probe, the device might also be used to nucleic acids analysis and biosensors ( 47 )."
} | 5,367 |
40127102 | PMC11979929 | pmc | 7,469 | {
"abstract": "Summary Hydrogels are prone to dehydration over prolonged use, leading to changes in their properties. Inspired by the human skin’s stratum corneum, we present a protocol for the fabrication and application of a low-water-content polyelectrolyte hydrogel (L-hydrogel) that maintains mechanical and electrical properties over time. We describe steps for preparing a hygroscopic monomer through an ionic exchange reaction and a low-water-content hydrogel by UV crosslinking reaction and testing the material behaviors of L-hydrogel. We then detail procedures for preparing and testing L-hydrogel-based devices. For complete details on the use and execution of this protocol, please refer to Shen et al. 1"
} | 175 |
34067748 | PMC8155992 | pmc | 7,470 | {
"abstract": "Reciprocating motion is a widely existing form of mechanical motion in the natural environment. Triboelectric nanogenerators (TENGs) that work in sliding mode are ideal for harnessing large-distance reciprocating motion, and their energy conversion efficiency could be greatly enhanced by adding springs to them. Herein, we focused on investigating the design and optimization principles of sliding mode TENGs by analyzing the effects of spring parameters and vibration frequency on the triboelectric output performance of typical cylindrical sliding TENGs (CS-TENGs). Experimental study and finite elemental analysis were carried out based on a CS-TENG model assembled using a polytetrafluoroethylene (PTFE) film as the negative layer and an aluminum film as the positive layer. The energy output was found to be mainly affected by the change of relative displacement between the two friction layers, rather than the reactive force applied by the springs or the velocity of the sliding motion. However, the frequency of the output signals could be improved when the stiffness coefficient of the springs and the CS-TENG vibration frequency were increased. This study provides valuable directions for the design and optimization of sliding mode TENGs containing springs, and will motivate in-depth research on the fundamental principles of TENG operation.",
"conclusion": "4. Conclusions In this work, we developed a typical CS-TENG working in one-dimensional sliding mode and focused on investigating the effects of the stiffness coefficient of the springs, and the vibration frequency, on the output performance of the CS-TENG. Electrostatic potential simulation results revealed that the relative displacement between the PTFE film attached to the inner cylinder and the aluminum film stuck to the inner surface of the outer tube was the main cause of the potential difference and the current flow in the external circuit. The springs fixed on both ends of the CS-TENG played a pivotal role in affecting the bouncing frequency and motion distance of the inner cylinder. Springs with a lower stiffness coefficient resulted in higher output because of their greater relative displacement between the PTFE film and the aluminum film. In addition, the output energy was increased when the operation frequency was enhanced. However, the improvement became insignificant when the frequency exceeded 5 Hz, indicating that an optimal level could be reached. In addition, we found that the peak output voltage could not be enhanced by further improving the sliding speed, but the increase in the device’s vibration frequency could greatly enhance the frequency of the energy output. This study revealed critical factors influencing the output performance of sliding mode TENGs, and offers design and optimization principles for sliding mode TENGs containing springs.",
"introduction": "1. Introduction The main problem related to the energy crisis is the worldwide growing demand for natural resources to power industrial societies. Green energy harvesting technology has become a hot topic due to the fuel shortages and the environmental problems caused by the use of fossil-fuel-based energy [ 1 , 2 ]. Recently, with the rapid development of portable devices, harnessing environmental mechanical energy by using small devices is recognized as one of the new green energy sources of the era, and has attracted tremendous attention [ 3 , 4 , 5 , 6 , 7 ]. Such devices are usually called energy nanogenerators [ 8 , 9 ]. Nanogenerators based on different mechanisms—including piezoelectric effects, [ 10 , 11 , 12 ] electrostatic effects [ 13 , 14 ], and electromagnetic effects [ 15 , 16 ]—have been developed. Meanwhile, nanogenerators based on triboelectric effects have provided a new paradigm for developing cost-effective, high-output, flexible, and durable portable energy harvesters [ 17 ]. The triboelectric nanogenerator (TENG) is based on the coupled effect of triboelectrification and electrostatic induction, and is a new application of Maxwell’s displacement current in energy harvesting [ 18 ]. It can be designed in various forms using a wide range of materials, and has shown a powerful capability to harvest energy from almost all kinds of mechanical motion, such as random vibrations [ 19 ], wind flow [ 20 ], ocean waves [ 21 ], air pressure [ 22 ], and body movement [ 23 ]. There are essentially four working modes of TENGs, including vertical contact-separation mode, linear sliding mode, single-electrode mode, and freestanding triboelectric-layer mode [ 24 ]. Among them, the vertical contact-separation mode has been investigated most, while the linear sliding mode has unique advantages in collecting energy generated by long-distance reciprocating motion because of its excellent structure design and stable output performance [ 25 ]. In order to adapt the reciprocating motion, springs or rubber bands are often used in sliding mode TENGs. Springs and rubber bands were used in recent studies to store elastic potential by constructing a pendulum-like structure, which could reduce energy loss during the TENG’s operation so as to realize continuous swing after mechanical triggering, and amplify the operation frequency while enhancing the energy conversion efficiency [ 26 , 27 , 28 ]. However, the majority of studies have focused on the assembly, materials, and application of the TENGs [ 29 , 30 , 31 , 32 , 33 ]. The effects of spring properties and the optimization principle of the spring-based sliding mode TENG were rarely reported. In this work, cylindrical sliding TENGs (CS-TENGs) were developed as a typical model in order to investigate the pivotal factors and the optimization principles of sliding mode TENGs equipped with springs. The TENG was designed to vibrate periodically in a vertical direction in order to convert mechanical energy into electrical energy. The inner cylinder and the outer tube, moving in coaxial directions, were assembled, together with a polytetrafluoroethylene (PTFE) film that served as the negative layer and an aluminum film that served as the positive layer. The motion behavior of the CS-TENG devices was simulated by using the finite element analysis method to investigate the effects of the springs’ stiffness coefficient and the vibration frequency on the triboelectric output performance. The working mechanism was elaborated in detail and validated by the experimental results. In addition, the optimized vibration state and the dependence of the output on the relative displacement of the CS-TENG were obtained from the experiments and verified by the simulation.",
"discussion": "3. Results and Discussion The structural configuration of the CS-TENG is depicted in Figure 1 a. An inner cylinder was coaxially assembled with an outer tube, and the inner cylinder could slide freely within the tube. Two springs were fixed on both ends of the outer tube so that the inner cylinder could be bounced back and forth when the CS-TENG was in operation. AO-treated aluminum film was stuck onto the inner wall of the outer polypropylene tube, and served as the positive electrode. The AO treatment could increase the roughness of the aluminum film effectively, as indicated by the inset SEM image in Figure 1 a. The AO approach has been widely used in previous studies to modify aluminum foils [ 34 , 35 , 36 ]. PTFE film was pasted onto the outer wall of the inner aluminum cylinder, and served as the negative electrode. The working principle of the CS-TENG was based on sliding triboelectrification at the interface between the PTFE layer and aluminum layer. The inner diameter of the outer tube was 34 mm, the outer diameter of the inner cylinder was 32 mm, the thickness of the PTFE film was 0.2 mm, and the thickness of the aluminum film was 0.4 mm. Thus, the two layers were controlled to have a tiny gap (about 0.4 mm), in order to allow the cylinders to move freely in a coaxial 1D motion and still be able to abrase with one another during the movement. Two copper wires were connected to the inner cylinder and the aluminum film was attached to the outer tube as current leads, in order to direct the generated current to the outer circuit. Figure 1 b shows the overall size and appearance of the assembled CS-TENG device from the side view and the top view. Figure 1 c illustrates the working mechanism of the CS-TENG device and its connection to the external circuit. The whole process can be divided into six stages. In the neutral state, the PTFE film completely overlaps with the aluminum film, and opposite charges would generate due to the coupling effects of contact electrification and electrostatic induction. An equal amount of positive and negative charges would be generated on the aluminum film and the PTFE film, respectively ( Figure 1 c-I). As the device moved downward, the two surfaces of the cylinder and the tube would slide under external mechanical excitation, and a potential difference would form accordingly, which triggered the flow of electrons from the inner aluminum cylinder stuck with the PTFE film to the aluminum film attached to the inner surface of the outer tube; thereby, a forward current flow was formed in the external circuit ( Figure 1 c-II). When the inner aluminum cylinder was bounced back by the spring, the mismatched area would become smaller as the inner cylinder slid towards the center of the outer tube. This process created another current flow in the opposite direction in the external circuit ( Figure 1 c-III). After a short neutral state ( Figure 1 c-IV), the inner aluminum cylinder would slide to the other end of the outer tube, and the PTFE film would move away from the aluminum film, creating another current flow from the inner aluminum cylinder to the aluminum film in the external circuit ( Figure 1 c-V). Similarly, when the inner cylinder was bounced back by the bottom spring, an opposite current flow would be generated ( Figure 1 c-VI). Through a series of vibrational processes, the developed CS-TENG could repetitively convert the mechanical energy to alternating current (AC) energy by creating a current flow in the circuit between the two electrodes. So far various TENGs working in the sliding mode have been developed. Most of them work in the rotation mode [ 8 ], but some are based on the reciprocating mode [ 37 ]. Springs and rubber bands have been used in the design of these sliding TENGs [ 38 , 39 ]. However, there is no literature focused on investigating the motion behavior and the effects of spring properties on the performance of reciprocating sliding TENGs. This study mainly focused on revealing the motion behavior of the two friction materials and the effect of the spring stiffness coefficient on the energy output performance of a typical cylindrical sliding TENG (CS-TENG), as developed above. Intuitively, the energy generated by the CS-TENG is related to the relative displacement of the two electrodes when the whole device undergoes reciprocated motion. As illustrated in Figure 2 a, the movement of the inner cylinder had a lag compared with the outer tube due to inertia. The springs fixed on the two ends would give a counterforce to the inner cylinder to accelerate its motion. As expected, the movement and the mismatched area of the inner cylinder relative to the outer tube greatly affected the performance of the whole CS-TENG device. Given a certain input power, the stiffness coefficient of the spring was a critical factor determining the movement of the inner cylinder relative to the outer tube and the triboelectric output performance. A 3D model of the CS-TENG device was established using ANSYS software to perform a 3D finite element simulation. The geometry, size, and mass of each component were modeled to match the developed CS-TENG, and three different spring stiffness coefficients (k1 = 60 N/mm, k2 = 120 N/mm, and k3 = 180 N/mm) were used in the simulation. Since TENG devices generally work in a simple harmonic motion, a motion function f ( x ) = A sin (2 π ωt ) ( A = 100, f = 0.5) was used as the input function in order to mimic the movement of the CS-TENG device. The motion direction of the inner cylinder was restricted to the same direction as the whole device, in order to reflect the confined environment in the real case. The motion trails of the inner cylinder and the outer tube over time could be simulated after meshing and running the calculation. The motion trajectories (displacements) of the CS-TENG device and the inner cylinder are shown in Figure 2 b–d for the use of springs with different stiffness coefficients. The simulated results revealed that the inner cylinder, which was wrapped in the PTFE film, exhibited an oscillating behavior, while the whole device underwent a sinusoidal motion. It was found that the overall displacement of the whole device remained the same when using different springs, but the reciprocating motion frequency of the inner cylinder was gradually increased as the stiffness coefficient of the springs was enhanced from 60 N/mm to 180 N/mm. By normalizing the displacement of the inner cylinder with the displacement of the whole device, it was easier to observe the changing trends of the inner cylinder as the stiffness coefficient of the springs was increased. As shown in Figure 2 d–f, the inner cylinder showed a periodic movement pattern with little fluctuation. The normalized displacement represents the distance between the center of gravity of the inner cylinder and the outer tube. It was found that this displacement gradually decreased with the increase of the stiffness coefficient of the springs. This was because greater counterforce was exerted on the inner cylinder when it impacted on a stiffer spring with a certain momentum, leading to smaller compression of the spring and a greater adverse acceleration speed. Therefore, stiffer springs in the CS-TENG would cause a faster motion but a shorter motion distance to the inner cylinder. It is believed that this movement phenomenon would greatly affect the triboelectric output performance of the CS-TENGs. The following section provides a concise and precise description of the experimental results, their interpretation, and the experimental conclusions that can be drawn from them. In the experiment, we were able to maintain the motion of the CS-TENG in a vertical direction, so that the inner cylinder could maintain a 1D coaxial motion relative to the outer tube. In order to investigate the effect of the stiffness coefficient of the springs, we assembled four CS-TENGs equipped with different numbers of springs on each end of the device, as illustrated in Figure 3 a. The stiffness coefficient of the springs was calculated to be 4.8 N/mm, and the stiffness coefficient would be multiplied by the increase of the number of springs from 1 to 4. The CS-TENGs were moved periodically in a vertical direction until stable signals were obtained, and the output voltage and current signals were recorded using an oscilloscope and an electrochemical working station. Figure 3 b–e shows the output voltage of 4 CS-TENGs when they were operated vertically at a frequency of 4 Hz. Although all of the CS-TENGs could produce stable electrical output, under the same excitation, their output voltage displayed significant differences, and the voltage demonstrated an increasing trend as the stiffness coefficient of the springs in the TENGs was increased. The output current results ( Figure 3 f–i) showed the same trend as the output voltage results. The output voltage and current statistical results of four CS-TENGs are compared in Figure 4 a, from which it is clear that the output voltage and current for the CS-TENG equipped with one spring on each end were the highest, at 150 V and 15 µA, respectively. As the number of springs increased, the output power of the CS-TENGs rapidly decreased. When the number of springs reached 4, the output voltage and current dropped to 20 V and 1.2 µA, respectively. This is because the stiffness coefficient of the springs increased in the form of superposition when the springs were arranged in a parallel state. As verified by the simulation results, the compression deformation of the springs by the inner cylinder was smaller when there were more springs, while the frequency and vibration amplitude remained constant. This resulted in a decrease in the relative displacement between the inner cylinder (negative PTFE film) and the outer tube (positive aluminum film), which was reflected by the shift in the center of gravity to the inner cylinder from the outer tube, as shown in Figure 4 b. The relative displacement between the inner cylinder and the outer tube during the CS-TENGs’ operation caused changes to the mismatched area between the PTFE film and the aluminum film, which was the main cause of the generation of current flow in the external circuit. The mismatched area was also gradually reduced as the spring stiffness coefficient increased, explaining the decreased output performance as the number of springs assembled in the CS-TENG was increased. In addition, COMSOL Multiphysics software was used to simulate the potential field distribution of the CS-TENGs in different operating states. As shown in Figure 5 , the potential difference between the top region and the bottom region was only 7 V when the CS-TENG devices were in the matched state ( Figure 5 a). Although the electrostatic field was insignificant, charge transfer occurred when the aluminum film was in contact with the PTFE film. Hence, a great potential difference could be generated when the inner cylinder and the outer tube moved away from one another. It was found that a high voltage difference of 136 V could be generated when the PTFE film attached to the inner cylinder was moved upward such that it departed from the aluminum film stuck to the inner surface of the outer tube ( Figure 5 b). The CS-TENG devices would adopt a relatively neutral state when the inner cylinder moved back to the matched state ( Figure 5 c), and the electrostatic field would be reversed when the inner cylinder was moved downward beneath the aluminum film ( Figure 5 d). The simulation results verified that a great electrostatic field would be created periodically as the inner cylinder slid up and down in the region confined by the outer tube, due to the triboelectric difference between the PTFE film and the aluminum film attached to the inner cylinder and the outer tube, respectively. One of the most important external factors in the CS-TENGs’ operation is the vibration frequency of the devices, while the effect of sliding mode TENGs’ vibration frequencies on their energy output performance is not yet fully elaborated. In this study, the CS-TENGs’ operation under different frequencies was also simulated using ANSYS finite element analysis. In the simulation, the motion frequency of the virtual model was set at 3 Hz, 4 Hz, 5 Hz, and 6 Hz; the motion amplitude was fixed for all cases, and the total displacement and the relative displacement results were analyzed accordingly. As shown in Figure 6 a–c, it was clear that the motion frequency of both the CS-TENG devices and the inner cylinder was greatly enhanced with the increase of frequency from 3 Hz to 5 Hz. Interestingly, it was found that although the displacement of the CS-TENGs was fixed at 100 mm for all cases, the relative displacement between the aluminum film and PTFE film gradually increased as the operating frequency increased ( Figure 6 e–g). However, when the motion frequency was increased to 6 Hz, the relative displacement between the aluminum film and the PTFE film was approximately the same as that at 5Hz, while the motion frequency of the inner cylinder was clearly increased ( Figure 6 h). This implies that the compression degree of the springs might have reached its maximum level when the vibration frequency of the CS-TENGs exceeded 5 Hz. Therefore, further increases in vibration frequency would not enhance the relative displacement, but would increase the motion frequency and velocity of the inner cylinder. The influence of the vibration frequency of the CS-TENG devices on their output performance was studied by vertically shaking the CS-TENGs at different frequencies of 3 Hz, 4 Hz, 5 Hz, and 6 Hz, at a constant vibration amplitude of 100 mm. The output voltage and current results were measured and compared, as shown in Figure 7 . It was found that, as the vibration frequency increased, both the voltage signal frequency and the output voltage ( Figure 7 a–c) increased significantly. The output current signal ( Figure 7 e–f) showed the same trend. These results indicate that the CS-TENGs can generate a stable output signal under various frequencies, and that the operating frequency is highly related to the energy output of the CS-TENGs. In addition, we found that the voltage and current signals could not be further enhanced when the voltage reached over 150 V, even when the vibration frequency was further increased. This was due to the fact that the relative displacement between the aluminum film and the PTFE film almost reached the maximum distance because of the size limitation of the CS-TENG devices. The PTFE film and the aluminum film reached their maximum relative displacement, beyond which no significant changes in the charge transfer and the potential difference would occur. For easy comparison, the statistical data is plotted in Figure 8 a. We found that the output voltage increased from 97 V to 153 V and the output current increased from 3.7 μA to 16.2 μA when the vibration frequency was enhanced from 3 Hz to 5 Hz. The voltage and current remained at the same levels when the vibration frequency was increased from 5 Hz to 6 Hz. The finite element simulation results ( Figure 8 b) revealed that the relative displacement and the mismatched area between the inner cylinder and the outer tube increased when the frequency was increased from 3 Hz to 5 Hz, while it remained about the same when the frequency was further enhanced to 6 Hz, which is consistent the experimental results. These results demonstrate that the peak output voltage and current of the CS-TENGs were mainly affected by the relative displacement between the negative PTFE film and the positive aluminum film, rather than the frequency or the motion velocity. However, the energy generation frequency could be significantly increased with the increase in operating frequency, due to the reactive force applied by the springs. Therefore, the results and basic conclusions obtained from this study may provide valuable directions for the design of sliding mode TENGs containing springs, as well as guidance for the optimization of their output performance."
} | 5,695 |
25112424 | null | s2 | 7,471 | {
"abstract": "The thioredoxin system, composed of thioredoxin reductase (TrxR) and thioredoxin (Trx), is widely distributed in nature, where it serves key roles in electron transfer and in the defense against oxidative stress. Although recent evidence reveals Trx homologues are almost universally present among the methane-producing archaea (methanogens), a complete thioredoxin system has not been characterized from any methanogen. We examined the phylogeny of Trx homologues among methanogens and characterized the thioredoxin system from Methanosarcina acetivorans. Phylogenetic analysis of Trx homologues from methanogens revealed eight clades, with one clade containing Trxs broadly distributed among methanogens. The Methanococci and Methanobacteria each contain one additional Trx from another clade, respectively, whereas the Methanomicrobia contain an additional five distinct Trxs. Methanosarcina acetivorans, a member of the Methanomicrobia, contains a single TrxR (MaTrxR) and seven Trx homologues (MaTrx1-7), with representatives from five of the methanogen Trx clades. Purified recombinant MaTrxR had 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase and oxidase activities. The apparent Km value for NADPH was 115-fold lower than that for NADH, consistent with NADPH as the physiological electron donor to MaTrxR. Purified recombinant MaTrx2, MaTrx6 and MaTrx7 exhibited dithiothreitol- and lipoamide-dependent insulin disulfide reductase activities. However, only MaTrx7, which is encoded adjacent to MaTrxR, could serve as a redox partner to MaTrxR. These results reveal that M. acetivorans harbors at least three functional and distinct Trxs, and a complete thioredoxin system composed of NADPH, MaTrxR and at least MaTrx7. This is the first characterization of a complete thioredoxin system from a methanogen, which provides a foundation to understand the system in methanogens."
} | 472 |
20529914 | PMC2881368 | pmc | 7,472 | {
"abstract": "Motivation: The availability of modern sequencing techniques has led to a rapid increase in the amount of reconstructed metabolic networks. Using these models as a platform for the analysis of high throughput transcriptomic, proteomic and metabolomic data can provide valuable insight into conditional changes in the metabolic activity of an organism. While transcriptomics and proteomics provide important insights into the hierarchical regulation of metabolic flux, metabolomics shed light on the actual enzyme activity through metabolic regulation and mass action effects. Here we introduce a new method, termed integrative omics-metabolic analysis (IOMA) that quantitatively integrates proteomic and metabolomic data with genome-scale metabolic models, to more accurately predict metabolic flux distributions. The method is formulated as a quadratic programming (QP) problem that seeks a steady-state flux distribution in which flux through reactions with measured proteomic and metabolomic data, is as consistent as possible with kinetically derived flux estimations. Results: IOMA is shown to successfully predict the metabolic state of human erythrocytes (compared to kinetic model simulations), showing a significant advantage over the commonly used methods flux balance analysis and minimization of metabolic adjustment. Thereafter, IOMA is shown to correctly predict metabolic fluxes in Escherichia coli under different gene knockouts for which both metabolomic and proteomic data is available, achieving higher prediction accuracy over the extant methods. Considering the lack of high-throughput flux measurements, while high-throughput metabolomic and proteomic data are becoming readily available, we expect IOMA to significantly contribute to future research of cellular metabolism. Contacts: kerenyiz@post.tau.ac.il ; tomersh@cs.technion.ac.il",
"introduction": "1 INTRODUCTION Modern genome-sequencing capabilities have led to the generation of genome-scale metabolic network reconstructions for many microorganisms, giving rise to more than 50 highly curated metabolic reconstructions that have been published to date (Duarte et al. , 2004 ; Feist and Palsson, 2008 ). A metabolic network reconstruction is composed of a set of biochemical reactions, and the associations between these reactions and their enzyme-coding genes. The constraint-based modeling (CBM) computational framework serves to analyze the functionality of these genome scale models, enabling the prediction of various metabolic phenotypes in microorganism such as growth rates, nutrient uptake rates, by-product secretions and gene essentiality (Price et al. , 2004 ). CBM has been used for a variety of applications including the comparative metabolic analyses over multiple organisms (Blank et al. , 2005 ; Lee et al. , 2009 ), drug discovery (Gordana et al. , 2005 ), metabolic flux analysis (Rantanen et al. , 2008 ), studies of network evolution (Fong et al. , 2005 ) and metabolic engineering tasks (Pharkya et al. , 2004 ). Using metabolic models as scaffolds for the analysis of high throughput data such as transcriptomics, proteomics and metabolomics suggests the possibility of inferring condition-dependent changes in the metabolic activity of an organism. Developing computational methods capable of predicting metabolic flux by integrating these data sources with a metabolic network is hence a major challenge of metabolic network modeling. Previous investigations have already utilized CBM to integrate high-throughput molecular datasets with a metabolic network in a qualitative manner: The methods developed by Åkesson et al. ( 2004 ) and Becker and Palsson ( 2008 ) use gene expression data to identify genes that are absent or likely to be absent in certain contexts. They then search for metabolic states that prevent (or minimize) the flux through the associated metabolic reactions. A recent method by Shlomi et al. ( 2008 ) considers data on both lowly and highly expressed genes in a given context, as cues for the likelihood that their associated reactions carry metabolic flux and employs CBM to accumulate these cues into a global, consistent prediction of the metabolic state. The lack of dependency on a cellular objective is a marked advantage of this approach as the latter is difficult to define for multi-cellular organisms. While transcriptomics and proteomics data provides important insight into hierarchical regulation of metabolic flux (representing the control over the maximum activity of enzymes—i.e. vmax), metabolomics may provide information on an additional level of regulation called, metabolic regulation (Rossell et al. , 2006 ). The latter denotes the effect of metabolite concentrations on actual enzyme activity through mass action, kinetic and allosteric effects. A previous CBM method for integrating metabolomic data with a metabolic network model, thermodynamic-based metabolic flux analysis (TMFA) (Henry et al. , 2007 ), derives constraints on reaction directionality from metabolite concentration data based on thermodynamic principles. Another method by Cakir et al. ( 2006 ) integrates quantitative metabolome data with genome-scale models to identify reporter reactions, defined as the set of reactions that respond to genetic or environmental perturbations through coordinated variations in the levels of surrounding metabolites. Currently, however, there is no CBM method that enables the integration of quantitative metabolomic data with a metabolic network model to directly infer the metabolic fluxes themselves. In this article we introduce a novel CBM method, integrative omics-metabolic analysis (IOMA), for integrating quantitative proteomic and metabolomic data with genome-scale metabolic network models to predict metabolic flux. This is achieved primarily by considering a mechanistic model of reaction rates. The method is formulated as a quadratic programming (QP) problem (Nocedal and Wright, 2006 ) geared to find a feasible, steady-state flux distribution, such that: (i) a set of stoichiometric mass-balance and enzymatic directionality constraints are satisfied; (ii) the flux through a core set of reactions for which measured proteomic and metabolomic data exists is as consistent as possible with flux estimations derived via Michaelis Menten-like kinetic rate equations for these reactions. The latter involves the estimation of missing enzyme kinetic constants, by searching for optimal parameters as part of the optimization problem, as described below. To examine the predictive performance of IOMA, we applied it to predict metabolic flux for red blood cells (RBC) for which a detailed kinetic model is available for validation (utilizing it to simulate metabolic flux changes following gene knockouts based on artificially generated proteomic data). As a further validation, we applied IOMA to predict metabolic flux for Escherichia coli under different gene knockouts, utilizing available metabolomic and proteomic data as input, and available experimental fluxes for validation (Ishii, 2007 ). A comparison of IOMA's performance to that of the commonly used methods of flux balance analysis (FBA) (Fell and Small, 1986 ; Kauffman et al. , 2003 ; Varma and Palsson, 1994 ) and minimization of metabolic adjustment (MOMA) (Segre et al. , 2002 ) shows the significant advantage of IOMA in both validation tests.",
"discussion": "4 DISCUSSION This study presents a novel approach for integrating quantitative proteomic and metabolomic data with a genome-scale metabolic network model to predict flux alterations under different perturbations, based on a mechanistic model for determining reaction rate. The method predicts feasible flux distributions while accounting for missing concentration levels of substrate and product metabolites for some enzymes, for potential noise in both the proteomic and metabolomic data, and for the simplifying rate equation formalism used. IOMA is shown to successfully predict changes in fluxes both in E.coli's central metabolism under various genetic perturbations and in a simulated RBC kinetic model, displaying a significant improvement versus the commonly used FBA and MOMA methods. Metabolic fluxes are the most informative and direct indices of the metabolic and physiological state of cells/tissues, and hence, inferring their state in different biological contexts is probably the holy grail of metabolic modeling. However, in a somewhat spiteful way, while we are facing an ever increasing availability of numerous pertaining high-throughput ‘omics’ data including transcriptomic, proteomic and metabolomic measurements, the measurement of fluxes is still very challenging and limited to a small fraction of the reactions present in cells. Hence, there is an emerging need to continue and develop new computational methods for robustly inferring the flux state, while capitalizing on these other available ‘omics’ measurements. In this context, IOMA presents an important step forward in this direction, which hopefully will be followed upon by others. IOMA profits from the absolute quantification of metabolites levels (in contrast to fold changes), that are becoming available, while absolute quantification of proteins is not necessary. Apart from the specific kind of reaction rate laws utilized in this work, IOMA can be used with a variety of rate laws including different types of regulation or enzyme saturation. The only restriction is that the rate laws can be represented in the form of Equation ( 5 ), where estimates of the terms a + and a − can be recomputed based on available data. Future work for improving flux predictions, could possibly utilize existing information on the thermodynamic constants of reactions to further constraint the model's solution space, following Henry et al. ( 2007 ). Another potential application of IOMA is the prediction of metabolic flux alterations associated with human metabolic disorders (as means for predicting potential clinical biomarkers). Encouragingly, genome-scale human metabolic models have already shown their value in this highly important clinical task [e.g. in the case of inborn errors of metabolism (Shlomi and Cabili, 2009 )], but as the methods used up until now have been simple and straightforward, there is certainly much room for improvement ahead, to which methods like IOMA are bound to serve as solid starting points. Funding : Grant from the Israel Science Foundation (ISF) to T.S.; European Commission [BaSysBio, grant number LSHG-CT-2006-037469] to W.L.; Fellowship from the Edmond J. Safra Bioinformatics program at Tel-Aviv University. Conflict of Interest : none declared."
} | 2,672 |
20057383 | null | s2 | 7,476 | {
"abstract": "Network reconstructions are a common denominator in systems biology. Bottom-up metabolic network reconstructions have been developed over the last 10 years. These reconstructions represent structured knowledge bases that abstract pertinent information on the biochemical transformations taking place within specific target organisms. The conversion of a reconstruction into a mathematical format facilitates a myriad of computational biological studies, including evaluation of network content, hypothesis testing and generation, analysis of phenotypic characteristics and metabolic engineering. To date, genome-scale metabolic reconstructions for more than 30 organisms have been published and this number is expected to increase rapidly. However, these reconstructions differ in quality and coverage that may minimize their predictive potential and use as knowledge bases. Here we present a comprehensive protocol describing each step necessary to build a high-quality genome-scale metabolic reconstruction, as well as the common trials and tribulations. Therefore, this protocol provides a helpful manual for all stages of the reconstruction process."
} | 288 |
26818854 | PMC5815134 | pmc | 7,477 | {
"abstract": "In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald − strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald − A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity—V max /K m for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.",
"introduction": "INTRODUCTION Introduction and optimization of heterologous pathways in industrial microorganisms by means of synthetic biology, provides viable biotechnological alternatives for petrochemistry-based production. Products from engineered microorganisms range from pharmaceuticals and pharmaceutical precursors (e.g. taxadiene and artemisinic acid) to bulk chemicals (e.g. lactate and 1, 3-propanediol) and biofuels (e.g. ethanol, isobutanol and farnesene) (Aristidou and Penttila 2000 ; Lee et al . 2012 ). The robustness of Saccharomyces cerevisiae in industrial fermentation processes, combined with rapid developments in yeast synthetic biology, has increased its popularity as a versatile metabolic engineering and industrial production platform (Hong and Nielsen 2012 ). Engineered yeast strains are already capable of producing a wide range of compounds from glucose (Nevoigt 2008 ). Moreover, metabolic engineering has expanded its substrate range to include pentose sugars and cellobiose derived from lignocellulosic biomass (van Maris et al . 2007 ; Wisselink et al . 2007 , 2009 ; Eriksen et al . 2013 ). Transport and storage of lignocellulosic feedstocks are more challenging than that of sucrose, starch and starch-based sugars. The low packing density of lignocellulosic biomass, its high water content and disadvantageous rheological properties, causes high transport costs and spoilage risks (Miao et al . 2012 ). If lignocellulosic biomass is fermented to ethanol close to the agricultural source, this yields a compound with high energy and physical density that is, moreover, stable, pumpable, does not contain inhibitors and is essentially free of water. Ethanol might then be transported and used as a substrate for the production of wide range of fuels and chemicals in microbial processes. In addition to efficient conversion of the lignocellulosic feedstocks to ethanol, this approach requires efficient conversion of ethanol into the desired products. S. cerevisiae can convert ethanol to a wide range of (heterologous) products of industrial interest and is naturally tolerant to high ethanol concentrations. Moreover, since ethanol is non-fermentable, ethanol-grown S. cerevisiae generally produce less by-products than sugar-grown cultures (Nielsen et al . 2013 ). Ethanol metabolism by S. cerevisiae is initiated by its conversion to cytosolic acetyl-CoA, which is an important precursor for a wide range of industrially relevant products. This conversion involves the concerted activity of alcohol dehydrogenase (ADH, encoded by ADH1 , 2 , 3 , 4 and 5 ), acetaldehyde dehydrogenase (ALD, encoded by ALD2, 3, 4, 5 and 6 ) and acetyl-CoA synthetase (ACS, encoded by ACS1 and 2 ). Conversion of acetate to acetyl-CoA in the latter reaction involves hydrolysis of ATP to AMP and pyrophosphate, and subsequent hydrolysis of pyrophosphate makes this equivalent to hydrolysis of 2 ATP to 2 ADP and 2 P i . This high ATP cost for acetate activation not only limits the maximum biomass yield of S. cerevisiae on ethanol but a low ATP yield from substrate dissimilation also constrains yields of products whose biosynthesis requires ATP (de Kok et al . 2012a ). Optimization of the ATP stoichiometry of cytosolic acetyl-CoA synthesis in S. cerevisiae has been intensively studied in sugar-grown cultures. Under such conditions, cytosolic acetyl-CoA is formed via the ‘pyruvate-dehydrogenase bypass’, which involves pyruvate decarboxylase (Pdc1, 5 and 6), ALD and ACS (Pronk, Steensma and van Dijken 1996 ). Synthesis of many cytosolic acetyl-CoA-derived compounds from glucose, including flavonoids, lipids, n -butanol, isoprenoids, artemisinic acid and fatty acids by engineered S. cerevisiae strains, has already been demonstrated (Dyer et al . 2002 ; Veen and Lang 2004 ; Shiba et al . 2007 ; Steen et al . 2008 ; Koopman et al . 2012 ; Chen et al . 2013 ; Paddon et al . 2013 ; Tang, Feng and Chen 2013 ). Previous studies have explored strategies to increase availability of cytosolic acetyl-CoA in glucose-grown cultures, both by improving the capacity of the native yeast pathway and by expressing heterologous pathways (Shiba et al . 2007 ; Chen et al . 2013 ; Kocharin, Siewers and Nielsen 2013 ; Tang et al . 2013 ). Amongst these strategies, replacement of the native pathway for cytosolic acetyl-CoA formation with ATP-independent acetylating acetaldehyde dehydrogenase (A-ALD), pyruvate-formate lyase (PFL) or a heterologous cytosolic pyruvate dehydrogenase (PDH) complex resulted in viable yeast strains (Kozak et al . 2014a , b ). Since PFL and PDH both convert pyruvate to acetyl-CoA, of these three strategies only the A-ALD-dependent pathway has, theoretically, the potential to improve the ATP efficiency of the conversion of ethanol to acetyl-CoA. Expression of five different heterologous A-ALDs has been shown to complement the growth defect of acs1Δ acs2Δ strains on glucose (Kozak et al . 2014a ). Specific growth rates on glucose of the engineered strains reached up to 79% of that of the reference strain. However, the biomass yield on glucose of the best performing A-ALD-dependent strain was 14% lower than that of the reference strain, probably due to toxic effects of elevated concentrations of acetaldehyde. However, even without the toxic effect of acetaldehyde, the benefit of the 2 ATP saved in the process of cytosolic acetyl-CoA synthesis would be difficult to demonstrate during respiratory growth of S. cerevisiae on glucose. In respiratory, glucose-grown cultures, ca. 16 ATP are formed per molecule of glucose converted to CO 2 (assuming a P/O ratio of 1; Verduyn 1991 ) and cytosolic acetyl-CoA is only required for biosynthesis of compounds such as lipids, lysine and sterols, corresponding to a requirement of 1.04 mmol acetyl-CoA per g biomass (Flikweert et al . 1999a ). Therefore, the expected increase of the biomass yield on glucose of A-ALD-dependent strains, relative to wild-type yeast, is predicted to be only 0.5%, which falls within the experimental variation of biomass yield measurements. In contrast, when ethanol serves as the sole carbon source, it is first converted to cytosolic acetyl-CoA, leading to a potential saving of two moles of ATP for every mole of ethanol that is metabolized through A-ALD instead of ALD/ACS. This study aims to investigate the potential of A-ALD-dependent cytosolic acetyl-CoA synthesis to replace the native pathway for growth on ethanol and, thereby, to increase biomass yields on ethanol of S. cerevisiae . If successful, such a replacement would strongly increase the attractiveness of ethanol as a feedstock for the production of acetyl-CoA-derived products by yeast and other eukaryotic cell factories. To identify potential bottlenecks in this metabolic engineering strategy, a set of strains that were previously engineered for A-ALD-dependent growth on glucose (Kozak et al . 2014a ) was tested for growth on ethanol as the sole carbon source. Laboratory evolution was then applied to enable and improve growth of the engineered strains on ethanol. Subsequently, the resulting strains were characterized physiologically and the genetic basis for their improved growth was studied by reverse engineering of mutations identified by whole-genome sequencing.",
"discussion": "DISCUSSION Based on reaction stoichiometries, replacement of the native S. cerevisiae acetaldehyde dehydrogenases by a heterologous A-ALD appears to provide a straightforward, ATP-independent alternative pathway from ethanol to acetyl-CoA. Previous work has already shown that expression of A-ALD can functionally replace the native pathway for cytosolic acetyl-CoA synthesis in glucose-grown S. cerevisiae cultures (Kozak et al . 2014a ). However, prolonged laboratory evolution was required to enable growth on ethanol of S. cerevisiae strains in which its native ALD genes had been deleted and replaced by a prokaryotic A-ALD gene. Whole-genome sequencing of multiple laboratory-evolved yeast strains, derived from parallel independent evolution experiments, followed by reconstruction of the observed mutations in naïve, non-evolved strains, is a powerful approach to gain a deeper understanding of yeast biology and to (re)construct industrially relevant phenotypes (Oud et al . 2012 ). Resequencing of evolved Ald − A-ALD strains that had acquired the ability to grow on ethanol, revealed that three genes ( ACS1 , ERG5 and RIM11 ) were affected by mutations in multiple evolved strains. Deletion of ACS1 was sufficient to enable slow growth on ethanol as the sole carbon source of the Ald − A-ALD strains. ACS1 encodes one of two acetyl-CoA synthetases (ACS) present in the cytosol of S. cerevisiae . In contrast to its paralog ACS2 , ACS1 is glucose repressed and encodes an ACS with a higher affinity for acetate (van den Berg et al . 1996 ; de Jong-Gubbels et al . 1997 ). Elevated acetate concentrations in ethanol-limited chemostat cultures of the evolved strains (Table 6 ), the amplification of the acetate-transporter-encoding ADY2 gene in three evolved strains, and the positive effect of acetate supplementation on growth of Ald − A-ALD strains, all indicates that acs1 mutations stimulate growth by conserving cellular acetate pools. In the absence of the known ALD (Ald2, Ald3, Ald4, Ald5 and Ald6), the metabolic origin of acetate is unclear. It is however conceivable that some acetate is generated by, for example, protein deacetylation and by chitin deacetylases (Cda1 and Cda2; Christodoulidou et al . 1999 ). This leads to the question why Ald − A-ALD strains require acetate for growth. An interesting possibility is related to synthesis of intramitochondrial acetyl-CoA, which is the precursor for mitochondrial synthesis of lipids and, in particular, for synthesis of lipoate. Ach1 is a mitochondrial enzyme (Fig. 2 ) that can use the TCA-cycle intermediate succinyl-CoA as a CoA donor to activate acetate to acetyl-CoA (Fleck and Brock 2009 ). The significantly increased intracellular concentration of succinate in the evolved Ald − A-ALD strain IMS456 relative to that of a reference strain (Table 8 ), may reflect increased conversion of succinyl-CoA to succinate. Figure 2. Schematic representation of the selected reactions of central metabolism of S. cerevisiae growing on ethanol as the only carbon source. Abbreviations: ACS—acetyl-CoA synthetase, ALD—acetaldehyde dehydrogenase, A-ALD—acetylating acetaldehyde dehydrogenase, Ach1—CoA-transferase, Idp2—isocitrate dehydrogenase, PPP—pentose phosphate pathway and TCA—tricarboxylic acid cycle. If Ach1 is required to provide intramitochondrial acetyl-CoA in S. cerevisiae during growth on ethanol of the Ald − A-ALD-dependent strains, this would indicate that the mitochondrial PDH complex cannot provide sufficient mitochondrial acetyl-CoA to sustain growth on ethanol. An insufficient flux through the PDH complex might be caused by regulation of the mitochondrial pyruvate transporters ( MPC1 , MPC2 and MPC3 ; Herzig et al . 2012 ), post-translational regulation of the PDH complex itself by phosphorylation (Gey et al . 2008 ) or changes in the intracellular pyruvate pools. The latter hypothesis is supported by the observation that the intracellular pyruvate concentration in the evolved A-ALD-dependent Ald − strain IMS456 was over 4-fold lower than in the reference strain CEN.PK113-7D (Acs + Ald + ) (Table 8 ). Moreover, supplementation of growth media with alanine (as a nitrogen source), which directly feeds pyruvate into the metabolic network, stimulated growth on ethanol of Ald − A-ALD acs1Δ strains (Fig. 1 ). These results are consistent with a role of Ach1 in mitochondrial acetyl-CoA synthesis in ethanol-grown Ald − A-ALD-dependent S. cerevisiae strains. This study therefore suggests intramitochondrial acetyl-CoA synthesis as a key target for further optimization of the growth of Ald − A-ALD strains on ethanol, for example via engineering mitochondrial pyruvate metabolism or via engineering of acetyl-CoA shuttles across the mitochondrial inner membrane. Mutations in the heterologous A-ALD genes that occurred during laboratory evolution of Ald − A-ALD strains on ethanol could be linked to changes in their kinetic properties and, in particular, in their affinity (V max /K M ) for acetaldehyde. Although a contribution of copy number changes of the A-ALD plasmids to the improvement in V max /K M cannot be excluded, the fact that the increase of the enzymatic activity in the acetyl-CoA forming direction was significantly larger than the increase in the reverse reaction points to a significant contribution of the mutations to the enzyme kinetics in the evolved strains. Simultaneously, the fact that the expression level of TDH3 , of which the promotor was used to express A-ALD, is generally lower on ethanol (Peng et al . 2015 ), might have led to an underestimation of the V max /K M differences. The most pronounced changes of V max /K M ratio were observed for Pseudomonas sp. DmpF. Prior to evolution, assays in cell extracts of an engineered yeast strain expressing DmpF showed an up to 125-fold lower V max /K M ratio than was observed for the corresponding strains expressing EutE from E. coli and Lin1129 from L. innocua . After evolution, this difference decreased to less than 16-fold (Table 5 ). The much less drastic increase of V max /K M of EutE and the absence of mutations in lin1129 , suggests that the affinities of EutE and Lin1129 were sufficient to support growth of the A-ALD-dependent strains on ethanol. A high V max /K M ratio of A-ALD enables the enzyme to achieve high fluxes at relatively low intracellular acetaldehyde concentrations. Acetaldehyde is a toxic molecule that can cause protein and DNA damage (Tuma and Casey 2003 ). Despite the improved affinity of its A-ALD, elevated acetaldehyde concentrations were observed in ethanol-limited chemostat cultures of the evolved strain IMS456 compared to the reference strain CEN.PK113-7D. To drive the A-ALD reaction (ΔG 0′ = −13.7 kJ mol −1 at pH 7, ionic strength of 0.2 M (Flamholz et al . 2012 ) in the oxidative direction high intracellular acetaldehyde concentrations may be required. The observed increased levels of ethanol are in line with these higher levels of intracellular acetaldehyde. This is further supported by the observation that the residual ethanol concentrations are higher in the chemostat cultures of strains expressing A-ALDs with lower affinities for acetaldehyde (Tables 5 and 6 ). The ΔG R 0 of the native S. cerevisiae pathway for conversion of ethanol into acetyl-CoA is more negative than that of the introduced pathway. To investigate the impact of this decreased thermodynamic ‘pull’, it will be of interest to determine the in vivo ΔG R of the A-ALD reaction in combination with the introduction of acetyl-CoA consuming reactions, for example by overexpression of the extramitochondrial citrate synthase Cit2 or by coupling A-ALD to a product pathway. In addition to its role in the linear oxidation pathway from ethanol to acetate, the native NADP + -dependent acetaldehyde dehydrogenase contributes to the provision of NADPH for assimilation. The increased intracellular concentration of isocitrate in strain IMS456 may reflect an increased contribution of cytosolic NADP + -dependent isocitrate dehydrogenase to NADPH generation to compensate for the absence of Ald6. Similarly, the decreased concentrations of the lower-glycolytic metabolites and the increased concentrations in upper glycolysis (Table 8 ), may reflect an increased role of the oxidative pentose-phosphate pathway in NADPH generation. The highest specific growth rate on ethanol of an evolved Ald − A-ALD strain (IMS459) of 0.11 h −1 corresponded to almost 70% of that of the reference strain CEN.PK113-7D (0.16 h −1 ) (Flikweert et al . 1999b ). The specific growth rate of reverse engineered strains in which deletion of ACS1 was combined with the expression of evolved alleles of A-ALD genes, did not exceed 0.035 h −1 . Unfortunately, low growth rates and poor genetic accessibility of Ald − A-ALD strains, resulting in very low transformation efficiency precluded a combinatorial analysis of the mutations that occurred in multiple strains. However, our results clearly indicate that, in addition to acs1 inactivation and improved affinity of A-ALD, other mutations contributed to the improved growth of the evolved strains. Only one of five evolved Ald − A-ALD strains (IMS456) showed a small but significant increase of its biomass yield on ethanol relative to an Ald + ACS1 reference strain (Table 7 ). However, the observed biomass yield of 0.60 g per g ethanol of this evolved strain remained far below the theoretical maximum of 0.80 g biomass g ethanol −1 calculated for Ald − A-ALD strains. This discrepancy could only partly be explained by carbon loss due to the increased formation of byproducts, such as acetaldehyde. Based on transcriptome analyses, a decrease in the biomass yield in glucose-limited cultures of metabolically engineered S. cerevisiae strains in which cytosolic acetyl-CoA synthesis had been rerouted via A-ALD was attributed to acetaldehyde toxicity (Kozak et al . 2014a ). Cellular mechanisms to combat toxicity of acetaldehyde (Tuma and Casey 2003 ) in the Ald − A-ALD strains may have led to increased ATP costs and, thereby, a lower biomass yield. For example, increased turnover of damaged proteins and regeneration of glutathione, which promotes tolerance by reacting with acetaldehyde, are both ATP-dependent processes. This study demonstrates, for the first time, that it is possible to replace the native ALD/ACS-based route for ethanol dissimilation with an A-ALD-dependent pathway that has the potential to enable strongly increased product yields on ethanol. Furthermore, compartmentation of acetyl-CoA metabolism and the regulation of intracellular acetaldehyde concentrations were identified as high priority targets for further research, which is required to harvest the full stoichiometric potential of the introduced modifications."
} | 4,926 |
36716369 | PMC9963307 | pmc | 7,478 | {
"abstract": "Significance Many engineering applications request soft materials to maintain tough under monotonic loads, and fatigue-resistant under cyclic loads. While fracture toughness of a soft material can be enhanced by orders of magnitude, its fatigue threshold remains insusceptible. Here we demonstrate a crack tip softening (CTS) concept to break this dilemma. Experiments show that the CTS concept enhances both toughness and threshold of polyacrylamide hydrogels by about four times. This simultaneous enhancement comes from stress de-concentration and elastic shielding at the crack tip. The CTS concept is generic to many material systems, geometrical singularity regions, and external loads. Our paper provides a crack retardation method during material application.",
"conclusion": "Conclusion In this work, we demonstrate a CTS concept to simultaneously improve the toughness and fatigue threshold of a single polymeric network. We applied monotonic loads and cyclic loads to polyacrylamide hydrogels and characterized the mechanical properties of pristine samples and CTS samples. A light-treated hydrogel is softer and more elastic than an un-treated one. Experimental results demonstrate that the CTS concept enhances both the toughness and fatigue threshold of polyacrylamide hydrogels by about four times. We attribute this enhancement to stress de-concentration and elastic shielding at the crack tip. The CTS concept is generic to many material systems, applicable to different geometrical singularity regions, and to various external loads (monotonic or cyclic). This paper provides a crack retardation method during material application.",
"discussion": "Discussion The degradation of ONB molecules reduces the crosslink density around the crack tip. Consequently, the crack tip has a lower shear modulus compared with the bulk material. Also, the crack tip becomes more stretchable than the bulk as the extensibility is approximately proportional to n , where n can be regarded as the chain length. These two effects make the crack blunt under both monotonic and cyclic loads. The degradation of ONB molecules at the crack tip would also cause chain entanglement and further affects the fracture and fatigue of polymers ( 38 , 40 , 41 ). In order to investigate the effects of chain entanglement in our polyacrylamide hydrogels, we perform three kinds of experiments: 1) monotonic tension tests at various stretch rates, 2) rheology experiments at various shear rates, and 3) swelling experiments of three kinds of hydrogel samples. Details can be found in SI Appendix , Figs. S4–S6 . The experimental results show that the polyacrylamide hydrogels used in this work are stretch rate-independent, shear rate-independent, and swelling-consistent. The effect of entanglement on the enhancement of fracture toughness and fatigue threshold is minimized. The CTS concept bears two mechanisms for fracture and fatigue: stress de-concentration and elastic shielding. The crack tip becomes softer and tougher after light treatment. A soft crack tip becomes blunt under external load, re-distributing the stress along a large amount of material. A tough crack tip raises the crack growth resistance. Consequently, the barrier between the driving force and resistance at the crack tip becomes greater, and the bulk material becomes fracture resistant under a monotonic load. The fatigue threshold of a CTS sample is much larger than that of a pristine sample. A crack will not grow under cyclic load in a CTS sample even when the energy release is great than the threshold of a pristine sample. This can be explained in Fig. 5 . The yellow region in the figure represents a treated region where the material is softer and possesses longer chains. This region becomes nearly elastic after treatment. Under cyclic load, only the small red region becomes higher stretched than the bulk. However, this stretch is not high enough to cause any damage around the crack tip due to stress de-concentration, and the material behaves elastically. In other words, the light-treated material acts as an elastic shielding under cyclic load, and the material becomes fatigue-resistant. The concept of elastic shielding is similar to the overload protection concept in hard materials like metals ( 42 – 44 ). However, when the cyclic load increases further, a crack extends by breaking one layer of chains based on the Lake-Thomas model. The light treatment introduces chain scission and increases the average chain length at the crack tip region. When the crack extends, each chain acts as an elastic dissipater ( 25 , 29 ), releases the energy along the whole chain, and contributes to the threshold. As a result, the CTS concept also enhances the threshold. Fig. 5. Schematic of elastic shielding makes the sample more fatigue-resistant. Softening makes the materials in the yellow region softer and more fatigue-resistant. This region could provide an elastic shield to retard the crack propagation under cyclic loads. A limiting case in the CTS concept is that the crack tip region has zero moduli, i.e., the material at the crack tip vanishes after light treatment. In this case, the crack tip has a finite radius. The geometric singularity is greatly avoided, and the material behaves approximately the same as an unnotched one. Our experiments show that the stretchability and strength of a notched sample with CTS treatment ( Fig. 3 B , green curve) are comparable with that of an unnotched sample ( Fig. 3 A , red curve). By employing the CTS concept, the effect of crack is minimized. In this sense, the CTS concept is analogous to drilling a stop hole at the crack tip in hard materials to arrest crack propagation ( 45 – 47 ). There are two representative concepts to improve both the toughness and threshold of a polymer network. One is the fiber/matrix composite concept ( 25 , 27 , 48 , 49 ). In this concept, the crack is arrested by a macroscopic fiber, which is fabricated vertically to the crack propagation direction during sample preparation and has a characteristic length of sample size. When the crack extends to the fiber/matrix interface, stress de-concentration is accomplished by the blocking of crack tip by the stiff fiber. To further grow the crack under a monotonic load or cyclic load, the fiber needs to be broken. The fiber acts as an elastic dissipater and a giant “polymer chain,” which releases a large amount of energy upon crack growth and contributes to both the toughness and fatigue threshold. The other way to improve the toughness and threshold is the crystallization concept ( 30 , 31 ). In this concept, the material is trained by a certain method, e.g., mechanically cyclic load, to introduce hard crystallization in a soft matrix. The hard phase can block crack growth. The characteristic length of the crystalline grain is much larger than that of a chain length. Similarly, crack propagation needs to break this crystallization phase. The energy stored in this hard phase contributes to the toughness and fatigue threshold. The CTS concept is fundamentally different from the above-mentioned approaches. One major difference is that both approaches toughen a material by a hard phase, while the CTS method employs a soft phase at the crack tip. The other difference is that both approaches need a material to be modified or\ndesigned before any application, either during fabrication or training after fabrication. On the other hand, the CTS concept allows a material during application to be toughened. In this sense, the CTS concept has a more realistic practice meaning. The CTS concept is generic to many material systems, geometrical singularity regions, and external loads. This paper uses polyacrylamide hydrogels as model materials. In fact, most polymer networks are imperfect ( 35 ). Short chains and long chains co-exist in the network. Short chains at the crack tip can always be broken by external loads to achieve softening. In this sense, the CTS concept is generic to most polymer systems. The CTS concept can be applied to stress concentration regions, not necessarily the crack tip. For example, notches, holes, grooves, and soft/rigid boundaries can be softened to prevent crack growth. We fabricate a hydrogel membrane and perform punctuation tests using a metal bar ( SI Appendix , Fig. S9 ). The as-prepared membrane fails when the loading displacement reaches 80 mm. By comparison, the central-softened sample sustains a load over 100 mm. In this test, stress concentration occurs in a metal-hydrogel contact region instead of a crack. In the present work, we demonstrate the CTS concept by using a light-degradable crosslinker. This softening method is non-contact, benefiting various practical applications. Light is an economic, easy-to-control, and readily available resource. Other stimuli such as thermal heat, electric, or mechanical load can be used to achieve CTS in practice. For example, as we demonstrated in a previous work ( 50 ), a dielectric elastomer membrane with a hole can sustain more mechanical stretch when the hole edge is softened by an electric field. All these examples prove that CTS concept makes a structure strong under external loads. In engineering applications, one does not need to clearly locate or predict the region where stress concentration occurs. A possible practice is to regard these defects as a black box and apply a load higher than the designed value on the sample for certain cycles. During this overload, the stress concentration region becomes softer and the material becomes more fatigue-resistant when the external load recovers to the designed value. These materials, geometries, and load aspects deserve further study."
} | 2,426 |
27408906 | null | s2 | 7,481 | {
"abstract": "To many, the poster child for David Marr's famous three levels of scientific inquiry is reinforcement learning-a computational theory of reward optimization, which readily prescribes algorithmic solutions that evidence striking resemblance to signals found in the brain, suggesting a straightforward neural implementation. Here we review questions that remain open at each level of analysis, concluding that the path forward to their resolution calls for inspiration across levels, rather than a focus on mutual constraints."
} | 131 |
20529691 | null | s2 | 7,482 | {
"abstract": "Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau approximately 12 micros, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau approximately 14 ns, 14 micros, and 280 micros. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-micros component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in approximately 4% of the enzyme population. The final, 280-micros component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium."
} | 510 |
35496619 | PMC9050787 | pmc | 7,484 | {
"abstract": "The development of passively driven microfluidic labs on chips has been increasing over the years. In the passive approach, the microfluids are usually driven and operated without any external actuators, fields, or power sources. Passive microfluidic techniques adopt osmosis, capillary action, surface tension, pressure, gravity-driven flow, hydrostatic flow, and vacuums to achieve fluid flow. There is a great need to explore labs on chips that are rapid, compact, portable, and easy to use. The evolution of these techniques is essential to meet current needs. Researchers have highlighted the vast potential in the field that needs to be explored to develop rapid passive labs on chips to suit market/researcher demands. A comprehensive review, along with patent analysis, is presented here, listing the latest advances in passive microfluidic techniques, along with the related mechanisms and applications.",
"conclusion": "Conclusion and future directions Microfluidics is articulated as a multidisciplinary research field that requires basic knowledge in fluidics, micromachining, electromagnetics, materials, and chemistry to find their relevance in the pharmaceutical industry, diagnosis, healthcare, and life science research. LOC is one of the essential applications of microfluidics, and is also a revolutionary tool for many varieties of applications in chemical and biological analyses due to its fascinating advantages (speed, low cost, simplicity, and self-testing) over conventional chemical or laboratory equipment. Microfluidics covers the science of fluidic behaviours at micro/nanoscales employed in design engineering, simulation, and fabrication of fluidic devices. It is the backbone of biological or biomedical microelectromechanical systems (BioMEMS) and the LOC concept, as most biological analysis involves fluid transport and reaction. MFDs have been operated in inkjet printing, blood analysis, biochemical detection, chemical synthesis, drug screening/delivery, protein analysis, DNA sequencing, and so on. Several different MFDs have been developed with basic structures analogous to macroscale fluidic devices. Such devices include microfluidic valves, microfluidic pumps, and microfluidic mixers. Active devices are usually more expensive, due to their functional and fabrication complexities. However, it has been very challenging to implement these actuation schemes fully at the microscale, owing to the requirements for high voltages, electromagnets, etc. Typically, passive MFDs do not require an external power source, where control is instituted by the energy drawn from the working fluid, or based purely on surface effects, such as surface tension or fluid pressure, with high reliability due to the lack of mechanical wear and tear. Hence, upcoming industrial sectors rely on passive methods to achieve better results in a composed way. MFDs offer very repeatable performance, once the underlying phenomena are well understood and characterized. They are well suited to bioMEMS applications, as they can handle several microfluidic manipulation sequences. They are also well suited to the low-cost mass production of disposable MFDs to specifically work with blood. In addition, inexpensive and simple fabrication techniques can promote the use of paper-based MFD in various research fields, such as drug testing and viral detection. Passive MFDs with high throughput, high flow rate, reduced operation time and easy equipment handling are expected to have profitable outcomes. Therefore, MFDs with passive methods are designed in such a way as to offer a wide range of optimizations that can be performed within the channel and reservoir dimensions for employment to certain specific applications with better flow rates. In the future, device progress will focus on developing new materials for substrates in such a way as to overcome the drawbacks of currently existing devices. Passive systems cannot maintain constant flow rates. Hence, systems with constant flow rates are expected to be developed as an advantageous method. In addition, a low operation time remains an essential parameter. A reduction in running time can be demonstrated to be the crucial factor on which to focus during upcoming developments. Future work is expected to focus on exploring these listed areas, so that microfluidics can find application in many other fields to satisfy growing demands and needs.",
"introduction": "Introduction Handling small volumes of fluids is very important in high-throughput screening, diagnosis, and research applications. 1 Microfluidics is one way to handle small volumes of fluids between microlitres (10 −6 ) and picolitres (10 −12 ). 2–4 Hundreds of simultaneous biochemical reactions can be performed in a collection of microarrays arranged on a solid substrate which acts as laboratories, embedded in which are chips known as biochips. 5,6 There are three main types of biochips: lab on chips (LOCs), DNA chips, and protein chips. LOCs employ a combination of one or more laboratory functions within a single integrated chip. 7 Some fields utilizing LOCs, such as sub-micrometer and nano-sized channels, DNA labyrinths, single-cell detection and analysis, 8 and nano-sensors, might become feasible, allowing new ways to interact with biological species and large molecules. In addition, a large number of biochemical analyses can be screened at a faster rate in disease diagnosis and the detection of bioterrorism agents. 9 Several reports have been published on the various aspects of these devices, including fluid transport, system properties, sensing techniques, 10 and bioanalytical applications. Advantages 11,12 include lower fabrication costs, allowing cost-effective disposable chips and mass production. Simple tests that could be performed by the bedside are known as point-of-care (POC) testing. 13 The ultimate aim of this technique is to obtain results in a concise period at or near the location of the patient, so that the treatment plan can be adjusted. 14 Microfluidics can be used for various lab experiments, such as drug testing and discovery, 15–23 filtration and separation of particles, 24 cell sorting and counting, 25–31 cell culture, 32–40 point-of-care diagnosis, 41,42 3D printing, stoichiometry, and flow synthesis. 43,44 Due to their simplicity with high throughput and very low reagent consumption, 45 microfluidic chips are vital components in research, for the delivery of accurate results. 46 Microfluidic chips are mostly made up of PDMS (polydimethylsiloxane). PDMS is commonly used because it is a transparent elastic polymer, permeable to oxygen and carbon dioxide. 47–51 Additionally, PDMS is now becoming a standard material as it can be easily fabricated for microfluidic devices (MFDs), and its high gas solubility, which obeys Henry's law, is a significant advantage of using PDMS material. 52 Microfluidic operation techniques The techniques assisting fluid flow in an MFD are generally classified as active or passive. 53,54 Active microfluidics 55,56 involves the movement or transport of biological samples and analysis of those samples through an external power source/field 57–59 or actuators, 60 such as peristaltic pumps, 61 electro-kinetics, 62,63 electro-wetting, 64 electro-osmotic pumps, 65 electrostatics, 66,67 centrifugal and magnetic pumps 68 and some other large power sources to power the pumps and actuators. 69–71 Thus the complexity of structure and size is increased, which requires additional human resources. Hence, the probability of integrating active microfluidics with LOCs and (POC) applications has dropped off. To counter these drawbacks, 72–74 the movement of the test sample is achieved either using fluid properties or passive mechanisms without any external supporting power sources. Hence, passive microfluidics 75 has been adopted and used to a large extent in modern-day research projects to avoid the use of external supporting power devices. This method is simple, easy to manufacture, and does not need any actuators or external power supplies, as it employs basic laboratory instruments, like micropipettes, and medical devices, such as syringe pumps.",
"discussion": "Critical discussions and limitations The evolution of new microfluidic tools for genomics, proteomics, and metabolomics is progressing swiftly in research laboratories and will provide the motive for large-scale production. Passive flow methodologies, such as surface-tension-driven flow, capillary-based flow, gravity-driven flow, hydrostatic-pressure-driven flow, and osmosis-driven flow techniques are found to be suitable in assisting the flow without any external sources. Despite the beneficial properties of PDMS that permitted its fast enactment in applied fields, there are several limitations in using the material in biomedical research. 226 The control of fluid flow over space and time with sufficient accuracy was the fundamental challenge in designing MFDs. 227 The initial fabrication of MFDs required clean-room facilities for silicon or glass devices which were eliminated by polymers and elastomers devices. However, PDMS was limited by its ability to withstand high temperature, difficulty in developing complex multi-layered 3D-configured devices, and incompatibility with organic solvents and low molecular weight organic solutes due to their surface chemistry. 228 The usage of inert chemicals and utilization of high-throughput methods for manufacturing and permeability limit the application of PDMS devices in a few cases. 229 Evaporation affects the application of microfluidics in various domains. 230 This could be overridden by maintaining a uniform surface throughout the entire channel area. In pressure-driven techniques, variation in pressure is noted due to the difference in channel length and utilization of different fingers at the inlet. 119,127 This unbalanced pressure may lead to backflow, followed by clogging or the entire destruction of the device. Hence, a uniform pressure needs to be maintained for a long period. This can be achieved by osmosis-driven MFDs. However, in osmosis-driven MFDs, the osmotic reagent had to be refreshed at regular intervals, which restricted their applications. 145 In addition to that, selection of a suitable membrane is necessary to maintain the pH and to avoid clogging when biological cells or biomolecules are used. 143 Hence, capillary-based passive flow techniques are used as they depend only on the design and material characteristics of the MFD. On the other hand, in some instances, due to a drop in flow rate, essential pressure prediction at each step decreased sensitivity in capillary-based passive flow techniques. 151,165 This could be averted by selection of suitable materials with the ability of spontaneous sample filling in the column. 231 Because of this, under many circumstances, gravity-driven techniques are adopted, but they generate an unstable or decreasing flow rate, which was further eliminated by maintaining an appropriate reservoir volume and proper inlet pressure. 22,198 Whereas hydrostatic-pressure-driven techniques are limited by the development of Laplace pressure at the air–liquid interface due to the affinity between the liquid, the atmosphere, and the reservoir parameters. 232 Another listed shortcoming was the linear pressure drop with time. These errors could be avoided, if the pressure difference was obtained by varying the altitude of the liquid to the atmospheric interface in the proper way. 197 They could be eliminated by using a vacuum MFD; nevertheless evacuation is necessary to drive the fluid in order to reduce the pressure within the channel. Due to the presence of high pressure initially, the suction of the fluid is higher and gradually decreases as the pressure decreases. Hence a syringe pump is used to maintain a constant flow in the device. This additional requirement can be neglected by the use of simple capillary force. 211 Based on the study performed, the vacuum-driven techniques with complex construction were not suitable for continuous operation due to the evaporation effect and the loss of sample in a few cases. 75,208,216 Besides the above-mentioned limitations, these methods found numerous clinical applications, because they use ultra-low volumes of biofluids for processing and can be accomplished quickly and efficiently. Industrial perspectives One of the promising types of assistance of microfluidics is found in fast, reliable, and accurate POC diagnostic devices. Detection and diagnostics of communicable diseases can decrease the mortality rate in many developing and less developed countries. As reported by the World Health Organization (WHO), nearly 46% of new tuberculosis case are not detected, causing over 3 million incidences to be missed annually. 233,234 Also, according to a recent report by WHO, the lack of proper diagnostic capacity is a significant challenge in monitoring effective service coverage. As an example, cholera was estimated to have infected 1.3–4 million people annually and to have caused 21 000–143 000 deaths each year during 2008–2012. But the average annual numbers of cases and deaths reported to WHO were only 313 000 and 5700, respectively. One of the major issues is the lack of diagnostic capacity, which results in the exact burden of the disease being unknown, thus making it difficult to take preventive measures. 235 Although many researchers in the microfluidic domain are focusing on developing diagnosis and detection devices – searching for a few related keywords such as diagnosis, detection, and pathogen detection in LOC, bio-microfluidics, and biomedical microdevices resulted in nearly 3000 papers – many of the products do not end up on the market. One major issue in this regard is transforming common expensive sample delivery systems in laboratories, such as syringe pumps, into passive and low-cost delivery systems to meet the needs of the market in developing and less developed countries. To analyse the status of current commercialized MFDs, we delved into the circle database of uFluidix Inc ( http://circle.ufluidix.com ). The database includes a list of registered start-ups offering microfluidic products. Nearly 85% of the analysed start-ups in the diagnostics industry were utilizing a passive method for sample delivery. This clearly emphasizes the importance of passive techniques for industrial sectors."
} | 3,616 |
33519743 | PMC7843935 | pmc | 7,487 | {
"abstract": "Quorum sensing (QS) is a density-dependent communicating mechanism that allows bacteria to regulate a wide range of biogeochemical important processes and could be inhibited by quorum quenching (QQ). Increasing researches have demonstrated that QS can affect the degradation of particulate organic matter (POM) in the photic zone. However, knowledge of the diversity and variation of microbial QS and QQ systems in sinking POM is scarce. Here, POM samples were collected from surface seawater (SW), bottom seawater (BW), and surficial sediment (SS) in the Yellow Sea of China. 16S rRNA gene amplicon and metagenome sequencing were performed to analyze the community structure of particle-associated microorganisms and distribution of QS genes [acylated homoserine lactone (AHL) synthesizing gene luxI and AHL sensing gene luxR ] and QQ genes (genes encoding for AHL lactonase and acylase) in POM. Shifting community structures were observed at different sampling depths, with an increase of microbial abundance and diversity from SW to BW. Along with the variation of microbial communities, the abundances of luxI and luxR decreased slightly but were restored or even exceeded when POM arrived at SS. Comparatively, abundances of AHL lactonase and acylase remained constant during the transportation process from SW to BW but increased dramatically in SS. Correlation tests indicated that abundances of luxI and luxR were positively correlated with temperature, while those of AHL acylase were positively correlated with depth, SiO 4 2– , PO 4 3– , and NO 3 – , but negatively correlated with temperature and pH. According to phylogenetic analyses, the retrieved QS and QQ genes are more diverse and distinctive than ever experimentally identified. Besides, the vertical transmission of QS and QQ genes along with POM sinking was observed, which could be one of the key factors leading to the prevalence of QS and QQ genes in marine ecosystems. Overall, our results increase the current knowledge of QS and QQ metabolic pathways in marine environment and shed light on the intertwined interspecies relationships to better investigate their dynamics and ecological roles in POM cycling.",
"conclusion": "Conclusion Microbial abundances and diversities increased along with POM sinking from SW to BW, and the diversities reached the highest when POM was sinking to SS. The abundances and diversities of QS ( luxI and luxR ) and QQ (AHL lactonase and AHL acylase) genes varied in seawater and SS. The abundance of luxI was the highest in SW, while that of LuxR and two QQ enzymes reached the highest in SS, showing the prevalence of QS signaling and interfering in SS. In addition to bacteria, luxR and QQ genes were further detected in archaea and eukaryotes. We assumed that AHL-based QS and QQ might be responsible for cross-kingdom interactions in POM. Our results provide support for future research on microbial cooperation or competition activity, and interaction mechanisms of bacteria in POM. Nonetheless, the presence or absence of QS and QQ genes only suggests the potential of QS and QQ activities in POM, and the expression level of QS or QQ genes and whether the associated reactions are active need further detection. In the future, investigations combining metagenomic, metatranscriptomic, and metabolomic analyses will be performed to restore the expression of QS and QQ genes and their regulatory roles in the biogeochemical cycle.",
"introduction": "Introduction Particulate organic matter (POM) is prevalent in marine environment and believed to be the main vehicle for vertical material transport in the ocean ( Fowler and Knauer, 1986 ). They are the foundation of the marine food web and primary food sources for creatures living in the aphotic zone ( Azam and Malfatti, 2007 ). POM are composed of diverse concentrated organic components including pellets, phytoplankton and zooplankton debris, plant secretions, microorganisms, and part of inorganic elements such as dust and detritus ( Alldredge and Silver, 1988 ) that attract quantities of microorganisms to colonize. In return, hydrolytic enzymes secreted by POM-associated microorganisms accelerate the degradation of POM ( Smith et al., 1992 ; Kiørboe et al., 2002 ). Previous research has revealed that bacterial abundances, community diversities, and the extracellular enzyme activities (EEAs) were higher in POM compared to those in ambient seawater ( Smith et al., 1992 ). The upregulated EEAs in POM were firstly explained using a bacterial density-dependent regulatory mechanism, named quorum sensing (QS), by Hmelo and his colleagues ( Hmelo et al., 2011 ). Quorum sensing triggers synchronous expression of multiple genes in a microbial population, initiating a coordinated action when high cell densities are reached, e.g., flagella formation ( Zan et al., 2012 ), biofilm formation ( Sun et al., 2015 ), and extracellular enzyme (EE) production ( Jatt et al., 2015 ; Su et al., 2018 ). QS-based communications are performed based on the production, release, and population-wide detection of several types of QS signaling molecules, named autoinducers (AIs). To date, more than 200 AIs have been identified from a variety of bacteria ( Rajput et al., 2016 ), most of which were classified as types of N -acyl homoserine lactones (AHL) ( Williams, 2007 ; Case et al., 2008 ). AHL molecules have been detected and extracted from various marine environments, such as POM ( Hmelo et al., 2011 ), the Trichodesmium phycosphere ( Van Mooy et al., 2012 ), and microbial mats ( Decho et al., 2009 ), implying a prevalence of AHL-based QS in marine ecosystems. AHL-mediated QS is the best characterized QS system and is commonly found in Gram-negative bacteria ( Case et al., 2008 ; Papenfort and Bassler, 2016 ). AHLs are produced by LuxI-type AHL synthases and sensed by LuxR-type receptors. Most AHL-producing bacteria that have been isolated from POM belong to Rhodobacterales , Sphingobacteriales , and Vibrionales ( Gram et al., 2002 ; Doberva et al., 2015 ; Su et al., 2018 ). Nonetheless, the abundance and diversity of microbial species possessing QS systems in marine environments are greatly underestimated since most of marine microorganisms have not been isolated and cultured. With the employment of metagenomic techniques, novel AHL synthases have been gradually discovered in other clades of bacteria, including the Gram-positive Exiguobacterium spp. ( Muras et al., 2018 ) and nitrite oxidizing bacteria Nitrospirae spp. ( Nasuno et al., 2012 ). Metagenome analyses, which avoid cultivation biases, have great probability to find new QS systems in marine environments. Bacterial QS signaling can be disrupted by another mechanism, which is termed quorum quenching (QQ). QQ-based mechanisms are diverse, for example, inhibition of signal reception by secreting inhibitors or antagonists ( Manefield et al., 2002 ) and enzymatic hydrolysis of AIs by QQ enzymes ( Dong and Zhang, 2005 ). Synthesizing QQ enzymes is a popular method for bacteria to interfere with QS-mediated processes, and AHL degradation enzymes have been extensively studied. Two types of AHL degradation enzymes (AHL lactonase and AHL acylase) have been identified so far. The lactonases hydrolyze the HSL ring of the AHL molecule generating the corresponding acyl homoserines ( Dong et al., 2007 ), while the acylases cleave the AHL amide bond to generate the free fatty acid and HSL ring ( Romero et al., 2008 ). QQ has been recommended as a promising strategy for anti-virulence therapy, since it only inhibits QS-regulated virulence instead of cell growth and division, which results in little selective pressure for the evolution of resistance ( Tang and Zhang, 2014 ; Tang et al., 2015 ). Until now, the most studied QQ enzymes are originated from genus Bacillus ( Dong et al., 2002 ; Lee et al., 2002 ), Agrobacterium ( Zhang et al., 2002 ; Uroz et al., 2009 ), and Pseudomonas ( Huang et al., 2003 ; Sio et al., 2006 ), which are mainly isolated from soil. In contrast, only a few novel QQ enzymes have been identified from marine environments ( Tang et al., 2015 ), which harbor unprecedented diverse microorganisms. In recent years, the existence and roles of QS and QQ in marine environments have been gradually verified. Diverse QS and QQ bacteria have been isolated from POM ( Jatt et al., 2015 ; Su et al., 2018 ), corals ( Gram et al., 2002 ; Golberg et al., 2011 ), dinoflagellates ( Wagner-Dobler et al., 2005 ), and the Trichodesmium phycosphere ( Van Mooy et al., 2012 ), while novel QS and QQ genes have been found using metagenomic techniques. According to Doberva and colleagues, genes coding for LuxI, AHL lactonase, and AHL acylase homologs were found in all samples obtained from the Global Ocean Sampling (GOS) database, which implied a prevalence of QS and QQ in the global ocean ( Romero et al., 2012 ; Doberva et al., 2015 ). Nevertheless, due to the limitation of GOS sampling, little is known about QS and QQ metabolic pathways in vertical distributed POM from Chinese marginal seas. In the present study, the microbial communities associated with POM were surveyed based on 16S rRNA gene and metagenome sequencing. Furthermore, metagenomic sequencing and analyses were conducted to analyze the abundances and diversities of QS and QQ genes in vertical distributed POM. Our results here expand the current knowledge of QS and QQ metabolic pathways in sinking POM, and help to understand the intertwined interspecies communications in marine ecosystems.",
"discussion": "Discussion In this study, we analyzed the microbial community and the pivotal genes related to QS and QQ mechanisms in the distributed POM collected from the YS. Microbial abundances and diversities increased along with POM sinking from SW to BW, and the diversities reached highest when POM was sinking to SS. However, the abundances of luxI and luxR even decreased slightly. Except for that, abundances of encoding genes for AHL lactonases and acylases held steadily in water column, but increased dramatically in SS, which indicated higher possibilities of AHL-degradation capabilities in POM from SS. According to the results of phylogenetic analyses, we assumed that the vertical transmission of QS and QQ genes exist in POM, which might be one of the key factors resulting in the high abundance and diversity of QS and QQ genes in POM. Our results add to the current knowledge of QS and QQ metabolic pathways in POM, and shed light on the intertwined interspecies relationships to better investigate their dynamics and ecological roles in POM cycling. Different Microbial Assemblage in Vertical Distributed POM The YS is a typical semi-enclosed marginal sea in China. It is an active area for POM processing and recycling with active land–ocean interaction, which is strongly influenced by rivers, terrestrial input, human activities, and complex current ( Lin et al., 2005 ; Zhu et al., 2018 ). The sampling sites HS5 and H12 are located at the same latitude, but HS5 is closer to the coast, where it is more affected by terrigenous materials. However, much more discrepancies of microbial communities resulted from variant depth, instead of the horizontal distance ( Figure 2 and Supplementary Figure 3 ). We therefore segregated the 10 samples into three groups (SW, BW, and SS) according to the sampling depth. Bacterial colonization was more likely impacted by the particle size in SW rather than in other sampling depth. Photoautotrophic Cyanobacteria ( Synechococcus spp., and P. mitra ) and E. huxleyi were more enriched in larger POM collected from SW ( Figure 2 ) for sufficient light energy and nutrients, which is in coincidence with the previous observations ( Cottrell and Kirchman, 2009 ; Thiele et al., 2015 ). Conversely, the SAR clusters (including SAR11, SAR86, and SAR116) preferred smaller particles in SW. They were also numerically significant in the Chinese marginal seas, which were of considerable interest to transport and metabolize the POM compounds ( Giovannoni and Rappé, 2000 ). Bacteria belonging to Rhodobacteraceae are another predominant member colonizing marine particles ( Dang et al., 2008 ; Geng and Belas, 2010 ). Many of them possess dual-particle-associated and free-living lifestyles ( Geng and Belas, 2010 ; Zan et al., 2012 , 2015 ), which could be switched by QS-regulated mechanisms, such as flagellar motility and biofilm formation ( Zan et al., 2012 ; Su et al., 2018 ), antimicrobial indigoidine biosynthesis ( Cude et al., 2015 ), TDA production ( Berger et al., 2011 ), and hydrolytic enzyme production ( Su et al., 2018 ). That might be the reason for the even distribution of Rhodobacteraceae on different size particles. Moreover, some specific species, like Alteromonas spp. and Puniceicoccaceae spp., were predominant at HS5 and H12, respectively. Both of them were pivotal polysaccharide degraders in marine systems with diverse CAZyme repertoires, which play important roles to remineralization of chemically diverse POM ( Martinez-Garcia et al., 2012 ; Koch et al., 2019 ). Oceanospirillales , Candidatus Actinomarina, and Marinimicrobia were distinctive and predominant species in BW. Many studies have revealed their roles in carbon utilization ( Bull et al., 2005 ; Cao et al., 2014 ; De Corte et al., 2018 ); therefore, they might be the key participants involved in the hydrocarbon utilization in a deeper water layer. Moreover, archaea belonging to Candidatus Nitrosopumilus and Thermoplasmatales accounted for more than 20% in BW microbiome and positively correlated with concentrations of SiO 4 2– , PO 4 3– , and NO 3 – . Specifically, these archaea might be highly involved in carbon ( Poulsen et al., 2013 ), nitrogen ( Lloyd et al., 2013 ), and sulfur cycling processes ( Zhang et al., 2015 ). The recalcitrant organic components in POM accumulate with depth during the sinking from SW to seafloor, given that the biodegradable components are continuously consumed by surrounding bacteria. Nonetheless, hydrolysis rates were found highest in surface or near-surface sediments ( Meyer-Reil, 1986 ; Poremba and Hoppe, 1995 ), which may result from the associated microbial communities with versatile hydrolytic enzymes. Xanthomonadales , Desulfuromonadales , and Nitrospirales were usually observed in areas with adequate organic matter, e.g., coastal seawater ( Dang et al., 2011 ), metal-contaminated soils ( Hemmat-Jou et al., 2018 ), and crude oil field soil ( Abbasian et al., 2016 ), which is in correspondence with results in this study. Moreover, part of dominant microorganisms in SS might be inherited from BW (e.g., Candidatus Nitrosopumilus spp. and Oceanospirillales ), and even from SW (e.g., Rhodobacteraceae , SAR86 clade and Pseudoalteromonas spp.). The migration of microorganisms along with sinking POM might be a common process in marine ecosystem. Besides, there was no absolutely superior species in SS compared with those in SW and BW, from where the microbial diversities were the highest. Distribution and Microbial Composition of QS Genes in POM Quorum sensing widely existed in marine environments and is of great significance to marine ecosystems by regulating POM degradation ( Hmelo et al., 2011 ; Su et al., 2018 ), maintaining a healthy and stable state of coral environment ( Golberg et al., 2011 ), and promoting organic phosphorus cycling ( Van Mooy et al., 2012 ). Therefore, revealing the abundance and distribution of QS in POM will facilitate our knowledge of microbial roles in marine environments. In previous studies, the QS systems were mainly characterized from the cultivated bacteria, while lots of information from the uncultivated microorganisms were missed. Here we utilized the metagenomic analyses to reveal the distribution pattern of QS and QQ systems in POM. Genes encoding for LuxI, LuxR, and the two QQ enzymes, AHL lactonase and AHL acylase did not exhibit the highest abundance in the same fraction. LuxI homologs were constrained in bacteria, especially in Proteobacteria ; thus, the high abundance of LuxI in the surface POM seemed largely dependent on the portion of specific species, such as Alteromonas spp. Conversely, LuxR and the two QQ enzymes are more widely distributed in diverse species across bacteria, archaea, viruses, and eukaryotes ( Supplementary Table 3 ); thus, their abundances rely on the structure of microbial community more than a single species. Therefore, the abundance of LuxI is likely to be capricious, but that of LuxR and the two QQ enzymes could resist slight fluctuations caused by succession of microbial community. To date, genes encoding for LuxI ( Doberva et al., 2015 ), AHL lactonase, and AHL acylase ( Romero et al., 2012 ) have been found ubiquitous in global SW using metagenomic analyses. Nonetheless, the abundance and diversity of QS and QQ genes in marine environment could be more abundant than ever expected, which may result from the vertical transmission of QS and QQ genes along with the sinking of POM. In this study, we found that the averaging abundance of luxI in POM from YS (ca. 0.02) is significantly higher than that in the GOS dataset (0.007). The discrepancy may result from the different sampling sites and screening methods. The GOS metagenomic dataset was mainly collected from the surface water from the Atlantic, Pacific, and Indian Oceans. A wide range of oceans were sampled in GOS project except the marginal seas of China, which possess large input volume of POM and diverse microorganisms. It seems like QS potentials in POM of marginal seas are underestimated. Besides, Doberva and his colleagues used a quite strict selection criteria for sequence screening, and only the luxI homologs in Alphaproteobacteria were detected ( Doberva et al., 2015 ). Comparatively, we conducted BLASTP with relatively loose parameters, and discarded sequences without functional domains manually to secure the coverage and accuracy of screening. The activities of LuxI homologs have been confirmed in Actinobacteria , Alphaproteobacteria , and Gammaproteobacteria ( Jatt et al., 2015 ; Su et al., 2018 ), which was in correspondence with the results here. It was verified that the screening method used in this study was reliable for the retrieval of QS genes. The relative abundance of luxR (0.53) was much higher than that of luxI , which attributed to quantities of solo luxR genes in bacteria ( Hudaiberdiev et al., 2015 ), archaea ( Pérez-Rueda et al., 2004 ), and algae ( Figure 3 ). Furthermore, the abundance of luxR in POM from SS was seven times higher than that from seawater, which indicated that microbiota in POM from SS may have greater potential receiving AHL signals to regulate community behaviors. Considering the enormous biological diversity present in an ecological niche, it seems logical that bacteria would produce or receive signals enabling communication with fungi, plants, and animals. Distribution and Microbial Composition of QQ Genes in POM Quorum quenching bacteria have been isolated from both eutrophic marine niches, e.g., POM and Trichodesmium phycosphere ( Van Mooy et al., 2012 ) and oligotrophic seas ( Romero et al., 2011 ), which are more universal than QS bacteria in marine environments. It was reported that the abundance of marine cultivable bacteria with QQ activity ( Romero et al., 2011 ) and the frequency of QQ genes in marine metagenomes ( Romero et al., 2012 ) were higher than that of QS bacteria and QS genes. In our previous study, 16 and 51% cultivable species isolated from POM have been experimentally verified possessing AHL synthesizing and degrading activities, respectively ( Su et al., 2018 ). Moreover, in our present study, the abundances and diversities of QQ genes in POM from the YS were also higher than that of QS genes, which was consistent with the previous studies. It was implied that microbial QQ activities were more prevalent in marine environments and might have more important ecological roles than ever expected. Quorum quenching mechanisms are beneficial to microbial competition by limiting the growth and the coordination of bacteria engaged in QS communication ( Defoirdt et al., 2004 ; Rasmussen and Givskov, 2006 ), which help keep the homeostasis of microbial communities. Moreover, microorganisms in open sea might use QQ enzymes to degrade AHL signaling molecules for additional energy supply. Therefore, the QQ process could serve as a universal adaptive strategy for microorganisms in marine environment. The production of virulence factors in most pathogens were regulated by QS systems, such as Pseudomonas aeruginosa , Vibrio harveyi , and Legionella pneumophila ( Tiaden et al., 2007 ; Nackerdien et al., 2008 ; Jimenez et al., 2012 ). Therefore, interfering with QS metabolic processes would attenuate their pathogenicity and be developed as new therapies combating pathogens. In recent years, more researches focused on developing novel QQ agents derived from marine environments for combating antibiotic-resistant bacteria in aquaculture, agriculture, and anti-biofouling ( Tang et al., 2015 ; Grandclément et al., 2016 ). Our results revealed that a large number of QQ strains are unexplored in the POM from the YS, especially in SS, thus provide invaluable information and inspiration for the study of marine-derived QQ agents in the future."
} | 5,366 |
34675325 | PMC8531344 | pmc | 7,488 | {
"abstract": "The soil microbial community plays a vital role in the biogeochemical cycles of bioelements and maintaining healthy soil conditions in agricultural ecosystems. However, how the soil microbial community responds to mitigation measures for continuous cropping obstacles remains largely unknown. Here we examined the impact of quicklime (QL), chemical fungicide (CF), inoculation with earthworm (IE), and a biocontrol agent (BA) on the soil microbial community structure, and the effects toward alleviating crop yield decline in lily. High-throughput sequencing of the 16S rRNA gene from the lily rhizosphere after 3 years of continuous cropping was performed using the Illumina MiSeq platform. The results showed that Proteobacteria , Acidobacteria , Bacteroidetes , Actinobacteria , Chloroflexi and Gemmatimonadetes were the dominant bacterial phyla, with a total relative abundance of 86.15–91.59%. On the other hand, Betaproteobacteriales , Rhizobiales , Myxococcales , Gemmatimonadales , Xanthomonadales, and Micropepsales were the dominant orders with a relative abundance of 28.23–37.89%. The hydrogen ion concentration (pH) and available phosphorus (AP) were the key factors affecting the structure and diversity of the bacterial community. The yield of continuous cropping lily with using similar treatments decreased yearly for the leaf blight, but that of IE was significantly ( p < 0.05) higher than with the other treatments in the same year, which were 17.9%, 18.54%, and 15.69% higher than that of blank control (CK) over 3 years. In addition, IE significantly ( p < 0.05) increased organic matter (OM), available nitrogen (AN), AP, and available potassium (AK) content in the lily rhizosphere soil, optimized the structure and diversity of the rhizosphere bacterial community, and increased the abundance of several beneficial bacterial taxa, including Rhizobiales, Myxococcales, Streptomycetales and Pseudomonadales. Therefore, enriching the number of earthworms in fields could effectively optimize the bacterial community structure of the lily rhizosphere soil, promote the circulation and release in soil nutrients and consequently alleviate the loss of continuous cropping lily yield.",
"conclusion": "Conclusion Earthworms inoculation (IE) significantly increased the yield of continuous cropping lily, and alleviated the decline in yield. Because IE significantly increased the content of OM, AN, AP, and AK in the rhizosphere, and the pH, and AP are key factors that influence the structure and diversity of bacterial communities. They optimized the structure and diversity of the bacterial community, improving the abundance of some beneficial bacteria, including members of Rhizobiales , Myxococcales , Streptomycetales and Pseudomonadales . These beneficial bacteria are helpful to reduce the occurrence of soil-borne diseases. The exotic earthworms inoculation and additional application of cow dung were helpful to understand the effect of earthworms on relieving the continuous cropping obstacle of lily. The results may guide farmers the application of OM (the food of native earthworms) to enrich the native earthworms in the soil, thereby reducing the barriers to continuous cropping.",
"introduction": "Introduction Lily is a perennial herb of the Lilium genus, monocotyledons subclass. It is widely cultivated in east Asia, Europe and North America benefiting from its high medicinal, edible, and ornamental value 1 – 3 . Lily cultivation in China has obvious regional characteristics, and has a long planting history. However, this crop is usually cropped for several years on the same arable land 4 , which will changes physical and chemical soil properties, resulting in self-toxic allelochemicals 4 , 5 . These chemical compounds are usually secreted by the roots or produced by the decomposition of root residues, which tend to cause a direct rhizosphere microorganisms selection 6 , 7 and lead to a soil microbial community structure imbalance 8 , to be the main cause of continuous cropping obstacles. These will subsequently result in decreasing of the diversity and richness indices of bacterial community 9 . The main manifestations include increasing in fungal pathogens 10 , and decreasing in beneficial bacteria 11 and ratio of bacteria to fungi. Consequently, bacteria-type soil shifts to fungi-type soil 10 . In particular, the number of pathogenic microorganisms such as Fusarium increases and causes the occurrence of lily leaf blight disease, then leads to a decline in lily yield and quality 9 , 11 , 12 . In order to effectively alleviate the decline in the yield of continuous crop, physical, chemical, and biological methods have been formulated to ameliorate this soil characteristic. Lime and ammonium bicarbonate are usually applied as physical agents to ameliorate acidic or alkaline soil 13 . In contrast chemical methods include the use of trifloxystrobin, carbendazim, mancozeb, thiram and chlorothalonil 14 , 15 , and biological methods include using biocontrol bacteria 16 , biological organic fertilizer 17 , and earthworm activities (wormcast) 18 , 19 . All of these treatments are beneficial for optimizing the microbial community structure of continuous cropping soils, and adjust the unfavorable factors affecting plant growth and directly inhibiting the rapid growth of soil fungi. Because the bacterial community and the interactions among different bacterial taxa play essential roles in continuous cropping fields 8 , the composition and structure of the soil bacterial community could be used as a new indicator of soil ecosystem health, productivity and environmental disturbance 20 , 21 . As an important component of the soil ecosystem, soil bacteria participates in nutrient cycling, organic matter (OM) decomposition, energy conversion 22 , 23 , and plays an important role in inhibiting soil-borne diseases 24 . Shen et al. 25 found that an increased abundance of Pseudomonas significantly positively correlates with soil available phosphorus (AP) and disease inhibition. Pearson's correlation demonstrated that the abundance of Arthrobacter and Lysobacter , which are considered to be beneficial bacteria, had a significant negative correlation with bacterial wilt disease 24 . However, the bacterial community changes with continuous cropping lily rhizosphere remains largely unknown. In this study, the responses of the soil microbial community were investigated for continuous cropping lily ( Lilium lancifolium Thunb.) for 3 years when the mitigation measures were applied for continuous cropping obstacles. Quicklime, thiram, earthworm inoculation, and a biocontrol agent were used to alleviate the characters of continuous cropping lily. Leaf blight and yield, soil properties, and the rhizosphere bacterial community were investigated to provide a theoretical basis for exploring an efficient cultivation technique for continuous cropping lily.",
"discussion": "Discussion Continuous cropping affects lily leaf blight and bulbs yields Continuous cropping obstacles related to the deteriorating of crop growth condition, yield and quality reduction, and aggravation of diseases due to the continuous planting of the same crop in the same plot over many years. Lily can be used as a medicinal plant, vegetable and cut flower variety. This crop is usually planted around September to October each year, and harvested around the July of the next year, so only one crop is cultivated in the same field every year. Restricted to farmers' planting habits and land resource, lily cultivation has obvious regional characteristics in China 28 . The economic value results in common continuous cropping, which leads to a severe decline in lily yield and quality 29 . In this study, we used different treatments (QL, CF, IE, and BA) to mitigate the continuous cropping obstacles for lily, demonstrating that all these four treatments increased the output of continuous cropping lily compared to CK. However, the lily yield in the same treatment still decreased annually (Fig. 2 ), which means that physical, chemical, and biological treatments for continuous cropping lily soil can relieve the declining yield. On the contrary, the incidence and disease index of continuous cropping lily in the same treatment increased annually (Fig. 1 A,B), resulting in a negative correlation between the bulb yields and disease index or incidence (Fig. S1 ). These results suggested that the continuous cropping obstacles of lily were remains obscure. Among different treatments, IE was the most effective one to alleviate the loss of continuous cropping lily yield. Earthworms activity affect the soil physical and chemical properties to increase the yield of continuous cropping lily Earthworms are known as “ecosystem engineers” 30 , and they constitute the largest biomass in soil fauna and play an important role in maintaining the structure and function of soil ecosystem. Earthworms are often used as an important indicator of soil health 31 , The excavation and excretion activities of earthworms can raise soil porosity and the formation of soil aggregate 32 , 33 , increase soil mineral-N and microbial biomass N 34 , and improve soil water infiltration rate 33 . These have been demonstrated to promote lily growth. Long-term continuous cropping can usually causes soil acidification 11 , and then greatly influence the soil microbial community structure 35 . The decrease of bacterial diversity and the increase of fungi in acidified soil will promote the proliferation of soil-borne pathogens and the occurrence of crop diseases 36 , so the pH was a driver of the microbial community (Fig. 7 ; Table 3 ). Literature reported that the number and the biomass of fungi decreased by 50% and by 42%, respectively, when soil pH increase from 4.5 to 7.0 37 . Therefore, alleviating soil acidification is beneficial to reduce the occurrence of continuous cropping lily leaf blight. Earthworms secretions, including calcium carbonate substances and nitrogen substances can increase the pH of acidic soil and alleviate soil acidification 38 , 39 . The inoculated E. fetida were fed with cow dung, which boosted their own reproduction, and increased 22.41-times the original input of E. fetida in the soil (the number of earthworms was 421.33 m −2 in the inoculum, and the number of E. fetida input was 18 m −2 ) (Fig. 3 ). The activities of earthworms, including digging, swallowing and excreting, can effectively increase soil porosity and promote the transformation of organic matter, subsequently increase the organic matter content in the rhizosphere of lily (Table 1 ), which will then affect soil microbial community structure and diversity of function 40 , and also retard soil acidification 41 . These are advantageous to soil nutrient cycling 42 , increasing the effectiveness of soil nutrients (such as AN, AP, and AK; Table 1 ), and the nutrient turnover rate 43 , 44 . In addition, earthworms can secrete humic acids, which are similar to plant hormones 45 . These compounds could promote the growth of lily roots, facilitate the absorption and utilization of nutrients, which is beneficial to the development of continuous cropping lily, and enhances disease resistance and the yield (Fig. 2 ). Earthworms activity affect the soil microbial community structure and diversity to reduce the disease of continuous cropping lily Earthworms influence the ecological processes mainly through the soil microbial communities 46 , 47 . They can directly influence microbial community structure through excavation and digestion 48 , or indirectly influence microbial community structure through changing the soil physical and chemical properties, including soil porosity, aggregate stability, pH, and availability nutrient 49 . Earthworms reduced the number of pathogenic fungi by selectively feeding on fungi in soil, destroying fungal hyphae, and competing with fungi for food resources 50 , 51 . In particular, the decline in the number of Fusarium effectively reduced the occurrence of lily leaf blight (Fig. 1 A,B), which is consistent with the results of Bi et al. 17 . The microenvironment of earthworm intestinal tract (high humidity, neutral pH, appropriate C/N) is suitable for microbial reproduction. Because the intestinal mucus of earthworm is an unstable substance with high nitrogen content, which can stimulate the growth and reproduction of bacteria 52 . According to available published researches, it can be deduced that the regulation of microbial community structure by earthworms would have enhanced soil microbial activity and enzyme activity 53 , 54 , improved soil microecological environment and promoted nutrient cycling, which were conducive to plant root growth and nutrients absorption, and improved the yield of lily (Fig. 2 ). The soil structure and function regulation are mainly because of the close relationship between earthworms and soil microorganisms 55 . Earthworms influenced the abundance and diversity of the rhizosphere bacterial community through their activities and wormcast, which resulted in the apparent aggregation of the rhizosphere bacterial community among different replicates in IE treatment (Fig. 4 ). IE mainly reduced the bacterial community diversity (Simpson Index, Table 2 ) and an increase in the abundance of some important bacterial species (Figs. 5 , 6 A,B), which are consistent with the results of Jayasinghe et al. 56 . At the phylum level, IE increased the relative abundance of Proteobacteria and Actinobacteria , but reduced the relative abundance of Acidobacteria , compared to the other treatments. Previous studies suggest that members of Proteobacteria are eutrophic bacteria and positively correlate with soil nutrients 57 . Actinobacteria play key roles in OM decomposition and humus formation processes 58 , 59 . The relative abundance of Streptomycetales (phylum Actinobacteria ) was higher in IE than in the other treatments (Fig. 6 A,B), and these taxon can produce a variety of antibiotics (secondary metabolites) 60 to protect roots from other pathogenic microorganisms 61 . The activity of earthworms can enhance soil porosity and aeration, promote the growth of aerobic bacteria, and reduce anaerobic bacteria (e.g., Acidobacteriales ). In this study, the relative abundance of Acidobacteriales (phylum Acidobacteria ) significantly negatively correlated with soil pH (Fig. S4 ), which is in line with the results of Jones et al. 62 . Because the IE treatment increased the number of earthworms, more earthworms dug in the soil and more wormcast was excreted, which then heightened OM diffusion, reduced the soil bulk density, increased the soil porosity and permeability, promoted the growth and reproduction of aerobic microorganisms, and affected the reproduction of anaerobic Acidobacteriales . This then led to a lower relative abundance of Acidobacteriales in the rhizosphere of IE treated soil compared to the other treatments (Fig. 6 A,B) and raised the pH (Table 3 ). Furthermore, the relative abundance of Rhizobiales was the highest in the IE group (Fig. 6 A,B). Liu et al . 63 reported a symbiotic relationship between lilies and Rhizobiales , which plays an important role in nitrogen fixation, increasing the content of TN and AN in the rhizosphere of continuous cropping lily. The increase in Flavobacteriales, Streptomycetales and Pseudomonadales by IE treatment should activate the fixed phosphorus and then increase the AP content in the rhizosphere 64 (Table 1 ). Besides, the higher abundance of Myxococcales in IE-treated soil enhanced the resistance to soil-borne pathogens 65 . Simultaneously, Pseudomonadales produced antibacterial metabolites that can effectively inhibit the occurrence of soil-borne pathogens 66 from diseases in continuous cropping lily. These results suggest that the increased abundance of these beneficial bacteria in IE treatment is helpful by promoting nutrient cycling and improving stress resistance in the rhizosphere 67 , positively affecting the growth and yield of continuous cropping lily, and alleviating the decline in yield. Relationship among earthworms and continuous cropping lily According to the feeding habits and life in different soil depths, earthworms can be divided into three ecological classes, including the epigeic species, the endogeic species, and the anecic species 68 , 69 . The application of exotic E. fetida (epigeic species) was beneficial to observe their abundance and cow dung conversion efficiency under the same organic material conditions, and distinguish the growth and reproduction from that of the native earthworms. In this study, local earthworms of the continuous cropping fields included one epigeic species, four endogeic species, and three anecic species. The epigeic species mainly live in surface soil, feeding on humus, with small size, short reproduction cycle, and can move quickly when disturbed. The endogeic species mainly live in the subsurface soil and feed on the soil organic matter. They have larger size and stronger digging capacity, moving slowly when the soil is disturbed. Anecic species have the largest size and strongest dragging capacity. They eat the humus in the surface soil and draw them into the hole in deep soil 70 . In comparison, E. fetida (epigeic species) is a compost earthworm, they live in organic matter and have a strong ability to convert fresh manure into vermicompost 53 . There were more E. fetida in IE group than the others (Fig. 3 ), which would promoted the degradation and diffusion of the organic matter by digging and excreting in the surface soil, and provide an additional food resource for the endogeic and anecic species (Table 1 ), and stimulated the activity of them 71 . Subsequently, more and more earthworms activity further improved the physical and chemical properties of the underlying soil (Table 1 ), provided available nutrient for lily growth, optimized the bulb roots rhizosphere bacterial community (Fig. 6 A,B), and also alleviated the yield loss of continuous cropping lily. At the same time, the stem roots of lily usually grow in the surface soil, where E. fetida active (IE). The surface-applied cow manure provided a food source for E. fetida and therefore improved the physical and chemical properties of the surface soil, also provides available nutrient for lily growth, and better increased the yield of continuous cropping lily. Compared to E. fetida of IE, local earthworms in the other trentments weakly transform the surface cow manure to soil organic matter and available nutrients (OM, AN, AP, AK; Table 1 ), causing that the nutrients of cow manure were not easily absorbed and utilized by lily. However, the rhizosphere fungal community, especially the pathogenic fungi (e.g., Fusarium , Alternaria ) and beneficial fungi (e.g., Mycorrhiza , Trichoderma ) should also play crucial roles in alleviating the decline in yield of continuous cropping lily . Therefore, whether earthworm inoculation affects the diversity and composition of rhizosphere fungal communities should be investigated in the future."
} | 4,800 |
26016911 | PMC4507646 | pmc | 7,489 | {
"abstract": "This paper presents the methodology and challenges experienced in the microfabrication, packaging, and integration of a fixed-fixed folded spring piezoelectric energy harvester. A variety of challenges were overcome in the fabrication of the energy harvesters, such as the diagnosis and rectification of sol-gel PZT film quality and adhesion issues. A packaging and integration methodology was developed to allow for the characterizing the harvesters under a base vibration. The conditioning circuitry developed allowed for a complete energy harvesting system, consisting a harvester, a voltage doubler, a voltage regulator and a NiMH battery. A feasibility study was undertaken with the designed conditioning circuitry to determine the effect of the input parameters on the overall performance of the circuit. It was found that the maximum efficiency does not correlate to the maximum charging current supplied to the battery. The efficiency and charging current must be balanced to achieve a high output and a reasonable output current. The development of the complete energy harvesting system allows for the direct integration of the energy harvesting technology into existing power management schemes for wireless sensing.",
"conclusion": "4. Conclusions The methodologies used and challenges encountered in the microfabrication and integration of fixed-fixed folded spring energy harvesters designed and characterized in [ 5 ] have been discussed in detail. A variety of challenges were overcome in the fabrication of the energy harvesters, such as the diagnosis and rectification of sol-gel PZT film quality and adhesion issues, including the use of more robust adhesion materials. Additionally, the use of a carrier wafer allowed for the use of a carrier wafer to promote non-invasive cleaving of the harvesters. A complete packaging and integration methodology was developed to facilitate testing the harvesters under a base vibration. This included developing a packaging methodology using printed circuit boards and wirebonding to allow for the electrical characterization of the harvester without relying on a traditional probe station. Two conditioning circuits were developed to electrically characterize the harvester and to store the harvested energy in a NiMH battery. The initial simple conditioning circuit was useful to measure the total available power from the harvester. The second conditioning circuit developed allowed for the creation of a complete energy harvesting system, consisting of the harvester, a voltage doubler, a voltage regulator and a NiMH battery. The biggest challenge in the development of the conditioning circuitry is maximizing the efficiency and current stored in the battery. The maximum circuit efficiency is 27.5%, at an input frequency of 100 Hz, input voltage of 3 V p-p, battery voltage of 1.15 V, and current limiting resistance of 5 kΩ. The battery charging current for this case is 27.9 µA, with voltage doubler efficiency of 47.5%, and voltage regulator efficiency of 67.9%. However, the maximum battery charging current was found to be 51.7 µA at an input frequency of 100 Hz, input voltage of 4 V p-p, battery voltage of 1 V, and current limiting resistance of 5 kΩ. The overall efficiency for this case is 20.32%. The voltage doubler efficiency is 41.9% and the voltage regulator efficiency is 62.9%. It can be concluded that the maximum efficiency does not correlate to the maximum charging current supplied to the battery. Conversely, the maximum charging current can be achieved at the expense of the overall efficiency. The charging current supplied to the battery is only effected by the battery voltage, increasing with decreasing battery voltage. The efficiency of the designed conditioning circuit is increased with decreasing input voltage, input frequency, and battery voltage. The efficiency and charging current must be balanced to achieve a high output and a reasonable output current. Transitioning to active conditioning circuitry in future iterations of the energy harvestings system should increase efficiency and current supplied to the battery. The development of the complete energy harvesting system allows for the direct integration of the energy harvesting technology previously developed into existing power management schemes for wireless sensing.",
"introduction": "1. Introduction The focus of the majority of Microelectromechanical (MEMS)-based energy harvesting research is the optimization of the power output of a specific energy harvester. Regardless of the harvesting methodology or design [ 1 , 2 , 3 , 4 ], significant challenges exist in the design, fabrication, and implementation of the energy harvester. This paper will focus on the challenges encountered in the microfabrication; packaging and integration of a fixed-fixed folded spring vibration-based piezoelectric energy harvesting system [ 5 ]. The focus of this specific design of the folded spring energy harvester was to controllably and reliably reduce the natural/operational frequency of the harvester while remaining insensitive to out of plane static deformations due to residual stresses [ 5 , 6 , 7 , 8 , 9 ]. The folded spring allows for the effective length of the spring to be increased while allowing for free expansion, contraction, and rotation of individual beam elements of the spring to dissipate residual stresses. In general, there are two methodologies available to fabricate the released structures required for the piezoelectric energy harvesters. The first method relies upon surface micromachining techniques to undercut the released structures [ 4 , 10 , 11 , 12 , 13 , 14 ]. Typically, this method uses a sacrificial material and an etch stop, such as a Silicon-On-Insulator (SOI) wafer, to release the structural members of the device. This fabrication methodology is capable of producing thin structural beams with high accuracy, which is desirable for low frequency energy harvesting applications. The second method relies on bulk micromachining to define the geometry and release the structure of the harvesters [ 4 , 15 , 16 , 17 , 18 ]. Typically, this method uses Deep Reactive Ion Etching (DRIE) to remove the structural silicon from both sides of the wafer to define the structural beams and proof masses required by the energy harvesters. Although the bulk micromachining methodology may introduce difficulties in fabricating thin structural members, integrated proof masses are difficult to achieve with surface micromachining techniques. A release methodology was developed for the energy harvesters designed in this research [ 5 , 9 ], based upon the bulk micromachining techniques, to allow for the required large proof masses and thin structural beams. Several difficulties encountered with this chosen method of releasing the energy harvester structure will be discussed at length in the following sections. Throughout the remaining microfabrication process flow, several obstacles were encountered, including difficulties caused by titanium residues from lower electrode etching, uneven and non-continuous films deposited by a simultaneous sol-gel and lift-off process, and wafer handling issues caused through the backside etching required to release the harvesters from the silicon wafer [ 9 , 19 ]. Additionally, the harvester operates under a base vibration, wafer-level testing was not possible. Therefore, full packaging of the harvesters were required, leading to several challenges dealing with adhesion of wirebonds and the overall design and implementation of suitable packaging [ 9 , 19 ]. Finally, significant challenges were encountered in designing and fabricating suitable conditioning circuitry including maximizing the efficiency and charging current delivered to the storage medium. Additionally, the relationship between the efficiency and battery charging current of the conditioning circuitry in response to variations in the input voltage, input frequency, and battery voltage level will be examined with a feasibility study. In the following sections, each of the microfabrication, packaging and integration and conditioning circuitry-based challenges will be examined in detail."
} | 2,039 |
21841845 | null | s2 | 7,491 | {
"abstract": "Computational Cognitive Neuroscience (CCN) is a new field that lies at the intersection of computational neuroscience, machine learning, and neural network theory (i.e., connectionism). The ideal CCN model should not make any assumptions that are known to contradict the current neuroscience literature and at the same time provide good accounts of behavior and at least some neuroscience data (e.g., single-neuron activity, fMRI data). Furthermore, once set, the architecture of the CCN network and the models of each individual unit should remain fixed throughout all applications. Because of the greater weight they place on biological accuracy, CCN models differ substantially from traditional neural network models in how each individual unit is modeled, how learning is modeled, and how behavior is generated from the network. A variety of CCN solutions to these three problems are described. A real example of this approach is described, and some advantages and limitations of the CCN approach are discussed."
} | 253 |
27415783 | PMC4944945 | pmc | 7,492 | {
"abstract": "In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of biological materials with diverse morphologies. The SilkSlider predictor software developed here is available at https://github.com/wwood/SilkSlider .",
"conclusion": "Conclusions Nature is capable of producing materials that far exceed the current technical capabilities of humankind. In this study we reveal that a class of proteins, the SLSPs, are components of these materials in a wide range of taxa. These proteins can be defined as possessing a signal peptide and a domain in which at least a quarter of the residues are glycine. Despite these common features, these proteins have evolved numerous times independently in different lineages. The glycine-rich domains likely confer elasticity and toughness to the materials these proteins form, and/or facilitate their construction by the formation of amorphous gels. This research suggests that common principles underlie the construction of divergent biological materials, despite them being made from evolutionarily distinct proteins.",
"introduction": "Introduction Animals produce a diverse array of materials to aid with the multiple functions of life, including support, defence, feeding and reproduction. These structures are made from substances produced by the animal itself, which thus are ultimately encoded and/or regulated by the genome, sometimes with the inclusion of inorganic elements (e.g., calcium carbonate in the skeletons of many invertebrates). Their expression is precisely controlled at the nanoscale to produce structures with outstanding mechanical properties, such as silk, shells, bones, teeth, and hair, however the process by which this control is achieved is not yet fully understood. These biological materials are the inspiration for materials scientists, who are yet to fully emulate the properties of these substances, or to generate them at ambient temperatures and atmospheric pressure. Arguably the best-studied biological material is silk, a fibre produced by a number of arthropods including spiders and silkworms that possesses remarkable properties including high elasticity and strength [ 1 , 2 ]. Underlying the exceptional properties of silk fibroins are a set of extraordinarily large proteins with a modular, repetitive design. The repetitive regions are made up of distinct protein motifs including poly-A or poly-GA stretches, GPGXX/GPGQQ repeats and collagen-like GGX repeats (A = alanine, G = glycine, Q = glutamine, X = alanine, serine, valine, tyrosine or threonine) [ 3 – 6 ] that produce a modular protein consisting of crystalline domains interspersed with amorphous regions [ 1 , 5 , 7 ]. These high-performance fibres are utilized for essential biological processes such as reproduction and feeding, and are critical for the success of the organisms that produce them. The different types of silks produced by these animals are finely tuned at the molecular level for their particular purpose. For example, spider flagelliform silks,which comprise the capture spiral of the web, are highly elastic but have lower tensile strength due to the inclusion of proline residues within the typical glycine-rich repeats, whereas major ampullate silks, which form the framework of the web, are much stronger but less elastic due to the inclusion of increased poly-A and GGX repeats [ 3 ]. Therefore, the amino acid content and arrangement–both of which are mutable and therefore under natural selection—directly influences the physical properties of the protein. Interestingly, the sequence features that are critical for the function of spider silks can also be found in proteins that are core components of tough, extracellular structures in other organisms. Proteins with this architecture have been described from mollusc shells [ 8 – 11 ], mussel byssus [ 12 – 15 ], lamprey cartilage [ 16 ], scallop hinge ligaments [ 17 ], polychaete tube cement [ 18 ], carp fertilisation envelopes [ 19 ], trematode eggshells [ 20 , 21 ], human epidermal cell envelopes [ 22 ], cnidarian nematocysts [ 23 ] and even in plant cell walls [ 24 , 25 ]. These proteins exhibit low sequence complexity, possessing single amino-acid tracts or sequence repeats of differing lengths [ 26 – 29 ]. They also often display modularity, containing one or more repetitive, low-complexity regions interspersed with other functional domains [ 13 , 30 , 31 ]. The practice of describing a non-silk structural protein as ‘silk-like’, based on these characteristics, is now common in the literature, despite the lack of primary sequence homology between these sequences and silk proteins. The term has been used to describe proteins with low-complexity glycine-rich regions, poly-alanine repeats, or both [ 11 – 15 , 19 , 23 , 32 – 34 ], thus the true nature and extent of the proposed similarity remains undefined. It is not clear whether the silk-like proteins described in the literature perform similar functions within the biological materials they form, although some insights can be garnered from a number of these proteins that have been the subject of biochemical and physical analyses due to their unusual mechanical properties and relevance for biomaterial design. Mussel byssal threads are the means by which these molluscs adhere to rocks against heavy wave action. They are primarily composed of preCol proteins, which are highly modular in nature and contain a central collagen domain [ 15 ]. However, the performance of byssal fibres significantly outperforms that of collagen itself, purportedly due to histidine-rich domains thought to mediate cross-linking between the proteins, poly-alanine rich domains that stiffen the fibre by the formation of crystalline beta sheets, and amorphous glycine-rich flanking domains which absorb stress and assist refolding of the protein once load is released [ 13 ]. Similarly, a number of silk-like proteins have been described from the organic matrix of molluscan shells [ 10 , 11 , 31 , 35 , 36 ]. These proteins also possess glycine-rich and/or poly-alanine rich regions, are localised within the organic matrix that surrounds the calcium carbonate tablets (possibly in the form of an amorphous gel) [ 32 , 33 , 37 ], and are thought to contribute to the strength, elasticity, and fracture toughness of the shell by absorbing strain applied to the shell that would otherwise cause it to crack [ 31 , 38 ]. Therefore, these silk-like proteins possess sequence characteristics that increase the strength and elasticity of the materials which they form, and have the propensity to be amorphous in nature (notably, silk fibroins exist in a hydrated, disordered state within silk glands prior to spinning [ 39 ]). Interestingly, silk fibroins have been found to induce and regulate the mineralisation of CaCO 3 and hydroxyapatite in vivo [ 40 – 43 ], providing further evidence that the similarities in amino acid sequences between silks and biomineralization proteins may be functionally significant. The description of a number of silk-like proteins with functions in the production of biological materials raises a number of questions. First, exactly which sequence features (modularity, repetitiveness, poly-alanine motifs, and/or glycine-rich regions) contribute to this similarity, and how widespread is it across metazoan taxa and the materials they produce? Second, is the similarity within these sequences due to descent from an ancestral (presumably structural) protein, or have similar proteins arisen multiple times throughout metazoan evolution? And, finally, given the likely conserved functions of these proteins, can careful characterisation of sequence similarity reveal how the advanced mechanical properties of these biological materials are dictated by the sequence of the proteins that comprise them? To answer these questions, we set out to systematically characterise these proteins and assess how widely they are distributed in metazoans that fabricate external biological materials. To do so, we identified defining sequence characteristics of proteins with silk-like or glycine-rich repeats that are known to contribute to tough, extracellular structures. We then used these sequence characteristics to develop a bioinformatic predictor for these proteins, which we named SilkSlider. This predictor was then used to survey the transcriptomes and genomes of a range of metazoan species that produce a diversity of biological materials. Using this method we identified genes encoding proteins with silk-like characteristics, which we call silk-like structural proteins (SLSPs), that are known components of biological materials in cnidarians, arthropods, nematodes, molluscs, echinoderms and chordates, as well as a large number of uncharacterized proteins from these taxa as well as from poriferans and annelids. To determine whether these uncharacterised proteins potentially represent hitherto unknown components of biological materials, we assessed their likely function in two distantly-related animals that produce well-studied biological materials, the abalone (a mollusc), and the sea urchin (an echinoderm), and found that a high proportion of predicted genes are associated with the production of shell or spicules, respectively. Our results indicate that the presence of SLSPs is widespread within biological materials produced by disparate metazoans. Interestingly, the genes encoding these proteins appear to have evolved multiple times independently in a number of lineages. The recurrent evolution of proteins with similar traits indicates that they perform common functions within biological materials, and that common principles underlie the formation of widely divergent biologically produced structures.",
"discussion": "Discussion Proteins containing glycine-rich domains are involved in biological material production in diverse metazoans In this study, we have confirmed that many proteins involved in the production of biological materials possess similarity to silks, and that this similarity is primarily due to a high proportion of glycine in at least part of the protein. These SLSPs are widespread throughout metazoans, being found in the transcriptomes and genomes of all animals investigated here (although the predictor identified a single gene, collagen, in the placozoan Trichoplax ; this organism does not appear to produce any tough, extracellular structures). The predictor developed during the course of this study identified a number of proteins to which no functions have yet been ascribed. To determine whether they are potentially undescribed components of biological materials, we assessed the localization of expression of the genes corresponding to several of these proteins in two animals, the gastropod mollusc H . asinina and the sea urchin S . purpuratus . Two uncharacterized proteins were identified by the predictor in the abalone H . asinina as being silk-like, and both had expression patterns consistent with a structural role in the nacreous layer of the shell. Sp-Cara7LA is an uncharacterized S . purpuratus gene that encodes both glycine-rich and carbonic anhydrase domains, and is expressed in the PMCs, consistent with a role in biomineralization in the sea urchin. These functional predictions are further supported by the extraction and characterisation of Has-CL10Contig2 and Sp-Cara7LA proteins from the abalone shell [ 87 ] and sea urchin calcified structures [ 61 – 63 ], respectively. Sequences identified by the predictor that have not been detected in proteomic analyses of sea urchin calcified parts may be minor (and thus undetected) components of these structures, or be involved with the production of other biological materials within this animal. Recurrent evolution of SLSPs in animals SLSPs can be found throughout the animal kingdom and are often components of structures that are morphological novelties for that particular taxon, such as the shells of molluscs, the nematocysts of cnidarians and the tests of sea urchins. It is therefore unlikely that they were inherited from a common ancestor, as there was no common precursor to these structures. The lack of primary sequence conservation between silk-like proteins (outside the presence of glycine-rich domains) also suggests that they arose independently multiple times. Consistent with this, very little similarity has been observed between shell-forming proteomes of various molluscs [ 27 , 89 ], and the spicule matrix (SM) gene family, which is crucial for the formation of sea urchin larval spicules [ 90 , 91 ], is completely absent from the genome of the closely-related hemichordates that also produce biomineralized structures [ 92 ]. The apparent convergent evolution of silk-like proteins in the production of biological materials suggests that high glycine content is functionally advantageous for this class of proteins. SLSPs appear to fall into two broad classes: those that likely evolved from existing functional protein coding sequences and those that appear to have evolved de novo . Several important biomineralization genes, such as sea urchin Cara7LA , encode glycine-rich regions in combination with other domains with biomineralization-related roles. In Cara7LA, a glycine-rich domain is combined with a carbonic anhydrase domain, an arrangement also seen in pearl oyster nacrein proteins [ 93 ]. Similarly, some sea urchin SM family genes encode both C-type lectin [ 94 ] and glycine-rich domains. Nematogalectins, core components of the nematocyst tubule in cnidarians, combine glycine-rich and galectin domains [ 77 ]. The occurrence of glycine-rich domains in proteins that likely already had a function within biological materials is consistent with silk-like properties evolving in some genes that were already part of the biological material regulatory networks in these animals. On the other hand, the expression of numerous lineage-specific silk-like genes in animal tissues responsible for fabricating external structures, such as the lysine (K)-rich mantle protein ( KRMP ) and shematrin genes in pearl oysters [ 24 ], suggests that some silk-like genes evolved de novo and were subsequently incorporated into a role in the formation of these structures. Sequence similarities within SLSPs provide insight into the function of these proteins and the mechanism underlying the production of biological materials The predictor developed in this study is able to detect proteins that are involved in biological material production from large-scale sequence databases, demonstrating a correlation between sequence characteristics and functional roles. The sequence characteristics found to be important for the identification of these proteins (i.e., common to this class of protein) are the possession of a signal peptide (most biological materials are extracellular) and a glycine-rich domain. The importance of glycine is likely because of the properties it confers to the secondary structure of the protein; glycine has smaller side-chains than other amino acids, and appropriate spacing of glycine residues in a sequence is critical for the formation of various ordered structures such as beta-sheets, coiled-coils and collagen-like triple helices, with the final secondary structure being determined by other amino acid residues within the sequence [ 95 ]. Proteins with these ordered structures are known to be important components of biological materials such as spider silks and mollusc shells [ 95 , 96 ]. However not all proteins that are identified by the predictor, including some that are known components of biological materials, have the regular arrangement of glycine residues necessary for the generation of these secondary structures. High glycine content is also known to be important for the flexibility of disordered protein domains; such disordered proteins confer elastomeric properties to biological materials and are also known to be components of spider silks and mollusc shells [ 66 , 68 ]. The glycine-rich domain common to these sequences could therefore facilitate the formation of either folded secondary structures or disorder within the proteins they are found in. It is possible that both of these configurations are important, given that SilkSlider outperforms disorder-based predictors in identifying proteins with biomaterial-related roles. The ability to predict biological material-related proteins based upon primary sequence indicates that common mechanisms may underlie the fabrication of biological materials in different animals. It is possible that proteins with glycine-rich regions provide increased elasticity and toughness to the structures they form, as proposed for several SLSPs [ 13 , 23 , 31 ]. Alternatively, glycine-rich regions may be important for the assembly of the structures. Molluscan biomineralized materials, in particular, are thought to be constructed within a gel-like protein matrix, and the intrinsic disorder of the components is essential for the formation of the gel itself [ 32 , 97 ]. Additionally, the amorphous, glycine-rich domains within mussel byssal threads are thought to facilitate the reformation of bonds after stress [ 13 ]. Additional clues may be provided by the apparent threshold of glycine content for SLSPs (25% glycine within an 80 residue window). Manipulation of the glycine content of SLSP-inspired peptides based upon this threshold and observation of the effects on the formation of biomaterials and their properties will reveal the functional relevance of these glycine-rich sequences."
} | 4,872 |
28245292 | PMC5357065 | pmc | 7,493 | {
"abstract": "RNA editing is a rare post-transcriptional event that provides cells with an additional level of gene expression regulation. It has been implicated in various processes including adaptation, viral defence and RNA interference; however, its potential role as a mechanism in acclimatization has just recently been recognised. Here, we show that RNA editing occurs in 1.6% of all nuclear-encoded genes of Symbiodinium microadriaticum , a dinoflagellate symbiont of reef-building corals. All base-substitution edit types were present, and statistically significant motifs were associated with three edit types. Strikingly, a subset of genes exhibited condition-specific editing patterns in response to different stressors that resulted in significant increases of non-synonymous changes. We posit that this previously unrecognised mechanism extends this organism’s capability to respond to stress beyond what is encoded by the genome. This in turn may provide further acclimatization capacity to these organisms, and by extension, their coral hosts.",
"introduction": "Introduction RNA editing is a collection of co- or post-transcriptional processes that produce RNA sequences that differ from their DNA templates (excluding mRNA splicing, capping and polyadenylation). These processes provide organisms with an additional layer of post-transcriptional control, and typically manifest as the production of tissue-specific protein isoforms [ 1 ], play a role in caste determination [ 2 ], or to adapt to a new environment [ 3 ]. In contrast to these well-characterised editing events, a potential role for RNA editing in response to short-term environmental change is just emerging. For instance, studies in human monocytes show specific induction of C-to-U edits in the human SDHB gene in response to hypoxic conditions [ 4 ]; in Drosophila , A-to-I edit frequencies have been shown to respond to heat stress [ 5 ]. However, for the latter, the changes were tightly linked to transcriptional silencing of the sole RNA editing enzyme encoded in the genome. Dinoflagellates are a highly diverse group of protists that thrive in both freshwater and marine environments. As one of the major primary producers in the world’s oceans, they are an integral part of the marine food web [ 6 ]. Dinoflagellates of the genus Symbiodinium are best known for being a key symbiont in many marine invertebrates, including reef-building corals [ 7 ]. As such, they also contribute to the acclimatization potential of their host [ 8 , 9 ]. In dinoflagellates, RNA editing has been described in organelles since the turn of this century. The combined work of [ 10 – 15 ] established the presence of base-substitution edits in mitochondrial cox1 , cob and cox3 genes; similarly, other studies [ 16 – 20 , 75 , 76 ] showed that numerous plastid-encoded genes are also subject to RNA editing. Evidence for editing on nuclear genes is, however, lacking.",
"discussion": "Discussion In this work, we are the first to report RNA editing in genome-encoded transcripts of a dinoflagellate, which supplements similar observations made for dinoflagellate organellar genomes. Unlike metazoans and plants, where the vast majority of edits are A-to-I and C-to-U respectively, there is a greater variation of edit types in dinoflagellates. Previous work in dinoflagellate organelles reported all edit types are possible; similarly, in our data, we could observe all N-to-N edit types. These edit types were not distributed uniformly: the most frequently edited type (C-to-T) was approximately 20-fold more common than the rarest edit type (T-to-G). This is somewhat similar to previous reports from dinoflagellate organelles, where C-to-T, A-to-G and T-to-C edits were among the most frequent; however, exceptions such as A-to-T (19% in S . microadriaticum genome, 0–3% in organelles) were evident as well. While the presence of all edit types and overall spread of edit type frequencies suggest that the editing machinery might be shared between the host and its organelles, the exceptions would indicate that the latter utilises a subset of the RNA editing machinery available, or perhaps the more restricted sequence contexts in these organelles strongly favour certain edits and disfavour others. A confounding factor to the observed condition-specific RNA editing was the dysregulation of the editing machinery, as observed in Drosophila [ 5 ]. To assess that possibility, we performed in silico identification of candidate S . microadriaticum RNA editing proteins (PPR-like and ADAR-like) via sequence similarity. While these candidates appear contentious, especially since ADARs are thought to be restricted to metazoans [ 32 ], further in vivo verification of these candidates is stymied by the dearth of reliable molecular techniques, e.g. transformation and gene knockdowns in dinoflagellates. Regardless, two observations indirectly suggest that some of these deaminases retained similar functionality in S . microadriaticum . Firstly, edited sites in nuclear-encoded genes of S . microadriaticum appear more clustered than expected, similar to edits in dinoflagellate organelles [ 36 ] and A-to-I edits in humans [ 28 ]. Secondly, sequence motifs have been reliably discerned in the vicinity of C-to-T edits. These motifs are, however, unlike any that have been identified in metazoans [ 37 – 39 ] or plants [ 30 , 40 ], perhaps due to the evolutionary distance of dinoflagellates from metazoans and plants [ 41 ]. Based on our data, we argue that Symbiodinium is able to shift its RNA editome in response to environmental stress. The 114 genes that we identified in S . microadriaticum were differentially edited in response to at least one of three bleaching-relevant stressors: cold, heat or dark. This subset of genes had significantly more non-synonymous amino acid changes, and our observations are in line with the non-synonymous change in a K + channel of the octopus Pareledone sp . that allows it to adapt to colder waters [ 3 ]. The prevalence of non-synonymous edits has similarly been demonstrated in other organisms such as Drosophila [ 22 ] and the squid Doryteuthis pealeii [ 42 ]. For non-synonymous mutations, larger structural or functional changes are possible through the introduction of amino acids with very different properties. This translational flexibility may represent a previously unrecognised mechanism of acclimatization that provides additional phenotypic plasticity to the organism, and thereby, increases its ability to respond to environmental change in comparison to the genome-encoded gene product [ 43 ]. As more research is done on dinoflagellate biology, it becomes more and more apparent that dinoflagellates do not seem to play by the same rules as metazoans or plants. Few genes are commonly observed to respond to stress with a change in expression [ 44 – 46 ], possibly linked to the relative paucity of proteins harbouring transcription factor domains [ 47 ]. Rather, expression profiles seem to be fixed between different clades, species, or even strains within species, irrespective of physiological conditions [ 45 , 48 ]. Our data concurred with these observations: roughly 2% of all genes are differentially expressed under any of the three stressors, and this proportion is independent of editing state. How, then, does the organism respond to stress? One possible answer to this conundrum is the use of post-transcriptional control mechanisms, previously illustrated through the identification of an RNAi machinery and a diverse set of miRNAs in two Symbiodinium species [ 44 , 49 ]. We suggest that RNA editing is an additional genome-wide, post-transcriptional mechanism that modulates gene expression through shifts in the RNA editome. What, then, would be the biological purpose of RNA editing in Symbiodinium ? We postulate that RNA editing serves a dual function—firstly, it serves to regulate gene expression on a genome-wide level, as evidenced by a substantial number of S . microadriaticum genes (774 genes, 1.6% of all genes) being edited. Within genes, edited sites have a propensity to be at the 5’ end of genes. Furthermore, we find that differentially edited genes that respond to environmentally relevant stressors show higher frequencies of non-synonymous changes. These observations might be tightly linked to the biological functions of these edits. Synonymous changes might affect gene expression via codon bias [ 50 ]; non-synonymous changes at the N-terminus signal peptide region will disrupt protein localisation, while changes in structural regions could affect the stability of the peptide. It is also likely that these edits exert larger effects when they are closer to the 5’ end of the gene, and our data substantiates this postulate: the initial 5% of the gene contains 126 of 859 (14.2%) non-synonymous changes; comparatively, 26 of 263 (11.2%) synonymous edits are in the same region. Secondly, RNA editing putatively provides a low evolutionary cost system that introduces additional genetic variation above the coding capacity of the genome on which selection can act without the risk of generating lethal variations. This is especially important as Symbiodinium is haploid in the vegetative state [ 21 ], thus deleterious mutations are more likely to be costly. In conclusion, it is a challenging prospect to identify true edits in an organism with a draft genome and unclear mechanistic origins of these edits. However, our conservative comparative transcriptomics of cultures under bleaching-relevant stressors have revealed bona fide RNA edits in the nuclear genome of S . microadriaticum . Our work highlights a novel role for RNA editing in providing a mechanism for dinoflagellates—and, by extension, their coral hosts—to acclimatize, and potentially adapt to, changing environments."
} | 2,467 |
40348765 | PMC12065812 | pmc | 7,494 | {
"abstract": "Manipulating floating objects, whether solid or liquid, from microscopic to mesoscopic sizes, is crucial in various microfluidics and microfabrication applications. While capillary menisci naturally self-assemble and transport floating particles, their shapes and sizes are limited by the properties of the fluid and the objects involved. We herein harness the superposition of capillary menisci to curve liquid interfaces controllably. By using 3D-printed spines piercing the interface, we can finely adjust height gradients across the liquid surface to create specific liquid topographies. Thus, our method becomes a powerful tool for manipulating floating objects into programmable paths. Combining experimental demonstrations, numerical simulations, and theoretical modeling, we study the liquid elevation created by specific spine dispositions and the three-dimensional manipulation of submillimetric particles. Multiple examples showcase the method’s potential applications, including sorting and capturing particles, which could pave the way for cleaning fluid interfaces.",
"introduction": "Introduction In nature, capillary menisci serve various purposes. They enhance the aggregation of objects at liquid interfaces, like mosquito eggs 1 , bubbles 2 , or cereals 3 . Water-walking insects use menisci to reach the shore 4 , while surface-piercing vegetation captures particles on water surfaces 5 , 6 . Such natural phenomena have long inspired scientists to exploit menisci or curved interfaces to self-assemble 7 – 19 , transport 20 – 22 , or manipulate 23 – 25 floating objects. Pillars, in particular, have been used to curve the interface by pinning it to the pillar cross-section, enabling particle transport 26 – 28 . However, these pillar-based strategies typically focus on single pillars, resulting in axisymmetric deformations limited by the capillary length λ , around 2.7 mm for the water-air interface. Peng et al. notably created a meniscus gradient for bubble transport using multiple slippery oil-infused pillars with height gradients 20 . In this study, we extend these concepts by exploring the superposition of capillary menisci generated by regularly arranged 3D-printed conical spines. By leveraging the interplay between spine geometry and spacing, we demonstrate control of liquid interface topographies over larger scales than the capillary length. Experimental results, supported by theoretical modeling and numerical simulations, show that specific liquid landscapes and artistic topographies can be programmed by tuning spine parameters. These tailored liquid interfaces enable precise manipulation of floating particles. For instance, we illustrate how objects of different sizes can be directed along programmable paths or trapped at predetermined locations. Additionally, we provide an example of time-dependent manipulation by incorporating structural subfeatures on the spines, where the interface is dynamically pinned as the liquid level decreases. Finally, we highlight the broad range of applications for this work, including particle sorting, micromanipulation, and interface cleaning. This offers a versatile and scalable platform for future advancements in microfluidic systems.",
"discussion": "Discussion The innovative approach presented herein demonstrates using arrays of 3D-printed conical spines to manipulate liquid surfaces precisely. By controlling the geometry, arrangement, and spacing of the spines, we achieved a wide variety of liquid topographies, ranging from inclined surfaces and sinusoidal patterns to complex artistic structures. These results confirm the ability to create tailored liquid landscapes over scales much larger than the capillary length, overcoming the limitations of traditional single-pillar systems. Through these programmable liquid interfaces, we showcased precise manipulation of floating particles, including directional transport, size-based sorting, and controlled trapping at predetermined locations. Moreover, by introducing structural subfeatures on the spines, we demonstrated time-dependent manipulation, where the interface dynamically evolves as the liquid level decreases. The versatility and scalability of this approach open promising avenues for practical applications, such as particle sorting, micromanipulation, and cleaning liquid interfaces from microscopic debris or oil droplets. Future work could explore the dynamic actuation of the spines using adaptive magnetic 49 or magnetoelastic 50 materials, shape-shifting materials 35 , shape memory polymers 43 , or mechanical systems to achieve real-time control of the liquid surface curvature. These advancements would further enhance the potential of this method for innovative microfluidic technologies and capillary-driven systems."
} | 1,188 |
23785373 | PMC3682122 | pmc | 7,495 | {
"abstract": "An emphasis is made on the diversity of nutrients that rhizosphere bacteria may encounter derived from roots, soil, decaying organic matter, seeds, or the microbial community. This nutrient diversity may be considered analogous to a buffet and is contrasting to the hypothesis of oligotrophy at the rhizosphere. Different rhizosphere bacteria may have preferences for some substrates and this would allow a complex community to be established at the rhizosphere. To profit from diverse nutrients, root-associated bacteria should have large degrading capabilities and many transporters (seemingly inducible) that may be encoded in a significant proportion of the large genomes that root-associated bacteria have. Rhizosphere microbes may have a tendency to evolve toward generalists. We propose that many genes with unknown function may encode enzymes that participate in degrading diverse rhizosphere substrates. Knowledge of bacterial genes required for nutrition at the rhizosphere will help to make better use of bacteria as plant-growth promoters in agriculture.",
"conclusion": "CONCLUDING REMARKS After considering the large diversity of potential nutrients (from rhizodeposits, root exudates, seeds, decaying organic matter, soil, and the rhizosphere community itself) for microbes at the rhizosphere we propose a hypothesis for bacterial nutrition at the rhizosphere: a buffet hypothesis where commensals choose their food from a diversity of options. This is in contrast to the proposal of oligotrophy at the rhizosphere ( Ramachandran et al., 2011 ). Copiotrophic rhizobia are very successful rhizosphere colonizers ( Gutiérrez-Zamora and Martínez-Romero, 2001 ). Microbial respiration is not carbon limited in the rhizosphere ( Cheng et al., 1996 ). Rhizosphere is a complex environment with substitutable resources. In experimental evolution in complex environments with substitutable resources, Pseudomonas lineages evolved as imperfect generalists that differentiate to assimilate a certain range of substrates but not all ( Barrett et al., 2005 ), this seems to happen with microbes at the rhizosphere.",
"introduction": "INTRODUCTION Ecophysiology of root systems cannot be understood without the microbiota that colonize outside and inside roots. Bacteria and fungi may impact root physiology, produce hormones, stimulate root growth or alter its morphology. Microbes provide protection against pathogens, tolerance to abiotic stresses, resistance to insect or herbivore attack; even allelopathy may be due to root-associated microorganisms. An extensive review on the ecophysiological contributions of microorganisms to plants has been published ( Friesen et al., 2011 ) and reviews on rhizospheric bacteria also highlight their effects on plants ( van Loon et al., 1998 ; Bais et al., 2006 ; de Bruijn, 2013 ). Microbial endophytes (meaning residing inside the roots) may contribute to nutrient assimilation and other plant traits, however, they are normally in lower numbers than rhizospheric bacteria ( Rosenblueth and Martínez-Romero, 2006 ; Hirsch and Mauchline, 2012 ) and we will focus only on the latter. Over the years, studies on root microbiota have addressed several questions such as: How are microbes selected or maintained in roots? What are the sources and resources for root microbes? How do bacteria or fungi affect root physiology? Are there key species that have a larger impact on plants? Is nutrient competition driving bacterial evolution? There are still questions without answer. The term rhizosphere was proposed by Hiltner (1904) and refers to 1–7 mm of soil from the root surface. The rhizosphere effect is the enrichment of microbial populations at the root–soil interface. Outside roots there is a heavy colonization of bacteria (for example, 10 9 \n Rhizobium phaseoli cells per gram of fresh maize root; Gutiérrez-Zamora and Martínez-Romero, 2001 ) mainly stimulated by root-derived nutrients. The microbial community itself may modify root nutrients and may contribute with resources by transforming soil material ( Baelum et al., 2008 ), by fixing nitrogen ( Fischer et al., 2012 ) or producing vitamins ( Phillips et al., 1999 ; Ramírez-Puebla et al., 2013 ). Rhizosphere nutrients may be very variable depending on the plant ( Brown et al., 2008 ; Haichar et al., 2008 ; Badri et al., 2013 ) and the soil biotic and abiotic conditions. There are bacterial species commonly encountered as rhizosphere colonizers but each plant species may harbor particular microbes at the rhizosphere ( Lundberg et al., 2012 ). A complex rhizosphere community may be structured in relation to the microbial specialization for different nutrients. The diversity of nutrients available at the rhizosphere may be equated to a buffet, and distinct microbes may have preferences for some of them. Furthermore, we propose that a large proportion of products from genes highly expressed by bacteria at the rhizosphere are involved in the transport and catabolism of the various buffet entries."
} | 1,247 |
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