pmid stringlengths 8 8 | pmcid stringlengths 8 11 ⌀ | source stringclasses 2
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26200335 | PMC4862617 | pmc | 1,679 | {
"abstract": "Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigated such an internal conflict within a microbial biofilm community: Cells in the biofilm periphery not only protect interior cells from external attack, but also starve them through nutrient consumption. We discovered that this conflict between protection and starvation is resolved through emergence of long-range metabolic codependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth.",
"introduction": "Introduction Cooperation and competition are complex social interactions that can play critical roles in biological communities. Cooperative behavior often increases the overall fitness of the population through processes such as division of labor and production of common goods 1 – 4 . At the same time, individuals in a community compete with each other for limited resources, such as nutrients 5 – 6 . Here we investigated bacterial biofilms 7 – 10 to determine how the conflict between the opposing social behaviors of cooperation and competition could be resolved at the community level to increase overall fitness. Biofilms typically form under environmental stress conditions, such as nutrient limitation 11 – 13 . As these bacterial communities grow larger, the supply of nutrients to interior cells becomes limited due to an increase in nutrient consumption associated with the growth of multiple layers of cells in the biofilm periphery. Severe nutrient limitation for interior cells is detrimental to the colony, since the sheltered interior cells are critical to the survival of the biofilm community in the event of an external challenge. This defines a fundamental conflict between the opposing demands for biofilm growth and maintaining the viability of protected (interior) cells ( Fig. 1a ). The identification of possible mechanisms that ensure the viability of the protected interior cells is fundamental to understanding biofilm development 14 , 15 . In order to directly investigate how Bacillus subtilis biofilms continue expanding while sustaining interior cells, we converted the potentially complex three-dimensional problem to a simpler two-dimensional scenario using microfluidics. Specifically, we used growth chambers that are unconventionally large in the lateral, x-y dimensions (3 × 3 mm), while confining biofilm thickness (z-dimension) to only a few micrometers ( Fig. 1b ). Therefore, biofilm expansion in this device is predominantly limited to two dimensions, creating a “pancake-like” configuration. In fact, biofilms often form in confined aqueous environments and thus this microfluidic chamber may better mimic those growth conditions 11 – 13 . This experimental set-up is thus ideal to interrogate how biofilms can reconcile the opposing benefits of growth and protection during biofilm development.",
"discussion": "Discussion The data presented here reveal that intracellular metabolic activity within biofilms is organized in space and time, giving rise to codependence between interior and peripheral cells. Even though bacteria are single-celled organisms, the metabolic dynamics of individual cells can thus be regulated in the context of the community. This metabolic codependence can in turn give rise to collective oscillations that emerge during biofilm formation and promote the resilience of biofilms against chemical attack. The community-level oscillations also support the ability of biofilms to reach large sizes, while retaining a viable population of interior cells. Specifically, periodic halting of peripheral cell growth prevents complete starvation and death of the interior cells. This overcomes the colony size limitation for a viable biofilm interior that would otherwise be imposed by nutrient consumption in the biofilm periphery. Metabolic codependence in biofilms therefore offers an elegant solution that resolves the social conflict between cooperation (protection) and competition (starvation) through oscillations. The intriguing discovery of biofilm oscillations presented here also provokes new questions. While cellular processes such as swarming or expression of extracellular matrix components are not required for the observed biofilm oscillations ( Extended Data Fig. 10 ), it will be interesting to pursue whether such cellular processes are influenced by oscillatory dynamics 29 . Another question worth pursuing is whether metabolic codependence can also arise in other biofilm-forming species. Perhaps other metabolic branches where metabolites can be shared among cells could also give rise to oscillations in biofilm growth. It will be exciting to pursue these questions in future studies to obtain a better understanding of biofilm development. Our observations also suggest future strategies to cope with the intriguing resilience of biofilms in the face of environmental stresses, such as antibiotic exposure. In particular, our findings show that straightforward application of stress (such as H 2 O 2 or chloramphenicol) to the biofilm counterintuitively promotes growth, effectively rejuvenating the biofilm. Death of the colony periphery relieves the repression on the growth of interior cells, allowing them to regenerate a new biofilm periphery and interior. In contrast, manipulation of the metabolic codependence may yield a more effective approach to control biofilm formation. Specifically, promoting continuous growth of peripheral cells can starve the biofilm interior, leaving behind the exposed peripheral cells that can more easily be targeted by external killing factors. Therefore, the metabolically driven collective oscillations in biofilm expansion described here not only reveal fundamental insights into the principles that govern formation of multicellular communities, but also suggest new strategies for manipulating the growth of biofilms."
} | 1,589 |
37307488 | PMC10288591 | pmc | 1,680 | {
"abstract": "Significance This work investigates the impact of the xanthophyll cycle in marine microalgae on the trade-off between photoprotection and light-use efficiency. Our results demonstrate that while zeaxanthin is essential for photoprotection upon exposure to strong illumination, it leads to unnecessary energy losses in light-limiting conditions and thus accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae in dense cultures.",
"discussion": "Discussion Biological Role of Zeaxanthin in Nannochloropsis . Nannochloropsis gaditana cells upon exposure to excess light show the ability to convert violaxanthin into zeaxanthin ( Fig. 1 ), as in many other photosynthetic eukaryotes ( 17 ). Nannochloropsis has a peculiar pigment composition, with violaxanthin being the most abundant carotenoid in this species, accounting for approx. 50% of the total ( 31 – 33 ). Likely because of this large reservoir of substrate, in contrast to plants and other microalgae ( 34 , 35 ), zeaxanthin synthesis in Nannochloropsis continues even upon prolonged exposure to extreme irradiances with no visible saturation ( Fig. 1 ). Considering the light intensities tested in this work, which went well beyond physiologically relevant conditions, our results also suggest that zeaxanthin synthesis is unlikely to ever reach saturation in the natural environment, meaning that Nannochloropsis cells are capable of additional zeaxanthin synthesis whenever needed even if they have already been exposed to strong illumination. The large capacity of zeaxanthin synthesis is accompanied by a strong impact of this pigment on the protection of the photosynthetic apparatus. The phenotype of both vde KO and WT cells treated with the VDE inhibitor DTT demonstrates that zeaxanthin synthesis has a major impact on NPQ in Nannochloropsis ( Fig. 3 B and SI Appendix , Fig. S1 ), as also observed in ref. 30 . Zeaxanthin synthesis impacts NPQ from the first few seconds of illumination ( Fig. 2 ), while HPLC analysis shows that a few minutes of illumination are needed before detecting a significant accumulation of molecules ( Fig. 1 ). This observation suggests that a small number of zeaxanthin molecules can activate NPQ in a few seconds after an increase of illumination, likely by associating to specific binding sites in light-harvesting complexes. Considering that also lhcx1 KO strain shows a major decrease in NPQ capacity, and that its full activation requires the presence of both zeaxanthin and LHCX1, it is likely that zeaxanthin activity in NPQ requires its association to the LHCX1 protein in N. gaditana , as previously suggested for N. oceanica ( 30 ). Similarly, in diatoms, NPQ is provided by a concerted action between LHCX proteins and diatoxanthin ( 36 ), a xanthophyll molecule part of the diadinoxanthin–diatoxanthin cycle, which is analogous to the VAZ cycle observed in Nannochloropsis ( 37 ). LHCX1 is the main NPQ effector also in diatoms, although additional LHCX proteins, namely LHCX2 and LHCX3, are involved when cells are exposed to prolonged high light, providing flexibility of quenching site but most likely with a similar mechanism ( 36 , 38 , 39 ). Pigment data of the lhcx1 KO strain also show that the absence of LHCX1 has a measurable impact on the xanthophyll cycle dynamics with a larger accumulation of zeaxanthin than that in WT, but also a faster conversion back to violaxanthin. This can be explained knowing that a large fraction of violaxanthin is bound to antenna proteins and it needs to be released into the thylakoid membrane to be converted into zeaxanthin. This exchange from antenna proteins limits the rate of xanthophyll conversion, as demonstrated in plants ( 40 ). lhcx1 KO is depleted of one of the most abundant antenna proteins in Nannochloropsis ( 41 ), and this is likely to accelerate zeaxanthin synthesis and degradation because of a larger presence of carotenoids not bound to antenna proteins, but free in the thylakoid membranes and thus more available to VDE. In N. gaditana , even though NPQ slowly continues to increase after 10 min induction, suggesting the presence of a qZ-type contribution associated with the progressive accumulation of zeaxanthin, the largest fraction of NPQ capacity reaches saturation in this time frame ( Fig. 2 ). Since zeaxanthin synthesis continues much longer without showing signs of saturation ( Fig. 1 ), this suggests that the influence of zeaxanthin molecules on NPQ reaches saturation, likely because of saturation of the potential binding sites for zeaxanthin in LHCX1. A second, larger, pool of zeaxanthin molecules continues to be synthesized upon prolonged exposure to strong light, but it does not contribute to NPQ and likely plays other roles in photoprotection such as direct scavenging of Chl triplets and ROS ( 9 ). While the zeaxanthin molecules active in NPQ are quickly synthesized, their impact on NPQ remains for a prolonged time. This is evidenced by the fact that NPQ induction kinetics are faster if cells have already been exposed to a previous light treatment ( Fig. 2 ). This effect is already visible after exposing cells to light for 2 min and it is still detectable after a 90-min dark relaxation, demonstrating that this time is not sufficient to reconvert all zeaxanthin synthesized in 8 min illumination ( Fig. 2 ). This effect can be modulated by overexpressing ZEP since cells are faster in reconverting zeaxanthin into violaxanthin during the 90-min dark relaxation, as demonstrated by the reduction in NPQ induction during the second kinetic with respect to the parental strain ( SI Appendix , Fig. S11 ), supporting the HPLC data of Fig. 3 . Zeaxanthin Plays an Essential Photoprotective Role in Nannochloropsis , beyond NPQ. Both vde KO and lhxc1 KO strains show sensitivity to saturating illumination, supporting the role of NPQ on protection of Nannochloropsis from light stress ( Fig. 4 ). When cells are cultivated in dense cultures, however, the results between the two genotypes are very different. In this context, some cells are exposed to full illumination, while the others, because of shading, are in limiting light or even dark ( 28 ). In the experimental system employed here, approx. 60% of incident radiation is absorbed by the 1st cm of culture depth ( 18 ). If the culture is exposed to a strong external illumination (1,200 µmol photons m −2 s −1 ), vde KO cells show a clear decrease in maximum quantum yield of PSII ( SI Appendix , Table S4 ), suggesting that more exposed cells are extensively damaged by illumination. This damage cannot even be counterbalanced by cells deeper in the culture volume and eventually it impairs the growth of the whole culture under strong illumination ( SI Appendix , Fig. S10 ). The inability of the vde KO strain to grow at higher illumination depends on its stronger photosensitivity as a consequence of the absence of both the NPQ response and the activation of the xanthophyll cycle upon exposure to saturating irradiance, as demonstrated in Figs. 3 – 5 . While vde KO and lhcx1 KO strains are similarly defective in NPQ ( Fig. 3 ), the latter retains ability to growth under strong illumination in very different conditions, such as agar plates ( Fig. 4 ) and laboratory-scale photobioreactors ( Fig. 5 ), while vde KO shows strong impairment in both. Moreover, the lhcx1 KO in those conditions does not show any additional photodamage with respect to WT ( Fig. 4 ), clearly demonstrating that the impact of zeaxanthin biosynthesis on photoprotection goes well beyond its role in enhancing NPQ and that its ability to increase scavenging of Chl triplets and ROS ( 9 , 42 ) is essential even in dense cultures. Xanthophyll Cycle Dynamics Has a Major Impact on Microalgae Biomass Productivity in Photobioreactor. Microalgae at industrial scale are cultivated at high concentration to maximize biomass productivity. Such dense cultures are also continuously mixed to maximize the exposure of cells to incident light and avoid nutrient and carbon limitation, causing cells to suddenly move between limiting and excess illumination, further increasing the complexity of the light environment. In these environmental conditions, more exposed cells need photoprotection mechanisms to withstand strong illumination, but the same mechanisms become detrimental for productivity once the cells move to light limitation of deeper layers. The trade-off between photoprotection and photochemical efficiency, which must be balanced by all photosynthetic organisms ( 3 ), is thus particularly challenging in such a complex and dynamic environmental context. It is not surprising that strategies for the optimization of photosynthetic productivity have generated mixed results so far ( 43 , 44 ), with the only reasonable conclusion being that the complexity of the natural and artificial changes experienced by microalgae during industrial cultivation has a major influence on productivity that cannot be underestimated ( 45 ). Strains with altered xanthophyll cycle analyzed in this work demonstrate that an efficient photoprotection is essential for microalgae fitness in dense cultures to ensure growth under full sunlight, as shown by the strong photosensitivity of vde KO. On the contrary, we observed that lhxc1 KO in dense cultures shows a positive impact on biomass productivity. This strain differs from WT not only because of its reduction in NPQ activation, but also for a reduced PSII antenna size and Chl content per cell ( 46 ) and a higher zeaxanthin content, observed in this work. Mathematical models suggest that the reduction in Chl content per cell should have the largest impact in improving biomass productivity ( 46 ), but it is also possible that the higher zeaxanthin content observed in lhcx1 KO can be beneficial to compensate for any eventual extra damage due to NPQ inactivation. Overall, this work demonstrates that an indiscriminate reduction of photoprotection mechanisms is detrimental for growth even in dense cultures typical of industrial cultivation systems. In fact, cells most exposed to illumination experience light saturation and eventually damage. While the zeaxanthin impact on photoprotection is clear, the impact of NPQ is more complex. The lhcx1 KO strain is impaired in NPQ but also has a reduction of both Chl content/cell and PSII antenna size (ASII) that are beneficial for cultivation in dense cultures. Overall, these results confirm that the reduction of NPQ alone has a limited impact on biomass productivity in dense microalgae cultures, at least in Nannochloropsis ( 46 , 47 ). Energy losses due to natural kinetics of photoprotection can however be detrimental for productivity in low light conditions, and accelerating zeaxanthin conversion to violaxanthin can be advantageous in this context. The impact on the overall xanthophylls pool that we observed because of the overaccumulation of the ZEP protein was quite modest and when we measured the relaxation kinetic at earlier time points (i.e., 5, 10, and 15 min of recovery in the dark), the differences between the ZEP OE and the WT strains were even smaller ( SI Appendix , Fig. S12 ). On the contrary, also these data highlight a difference in the biological role of zeaxanthin, depending on its binding localization, with a relatively small pool hypothetically binding LHCX1 and involved in NPQ activation/relaxation. The overexpression of ZEP likely shows a much stronger effect on this latter pool of zeaxanthin with the largest impact on NPQ, explaining why differences are observable by measuring NPQ kinetics, but they are too small to be assessable by HPLC. In this work, we also simulated the light fluctuation experienced by microalgae in dense cultures of industrial systems ( Fig. 6 B ) as a consequence of mixing and observed that WT cells showed a substantial reduction of photosynthetic functionality in light limitation after only a few fluctuation cycles ( Fig. 6 D ). This decrease could be due to multiple phenomena, such as the activation of photoprotection or photoinhibition. The lhcx1 KO strain does not show the same reduction of WT, suggesting that NPQ is the major factor responsible for the loss of activity observed in the parental strain in dense cultures. On the contrary, the vde KO strain showed an even larger reduction of photosynthetic functionality in light limitation ( Fig. 7 F ), suggesting that photoinhibition can also play a major role, as measured in Fig. 7 H . In the case of the ZEP OE, cells maintain the ability to activate NPQ but also have faster recovery, suggesting that increasing the rate of violaxanthin biosynthesis alone has a beneficial effect on productivity. This is achieved because cells have a faster reconversion rate to violaxanthin when light becomes limiting, which likely provides an advantage when cells move from external to internal, light-limited positions in the dense culture, where they remove zeaxanthin faster and can therefore channel more energy toward photochemistry. These properties, however, also represent a potential cost, in terms of lower photoprotection, when cells move in the opposite direction from internal to external layers. This cost, however, is lower than the advantage, because of two main reasons. One is that the cells still maintain the ability to synthesize zeaxanthin when needed for photoprotection ( Fig. 3 ) and two, in all tests run here, there were no major signs of increased damage. It is also worth noting that light-limited layers represent the major fraction of the volume in dense cultures of industrial systems (an estimated >70% of the culture volume for the setup schematized in SI Appendix , Fig. S6 A ) ( 18 ), and therefore it can be expected that a large fraction of the culture benefits from this genetic optimization, while there is a smaller fraction that might eventually have a modest disadvantage. Consequently, even a modest increase in the speed of reconversion of zeaxanthin into violaxanthin can provide a substantial effect on biomass productivity. This is consistent with the observation that lhcx1 KO and ZEP OE, the two strains that show the smaller reduction in photosynthetic activity upon exposure to light fluctuations, also showed an increase in biomass productivity in dense cultures ( Fig. 5 ). This suggests that the optimization of the xanthophyll cycle is a valuable strategy in photosynthesis engineering, yet a fine-tuning is preferable to an indiscriminate activation, likely because in the latter case, the improvement in cell fitness cannot fully compensate the metabolic burden of a hyperactive xanthophyll cycle. Optimization of Xanthophyll Dynamics in Microalgae vs. Plants. The genetic modification of NPQ and xanthophyll cycle has already been demonstrated to be effective to improve biomass productivity in crop plants in the field ( 14 , 15 ). In our current work, effects are observed in Nannochloropsis by overexpressing only ZEP. It is in fact worth mentioning that VDE activity remains strong in the ZEP OE strain, such that it is still fully capable of producing zeaxanthin upon excess light exposure. This is likely also connected with a high violaxanthin content of N. gaditana with respect to plants, suggesting that this organism likely also has high endogenous VDE activity. However, when metabolic engineering is applied to photosynthesis, the complexity of the environmental conditions of the intended cultivation system should also be considered, as well as the physiology of the species targeted for improvement. For instance, in plants of Nicotiana benthamiana , Arabidopsis thaliana, and Solanum tuberosum , VDE, ZEP, and PSBS overexpression did not show the same effects ( 48 , 49 ), indicating that species-specific physiological or morphological features are highly influential on the homeostasis of the photosynthetic metabolism. In the environment of photobioreactors, most of the culture is light limited, while only a small layer of cells is exposed to full sunlight. The design of photobioreactors, as well as operational conditions (e.g., culture concentration), strongly affects the percentage of cells that are in light-limiting conditions or excess light, affecting the optimal balance between photoprotection and photochemical efficiency. Culture mixing is also expected to play a major role on this balance. It is then worth noting that the complexity of the natural and artificial changes experienced by microalgae in dense cultures of industrial systems is likely to prevent the identification of ideal strains more productive in all operational conditions, suggesting that photosynthesis optimization efforts should be tuned to the specific operational conditions in use."
} | 4,213 |
37206669 | null | s2 | 1,682 | {
"abstract": "DNA origami has emerged as a powerful method to generate DNA nanostructures with dynamic properties and nanoscale control. These nanostructures enable complex biophysical studies and the fabrication of next-generation therapeutic devices. For these applications, DNA origami typically needs to be functionalized with bioactive ligands and biomacromolecular cargos. Here, we review methods developed to functionalize, purify, and characterize DNA origami nanostructures. We identify remaining challenges, such as limitations in functionalization efficiency and characterization. We then discuss where researchers can contribute to further advance the fabrication of functionalized DNA origami."
} | 173 |
40114721 | PMC11924041 | pmc | 1,687 | {
"abstract": "The collection and transportation of underwater bubbles has attracted significant attention due to their wide range of applications in the mining, petroleum, and chemical industries. In this study, robust superhydrophobic tapered needles were successfully fabricated by spraying a superhydrophobic coating prepared by an organic–inorganic hybrid method. The prepared tapered needles present excellent surface stability and good superhydrophobicity with a contact angle (CA) of about 156°. The fabricated tapered needles demonstrate excellent performance in collection and transportation of underwater bubbles and the working mechanism was also thoroughly studied. The prepared robust superhydrophobic tapered needles provide a simple, efficient and economical way for collection and transportation of underwater bubbles.",
"conclusion": "4 Conclusions In this article, a fast and simple method for preparing superhydrophobic coating by spraying perfluoro thiol/acrylate resin containing hydrophilic silica nanoparticles was introduced. The hydrophobic angle reaches 156°, and it has excellent mechanical properties and acid resistance. The spray deposition method of the nanoparticle-loaded resin provides the coating with a layered rough morphology on a wide range of substrates. Combined with copper cones, it can be used for gas collection and transportation in gas–water–solid three-phase systems. The superhydrophobic copper cone continuously collects microbubbles from the carbonated water, and then directionally transport them to the bottom surface driven by the gradient of Laplace pressure and surface free energy. The transport direction of the microbubbles can be controlled by the superhydrophobic copper cone.",
"introduction": "1 Introduction Micro-bubbles, characterized by their small diameter ranging from 10 to 50 μm, have garnered significant interest for their diverse applications, 1–3 including heavy medium coal preparation, 4 pipeline transportation, 5–9 drag reduction, 5,10–14 and more. In heavy-medium coal preparation, micro-bubbles alter the surface properties of target minerals, facilitating efficient fine coal separation. In pipeline transport, they increase the mass transfer area, effectively mitigating clogging issues. Additionally, when applied to the underwater surfaces of ships, micro-bubbles reduce skin friction between the hull and water, leading to a significant decrease in hydrodynamic resistance. On the other hand, micro-bubbles may also be harmful. For example, bubbles in crude oil will seriously affect the measurement accuracy of crude oil storage and transportation, resulting in reduced transportation and separation efficiency. 15–17 At the same time, micro-bubbles in crude oil also tend to absorb small solid impurities, which can cause blockages in pipelines and damage the transport and separation equipment. The presence of micro-bubbles in a water-based coating will produce micro pores, causing cavitation erosion and seriously affecting the protective effect of coatings. 15,18 Above all, it is meaningful to explore effective methods for collection and transportation of underwater bubbles. Traditional methods are mainly divided into physical methods and chemical methods, 19,20 such as the defoaming method, mechanical method, heating method, ultrasonic method, and more. However, these methods are either energy-intensive or necessitate the installation of additional costly specialized equipment. Furthermore, they exhibit low efficiency, particularly when dealing with small-diameter micro-bubbles. Consequently, there is an urgent need to develop a cost-effective and highly efficient method for the collection and transportation of underwater bubbles. In recent years, super-wetting surfaces have garnered significant attention from researchers due to their broad applicability and ease of fabrication. Surfaces with tailored super-wetting properties have been engineered for a variety of applications, including corrosion protection, 21,22 water vapor collection, 23 and droplet directional movement, 24,25 among others. Some researchers have begun to explore the potential of super-wetting surfaces as a solution for manipulating underwater bubbles. 26–33 Ma Rui et al. reported a superhydrophobic sponge, which achieves rapid collection and stable storage of underwater bubbles. However, the superhydrophobic sponge did not achieve efficient control over the dynamic behavior of underwater bubbles, such as their movement or size distribution. Pei et al. reported a Janus membrane with superhydrophobic and superhydrophilic surfaces on opposite sides. It shows excellent unidirectional bubble permeability, and the results prove its effectiveness in underwater bubble manipulation. Yin et al. reported a trapezoidal platform featuring a micro/nano – structure through femtosecond laser direct cutting. The surface of this gradient platform exhibits superhydrophobicity in the air. Notably, this platform can gather bubbles in water and achieve the directional transport of bubbles without external forces. Cao et al. reported a superhydrophobic copper helix capable of achieving controlled, directional bubble transport in water. The bubble velocity can be adjusted based on the helix distance. Yu et al. reported a lubricant-infused slippery (LIS) surface with exceptional water repellency, enabling efficient manipulation of bubbles in aqueous environments. However, its poor surface strength and high consumption significantly limit applications. Our group has been paying much attention to the preparation and application of super-wetting films for a long time. 34–36 Ye et al. prepared a superhydrophobic coating with excellent mechanical durability, self-cleaning performance, and corrosion resistance through a novel organic synthesis and organic–inorganic composite method. Liang et al. prepared a composite coating for wax prevention in crude oil transportation. Yang et al. used the hydrolysis and condensation of tetraethoxysilane (TEOS) to form a silica sol–gel film while achieving controllable film material wettability. The thiol synthesis method based on click reaction has become a research hotspot in recent years due to its advantages such as low oxygen resistance, commercially accessible raw materials, few side reactions, simple operating conditions, and no use of metal catalysts. 37–45 In this study, a facile click reaction was utilized to fabricate robust superhydrophobic coatings on tapered copper needles. Organic–inorganic composite coatings were fabricated via thiol-ene click reaction, leveraging its rapid kinetics and robust bonding characteristics. The influence of the organic-to-inorganic ratio on hydrophobicity was investigated, revealing that a composition of 0.4 g resin and 0.2 g SiO 2 achieved a water contact angle of 156°, indicating superior hydrophobicity. The durability of coatings was assessed via cyclic abrasion tests. A water contact angle of 147° persisted after 20 friction cycles, confirming mechanical robustness. The superhydrophobic tapered copper needles demonstrated efficient underwater bubble manipulation, attributed to synergistic effects of hierarchical microstructures and surface chemistry. The superhydrophobic surface stabilizes an air plastron (Cassie–Baxter state) on the tapered copper needle submerged underwater, creating interfacial pathways for sustained directional transport of microbubbles. Due to the tapered shape, the needles will produce a gradient of Laplace pressure, which can derive a directional driving force and transport droplets directionally. 46–48 The collection and transportation of bubbles by superhydrophobic copper needles under reverse gravity was also studied. This method will provide a simple and effective method to collect and transport micro-bubbles.",
"discussion": "3 Results and discussion 3.1 Reaction mechanism and surface morphology analysis of prepared organic–inorganic composite coating The thiol-ene click reaction mechanism and its kinetic behavior have garnered significant attention from the global materials science community since their discovery. This surface modification strategy enables simultaneous introduction of functional groups and enhancement of critical material properties including adhesion and hydrophobicity. As illustrated in Fig. 1(a) , the reaction mechanism bifurcates based on olefin configuration types. The thiol-alkene system operates through a radical chain-transfer mechanism, where thiyl radicals (–S·) initiate nucleophilic attack on the carbon–carbon double bond (C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n C), subsequently propagating the active site through chain transfer processes. The radical-mediated thiol-ene addition proceeds through iterative chain propagation, while thiol-methacrylate systems with conjugated olefins follow a Michael addition pathway. In the latter mechanism, the nucleophilic thiolate anion (S − ) attacks the β-carbon of the electron-deficient C C bond, forming a carbanion intermediate that regenerates active species via proton abstraction from either –SH groups or alkali cations. This chain-propagation mechanism significantly enhances reaction kinetics. Subsequent integration of –Si(OCH 3 ) 3 low-surface-energy groups into the resin matrix, coupled with TEOS as a curing agent and dibutyltin dilaurate catalysis, promotes siloxane (Si–O–Si) network formation through hydrolytic condensation. As illustrated in Fig. 1(b) , this cross-linked architecture synergistically optimizes both hydrophobicity and mechanical strength in the final coating. Fig. 1 Schematic diagram of organic–inorganic composite coating preparation. (a) Thiol-ene click chemistry reaction based on free radical mechanism; (b) schematic diagram of silane hydrolysis to form a network structure. \n Fig. 2 characterizes the surface morphology and chemical composition of modified SiO 2 particles prepared via a photoinitiated fluorosilicone resin synthesis. FT-IR analysis in Fig. 2(a) reveals significant spectral changes post-modification: the broadened and attenuated –OH stretching vibration at 3433 cm −1 confirms successful silanol group consumption through chemical grafting. Characteristic perfluoroalkyl absorptions at 1204 cm −1 and 1149 cm −1 , combined with the absence of residual C C (1636 cm −1 ) and –SH (2564 cm −1 ) signals, demonstrate complete thiol-ene conversion and successful integration of fluorinated moieties into the resin matrix. SEM imaging at multiple magnifications in Fig. 2(b and c) reveals inherent micro/nanoscale hierarchical structures formed during spray deposition. High-resolution imaging confirms the coexistence of micron-sized aggregates and nanoscale surface asperities from fumed SiO 2 , collectively establishing a porous multilevel morphology. EDS elemental mapping in Fig. 2(d) verifies uniform surface distribution of F (17.58 ± 0.13 wt%), S, and Si – with fluorine enrichment ( vs. bulk 11%) indicating surface-segregation of low-surface-energy fluoropolymer chains. The detected sulfur and silicon derive respectively from PETMP crosslinkers and SiO 2 nanoparticles, consistent with the designed formulation. Fig. 2 (a) FT-IR images of FCS-SiO 2 particles before and after modification; SEM micrographs of modified SiO 2 particles at (b) low and (c) high magnification; (d) EDS spectrums of modified SiO 2 particles and corresponding element distribution images of F, S and Si. 3.2 Influence of surface composition on hydrophobicity Coatings featuring micro-nano-scale roughness structures often exhibit mechanical fragility and susceptibility to abrasion, particularly on rigid substrates. Organic–inorganic nanocomposites have emerged as a promising strategy to address this limitation, achieved either through polymer-grafted nanoparticles or dispersion of surface-modified nanoparticles within polymeric matrices. Crucially, the organic/inorganic mass ratio critically determines the hydrophobic performance during composite fabrication, necessitating systematic optimization. Table 1 shows 9 formulations for follow-up research. Table 1 Hydrophobic coatings of different fluorosilicone and silica resins Group Fluor silicone/g SiO 2 /g Acetone/g A 0.0 0.2 5.0 A 0.2 0.2 5.0 A 0.4 0.2 5.0 A 0.6 0.2 5.0 B 0.4 0.0 5.0 B 0.4 0.05 5.0 B 0.4 0.1 5.0 B 0.4 0.2 5.0 B 0.4 0.4 5.0 During the preparation process, the ratio of SiO 2 particles to resin plays a great role in the hydrophobic effect. In order to determine the most suitable ratio, two sets of samples were prepared: the SiO 2 particles of group A were fixed at 0.2 g, and the resin was 0 g, 0.2 g, 0.4 g, and 0.6 g. The resin of group B was fixed at 0.4 g, and the SiO 2 particles were 0 g, 0.05 g, 0.1 g, 0.2 g, and 0.4 g. The solvent of the two groups is 5 g of acetone, and the curing agent is 0.2 g. 3.2.1 Effect of fluorosilicone on hydrophobicity The hydrophobicity of coatings is principally governed by two critical factors: low surface energy characteristics and hierarchical surface roughness. To systematically investigate the SiO 2 /resin mass ratio's impact on water-repellent performance, FT-IR analysis was conducted on component B formulations in Fig. 3(c) . Spectral analysis reveals characteristic C–F stretching vibrations within the 1298–1162 cm −1 range, demonstrating successful perfluoroalkyl group incorporation. Notably, the progressive intensification of these absorption bands with increasing resin content confirms enhanced surface fluorination – a critical determinant of surface energy reduction. Concurrently, emerging hydroxyl group signals near 3000 cm −1 suggest competing hydrophilic interactions, though their relative intensity remains subordinate to dominant hydrophobic functionalities. Optimal hydrophobicity was achieved at 0.4 g resin loading, evidenced by water contact angles exceeding 150° in Fig. 3(a and b) . This performance maximum correlates with the synergistic balance between fluorocarbon coverage and surface roughness preservation. Fig. 3 (a) WCAs with different resin content. (b) Hydrophobic angles of samples with different SiO 2 particle masses. (c) Infrared spectra of different resin contents. 3.2.2 Effect of SiO 2 on hydrophobic effect In order to explore the influence of SiO 2 particles on wettability, the SEM was used to characterize the coatings of component B. The surface morphology of samples with different SiO 2 particle masses is shown in Fig. 4(a–d) . When the mass of SiO 2 particles is 0.05 g, the coating surface is relatively smooth. The irregular pattern in the figure is fluorosilicone resin, and the SiO 2 particles do not provide enough roughness, Fig. 3(b) shows its water contact angle is only 73.5°, which fails to meet the requirement of hydrophobic; when the mass of SiO 2 particles is 0.1 g, the roughness of the coating increases significantly, but there are obvious cavities on the surface, and the distribution of SiO 2 particles is uneven, its hydrophobic angle is slightly increased to 90.9°, barely meeting the requirement of hydrophobic; when the mass of SiO 2 particles is 0.4 g in Fig. 4(d) , even though the hydrophobic angle is 160°, but it will be caused by too many SiO 2 particles, which can cause multi-initiated reunion and poor film formation. Therefore, the effect is best when the mass of SiO 2 particles is 0.2 g. It can be seen from the Fig. 4(c 1 ) shows that the SiO 2 particles are evenly distributed, with less agglomeration and voids, and provide sufficient roughness to the coating surface, and the hydrophobic coating will be more hydrophobic and the effect is best, its water contact angle is 159.3°, which meets the requirement of superhydrophobic. Fig. 4 The surface macro morphology, micro morphology and roughness of samples with different SiO 2 particle quality (a) 0.05 g (b) 0.1 g (c) 0.2 g (d) 0.4 g. 3.3 The superhydrophobic property of the prepared coating The fluorosilicone resin–SiO 2 superhydrophobic composite coating exhibits excellent superhydrophobicity, with a static water contact angle of up to 156° on the glass substrate. To evaluate its self-cleaning potential, dynamic tests were conducted. As shown in Fig. 5(a) , a 10 μL water droplet rapidly slides off the inclined surface (tilt angle: 3°) within 273 ms, indicating the coating's low adhesion properties, which facilitate bubble transportation. Fig. 5(b) further characterizes the dynamic adhesion behavior: when a 5 μL water droplet on a needle contacts the coating surface, a defined pressure is applied to deform the droplet. Subsequently, the droplet detaches without leaving any residue, confirming the coating's exceptional superhydrophobicity. Fig. 5 Superhydrophobic properties (a) rolling angle (b) dynamic adhesion behavior. 3.4 Collect transport bubbles Numerous studies have demonstrated that a structurally modified conical surface generates gradients of Laplace pressure and surface free energy, creating a directional driving force for droplet collection and transport. Inspired by this mechanism, we aim to introduce analogous gradients onto conical surfaces to enable continuous collection and directional transport of microbubbles in aqueous media. In this work, a superhydrophobic coating was integrated onto copper needles to achieve efficient microbubble manipulation in deionized water. Upon immersion, the superhydrophobicity of the coated needle induces a stable air layer underwater, providing a low-resistance pathway for microbubble transportation. The Laplace pressure gradient arising from the conical geometry serves as the primary driving force, directing bubbles along the needle surface. This approach offers a simple yet effective strategy for microbubble elimination in liquid systems. As shown in Fig. 6 , on the surface of the horizontally placed superhydrophobic copper needle, there are four forces acting on the moving bubbles, namely Laplace pressure ( F L ), synthetic resistance ( F R ) generated by the liquid and lubricating layer, buoyancy ( F B ), and the adhesion of the superhydrophobic copper needle surface to the air bubbles ( F A ). F L is the driving force for the directional movement of bubbles induced by the cone shape of a superhydrophobic copper cone. 49–52 1 where R 1 and R 2 are the local radii on opposite sides of the bubble, S is the area of the area surrounded by the bubble, and α is the half-vertex angle of the cone. Since the bubble is attached to the surface of the superhydrophobic copper cone, there will be a difference in radius between the two opposite sides of the bubble. Therefore, the F L directionally drives the bubbles on the surface of the superhydrophobic copper needle from its tip to its bottom. When air bubbles move on a smooth surface in an aqueous environment, the resistance generated by the liquid and the viscous layer must be considered. The resultant resistance ( F R ) can be calculated as follows: 53–55 2 where C D and ρ are the resistance coefficient and the density of the aqueous medium, respectively; v is the transmission speed of the bubble; A is the cross-sectional area of the bubble; a and k are uncertain parameters derived from the resistance of the lubricating layer. F B is determined by the volume of the bubble ( V ), the density of the liquid ( ρ ), and the acceleration of gravity ( g ), and can be expressed as follows: 3 F B = ρgV Fig. 6 Schematic diagram of Laplace driving force. \n F \n A can be derived as follows: 56,57 4 F A = γ l 1 l 2 L TCL sin β where L is the length of the three-phase contact line and β is the effective contact angle of the bubble on the smooth surface. For the case where the bubble moves horizontally on the surface of the superhydrophobic copper cone, F A can resist F B , and its resultant force is equal to zero in the vertical direction. The collection and transport processes of microbubbles in water by superhydrophobic copper needles were studied. To prevent carbonic acid interference from dissolved CO 2 , a closed pipe flow system with a flow rate of 0.5 μL s −1 was utilized. The copper needle was positioned horizontally in deionized water to suppress buoyancy effects on bubble dynamics. As shown in Fig. 7 , microbubbles contacting the needle surface spread rapidly, confirming its superaerophilic behavior in water. These bubbles migrated from the needle's tip to its tail at 20.5 ± 0.5 mm s −1 , merging into a single large bubble. At 97 s, buoyancy exceeded the adhesive force, causing detachment of the 48 μL bubble. This cycle repeated continuously, enabling sustained microbubble collection and transport. The superhydrophobic copper needle was taken out, stood at room temperature for 24 h, and then immersed in water again, it still had the ability to collect and transport bubbles. The application of superhydrophobic copper needles in crude oil exploitation can reduce the bubble content and improve the efficiency of crude oil exploitation; when applied to waterborne coatings, it can improve the film-forming property and make the coating more uniform. Fig. 7 Microbubble collection and transportation experiment. \n Fig. 8 investigates bubble collection and transport by the superhydrophobic copper needle under reverse gravity conditions. At a tilt angle of 20°, the average transport velocity is 17.4 ± 0.5 mm s −1 with a transport distance of 0.6 cm. When the tilt angle increases to 90°, the velocity decreases to 6.5 ± 0.5 mm s −1 and the distance shortens to 0.5 cm. These results confirm that the needle can collect and transport bubbles against gravity. Comparing these data with the 0° tilt angle case (horizontal placement, Fig. 7 ), we observe a clear trend: as the tilt angle increases, buoyancy increasingly opposes the adhesive force, leading to slower transport speeds and shorter distances. Nevertheless, the needle maintains functionality even at 90°, demonstrating its robustness in overcoming gravitational effects. Fig. 8 Superhydrophobic copper needles collect and transport air bubbles against gravity. (a) 20° (b) 90°. 3.5 Stability The limited mechanical durability of superhydrophobic surfaces poses a significant challenge for their real-world applications. To address this, we systematically evaluated the robustness of an organic–inorganic composite coating through standardized sandpaper abrasion tests and knife scratch experiments. 58,59 As illustrated in Fig. 9(a) , the abrasion protocol involved cyclically moving the coating (under a 100 g load) across 800# SiC sandpaper—10 cm laterally, followed by a 90° rotation and another 10 cm longitudinal movement—to simulate severe mechanical wear. Remarkably, after 20 such cycles, the coating retained a water contact angle of 147° in Fig. 9(b) , indicating robust mechanical resistance. This durability stems from the coating's “bulk superhydrophobicity”, a structural design where hydrophobic SiO 2 nanoparticles and interconnected cavities are distributed not only on the surface but also throughout the interior. Consequently, even if the top layer is abraded, the underlying hydrophobic network maintains non-wettability. Comparative analysis with the DMS/ODA-PDA@PI nanofibrous membrane reported by Wenjing Ma et al. , 58 revealed that our coating achieved a higher post-abrasion water contact angle (147° vs. 135° after 20 cycles), underscoring its superior mechanical resilience. Furthermore, the coating's ability to repel impacting droplets—causing them to retract or rebound—significantly reduces ice nucleation sites, suggesting strong potential for anti-icing applications in harsh environments. These results collectively demonstrate that the synergy of structural design and material composition can effectively mitigate the mechanical fragility of superhydrophobic coatings. Fig. 9 (a) Wear diagram (b) the relationship between the number of sandpaper abrasion test cycles and the wettability of the coating surface (c) scratch test (d) the static contact angle of water droplets with different pH values on the surface of the composite coating. As shown in Fig. 9(c) , in the blade scratch test, 60 after the blade is scratched according to the preset route, the coating still maintains superhydrophobic properties. After the test, when the dyed water droplets were dropped onto the surface of the scribed coating, they rolled off quickly without any adhesion, indicating the excellent anti-scratch stability of the composite coating. In the actual application process, the coating may come into contact with robust acid or alkaline substances. In order to evaluate the chemical stability of the composite coating in an acid-base environment, this section adopts two methods, using aqueous solutions of different pH values to test the static contact angle of the coating surface and immersing the coating in aqueous solutions of different pH values. After a certain period of time, take out the coating and measure the static contact angle for evaluation. Fig. 9(d) shows the static contact angles of aqueous solutions with different pH values on the coating surface. It can be seen from the figure that solutions with different acidity and alkalinity all exhibit excellent superhydrophobicity on the surface of the composite coating. The contact angle of acidic droplets is generally higher and more stable than that of alkaline droplets. By comparing with the DMS/ODA-PDA@PI nanofibrous membrane prepared by Wenjing Ma et al. , 58 the performance of this coating is better in acidic environment, and the hydrophobic angle is more than 154°. However, in alkaline environment, it is slightly insufficient and fluctuates greatly. This may be due to the high affinity of the nanoparticles on the surface of the coating with the sodium hydroxide solution, which allows the alkaline aqueous solution to diffuse into the porous micro–nano structure with air hidden on the surface of the coating. Further, the composite coating is immersed in a solution of hydrochloric acid and sodium hydroxide with pH of 1 and 14. The experimental results show that after immersing in a hydrochloric acid solution with a pH of 1 for 10 hours, there is no change on the surface of the coating, and the surface remains dry. After removal from the solution, the surface of the coating remains dry and does not stick to the solution. These results indicate that the superhydrophobic composite coating has outstanding stability in a robust acid environment. However, for a robust alkaline sodium hydroxide solution with a pH of 14, the surface of the coating was partially wetted and lost its mirror effect after 1 hour of soaking. After soaking for 10 hours, the surface of the composite coating was completely wetted. This indicates that the composite coating has poor resistance to robust alkaline environments."
} | 6,837 |
32937458 | PMC7458436 | pmc | 1,688 | {
"abstract": "Ion gels enable temperature-stable and high-performance organic memories for integration into hardware artificial neural networks.",
"introduction": "INTRODUCTION Novel materials and neuromorphic devices are required to address the inability of complementary metal-oxide-semiconductor (CMOS) transistor scaling to meet the increasingly demanding computational density and energy requirements of artificial neural networks (ANNs), particularly in embedded platforms with limited area and power budget ( 1 ). While substantial progress has been made using crossbar arrays of emerging nonvolatile memories, such as phase-change memory (PCM) ( 2 ) and resistive random-access memory (ReRAM) ( 3 ), these technologies present fundamental limitations. The inherently stochastic switching in PCMs (melting and recrystallization) and ReRAMs (filament formation) inevitably causes nonlinear and asymmetric resistance tuning, leading to large write errors that are detrimental for accelerating training in hardware ANNs ( 4 , 5 ). Electrochemical random-access memories (ECRAMs) ( 6 – 9 ) on the other hand, where resistive switching is instead controlled by ion insertion from the electrolyte into a semiconductor channel, enable linear resistance switching with low write noise ( 9 ). While linear resistance switching is not essential for high ANN performance, since symmetry in resistance tuning for potentiation/depression is more critical ( 4 ), device linearity remains the most straightforward way to fulfill the symmetry requirement. Because of the nearly ideal switching characteristics of ECRAM devices, they have therefore emerged as a promising alternative for analog ANN accelerators. ECRAMs have been demonstrated using several materials classes: Two-dimensional (2D) materials such as graphene or α-phase molybdenum oxide (α-MoO 3 ), as well as metal oxides such as lithium titanium oxide (Li x TiO 2 ), tungsten oxide (WO 3 ), or lithium cobalt oxide (Li 1− x CoO 2 ), have all been used as channel materials. These demonstrations relied on conventional Li-based battery electrolytes, e.g., lithium phosphorous oxynitride (LiPON) ( 7 , 8 ) or lithium perchlorate (LiClO 4 ) mixed with polyethylene oxide (PEO) ( 10 – 12 ). While Li-based ECRAMs have shown promising characteristics, most devices are limited to millisecond write speeds. As an exception, a WO 3 channel combined with LiPON electrolyte was shown to respond to 5-ns write pulses but required 1.5-s write-read delays ( 8 ), thereby severely limiting the overall speed of the device. While the ultimate speed of Li-based devices is being investigated, the relatively sluggish kinetics of Li intercalation poses a fundamental challenge. In addition, Li reactivity is a concern for compatibility with semiconductor fabrication processes. In contrast, previously reported organic ECRAMs ( 6 , 9 ), which relied on proton exchange membranes, such as Nafion ( 13 ), demonstrated 200-ns switching and <1-μs write-read cycles with endurance to >10 8 write-read events ( 9 ). Nevertheless, despite promising initial demonstrations ( 6 , 9 ), organic ECRAMs face several challenges. Proton exchange membranes require extensive hydration to conduct protons ( 13 , 14 ). Organic ECRAMs ( Fig. 1A ) made using poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) ( Fig. 1B ) as the channel/gate material and a commercially available perfluorosulfonic acid ionomer membrane (Aquivion; see Materials and Methods) as electrolyte do not exhibit any conductance modulation when operated in moderate (2 × 10 −4 mbar) vacuum ( Fig. 1C ). This limitation renders the use of conventional proton exchange membranes incompatible with dry environments and thus integration into ANN accelerators, which is the first technological challenge. The second technological challenge stems from water evaporation from the proton exchange membrane at the temperatures generated in conventional electronic packaging during device operation (up to ~90°C), which, similarly to vacuum, would impair organic ECRAM functionality. While one could argue that this is not a major impediment as biological neurons are temperature sensitive, microelectronics requires temperature-resilient devices. Fig. 1 Organic ECRAMs using ion gels enable submicrosecond switching in vacuum. ( A ) ECRAM device schematic. ( B ) Chemical structures of the channel/gate (left) and electrolyte (right) materials. The blue circle on 1-ethylimidazolium bis (trifluoromethylsulfonyl)imide (EIM:TFSI) highlights the hydrogen that renders EIM:TFSI protic.( C ) Resistive switching characteristics of ECRAM with PEDOT:PSS as the channel/gate material and Aquivion as the electrolyte rapidly deteriorate when going from 20% relative humidity (RH) in N 2 atmosphere (black) to 2 × 10 −4 mbar vacuum (gray). ( D ) Cycling of ECRAM with PEDOT:PSS as the channel/gate material and EIM:TFSI poly(vinylidene fluoride- co -hexafluoropropylene) (PVDF-HFP) as the electrolyte operating in vacuum. ( E ) Cycling of ECRAM with poly(2-(3,3- bis (2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-[2,2-bithiophen]-5-yl)thieno[3,2- b ]thiophene) [p(g2T-TT)] as the channel/gate material and 1-ethyl-3-methylimidazolium bis (trifluoromethylsulfonyl)imide (EMIM:TFSI) PVDF-HFP as the electrolyte operating in vacuum. Inset shows normalized channel conductance Δ G SD / G min for PEDOT:PSS-based (blue) and p(g2T-TT)-based (red) ECRAMs. In addition, previously reported organic ECRAMs suffer from a limited dynamic range (<2×) between the highest/lowest conductance states when using short (~1 μs or less) write pulses ( 9 ). While operating the devices over a small dynamic range is desirable for neuromorphic computing, since it prevents large currents from saturating neurons (e.g., stuck ON cells in PCM arrays), the device dynamic range should be inherently large for improved tolerance to device-to-device variations. For example, if the median ECRAM channel conductance varies across devices in an array, then the lack of conductance range overlap between ECRAMs with a limited dynamic range would be detrimental for ANN computation. In contrast, ECRAMs with a larger dynamic range can be operated over a conductance range common to all devices. A larger dynamic range also improves ECRAM tolerance to write noise, since each state can span a larger conductance range relative to the write noise level. For high ANN accuracy, the targeted dynamic range is >330 times larger than the device write noise ( 4 ). The improved dynamic range, however, must be attained under submicrosecond write speeds to be relevant for ANN computation. If organic ECRAMs are to be integrated with Si, then these limitations must be overcome while retaining all of the other favorable device metrics, such as write linearity, low write noise, high speed, low switching energy, and high endurance.",
"discussion": "DISCUSSION In summary, we fulfill the large number of requirements for efficient neuromorphic computing in a single organic ECRAM device: linear resistance tuning with a >2× dynamic range, fast switching (20 ns), submicrosecond write-read cycling, low-voltage (±1 V) and low-energy (~80 fJ per write) operation, low noise (Δ G 2 /σ 2 > 100), and excellent endurance both at room temperature and at elevated temperature (>10 9 write-read operations at 90°C). Our materials selection strategy proves to be quite general, as it can be effectively implemented using various ionic liquids and other block copolymers (fig. S7). In contrast, it has been proven difficult to optimize PCMs and ReRAMs across all metrics, such as attaining both low write noise and low current operation, which are needed for scaling to large arrays. While this work represents a fundamental step toward designing organic ECRAMs to be compatible with fabrication into high-density synaptic arrays, some important challenges remain due to the strict temperature and contamination requirements for integration with CMOS. Alternative fabrication pathways exist for integrating emerging nonvolatile memories with CMOS, such as low-temperature monolithic 3D integration of carbon nanotubes and ReRAMs ( 29 ), and remain an active area of pursuit to improve such compatibility. Last, we emphasize that the ECRAM concept is not limited to organic materials. The lessons learned here, such as the importance of mobile species infiltrating the channel before electrochemical gating and possibly relying on proton conduction for fast switching, can also benefit ECRAMs made with other ion intercalation materials such as 2D materials, van der Waals heterostructures, or MXene composites."
} | 2,152 |
32574214 | PMC7310705 | pmc | 1,689 | {
"abstract": "Crustose coralline algae (CCA) are vital to coral reefs worldwide, providing structural integrity and inducing the settlement of important invertebrate larvae. CCA are known to be impacted by changes in their environment, both during early development and adulthood. However, long-term studies on either life history stage are lacking in the literature, therefore not allowing time to explore the acclimatory or potential adaptive responses of CCA to future global change scenarios. Here, we exposed a widely distributed, slow growing, species of CCA, Sporolithon cf. durum , to elevated temperature and p CO 2 for five months and their first set of offspring (F 1 ) for eleven weeks. Survival, reproductive output, and metabolic rate were measured in adult S . cf. durum , and survival and growth were measured in the F 1 generation. Adult S . cf. durum experienced 0% mortality across treatments and reduced their O 2 production after five months exposure to global stressors, indicating a possible expression of plasticity. In contrast, the combined stressors of elevated temperature and p CO 2 resulted in 50% higher mortality and 61% lower growth on germlings. On the other hand, under the independent elevated p CO 2 treatment, germling growth was higher than all other treatments. These results show the robustness and plasticity of S . cf. durum adults, indicating the potential for them to acclimate to increased temperature and p CO 2 . However, the germlings of this species are highly sensitive to global stressors and this could negatively impact this species in future oceans, and ultimately the structure and stability of coral reefs.",
"conclusion": "Concluding remarks The results from this study show polarising responses from adult Sporolithon and their F 1 germlings to increased temperature and p CO 2 , suggesting life history stage plays an important role in resilience to global change variables and ability to exhibit a plastic response. Adult Sporolithon were largely resilient to increased temperature and p CO 2 suggesting a robust potential for acclimation, whereas the results from the F 1 germling growth and survival suggest that the germlings of Sporolithon are more sensitive to changes in their environment. The mechanisms explaining the different acclimatory response between life history stages are not well understood, but may be related to 1) variable carbon uptake physiologies (e.g. with germlings lacking or having low affinity CCM as growth increased under high p CO 2 alone); 2) variable skeletal mineralogy (e.g. nature, composition, or skeletal protection by living tissue); 3) different composition and/or quantities of cell-wall organic constituents, or 4) different nutrient requirements. In particular, CCA tissue organisation varies throughout ontogenesis [ 82 , 83 ] and it is likely that different tissues that develop following germination to the formation of adult, thick crusts may have different susceptibilities to pH reduction. For example, CCA spores mainly germinate developing a basal hypothallial tissue [ 82 ] and recent studies show the hypothallus of a number of CCA species has different mineralogy from mature (perithallial) tissue (e.g. in Phymatolithon , perithallial cell walls have mean 13.4 mol% MgCO3 while the hypothallus has 17.1 mol% MgCO3) [ 13 ], and this may influence early germling development. There is very little information on Sporolithon germination [ 84 ] and further work should elucidate the contribution of these processes to the different responses to climate stressors between life history stages of CCA. Our study also suggests a mechanism by which adult Sporolithon is able to cope with ocean acidification and warming conditions. A decline in O 2 production (with no thallus mortality) is a possible indicator of an acclimatory response in this species, and this observation has not been documented previously in other coralline algae [ 43 , 85 ], but is common in some terrestrial plants [ 55 , 56 ]. Although there may be a positive outlook for adult Sporolithon to acclimate and possibly eventually adapt to future ocean conditions, the sensitivity of Sporolithon germlings to combined environmental stressors complicates this species persistence in future oceans. A previous study showed OA considerably reduced net calcification of S . durum rhodoliths, suggesting adults of this species may also be sensitive to pH decline [ 40 ]. Although we did not measure calcification in our Sporolithon , the negative net calcification rate (i.e. dissolution) observed in the rhodoliths could have been due to the use of this growth form (i.e. rhodoliths, as opposed to crusts), which have considerable internal porosity, potentially facilitating skeletal dissolution. Future studies are needed to determine the effects of OW and OA on gross and net calcification of Sporolithon to obtain a more complete picture of the adult’s sensitivity to climate stressors. Our study began as a novel attempt to conduct a multi-generational study with a slow growing, reef building species of CCA. However, multiple generations were not achieved (cf. Cornwall et al. [ 35 ]), therefore, the experiment was adapted to look at the long-term effects of global change factors across lifecycles. Acclimation history of adults prior to collection may have played a role in responses seen in this study, therefore future studies are necessary to more fully compare response to global change across life history stages. These future studies should also further investigate the adaptation potential of this slow growing, ancient group of CCA, specifically looking at the possibility for gained tolerance to both increasing temperature and OA.",
"introduction": "Introduction Coral reefs depend on a multiplicity of organisms to ensure their success. Crustose coralline algae (CCA) are integral inhabitants of coral reef ecosystems, building and stabilising the carbonate framework of coral reefs by deposition of calcium carbonate (CaCO 3 ) [ 1 , 2 ], providing food to herbivorous fish and invertebrate grazers [ 1 , 3 ], and inducing the settlement of coral larvae and other invertebrate larvae [ 4 – 6 ]. CCA’s part in facilitating the induction of coral larvae to a reef give them a crucial role in helping coral reefs withstand and recover from disturbances, and therefore their overall resilience to future changes in our oceans [ 6 , 7 ]. Coral reefs, which have already experienced warming and are predicted to experience more [ 8 ], are among the most threatened ecosystems by environmental changes brought on by the increase in atmospheric CO 2 , namely the increase in ocean temperature, also termed ocean warming (OW), and ocean acidification (OA), both of which threaten key, calcifying species that make up the framework of reefs, such as CCA. The responses of CCA to changes in their environment are variable, having been found to be mostly either vulnerable or insensitive to both increasing ocean temperature and/or OA [ 9 – 12 ]. CCA secrete the most soluble polymorph of CaCO 3 , high Mg-calcite [ 13 ] and their process of calcification is suggested to be biologically induced rather than controlled [ 14 ], and therefore are thought to be particularly sensitive to changes in seawater carbonate chemistry that occur with OA [ 15 – 19 ]. In saying this, however, there are examples of CCA taxa that are well adapted to extreme environments, such as the freshwater CCA Pneophyllum cetinaensis [ 20 ], and the arctic-subarctic Clathromorphum genus [ 21 , 22 ], which is able to calcify under dark conditions [ 23 ]. Moreover, some CCA taxa, particularly those from high energy environments, have areas of the thallus which are rich in dolomite, resulting in lower dissolution rates under high-CO 2 treatment [ 23 , 24 ]. Increases in p CO 2 have been found to negatively affect the abundance and community structure of CCA [ 9 , 10 ]. While, when examining the photophysiology of CCA, the effect of p CO 2 on metabolic rate (e.g. net photosynthesis, gross photosynthesis, and respiration) shows species of adult CCA studied to be largely insensitive [ 11 , 12 ], although there is considerable variability across taxa. No change or increase in O 2 production in response to increased p CO 2 in algae may be attributed to the presence of carbon concentrating mechanisms or CCMs [ 25 ]. A study on the CCA species Porolithon onkodes found gross photosynthesis and respiration to be unaffected by increased p CO 2 but net photosynthesis was negatively affected by increased p CO 2 [ 12 ]. Another study found no effect of p CO 2 on net photosynthesis, gross photosynthesis, or respiration [ 11 ]. The combined effects of increased temperature and OA have been found to decrease calcification [ 26 ] and increase mortality and dissolution [ 16 ] in adult CCA species. Much of the work on CCA and environmental change has been done with adult algae, with fewer studies investigating the combined and independent effects of increased temperature and p CO 2 on early life history stages (i.e. spores and germlings). Early life history stages of organisms are thought to be the most influenced by changes in their environment [ 27 , 28 ]. Our knowledge on offspring or early life history stages of CCA is limited and variable, with studies finding early life history stages to be highly susceptible to combined stressors of increases in temperature and OA [ 29 – 33 ] or largely unaffected by p CO 2 when it is an independent stressor [ 34 ]. A more recent study even suggests CCA can gain tolerance to OA over multiple generations [ 35 ]. A study done examining the combined and independent effects of increased temperature, p CO 2 , and irradiance on germination success of the CCA species Porolithon cf. onkodes found elevated p CO 2 was the main driver behind a reduction in the rate of spore germination and a higher rate of abnormalities in developing spores, with the magnitude of the effect being enhanced by elevated temperature [ 29 ]. Increased p CO 2 also resulted in reduced vertical and marginal growth of germlings [ 29 ]. However, an increase in p CO 2 was found to increase gross photosynthesis and respiration of germlings from P . cf. onkodes [ 30 ]. When the effect of elevated p CO 2 was studied on the germlings from another species of coralline algae, Phymatolithon lenormandii , an increase in mortality and abnormalities were found under more acidic conditions, with as little as a 0.1 pH unit change negatively impacting both survival and development [ 33 ]. Population persistence of macroalgae rely on spores settling and adhering onto substratum, to continue the algal lifecycle. In a study done by Guenther et al. [ 31 ], reduced pH, or the increase of p CO 2 , resulted in a 40–52% delay in spore attachment and weakened the attachment strength in the coralline alga species, Corallina vancouveriensis . The previously mentioned studies examined shorter-term exposure (1 month or less) to OW and OA, either in combination or independently, and found mostly negative responses over a number of genera. However, a recent study looking at the independent effect of OA on a species of CCA, Hydrolithon reinboldii , found that this species can gain tolerance to OA alone over multiple generations [ 35 ], which suggests it’s possible for other species of CCA and corallines alike to exhibit resistance or transgenerational acclimation to OA. It is unknown, however, if and how increased temperature will negate this, and whether other species show similar outcomes. Here, our study sought to examine the potential for a widely distributed, slow growing, reef building species of CCA, Sporolithon cf. durum ( Sporolithon hereafter), to acclimate to global change related stressors across lifecycles. Although Sporolithon is not one of the major reef building species of CCA in tropical reefs, like, but not limited to, P . onkodes [ 1 ], Sporolithon is an important reef benthic component [ 36 ] (pers. obsv) and a dominant coralline alga of rhodolith beds in the topics and subtropics, particularly in mid-to deep water environments [ 37 – 39 ]. Sporolithon spp. mineralise high Mg-calcite within their cell walls like the primary reef building CCA species [ 14 ]. Therefore, furthering our knowledge on the responses of Sporolithon to elevated temperature and OA [ 40 ] is not only relevant to, but vital for understanding the effects of such stressors on benthic reef communities more broadly and those species that function to cement the framework of coral reefs. Ability to acclimate to environmental conditions through phenotypic plasticity has been seen in previous studies across a range of organisms, partially or fully ameliorating the negative effects of increased temperature and p CO 2 [ 35 , 41 , 42 ]. In this study, we present data on the performance of adults (survival and metabolic rates, and reproductive output) and their F 1 germlings (growth and survival) from the species Sporolithon exposed to increased temperature and p CO 2 over five months and eleven weeks, respectively. Long-term ocean warming (OW) and OA experiments (months to years) are less common than short-term exposure (days to weeks) experiments [ 43 , 44 ], therefore, the longevity of this experiment enables us to examine the acclimatory potential of this species. It was hypothesised that p CO 2 would negatively affect the growth and survival of the germlings and this negative effect would be enhanced by increased temperature. The order that Sporolithon belongs to, Sporolithales, has basal evolutionary origins having diverged and persisted during times of elevated temperature and p CO 2 (relative to current levels) [ 45 – 47 ], due to this, it was hypothesised that the adult S . cf. durum , although a more recently diverged species from the genus Sporolithon , would acclimate more readily than their germlings to prolonged exposure to increased temperature and p CO 2 , resulting in low mortality and no effect on metabolic rate across all treatments.",
"discussion": "Discussion Our study shows a contrasting response in life history stages to environmental stressors, with adult Sporolithon being remarkably resistant and early life history stages being very sensitive to OW and OA. These data contribute essential knowledge to understanding the longer-term effects of global change stressors across lifecycles of an important and abundant species of CCA and investigates the potential for acclimation in adult Sporolithon and survivorship and growth of germlings of the first generation (F 1 ). Adult Sporolithon ultimately did well throughout the entirety of this experiment, showing no mortality, no significant change in reproductive output or respiration, and photosynthesis was slightly decreased in response to increased p CO 2 (low pH). The results from the adult Sporolithon support the hypothesis that this species is robust, and the divergence time of the genus of Sporolithon , and more broadly the family, may play a role in this [ 45 – 47 ]. The slight decrease in O 2 production in response to increased p CO 2 without any impact on survivorship indicates a potential acclimatory or plastic response to new CO 2 conditions [ 55 , 56 ]. The success of adult life stages is dependent on the health of their earlier life history stages. Here, the response of Sporolithon germlings to global change variables may present an issue for their subsequent life history stages. There were strong and complex interactions between temperature and p CO 2 . In fact, elevated p CO 2 may benefit growth, compared to low p CO 2 , but, when combined with elevated temperature, there was a strong interaction leading to reduced survival and growth under the combined environmental stressors. Although the genus Sporolithon is not a primary reef builder, they have a similar calcification process to reef building CCA species (e.g. Nash et al. [ 14 ]) and are important members of the benthic community in tropical reefs, and the negative response of the germlings to combined environmental stressors may impact some essential functions of CCA in coral reefs under future global change scenarios. Adult survival & metabolic rates After five months of exposure to elevated temperature and p CO 2 , adults of Sporolithon survived and remained visibly healthy, with no thallus mortality observed, across all treatments. However, O 2 production was significantly reduced under the high p CO 2 treatments at both ambient and high temperature, with an average reduction of 25.22%. The lack of mortality and the reduction in O 2 production may be interpreted as a plastic or possibly an acclimatory response to prolonged exposure to elevated p CO 2 , in which the alga downregulates the photosynthetic activity without significant implication for survivorship [ 56 , 57 ]. This response has been commonly observed in terrestrial plants, particularly when plants experience nutrient limitation [ 56 , 57 ]. Photosynthetic acclimation to elevated CO 2 may be attributed to an overabundance of additional carbohydrates that photosynthesis in elevated CO 2 provides, suppression of nitrogen assimilation, or at elevated CO 2 a reallocation of resources occurs within the photosynthetic apparatus, requiring less Rubisco for photosynthesis [ 55 , 56 , 58 , 59 ]. Like in terrestrial plants, nutrients may have played a role in the acclimatory response of Sporolithon to prolonged increased p CO 2 , and had nutrients been added, we may have seen a “positive” or increase in metabolic rate instead of a “negative” or acclimatory response (as seen in terrestrial plants) [ 57 , 59 ], however, this hypothesis needs experimental testing. This is a realistic possibility as anthropogenic nutrification is occurring on reefs due to increased agricultural runoff and changing land-use [ 60 , 61 ]. We acknowledge there are more ways of acclimation to increased p CO 2 , however, suppression of O 2 production, seen in this study, in response to prolonged p CO 2 exposure in Sporolithon is likely their acclimatory response, similar to terrestrial plants, in which resources and activities are modulated in order to benefit the plant [ 56 – 58 , 62 ]. If observing only the effect of p CO 2 on the O 2 production of species of coralline algae, contrasting responses have been seen, where elevated p CO 2 alone hasn’t influenced significant changes in O 2 production. This was observed in the tropical CCA species, P . onkodes , after 14 days in treatment there was no effect of elevated p CO 2 on stable environment samples [ 12 ]. A lack of strong response was also seen in three temperate coralline algae species when exposed to increasing p CO 2 [ 53 ]. Sporolithon reacted conversely to the above studies, showing a decrease in net production driven by increased p CO 2 , most likely as an acclimatory response to sustained increased p CO 2 . The CCMs most likely present in Sporolithon , like in many other macroalgae [ 63 – 65 ], may also play a role in why a positive response, or increase in photosynthesis, was not seen in adult Sporolithon . Algae maintaining high affinity CCMs are predicted to not respond to or benefit considerably from increased p CO 2 in terms of metabolic processes [ 25 ]. Ultimately, the lack of mortality and decrease in photosynthesis suggests this species of CCA is quite robust and able to acclimate to changes in their environment (e.g. increased temperature and p CO 2 ), suggesting a positive outlook for their persistence and function in coral reef ecosystems under future global change scenarios. Reproductive output Reproductive output is an essential fitness measurement across organisms and a reduction in this process could negatively impair the persistence of species. However, in this study reproductive output was not found to be significantly influenced by experimental treatment, with only a slight increase in mean number of spores released under the most stressful treatment (high temperature + high p CO 2 ) when compared to the ambient treatment. To our knowledge, no past studies have looked into the reproductive output of CCA after exposure to warming and acidification, however, reproductive output from other calcifying macroalgae [ 66 ] and seagrasses [ 67 , 68 ] has been measured in response to OA. For a species of coralline algae, Arthrocardia corymbosa , altered pH did not significantly alter reproductive output [ 66 ]. Elevated p CO 2 has been found to increase reproductive output of seagrass [ 67 , 68 ], and it is suspected that seagrass will be impacted by the interaction of elevated temperature and p CO 2 [ 67 ]. The slight increase of number of spores in the combined high temperature and p CO 2 treatment could have been a stress response and final push to release as many spores as possible after the additional stress of a temperature spike to induce spore release. CCA can reproduce asexually or sexually and maintain different reproductive structures based on this. For Sporolithon the asexual (tetrasporophytic) reproductive structures, sori, are much smaller than the general conceptacle, or asexual reproductive structure on other CCA species [ 69 ], which are sometimes even visible by the naked eye [ 48 ]. Due to their small size and the differing amount of area sorus take up on each individual crust [ 69 ], there was likely variability from adult to adult in each treatment and that may have influenced the variable response in reproductive output across treatments. Although the sori area was kept as constant as possible, large variability was inevitable. Additionally, there is no, non-invasive way to count the number of sori that contain tetraspores. We acknowledge the limitation in completely quantifying reproductive output by measuring number of spores released, and this should be further investigated. Despite the limitations and the variability observed in Sporolithon spore release across treatments, reproductive output was not negatively influenced by temperature and/or CO 2 factors, suggesting that the reproductive process is robust to warming and acidification. Germling survival & growth The sensitivity of the early life history stages to the combined effects of environmental change stressors supports multiple studies that have found early life history stages to be more vulnerable to global change stressors than adults of the same species [ 27 ]. In the current study, high p CO 2 in combination with increased temperature resulted in a significant decrease in both survival probability and germling growth, consistent with past studies on the germination success and growth of germlings from the CCA Porolithon cf. onkodes [ 29 ]. Coralline algae species differ in their responses to environmental change [ 9 – 12 ] and this is most likely consistent across their different lifecycles as well. Similarly, other early life history stages of different taxa are highly sensitive to exposure to elevated temperature and p CO 2 , with reduction in survival found in the juvenile bivalve, Argopecten irradians , after 45 days in treatment [ 70 ], and a total die off in the larvae of the brittle star, Ophiothrix fragilis , at reduced pH (7.9 and 7.7) after only 8 days in treatment [ 71 ]. Early life history stages of the coral Porites panamensis [ 72 ] and the tropical sea hare Stylocheilus striatus [ 73 ] had reduced growth under simultaneous high temperature and p CO 2 . Responses of early life history stages of Sporolithon found in the current study suggest a similarly found [ 70 – 73 ] sensitivity to environmental change that could ultimately impact their persistence in future oceans. Decreased growth rate in our Sporolithon germlings, particularly under combined high temperature and p CO 2 , could be linked to skeletal dissolution, as documented in other CCA species [ 29 ] and a variety of marine calcifiers across life history stages [ 27 , 74 , 75 ]. Skeletal dissolution may be partially explained by undersaturation of seawater with respect to high Mg-calcite under high p CO 2 and high temperature ( S1 Table , Ω high Mg-calcite = 0.79), although the high p CO 2 but low temperature treatment also had a Ω high Mg-calcite < 1, yet germlings experienced comparable growth rates to ambient p CO 2 . Declined Ω high Mg-calcite in the calcifying fluid (as induced by low pH) has also been proposed to influence calcification in adult Sporolithon [ 40 ] and may be a possibility in our experimental germlings. It is also worth considering the influence of not only the carbonate chemistry (i.e. omega) of the experimental seawater and calcifying fluid on germling calcification and growth, but also the interactions of the germlings with the benthos. Germlings were grown on an acrylic substrate in the laboratory environment, whereas, in the field germlings would most likely be growing on porous carbonate substrate with diverse endolithic and epilithic algal communities that modify carbonate chemistry via metabolic processes. Past studies have found substrate to not be critical for CCA community structure [ 76 ], however, if substrate plays a role in germling calcification and growth in the presence of lowered pH has not been investigated and it is possible that the carbonate substrate in the field could act as a buffer to changes in carbonate chemistry surrounding the germling. It is important to note that under all treatments, there was mortality and reductions in growth rate. However, the survival probability was still significantly reduced in the combined stressor treatment, whereas the other treatments maintained similar survival probability. Additionally, this can be said with growth, seeing a significant increase under increased p CO 2 and a significant decrease in the combined stressor treatment. The negative response of the F 1 germlings to the combined stressor treatment suggests a moderately low likelihood for Sporolithon germlings to exhibit a plastic or an acclimatory or adaptive response to combined future environmental changes, in turn greatly reducing the success of the offspring of this species in future oceans. However, the results in the present study somewhat contradict findings from a multigenerational study done with CCA species H . reinboldii , where juvenile resistance to OA was found, recorded as a lessening of difference in growth across generations [ 35 ]. In this study, the second-generation of CCA grew 56.1% more slowly across OA treatments relative to the ambient/control treatment, whereas the final generation (7) had only a 0.6% difference in growth when comparing OA treatments to the ambient/control treatment, indicating that sustained exposure to OA across multiple generations enables H . reinboldii to gain a tolerance [ 35 ]. The current study suggests that Sporolithon germlings may not be able to exhibit resistance to global change stressors, unlike juveniles of H . reinboldii [ 35 ], however, our results may have been more similar should this study have been conducted over multiple generations, and only investigating the response to OA. Alternatively, differences in germling responses between H . reinboldii and Sporolithon may be attributed to species-specific optima for calcification and growth [ 40 ]. The data in this study suggest that temperature is a critical factor influencing survival and growth of Sporolithon germlings. On the other hand, the increase of p CO 2 in isolation of the increase in temperature (i.e. in ambient temperature conditions) enhanced germling growth. As indicated earlier, however, when combined with increased temperature, growth was significantly decreased. Increased p CO 2 at ambient temperature has been found to positively affect growth of some algal species [ 77 ], possibly by alleviating CO 2 limitation of photosynthesis, or downregulation of CCMs [ 25 ]. On the other hand, studies have shown negative growth responses of some coralline algal species to elevated p CO 2 at ambient temperature (e.g. P . cf. onkodes recruits/germlings [ 30 ] and Arthrocardia corymbose [ 78 ]). In our study, temperature played a role in decreasing growth, which has been previously observed in a temperate species of coralline algae [ 79 ]. However, in a study with a tropical CCA species, P . cf. onkodes , temperature had a positive effect on growth rate [ 29 ]. The differences in responses to the independent stressors of temperature and p CO 2 between P . cf. onkodes and Sporolithon could be attributed to a number of reasons including differences between the divergence times of these genera [ 47 , 80 ], their habitat preferences [ 81 ], or other unknown factors. The genus Sporolithon diverged during previous times of high temperature and CO 2 , possibly suggesting that if more CO 2 is present, Sporolithon germlings will positively respond as energy usage for CO 2 uptake is reduced and the germlings are able to utilise the additional CO 2 instead of relying on CCMs. Of course, increased growth would be highly dependent on temperature (and possibly nutrients as discussed earlier), but it is likely that this increased growth may be realised in habitats exposed to comparatively lower temperatures and light regimes, such as relatively deep-water reefs. Our study illustrates the considerable variability in germling responses to global change among CCA species (e.g. P . onkodes [ 29 , 30 ], H . reinboldii [ 35 ], P . lenormandii [ 33 ])."
} | 7,436 |
28512304 | PMC5434029 | pmc | 1,690 | {
"abstract": "Fabrication of superhydrophobic surfaces is an area of great interest because it can be applicable to various engineering fields. A simple, safe and inexpensive fabrication process is required to fabricate applicable superhydrophobic surfaces. In this study, we developed a facile fabrication method of nearly perfect superhydrophobic surfaces through plasma treatment with argon and oxygen gases. A polytetrafluoroethylene (PTFE) sheet was selected as a substrate material. We optimized the fabrication parameters to produce superhydrophobic surfaces of superior performance using the Taguchi method. The contact angle of the pristine PTFE surface is approximately 111.0° ± 2.4°, with a sliding angle of 12.3° ± 6.4°. After the plasma treatment, nano-sized spherical tips, which looked like crown-structures, were created. This PTFE sheet exhibits the maximum contact angle of 178.9°, with a sliding angle less than 1°. As a result, this superhydrophobic surface requires a small external force to detach water droplets dripped on the surface. The contact angle of the fabricated superhydrophobic surface is almost retained, even after performing an air-aging test for 80 days and a droplet impacting test for 6 h. This fabrication method can provide superb superhydrophobic surface using simple one-step plasma etching.",
"introduction": "Introduction The wettability of a solid surface is important in a wide range of academic science and engineering applications. As an extreme state of surface wettability, superhydrophobicity has received considerable attention because of its strong potential in various fields including self-cleaning 1 , 2 , anti-fogging 3 , 4 , anti-frosting 5 – 10 , anti-icing 11 – 13 , condensed microdrop self-removal 14 – 19 , water collection 20 , 21 and enhancing condensation heat transfer 22 – 25 . Various fabrication methods of superhydrophobic surfaces have been introduced in the past few years, including the growth and synthesis of nanostructures 26 , 27 , coating of nanoparticles or nanofilaments 28 , 29 , wet-etching with chemicals 30 – 33 , and dry-etching with SF 6 and CHF 3 gases 34 . Despite technical advances in fabrication methods of superhydrophobic surfaces, many of these methods require multi-step processes 29 , 30 and post-chemical treatments 27 , 35 . Thus, the development of a simple, cost-effective and environmentally friendly fabrication method of superhydrophobic surface is strongly and timely required. Plasma etching treatment has been proven as a simple method for modifying the surface properties. Surface structure is modified by the bombardment of excited ions, which are generated from the plasma, to the substrate 36 . This fabrication method can alter the wettability of substrate by increasing roughness 37 or changing functional group of the surface 38 . When the plasma was treated with only argon gas on a polytetrafluoroethylene (PTFE) substrate, the surface attained hydrophilic property due to formation of the peroxy radical bond to carbon as the cross-linked structure 39 . The hydrophobic surface could be obtained by applying reactive plasma generated from argon and oxygen mixture on the PTFE substrate 36 , 40 . The surface morphology was changed to have microstructures with various surface roughness. However, they did not mention the sliding behavior of water droplets on the fabricated surface. In addition, formation of microstructures was studied only against treatment time. Thus, a systematic investigation on the important parameters of plasma treatment is strongly required for better understanding about the relationship between treatment parameters and surface wettability. In this study, we propose a simple one-step plasma treatment method for fabricating superb superhydrophobic surface on a fluorocarbon-based polymer, which has a low surface energy of 20 mN m −1 at 20 °C. We optimize the fabrication parameters of superhydrophobic surfaces through plasma treatment with argon and oxygen gases by adopting the Taguchi method. The performance of the fabricated surfaces is examined by measuring their wetting properties, such as static contact angle, sliding angle, and self-cleaning effect. In addition, we demonstrate superior stability in the superhydrophobicity of the fabricated PTFE surface.",
"discussion": "Discussion In this study, nearly perfect superhydrophobic PTFE specimens with a high contact angle up to 178° and an extremely low sliding angle less than 1° were fabricated via a simple one-step plasma treatment using only argon and oxygen, both of which are inexpensive and safe reactive gases. The fabricated superhydrophobic surfaces exhibit excellent superhydrophobicity in the aging test; they almost maintain the initial wettability, even after prolonged exposure to air for 80 days, and exhibit good water repellency with maintaining a high contact angle, even after the repetitive water droplet impact experiments. This fabrication method can be equally applicable to a thin film of PTFE. We believe that the superhydrophobic surface fabricated via plasma etching method using safe gases can be a good candidate to develop tip-like polymer nanoarrays with special performances in a simple process."
} | 1,300 |
29090193 | PMC5650985 | pmc | 1,691 | {
"abstract": "Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP content compared with cells with higher c-di-GMP content. The induced expression of pqs and rhl QS required a functional PqsR, the transcriptional regulator of pqs QS. Next, we observed increased production of pqs and rhl -regulated virulence factors, such as pyocyanin and rhamnolipids, in P. aeruginosa cells with low c-di-GMP levels, conferring them with increased intracellular survival rates and cytotoxicity against murine macrophages. Hence, our data suggested that low intracellular c-di-GMP levels in bacteria could induce QS-regulated virulence, in particular rhamnolipids that cripple the cellular components of the innate immune system.",
"introduction": "Introduction Pseudomonas aeruginosa can cause opportunistic infections in humans, such as cystic fibrosis lung infections, burn wounds and urinary tract infections (Bodey et al., 1983 ). This is attributed to its ability to form biofilms and produce an abundance of virulence factors to impair the host immune response (Bjarnsholt et al., 2009 ; Fazli et al., 2011 ). Similar to many Gram-negative bacteria species, the biofilm and planktonic lifestyles in P. aeruginosa are controlled by the secondary messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP) (Romling et al., 2005 ). C-di-GMP is synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) (Hengge, 2009 ). High intracellular c-di-GMP content enhances biofilm formation, whereas low intracellular c-di-GMP content leads to biofilm dispersal and the return to planktonic phase (Hisert et al., 2005 ; Romling et al., 2005 ; Kulasakara et al., 2006 ; Chua et al., 2014 ; Yu et al., 2015 ). The redundancy of DGC and PDE genes in the genome confers P. aeruginosa the survival advantage to respond to various stresses from the environment. For instance, the wspR DGC is important in the sensing of reactive oxygen species (ROS) and formation of biofilms resilient to ROS stress (Chua et al., 2016a ). Another system that plays important roles in biofilm formation and virulence is quorum sensing (QS), which is the intercellular communication system positively dependent on cell density and QS autoinducer (AI) concentrations (Fuqua et al., 1994 ; Whitehead et al., 2001 ; Ng and Bassler, 2009 ). P. aeruginosa possesses four major QS systems, encoded by the las, rhl, pqs and iqs systems, with the las and rhl systems employing homoserine lactones, namely the N -(3-oxododecanoyl)-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively as their AIs (Gambello and Iglewski, 1991 ; Passador et al., 1993 ; Ochsner and Reiser, 1995 ; Pearson et al., 1995 ), while pqs and iqs sytems using the 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde respectively (Cao et al., 2001 ; Diggle et al., 2007 ; Lee et al., 2013 ). The AIs will bind and activate the transcriptional regulators, resulting in the transcription of downstream QS operons. The QS systems interregulate one another, notably the las system and pqs system activate the rhl system (Pesci et al., 1997 ; McKnight et al., 2000 ; Farrow et al., 2008 ). The QS systems control the production of many virulence factors, such as pyocyanin (by the pqs operon) and rhamnolipids (by the rhl operon) (Pearson et al., 1997 ; Xiao et al., 2006 ). The rhamnolipids are biosurfactants which are highly cytotoxic to eukaryotic cells, as previously demonstrated by the induction of rhl operon- controlled gene expression in biofilm bacteria exposed to polymorphonuclear leukocytes (PMNs) and subsequent destruction of these important defensive immune cells (Alhede et al., 2009 ). While the impact of high c-di-GMP content on biofilm formation is well understood, the consequences of low intracellular c-di-GMP content other than biofilm dispersal remain unclear. Our previous study showed that freshly dispersed cells, during the short-term liberation process, appeared to be highly virulent as compared to biofilm cells (Chua et al., 2014 ). It remains elusive whether reduced c-di-GMP content may have a long-term impact on bacterial physiology and virulence. Hence, we aimed to investigate the impact of low vs. high c-di-GMP concentrations on P. aeruginosa virulence mechanisms. We compared the transcriptomes of P. aeruginosa PAO1 cells “locked” in a condition with high c-di-GMP content (by using the wspF mutation to induce constitutive expression of WspR) and the cells “locked” in a condition with low c-di-GMP content (by over expressing the YhJH PDE) cultivated under the similar growth conditions. As the WspF protein is the inhibitor of the WspR DGC, the wspF mutation will cause expression of WspR, thereby promoting the synthesis of c-di-GMP leading to high internal levels (Hickman et al., 2005 ). The PAO1/p lac - yhjH strain contains the constitutively expressed PDE gene yhjH leading to low internal levels of c-di-GMP, a condition important in swarming and swimming motility (Pesavento et al., 2008 ; Chua et al., 2013 ). We found that low intracellular c-di-GMP content induced expression of the QS systems, specifically the rhl and pqs systems, which led to increased production of several virulence factors, such as rhamnolipids and pyocyanin. This was correlated to increased killing of macrophages. We showed that the induction of rhl and pqs QS under conditions of low c-di-GMP levels, was mediated by PqsR, the transcriptional regulator of pqs QS. Hence, our present study suggested that c-di-GMP-governed biofilm dispersal might liberate bacteria capable of producing virulence factors, so as to survive and protect themselves from the phagocytic immune cells in the host. Hence, as a strategy to prevent the dissemination of biofilm infections, the use of QS inhibitors (Hentzer et al., 2003 ) can potentially reduce the production of QS-related virulence factors.",
"discussion": "Discussion While most studies focused on the effects of high c-di-GMP levels on biofilm formation, there is a paucity of research on the physiology of cells undergoing conditions of low c-di-GMP signaling. Our previous study had shown that cells freshly dispersed from biofilms contained lower c-di-GMP levels than planktonic cells and biofilm cells (Chua et al., 2013 ), implying that dispersed cells possess a different physiology from biofilm and planktonic cells. This raised the question of how differing c-di-GMP levels impact the physiology in P. aeruginosa . As biofilm cells and dispersed cells had high physical and physiological heterogeneity (Stewart and Franklin, 2008 ), we used the Δ wspF and PAO1/p lac -yhjH mutants to imitate biofilm and dispersed cells respectively, and cultivated them as planktonic cultures which were easy to manipulate in controlled conditions. In this work, we compared the transcriptomics of cells with high and low c-di-GMP levels. Other than biofilm dispersal, we had shown using transcriptomics that low c-di-GMP levels could lead to the induction of the pqs and rhl QS, with PqsR acting as mediator to activate both QS systems. Although we do not show that conditions of low c-di-GMP mediate increased PqsR, a previous study had shown that RsmA from the c-di-GMP-mediated Gac/Rsm pathway, was important in pqs and rhl QS (Burrowes et al., 2006 ). Hence, the result of activating both QS systems was the increased production of pyocyanin and rhamnolipids, which were correlated to higher virulence to immune cells. Interesting, it was previously observed that rhamnolipids acted as surfactant to facilitate biofilm cells to disperse from biofilms (Bhattacharjee et al., 2016 ). Several research groups, such as ours (Chua et al., 2015 ; Yu et al., 2015 ) are currently investigating the possibility of exploiting the lowering of the c-di-GMP content in bacteria and dispersal as a biofilm control strategy. Our study had several implications for clinical and environmental applications of this biofilm dispersal strategy. Firstly, liberated bacterial cells could attain a unique physiological state if the c-di-GMP content is maintained at a lower level than planktonic cells and biofilm cells. This state could be reached after long-term growth of dispersed biofilm cells in the presence of agents that cause biofilm dispersal, thus warranting further studies on the biofilm-dispersed cells. Secondly, it appeared that a constitutively low c-di-GMP content renders the bacterial cells highly virulent, which might be essential for dispersed cells to survive the encounter with immune cells and cause development of sepsis. Hence, it is important to evaluate the potential virulence outcome which applying c-di-GMP mediated biofilm dispersal during the eradication of biofilms, especially in infections. The use of QS inhibitors (Hentzer et al., 2003 ) can effectively negate the induction of QS pathways and production of virulence factors, to be used concurrently with c-di-GMP-mediated biofilm dispersal."
} | 2,478 |
32097412 | PMC7041794 | pmc | 1,692 | {
"abstract": "The biomineralization protein Mms6 has been shown to be a major player in the formation of magnetic nanoparticles both within the magnetosomes of magnetotactic bacteria and as an additive in synthetic magnetite precipitation assays. Previous studies have highlighted the ferric iron binding capability of the protein and this activity is thought to be crucial to its mineralizing properties. To understand how this protein binds ferric ions we have prepared a series of single amino acid substitutions within the C-terminal binding region of Mms6 and have used a ferric binding assay to probe the binding site at the level of individual residues which has pinpointed the key residues of E44, E50 and R55 involved in Mms6 ferric binding. No aspartic residues bound ferric ions. A nanoplasmonic sensing experiment was used to investigate the unstable EER44, 50,55AAA triple mutant in comparison to native Mms6. This suggests a difference in interaction with iron ions between the two and potential changes to the surface precipitation of iron oxide when the pH is increased. All-atom simulations suggest that disruptive mutations do not fundamentally alter the conformational preferences of the ferric binding region. Instead, disruption of these residues appears to impede a sequence-specific motif in the C-terminus critical to ferric ion binding.",
"conclusion": "Conclusion In conclusion, the development of a convenient luminescence based binding assay has allowed for the rapid parallel screening of a range of amino acid substitutions of Mms6 for binding to ferric ions with a number of key residues now identified. Namely, the three mutants E44A, E50A and R55A showed significantly reduced ferric binding, revealing E44, E50 & R55 are important for ferric ion binding. This is the first time residue specific ferric binding sites have been identified in Mms6. The cumulative effect of all three significant mutations were explored in a surface-bound nanoplasmonic assay and showed distinct differences between the binding and precipitation of iron-oxide on the surface of each protein. Mms6MM showed reduced binding to iron ions and did not show a dramatic shift when the pH was raised when compared to native Mms6 on the surface that did. We suggest the reduced binding of Mms6MM is due to the inhibition of ferric ion binding and thus only ferrous ions are bound in this experiement. Ferrous ions will not precipitate to the oxide when the pH is raised to ultra pure water. In the Mms6 case where both ferric and ferrous ions can bind iron-oxide precipitation is then possible and we suggest is the responsible for the large signal shift. It is interesting to note the whole DEEVE region is implicated in ferrous binding with particular affinity for E51 [ 19 ], while E50 and E44 are involved in ferric binding, suggesting Mms6 C-terminal contains specific ferric and ferrous binding sites which are orthogonal to one another ( Fig 2D ) and supports the hypothesis that Mms6 is a specific magnetite nucleation protein, functioning to specifically bind ferric and ferrous ions in a specific conformation to favour magnetite formation [ 24 ]. All-atom simulations agree with extant sequence-based predictions and NMR results that the C-terminal acidic region is intrinsically disordered. Previous NMR results observed a minimal change in helicity upon metal ion binding, implying binding is not strongly coupled to a specific structural changes [ 19 ]. In agreement with this, simulations found that changes to the intrinsic helicity driven by mutations had no obvious relationship to the binding affinity as measured experimentally. The simulations and previous NMR address a similar question in entirely independent manners yet arrive at the same conclusion. These results imply that Mms6 binding is likely mediated through a structurally heterogeneous binding interface than can bind equally well through a number of different conformations. Our results provide an example of how intrinsically disordered regions can be used by bacteria to achieve biological function, further challenging the decaying assumption that bacteria lack biologically important disordered regions.",
"introduction": "Introduction Biomineralization is the process of forming inorganic minerals under biological control and encompasses the production of calcium carbonates, calcium phosphates, and silicates amongst others [ 1 – 4 ]. One example is magnetic nanoparticles (MNP) synthesised by magnetotactic bacteria [ 5 ]. This diverse range of aquatic bacteria share the capability to synthesise single crystals of the iron oxide magnetite inside dedicated organelles termed magnetosomes [ 6 – 8 ], Fig 1 . 10.1371/journal.pone.0228708.g001 Fig 1 (a) Transmission electron microscopy image of Magnetospirillum magneticum AMB-1, with schematic of the magnetosome shown. (b) Sequence of Mms6 alongside residue numbering used in this paper. The hydrophobic part in green, Gly-Leu repeat motif underlined and the acidic C-terminal region in yellow. The magnetosome comprises a lipid bilayer vesicle that surround the MNP as shown in Fig 1 , and harbours a large number of specialised proteins. These function to load the vesicle with soluble iron ions, to nucleate the growth of the crystal and ensure adequate maturation of the particle to produce not only the appropriate iron oxide, magnetite, but also with a species specific size and morphology [ 8 – 10 ]. Four magnetosome membrane specific (Mms) proteins were identified as being tightly associated with the magnetite nanoparticles in Magnetospirillum magneticum AMB-1 [ 11 ]. Designated Mms5, Mms6, Mms7, and Mms13 these proteins, due to their close interaction with the nanoparticles, were considered likely candidates for controlling particle formation in vivo . An Mms6 deletion mutant displays an irregularly sized and misshapen nanoparticle phenotype [ 12 ], indicating a key role in the formation of the nanoparticles, although other deletions using different methods show less effect, demonstrating it is likely Mms6 works in concert with other Mms proteins in vivo [ 13 ]. Proteins such as MmsF have recently been shown to also have a significant role in this process and are closely located in the Mms6 gene cluster [ 13 , 14 ]. The addition of purified Mms6 to simple synthetic magnetite precipitation reactions improves the homogeneity of the resulting nanoparticles [ 11 , 15 , 16 ]; a prerequisite for use in biomedicine [ 17 ]. Mms6 is a small membrane interacting protein with a low complexity hydrophobic N -terminal region, a predicted central transmembrane helix, and a hydrophilic, acid rich C-terminal region, predicted to present on the interior of the magnetosome lumen, so will be exposed to the forming magnetite [ 11 ]. Unlike the majority of bacterial proteins, with the exception of the transmembrane helix, the remainder of the protein is predicted to be intrinsically disordered, meaning it does not adopt a stable three-dimensional structure, but instead exists in an ensemble of different conformations [ 18 ] (S1). This prediction is supported by previous NMR experiments that showed random-coil chemical shifts in the C-terminal region, but there is also some evidence for the formation of transient helicity, a common structural feature utilized by intrinsically disordered regions when binding [ 10 , 19 ]. Although predicted to be an integral membrane protein, it is possible to purify overexpressed Mms6 from E . coli using protein refolding strategies [ 11 , 15 ] which result in soluble Mms6 oligomers. Previous studies show this amphiphilic protein self-assembles into 10 nm sized micelles in aqueous solution [ 20 , 21 ]. With a hydrophobic core, it displays the hydrophilic C-terminus on the micellar surface and demonstrates the ability to bind ferric [ 11 , 20 , 21 ] and ferrous [ 19 ] ions with high affinity, therefore implicating the C-terminal acid rich region as the site of iron binding [ 19 – 24 ]. This activity, coupled with the self-assembly (also thought to aggregate within the native magnetosome membrane [ 25 ]) may act to display a negatively charged iron binding surface to produce a locally high iron concentration that is predicted to be the magnetite nucleation site. This process has been observed during in situ transmission electron microscopy analysis of Mms6 iron complexes [ 23 ]. Likely due to a combination of the protein’s intrinsically disordered nature coupled with the presence of a transmembrane helix, crystallisation has not been possible to provide full structural characterisation. Recently, NMR was used to obtain amino acid specific structural information about the acidic C-terminal region when bound to various metal ions (ferric, ferrous, calcium and zinc) [ 19 ]. A significant structural change was produced in all four acidic residues of the DEEVE (residues 49–53) motif when this peptide was introduced to ferrous ions, with computational modelling highlighting interactions with the E51 and the carbonyl backbone between E50/E51, but surprisingly, no significant conformational change was seen on binding ferric ions [ 19 ]. In vivo studies of mutations within the C-terminal region of Mms6 have also implicated this DEEVE motif as important for morphology control of the resulting magnetosome particles [ 26 ]. Therefore, while there is now some insight into the binding site of ferrous ions, the precise residues involved in ferric binding have not yet been determined.",
"discussion": "Results and discussion Ferric iron binding investigation of Mms6 and mutants A range of soluble SUMO-tagged Mms6 mutants were designed to probe the contribution of specific residues to the overall ferric binding activity of the protein. Amino acids with acidic side chains (glutamic and aspartic acid) were targeted as these will typically be negatively charged at neutral to basic pH, which is required for magnetite formation and stability, and would therefore be able to coordinate positively charged iron ions. Other commonly associated iron binding residues such as histidine, cysteine, and tyrosine are not present within the acidic region of Mms6. Aspartic acids (D) at positions 42, 49 and 56, and Glutamic acids (E) at positions 44, 50, 51 and 53 were all individually substituted to alanine giving mutants designated ‘D42A’ for instance. Additionally, a double mutant EE50AA was created by mutating two adjacent glutamic acids to alanine residues. The arginine residue located at position 55 is conspicuous as being a lone residue of the opposing charge state within this highly acidic region and its role was also investigated by substitution to alanine. In order to probe how Mms6 iron binding was affected by the introduced substitutions we used a radiolabel-free iron binding assay based on a system developed by Hogbom et al [ 28 ] ( Fig 2A and 2B ), which uses Fenton chemistry and luminol to generate a luminescent signal, demonstrating an effective quick screening methodology for our range of Mms6 mutants. GFP was also produced to act as a negative control protein which does not interact with iron ions but has a poly-histidine tag as Mms6. 10.1371/journal.pone.0228708.g002 Fig 2 (a) Overall assay scheme. (I) De-metallized Mms6 is incubated with excess ferric citrate (II), before the excess is removed by a desalting procedure (III). (b) The iron bound protein from (III) is mixed with luminol, hydrogen peroxide, urea and hydroxide which releases iron from the protein and causes subsequent reaction of the luminol and emission of light. (c) Averaged luminescence intensity obtained from three replicates of each protein sample with error bars indicated (standard error of mean). Statistically significant values are identified by an asterisk using 1 way Anova (p = 0.05). (d) C-terminal sequence of Mms6 with the sites that are critical to ferric binding highlighted in red, and the sites found critical in ferrous binding in green (from a previous study [ 19 ]). Mms6 and the Mms6 variants were expressed in E . coli as a fusion to an N-terminal His6-SUMO tag; the His6 tag to enable purification by nickel affinity chromatography, and the SUMO to maintain protein solubility. It should be noted that these tags are likely to affect/inhibit Mms6 self-assembly. Size exclusion chromatography analysis of SUMO-Mms6 reveals two species. The major species has a retention volume equivalent to monomeric SUMO-Mms6, and the minor species elutes at a volume consistent with dimeric SUMO-Mms6. Similar results are observed in SDS-PAGE (S4, S5). The proteins were de-metallized to reduce background luminescence by dialysis against EDTA then Chelex treated ultrapure water. Protein concentrations were normalized based on their absorbance at 280 nm. Each protein was incubated with ferric citrate before being rapidly desalted (Zeba desalting spin columns, Thermo-Pierce) to remove unbound metal ions. Proteins were then denatured to release bound metal ions and luminol based detection reagent applied to each sample before measuring the luminescence intensity. As expected, the GFP negative control gave the lowest luminescent signal indicating that the desalting procedure sufficiently removed most unbound ferric ions from the protein incubation step, generating a negligible background luminescence. Mms6 gave a significantly higher value indicating that ferric ions bound to and were retained by the protein ( Fig 2C ). To sequester iron from the citrate complex Mms6 must have a higher binding affinity than citrate, which is consistent with previous Mms6 studies [ 20 ]. Using this assay several of the Mms6 variants gave luminescent intensities close to that of wild type Mms6, suggesting that the substitution of those particular residues to alanine did not significantly impair the iron binding capability on their own. These residues include glutamate 51 and 53, and all the aspartate residues in the C-terminus: 42, 49 and 56 ( Fig 2D ). Four of the variants E44A, E50A, EE50AA and R55A gave a luminescence intensity below wild type Mms6, indicating impaired ferric ion binding. However, neighbouring E51 does not appear to significantly affect ferric binding in this study either alone or when combined in the double substitution, suggesting the effect of the double mutant is solely due to the effect of E50A. Our studies also implicate R55 in ferric binding. Although unlikely to interact directly with the ferric ions, we speculate that as one of the few basic residues in this region of the protein it may play a structural role by electrostatically mediating the geometry of the binding site, or in the coordinating of a multimeric Mms6 binding complex. Nanoplasmonic surface investigation of Mms6 triple mutant Overall, the luminol screening assay has shown E44, E50 and R55 to be amino acid sites implicated in ferric binding. We produced an Mms6 triple mutant comprising alanine substitutions of the three key residues identified EER44,50,55AAA Mms6 (designated Mms6MM for Mms6 multi-mutant) to investigate the cumulative effect of all three mutations. It should also be noted that the deletion of the more basic arginine with the two glutamic acids, goes some way to ensuring the overall charge of this variant is only + 1 different, so the same as the other acidic single mutants. Unfortunately, the aqueous stability of this variant was extremely low, making purification challenging, and characterisation within the ferric iron binding assay impossible. However, we were able to use a surface based nanoplasmonic sensing experiment (using the Xnano (Insplorion) [ 32 ]) to probe Mms6MM and compare it to native Mms6 in terms of their interaction with iron ions and their ability to promote/ impede the precipitation of iron-oxide in vitro . Previous research has demonstrated how Mms6 is able to promote the formation of magnetite when assembled on a gold surface as this surface assembly better mimics the native environment on the membrane within the magnetosome [ 25 , 33 , 34 ]. The absence of the SUMO tag thus ensures any self-assembly is not restricted. Using bare gold sensors allowed us to immobilise Mms6 and Mms6MM via a single cysteine residue incorporated in the N-terminal tag (as we have shown previously [ 25 , 33 , 34 ]). The change in wavelength of the plasmon peak of the gold sensor was monitored whilst immobilising the protein, applying a mixed valence iron solution, and washing with ultrapure water ( Fig 3 ). Shifts in wavelength can be interpreted as changes in the species bound to the sensor [ 32 ]. It appears that both proteins attached in a similar manner to the gold surface, as evidenced by comparable shifts in the plasmon peak position showing similar levels of surface coverage to near saturation after approximately 5 minutes ( Fig 3A ). In the case of the protein free experiment, buffer alone was applied to the sensor in place of the protein. Any excess protein in the sensor chamber was removed by application of ultrapure water to the sensor surface before application of an iron solution. This resulted in a small shift in wavelength for both protein experiments compared to the protein free sensor. We interpret this as either an interaction between the iron ions and the protein, a rearrangement of the protein structure upon application of the iron, or a combination of the two factors. This latter interpretation is consistent with previous reports of Mms6 iron binding and structural rearrangement [ 19 – 24 ]. It is noteworthy that the signal for Mms6MM decays over time. This suggests that the strength of binding to iron ions in Mms6MM is weaker than the native Mms6 interaction, and points to differences in behavior between the two proteins under the same conditions. It is predicted the binding in Mms6MM is of ferrous ions only (due to ferric binding inhibition), or weaker unspecific general binding ( Fig 3B ). 10.1371/journal.pone.0228708.g003 Fig 3 Nanoplasmonic sensing of: a) protein adsorption, b) iron binding, and c) iron oxide precipitation. Red line is Mms6, blue dotted line is the Mms6MM, and the black line is the protein free experiment. Each panel depicts the transition between two solutions in the experiment, with time zero being the transition point in each case The change in the wavelength of the plasmon peak for each experiment is shown. Following application of the iron ions, the system was returned to ultrapure water. In the Mms6 experiment this resulted in a large shift in wavelength, much more significant than that observed for application of either protein or iron ions. A similar response was observed in the protein free condition. Such a large shift in the position of the plasmon peak indicates a highly significant change in the environment close to the sensor surface. A potential explanation is precipitation of iron oxide due to the increase of pH from the acidic iron solution to the pH of neutral ultrapure water. Ferric iron oxides can precipitate at pH ≤ 6 whereas, ferrous iron is more soluble with ferrous iron oxides only precipitating at pH > 6 [ 19 , 35 ]. Interestingly, the shift was not observed in the Mms6MM experiment ( Fig 3C ). These results show Mms6 and Mms6MM coated sensors behave differently when the pH of the iron solution is increased to return to ultrapure water, whereby Mms6MM appears to inhibit the significant environmental change observed in the Mms6 and protein free experiments. At this pH ferric ions should precipitate as the oxide but ferrous ion should stay soluble. If, as suggested by the iron binding assay Mms6MM ferric iron binding is inhibited, then this would block the formation of an iron oxide precipitate on the surface of the protein on the sensor at this pH. However, we cannot exclude the possibility that the substitution of the three residues in Mms6MM has altered the protein structure, leading to a conformationally inactive form of Mms6 which may just block the sensor surface. Monte Carlo simulation of Mms6 and mutants The mutations that reduce ferric binding may exert their influence through two non-mutually exclusive mechanisms: they may disrupt specific amino acids that engage in direct interaction with the ferric ion (disrupting inter- molecular interactions), or they may lead to significant changes in the underlying structure and conformational preferences of the C-terminal region (disrupting intra -molecular interactions) indirectly rendering this region less binding competent. To differentiate between these two scenarios, we performed extensive all-atom simulations of the C-terminal 21 residues for the WT sequence and all mutants. Given this region is predicted to be intrinsically disordered (S1), simulations were performed with the ABSINTH implicit solvent paradigm and CAMPARI Monte Carlo simulation engine [ 30 ]. The combination of ABSINTH and CAMPARI has been used extensively to characterize atomistic ensembles of intrinsically disordered regions and proteins [ 36 – 39 ]. All simulations led to expanded and conformationally heterogeneous ensembles, consistent with this region being intrinsically disordered (S1, S7, S8 and movie SM1). In agreement with previous predictions, simulations of all the constructs consistently identified transient helicity (10–15%) in the C-terminal region of the peptide ( Fig 4B ). Bioinformatic predictions using MoRFchibi SYSTEM identified two putative molecular recognition features (MORFs) in this region ( Fig 4A ) hinting at possible binding sites [ 40 ]. With these predictions in mind, it might be expected that mutations inside these MORFs would have a greater impact on the conformational behaviour of the peptide than mutations in surrounding residues. To assess this, we quantified the global and local conformational preference for each construct compared with wildtype, as well as the conformational heterogeneity for each ensemble ( Fig 4 , see methods for details). We also considered if mutations that significantly attenuated iron binding (E44A, E50A, R55A, or the triple mutant) seemed more similar to one another in terms of their impact on the conformational behaviour when compared to those that had no effect. 10.1371/journal.pone.0228708.g004 Fig 4 All-atom simulations of C-terminal residues 38–59. a) Bioinformatics analysis of the primary sequence identified two putative molecular recognition features (MORFs) in the C-terminal region. b) All mutations except E53A and D56A had a minimal impact on residual helicity. The mutation position is shown as a red circle, darker shades represent enhanced helicity. c) Local conformational differences are calculated as the deviation from wildtype in terms of all pairwise intramolecular distances (see also S2 Fig ). The mutants that show the smallest (E50A) and largest (triple) deviation from WT both significantly reduce iron binding, suggesting local interactions are not a useful metric for assessing the determinants of iron binding. d) Global conformational preferences are calculated in terms of the deviation from ensemble average shape (asphericity) and size (radius of gyration) 2D distributions (see also S3 Fig ). Global conformational behaviour is again not predicting of iron binding with no discernible correlations identified. e) Global heterogeneity quantifies the extent of conformational disorder in terms of the distribution of structurally dissimilar conformations [ 49 ]. Larger values indicate higher heterogeneity. There is no correlation between heterogeneity and iron binding ability. Irrespective of the impact on ferric binding, all the mutations examined led to relatively minor and similar changes in the conformational properties of the ensemble (Figs 4B–4E and S7 and S8 ). More importantly, we did not identify any trends between the extent of global or local conformational change and the impact on iron binding. For example, the mutants with the smallest and greatest deviation from wildtype conformational behaviour ( Fig 4C , see also S8 Fig ) were E50A and the triple mutant, respectively, both of which also reduced ferric binding. This suggests that across the mutants tested, the intrinsic conformational ensembles are not predictive of the ferric binding capacity of a given mutant. Given the challenges associated with parameterizing fixed-charge models for metal ions and the absence of current support in the ABSINTH implicit solvent model we did not pursue simulations that included ferric ions. These results support a model in which the C-terminal region of Mms6 acts as a conformationally malleable binding interface, whereby specific residues engage in direct interactions with iron. Considering many disordered regions undergo helix formation upon binding to a cognate partner [ 41 , 42 ] we were surprised to notice that changes to intrinsic helicity appeared to have a minimal impact on binding. For example, the neutral mutation D56A led to a substantial enhancement in C-terminal helicity, while another neutral mutation (E53A) extended the helix towards the N-terminus of the peptide. Given prior NMR results did not detect any increase in helicity upon binding to distinct metals, these results agree with a model in which binding is likely mediated by binding interface that lacks any strong structural biases, in contrast to the folding-upon-binding mechanism often associated with disordered regions [ 43 ]. These results are reminiscent of examples in which highly charged intrinsically disordered regions bind without the acquisition of structure in polyelectrolyte complexes, although the mechanism invoked here may be better described in terms of counterion condensation, as has been explored extensively in the context of nucleic-acid:cation interactions [ 44 – 46 ]. Despite this, apparent net charge appears to be a poor predictor of the impact (or lack thereof) of mutations. One explanation for this may reflect the fact that the local electrostatic environment may shift the pKa values of negatively charged residues such that, even at a pH of 7.4 in the absence of ferric iron, some of the ostensibly charged residues may be neutralized due to upshifted pKa values [ 47 , 48 ]."
} | 6,547 |
33042731 | null | s2 | 1,693 | {
"abstract": "Due to their unique functionality, superomniphobic surfaces that display extreme repellency toward virtually any liquid, have a wide range of potential applications. However, to date, the mechanical durability of superomniphobic surfaces remains a major obstacle that prevents their practical deployment. In this work, a two-layer design strategy was developed to fabricate superomniphobic surfaces with improved durability via synergistic effect of interconnected hierarchical porous texture and micro/nano-mechanical interlocking. The improved mechanical robustness of these surfaces was assessed through water shear test, ultrasonic washing test, blade scratching test, and Taber abrasion test."
} | 174 |
35631870 | PMC9144647 | pmc | 1,694 | {
"abstract": "Alongiside the growing demand for wearable and implantable electronics, the development of flexible thermoelectric (FTE) materials holds great promise and has recently become a highly necessitated and efficient method for converting heat to electricity. Conductive polymers were widely used in previous research; however, n -type polymers suffer from instability compared to the p -type polymers, which results in a deficiency in the n -type TE leg for FTE devices. The development of the n -type FTE is still at a relatively early stage with limited applicable materials, insufficient conversion efficiency, and issues such as an undesirably high cost or toxic element consumption. In this work, as a prototype, a flexible n -type rare-earth free skutterudite (CoSb 3 )/poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT:PSS) binary thermoelectric film was fabricated based on ball-milled skutterudite via a facile top-down method, which is promising to be widely applicable to the hybridization of conventional bulk TE materials. The polymers bridge the separated thermoelectric particles and provide a conducting pathway for carriers, leading to an enhancement in electrical conductivity and a competitive Seebeck coefficient. The current work proposes a rational design towards FTE devices and provides a perspective for the exploration of conventional thermoelectric materials for wearable electronics.",
"conclusion": "4. Conclusions In this study, a CoSb 3-x Te x /PEDOT:PSS hybrid film was produced using a simple method. First, we succeeded in our preliminary step to obtain a submicron to several micron particle size suitable for hybridization with the organic counterpart. Then, by suction-filtering the mixed solution with the PEDOT:PSS aqueous dispersion to an MCE membrane, we achieved the production of a uniform and flexible n -type FTE film. As for the TE performance, the Seebeck coefficient of the hybrid film increased as the amount of CoSb 3-x Te x in the hybrid film increased and the doping rate of CoSb 3-x Te x decreased. As a result, the CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film obtained the largest negative Seebeck coefficient of −161.7 µV/K at 98 wt% at room temperature. As it is more challenging to obtain a large negative Seebeck coefficient in the hybrid thin films than improving the electrical conductivity of these type of films, wherein few approaches have been attempted, our work established a useful method of improving the power factor by raising the Seebeck coefficient. In addition, the 98 wt% hybrid film retained the flexibility to maintain a certain degree of electrical conductivity even after being bent 1000 times. It was proved that it is relatively easy to fabricate a flexible film with a very small amount of PEDOT:PSS and obtain moderate properties by finely tuning the composition of the TE films. The same method in this study can be applied to other inorganic materials. Therefore, it is expected that the selection of inorganic TE materials used for the development of n -type flexible thermoelectric materials will be expanded in the future.",
"introduction": "1. Introduction Out of the primary energy sources such as feedstocks, oil, natural gas, etc., there has been an increasing demand for the development of renewable and sustainable sources of energy [ 1 , 2 , 3 ]. Heat conversion via thermoelectric (TE) devices represents a promising avenue for generating electricity and clean energy in a renewable and sustainable way for future energy development [ 4 , 5 , 6 ]. Various ongoing efforts in experiments and theories have been attempting to improve the TE properties and conversion efficiency of relevant materials [ 7 , 8 , 9 , 10 ]. The efficiency of TE materials is described by a dimensionless figure of merit ZT = S 2 σTκ −1 , where S represents the Seebeck coefficient, σ represents electrical conductivity, κ represents thermal conductivity, and T represents absolute temperature. To improve the figure of merit, the majority of recent research is focused on two aspects: the enhancement of the power factor ( S 2 σ ) [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ] and the reduction in thermal conductivity κ [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. In recent years, a variety of novel flexible electronic devices, ranging from wearable smart electronics to printable circuit boards, have steadily been developed in line with the concept of the Internet of things (IoT) society [ 28 , 29 ]. The growing demand for wearable and implantable electronics and sensors that use body heat advances the development of flexible thermoelectric (FTE) devices [ 30 , 31 , 32 , 33 , 34 , 35 ]. Polymers are one of the promising candidates for FTE conversion materials. Most of the advances achieved in FTE materials so far have been focused on conductive polymer-based TE materials. Typical conductive polymers including Polyaniline (PANI) [ 36 ], poly(3,4-ethylenedioxythiophene) (PEDOT) [ 37 ], Polypyrrole (PPy) [ 38 ], etc., show p -type TE performance. Benzodifurandione-based polyphenylene vinylene (BDPPV) [ 39 ] and poly(nickel-1,1,2,2-ethenetetrathiolate) (poly(Kx(Ni-ett)) [ 40 ] as conductive polymers exhibit n -type TE performance. However, the n -type conductive polymers have a lower TE performance and stability in air compared to p -type polymers due to their unstable dopants [ 33 ]. Therefore, p -type polymers are still the mainstream in current FTE development. Among the various polymers, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) stands out in particular because of its high electrical conductivity of up to 4839 S/cm [ 41 ], and the possibility of further increasing its TE property via post-treatment with acids or reducing agents [ 42 , 43 ]. Despite its high electrical conductivity and flexibility to be easily tailored into various shapes, the Seebeck coefficient of PEDOT:PSS has remained limited with a value lying in the 10.35–67 µV/K range [ 41 , 44 ], which is far from satisfactory when compared to conventional inorganic TE materials. To overcome the rigidity of the conventional inorganic TE materials and the low performance of polymers, the polymers and inorganic TE materials have been hybridized into an assembly. Various methods have been conducted to build a hybrid composed of inorganic TE materials and polymers. For example, polyethyleneimine (PEI) has been used as a dopant in carbon nanotube yarn (CNTY) by donating electrons to fabricate an n -type FTE film [ 45 ]. Furthermore, depositing the specific n -type inorganic TE materials on flexible substrates such as nylon and/or PI substrates by suction filtration [ 46 ] or magnetron sputtering [ 47 ] could also effectively provide flexibility comparable to that of organic materials. However, TE materials that exhibit high performance usually contain relatively expensive and rare elements such as Ag and Te, or toxic elements such as Se. In addition, to produce uniform films, most inorganic materials are synthesized into nanoparticles using chemical processing, but the number of inorganic materials that can be obtained using the same method is limited. Recently, the hybridized FTE films between the ball-milled chalcopyrite (Cu x Zn 1-x FeS 2 ) and PEDOT:PSS on the polytetrafluoroethylene (PTFE) membrane have been reported and exhibit outstanding flexibility and performance [ 48 ]. This versatile and promising approach to obtaining FTE film could be extended to various inorganic TE materials that present natively attractive TE performance. Skutterudite, with a general formula of CoSb 3 , is another family of promising TE candidates which shows high TE performance in the medium to high-temperature range due to its high electrical conductivity [ 49 ]. By doping CoSb 3 with Te to form a ternary solid solution alloy, Te provides electrons as a donor, and the electrical conductivity increases rapidly due to the increase in the carrier concentration [ 50 ]. Since CoSb 3-x Te x has been mainly studied as a bulk TE material, the study of CoSb 3-x Te x as an FTE film will provide new insights and is expected to broaden the selection of inorganic materials used in the development of FTE materials in the future. In this study, an n -type flexible CoSb 3-x Te x /PEDOT:PSS TE film was produced using a facile method. CoSb 3-x Te x ingots were prepared using a top-down method by breaking down the size of the bulk counterpart via ball-milling that does not require chemical treatment, and the powder was mechanically mixed with PEDOT:PSS, followed by suction filtering onto a membrane. The TE properties (electric conductivity, Seebeck coefficient, power factor, etc.) with different compositions and Te doping of the hybrid films were evaluated and discussed, which demonstrated the potential for CoSb 3-x Te x to be developed into an FTE component.",
"discussion": "3. Results and Discussion Figure S1a shows the morphology of the representative as-synthesized CoSb 2.95 Te 0.05 powder with the corresponding EDX spectrum presenting the elemental Co, Sb, and Te distribution in Figure S1b . The corresponding XRD patterns of the as-synthesized CoSb 3-x Te x powders with different amounts of Te dopants are shown in Figure S2 . The ball-milled powder is consistent with the CoSb 3 composition, which confirms the purity of the CoSb 2.95 Te 0.05 prepared in this study. All the patterns can be indexed to the skutterudite structure (JCPDS no.01-083-0055) with a space group of Im -3 (No.204) . The Rietveld refinement results are summarized in Table S1 and exhibit low-reliability factors demonstrating evidence of how the CoSb 3 structure purity is exempt from structural defects. Moreover, the lattice size varied linearly with the Te content attesting to the successful substitution of the Sb by the Te within the structure. Figure 1 shows the morphology and microstructural information of the CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film. According to the digital photo image of the 98 wt% CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film shown in Figure 1 a, the color of the hybrid film is black, similar to the color of CoSb 2.95 Te 0.05 powder. As displayed in Figure 1 b, the hybrid film retains its flexibility without obvious cracks in the bent state even though the content of the CoSb 2.95 Te 0.05 powder is very high. The morphology of the hybrid CoSb 2.95 Te 0.05 /PEDOT:PSS films with different fractions of PEDOT:PSS can be found in the SEM images in Figure 1 c–i, from which it is noteworthy that the PEDOT:PSS significantly affects the homogeneity of the hybrid film. The contours of the CoSb 2.95 Te 0.05 particles become more obvious with the increase in the weight percentage of the inorganic components. From the top view image of the 98 wt% CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film shown in Figure 1 i, CoSb 2.95 Te 0.05 particles can be observed with sizes ranging from submicron to several microns spread on the surface of the hybrid film. In the EDX mapping ( Figure 1 j) corresponding to the SEM image shown in Figure 1 i, the uniform distribution of the elements Co, Sb, and Te, which constitute CoSb 2.95 Te 0.05 , and the C, O, and S contained in PEDOT:PSS reveal how the TE materials and PEDOT:PSS are evenly dispersed within each other. The cross-sectional image in Figure 1 k indicates that the typical film in our experiment has a thickness of 53.0 ± 7 μm. According to the TGA results shown in Figure 2 a, even in the hybrid film with 98 wt% CoSb 2.95 Te 0.05 where the ratio of the PEDOT:PSS is extremely small, it is evident that a mass loss occurrs corresponding to the decomposition of the PEDOT:PSS when increasing the measurement temperature, confirming the successful assembly of the PEDOT:PSS and the CoSb 2.95 Te 0.05 powders . Figure 2 b shows the FT-IR spectra of the CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid films. It is known that PEDOT:PSS has several distinct absorption bands between 800 cm −1 and 1600 cm −1 [ 53 , 54 ], and a similar spectrum was observed for the PEDOT:PSS used in this study. As the amount of CoSb 2.95 Te 0.05 increases, the signature peaks of PEDOT:PSS are weakened, but the peak position of C-S at wavenumber 977 cm −1 and S-O at wavenumber 1141 cm −1 are not shifted, indicating that the structure of the PEDOT:PSS has not changed. The properties of the hybrid films in Figure 3 containing a lower content (<95 wt%) of CoSb 2.95 Te 0.05 show much lower TE properties than the hybrid films containing a CoSb 2.95 Te 0.05 content above 95 wt%. Our experiments in the later section are mainly focused on the hybrid films with the CoSb 3-x Te x weight fraction over 95 wt%. The XRD patterns of the hybrid films with 95 wt%, 97 wt%, and 98 wt% of CoSb 2.95 Te 0.05 are shown here in Figure 2 c, which are very consistent with the XRD patterns of the original powders in Figure S2 . The structure of CoSb 2.95 Te 0.05 did not change, indicating that no mutual interference on the microstructure occurred between the CoSb 2.95 Te 0.05 and the PEDOT:PSS during the hybridization of the two materials. The lattice sizes of all the CoSb 2.95 Te 0.05 films are comparable to the reference powder, attesting to the non-degradation of the powder during the hybridization with PEDOT:PSS. The same trend was also observed when the doping concentration of CoSb 3-x Te x in the hybrid film was varied ( Figure 2 d). The TE properties of the hybrid films were first investigated containing various weight fractions of CoSb 2.95 Te 0.05 . As shown in Figure 3 , there was no significant difference in the Seebeck coefficient of the hybrid films with CoSb 3-x Te x percentages ranging from 0 wt% to 90 wt% except for a small fluctuation at 80 wt%. The positive signs of the Seebeck coefficient of the CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film showed p -type features until the content of CoSb 2.95 Te 0.05 increased up to 90 wt%, attesting to the dominant properties of the PEDOT:PSS. The hybrid film started to show the n -type features with the CoSb 2.95 Te 0.05 weight fraction more than 95 wt%. The Seebeck coefficient of the hybrid film is a compromise of the n -type property of CoSb 3-x Te x and the p -type property of PEDOT:PSS. Thus, with an excessive amount of PEDOT:PSS, the dominant charge carriers in the hybrid film are holes. In order to fabricate the n -type film, it is necessary to increase the fraction of skutterudite to above 95 wt%. The electrical conductivity decreases gradually while increasing the amount of CoSb 3-x Te x powder. In comparison, the CoSb 2.95 Te 0.05 film was also fabricated using the same method for the hybrid film without the addition of PEDOT:PSS, which only showed 0.031 S/cm as displayed in Table S2 . The very low electrical conductivity can be attributed to the skutterudite particles aggregating in a random way to form a loose structure on the film where there is no effective bonding between the CoSb 2.95 Te 0.05 particles ( Figure S3 ). While improving the amount of PEDOT:PSS to bridge the particles, the electrical conductivity shows a drastic upward trend. According to the previous method [ 55 ], we analyzed the composite method between CoSb 2.95 Te 0.05 and PEDOT:PSS. As shown in Figure 3 a, the red line is fitted in a parallel-connected model with S and σ as in the follow the Equation: (1) S p a r a l l e l = x s σ s S s + ( 1 − x s ) σ p S p x s σ s + ( 1 − x s ) σ p \n (2) σ p a r a l l e l = x s σ s + ( 1 − x s ) σ p \nwhere S parallel and σ parallel are the Seebeck coefficient and the electrical conductivity of the parallel-connected composite; S s and S p are the Seebeck coefficients of CoSb 2.95 Te 0.05 and PEDOT:PSS, respectively; σ s and σ p are the electrical conductivities of CoSb 2.95 Te 0.05 and PEDOT:PSS, respectively; and x s is the volume fraction of CoSb 2.95 Te 0.05 . The blue line is fitted in a series-connected model with S and σ as in the following equation: (3) S s e r i e s = x s κ p S s + ( 1 − x s ) κ s S p x s κ p + ( 1 − x s ) κ s \n (4) σ s e r i e s − 1 = x s σ s − 1 + ( 1 − x s ) σ p − 1 \nwhere S series and σ series are the Seebeck coefficient and the electrical conductivity of the series-connected composite, and κ s and κ p are the thermal conductivities of CoSb 2.95 Te 0.05 and PEDOT:PSS, respectively. The experimental values of the Seebeck coefficient and electrical conductivity show properties closer to those of the series-connected model. To optimize the CoSb 3 -based FTE film, the doping level of the native inorganic powder was modulated to promote a larger negative Seebeck coefficient. Different doping ratios of CoSb 3-x Te x (x = 0.05, 0.10, 0.15) and of skutterudite hybridized with different mass ratios of X CoSb 3-x Te x /1-X PEDOT:PSS (X = 95, 97, 98 wt%) have been developed, and the influence on the FTE properties was investigated as shown in Figure 4 . With the same fraction of skutterudite, the Seebeck coefficient of the hybrid film increases with a decreasing CoSb 3-x Te x doping ratio and obtained a largest negative value of −161.7 μV/K at 98 wt%, considering an uncertainty of 6% from the measurement [ 56 ]. This trend is also observed in the CoSb 3-x Te x bulk sample, where the Seebeck coefficient increases with a decreasing doping rate due to the charge carrier tuning ( Table S3 ). In other words, a larger Seebeck coefficient in the native powder likely helps to reach a higher Seebeck coefficient in the hybrid FTE film. The contribution of CoSb 3-x Te x to the Seebeck coefficient is dominant in the n -type film with a high CoSb 3-x Te x content. However, there is only a small variation in the electrical conductivity of the hybrid film when decreasing the CoSb 3-x Te x doping ratio, and the electrical conductivity generates a different trend with the variation in the Te doping ratio in the CoSb 3-x Te x . The skutterudite grains of micrometer size synthesized under high vacuum are very stable with a negligible effect from the surface oxidation; this is thought to be due to the electrical conduction of the hybrid film being modulated by the interaction of the PEDOT:PSS and the bulk CoSb 3-x Te x particles. The electrical conductivity at various doping levels of Te showed a decreasing trend when the weight fraction increased from 90 wt% to 97 wt%. However, at 98 wt%, the electrical conductivity slightly increased compared to the 97 wt%, which might be due to the slightly larger compacity of CoSb 3-x Te x powders within the film. In addition, we compared the electrical conductivity with different doping levels at the same weight fraction. The CoSb 2.85 Te 0.15 /PEDOT:PSS film shows a much higher electrical conductivity than the CoSb 2.90 Te 0.10 /PEDOT:PSS and CoSb 2.95 Te 0.05 /PEDOT:PSS at 95 wt%, but it becomes the lowest at 98 wt%. At the same time, CoSb 2.95 Te 0.05 /PEDOT:PSS becomes the best hybrid film when improving the weight fraction above 97 wt%, while CoSb 2.85 Te 0.15 /PEDOT:PSS and CoSb 2.90 Te 0.10 /PEDOT:PSS generate similar electrical conductivities. The exact mechanism is quite complex but might be related to the drastic decrease of the carrier mobility with doped Te incremented as indicated previously in the reference. As a result, the hybrid film with the smallest doping ratio (x = 0.05) of Te, CoSb 2.95 Te 0.05 , at 98 wt% shows the largest power factor of 6.47 µW/m K 2 at room temperature. Table 1 shows the Seebeck coefficient, electrical conductivity, and power factor of the n -type flexible thermoelectric materials reported so far. In this study, CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film records the largest negative Seebeck coefficient value of −161.7 µV/K at ambient temperature. However, the electrical conductivity and the power factor are much lower compared to the previous work. We will improve our efforts in the future by downsizing the skutterudites and uniformizing the grain size. In addition, we measured the temperature-dependent TE properties of the 98wt% CoSb 2.95 Te 0.05 hybrid film as shown in Figure S4 . Both the electrical conductivity and the absolute value of Seebeck coefficient increased with an increase in temperature. This is a typical metallic behavior dependence observed in Te-doped CoSb 3 , which agrees with a large carrier concentration and supports the idea that the inorganic component led the electrical transport properties rather than the organic component. The 98 wt% CoSb 2.95 Te 0.05 /PEDOT:PSS hybrid film, which has the highest thermoelectric performance, was subjected to a bending test to measure its flexibility as shown in Figure 5 . The Seebeck coefficient and the electrical conductivity decreased from the original value promptly in the first 250 bending cycles along a glass rod with a 4 mm radius, but the rate of decrease became much slower after 250 bending cycles, demonstrating the film’s flexibility to a certain degree considering that the film contains a large weight of powder and the facile method for breaking down the size of rigid TE materials."
} | 5,266 |
34348898 | PMC8336957 | pmc | 1,695 | {
"abstract": "Cointegration of single-transistor neurons and synapses for highly scalable neuromorphic hardware is demonstrated.",
"introduction": "INTRODUCTION Although software-based artificial neural networks (ANNs) have led to breakthroughs in a variety of intelligent tasks, they inevitably have inherent delays and energy consumption because the hardware structure to support the ANNs is still based on the von Neumann architecture ( 1 – 3 ). To overcome these limitations, hardware-based ANNs, known as brain-inspired neuromorphic systems, have been intensively studied ( 4 – 6 ). The human brain consists of neurons for the information encoding and synapses for the memory and learning function, as shown in Fig. 1A . There are about 10 11 neurons and 10 15 synapses, and thus, it is important to implement neurons and synapses with high density and low power to mimic the brain in hardware, especially for mobile devices and Internet of Things applications ( 7 , 8 ). Fig. 1 Concept of cointegrated single-transistor neurons and synapses. ( A ) Schematic of biological neuron and synapse. About 10 11 neurons and 10 15 synapses are densely interconnected in human brain. ( B ) Schematic of cointegrated single-transistor neurons and synapses. They have exactly the same SONOS structure, which includes a charge trap layer (Si 3 N 4 ) in the gate dielectrics as shown in the cross-sectional transmission electron microscopy (TEM) image. They are fabricated with the same fabrications and connected through metallization. ( C ) Operation scheme of the neuron and synapse. The input and output of the neuron are current and voltage, respectively, while those of the synapse are voltage and current. ( D ) Fabricated 8-inch wafer in which single-transistor neurons, synapses, and additional CMOS circuits were cointegrated. It was fabricated with 100% standard Si CMOS fabrications. Photo Credit: J.-K. Han, Korea Advanced Institute of Science and Technology (KAIST). Neurons are mainly composed of complementary metal-oxide semiconductor (CMOS)–based circuits, while synapses primarily comprise memristors ( 9 – 15 ). However, circuit-based neurons are problematic for high packing density and power consumption with low cost because they are composed of a capacitor, integrator, and comparator including many transistors ( 16 , 17 ). To overcome the limitations of circuit-based neurons, few works to cointegrate memristor-based artificial neuron devices and synaptic devices in a single crossbar array for a fully memristive neural network have been reported ( 18 – 20 ). Memristor-based neurons were realized with a single device, diffusive memristor (SiO x :Ag), or metal-insulator transition materials (NbO x and VO x ). Meanwhile, neuronal inhibition and tunability of firing threshold voltage are important for an energy-efficient and reliable neural network. The inhibitory function of the neuron related to biological lateral inhibition can improve energy efficiency by firing only specific neurons and enhance learning efficiency by enabling winner-takes-all (WTA) mechanism ( 21 – 23 ). In addition, the tunable firing threshold voltage related to biological homeostasis can allow reliable computation even when some neurons and synapses fail by process variations and endurance problems ( 24 – 26 ). However, the memristor-based neurons could not self-function for control of neuronal inhibition and firing threshold voltage because of the lack of controllability. On the other hand, it is advantageous that neuron devices and synaptic devices have the same homotypic structures and materials because simultaneous integration of neurons and synapses in a single chip with the same fabrication process is possible. Specific interconnections owing to inherent heterotypic structures and materials can impose constraints on reducing packing density and simplifying process complexity. Also, extra energy consumption cannot be avoided at the interface between the neurons and the synapses. However, there was no work that neuron devices and synapse devices are cointegrated with having exactly the same structures and materials. The metal-oxide semiconductor field-effect transistor (MOSFET) structure is attractive for commercialization because it has been verified for more than 60 years. In addition, the neuromorphic hardware should contain additional CMOS circuits to support processing units, peripheral interfaces, memory, clocking circuits, and input/output (I/O) for a complete application, as well as neurons and synapses ( 27 – 30 ). Therefore, if both neuron devices and synaptic devices can be realized with the same MOSFET structures, then commercialization of highly scalable neuromorphic system can be boosted by cointegration of neurons, synapses, and additional CMOS circuits on the same plane with commercial CMOS fabrication. In this work, highly scalable neuromorphic hardware was implemented by simultaneously integrating multistate single-transistor neurons and synapses on the same plane, in which both devices have the same homotypic MOSFET structure. In detail, the MOSFET for a neuron and a synapse encloses a charge trap layer in gate dielectrics with the same manner as a commercial flash memory based on a silicon-oxide-nitride-oxide-silicon (SONOS) structure that comprise a gate polycrystalline Si (S), blocking SiO 2 (O), charge trap Si 3 N 4 (N), tunneling SiO 2 (O), and channel single-crystalline Si (S). Because of this CMOS compatibility, they were fabricated and integrated on the same plane using the standard Si CMOS fabrication. It is possible to cointegrate single-transistor neurons and synapses with CMOS circuits for processing units, peripheral interfaces, memory, clocking circuits, and I/O at the same time, and thus, cointegration of the entire neuromorphic system is available. Therefore, a highly scalable neural network can be implemented in a single chip, which can enhance packing density, reduce chip cost, and simplify fabrication procedures. Neuron devices and synaptic devices were fabricated and directly interconnected, and their connection properties were analyzed. The abovementioned charge trap Si 3 N 4 in the MOSFET can allow multistates. The multistates according to trapped charges control the excitatory/inhibitory function or change the firing threshold voltage in the neuron, while they regulate synaptic weight in the synapse. Although the applicability of charge trap flash memory as a synapse has already been confirmed by taking advantage of its maturity of device technologies, stable multistate operations, high ratio of on/off conductance, and superior retention characteristics ( 31 , 32 ), cointegration of a Si-based single-transistor neuron with Si-based synapses has not been reported ever as a homotypic configuration. Homotypic neurons and synapses were directly connected to realize spatiotemporal neural computations. At the same time, CMOS circuits such as a current mirror and inverter, which are key elements for analog and digital circuits, were fabricated on the same plane to show the feasibility of cointegration of the interface and control circuits. In addition to real device fabrication, image recognition was successfully implemented with the aid of experimental-based simulations.",
"discussion": "DISCUSSION Completely CMOS-based neuromorphic hardware with high scalability was fabricated by the cointegration of single transistor–based neurons and synapses that are homotypic. The charge-trapping layer intercalated in the neurons and synapses allows multistates. They were used to control the excitatory/inhibitory function and to modulate the firing threshold voltage for the neurons, which were not accomplished at memristor-based neurons (table S1). They were also used to determine the weight for the synapses. A footprint area of the single-transistor neuron could be reduced to 6 F 2 , and its power consumption can be smaller than 1.5 μW (table S2). Because the neuron and the synapse have exactly the same structure, they were simultaneously integrated on the same plane at the same time with the same fabrications. This feature permits improvement of packing density, reduction of chip cost, and simplification of the fabrication procedures. In addition, it is possible to cointegrate with additional CMOS circuits for processing units, peripheral interfaces, memory, clocking circuits, and I/O because of the same in situ CMOS fabrications."
} | 2,105 |
39436143 | PMC11575353 | pmc | 1,697 | {
"abstract": "ABSTRACT High molecular weight (HMW; >1 kDa) carbohydrates are a major component of dissolved organic matter (DOM) released by benthic primary producers. Despite shifts from coral to algae dominance on many reefs, little is known about the effects of exuded carbohydrates on bacterioplankton communities in reef waters. We compared the monosaccharide composition of HMW carbohydrates exuded by hard corals and brown macroalgae and investigated the response of the bacterioplankton community of an algae-dominated Caribbean reef to the respective HMW fractions. HMW coral exudates were compositionally distinct from the ambient, algae-dominated reef waters and similar to coral mucus (high in arabinose). They further selected for opportunistic bacterioplankton taxa commonly associated with coral stress (i.e., Rhodobacteraceae , Phycisphaeraceae , Vibrionaceae , and Flavobacteriales ) and significantly increased the predicted energy-, amino acid-, and carbohydrate-metabolism by 28%, 44%, and 111%, respectively. In contrast, HMW carbohydrates exuded by algae were similar to those in algae tissue extracts and reef water (high in fucose) and did not significantly alter the composition and predicted metabolism of the bacterioplankton community. These results confirm earlier findings of coral exudates supporting efficient trophic transfer, while algae exudates may have stimulated microbial respiration instead of biomass production, thereby supporting the microbialization of reefs. In contrast to previous studies, HMW coral and not algal exudates selected for opportunistic microbes, suggesting that a shift in the prevalent DOM composition and not the exudate type (i.e., coral vs algae) per se , may induce the rise of opportunistic microbial taxa. IMPORTANCE Dissolved organic matter (DOM) released by benthic primary producers fuels coral reef food webs. Anthropogenic stressors cause shifts from coral to algae dominance on many reefs, and resulting alterations in the DOM pool can promote opportunistic microbes and potential coral pathogens in reef water. To better understand these DOM-induced effects on bacterioplankton communities, we compared the carbohydrate composition of coral- and macroalgae-DOM and analyzed the response of bacterioplankton from an algae-dominated reef to these DOM types. In line with the proposed microbialization of reefs, coral-DOM was efficiently utilized, promoting energy transfer to higher trophic levels, whereas macroalgae-DOM likely stimulated microbial respiration over biomass production. Contrary to earlier findings, coral- and not algal-DOM selected for opportunistic microbial taxa, indicating that a change in the prevalent DOM composition, and not DOM type, may promote the rise of opportunistic microbes. Presented results may also apply to other coastal marine ecosystems undergoing benthic community shifts.",
"conclusion": "Conclusion Coral HMW DOM was compositionally distinct from ambient reef water and enriched opportunistic microbial taxa commonly associated with coral stress, significantly increasing the predicted metabolic potential for energy-, amino acid-, carbohydrate-, fatty acid and lipid-, and secondary metabolism ( Fig. 7 ). In contrast, brown macroalgae HMW DOM was similar to ambient reef water and did not induce any effects on the bacterioplankton community composition. We propose two not mutually exclusive explanations for these results. A greater alteration in HMW DOM composition through coral compared to algal exudates Our results indicate that whether coral or macroalgae DOM exerts stronger effects on the bacterioplankton community composition depends on local DOM and bacterioplankton characteristics which are at least partly shaped by the local (benthic) DOM-producing community. We hypothesize that a change in DOM away from the ambient composition acts as a disturbance, thus resulting in the dominance of opportunistic microbes that are able to adapt fast to environmental change. A higher bacterial growth efficiency on coral compared to algal HMW exudates The strong effects of a small addition of coral HMW DOM to the bacterioplankton community suggest an efficient transformation of coral HMW DOM into microbial biomass, an important characteristic of nutrient cycles in healthy coral reefs. Brown macroalgae HMW exudate addition revealed no effects on the bacterioplankton community, indicating a low bacterial growth efficiency on algae exudates (i.e., more respiration). Brown algae HMW exudates, especially complex fucose-containing polysaccharides, could have additionally resisted microbial degradation (i.e., reduced bioavailability). Overall, our results suggest that changes in HMW DOM composition support the rise of opportunistic microbes in coral reefs. Inefficient and/or incomplete degradation of HMW macroalgae exudates could ultimately lead to a reduced transfer of energy and nutrients stored in algal DOM to higher trophic levels, thereby supporting the proposed reef microbialization.",
"introduction": "INTRODUCTION Corals are the main ecosystem engineers of tropical coral reefs, as they provide habitat and nutrients ( 1 ) to one of the most diverse and productive ecosystems on the planet ( 2 , 3 ). However, coral cover is declining on many reefs worldwide due to global and local human stressors ( 4 ), often leading to overgrowth by fleshy algae ( 5 – 8 ). Particularly in the Caribbean widespread shifts toward stages of fleshy macroalgae dominance have been reported ( 7 , 9 , 10 ). These shifts have been associated with changes in coral reef community metabolism caused by benthic primary producer-specific differences in the exudation of dissolved organic matter (DOM) ( 11 ). Algae usually exude more DOM than corals which increases the bacterioplankton abundance in reef water ( 12 , 13 ). In addition, algae DOM stimulates microbial respiration ( 11 ) which can lead to deoxygenation of reefs ( 14 – 16 ). Concomitantly, less energy is transformed into microbial biomass (i.e., a shift from biomass generation to respiration), reducing the transfer of energy to higher trophic levels ( 17 , 18 ). This shift in ecosystem-wide energy allocation from heterotrophic macrobes (e.g., fish and invertebrates) to foremost microbes was termed the microbialization of reefs and was proposed to occur globally on degraded, algae-dominated reefs ( 17 , 19 ). Algae DOM also appears to select for putative opportunistic and pathogenic microbes ( 17 , 20 – 22 ). Combined, these indirect effects of algae DOM on the microbial community can lead to coral mortality through hypoxia and disease ( 23 – 25 ). Coral mortality opens up space on the reef for algae growth, thus resulting in the DDAM positive feedback loop (DOC, disease, algae, and microorganisms) which is considered to facilitate reef degradation ( 26 , 27 ). These contrasting responses of microbial communities to coral- vs algae DOM suggest underlying differences in DOM composition. Indeed, liquid chromatography-tandem mass spectrometry has recently revealed a great compositional diversity in coral and algae exudates ( 28 , 29 ). However, this method is mostly limited to low molecular weight (LMW) components (e.g., organic acids, lipids, and lipid-like molecules) which efficiently elute from solid phase extraction columns ( 30 , 31 ), thus not capturing most carbohydrates. Carbohydrates are the most abundant biomolecules of high molecular weight (HMW; i.e., >1 nm or 1,000 Dalton) DOM in surface oceans ( 32 , 33 ) and play a major role in shaping bacterioplankton communities ( 34 , 35 ). Only very few studies have investigated the carbohydrate composition of the coral reef DOM so far. Nelson et al. ( 22 ) found increased concentrations of fucose, galactose, and rhamnose in bulk macroalgae-DOM (i.e., HMW + LMW), which exerted strong effects on the composition of natural bacterioplankton communities ( 22 ). Particularly the growth of copiotrophs and putative pathogens belonging to Gammaproteobacteria was stimulated. In contrast, bulk DOM exuded by the coral Porites lobata mainly consisted of glucose, mannose, and xylose and was similar in composition to the ambient reef water. This coral DOM selected for oligotrophic microbes of the Alphaproteobacteria and exerted only a small effect on the bacterioplankton community composition. Most carbohydrates exuded by corals ( 36 ) and macroalgae ( 37 – 39 ) belong to the HMW size fraction (i.e., glycoproteins and polysaccharides, respectively), which can be extracted from seawater with ultrafiltration (UF ;~1 nm pore size) ( 40 ). The HMW carbohydrates exuded by corals and macroalgae can thus be concentrated using UF and subsequently added to ambient seawater with minimal dilution of the bacterioplankton community while, at the same time, keeping DOM concentrations within a natural range. The majority of studies investigating the effects of primary producer exudates on bacterioplankton apply dilution culture experiments ( 41 ), where prefiltered exudate-enriched water is inoculated with unfiltered ambient reef water resulting in a reduction of microbial cell abundance by at least 40% ( 11 , 22 , 28 , 42 ). This approach alleviates density-dependent effects and allows to determine exponential growth rates as a measure of substrate quality. However, dilution also influences the community composition of bacterioplankton ( 43 , 44 ) and affects competitive outcomes ( 45 , 46 ). Differences in the amount of exuded DOM between corals and macroalgae in previous studies further resulted in higher starting concentrations for macroalgae compared to coral DOM ( 22 , 42 ), which makes it difficult to untangle the effects of DOM concentration (i.e., quantity) and DOM composition (i.e., quality) on bacterioplankton communities. Thus, the question remains whether coral and macroalgae exudates, added at similar concentrations, also differentially influence an undiluted microbial community. All in all, interactions of coral- and macroalgae-derived DOM with bacterioplankton received much attention over the last decades ( 47 ) due to increasing macroalgae dominance, especially in the Caribbean ( 10 ), and global evidence that algae DOM supports reef microbialization ( 17 ). Nevertheless, it remains unclear how much of the previously observed effects on bacterioplankton were due to differences in composition vs concentration of coral- vs macroalgae-derived DOM ( 22 ). Finally, no study has yet assessed how the most abundant part of exuded carbohydrates, the HMW fraction, affects undiluted coral reef bacterioplankton communities. To address these knowledge gaps, we investigated the exudation of HMW carbohydrates by corals and macroalgae, and how this particular DOM size fraction influences bacterioplankton communities from a Caribbean reef. We hypothesized that (i) coral and macroalgae HMW DOM differ in carbohydrate compositions and (ii) that these exudates enrich different taxa of the bacterioplankton community relative to seawater controls, with algae exudates exerting a stronger effect. We conducted a two-part experiment where we first incubated four hard coral species, two brown macroalgae genera, and seawater controls in aquaria to collect the exudates (see experimental design in Fig. 1 ). Subsequently, we concentrated HMW exudates from the incubation water and analyzed the monosaccharide composition of hydrolyzable carbohydrates. Finally, we added the concentrated HMW DOM to ambient seawater in 4-day dark incubations to elucidate effects on the growth and community composition of the heterotrophic bacterioplankton community to coral and macroalgae HMW DOM. By investigating bacterioplankton dynamics from a macroalgae-dominated reef in response to primary producer-specific HMW DOM-carbohydrate compositions, our study may help to understand the functioning of changing reef communities from a microbial ecology perspective. Fig 1 Experimental design divided into (a) coral and macroalgae incubations for exudate collection and (b) bacterioplankton incubations for exudate remineralization by microbes from ambient reef water. DOC = dissolved organic carbon; HMW DOM = high molecular weight (>1,000 Dalton) dissolved organic matter. Concentrated HMW DOM (concentration factor of 100) enriched with exudates from coral and macroalgae incubations was diluted with ambient reef water in dark incubations (dilution factor of 100). Flowchart detailing the incubation of corals and macroalgae, followed by bacterioplankton incubations. It outlines sample collection methods for different parameters, including carbohydrate- and microbial community composition.",
"discussion": "DISCUSSION Our results (summarized in Fig. 7 ) revealed that brown macroalgae, as well as scleractinian corals, exude significant amounts of HMW carbohydrates ( Fig. 2d ) which differ in composition ( Fig. 3 ). The compositional differences in exuded HMW DOM, added at low concentrations (<1% of ambient DOC), had no effect on the overall bacterioplankton cell density or dissolved nutrient concentrations (Fig. S3). However, coral HMW DOM significantly affected the bacterioplankton community composition ( Fig. 5 ) and increased the predicted potential for specific metabolic functions relative to controls ( Fig. 6 ). In contrast, algae HMW DOM addition induced no significant differences compared to seawater controls. Fig 7 Summary of HMW DOM compositions of coral and macroalgae exudates and microbial community responses. Pie charts display mean mole percent compositions of control-corrected fluxes or ambient seawater composition. Values >10% are shown in the figure. Gray italics indicate possible explanations for the lack of microbial response to algal HMW exudates. Icon attribution: Integration and Application Network (ian.umces.edu/media-library). A schematic diagram illustrating combined results and possible explanations. Pie charts display carbohydrate composition of HMW DOM and the microbial community response to coral and algae exudate treatments compared to controls is summarized. Coral exudates reflected HMW carbohydrate composition of coral mucus and enriched opportunistic coral-associated microbes The HMW carbohydrates released by corals were enriched in arabinose, glucosamine, mannose, and galactosamine ( Fig. 3 ) and mostly reflected the monosaccharide composition of hydrolyzed mucus from A. cervicornis ( Fig. 4 ). The main carbohydrate-containing macromolecules in coral mucus are mucin glycoproteins (i.e., 0.5–50 mDa) ( 82 ), and carbohydrate side chains of mucins isolated from Acropora formosa were rich in arabinose, glucosamine, and mannose ( 83 ), supporting the presence of mucins in HMW coral-DOM. Furthermore, although the prediction of metabolic functions from 16S rRNA data is limited in accuracy ( 76 , 77 ) (see Materials and Methods for details), the increase in predicted carbohydrate, amino acid, and fatty acid and lipid metabolism in bacterioplankton communities ( Fig. 6 ) is consistent with degradation of coral mucus, which mainly consists of carbohydrates, proteins, and lipids ( 84 , 85 ). The minor addition of HMW coral DOM (~0.6 µM C; <1% of DOC) to an ambient bacterioplankton community significantly increased the relative abundance of several genera belonging to the Alphaproteobacteria ( Rhodobacterales ), Planctomycetes ( Phycisphaerales , OM190 class), Gammaproteobacteria ( Vibrionales and Thiotrichales ), and Bacteroidetes ( Flavobacteriales and Saprospirales ; Fig. 5 ). However, the increase in microbial cell densities over time was similar to those observed in HMW macroalgae- and seawater control DOM incubations (Fig. S3). Bacterioplankton growth kinetics in dilution cultures usually include an exponential growth phase followed by a stationary phase ( 41 ), where the stationary abundance at least partially depends on the amount of substrate added ( 22 , 86 ). As we only diluted the microbial community by ~1% and added low and similar amounts of substrate for treatments (2.8 µM DOC) and controls (2.2 µM DOC), we did not expect differences in final microbial cell abundances. Rhodobacterales , Thiotrichales , Vibrionales , Flavobacteriales , and OM190 have been previously reported in coral mucus ( 87 – 91 ) and/or increased in seawater when coral mucus ( 92 – 94 ) or coral exudates ( 22 , 28 ) were added (Table S2). Phycisphaeraceae were previously found in Acropora hyacinthus ( 95 ), deep-sea corals ( 96 ), and in association with cultured Symbiodiniaceae ( 97 ). Thus, HMW coral-DOM enriched bacterioplankton taxa are commonly associated with corals. Rhodobacterales , Vibrionales , and Flavobacteriales were previously found to increase with macroalgae DOM addition and are considered to be opportunistic heterotrophic bacteria adapted to fast growth on DOM ( 22 ). Furthermore, they can increase in coral holobionts under stress ( 98 , 99 ) and disease ( 100 , 101 ) and were therefore suggested as indicators for poor reef health ( 102 ). Additionally, Thiotrichaceae ( 103 ) and Phycisphaeraceae ( 95 , 104 ) can be associated with coral disease, and OM190 increased in bacterioplankton during a marine heatwave ( 105 ). Thus, HMW coral DOM mostly selected for coral-associated microbes with the ability for opportunistic growth, which is also supported by the increase in predicted carbohydrate-, amino acid-, and fatty acid metabolism ( Fig. 6 ) ( 106 ). Algae exudates reflected algae tissue and ambient reef water HMW carbohydrate composition and did not affect the bacterioplankton community composition HMW carbohydrates released by the two brown macroalgae Dictyota spp. and Lobophora spp. were enriched in fucose, galactose, galacturonic acid, and rhamnose ( Fig. 3 ) and were similar in composition to tissue extracts from both species ( Fig. 4 ). Furthermore, the HMW carbohydrate composition of ambient reef water mostly reflected algae exudate compositions ( Fig. 4 and 7 ). In contrast, the previously reported carbohydrate composition of ambient reef water from Moorea in the central Pacific was most similar to that of coral exudates ( 22 ). This suggests that the composition of HMW DOM in the reef may depend on the community of benthic primary producers fueling and shaping the local DOM pool ( 17 , 107 ). Indeed, our study site is characterized by high algae (i.e., 17% macroalgae and 13% turf algae) and low coral (i.e., 7%) cover ( 108 ), with Dictyota and Lobophora being both highly abundant and releasing substantial amounts of DOM ( 12 , 13 , 109 ). On the other hand, the DOM pool of the rapidly flushed backreef of Moorea appears to be at least partly fueled by dense coral communities on the outer reefs ( 22 , 110 , 111 ). Microbial communities growing on HMW macroalgae DOM did not reveal any differences to seawater control incubations, thus not confirming the hypothesis that algae HMW exudates exert stronger effects on bacterioplankton communities. However, our addition of DOM was different from previous studies in several ways. We enriched the microbial community exclusively with HMW DOM and thus removed any molecules <1 kDa from the exuded fraction. Freshly produced exudates can contain LMW DOM with high bioavailability like free monosaccharides and amino acids, which are taken up rapidly by microbes ( 112 ) and could have contributed to the microbial response in previous studies. Further studies using undiluted bacterioplankton communities in combination with a natural ratio of HMW and LMW coral and macroalgal DOM are required to fully unravel the control of DOM source (and thereby composition) on bacterioplankton communities. Furthermore, the DOC addition in the present study (~2–3 µM DOC) is more than one order of magnitude lower compared to previous studies (~40–100 µM DOC [ 22 , 42 ]). These comparable high DOC additions allowed for high DOC uptake rates to compensate for low bacterial growth efficiencies (i.e., increased respiration at the expense of biomass formation) of algal-DOM, resulting in higher microbial growth rates on algal- vs coral-DOM ( 19 , 22 , 42 ). In the present study, there were neither differences in initial DOC concentrations between coral and macroalgae exudates ( Fig. 2a and b ) nor differences in microbial growth rates (Fig. S3a). Our approach thereby allowed a decoupling of DOM concentration-dependent from DOM composition-dependent effects, which suggest that the increased DOC concentration component at least partly (excluding potential effects from LMW DOM) explained previously reported differences in bacterial growth rates between coral and algae DOM. HMW macroalgae DOM could have also resisted microbial degradation throughout the 4-day incubations. Brown macroalgae can exude large quantities of fucoidan ( 38 ), a complex fucose-rich polysaccharide that forms an extracellular matrix and prevents desiccation of algal thalli ( 113 , 114 ). Fucose was the most abundant monosaccharide in HMW DOM of the brown macroalgae in the present study, as well as for the brown algae Turbinaria on reefs in French Polynesia ( 22 ). Using monoclonal antibodies (see supplemental methods), we detected three epitopes present in sulfated fucan (BAM1, BAM3, and BAM4) in macroalgae tissue (Fig. S7a), which is consistent with previous findings of fucoidan in the tissue of both genera ( 113 , 115 ). Additionally, relative proportions of fucose, galactose, xylose, and mannose were similar in fucoidan extracted from Dictyota spp. and Lobophora spp. compared to macroalgae exudates (Fig. S7b), suggesting that fucoidan contributed to macroalgae HMW DOM. Microbial degradation of brown algae fucoidans is energetically costly because it can require hundreds of different enzymes to degrade its complex structure ( 116 ). This could also explain why bacterioplankton growing on brown macroalgae exudates incorporate less carbon and have higher respiratory costs compared to green algae exudates ( 22 , 42 ), as green algae do not contain fucoidan in their cell walls ( 117 ). Similarly, the high contribution of fucose, galactose, xylose, and mannose in ambient reef water (combined making up 54% of HMW carbohydrates, Fig. 4 and 7 ) suggests a considerable abundance of fucoidan in the HMW fraction of reef water, which supports the high resistance of brown macroalgae HMW DOM to microbial degradation. Ecological implications Previous studies were mainly conducted on coral-dominated reefs and found that coral exudates support a diverse oligotrophic bacterioplankton community, while algae exudates promote opportunistic microbial taxa ( 22 ) and less energy-efficient nutrient cycling ( 17 ). A shift toward macroalgae dominance can thus reduce ecosystem productivity by enhancing microbial respiration ( 11 , 118 ) and decrease the transfer of energy to higher trophic levels ( 17 , 18 ). The strong effects observed here from a small addition of coral exudates on the bacterioplankton community composition support the energy-efficient transformation of coral exudates into microbial biomass ( 11 , 17 ). However, the increase in mainly opportunistic microbial taxa with coral exudates seems to contradict previous results. A change in carbon substrates can act as a disturbance on microbial communities ( 119 ). Thus, the addition of coral exudates to a microbial community from a reef with macroalgae DOM dominating the local DOM pool could be considered a disturbance of the alternative stable state (i.e., the algae-dominance [ 120 ]). A common ecosystem response to stress is a shift toward opportunists which are less specialized but respond rapidly to perturbations by adapting to new environmental conditions ( 121 , 122 ). We, therefore, propose that the increase of some opportunistic microbial taxa on reefs may not be a direct response to the exudates of a specific primary producer but rather to a disturbance in the form of a change in the availability of DOM producer-specific carbon substrates. Brown macroalgae HMW DOM did not appear to support microbial growth, which could be explained by increased respiration instead of biomass production (not measured here but shown previously [ 11 , 17 , 19 ]). An additional explanation could be that resistant HMW molecules form brown macroalgae such as fucoidan defied degradation during the 4 days of dark incubations. Previous studies revealed resistance of brown algae exudates to microbial degradation for up to 5 months, which leads to a net export of DOM from brown macroalgae beds ( 123 – 126 ). Water residence times of fringing reefs can range from hours to days ( 127 , 128 ). Thus, brown algae exudates could be exported from coral reefs. Release of refractory DOM by brown macroalgae which replace corals on many reefs could be an additional pathway by which the transfer of energy to higher trophic levels declines on degraded reefs. This hypothesis could be tested by measuring fucose concentrations at gradients away from algae-dominated reefs, as fucose can function as a biomarker for brown algae origin ( 129 ). Effects of changing DOM compositions beyond coral reefs Our results indicate that opportunistic microbial taxa increase in bacterioplankton communities of coral reefs following a change in the main DOM substrates (here induced by addition of coral DOM to macroalgae DOM-dominated ambient reef water; Fig. 7 ). This hypothesis is based on the r - and K -selection framework, where copiotrophic r- strategists can grow faster on new carbon sources, thus outcompeting the oligotrophic K- strategists ( 121 ). Changes in the main benthic DOM producer are not exclusive to coral reefs and have been reported for coastal ecosystems worldwide, including macroalgae beds ( 130 ), kelp forests ( 131 ), and seagrass meadows ( 132 ). These macrophytes release significant amounts of their photosynthetically fixed carbon as DOM ( 38 , 133 , 134 ). Changes in benthic composition and resulting alterations of the local DOM pool may disrupt the stable microbial community states, inducing the rise of opportunistic heterotrophic microbes until a new equilibrium has been reached following the perturbation. Conclusion Coral HMW DOM was compositionally distinct from ambient reef water and enriched opportunistic microbial taxa commonly associated with coral stress, significantly increasing the predicted metabolic potential for energy-, amino acid-, carbohydrate-, fatty acid and lipid-, and secondary metabolism ( Fig. 7 ). In contrast, brown macroalgae HMW DOM was similar to ambient reef water and did not induce any effects on the bacterioplankton community composition. We propose two not mutually exclusive explanations for these results. A greater alteration in HMW DOM composition through coral compared to algal exudates Our results indicate that whether coral or macroalgae DOM exerts stronger effects on the bacterioplankton community composition depends on local DOM and bacterioplankton characteristics which are at least partly shaped by the local (benthic) DOM-producing community. We hypothesize that a change in DOM away from the ambient composition acts as a disturbance, thus resulting in the dominance of opportunistic microbes that are able to adapt fast to environmental change. A higher bacterial growth efficiency on coral compared to algal HMW exudates The strong effects of a small addition of coral HMW DOM to the bacterioplankton community suggest an efficient transformation of coral HMW DOM into microbial biomass, an important characteristic of nutrient cycles in healthy coral reefs. Brown macroalgae HMW exudate addition revealed no effects on the bacterioplankton community, indicating a low bacterial growth efficiency on algae exudates (i.e., more respiration). Brown algae HMW exudates, especially complex fucose-containing polysaccharides, could have additionally resisted microbial degradation (i.e., reduced bioavailability). Overall, our results suggest that changes in HMW DOM composition support the rise of opportunistic microbes in coral reefs. Inefficient and/or incomplete degradation of HMW macroalgae exudates could ultimately lead to a reduced transfer of energy and nutrients stored in algal DOM to higher trophic levels, thereby supporting the proposed reef microbialization."
} | 7,084 |
35382303 | PMC8973109 | pmc | 1,698 | {
"abstract": "Design and fabrication\nof functional materials for anti-icing and\ndeicing attract great attention from both the academic research and\nindustry. Among them, the study of fish-scale-like materials has proved\nthat enabling sequential rupture is an effective approach for weakening\nthe intrinsic interface adhesion. Here, graphene platelets were utilized\nto construct fish-scale-like surfaces for easy ice detachment. Using\na biomimicking arrangement of the graphene platelets, the surfaces\nwere able to alter their structural morphology for the sequential\nrupture in response to external forces. With different packing densities\nof graphene platelets, all the surfaces showed universally at least\n50% reduction in atomistic tensile ice adhesion strength. Because\nof the effect of sequential rupture, stronger ice–surface interactions\ndid not lead to an obvious increase in ice adhesion. Interestingly,\nthe high packing density of graphene platelets resulted in stable\nand reversible surface morphology in cyclic tensile and shearing tests,\nand subsequently high reproducibility of the sequential rupture mode.\nThe fish-scale-like surfaces built and tested, together with the nanoscale\ndeicing results, provided a close view of ice adhesion mechanics,\nwhich can promote future bioinspired, stress-responsive, anti-icing\nsurface designs.",
"conclusion": "4 Conclusions In summary, three fish-scale-like surfaces were built by assembling\ngraphene platelets in a uniform ordered orientation. By using cyclic\ntensile and shearing deicing tests, surfaces with a high packing density\nof graphene platelets exhibited stable and reversible surface morphology\nfor the reproducibility of sequential ice rupture and the subsequent\nlow atomistic ice adhesion strength. Despite varied ice–substrate\ninteractions resulting from different graphene platelet packing densities,\nall the surfaces showed a 50% reduction in ice adhesion strength.\nThe low tensile ice adhesion strength on different fish-scale-like\nsurfaces was in a similar range, which signified that the sequential\nrupture mode was the dominating factor for the reduction of ice adhesion.\nThe high packing density of graphene platelets was key to the full\nand reversible coverage of the surface, which remained the integrality\nof the effective structure for deicing. Furthermore, the high packing\ndensity was crucial for maintaining a uniform graphene platelet orientation\nunder shearing stress. Motivated by natural surface examples, this\nwork supplied a low intrinsic ice adhesion surface design strategy\nof deicing, which further verified the sequential rupture as an effective\napproach for lowering atomistic ice adhesion and at the same time\nshed light on new icephobic materials that are responsive to external\nstimuli.",
"introduction": "1 Introduction Unwanted icing is one of the major challenges to infrastructure\nand human activities in environments below water freezing temperature. 1 , 2 For instance, atmospheric icing including precipitation, in-cloud,\nand frost, directly results in problems of electrical failure, overproduction,\npower losses, measurement errors, and safety hazards on wind turbines 3 , 4 at high altitudes. Ice accretion is also a lethal hazard to aircraft 5 , 6 due to its icing effects on the handling and performance of the\nwings. Icing combined with wind could cause damage and power outages\non power networks. Highly relevant to our daily life during winter\nor in cold regions, icing could blot out the visual field from the\nwindshield, causing inconvenience to drivers or passengers. Many applications\nof anti-icing or deicing have been used to prevent or minimize icing\neffects, aiming at lifetime extension, energy saving, and cost reduction. 4 , 5 , 7 − 12 Materials with super-low ice adhesion strength are highly desired\nin addressing the icing problem and are under active development today. 13 − 15 After identifying the determinants of ice adhesion, it is recognized\nthat intrinsic ice adhesion is a key factor for the firm attachment\nof ice on different surfaces. 1 , 16 , 17 Specifically, for a seemingly ice-covered area on a rough surface\non the macroscale, termed apparent adhesion, only the truly effective\ncontacting points or areas and interlockings on the nanoscale, termed\nintrinsic adhesion, are responsible for the observed ice adhesion\nstrength. 1 Seeking low intrinsic ice adhesion\nstrength can rely not only on physical chemistry level atomistic interactions, 8 for instance, using superhydrophobic\nmaterials, but also on the design of the stress-responsive rupture\nmode of atomistic ice–substrate interactions. 19 , 20 Considering the full detachment of an intrinsic contacting area\nas depicted in Figure 1 , the sequential rupture between the ice and its substrate leads\nto much lower rupture force, and thus stress, than the concurrent\nbreakage of all the atomistic interactions at once. 19 Figure 1 Sequential and concurrent rupture between the ice and its adhering\nsubstrate. Atomistic interactions, indicated as red dashed lines,\nare broken in an incremental manner in sequential rupture (left panel),\nwhile all at once in the concurrent rupture mode (right panel). Given\nthe same number and strength of atomistic interactions, the sequential\nrupture mode leads to a lower rupturing force. Inspiring by the application of biomaterials 21 , 22 to designing materials with low intrinsic ice adhesion, natural\nsurfaces can provide inspiring guidelines. There are many natural\nsurfaces with topography for tailored mechanical functions, especially\nthe ones that are able to respond to external stress in wetting and\nadhesion. 23 Two outstanding examples in\nthis regard are the hierarchical surfaces of water strider legs and\ngecko toes. 24 , 25 These two nature-designed surfaces\nconsist of hierarchical structures of small, flexible, and organized\nunits for realizing tailored properties. Specifically, the uniquely\noriented needle-like microsetae on water strider legs enable superior\nwater repellence, and well-organized setae on gecko toes enable fast\nswitching between strong attachment and easy detachment. The microsetae\non water strider legs are superhydrophobic, namely having super-low\nadhesion to water. 26 In comparison, the\nsetae on gecko toes can on the one hand, apply strong van der Waals\nforces on different surfaces, 27 , 28 and, on the other hand,\ncan easily detach from solid surfaces by the rolling of the gecko\nfeet, namely by sequential rupture of the setae–substrate interactions. 25 , 29 The ordered oriented microsetae and setae on the two surfaces are\nmade for sequential rupture of any adhesion, which is demonstrated\nin the detachment process of gecko toes from different surfaces. Most\nimportantly, the weak adhesion of gecko toes to solid substrates is\nhighly reusable, which is enabled by the optimized packing of the\nsetae in the surface hierarchical structures. Mimicking the organization\nand the mechanical functions of such natural surfaces by featuring\ntheir surface topography can serve as a practical approach for lowering\nintrinsic ice adhesion. 19 Former\nstudies have illustrated that sequential rupturing of atomistic\ninteractions can lead to weaker adhesion, which was also applied to\nlow intrinsic ice adhesion strength. 19 Using\ngraphene platelets for constructing a fish-scale-like surface as shown\nin Figure 2 a,b, the\nprevious study realized the two rupture modes of sequential and concurrent\nice detachment from its adhering substrates. Strikingly, the sequential\nrupture mode of ice detaching can lead to a ∼60% reduction\nin ice adhesion strength. Figure 2 Atomistic models and cyclic deicing. (a) Tip4p/ice\nwater model\nand the graphene platelet. Atoms on the graphene platelets that are\nfixed to enable sequential rupture are highlighted in red. (b–d)\nC4, C5, and C8 fish-scale-like surfaces from left to right, with a\nlow to high graphene packing density. (e) Adopted cyclic deicing procedure,\nincluding ice equilibration adhesion on the surfaces, first round\ndetachment, re-adhesion, and re-detachment. Given the role of the sequential rupture mode in lowering ice adhesion,\nan interesting question that awaits an answer is how the arrangement\nor packing of graphene platelets in the fish-scale-like surface affects\nthe intrinsic ice adhesion in attaching-and-detaching cycles. Addressing\nthis question can further verify the fish-scale-like surface for successful\nanti-icing and guide the pattern design. As such, this work modeled\nand systematically compared ice adhesion and friction on fish-scale-like\nsurfaces with varied packing densities of graphene platelets. This\nstudy aims at icephobic surface design for low intrinsic ice adhesion,\nand it also serves as a reference for the nanoscale interface tribology\nof snow and ice on solid surfaces.",
"discussion": "3 Results and Discussion 3.1 Cyclic Tensile Deicing\non the Fish-Scale-Like\nSurfaces The most interesting mechanical properties of the\nfish-scale-like surfaces are their ability to enable the sequential\nrupture for the purpose of weakening adhesion strength. 19 Because of the different packing densities of\nthe graphene platelets, the surface area contacting the ice in the\nthree systems varied, as shown in Figure S1 . Specifically, the C4 and C5 surfaces with a low packing density\nshowed a larger surface area than the C8 surface with a high packing\ndensity, as shown by Figure 2 b–d and Supporting Information Figure S2 . Correspondingly, the equilibrated ice adhering interfaces\nalso differed on the three surfaces, namely the large rough ridges\nobserved on the equilibrated ice interface on C4 and C5 but small\ntips on C8, as shown in Supporting Information Figure S2 . By comparing the interaction potential between the\nice and the three surfaces, the C5 surface had the strongest interaction\nthanks to a large amount of water/ice molecules trapped in the grooves\nof the surface, while the C8 had the weakest, as depicted in Supporting Information Figure S2 . Thus, the C5\nsurface exhibited the best complementing matching with the ice layer,\nwhich is highly likely to lead to strong interlocking if the graphene\nsurface was positionally fixed. The atomistic ice adhesion strength\nof the three fish-scale-like surfaces was first compared in a cyclic\ndeicing test in order to verify the effect of sequential rupture.\nTo do so, the tensile detaching process of the ice layer from the\nsurfaces was carried out using the same procedure as in the former\nstudies. 19 , 31 , 36 As the representative\nice detaching event given in Supporting Information Movie (pulling-process.mp4), the ice layer was first slowly\nlifted from the surface under the increasing pulling force and finally\ndetached from the substrate, as shown in Figure 3 a. All the tensile stress on the ice layer\nfeatured a steady increase owing to the constant pulling speed of\nthe harmonic spring and the slow movement of the ice, as shown in Figure 3 b. Because the pulling\nforce was applied on the COM of the ice layer, the counterforce came\nfrom the interaction between the ice and the surfaces. When the tensile\nstress reached the critical value σ r , the ice layer\nwas fully detached from the surfaces, resulting in a sudden drop at\nthe end of the tensile stress curve. Under the concurrent and sequential\nrupture modes, the fish-scale-like surfaces reacted to external pulling\nstress in remarkably different manners. Because all the graphene platelets\nwere fixed, the three surfaces showed no structural change throughout\nthe deicing process in concurrent rupture. In contrast, the average\nthickness of the graphene platelet layer in the sequential rupture\nmode first increased due to the opening of the graphene platelets\nand then decreased after ice detachment, as indicated also in Figure 3 a. The concurrent\nrupture modeled to strong ice adhesion, with tensile ice adhesion\nstrength σ of 330.2 ± 7.5, 348.3 ± 4.8, and 281.9\n± 5.6 MPa for the C4, the C5, and the C8 surfaces, respectively.\nThe difference in the concurrent σ on the three surfaces was\ncorrelated with the combined effects of the local structures of the\nice–surface interface and the atomistic interactions ( Supporting Information Figures S2 and S3 ). Better\naccommodation of water/ice molecules in the surface roughness grooves\nand the resulting stronger interfacial interaction between the ice\nand the surface have resulted in higher ice adhesion strength, with\nC5 showing the strongest ice–surface interaction and ice adhesion.\nStrikingly, the sequential rupture mode in cyclic deicing tests on\nthe three surfaces resulted in around a 50% reduction in σ r as shown in Figure 3 c, which was 122.2 ± 3.7, 169.1 ± 7.4, and 152.9\n± 6.9 MPa for the C4, C5, and C8 surfaces, respectively. The\nresult of a significant decrease in σ r further confirmed\nthe effect of the sequential rupture in lowering ice adhesion. 19 The σ r by the sequential rupture\nmode obtained in cyclic deicing tests on each surface was stable,\nas demonstrated in Figure 3 c by the similar σ r values monitored in the\nfirst and second rounds of deicing on each of the three surfaces.\nAlthough the local structure of the ice–substrate interface\nhad changed after the first detaching event and the subsequent re-adhesion\nof ice (see below), the σ r by sequential rupture\nwas not significantly affected. The key determinant of σ r by sequential rupture was the rupture mode of detachment\nrather than the local structure of the ice–substrate interface. Figure 3 Deicing\ntesting on the fish-scale-like surfaces. (a) Representative\nsystem snapshots in the sequential rupture process of cyclic tensile\ndetaching from the C5 surface. The pulling force is indicated by the\narrow. The changes in the thickness of the surfaces are highlighted\nby the red bar. (b) Pulling stress profiles in cyclic deicing tests\non the C5 surface. The sudden drop in the pulling stress represents\nice detaching events in each independent simulation. Pulling stress\nresponses obtained in the concurrent rupture mode on the C5 surface\nare plotted for comparison (bottom). (c) Ice rupture stress observed\non the three surfaces, including cyclic sequential and concurrent\nrupture stress. The error bars denoted standard deviations of five\nindependent runs. The reversibility of\nthe hierarchical morphologies on biological\nsurfaces such as gecko toes and water strider legs is crucial for\nthe reproducibility of their special surface mechanical properties. 24 , 25 For these natural surfaces, the arrangement of the surface units,\nespecially the pattern and packing density, is key to the surface\ndurability. It is critical for these natural surfaces to resist any\nmechanical damage and, in the worst scenario, to recover rapidly from\ndamage. Mimicking the abilities of damage resistance is an important\naim of the fish-scale-like surfaces. The reversibility of the hierarchical\nstructure of the three fish-scale-like surfaces in the cyclic deicing\ntests was put together for comparison, as shown in Figure 4 . Specifically, the C8 surfaces\ndemonstrated excellent reversibility of graphene platelet orientation\nand surface coverage after the cyclic deicing tests. Thanks to the\nclose packing of the graphene platelets, the top half of the graphene\nplatelets responded to the ice adhesion and detachment events, while\nthe lower half of the graphene platelets maintained a close-packed\nstructure not affected by the deicing forces. At the end of the deicing\ntests, the graphene platelets showed a re-adjusted position and yet\nsimilar morphology as before the deicing test. Importantly, the C8\ngave full coverage of the XZ -plane, which indicated\nan ability to produce a sequential rupture mode. In contrast, the\nsurface morphology of C5 and C4 was significantly altered in the cyclic\ndeicing test. The C5 surface was already partially damaged after the\nfirst round of deicing, as shown in Figure 4 . Although the fish-scale-like structure\nof C5 was slightly restored in the ice re-adhesion equilibration simulation,\nthe final arrangement of the graphene platelets was completely distorted\nif compared to the original state. The surface area of C5 was not\nfully covered by the graphene platelets after the second round of\ndeicing, which was an indication of breakages. The C4 showed the least\nreversibility in morphology. Not only was the fish-scale-like structure\nlost but also a large area of open space was not covered by the graphene\nplatelets (top row in Figure 4 ). Given that the three surfaces consist only of the graphene\nplatelets, the open space without coverage thus becomes the contact\narea between the ice and the solid substrate below the graphene platelets\nin reality. Such areas can serve as large interlocking points for\nenhanced ice adhesion, 41 , 42 which can greatly weaken the\nanti-icing properties of the surfaces. Here, the open areas did contribute\nto the low ice adhesion strength in the second round of the deicing\ntest observed on the C4 and C5 because there were no atomistic interactions\nbetween the ice and the surface. The low ice adhesion resulting from\nthe sequential rupture on the C4 and C5 surfaces thus was not sustainable,\ngiven the surface structures easily destroyed by external forces. Figure 4 Top view\nof the three fish-scale-like surfaces in the cyclic deicing\ntests. The packing morphologies of the three surfaces, C4, C5, and\nC8, after the first equilibrated ice adhesion, first-time deicing,\nsecond-time ice adhesion, and second-time deicing, were shown in subsequence\nhorizontally. Each graphene platelet on the surfaces is colored differently\nfor better visualization. It is known that a higher loading rate, determined by the spring\ntensile pulling speed, can lead to higher rupture stress. 19 , 31 The loading rate thus influences the deicing behavior and morphology\nreversibility of the surfaces. For comparison, two extra tensile pulling\nspeeds of 0.2 and 1 nm/ns were chosen for the deicing test on the\nC5 surface. As the surface snapshots after first-round deicing are\nshown in Figure 5 a,\nthe morphologies showed no obvious differences after deicing with\nthe three tensile pulling speeds. The main characteristic of the fish-scale-like\narrangement of the graphene platelets remained after the ice detachment\nevent, with random areas not covered by graphene platelets. Because\nthe pulling rates tested here ranged only 1 order of magnitude, the\nresulting rupture stress monitored was 161.7, 164.6, and 171.0 MPa\nfor the loading rates of 0.2, 0.5, and 1 nm/ns, respectively, as shown\nin Figure 5 b. The difference\nin the resulting rupture stress was less than 10 MPa. As it is known\nthat rupture stress increases logarithmically with the loading rate, 43 a much higher pulling loading rate is needed\nto generate an obvious difference in rupture stress, which is beyond\nthe scope of this work. The increase in rupture stress was ∼6%\nfrom the lowest to the highest pulling rate (from 161.74 to 170.96\nMPa). In accordance with the rupture stress, the rupture work needed\nfor complete deicing also increased with the pulling rate, as depicted\nin Figure 5 c. Surprisingly,\nthe rupture work showed an increase of ∼25% with the increasing\npulling rate from 0.2 to 1 nm/ns, in contrast to the slight increase\nin the rupture stress. The obvious increase in rupture work indicated\nthe surface had varied sequential opening distances under different\ntensile pulling rates. Indeed, as shown by the ice displacement distances\nbefore the rupturing event in Figure 5 d, a lower tensile pulling rate led to lower ice upward\nmovement and thus the sequential opening distance of the surface.\nBecause the sequential opening distance is correlated with the displacement\nof the graphene platelets from their original position, a higher pulling\nrate can result in large displacement and thus an increased probability\nof distortion of the graphene platelets and damage to the surfaces. Figure 5 Deicing\non the C5 surface with different pulling rates. (a) Surface\nmorphologies of the C5 before and after deicing tests with different\npulling rates. Random areas not covered by graphene platelets after\ndeicing are highlighted by red dashed circles. The corresponding pulling\nrate of each resulting morphology is highlighted in the figure. (b)\nTypical pulling stress profiles observed in the deicing with varied\npulling rates. (c) Loading-dependent rupture work of the ice from\nthe C5 surface. (d) Ice displacement under pulling force with corresponding\npulling speed given. The displacement of ice where the detaching event\nhappened is marked in blue. 3.2 Shearing Ice on the Fish-Scale-Like Surfaces Because of the anisotropic structure of the fish-scale-like surfaces,\nshear stress was expected to exhibit different features along and\nagainst the graphene platelet orientation direction. 19 Furthermore, the different packing densities of graphene\nplatelets on the three surfaces led to significant variation in the\nlocal structure of the ice surface and interaction potential, as shown\nin Supporting Information Figures S2 and S3 , which also contributed to the differences in the shearing of the\nice on the three surfaces. For a detailed comparison, ice was sheared\non all the three surfaces along and against the graphene platelet\norientation, under both the concurrent mode and sequential mode. In\ncontrast to the no structural changes of the surfaces under the concurrent\nmode, the morphology of graphene platelets can be opened and overthrown\nin a different direction by external shearing force in the sequential\nrupture mode. As shown in Figure 6 , all the three surfaces in the sequential rupture\nmode can be easily altered by shearing stress against the ordered\ndirection of the graphene platelets due to the flexibility of the\ngraphene platelets. As such, the fish-scale-like organization of the\ngraphene platelets was maintained in shearing, which was important\nfor deicing. As shown by the shearing movie in Supporting Information Movie (shearing-process.mp4), the sequential\nrupture mode was able to keep the hierarchical structure of the surfaces. Figure 6 Top view\nof the morphology of graphene platelets on the three surfaces\nafter ice shearing along and against the ordered direction of the\nplatelet in the sequential mode, with (a,b) for the C4, (c,d) for\nthe C5, and (e,f) for the C8, respectively. Here, the red arrow indicates\nthe direction of ice motion. The anisotropic surfaces, namely the uniform arrangement of the\ngraphene platelet orientation, resulted in anisotropy in the observed\nice shear stress. The patterns of ice shear stress profiles were similar\nto the results reported in the previous study, as shown in Figure 7 . 19 Specifically, high shear stress values were observed if\nthe ice was sheared against the graphene platelet orientation, owing\nto the intercalating ice adhering interface with the fish-scale-like\nsurface. Otherwise, the shear stress was relatively low with saw-teeth-like\nfluctuations if the ice was sheared along with the graphene platelet\norientation. The saw-teeth-like stress pattern was caused by the ice\nattaching/detaching at the fish-scale-like surface. Because the equilibrated\nice adhesion led to the matching of the ice with the periodic low\nand high repeated surface topography, low shear resistance along the\ngraphene platelet orientation facilitated sliding of the whole ice\nlayer and repeated re-matching between the ice and the surface. The\npeak value of the shear stress was in the range of 60–80 MPa,\nas depicted in Figure 7 . For all the surfaces, the highest stress was monitored during shearing\nagainst the ordered direction of the graphene platelet and with all\nthe platelets fixed in position (the concurrent rupture mode). The\npeak stress value was close to 140 MPa for all the three surfaces,\ndespite the differences in the platelet packing density. Because the\nflexibility and re-orientation of the graphene platelets in the sequential\nrupture mode could relax stress in shearing against the platelet orientation,\nthe corresponding shearing stress decreased gradually in the shearing\ntest. In contrast, constant high shearing stress was observed in ice\nshearing against the platelet orientation direction in the concurrent\nmode on all the three surfaces. Figure 7 Ice shearing stress profiles on the fish-scale-like\nsurfaces. Shearing\nstress labeled with “concurrent” legends were obtained\nwith all the graphene platelets fixed, and those labeled with “sequential”\nlegends were obtained with the sequential rupture mode enabled. All\nthe stress profiles shared the same color code in the three plots.\nAbnormal drops in shearing stress monitored on the C5 surface were\nhighlighted in red dashed circles. Given that the graphene platelets of all the three surfaces were\nfixed in the concurrent mode, the interface matching of the ice and\nthe surfaces became strong interlocking against shearing, especially\non the C5 surface. As highlighted in Figure 7 , abnormal drops in shear stress were observed\nin shearing against the platelet orientation, which indicated abrupt\nchanges in the ice structure under high shearing stress. As shown\nin Figure 8 , the observed\nabnormal drops indeed indicated breakthroughs in the interlocking\nbetween the ice layer and the surface. Namely, with the building up\nof shearing stress, the interaction potential of the system also steadily\nincreased, as shown in Figure 8 b. Because the surface structure was fixed in the concurrent\nmode, the increase in the system potential can be attributed to the\nchanges in the ice structure. The structural root-mean-squared deviation\n(rmsd) of the ice structure agreed with such an assumption, as shown\nin Figure 8 c. The ice\nlayer buckled under the shearing, as shown in Figure 8 d. Under the highest peak of the shear stress,\nthe interlocking between the ice and the surface was destroyed, where\nthe whole ice layer took off from the surface and re-adhered back\nto the surface in a short time of several picoseconds. The process\nis captured by system snapshots shown in Figure 8 d. The abrupt structure change in the ice\nlayer in interlocking breakthrough events can be expected in ice shearing\ntests on hard surfaces in experiments. Figure 8 Interlocking breakthrough\non the C5 surface. (a) Shear stress profile\nwith abnormal drops observed in shearing ice against the graphene\nplatelet orientation direction, as taken from Figure 7 . (b) Corresponding system interaction potential\nprofile in the shearing process. (c) rmsd of the ice layer structure\nin the whole shear test. (d) System snapshots in an interlocking breakthrough\nevent. The circle number of each snapshot indicated its corresponding\nposition in the stress, potential, and rmsd profile showed in (a–c).\nThe bending of the ice layer was further sketched for better visualization\neffect."
} | 6,717 |
36456707 | PMC9715565 | pmc | 1,699 | {
"abstract": "Iron (Fe) is an essential trace element for life. In the ocean, Fe can be exceptionally scarce and thus biolimiting or extremely enriched causing microbial stress. The ability of hydrothermal plume microbes to counteract unfavorable Fe-concentrations up to 10 mM is investigated through experiments. While Campylobacterota ( Sulfurimonas ) are prominent in a diverse community at low to intermediate Fe-concentrations, the highest 10 mM Fe-level is phylogenetically less diverse and dominated by the SUP05 clade (Gammaproteobacteria) , a species known to be genetically well equipped to strive in high-Fe environments. In all incubations, Fe-binding ligands were produced in excess of the corresponding Fe-concentration level, possibly facilitating biological Fe-uptake in low-Fe incubations and detoxification in high-Fe incubations. The diversity of Fe-containing formulae among dissolved organics (SPE-DOM) decreased with increasing Fe-concentration, which may reflect toxic conditions of the high-Fe treatments. A DOM-derived degradation index (I DEG ) points to a degradation magnitude (microbial activity) that decreases with Fe and/or selective Fe-DOM coagulation. Our results show that some hydrothermal microbes (especially Gammaproteobacteria) have the capacity to thrive even at unfavorably high Fe-concentrations. These ligand-producing microbes could hence play a key role in keeping Fe in solution, particularly in environments, where Fe precipitation dominates and toxic conditions prevail.",
"introduction": "Introduction Iron (Fe) is a fundamental trace nutrient regulating phytoplankton productivity in the upper water column and thus affecting the biological carbon pump 1 . Although Fe is considerably scarce in most parts of the ocean, in certain habitats (e.g. hydrothermal vents) its concentrations can exceed levels that stress microbial life, where minerals precipitate in the cell’s periplasm or on the cell surface resulting in irreversible encrustation and cell death 2 , 3 . Some microbes have evolved mechanisms to bind Fe, e.g., via active production of organic molecules, so called ligands, to condition the environment in their favor. Organic ligands include e.g., siderophores, polyphenols, hemophores and heme and can either enhance Fe-bioavailability or be used as detoxification tools via complexation 4 – 7 . Fe-complexation with organic ligands also effectively prevents rapid precipitation in seawater. The thereby resulting increased residence time enables Fe-export over greater distances and thus enhances Fe-transfer to the deep sea or the surface ocean 8 – 10 . In addition, the systematics of coupled Fe-oxidation and Fe-organic complexation can influence toxicity of other metals and metalloids (e.g. sorption of cadmium or arsenic to Fe-precipitates) as well as the accumulation of toxic nitrite (if Fe(II) is not complexed) 11 . Besides dust, rivers, and sediments, hydrothermal vents are one of the dominant sources of Fe to the ocean (see Fig. 1 ) 9 , 12 – 15 . For a long time, it was assumed that Fe precipitates nearby vent outlets. However, in recent years, several studies have shown that organically bound Fe can be stabilized and transported over long distances of up to 4000 km in the open ocean 10 , 16 . Hydrothermal vents are also sources of dissolved organic matter (DOM) 17 , 18 , which potentially include Fe-binding ligands 19 , 20 , and host specialized microbial communities 21 that might be able to reduce Fe-toxicity by actively producing Fe-binding organic ligands. An analogous ability has been demonstrated by vent microbes of the Mid-Atlantic Ridge in laboratory-based experiments with copper (Cu) 22 at levels above the required threshold, and similar processes could be assumed for Fe. This assumption is supported by field studies quantifying Fe-binding ligands in hydrothermal plumes and geochemical models, both confirming that organic ligands are crucial for the distribution of hydrothermal Fe in the water column and for mediating Fe-availability to marine microbes 8 , 23 , 24 . Figure 1 Schematic sketch of the importance of Fe within a plume environment including parameters considered in this study. Shown is a hot, focused Fe-enriched hydrothermal fluid vent with associated buoyant plume. Within the vent and plume, endemic microbes interact with dissolved organics (DOM) and potentially produce Fe-binding ligands. These interactions are crucial for the potential of Fe being exported away from the vent source. The bulk plume water sample (yellow star) used to set up the incubated dilutions (50 mL plume sample + 450 mL artificial seawater medium + individual Fe amendment) was collected using a rosette water sampler equipped with 20 L bottles and a CTD sensor from the deep plume maximum at 1549 m water depth at Brothers volcano. Figure 2 Design of the conducted 7-day incubation experiments. Triplicates (one replicate was lost for 1 mM Fe-level) consisting of 50 mL plume sample plus 450 mL artificial seawater medium (ASW) were set up for a range of spiked Fe-concentrations (Fe(II)SO 4 , 0 µM, 0.1 µM, 1 µM, 10 µM, 100 µM, 1 mM, and 10 mM Fe). Pure 500 mL ASW triplicates were set up as controls. After 7 days of incubation at 4 °C in the dark, the bottles were sampled for characterization of the indicated parameters. To date, studies on hydrothermal plumes have either dealt with identifying the dominant microbial lineages and relating that with general chemistry 25 , assessing the genomic and/or transcriptomic potential 26 , describing and characterizing Fe-binding ligand concentrations/compositions, or measuring Fe-concentrations 27 . However, a holistic experimental approach combining microcosm experiments along Fe-gradients with spiked plume water samples, where microbial community changes are monitored along with evolving molecular characteristics of DOM and Fe-binding organic ligands, is still lacking. In this pilot study, we describe such a systematic experiment and evaluate how variable Fe-concentrations in an incubation solution using natural plume samples affect the structure of microbial vent communities, the composition of related DOM, and excess Fe-binding ligand concentrations.",
"discussion": "Discussion This pilot experimental study provides first insights into how plume microbial communities might respond to variable Fe-enriched environments. Microbial community structure was assessed in conjunction with Fe, Fe-binding ligands, and SPE-DOC levels, as well as the SPE-DOM composition along a broad Fe-gradient. The plume microbes, dominated by some Campylobacterota ( Sulfurimonas ) and Gammaproteobacteria (SUP05 clade), react to Fe-addition possibly by producing organic complexes, i.e. ligands, however, a small proportion of passively released Fe-binding ligands from virus induced cell lysis cannot be excluded 27 , 44 . The excess of ligands found in each sample might point to microbial strategies for managing Fe-availability in low-Fe conditions and avoiding cell encrustation in Fe-replete conditions which allows these plume microbes to survive in dynamic habitats with various Fe-gradients such is the case for hydrothermal plumes or vents 2 , 3 , 11 . While a mixture of Sulfurimonas and SUP05 clade (± Arcobacteraceae , Oleispira and Massilia ) coexist at low to intermediate Fe-levels (0–100 µM), in the extreme 10 mM Fe-amended incubations, the bacterial community clearly shifted to include a dominance (~ 93%) of SUP05 (± Massilia ) affiliates (see Fig. 4 A). This shift is in line with another recent study finding that different plume adapted specialized Gammaproteobacteria of SUP05 clade are characterized by a high expression of heavy-metal resistance (e.g., for avoiding cell encrustation) and Fe-acquisition genes and hence might be exclusively capable of dealing with such high Fe-levels 35 . The clear dominance of SUP05 in the original plume sample despite it’s relatively low Fe contents (0.072 µM) could be interpreted to mean that most of the microbes in the plume were actually exported from near the vent, where mM Fe concentrations levels are frequently encountered in the dynamic environment at Brothers 45 , 46 . In addition, the anoxic conditions set in the incubations (H 2 :CO 2 purging of the solution and replacement of the bottle headspace) could have favored Sulfurimonas compared to the more oxygenated environment of the plume (> > 150 µmol/kg O 2 ). While both bacteria are found in similar environments (suboxic, anoxic and sulfidic), Sulfurimonas was found to dominate over SUP05 under more anoxic conditions (anoxic and sulfidic) 31 . While the total SPE-DOC concentrations increase is rather indistinguishable over all incubations, the SPE-DOM degradation index I DEG indicates a considerably more degraded DOM composition at lower Fe-concentrations (see Figs. 3 B, 4 B and Table 2 ). It is likely that as Fe content increased, the microbes had to produce more and more ligands to avoid cell encrustation and overall microbial growth was inhibited, ultimately resulting in a less diverse SPE-DOM composition. Further support for this hypothesis is provided by evaluation of Fe-containing formulae within the SPE-DOM mixture (see Fig. 3 C). A considerably higher diversity of individual Fe-formulae (~ 100) in the low-Fe-incubations (0 and 0.1 µM) may reflect a more versatile microbial community structure. At higher Fe-concentrations (> 10 µM) only very few Fe-containing formulae remain (~ 10) and most of the ones with N-heteroatoms disappear (see Supplemental Table S2 ). Synthesizing compounds with heteroatoms requires more effort and thus the absence of these compounds would be in line with a greater stress level caused by toxic Fe-concentrations. Simultaneously, part of the SPE-DOM compositional variability clearly appears subjectable to a selective DOM-Fe-coagulation that increases with Fe-concentration. The nature of the related changes is mostly in agreement with previous findings for Fe-coprecipitation with more terrigenous DOM (preferential removal of larger oxygen-rich unsaturated high NOSC compounds) 40 . But in contrast to these findings, SPE-DOM aromaticity increases with Fe and the bimodal split (with SUP05 and Massilia clades dominating at 10 mM Fe) is reflected in the reactivity related functional diversity index D F (NOSC). Overall, SPE-DOM variability depicts noticeable trends that are in agreement with broader microbial activity as well as the selective DOM-Fe-coagulation, and even suggests a relationship between specific microorganisms and certain formulae (Fe- and non-Fe-containing). In conjunction with Fe, ligand and microbial systematics, the broad range of parameters monitored in this pilot experimental study delivered a multitude of new potential implications regarding the significance of Fe in a hydrothermal plume environment. Future targeted experiments are needed to unequivocally differentiate and quantify the feedbacks between microbial community, dissolved organics including organic ligands, abiotic ligands and Fe-organic coagulation under different Fe-enriched conditions."
} | 2,776 |
36188001 | PMC9517587 | pmc | 1,701 | {
"abstract": "In this study, electrogenic microbial communities originating from a single source were multiplied using our custom-made, 96-well-plate-based microbial fuel cell (MFC) array. Developed communities operated under different pH conditions and produced currents up to 19.4 A/m3 (0.6 A/m2) within 2 days of inoculation. Microscopic observations [combined scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS)] revealed that some species present in the anodic biofilm adsorbed copper on their surface because of the bioleaching of the printed circuit board (PCB), yielding Cu2 + ions up to 600 mg/L. Beta- diversity indicates taxonomic divergence among all communities, but functional clustering is based on reactor pH. Annotated metagenomes showed the high presence of multicopper oxidases and Cu-resistance genes, as well as genes encoding aliphatic and aromatic hydrocarbon-degrading enzymes, corresponding to PCB bioleaching. Metagenome analysis revealed a high abundance of Dietzia spp., previously characterized in MFCs, which did not grow at pH 4. Binning metagenomes allowed us to identify novel species, one belonging to Actinotalea , not yet associated with electrogenicity and enriched only in the pH 7 anode. Furthermore, we identified 854 unique protein-coding genes in Actinotalea that lacked sequence homology with other metagenomes. The function of some genes was predicted with high accuracy through deep functional residue identification (DeepFRI), with several of these genes potentially related to electrogenic capacity. Our results demonstrate the feasibility of using MFC arrays for the enrichment of functional electrogenic microbial consortia and data mining for the comparative analysis of either consortia or their members.",
"introduction": "Introduction Microbial fuel cells (MFCs) are a type of chemical fuel cell in which the anodic reaction is catalyzed by various microorganisms that oxidize organic matter. When coupled with the cathodic reduction of oxygen, this system yields energy in the form of electricity. Given the exponential growth of studies focused on MFCs and electrogenic bacteria in general ( Santoro et al., 2017 ), numerous reactor designs have been developed. However, the singularities of these systems render the reproducibility of experiments extremely difficult. Prior focus on unifying reactor conditions to study extracellular electron transfer (EET) has been on manufacturing multiple stand-alone microbial reactors, such as those based on small glass vials ( Call and Logan, 2011a , b ). The reactor design, materials used, dimensions, and electrochemical properties (e.g., internal resistance) have differed between research groups. Hou et al. (2009 , 2011 , 2012) demonstrated the use of 24-well plate arrays comprising microfabricated gold electrodes with ferricyanide ( Hou et al., 2009 ), air-cathodes ( Hou et al., 2011 ), or microfluidic channels with continuous anolyte and catholyte replenishment ( Hou et al., 2012 ), which increased the power output by a factor of three. These reactors were used to screen previously selected electrochemically active environmental isolates. Another example of a well plate array implementation was demonstrated by Yuan et al. (2011) , in which EET was coupled to the color change of the probe. Recently, Molderez et al. (2021) constructed a 128-channel potentiostat connected to a printed circuit board (PCB) microarray. The entire microarray was immersed in an anolyte solution and supplied with a reference electrode to perform a high-throughput investigation pertaining to the effect of the anodic potential on electroactive biofilm growth. Zhou et al. (2015) proposed a well-plate-based, high-throughput colorimetric assay for microbial electrochemical respiration to indicate EET. Alternatively, a 48-well plate with a hydrophobic wax layer separating electrodes was developed by Choi et al. (2015) , followed by a single-sheet paper-based electrofluidic array (eight cells) developed by Gao et al. (2017) . Tahernia et al. (2019) developed paper-based, disposable 64-well arrays yielding power densities of up to 23 μ W/ cm 2 . The device has been successfully used to characterize the electrochemical properties of various Shewanella oneidensis and Pseudomonas aeruginosa strains. They later developed, a 96-well electrofluidic array using the same fabrication method ( Tahernia et al., 2020 ). Recently, another 96-microwell device (four 24-well modules) was demonstrated, with three electrodes and gas outlets to maintain anaerobic environment ( Kuchenbuch et al., 2022 ). These newly developed MFC platforms allow higher precision in comparative studies of electrogenic biofilms. Metagenomes derived from electrogenic communities encode many unique genes that help with EET. Moreover, metabolic pathways within electrogenic communities can specialize in the degradation of toxic compounds and precipitation of heavy metals, thereby offering energetically favorable alternatives to existing bioprocesses. Metagenomic assembly and annotation have become increasingly informative with the constant growth of databases. For example, the number of reference human gut bacterial genomes increased from 194 in 2010 ( Qin et al., 2010 ) to 204938 in 2021 ( Almeida et al., 2021 ). Advances in sequencing technology now allow single genomes to be assembled directly from the metagenome, creating metagenome- assembled genomes (MAGs)( Tyson et al., 2004 ). Despite being more abundant than human microbiomes, environmental metagenomes are less resolved, with only 52,515 MAGs ( Nayfach et al., 2021 ). MAGs have allowed for the discovery of novel metabolic pathways, such as commamox ( Daims et al., 2015 ). However, as the majority of MAGs are uncultured organisms, it is not surprising that a substantial proportion of genes lacks functional annotation. To make a comparative analyses of metagenomes easier, several tools for de novo annotation have been developed ( Chen et al., 2020 ; Almeida et al., 2021 ; Beghini et al., 2021 ; Nayfach et al., 2021 ). In this study, we present a 30-day enrichment of electrogenic consortia derived from a single inoculum [air-conditioning (AC) outflow] fed with an identical substrate. We applied different pH conditions to those consortia within identical reactors to compare their electrochemical performance in relation to changes in microbial community structure. We sampled anodic biofilms from all tested groups and visualized them under an electron microscope. Furthermore, we performed metagenomic sequencing and subsequent genome assembly and annotation from different compartments of the MFCs [anodes, cathodes, and open circuit potential (OCP) controls], which allowed for the identification of new species from the MAG isolates. Unannotated, unique genes present in novel electrogenic MAGs were further investigated using in silico analysis tools such as DeepFRI ( Gligorijevic et al., 2021 ) and AlphaFold ( Jumper et al., 2021 ).",
"discussion": "Discussion Our analysis identified the novel electrogenic bacterium Actinotalea sp. nov. has been identified in the anodic community of our MFC. Actinotalea is a high GC, gram-positive, facultative anaerobic genus derived from Cellulomonas ; the main taxonomic difference is the major respiratory quinone menaquinone, with MK-10(H4) in the former and MK-9(H3) in the latter ( Yi et al., 2007 ; Jin et al., 2017 ). Actinotalea can survive across a wide pH spectrum (4–11), although the most optimal conditions for its growth was pH 7 in our test. Other species from this genus have been found in soil ( Suman et al., 2014 ; Yan et al., 2018 ), iron mine ( Li et al., 2013 ), and biofilm reactors ( Jin et al., 2017 ). The presence of numerous sequences related to electron transfer in its genome, e.g., the type IV pilus biosynthesis gene pilB , confirmed that this organism can respire through anodes, just like the well-studied electrogen Geobacter ( McCallum et al., 2017 ). It would be interesting to compare the expression levels of the Actinotalea ’s unique genes while grown in the MFC wrt OCP. Moreover, the presence of novel and unique enzymes, such as NADH translocases, could provide resilience to high Cu content. Many of the unannotated sequences obtained from the Actinotalea MAG received medium DeepFRI prediction scores, which may indicate the presence of a novel homologue; however, this must be experimentally validated. Overall, the metagenomes analyzed in this study contained many genes encoding pathways related to anaerobic respiration. The inoculum derived from the AC outflow also consisted of marine bacterial species originating from the deep sea. Abrevaya et al. (2015) previously found Dietzia to be enriched in MFCs, although it is mostly found in oil-contaminated marine environments ( Yumoto et al., 2002 ). Interestingly, despite its alleged alkalinity, we found the abundance of Dietzia to be similar in both pH 4 and pH 7 anodic samples ( Figure 3 ), and binned contigs from both samples revealed 99.9% sequence similarity between the MAGs. This suggests that Dietzia can withstand periods of lower pH, since the pH increased from 4 to 7 in our pH 4 group. Moreover, its abundance in anodic biofilms may be explained by its ability to degrade numerous hydrocarbons ( Yumoto et al., 2002 ). Owing to bioleaching of the PCB, Cu together with the organic epoxy coating become oxidized, thus creating compounds toxic to many bacteria. Since Dietzia can degrade these compounds, its abundance in anodic consortia is not surprising. The high abundance of EET-related and hydrocarbon degrading genes in pH 7 OCP, as seen in Table 1 , as well as the total number of unique annotations may reflect higher metabolic diversity of this community due to the lack of external electron circuit. Indeed previous studies comparing MFC communities ( Ishii et al., 2018 ; Kouzuma et al., 2018 ; Szydlowski et al., 2020 ) reveal higher functional diversity in communities grown at OCP, whereas the presence of external circuit applies selective pressure on the microbial metabolism. Despite our efforts to separate the PCB from electrogenic bacteria, our results revealed that chemically deposited Au on the electrodes did not provide a sufficient barrier for the PCB and ultimately led to its corrosion. Such problems could be avoided by using PCBs with the so-called hard gold finish (electrochemically plated gold) or by covering the PCB anode with other conductive materials, such as stainless steel sheets, thus protecting the underlying Cu from being oxidized. Indeed, the electrochemical plating of the PCB successfully prevented Cu leakage in our modified well plate ( Szydlowski et al., 2022 ). Owing to this unexpected leakage of Cu ions, we successfully enriched the Cu-resistant communities using our 96-well platform. Through SEM analysis, we observed that the anodic biofilm comprised species that concentrated Cu on their surfaces, leaving other parts of the biofilm relatively Cu-free. Therefore, it is tempting to suggest that this mechanism can be mediated by direct interspecies EET or electron bifurcation using Cu ions, as in the FixABCX ( Ledbetter et al., 2017 ) or Rnf ( Kuhns et al., 2020 ) complexes. Especially Rnf-encoding genes may be particularly interesting since unique genes were identified in the Actinotalea MAG that are putative members of the rnf family ( Figure 5 ). Rnf enable difficult reductions (reviewed in Buckel and Thauer, 2018 ), which may indicate the ongoing degradation of the PCBs’ organic compounds, such as epoxy resins. Based on the studies describing Cu-bioleaching from PCB waste ( Yang et al., 2009 ; Pant et al., 2012 ; Wu Y. et al., 2018 ), it can be concluded that Cu mobilization from its zero-valent state may be due to the bioelectrochemical cycle, which is also depicted in the model proposed by Becci et al. (2021) . Copper can be usually oxidized by the simultaneous reduction of Fe 3+ to Fe 2+ and its efficiency depends on the reoxidation of iron back to Fe 3+ . Iron oxidation may be catalyzed by bacteria in the presence of oxygen, as well as alternative electron acceptors, such as electrodes, which in turn may explain the high current densities observed in our reactors. Moreover, simultaneous proton consumption that accompanies Fe reoxidation ( Becci et al., 2021 ), can explain the pH shift observed in our study, as well as the differences in Cu content ( Figure 1E ). Wu W. et al. (2018) demonstrated a bacterial-free supernatant derived from Fe/S-oxidizing bacteria, also known for their electrogenic activity, resulting in complete Cu recovery from wasted PCBs. Other studies on the metallurgic, Cu-removing MFCs demonstrated that electrogenic consortia can reduce Cu from aqueous solutions and precipitate it on electrodes. The efficiency of this process depends on various factors, such as the use of pH separators (membranes), presence/absence of oxygen, and initial copper concentration ( Heijne et al., 2010 ; Tao et al., 2011a , b ; Motos et al., 2015 ; Miran et al., 2017 ). Given that Cu was cycling from solid state (PCB) to the solution and precipitated back to the anodic biofilm, the microbial communities enriched in our well plate exhibit a combination of oxidative and reductive bioleaching ( Brar et al., 2021 ), which may also indicate the fluctuations in the current densities ( Figure 1D ). Our results illustrate the extent of selective enrichment of our MFC array, which yielded electrogenic microbial communities capable of adapting to different physical conditions, such as pH and electric circuits, and resulted in bioleaching of the PCB. Through a combination of phylogenetic analysis and metagenome binning with in silico functional assays, we were able to identify, characterize and compare the microbial communities and link their features to the electrochemical performance that was measured in unified array conditions. This platform is a rapid, high-throughput system that offers parallelization for the screening of electrochemical microorganisms, as well as comparative analysis of functional metagenomes."
} | 3,547 |
24728373 | PMC3984134 | pmc | 1,702 | {
"abstract": "The capacity of reef-building corals to associate with environmentally-appropriate types of endosymbionts from the dinoflagellate genus Symbiodinium contributes significantly to their success at local scales. Additionally, some corals are able to acclimatize to environmental perturbations by shuffling the relative proportions of different Symbiodinium types hosted. Understanding the dynamics of these symbioses requires a sensitive and quantitative method of Symbiodinium genotyping. Electrophoresis methods, still widely utilized for this purpose, are predominantly qualitative and cannot guarantee detection of a background type below 10% of the total Symbiodinium population. Here, the relative abundances of four Symbiodinium types (A13, C1, C3, and D1) in mixed samples of known composition were quantified using deep sequencing of the internal transcribed spacer of the ribosomal RNA gene (ITS-2) by means of Next Generation Sequencing (NGS) using Roche 454. In samples dominated by each of the four Symbiodinium types tested, background levels of the other three types were detected when present at 5%, 1%, and 0.1% levels, and their relative abundances were quantified with high (A13, C1, D1) to variable (C3) accuracy. The potential of this deep sequencing method for resolving fine-scale genetic diversity within a symbiont type was further demonstrated in a natural symbiosis using ITS-1, and uncovered reef-specific differences in the composition of Symbiodinium microadriaticum in two species of acroporid corals ( Acropora digitifera and A. hyacinthus ) from Palau. The ability of deep sequencing of the ITS locus (1 and 2) to detect and quantify low-abundant Symbiodinium types, as well as finer-scale diversity below the type level, will enable more robust quantification of local genetic diversity in Symbiodinium populations. This method will help to elucidate the role that background types have in maximizing coral fitness across diverse environments and in response to environmental change.",
"conclusion": "Conclusions In summary, this study is the first to evaluate the ability of NGS to quantitatively analyze samples with known densities of Symbiodinium types. We demonstrate here that NGS of Symbiodinium diversity is sensitive and quantitative, with a detection threshold at 0.11% of 1×10 6 cells. Importantly, we also show that NGS is highly applicable for discerning haplotype-level diversity in natural coral populations. These results demonstrate that NGS has the potential to elucidate the diversity and abundances of background Symbiodinium types, either when endosymbiotic within coral hosts or possibly free-living in the environment. Further in-depth profiling of total Symbiodinium complements within host corals, now possible with this technique, will provide new insights into the relative abundance of Symbiodinium -type specialist and generalist corals [14] , [97] , and will enable the development of better models to predict host susceptibility to stress events. Our results demonstrate that this new methodology will significantly advance the evolutionary and ecological understanding of this important photosymbiont.",
"introduction": "Introduction Coral reefs are one of the most biodiverse ecosystems on earth [1] , largely as a consequence of the symbiosis that exists between scleractinian corals and endosymbiotic dinoflagellates within the genus Symbiodinium \n [2] . The physiology and health of the coral host relies heavily on carbon translocation from these symbionts [3] , [4] , which enhances calcification of the coral host and leads to accretion of present day coral reefs [5] . The stability of this symbiosis is threatened by many factors, such as chronic and acute changes in CO 2 \n [6] , temperature [7] , and irradiance [8] . These factors elicit stress responses from the coral holobiont (cnidarian host and associated dinoflagellate, bacterial and viral communities), causing the breakdown of symbiosis and loss of Symbiodinium from host tissues, a phenomenon known as bleaching [9] . Predictions of increased frequency and intensity of bleaching events represent a major threat to reef biodiversity and long-term viability of this important ecosystem [10] , highlighting the need to fully understand the diversity and population dynamics of Symbiodinium types associated with corals. Currently, nine genotypic clades are recognized within the genus Symbiodinium (A through I), with a range of types recognized within each clade (e.g. C1, C2, C3) [2] , [11] . The relationship between sequence and physiological diversity of these dinoflagellates is still being investigated [12] . At least some coral species have been shown to harbor multiple Symbiodinium clades and types [13] , [14] in abundances ranging from high (dominant) to low (background or rare) proportions of the endosymbiont community [15] , [16] , in associations that can vary in time and space. Uptake of novel types from the environment by adult corals (“symbiont switching” [2] ) has received little experimental support (but see [17] ), however, the relative abundances of pre-existing symbiont types can change substantially within the coral host as a result of environmental stressors (“symbiont shuffling”) [18] , [19] , [20] . These changes in Symbiodinium type complements can strongly influence holobiont fitness characteristics. For example, specific types within clade D (ITS-1 and 2) provide tolerance to higher temperatures [16] , [18] , [20] , [21] , C1 (ITS-1) can enhance coral growth rates [4] , [22] , type B2 (ITS-2) can enable recovery particularly rapidly after cold stress [23] , and an unspecified type within clade A (ITS-2) confers high tolerance to elevated light [8] . Furthermore, holobiont physiological parameters can vary, not only with respect to the major clade of Symbiodinium hosted [21] , [22] , [24] , [25] , but also as a consequence of Symbiodinium type [26] and population within type [27] . The consequences of hosting functionally different clades, types and populations for coral physiology are increasingly appreciated [28] , however, the full extent of this variation and its impacts on holobiont health and resilience remain open questions [29] . Standard techniques such as Single Strand Conformation Polymorphism (SSCP) and Denaturing Gradient Gel Electrophoresis (DGGE) are commonly used in Symbiodinium research to determine symbiont distributions or changes in dominant types after stress events [30] , [31] . Briefly, SSCP and DGGE are gel-based electrophoresis techniques that allow for PCR products to be distinguished based on variation in nucleotide sequence [32] , [33] . The dominant bands retrieved from DGGE accurately represent members of the Symbiodinium community in high abundance [34] , and this coarse detection limit may make it immune to the accidental retrieval of non-symbiotic Symbiodinium found within the mucus or coral gastrovascular cavity. However, DGGE offers limited information about potentially crucial background Symbiodinium types due to poor detectability of low-abundant Symbiodinium types [16] and the generally qualitative rather than quantitative nature of this method. This has led to a major knowledge gap concerning the role of background types during ambient and stressful conditions [35] , despite their potentially critical role in reef resilience during times of stress [36] . This is especially important as stressors such as increased temperature and CO 2 may promote a change in the relative abundance of symbiont types through shuffling following bleaching [19] . Directional changes in relative symbiont abundances could change the holobiont's response to a bleaching stressor, representing relatively rapid acclimatization to increasingly stressful conditions. On longer time scales, symbiosis can also affect host speciation and niche diversification [37] . However, the extent to which Symbiodinium diversity and abundance influence a coral's capacity to adapt or acclimatize to warming and acidifying oceans is not yet fully understood and remains an area of active research [38] . Similar knowledge gaps concerning the role of low-abundance and/or uncultured marine microbial strains in other symbiotic organisms have been addressed using deep sequencing of DNA barcoded amplicons [39] . This research has highlighted the presence of core holobiont bacterial diversity and transient states associated with environmental stress [40] , [41] . NGS in particular has uncovered vast assemblages of low-abundance bacteria, termed the rare bacterial biosphere, in the water column and associated with various marine invertebrate hosts and has elucidated the function of low-abundance bacteria within the holobiont, for example, in nitrogen cycling [42] . Although the detection of low-abundance microbes and Symbiodinium in corals has improved vastly with the much higher detection rate and quantitative accuracy offered by quantitative polymerase chain reaction (qPCR) relative to gel fingerprinting [15] , [43] , this method may still be biased by primer efficiencies and exclude symbiont diversity that is not recognized by specific priming sequences. While great promise is afforded by NGS methods, care must be taken to identify sequencing error and partition sequence variation within and among genomes (i.e., inter- and intra-genomic variants) to provide meaningful data on the distribution and abundance of in hospite and free-living Symbiodinium , especially those in low abundance. Cultured genetic stocks of Symbiodinium offer an opportunity to benchmark sequencing reads to cell numbers to reveal the presence of bias favoring dominant species [44] and its ability to distinguish real sequence variants from sequencing errors [45] . If quantitative and sensitive, NGS marker gene survey techniques [45] offer great potential for the detailed characterization of Symbiodinium diversity in time and space, including an examination of the rare Symbiodinium biosphere. The utilization of the rDNA operon (which includes the ITS-1 and ITS-2 loci) has been pivotal to understanding Symbiodinium molecular diversity [46] . However, the high intra-genomic variation of this region and the tandem array of multiple ITS copies (multicopy) mean that assigning natural diversity is problematic [34] , [47] , [48] . For example, ITS-2 can be more variable, at times, between cells of the same Symbiodinium type than between types [48] . Furthermore, Symbiodinium types contain multiple and variable copy numbers of ITS loci, for example, clade D has an approximately 3-fold higher ITS-1 copy number than clade C (D: 3,181±69, [15] ). Identifying real unique taxonomic units from variable intra-genomic copies exhibiting sequence variation remains a challenge in Symbiodinium research [34] . 454 NGS has been used to explore intra-genomic variation along the ITS-2 region for hundreds of plant species, and shown that only a few intra-genomic variants make up the vast majority (91%) of sequence reads retrieved per species [49] . NGS data may similarly allow for the inspection of intra-genomic variation within single types of Symbiodinium \n [50] , however, further data are required before these analyses can be validated. Here, we investigate the sensitivity and accuracy of marker gene survey techniques using the Roche 454 method to detect and quantify both dominant and background Symbiodinium types in mixed samples of known Symbiodinium type composition. This study also explores whether different symbiont clades and types are equally detectable using 454 NGS. We then demonstrate the potential of the new method for studies of natural coral populations by uncovering finer-scale, population-level differences in the genotypic composition of Symbiodinium within the same type hosted by corals from two reef sites in Palau.",
"discussion": "Discussion The capacity to detect and quantify the abundance of Symbiodinium types associated with corals is essential for studies aimed at understanding holobiont physiology, susceptibility to stress and, ultimately, the resilience of corals to environmental change. Our results confirm that sequencing of the ITS-2 region using 454 NGS is able to detect the presence of co-occurring Symbiodinium types D1, C1, C3 and A13 at abundances as low as 0.1% of 1×10 6 cells i.e., 1000 cells per sample. Amplicon sequencing of the ITS-1 region for Symbiodinium types associated with acroporid corals from Palau also demonstrated that this NGS approach can detect haplotype variants of Symbiodinium microadriaticum ITS-1 populations when in hospite , and distinguish differences in their frequencies among colonies and between sites that are less than 28 km apart. Our method is therefore well placed to detect and quantify rare, low-abundant haplotype variation within symbiont types that are likely under-represented by current methods of Symbiodinium detection (i.e DGGE and SSCP). We conclude that next generation sequencing will play an important role in providing a clearer understanding of microbial diversity and interactions between symbionts and marine metazoan hosts, including important groups like scleractinian corals. 454 NGS was able to quantify the abundances of types at low background levels (0–5%), whether they originated from cultured material or from freshly extracted DNA from frozen or ethanol-preserved tissue. This can be attributed to the 99.75% sequencing accuracy after clean-up and the high depth and coverage afforded by next generation sequencing [69] . For type C3, however, the degree of correlation between expected and observed background abundances was much weaker than for the three other symbiont types and not statistically significant. This may be due to high sequence similarity between C1 and C3, which led to more variable data for observed abundances (normalized read counts) compared to the other types. It is likely that small SNP PCR errors impact highly similar sequences differentiated by only one bp (as with C1 and C3 NCBI sequences), affecting clustering and/or mapping during bioinformatics processing and accounting for the detection of high numbers of C3 sequences in the pure 100% C1 sample. However, this method represents a significant improvement on those currently available for quantifying de novo background abundances of Symbiodinium . Although no overall trend in inflation was found in residual plots from 0.1–5%, symbiont abundances were marginally inflated when they comprised 0.1% of cells in mixed Symbiodinium samples. Estimates for types C1 and C3 were 1.8–3.9% higher than expected, and those for D1 and A13 only 0.15–0.26% higher than expected. The largest deviations of observed from expected abundances occurred when C1 or C3 were in high abundance (for example, 60.7% C1 observed in a sample expected to be 85% C1). Such deviations were evident in most C1-dominated samples (3, 8, 11, 15), and especially in Sample 15, where 15.6% (1032 reads) of sequences were annotated as C3 in the 100% C1 sample. PCR or sequencing errors and uncertainty in clustering and mapping caused by high sequence similarity between C1 and C3 are likely to have contributed to these contradictory values, increasing the number of sequences identified as C1 at the expense of C3, as suggested by the C3 gamma correlation. Interestingly, our C3 population analyzed here may also include cells that contain sequences intermediary between those of other C3 and C1 populations, caused by the presence of both C3 and C1 sequences within the ITS-2 operon of this particular C3 population. Intermediary types, like C3h between C3 and C21, or type C3i between C3 and C1, have been documented and may have arisen through sexual recombination between the two types [70] , [71] , [72] . Hypotheses exploring the ecology and evolution of Symbiodinium can therefore be tested with NGS data. Detection of false-positives in pure samples 454 NGS detected low-abundant symbiont types in pure samples that were not expected in these single-type samples. Most pure samples returned 1–15 reads (4.83 reads ±2.1 SE), equivalent to 0.07%±0.02 SE, annotated as non-pure types. The exception was the C1 pure sample (Sample 15), which returned 1032 reads that matched C3 reference sequences C3 e (1.9%), h (81.8%), and x (16.3%). Unexpected reads may have occurred for a number of biological or technical reasons, including the presence of both C3 and C1 sequences within the same genome, contamination of other types or haplotypes that escaped SSCP detection during the genotyping of type C3 from frozen coral samples, clustering/mapping errors in bioinformatics processing or contamination at the cell culture or mixture stages. If we assume that the most likely explanation is that non-C3 reads found in pure samples signify contamination or PCR/454 error, the overabundance of C3 in Sample 15 (15.6%) becomes a biological outlier, which can be discounted. Accordingly, we propose a conservative detection limit cut-off at >0.11% ± two SEs (0.02). Read depth and coverage The low number of sequence reads found for background types at expected abundances of 0.1% (1–74 sequences) raises questions about the number of sequences that are sufficient to confirm the identification of a symbiont type in a sample. Here, we use the depth of sequencing to discern the number of positive sequences required to parse out signal from noise and therefore set the detection limit of our assay. We set a 10,000 sequence minimum read number per sample for the mixed dilution samples, which would allow detection of minor types in 0.1% abundance with a coverage of 10. Nevertheless, some samples ended up with more or less reads representing minor types (1 to 74 reads). A single mapped read in a sample may be indicative of actual diversity; however, it is important to distinguish between single reads per sample and single reads across the whole data set. A single read in the data set with a unique identity (a unique haplotype or reference sequence) may equally represent a rare read variant (true diversity) or a PCR/sequencing error (false diversity). However, one read in a single sample may be more likely to represent true diversity if it is retrieved across multiple samples many times. Reads in our samples were only mapped to reference sequences if clusters had more than 10 reads in the combined dataset, thus eliminating singletons and many rare reads that had a high probability of being false positives. Robustness in detection may be increased by: 1) using biological replicates in the experimental design, 2) sampling greater than 1 million cells, i.e. more than one cm 2 of coral tissue [73] , or 3) sequencing at a higher coverage, albeit at an increased price per sample. These strategies will not only enable ecologically relevant distinctions in symbiont presence to be made, but will also increase detection of low abundance types. Quantifying sequence reads using NGS A common issue in NGS marker gene surveys is how to relate read abundance with taxon abundance [44] , [74] . The use of both multicopy and intragenomically variable loci for sequencing, in addition to biases associated with DNA extraction, PCR and 454 NGS, have led to a debate concerning whether read counts can be used for quantification purposes [44] , [74] , [75] . For example, NGS surveys of known dilution mixtures of fungal [44] and algal species [76] found order of magnitude differences in abundance estimates between species, and significant differences after filtering/clean-up steps in the number of reference sequences retrieved per species, however, intra-genomic variants or copy number were not accounted for in these studies. Alternatively, bacterial sequencing trials show 454 NGS to be both reproducible and quantitative [75] , although some authors suggest that differences in read abundances between samples should only be compared within species [44] . It is likely that errors in quantifying type C3 in our study are related to copy number issues or sequence similarities with C1 at this locus. New computational methods employing locus copy numbers are now able to more accurately detect diversity and quantify species within environmental data sets [74] . 454 NGS detects and quantifies fine-scale variation in Symbiodinium populations in hospite Our results demonstrate the presence of variation in Symbiodinium diversity and population composition at much finer scales than previously detected. At the level of Symbiodinium type, symbiont diversity has been shown to vary with host species and biogeographic region, and in response to reef environment and depth [61] , [70] , [77] , [78] , [79] , [80] . Detection of variation in Symbiodinium diversity between sites at the haplotype level, using a small sample size (N = 3 corals per site), highlights the levels of symbiont diversity the NGS approach is able to uncover. In addition, use of cloning in previous studies has restricted the number of sequences analyzed (e.g. [61] ) compared to NGS. Differences in the proportion of symbiont haplotypes between the two reef sites, which are separated by ∼28 km, might reflect environmental specialization, perhaps to differences in wave exposure at the two sites (WC is more exposed than LH), indicative of local adaptation of the holobiont. Further research into variation in Symbiodinium population composition with environmental variation and manipulative experiments are needed to test this hypothesis. Detection and quantification thresholds for 454 NGS as compared to DGGE and qPCR Two methods, DGGE and qPCR, are typically used to detect and quantify Symbiodinium ; the former generally accepted as a non-quantitative technique [81] , with detection thresholds of 10–30% of total symbiont abundance [30] , [82] , [83] . More recently, the high sensitivity of qPCR, which has 1000-fold greater detection ability than gel fingerprinting [15] , [36] , has made it a popular technique for detecting intra-clade types and for quantifying Symbiodinium . The detection limit for qPCR has been suggested to be roughly 7,000 cells per 1.5×10 6 sample if using a single copy marker [36] , equivalent to a 0.46% detection threshold. Despite this benchmark for detection, further experimental work is needed to determine the number of cells required for accurate and precise quantification of Symbiodinium abundance using qPCR because variability in amplification exists between clades D, A, B and C [36] . We did not detect amplification bias toward any clade or type with our NGS method, however, we did encounter bioinformatics challenges in separating types with high sequence similarity. With enhanced bioinformatics pipelines, sequencing using NGS has a greater capacity to detect and quantify Symbiodinium abundance when present at densities as low as ∼1,000 cells in 1×10 6 (0.1%) than either qPCR or DGGE. Finally, it is important to note that, unlike DGGE, 454 NGS does not appear to be a subjective technique. DGGE bands must be identified for each symbiont type and compared to other single bands or combinations of bands in adjacent lanes, introducing subjectivity in identifying their presence or absence. In comparison, the bioinformatic steps involved in NGS, which compare individual bases in each sequence using a standardized algorithm, remove such subjectivity. Furthermore, the sequencing data from NGS is able to differentiate intra-type variation (i.e. individual haplotypes) as well, as suggested by the numerous positively correlated haplotypes found for all symbiont types tested here. Thus far, only microsatellites have exhibited the ability to discern below the type level [84] , however, specific microsatellites must be developed for most clades and types [52] and detection is limited to targeted loci, eliminating the possibility of finding novel diversity. The development of new loci for amplicon sequencing [85] , possibly applied together with historically used markers such as ITS, will enable enhanced resolution to differentiate both clades and types. For example, the chloroplast DNA psbA ncr locus is able to distinguish closely related types, but has limited resolution across clades [86] . The difficulty of differentiating between C3 and C1 in this study may therefore be ameliorated with the application of new and/or additional markers for NGS. \n Symbiodinium copy number and intra-genomic variation at the ITS-1 and ITS-2 loci Many genes utilized for resolving Symbiodinium taxonomies are multicopy [16] , possibly resulting from numerous complete and partial duplications of genes and genomes, as commonly seen in dinoflagellates [87] , or through the integration of foreign DNA [88] . Attempts have been made to find single copy loci and determine copy number of known markers [85] and at least six other commonly used loci are multicopy: PsbA, Cp23S, 28S/5.8S, ITS-2, 18S [15] , [89] , [90] , [91] , [92] . For example, the actin region has seven copies in the clade C genomes tested and ∼1 copy in clade D genomes [16] . Pairwise correlations shown here suggest that the ITS-2 regions of types D1, C3 and C1 consist of multiple intra-genomic variants, results that may reflect the multicopy nature for the clades to which these types belong [16] . Therefore, finding a single-copy marker is essential to eliminate ambiguity stemming from Symbiodinium intra-genomic variability. This is particularly true in the context that ecologically relevant diversity likely exists on a continuum, from every read retrieved representing a unique haplotype (option 1), to the grouping of all read variants as intra-genomic variants of the same single symbiont type (option 2) [61] . The expected abundances presented here account for ITS-2 copy number by equating different reference sequences to represent intra-genomic variants, an approach that parallels option 1 of Stat and colleagues [61] . Further use of NGS data in conjunction with Symbiodinium genomic databases will play an important role in the identification and confirmation of intra-genomic variation across types. As the mixture data presented here were of known diversity, read variants remaining after quality-control measures were assumed to be intra-genomic variants, and thus were pooled, enabling estimates of both diversity and abundances. However, the main challenges for applying this technique to natural samples with multicopy regions that exhibit intra-genomic variability will be: 1) distinguishing between variant sequences that represent intra- versus inter-species diversity; and 2) quantifying abundances. The use of NGS with a single-copy marker (and therefore no intra-genomic variation) that is able to detect equally well across Symbiodinium type diversity would clarify both detection and quantification problems. However, as ITS-2 is the predominant marker currently used to assign Symbiodinium diversity, intra-genomic variation in environmental data sets may be discerned using secondary structures and homology modeling [93] , [94] . Indeed, the use of secondary structure analysis has been used previously in the construction of Symbiodinium and coral phylogenies [34] , [95] . Developing more strategies in addition to pairwise comparisons [96] to account for Symbiodinium multicopy nature will improve both the precision and quantification of this method. Conclusions In summary, this study is the first to evaluate the ability of NGS to quantitatively analyze samples with known densities of Symbiodinium types. We demonstrate here that NGS of Symbiodinium diversity is sensitive and quantitative, with a detection threshold at 0.11% of 1×10 6 cells. Importantly, we also show that NGS is highly applicable for discerning haplotype-level diversity in natural coral populations. These results demonstrate that NGS has the potential to elucidate the diversity and abundances of background Symbiodinium types, either when endosymbiotic within coral hosts or possibly free-living in the environment. Further in-depth profiling of total Symbiodinium complements within host corals, now possible with this technique, will provide new insights into the relative abundance of Symbiodinium -type specialist and generalist corals [14] , [97] , and will enable the development of better models to predict host susceptibility to stress events. Our results demonstrate that this new methodology will significantly advance the evolutionary and ecological understanding of this important photosymbiont."
} | 7,187 |
38891516 | PMC11174759 | pmc | 1,703 | {
"abstract": "Poly(dimethylsiloxane) (PDMS) coatings are considered to be environmentally friendly antifouling coatings. However, the presence of hydrophobic surfaces can enhance the adhesion rate of proteins, bacteria and microalgae, posing a challenge for biofouling removal. In this study, hydrophilic polymer chains were synthesised from methyl methacrylate (MMA), Poly(ethylene glycol) methyl ether methacrylate (PEG-MA) and 3-(trimethoxysilyl) propyl methacrylate (TPMA). The crosslinking reaction between TPMA and PDMS results in the formation of a silicone-based amphiphilic co-network with surface reconstruction properties. The hydrophilic and hydrophobic domains are covalently bonded by condensation reactions, while the hydrophilic polymers migrate under water to induce surface reconstruction and form hydrogen bonds with water molecules to form a dense hydrated layer. This design effectively mitigates the adhesion of proteins, bacteria, algae and other marine organisms to the coating. The antifouling performance of the coatings was evaluated by assessing their adhesion rates to proteins ( BSA-FITC ), bacteria ( B. subtilis and P. ruthenica ) and algae ( P. tricornutum ). The results show that the amphiphilic co-network coating (e.g., P-AM-15) exhibits excellent antifouling properties against protein, bacterial and microalgal fouling. Furthermore, an overall assessment of its antifouling performance and stability was conducted in the East China Sea from 16 May to 12 September 2023, which showed that this silicon-based amphiphilic co-network coating remained intact with almost no marine organisms adhering to it. This study provides a novel approach for the development of high-performance silicone-based antifouling coatings.",
"conclusion": "4. Conclusions We present a silicone-based amphiphilic co-network coating with excellent antifouling and underwater surface restoration properties. TPMA was used as the crosslinking agent to form a covalently bonded network of hydrophilic and hydrophobic regions, effectively preventing macroscopic phase separation between the two incompatible components. In the amphiphilic coating, hydrophilic polymers undergo rapid migration to the surface in an underwater environment, resulting in structural changes to the surface. A dense hydration layer is formed with water molecules through hydrogen bonding, which further enhances the resistance to protein, bacterial and algal adhesion. Our experimental results show the excellent antifouling properties of this coating. Due to its exceptional properties and unique surface characteristics, our proposed amphiphilic co-network coating has great potential for marine antifouling applications and contributes to the advancement of amphiphilic polymer materials.",
"introduction": "1. Introduction Marine biofouling refers to the undesirable accumulation of microorganisms, algae and macrofouling organisms (e.g., barnacles, mussels, etc.) that attach to marine structures [ 1 , 2 , 3 ]. This process results in increased hull resistance to navigation, increased maintenance costs, increased fuel consumption and greenhouse gas emissions, and potential species invasions with consequent impacts on local ecosystems [ 4 ]. Antifouling coatings are recognised as one of the most cost-effective strategies to combat marine fouling [ 5 , 6 ], with fouling release coatings (FRCs) being non-toxic, environmentally friendly coatings that use hydrodynamics to prevent macrofouling organisms from adhering to their surfaces [ 7 , 8 , 9 , 10 ]. Poly(dimethylsiloxane) (PDMS) is a highly effective FRC due to its low surface energy and modulus of elasticity [ 8 , 11 ], which facilitates the efficient removal of macrofouling organisms such as barnacles by seawater washing. However, the effectiveness of PDMS-based coatings in preventing fouling organism settlement is limited by non-specific binding effects. In particular, PDMS exhibits inadequate resistance to the accumulation and growth of marine slime layers composed of bacteria, algae and the secretion of extracellular polymers [ 12 , 13 ]. Researchers have found that the formation of a hydrated layer by hydrophilic polymers (e.g., PEG-based coatings) effectively prevented fouling organisms from settling in the early stages [ 14 ]. However, once attached to the surface, the release of fouling organisms became difficult due to the properties of the coating [ 15 ]. Therefore, a more promising antifouling strategy is to create amphiphilic polymers by physically mixing or chemically crosslinking hydrophilic and hydrophobic polymers [ 16 , 17 ]. The amphiphilic modification of PDMS is the most commonly used strategy to improve its antifouling ability [ 18 ]. However, due to the thermodynamic incompatibility between hydrophobic and hydrophilic regions, this can lead to the macroscopic phase separation of PDMS from the hydrophilic polymer, compromising material stability [ 19 ]. Therefore, the formation of silicon-based amphiphilic co-network via a forced chemical crosslinking approach is an effective solution to address this issue. Zhao et al. [ 20 ] prepared a PVP-based hydrophilic polymer in which an antifouling silicone-based amphiphilic polymer for marine applications with excellent mud resistance was obtained by crosslinking between the reactive hydroxyl functional group of 2-hydroxyethyl methacrylate (HEMA) and PDMS. The use of silane coupling agents containing both siloxane groups and reactive functional groups as crosslinking monomers is an innovative strategy. Guo et al. [ 21 ] and Zeng et al. [ 22 ] successfully synthesised two novel silicone-based amphiphilic polymers using 3-isocyanatopropyltriethoxysilane and 3-mercaptopropyltrimethoxysilane, respectively, which exhibited exceptional antifouling properties. HEMA forms hydrophilic chains with antifouling active monomers through radical polymerisation, which can effectively increase the crosslink density of the crosslinked network. The hydrophilic polymers are supplemented with silane coupling agents as capping agents to facilitate crosslinking with PDMS, and dehydration condensation between silanols promotes the crosslinking reaction. The incorporation of crosslinking monomers is therefore essential for the preparation of silicone-based amphiphilic co-network. In order to develop amphiphilic co-network polymers with anti-adhesive properties, we selected Poly(ethylene glycol) methyl ether methacrylate (PEG-MA) as a hydrophilic monomer due to its low interfacial energy (water-PEG, <5 mN/m) [ 14 , 23 ], which allows for the formation of a dense hydration layer with water molecules and imparts excellent anti-adhesive properties to the coatings. In addition, hydroxyl-terminated PDMS (HO-PDMS-OH) was used as the hydrophobic monomer, while 3-(trimethoxysilyl) propyl methacrylate was used as the crosslinker [ 24 ]. 3-(trimethoxysilyl) propyl methacrylate has an ethylene linkage and reacts with methacrylate monomer to form polymer chains. Compared with KH590 or other silane coupling agents, it not only forms a crosslinked network by dehydration condensation with PDMS-OH, but also improves the crosslink density and stability of amphiphilic polymers. In this study, we successfully synthesised a silicone-based amphiphilic gel marine antifouling coating incorporating polyethylene glycol ( Scheme 1 ). Firstly, p(MMA-PEG-MA-TPMA) was synthesised using methyl methacrylate (MMA), polyethylene glycol monomethyl ether methacrylate (PEG-MA) and 3-(trimethoxysilyl)propyl methacrylate (TPMA) as reaction monomers, denoted as AM. Subsequently, at ambient temperature, TPMA acts as a crosslinking agent to facilitate the crosslinking of AM, PDMS and methyltriethoxysilane to form a silicone-based amphiphilic gel coating. Finally, the antifouling performance was evaluated in laboratory and natural marine environments. The amphiphilic coating exhibited exceptional antifouling performance in both environments, highlighting its potential as an environmentally friendly marine antifouling coating.",
"discussion": "3. Results and Discussion 3.1. Chemical Characterization of P-AM Coatings The preparation process of a silicone-based amphiphilic gel coating is shown in the Scheme 1 c. PEG-MA was used as the hydrophilic monomer, while TPMA served as the crosslinking agent to generate a non-crosslinked polymer chain by radical polymerisation [ 24 ]. To mitigate excessive expansion of the hydrophilic polymers in aqueous environments, high-molecular-weight HO-PDMS-OH was incorporated. The PDMS, METES and non-crosslinked polymer chains were mixed together, followed by the hydrolysis of the silane to produce silanols. These silanols were then subjected to dehydration condensation to form the silicon-based amphiphilic gel coating. The FT-IR spectrum of AM is shown in Figure 1 a and shows the presence of absorption peaks corresponding to C=O (1725 cm −1 ), -CH 3 (1448 cm −1 ) and C-O-C (1145 cm −1 ). Furthermore, the absorption peaks at 1078 cm −1 and 816 cm −1 could be attributed to the Si-O-C and Si-C groups, respectively. Notably, no absorption peak associated with the C=C group (1637 cm −1 ) was observed in the spectra, providing evidence for the successful synthesis of AM polymers by free radical mechanisms. The chemical structure of AM was characterised by 1 H NMR spectroscopy ( Figure S1 ). δ 0.77–0.96 (Si-CH 2 ) were the methylene proton absorption peaks of Si-CH 2 -CH 2 in TPMA. δ 3.50–3.60 (Si-O-(CH 3 ) 3 ) was attributed to the proton peaks of the three methyl groups of the TPMA end group. δ 3.60–3.65 (O-CH 2 -CH 2 -O) were the methylene proton peaks on the PEG-MA segment [ 31 ]. The signals at δ 4.10–4.20 and δ 4.30–4.35 were assigned to the methylene protons (C-O-CH 2 ) originating from TPMA and PEG-MA, respectively. Combining δ 1.88–1.97 (Si-CH 2 -CH 2 ), δ 3.30–3.48 (O-CH 3 ) and δ3.65–3.80 (O-CH 3 ) [ 32 , 33 ], it is evident that the non-crosslinked polymer chain segment polymers were successfully prepared by radical polymerisation reactions with the three monomers. Subsequently, by introducing a minute amount of catalyst, the siloxane on the AM branched chain undergoes dehydration condensation with PDMS and METES, forming an amphiphilic crosslinked network by curing. Four different AM/PDMS ratios were prepared and characterised by ATR-IR ( Figure 1 b). The characteristic absorption peaks at 861 cm −1 and 1260 cm −1 indicate the Si-C stretching vibration, while the characteristic peak of -CH 3 was shown at 2960 cm −1 . Furthermore, the characteristic peak of the Si-O-Si group at 1003 cm −1 confirmed the occurrence of a condensation reaction during the curing process of the coating. Interestingly, with increasing AM addition, the characteristic absorption peak of ν C=O can be observed at 1730 cm −1 on P-AM-15, indicating the successful formation of a co-network between AM and PDMS by condensation, accompanied by migration to the surface of the coating. Amphiphilic polymers play a key role in the design of antifouling/foul release coatings. By strategically combining different monomers, one can achieve microphase separation and localised swelling within the coating, thereby creating a heterogeneous surface with a precise chemical composition at the micro–nano scale [ 34 , 35 ]. The surface morphology of the silicone-based amphiphilic gel coating was examined by SEM to determine its microscopic features ( Figure 1 c). Convex vesicles with a diameter of 1–4 microns were observed on the P-AM-5, P-AM-10 and P-AM-15 coatings compared to the PDMS coating. During the crosslinking reaction, phase separation occurred due to differences in hydrophilicity and hydrophobicity between the monomers, resulting in a thermodynamically unstable system where components with different properties tended to separate. However, macroscopic phase transitions were prevented by covalent bond limitations between different monomers occurring at the micro–nano scale, leading to the formation of convex vesicles on the surface. The silicon-based amphiphilic gel coating was immersed in ASW for 3 days and then observed by laser confocal microscopy to study its surface structure and stability. A comparison of the samples before and after immersion ( Figure 1 d) shows no significant changes in PDMS, P-AM-5 and P-AM-10. However, it is noteworthy that a large number of vesicle-like structures were observed on the surface of P-AM-15 before immersion, but their number decreased and changed to a densely packed mountain-like structure after immersion. As a result, a microdomain structure with more uniformly distributed hydrophilic and hydrophobic properties was formed. The research results suggest that this phenomenon can be attributed to surface reconstruction resulting from different degrees of chain segment movement exhibited by the hydrophilic and hydrophobic components within the amphiphilic coating under different environmental conditions. Furthermore, when comparing the Ra surface roughness of the samples before and after immersion ( Table S1 ), it was observed that all the samples exhibited a change in roughness within 1 μm. The hydrophobic nature of PDMS prevents the coating from swelling due to excessive water absorption. In addition, TPMA acts as a strong crosslinking agent with other silanols, thereby limiting the expansion of the hydrophilic polymers on the surface of the coating. The demonstrated behaviour indicates that the coating exhibits exceptional resistance to swelling and deformation during the formation of a hydrated layer, ensuring its stability for use in marine environments. In this study, the wettability and surface energy of these coatings are investigated in Figure 2 a,b. Figure 2 a shows that the PDMS coating has a water contact angle of 108.99°. Its highly hydrophobic nature is attributed to the rapid orientation of methyl groups on the surface, resulting in a low surface tension and achieving a hydrophobic effect. The contact angle of the coating decreases slightly as the AM content increases. This phenomenon can be attributed to the preferential migration of high-molecular-weight PDMS towards the air–polymer interface during film formation, thereby maintaining the hydrophobicity of the coating. The surface energy of each sample was determined using the Owens–Winter method ( Figure 2 b). The γ D and γ P are summarised in Table S2 . The surface energy of the coating is directly proportional to the content of polymer chain segments. It is known that γ P is determined by the intermolecular forces between polar molecules in the material [ 27 ]. Increasing the content of the AM will result in a corresponding increase in γ P , thereby inducing an increase in surface energy. To further investigate the surface properties of the coating, the water dynamic contact angle was tested and shown in Figure 2 c,d. Neither PDMS nor P-AM-5 show significant changes in the dynamic contact angle, with a value still above 90° after 10 min. Both P-AM-10 and P-AM-15 show a rapid decrease over time, particularly P-AM-15, which decreases from 89° to 74° in 3–10 min. The hydrophilic chain of P-AM-15 gradually migrates from the interior to the surface of the coatings after 3 min. This phenomenon means that the coating accumulates hydrophilic groups on its surface after immersion in seawater, forming a hydrated layer [ 28 ]. At the same time, PEG-MA exhibits remarkable anti-protein properties, reducing non-specific protein binding to the hydrophobic surface and giving the coating antifouling properties. 3.2. Antifouling Properties of the P-AM Coatings 3.2.1. FITC-BSA Adsorption Tests Marine fouling is a continuous and stochastic process, typically initiated by the rapid accumulation of dissolved organic molecules in seawater, such as polysaccharides, proteins and lipids [ 27 , 36 ]. The hull surface generates abundant carbon sources that are used for reproduction by subsequent fouling organisms. Therefore, the representative FITC-BSA was chosen to assess the protein resistance of the coating. As shown in Figure 3 a, the BSA adhesion rate on the PDMS surface is 1, and the protein adhesion rate on the coating surface is normalised, and the corresponding fluorescence image is shown in Figure S2 . The PDMS coating surface showed a substantial amount of adhesion by FITC-BSA, whereas the adhesion on the P-AM-x surface was significantly reduced. Among the P-AM-x coatings, P-AM-15 in particular showed the lowest rate of surface protein adhesion. These results demonstrate that hydrophilic modification confers increased resistance to protein adhesion to the coating as a whole. As a typical hydrophobic material, PDMS exhibits non-specific protein binding [ 37 ], which can induce conformational changes and denaturation of proteins, facilitating their adhesion to surfaces. With the incorporation of hydrophilic polymers, PEG will form a compact hydration layer on the coating surface, effectively hindering physical protein adsorption and creating an energy barrier. The rapid migration of the hydrophilic component in P-AM-x under seawater immersion provides exceptional resistance to protein adhesion. 3.2.2. Bacterial Adhesion Test Biofilms of bacteria and organic matter form on the surface of materials exposed to the marine environment and are an essential factor in the adhesion of macrofouling organisms such as barnacles and mussels [ 38 , 39 ]. Therefore, improving the antibacterial adhesion of the coating can also improve the antifouling efficiency of the coating. In this paper, two representative marine bacteria, a Gram-positive bacterium ( B. subtilis ) and a Gram-negative bacterium ( P. ruthenica ), were selected to test the performance of the coating. By testing the anti-adhesion performance of four coating materials, we obtained data on the coverage and anti-adhesion rate of two bacteria on different coating surfaces ( Figure 3 b,c). The anti-adhesion rate was determined from the amount of adhesion observed on PDMS-coated surfaces. Specifically, B. subtilis was reduced by approximately 98.09%, while P. ruthenica was reduced by approximately 99.48% ( Table S3 ). These results indicate that P-AM-x coatings have some antibacterial adhesion ability (the fluorescence image depicting bacterial adhesion is shown in Figure S2 ). 3.2.3. Alga Resistance Tests Microalgae are an integral part of the marine biological membrane. The biofilm is the early stage of the marine fouling process, followed by the topography of large organisms on the biofilm. Therefore, the representative Phaeodactylum tricornutum ( P. tricornutum ) was chosen as a focus of this research to study the adhesion of algae to the coating surface. Figure 3 d shows the coverage and anti-adhesion rate of P. tricornutum cultivated on the coating surface for seven days after the washing treatment. The algae attachment area decreased for PDMS, P-AM-5, P-AM-10 and P-AM-15, with values of 32.53%, 21.11%, 0.95% and 0.25%, respectively, indicating excellent algae resistance when the amphiphilic P-AM content exceeds 10 wt% (the fluorescence image of surface adhesion is shown in Figure S2 ). In the initial phase of attachment, diatoms secrete extracellular polymers (EPS), composed of complex proteins and glycans, to adhere to the substrate surface. The EPS induces non-specific binding to PDMS, thereby increasing the adhesion strength of the diatoms. As an excellent anti-protein monomer, PEG-MA can effectively reduce protein adsorption and decrease diatom adhesion on P-AM surfaces. In addition, silicone-based amphiphilic gel coating improves the fouling release performance of ambiguous surfaces exposed to water [ 40 , 41 , 42 ]. 3.2.4. Marine Field Test The antifouling performance of the P-AM coatings was further evaluated in a marine environment (Xiamen, China, 24°56′ N, 118°10′ E). Figure 4 shows the photographic images of the PDMS and P-AM-X coatings before and after immersion in seawater for 4 months. The coatings showed excellent durability and adhesion throughout the test period, as evidenced by their intact surfaces, free of any damage or peeling. The PDMS coating was found to be heavily covered with sea silt, accompanied by the presence of fresh algae, barnacles and their larvae. The surface of P-AM-5 showed a significant reduction in marine silt adhesion and a reduction in barnacle and algae adhesion. The surface of P-AM-10 showed no apparent fouling organisms, although there were minor accumulations of algae and marine sediment. The surface of the P-AM-15 coating showed minimal fouling organisms and only a negligible amount of marine silt deposition, indicating its exceptional antifouling biological properties. These results further demonstrate that the amphiphilic modification of PDMS can effectively enhance the antifouling performance of silicone coatings, giving them long-term potential for marine antifouling. Based on the above experimental results, a potential mechanism can be proposed to explain the antifouling performance of the coating ( Figure 5 ): The surface of amphiphilic coatings consists of both hydrophobic and hydrophilic components that undergo surface reconstruction in the marine environment. Combined with their inherent hydrophilic and hydrophobic properties, they form a unique “mosaic” blurred surface structure [ 20 , 28 ]. This distinctive surface architecture enables the silicone-based amphiphilic gel coating to exhibit an excellent antifouling performance. At the same time, the coating retains the foul release (FR) properties of PDMS, while also possessing the foul resistance properties of the hydrated hydrophilic layer. In marine environments, the rapid formation of a water layer at the seawater interface by the hydrophilic structural domains on the surface prevents adhesion proteins or extracellular polymers produced by fouling organisms from adhering to the surface of the coating. During ship operation, shear erosion caused by seawater helps to effectively remove fouling organisms adhering to the surface, thereby achieving efficient fouling release."
} | 5,521 |
23437379 | PMC3577713 | pmc | 1,705 | {
"abstract": "Background Bacterial communities that are associated with tropical reef-forming corals are being increasingly recognized for their role in host physiology and health. However, little is known about the microbial diversity of the communities associated with temperate gorgonian corals, even though these communities are key structural components of the ecosystem. In the Northwestern Mediterranean Sea, gorgonians undergo recurrent mass mortalities, but the potential relationship between these events and the structure of the associated bacterial communities remains unexplored. Because microbial assemblages may contribute to the overall health and disease resistance of their host, a detailed baseline of the associated bacterial diversity is required to better understand the functioning of the gorgonian holobiont. Methodology/Principal Findings The bacterial diversity associated with the gorgonian Paramuricea clavata was determined using denaturing gradient gel electrophoresis, terminal-restriction fragment length polymorphism and the construction of clone libraries of the bacterial 16S ribosomal DNA. Three study sites were monitored for 4 years to assess the variability of communities associated with healthy colonies. Bacterial assemblages were highly dominated by one Hahellaceae -related ribotype and exhibited low diversity. While this pattern was mostly conserved through space and time, in summer 2007, a deep shift in microbiota structure toward increased bacterial diversity and the transient disappearance of Hahellaceae was observed. Conclusion/Significance This is the first spatiotemporal study to investigate the bacterial diversity associated with a temperate shallow gorgonian. Our data revealed an established relationship between P. clavata and a specific bacterial group within the Oceanospirillales . These results suggest a potential symbiotic role of Hahellaceae in the host-microbe association, as recently suggested for tropical corals. However, a transient imbalance in bacterial associations can be tolerated by the holobiont without apparent symptoms of disease. The subsequent restoration of the Hahellaceae -dominated community is indicative of the specificity and resilience of the bacteria associated with the gorgonian host.",
"introduction": "Introduction The interactions between microbial communities and sessile marine invertebrates such as sponges or corals are increasingly recognized as a critical component of the overall biology of these organisms. Recent research on the microbiology of scleractinian corals has demonstrated close relationships between the hexacoral animal and numerous prokaryotic organisms, and studies on coral reef ecosystems should consider the coral host with its associated microbiota as a unique evolutionary unit termed the “holobiont” [1] – [3] . Corals harbor highly diverse bacterial communities, and several reports have highlighted the existence of specific coral-bacteria associations that are mostly maintained among colonies from the same locality or even across distinct geographical locations [2] , [4] – [7] . These bacterial assemblages may play important roles in the host's physiology, mainly through their functions in nutrient cycling and health status [8] . In particular, there is increasing evidence for a relationship between coral-associated bacteria and disease resistance, which is likely the result of antibiotics produced by the resident microbiota and/or niche competition with potential pathogens [1] , [9] , [10] . In contrast to scleractinian reef-forming corals, information on the microbial diversity associated with gorgonians (octocorals) is limited. A few studies have investigated bacterial associations with tropical or cold-water species [11] – [13] , but to our knowledge, the bacterial diversity of shallow temperate octocorals has not yet been explored. In the Northwestern (NW) Mediterranean Sea, gorgonians play an important ecological role in highly diverse coralligenous outcrops [14] . During the last few decades, coralligenous benthic invertebrates have suffered from large-scale disease and mortality [15] – [18] . These events are linked to unusual positive anomalies of seawater temperature in the summer and have affected a wide range of macro-benthic species over hundreds of kilometers, from the Italian to Spanish coasts [19] . Gorgonians populations have undergone extensive damage, although differences in the degree of impact have been observed in various geographic areas. One of the most affected species is the red gorgonian Paramuricea clavata (Risso, 1826), a long-lived, aposymbiotic colonial octocoral that is considered a key species within coralligenous assemblages [20] . During the summer mortality outbreaks, P. clavata colonies exhibited symptoms of necrosis that may have been caused by multiple factors acting in synergy with thermal stress, including food limitation, metabolic constraints and microbial virulence [15] , [21] . Colonies that were affected during mass mortality events in 2003 and 2008 harbored culturable isolates of Vibrio coralliilyticus , a pathogenic bacterium that can induce host tissue damage at elevated temperatures [22] , [23] . Members of this Vibrio clade were previously identified as the etiologic agents of bleaching in the Indo-Pacific coral Pocillopora damicornis and White Syndrome disease in other coral species, thus satisfying Koch's postulate [24] , [25] . This finding suggests that at least some mechanisms underlying the disease process in P. clavata are comparable to the mechanisms involved in tropical coral outbreaks. Several studies have concluded that coral pathogens take advantage of changes in the bacterial community structure during stressful conditions to proliferate and cause tissue damage [4] , [26] , [27] . In support of this hypothesis, compositional shifts in bacterial assemblages have been observed in diseased coral colonies [28] , [29] . Similarly, the recurrent disease and mortality of P. clavata may be promoted by a disturbance of the resident microbiota in response to anomalous high-temperature conditions during the summer. To evaluate the contribution of microbial communities to temperate gorgonian health and disease, a detailed knowledge of the structure of the bacterial assemblages in healthy colonies is required. The objective of the present study was to provide a baseline of the bacterial communities associated with P. clavata populations in the NW Mediterranean basin. Given the predicted increase in extreme climatic events in this region, new mortality outbreaks are expected [30] , [31] . Thus, tracking potential modifications of this baseline during future high-temperature stress could greatly aid our understanding of the role of bacterial associations in maintaining gorgonian health. In this work, we conducted a seasonal sampling over 4 years (2007–2010) in 3 distinct geographical locations separated by hundreds of kilometers (Provence, Corsican and Catalan coasts), which allowed us to characterize the bacterial communities associated with P. clavata and the spatiotemporal variability of their structure. Three different molecular culture-independent approaches (denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) and 16S RNA gene clones library construction) were used to determine which bacteria might be conserved across geographically remote P. clavata populations. Our findings reveal the presence of host-specific bacterial associations in the gorgonian holobiont, although transient and reversible variations in microbiota composition were also recorded during our seasonal survey.",
"discussion": "Discussion This study provides the first description of the spatial and temporal patterns of the structure of the microbial communities associated with a temperate gorgonian in the Mediterranean Sea. The analysis of the DGGE fingerprint data indicated that the bacterial diversity hosted by P. clavata is broadly similar within and between individual colonies from a population and differs from the community composition within the surrounding water. In addition, the MDS ordination of the T-RFLP diversity profiles revealed a lack of clustering by location for the gorgonian populations sampled among our 3 study sites, which are separated by hundreds of kilometers. These results clearly suggest that the structure of bacterial communities that inhabit P. clavata does not rely on local environmental drivers or discrete gorgonian populations. Most of the T-RFLP profiles from the 48 colonies sampled between March 2007 and September 2010 grouped together, thus supporting the notion of specific bacterial-host associations. Taken together, data from the T-RFLP analysis and 16S rRNA gene clone libraries indicate that P. clavata colonies are associated with an almost permanent microbial population that is highly dominated by a unique gammaproteobacterial ribotype. However, we detected a transient compositional shift between this relatively stable bacterial assemblage that was observed throughout 7 of the 8 investigated seasons, with a strikingly different diversity pattern in summer 2007. The dominant identified ribotype is affiliated with the family Hahellaceae within the order Oceanospirillales and is related to Endozoicomonas and Spongiobacter spp. Interestingly, a meta-analysis of coral-associated bacterial assemblages revealed that Oceanospirillales- affiliated sequences were among the most frequent ribotypes found in healthy hexacorals [50] , and several recent reports indicated that Hahellaceae members may represent a common group of coral-associated Oceanospirillales . Spongiobacter sequences have been retrieved from the hexacoral Acropora millepora, in which they represent the largest proportion of bacterial clone libraries [28] , [51] . In 2 other hard coral species, Acropora hyacinthus and Stylophora pistillata , Kvennefors et al. \n [29] identified a cluster of ribotypes that are closely related to the genus Endozoicomonas . This so-named “Type A Associates” cluster includes several ribotypes that were previously observed in different tropical coral species or specifically associated with benthic marine invertebrates. Spongiobacter -related 16S ribosomal sequences were also detected in the cold-water coral Madrepora oculata and dominated the DGGE banding pattern of the associated bacterial community in this species [52] . Very recently, Morrow et al. \n [53] found that Endozoicomonas represented the most abundant bacterial genus in communities of the reef-building coral Porites astreoides , representing up to 99% of the 16S rRNA sequences recovered from colonies in one of the studied sites. The presence of Hahellaceae as a major component of the bacterial communities in both hexacorals and gorgonians (octocorals) suggests that these associates have differentiated to form a stable symbiotic complex specific to a cnidarian taxon. Further research focusing on the patterns of diversity of this bacterial group will certainly help to understand the evolutionary forces that may drive host-associated community composition in divergent phylogenetic lineages of cnidarians. In contrast to the unique Hahellaceae ribotype revealed by our study in P. clavata , several distinct Endozoicomonas-Spongiobacter sequences coexist in hexacoral species. This difference suggests that distinct hexa- and octocoral hosts may exert different selective controls on their microbial partners, resulting in various diversity patterns of Hahellaceae associates. Consistent with this hypothesis of host-driven control on associated Hahellaceae bacteria, the ribotype identified in P. clavata did not closely match the Spongiobacter-Endozoicomonas sequences found in hexacorals (<93% similar) but was more closely related (>96% similar) to bacterial sequences retrieved from the tropical gorgonian Gorgonia ventalina (GenBank accession number GU118518; [13] ), suggesting that the species associated with P. clavata may belong to a new Hahellaceae genus that is adapted to gorgonian hosts. To date, very little is known about the potential contribution of Hahellaceae bacteria to the functioning of the holobiont. One study has shown that Spongiobacter -related organisms isolated from A. millepora tissues possess the ability to metabolize the organosulfur compound dimethylsulfoniopropionate (DMSP), suggesting a role in sulfur cycling in scleractinian corals [51] . However, because DMSP production in corals is believed to rely on the presence of endosymbiotic zooxanthellae [51] , a similar hypothesis for Hahellaceae -affiliated bacteria in P. clavata seems unlikely because this gorgonian species is devoid of photosynthetic symbionts [20] . Despite multiple trials, we failed to isolate culturable Hahellaceae bacteria from P. clavata tissues; consequently, their metabolic potential could not be investigated. Another interesting property of the members of the Oceanospirillales clade is the production of extracellular hydrolytic enzymes that are involved in the degradation of various complex organic substrates, providing a potential nutritional integration between bacterial associates and their host, as suggested for the endosymbiotic Oceanospirillales found in the deep-sea worm Osedax \n [54] . In the case of coral-associated bacteria, one might speculate that heterotrophic Hahellaceae help supply the host with macromolecular nutrients that are not directly assimilated under a complex form. The previous identification of Gammaproteobacteria aggregates within the gastrodermis tissue layer in the digestive cavity of several reef-building corals suggests that these bacteria may play such a role in the coral diet [55] . However, the available data on the 16S rRNA gene sequences of these intracellular bacteria are restricted to the Gammaproteobacteria FISH probe regions, thus precluding phylogenetic affiliation at the genus or family levels and hindering comparisons with Hahellaceae sequences. Further investigations, including in situ localization of bacteria in P. clavata, are clearly required to provide information on their possible role in the functioning of the holobiont. Hopefully, the 16S rRNA gene sequence retrieved from the dominant Hahellaceae ribotype in our study will allow us to design oligonucleotide probes for their specific detection in gorgonian tissues. While our T-RFLP and clone libraries analysis indicated the existence of a relatively stable Hahellaceae -dominated microbiota in P. clavata populations across a large geographic range and throughout the 4-year survey, we identified a simultaneous and deep compositional shift of communities in summer 2007 at a regional scale. During this summer, the Hahellaceae sequences were not detected by either technique and were transiently replaced by more diverse assemblages with prominent Paenibacillus- and Propionibacterium -related sequences, 2 bacterial genera that respectively belong to Firmicutes and Actinobacteria . Members of Firmicutes and Actinobacteria have previously been detected in cold-water and tropical scleractinian corals [4] , [29] , [56] , [57] , where they are generally found in low abundance in healthy and/or bleached colonies [50] although representatives of Paenibacillus and Propionibacterium spp. were among the most commonly recovered bacteria from a Brazilian coral species [58] . Because we did not observe a similar transition in any of the other summers that were analyzed, we suggest that an abnormal and transient disruption of the host- Hahellaceae association occurred in summer 2007. However, ordination of the T-RFLP Msp I data set revealed that the profiles of all summer 2007 samples grouped with profiles from several colonies sampled in other seasons ( Figure 5B ). Significantly, the Paenibacillus TRF (TRF-100.1) was observed together with the dominant Hahellaceae TRF (TRF-105.1) in the latter profiles ( Figure 4 ). These observations indicate a possible overlap between the 2 communities and suggest that bacteria related to Paenibacillus could be normal but infrequently detected associates, except during periods of anomalous abundance. Therefore, the shift in dominant ribotypes from Hahellaceae to Paenibacillus may result from a transient imbalance of endogenous bacterial populations that naturally reside in the holobiont rather than opportunistic colonization by Paenibacillus from the surrounding water concomitant with a decrease in the Hahellaceae community. The subsequent shift back toward the initial Hahellaceae -dominated community further supports the hypothesis that the holobiont strongly regulates its microbial diversity and indicates that Hahellaceae most likely represent host-specific bacterial associates of P. clavata . The causes of the transition in P. clavata bacterial diversity from Hahellaceae - to Paenibacillus- dominant ribotypes during summer 2007 are not clear. As mentioned above, the shift occurred at the 3 studied areas in an almost synchronous manner, suggesting the involvement of environmental and/or biological factors acting on a large geographical scale. Several previous studies have identified shifts in coral-associated bacterial communities during bleaching and disease outbreaks or under conditions of environmental stress, such as increased temperature and organic matter enrichment [55] , [59] , [60] . For instance, changes in microbial associates were observed in the stony coral A. millepora during a mass bleaching event, and occurred prior to visual signs of bleaching on the sampled colonies [28] . Major shifts in bacterial communities were also observed in diseased colonies of 2 coral species that were affected by White Syndrome disease [29] . Interestingly, the Hahellaceae -related bacteria found in healthy colonies were replaced by other bacterial groups in bleached or diseased individuals. Whether the shift in bacterial assemblages is the cause or the effect of the disease is unclear, but these observations indicate that changes in Hahellaceae -host associations are related to an altered physiological state of the holobiont during stress conditions. Regarding the potential causes of stress within the gorgonian populations in summer 2007, the available temperature time series data recorded at the study areas during the survey period (T-MedNet network; http://t-mednet.org ) did not reveal any temperature anomaly. In addition, surveys of P. clavata populations conducted during and after summer 2007 in the 3 study areas did not reveal any symptoms of disease (J. Garrabou et al. , unpublished data). Notably, we did not detect the presence of Vibrio coralliilyticus 16S rDNA in the Paenibacillus -dominated summer 2007 clone libraries, although this Vibrio has been implicated in recent disease outbreaks in P. clavata populations during climatic anomalies [22] , [23] . Several other Vibrio species were also involved in P. clavata tissue necrosis in Mediterranean [17] , [61] , or recovered from healthy and diseased cold-water gorgonians [62] . Altogether, this suggested that unhealthy conditions of the host are allowing colonization by potentially pathogenic Vibrio strains that can also be a natural component of the holobiont and may exploit the disturbance of the normal microbiota [63] . By contrast, we detected only 2 Vibrionaceae sequences among the 523 clones that were analyzed from the summer 2007 libraries (data not shown). This result suggests a very moderate abundance of Vibrio and indicates that the shift observed in P. clavata was likely not related to the onset of a disease. However we cannot rule out that other bacterial groups among the taxon diversity in summer 2007 may represent potential pathogens or detrimental associates that overcome the Hahellaceae -dominated natural assemblage. For instance, although Paenibacillus and Propionibacterium spp. are not considered as coral pathogens and can be normal components of the associated microbiota, their overwhelming dominance in summer 2007 might have resulted in transiently compromised holobiont homeostasis without expression of disease symptoms. Besides climate-related stresses and disease, regional oceanographic perturbations including land run-off and anthropogenic disturbance have also been shown to cause changes in microbial communities associated with corals [56] , [64] , [65] . Degraded ecological conditions may facilitate the existence of alternate states of microbial community structure and overgrowth of opportunistic bacteria [59] . However, an exposure of P. clavata colonies to a pollutant or effluent in summer 2007 is very unlikely considering the large geographical area in which this bacterial shift was observed. In addition, our studies sites are variably impacted by anthropogenic effects as the 3 regions are subjected to different levels of protection. Riou site was located in a non-protected region, a few kilometers away from Marseille, the second major French city. Medes and Scandola sites are both located in marine protected areas, Scandola being a no-take area submitted to very limited human pressure. Therefore, local anthropogenic impacts do not seem to be the cause for the bacterial shifts simultaneously observed in the 3 sites in summer 2007, although a monitoring of environmental parameters and microbial community structure in the water column would be required to better evaluate differences between the sites. Finally, the factors that could have directly or indirectly caused a transient change in the natural bacterial community remain unclear and several other hypotheses cannot be ruled out, such as a bacteriophage infection targeting Hahellaceae and causing microbial mortality [66] , or the occurrence of subtle alterations in gorgonian physiology in summer [67] that would disrupt the host-bacteria relationship without macroscopic disease signs. In conclusion, the present work provides a reliable evaluation of the structure of and variations in gorgonian-associated bacterial communities. To our knowledge, this is the first spatiotemporal study demonstrating that transient but dramatic shifts in the natural baseline of these assemblages can arise in apparently healthy octocorals. In light of our results, it is important to encourage multi-year monitoring to avoid erroneous or incomplete descriptions of the bacterial assemblages, as we might have drawn by investigating the P. clavata microbiota during the phase shift. Although this shift was only observed once in the 4-year survey and thus presumably corresponds to an abnormal or stressful event, it indicates that transient bacterial populations may replace the natural Hahellaceae -dominated community without visible evidence of deleterious effects. Further studies are now required to address the question of whether the phase shift in the microbial community may represent a potential monitoring tool for determining the health state of gorgonians and the ecological condition of their environment. This research may aid in understanding the causes of recurrent mass mortalities and designing effective management strategies to preserve one of the most emblematic Mediterranean species."
} | 5,872 |
32005817 | PMC6994492 | pmc | 1,710 | {
"abstract": "The honeybee (Apis mellifera) dance communication system is a marvel of collective behaviour, but the added value it brings to colony foraging efficiency is poorly understood. In temperate environments, preventing communication of foraging locations rarely decreases colony food intake, potentially because simultaneous transmission of olfactory information also plays a major role in foraging. Here, we employ social network analyses that quantify information flow across multiple temporally varying networks (each representing a different interaction type) to evaluate the relative contributions of dance communication and hive-based olfactory information transfer to honeybee recruitment events. We show that virtually all successful recruits to novel locations rely upon dance information rather than olfactory cues that could otherwise guide them to the same resource. Conversely, during reactivation to known sites, dances are relatively less important, as foragers are primarily guided by olfactory information. By disentangling the contributions of multiple information networks, the contexts in which dance communication truly matters amid a complex system full of redundancy can now be identified.",
"introduction": "Introduction During a waggle dance, a honeybee forager provides location information about a high-quality resource (e.g., foraging or nest sites) by producing multiple waggle runs, the duration and orientation of which correspond to spatial coordinates in the field 1 . That other foragers can use this spatial information to locate the indicated site 2 , 3 remains one of the most astounding discoveries of the last century within the field of animal behaviour 4 . Yet efforts to quantify the value that this spatial communication system brings to a colony’s foraging operation have generally failed to find clear net benefits, repeatedly concluding that rendering the dance’s directional component meaningless only rarely compromises colony foraging efficiency 5 – 9 . Concurrent work has emphasised that even for undisrupted dances, the spatial information provided is often of secondary importance to the dance’s role as a trigger of navigational memories in dance followers 10 – 14 , or to the olfactory information about profitable food sources that dancers provide through trophallactic nectar donations and scents carried on their bodies 10 , 13 – 18 . Since dance communication is integral to nest-site selection in honeybees 19 , 20 , these findings have led to the suggestion that the role of dancing in foraging could in fact be a secondary one that is less critical than commonly supposed 21 . However, establishing whether foraging bees are responding to dance information, to alternative recruitment mechanisms, or to a combination of these, is challenging given that all information sources are typically available simultaneously within the hive. To overcome this difficulty, we use network-based diffusion analysis (NBDA) 22 , 23 to tease apart the relative importance of dance-based communication networks, in relation to individual search and olfactory information transfer, in guiding honeybees to both novel and known foraging sites. NBDA has enabled in-depth study of social transmission processes across a range of vertebrate taxa 24 – 29 . Its core assumption is that if a learnt behaviour or piece of information—such as discovery of a novel foraging site—spreads via social transmission, then its diffusion will follow a social network 30 . Here, we extend this approach to allow for the simultaneous inclusion of multiple, time-varying social networks in order to identify the key information pathways amongst honeybee foragers. By training cohorts of bees to novel feeders, we mimic natural foraging events and observe the in-hive interactions that follow in order to build alternative social networks based on dance interactions, trophallactic nectar donations and antennation of returning foragers. All three interaction types are known to motivate honeybees to search out known or novel sites in the field 1 – 3 , 11 , 13 , 31 – 33 . However, only the dance additionally provides navigational information 2 , 3 , whereas trophallaxis and antennation (both of which can also occur during dance following) lead to learning about the scent of the target foraging site 15 , 16 , 18 . We estimate the power of each network to predict the order of arrival at the feeders, and find that in a context mimicking natural depletion of one patch of a flower species followed by discovery of another, waggle dance communication is the dominant mode of transmission. Neither of the two main olfactory information pathways—antennation and trophallaxis—are important for discovering foraging sites in this particular context. Conversely, all three pathways contribute to motivating temporarily unemployed foragers to resume collecting from a known, familiarly scented site, but antennation is especially important in this regard. By revealing how alternative information pathways combine to shape behaviour, NBDA offers a promising approach for identifying the contexts in which dance communication matters most in driving honeybees to food.",
"discussion": "Discussion That odour-based and dance-based recruitment operate in parallel within honeybee hives has been recognised for many years 1 , and was central to a major challenge to Karl von Frisch’s original description of the dance recruitment system that played out in the latter half of the 20th century 37 , 46 . While James Gould convincingly dispelled claims that odour-based recruitment could be the only means by which dance-followers located foraging sites—performing an experimentum crucis in which followers were recruited to sites that had never been visited by the dancers that indicated them—he nonetheless concluded that “the inherently ‘spectacular’ nature of the dance language may have helped to emphasise it out of proportion to its actual place in the ecology and dynamics of foraging”, and that “only further work can establish whether the dance-language is common or rare under normal circumstances” 2 . We have shown that NBDA offers a means to meet this challenge by identifying those contexts in which dances are the major driver of arrivals at foraging sites. In our experiment, dance following was the major elicitor of discovery of new patches of a target flower species. Conversely, although bees also follow dances before reactivating to known food sites, trophallaxis events explained almost the same number of feeder arrivals during reactivation as dance following, and antennation explained over twice as many. By disentangling the effects of alternative social networks, the NBDA approach thus offers a broadly applicable means of identifying the key social learning pathways that operate simultaneously within freely interacting animal groups. That dance-conveyed information may simultaneously guide followers engaged in distinct foraging activities—a property known as pluripotentiality in systems science 47 , 48 —may go some way towards explaining why studies have often failed to detect clear benefits to this communication system 5 – 9 . The costs of producing spatial information under ecological contexts in which it is less valuable may be offset by the benefits gained through the dance’s motivational, reactivation and reaffirmation functions 1 , 12 , 13 , 49 . At the same time, because reactivation is also elicited by antennation and trophallaxis, these other transmission pathways may aid in maintaining the dance communication system through such overlapping functionality (i.e., degeneracy 47 , 48 , 50 ). In other words, responses to selection pressure favouring the transmission of spatial information may be enhanced, as the dance’s reactivation function can be fulfilled through alternative information networks. Indeed, honeybee colonies may only gain weight during brief periods of intense nectar flow each year 51 , during which time the ability of the dance to direct foragers towards highly profitable foraging sites may be crucial for colony survival. While our results confirm the key role of the dance’s spatial information during recruitment to novel sites, they also suggest that previous studies may have underestimated the importance of non-dance interactions for honeybee foraging. For instance, foragers that leave the hive without having followed any dances have often been assumed to be scouting for novel resources 12 , 14 , 52 . Yet many of these bees are likely to have engaged in olfactory interactions in the hive, which could motivate them to either revisit known sites 10 , 13 , 45 or search for a particular flower species 2 , 31 , 33 . Trophallactic exchange has long been considered potentially key to this process 15 , 16 . More recently, it has been demonstrated that even without experiencing a sucrose reward, simply detecting floral scents during antennation can be sufficient for honeybees to learn food-odour pairings 18 , and to preferentially select food sources bearing that scent when encountered in the field 33 . Our findings support the importance of this network as a driver of reactivation, and reveal that under certain circumstances, information transferred during such interactions can actually be of greater importance for organising honeybee collective foraging than that gained from following waggle dances. Intriguingly, as antennation occurs within all social bees (including honeybees, bumblebees and stingless bees), similar transmission pathways may be present across species and play a similar role in coordinating forager efforts. Although detection of familiar odours seems the most likely explanation for our finding that antennal contact was the single most important driver of reactivation, it is also possible that simply coming into contact with employed foragers was sufficient to elicit reactivation 49 . Future work should therefore explicitly examine how food odour familiarity influences the relative importance of these alternative information networks. In addition, despite its well-established ability to facilitate olfactory learning 15 , 16 and rapidly distribute collected nectar through the hive 53 , 54 , we found that trophallaxis contributed little towards forager recruitment. The experimental design used here may have limited the importance of trophallaxis for successful recruitment, as recruits had learned the food odour prior to the trial. Thus, after being guided to the feeder’s general proximity via the dance, it is likely that they could simply home in on the familiar scent without having needed to experience it first in the hive 38 . In contrast, during recruitment to an unfamiliarly scented resource, trophallaxis may enable foragers to rapidly learn the novel odour, which could then be used in tandem with spatial communication to successfully locate the advertised site. It has also been hypothesised that brief trophallactic contacts facilitate assessment of the relative quality of available resources 55 – 57 , such that deteriorating foraging options can be abandoned more rapidly. Perhaps the most pressing question, however, is whether honeybees do in fact rely more heavily on dance information when resources are scarce and patchily distributed, as has previously been suggested 5 – 7 , 9 , 58 , 59 . Identifying the circumstances under which honeybees use information gained through dance communication is crucial if we are to understand the selective pressures that may have shaped its evolution. Automated behavioural tracking technologies are rapidly advancing to the point that researchers will soon be able to construct detailed interaction networks involving multiple interaction types and encompassing the entire hive 60 – 63 . Combining such data with NBDA may finally provide the means of answering one of the most enduring and fascinating mysteries in the field of animal behaviour: what is the adaptive value of honeybee dance communication, given that this system has failed to evolve in even a single other social insect group?"
} | 3,016 |
39394388 | PMC11470012 | pmc | 1,712 | {
"abstract": "Honeybee ( Apis mellifera ) colonies use a unique collective foraging system, the waggle dance, to communicate and process the location of resources. Here, we present a means to quantify the effect of recruitment on colony forager allocation across the landscape by simply observing the waggle dance on the dancefloor. We show first, through a theoretical model, that recruitment leaves a characteristic imprint on the distance distribution of foraging sites that a colony visits, which varies according to the proportion of trips driven by individual search. Next, we fit this model to the real-world empirical distance distribution of forage sites visited by 20 honeybee colonies in urban and rural landscapes across South East England, obtained via dance decoding. We show that there is considerable variation in the use of dancing information in colony foraging, particularly in agri-rural landscapes. In our dataset, reliance on dancing increases as arable land gives way to built-up areas, suggesting that dancing may have the greatest impact on colony foraging in the complex and heterogeneous landscapes of forage-rich urban areas. Our model provides a tool to assess the relevance of this extraordinary behaviour across modern anthropogenic landscape types.",
"introduction": "Introduction In group living animals, collective decisions can be taken by integrating information from multiple individuals to produce behaviour that extends beyond that of the individual 1 . Collective behaviour thus emerges from simple behavioural ‘rules’ which filter social information 2 , 3 . In honeybees, and other eusocial insects, the behavioural architectures that produce emergent behaviours have become particularly complex. Honeybee colony foraging is coordinated via the waggle dances of individual foragers, which communicate food source locations to nestmates (Fig. 1 ). A series of behavioural rules that determine when, and how much, bees dance mean that choices between feeding sites can be made by the group 4 – 6 . For example, the number of dance circuits performed by a forager on returning from a food source reflects the net energetic benefits of the trip 6 . As a result, more of the colony’s workforce will be recruited to the closer of two equally rich sources 7 or the richer of two equidistant sources 5 . This filtered recruitment mechanism allows a colony to allocate collective foraging effort, effectively choosing which of the available forage sites to focus upon, without the need for any individual to compare resources. Fig. 1 The honeybee waggle dance carries information about the location of a resource. a The honeybee waggle dance communicates the direction of the resource, relative to the direction of the sun through the angle of the dance relative to the vertical. The duration of the waggle run indicates the distance to the resource. b This information, in the form of a bearing and a distance, allows other foragers to locate the resource in the landscape (blue circle). Dancing is a universal feature of honeybee behaviour and is commonly observed in all Apis mellifera colonies. However, individual bees only respond to the spatial directions provided by dances under particular circumstances 8 , 9 . For example, foragers that have current knowledge of a resource site are rarely influenced by dances for alternative sites 10 , even if those alternatives are more rewarding 11 . In contrast, dances are highly influential for foragers whose known sites become depleted 8 , 12 , 13 , or after temporal gaps in foraging. As a result, the importance of dance communication—and therefore collective decision-making—for a colony’s choice of forage sites should depend on resource distribution in the landscape. Environments where recruitment is influential are intriguing, because they are likely to have been important in driving the evolution of the waggle dance, yet empirical attempts to identify them have produced mixed results 14 – 21 . Initial work, in which dances were rendered meaningless by preventing bees from referencing the sun’s position (Fig. 1 ), tentatively linked the benefits of collective foraging to landscape characteristics such as heterogeneity 21 . However, empirical attempts to systematically test this relationship have failed to provide support 14 , 15 , and dance disruption has sometimes even been associated with higher, rather than lower, foraging success 19 . Consequently, no clear pattern has yet emerged with respect to the ecological conditions that determine whether colonies forage collectively, or as a group of individuals 22 . Here, we present a methodology to infer the influence of recruitment on colony foraging, and to quantify differences in this influence across landscape types, simply by observing the dances on a colony’s dancefloor. Using this method, we will show that the importance of dancing for colony foraging patterns is typically high in forage-rich, complex urban areas, but more variable at rural sites that are dominated by intensive agriculture. We first develop a theoretical model to establish how colony foraging patterns should appear when recruitment is used to varying degrees, compared to cases where all bees search for food individually. We find that recruitment leaves a characteristic “humped” imprint (see Results section “Recruitment creates a characteristic pattern in the distribution of foraging distances”) on the cumulative distribution of distances reported on the dance floor, the magnitude of which correlates with the use of the waggle dance for collective foraging. We then fit these theoretical distributions to an empirical data set consisting of observations of waggle dances from 20 real-world hives in two different landscape types—urban and agri-rural—to quantify the relative contribution of waggle dance recruitment to colony foraging decisions in each case. Finally, we relate the variation in waggle dance use that we identify to local land-use patterns.",
"discussion": "Discussion Our results show that there is considerable variation in the impact that waggle dance recruitment has on the distribution of forage sites chosen by honeybee colonies foraging naturally across anthropogenic landscapes 8 , 9 . We do this by using a purely observational approach that does not disrupt the natural foraging behaviour of the colony: by simply decoding the dances produced by a colony’s workforce and fitting our model to the foraging site distances obtained as a result, it is possible to see that the foraging patterns of some colonies bear a clear hallmark of recruitment, whereas others do not. What causes this variation? Our metric for assessing the impact of dance recruitment is the proportion of bees that follow a “recruit” strategy. Bees that use this strategy sample a dance from the dance floor before leaving the hive to find the site communicated by the dance, rather than searching independently. At the proximate level, the factors that underlie the choice to act as scout or recruit are not well understood, although previous work has found that certain gene expression profiles are predictive of scouting behaviour 32 and that the tendency to act as a recruit is greater in younger bees 13 , 33 . But it is clear that these strategies are sufficiently flexible to allow changes in the proportion of scouts with local foraging conditions, and Seeley 33 observed through intensively tracking individual bees in observation hives that the proportion of scouts decreases dramatically with forage availability, as the number of dances increases 33 . Thus, there is reason to expect that the hives we identified as relying heavily on dance recruitment are those that are in forage-rich areas. Accordingly, we found patterns in the importance of waggle dance recruitment that reflected landscape structure, such that variation was higher in agri-rural than urban environments, and within the agri-rural category, increased along an axis that reflected a transition from forage-poor agricultural land towards increasing residential development. Agricultural land in the UK is typically considered nutritionally poor for bees, with large areas of limited food availability punctuated by brief availability of rich mass-flowering crops in some areas, while more urbanized residential areas that contain gardens are relatively forage rich, with many diverse small patches of flowers in residential gardens and allotments 29 , 34 , 35 . We speculate that the dancefloors of hives in these urbanized areas may contain dances advertising multiple alternative sites, such that when rewards decrease at one site, dances advertising others are quickly encountered. At sparser agri-rural sites dominated by arable land, where food is potentially more challenging to find, representation of multiple alternatives on the dancefloor may be rarer. Our current dataset lacks the temporal resolution to explore whether recruitment is more common at times when greater diversity is likely within such landscapes, such as late spring, but further work could focus on rural locations systematically chosen to represent a range of floral diversity and abundance (e.g. ref. 36 ), with the temporal resolution to focus on specific periods of the year, to identify those ecological contexts in which dance communication has a detectable impact on colony foraging. It is widely assumed that certain aspects of the tropical forests in which Apis evolved favoured the evolution of the dance 22 . Current attempts to identify those critical features rely upon elegant but labour-intensive dance-disorientation protocols, whereby dance communication is actively prevented and the consequences for foraging efficiency monitored 16 , 17 , 21 , 36 , 37 . Our method complements and builds upon these studies. On the one hand, our findings contribute to explaining why dance disorientation sometimes has little effect on foraging efficiency in some environments 14 , 15 , 17 , 19 – 21 : although dancing takes place, recruitment is relatively rare. Rather than collective foraging being an inflexible adaptation to a landscape type, this suggests that the feedback loops that regulate collective foraging also provide the mechanism to fine-tune the foraging to fine-grained spatial or temporal variation in food availability. On the other, this method has the potential for expansion across multiple landscapes and land-use types. We have described a means to quantify the extent to which honeybee colonies rely on recruitment that functions simply by observing and decoding the dances on the dancefloor, and although we were limited by the need to manually decode dances, further study will not be. Given the recent development of automated dance-decoding protocols 38 , 39 , now also validated for field-based videos 40 , our inferential method has broad-ranging potential to provide a time and labour efficient means to identify the environments in which this iconic example of animal communication shapes collective behaviour."
} | 2,752 |
35211696 | PMC8861933 | pmc | 1,713 | {
"abstract": "Triboelectric nanogenerators\n(TENGs) that utilize triboelectrification\nand electrostatic induction to convert mechanical energy to electricity\nhave attracted increasing interest in the last 10 years. As a universal\nphysical phenomenon, triboelectrification can occur between any two\nsurfaces that experience physical contact and separation regardless\nof the type of material. For this reason, many materials, including\nboth organic and inorganic materials, have been studied in TENGs with\ndifferent purposes. Although organic polymers are mainly used as triboelectric\nmaterials in TENGs, the application of inorganic nanomaterials has\nalso been intensively studied because of their unique dielectric,\nelectric, piezoelectric, and optical properties, which can improve\nthe performance of TENGs. A review of how inorganic nanomaterials\nare used in TENGs would help researchers gain an overview of the progress\nin this area. Here, we present a review to summarize how inorganic\nnanomaterials are utilized in TENGs based on the roles, types, and\ncharacteristics of the nanomaterials.",
"conclusion": "4 Conclusions and Perspectives The applications\nof TENGs include energy harvesting, a variety\nof sensor types, biomedical applications, the Internet of Things (IoT),\nand human–computer interactions. Different applications have\ndifferent requirements for the materials and production technologies. 170 , 171 For example, a wearable TENG requires flexible materials. Many of\nthe requirements could not be fulfilled by macroscale materials but\nby nanosized materials. Therefore, different types of nanomaterials\nhave been studied to determine their applications in TENGs. The utilization\nof inorganic nanomaterials in TENGs has been proven very successful.\nThe benefits of inorganic nanomaterials include the following: (1) Increase the contact\narea of the triboelectric\nsurfaces. The high surface area to volume ratio significantly increases\nthe contact area. (2) Tune\nthe dielectric properties of\nthe composited triboelectric materials. Inorganic nanomaterials include\nmany types of materials with dielectric properties, such as dielectric\nconstants, spread in a very large range. By selecting the type, size,\nand content of the inorganic nanomaterials, the dielectric properties\nof the triboelectric materials could be tuned as expected. (3) Enhance the charge transportation.\nThe interface between a triboelectric material and an electrode may\nhave a barrier due to the misalignment of the band structure. The\npresence of the nanomaterials could reduce the barrier so that the\ncharges could be transported more conveniently. (4) Tune the optical properties of the\nTENGs. Networks of nanowires such as silver and CNT nanowires can\nbe used to fabricate transparent electrodes for use in TENGs. (5) Mechanical properties:\nflexibility\nand stretchability. The network structures of the nanowires enabled\nthe triboelectric films to be flexible and stretchable but retained\ntheir electrical properties. (6) Hybrid nanogenerators. Some inorganic\nnanomaterials, such as ferroelectric nanomaterials, have piezoelectric\nproperties that allow the fabrication of hybrid nanogenerators. Although many works reviewed here have explored\nthe advances of\ninorganic nanomaterials in TENGs, there are still some issues that\nneed to be addressed in the future: (1) Quantitative understanding of the\nrelationship between the performances of the TENGs and the sizes of\nthe inorganic nanomaterials. Theoretical models need to be developed\nand optimized in the future. (2) Theoretical models of how the size\nand percentage of inorganic nanomaterials change the dielectric properties\nof the composites. (3) Applications of new types of inorganic\nnanomaterials 172 in TENGs. Only a small\nportion of inorganic nanomaterials have been studied in the last several\nyears, requiring more effort. In summary,\nwe have reviewed the recent advances of inorganic nanomaterials\nin triboelectric nanogenerators based on the roles, types, and characteristics\nof nanomaterials. The advantages of inorganic nanomaterials and the\nperformance of TENGs promoted by inorganic nanomaterials have been\nreviewed. Some prospective studies have been suggested that could\ninspire future research in the area. This Review provides an overview\nof how and why inorganic nanomaterials are utilized in TENGs, which\noffers guidance for future studies.",
"introduction": "1 Introduction The invention of triboelectric\nnanogenerators (TENGs) in 2012 1 turned\nthe historic physical phenomenon of triboelectrification\n(contact electrification) 2 into a working\nprinciple for energy conversion. With the development of TENGs, additional\napplications 3 to energy conversion have\nbeen discovered, including sensors, 4 − 8 control interfaces, 9 functional systems, 10 and biomedical applications. 11 , 12 The nature of triboelectrification implies that it can occur between\nany two materials that have physical contact and separation, as charges\ncan transfer between the surfaces. For this reason, diverse materials\nhave been studied for use in TENGs with different purposes, utilizing\nunique physical and chemical properties. There are many review\narticles 13 − 24 that have described the working modes, mechanisms, and applications\nof TENGs. Zheng et al. have reviewed the application of TENGs in biomedical\napplications. 13 Chen and co-workers 14 and Wang et al. 20 have reviewed energy harvesting and self-powered sensing using TENGs.\nWang 15 , 16 has reviewed the theoretical progress of\ntriboelectrification. Zhang and Olin 17 and\nBai et al. 19 have reviewed the materials\nthat are utilized in TENGs. Pan and co-workers 25 have reviewed the applications of TENGs for future soft\nrobots and machines. In this Review, we skip this part of the information\nand focus solely on how inorganic nanomaterials are used to improve\nthe performance of TENGs. The performance of TENGs is highly\ndependent not only on the triboelectric\nmaterials and their dielectric properties but also on how to pair\ntwo triboelectric materials. With the aim of boosting performance,\nnanomaterials have been introduced to TENGs. Nanomaterials can serve\nas either electrode or triboelectric materials, depending on the types\nof nanomaterials and how they are utilized. Nanomaterials in all dimensions\nhave been applied to TENGs with significant performance improvements. This Review summarizes the utilization of nanomaterials via several\naspects: (1) the roles of inorganic nanomaterials, (2) the types of\ninorganic nanomaterials, and (3) the composition of the nanomaterials.\nPerspectives of future studies are also given after the summary. This\nReview provides an overview of how inorganic nanomaterials that are\nused in TENGs can lead to further development in this area."
} | 1,689 |
32561727 | PMC7305114 | pmc | 1,715 | {
"abstract": "Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum , changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L −1 . Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.",
"introduction": "Introduction Hyaluronan (hyaluronic acid or HA) is a negatively charged, non-sulfated glycosaminoglycan comprising repeating uridine diphosphate glucuronate (UDP-GlcA) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) disaccharide units. It is a natural substance in vertebrates and mainly found in the eyes, joints and skin, where it absorbs large amounts of water for joint lubrication 1 , cell coating or repair of damaged skin tissue 2 . In addition to vertebrates, some pathogenic microorganisms, such as group A Streptococcus 3 and Pasteurella multocida type A strains 4 , produce HA as the major component of their capsules for protection against exterior damage. Due to its biocompatibility, hygroscopicity and non-immunogenicity, HA and its derivatives are important to the cosmetics, pharmaceutical, biomedical, and food industries 5 – 8 . Currently, commercial production of HA is mainly dependent on fermentation of group A Streptococcus 9 ; however, high risk of pathogenicity to livestock and contamination by exotoxins in the HA product hinder its broader application. The development of metabolic engineering allows the engineering of non-pathogenic Escherichia coli strains for heterologous HA production 10 , 11 . To further eliminate potential safety concerns, many generally recognized as safe (GRAS) strains, such as Bacillus subtilis 12 – 16 , Lactococcus lactis 17 – 20 , Corynebacterium glutamicum 21 , 22 , and Pichia pastoris 23 , have been engineered as alternative producers of HA. Additionally, cell-free systems have been exploited to produce HA at specific molecular weight (MW) 24 , 25 . Various strategies have been adopted to construct these HA producers, with selection of HA synthases and host strains representing the most critical step, as variations in HA synthase sequence, structural conformation 26 , or host cell metabolic capacities make differences in HA yield or MW. HA synthase activities have been improved by protein engineering 24 , 27 or modification of the microenvironment of the enzymatic reaction, including the membrane lipid composition 28 . Moreover, metabolic engineering strategies such as overexpressing enzymes of intermediate metabolic pathways (e.g., UDP-glucose 6-dehydrogenase or glucosamine-1-phosphate N-acetyltransferase) or blocking the synthesis of unwanted metabolites (e.g., l -lactate) were adopted to drive the generation of intermediate metabolites required for HA synthesis 29 , 30 . In view of the GRAS status and metabolic capacity, C. glutamicum as the Gram-positive model organism has been engineered as an HA producer. In 2014, Hoffmann et al. 21 first constructed the HA biosynthetic pathway by expressing Streptococcus equi subsp. zooepidemicus HA synthase (seHasA) in C. glutamicum . The engineered strain produced 1.2 g L −1 of HA. In 2016, Cheng et al. 31 co-expressed the codon-optimized Streptococcus dysgalactiae subsp. equisimilis ssehasA gene along with endogenous ugdA in C. glutamicum to produce 8.3 g L −1 HA in fed-batch cultivation. Additionally, deletion of ldh , encoding lactate dehydrogenase, increased HA production to 21.6 g L −1 in fed-batch culture 29 . A follow-up study enhanced the HA yield to 28.7 g L −1 by attenuating the glycolysis pathway, pentose phosphate pathway and the dehydrogenation of pyruvate 22 . In the present study, we engineer C. glutamicum for high-yield HA production by selecting the most productive HA synthase, overexpressing enzymes of the intermediate pathways to convert glucose into the HA building blocks UDP-GlcA and UDP-GlcNAc and decreasing endogenous extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 HA in fed-batch cultures. Our analysis of cell morphology reveals that the secreted HA forms an HA capsule-like layer, which is subsequently found to restrict nutrient uptake and inhibit HA synthesis. To relieve this inhibition effect, we supplement leech hyaluronidase (LHYal, hydrolase) 32 to the fed-batch culture to disrupt cell encapsulation and decrease broth viscosity. This strategy significantly promotes glucose uptake and HA production.",
"discussion": "Discussion Construction of biosynthesis pathways of HA and other glycosaminoglycans in genetically engineered microbes represents a green and safe method for production of these invaluable compounds. To construct an efficient HA-producing cell factory, selection of a proper HA synthase is critical, given that HA productivity and MW are largely determined by synthase activity 19 . Most HA synthases are found in vertebrates, whereas bacterial HA synthases are mainly found in the group A Streptococcus 3 . Streptococcal HasA has four transmembrane helices and two membrane-associated helices 33 – 35 ; therefore, it is conceivable that the membrane microenvironment influences HasA activity. In the present study, we found that spHasA displayed stronger HA-synthesis activities in C. glutamicum and resulted in higher HA accumulation relative to HasA from the other species (Fig. 1b ). The differences in the sequences of these enzymes were primarily found in the transmembrane region (Supplementary Fig. 2a ), suggesting that spHasA might be better suited to the C. glutamicum cytoplasmic membrane. Recently, Westbrook et al. 27 successfully enhanced the activity of Streptococcus equisimilis HasA (sseHasA) in B. subtilis by regulating membrane cardiolipin content and distribution. Additionally, previous studies engineered E. coli species as alternative HA producers 10 , 11 , 43 . However, E. coli exhibit a low degree of activity by heterologously expressed HA synthases, possibly because of the inherent incompatibility between HA synthase and the Gram-negative E. coli cell structure: E. coli cells have two membranes, whereas the type I HA synthase from Gram-positive Streptococcus secretes HA chains only through the inner membrane. Protein engineering of HasA 44 , especially changes in the transmembrane region, might represent a possible direction for further improvements in HA production (Supplementary Fig. 2b, c ). In addition to overexpressing UgdA and GlmS for precursor generation (Fig. 1a ), deletion of the competing pathway to increase the supply of UDP-glucuronate and UDP-GlcNAc is a commonly used strategy 22 , 29 . In C. glutamicum , there is less availability of UDP-GlcA relative to UDP-GlcNAc, given that overexpression of UgdA improved HA yield by 2-fold (Fig. 1c ). Additionally, UDP-GlcNAc is naturally produced for peptidoglycan biosynthesis (Fig. 1a ), which is critical for cell survival. UDP-glucose, the precursor of UDP-GlcA, is used to construct the outmost layer of the C. glutamicum cell wall 38 , which should be less important to the cell than peptidoglycans for maintaining the cell-envelope structure. A previous report showed that cg0420 expression enhanced the biosynthesis of extracellular polysaccharides 40 . In the present study, we selected four putative genes encoding glycosyltransferases ( cg0420 , c g0424 , cg0419 , and cg0438 ) and located in close proximity to galU and ugdA for deletion, finding that loss of cg0424 and cg0420 reduced cell-surface polysaccharides such as mannan and arabinomannan (Fig. 2d ) and enhanced HA production (Table 1 ) without impairing cell growth (Fig. 2c ). These results demonstrated that the enzymes encoded by cg0420 40 and cg0424 are involved in extracellular polysaccharide synthesis and that the outmost layer of the C. glutamicum cell wall 38 comprising extracellular polysaccharides is likely not essential to cell growth. The feedback effects of HA accumulation on cell morphology and metabolism have previously been ignored. Here, we found that during the later period of fermentation, cells of the engineered C. glutamicum strain were encapsulated following HA accumulation. The formation of a capsule-like layer (Fig. 3a ) inhibited cell metabolism and growth (Fig. 4b ). The morphology of the C. glutamicum CgspH-7 cells resembled the chain shape of native HA-producing Streptococcus species (Fig. 4a ). Moreover, our results suggest that the capsule of pathogens not only protects cells against various stressors or phages or acts as a virulence factor, but also could adversely affect cell metabolism (Fig. 4b ). This could explain why capsule formation occurs under so complex regulatory processes 45 . In conclusion, optimization of the HA-biosynthesis pathway, inactivation of extracellular polysaccharide biosynthesis, destruction of cell encapsulation and resolving the mass-transfer bottleneck by supplementation with LHYal resulted in an efficient C. glutamicum cell factory and a fermentation approach. This engineered strain allowed generation of an average of 74.1 g L −1 HA with an MW of 53 kDa in 5-L fed-batch cultures (Fig. 6c and Table 2 ). The results and strategies reported here are likely applicable to the production of other biopolymers."
} | 2,535 |
39253440 | PMC11383321 | pmc | 1,716 | {
"abstract": "SUMMARY Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina : nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.",
"introduction": "INTRODUCTION Endosymbiotic organelles are uniquely eukaryotic innovations for the acquisition of complex cellular functions, including aerobic respiration, photosynthesis, and nitrogen fixation. Endosymbioses contributed to expansive eukaryotic diversity 1 . An important question in cell evolution and engineering is: how do intermittent, facultative endosymbioses evolve into permanent integral cell compartments, i.e. organelles? With the recognition of the bacterial origin of mitochondria and chloroplasts in the 1980s, Cavalier-Smith & Lee proposed that the key distinction between a transient endosymbiont and an organelle was that organelles do not synthesize all their own proteins 2 , 3 . Instead, in organelles, some genes are transferred from the endosymbiont genome to the eukaryotic nucleus in a process called endosymbiotic gene transfer (EGT). These and other gene products, now under the control of host gene expression, are imported back into the endosymbiotic compartment to regulate endosymbiont growth and division. This definition has since been commonly applied 4 , 5 . However, the underlying hypothesis for organelle evolution — that genetic integration resulting from EGT and/or import of host-encoded gene products is essential for maintaining the endosymbiont as an integral cellular compartment — has not been rigorously tested. In the decades since, increased sampling of eukaryotic diversity has uncovered evidence that, amongst microbes, endosymbioses are a common strategy for acquisition of new functions. Based on observations of EGT and host protein import, new organelles have been recognized: the chromatophore in Paulinella chromatophora 6 – 9 and UCYN-A in Braarudosphaera bigelowii 10 . EGT and host protein import have also been observed in obligate, vertically-inherited nutritional endosymbionts of the parasite Angomonas deanei and insects, which are not formally recognized as organelles 11 , 12 . With the benefit of these newer models, our understanding of genetic integration has become more nuanced 13 , 14 . For example, the majority of host proteins imported into the Paulinella chromatophore do not originate from EGT but rather horizontal gene transfer (HGT) from other bacteria or eukaryotic genes 15 , showing that a host’s repertoire of pre-existing genes may play an outsized role in facilitating genetic integration 16 . UCYN-A was initially described as having an unstable relationship with its host as it is often lost from host cells during isolation and in culture, suggesting environmental conditions can affect endosymbiont stability despite genetic integration 17 . There have been bigger surprises: Some organisms temporarily acquire plastids from partially-digested prey algae 18 , 19 . The retained chloroplasts, called kleptoplasts, perform photosynthesis and, in several species, depend on imported host proteins to fill gaps in their metabolic pathways. Despite their genetic integration, these kleptoplasts cannot replicate in the host cell and are not required for host cell survival, indicating that genetic integration is not sufficient to achieve stable integration of the endosymbiont 20 – 22 . These findings highlight the importance of studying biodiverse organisms to inform new hypotheses for endosymbiotic evolution. Amongst new model systems, Epithemia spp. diatoms offer a unique perspective on organellogenesis. These photosynthetic microalgae contain diazotroph endosymbionts (designated diazoplasts) that perform nitrogen fixation, a biological reaction that converts inert atmospheric nitrogen to bioavailable ammonia 23 – 28 . The ability to fix both carbon and nitrogen fulfills a unique niche in ecosystems. Numerous Epithemia species are globally widespread in freshwater habitats and have recently been isolated from marine environments 29 – 31 . The Epithemia endosymbiosis is very young relative to mitochondria and chloroplasts, having originated ~35 Mya, based on fossil records 32 . Nonetheless, diazoplasts are obligate endosymbionts which are coordinately inherited during host cell division and present in all Epithemia species described so far, indicating co-evolution of diazoplasts and their host algae. Finally, Epithemia diazoplasts are closely related to UCYN-A, the diazotroph endosymbiont of B. bigelowii which was recently designated the first nitrogen-fixing organelle, or nitroplast 10 , 28 , 33 . Both Epithemia diazoplasts and UCYN-A evolved from free-living Crocosphaera cyanobacteria that have engaged in endosymbioses with several host microalgae. The independent evolution of free-living Crocosphaera into diazotroph endosymbionts in multiple host lineages enables comparisons that can lead to powerful insights. If the significance of organelles lies in their function as integral cellular compartments, then metabolic and cellular integration with the host cell are paramount 34 . By these criteria, diazoplasts show a level of host-symbiont integration comparable to UCYN-A. Nitrogen fixation requires large amounts of ATP and reducing power, energy that can be supplied by photosynthesis. Yet nitrogenase, the enzyme that catalyzes nitrogen fixation, is exquisitely sensitive to oxygen produced during oxygenic photosynthesis. In free-living Crocosphaera , photosynthesis and nitrogen fixation is temporally separated such that fixed carbon from daytime photosynthesis is stored as glycogen to fuel exclusively nighttime nitrogen fixation. Diazoplasts have lost all photosystem genes and depend entirely on host photosynthesis for fixed carbon 27 , 35 . Recently, we showed that host and diazoplast metabolism are tightly coupled to support continuous nitrogenase activity throughout the day-night cycle in E. clementina : Diatom photosynthesis is required for daytime nitrogenase activity in the diazoplast, while nighttime nitrogenase activity also depends on diatom, rather than diazoplast, carbon stores 28 . In comparison, UCYN-A has lost only photosystem II and is dependent on both host photosynthesis and, likely, its own photosystem I, restricting it to daytime nitrogen fixation 36 , 37 . Epithemia spp. described so far typically contain 1–2 diazoplasts per cell that are vertically inherited during asexual cell division 25 , 28 , 30 . Diazoplasts have further been shown to be uniparentally inherited during sexual reproduction, similar to mitochondria and chloroplasts 38 . Coordinated replication of UCYN-A with host cell division has been observed to maintain a single endosymbiont per cell 10 . Similar mechanisms are likely in place to coordinate diazoplast inheritance with diatom division. In fact, the presence of diazoplasts in diverse Epithemia species globally widespread in freshwater and marine ecosystems demonstrates that the mechanisms of inheritance are robust through speciation events. Diazoplasts effectively serve as dedicated nitrogen-fixing compartments in Epithemia , whether or not genetic integration has occurred. An important question emerges from these observations: is EGT and/or host protein import required to achieve the level of host-symbiont integration observed between diazoplasts and host Epithemia ? Based on the similarity of diazoplasts to UCYN-A, the assumption is yes. However, there is evidence that metabolite exchange via endosymbiont-encoded transporters 39 and division coordinated by host proteins outside the endosymbiotic compartment 11 , 40 , 41 could form a stable compartment without genetic integration. We previously established freshwater E. clementina as a laboratory model for functional studies and herein performed de novo assembly and annotation of its genome. The genome sequence for E. pelagica , a recently-discovered marine species, was publicly released by the Wellcome Sanger Institute 30 , 42 . To facilitate comparison between these species, we also performed de novo genome annotation of E. pelagica . Notably, no genomes of B. bigelowii (which hosts UCYN-A) nor the eukaryotic host in any other diazotroph endosymbiosis have been available. We report genome and transcriptome analyses of these two Epithemia species as well as proteome analyses of E. clementina with the goals of 1) providing a necessary resource to accelerate investigation of this model and 2) elucidating the role of genetic integration in this very young, stably integrated endosymbiont.",
"discussion": "DISCUSSION EGT and host protein import have been held as a necessary to achieve the “permanent” integration of organelles 2 , 5 . But this hypothesis for organelle evolution has been challenged by findings in young endosymbionts from diverse organisms 13 , 14 , 34 . We report analysis of two genomes of Epithemia diatoms and evaluate the extent of their genetic integration with their nitrogen-fixing endosymbionts (diazoplasts), thereby adding this very young endosymbiosis to existing model systems that can elucidate the integration of two cells into one. A window into the early dynamics of nuclear gene transfers. Our first significant finding was the detection of active diazoplast-to-nucleus DNA transfers but, as yet, no functional EGT in Epithemia . Our observations support findings in the chromatophore and UCYN-A that EGT is not necessary for genetic integration 10 , 15 , 53 . Given that EGT does not necessarily precede evolution of host protein import pathways, it may be a suboptimal solution for the inevitable genome decay in small asexual endosymbiont populations as a consequence of Muller’s ratchet 54 , 55 . Instead, the decayed nature of the NUDTs we detected in E. clementina is consistent with stochastic, transient, ongoing DNA transfer. Nonfunctional DNA transfers were previously only described from mitochondria or plastids with far more reduced genomes. The status of nuclear transfers from more recently-acquired organelles is unknown, as only protein-coding regions were used as queries to identify chromatophore transfers in Paulinella and only a transcriptome is available for the UCYN-A host, B. bigelowii 10 , 15 , 56 . NUDTs in Epithemia genomes therefore provide a rare window into the early dynamics of DNA transfer. For example, using the same homology criteria, we identified 5 NUMTs but no NUPTs in E. clementina . The NUMTs were significantly shorter than NUDTs and did not show rearrangement, which may suggest different mechanisms of transfer for NUDTs, NUMTs, and NUPTs in the same host nucleus. In addition, between-species differences may identify factors that affect transfer rates. The lack of observed NUDTs in E. pelagica suggest constraints on diazoplast-to-nucleus transfers. Previous observations in plant chloroplasts supported the limited transfer window hypothesis, which proposes the mechanism of gene transfer requires endosymbiont lysis and therefore the frequency of gene transfers correlates with the number of endosymbionts per cell 57 – 59 . However since E. pelagica and E. clementina contain similar numbers of diazoplasts per cell (1–2) 28 , 30 , the limited transfer window hypothesis does not explain the observed differences. Instead, there may be additional constraints imposed such as lower tolerance to DNA insertions in the comparatively smaller, non-repetitive genome of E. pelagica . Finally, the lack of NUDT gene expression, even with transfer of a full-length unmutated tusA gene, points to barriers to achieving eukaryotic expression from bacterial gene sequence. Epithemia is an apt model system to interrogate how horizontal gene transfer impacts eukaryotic genome evolution with at least 20 species easily obtained from freshwater globally and consistently adaptable to laboratory cultures 28 – 30 , 33 , 35 . Epithemia diazoplasts as a counterpoint to existing models of organellogenesis. A second unexpected finding was the detection of only 6 host protein candidates in the diazoplast proteome, much fewer and with less clear functional significance than in comparable endosymbionts that have been designated organelles. Methods for validating the localization of these import candidates are unavailable in Epithemia. Even if confirmed to target to the diazoplast, the candidates lack conserved domains or homology with cyanobacterial proteins to indicate they replace or supplement diazoplast metabolic function, growth, or division. Our findings are not explained by current models of organellogenesis that propose import of host proteins as a necessary step to establish an integral endosymbiotic compartment. In the traditional model described in the introduction, host protein targeting is a “late” bottleneck step required for the regulation of the endosymbiont growth and division. More recently, “targeting-early” has been proposed to account for establishment of protein import pathways prior to cellular integration as observed in kleptoplasts 19 , 20 . In this model, protein import is selected over successive transient endosymbioses, possibly driven by the host’s need to export metabolites from the endosymbiont via transporters or related mechanisms 60 . The establishment of protein import pathways then facilitates endosymbiont gene loss with metabolic functions fulfilled by host proteins leading to endosymbiont fixation. Contradicting both models, we observed minimal evidence for genetic integration despite millions of years of co-evolution resulting in diverse Epithemia species retaining diazoplasts, indicating that genetic integration is not necessary for its stable maintenance. At a minimum, the unclear functions of the few host proteins identified in the diazoplast proteome, if imported, suggest that the genesis of host protein import in Epithemia is very different than would be predicted by current models. Diazotroph endosymbioses are fundamentally different from photosynthetic endosymbioses that are the basis for current organellogenesis models. First, the diazoplast is derived from a cyanobacterium that became heterotrophic by way of losing its photosynthetic apparatus. Regulation of endosymbiont growth and division by the availability of host sugars (without requiring an additional layer of regulation via import of host metabolic enzymes) may be more facile with heterotrophic endosymbionts maintained for a nonphotosynthetic function compared to autotrophic endosymbionts. It will be interesting to see how integration of the diazoplast differs from the endosymbiont of Climacodium freunfeldianum , another diazotrophic endosymbiont descended from Crocosphaera that likely retains photosynthesis 61 , 62 . Second, ammonia, the host-beneficial metabolite in diazotroph endosymbioses, can diffuse through membranes in its neutral form and does not require host transporters for efficient trafficking 63 . Previously, we observed efficient distribution of fixed nitrogen from diazoplasts into host compartments following 15 N 2 labeling 28 . Ammonia diffusion may have reduced early selection pressure for host protein import as posited by the targeting-early model. Finally, the eukaryotic hosts in most diazotroph endosymbioses are already photosynthetic, in contrast to largely heterotrophic hosts that acquired photosynthesis by endosymbiosis. For instance, cellular processes that enabled intracellular bacteria to take up residence in the ancestor of Epithemia spp. were likely different than those of the bacterivore amoeba ancestor of Paulinella chromatophora . Autotrophy and lack of digestive pathways would reduce the frequency by which bacteria might gain access to the host cell, such that the selection of host protein import pathways over successive transient interactions would be less effective. Overall, a universal model of organellogenesis is premature given the limited types of interaction that have been investigated in depth, highlighting instead the importance of increasing the diversity of systems studied. Are diazoplasts “organelles”? As detailed in the introduction, diazoplasts show metabolic and cellular integration with their host alga comparable to that of UCYN-A, the first documented nitrogen-fixing organelle 10 , 17 . However, while hundreds of host proteins were detected in the UCYN-A proteome, including many likely to fill gaps in its metabolic pathways, a handful of host proteins with unknown function were detected in the diazoplast proteome. Based on the conventional definition which specifies genetic integration as the dividing line between endosymbionts and organelles, diazoplasts would not qualify 2 , 5 . However, over a decade ago, Keeling and Archibald 34 suggested that “if we use genetic integration as the defining feature of an organelle, we will never be able to compare different routes to organellogenesis because we have artificially predefined a single route.” They further hypothesized that if an endosymbiont became fixed in its host absent genetic integration, “it might prove to be even more interesting… by focusing on how it did integrate, perhaps we will find a truly parallel pathway for the integration of two cells.” The diazoplast appears to be such a parallel case in which non-genetic interactions were sufficient to integrate two cells. If not gene transfer and host protein import, then what is the “glue” that holds this endosymbiosis together? The loss of cyanobacterial photosystems and dependence on host photosynthesis indicate that diazoplasts acquired new transporters for host sugars 28 . There are several examples of cyanobacteria that express genes for sugar transport 39 , 64 , 65 . Therefore, acquisition via horizontal gene transfer from another bacteria to the diazoplast ancestor, prior to the endosymbiosis, rather than targeting of a eukaryotic transporter post-endosymbiosis, seems more likely. Consistent with this hypothesis, potential transporters were not detected amongst host protein import candidates detected in UCYN-A 10 . In fact, mixotrophy may have facilitated the adoption of an endosymbiotic lifestyle by the free-living cyanobacterium. Regardless of the timing of acquisition, sugar transport function could allow diazoplast growth to be regulated by nutrient availability from the host. Similarly, cytosolic host proteins may coordinate diazoplast division from outside the endosymbiotic compartment 11 , 41 . Eukaryotic dynamins required for mitochondria and chloroplast fission localize to the surface of the organellar outer membrane, acting coordinately with bacterial fission factors located in the organelle 40 , 66 . Diazoplasts appear to be surrounded by a host-derived membrane 26 , 67 (which may be lost during diazoplast purification) ( Figure 4A ). In analogy to dynamins, host proteins localized to this outer membrane may mediate diazoplast fission without requiring protein import pathways. Finally, cell density 68 and mechanical confinement 69 have been demonstrated to limit the growth of cyanobacteria, suggesting that host regulation of the volume of the endosymbiotic compartment could also be an effective mechanism. The mechanisms for the robust metabolic exchange and coordinated division observed in diazoplasts will be the focus of future studies. Application of cell evolution models to bioengineering. Diazoplasts provide another example that the current organelle definition does not account for observations in many biological systems and may be overdue for revision to reflect biological significance in the spectrum of endosymbiotic interactions. At a minimum, it is time to disentangle the current definition of an organelle from models that elucidate the formation of integral cellular compartments. Identifying mechanisms to integrate cells is more than an academic exercise. The ability to engineer bacteria as membrane compartments to introduce new metabolic functions in eukaryotes would be transformative 70 , 71 . For example, nitrogen-fixing crop plants that could replace nitrogen fertilizers is a major goal for sustainable agriculture. Efforts to transfer the genes for nitrogen fixation to plant cells have been slow, hampered by the many genes required as well as the complex assembly, high energy requirements, and oxygen sensitivity of the reaction. We previously proposed an alternative strategy inspired by diazotroph endosymbioses: introducing nitrogen-fixing bacteria into plant cells as an integral organelle-like compartment 28 . This approach has the advantage that diazotrophs express all required genes with intact regulation, coupled to respiration, and in a protected compartment. Diazoplasts, which achieve stable integration without significant genetic integration, are an important alternative to UCYN-A and other organelles, which are defined by their genetic integration, to inform this strategy. Identifying the nongenetic interactions that facilitated diazoplast integration with Epithemia will be critical for guiding bioengineering. Ongoing genome reduction may drive genetic integration in diazotroph endosymbioses. The fewer number of host protein candidates and their lack of clear function in diazoplasts versus UCYN-A is not associated with differences in their function as nitrogen-fixing cellular compartments. Rather, an alternative explanation points towards differences in the extent of genome reduction in diazoplasts, which encode 1585–1848 protein-coding genes, compared to UCYN-A, which encode 1200–1246 protein-coding genes 72 . Among the genes missing from the UCYN-A genome are cyanobacterial IspD, ThrC, PGLS, and PyrE; for each, an imported host protein was identified that could substitute for the missing function 10 . In contrast, these genes are retained in diazoplast genomes, including those of E. clementina and E. pelagica , obviating the need for host proteins to fulfill their functions ( Figure S4 ). Consistent with diazoplasts and UCYN-A being at different stages of genome reduction, diazoplast genomes contain >150 pseudogenes compared to 57 detected in the UCYN-A genome, suggesting diazoplasts are in a more active stage of genome reduction 27 , 33 , 35 . Interestingly, even genes retained in UCYN-A, namely PyrC and HemE, have imported host-encoded counterparts 10 . The endosymbiont copies may have acquired mutations resulting in reduced function, necessitating import of host proteins to compensate. Alternatively, once efficient host protein import pathways were established, import of redundant host proteins may render endosymbiont genes obsolete, further accelerating genome reduction. Genetic integration may in fact be destabilizing for an otherwise stably integrated endosymbiont, at least initially, as it substitutes essential endosymbiont genes with host-encoded proteins that may not be functionally equivalent and require energy-dependent import pathways. Comparing these related but independent diazotroph endosymbioses yields valuable insight, which otherwise would not be apparent. Diazoplasts at 35 Mya may represent an earlier stage of the same evolutionary path as ~140 Mya UCYN-A, in which continued genome reduction will eventually select for protein import pathways. Alternatively, diazoplasts may have evolved unique solutions to combat destabilizing genome decay, for example through the early loss of mobile elements. 27 , 33 , 73 Whether they represent an early intermediate destined for genetic integration or an alternative path, diazoplasts provide a valuable new perspective on endosymbiotic evolution. Limitations of the study Accurate gene family analysis is dependent on species sampling. While we sought to sample species representative of diatom diversity, some of our reported Epithemia -specific gene families may be shared by non- Epithemia species not present in the data set. While we included free-living Crocosphaera relatives for our homology search for NUDTs, we cannot eliminate the possibility that there are NUDTs or EGTs derived from sequences that were once in diazoplast genomes but have been lost. We did not observe expression associated with genes contained in NUDTs, however, we cannot exclude that they may be expressed under untested conditions. While differential centrifugation was an effective means of diazoplast enrichment, there may still be contamination by other cellular compartments. Low-abundance proteins may fall below the detection threshold in label-free proteomics. While our proteome coverage was comparable to previously reported studies, we cannot eliminate the possibility that there are undetected host proteins enriched in the diazoplast fraction. Tools such as immunofluorescence and genetics have not yet been established in Epithemia diatoms. We are therefore unable to confirm the localization or function of any Epithemia proteins."
} | 6,488 |
39253440 | PMC11383321 | pmc | 1,716 | {
"abstract": "SUMMARY Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina : nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.",
"introduction": "INTRODUCTION Endosymbiotic organelles are uniquely eukaryotic innovations for the acquisition of complex cellular functions, including aerobic respiration, photosynthesis, and nitrogen fixation. Endosymbioses contributed to expansive eukaryotic diversity 1 . An important question in cell evolution and engineering is: how do intermittent, facultative endosymbioses evolve into permanent integral cell compartments, i.e. organelles? With the recognition of the bacterial origin of mitochondria and chloroplasts in the 1980s, Cavalier-Smith & Lee proposed that the key distinction between a transient endosymbiont and an organelle was that organelles do not synthesize all their own proteins 2 , 3 . Instead, in organelles, some genes are transferred from the endosymbiont genome to the eukaryotic nucleus in a process called endosymbiotic gene transfer (EGT). These and other gene products, now under the control of host gene expression, are imported back into the endosymbiotic compartment to regulate endosymbiont growth and division. This definition has since been commonly applied 4 , 5 . However, the underlying hypothesis for organelle evolution — that genetic integration resulting from EGT and/or import of host-encoded gene products is essential for maintaining the endosymbiont as an integral cellular compartment — has not been rigorously tested. In the decades since, increased sampling of eukaryotic diversity has uncovered evidence that, amongst microbes, endosymbioses are a common strategy for acquisition of new functions. Based on observations of EGT and host protein import, new organelles have been recognized: the chromatophore in Paulinella chromatophora 6 – 9 and UCYN-A in Braarudosphaera bigelowii 10 . EGT and host protein import have also been observed in obligate, vertically-inherited nutritional endosymbionts of the parasite Angomonas deanei and insects, which are not formally recognized as organelles 11 , 12 . With the benefit of these newer models, our understanding of genetic integration has become more nuanced 13 , 14 . For example, the majority of host proteins imported into the Paulinella chromatophore do not originate from EGT but rather horizontal gene transfer (HGT) from other bacteria or eukaryotic genes 15 , showing that a host’s repertoire of pre-existing genes may play an outsized role in facilitating genetic integration 16 . UCYN-A was initially described as having an unstable relationship with its host as it is often lost from host cells during isolation and in culture, suggesting environmental conditions can affect endosymbiont stability despite genetic integration 17 . There have been bigger surprises: Some organisms temporarily acquire plastids from partially-digested prey algae 18 , 19 . The retained chloroplasts, called kleptoplasts, perform photosynthesis and, in several species, depend on imported host proteins to fill gaps in their metabolic pathways. Despite their genetic integration, these kleptoplasts cannot replicate in the host cell and are not required for host cell survival, indicating that genetic integration is not sufficient to achieve stable integration of the endosymbiont 20 – 22 . These findings highlight the importance of studying biodiverse organisms to inform new hypotheses for endosymbiotic evolution. Amongst new model systems, Epithemia spp. diatoms offer a unique perspective on organellogenesis. These photosynthetic microalgae contain diazotroph endosymbionts (designated diazoplasts) that perform nitrogen fixation, a biological reaction that converts inert atmospheric nitrogen to bioavailable ammonia 23 – 28 . The ability to fix both carbon and nitrogen fulfills a unique niche in ecosystems. Numerous Epithemia species are globally widespread in freshwater habitats and have recently been isolated from marine environments 29 – 31 . The Epithemia endosymbiosis is very young relative to mitochondria and chloroplasts, having originated ~35 Mya, based on fossil records 32 . Nonetheless, diazoplasts are obligate endosymbionts which are coordinately inherited during host cell division and present in all Epithemia species described so far, indicating co-evolution of diazoplasts and their host algae. Finally, Epithemia diazoplasts are closely related to UCYN-A, the diazotroph endosymbiont of B. bigelowii which was recently designated the first nitrogen-fixing organelle, or nitroplast 10 , 28 , 33 . Both Epithemia diazoplasts and UCYN-A evolved from free-living Crocosphaera cyanobacteria that have engaged in endosymbioses with several host microalgae. The independent evolution of free-living Crocosphaera into diazotroph endosymbionts in multiple host lineages enables comparisons that can lead to powerful insights. If the significance of organelles lies in their function as integral cellular compartments, then metabolic and cellular integration with the host cell are paramount 34 . By these criteria, diazoplasts show a level of host-symbiont integration comparable to UCYN-A. Nitrogen fixation requires large amounts of ATP and reducing power, energy that can be supplied by photosynthesis. Yet nitrogenase, the enzyme that catalyzes nitrogen fixation, is exquisitely sensitive to oxygen produced during oxygenic photosynthesis. In free-living Crocosphaera , photosynthesis and nitrogen fixation is temporally separated such that fixed carbon from daytime photosynthesis is stored as glycogen to fuel exclusively nighttime nitrogen fixation. Diazoplasts have lost all photosystem genes and depend entirely on host photosynthesis for fixed carbon 27 , 35 . Recently, we showed that host and diazoplast metabolism are tightly coupled to support continuous nitrogenase activity throughout the day-night cycle in E. clementina : Diatom photosynthesis is required for daytime nitrogenase activity in the diazoplast, while nighttime nitrogenase activity also depends on diatom, rather than diazoplast, carbon stores 28 . In comparison, UCYN-A has lost only photosystem II and is dependent on both host photosynthesis and, likely, its own photosystem I, restricting it to daytime nitrogen fixation 36 , 37 . Epithemia spp. described so far typically contain 1–2 diazoplasts per cell that are vertically inherited during asexual cell division 25 , 28 , 30 . Diazoplasts have further been shown to be uniparentally inherited during sexual reproduction, similar to mitochondria and chloroplasts 38 . Coordinated replication of UCYN-A with host cell division has been observed to maintain a single endosymbiont per cell 10 . Similar mechanisms are likely in place to coordinate diazoplast inheritance with diatom division. In fact, the presence of diazoplasts in diverse Epithemia species globally widespread in freshwater and marine ecosystems demonstrates that the mechanisms of inheritance are robust through speciation events. Diazoplasts effectively serve as dedicated nitrogen-fixing compartments in Epithemia , whether or not genetic integration has occurred. An important question emerges from these observations: is EGT and/or host protein import required to achieve the level of host-symbiont integration observed between diazoplasts and host Epithemia ? Based on the similarity of diazoplasts to UCYN-A, the assumption is yes. However, there is evidence that metabolite exchange via endosymbiont-encoded transporters 39 and division coordinated by host proteins outside the endosymbiotic compartment 11 , 40 , 41 could form a stable compartment without genetic integration. We previously established freshwater E. clementina as a laboratory model for functional studies and herein performed de novo assembly and annotation of its genome. The genome sequence for E. pelagica , a recently-discovered marine species, was publicly released by the Wellcome Sanger Institute 30 , 42 . To facilitate comparison between these species, we also performed de novo genome annotation of E. pelagica . Notably, no genomes of B. bigelowii (which hosts UCYN-A) nor the eukaryotic host in any other diazotroph endosymbiosis have been available. We report genome and transcriptome analyses of these two Epithemia species as well as proteome analyses of E. clementina with the goals of 1) providing a necessary resource to accelerate investigation of this model and 2) elucidating the role of genetic integration in this very young, stably integrated endosymbiont.",
"discussion": "DISCUSSION EGT and host protein import have been held as a necessary to achieve the “permanent” integration of organelles 2 , 5 . But this hypothesis for organelle evolution has been challenged by findings in young endosymbionts from diverse organisms 13 , 14 , 34 . We report analysis of two genomes of Epithemia diatoms and evaluate the extent of their genetic integration with their nitrogen-fixing endosymbionts (diazoplasts), thereby adding this very young endosymbiosis to existing model systems that can elucidate the integration of two cells into one. A window into the early dynamics of nuclear gene transfers. Our first significant finding was the detection of active diazoplast-to-nucleus DNA transfers but, as yet, no functional EGT in Epithemia . Our observations support findings in the chromatophore and UCYN-A that EGT is not necessary for genetic integration 10 , 15 , 53 . Given that EGT does not necessarily precede evolution of host protein import pathways, it may be a suboptimal solution for the inevitable genome decay in small asexual endosymbiont populations as a consequence of Muller’s ratchet 54 , 55 . Instead, the decayed nature of the NUDTs we detected in E. clementina is consistent with stochastic, transient, ongoing DNA transfer. Nonfunctional DNA transfers were previously only described from mitochondria or plastids with far more reduced genomes. The status of nuclear transfers from more recently-acquired organelles is unknown, as only protein-coding regions were used as queries to identify chromatophore transfers in Paulinella and only a transcriptome is available for the UCYN-A host, B. bigelowii 10 , 15 , 56 . NUDTs in Epithemia genomes therefore provide a rare window into the early dynamics of DNA transfer. For example, using the same homology criteria, we identified 5 NUMTs but no NUPTs in E. clementina . The NUMTs were significantly shorter than NUDTs and did not show rearrangement, which may suggest different mechanisms of transfer for NUDTs, NUMTs, and NUPTs in the same host nucleus. In addition, between-species differences may identify factors that affect transfer rates. The lack of observed NUDTs in E. pelagica suggest constraints on diazoplast-to-nucleus transfers. Previous observations in plant chloroplasts supported the limited transfer window hypothesis, which proposes the mechanism of gene transfer requires endosymbiont lysis and therefore the frequency of gene transfers correlates with the number of endosymbionts per cell 57 – 59 . However since E. pelagica and E. clementina contain similar numbers of diazoplasts per cell (1–2) 28 , 30 , the limited transfer window hypothesis does not explain the observed differences. Instead, there may be additional constraints imposed such as lower tolerance to DNA insertions in the comparatively smaller, non-repetitive genome of E. pelagica . Finally, the lack of NUDT gene expression, even with transfer of a full-length unmutated tusA gene, points to barriers to achieving eukaryotic expression from bacterial gene sequence. Epithemia is an apt model system to interrogate how horizontal gene transfer impacts eukaryotic genome evolution with at least 20 species easily obtained from freshwater globally and consistently adaptable to laboratory cultures 28 – 30 , 33 , 35 . Epithemia diazoplasts as a counterpoint to existing models of organellogenesis. A second unexpected finding was the detection of only 6 host protein candidates in the diazoplast proteome, much fewer and with less clear functional significance than in comparable endosymbionts that have been designated organelles. Methods for validating the localization of these import candidates are unavailable in Epithemia. Even if confirmed to target to the diazoplast, the candidates lack conserved domains or homology with cyanobacterial proteins to indicate they replace or supplement diazoplast metabolic function, growth, or division. Our findings are not explained by current models of organellogenesis that propose import of host proteins as a necessary step to establish an integral endosymbiotic compartment. In the traditional model described in the introduction, host protein targeting is a “late” bottleneck step required for the regulation of the endosymbiont growth and division. More recently, “targeting-early” has been proposed to account for establishment of protein import pathways prior to cellular integration as observed in kleptoplasts 19 , 20 . In this model, protein import is selected over successive transient endosymbioses, possibly driven by the host’s need to export metabolites from the endosymbiont via transporters or related mechanisms 60 . The establishment of protein import pathways then facilitates endosymbiont gene loss with metabolic functions fulfilled by host proteins leading to endosymbiont fixation. Contradicting both models, we observed minimal evidence for genetic integration despite millions of years of co-evolution resulting in diverse Epithemia species retaining diazoplasts, indicating that genetic integration is not necessary for its stable maintenance. At a minimum, the unclear functions of the few host proteins identified in the diazoplast proteome, if imported, suggest that the genesis of host protein import in Epithemia is very different than would be predicted by current models. Diazotroph endosymbioses are fundamentally different from photosynthetic endosymbioses that are the basis for current organellogenesis models. First, the diazoplast is derived from a cyanobacterium that became heterotrophic by way of losing its photosynthetic apparatus. Regulation of endosymbiont growth and division by the availability of host sugars (without requiring an additional layer of regulation via import of host metabolic enzymes) may be more facile with heterotrophic endosymbionts maintained for a nonphotosynthetic function compared to autotrophic endosymbionts. It will be interesting to see how integration of the diazoplast differs from the endosymbiont of Climacodium freunfeldianum , another diazotrophic endosymbiont descended from Crocosphaera that likely retains photosynthesis 61 , 62 . Second, ammonia, the host-beneficial metabolite in diazotroph endosymbioses, can diffuse through membranes in its neutral form and does not require host transporters for efficient trafficking 63 . Previously, we observed efficient distribution of fixed nitrogen from diazoplasts into host compartments following 15 N 2 labeling 28 . Ammonia diffusion may have reduced early selection pressure for host protein import as posited by the targeting-early model. Finally, the eukaryotic hosts in most diazotroph endosymbioses are already photosynthetic, in contrast to largely heterotrophic hosts that acquired photosynthesis by endosymbiosis. For instance, cellular processes that enabled intracellular bacteria to take up residence in the ancestor of Epithemia spp. were likely different than those of the bacterivore amoeba ancestor of Paulinella chromatophora . Autotrophy and lack of digestive pathways would reduce the frequency by which bacteria might gain access to the host cell, such that the selection of host protein import pathways over successive transient interactions would be less effective. Overall, a universal model of organellogenesis is premature given the limited types of interaction that have been investigated in depth, highlighting instead the importance of increasing the diversity of systems studied. Are diazoplasts “organelles”? As detailed in the introduction, diazoplasts show metabolic and cellular integration with their host alga comparable to that of UCYN-A, the first documented nitrogen-fixing organelle 10 , 17 . However, while hundreds of host proteins were detected in the UCYN-A proteome, including many likely to fill gaps in its metabolic pathways, a handful of host proteins with unknown function were detected in the diazoplast proteome. Based on the conventional definition which specifies genetic integration as the dividing line between endosymbionts and organelles, diazoplasts would not qualify 2 , 5 . However, over a decade ago, Keeling and Archibald 34 suggested that “if we use genetic integration as the defining feature of an organelle, we will never be able to compare different routes to organellogenesis because we have artificially predefined a single route.” They further hypothesized that if an endosymbiont became fixed in its host absent genetic integration, “it might prove to be even more interesting… by focusing on how it did integrate, perhaps we will find a truly parallel pathway for the integration of two cells.” The diazoplast appears to be such a parallel case in which non-genetic interactions were sufficient to integrate two cells. If not gene transfer and host protein import, then what is the “glue” that holds this endosymbiosis together? The loss of cyanobacterial photosystems and dependence on host photosynthesis indicate that diazoplasts acquired new transporters for host sugars 28 . There are several examples of cyanobacteria that express genes for sugar transport 39 , 64 , 65 . Therefore, acquisition via horizontal gene transfer from another bacteria to the diazoplast ancestor, prior to the endosymbiosis, rather than targeting of a eukaryotic transporter post-endosymbiosis, seems more likely. Consistent with this hypothesis, potential transporters were not detected amongst host protein import candidates detected in UCYN-A 10 . In fact, mixotrophy may have facilitated the adoption of an endosymbiotic lifestyle by the free-living cyanobacterium. Regardless of the timing of acquisition, sugar transport function could allow diazoplast growth to be regulated by nutrient availability from the host. Similarly, cytosolic host proteins may coordinate diazoplast division from outside the endosymbiotic compartment 11 , 41 . Eukaryotic dynamins required for mitochondria and chloroplast fission localize to the surface of the organellar outer membrane, acting coordinately with bacterial fission factors located in the organelle 40 , 66 . Diazoplasts appear to be surrounded by a host-derived membrane 26 , 67 (which may be lost during diazoplast purification) ( Figure 4A ). In analogy to dynamins, host proteins localized to this outer membrane may mediate diazoplast fission without requiring protein import pathways. Finally, cell density 68 and mechanical confinement 69 have been demonstrated to limit the growth of cyanobacteria, suggesting that host regulation of the volume of the endosymbiotic compartment could also be an effective mechanism. The mechanisms for the robust metabolic exchange and coordinated division observed in diazoplasts will be the focus of future studies. Application of cell evolution models to bioengineering. Diazoplasts provide another example that the current organelle definition does not account for observations in many biological systems and may be overdue for revision to reflect biological significance in the spectrum of endosymbiotic interactions. At a minimum, it is time to disentangle the current definition of an organelle from models that elucidate the formation of integral cellular compartments. Identifying mechanisms to integrate cells is more than an academic exercise. The ability to engineer bacteria as membrane compartments to introduce new metabolic functions in eukaryotes would be transformative 70 , 71 . For example, nitrogen-fixing crop plants that could replace nitrogen fertilizers is a major goal for sustainable agriculture. Efforts to transfer the genes for nitrogen fixation to plant cells have been slow, hampered by the many genes required as well as the complex assembly, high energy requirements, and oxygen sensitivity of the reaction. We previously proposed an alternative strategy inspired by diazotroph endosymbioses: introducing nitrogen-fixing bacteria into plant cells as an integral organelle-like compartment 28 . This approach has the advantage that diazotrophs express all required genes with intact regulation, coupled to respiration, and in a protected compartment. Diazoplasts, which achieve stable integration without significant genetic integration, are an important alternative to UCYN-A and other organelles, which are defined by their genetic integration, to inform this strategy. Identifying the nongenetic interactions that facilitated diazoplast integration with Epithemia will be critical for guiding bioengineering. Ongoing genome reduction may drive genetic integration in diazotroph endosymbioses. The fewer number of host protein candidates and their lack of clear function in diazoplasts versus UCYN-A is not associated with differences in their function as nitrogen-fixing cellular compartments. Rather, an alternative explanation points towards differences in the extent of genome reduction in diazoplasts, which encode 1585–1848 protein-coding genes, compared to UCYN-A, which encode 1200–1246 protein-coding genes 72 . Among the genes missing from the UCYN-A genome are cyanobacterial IspD, ThrC, PGLS, and PyrE; for each, an imported host protein was identified that could substitute for the missing function 10 . In contrast, these genes are retained in diazoplast genomes, including those of E. clementina and E. pelagica , obviating the need for host proteins to fulfill their functions ( Figure S4 ). Consistent with diazoplasts and UCYN-A being at different stages of genome reduction, diazoplast genomes contain >150 pseudogenes compared to 57 detected in the UCYN-A genome, suggesting diazoplasts are in a more active stage of genome reduction 27 , 33 , 35 . Interestingly, even genes retained in UCYN-A, namely PyrC and HemE, have imported host-encoded counterparts 10 . The endosymbiont copies may have acquired mutations resulting in reduced function, necessitating import of host proteins to compensate. Alternatively, once efficient host protein import pathways were established, import of redundant host proteins may render endosymbiont genes obsolete, further accelerating genome reduction. Genetic integration may in fact be destabilizing for an otherwise stably integrated endosymbiont, at least initially, as it substitutes essential endosymbiont genes with host-encoded proteins that may not be functionally equivalent and require energy-dependent import pathways. Comparing these related but independent diazotroph endosymbioses yields valuable insight, which otherwise would not be apparent. Diazoplasts at 35 Mya may represent an earlier stage of the same evolutionary path as ~140 Mya UCYN-A, in which continued genome reduction will eventually select for protein import pathways. Alternatively, diazoplasts may have evolved unique solutions to combat destabilizing genome decay, for example through the early loss of mobile elements. 27 , 33 , 73 Whether they represent an early intermediate destined for genetic integration or an alternative path, diazoplasts provide a valuable new perspective on endosymbiotic evolution. Limitations of the study Accurate gene family analysis is dependent on species sampling. While we sought to sample species representative of diatom diversity, some of our reported Epithemia -specific gene families may be shared by non- Epithemia species not present in the data set. While we included free-living Crocosphaera relatives for our homology search for NUDTs, we cannot eliminate the possibility that there are NUDTs or EGTs derived from sequences that were once in diazoplast genomes but have been lost. We did not observe expression associated with genes contained in NUDTs, however, we cannot exclude that they may be expressed under untested conditions. While differential centrifugation was an effective means of diazoplast enrichment, there may still be contamination by other cellular compartments. Low-abundance proteins may fall below the detection threshold in label-free proteomics. While our proteome coverage was comparable to previously reported studies, we cannot eliminate the possibility that there are undetected host proteins enriched in the diazoplast fraction. Tools such as immunofluorescence and genetics have not yet been established in Epithemia diatoms. We are therefore unable to confirm the localization or function of any Epithemia proteins."
} | 6,488 |
39253440 | PMC11383321 | pmc | 1,717 | {
"abstract": "SUMMARY Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina : nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.",
"introduction": "INTRODUCTION Endosymbiotic organelles are uniquely eukaryotic innovations for the acquisition of complex cellular functions, including aerobic respiration, photosynthesis, and nitrogen fixation. Endosymbioses contributed to expansive eukaryotic diversity 1 . An important question in cell evolution and engineering is: how do intermittent, facultative endosymbioses evolve into permanent integral cell compartments, i.e. organelles? With the recognition of the bacterial origin of mitochondria and chloroplasts in the 1980s, Cavalier-Smith & Lee proposed that the key distinction between a transient endosymbiont and an organelle was that organelles do not synthesize all their own proteins 2 , 3 . Instead, in organelles, some genes are transferred from the endosymbiont genome to the eukaryotic nucleus in a process called endosymbiotic gene transfer (EGT). These and other gene products, now under the control of host gene expression, are imported back into the endosymbiotic compartment to regulate endosymbiont growth and division. This definition has since been commonly applied 4 , 5 . However, the underlying hypothesis for organelle evolution — that genetic integration resulting from EGT and/or import of host-encoded gene products is essential for maintaining the endosymbiont as an integral cellular compartment — has not been rigorously tested. In the decades since, increased sampling of eukaryotic diversity has uncovered evidence that, amongst microbes, endosymbioses are a common strategy for acquisition of new functions. Based on observations of EGT and host protein import, new organelles have been recognized: the chromatophore in Paulinella chromatophora 6 – 9 and UCYN-A in Braarudosphaera bigelowii 10 . EGT and host protein import have also been observed in obligate, vertically-inherited nutritional endosymbionts of the parasite Angomonas deanei and insects, which are not formally recognized as organelles 11 , 12 . With the benefit of these newer models, our understanding of genetic integration has become more nuanced 13 , 14 . For example, the majority of host proteins imported into the Paulinella chromatophore do not originate from EGT but rather horizontal gene transfer (HGT) from other bacteria or eukaryotic genes 15 , showing that a host’s repertoire of pre-existing genes may play an outsized role in facilitating genetic integration 16 . UCYN-A was initially described as having an unstable relationship with its host as it is often lost from host cells during isolation and in culture, suggesting environmental conditions can affect endosymbiont stability despite genetic integration 17 . There have been bigger surprises: Some organisms temporarily acquire plastids from partially-digested prey algae 18 , 19 . The retained chloroplasts, called kleptoplasts, perform photosynthesis and, in several species, depend on imported host proteins to fill gaps in their metabolic pathways. Despite their genetic integration, these kleptoplasts cannot replicate in the host cell and are not required for host cell survival, indicating that genetic integration is not sufficient to achieve stable integration of the endosymbiont 20 – 22 . These findings highlight the importance of studying biodiverse organisms to inform new hypotheses for endosymbiotic evolution. Amongst new model systems, Epithemia spp. diatoms offer a unique perspective on organellogenesis. These photosynthetic microalgae contain diazotroph endosymbionts (designated diazoplasts) that perform nitrogen fixation, a biological reaction that converts inert atmospheric nitrogen to bioavailable ammonia 23 – 28 . The ability to fix both carbon and nitrogen fulfills a unique niche in ecosystems. Numerous Epithemia species are globally widespread in freshwater habitats and have recently been isolated from marine environments 29 – 31 . The Epithemia endosymbiosis is very young relative to mitochondria and chloroplasts, having originated ~35 Mya, based on fossil records 32 . Nonetheless, diazoplasts are obligate endosymbionts which are coordinately inherited during host cell division and present in all Epithemia species described so far, indicating co-evolution of diazoplasts and their host algae. Finally, Epithemia diazoplasts are closely related to UCYN-A, the diazotroph endosymbiont of B. bigelowii which was recently designated the first nitrogen-fixing organelle, or nitroplast 10 , 28 , 33 . Both Epithemia diazoplasts and UCYN-A evolved from free-living Crocosphaera cyanobacteria that have engaged in endosymbioses with several host microalgae. The independent evolution of free-living Crocosphaera into diazotroph endosymbionts in multiple host lineages enables comparisons that can lead to powerful insights. If the significance of organelles lies in their function as integral cellular compartments, then metabolic and cellular integration with the host cell are paramount 34 . By these criteria, diazoplasts show a level of host-symbiont integration comparable to UCYN-A. Nitrogen fixation requires large amounts of ATP and reducing power, energy that can be supplied by photosynthesis. Yet nitrogenase, the enzyme that catalyzes nitrogen fixation, is exquisitely sensitive to oxygen produced during oxygenic photosynthesis. In free-living Crocosphaera , photosynthesis and nitrogen fixation is temporally separated such that fixed carbon from daytime photosynthesis is stored as glycogen to fuel exclusively nighttime nitrogen fixation. Diazoplasts have lost all photosystem genes and depend entirely on host photosynthesis for fixed carbon 27 , 35 . Recently, we showed that host and diazoplast metabolism are tightly coupled to support continuous nitrogenase activity throughout the day-night cycle in E. clementina : Diatom photosynthesis is required for daytime nitrogenase activity in the diazoplast, while nighttime nitrogenase activity also depends on diatom, rather than diazoplast, carbon stores 28 . In comparison, UCYN-A has lost only photosystem II and is dependent on both host photosynthesis and, likely, its own photosystem I, restricting it to daytime nitrogen fixation 36 , 37 . Epithemia spp. described so far typically contain 1–2 diazoplasts per cell that are vertically inherited during asexual cell division 25 , 28 , 30 . Diazoplasts have further been shown to be uniparentally inherited during sexual reproduction, similar to mitochondria and chloroplasts 38 . Coordinated replication of UCYN-A with host cell division has been observed to maintain a single endosymbiont per cell 10 . Similar mechanisms are likely in place to coordinate diazoplast inheritance with diatom division. In fact, the presence of diazoplasts in diverse Epithemia species globally widespread in freshwater and marine ecosystems demonstrates that the mechanisms of inheritance are robust through speciation events. Diazoplasts effectively serve as dedicated nitrogen-fixing compartments in Epithemia , whether or not genetic integration has occurred. An important question emerges from these observations: is EGT and/or host protein import required to achieve the level of host-symbiont integration observed between diazoplasts and host Epithemia ? Based on the similarity of diazoplasts to UCYN-A, the assumption is yes. However, there is evidence that metabolite exchange via endosymbiont-encoded transporters 39 and division coordinated by host proteins outside the endosymbiotic compartment 11 , 40 , 41 could form a stable compartment without genetic integration. We previously established freshwater E. clementina as a laboratory model for functional studies and herein performed de novo assembly and annotation of its genome. The genome sequence for E. pelagica , a recently-discovered marine species, was publicly released by the Wellcome Sanger Institute 30 , 42 . To facilitate comparison between these species, we also performed de novo genome annotation of E. pelagica . Notably, no genomes of B. bigelowii (which hosts UCYN-A) nor the eukaryotic host in any other diazotroph endosymbiosis have been available. We report genome and transcriptome analyses of these two Epithemia species as well as proteome analyses of E. clementina with the goals of 1) providing a necessary resource to accelerate investigation of this model and 2) elucidating the role of genetic integration in this very young, stably integrated endosymbiont.",
"discussion": "DISCUSSION EGT and host protein import have been held as a necessary to achieve the “permanent” integration of organelles 2 , 5 . But this hypothesis for organelle evolution has been challenged by findings in young endosymbionts from diverse organisms 13 , 14 , 34 . We report analysis of two genomes of Epithemia diatoms and evaluate the extent of their genetic integration with their nitrogen-fixing endosymbionts (diazoplasts), thereby adding this very young endosymbiosis to existing model systems that can elucidate the integration of two cells into one. A window into the early dynamics of nuclear gene transfers. Our first significant finding was the detection of active diazoplast-to-nucleus DNA transfers but, as yet, no functional EGT in Epithemia . Our observations support findings in the chromatophore and UCYN-A that EGT is not necessary for genetic integration 10 , 15 , 53 . Given that EGT does not necessarily precede evolution of host protein import pathways, it may be a suboptimal solution for the inevitable genome decay in small asexual endosymbiont populations as a consequence of Muller’s ratchet 54 , 55 . Instead, the decayed nature of the NUDTs we detected in E. clementina is consistent with stochastic, transient, ongoing DNA transfer. Nonfunctional DNA transfers were previously only described from mitochondria or plastids with far more reduced genomes. The status of nuclear transfers from more recently-acquired organelles is unknown, as only protein-coding regions were used as queries to identify chromatophore transfers in Paulinella and only a transcriptome is available for the UCYN-A host, B. bigelowii 10 , 15 , 56 . NUDTs in Epithemia genomes therefore provide a rare window into the early dynamics of DNA transfer. For example, using the same homology criteria, we identified 5 NUMTs but no NUPTs in E. clementina . The NUMTs were significantly shorter than NUDTs and did not show rearrangement, which may suggest different mechanisms of transfer for NUDTs, NUMTs, and NUPTs in the same host nucleus. In addition, between-species differences may identify factors that affect transfer rates. The lack of observed NUDTs in E. pelagica suggest constraints on diazoplast-to-nucleus transfers. Previous observations in plant chloroplasts supported the limited transfer window hypothesis, which proposes the mechanism of gene transfer requires endosymbiont lysis and therefore the frequency of gene transfers correlates with the number of endosymbionts per cell 57 – 59 . However since E. pelagica and E. clementina contain similar numbers of diazoplasts per cell (1–2) 28 , 30 , the limited transfer window hypothesis does not explain the observed differences. Instead, there may be additional constraints imposed such as lower tolerance to DNA insertions in the comparatively smaller, non-repetitive genome of E. pelagica . Finally, the lack of NUDT gene expression, even with transfer of a full-length unmutated tusA gene, points to barriers to achieving eukaryotic expression from bacterial gene sequence. Epithemia is an apt model system to interrogate how horizontal gene transfer impacts eukaryotic genome evolution with at least 20 species easily obtained from freshwater globally and consistently adaptable to laboratory cultures 28 – 30 , 33 , 35 . Epithemia diazoplasts as a counterpoint to existing models of organellogenesis. A second unexpected finding was the detection of only 6 host protein candidates in the diazoplast proteome, much fewer and with less clear functional significance than in comparable endosymbionts that have been designated organelles. Methods for validating the localization of these import candidates are unavailable in Epithemia. Even if confirmed to target to the diazoplast, the candidates lack conserved domains or homology with cyanobacterial proteins to indicate they replace or supplement diazoplast metabolic function, growth, or division. Our findings are not explained by current models of organellogenesis that propose import of host proteins as a necessary step to establish an integral endosymbiotic compartment. In the traditional model described in the introduction, host protein targeting is a “late” bottleneck step required for the regulation of the endosymbiont growth and division. More recently, “targeting-early” has been proposed to account for establishment of protein import pathways prior to cellular integration as observed in kleptoplasts 19 , 20 . In this model, protein import is selected over successive transient endosymbioses, possibly driven by the host’s need to export metabolites from the endosymbiont via transporters or related mechanisms 60 . The establishment of protein import pathways then facilitates endosymbiont gene loss with metabolic functions fulfilled by host proteins leading to endosymbiont fixation. Contradicting both models, we observed minimal evidence for genetic integration despite millions of years of co-evolution resulting in diverse Epithemia species retaining diazoplasts, indicating that genetic integration is not necessary for its stable maintenance. At a minimum, the unclear functions of the few host proteins identified in the diazoplast proteome, if imported, suggest that the genesis of host protein import in Epithemia is very different than would be predicted by current models. Diazotroph endosymbioses are fundamentally different from photosynthetic endosymbioses that are the basis for current organellogenesis models. First, the diazoplast is derived from a cyanobacterium that became heterotrophic by way of losing its photosynthetic apparatus. Regulation of endosymbiont growth and division by the availability of host sugars (without requiring an additional layer of regulation via import of host metabolic enzymes) may be more facile with heterotrophic endosymbionts maintained for a nonphotosynthetic function compared to autotrophic endosymbionts. It will be interesting to see how integration of the diazoplast differs from the endosymbiont of Climacodium freunfeldianum , another diazotrophic endosymbiont descended from Crocosphaera that likely retains photosynthesis 61 , 62 . Second, ammonia, the host-beneficial metabolite in diazotroph endosymbioses, can diffuse through membranes in its neutral form and does not require host transporters for efficient trafficking 63 . Previously, we observed efficient distribution of fixed nitrogen from diazoplasts into host compartments following 15 N 2 labeling 28 . Ammonia diffusion may have reduced early selection pressure for host protein import as posited by the targeting-early model. Finally, the eukaryotic hosts in most diazotroph endosymbioses are already photosynthetic, in contrast to largely heterotrophic hosts that acquired photosynthesis by endosymbiosis. For instance, cellular processes that enabled intracellular bacteria to take up residence in the ancestor of Epithemia spp. were likely different than those of the bacterivore amoeba ancestor of Paulinella chromatophora . Autotrophy and lack of digestive pathways would reduce the frequency by which bacteria might gain access to the host cell, such that the selection of host protein import pathways over successive transient interactions would be less effective. Overall, a universal model of organellogenesis is premature given the limited types of interaction that have been investigated in depth, highlighting instead the importance of increasing the diversity of systems studied. Are diazoplasts “organelles”? As detailed in the introduction, diazoplasts show metabolic and cellular integration with their host alga comparable to that of UCYN-A, the first documented nitrogen-fixing organelle 10 , 17 . However, while hundreds of host proteins were detected in the UCYN-A proteome, including many likely to fill gaps in its metabolic pathways, a handful of host proteins with unknown function were detected in the diazoplast proteome. Based on the conventional definition which specifies genetic integration as the dividing line between endosymbionts and organelles, diazoplasts would not qualify 2 , 5 . However, over a decade ago, Keeling and Archibald 34 suggested that “if we use genetic integration as the defining feature of an organelle, we will never be able to compare different routes to organellogenesis because we have artificially predefined a single route.” They further hypothesized that if an endosymbiont became fixed in its host absent genetic integration, “it might prove to be even more interesting… by focusing on how it did integrate, perhaps we will find a truly parallel pathway for the integration of two cells.” The diazoplast appears to be such a parallel case in which non-genetic interactions were sufficient to integrate two cells. If not gene transfer and host protein import, then what is the “glue” that holds this endosymbiosis together? The loss of cyanobacterial photosystems and dependence on host photosynthesis indicate that diazoplasts acquired new transporters for host sugars 28 . There are several examples of cyanobacteria that express genes for sugar transport 39 , 64 , 65 . Therefore, acquisition via horizontal gene transfer from another bacteria to the diazoplast ancestor, prior to the endosymbiosis, rather than targeting of a eukaryotic transporter post-endosymbiosis, seems more likely. Consistent with this hypothesis, potential transporters were not detected amongst host protein import candidates detected in UCYN-A 10 . In fact, mixotrophy may have facilitated the adoption of an endosymbiotic lifestyle by the free-living cyanobacterium. Regardless of the timing of acquisition, sugar transport function could allow diazoplast growth to be regulated by nutrient availability from the host. Similarly, cytosolic host proteins may coordinate diazoplast division from outside the endosymbiotic compartment 11 , 41 . Eukaryotic dynamins required for mitochondria and chloroplast fission localize to the surface of the organellar outer membrane, acting coordinately with bacterial fission factors located in the organelle 40 , 66 . Diazoplasts appear to be surrounded by a host-derived membrane 26 , 67 (which may be lost during diazoplast purification) ( Figure 4A ). In analogy to dynamins, host proteins localized to this outer membrane may mediate diazoplast fission without requiring protein import pathways. Finally, cell density 68 and mechanical confinement 69 have been demonstrated to limit the growth of cyanobacteria, suggesting that host regulation of the volume of the endosymbiotic compartment could also be an effective mechanism. The mechanisms for the robust metabolic exchange and coordinated division observed in diazoplasts will be the focus of future studies. Application of cell evolution models to bioengineering. Diazoplasts provide another example that the current organelle definition does not account for observations in many biological systems and may be overdue for revision to reflect biological significance in the spectrum of endosymbiotic interactions. At a minimum, it is time to disentangle the current definition of an organelle from models that elucidate the formation of integral cellular compartments. Identifying mechanisms to integrate cells is more than an academic exercise. The ability to engineer bacteria as membrane compartments to introduce new metabolic functions in eukaryotes would be transformative 70 , 71 . For example, nitrogen-fixing crop plants that could replace nitrogen fertilizers is a major goal for sustainable agriculture. Efforts to transfer the genes for nitrogen fixation to plant cells have been slow, hampered by the many genes required as well as the complex assembly, high energy requirements, and oxygen sensitivity of the reaction. We previously proposed an alternative strategy inspired by diazotroph endosymbioses: introducing nitrogen-fixing bacteria into plant cells as an integral organelle-like compartment 28 . This approach has the advantage that diazotrophs express all required genes with intact regulation, coupled to respiration, and in a protected compartment. Diazoplasts, which achieve stable integration without significant genetic integration, are an important alternative to UCYN-A and other organelles, which are defined by their genetic integration, to inform this strategy. Identifying the nongenetic interactions that facilitated diazoplast integration with Epithemia will be critical for guiding bioengineering. Ongoing genome reduction may drive genetic integration in diazotroph endosymbioses. The fewer number of host protein candidates and their lack of clear function in diazoplasts versus UCYN-A is not associated with differences in their function as nitrogen-fixing cellular compartments. Rather, an alternative explanation points towards differences in the extent of genome reduction in diazoplasts, which encode 1585–1848 protein-coding genes, compared to UCYN-A, which encode 1200–1246 protein-coding genes 72 . Among the genes missing from the UCYN-A genome are cyanobacterial IspD, ThrC, PGLS, and PyrE; for each, an imported host protein was identified that could substitute for the missing function 10 . In contrast, these genes are retained in diazoplast genomes, including those of E. clementina and E. pelagica , obviating the need for host proteins to fulfill their functions ( Figure S4 ). Consistent with diazoplasts and UCYN-A being at different stages of genome reduction, diazoplast genomes contain >150 pseudogenes compared to 57 detected in the UCYN-A genome, suggesting diazoplasts are in a more active stage of genome reduction 27 , 33 , 35 . Interestingly, even genes retained in UCYN-A, namely PyrC and HemE, have imported host-encoded counterparts 10 . The endosymbiont copies may have acquired mutations resulting in reduced function, necessitating import of host proteins to compensate. Alternatively, once efficient host protein import pathways were established, import of redundant host proteins may render endosymbiont genes obsolete, further accelerating genome reduction. Genetic integration may in fact be destabilizing for an otherwise stably integrated endosymbiont, at least initially, as it substitutes essential endosymbiont genes with host-encoded proteins that may not be functionally equivalent and require energy-dependent import pathways. Comparing these related but independent diazotroph endosymbioses yields valuable insight, which otherwise would not be apparent. Diazoplasts at 35 Mya may represent an earlier stage of the same evolutionary path as ~140 Mya UCYN-A, in which continued genome reduction will eventually select for protein import pathways. Alternatively, diazoplasts may have evolved unique solutions to combat destabilizing genome decay, for example through the early loss of mobile elements. 27 , 33 , 73 Whether they represent an early intermediate destined for genetic integration or an alternative path, diazoplasts provide a valuable new perspective on endosymbiotic evolution. Limitations of the study Accurate gene family analysis is dependent on species sampling. While we sought to sample species representative of diatom diversity, some of our reported Epithemia -specific gene families may be shared by non- Epithemia species not present in the data set. While we included free-living Crocosphaera relatives for our homology search for NUDTs, we cannot eliminate the possibility that there are NUDTs or EGTs derived from sequences that were once in diazoplast genomes but have been lost. We did not observe expression associated with genes contained in NUDTs, however, we cannot exclude that they may be expressed under untested conditions. While differential centrifugation was an effective means of diazoplast enrichment, there may still be contamination by other cellular compartments. Low-abundance proteins may fall below the detection threshold in label-free proteomics. While our proteome coverage was comparable to previously reported studies, we cannot eliminate the possibility that there are undetected host proteins enriched in the diazoplast fraction. Tools such as immunofluorescence and genetics have not yet been established in Epithemia diatoms. We are therefore unable to confirm the localization or function of any Epithemia proteins."
} | 6,488 |
39253440 | PMC11383321 | pmc | 1,717 | {
"abstract": "SUMMARY Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina : nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.",
"introduction": "INTRODUCTION Endosymbiotic organelles are uniquely eukaryotic innovations for the acquisition of complex cellular functions, including aerobic respiration, photosynthesis, and nitrogen fixation. Endosymbioses contributed to expansive eukaryotic diversity 1 . An important question in cell evolution and engineering is: how do intermittent, facultative endosymbioses evolve into permanent integral cell compartments, i.e. organelles? With the recognition of the bacterial origin of mitochondria and chloroplasts in the 1980s, Cavalier-Smith & Lee proposed that the key distinction between a transient endosymbiont and an organelle was that organelles do not synthesize all their own proteins 2 , 3 . Instead, in organelles, some genes are transferred from the endosymbiont genome to the eukaryotic nucleus in a process called endosymbiotic gene transfer (EGT). These and other gene products, now under the control of host gene expression, are imported back into the endosymbiotic compartment to regulate endosymbiont growth and division. This definition has since been commonly applied 4 , 5 . However, the underlying hypothesis for organelle evolution — that genetic integration resulting from EGT and/or import of host-encoded gene products is essential for maintaining the endosymbiont as an integral cellular compartment — has not been rigorously tested. In the decades since, increased sampling of eukaryotic diversity has uncovered evidence that, amongst microbes, endosymbioses are a common strategy for acquisition of new functions. Based on observations of EGT and host protein import, new organelles have been recognized: the chromatophore in Paulinella chromatophora 6 – 9 and UCYN-A in Braarudosphaera bigelowii 10 . EGT and host protein import have also been observed in obligate, vertically-inherited nutritional endosymbionts of the parasite Angomonas deanei and insects, which are not formally recognized as organelles 11 , 12 . With the benefit of these newer models, our understanding of genetic integration has become more nuanced 13 , 14 . For example, the majority of host proteins imported into the Paulinella chromatophore do not originate from EGT but rather horizontal gene transfer (HGT) from other bacteria or eukaryotic genes 15 , showing that a host’s repertoire of pre-existing genes may play an outsized role in facilitating genetic integration 16 . UCYN-A was initially described as having an unstable relationship with its host as it is often lost from host cells during isolation and in culture, suggesting environmental conditions can affect endosymbiont stability despite genetic integration 17 . There have been bigger surprises: Some organisms temporarily acquire plastids from partially-digested prey algae 18 , 19 . The retained chloroplasts, called kleptoplasts, perform photosynthesis and, in several species, depend on imported host proteins to fill gaps in their metabolic pathways. Despite their genetic integration, these kleptoplasts cannot replicate in the host cell and are not required for host cell survival, indicating that genetic integration is not sufficient to achieve stable integration of the endosymbiont 20 – 22 . These findings highlight the importance of studying biodiverse organisms to inform new hypotheses for endosymbiotic evolution. Amongst new model systems, Epithemia spp. diatoms offer a unique perspective on organellogenesis. These photosynthetic microalgae contain diazotroph endosymbionts (designated diazoplasts) that perform nitrogen fixation, a biological reaction that converts inert atmospheric nitrogen to bioavailable ammonia 23 – 28 . The ability to fix both carbon and nitrogen fulfills a unique niche in ecosystems. Numerous Epithemia species are globally widespread in freshwater habitats and have recently been isolated from marine environments 29 – 31 . The Epithemia endosymbiosis is very young relative to mitochondria and chloroplasts, having originated ~35 Mya, based on fossil records 32 . Nonetheless, diazoplasts are obligate endosymbionts which are coordinately inherited during host cell division and present in all Epithemia species described so far, indicating co-evolution of diazoplasts and their host algae. Finally, Epithemia diazoplasts are closely related to UCYN-A, the diazotroph endosymbiont of B. bigelowii which was recently designated the first nitrogen-fixing organelle, or nitroplast 10 , 28 , 33 . Both Epithemia diazoplasts and UCYN-A evolved from free-living Crocosphaera cyanobacteria that have engaged in endosymbioses with several host microalgae. The independent evolution of free-living Crocosphaera into diazotroph endosymbionts in multiple host lineages enables comparisons that can lead to powerful insights. If the significance of organelles lies in their function as integral cellular compartments, then metabolic and cellular integration with the host cell are paramount 34 . By these criteria, diazoplasts show a level of host-symbiont integration comparable to UCYN-A. Nitrogen fixation requires large amounts of ATP and reducing power, energy that can be supplied by photosynthesis. Yet nitrogenase, the enzyme that catalyzes nitrogen fixation, is exquisitely sensitive to oxygen produced during oxygenic photosynthesis. In free-living Crocosphaera , photosynthesis and nitrogen fixation is temporally separated such that fixed carbon from daytime photosynthesis is stored as glycogen to fuel exclusively nighttime nitrogen fixation. Diazoplasts have lost all photosystem genes and depend entirely on host photosynthesis for fixed carbon 27 , 35 . Recently, we showed that host and diazoplast metabolism are tightly coupled to support continuous nitrogenase activity throughout the day-night cycle in E. clementina : Diatom photosynthesis is required for daytime nitrogenase activity in the diazoplast, while nighttime nitrogenase activity also depends on diatom, rather than diazoplast, carbon stores 28 . In comparison, UCYN-A has lost only photosystem II and is dependent on both host photosynthesis and, likely, its own photosystem I, restricting it to daytime nitrogen fixation 36 , 37 . Epithemia spp. described so far typically contain 1–2 diazoplasts per cell that are vertically inherited during asexual cell division 25 , 28 , 30 . Diazoplasts have further been shown to be uniparentally inherited during sexual reproduction, similar to mitochondria and chloroplasts 38 . Coordinated replication of UCYN-A with host cell division has been observed to maintain a single endosymbiont per cell 10 . Similar mechanisms are likely in place to coordinate diazoplast inheritance with diatom division. In fact, the presence of diazoplasts in diverse Epithemia species globally widespread in freshwater and marine ecosystems demonstrates that the mechanisms of inheritance are robust through speciation events. Diazoplasts effectively serve as dedicated nitrogen-fixing compartments in Epithemia , whether or not genetic integration has occurred. An important question emerges from these observations: is EGT and/or host protein import required to achieve the level of host-symbiont integration observed between diazoplasts and host Epithemia ? Based on the similarity of diazoplasts to UCYN-A, the assumption is yes. However, there is evidence that metabolite exchange via endosymbiont-encoded transporters 39 and division coordinated by host proteins outside the endosymbiotic compartment 11 , 40 , 41 could form a stable compartment without genetic integration. We previously established freshwater E. clementina as a laboratory model for functional studies and herein performed de novo assembly and annotation of its genome. The genome sequence for E. pelagica , a recently-discovered marine species, was publicly released by the Wellcome Sanger Institute 30 , 42 . To facilitate comparison between these species, we also performed de novo genome annotation of E. pelagica . Notably, no genomes of B. bigelowii (which hosts UCYN-A) nor the eukaryotic host in any other diazotroph endosymbiosis have been available. We report genome and transcriptome analyses of these two Epithemia species as well as proteome analyses of E. clementina with the goals of 1) providing a necessary resource to accelerate investigation of this model and 2) elucidating the role of genetic integration in this very young, stably integrated endosymbiont.",
"discussion": "DISCUSSION EGT and host protein import have been held as a necessary to achieve the “permanent” integration of organelles 2 , 5 . But this hypothesis for organelle evolution has been challenged by findings in young endosymbionts from diverse organisms 13 , 14 , 34 . We report analysis of two genomes of Epithemia diatoms and evaluate the extent of their genetic integration with their nitrogen-fixing endosymbionts (diazoplasts), thereby adding this very young endosymbiosis to existing model systems that can elucidate the integration of two cells into one. A window into the early dynamics of nuclear gene transfers. Our first significant finding was the detection of active diazoplast-to-nucleus DNA transfers but, as yet, no functional EGT in Epithemia . Our observations support findings in the chromatophore and UCYN-A that EGT is not necessary for genetic integration 10 , 15 , 53 . Given that EGT does not necessarily precede evolution of host protein import pathways, it may be a suboptimal solution for the inevitable genome decay in small asexual endosymbiont populations as a consequence of Muller’s ratchet 54 , 55 . Instead, the decayed nature of the NUDTs we detected in E. clementina is consistent with stochastic, transient, ongoing DNA transfer. Nonfunctional DNA transfers were previously only described from mitochondria or plastids with far more reduced genomes. The status of nuclear transfers from more recently-acquired organelles is unknown, as only protein-coding regions were used as queries to identify chromatophore transfers in Paulinella and only a transcriptome is available for the UCYN-A host, B. bigelowii 10 , 15 , 56 . NUDTs in Epithemia genomes therefore provide a rare window into the early dynamics of DNA transfer. For example, using the same homology criteria, we identified 5 NUMTs but no NUPTs in E. clementina . The NUMTs were significantly shorter than NUDTs and did not show rearrangement, which may suggest different mechanisms of transfer for NUDTs, NUMTs, and NUPTs in the same host nucleus. In addition, between-species differences may identify factors that affect transfer rates. The lack of observed NUDTs in E. pelagica suggest constraints on diazoplast-to-nucleus transfers. Previous observations in plant chloroplasts supported the limited transfer window hypothesis, which proposes the mechanism of gene transfer requires endosymbiont lysis and therefore the frequency of gene transfers correlates with the number of endosymbionts per cell 57 – 59 . However since E. pelagica and E. clementina contain similar numbers of diazoplasts per cell (1–2) 28 , 30 , the limited transfer window hypothesis does not explain the observed differences. Instead, there may be additional constraints imposed such as lower tolerance to DNA insertions in the comparatively smaller, non-repetitive genome of E. pelagica . Finally, the lack of NUDT gene expression, even with transfer of a full-length unmutated tusA gene, points to barriers to achieving eukaryotic expression from bacterial gene sequence. Epithemia is an apt model system to interrogate how horizontal gene transfer impacts eukaryotic genome evolution with at least 20 species easily obtained from freshwater globally and consistently adaptable to laboratory cultures 28 – 30 , 33 , 35 . Epithemia diazoplasts as a counterpoint to existing models of organellogenesis. A second unexpected finding was the detection of only 6 host protein candidates in the diazoplast proteome, much fewer and with less clear functional significance than in comparable endosymbionts that have been designated organelles. Methods for validating the localization of these import candidates are unavailable in Epithemia. Even if confirmed to target to the diazoplast, the candidates lack conserved domains or homology with cyanobacterial proteins to indicate they replace or supplement diazoplast metabolic function, growth, or division. Our findings are not explained by current models of organellogenesis that propose import of host proteins as a necessary step to establish an integral endosymbiotic compartment. In the traditional model described in the introduction, host protein targeting is a “late” bottleneck step required for the regulation of the endosymbiont growth and division. More recently, “targeting-early” has been proposed to account for establishment of protein import pathways prior to cellular integration as observed in kleptoplasts 19 , 20 . In this model, protein import is selected over successive transient endosymbioses, possibly driven by the host’s need to export metabolites from the endosymbiont via transporters or related mechanisms 60 . The establishment of protein import pathways then facilitates endosymbiont gene loss with metabolic functions fulfilled by host proteins leading to endosymbiont fixation. Contradicting both models, we observed minimal evidence for genetic integration despite millions of years of co-evolution resulting in diverse Epithemia species retaining diazoplasts, indicating that genetic integration is not necessary for its stable maintenance. At a minimum, the unclear functions of the few host proteins identified in the diazoplast proteome, if imported, suggest that the genesis of host protein import in Epithemia is very different than would be predicted by current models. Diazotroph endosymbioses are fundamentally different from photosynthetic endosymbioses that are the basis for current organellogenesis models. First, the diazoplast is derived from a cyanobacterium that became heterotrophic by way of losing its photosynthetic apparatus. Regulation of endosymbiont growth and division by the availability of host sugars (without requiring an additional layer of regulation via import of host metabolic enzymes) may be more facile with heterotrophic endosymbionts maintained for a nonphotosynthetic function compared to autotrophic endosymbionts. It will be interesting to see how integration of the diazoplast differs from the endosymbiont of Climacodium freunfeldianum , another diazotrophic endosymbiont descended from Crocosphaera that likely retains photosynthesis 61 , 62 . Second, ammonia, the host-beneficial metabolite in diazotroph endosymbioses, can diffuse through membranes in its neutral form and does not require host transporters for efficient trafficking 63 . Previously, we observed efficient distribution of fixed nitrogen from diazoplasts into host compartments following 15 N 2 labeling 28 . Ammonia diffusion may have reduced early selection pressure for host protein import as posited by the targeting-early model. Finally, the eukaryotic hosts in most diazotroph endosymbioses are already photosynthetic, in contrast to largely heterotrophic hosts that acquired photosynthesis by endosymbiosis. For instance, cellular processes that enabled intracellular bacteria to take up residence in the ancestor of Epithemia spp. were likely different than those of the bacterivore amoeba ancestor of Paulinella chromatophora . Autotrophy and lack of digestive pathways would reduce the frequency by which bacteria might gain access to the host cell, such that the selection of host protein import pathways over successive transient interactions would be less effective. Overall, a universal model of organellogenesis is premature given the limited types of interaction that have been investigated in depth, highlighting instead the importance of increasing the diversity of systems studied. Are diazoplasts “organelles”? As detailed in the introduction, diazoplasts show metabolic and cellular integration with their host alga comparable to that of UCYN-A, the first documented nitrogen-fixing organelle 10 , 17 . However, while hundreds of host proteins were detected in the UCYN-A proteome, including many likely to fill gaps in its metabolic pathways, a handful of host proteins with unknown function were detected in the diazoplast proteome. Based on the conventional definition which specifies genetic integration as the dividing line between endosymbionts and organelles, diazoplasts would not qualify 2 , 5 . However, over a decade ago, Keeling and Archibald 34 suggested that “if we use genetic integration as the defining feature of an organelle, we will never be able to compare different routes to organellogenesis because we have artificially predefined a single route.” They further hypothesized that if an endosymbiont became fixed in its host absent genetic integration, “it might prove to be even more interesting… by focusing on how it did integrate, perhaps we will find a truly parallel pathway for the integration of two cells.” The diazoplast appears to be such a parallel case in which non-genetic interactions were sufficient to integrate two cells. If not gene transfer and host protein import, then what is the “glue” that holds this endosymbiosis together? The loss of cyanobacterial photosystems and dependence on host photosynthesis indicate that diazoplasts acquired new transporters for host sugars 28 . There are several examples of cyanobacteria that express genes for sugar transport 39 , 64 , 65 . Therefore, acquisition via horizontal gene transfer from another bacteria to the diazoplast ancestor, prior to the endosymbiosis, rather than targeting of a eukaryotic transporter post-endosymbiosis, seems more likely. Consistent with this hypothesis, potential transporters were not detected amongst host protein import candidates detected in UCYN-A 10 . In fact, mixotrophy may have facilitated the adoption of an endosymbiotic lifestyle by the free-living cyanobacterium. Regardless of the timing of acquisition, sugar transport function could allow diazoplast growth to be regulated by nutrient availability from the host. Similarly, cytosolic host proteins may coordinate diazoplast division from outside the endosymbiotic compartment 11 , 41 . Eukaryotic dynamins required for mitochondria and chloroplast fission localize to the surface of the organellar outer membrane, acting coordinately with bacterial fission factors located in the organelle 40 , 66 . Diazoplasts appear to be surrounded by a host-derived membrane 26 , 67 (which may be lost during diazoplast purification) ( Figure 4A ). In analogy to dynamins, host proteins localized to this outer membrane may mediate diazoplast fission without requiring protein import pathways. Finally, cell density 68 and mechanical confinement 69 have been demonstrated to limit the growth of cyanobacteria, suggesting that host regulation of the volume of the endosymbiotic compartment could also be an effective mechanism. The mechanisms for the robust metabolic exchange and coordinated division observed in diazoplasts will be the focus of future studies. Application of cell evolution models to bioengineering. Diazoplasts provide another example that the current organelle definition does not account for observations in many biological systems and may be overdue for revision to reflect biological significance in the spectrum of endosymbiotic interactions. At a minimum, it is time to disentangle the current definition of an organelle from models that elucidate the formation of integral cellular compartments. Identifying mechanisms to integrate cells is more than an academic exercise. The ability to engineer bacteria as membrane compartments to introduce new metabolic functions in eukaryotes would be transformative 70 , 71 . For example, nitrogen-fixing crop plants that could replace nitrogen fertilizers is a major goal for sustainable agriculture. Efforts to transfer the genes for nitrogen fixation to plant cells have been slow, hampered by the many genes required as well as the complex assembly, high energy requirements, and oxygen sensitivity of the reaction. We previously proposed an alternative strategy inspired by diazotroph endosymbioses: introducing nitrogen-fixing bacteria into plant cells as an integral organelle-like compartment 28 . This approach has the advantage that diazotrophs express all required genes with intact regulation, coupled to respiration, and in a protected compartment. Diazoplasts, which achieve stable integration without significant genetic integration, are an important alternative to UCYN-A and other organelles, which are defined by their genetic integration, to inform this strategy. Identifying the nongenetic interactions that facilitated diazoplast integration with Epithemia will be critical for guiding bioengineering. Ongoing genome reduction may drive genetic integration in diazotroph endosymbioses. The fewer number of host protein candidates and their lack of clear function in diazoplasts versus UCYN-A is not associated with differences in their function as nitrogen-fixing cellular compartments. Rather, an alternative explanation points towards differences in the extent of genome reduction in diazoplasts, which encode 1585–1848 protein-coding genes, compared to UCYN-A, which encode 1200–1246 protein-coding genes 72 . Among the genes missing from the UCYN-A genome are cyanobacterial IspD, ThrC, PGLS, and PyrE; for each, an imported host protein was identified that could substitute for the missing function 10 . In contrast, these genes are retained in diazoplast genomes, including those of E. clementina and E. pelagica , obviating the need for host proteins to fulfill their functions ( Figure S4 ). Consistent with diazoplasts and UCYN-A being at different stages of genome reduction, diazoplast genomes contain >150 pseudogenes compared to 57 detected in the UCYN-A genome, suggesting diazoplasts are in a more active stage of genome reduction 27 , 33 , 35 . Interestingly, even genes retained in UCYN-A, namely PyrC and HemE, have imported host-encoded counterparts 10 . The endosymbiont copies may have acquired mutations resulting in reduced function, necessitating import of host proteins to compensate. Alternatively, once efficient host protein import pathways were established, import of redundant host proteins may render endosymbiont genes obsolete, further accelerating genome reduction. Genetic integration may in fact be destabilizing for an otherwise stably integrated endosymbiont, at least initially, as it substitutes essential endosymbiont genes with host-encoded proteins that may not be functionally equivalent and require energy-dependent import pathways. Comparing these related but independent diazotroph endosymbioses yields valuable insight, which otherwise would not be apparent. Diazoplasts at 35 Mya may represent an earlier stage of the same evolutionary path as ~140 Mya UCYN-A, in which continued genome reduction will eventually select for protein import pathways. Alternatively, diazoplasts may have evolved unique solutions to combat destabilizing genome decay, for example through the early loss of mobile elements. 27 , 33 , 73 Whether they represent an early intermediate destined for genetic integration or an alternative path, diazoplasts provide a valuable new perspective on endosymbiotic evolution. Limitations of the study Accurate gene family analysis is dependent on species sampling. While we sought to sample species representative of diatom diversity, some of our reported Epithemia -specific gene families may be shared by non- Epithemia species not present in the data set. While we included free-living Crocosphaera relatives for our homology search for NUDTs, we cannot eliminate the possibility that there are NUDTs or EGTs derived from sequences that were once in diazoplast genomes but have been lost. We did not observe expression associated with genes contained in NUDTs, however, we cannot exclude that they may be expressed under untested conditions. While differential centrifugation was an effective means of diazoplast enrichment, there may still be contamination by other cellular compartments. Low-abundance proteins may fall below the detection threshold in label-free proteomics. While our proteome coverage was comparable to previously reported studies, we cannot eliminate the possibility that there are undetected host proteins enriched in the diazoplast fraction. Tools such as immunofluorescence and genetics have not yet been established in Epithemia diatoms. We are therefore unable to confirm the localization or function of any Epithemia proteins."
} | 6,488 |
27189979 | PMC4943185 | pmc | 1,718 | {
"abstract": "Methanogenesis coupled to the Wood–Ljungdahl pathway is one of the most ancient metabolisms for energy generation and carbon fixation in the Archaea. Recent results are sensibly changing our view on the diversity of methane-cycling capabilities in this Domain of Life. The availability of genomic sequences from uncharted branches of the archaeal tree has highlighted the existence of novel methanogenic lineages phylogenetically distant to previously known ones, such as the Methanomassiliicoccales. At the same time, phylogenomic analyses have suggested a methanogenic ancestor for all Archaea, implying multiple independent losses of this metabolism during archaeal diversification. This prediction has been strengthened by the report of genes involved in methane cycling in members of the Bathyarchaeota (a lineage belonging to the TACK clade), representing the first indication of the presence of methanogenesis outside of the Euryarchaeota. In light of these new data, we discuss how the association between methanogenesis and the Wood–Ljungdahl pathway appears to be much more flexible than previously thought, and might provide information on the processes that led to loss of this metabolism in many archaeal lineages. The combination of environmental microbiology, experimental characterization and phylogenomics opens up exciting avenues of research to unravel the diversity and evolutionary history of fundamental metabolic pathways."
} | 361 |
37836031 | PMC10574912 | pmc | 1,719 | {
"abstract": "Slippery coatings, such as the slippery liquid-infused porous surface (SLIPS), have gained significant attention for their potential applications in anti-icing and anti-fouling. However, they lack durability when subjected to mechanical impact. In this study, we have developed a robust slippery coating by blending polyurethane acrylate (PUA) with methyltriethoxysilane (MTES) and perfluoropolyether (PFPE) in the solvent of butyl acetate. The resulting mixture is homogeneous and allows for uniform coating on various substrates using a drop coating process followed by drying at 160 °C for 3 h. The cured coating exhibits excellent water repellency (contact angle of ~108° and sliding angle of ~8°), high transparency (average visible transmittance of ~90%), exceptional adherence to the substrate (5B rating according to ASTMD 3359), and remarkable hardness (4H on the pencil hardness scale). Moreover, the coating is quite flexible and can be folded without affecting its wettability. The robustness of the coating is evident in its ability to maintain a sliding angle below 25° even when subjected to abrasion, water jetting, high temperature, and UV irradiation. Due to its excellent nonwetting properties, the coating can be employed in anti-icing, anti-graffiti, and anti-sticking applications. It effectively reduces ice adhesion on aluminum substrates from approximately 217 kPa to 12 kPa. Even after 20 cycles of icing and de-icing, there is only a slight increase in ice adhesion, stabilizing at 40 kPa. The coating can resist graffiti for up to 400 cycles of writing with an oily marker pen and erasing with a tissue. Additionally, the coating allows for easy removal of 3M tape thereon without leaving any residue.",
"conclusion": "4. Conclusions A multifunctional and robust slippery coating was developed using polyurethane acrylate, methyltriethoxysilane, and perfluoropolyether. The smooth surface morphology of the coating resulted in impressive slipperiness, with a contact angle of approximately 108° and a sliding angle of around 8°. Significantly, the coating maintained its effectiveness even after exposure to challenging conditions, such as high temperatures or low humidity. It also exhibited exceptional hardness (4H) and substrate adherence (5B), making it highly resistant to water-jet impacting and abrasion, showcasing its durability. The coating demonstrated excellent optical transparency and remarkable performance in terms of graffiti resistance and prevention of sticking. Additionally, its low shear strength against ice made de-icing effortless. This versatile coating holds great potential for applications in anti-icing and anti-smudge scenarios. Overall, this work represents a significant step forward in the development of a versatile and durable slippery coating with multiple benefits for various applications. Future directions for this work may include the development of such coatings through room temperature curing, which could lead to more efficient and environmentally friendly manufacturing processes.",
"introduction": "1. Introduction Bioinspired surfaces, such as a superhydrophobic surface [ 1 ] and slippery liquid-infused porous surface (SLIPS) [ 2 , 3 , 4 ], exhibit similarities in their water-repellent properties. Aizenberg et al. drew inspiration from nepenthe and developed the SLIPS by introducing a low-surface-energy lubricating liquid into porous surface structures. This process resulted in the formation of a continuous and smooth liquid lubricant film, enabling water droplets to effortlessly slide off, even at a small contact angle [ 5 , 6 , 7 ]. Due to the excellent water repellency and low ice adhesion strength, the SLIPS demonstrated great potential in anti-icing applications [ 8 , 9 , 10 , 11 ]. For instance, Wilson et al. [ 12 ] found that the SLIPS could significantly reduce the nucleation temperature of supercooled water in contact, with statistical significance, and showed no deterioration or change in the coating performance even after 150 freeze–thaw cycles. Liu et al. [ 13 ] proposed a novel self-assembly method of fabricating an electric heating SLIPS, which possessed ultra-low ice adhesion. Mahmut et al. [ 14 ] fabricated three kinds of SLIPS with the lubricants polychlorotrifluoroethylene oil, silicone oil, and liquid paraffin. All the SLIPS revealed extremely low ice adhesion of lower than 1 kPa. The fabrication of the SLIPS typically involves three primary steps: (1) creating rough surface structures; (2) applying a layer of low-surface-energy molecules to passivate the rough surface; and (3) impregnating the surface with liquid lubricants [ 2 , 15 , 16 ]. The rough surface structures generated in the first step serve as a physical reservoir for the liquid lubricant, while the second step employs low-surface-energy molecules to chemically attract the lubricant. For instance, Sun et al. [ 17 ] used electrochemical etching and anodization to create a hierarchical porous structure on the substrate Al. After lowering the surface energy and infusing silicone oil, the SLIPS was obtained, showing a water roll-off angle of 3°. Yan et al. [ 18 ] synthesized titanium dioxide nanotube arrays on titanium substrate through the anodic oxidation method, which were further modified and infused with perfluoropolyether lubricant to form the SLIPS. The contact angle hysteresis and sliding angles of water droplets on the TiO 2 SLIPS were as low as 1° and 4°, respectively, indicating the excellent slippery property. The inherent weakness of the SLIPS is that the lubricating oil can be lost easily, leading to a decline in sliding performance. To enhance the durability of the SLIPS, Tan et al. [ 19 ] fabricated a fractal surface with micro-sized pyramid holes and porous nanostructures. The porous nanostructures served to retain the lubricant perfluoropolyether whilst the robust micro-pyramidal holes provided protection for the nanostructures. The other solution to enhance the durability of SLIPS is to bond the lubricant to the substrate via affinity bonding. For example, Wu et al. [ 20 ] developed a liquid-attached SLIPS via a one-step equilibration reaction by tethering methoxy-terminated polydimethylsiloxane polymer brushes onto a substrate to form a transparent “liquid-like” layer. The so-obtained surface demonstrated superior abrasion resistance and maintained its excellent self-cleaning capacity even after being subjected to abrasion with water or sand particles. Solid lubricant such as paraffin wax was also used for the robust slippery coating. For instance, Meng et al. [ 21 ] developed a coating by incorporating paraffin wax into a porous polystyrene structure. This solid slippery surface offered remarkable stability, even when exposed to various pH solutions or contact with other materials. Additionally, it demonstrated rapid self-healing capabilities under the heating–cooling process. However, the fabrication procedure was complex and not conducive to large-scale production. Moreover, the use of porous polymers restricted the choice of substrate materials to polymers, thereby limiting the application range. This study introduces a novel robust slippery coating that is synthesized using a one-pot method. To form the coating film, polyurethane acrylate is chosen as the film-forming material. In order to enhance water repellency and stability, a combination of methyltriethoxysilane (MTES) and perfluoropolyether (PFPE) is incorporated. The resulting coating exhibits exceptional durability, making it suitable for long-term use. Its low surface energy also allows it to effectively resist ice formation and repel stains.",
"discussion": "3. Results and Discussion 3.1. Component Optimization and Surface Characterization The C=C bonds within the acrylate moiety dissociated under heat, leading to the formation of a polymer structure (as shown in Figure 1 a). Simultaneously, heat-induced dehydration condensation took place among the hydrolyzed MTES molecules, resulting in the formation of a closely linked Si-O-Si network (as shown in Figure 1 b) [ 22 ]. This network significantly enhances the crosslinking density of the coating and improves its durability and overall performance, which will be elaborated upon in the upcoming sections. The incorporation of PFPE into the coating formulation allowed it to intertwine among the polymer chains, resulting in increased slipperiness to water droplets. This combination of robustness and slipperiness contributed to the achievement of a coating that was both durable and offered improved slip behavior. The results obtained from the infrared spectroscopy (FTIR) analysis ( Figure 2 a) provide valuable insights into the chemical composition of the coatings. The stretching vibration peaks located at 3372 cm −1 (N-H) and 1682 cm −1 (C=O) indicate the presence of urethane functional groups. The absorption peaks at 2934 cm −1 and 1462 cm −1 were attributed to the stretching and bending vibrational features of C-H and -CH 3 groups, respectively. Additionally, the stretching vibrational absorption peak observed at 1059 cm −1 indicated the successful incorporation of MTES, as it was assigned to Si-O-Si bonds. Furthermore, the asymmetric stretching vibrational absorption peak at 1158 cm −1 was linked to C-F bonds, confirming the successful introduction of PFPE into the coating formulation. Overall, the FTIR analysis provides evidence of the presence of specific functional groups and the successful incorporation of key components within the coating. The transmittance of the coating on the glass was measured using a UV-VIS spectrophotometer, revealing an average transmittance of 89.7% within the wavelength range of 400–800 nm. This value was quite similar to that of the bare glass (90%, Figure 2 b). The coating’s transparency allowed for clear visibility of the school emblem of Jiangsu University of Technology, which was placed under the coating on the glass substrate (inset in Figure 2 b). An atomic force microscope was used to measure the surface morphology of the coating. The coating exhibited a smooth and compact surface with minimal height differences between peaks, as demonstrated by a surface roughness of only 1.02 nm ( Figure 2 c). Furthermore, SEM analysis revealed a thickness of approximately 10 μm when performing cross-sectional analysis ( Figure 2 d). The influence of MTES content on the sliding performance of the coating was investigated ( Figure 3 a). The weight of PFPE in the mixture was kept constant at 0.18 g. In the absence of MTES, the sample contained only PUA and PFPE, exhibiting hydrophobic properties with a contact angle of 100° and a sliding angle of approximately 17°. As the MTES content increased, there was an initial rise followed by a decline in the contact angle. Similarly, the sliding angle initially decreased and then increased. This behavior can be attributed to the presence of two types of groups in MTES: the non-polar CH 3 group and the polar -OC 2 H 5 group. The latter group can be hydrolyzed in the presence of water, giving rise to hydrophilic −OH groups and Si-O-Si groups ( Figure 1 b). Thus, in the initial stage of increasing the MTES amount, the non-polar CH 3 group dominates, resulting in an increase in the contact angle. However, once the MTES amount exceeds 4.5 g, the hydrophilic groups become dominant, causing a decrease in the contact angle. Following the optimization of the MTES content at 4.5 g, the effect of varying the PFPE content on the water droplet sliding performance of the solid slippery coating was examined. The optimal performance was achieved when the weight of PFPE was 0.18 g, resulting in a contact angle of approximately 108° and a sliding angle of approximately 8° ( Figure 3 b). Subsequently, sliding speed tests were conducted on the coating, demonstrating that water droplets could easily slide off, with the sliding speed increasing as the tilt angle increased ( Figure 3 d). 3.2. Durability and Protection Performance The durability of the slippery coating on the glass substrate was assessed using various tests [ 23 ]. Rubbing tests were employed to evaluate its mechanical durability. The results showed that, after 500 cycles of rubbing against medical gauze at a load of 1.25 kPa, the contact angle of the coating decreased slightly from around 108° to approximately 105° ( Figure 4 a) and the sliding angle increased from 8° to 17.5° ( Figure 4 b). It is worth noting that despite these changes, there were no visible wear scars on the coating’s surface; only scattered fine particles were observed after 500 cycles ( Figure 4 c-2). When subjected to a higher load of 5 kPa, the contact angle remained relatively high at around 95°, while the sliding angle increased to approximately 25°. After 500 friction cycles at this load, the coating exhibited slight wear scars along with debris ( Figure 4 d). Although there was a slight alteration in the microscopic morphology of the coating due to rubbing, there were no observable changes in its macroscopic appearance. In summary, the slippery coating demonstrated satisfactory mechanical durability during the rubbing tests, as there were minimal visible wear scars and particle accumulation even after a significant number of cycles. Key parameters for the water jetting test are shown in Figure 5 a. After 9 min, a water droplet (20 μL) could still easily slide off the tiled sample at a 30° angle to the horizontal surface ( Figure 5 b). The slippery coating was highly resilient, exhibiting a pencil hardness level of 4H. To assess the adhesive strength of the slippery coating, we followed the ASTM D 3359 standard and employed a cross-cut tester to create a grid pattern on the slippery coating. Subsequently, we applied adhesive tape to the grid and peeled it off, ultimately observing no detachment of the coating from the substrate. This outcome indicated a robust adhesive strength of 5B between the coating and the substrate ( Figure 5 c). We performed tests to evaluate the flexural resistance of the coating on PET films and aluminum sheets. In the case of PET films, a “U” shape was formed by bending the film and moving it back and forth to assess its flexural resistance, as depicted in Figure 6 a [ 24 ]. After bending, the sliding angle and sliding velocity of a 20 μL water droplet, tilted at an angle of 20°, were measured. Figure 6 c illustrates that after 10 bending cycles, there was only a slight increase in the sliding angle of the coating (13°), while the sliding speed remained relatively fast (12 mm/s). After 50 bending cycles, the coating remained intact and the sliding angle increased to 19°, while the sliding speed decreased to 1.8 mm/s. The flexural resistance of the coating on aluminum was assessed by folding the aluminum sheets at a 90° angle, followed by restoration. After 10 bending–restoration cycles, no cracking was observed on the bent area of the coating. In Figure 6 d, we present the results of tests examining the ability of water droplets to smoothly slide off the crease. The coating exhibited a sliding angle lower than 20° and a sliding velocity of approximately 1 mm/s, indicating its favorable flexural resistance. These findings demonstrate the durability and performance of the coating on PET films and aluminum sheets under bending conditions, making it suitable for various applications. After storage in a furnace for 1 h at 350 °C, the contact angle of the slippery coating slightly decreased to approximately 102°, and the sliding angle increased to approximately 18° ( Figure 7 a). Upon continuous heating to 400 °C, the contact angle of the slippery coating surface sharply dropped to 42°, and the sliding angle increased to be higher than 90°. The high sliding angle indicated that the water droplet could not slide off, even when the slippery coating was vertically placed. When the slippery coating was exposed to UV irradiation (313 nm, 30 W) at a distance of 5 cm, the contact angle decreased to 103° and the sliding angle increased to approximately 20° after 7 days. To simulate the impact of acid rain and alkaline rain, the slippery coating was subjected to water droplets of an aqueous solution of HCl and NaOH [ 25 ]. After seven cycles, the contact angle remained at a high level of approximately 95°. However, the water droplets could not easily slide off the surface. The results obtained from the experiments were consolidated into Table 1 and compared with typical reference samples [ 9 , 10 , 23 , 24 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. The findings revealed that the slippery coating developed in this study demonstrates exceptional performance in various aspects, including optical transparency, mechanical durability, flexibility, chemical robustness, and UV resistance. These remarkable characteristics render it ideal for practical applications, such as anti-ice, anti-graffiti, and anti-sticking purposes, which will be discussed in detail shortly. 3.3. Applications: Anti-Icing, Anti-Graffiti and Anti-Sticking The accumulation of ice on surfaces can cause significant problems, and current de-icing methods have limitations in effectively removing ice. To address this issue, a slippery coating was utilized to reduce ice adhesion. Without the coating, the glass surface demonstrated a high ice adhesion strength of approximately 217 kPa ( Figure 8 a). However, when the slippery coating was applied, the ice adhesion strength dropped significantly to 12 kPa. It is worth noting that ice adhesion below 20 kPa allows for easy removal [ 33 ]. Although similar low ice adhesion has been observed in previous studies on SLIPS [ 34 ], ensuring long-term durability for an anti-icing SLIPS remains a challenge. To evaluate the durability of the slippery coating, multiple ice formation/removal cycles were conducted. Even after 20 cycles, the ice adhesion strength of the coating remained between 30 and 40 kPa ( Figure 8 b). This indicates that the slippery coating exhibits exceptional mechanical strength, enabling it to withstand the expansion stress caused by freezing. Furthermore, compared to previously reported slippery coatings [ 8 , 9 , 11 , 35 , 36 ], our slippery coating demonstrates comparable anti-icing performance and superior durability ( Table 2 ). The anti-smudge properties of the developed slippery coating were evaluated, particularly with regards to oily markers. It should be noted that high contact angles and low sliding angles of water on the coating surface do not necessarily indicate anti-smudge capabilities, as the contraction of oil-based ink on the coating poses a greater challenge. In the case of an insufficiently cross-linked coating surface, the ink may exhibit minimal or no contraction behavior, resulting in persistent and noticeable marks even after wiping. When an oily marker was used on glass, visible lines remained on the surface that were not easily removed by simple wiping with a dry tissue ( Figure 9 a-1). However, the slippery coating demonstrated excellent anti-graffiti properties. The weak interaction between the ink and the coating caused the ink to form a discontinuous line, allowing for easy erasure with a tissue [ 37 ]. Even after 400 cycles of writing and erasing (as shown in Figure 9 a-2), the anti-graffiti performance remained unaffected, making it highly practical for real-world applications. Additionally, the slippery coating proved highly effective in resisting adhesion to solid adhesives. As illustrated in Figure 9 b, the 3M adhesive tape adhered firmly to the bare glass, making it difficult to peel off. However, when applied to the slippery coating, the 3M tape could be effortlessly removed without leaving any residue on the sample."
} | 4,933 |
39807158 | PMC11727649 | pmc | 1,720 | {
"abstract": "With the expansion of the mining industry, environmental pollution from microelements (MP) and red mud (RM) has become a pressing issue. While bioremediation offers a cost-effective and sustainable solution, plant growth in these polluted environments remains difficult. Arundo donax is one of the few plants capable of surviving in RM-affected soils. To identify endophytic fungi that support A. donax in different contaminated environments and to inform future research combining mycorrhizal techniques with hyperaccumulator plants, we conducted a field experiment. The study compared endophytic fungal communities in A. donax grown in uncontaminated, MP soils contaminated with cadmium (Cd), arsenic (As), and lead (Pb), and RM-contaminated soils. Our findings showed that soil nutrient profiles differed by contamination type, with Cd concentrations in MP soils exceeding national pollution standards (GB 15168-2018) and RM soils characterized by high aluminum (Al), iron (Fe), and alkalinity. There were significant differences in the endophytic fungal community structures across the three soil types ( p < 0.001). Co-occurrence network analysis revealed that endophytic fungi in MP soils exhibited competitive niche dynamics, whereas fungi in RM soils tended to share niches. Notably, Pleosporales sp., which accounted for 18% of the relative abundance in RM soils, was identified as a dominant and beneficial endophyte, making it a promising candidate for future bioremediation efforts. This study provides valuable insights into the role of endophytic fungi in phytoremediation and highlights their potential as resources for improving plant-microbe interactions in contaminated environments.",
"conclusion": "Conclusions The endophytic fungal community in A. donax roots is predominantly composed of Basidiomycetes, Ascomycetes, unclassified fungi, and Zygomycetes, with varying abundance and diversity across different contaminated soil types. Notably, higher concentrations of trace elements and elevated pH levels, coupled with lower nutrient availability in trace element and red mud-contaminated soils, tend to suppress Basidiomycete populations while promoting Ascomycete proliferation. Furthermore, A. donax may adapt to extreme environments, such as red mud, by colonizing novel (unknown) species and facilitating interactions among endophytic fungi. Remarkably, Pleosporales sp. emerges as a highly promising endophytic fungus for remediation in contaminated soils. Future research should focus on identifying the functional roles of dominant unknown species and evaluating the remediation potential of Pleosporales sp. to investigate the synergistic effects of endophytic fungi and hyperaccumulator A. donax in the remediation of trace element and red mud contamination.",
"introduction": "Introduction Soil pollution resulting from human mining activities poses a significant environmental challenge. Mining operations increase the concentration of microelements in soils, leading to soil acidification or alkalization, which can be detrimental to plants, animals, and microorganisms ( Rossi et al., 2017 ; Paez-Osuna et al., 2024 ). One of the most severe consequences of soil microelement pollution is its impact on ecosystem processes, notably by hindering biological degradation due to altered microbial biodiversity and soil properties ( Hontoria et al., 2019 ). Alarmingly, this pollution does not diminish over time; for instance, in Sidi Village (Guangxi, China) elevated concentrations of cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) persist in agricultural soils near an abandoned Pb-Zn mine, even after 40 years ( Cao et al., 2018 ). Additionally, waste from the mining industry, such as red mud—an industrial solid waste generated during aluminum (Al) extraction—exacerbates environmental pollution. Approximately 1.0 to 1.8 tons of red mud are produced for every ton of alumina extracted, depending on ore grade and production methods ( Oprčkal et al., 2020 ). The increasing accumulation of red mud, coupled with its severe environmental impact, necessitates effective remediation strategies. Current research primarily focuses on resource recovery, such as utilizing red mud for ceramics and iron production ( Liao, Zeng & Shih, 2015 ; Xiao et al., 2022 ). However, these methods often lack appropriate technology, incur high costs, and fail to fully address red mud pollution. Thus, there is an urgent need for eco-friendly and cost-effective remediation methods. Bioremediation relies primarily on the synergistic effects of plants and microorganisms. Our research group has previously identified several hyperaccumulator plants, including Sedum , and Solanum nigrum , with Arundo donax ( A. donax ) emerging as a highly adaptable and resilient hyperaccumulator capable of suviving even in red mud, albeit with low biomass ( Pu et al., 2020 ). To enhance the phytoremediation potential of A. donax in environments contaminated by trace elements and red mud, we aim to leverage mycorrhizal technology. By establishing symbiotic relationships between microorganisms and plants, we can effectively improve the host plant’s tolerance and accumulation abilities ( Tedersoo, Bahram & Zobel, 2020 ; Rich et al., 2021 ). Thus, this study investigates the endophytic fungi associated with A. donax in polluted environments as a foundation for mycorrhizal technology. The diversity and composition of plant endophytic fungi are influenced by various environmental factors, including pollution types, plant species, and soil characteristics ( Chen et al., 2023 ). Endomycorrhizal fungi are often considered to be key factors in the bioavailability of metals to plants since they play a vital role in the activation and solidification of metals in soil ( Liu et al., 2019 ). Therefore, it is essential to investigate the composition of endophytic fungi in relation to different types of pollution and soil properties. This study aimed to identify the ecotypes of endomycorrhizal fungi of A. donax under unpolluted, trace element-contaminated, and red mud-contaminated conditions to develop effective phytoremediation strategies. Specifically, we conducted a field experiment focused on A. donax to (1) assess the diversity of endomycorrhizal fungi in its roots; (2) investigate the impact of different soil pollution types on the structure of endomycorrhizal fungi communities, and (3) analyze the changes in endophytic fungi diversity and the environmental factors driving these changes. This field-based approach provides valuable insights into the ecological interactions at play and highlights the potential of A. donax for phytoremediation in polluted environments.",
"discussion": "Discussion Composition of endophytic fungal community in the roots of A. donax The endophytic fungal community of A. donax is primarily composed of Basidiomycota, Ascomycota, unclassified_k__Fungi, and Zygomycota. Similarly, Dang et al. (2021) found that the endophytic fungi in the main root of Glycyrrhiza uralensis were predominantly Ascomycota and Basidiomycota. However, the richness and composition of these fungi vary significantly across different contaminated environments, with specific indicator taxa identified in each soil type. For instance, Basidiomycota dominated in UP treatment, while Ascomycota were more prevalent in MP and RM treatments. The correlation analysis suggests that increases in Cd and pH, or decreases in nutrient levels (C, N, P, and K), may drive a reduction in the abundance and diversity of Basidiomycota and an increase in Ascomycota. Moreover, the dominant endophytic species in A. donax across different treatments were primarily pathogenic fungi, such as Marasmiellus (a plant pathogen), Fusarium sp. (associated with citrus gummosis), and Phialemoniopsis (linked to various human infections) ( Almaliky et al., 2013 ; Ito et al., 2017 ; Zakaria, 2023 ). One notable exception is Pleosporales sp., which has been reported to produce azaphilone compounds that show moderate inhibitory activity against three agricultural pathogens: Thielaviopsis paradoxa , Pestalotia calabae , and Glorosprium musarum ( Cao et al., 2016 ). Thus, Pleosporales sp. may serve as a candidate strain for future bioremediation studies involving A. donax . Interestingly, the three most abundant endophytic fungi colonizing the roots of A. donax in RM soil are unknown species. This could be attributed to both the extreme environment and potential technical limitations. RM soil represents an extreme environment with high potassium, salinity, alkalinity, and heavy metal concentrations. Microorganisms may undergo genetic mutation over long period of adaptation and evolution, leading to the emergence of unknown species better suited to such conditions. Previous studies have shown that fungal communities can adapt to extreme environments through traits like asexual reproduction, melanin-like pigment production, and flexible morphology ( Gostincar et al., 2010 ). Additionally, Wang et al. (2023a) noted that the presence of unknown species in the identification of arbuscular mycorrhizal fungi in the rhizosphere of artificially planted pine forests might result from technological limitations in genetic sequencing. Future research should focus on identifying the functions of these unknown species to explore the potential applications of endophytic fungi that can tolerate extreme pollution. Different soil types affect the diversity of endophytic fungal communities in the roots of A. donax The composition and abundance of endophytic fungi in A. donax roots vary across different soil types, likely due to site-specific effects. Microbial communities in the rhizosphere are influenced by contaminants such as Pb, Cd, Zn, Mn, Fe, and S ( Pfendler et al., 2024 ). Plants selectively filter specific microbial taxa from the soil, enriching them in the root zone ( Cui et al., 2023 ). Stressful environments, such as contaminated soils, generally reduce the abundance and diversity of root endophytic fungi, while the abundance of pathogenic fungi tends to increase with greater levels of contamination ( Wiewióra & Zurek, 2021 ; Cui et al., 2023 ). This may be due to the toxic effects of trace elements like Cd, As, and Pb, as well as high salinity and alkalinity, which decrease the abundance and diversity of heavy metal-sensitive endophytic fungi. Additionally, the negative impacts of pollution on plant growth hinder fungal colonization in the roots, with the severity of this limitation increasing with higher soil contamination levels ( Becerra et al., 2023 ). Our study also observed that A. donax roots in MP and RM soils were enriched with plant pathogenic fungi, potentially altering the interactions among microorganisms across different sites. Microelements and red mud contamination reduced the stability of the root endophytic fungal network of A. donax . The interactions among microorganisms influence the ecological niches and competitive dynamics of endophytic fungi ( Huang et al., 2023 ; Zhang et al., 2024 ). Microelement pollution shifted many positive correlations among root endophytic fungi to negative, while the root endophytic fungi in the RM soil were closely and positively associated with each other. This may be due to the Cd, As, and Pb in microelement pollution will intensify the competition among root endophytic fungi on space and resource, and generally the diversity of root endophytic fungi decreasing with an increasing microelement concentrations ( Li et al., 2016 ). However, except with high heavy metals, RM soil is also an extreme environment with high K and alkalinity ( Ren et al., 2018 ). Plants and associated endophytic fungi in this environment may undergone adaptive evolution to cope with these specific stressors ( Xie et al., 2024 ; Zeng et al., 2024 ). In summary, during the phytoremediation process, A. donax adjusts not only the composition of root endophytic fungi but also the relationships among microorganisms to adapt to varying stress conditions. Environmental factors regulate the diversity and function of endophytic fungi in A. donax roots The Cd content in MP contaminated soil exceeded the standard content of Class II soil (general land, GB 15168-2018) by about 12 times, and the K and P contents were also quite high. This may be due to excessive leaching of heavy metals from nearby mines or improper use of inorganic phosphate fertilizers over many years ( Tian et al., 2023 ). According to the survey results of the National Bureau of Statistics ( https://data.stats.gov.cn/ ), the pure volume of agricultural phosphate fertilizer application in Guangxi Zhuang Autonomous Region is 267,600 tons in 2022. The production process of phosphate fertilizer may cause heavy metal residues, leaving excess heavy metals and phosphorus in the soil with microelements ( Lei et al., 2023 ). Moreover, this study found that arbuscular mycorrhizal fungi are not the main root endosymbiotic fungi in the three soil types. Studies have shown that some functions of arbuscular mycorrhizal fungi may be similar to the recruiting phosphorus-solubilizing bacteria and heavy metal-tolerant bacteria ( Gutjahr et al., 2015 ). Therefore, in this study soil phosphorus may be not effectively utilized and remains. In the MP soil, soil total nitrogen rather than other environmental factors has the biggest impact on the endophytic fungal community of A. donax roots. The function undefined saprotrophs has a high proportion in all soil types, and its relative abundance decreased following the order of the UP, MP, and RM soil. Saprophytic fungi is indispensable in the decomposition of soil organic matter, and the fungi is beneficial to soil nutrient recycling, plant growth, and ecosystem balance ( Wang et al., 2021 ). Saprophytic fungi may also improve the plant’s ability to absorb water and mineral nutrients by forming a symbiotic relationship with plants ( Cruz et al., 2022 ). Therefore, the reduction in the proportion of undefined saprotrophs may detrimentally affect soil nutrient cycling, which may be the main reason for the low organic carbon and nitrogen contents in the MP and RM soils. Meanwhile, in the RM soil, the soil pH rather than other environmental factors has the biggest impact on the diversity of endophytic fungi in A. donax roots. Generally, the RM pollution is formed from sedimentation and stratification in areas with rich iron contents. During this process, abundant elements such as aluminum, iron, and potassium are accumulated, and the accumulation of soil potassium may contribute to a high-level soil pH in the RM soil ( Wang et al., 2023b ). In addition, soil nutrient contents (C, N, K, P) was negatively correlated with soil pH and Cd. The Basidiomycota was mainly positively correlated with C, N, K, and P, but negatively correlated with Cd, Pb, and soil pH. Previous studies found the Basidiomycota fungal populations related to macromolecule degradation are beneficial to inhibiting soil Cd activity ( Zhao et al., 2023 ). Besides, the correlation between Ascomycota and environmental factors is bidirectional. In polluted environments, Ascomycota may be the most abundant fungal group ( Cai et al., 2024 ). Although the Ascomycota species are diverse, generally these species ultimately contribute to the collaboration of microorganisms and plants, and thus promote the improvement of cadmium/chromium removal efficiency by shifting the proportion of dominant microbial-resistant taxa ( Zhang et al., 2023 ). Overall, A. donax may assemble root endophytes according to soil types to deal with different soil pollutants."
} | 3,923 |
35255531 | PMC9543053 | pmc | 1,721 | {
"abstract": "ABSTRACT Kelp forest ecosystems and their associated ecosystem services are declining around the world. In response, marine managers are working to restore and counteract these declines. Kelp restoration first started in the 1700s in Japan and since then has spread across the globe. Restoration efforts, however, have been largely disconnected, with varying methodologies trialled by different actors in different countries. Moreover, a small subset of these efforts are ‘afforestation’, which focuses on creating new kelp habitat, as opposed to restoring kelp where it previously existed. To distil lessons learned over the last 300 years of kelp restoration, we review the history of kelp restoration (including afforestation) around the world and synthesise the results of 259 documented restoration attempts spanning from 1957 to 2020, across 16 countries, five languages, and multiple user groups. Our results show that kelp restoration projects have increased in frequency, have employed 10 different methodologies and targeted 17 different kelp genera. Of these projects, the majority have been led by academics (62%), have been conducted at sizes of less than 1 ha (80%) and took place over time spans of less than 2 years. We show that projects are most successful when they are located near existing kelp forests. Further, disturbance events such as sea‐urchin grazing are identified as regular causes of project failure. Costs for restoration are historically high, averaging hundreds of thousands of dollars per hectare, therefore we explore avenues to reduce these costs and suggest financial and legal pathways for scaling up future restoration efforts. One key suggestion is the creation of a living database which serves as a platform for recording restoration projects, showcasing and/or re‐analysing existing data, and providing updated information. Our work establishes the groundwork to provide adaptive and relevant recommendations on best practices for kelp restoration projects today and into the future.",
"conclusion": "VII. CONCLUSIONS \n Kelp forest restoration has a long history, spanning 16 countries and over 300 years of practice. The field is diverse with representation in many sectors of society, including academia, governments, communities, indigenous groups, and businesses. The field is accelerating with more projects in the 10 years between 2009 and 2019 than ever before. While a global field, more restoration projects have occurred in Japan than the rest of the world combined, but access to the results of those projects remains limited. To date, most restoration projects have been small in size, short in duration, and focused on a few genera ( Macrocystis , Ecklonia , Cystoseira , and Sargassum ). Six recorded projects have achieved large‐scale success (100 and 1000 s of ha) in restoring kelp forests. This success shows that large‐scale restoration is currently possible and a reasonable goal to strive for. The most successful restoration projects are those that are near existing kelp forests. Preventing kelp forest decline aids kelp recovery, therefore actions to ensure that kelp is not lost from a system are critical. Urchin grazing is the most frequent reason that kelp restoration is needed and also the most common cause of project failure. Projects should work to mitigate this stress prior to restoration and maintain low grazer densities to achieve success. Although not necessarily acceptable due to potential ecological risks, quicklime maybe a technically viable solution to remove large numbers of sea urchins at low financial cost. Urchin fisheries and/or urchin ranching are other options which may profitably remove urchins. Transplanting kelps should work to establish significant population sizes for the best chance of success, particularly if they are adjacent to existing kelp beds. Artificial reefs are a common but expensive and contentious tool for afforestation and restoration. Projects need to carefully consider the economic and environmental costs and benefits before deploying artificial reefs. Further work is needed to investigate seeding methods for restoration. If successful, this method could help scale up kelp restoration projects to larger sizes at reasonable costs. Projects have been very expensive to date, but costs are reducing, and the social and economic benefits of kelp restoration are high. Future methods for restoration (genetic manipulation, kelp aquaculture, autonomous technology) have the potential to address barriers to restoration (warming oceans, low abundance of existing kelp, high urchin populations), but risks and benefits must be weighted, and considered in context of holistic ocean management. Legal frameworks are often inappropriate for kelp restoration and may need to be reconsidered to allow for careful manipulation of ocean spaces for restoration where needed (e.g. transplanting, seeding, herbivore removal). Kelp restoration initiatives present opportunities for rich collaborations among individuals, organisations, and countries, to reforest the ocean, achieve benefits for multiple user groups, and link into the UN Sustainable Development Goals. Global efforts to consolidate and share experiences and learning, such as the Kelp Forest Alliance ( kelpforestalliance.com ), represent concrete steps towards advancing future efforts.",
"introduction": "I. INTRODUCTION (1) The need to restore kelp forests Kelp forests, defined here as habitat‐forming brown algae in the orders Laminariales, Fucales, and Desmarestiales (Wernberg & Filbee‐Dexter, 2019 ), are globally distributed habitats which have declined around the world (Thibaut et al ., 2005 ; Fujita, 2011 ; Johnson et al ., 2011 ; Vásquez et al ., 2014a ; Blamey & Bolton, 2018 ; Rogers‐Bennett & Catton, 2019 ). The causes of these declines range from local stressors such as pollution to global impacts, such as climate change (Wernberg et al ., 2019 ). Early and persistent declines of kelp forests in the 1800s were linked to population expansion of sea urchins, most often facilitated by the removal of urchin predators from the ecosystem (Roberts, 2007 ). Subsequent kelp population declines in the 20th century were driven by threats such as direct harvest of kelp or high levels of water pollution from urban areas (Wilson & North, 1983 ; Vogt & Schramm, 1991 ; Coleman et al ., 2008 ; Connell et al ., 2008 ). These stressors are still relevant to contemporary kelp ecosystem management but now interact with climate change, a phenomenon that has multiple consequences for kelp forests (Smale, 2020 ). Increasing water temperatures and marine heatwaves have resulted in large contractions of kelp populations as they are pushed past their physiological preferences and limits (Tegner & Dayton, 1991 ; Kang, 2010 ; Wernberg et al ., 2016a ; Arafeh‐Dalmau et al ., 2019 ; Rogers‐Bennett & Catton, 2019 ). Warmer sea water temperatures have also facilitated the range expansion of herbivorous sea urchins which can overgraze entire forests and create urchin barrens, a phenomenon identified in most countries that contain kelp (Fujita, 2010 ; Filbee‐Dexter & Scheibling, 2014 ; Ling et al ., 2015 ). More recently, temperature‐driven shifts in the ranges of herbivorous fishes are also causing similar declines in kelp forests near the warm edge of their distribution (Vergés et al ., 2014 ; Zarco‐Perello et al ., 2017 ). Such extensive losses have dramatic ecological and economic impacts. For instance, kelp losses have caused the closure of lobster, abalone, sea urchin, and kelp fisheries in several regions around the globe (Steneck et al ., 2013 ; Bajjouk et al ., 2015 ; Rogers‐Bennett & Catton, 2019 ). (2) History of kelp forest management Managing kelp forests and their declines has a lengthy global history. Traditionally, kelp forest management has been a passive activity whereby managers focused on improving environmental or physical conditions, for instance, by improving water quality (Foster & Schiel, 2010 ), limiting kelp harvest (Fujita, 2011 ; Frangoudes & Garineaud, 2015 ), or protecting species that facilitate kelp forests (Caselle et al ., 2015 ). These methods can be successful, and low‐level exploitation in Chile, Norway, Ireland, and France have ensured that sustainable kelp harvesting continues to exist in those countries (Werner & Kraan, 2004 ; Lorentsen, Sjotun & Gremillet, 2010 ; Buschmann et al ., 2014 ; Frangoudes & Garineaud, 2015 ). Marine protected areas (MPAs) have also worked to increase populations of species that facilitate kelp forests and reduce human pressures (Caselle et al ., 2015 ). For example New Zealand created the Cape Rodney to Okakari Point Marine Reserve (i.e. ‘Leigh Reserve’) in 1976 and this MPA now maintains healthy kelp forests ( Ecklonia radiata , J. Agardh, and Fucales species) relative to areas outside the reserve, which are dominated by urchin barrens (Shears & Babcock, 2003 ). Despite successes with other conservation objectives such as restoring predator populations, (Lester et al ., 2009 ), many passive measures (i.e. those that do not manipulate kelp or their consumers) have failed to re‐establish lost kelp populations (Wernberg et al ., 2019 ). For instance, improvements in water quality in Sydney, Australia (Scanes & Philip, 1995 ) did not lead to the re‐establishment of the locally extinct fucoid, crayweed ( Phyllospora comosa , C. Agardh) (Coleman et al ., 2008 ; Vergés et al ., 2020 ). Transplant experiments demonstrated that while the environment was now suitable for P. comosa , propagule supply and/or post‐settlement survival was likely insufficient for the species to re‐establish populations naturally (Campbell et al ., 2014 ). While other passive approaches like MPAs can succeed in restoring predator species and kelp forests (Eger & Baum, 2020 ), they can also fail to facilitate the re‐establishment of a kelp forest (Leung, Yeung & Ang, 2014 ). As a result, managers are increasingly considering active restoration approaches in combination with removing or mitigating the causes of decline (Morris et al ., 2020 ; Layton et al ., 2020b ). Restoration is defined by the Society for Ecological Restoration (SER) as ‘the process of assisting the recovery of an ecosystem that has been degraded, damaged or destroyed’ (SER, 2004 , p. 3). Active restoration is attempted by introducing or removing biotic or abiotic materials from the environment. If kelp reproduction is limited, reproductive individuals are introduced, either by adding spores or gametophytes and/or by transplanting mature plants that act themselves as the spore source (Layton et al ., 2021 ). If herbivory is an issue, it can be mitigated by culling, transporting, or harvesting grazers such as urchins or herbivorous fish (Fujita, 2010 ; Watanuki et al ., 2010 ; Tracey et al ., 2015 ; Strand et al ., 2020 ; Lee et al ., 2021 ). Thus, restoration as defined by SER requires that the activity improves or brings back previously existing species or habitats, regardless of the restoration methods used. Restoration as defined above is distinguished from ‘afforestation’ (e.g. habitat offsetting) which is the process of creating new kelp habitat in areas that did not previously have kelp forests and is therefore not considered ‘true’ restoration. Artificial reef deployment is the most common form of afforestation, which creates kelp habitat by adding new rocky reef substrate that can enhance the settlement and growth of existent kelp propagules or can act as a base for transplanting or seeding (Schroeter, Reed & Raimondi, 2018 ; Shelamoff et al ., 2020 ). (3) Motivations for restoring kelp forests in the 21st century Restoring kelp forests provides society with many benefits. Healthy kelp forests directly support United Nations Sustainable Development Goals 2 (zero hunger), 8 (work and economic growth), 13 (climate action), and 14 (life under water; Cormier & Elliott, 2017 ). By conserving and restoring kelp ecosystems, we maintain a foundational marine habitat and ensure access to key ecosystem services such as habitat provisioning (Teagle et al ., 2017 ), nutrient cycling (Kim, Kraemer & Yarish, 2015 ) and carbon sequestration (Chung et al ., 2013 ; Filbee‐Dexter & Wernberg, 2020 ). Kelp forests also underpin harvest services, for example, supporting direct kelp harvest (Buschmann et al ., 2014 ) or fisheries through the species that they support (Smale et al ., 2013 ). The services provided by these underwater forests are currently estimated at millions of dollars per km of coastline and billions of dollars per country (Smale et al ., 2013 ; Vásquez et al ., 2014a ; Bennett et al ., 2016 ; Blamey & Bolton, 2018 ; Eger et al ., 2021 ), and provide livelihoods for coastal communities around the world. In addition to their economic values, kelp forests also hold significant cultural and aesthetic value to their local community (Thurstan et al ., 2018 ; Turnbull et al ., 2020 ). International interest and recognition of marine ecosystem restoration is increasing, yet kelp forests are often excluded from these agendas despite their potential contributions to international goals and targets (Feehan, Filbee‐Dexter & Wernberg, 2021 ). The largest initiatives are led by the United Nations (UN), which has declared 2021–2030 as the ‘Decade of Ecosystem Restoration’ as well as the ‘Decade of Ocean Science for Sustainable Development’. These independent but complementary initiatives are calling for a global focus on renewing marine and other ecosystems (Waltham et al ., 2020 ), while also providing needed ecosystems services, helping combat climate change and safeguarding biodiversity and food security (Claudet et al ., 2020 ). Kelp forest restoration has the potential to meet the objectives of both UN initiatives. If carbon credits are verified and established, kelp forest restoration also provides a means for countries to work toward their ‘Nationally Determined Contribution’ (NDC) to mitigate carbon emissions under the Paris Agreement, in addition to European Union agreements to restore set amounts of habitat. These contributions could then also be commodified as carbon credits, while other services such as nutrient removal could also be commodified and provide further incentives to restore kelp forests (Platjouw, 2019 ; Seddon et al ., 2019 ; Vanderklift et al ., 2019 ). While there are clear benefits from restoring kelp forests and global interest is accelerating, the path forward is uncertain. This uncertainty is in part because despite similarities in the causes of decline and restoration methodologies, very little information has been shared between projects within and among countries. The most recent analyses provide useful qualitative assessments of past restoration projects, but focus on work published in English‐speaking countries and in the peer‐reviewed literature (Morris et al ., 2020 ; Layton et al ., 2020b ). Most restoration projects, however, are not formally published in peer‐reviewed journals and occur in non‐English speaking countries (Bayraktarov et al ., 2020 ; Eger et al ., 2020c ). As a result, projects have typically learned and applied methodologies independently. Addressing this limitation will help ensure that lessons learned from 60 to 300 years of history in kelp restoration contribute to a more rapid rate of restoration successes. (4) Study objectives This review aims to provide a comprehensive history of kelp forest restoration, assess the current state of the field, and provide recommendations for how this field can advance. We achieve this by reviewing the global history of kelp restoration, analysing past projects, examining the determinants of success, and describing solutions to barriers to future restoration projects. This comprehensive, multi‐language project first reviews the history of kelp restoration in independent geographic clusters around the world. Following this qualitative overview, we present the results of a new kelp restoration project database ( kelpforestalliance.com ) and describe the global state of the field, what factors have resulted in success, and which in failure. Finally, we discuss the methodologies, costs, motivations, and legal frameworks currently related to kelp restoration and how we can enhance the factors that can lead to success in restoration and mitigate potential barriers in future."
} | 4,130 |
27582875 | PMC5006580 | pmc | 1,722 | {
"abstract": "Background Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. Results 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1–S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l −1 day −1 (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l −1 day −1 ). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. Conclusions All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale , without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production. Graphical abstract",
"conclusion": "Conclusions In this work, we presented an approach to select microalgae cells with increased lipid productivity under N-starvation. Our approach was successful because we successfully selected new cell lines with increased lipid content (under N-starvation) with no effect on growth (N supplied), resulting in progressive improved TAG productivities. S5, sorted after 5 cycles of starvation-sorting, showed 1.9× higher TAG productivity than the Wt. Hence, S5 and original were compared under simulated summer conditions in a flat-panel photobioreactor. The results from comparing S5 and original confirmed the results of the first experiment: S5 showed an 2× higher TAG productivity (from 0.2 to 0.4 g l −1 day −1 ) because we have removed cells with low TAG yield (on light) in comparison to Wt. The experiments comparing S5 and original were done 1.5 year after the sorting, therefore, indicating a stable phenotype. S5 showed superior TAG yields on light when compared with other Wt-high-lipid green microalgae (0.32 against 0.16–0.20 g mol −1 ), being comparable to the highest yield registered with a starchless mutant (0.32 against 0.37 g mol −1 ). Biomass composition indicates that S5 has no alterations in the carbon partitioning between TAG and starch, but only an doubled TAG yield on light when compared to the original population. All combined, our results present a successful strategy to improve the TAG productivity of C. littorale , without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production.",
"discussion": "Results and discussion Gate set-up to sort lipid-rich cells Our approach started by choosing the sorting criteria. Besides the obvious choice of lipid-dependent fluorescence (Bodipy, BP) we also included chlorophyll-dependent fluorescence (Autofluorescence, AF). We included AF for two reasons: the first reason is because AF correlates to cell size, hence it can be used to estimate the ratio lipid/cell. Secondly, we hypothesized that cells that could keep their levels of AF after a period of N-starvation would be cells with low levels of chlorophyll degradation, hence cells that could grow again if N is re-supplied. We were surprised to observe an almost absence of chlorophyll fluorescence degradation after long periods of N starvation. This has been observed in our previous publication [ 22 ] and might be due to the fact that C. littorale is a highly resilient strain under abiotic stress [ 23 – 25 ]. To establish the sorting gate we first carried out a screening test in which N-starved cells (under the same conditions to be used in the experiments to follow) were analyzed with flow cytometry. The scatter-plot between autofluorescence (chlorophyll-dependent, AF) and Bodipy fluorescence (lipid-dependent, BP) (Fig. 1 ) shows that there are some regions of the population in which these two variables correlate more. Hence, we included the BP/AF ratio as a criterion to estimate the amount of lipids (BP) per cell size (AF). Our analyses of the population of N-starved cells pointed to two interesting sub-populations, as showed in Fig. 1 . The first sub-population to be analyzed were the cells that presented the highest values of lipid fluorescence (High BP). These cells, however, show high values of BP because they are also the biggest cells within the population, which can be evidenced by its bigger size in comparison with the whole population (8.11 against 6.66 µm; Table in Fig. 1 ). Cell size could be a misleading variable since bigger cells might have, proportionally, the same lipid content as the population’s median. When normalizing the median BP fluorescence by the median cell volume of each population it is clear that the sub-population marked as High BP doesn’t have more fluorescence per cell volume than the whole population (4.04 against 4.37E+03 BP/cell; Fig. 1 ). The other sub-population was chosen using the BP/AF ratio as criterion (High BP/AF, Fig. 1 ). We hypothesized that BP/AF ratio would lead to a sub-population of cells with a higher lipid content per cell. At the same time this sub-population selects the top lipid producers while keeping the median AF value, hence avoiding the AF as a cofounding variable (either due to cell size or relative chlorophyll content). The normalized BP/cell volume show us that indeed the High BP/AF sub-population has a higher value of BP/cell volume in comparison with the whole population (4.54 versus 4.37E+03, Fig. 1 ). Thus, the gate depicted in red on Fig. 1 as BP/AF was chosen as sorting criteria. Sorting lipid-rich cells The sorting’s were done as depicted in the graphical abstract. After each round of sorting the cells were transferred to shake flasks to produce new inoculum to start a new N-runout (and consequently a new round of sorting). Additionally, an aliquot of the sorted cells was transferred to an agar plate and kept under low light conditions for long term storage and further use for comparing all sorted populations. All sorting rounds had a period of 1 month in between to grow the inoculum after sorting. Hence, each new round of N-runout was started with cells that came from different acclimation conditions than the Wt. Therefore, we produced inoculum from original and all sorted populations that were kept on plate, at the same time and started parallel runs with an independent reactor for each population. The population from the first round of sorting (S1) was kept out of the parallel runs because preliminary data from flow cytometry showed that the cells of S1 had the same distribution of lipid fluorescence as the original (data not shown). Original was run in parallel with S2–S5 to compare growth rates, biomass and lipid productivities and biomass composition. Figure 2 shows the overlapped growth curves for all five populations with a clear similarity among all populations during the growth phase (during the first 2 days N was available, green area). Table 1 gives the values of specific growth rates (µ) and biomass productivities ( P X ) among all sorted populations, which were similar to each other. Since cells were sorted based on their lipid content per cell size, the first parameter to be investigated was the relative lipid accumulation rate (BP r , day −1 ), showing that all sorted populations presented higher accumulation of lipids in comparison with the original (1.15–1.20 against 1.09 day −1 , Table 1 ). Next, the median fluorescence values (both AF and BP) were compared, revealing an progressive increase in median BP fluorescence while the AF was reduced (Fig. 3 a). This resulted in a progressive increase in the BP/AF ratio, the aim of our sorting criteria. The parameter BP/cell was calculated dividing the median value of BP-fluorescence by the total cell number (at the end of the N-runout, thus at day 8), resulting in a similar trend as the BP r . These parameters were used to decide to continue/stop the sorting in the early stage of the research as an indication of the lipid productivity. It should be highlighted that they are relative measurements, hence not capable of replacing the actual measurement of lipid productivity, which can only be determined after sorting a new population when enough biomass is produced. Biomass samples of all populations were taken at the end of the comparison runs to measure the TAG productivity. Fig. 2 All sorted populations are similar to the wildtype (Wt) in the growth phase. The graphs shows the biomass concentration per day (g l −1 ). The green area (days 0–2) marks the growth phase, hence both populations consumed nitrogen at the same rate. After day 2 all nitrogen was taken up by the cells. From day 2 onwards it should be considered as starvation phase (N−) Table 1 Comparing kinetic parameters and biomass composition between original and sorted populations Kinetic parameters µ (day −1 ) \n P \n x (g l −1 day −1 ) P TAG (g l −1 day −1 ) BP r (day −1 ) BP/cell (RFU cell −1 ) Original 0.69 1.54 0.18 1.09 1.09 S2 0.67 1.48 0.20 1.15 1.12 S3 0.73 1.66 0.20 1.22 1.14 S4 0.64 1.39 0.25 1.19 1.20 S5 0.67 1.48 0.34 1.20 1.23 Biomass composition Starch (g g −1 ) Carbs (g g −1 ) Proteins (g g −1 ) PL (g g −1 ) TAG (g g −1 ) Original 0.24 0.10 0.14 0.05 0.23 S2 0.24 0.12 0.15 0.06 0.27 S3 0.25 0.12 0.14 0.05 0.26 S4 0.24 0.13 0.16 0.06 0.29 S5 0.25 0.12 0.16 0.06 0.34 The kinetic parameters are: specific growth rate (µ) and biomass productivity ( P \n x ), both under growth conditions (between t = 0 and t = 2). TAG productivity (P tag ) and lipid fluorescence accumulation rate (BP r ) consider the whole N-starvation period ( t = 2 to t = 8), while lipid fluorescence per cell was measured considering the values at the end of the starvation phase ( t = 8). All analyzed biomass components are expressed as relative to biomass dry weight (g g −1 DW −1 ). Standard variation of measurements is not depicted because the technical error was always around 5 % Fig. 3 A progressive increase in lipid fluorescence leads to a progressive increase in BP/AF ratio without changes in the saturation degree of TAG’s. Fluorescence measurements of both Bodipy (BP) and autofluorescence (AF) on the primary y -axis and the BP/AF ratio on the secondary y -axis in all populations ( x -axis) ( a ). All populations showed similarities in the TAG’s saturation degree (SFA, MUFA and PUFA’s as % of total TAG’s) ( b ) TAG productivity (P TAG ) was measured for all populations, showing increased P TAG values among all sorted populations (Table 1 ). Compared to original, S2 and S3 showed a discrete increase (from 0.18 in original to 0.20 g l −1 day −1 in S2 and S3) and S4 showed an increase of 38 % (from 0.18 to 0.25 g l −1 day −1 ). S5 showed the highest P TAG0 , resulting in an 88 % increase in P TAG when compared with original (from 0.18 to 0.34 g l −1 day −1 ). The biomass produced by all sorted populations was analyzed for its content as total starch, total carbohydrates (minus starch), total proteins, polar lipids and TAGs (Table 1 ). The biomass composition of all sorted populations and the original population was similar among each other, with the exception of TAGs. The analyses of biomass composition showed that the extra biomass produced by the sorted populations was caused only by an overproduction of TAGs (Table 1 ). We would like to highlight that the sorted criteria used in the current research didn’t affect the TAGs composition of all sorted populations, which showed the same saturation degree (Fig. 3 b). This finding was intentional and expected, considering the staining used in the current research. Bodipy (505/515) is a lipophilic fluorophore that binds to neutral lipids, being a relative measurement of total TAGs [ 26 , 27 ]. Hence, in our work cells were selected based on total TAGs content per cell, without applying any pressure that would favor a change in TAGs composition. Two previous works on microalgae strain improvement with cell sorting have reported a change in lipid composition [ 7 , 10 ]. Both works, however, have used random mutagenesis, which could explain the change in lipid composition. Flow cytometry data were used to see the relationship at cellular level, between relative lipid content and autofluorescence (Fig. 4 , scatter plots). From such relation we can see that the sorted cells don’t show fluorescence values above the maximum already exhibited by the Wt. We hypothesized that our sorted cells would achieve higher values of BP-fluorescence than the Wt, similar to what has been reported by Thi-Thai Yen Doan and Obbard [ 8 ] and by Montero et al. [ 13 ]. What we see, however, is that we increased the lipid/cell ratio by progressively removing the cells with a low lipid/cell ratio, hence increasing the median BP fluorescence of the population. This effect can be visualized in numbers using the coefficient of variation (CV) of both auto and BP-fluorescence, which were progressively reduced in all sorted populations (values available on the scatter plots, from 0.11 (Wt) to 0.06 (S5), Fig. 4 ). The results from the scatter plots (Fig. 4 ) can be combined with the histograms of frequency of BP (right side, Fig. 4 ). The histograms show that the populations have a higher median TAG content because they show a higher proportion of the population composed of lipid-rich cells (percentage values on the charts show the proportion of cells within the population that have fluorescence values above 10 2 RFUs). We can conclude, combining all results above, that the sorting carried out in the present research was successful in producing new cell lines with increased lipid productivity. Fig. 4 Sorted populations show a progressive increase in lipid productivity due to an increase in the proportion of lipid-rich cells within the populations. Scatter plots represent the relation between autofluorescence (AF, x -axis) and lipid fluorescence (BP, y -axis). On all scatter-plots the coefficient of variation (CV) of both AF and BP are depicted. Each populations has a histogram plotted showing the frequency distribution of lipid fluorescence (BP, x -axis). Additionally, each histogram is market to show the percentage of cells that have fluorescence signal above 10^2. All graphs represent the readings of 1000 cells Previous research articles have also reported successful sorting of microalgae cells to produce populations with increased lipid content [ 7 , 8 , 10 , 13 , 16 ]. All these publications, however, do not measure the impact of the sorting on biomass or lipid productivity. Furthermore, all these publications were done at small laboratory scale, many using well-plates or shake flasks, hence the estimation of industrial performance of strains is limited. One exception was the work of Beacham and co-authors, which assessed both growth rate and lipid productivity of Nannochloropsis salina and sorted mutants [ 15 ]. The combination of mutagenesis and cells sorting lead to 4 mutants, all exhibiting an overproduction of lipids, but all mutants also exhibited a reduction in biomass productivity (from −18 to −95 % growth). In their results only one mutant showed a final lipid productivity higher than the original strain (from 0.40 to 0.49 × 10 −4 g ml −1 day −1 ). Another important remark when comparing sorted cells/mutants is the timing in between sorting rounds, which may cause an effect on growth and lipid kinetics of sorted populations. Most of other works above mentioned compared the lipid content of cell populations cultivated immediately after being sorted, hence ignoring a possible effect of the post-sorting period of re-growth on the physiological response of microalgae [ 7 , 8 , 14 – 16 ]. Previous research has found a 3.2 times longer growth phase in sorted populations in comparison with the original [ 14 ]. Since the growth was measured immediately after sorting, it is impossible to say if the slower growth was a feature of the new population or a longer lag phase caused by the sorting process. The effects of the sorting process itself could decrease growth rate and/or affect the lipid metabolism response since it represents a source of stress and the cellular response is strain-specific [ 17 , 28 , 29 ]. The above discussed results highlight the importance of assessing cellular growth after a long period post-sorting, to guarantee the stability of the sorted population to industrial cultivation. Comparing original and S5 under simulated Dutch summer conditions The parallel runs with sorted populations and original population showed S5 as the population with the highest TAG productivity. The next step was to cultivate original and S5 under simulated outdoor conditions. This is an important step since part of the biomass (carbons sources, e.g. starch and TAGs) can be respired during dark periods for cellular maintenance, leading to possible changes in the productivity of different biomass components when comparing to experiments under continuous light [ 30 ]. The goal of this comparison was to evaluate how superior the productivity of S5 was in comparison with original under day/night cycles, hence we simulated an average Dutch summer day in an indoor flat panel reactor. We also used the comparison experiment between S5 and original to estimate the stability of the sorted phenotype as the comparison experiments were carried out 1.5 years after the population had been sorted. Original and S5 were kept under low light in agar plates containing growth medium in the time between the sorting and the comparison experiments (cultures were transferred to new plates every 3 months). Therefore, original and S5 were compared using inoculum that came from similar conditions, and both were done in biological replicates, all combined to make the comparison more accurate. Figure 5 a shows the growth (as biomass DW, g l −1 ) of original and S5, confirming our results from the parallel runs: under growth conditions (days 0–2, Fig. 5 ) S5 shows similar growth kinetics as the original (µ and P x, Table 2 ). Previous research articles have reported that cells with improved lipid content presented also a reduction in growth [ 15 , 10 ]. Our results, however, highlight that the sorting criteria used in the current work didn’t affect cell growth. One explanation could be the inclusion of AF as part of the selection criterion. We hypothesized that cells that could keep their levels of AF after a period of N-starvation would be cells with low levels of chlorophyll degradation, hence cells that could grow again if N is re-supplied. Fig. 5 Comparing growth and lipid accumulation of original and S5 under simulated Dutch summer conditions. Graph \n a shows that the S5 strain produces more biomass under nitrogen starvation than the Wt. The grey area represents the period of the cultivation in which N was available, hence both populations consumed nitrogen at the same rate. From day 2 onward the N-starvation phase (N−) is considered . Error bars are depicted at each time point (standard deviation, three replicates), although for most of the points the bars are too small to be visualized. Graph \n b shows the difference between S5 and original at cellular level (at the end of the N-starvation period): the S5 population has higher values of Bodipy fluorescence than the Wt, leading to an increase in the BP/AF ratio. Both populations had the same number of cells analyzed ( n = 500). Error bars at figure b represent the 95 % confidence intervals Table 2 Comparing kinetic parameters and biomass composition between original and S5 under indoor simulated Dutch summer conditions Populations µ (day −1 ) P X (g l −1 day −1 ) P TAG (g l −1 d −1 ) Y TAG (g mol −1 ) Original 0.52 ± 0.08 a \n 1.03 ± 0.19 a \n 0.2 ± 0.02 a \n 0.16 ± 0.01 a \n S5 0.48 ± 0.03 a \n 1.08 ± 0.08 a \n 0.4 ± 0.06 b \n 0.32 ± 0.03 b \n Starch (g g −1 ) Carbs (g g −1 ) Polar lipids (g g −1 ) TAG (g g −1 ) Original 0.25 ± 0.04 a \n 0.20 ± 0.04 a \n 0.07 ± 0.01 a \n 0.21 ± 0.02 a \n S5 0.29 ± 0.01 a \n 0.19 ± 0.05 a \n 0.06 ± 0.01 a \n 0.30 ± 0.04 b \n The kinetic parameters are: specific growth rate (µ), biomass productivity ( P \n x ), TAG productivity (P tag ) and TAG yield (Y TAG ). All analyzed biomass components are expressed as relative to biomass dry weight (g g −1 DW −1 ). Error bars in the table indicate the standard deviation. Superscript letters represent the results of the statistical analyses between Original and S5. Different letters indicate statistical significance (ANOVA, p < 0.05) Differences, however, were observed after the start of the N-runout (Fig. 5 , after day 2). Biomass composition shows again that TAGs were the biochemical fraction that explained the increase in biomass accumulation in the N-starvation period (Table 2 ). The TAG content of S5 was 1.42 × higher than original (0.30 against 0.21 g/g DW, Table 2 ), while other biomass components (starch, carbohydrates (minus starch) and polar lipids) were similar between original and S5. The increase in TAG content resulted in a twofold increase in the P TAG of S5 when compared with original (Table 2 ), thus confirming the results of the first experiments. Cytometry data were used to evaluate the cause for increase in lipid productivity at cellular level. Flow cytometry data from the cells at the end of the cultivation corroborate the previous results since we observe once again an increased median lipid fluorescence (BP) being the responsible variable that results in an increased BP/AF ratio (Fig. 5 b). Additionally, Fig. 6 shows that S5 has a higher percentage of lipid-rich cells under similar conditions when compared with Wt. The amount of lipid-rich cells in S5 was 99 % while in the original population it was 66 % (arbitrarily chosen as cells with fluorescence signals above the half of the scale; numbers derived from the histograms on Fig. 6 ). Added to that, biomass composition indicates that S5 has no alterations in the carbon partitioning between TAG and starch (Tables 1 , 2 ), but only an doubled TAG yield on light when compared to the original population. Hence, the strategy here presented was successful in selecting microalgae cells with increased TAG yield without affecting the metabolism of other cellular components. Fig. 6 S5 population has a higher percentage of lipid-rich cells than the original population. Graph \n a shows a scatter-plot between autofluorescence and Bodipy fluorescence at the end of the N-runout. Each dot represents one-cell measurement. Graph \n b shows the distribution of frequency of Bodipy-fluorescence of both original and S5. In both graphs a total number of 500 cells were analyzed per sample The results from our experiments were compared with other researches with batch-wise TAG production. A review of TAG production is given by Benvenuti et al. [ 31 ], comparing different microalgae strains using the yields of TAG on light to compensate for different growth/stress conditions. We calculated the TAG yields on light of both original and S5 as being 0.16 and 0.32 g mol −1 , respectively (under simulated summer conditions). First it is important to highlight that original C. littorale showed an TAG yield close to other high-lipid microalgae, such as: Scenedesmus obliquus ; 0.22 g mol −1 ; Nannochloropsis oculat a, 0.17 g mol −1 ; Chlorella zofingiensis , 0.19 g mol −1 and Nannochloropsis sp., 0.14 g mol −1 [ 32 – 35 ]. The work of Breuer et al. [ 33 ] also presents the TAG yield of an starchless mutant of S. obliquus at 0.37 g mol −1 , close to the yields measured for the S5 in the current work (0.32 g mol −1 )."
} | 6,161 |
34618081 | PMC8566233 | pmc | 1,724 | {
"abstract": "Abstract The enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins. Here, we analyzed transgenic poplars ( Populus tremula × Populus alba ) expressing the Brachypodium ( Brachypodium distachyon ) p -coumaroyl-Coenzyme A monolignol transferase 1 ( BdPMT1 ) gene driven by the Arabidopsis ( Arabidopsis thaliana ) Cinnamate 4-Hydroxylase ( AtC4H ) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase ( AtF5H ). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p- coumaric acid ( p CA). Several BdPMT1 -OE/WT and BdPMT1 -OE /AtF5H -OE lines were grown in the greenhouse, and BdPMT1 expression in xylem was confirmed by RT-PCR. Analyses of poplar stem cell walls (CWs) and of the corresponding purified dioxan lignins (DLs) revealed that BdPMT1 -OE lignins were as p -coumaroylated as lignins from C3 grass straws. For some transformants, p CA levels reached 11 mg·g −1 CW and 66 mg·g −1 DL, exceeding levels in Brachypodium or wheat ( Triticum aestivum ) samples. This unprecedentedly high lignin p- coumaroylation affected neither poplar growth nor stem lignin content. Interestingly, p -coumaroylation of poplar lignins was not favored in BdPMT1 -OE/ AtF5H -OE transgenic lines despite their high frequency of syringyl units. However, lignins of all BdPMT1 -OE lines were structurally modified, with an increase of terminal unit with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1 -OE lignins and makes them more soluble in cold NaOH solution. The p -coumaroylation of poplar samples improved the saccharification yield of alkali-pretreated CW, demonstrating that the genetically driven p -coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to alkaline treatments used during the industrial processing of lignocellulosics.",
"conclusion": "Conclusion In this study, we have shown that p -coumaroylating poplar lignins up to the level of grass lignins has consequences that go far beyond a simple lignin decoration and that deeply change not only lignin structural traits, but also important industrial potentialities of lignified CW. Remarkably enough the expression of BdPMT1 under the control of the AtC4H promoter introduced neither any growth penalty, nor reduced lignin content in the various transgenic greenhouse-grown poplar lines that were obtained in two genetic backgrounds. In agreement with a recent study ( Sibout et al., 2016 ), choosing the lignin-specific AtC4H promoter to drive the heterologous expression of BdPMT1 in dicot CW had very likely a key role in changing wood properties. Since the last decades and with the objective to facilitate the industrial conversion of lignocellulosics into pulp or into bioethanol, many approaches have been used to genetically modify lignin content and/or structure (reviewed in Boerjan and Ralph, 2019 ; Halpin, 2019 ; Mahon and Mansfield, 2019 ; Ralph et al., 2019 ). Among the lignin structural traits that can be affected by the genetic transformation of angiosperm species, the S/G ratio is probably the most systematically scrutinized one ( Chanoca et al., 2019 ). In contrast, the relative frequency of free phenolic units in native lignins is a key structural trait, which is surprisingly overlooked despite its biological significance and its major effect on the susceptibility of lignins to alkaline or oxidative treatments. In past studies, redesigning native lignins with more free phenolic groups (and therefore with increased alkali-solubility) could be obtained with other genetic transformations, such as CCR or CAD downregulation ( O'Connell et al., 2002 ; Lapierre et al., 2004 ). In this work, we provide another compelling evidence that the genetically driven increase of free phenolic units in lignins is an efficient strategy for the rational design of lignocellulosics more adapted to industrial biorefineries.",
"introduction": "Introduction Wood appears as a major feedstock for traditional or innovative biorefineries producing pulp, chemicals, or fermentable sugars. However, most industrial fractionations of lignocellulosics are detrimentally affected by lignins. For instance, the enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins that hinder the accessibility of enzymes to cell wall (CW) polysaccharides. Indeed, the economically effective production of cellulosic ethanol necessitates costly, polluting and energy-intensive pretreatments that most often aim at reducing the lignin shield effect ( Yang and Wyman, 2008 ; Sun et al., 2016 ). Since the last decades, lignin engineering in trees has been the subject of intensive studies to produce tailor-made wood more amenable to efficient deconstruction by milder processes ( Pilate et al., 2012 ; Chanoca et al., 2019 ; Mahon and Mansfield, 2019 ). However, lignins play key roles in wood and sufficient lignin amounts are required to warrant tree growth, development and defense. On this basis, reducing lignin content may result in impaired tree growth and redesigning lignin structure appears as a better strategy to obtain wood biomass more adapted to industrial deconstruction without yield penalty. Lignins primarily result from the enzymatically driven oxidation of monolignols, mainly coniferyl alcohol and sinapyl alcohol that give rise to guaiacyl (G) and syringyl (S) units, respectively. It is now well established that lignin biosynthesis is very plastic and that, besides the main monolignols, a number of other molecules may participate in the formation of lignin polymers ( Mottiar et al., 2016 ; del Río et al., 2020 ). For instance, p -coumaroylated sinapyl alcohol and, to a lower extent, p -coumaroylated coniferyl alcohol, are naturally incorporated into grass lignins ( Grabber et al., 1996 ; Lu and Ralph, 1999 ; Hatfield et al., 2009 ; Ralph, 2010 ). This p -coumaroylation of grass monolignols is specifically catalyzed by a p -coumaroyl-coenzyme A monolignol transferase (PMT) studied in various grass species ( Hatfield et al., 2009 ; Withers et al., 2012 ; Marita et al., 2014 ; Petrik et al., 2014 ). The p -coumaroylation of dicot lignins was recently achieved by introducing the rice ( Oryza sativa ) PMT gene into poplar and Arabidopsis plants ( Smith et al., 2015 ), but the p -coumaroylation level of transgenic dicot CW reported in this study was modest (varying from 1 to 3.5 mg·g − 1 CW) and much lower than that of lignified grass stems ( p -coumaric acid [ p CA] ranging from 6 to 39 mg·g − 1 CW; Hatfield et al., 2009 ). In contrast, the introduction of two different Brachypodium PMT genes ( BdPMT1 or BdPMT2 ) under the control of the AtC4H promoter into various Arabidopsis lines boosted the p -coumaroylation of mature stem lignins up to the grass lignin level ( Sibout et al., 2016 ). In addition to a high p CA content, the Arabidopsis BdPMT1 -OE lignins displayed other traits specific to grass lignins, i.e. a high frequency of free phenolic units in lignins and an increased solubility in cold alkali. In this work, we explored the potential of introducing the proAtC4H::BdPMT1 construct into poplar in order to beneficially tailor lignin structure without biomass penalty. To this end, BdPMT1 was expressed not only in the poplar wild-type (WT) background, but also in a transgenic poplar line overexpressing the AtF5H gene ( AtF5H -OE). By so doing, we obtained several independent transformants that were grown in the greenhouse together with the corresponding controls during 3 months. In this study, we first evaluated the growth of the BdPMT1 -OE lines and the p -coumaroylation of their stem lignins, as compared to control trees. We then investigated the effect of the BdPMT1 expression on lignin content and structure before subjecting the transgenic and control poplar stems to alkali-solubilization assays and saccharification tests.",
"discussion": "Results and discussion The expression of heterologous BdPMT1 gene under the control of the AtC4H promoter does not alter poplar growth The BdPMT1 acyltransferase (referred to as Bradi2g36910) has been shown to be specific to monolignol p -coumaroylation ( Petrik et al., 2014 ). BdPMT1 is a close homolog of the rice OsPMT that was introduced by Smith et al. (2015) into poplar and Arabidopsis plants. We used the AtC4H promoter to drive the expression of BdPMT1 as it is highly expressed in Arabidopsis xylem tissues during lignification ( Bell-Lelong et al., 1997 ) and is also efficient to drive transgene expression in poplar wood ( Franke et al., 2000 ). The transformation was performed in two poplar genetic backgrounds, the WT line and a transgenic line overexpressing the AtF5H gene. The AtF5H expression was driven by a poplar cellulose synthase A4 promoter, known to be highly active in the fibers and vessels of poplar developing xylem ( Hai et al., 2016 ). The AtF5H -OE poplar line was chosen to test the hypothesis that the p -coumaroylation of poplar lignins may be favored by a high frequency of S units based on the two following published data: (1) the p -coumaroylation of grass lignins mostly occurs on S units (reviewed in Ralph, 2010 ; Karlen et al., 2018 ) and (2) overexpressing the AtF5H gene in poplar substantially increases the frequency of S lignin units ( Franke et al., 2000 ). The Agrobacterium tumefaciens -mediated transformation yielded several independent transformants in the WT background (referred to as BdPMT1 -OE/WT lines) and in the AtF5H- overexpressing background (referred to as BdPMT1 -OE /AtF5H -OE lines). Three BdPMT1 -OE/WT lines and two BdPMT1 -OE/ AtF5H -OE lines were randomly chosen for further analyses: They were acclimatized and grown for 3 months in the greenhouse together with corresponding control plants ( Supplemental Figure S1A ). Semi-quantitative RT-PCR with BdPMT1 specific primers revealed a substantial BdPMT1 transcript abundance in developing xylem of BdPMT1 -OE lines, with some variations between lines, whereas no BdPMT1 expression could be detected in the WT or AtF5H -OE control trees ( Supplemental Figure S1B ). Likewise, when using primers directed to AtF5H , a strong RT-PCR signal was observed in the AtF5H -OE transgenic lines ( Supplemental Figure S1C ). Relative to the control trees, the BdPMT1 -OE did not induce any significant difference in height and diameter ( Figure 1 ) and the transgenic poplar plants did not show any obvious phenotype difference when compared to WT trees. Figure 1 Growth response to the introduction of the proAtC4H::BdPMT1 construct into the poplar WT background (black bars) and into the AtF5H -OE background (gray bars), as compared to control (Ctrl) trees. The basal diameter (A) and the tree height (B) were measured on 3-month-old greenhouse-grown trees. Data are means ( sd ) values of three or four biological replicates. Duncan tests (at P < 0.05) did not reveal any significant differences between poplar lines. The BdPMT1 -OE poplar stems and their corresponding purified dioxane lignin fractions are p -coumaroylated to the levels of C3 grass samples CW samples from the stems of 3-month-old greenhouse-grown poplar trees were subjected to mild alkaline hydrolysis to quantify p -hydroxybenzoic acid (Bz), p CA, and ferulic acid (FA) ester-linked to CW polymers. Poplar wood is typified by the occurrence of Bz ester-linked to lignins ( Smith, 1955 ; Venverloo, 1969 ) and preferentially to the γ position of S lignin units ( Lu et al., 2004 ; Morreel et al., 2004 ). Most BdPMT1 -OE poplar samples displayed similar p -hydroxybenzoylation levels as their corresponding controls ( Table 1 ). In addition to Bz, mild alkaline hydrolysis of poplar samples released small amounts of FA consistently and significantly in slightly smaller quantities in all BdPMT1 -OE/WT lines compared to the WT ( Table 1 ). In plant CW, FA preferentially acylates noncellulosic polysaccharides ( Ishii, 1997 ). Some of these FA esters can be oxidatively coupled to monolignols and act as lignin nucleation sites ( Ralph, 2010 ). The small differences of FA esters between poplar lines might reflect some variations in the feruloylation degree of CWs polymers and/or in their ferulate mediated cross-linking. Table 1 Amount of Bz, p CA, and FA released by mild alkaline hydrolysis of extract-free poplar stems (referred to as CWs) from BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds, as compared to their respective controls Line ( n Replicates) Bz \n p CA FA mg·g −1 CW mg·g −1 CW mg·g −1 CW WT control (3) 3.86 (0.10) a,b 0.01 (0.00) f 0.22 (0.00) a \n BdPMT1 -OE / WT line 9 (3) 3.21 (0.51) b 7.12 (0.49) b 0.15 (0.02) c \n BdPMT1 -OE / WT line 17 (3) 3.66 (0.28) a,b 10.69 (0.49) a 0.18 (0.01) b \n BdPMT1 -OE / WT line 31 (3) 3.57 (0.11) ab 3.63 (0.42) d 0.10 (0.00) d \n AtF5H -OE control (4) 3.29 (0.14) b 0.01 (0.00) f 0.06 (0.00) e \n BdPMT1 -OE /AtF5H -OE line 1 (4) 3.06 (0.56) b 0.76 (0.04) e 0.07 (0.01) de \n BdPMT1 -OE /AtF5H -OE line 21 (3) 4.35 (0.08) a 4.92 (0.19) c 0.13 (0.02) c The data represent mean ( sd ) values from n biological replicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01). While expressing the BdPMT1 gene both in the WT and the AtF5H -OE backgrounds had no effect on Bz units ester-linked to poplar CW, this transformation dramatically increased CW p -coumaroylation with up to 1,000-fold higher levels compared to the trace amounts of the controls ( Table 1 ). Remarkably enough, this quantity was boosted up to about 11 mg·g − 1 CW in BdPMT1 -OE/WT line 17. As compared to grass mature stems, the p CA levels of this poplar line exceeded those of most C3 grass CW, but remained lower than those of C4 grass CW ( Supplemental Table S1 ). With the exception of line 1, the obtained BdPMT1 -OE poplar lines were as p -coumaroylated as extract-free proAtC4H::BdPMT1 Arabidopsis mature stems ( p CA amounts ranging between 3.5 and 12.6 mg·g − 1 CW; Sibout et al., 2016 ). In contrast, these levels were much higher than the values reported for OsPMT -OE poplar lines ( p CA range: 1.2–3.5 mg·g − 1 CW) or for OsPMT -OE Arabidopsis lines ( p CA range: 1.0–2.0 mg·g − 1 CW) when OsPMT expression was driven by the 35S CAMV promoter or by the CELLULOSE SYNTHASE7 promoter ( Smith et al., 2015 ). In agreement with Smith et al. (2015) , the p -coumaroylation of poplar CW did not affect their p -hydroxybenzoylation ( Table 1 ). The high p -coumaroylation of poplar CWs obtained in the present work is very likely related to the efficiency of the AtC4H promoter, in agreement with recent data obtained with BdPMT1 -OE Arabidopsis lines ( Sibout et al., 2016 ). Isolation of dioxan lignin (DL) fractions followed by their mild alkaline hydrolysis recently proved to be an efficient strategy to demonstrate that p CA units introduced in BdPMT1 -transformed Arabidopsis plants are ester-linked to lignins ( Sibout et al., 2016 ). The isolation method consists in mild acidolysis (refluxing CW samples in dioxane/0.2 M aq. HCl for 30 min under N 2 ), which provides a rough lignin extract then purified to recover DL fractions. This isolation method relies on the hydrolysis of some ether bonds in lignins to make the insoluble native lignin polymers partially soluble into the reaction medium. The purified DL fractions contain a low amount of sugar contaminants (<10% by weight) and the mild isolation procedure mostly preserves lignin-linked p CA esters, if present ( Chazal et al., 2014 ). Purified poplar DL fractions were isolated from a few control and BdPMT1 -OE poplar lines and then subjected to mid-infrared (IR) spectroscopy. Their mid-IR spectra not only confirmed their low contamination by sugar components, but also suggested that the lignin fractions isolated from BdPMT1 -OE / WT and BdPMT1 -OE /AtF5H -OE lines were enriched in p CA esters ( Supplemental Figure S2 ). Relative to their respective controls, the IR spectra from BdPMT1 -OE lines displayed increased signals at 1,604, 1,164, and 833 cm −1 , which can be assigned to the occurrence of p CA units ( Chazal et al., 2014 ). More importantly, high p CA amounts (from 31 to 66 mg·g − 1 DL, Table 2 ) were released by mild alkaline hydrolysis of the purified DL fractions isolated from BdPMT1 -OE poplar lines, as confirmed by both high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analyses ( Supplemental Figure S3 ). The upper values were similar to the p CA levels of DL fractions isolated from C3 grass CW, but remained lower than those of DL fractions isolated from C4 grass species ( Supplemental Table S1 ). Alkaline hydrolysis of the DL fractions isolated from control samples released very low amounts of p CA units ( Table 2 ), which reveals that p CA acylates poplar lignins to a weak extent and is in agreement with results obtained for Arabidopsis lignins ( Sibout et al., 2016 ). The p CA contents of DL fractions from BdPMT1 -OE poplar line were found to be 6- to 10-fold higher than those from the corresponding CW ( Table 1 ). Such an outstanding enrichment further establishes that most p CA units introduced in the transgenic poplars are ester-linked to lignins. Table 2 Amount of p CA released by mild alkaline hydrolysis of DL fractions isolated from control and BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds Line \n p CA mg·g −1 DL WT control 3.21 (0.17) \n BdPMT1 -OE / WT line 9 50.00 (0.77) \n BdPMT1 -OE / WT line 17 66.52 (0.47) \n BdPMT1 -OE / WT line 31 31.36 (0.43) \n AtF5H -OE control 0.87 (0.04) \n BdPMT1 -OE /AtF5H -OE line 21 33.98 (0.53) The data represent mean ( sd ) values from technical duplicates. Analytical pyrolysis further confirms the high p -coumaroylation of BdPMT1- OE poplar lines The main advantages of the pyrolysis-GC/MS (Py-GC/MS) method is its high-throughput screening capabilities together with its low sample demand ( Ralph and Hatfield, 1991 ; Lapierre, 1993 ). When subjected to this method, lignified CW samples provide lignin-derived phenolics originating from G and S lignin units. In addition, during pyrolysis, ester-linked Bz and p CA units (if present) are decarboxylated to produce phenol and 4-vinylphenol, respectively. The relative abundances (area %) of the main G and S pyrolysis products and of phenol and 4-vinylphenol generated from the poplar CW samples are listed in Table 3 . Table 3 Relative percentage values of the peaks assigned to the main phenolics released by Py-GC/MS of poplar CWs from BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds, as compared to their respective controls Line ( n Replicates) Phenol 4-Vinylphenol G Compounds a S Compounds b S/G Ratio WT control (4) 5.33 (0.46) a , b 0.21 (0.07) e 24.76 (1.31) a 69.71 (1.57) b 2.82 (0.21) b \n BdPMT1 -OE / WT line 9 (4) 4.60 (0.94) a , b 10.73 (0.35) b 21.84 (0.80) b 62.83 (1.11) c 2.88 (0.13) b \n BdPMT1 -OE / WT line 17 (4) 5.43 (0.43) a , b 15.44 (0.71) a 20.69 (0.78) b c 58.44 (0.43) d 2.83 (0.12) b \n BdPMT1 -OE / WT line 31 (4) 5.06 (0.56) a , b 5.40 (1.46) d 24.10 (0.89) a 65.44 (2.40) b c 2.72 (0.19) b \n AtF5H -OE control (4) 4.99 (0.70) a , b 0.10 (0.04) e 18.64 (0.94) cd 76.38 (1.53) a 4.11 (0.29) a \n BdPMT1 -OE /AtF5H -OE line 1 (4) 4.40 (1.17) b 1.15 (0.010) e 17.86 (1.69) d 76.59 (2.74) a 4.32 (0.52) a \n BdPMT1 -OE /AtF5H -OE line 21 (3) 6.21 (0.42) a 7.43 (0.25) c 16.40 (0.85) d 69.95 (0.56) b 4.27 (0.24) a These area values are expressed as percentage of the total area per sample (set to 100). The data represent mean (SD) values from n biological replicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01) a G compounds include: guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, 4-allylguaiacol (two isomers), vanillin, acetoguaiacone, guaiacylacetone. b S compounds include: syringol, 4-methylsyringol, 4-ethylsyringol, 4-vinylsyringol, 4-allylsyringol (two isomers), syringaldehyde, acetosyringone, syringylacetone. The pyrolysis S/G ratio calculated from the relative amount of lignin-derived S and G pyrolysis compounds was not significantly affected in the BdPMT1 -OE/WT lines ( Table 3 ). This result suggests that the proportion of G and S lignin unit is not affected by the introduction of BdPMT1 in poplar. In agreement with literature data ( Franke et al., 2000 ; Stewart et al., 2009 ), this ratio was substantially increased in the AtF5H- OE control line as well as in the BdPMT1 -OE /AtF5H -OE lines 1 and 21. The relative percentage of pyrolysis-derived phenol did not discriminate the various transgenic samples from their control. This result is quite consistent with mild alkaline hydrolysis which provided similar Bz amounts from most transgenic lines and their respective controls. In contrast, the relative amount of 4-vinylphenol was dramatically increased in the BdPMT1 -OE lines as compared to their controls. Such a relative increase concomitantly decreased the relative percentage of the lignin-derived pyrolysis G and/or S compounds ( Table 3 ). Even though the pyrolysis-derived 4-vinylphenol might originate from tyrosine residues of putatively present protein contaminants, it is essentially produced from the decarboxylation of CW-linked p CA units ( Ralph and Hatfield, 1991 ). The relative abundance of 4-vinylphenol was found to nicely echo the level of alkali-releasable p CA, as revealed by the positive correlation between p CA amount and the 4-vinylphenol % ( R 2 = 0.982; Supplemental Figure S4 ). In other words, the relative amount of pyrolysis-derived 4-vinylphenol may be viewed as a good signature of the CW p -coumaroylation level. To further confirm that 4-vinylphenol prominently originates from p CA decarboxylation, a few pyrolysis assays were carried out in the presence of tetramethylammonium hydroxyde (TMAH). The TMAH-Py-GC/MS method yields methyl 4-methoxybenzoate (Bz Me ) and methyl 4-methoxy- p -coumarate ( p CA Me ) from Bz and p CA units, respectively (Kuroda et al., 2001 , 2002 ). As shown in the pyrograms outlined in Figure 2 , the relative intensity of the Bz Me peak was similar in the BdPMT1 -OE and in their corresponding controls whereas the p CA Me peak was prominent in the BdPMT -OE poplar lines. Figure 2 Traces of poplar CWs after Py-GC/MS in the presence of TMAH. (A) WT control, (B) BdPMT1 -OE / WT line 9, (C) AtF5H -OE control, and (D) BdPMT1 -OE /AtF5H -OE line 21. Bz Me , 4-methoxybenzoate; p CA Me , methyl 4-methoxy- p -coumarate; peaks quoted G and S correspond to methylated G and S compounds, respectively. The expression of BdPMT1 transformation has no or little effect on the lignin content of poplar stems, but a strong impact on lignin structure The most p -coumaroylated transgenic poplar lines were analyzed for their lignin content, using both the Klason lignin (KL) and the acetyl bromide lignin (ABL) methods. As shown in Table 4 , the BdPMT1 transformation had no impact on the lignin content of the poplar stem CW. This result contrasts with those obtained for proAtC4H::BdPMT1 Arabidopsis transformants provided with similar p- coumaroylation levels as these poplar transgenics, but with 10%–30% lower lignin contents than their controls ( Sibout et al., 2016 ). Introducing the proAtC4H::BdPMT1 into Arabidopsis plants seemed to affect the metabolic flux to lignins and thereby the stem lignin content whereas such an effect was not observed in the BdPMT1 -OE poplar lines. Table 4 Lignin content of extract-free poplar stems from BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds, as compared to their respective controls Line KL (%) ABL (%) WT control 21.82 (0.21) a 19.18 (0.33) ab \n BdPMT1 -OE / WT line 9 21.22 (0.09) a 18.77 (0.50) b \n BdPMT1 -OE / WT line 17 21.60 (0.21) a 19.47 (0.44) ab \n BdPMT1 -OE / WT line 31 21.09 (0.43) a 19.27 (0.28) ab \n AtF5H -OE control 20.86 (0.23) a 19.95 (0.23) a \n BdPMT1 -OE /AtF5H -OE line 21 20.87 (0.69) a 19.86 (0.13) a The data represent mean ( sd ) values from biological triplicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01). The lignin content is expressed as weight percentage of the sample and was determined using the KL and the ABL methods A major structural trait of native lignins is their percentage of free phenolic groups, which has a strong impact on lignin susceptibility towards industrial alkaline or oxidative treatments. When thioacidolysis is performed on CW exhaustively permethylated with diazomethane or trimethylsilyldiazomethane, the percentages of free phenolic groups in β-O-4 linked G or S lignin units, referred to as %GOH or %SOH, can be evaluated. These percentages have been shown to nicely parallel that of the whole polymer ( Lapierre, 2010 ). With the objective to evaluate the impact of the BdPMT1 transformation on the structure of poplar native lignins, we employed this analytical approach, the principle of which is outlined in Figure 3 . Past studies have shown that the thioacidolysis yield is not affected by the mild permethylation procedure ( Lapierre et al., 1988 ; Lapierre, 2010 ). Whatever the sample, the p -hydroxyphenyl (H) thioacidolysis monomers were found to be obtained as trace components (<1% of the monomer yield) and, in consequence, these minor H units were not considered in the following. In agreement with the Py-GC/MS data, the thioacidolysis S/G ratio was not affected by the BdPMT1 transformation in the WT background ( Table 5 ). Consistently with the pyrolysis data ( Table 3 ) and as compared to the WT, the thioacidolysis S/G ratio was found to be drastically increased in the AtF5H -OE samples ( Table 5 ). Figure 3 Principle of the evaluation of free phenolic units in lignin by thioacidolysis of permethylated samples. Lignin units only involved in β-O-4 bonds give rise to thioacidolysis guaiacyl (R 2 = H) and syringyl (R 2 = OMe) monomers. Terminal G and S units with free phenolic group (R 1 = H) are first methylated at C4, then degraded to monomers 1 and 3 (erythro/threo mixture), respectively. Internal G and S units (R 1 = C β of another lignin sidechain) are degraded to monomers 2 and 4 , respectively (erythro/threo mixture), EtS = SEt = thio-ethyl. Table 5 Thioacidolysis of TMSD-methylated poplar CWs from BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds, as compared to their respective controls Line S/G Molar Ratio % Free Phenolic Units in β-O-4 Linked G or S Units ( 3 + 4 )/( 1 + 2 ) %GOH %SOH 100 × 1 /( 1 + 2 ) 100 × 3 /( 3 + 4 ) WT control 2.05 (0.03) b 19.45 (0.22) d 2.81 (0.07) e \n BdPMT1 -OE / WT line 9 2.09 (0.18) b 22.85 (0.12) b 3.65 (0.19) bc \n BdPMT1 -OE / WT line 17 2.12 (0.19) b 23.65 (0.48) a 4.44 (0.25) a \n BdPMT1 -OE / WT line 31 2.08 (0.06) b 21.09 (0.26) c 3.44 (0.10) cd \n AtF5H -OE control 3.12 (0.13) a 20.85 (0.44) c 3.26 (0.03) d \n BdPMT1 -OE /AtF5H -OE line 21 2.96 (0.15) a 23.10 (0.49) b 4.02 (0.21) bc The S/G molar ratio corresponds to the ratio of the S monomers ( 3 + 4 ) to the G monomers ( 1 + 2 ; monomers shown in Figure 3 ). The molar % of free phenolic groups in β-O-4 linked G or S units, referred to as %GOH or %SOH, is calculated according to the outlined formula. The data represent mean ( sd ) values from biological triplicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01). At this stage of the study and from the simultaneous examination of both thioacidolysis S/G ratio ( Table 5 ) and p CA level ( Table 1 ), our anticipated hypothesis that a high frequency of sinapyl alcohol in AtF5H -OE poplar lines might increase the BdPMT1 -induced acylation of poplar lignins is most likely to be ruled out. This conclusion is consistent with literature data reporting on the impact of F5H overexpression in plant species provided with acylated S lignin units. For instance, upregulating F5H in poplar increased the S frequency up to 97.5%, whereas the incorporation of p -hydroxybenzoic acid in lignins was two-fold lower than the control level ( Stewart et al., 2009 ). Upregulating F5H in rice also increased the frequency of S units up to 89%, whereas p CA levels were similar in the transgenic and control plants ( Takeda et al., 2017 ). In agreement with literature data ( Lapierre, 1993 , 2010 ), the control poplar samples displayed a %GOH and a %SOH close to 20% and 3%, respectively, which confirms that S units essentially are internal units. Even though the impact of F5H upregulation on poplar lignins is out of the main scope of this study, the data of Table 5 revealed that, in addition to the expected higher S/G ratio, lignins from AtF5H -OE control plants have more terminal units with free phenolic groups than WT lignins ( Table 5 ). This result is consistent with a published paper about lignin structure in F5H -upregulated poplars ( Stewart et al., 2009 ). In this study and relative to the WT samples, lignin fractions isolated from F5H -upregulated poplars were shown to concomitantly have a twice higher frequency of phenolic OH and a lower degree of polymerization ( Stewart et al., 2009 ). More strikingly and whatever the genetic background, both %GOH and %SOH were significantly increased in the p -coumaroylated lignins of the BdPMT1 -OE poplar lines ( Table 5 ). The increase in %GOH or in %SOH was found to be nicely correlated to the p CA level of the BdPMT1 -OE/WT lines (R 2 = 0.95 for %GOH and 0.93 for %SOH; Figure 4 ). This result means that the incorporation of p -coumaroylated monolignols in poplar lignins increases the frequency of free phenolic terminal units relative to internal units. Such a structural change may be accounted for by the occurrence of lignin polymers with lower polymerization degree and/or with a higher content of biphenyl or biphenyl ether branching structures. Figure 4 Relationships between the p CA amounts in poplar CWs and the percentage of G lignin units with free phenolic groups (%GOH, black circles, full line) or the percentage of S lignin units with free phenolic groups (%SOH, white circles, dotted lines). The lignin structural traits %GOH and %SOH are evaluated by thioacidolysis of permethylated samples for BdPMT1 -OE poplars and their WT controls The alkaline hydrolysis of the DL fractions isolated from the BdPMT1 -OE poplar lines revealed that their p CA units were primarily ester-linked to lignins. With the objective to more precisely localize these p CA esters on lignin units, we subjected the BdPMT1- OE/WT line 17 and the WT samples to 1-h long thioacidolysis experiments, followed by Raney nickel desulfuration in order to identify the syringylpropanol and/or guaiacylpropanol units acylated by p -dihydrocoumaric acid (diHCA). This short thioacidolysis time is necessary as p CA esters do not survive the standard 4-h long thioacidolysis method ( Lapierre, 1993 ; Sibout et al., 2016 ). When applied to the BdPMT1 -OE / WT line 17, the method provided substantial amount of syringylpropanol acylated by diHCA while this dimer could not be observed with a longer thioacidolysis duration ( Supplemental Figure S5 and Supplemental Table S2 ). Interestingly enough and in contrast to the results reported by Smith et al. (2015) , its G analogue could not be detected. Taken together and similarly to grass lignins, these results support the hypothesis that the p -coumaroylation of lignins in the BdPMT1 -OE/WT line 17 primarily involves S lignin units. The analysis of the lignin-derived dimers obtained with the standard thioacidolysis method followed by Raney nickel desulfuration was comparatively performed for the WT line and for the BdPMT1 -OE/WT line 17. With the caveat that the results were obtained from four technical replicates of WT and line 17 samples, this analysis supported the following conclusions. When expressed as relative percentage of the total area of the main dimers (set to 100; Supplemental Table S2 ), the relative amount of dimers with biphenyl or biphenyl ether bonds was not increased by the presence of BdPMT1 and suggests that BdPMT1 -OE/WT line 17 does not contain lignins more branched than the WT ones. In contrast, the relative percentage of the syringaresinol-derived dimers displayed a 1.4-fold increase in the case of the BdPMT1 -OE/WT line 17 sample relative to the WT level ( Supplemental Table S2 ). The syringaresinol structures exclusively originate from the dimerization of sinapyl alcohol and are thus starting points for lignin growth ( Ralph et al., 2004 ). Their higher relative recovery from the BdPMT1 -OE/WT line 17 further argues for the occurrence of lignin polymers with lower polymerization degrees than in the WT sample. The BdPMT1 -driven substantial p -coumaroylation of poplar samples makes their lignins more easily solubilized in cold alkali The enrichment in free phenolic G and S units is very likely to improve the lignin susceptibility to alkaline treatments that are employed in chemical pulping or in the cellulose-to-ethanol conversion process. The beneficial impact of lignin terminal units with free phenolic groups on the CW delignification induced by alkaline treatment has been established for a long time for grass samples ( Lapierre et al., 1989 ; Lapierre, 2010 ) and confirmed for poplar trees deficient in cinnamyl alcohol dehydrogenase (CAD) activity (Lapierre et al., 1999 , 2004 ; Van Acker et al., 2017 ), for tobacco ( Nicotiana tabacum ) plants deficient in cinnamoyl-coenzyme A reductase (CCR) activity ( O'Connell et al., 2002 ) and for BdPMT1 -transformed Arabidopsis lines ( Sibout et al., 2016 ). The results of a mild alkaline treatment applied to the poplar samples are shown in Table 6 . The residue recovered after this treatment, referred to as the saponified residue (SR), was obtained with similar yields whatever the line. The percentage of the alkali-soluble lignin (%Alk-L) was calculated from the SR recovery yield and the lignin amount of the CW and SR samples. From the data of Table 6 , we can see that the lignins from the AtF5H -OE control samples are more alkali-soluble than the lignins from the WT samples. This result is consistent with the higher frequency of terminal units with free phenolic groups in the AtF5H -OE control samples as compared to the WT samples ( Table 5 ). Whatever the genetic background, the percentage of the alkali-soluble lignin (%Alk-L) revealed that the BdPMT1 -OE lines are more easily delignified by the employed mild alkaline treatment compared with their control lines. Whereas 15%–20% of the lignin polymers were solubilized by cold alkali for the controls, the %Alk-L was substantially increased in the BdPMT1 -OE lines (up to 26%–28% in lines 9, 17, and 21, Table 6 ). As reported for transgenic CAD- or CCR-deficient plants ( Lapierre et al., 1999 ; O'Connell et al., 2002 ), increasing the percentage of lignin units with free phenolic groups has beneficial effects on the kraft pulping properties of the lignocellulosic biomass, thereby decreasing the energy and environmental costs of this industrial process. The introduction of BdPMT1 in trees would likely improve the pulping properties of poplar wood. Table 6 Impact of a mild alkaline treatment (aq. NaOH 1 M, overnight, room temperature) on extract-free poplar stems from control and BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds Line %SR %ABL in SR %Alk-L WT control 68.03 (0.88) a 23.69 (0.04) a 15.5 (2.1) d \n BdPMT1 -OE / WT line 9 67.17 (0.46) a 20.85 (0.47) b 25.5 (1.1) a,b \n BdPMT1 -OE / WT line 17 65.25 (0.73) b 21.52 (0.24) a,b 28.1 (1.7) a \n BdPMT1 -OE / WT line 31 68.17 (0.22) a 22.11 (0.75) a,b 21.7 (1.6) b,c \n AtF5H -OE control 69.02 (0.65) a 22.96 (0.28) a,b 20.5 (1.3) c \n BdPMT1 -OE /AtF5H -OE line 21 67.65 (1.28) a 21.52 (0.13) a,b 26.4 (1.7) a The percentage of the recovered saponified residue (%SR) is expressed relative to the initial sample. The lignin content of the SR sample is measured as acetyl bromide lignin (%ABL). The percentage of alkali-soluble lignins (%Alk-L) is calculated from the ABL content of the CW and from the %SR recovery yield. The data represent mean ( sd ) values from biological triplicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01). The relationship of the free phenolic groups in poplar lignins to their susceptibility towards cold alkaline treatment is further illustrated in Figure 5 . On this scheme, we have gathered the data from 17 different poplar lines, comprising the current BdPMT1 -OE / WT lines and CAD-deficient ones ( Lapierre et al., 2004 ), together with their respective controls. The effect of the %GOH structural property onto the solubility of poplar lignins in cold alkali is supported by the positive correlation between %GOH and %Alk-L (R 2 = 0.9513; Figure 5 ). Figure 5 Relationship between the percentage of G lignin units with free phenolic groups (%GOH) and the solubility of poplar lignins in cold alkali (%Alk-L). The data correspond to BdPMT1 -OE trees and their WT controls (black circles) as well as to CAD-deficient trees and their corresponding controls (white circles). The BdPMT1-driven p -coumaroylation of poplar samples results in improved saccharification after cold alkaline pretreatment It is well established that the detrimental role of lignins on the cost-effective enzymatic conversion of lignocellulosic polysaccharides into fermentable sugars makes necessary the use of pretreatments ( Yang and Wyman, 2008 ; Wang et al., 2015 ; Sun et al., 2016 ). Among these pretreatments, alkaline technologies with sodium hydroxide or with lime have emerged as major procedures for the conversion of lignocellulosic biomass ( Kim et al., 2016 ). Most alkaline pretreatments are carried out under mild conditions (temperature below 60°C) and with moderate alkaline charge (0.5%–10% NaOH w/v) and long reaction time (several hours to several days; Carvalho et al., 2016 ; Kim et al., 2016 ; Moreno and Olsson, 2017 ; Rezania et al., 2020 ). These simple processes improve saccharification by partially removing lignins and hemicelluloses and by cellulose swelling ( Carvalho et al., 2016 ). Sodium hydroxide pretreatments are more effective with grass feedstocks than on woody ones, which is related to the higher solubility of grass lignins in alkali ( Beckmann et al., 1923 ; Scalbert et al., 1986 ; Lapierre et al., 1989 ). From these various literature data about NaOH pretreatments and from the analytical results that we obtained so far on the BdPMT1 -OE poplar lines, we could anticipate that an alkaline pretreatment would be well suited to reduce the lignin-related recalcitrance of poplar wood to saccharification. Accordingly, the saccharification experiments run on the poplar samples were preceded by a cold alkaline pretreatment (aq. NaOH 1M, overnight, room temperature). Even though the optimization of this pretreatment was out of the scope of this study, we selected reaction duration and temperature as well as NaOH charge that were similar to literature data about alkaline pretreatment technologies ( Carvalho et al., 2016 ; Kim et al., 2016 ). The saccharification efficiency was evaluated both by the weight loss (%WL) and by the amount of released glucose (Glc; Table 7 ). In agreement with their higher level of alkali-soluble lignins, the alkali-pretreated AtF5H -OE control samples displayed a higher saccharification efficiency than the alkali-pretreated WT samples ( Table 7 ). More importantly, the saccharification efficiency was higher in the alkali-pretreated BdPMT1 -OE lines, compared with their corresponding control ( Table 7 ). In contrast, when the assays were carried out without any alkali pretreatment, low saccharification yields were observed and the transgenic samples were not significantly different from their controls ( Supplemental Table S3 ). Not unexpectedly, in the WT background, the best saccharification results from alkali-pretreated samples were obtained for the lines provided with the concomitant highest p CA level, %GOH and %Alk-L. The enrichment of poplar lignins in free and readily ionizable phenolic groups favored lignin solubilization in alkali, which consequently improved the saccharification of alkali-pretreated samples. Taken together, these results reveal that the lignins from the current BdPMT1 -OE poplar plants share common features with grass lignins. As compared to nongrass lignins from WT plants, these common features are (1) a substantial p -coumaroylation of S lignin units, (2) a higher level of free phenolic units, and (3) a higher solubility in cold alkali. At this point, we may hypothesize that, similar to grass lignins, lignins from the BdPMT1 -OE poplar lines obtained herein are distributed in the CWs as small lignin domains which are both rich in free phenolic groups and more easily extracted by cold alkali treatment ( Lapierre, 2010 ). Table 7 Saccharification of the poplar SR obtained after a mild alkaline treatment (aq. NaOH 1 M, overnight, room temperature) and corresponding to BdPMT1 -OE lines obtained in the WT and AtF5H -OE backgrounds, as compared to their respective controls SR from Line %WL Glc Glc mg·g −-1 SR mg·g −-1 CW WT control 39.8 (1.3) d 307.7 (16.0) e 210.1 (11.3) e \n BdPMT1 -OE / WT line 9 52.1 (1.6) a,b 417.5 (23.2) b,c 280.1 (17.4) b,c \n BdPMT1 -OE / WT line 17 55.1 (2.0) a 452.4 (17.7) a,b 294.2 (8.3) a,b \n BdPMT1 -OE / WT line 31 45.8 (0.3) c,d 369.3 (17.9) d 251.8 (11.9) d \n AtF5H -OE control 44.2 (2.2) c,d 401.4 (6.7) c,d 277.1 (6.6) c,d \n BdPMT1 -OE /AtF5H -OE line 21 49.4 (1.5) b,c 461.7 (22.4) a 312.4 (16.5) a The saccharification efficiency is evaluated both by the weight loss (%WL) and by the released glucose (Glc). Glc yields are expressed either relative to the SR samples or to the initial CW samples. The data represent mean ( sd ) values from biological triplicates. Different letters in columns indicate significant differences (Duncan test, P < 0.01)."
} | 10,591 |
30177923 | PMC6109780 | pmc | 1,725 | {
"abstract": "Quorum sensing (QS) is a central mechanism for regulating bacterial social networks in biofilm via the production of diffusible signal molecules (autoinducers). In this work, we assess the contribution of QS autoinducers to microbial extracellular electron transfer (EET) by Pseudomonas aeruginosa strain PAO1 and three mutants pure culture-inoculated in microbial electrolysis cells (MECs) and microbial fuel cells (MFCs). MECs inoculated with different P. aeruginosa strains showed a difference in current generation. All MFCs reached a reproducible cycle of current generation, and PQS-deficient pqsA mutant inoculated-MFCs obtained a much higher current generation than pqsL mutant inoculated-MFCs which overproduced PQS. lasIrhlI -inoculated MFCs produced a lower power output than others, as the strain was deficient in rhl and las . Exogenous N-butanoyl-l-homoserine lactone could remedy the electricity production by lasIrhlI mutants to a level similar to wild-type strains while signaling molecules had little effect on wild-type bacteria in MFCs. Meanwhile, experiments with the wild-type and pqsA , pqsL mutants indicated that the overexpression of PQS signaling molecules made no significant contribution to EET. QS signaling molecules therefore have dual-edged effects on microbial EET. These findings will provide favorable suggestions on the regulation of EET, but detailed QS regulatory mechanisms for extracellular electron transfer in pure- and mixed-cultures are yet to be elucidated.",
"conclusion": "Conclusion In summary, we investigated the effect of signal molecules on MFC performance and found that C4-HSL could increase the current generation of gene deficient variants to that of unmodified strains. PQS quorum sensing made no significant contribution to current output, and anodic biomass did not significantly alter electricity production when compared with QS. Thus, strategies to up-regulate the rhl QS system while limiting the PQS production are adoptable. However, the exogenous addition of signal molecules had little effect on the normal strain, which indicated that more efficient manipulation and utilization of QS needs to be further explored in mixed cultures MFCs. These findings will help provide suggestions for the enhancement of EET in MESs.",
"introduction": "Introduction Microbial electrochemical systems (MESs) are a versatile group of technologies with the potential to achieve sustainable bioenergy generation, biosensing and bioelectrosynthesis using organic or inorganic carbon sources ( Liu et al., 2016 ). Electroactive bacteria (EAB) function as biocatalysts and are able to exchange electrons between cells and electrodes via multiple processes of extracellular electron transfer ( Bond and Lovley, 2003 ; Rabaey et al., 2004 ; Reguera et al., 2005 ). The optimal reactor configurations, operating conditions and electrode materials for increased electron transfer in MES have been described previously ( Rinaldi et al., 2008 ; Karthikeyan et al., 2015 ; Logan et al., 2015 ; Mei et al., 2015 ; Xing et al., 2015 ; Zou et al., 2016 ; Bi et al., 2018 ). However, further improvement of electron transfer in MES is difficult due to inadequate understanding of electrode-biofilm formation in EAB. Low efficiency electron transfer at the anodic biofilm-electrode interface remains one of the major limitations for full-scale implementation of microbial fuel cells (MFCs) ( Logan, 2009 ). Manipulating exoelectrogenic biofilms to improve the efficiency of the electron transfer pathway is therefore a feasible strategy to improve MES performance. Quorum sensing (QS) is a cell-cell communication mechanism in which extracellular signal molecules called autoinducers are released by bacteria. The autoinducers pass on information about population density, and the bacteria population then collectively regulates the expression of related genes as a response ( Swift et al., 1996 ). QS has been found to regulate many bacterial physiological activities, including biofilm formation, in several bacterial species ( Davies et al., 1998 ; Zhu and Mekalanos, 2003 ; Boles and Horswill, 2008 ). In particular, relatively complex QS systems are found in Pseudomonas aeruginosa ( Juhas et al., 2005 ), a species commonly found in the biofilms of MESs. The las system of QS in P. aeruginosa is comprised of the transcriptional activator LasR and the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) signal molecule synthase LasI ( Gambello and Iglewski, 1991 ; Passador and Iglewski, 1993 ). Similarly, in the rhl system, the enzyme RhlI catalyzes the synthesis of the N-butyryl-L-homoserine lactone (C4-HSL) signal molecule, which is detected by the transcriptional activator protein RhlR ( Ochsner et al., 1994 ; Parsek et al., 1997 ). In addition to the two N-Acyl homoserine lactone (AHL) type signal molecules, a third autoinducer, 2-heptyl-3-hydroxy-4-quinolone (designated as Pseudomonas quinolone signal, PQS), provides a link between the las and rhl quorum-sensing systems ( Pesci et al., 1999 ; Mcknight et al., 2000 ). These QS systems constitute a hierarchical regulation network in P. aeruginosa ( Williams and Camara, 2009 ; Yong et al., 2015 ). Recent studies have shown that QS signaling molecules play crucial roles in electricity generation by MFCs ( Yong et al., 2011 , 2015 ; Chen et al., 2017 ). For example, genetic enhancement of the QS circuit was applied in MFCs to enhance electricity production ( Yong et al., 2011 ). The overexpression of rhlI and rhlR genes in wild-type strain lead to a significant increase in phenazine production, which directly resulted in an increase of current output in the rhlI overexpressed strain inoculated-MFCs. A PQS defective strain also produced higher concentrations of phenazines and exhibited increased current production when used in MFCs compared to the parent strain ( Wang et al., 2013 ). Electrochemical activity of the bio-anode was promoted by the addition of 3-oxo-hexanoyl-homoserine lactone and 3-oxo-dodecanoyl-homoserine lactone in microbial electrolysis cells (MECs), and a higher current was produced with the addition of short chain acyl-homoserine lactone ( Liu et al., 2015 ). In particular, these studies substantiated the observation that QS regulatory networks are involved in microbial extracellular electron transfer, and most recent studies focused on genetically engineering QS to improve the electricity output in MFCs. However, it is still unclear which signal molecules could be applied to improve MFC performances, and whether or not they could remedy the electricity production of mutants. In this study, P. aeruginosa strain PAO1 and its three mutants were pure culture-inoculated in MFCs and MECs to investigate the effect of QS on extracellular electron transfer. We subsequently assess the ability of QS to enhance attachment of anodic bacteria and current generation of MFCs by the addition of exogenous QS signals such as PQS and AHLs.",
"discussion": "Results and Discussion Electrochemical Performance of Pure-Culture MECs The small-scale MECs could provide high throughput operation to maintain pure culture conditions. MECs inoculated with different P. aeruginosa strains showed a difference in current generation ( Figure 2A ). Two cycles of current generation indicated that the current output of the pqsA strain with deficient production of PQS was higher than that of pqsL with overproduced PQS. The overexpression of PQS signaling molecules unexpectedly limited extracellular electron transfer. In comparison to the wild-type, the AHL-deficient lasIrhlI mutant showed lower EET (extracellular electron transfer) rate based on the average of duplicate reactors. FIGURE 2 Current generation of MECs (A) and MFCs (B) with the strains of PAO1 and three mutants. Electrochemical Performance of Pure-Culture MFCs Microbial fuel cells inoculated with four different strains of P. aeruginosa were operated simultaneously to further investigate the difference in electricity generation. MFC tests showed a similar and more obvious result compared to MECs. All MFCs attained reproducible cycles of current generation ( Figure 2B ). The maximum current density generated by wild-type P. aeruginosa PAO1 in MFC was 0.105 μA/cm 2 . It is at a similar level as a previous study with the same PAO1 strain, which generated approximately 0.150 μA/cm 2 in a MFC ( Wang et al., 2013 ). The coulombic efficiency was relative low, as it was not higher than 5%. Although the current generation and electron recovery from substrate was low, we could still analyze the effect of QS on the EET clearly. MFCs inoculated with pqsA mutants obtained the highest current density. The lasIrhlI -inoculated MFC produced lower current density than the other strains, due to its deficiency in rhlI and lasI . The AHLs QS systems induced cell lysis and extracellular DNA release which were responsible for biofilm structure formation in P. aeruginosa strains ( Allesen-Holm et al., 2006 ). Additionally, the production of pyocyanin, which functions as an electron shuttle to enhance electron transport ( Rabaey et al., 2005 ), is highly regulated by las and rhl ( Brint and Ohman, 1995 ; Latifi et al., 1995 ; Pesci et al., 1997 ). The rhl QS system directly affect the EET as the production of pyocyanin was overproduced by the overexpression of the rhl genes ( Yong et al., 2011 ). While the las QS system seemed to make indirect effect on the synthesis of pyocyanin, as it controls the rhl QS in two ways ( Pesci et al., 1997 ). Thus, the susceptible biofilm ( Allesen-Holm et al., 2006 ; Sakuragi and Kolter, 2007 ) and reduced production of pyocyanin may lead to the observed decrease in current generation of lasIrhlI -inoculated MFCs. The MFCs inoculated with PQS-deficient pqsA mutants obtained a much higher level of current generation than those inoculated with pqsL mutant, which overproduced PQS. PQS was demonstrated to inhibit bacteria growth ( Toyofuku et al., 2008 , 2010 ). Thus, large amount of PQS overproduced by the pqsL mutant might repress the production of electron shuttles, which limited the EET from EAB to the anode. However, to understand the regulation mechanism needs the more evidences of biofilm formation and transcriptome in the future. Conversely, PQS-deficient pqsA mutants could produce normal concentration level of electron shuttles under the directly regulation of rhl QS system and avoid negative effect of PQS, which promoted the EET with anode. Therefore, comparable experiments with the wild-type and mutant strains indicated that the overexpression of signaling molecules may limit the current generation in MFCs. The difference in performance between the MEC and MFC was mainly due to differences in operating conditions. The configuration is an important factor that could affect the performance of MFCs ( Logan et al., 2015 ) and MECs ( Kadier et al., 2016 ). Previous studies suggest that MFCs fed with acetate as substrate produce higher power densities in small scale reactors in comparison to large-scale ( Logan et al., 2015 ). In this study, the MFC volume (300 mL) was 60 folds larger than the MEC volume (5 mL). The MEC had a higher specific anode surface area (100 m 2 /m 3 ) than the MFC (24.2 m 2 /m 3 ). In addition, a higher ratio of inoculation was used in the MEC (10%) in comparison to the MFC (1.5%). Although the MEC could have higher current density and lower reagents costs because of its small size, the limited growth of P. aeruginosa under anaerobic conditions ( Toyofuku et al., 2008 ) and insufficient biofilm formation resulted in the MEC being unable to be operated as long as the MFC. The oxygen level was also an important factor for the PQS system ( Schertzer et al., 2010 ). Since oxygen was limited, the differences between different strains was not as visible in the MEC. Effects of Biomass on Current Generation Low production of currents in MFCs may be due to less enriched bacteria on the anode. To evaluate the hypothesis that anodic biomass may affect the current output, a measurement of protein concentration was made for biomass estimation during the third batch of MFC operation. Biomass of anode and solution in MFC were analyzed, respectively. Despite the highest anodic biomass, the lasIrhlI -inoculated MFC still had lower performance in electricity production ( Figure 3A ), while, the pqsL -inoculated MFC had a higher biomass in solution than other groups. This is largely due to the elevated release of extracellular DNA, as the QS-regulated DNA release can cause cell lysis ( Webb et al., 2003 ). These results indicate that anodic biomass did not significantly affect the electricity production compared to QS. FIGURE 3 The maximum and minimum measured biomass of anode and solution in MFCs (A) . Bacterial growth curves without signal molecules (B) and with PQS (C) and AHLs (D) . Error bars represent the standard deviation of triplicate (B – D) tests. Effects of QS Signals on Bacterial Growth To examine the effect of different signal molecules on the growth of P. aeruginosa , the bacteria were grown in 96-well plates and signal molecules were added separately until a final concentration of 10 μM was reached. First, we examined the growth curve of different strains. There were no significant differences between them, except for pqsA which had a higher stationary phase OD than other strains ( Figure 3B ). When PQS was added to the cultures of PAO1, pqsA , and pqsL , a longer lag phase was observed. In addition, the pqsA +PQS had a shorter stationary-phase OD than pqsA ( Figure 3C ). These results indicate that PQS suppressed cell density and affected bacterial growth, as has been observed previously ( Häussler and Becker, 2008 ; Toyofuku et al., 2008 ). Furthermore, the exogenous addition of 3-oxo-C12-HSL and C4-HSL increased the stationary phase OD of the lasIrhlI mutant strain ( Figure 3D ). The accumulation of extracellular DNA in the late-log phase was restored in the lasIrhlI mutant culture supplemented with signal molecules in a previous study ( Allesen-Holm et al., 2006 ), which indicated that AHL QS regulated bacterial growth. The addition of signal molecules did not significantly influence the growth of the wild type planktonic cultures. Effects of QS Signaling Molecules on the Performance of Pure-Culture MFCs Quorum sensing played important roles in mediating the bioelectrochemical characteristics in MFCs. The pyocyanin-mediated EET from EAB to the anode is crucial for electricity generation in Pseudomonas -based MFC and the biosynthesis is regulated by QS systems ( las , rhl , and PQS) ( Rabaey et al., 2005 ; Yong et al., 2011 ; Wang et al., 2013 ). The electron shuttle biosynthesis is directly regulated by the rhl QS system as the overexpression of rhl QS system could lead a significant improvement in electricity generation ( Yong et al., 2011 ). While the las QS system seemed to make indirect effect on the synthesis of pyocyanin, as it controls the rhl QS system and the mutations of las QS system decreased the pyocyanin production ( Pesci et al., 1997 ). Besides, the PQS signaling system also regulates electron shuttle biosynthesis but this regulatory network may be inactivated due to the oxygen level ( Schertzer et al., 2010 ). In order to further investigate the effect of QS on extracellular electron transfer regulation, the effect of the AHLs signal molecules on EET of wild-type and lasIrhlI mutant strains was studied in single-chamber air-cathode MFCs. 3-oxo-C12-HSL and C4-HSL were added until final concentration levels of 10 μM were reached. Results showed that adding 3-oxo-C12-HSL signal molecule caused no significant increase in EET of either wild-type or mutant strains, indicating that the las QS system may not directly affect EET in MFCs ( Figure 4A ). Although it showed that the las QS system could make indirect effect on the synthesis of pyocyanin by controlling the rhl QS system in P. aeruginosa , the rhl QS system could not be activated in the lasIrhlI mutant due to the deficiency of rhlI gene. However, a very significant increase was caused in lasIrhlI inoculated MFCs by the addition of C4-HSL signal molecule. This could increase the electricity production of mutant strains to a level similar to wild-type strain. In a previous related study, the engineered strains could attain much higher current than the wild-type parent strains by overexpressing the rhl QS system. This genetic manipulation resulted in an enhanced transcription level of the rhlI and rhlR genes, which increased the amount of signal molecules and transcriptional activators simultaneously and strengthened the current production when compared with wild-type ( Yong et al., 2011 ). Thus, separately compensating for C4-HSL signal molecule in lasIrhlI mutant-inoculated MFCs restored the current generation to normal levels. Meanwhile, both signal molecules had little effect on wild-type strain-inoculated MFCs. FIGURE 4 Current generation of MFCs after the addition of AHLs (A) and PQS (B) . Arrows indicate the time point for adding QS signal molecules. Microbial fuel cells inoculated with PAO1, pqsA and pqsL were supplemented with 10 μM of PQS. The maximum current decreased after adding PQS to pqsA -inoculated MFCs ( Figure 4B ). However, there was no significant effect on wild-type and pqsL mutants. In P. aeruginosa , PQS decreased growth rates under aerobic conditions and repressed anaerobic growth ( Häussler and Becker, 2008 ; Toyofuku et al., 2008 ). Previous studies showed that P. aeruginosa didn’t produce detectable PQS anaerobically ( Toyofuku et al., 2008 ; Schertzer et al., 2010 ). However, the special configuration of the air-cathodes formed a relatively micro-aerobic environment in the MFCs. Thus, the wild-type and pqsL mutants could both produce a considerable amount of PQS, which had negative effects on MFC performance. The addition of exogenous PQS to higher performance MFCs ( pqsA -inoculated) decreased the current generation. This result is similar to a previous study ( Wang et al., 2013 ), in which a PQS defective mutant inoculated MFC attained the lowest current generation. We hypothesized that this difference was likely caused by differences in reactor configuration. A higher oxygen level was available in the air-cathode MFCs than in dual chamber U-tube MFCs, which promoted bacterial growth ( Toyofuku et al., 2008 ) and pyocyanin biosynthesis ( Schertzer et al., 2010 )."
} | 4,662 |
36160950 | PMC9490189 | pmc | 1,726 | {
"abstract": "Although desert plants often establish multiple simultaneous symbiotic associations with various endophytic fungi in their roots, most studies focus on single fungus inoculation. Therefore, combined inoculation of multiple fungi should be applied to simulate natural habitats with the presence of a local microbiome. Here, a pot experiment was conducted to test the synergistic effects between three extremely arid habitat-adapted root endophytes ( Alternaria chlamydospora, Sarocladium kiliense , and Monosporascus sp.). For that, we compared the effects of single fungus vs . combined fungi inoculation, on plant morphology and rhizospheric soil microhabitat of desert plant Astragalus adsurgens grown under drought and non-sterile soil conditions. The results indicated that fungal inoculation mainly influenced root biomass of A. adsurgens , but did not affect the shoot biomass. Both single fungus and combined inoculation decreased plant height (7–17%), but increased stem branching numbers (13–34%). However, fungal inoculation influenced the root length and surface area depending on their species and combinations, with the greatest benefits occurring on S. kiliense inoculation alone and its co-inoculation with Monosporascus sp. (109% and 61%; 54% and 42%). Although A. chlamydospora and co-inoculations with S. kiliense and Monosporascus sp. also appeared to promote root growth, these inoculations resulted in obvious soil acidification. Despite no observed root growth promotion, Monosporascus sp. associated with its combined inoculations maximally facilitated soil organic carbon accumulation. However, noticeably, combined inoculation of the three species had no significant effects on root length, surface area, and biomass, but promoted rhizospheric fungal diversity and abundance most, with Sordariomycetes being the dominant fungal group. This indicates the response of plant growth to fungal inoculation may be different from that of the rhizospheric fungal community. Structural equation modeling also demonstrated that fungal inoculation significantly influenced the interactions among the growth of A. adsurgens , soil factors, and rhizospheric fungal groups. Our findings suggest that, based on species-specific and combinatorial effects, endophytic fungi enhanced the plant root growth, altered soil nutrients, and facilitated rhizospheric fungal community, possibly contributing to desert plant performance and ecological adaptability. These results will provide the basis for evaluating the potential application of fungal inoculants for developing sustainable management for desert ecosystems.",
"conclusion": "Conclusion This study explored the synergistic effects of three extremely arid habitat-adapted root endophytes, on the growth of non-host desert plant A. adsurgens , under drought and non-sterile conditions. Fungal inoculation markedly improved the root growth and biomass of A. adsurgens , and this favorable effect was dependent on specific fungal species and their combinations. However, the synergistic effect of fungal inoculation on the rhizospheric soil fungi community was inconsistent with that of plant growth. The three species combined inoculation that promoted the diversity of rhizospheric fungal community most had no effect on plant root growth when compared to the control treatments. This insinuates the differed responses between plant growth and rhizospheric fungal community to the multiple fungal interactions. Thus, the fungal species and the combined consortium used should be carefully evaluated owing to their differential effects on plant growth and soil microhabitat, as well as their complementary functional roles. Further investigation of interactions between host plants and rhizospheric soil microhabitat mediated by fungal combined inoculations are still needed to be elucidated.",
"introduction": "Introduction Global warming has intensified the appearance of the drought phenomenon, thus aggravating the adverse impact of drought stress, especially in arid desert areas (Zandalinas et al., 2021 ). As the most important threat to desert ecosystems, drought adversely influences plant growth, including its development and survival (Omer et al., 2020 ; Terletskaya et al., 2020 ; Barker et al., 2021 ). Plants respond to such drought environments both directly and indirectly; furthermore, indirect responses through intimate associations with endophytic fungi have recently received increased attention (Beckers et al., 2017 ; Trivedi et al., 2020 ). Therefore, identifying the most beneficial combinations between plant hosts and endophytes may be of particular value for improving the plant growth in desert habitats and mitigating the negative effects on soil ecosystems. The rhizosphere is known as the soil region surrounding the plant roots, as well as the reservoir of soil microorganisms (Li et al., 2021 ). Changes in microorganisms significantly influence plant productivity, survival, and stress resistance by acting on geochemical characteristics of subsurface soils, such as material cycling, decomposition rate, and pathogenicity (Bardgett and van der Putten, 2014 ; Zhang et al., 2020 ). The rhizospheric fungal community, in particular, plays a central role in improving soil nutrient recycling and availability, resulting in beneficial effects on plant growth and adaptation (Bardgett and van der Putten, 2014 ). Recent studies have shown that utilizing beneficial endophytic fungi can alter the composition of soil fungal communities and promote the abundance and beneficial interactions of soil fungi (Azcón et al., 2013 ; Nanjundappa et al., 2019 ; Li et al., 2020 ). Furthermore, endophytic fungi could influence plant–rhizosphere interactions to alleviate abiotic stress (Dimkpa et al., 2009 ). For example, increased production of fungal exopolysaccharides and microbial activity under water-deficit conditions can impact soil water retention and field performance of tomatoes (Le Gall et al., 2021 ). In this light, optimizing plant–fungal partnership to increase soil microbial abundance and functionality is also critical for the arid desert soil ecosystem. Fungal endophytes are ubiquitous in the roots of almost all plants, which co-evolved with hosts with their high genetic and functional diversity, endowing plants with diverse resistance and multiple evolutionary strategies (Barnes et al., 2018 ; Alzarhani et al., 2019 ; Leroy et al., 2021 ). Root endophytes isolated from desert habitats have been evidenced to improve plant drought tolerance and performance, enabling the establishment and survival of host plants in a stressful environment (He et al., 2021 ; Moghaddam et al., 2021 ). Rodriguez et al. ( 2008 ) suggested that the ability to confer drought tolerance to hosts may be a unique genetic resource of endophytic fungi. Under drought stress, endophytic fungi can improve the osmotic adjustment capacity of the host by increasing the production of antioxidant enzymes and active substances, thus reducing water consumption (Xu et al., 2017 ). Improved nutrition, phytohormones production, and acquired host resistance or immunity induction are also related to endophytic fungi stimulating plant growth and drought tolerance (Mandyam and Jumpponen, 2005 ; Kour et al., 2019 ; Fontana et al., 2021 ). Additionally, fungal endophytes have been found to increase soil fungal abundance and improve microbial community structure, thereby contributing to plant growth and ecological adaptability under water deficit conditions (He et al., 2019 ). Some beneficial endophytic fungi may even compensate for the low colonization of arbuscular mycorrhizal fungi (AMF) under natural conditions when plant nutrient consumption is challenged (Han et al., 2021 ). Compared with the well-studied AMF association, fungal endophytes are readily isolated and cultured in vitro , which are gradually regarded as promising reciprocal partners in plants (Gonçalves et al., 2021 ; Zhong et al., 2022 ). Most studies have focused on assessing the effects of single strains on plants under drought stress in essentially sterile conditions (González-Teuber et al., 2018 ; Li et al., 2019a ; Hereme et al., 2020 ). In natural habitats, plant roots are often colonized by multiple fungi species, which together perform complicated ecological functions, rather than a single fungus alone (Durán et al., 2018 ; Liu et al., 2020a ). Additionally, natural habitats are non-sterile environments due to the presence of local microbiota. Therefore, combined inoculation of multiple endophytic fungi simulates natural conditions better than single inoculations, and is thought to be more competitive and effective in a non-sterile natural environment (Baez-Rogelio et al., 2017 ; Li et al., 2021 ). Chen et al. ( 2017 ) evaluated the effects of combined inoculation of different AMF species on the growth, nutrient absorption, and photosynthesis of cucumber seedlings, which indicated that the AMF composition consists of distant AMF species showing a better synergistic effect than a single or closely related AMF spp. Nanjundappa et al. ( 2019 ) reported that under field conditions, the synergistic effect of AMF and Bacillus spp. on crop growth and soil nutrient uptake was much greater than that of inoculation with either AMF or Bacillus alone. He et al. ( 2022 ) suggested that the dual inoculation of dark septate endophytes and Trichoderma viride enriched beneficial microbiota, altered soil nutrient status, and might contribute to enhancing the cultivation of medicinal plants in dryland. Hence, it is essential to identify the combinations of different fungal species and determine their subsequent synergistic effects in non-sterile environments, especially with the inclusion of drought stress. Astragalus adsurgens Pall. (Leguminosae) is a native perennial herbaceous mainly distributed in the desert region of Northwest China (Liu et al., 2018 ). As typical desert herbage and fine forage, this species is a preferred plant for desert ecological restoration and desert grassland planting because it is strong drought and sandstorm resisting plant (Chen et al., 2013 ). Therefore, considering the concept of sustainability and the need to enhance the growth status and drought resistance of grassland plants, understanding the interaction between plants and beneficial microbes is crucial to receive benefit from the symbiotic mechanisms. In a previous study, we investigated the influences of several endophytic fungal strains alone from the roots of desert shrubs on the performance of Hedysarum scoparium under different soil water conditions with sterile substrates. These strains established a positive symbiosis with the host plant depending on fungal species and water availability (Li et al., 2019b ). In the current study, the effects of single and mixed inoculation of three desert endophytic fungi on A. adsurgens plants were studied in a greenhouse pot experiment with non-sterile substrates. Since A. adsurgens is a desert plant, our pot experiment was conducted under drought stress which simulated the natural conditions for the plant host and the root endophytes. We hypothesize that mixed inoculations of desert endophytic fungi could either promote plant growth or change the rhizospheric fungal community of A. adsurgens , and the synergistic effects between mixed inoculations would depend on the combination of different fungal species. Based on our conjecture, we investigated the effects of mixed inoculations on (1) plant biomass (shoot and root biomass), (2) the morphological parameters (plant height, leaf number, root length, surface area, and diameter), (3) soil physicochemical properties, and (4) the rhizospheric fungal community composition. Such data would display endophytic fungal groups that could withstand the drought conditions that impacted host growth, and their potential for improving the stress tolerance and symbiotic performance of A. adsurgens in drought-affected arid lands.",
"discussion": "Discussion Synergistic effect of combined fungal inoculation depending on fungal species In this study, we determined the effects of single or combined inoculation of three root endophytic fungi on promoting the growth of desert plant A. adsurgens under a non-sterile substrate simulating natural drought conditions. Meanwhile, the parallel experiment of well-watered treatment was carried out when the fungus was inoculated alone, and the reduced plant height of A. adsurgens seedlings under drought conditions evidenced the effectiveness of imposed drought stress in our experiment ( Supplementary Figure 5 ). Under the drought stress, we demonstrated the synergistic effects between different fungal combinations, which are consistent with that of He et al. ( 2022 ), who documented the increased dual inoculation effects of beneficial endophytic fungi between Paraboeremia putaminum and Trichoderma viride on the growth of medicinal plant Astragalus mongholicus under water stress conditions. However, not all fungal inoculations participated in plant growth promotion. Regardless of the single or combined inoculations, the effects mainly depended on different fungal species and combinations, which approved the previous reports that fungal species may be one of the factors influencing symbiotic relationships and plant performance (Wazny et al., 2018 ; Geisen et al., 2021 ). Liu et al. ( 2020b ) found that the synergistic effect of arbuscular mycorrhizal fungi and endophytic fungi on tall fescue depended on the species of arbuscular mycorrhizal fungi. Yang et al. ( 2021 ) also showed that root endophytes represented an important role in promoting the biological process of the host relying on the type of host and the species of endophytes. Therefore, the use of suitable fungal endophytes, either singly or in combination, remains a major challenge as different fungal species may play different roles in plant symbiosis. It is worth noting that in our study, the response of plant growth to fungal inoculation is different from that of the soil fungal community. The combined inoculation of ASM did not have positive effects on the growth of A. adsurgens , but most significantly promoted the diversity and abundance of soil fungal community. This suggests that combined fungal inoculation mediates the composition and ecological adaptation of the soil microbial community (Pang et al., 2020 ; He et al., 2022 ). Studies have shown that fungal inoculation may impact competition for niches in the fungal community, resulting in changes in species abundance (Wagg et al., 2015 ; Van Nuland and Peay, 2020 ). Due to the dependence of endophytes on plant habitat and nutrition, host plants may face tradeoffs in resource allocation among symbionts (Niwa et al., 2018 ). Under drought conditions, plants may allow more fungal species to coexist by controlling the abundance of dominant taxa; it may also increase or decrease certain microbial populations to alter the rhizospheric microbial community composition (Achouak et al., 2019 ). However, the high diversity of fungal species also means that more plant resources are needed to sustain microbial growth and reproduction, as well as more complex interspecific interactions (Voges et al., 2019 ; Wagg et al., 2019 ). Thus, the allocation of plant resources to microorganisms may be responsible for our failure to observe the fungal promoting plant growth under the combined inoculation of ASM. Additionally, the interactions between fungal species may influence plant performance by altering the way host nutrient acquisition, uptake, and metabolism (He et al., 2017 ; Liu et al., 2020a ). Therefore, various interactions of different fungal species in plant symbiosis and competition may have important implications for host plant growth and health. Fungal inoculation-mediated morphological growth of A. adsurgens plants Using biomass quantification as an indicator of fungal inoculation performance, we found that fungi mainly promoted the root biomass, but did not influence the shoot biomass, which may be related to the fungi-mediated morphological growth of roots (Li et al., 2019a ; Jabborova et al., 2021 ; Toppo et al., 2022 ). In this study, inoculation of all fungi alone decreased the plant height of A. adsurgens seedlings, but significantly increased the total root length and root surface area. This may be related to endophytic fungal symbiosis cost or resource allocation. Wäli et al. ( 2006 ) have documented that seedlings of Festuca ovina containing Epichloë endophytes grew smaller than those without endophytes. Alternatively, the theory of the optimal allocation of plant resources states that plants allocate biomass preferentially to obtain the most growth-limiting resources (Puglielli et al., 2021 ). Thus, plants tend to invest in root growth at the expense of reduced aboveground growth in an arid environment, resulting in a decrease in aboveground plant height and biomass (Schall et al., 2012 ; Azizi et al., 2021 ). However, the decreased root diameter indicated that the fungal symbionts promote the growth and development of fine roots of A. adsurgens seedlings. A well-developed root system and architecture are beneficial for desert plants to improve water and nutrient absorption in deep dry soil (Likar and Regvar, 2013 ; Li et al., 2019a ). This contributes to the ecological adaptation and resistance of host plants to adverse environments in desert habitats. Effects of fungal inoculation on soil properties and rhizospheric fungal community Soil fungi interact in complex ways and play important roles in soil nutrient cycling, transformation, and promotion of plant nutrient uptake (Yadav et al., 2015 ; Koshila Ravi et al., 2019 ). In our study, the decreased soil pH under inoculations of A. chlamydospora may be related to the enrichment of the acidophilic fungus Pluteaceae . The members of Pluteaceae have been reported to mainly adapted to grow in acidic soil with pH 4.5–4.99 with a strong ability to decompose organic matter, thus playing an important role in soil bioremediation (Mohammadi-Sichani et al., 2017 ; Kunca and Pavlik, 2019 ). However, the increase of soil organic carbon, especially under Monosporascus sp. inoculation, may be related to the carbon conversion process mediated by the increased abundance of soil fungi (Kohout et al., 2018 ). Alternatively, the dominant group Sordariomycetes may also be associated with the increased organic carbon, which has been reported to favor the mineralization of soil aggregate organic matter, thereby promoting the content of soil organic matter (Xu et al., 2021 ). Nevertheless, the reduced soil ammonia under fungal inoculations probably corresponded to the nitrogen fixation by rhizobia. In our experiment, due to the identity of the leguminous plant, nodules were indeed observed in the roots of A. adsurgens plants, especially under the fungal inoculations. Therefore, the presence of adequate nitrogen-fixing bacteria may reduce the dependence of plants on microbial nitrogen mineralization (Kakraliya et al., 2018 ). Meanwhile, the symbiosis of nitrogen-fixing bacteria may also be related to plant growth promotion, which in turn triggers the higher demands of the host plant for other nutrients, especially phosphorus demand in N-fixing legumes (Png et al., 2017 ). In our study, the increases in soil available P under fungal combined inoculation were indeed observed, which was inferred to be relevant to the secretion of various hydrolases by fungi to promote the transformation of insoluble phosphorus (Fabiańska et al., 2019 ). Thus, we speculate that symbiotic nitrogen-fixing bacteria and endophytic fungi may exhibit complementary benefits in terms of nutrient access to their common host (Tang et al., 2019 ; Primieri et al., 2021 ). Such tri-partite symbiosis of plant-endophytic fungi-N fixing bacteria should be further explored in future studies. In the current study, the enrichment of soil fungal communities was different under the combined inoculation treatments. Despite the acidified soil pH, inoculations of AM and AS mitigated the loss of fungal diversity and enrichment of acidophilic fungi caused by acid soils. In contrast, members of Acremonium, Entoloma cremeoalbum, Scolecobasidium , and Diatrypaceae were enriched under AS inoculation; while Scleroramularia and Gaeumannomyces were enriched under AM inoculation. Acremonium and Diatrypaceae have been widely reported to be isolated from desert halophytes and showed the ability to improve host resistance to biotic stress (Jalili et al., 2020 ; Ameen et al., 2021 ). However, Scleroramularia and Gaeumannomyces have been reported as pathogens of crops (Coombs et al., 2004 ; Li et al., 2011 ), which may be related to the transformation of endophytic fungal lifestyle. Studies have shown that the phase of endosymbiosis represents a balanced interaction between fungal virulence and host defense factors (Kuo et al., 2015 ). In the presence of appropriate environmental factors, saprophytic and pathogenic bacteria may transform into endophytes. Nevertheless, some beneficial fungal species, such as Aspergillus, Psilocybe , and Strophariaceae taxa, were enriched under the inoculation of ASM. Aspergillus has been reported to be the most common genus of desert medicinal plants and can stimulate the growth of desert medicinal plants (Ntemafack et al., 2021 ). Psilocybe was considered to be an important biological source for medical compounds and displays important research value in phytochemistry, antioxidant and cellular immunity (Nkadimeng et al., 2020 ). Strophariaceae were found as basidiomycetes, and their mycelia biomass had a significant effect on garlic scale and cucumber seed germination and seedling growth (Regeda et al., 2021 ). We speculate that the combined inoculation of the three fungi in this study may have promoted the increase of Basidiomycetes in the rhizospheric soil of A. adsurgens . This may also suggest that when inoculated with ASM, plants may reduce resource allocation for growth in order to recruit more beneficial microorganisms (Vandenkoornhuyse et al., 2015 ). According to previous studies, due to the different growth rates of fungal species, the symbiotic effects of slow-growing fungi may be delayed (Getachew et al., 2019 ; Vrabl et al., 2019 ). Consequently, considering the short duration of our pot experiment, the beneficial effects of the rhizospheric fungal microbes recruited by ASM mixed inoculation on plant growth will likely take a longer time to manifest. Moreover, the most enriched number of differential fungal species appeared in the inoculation of SM further indicating that fungal cross-inoculation plays a dominant role in the process of plant–soil ecological adaptation by adjusting the abundance and quantity of soil fungal taxa. The synergistic effect indicated that fungal combination inoculation can improve the rhizospheric ecological environment in natural habitat or the presence of a local microbiome, which is more than that of single strain inoculation (Vorholt et al., 2017 ). Interactions among plants growth, soil nutrients, and fungal community In natural ecosystems, endophytic fungi have complex interactions with host plants and are closely related to rhizospheric microorganisms (Trivedi et al., 2020 ). In our study, we found that fungal inoculation mainly influenced soil nutrients by changing soil fungal species diversity and richness, thereby indirectly impacting the number of stem branches, root diameter, and total biomass of A. adsurgens seedlings. Certain fungal species, such as Sordariomycetes, Chaetomium piluliferum , and Pleosporales , were directly linked to soil nutrient cycling. However, in addition to the indirect effects, the fungal community themselves could also directly influence plant growth. For example, Aspergillus, Humicola nigrescens, Pleosporaceae , and Lasiosphaeriaceae were highly correlated with root growth of A. adsurgens . This may be due to the formation of a stable symbiotic relationship between root fungi and host plants (Bouasria et al., 2012 ; Yeh et al., 2019 ). Plants can recruit target microbial communities through signaling molecules, and subsequently exert selective pressure through the immune system, specific nutrient supply or habitat type, etc., allowing the successful colonization of beneficial microorganisms (Foster et al., 2017 ; Martin et al., 2017 ; Cordovez et al., 2019 ). The interaction or synergistic effect among root endophytic fungi responds to plant growth by creating favorable microflora in the rhizospheric environment of A. adsurgens plants. Therefore, the enrichment of beneficial microbial communities in the rhizospheric soil is crucial for plant survival and development and can confer abiotic and biotic tolerance in plants to improve fitness. Potential application of fungal assemblages in desert area Promoting better growth of desert plants, improving the soil environment, and the maintenance of soil microbial diversity, are critical for the stability of arid ecosystems. In the present study, the enhanced stem branching and root growth A. adsurgens under S. kiliense and SM inoculations will help desert plants to grow better and adapt to the desert environment to promote wind prevention and sand fixation (Li et al., 2018 ; Zuo et al., 2020 ). However, soil acidification under A. chlamydospora and co-inoculations of AM and AS indicates that this species may be potentially applied to neutralize soil pH in alkaline areas. In contrast, Monosporascus sp. alone and its combined inoculation of AM and SM markedly promoted the accumulation of soil organic carbon, which is speculated to be important for the nutrient and fertility health of desert soil (Hammad et al., 2020 ). Most importantly, the synergistic effects of combined inoculation on the increased diversity and abundance of rhizospheric fungal community suggest that more fungal species than fewer might be conducive to regulating the composition and structure of microbial community (Mawarda et al., 2020 ; Cheng et al., 2021 ). This may be beneficial to the subsequent restoration of soil microbial functions in the desert. However, this needs to be interpreted with caution, as fungal communities do not independently in line with the plant growth-promoting effects. In desert habitats, the microbial community still includes bacteria, archaea, nematodes, and other groups, which may have a very distinct response to the endophytic fungi inoculation (Trivedi et al., 2020 ). Therefore, the changes in the entire microbial community may be in line with the plant growth-promoting effects and fully reflect the soil microbial functions. Based on our experimental results, inoculations of S. kiliense alone and SM were recommended for use in the desert plant A. adsurgens due to their promoted plant performance, improved soil fungal diversity, and soil organic carbon without the acidification of soil pH. Our research hints at the combined consortium of multiple fungi as a possible solution for promoting sustainable desert conservation and restoration. However, the optimal combination of strains and the complementary effects between different species should be emphasized when constructing a synthetic community (Li et al., 2021 )."
} | 6,884 |
35423722 | PMC8693268 | pmc | 1,727 | {
"abstract": "A Janus membrane/mesh is a type of functional membrane/mesh composed of opposing wetting properties formed into a single layer in order to achieve novel properties. Janus membranes/meshes have attracted increasing attention from materials scientists due to their promising applications in the fields of microfluid transportation, water–oil separation and cleaning energy applications. Herein, we report a simple method to fabricate a Janus mesh by combining opposite wettability functions into one copper mesh substrate. The superhydrophilicity is achieved by chemical etching and the superhydrophobicity is fabricated by hydrophobic SiO 2 nanoparticle spraying. Due to its special composition and structure, the prepared mesh demonstrates distinct wetting properties on its two sides. Meanwhile, aqueous fluids can pass through the mesh from the hydrophobic side to the hydrophilic side spontaneously, whilst being blocked by the mesh when coming from the other direction. This unique property can realize unidirectional transportation of water fluids. The mechanism of the unique property based on Janus wettability is proposed and the stability of the prepared Janus mesh was also tested. The prepared Janus mesh can be used in the fields of microtidal energy, the chemical industry and in astronautics, demonstrating promising practical prospects.",
"conclusion": "4. Conclusion In this work, we endowed a copper mesh substrate with opposite wettability on its two sides in order to fabricate a mesh with Janus wetting properties. We utilized potassium persulfate solution to oxidize one side of the mesh to fabricate hydrophilicity and sprayed hydrophobic-modified SiO 2 nanoparticles onto the other side to fabricate hydrophobicity. We sprayed one side of the mesh first and then floated the mesh on the interface of potassium persulfate solution so that the hydrophobic side could be kept away from the etching process. The prepared mesh could transport water fluid unidirectionally. Water passed through the mesh in the hydrophobic-to-hydrophilic direction, while it was blocked in the opposite direction. The possible principle for this based on force analysis is discussed in this work and the stability was recorded as being good enough. The interesting phenomenon in the way that the interface of the water fluid behaves was also discussed. The prepared Janus mesh has great potential in medical fields, energy fields and in the chemistry industry.",
"introduction": "1. Introduction Materials with superwettability have attracted the attention of researchers due to their unique chemical and physics properties as well as their extensive practical prospects. 1–4 In previous studies, researchers have found that superhydrophobic materials can be utilized in the fields of antifouling surfaces 5–7 and water droplet controlling, 8 while superhydrophilic materials can be used in the fields of fog harvesting 9–11 and in the chemical industry. 12 Recently, it has been found that a membrane/mesh with binary wettability, and is fabricated by combining hydrophilicity and hydrophobicity into one membrane/mesh, can achieve novel properties. 13–15 Janus was an ancient Roman god with two different faces. The Janus membrane/mesh obtained this interesting name due to its asymmetric composition with regards to its wettability. 13 Scientists combined opposite properties into one membrane/mesh and collectively named it as a “Janus membrane/mesh”. Amongst all types of Janus membranes/meshes, the combination of hydrophilicity and hydrophobicity is the most studied property. A Janus membrane/mesh has plenty of unique properties due to its binary wettability and porous structure. This type of membrane/mesh has promising prospects in the fields of oil/water separation, 16–25 microfluidic transportation, 3,13 demulsification, 26–29 and so on. 30 The behavior of water is mostly affected by interface tension when it comes into contact with the mesh surface. 1 However, the Laplace pressure that is generated at the interface influences the behavior of water in the pores. 13 The Laplace pressure that is generated by the interface can be calculated from the well-known Laplace equation: 1 where Δ p is the Laplace pressure, γ is the surface tension of the interface and R is the radius of the curvature of the interface. Water can maintain a stable shape on two sides of traditional hydrophilic surfaces or hydrophobic surfaces. 1 However, for a Janus mesh, the asymmetric wettability can cause distinct surface tensions on two sides of the mesh. When waterflow penetrates the mesh and enters the pores, the interface of the water can generate different Laplace pressures on the two sides of the mesh. The changes in Laplace pressure can influence the water, causing distinctive wetting behavior and achieving novel functions. 13 This type of mesh has promising prospects in the fields of oil/water separation, 16–25 microfluidic transportation, 3,13 demulsification 26–29 and so on. 30 Due to their unique properties and extensive practical prospects, Janus meshes have attracted the attention of materials scientists on a global scale. In previous work, scientists have studied methods of Janus membrane/mesh fabrication and have conducted much explorative research. The most studied property of a Janus membrane/mesh is water/oil separation. Yao fabricated a double-woven structure Janus membrane with hydrophobic yarns on one side and hydrophilic yarns on the other side. 25 The water contact angle on the hydrophobic surface was 152.8 ± 0.3°, and the contact angle on the hydrophilic side was almost 0° (water droplets were absorbed immediately by the membrane). The water droplets can penetrate the woven membrane from the hydrophobic to the hydrophilic side, whilst being blocked in the opposite direction. This phenomenon demonstrates the potential of the unidirectional water transporting property of a Janus membrane. The double woven system can be used for water/oil separation. Lin fabricated a Janus mesh on a stainless steel mesh substrate. 16 In the study, an acid solution was used to etch the mesh, endowing it with hydrophilicity. Then, hydrophobic particles were deposited onto one side of the mesh. The water contact angle of the hydrophobic side was 164 ± 0.5°, and the contact angle of the hydrophilic surface was almost 0°. Interestingly, the mesh showed oleophilicity on the hydrophobic side and oleophobicity on the hydrophilic side. This type of Janus wetting system can also be utilized in water/oil separation processes. The mesh showed excellent water/oil separation ability. Liang used electro-spun technology to fabricate a Janus membrane. 24 In the experiment, an electro-spun PS nanofiber demonstrated hydrophobicity while the electro-spun PAN nanofiber was hydrophilic. The contact angle of the hydrophobic PS membrane was 126 ± 0.2°. The electrospun Janus membrane showed outstanding demulsification ability and had a promising prospect in oil leaking situations. However, there are many drawbacks in the fabrication of these mentioned Janus membranes, such as a high fabricating expense, a complex preparation process, poor stability, and so on, and this greatly limits their practical application. It is therefore necessary to develop a simple and inexpensive method of constructing a stable Janus mesh. Our group has extensive experience in preparing superwetting materials. 31–36 In our previous work, several substrates with a special wetting property have been prepared and excellent performance of these substrates was achieved. In this work, we report a novel method to fabricate a new type of Janus mesh. We sprayed hydrophobic modified SiO 2 nanoparticles onto one side of the mesh for hydrophobic modification. 37,38 To fabricate the hydrophilic property on the mesh, we soaked the mesh into potassium persulfate solution, endowing the surface with hydrophilicity. 39,40 The water contact angle of the hydrophobic mesh that was fabricated by the spraying method was tested to be 162 ± 0.4°, and the water contact angle of the hydrophilic mesh that was treated by the etching process was less than 5°. The influence of mesh numbers on the surface wetting behavior was also studied. We found that a Janus mesh with a mesh number of 200 demonstrated the best wetting property. In order to combine the two opposite wetting properties, the mesh was floated on the interface of the potassium persulfate solution so that the hydrophobic side could be kept upwards without etching by the solution. The unidirectional water transportation property was studied in a mimic glass tube. The prepared Janus mesh could block the transport of water fluid in the hydrophilic-to-hydrophobic direction. Meanwhile, the water fluid could pass through the mesh from the hydrophobic side to the hydrophilic side. The mesh also presented excellent stability. A mechanism, based on the superwetting theory, was also proposed to explain the unique property. The unidirectional water transportation property shows wide application prospects in the energy industry and in vessel surgery.",
"discussion": "3. Results and discussion 3.1 Surface morphology and composition analysis \n Fig. 1 demonstrates the morphologies and wetting behaviors of the meshes that were treated with different modifications. In this experiment, we utilized a brass mesh as a substrate. Fig. 1(a1) shows the SEM image of the bare substrate surface. From the image, we can see that the brass substrate is rather smooth, with some irregular crystalline grains on it. The results match well with the typical SEM images of a copper surface. Fig. 1(a2) shows the image of a water droplet in contact with the brass mesh substrate. In this image, the original mesh substrate shows low hydrophobicity and the water contact angle is about 100°. Fig. 1(b1) shows the SEM image of a copper mesh treated with the hydrophobic modification. A hydrophobically modified SiO 2 nanoparticle solution was sprayed onto the copper mesh to fabricate the superhydrophobic coating. The diameter of the SiO 2 particles was about 100 nm. The hydrophobic group of –CH 3 was introduced onto the modified nano-SiO 2 particle, so that the surface energy of the mesh reduced dramatically. At the same time, the nano-size SiO 2 particles could form a microstructure on the surface. According to the Wenzel equation, the relationship between the contact angle and surface tension can be described as follows: 2 where γ SG , γ SL and γ LG stand for the surface tensions of the solid–gas interface, the solid–liquid interface and of the liquid–gas interface. r is a parameter that reflects the roughness of a surface ( r ≥ 1). From eqn (2) , we can conclude that the roughness can enhance the hydrophilicity of a hydrophilic surface and can also enhance the hydrophobicity of a hydrophobic surface. Therefore, the microstructure that is formed by the nano SiO 2 particles can increase the hydrophobicity of the surface. As Fig. 1(b2) shows, the water contact angle of the sprayed mesh is about 162 ± 0.4°, demonstrating the superhydrophobicity of the as-prepared mesh. For the hydrophilic modification, we utilized the oxidizing effect of potassium persulfate solution and created a hydrophilic layer on the copper mesh substrate. The oxidizing process can generate copper oxide on the mesh and can increase its surface energy. The etching process can form a flower-like microstructure on the wire surface, and this is shown in Fig. 1(c1) . This microstructure can increase the roughness and can also enhance the hydrophilicity of the surface according to eqn (2) . Water droplets were absorbed onto the mesh immediately and the static water contact angle was less than 10°, as shown in Fig. 1(c2) . A comparison of Fig. 1(c2) with Fig. 1(b2) indicates the great difference between the wetting behaviors of a hydrophilic mesh and a hydrophobic one. Fig. 1(d1) shows the infrared spectrum of the mesh treated with blank spraying, while Fig. 1(d2) shows the infrared spectrum of the mesh treated with hydrophobic spraying. There are obvious distinctions in the 2925 and 2854 cm −1 wavenumbers in Fig. 1(d2) , and this matches well with the characteristic peaks of a –CH 3 group. The appearance of the –CH 3 group confirms that the hydrophobic modifier has been attached onto the substrate successfully. Fig. 1 Surface morphology, water contact angles and composition of three types of meshes. (a1), (b1) and (c1) Show the SEM images of the original substrate, the surface after the hydrophobic spraying process and the surface after the hydrophilic etching process, respectively. (a2), (b2) and (c2) Show the wetting behaviors of the original mesh substrate, the hydrophobic mesh and the hydrophilic mesh, respectively. (d1) and (d2) Are the infrared spectra of the mesh after the blank spraying process and the mesh after the hydrophobic spraying process, respectively. 3.2 Analysis of the gradient of the mesh numbers The mesh number affects the wetting behavior of the mesh tremendously. In order to achieve the best unidirectional water transporting function, the influence of the mesh number is studied, and the result is shown in Fig. 2 . In our experiment, four different mesh numbers (50, 100, 200, and 300) were chosen. Optical images of the meshes with different mesh numbers in the same field of vision are shown in Fig. 2(a)–(d) . Fig. 2(a)–(d) show the optical images of the mesh substrates with different mesh numbers of 50, 100, 200 and 300, respectively. From the optical images, we can see that, in the same field of vision, the density of the holes increases with increasing mesh number. The water droplets also behave differently on the meshes with various mesh numbers. To determine the ideal mesh number of the substrate, we treated the meshes with the above-mentioned modification and tested their wettability. The water contact angles (WCAs) that were captured by the contact angle meter are shown. Fig. 2(a1)–(d1) show the WCA images of the hydrophobic meshes. In Fig. 2(a1) , we can see that the water contact angle is about 100 ± 0.5°, and the specimen shows low hydrophobicity. However, in Fig. 2(b1)–(d1) , the water contact angles clearly change when the mesh number increases to 100, 200 and 300. The contact angle of the specimen with a mesh number of 100 is about 145 ± 0.2°, and this can reach up to 162 ± 0.4° with mesh numbers of 200 and 300. The static contact angle results demonstrate the excellent hydrophobicity of the corresponding meshes. On the other hand, Fig. 2(a2)–(d2) show the images of the meshes etched by potassium persulfate, and all the meshes are hydrophilic. In Fig. 2(a2)–(c2) , which are for the specimens with mesh numbers 50, 100 and 200, respectively, the contact angles are less than 10°. Therefore, water droplets can penetrate the mesh easily. The water contact angle increases to about 30 ± 0.6° when the mesh number increases to 300, as shown in Fig. 2(d2) , and the water droplets can also pass through the mesh. The hydrophilicity of the mesh decreases with a mesh number of 300. Based on the above experiments, the substrate with a mesh number of 200 demonstrates the best wetting performance in terms of both hydrophilicity and hydrophobicity. Consequently, we chose the copper mesh with a mesh number of 200 as the substrate of the Janus mesh. Fig. 2 Images regarding analysis of the gradient of the mesh numbers. (a) to (d) Are the optical images of the meshes with mesh numbers of 50, 100, 200 and 300. (a1) to (d1) Show the static water contact angles of hydrophilic meshes with corresponding mesh numbers, and (a2) to (d2) demonstrate the static water contact angles of the hydrophilic meshes with corresponding mesh numbers. 3.3 Wetting behavior analysis In Fig. 1 and 2 , the static wetting behavior of the prepared surface is demonstrated. In order to further observe the wetting behaviors of the prepared mesh, the dynamic contact process is captured by a contact angle meter and the results are shown in Fig. 3 . The procedure for a water droplet coming into contact with the prepared hydrophobic mesh is demonstrated in Fig. 3(a1)–(f1) . The black arrow represents the direction of movement of the mesh. Fig. 3(a1) and (b1) show that the mesh approaches the droplet and is then in contact with the droplet. The mesh keeps moving upwards and then squeezes the droplet, and this is shown in Fig. 3(c1) . Afterwards, the mesh starts to move downwards. Fig. 3(d1) and (e1) show that the droplet has been drawn into a spindle-like shape by the hydrophobic mesh, instead of dropping on the surface. Finally, the water droplet detaches from the mesh, as shown in Fig. 3(f1) . The droplet was pulled into a spindle-like shape by the surface of the mesh, and this shows the excellent hydrophobic property of the surface. Fig. 3(a2)–(f2) show the process of a water droplet in contact with a hydrophilic mesh. Using the same testing procedure as for the hydrophobic mesh, the hydrophilic mesh first moves upwards to be in contact with the droplet, and this is shown in Fig. 3(a2) and (b2) . The water droplet was quickly absorbed by the mesh as soon as they came into contact, as shown in Fig. 3(c2) . When the syringe was pulled away from the mesh, the droplet detached from the syringe and spread out onto the mesh spontaneously, as shown in Fig. 3(d2)–(f2) . From this procedure, we found that the water droplet was absorbed by the mesh and spread immediately as soon as it came into contact with the hydrophilic surface, indicating the outstanding hydrophilicity of the oxidized mesh. Fig. 3 Dynamic process of a water droplet in contact with the prepared hydrophobic and hydrophilic mesh surfaces. The arrows represent the direction of movement through the mesh. (a1)–(f1) and (a2)–(f2) Demonstrate the contact processes of the hydrophobic surface and the hydrophilic surface, respectively. The spraying and etching treatments that are used in this work are two conventional modification methods that are used to generate different wettability effects on a surface. 37–40 However, the two methods cannot be conducted on one mesh simultaneously, in order to obtain their opposite effects. Due to the porous nature of meshes, the ingredients of the solution will inevitably infiltrate to the other side when spraying on one side. The spraying modification method finally fabricates a unified hydrophobic mesh. It is clear that immersing a mesh into potassium persulfate solution entirely will oxidize both sides of the mesh. In order to fabricate Janus wettability on one mesh substrate, the two methods must be effectively combined. In our experiment, we sprayed the hydrophobic SiO 2 nano particles on one side of the mesh first, then we floated the as-prepared mesh on the interface of potassium persulfate solution. The etching process can then endow the treated side with the hydrophilic property. At the same time, by remaining in the air phase, the sprayed modified side can be kept away from the liquid phase and maintain its hydrophobic property. Through the above-mentioned method, a Janus mesh with opposite wettability on different sides could be fabricated, as shown in Fig. 4 . Fig. 4 A schematic diagram of the fabrication process of the Janus mesh for unidirectional water fluid transportation. 3.4 Unidirectional water transportation analysis Water fluid unidirectional transportation of the as-prepared Janus mesh is demonstrated in Fig. 5 . In our experiment, we placed the Janus mesh in a mock tube vertically in order to test its asymmetry blocking/passing properties. The water fluid will be blocked by the mesh when the water moves in a hydrophilic-to-hydrophobic direction, and this process is shown in Fig. 5(a)–(d) . Fig. 5(a) shows the empty tube and Janus mesh system. The red arrows represent the direction of movement of the water fluid. Furthermore, in order to emphasize the location of the fluid, we used CuSO 4 to dye the water in this experiment. Fig. 5(b) shows that the water fluid movement from the hydrophilic side was blocked by the Janus mesh. Water was still blocked by the mesh when it carried on accumulating, as shown in Fig. 5(c) . Finally, water fluid was still blocked even when the water nearly filled the tube, as shown in Fig. 5(d) . The schematic diagrams are shown in Fig. 5(a1) and (b1) . On the other hand, if the water fluid moves in a hydrophobic-to-hydrophilic direction, it can pass through the mesh freely. The process is demonstrated in Fig. 5(e)–(h) . Water fluid comes from the hydrophobic side and goes to the hydrophilic side in the tube system, as shown in Fig. 5(e) . Instead of being blocked by the mesh, the water fluid can pass easily through the mesh, as shown in Fig. 5(f) . From Fig. 5(g) and (h) , we can see that the water fluid carries on passing through the mesh as water accumulates. Using this novel phenomenon, the as-prepared Janus mesh can realize the unidirectional water transporting function. The corresponding schematic diagrams are shown in Fig. 5(c1) and (d1) . Fig. 5 Illustration of the unidirectional water fluid blocking/passing through function in a tube of the as-prepared Janus mesh. (a–h) show the actual images and (a1–d1) show the schematic diagram. The unidirectional water blocking effect of a Janus membrane/mesh has been mentioned in a number of previous studies. 13,14,41 However, results from different articles appear to be rather confusing. Among the articles, a number of them have been similar to Yang's experiment. 14 Here, the authors used a hydrophilic cotton membrane as a substrate, and modified one side to be hydrophobic in order to fabricate a Janus membrane. The results showed that a water droplet could penetrate the membrane from the hydrophobic to the hydrophilic side, whilst be blocked in the opposite direction, and this is similar to our results. However, at the same time, there are also many different results that are similar with Liu's experiment. 41 They used a hydrophobic polymer membrane as the substrate and conducted a hydrophilic modifying process on one side of the membrane to fabricate a Janus membrane. In the article, the membrane could block water fluid in two directions. However, when the hydrophilic side of the membrane was placed downwards and the hydrophobic side was placed upwards, the membrane could maintain a higher water column. It seems that the blocking effect is more intense in the hydrophobic-to-hydrophilic direction, and this is opposite to our results. Based on these previous works, we proposed a possible mechanism for the unidirectional blocking phenomenon of the Janus membrane. The mechanism diagram is shown in Fig. 6 . Fig. 6 The mechanistic diagram for the unidirectional blocking phenomenon of the Janus mesh. (A) Is the schematic graphic and cross section of the Janus mesh. B (b1–b4) shows the process of water fluid moving in the hydrophobic to hydrophilic direction and passing through the mesh. C (c1–c4) demonstrates the process of the water fluid moving from the hydrophilic to hydrophobic side and being blocked by the mesh. \n Fig. 6A shows a schematic diagram of our Janus mesh, and the picture on the right-hand side is the cross section of the mesh. We conducted hydrophobic nano SiO 2 particle spraying on one side of the substrate, and then utilized the etching effect of potassium persulfate solution to endow the substrate with hydrophilic property. Therefore, as the schematic graphic shows, our Janus mesh has a thick hydrophilic part and a thin hydrophilic part. Fig. 6B shows the passing process of water fluid that comes from the hydrophobic side and goes to the hydrophilic side. Fig. 6(b1) demonstrates the schematic graphic of the cross section. In Fig. 6(b2) , we can see that the contact angle in the solid/liquid/gas three-phase point is rather large, and the surface free energy is extremely low at the hydrophobic part. The interface of the water forms a convex shape in the pores of the mesh. The Laplace pressure that is generated by the convex interface is in the reverse direction and has an impeding effect to the water fluid. However, the hydrophobic part of the mesh is so thin that the impeding effect cannot block the water fluid entirely. Water penetrates the hydrophobic part and comes into contact with the hydrophilic part of the mesh, as demonstrated in Fig. 6(b3) . When the hydrophobic part is immersed entirely in water, the convex interface will not be formed and the reversed Laplace pressure disappears as well. On the other hand, a concave water interface can be formed at the hydrophilic part, and the Laplace pressure generated by the interface is in the forward direction. This phenomenon can have a positive effect for water fluid passing through the mesh. Under these circumstances, the impeding effect of the mesh comes inherently from the reversed pressure of the mesh pores. However, the effect cannot counterbalance the hydraulic pressure and forwards Laplace pressure that is generated from the assembled interface. Our experiment therefore indicates that the hydrophilic mesh cannot block the water fluid. Fig. 6(b4) shows that water fluid finally penetrates the pore of the mesh and Fig. 6(b5) shows the force analysis of this process. \n Fig. 6C demonstrates that the process of water fluid moving in the hydrophilic-to-hydrophobic direction is blocked by the Janus mesh. The schematic graphic of the cross section is shown in Fig. 6(c1) . In Fig. 6(c2) , water fluid leaks into the pore and comes into contact with the hydrophilic part of the mesh. Pores of the mesh have an impeding effect to the water, but they cannot block the water fluid entirely. Therefore, water fluid moves forward and comes into contact with the hydrophobic part of the mesh, as shown in Fig. 6(c3) . The convex water interface is formed in the pore and reversed Laplace pressure is generated simultaneously. In this case, impeding pressure is composed by inherent reversed pressure of the mesh pores and the reversed Laplace pressure. The two impeding effects can overcome the hydraulic pressure so that the water fluid is blocked by the Janus mesh, as demonstrated in Fig. 6(c4) . Fig. 6(c5) shows the force analysis of this process. Based on this theory, the unidirectional water blocking phenomenon of a “thick hydrophilic part and thin hydrophobic part” Janus membrane/mesh can be perfectly explained. As for the “thick hydrophobic part and thin hydrophilic part” Janus membrane/mesh, when water comes into contact with the hydrophobic part directly, it cannot penetrate the hydrophobic part of the mesh, so the water fluid is blocked. On the other hand, contacting the hydrophilic part first can help the water fluid pass through the mesh and reduces the impeding ability of the mesh. In summary, for a “thick hydrophobic part and thin hydrophilic part” Janus mesh, the blocking ability is more intense when water fluid comes from the hydrophobic side and goes to the hydrophilic side. 3.5 Analysis of the interface of the water fluid When the Janus mesh is placed in a water tube, there is an interesting phenomenon of the water fluid that needs to be discussed, and this is described in Fig. 7 . The interfaces of water behave differently at the two sides of the Janus mesh, and this phenomenon can be attributed to the difference in surface tension at the liquid/solid/gas three-phase points. For an integrated hydrophobic mesh, the interface of the water is in a convex shape. A schematic diagram is shown in Fig. 7(c1) and the actual image is shown in Fig. 7(c2) . The water was dyed with CuSO 4 to display it more clearly. On the other hand, the interface of the water is in a concave shape if the mesh is integrated with hydrophilicity, as shown in Fig. 7(b1) and (b2) . As for the Janus mesh with distinct wettability on its two sides, the interface of the water fluid also behaves differently at the two sides, as demonstrated in Fig. 7(a1) and (a2) . On the hydrophobic side, the interface of the water fluid is in a convex shape, while on the hydrophilic side, the interface is in a concave shape. This novel phenomenon can be explained by the Young–Laplace equation. The interface of the water fluid is similar to a water droplet at the solid/liquid/gas three-phase point, as the well-known Young–Laplace equation demonstrates, 3 γ SG − γ SL = γ LG cos θ where γ SG , γ LG and γ SL stand for the surface tensions of the solid–gas phase interface, the liquid–gas phase interface and of the solid–liquid phase interface. θ represents the contact angle of the droplet. The contact angle can be large for a hydrophobic surface, while it can be rather small for a hydrophilic surface, and this is shown by eqn (3) . From the images, we can see that the convex interface at the hydrophobic side has a large contact angle. On the other hand, the concave interface at the hydrophilic side of the mesh has a small contact angle. This novel phenomenon fits Young's theory well. Fig. 7 Schematic diagram of the water fluid interface when in contact with the meshes with different wettability properties. (a1) and (a2) Show the water fluid surfaces of a Janus mesh. (b1) and (b2) Demonstrate the water fluid surfaces of a hydrophilic mesh, and (c1) and (c2) demonstrate the water fluid surfaces of a hydrophobic surface. In addition, for an as-prepared hydrophobic mesh surface, tiny water droplets may remain on the surface if they come into contact with the mesh first. The droplets that remain on the mesh can transform the hydrophobic surface into a hydrophilic one. Therefore, if a hydrophobic mesh blocks water fluid on one side first and then comes into contact with water again, the interface of the water can be different on two sides of the mesh, just as in a Janus mesh. 3.6 Stability of the prepared surface The stability of a prepared mesh plays a vital role in its practical applications, as it determines the life span of the mesh. In order to evaluate the stability of the prepared Janus mesh in this work, we tested the mesh from two aspects: the time it takes to hold the aqueous fluid stably (from the hydrophilic side to the hydrophobic side), and repeat circles of the mesh holding the water fluid (from the hydrophilic side to the hydrophobic side), as shown in Fig. 8 . Fig. 8 Schematic diagram of the stability of the mesh. A (a1–a6) demonstrates the cycle times that the mesh can hold the water fluid and B (b1–b4) demonstrates the holding times that the mesh can maintain. As for the cycle times, the experimental results are shown in Fig. 8A . As mentioned above, the aqueous fluid can be blocked by the mesh when moving in a hydrophilic-to-hydrophobic direction, while it can penetrate the mesh when moving from the hydrophobic side to the hydrophilic side. In our experiment, we chose a Janus mesh with a mesh number of 200 as the specimen to test the unidirectional blocking stability of the mesh. We injected water fluid from the hydrophilic side to see if the mesh could block it. Then, we removed the mesh, dried it, and repeated the process again. Fig. 8(a1) demonstrates the testing system. The display device next to the tube showed the cycle number that the mesh had been tested. As Fig. 8(a2)–(a6) show, the mesh could block the fluid after 1, 5, 10, 15 and 20 cycles, and so the mesh showed outstanding stability. It is also worth mentioning that during the stability experiment, water fluid could pass through the mesh in the hydrophobic-to-hydrophilic direction, maintaining its unidirectional blocking function in the process. When it came to the amount of time holding the aqueous fluid, the prepared Janus mesh also showed excellent stability. The testing process of the prepared Janus mesh is demonstrated in Fig. 8B . The clock in the image shows the time the mesh held the water fluid. The maximum number of the clock is 24 h. Fig. 8B(b1)–(b3) show that the Janus mesh could hold water without any leaking. After 32 h, water fluid finally leaked from the mesh, as (b4) shows. The results indicate good stability of the prepared mesh, greatly expanding its application potential. It is clear that, compared with static fluid, the collision effect between a stream current and the mesh can help the water to penetrate the Janus mesh. Our experiments also confirmed this point. The “Reynold numbers” factor contains the influence of varying viscosities and different diameters, and is the ideal factor for measuring the effect of a moving stream current. In our experiment, we adjusted the rate of the water flow and maintained the other factors in order to change the Reynold numbers of the aqueous fluid. The as-prepared Janus mesh could hold static water from the hydrophilic side to the hydrophobic side. For a moving water current, the mesh could hold water when the velocity of the fluid was slow. As we gradually increased the velocity, the moving water current finally passed through the mesh. The equation used to calculate the Reynold number is demonstrated as follows: where Re, ρ , v , d , and μ stand for the Reynold number, density of the liquid, the velocity of the flow, diameter of the tube and the viscosity of the liquid, respectively. In our experiment, the diameter of the tube was fixed at 5 micrometer, and the temperature of the environment was 20 °C. Under these circumstances, the viscosity of the water was 1.0050 mPa s. The aqueous fluid could penetrate the as-prepared Janus mesh when the velocity of the flow was larger than 0.90 m s −1 . The results of our experiment are shown in the following table ( Table 2 ). The Reynold numbers and other factors of the stream current test \n V (m s −1 ) \n ρ (kg m −3 ) \n μ (mPa s) \n T (°C) \n d (mm) Re Pass or blocked 0.90 1000 1.0050 20 5 4477.6 Pass 0.77 3837.9 Blocked 0.64 3198.3 Blocked 0.51 2558.6 Blocked 0.26 1279.3 Blocked The experiment confirms that the collision between the fluid and the mesh can help the water to penetrate the mesh. At the same time, the as-prepared mesh also demonstrated outstanding stability when blocking the stream current unidirectionally. As for the phenomenon of “leaking after 32 h”, we have proposed a theory to explain it. We conducted facile modification of the surface of the mesh to endow the surfaces with different wettability properties. However, as mentioned above, tiny droplets that remain on the surface can transform a hydrophobic surface into a hydrophilic one. During the stability test, water vapor could penetrate the holes of the mesh from the hydrophilic side and condense on the hydrophobic side, changing the wettability of the hydrophobic surface. After 32 hours, the mesh loses its Janus wettability and transforms into a hydrophilic one that can hardly hold the water fluid. In this case, water fluid can pass through the mesh, as demonstrated by the experiment."
} | 8,754 |
40000386 | PMC11860738 | pmc | 1,728 | {
"abstract": "Abstract Plant–arbuscular mycorrhizal fungal (AMF) mutualisms are crucial to ecosystem biodiversity and productivity. Yet, our understanding of the functional roles of plants as AMF generalists or specialists, and the consequences of these plant interaction traits for soil ecosystems are virtually unknown. We grew eight pasture plant species under two experimental conditions, sequencing their root AMF communities to assess interaction traits using a range of numeric and phylogenetic diversity metrics, thereby characterizing each plant species' interaction generalism with AMF. We used lipid analysis of rhizosphere soils and Bayesian modeling to explore how host interaction traits affected carbon allocation to AMF and bacteria. We found that plant interaction traits for AMF remained stable despite large variation in soil conditions and AMF pools. Host interaction generalism was linked to contrasting patterns in bacterial and AMF biomass: Phylogenetic diversity in plant interactions was positively associated with AMF biomass, while numeric diversity was negatively associated with bacterial biomass in rhizosphere soils. Explicit consideration of plant interaction niches may enhance understanding of how changes in biodiversity affect ecosystem carbon cycling.",
"introduction": "INTRODUCTION Species interactions are important for maintaining biodiversity, productivity, and resilience of ecosystems (McCann, 2000 ; Ratzke et al., 2020 ; Tylianakis et al., 2010 ). Many species depend on mutualistic relationships for crucial processes such as pollination, dispersal, resource acquisition, or stress alleviation (Allesina & Tang, 2012 ; Bascompte et al., 2006 ), such that these interactions comprise a key component of a species' niche (Carscadden et al., 2020 ). A species' interaction niche, the degree to which it interacts with members of another trophic guild, such as its mutualistic partners, is often described as a continuum between specialism and generalism and has important ecological implications for both guilds (Poisot et al., 2015 ). For example, generalist pollinators tend to positively affect plant production (Maldonado et al., 2013 ), while specialists can enhance coexistence by reducing competition (Bastolla et al., 2009 ). In nature, the presence of a range of interaction niches contributes to biodiversity and community stability (Dehling et al., 2021 ; Poisot et al., 2015 ). Despite this importance, the definition of a specialist and generalist is not always straightforward (Poisot et al., 2012 ; Rohr et al., 2014 ). For example, the term specialist is often applied to members of one guild that interact with few partners but also to those selectively interacting with phylogenetically related partners (Bascompte, 2009 ; Montesinos‐Navarro et al., 2015 ). By contrast, a generalist is commonly defined either as a species with many or diverse interactions. These differences in the definitions of specialism and generalism are problematic because they lead to the pooling of species interaction traits that may vary in their effects on the community. Additionally, mutualistic interactions can be predicted through phylogenetic relationships (Rezende et al., 2007 ) or by species traits (Eklöf et al., 2013 ; Vázquez et al., 2009 ) and generality can be conserved across a species' range (Emer et al., 2016 ), yet interactions (particularly those of generalists) can be determined by random encounter probability (related to species' abundances; Vázquez et al., 2009 ) and shaped by the local environment (Tylianakis et al., 2008 ). Thus, it remains unclear to what extent the local environment shapes species interaction generalism. Resolving the various facets of interaction traits of mutualistic species would improve understanding of the assembly and maintenance of ecological communities. Possibly the oldest mutualism among eukaryotes is that between plants and arbuscular mycorrhizal fungi (AMF), which occurs in more than three‐quarters of vascular plant species and most terrestrial ecosystems (Brundrett & Tedersoo, 2018 ). Arbuscular mycorrhizal (AM) plants allocate on average 6% of photosynthetic carbon (C) to obligately biotrophic soil fungi of the subphylum Glomeromycotina (Hawkins et al., 2023 ). In exchange, plants receive multiple benefits from AMF, including improved water and nutrient acquisition (Vogelsang et al., 2006 ) and pathogen and stress resistance (Begum et al., 2019 ; Lutz et al., 2023 ). Consequently, AM plants are significant sinks for atmospheric carbon dioxide (Parihar et al., 2020 ). While AMF abundance is highly correlated to soil C sequestration in field studies (Wilson et al., 2009 ), it is less clear how AMF diversity, largely mediated by plant hosts, influences C allocation to the soil microbial community. Enhanced understanding of plant interaction traits for AMF may provide insight into how host species affect the diversity and production of soil ecosystems (Bennett & Groten, 2022 ). Compared with other mutualisms, plant–AMF interactions are not well understood, partly due to the many stochastic, abiotic, and biotic filters that affect community assembly (HilleRisLambers et al., 2012 ; Vályi et al., 2016 ). For example, root AMF communities vary based on the available AMF pool (Šmilauer et al., 2020 ), which is context‐dependent (Šmilauer et al., 2021 ; Tylianakis et al., 2008 ) and influenced by soil properties (Gerz et al., 2016 ). Locally, host‐specific AMF assemblages suggest that host identity plays a key role in determining AMF composition and biomass (Leff et al., 2018 ; Veresoglou & Rillig, 2014 ). Increasing evidence points to the role of host traits in plant–AMF niche partitioning. AMF colonization rates correlate with AM plant root traits (Bergmann et al., 2020 ), shaping plant interaction niches for AMF. For instance, grasses tend to host more AMF taxa than forbs in grasslands and may also differ in AMF colonization rates and composition (Sepp et al., 2019 ; Šmilauer et al., 2020 ). AM hosts may adopt various strategies in selecting the number and taxonomic composition of their mutualists because AMF vary in root colonization patterns and nutrient transfer abilities (Horsch et al., 2023 ; Lendenmann et al., 2011 ). Generalist hosts may benefit from the complementary effects of multiple AMF (Jansa et al., 2008 ; Koide, 2000 ), but these benefits come with trade‐offs, such as higher carbon costs, especially when cheaters are present (Bever et al., 2009 ; Kiers & Denison, 2008 ). In some environments, forming specialized interactions with a few beneficial AMF may be advantageous (Werner & Kiers, 2015 ). Distinct plant interaction niches for AMF can influence ecosystem C cycling both directly by altering AMF communities and indirectly through AMF‐mediated effects on soil bacterial communities. Up to 40% of photosynthetic C is lost from plant roots as fatty acids (FAs), carbohydrates, and other metabolites, fueling the growth of the AMF mycelium and a complex, yet specific, community of rhizosphere bacteria (Jiang et al., 2017 ; Marschner & Baumann, 2003 ). Arbuscular mycorrhizal fungi also produce metabolites that alter the bacterial composition of their hyphospheres (Huang et al., 2023 ) and nutrient availability in soil (Zhang et al., 2020 ). Together, these processes create plant–soil feedback that shapes future plant community assembly (Crawford et al., 2019 ), ultimately influencing ecosystem carbon cycling on larger spatial and temporal scales. While plant interaction niches play a crucial role in structuring soil communities, detailed knowledge of their effects on C allocation to AMF and bacterial communities remains sparse. Here, we characterized plant interaction niches with AMF, which we define using a range of diversity metrics to encompass the various facets of specialism/generalism. We sought to understand how these interaction traits affect AMF and bacterial biomass in rhizosphere soil. Firstly, we generated different biotic and abiotic filters on AMF community assembly by growing eight plant species under two experimental conditions. We test the hypothesis (Hyp 1 ) that plant species' interaction roles as AMF generalists or specialists are stable to these changes, comparing the multidimensional plant interaction niches under different experimental conditions by Procrustes analyses. Secondly, we sought to learn how plant interaction niches affect AMF biomass in rhizosphere soil. We expected interaction generalist hosts to be capable of greater C allocation to AMF due to their enhanced nutrient supply resulting from complementarity effects of their AMF communities (Jansa et al., 2008 ; Koide, 2000 ). In turn, we expected that higher rhizosphere AMF biomass would lead to a greater root‐encounter probability and a greater proportion of the root system being colonized, increasing interaction generalism. We therefore test the hypothesis (Hyp 2 ) that host interaction generalism is positively associated with AMF biomass in rhizosphere soils. We quantified the abundance of the neutral lipid fatty acid (NLFA) 16:1ω5 as a proxy for AMF biomass and modeled its response to plant interaction generalism, while accounting for plant phylogeny, root, and shoot biomass in a Bayesian framework. Finally, we explore the effect of host interaction generalism with AMF on bacterial biomass in the rhizosphere. While plant and AMF species may have differential effects on bacterial communities (Scheublin et al., 2010 ; Söderberg et al., 2002 ), we expected a positive relationship between soil bacterial biomass and plant interaction generalism due to complementary effects of many AMF on bacterial species. We therefore test the hypothesis (Hyp 3 ) that soil bacterial biomass would increase in response to plants' interaction traits associated with host generalism for AMF. We estimate bacterial biomass using phospholipid fatty acid (PLFA) analysis of bacterial biomarkers and model the effect of plant interaction traits, accounting for plant phylogeny, root, and shoot biomass in a Bayesian framework. Our study reveals how plant interaction traits affect the productivity of soil ecosystems, contributing to the understanding of how changes in biodiversity affect ecosystem C cycling.",
"discussion": "DISCUSSION We found that pasture plant species exhibit stable interaction niches for AMF, even under varying environmental conditions. Our comprehensive characterization of plant–AMF interaction niches provides novel insight into how plant niche partitioning for interaction partners affects C allocation to soil microbial communities. We show that C allocation to AMF and bacteria is associated with different aspects of the plant interaction niche. Further, we show that interaction generalism had opposite effects on AMF and bacterial biomass in soils. Below, we discuss these results in detail and explore how plant interaction niches for AMF may impact ecosystem C cycling. We found remarkable similarity in plant species' interaction niches in experiments 1 and 2, supporting our first hypothesis, despite that different edaphic conditions and AMF inoculum pools used in the two experiments generated substantial differences in the taxonomic composition of AMF communities of plant species. Although rhizosphere AMF communities respond to edaphic conditions (Davison et al., 2021 ) and available AMF pools (Van Geel et al., 2018 ), the stability of plant interaction niches suggests that plants exhibit fundamental interaction niches for AMF. This observation is consistent with findings from plant‐pollinator networks, where species retain their interaction niches moving from their native to alien ranges (Emer et al., 2016 ). However, given that only eight plant species in two experiments were compared, further work is needed to confirm the general stability of plant interaction traits for AMF. Nonetheless, our findings suggest that plant interaction generalism for AMF could serve as a useful functional trait (Funk et al., 2017 ) for understanding how interactions with soil organisms drive ecosystem processes. Our multidimensional approach to interaction generalism allowed us to resolve niche partitioning among generalist hosts for AMF partners. We found that generalists partitioned interaction trait space through variation in numeric and phylogenetic AMF diversity, which likely involves distinct trade‐offs. Niche partitioning may occur as plants select the most beneficial AMF partners (Werner & Kiers, 2015 ) or interact with AMF exhibiting diverse nutrient acquisition strategies (Powell & Rillig, 2018 ). Thus, plant niche partitioning for AMF partners may significantly contribute to maintaining ecosystem functional diversity (Dehling et al., 2021 ), enhancing ecosystem resilience to environmental change (Turnbull et al., 2016 ). We found some support for our second hypothesis that plant interaction generalism for AMF is positively related to AMF biomass in the rhizosphere. However, the increase in soil AMF biomass was driven by the phylogenetic α‐diversity aspect of host interaction generalism. Phylogenetically diverse AMF communities are linked to higher variability in traits like hyphal growth (Hart & Reader, 2002 ) and nutrient acquisition (Horsch et al., 2023 ). This may suggest that complementarity among AMF taxa increased C allocation to the rhizosphere. Alternatively, interaction generalists hosting diverse AMF taxa may have been less able to downregulate C flow to less favorable mutualists (Grman, 2012 ) making them more susceptible to cheaters (Kiers & Denison, 2008 ). Indeed, the significant positive effect of β(CU), which reflects heterogeneity of AMF among replicates of a host species, supports the idea that generalist hosts may have been less selective for beneficial AMF. Root biomass, rather than AMF biomass in roots, was an important covariate. While root traits (e.g., diameter, branching) influence plant interaction niches for AMF (Bergmann et al., 2020 ; Ramana et al., 2023 ), our results likely reflect that plants with higher root biomass provide more habitat for AMF (Sweeney et al., 2021 ). Greater habitat availability can reduce competition, favoring higher AMF diversity (Bergmann et al., 2020 ; Mony et al., 2021 ). Given the role of AMF in C sequestration into the soil organic C pool (Zhu & Miller, 2003 ), the relationship between soil AMF biomass and the phylogenetic diversity aspect of plant interaction generalism highlights the importance of generalist plants in regulating C flux between the atmosphere and biosphere. Contrary to our third hypothesis, we found that interaction generalist plants were associated with lower bacterial biomass in rhizosphere soils. The interactions between plants, AMF, and bacteria in the hyphosphere and rhizosphere are complex, with both plants and AMF releasing compounds that can affect bacterial taxa either positively or negatively (Bharadwaj et al., 2012 ; Changey et al., 2019 ). Furthermore, AMF and soil bacteria often compete for resources, and AMF can outcompete bacteria in the rhizosphere as AMF hyphae can significantly reduce bacterial access to nutrients (Bukovská et al., 2018 ). Indeed, the effect of AMF on bacteria strongly depends upon the nutrient status of the host plant and AMF (Huang et al., 2023 ; Lanfranco et al., 2018 ). The positive effect of nutrient limitation on plant C allocation to mycorrhizas is well known (Huang et al., 2023 ). Under nutrient‐limited conditions, plants hosting large AMF communities may generate strong competitive effects on rhizosphere bacteria. In our study, nutrient limitation was likely, as mesocosms consisted primarily of sand with only small amounts of field soil as inoculum and no mineral nutrient supplementation. Despite ample light, the relatively small plant size at harvest suggests nutrient stress. Root and shoot biomass were significant covariates in our bacterial biomass model, indicating that larger plants were associated with larger bacterial communities. Together, these findings suggest that competition between AMF and bacteria for C limited bacterial biomass in our study. We sought a better understanding of plant interaction niches for AMF and their effects on soil microbial biomass. We demonstrate that, despite variation in environmental conditions, plant interaction niches for AMF were stable relative to other plants in their community. This aligns with niche theory and other studies of plant functional traits (Funk et al., 2017 ) and interaction traits in other types of networks (Emer et al., 2016 ). However, under field conditions, we expect realized plant–AMF interaction niches to be shaped by various filters on community assembly including biotic and stochastic factors like priority effects and plant–soil feedbacks (HilleRisLambers et al., 2012 ). Under the nutrient‐limited conditions of our experiments, we found that plants with high phylogenetic interaction generalism were associated with higher soil AMF biomass, while high numeric interaction generalism was linked to lower bacterial biomass, suggesting strong AMF‐bacterial competition for C in the rhizosphere. These findings align with well‐described patterns in community and ecosystem ecology, such as greater fungal‐to‐bacterial biomass (Wardle et al., 2004 ) and plant‐mycorrhizal dependence (Huang et al., 2023 ) under nutrient limitation. Nonetheless, over 50% of the variance in AMF and bacterial biomass remains unexplained, suggesting that other factors may also play important roles. We propose that plant interaction niches for AMF are a promising new avenue to enhance understanding of how plant traits alter key ecosystem functions, such as C cycling."
} | 4,449 |
37619982 | PMC10476156 | pmc | 1,729 | {
"abstract": "Abstract Microbial strategies for resource use are an essential determinant of their fitness in complex habitats. When facing environments with multiple nutrients, microbes often use them sequentially according to a preference hierarchy, resulting in well-known patterns of diauxic growth. In theory, the evolutionary diversification of metabolic hierarchies could represent a mechanism supporting coexistence and biodiversity by enabling temporal segregation of niches. Despite this ecologically critical role, the extent to which substrate preference hierarchies can evolve and diversify remains largely unexplored. Here, we used genome-scale metabolic modeling to systematically explore the evolution of metabolic hierarchies across a vast space of metabolic network genotypes. We find that only a limited number of metabolic hierarchies can readily evolve, corresponding to the most commonly observed hierarchies in genome-derived models. We further show how the evolution of novel hierarchies is constrained by the architecture of central metabolism, which determines both the propensity to change ranks between pairs of substrates and the effect of specific reactions on hierarchy evolution. Our analysis sheds light on the genetic and mechanistic determinants of microbial metabolic hierarchies, opening new research avenues to understand their evolution, evolvability, and ecology.",
"introduction": "Introduction When presented with an environment with multiple nutrients, many microbes tend to use them one at a time in a preferred order. This phenomenon of hierarchical substrate use was famously characterized by Monod in the decade of 1940 ( Monod 1942 ) when he coined the term “diauxie” to describe the experimentally observed double growth curve pattern. Despite the foundational role of Monod's work in molecular biology ( Jacob and Monod 1961 ; Belliveau et al. 2018 ), we still know surprisingly little about the evolution and diversity of resource hierarchies across bacterial species ( Perrin et al. 2020 ). For instance, are some hierarchies easier to evolve than others? And what determines how easy it is to evolve a preference for a given substrate? The questions about the ecology and evolution of metabolic hierarchies have received renewed attention in recent years ( Bajic and Sanchez 2020 ; Okano et al. 2021 ), as part of the ongoing effort to understand the drivers of microbial community assembly and coexistence ( Chang et al. 2022 ; Estrela et al. 2022 ; Gralka et al. 2022 ; Schäfer et al. 2023 ). Recent theoretical work ( Posfai et al. 2017 ; Goyal et al. 2018 ; Pacciani-Mori et al. 2020 ; Wang et al. 2021 ; Bloxham et al. 2023 ) and experiments with model communities ( Pacciani-Mori et al. 2020 ; Bloxham et al. 2022 ) have shown that differences in metabolic hierarchies can impact ecology, for example by allowing species to segregate their metabolic niches and avoid competition. Although these studies carry the implicit assumption that metabolic preferences will readily diversify provided the ecological opportunity (e.g., in an environment with multiple nutrients), it is unclear in which cases this assumption will hold true. Systematic empirical analyses are lacking, and available evidence remains scarce and anecdotal. For example, the deep conservation of some preferences, for example the almost universal preference for glucose in fermentative microbes ( Görke and Stülke 2008 ), would suggest that metabolic hierarchies are hard to rewire. If this is the case, we would expect metabolic hierarchies to be deeply conserved in the phylogenetic tree and act as a mechanism of coexistence only between distantly related species. However, other studies report divergent resource preferences in closely related species ( Tuncil et al. 2017 ), implying that metabolic hierarchies can quickly diversify. In this scenario, we might expect resource hierarchies to promote the coexistence of closely related strains and possibly also lead to eco-evolutionary feedbacks ( Bajić et al. 2018 ; Pacciani-Mori et al. 2020 ). Thus, in order to better understand the role of metabolic hierarchies in structuring coexistence within microbial communities, it is imperative to understand in a systematic way their potential to diversify. A central determinant of the evolution of biological systems is the underlying genotype–phenotype (G–P) map ( Fontana and Schuster 1998 ; Stadler et al. 2001 ). The architecture of this map determines the amount of phenotypic variation that can be accessed via mutations, which ultimately fuels evolution. Even in the presence of selection, evolution often follows lines of “least genetic resistance” ( Schluter 1996 ), which are in principle determined by the G–P map. In the case of metabolic traits, the structure of the metabolic network is a central determinant of this map. The structure of metabolism has been shown to influence the evolution of individual enzymes ( Papp et al. 2004 ; Vitkup et al. 2006 ; Notebaart et al. 2014 ; Aguilar-Rodríguez and Wagner 2018 ), metabolic innovation ( Barve and Wagner 2013 ), or eco-evolutionary interactions ( Bajić et al. 2018 ). Importantly, recent work has demonstrated that the structure of the metabolic network is also a key determinant of the strategy microbes adopt in mixed substrate environments, for example their choice to use them sequentially versus simultaneously ( Wang et al. 2019 ). Because preferential substrate use represents an optimal metabolic strategy ( Salvy and Hatzimanikatis 2021 ), the presence of one pathway or another will fundamentally determine which substrates an organism prefers, as different pathways have a different balance of benefits and costs when processing a substrate ( Noor et al. 2016 ; Waschina et al. 2016 ; Wortel et al. 2018 ). However, how precisely the structure of the metabolic networks determines and constrains the evolution of metabolic hierarchies remain unexplored. Here, we asked how the metabolic G–P map determines the evolutionary flexibility of microbial metabolic hierarchies using genome-scale metabolic modeling. Metabolic modeling techniques such as Flux Balance Analysis (FBA) enable accurate predictions of metabolic phenotypes from genotypes ( Orth et al. 2011 ; Bordbar et al. 2014 ; O’Brien et al. 2015 ) and are widely used as a workhorse for the comprehensive exploration of metabolic G–P maps ( Segrè et al. 2005 ; Barve and Wagner 2013 ; Notebaart et al. 2014 ; Szappanos et al. 2016 ; Goldford et al. 2017 ). Using FBA, we first show that a handful of “typical” resource hierarchies appear much more commonly across genotype space than other configurations. Hierarchies are easier to rewire for substrates that are more metabolically different. However, their evolutionary flexibility strongly depends on the presence or absence of a small number of central metabolic reactions that determine the behavior of the metabolic network across substrates. Our study provides the first systematic analysis of how metabolic networks determine the evolution of metabolic hierarchies, and we end by proposing new testable hypotheses, null expectations, and potential directions for future studies.",
"discussion": "Discussion The strategies that microorganisms use to metabolize resources have a significant impact on their interactions and coexistence ( Bajic and Sanchez 2020 ; Estrela et al. 2022 ). Theoretically, having different preferences for the same substrates could enhance biodiversity by allowing temporal niche segregation ( Goyal et al. 2018 ; Bloxham et al. 2023 ). However, how easily microbial populations evolve alternative metabolic hierarchies remains unclear. In this study, we utilized genome-scale metabolic modeling to investigate how the structure of empirical metabolic G–P maps affects the evolution and diversity in the hierarchical use of sugars by microbes. Our findings indicate that the architecture of the metabolic network only permits the evolution of a limited set of hierarchy configurations. Moreover, the evolution of alternative strategies is restricted to substrates that can be processed through substantially different reactions and pathways. Overall, these findings suggest that the diversity of optimal metabolic hierarchies in natural populations may be in general limited. The evolutionary flexibility of metabolic hierarchies (within the available alternatives) depended on a small set of reactions belonging to central metabolic pathways. From a genetic point of view, these reactions appear as strongly pleiotropic, as they are often active across different sugars. Because of this, they introduce strong genetic correlations in the growth of different sugars ( Falconer and Mackay 1996 ), preventing to an extent the independent variation in their growth rates and thus “locking” the evolution of metabolic hierarchies. At the same time, these central metabolic reactions act as evolutionary modifiers—capacitors and potentiators that modulate the ability of the genetic system to generate variability and fuel evolution ( Rutherford and Lindquist 1998 ; Bergman and Siegal 2003 ; Richardson et al. 2013 ; Geiler-Samerotte et al. 2019 ; Poyatos 2020 ). One prediction of this result is that sugar hierarchies will show different degrees of conservation across different clades, depending on the architecture of their central carbon metabolism. This suggests a possible explanation to why some phylogenetically distant species show similar metabolic preferences (e.g., the almost universal preference for glucose over other sugars [ Monod 1942 ; Görke and Stülke 2008 ]), whereas at the same time for some species and substrates, we find differences between closely related species ( Tuncil et al. 2017 ). Going beyond these anecdotal cases and understanding the evolution and conservation of resource hierarchies will require a more systematic empirical approach. From an ecological standpoint, our observation that some pairs of resources (e.g., glucose–fructose) are much more easily rewired than others (e.g., glucose–galactose), leads to the experimentally testable prediction that coexistence will evolve more often in environments containing the former than the latter ( Bloxham et al. 2022 ; Bloxham et al. 2023 ). Altogether, we believe that our computational results provide a useful guide and well-defined set of expectations for future empirical studies. A key assumption of our study is related to optimality in cell behavior. First, FBA operates under a strong assumption of optimality: phenotypes are predicted by assuming that the kinetic parameters of the metabolic enzymes and their regulation are optimal in a particular environment. Although this is generally accepted as a valid approximation ( Dykhuizen et al. 1987 ; Elena and Lenski 2003 ; Dekel and Alon 2005 ; Schuetz et al. 2007 ; Schuetz et al. 2012 ), it might not be accurate across all conditions ( Towbin et al. 2017 ). Additionally, we assumed that metabolic hierarchies mirror the hierarchy of growth rates. This is reasonable given previous empirical studies ( Aidelberg et al. 2014 ) and, more generally, fits the established view that sequential substrate use represents an optimal “economic” strategy that maximizes the benefit obtained from the investment of costly cellular resources in processing a substrate ( Beg et al. 2007 ; Kremling et al. 2015 ; de Groot et al. 2019 ; Wang et al. 2019 ; Salvy and Hatzimanikatis 2021 ). However, there are possible exceptions to this rule ( Okano et al. 2021 ), for example if cells have evolved mechanisms to “prepare” for environmental uncertainty at the cost of optimality in certain environments ( Schmidt et al. 2016 ; Balakrishnan et al. 2021 ). Deviations from optimality might be especially strong in organisms in which nonmetabolic functions (e.g., motility, biofilm formation, persistence) constitute major components of fitness. An important caveat of our method is the inability to consider the effect of regulatory mutations. For example, E. coli implements the preference of some substrates over others through repression of their respective operons at different cAMP (cyclic adenosine monophosphate) concentration thresholds ( Okano et al. 2020 ). Mutations in this regulatory system, for example promoter mutations changing the binding strength of the repressor, could therefore represent targets in the evolution of metabolic hierarchies. However, these repression thresholds typically evolve to implement a hierarchy matching the growth rates supported by the substrate. In other words, regulation does not define which metabolic strategy is optimal but evolves as a means to implement it. We might therefore expect that regulatory mutations driving the hierarchy away from growth optimality will be typically purged by selection (given that regulatory mechanisms evolve fast compared with their regulation targets ( Lozada-Chávez et al. 2006 ; Price et al. 2008 ; Aguilar-Rodríguez et al. 2017 ). However, exceptions to this rule may emerge under certain ecological contexts. For example, regulatory rewiring to prefer suboptimal resources may evolve when resources are supplied sequentially in a nonoptimal order, or when an ecological competitor is able to monopolize the most optimal resource. We want to note that a core result of this work, the strong bias toward a specific set of hierarchies in the genotype, was obtained using a method that relies only on stoichiometry. Our intuition is that other factors (e.g., thermodynamics, regulatory suboptimality) should overall only additionally constrain the available phenotypes but never “free” them from the yoke imposed by stoichiometry. If this logic is true, our work is providing only a null baseline for phenotypic bias in metabolic hierarchies, which might be in reality even stronger. In summary, our study describes with mechanistic detail how the metabolic G–P map influences and constrains the evolution of microbial metabolic hierarchies. This mechanistic perspective has proven to be essential in advancing the field of evolutionary biology, as well as other disciplines ( Wagner et al. 2000 ; de Visser et al. 2003 ). Future research will be required to explore how the patterns and mechanisms outlined in our study contribute to the phylogenetic and ecological distribution of microbial metabolic hierarchies, as well as their implications for natural and synthetic communities."
} | 3,611 |
35126046 | PMC8811373 | pmc | 1,732 | {
"abstract": "Realization of spiking neural network (SNN) hardware with high energy efficiency and high integration may provide a promising solution to data processing challenges in future internet of things (IoT) and artificial intelligence (AI). Recently, design of multi-core reconfigurable SNN chip based on resistive random-access memory (RRAM) is drawing great attention, owing to the unique properties of RRAM, e.g., high integration density, low power consumption, and processing-in-memory (PIM). Therefore, RRAM-based SNN chip may have further improvements in integration and energy efficiency. The design of such a chip will face the following problems: significant delay in pulse transmission due to complex logic control and inter-core communication; high risk of digital, analog, and RRAM hybrid design; and non-ideal characteristics of analog circuit and RRAM. In order to effectively bridge the gap between device, circuit, algorithm, and architecture, this paper proposes a simulation model—FangTianSim, which covers analog neuron circuit, RRAM model and multi-core architecture and its accuracy is at the clock level. This model can be used to verify the functionalities, delay, and power consumption of SNN chip. This information cannot only be used to verify the rationality of the architecture but also guide the chip design. In order to map different network topologies on the chip, SNN representation format, interpreter, and instruction generator are designed. Finally, the function of FangTianSim is verified on liquid state machine (LSM), fully connected neural network (FCNN), and convolutional neural network (CNN).",
"introduction": "Introduction The success of artificial intelligence technology represented by deep neural network (DNN) today depends heavily on the development of big data and chip technology. However, the problem of DNN lies in its massive parameter volume, leading to high energy consumption. Therefore, people are exploring energy-efficient artificial intelligence algorithms. Inspired by the characteristics of biological brain, such as asynchrony, and being event driven, spiking neural networks (SNNs) are considered to have the potential to realize ultra-low power intelligent computation ( Maass, 1997 ). At present, SNNs can achieve similar results with DNNs in some small-scale applications, based on various training algorithms, e.g., BP ( Esser et al., 2015 ), spike-timing-dependent plasticity (STDP) ( Neftci et al., 2014 ), and network conversion ( Diehl et al., 2015 ). In addition, compared with DNN, SNN is better at processing spatiotemporal information. For example, the performance of SNN in tasks, e.g., dynamic gesture recognition and language feature extraction, is equivalent to DNN with the same structure ( Blouw et al., 2019 ). In order to run SNN efficiently, dedicated chips with asynchronous operation and being event driven have been widely studied. In the early stage of SNN chip research, analog circuit was used to realize a neuron model ( Silver et al., 2007 ) and cooperate with digital communication systems, such as network-on-chip (NoC) to realize the chip ( Boahen, 2006 ; Schemmel et al., 2008 ; Qiao et al., 2015 ). Although this chip runs SNN with low power consumption, its function is very limited, and it is mostly used to study a small-scale brain model. In recent years, people use advanced semiconductor technology and advanced asynchronous circuit design technology to realize large-scale integrated SNN chips. These chips not only have high energy efficiency but can also realize complex neuron models and a variety of synaptic plasticity. Good results have been obtained in the fields of handwritten character recognition, dynamic vision sensor (DVS) gesture recognition, and small sample gas classification ( Akopyan et al., 2015 ; Davies et al., 2018 ; Deng et al., 2020 ). However, due to the limitations of complementary metal oxide semiconductor (CMOS) technology and circuit design methods, both the analog–digital hybrid SNN chip and SNN chip based on asynchronous circuit are far from the scale of biological brain. To further increase the energy efficiency and integration of the chip, researchers have focused on emerging RRAM ( Strukov et al., 2008 ). At present, small-scale SNN based on RRAM array has been verified ( Fang et al., 2020 ; Lu et al., 2020 ; Shi et al., 2021 ). Owing to its multi-level resistance states, RRAM is also used to realize the SNN chip with in situ learning ability ( Jo et al., 2010 ). However, current RRAM-based SNN chips are small-scale and designed for fixed network structures. It is still challenging to realize large-scale reconfigurable chips based on RRAM. The origins are the non-ideal characteristics of RRAM, and the complexity of cross-level design and optimization (device-circuit-architecture-algorithm) restricts the performance of networks. Solving the above problems not only depends on the continuous improvement of the RRAM device and circuit design but also heavily depends on the development of simulation tools for cross-level design and optimization. In order to break the barrier between SNN algorithm, hardware architecture, circuit and RRAM devices, some simulation tools have been developed, such as MNSIM for the behavior level modeling of device and circuit ( Xia et al., 2017 ). The simulator can estimate the area, power consumption, and delay of the RRAM chip according to the actual process, but this work lacks the research on the architecture and algorithm levels. NeuroSim and some works based on it ( Chen et al., 2018 ; Peng et al., 2019 ; Wu et al., 2019 ) and Neurosim+ ( Chen et al., 2017 ) provide modeling from device level to circuit and algorithm level. However, the tool directly jumps from circuit level to algorithm level. Although it complements the discussion of algorithm, it lacks the ability to analyze chip architecture. PIMSim ( Xu et al., 2018 ) provides a tool to understand configurable PIM. It supports a variety of PIM models, supports instruction execution, and simulates the PIM system from the system architecture level. However, these works still have two disadvantages. One is the lack of support for SNN algorithm, and the other is the inability to carry out clock cycle level simulation, so it is impossible to explore the impact of pulse transmission delay on the network. For some NPU simulators, the software and hardware collaborative design language SystemC ( Panda, 2001 ) is used to model the circuit architecture, and system architecture simulation at the clock level is realized, such as NN-Noxim ( Chen and Wang, 2018 ), Noxim ( Catania et al., 2015 ), etc. However, SNN and RRAM are not included in these works. According to the requirements of designing large-scale reconfigurable SNN chip based on RRAM, we provide a simulator FangTianSim that can conduct behavior level modeling for RRAM-based SNN architecture. The simulator can conduct behavior level modeling for NoC, spiking neurons, and RRAM arrays. Operation speed, delay, and function can be simulated in the digital domain with accuracy to the clock cycle level. This tool can save a lot of time and simulation resources and guide chip design effectively. The contributions of this paper mainly include a clock cycle level simulator for RRAM-based SNN chip architecture, power consumption analysis method based on actual process, and network parsers and instruction generation tools for a variety of SNN architectures."
} | 1,868 |
28280778 | PMC5324081 | pmc | 1,734 | {
"abstract": "Artificial cells\ncapable of both sensing and sending chemical messages\nto bacteria have yet to be built. Here we show that artificial cells\nthat are able to sense and synthesize quorum signaling molecules can\nchemically communicate with V. fischeri , V. harveyi , E. coli , and P. aeruginosa . Activity was assessed by fluorescence, luminescence, RT-qPCR, and\nRNA-seq. Two potential applications for this technology were demonstrated.\nFirst, the extent to which artificial cells could imitate natural\ncells was quantified by a type of cellular Turing test. Artificial\ncells capable of sensing and in response synthesizing and releasing N -3-(oxohexanoyl)homoserine lactone showed a high degree\nof likeness to natural V. fischeri under specific\ntest conditions. Second, artificial cells that sensed V. fischeri and in response degraded a quorum signaling molecule of P. aeruginosa ( N -(3-oxododecanoyl)homoserine\nlactone) were constructed, laying the foundation for future technologies\nthat control complex networks of natural cells.",
"conclusion": "Conclusions Our incomplete understanding of basic biochemical\nprocesses limits\nwhat can be built. Although we succeeded in assembling several different\nquorum pathways, the cycle of sensing and responding was only fully\nreconstituted for V. fischeri . One critical difficulty\nwas the reconstitution of active sensing systems, even if the sensing\nmechanisms of the transcriptional activators and repressors were thought\nto be known. 9 Conversely, every cell-free,\nquorum molecule synthesis pathway tested was functional. Although in vivo experiments are indispensable to the study of biology, in vivo experiments alone are often not sufficient to identify\nall of the molecular components needed for activity. Only by reconstituting\na fully functional system in vitro can we begin to\nunderstand how the pieces fit together. 36 − 39 Such an approach can extend beyond\nthe characterization of individual biomolecules and pathways to our\nunderstanding of cellular life. In other words, we likely will not\nunderstand what is needed to make something alive until we can build\na living cell from individual component parts. This requires an identification\nof the necessary genes and cytoplasmic components needed to synthesize\na functioning cell from DNA. 40 Impressive\nprogress has been made in synthetic genomics, 31 , 41 but the resulting living systems still depend on many genes with\nunknown function and many unidentified factors present in the living\ncell that receives the synthetic genome. The artificial cells described\nhere suffer from similar complications; extract compositions are not\nfully known, and it is not currently possible to express in\nvitro functioning translation machinery. 35 Removing these unknowns is necessary to build artificial\ncells that more fully break from the concept of vivum ex vivo . Building a fully defined artificial cell from scratch would lead\nto a much deeper understanding of life. A cellular Turing test can\nhelp guide progress toward such a goal.",
"introduction": "Introduction Artificial cells are encapsulated chemical\nsystems that mimic cellular\nlife. Most attempts at making artificial cells have focused on building\nsome type of self-replicating system. 1 , 2 Although self-replication\nis an important feature of life as we know it, self-replication alone\nis an insufficient criterion for assessing how lifelike a chemical\nsystem is. 3 For example, cross-catalytic\nribozyme ligases are capable of self-replication 4 but do not alone constitute a living system. What is lacking\nis some sort of metric by which progress can be measured. One solution\nmay be to describe chemical systems on a continuum where the typical\nbinary categorization of alive and not alive is replaced by states\nthat are increasingly lifelike. In this way, each iteration of constructing\nan artificial cell could be objectively and quantifiably evaluated\nin terms of likeness to a target natural cell. Such an approach is\nintuitive, because the emergence of life on Earth did not occur in\na single event, but likely encompassed a series of steps, each bringing\nthe chemical system closer to what is recognized as living today. 5 , 6 It was previously suggested that a type of imitation game\ncould\nbe used to guide the construction of artificial cells in a way that\nbypasses the problems associated with a lack of a definition of life. 7 In the original imitation game (or Turing test),\nthe ability of a machine to deceive a judge (or interrogator) through\ntextual communication into believing that the machine is a person\nwas used to circumvent the problem of defining intelligence. 8 In the cellular version, the ability of an artificial\ncell to deceive a natural cell is used to evaluate the artificial\ncell. Such a cellular Turing test is possible, because all cells communicate,\nfrom quorum sensing pathways in bacteria to pheromone responses in\nhigher organisms. 9 Further, artificial\ncells containing DNA and/or transcription–translation machinery\ncan express genes, 10 , 11 send chemical messages to bacteria, 12 , 13 and interact with each other. 14 Additionally,\ngenetic constructs in water-in-oil emulsion droplets are able to either\nsense or send quorum molecules. 15 Therefore,\nit should be possible to build genetically encoded artificial cells\nthat can chemically communicate with bacteria. Since chemical communication\nleads to measurable changes in gene expression, next generation sequencing\ntechnologies can be used to quantifiably evaluate the extent of mimicry\nin a manner that is neither subjective nor binary. In other words,\nthe cellular Turing test allows for the quantification of how lifelike\nthe artificial cells are in comparison to a target living cell in\na stratified manner.",
"discussion": "Results and Discussion Artificial Cells Can Sense\nBacteria To build artificial\ncells that mimic the ability of natural cells to chemically communicate,\nwe attempted to reconstitute the well characterized quorum sensing\npathways of Vibrio fischeri , Pseudomonas\naeruginosa , and Escherichia coli in vitro . Genetic constructs were assembled with genes coding\nfor the quorum responsive transcriptional activator or repressor plus\nadditional accessory factors, as needed, and a transcriptional regulator\nbinding site upstream of a gene encoding a fluorescent protein. In\nthis way, the activity of each pathway could be assessed by the fluorescence\narising from in vitro transcription–translation\nreactions. The N -3-(oxohexanoyl)homoserine lactone\n(3OC6 HSL) responsive system from V. fischeri was\nfunctional in vitro ( Figure S1a,b ). GFP expression in the presence of 10 μM 3OC6 HSL was 4-fold\ngreater than in the absence of this quorum signal. Since the same\ntranscriptional activator can sense another quorum molecule ( N -octanoyl- l -homoserine lactone or C8 HSL) secreted\nfrom V. fischeri, ( 16 ) responsiveness\nto C8 HSL was assessed. Although the affinity of the transcriptional\nregulator LuxR for C8 HSL was low, a higher affinity mutant version\nof the protein (T33A S116A S135I LuxR or LuxR*) 17 activated cell-free expression 7-fold in the presence of\nC8 HSL and 6-fold in the presence of 3OC6 HSL ( Figure S1a,b ). The ability to sense 3OC6 HSL could be removed\nby introducing an additional M65R substitution, as previously reported. 17 Next, two P. aeruginosa quorum\npathways were tested, including the N -(3-oxododecanoyl)homoserine\nlactone (3OC12 HSL) responsive LuxR and the N -butanoylhomoserine\nlactone (C4 HSL) responsive RhlR pathways. As previously observed, 18 the genetic construct containing lasR was responsive to the quorum signal 3OC12 HSL in vitro , showing a 2-fold increase in protein expression ( Figure S1c ). However, the RhlR dependent system showed indistinguishable\nactivity in the presence and absence of C4 HSL ( Figure S1d ). Finally, the autoinducer-2 (AI-2) system from E. coli was tested. While the expression of the transcriptional\nrepressor LsrR fully inhibited protein expression, none of the tested\nconstructs were derepressed by AI-2 ( Figure S1e ). The inclusion of the cAMP receptor protein (CRP) did not sufficiently\nimprove derepression ( Figure S1f ). In summary,\n3OC6 HSL, C8 HSL, and 3OC12 HSL were successfully detected by in vitro transcription–translation reactions. Each functioning quorum sensing\npathway was then encapsulated within\ncholesterol containing 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine\n(POPC) phospholipid vesicles to determine whether quorum molecules\ncould diffuse across the phospholipid membrane and activate gene expression\nof living cells. Here, activation resulted in the expression of firefly\nluciferase instead of GFP. Vesicles were incubated at 37 °C for\n5 h in the presence and absence of the quorum molecule. The vesicles\nwere then broken with Triton X-100 in the presence of luciferin and\nimmediately measured for luminescence. Only in the presence of 3OC6\nand C8 HSL was luminescence observed, indicating that the signaling\nmolecules crossed the phospholipid membrane and activated gene expression\n( Figure S2a,b ). 3OC6 HSL was previously\nshown to diffuse through the oil phase of water-in-oil emulsion droplets. 15 Together, these results suggested that artificial\ncells should be able to sense quorum molecules that are naturally\nsecreted from bacteria. To demonstrate that the sensing mechanism\nof artificial cells was capable of responding to V. fischeri , the supernatant of a V. fischeri culture was added\nto the suspension of vesicles. After 4 h of incubation, 69-, 19-,\nand 8-fold more luminescence was observed for artificial cells expressing\nLuxR, LuxR*, and M65R LuxR*, respectively, in response to the supernatant\nof V. fischeri than in the absence of the supernatant\n( Figure 1 ). The use\nof the supernatant of a V. fischeri culture removed\nthe confounding effects of the natural luminescent properties of the\nbacterium itself. The data supported the ability of artificial cells\nmade of phospholipid vesicles and transcription–translation\nmachinery to sense molecules secreted from natural cells. Figure 1 Artificial\ncells can sense quorum molecules released by natural\ncells. Artificial cells (AC) encoding either LuxR or LuxR* were able\nto sense the presence of V. fischeri . Negative control\nreactions were the artificial cells in the absence of the supernatant\nfrom V. fischeri ( n = 3 biological\nreplicates, mean ± SD). The schematic shows V. fischeri (teal, oblong) releasing quorum molecules that are sensed by artificial\ncells (gray, circle). RLU (relative luminescence units). Artificial Cells Can Synthesize and Send\nQuorum Molecules to\nNatural Cells Since communication requires the ability to\nboth receive and send messages, we next probed whether it was possible\nto build artificial cells that could send chemical messages to bacteria\nin the form of quorum molecules. Genetic constructs encoding the synthesis\nmachinery necessary to send chemical messages to V. fischeri , P. aeruginosa , and E. coli were\nassembled. V. fischeri synthesizes the N -acylhomoserine lactone 3OC6 HSL through the activity of LuxI, which\nuses S -adenosylmethionine and acyl chains donated\nfrom acyl carrier proteins as reactants. 19 Similarly, P. aeruginosa synthesizes 3OC12 HSL\nthrough the activity of the LuxI homologue LasI. Additionally, P. aeruginosa synthesizes C4 HSL through a similar pathway\nthat uses RhlI in place of LasI. 20 The\nfunctionality of each genetic construct was assessed with reporter E. coli strains engineered to express GFP in response to\na specific quorum molecule. After 6 h of transcription–translation\nat 37 °C of each genetic construct, an aliquot was added to the\nreporter strain and analyzed by flow cytometry. The 3OC6 HSL, 3OC12\nHSL, and C4 HSL synthesis systems individually activated the expression\nof GFP of 90%, 50%, and 87% of the cells of the corresponding reporter\nbacterial strain, indicating that each genetically encoded quorum\nsynthesis system was functional in vitro ( Figure S3a ). The AI-2 synthesis pathway used\nby E. coli is different and depends on the activity\nof three enzymes. 21 The SAM-dependent methyltransferase\nconverts S -adenosylmethionine to S -adenosylhomocysteine, which is in turn converted to S -ribosylhomocysteine by the enzyme Pfs. Lastly, LuxS produces AI-2\nand homocysteine in a 1:1 ratio from S -ribosylhomocysteine.\nPfs and LuxS can be fused together to form a larger polypeptide that\nefficiently synthesizes AI-2 in the presence of S -ribosylhomocysteine. 22 , 23 We demonstrated that this fusion\nprotein was active after in vitro transcription–translation\nby detecting synthesized AI-2 with the luminescent reporter Vibrio harveyi BB170 ( Figure S3b ). To ensure that each synthesized quorum molecule could escape\nlipid vesicles, the transcription–translation reactions were\nplaced inside of vesicles. The loaded vesicles were mixed with reporter\nbacterial strains at 37 °C and analyzed by flow cytometry. Encapsulated\ngenetic constructs for the synthesis of 3OC6 HSL and 3OC12 HSL resulted\nin approximately 90% and 35%, respectively, of fluorescent cells after\n6 h of incubation, while the encapsulated C4 HSL synthesis system\nfailed to induce detectable fluorescence of the reporter strain ( Figure 2 a). Two mutated versions\nof LuxI were also evaluated in an attempt to identify more active\nversions of this 3OC6 HSL synthesizing enzyme. 24 Vesicles containing DNA encoding wild type LuxI, E34G E63G\nLuxI, and E34G E40G E63G LuxI (hereafter referred to as LuxI*) were\nincubated with a dilute culture of V. fischeri , and\nthe induced luminescence of V. fischeri was evaluated.\nAll three of the tested versions of LuxI induced similar levels of\nluminescence from V. fischeri ( Figure 2 b). The encapsulation of the genetically\nencoded AI-2 synthesis system resulted in the induction of luminescence\nof the AI-2 reporter strain of V. harveyi ( Figure 2 c). Therefore, the\ndata indicate that artificial cells can be built to synthesize and\nrelease 3OC6 HSL, 3OC12 HSL, and AI-2. To ensure that the vesicles\nused to build the artificial cells could withstand the presence of\nbacteria, the release of encapsulated fluorophore from vesicles incubated\nwith different bacteria was monitored. V. fischeri , V. harveyi , and E. coli did not\ndegrade the vesicles under the conditions used for chemical communication\nwithin 6 h, whereas the presence of the opportunistic pathogen P. aeruginosa resulted in the degradation of the vesicles\n( Figure S4a,b ). Figure 2 Artificial cells can\nsynthesize and release quorum molecules to\nnatural cells. (a) Artificial cells (AC) carrying genetic constructs\nfor the synthesis of 3OC12 HSL, 3OC6 HSL, and C4 HSL were incubated\nwith E. coli sensor strains and quantified by flow\ncytometry. (b) Artificial cells that expressed either LuxI, LuxI*,\nor E34G E63G LuxI for the synthesis of 3OC6 HSL successfully induced\nthe production of luminescence in V. fischeri . (c)\nArtificial cells that expressed the AI-2 synthesizing fusion protein\nHLPT (His 6 -LuxS-PfS-Tyr 5 ) 22 were incubated with V. harveyi and monitored\nby luminescence. For all the experiments, n = 3 biological\nreplicates, mean ± SD. RLU/CFU (relative luminescence units per\ncolony forming unit per milliliter). Artificial Cells Can Establish New Communication Networks between\nNatural Cells After demonstrating that the sensing and sending\nmodules were functional inside of lipid vesicles, we next constructed\nartificial cells that were able to sense a quorum molecule and in\nresponse synthesize and release another quorum molecule. When properly\nengineered, such artificial cells would be able to mediate communication\nbetween two organisms that do not naturally communicate with each\nother. Further, the activity of the artificial cell would be easy\nto evaluate since the confounding influences of natural quorum pathways\nwould be diminished. A genetic device that allowed for the synthesis\nof 3OC12 HSL in response to the presence of 3OC6 HSL was constructed.\nAn engineered E. coli sensor strain for 3OC12 HSL\nwas used as the receiver cell to avoid the cytotoxic effects of P. aeruginosa . The supernatant of V. fischeri was mixed with artificial cells and the E. coli reporter strain for 3OC12 HSL. 20% of the reporter strain expressed\nGFP, indicating that E. coli received a chemical\nmessage from the artificial cells in response to the 3OC6 HSL secreted\nby V. fischeri ( Figure 3 a,c). In the absence of the supernatant of V. fischeri , the artificial cells showed no activity. When\nthe gene coding for the enzyme that synthesizes 3OC12 HSL was replaced\nby the fusion protein that produces AI-2, the resulting genetic circuit\ndid not mediate communication with V. harveyi ( Figure S5a,b ). Figure 3 Artificial cells mediate communication\nbetween two different cell\ntypes. (a, b) A schematic of the experimental setup. (c) Communication\nbetween V. fischeri and engineered E. coli mediated by artificial cells was assessed by flow cytometry. (d,\ne) Artificial cells sense V. fischeri and in response\ndegrade the 3OC12 HSL released by P. aeruginosa .\nQuantification was with an E. coli reporter strain\nby flow cytometry. For all the experiments, n = 3\nbiological replicates, mean ± SD. AC indicates artificial cells. Artificial cells can be designed\nto disrupt the natural quorum\npathways of P. aeruginosa . Acylhomoserine lactones\nare degraded by the Bacillus thuringiensis enzyme\nAiiA. 25 After confirming that in\nvitro expressed AiiA was functional ( Figure S6a ), artificial cells were built to constitutively\nexpress AiiA so that the quorum molecules secreted by P. aeruginosa would be degraded. The LasR sensor for 3OC12 HSL was not encoded\nwithin the genetic content of the artificial cells since the membrane\nitself could serve as the sensor, that is, the membrane was disrupted\nby P. aeruginosa . When artificial cells expressing\nAiiA were incubated with P. aeruginosa , the extracellular\nlevels of 3OC12 HSL were significantly reduced. In fact, in the absence\nof artificial cells, 90% of the E. coli reporter\nstrain sensed 3OC12 HSL, whereas, in the presence of the artificial\ncells, only 18% of the reporter cells were activated ( Figure S6b ). Next, a 3OC6 HSL and C8 HSL responsive\nversion of the artificial cells was prepared so that the signaling\nfrom one type of cell could result in the quenching of communication\nof another type of cell. A genetic construct expressing AiiA under\nthe control of LuxR* allowed the artificial cells to decrease extracellular\n3OC12 HSL by 95% in the presence of V. fischeri ( Figure 3 b,d,e). Although\nmore work would be needed to convert such artificial cells into a\nuseful technology, including the development of a membrane that can\nwithstand P. aeruginosa , the data show that artificial\ncells could be built to interfere with biofilm formation in response\nto chemical signaling from another natural cell, since biofilm formation\nis strongly influenced by quorum signaling. However, more is possible.\nEngineered living cells have already been embedded in the gut microbiota 26 and developed to treat inflammatory bowel disease 27 and psoriasis, 28 and\nto suppress appetite. 29 Such technologies\navoid flooding the organism with drug molecules, since therapeutic\nagents are only synthesized and released when and where needed. Artificial\ncells could do the same but within a more controllable chassis that\ndoes not replicate nor evolve. 13 Artificial\nCells Capable of Two-Way Communication Can Be Quantified\nby a Cellular Turing Test Having established that artificial\ncells can sense quorum molecules that are naturally secreted from\nbacteria, send chemical messages to natural bacteria, and mediate\ncommunication between two different bacterial species, we next sought\nto evaluate how lifelike such artificial cells are through a cellular\nTuring test. Therefore, artificial cells were constructed that could\nchemically communicate in a manner similar to V. fischeri . Four different genetic constructs that included the wild type or\nmutant versions of the receptor LuxR and the synthase LuxI were tested\n( Figure S7a ). Artificial cells were added\nto a low density culture of V. fischeri exhibiting\nlow luminescence and incubated for 3 h at 30 °C. The artificial\ncells containing DNA encoding LuxR* and LuxI* induced the greatest\nluminescent response per colony forming unit (CFU) and thus were best\nable to chemically communicate with V. fischeri ( Figure S7b,c ). Since the artificial cells could\nnot replicate, the CFU solely reflected the number of viable natural\ncells. The extent of communication was influenced by the lipid composition\nof the membrane of the artificial cells, consistent with the diffusion\nof molecules across intact membranes ( Figure S8 ). Further, identical reactions that were not encapsulated in vesicles\nwere not able to engage in chemical communication with V.\nfischeri under the experimental conditions employed ( Figure S8 ). The experiment was then repeated\nwith the optimized genetic sequence so that the same samples could\nbe evaluated by luminescence, RT-qPCR, and RNA sequencing. The luminescence\ndata ( Figure 4 b) was\nconfirmed by RT-qPCR ( Figure 4 c), which showed that the expression of luxA and luxB was similarly upregulated 5-fold both\nfor communication mediated by artificial cells and for natural V. fischeri-V. fischeri communication. luxA and luxB were previously shown to be upregulated\nby 3OC6 HSL. 30 Figure 4 Two-way chemical communication\nfor a cellular Turing test. (a)\nA schematic of the experimental setup showing chemical communication\nbetween V. fischeri and functional artificial cells\n(top, green), nonfunctional artificial cells (middle, black), and V. fischeri (bottom, magenta). Nonfunctional artificial\ncells could sense the presence of the quorum molecules released by V. fischeri and in response express T7 RNA polymerase, i.e.,\na response that had no bearing on V. fischeri . (b)\nLuminescence of V. fischeri in response to functional\nand nonfunctional artificial cells. (c) The activation of luxAB was assessed by RT-qPCR. Gene expression with respect\nto the negative control ( V. fischeri in the presence\nof nonfunctional artificial cells) is shown. (d) RNA-seq analysis\nof the lux operon for communication between V. fischeri and nonfunctional artificial cells, V. fischeri , and functional artificial cells. For all the\nexperiments, n = 6 biological replicates; mean ±\nSD. AC (artificial cells), FPKM (fragments per kilobase of transcript\nper million mapped reads), RLU/CFU (relative luminescence units per\ncolony forming unit per milliliter). RNA-seq can be used to quantify the extent to which artificial\ncells mimic natural cells. Although the luminescence and RT-qPCR data\ndemonstrated that the artificial cells behaved at some level as natural\ncells, such data were clearly not sufficient to determine if the artificial\ncells were alive or not. To more quantitatively assess the performance\nof the artificial cells, the gene expression profile of natural cells\nin response to the activity of artificial cells was evaluated. Six\nreplicates of the cellular Turing test were subjected to RNA-seq analysis.\nIncubation of V. fischeri with nonfunctional artificial\ncells resulted in 175 differently expressed coding sequences with\nrespect to the undiluted, V. fischeri – V. fischeri communicating sample ( Tables S1 and S2 ). Nonfunctional artificial cells contained transcription–translation\nmachinery plus DNA encoding LuxR and T7 RNA polymerase under the control\nof a LuxR-responsive promoter. That is, nonfunctional artificial cells\ncould sense quorum molecules but could not respond by synthesizing\nquorum molecules. The same experiment in the presence of functional\nartificial cells containing DNA encoding LuxR* and LuxI* showed 107\ndifferently expressed coding sequences ( Tables S1 and S3 ), meaning that the functional artificial cells better\nmimicked the influence of natural V. fischeri on V. fischeri than nonfunctional artificial cells. Although\nthe RNA sequencing analysis, after false discovery rate (FDR) p value adjustment, did not identify statistically significant\ndifferences in the expression of the lux operon in\nresponse to functional and nonfunctional artificial cells, the increase\nin the number of reads from the six RNA-seq samples ( Figure 4 d) was similar to the activation\nmeasured by RT-qPCR ( Figure 4 c). In other words, although all of the comparisons had a\nFDR adjusted p value >0.05, the data were consistent\nwith RT-qPCR data with p values of 0.0001 and 0.0006\nfor luxA and luxB , respectively.\nFurther, the expression over the entire lux operon,\nwith the exception of luxI and luxR , was more similar between natural V. fischeri and\nfunctional artificial cells than with nonfunctional artificial cells\n( Figure 4 d). The luxI and luxR data were more difficult\nto interpret since these two genes were present in both V.\nfischeri and the functional artificial cells. A correlation\nbetween the gene expression profile of V. fischeri in response to nonfunctional and functional artificial cells showed\nthat six of the seven genes of the lux operon fell\noff the correlation trend ( Figure S9a ),\nsuggesting that the critical difference between the two types of artificial\ncells was their effect on quorum signaling, as expected. Additionally,\nthe difference in the number of reads between V. fischeri – V. fischeri compared with V. fischeri –nonfunctional artificial cell and V. fischeri –functional artificial cell samples showed that the functional\nartificial cells better mimicked the effect on gene expression across\nthe entire genome than the nonfunctional artificial cells ( Figure S9b ). It is possible to calculate\nhow lifelike the artificial cells are\nfrom the RNA-seq data. The nonfunctional artificial cells changed\nthe expression of 175 coding sequences differently than V.\nfischeri . An artificial cell that functioned identically\nto V. fischeri would have induced zero differences\nin gene expression. If we consider the nonfunctional artificial cells\nas having 0% likeness to V. fischeri , then any reduction\nin the number of differences in gene expression would increase the\ndegree of likeness of the artificial cell to V. fischeri . Such a calculation would indicate that the artificial cells here\nwere 39% lifelike or V. fischeri -like ([(175 –\n107)/175] × 100), but this value is clearly an overestimation\nbecause only two of the necessary components of the artificial cell\nwere genetically encoded (LuxR* and LuxI*). The remaining components\ncame from an extract of E. coli that was used to\nmediate transcription and translation. Engineered and naturally reduced\nbacterial genomes require over 100 genes to produce their transcription–translation\nmachinery. In fact, the percentage of reduced genomes dedicated to\ngene expression is similar to the 39% lifelike value calculated here.\nFor example, 41% of the synthetically produced, reduced Mycoplasma\nmycoides genome (i.e., JCVI-syn3.0) is necessary for gene\nexpression. 31 Similarly, one-third of the\nnaturally reduced genomes of parasitic microorganisms, including Sulcia muelleri , Carsonella ruddii , and Buchnera aphidicola , are retained for gene expression. 32 − 34 In other words, the data only make sense when put into the context\nof the entire genetic system required to support the synthesis of\nRNA, protein, and the products of protein enzymes, in this case quorum\nmolecules. It thus follows that, even if it were possible to assemble\nan artificial cell containing a genome that can make its own transcription–translation\nmachinery 35 plus additional genes for quorum\nsignaling, this artificial cell would still not pass the 50% mark\nwith respect to V. fischeri . That is, it is more\naccurate to say that if the artificial cells used here were completely\ngenetically encoded, then these artificial cells would be 39% V. fischeri -like, according to the described cellular Turing\ntest. As the complexity of artificial cells increases, more\nstringent\nversions of a cellular Turing test that better capture lifelike activity\ncan be built. Here, the artificial cells were mixed with natural V. fischeri at an OD of 0.2–0.3 and incubated for\n3 h at 30 °C before analysis. Under these conditions, replication\nwas not required and the artificial cells did not need to survive\nfor very long. A more stringent version of the cellular Turing would\nmix artificial cells with more dilute cultures of V. fischeri , or another target cell type, and would be assessed for activity\nafter longer lengths of time. Such artificial cells would be capable\nof replication, which would also lead to daughter vesicles containing\na greater fraction of machinery encoded within its own genome, as\nopposed to components purified from bacteria. It should be emphasized\nthat such cellular Turing tests are not meant to function as a definition\nof life, but rather as a way to circumvent the problems associated\nwith defining life. The choice of quorum signaling may appear arbitrary,\nparticularly since not all organisms engage in quorum signaling, but\nall organisms do sense and respond to their chemical environment and\ninteract with each other in some way that is processed on a chemical\nlevel. A version of the cellular Turing test described here may not\nbe applicable to all organisms, but this test does provide an objective\nmetric that does not emerge from qualitative lists of lifelike properties."
} | 7,424 |
37893277 | PMC10608997 | pmc | 1,735 | {
"abstract": "Memristor crossbar arrays are a promising platform for neuromorphic computing. In practical scenarios, the synapse weights represented by the memristors for the underlying system are subject to process variations, in which the programmed weight when read out for inference is no longer deterministic but a stochastic distribution. It is therefore highly desired to learn the weight distribution accounting for process variations, to ensure the same inference performance in memristor crossbar arrays as the design value. In this paper, we introduce a design methodology for fault-tolerant neuromorphic computing using a Bayesian neural network, which combines the variational Bayesian inference technique with a fault-aware variational posterior distribution. The proposed framework based on Bayesian inference incorporates the impacts of memristor deviations into algorithmic training, where the weight distributions of neural networks are optimized to accommodate uncertainties and minimize inference degradation. The experimental results confirm the capability of the proposed methodology to tolerate both process variations and noise, while achieving more robust computing in memristor crossbar arrays.",
"conclusion": "6. Conclusions This paper proposes a novel design methodology based on a Bayesian neural network to improve the fault tolerance of neuromorphic computing systems. The proposed methodology creatively combines the variational Bayesian inference technique with a fault-aware variational posterior distribution to accommodate the underlying deviations in memristors through one BNN training process. The experimental results show that the proposed framework can effectively rectify accuracy losses of up to ∼40% due to process variations and stochastic noise without involving expensive retraining and remapping.",
"introduction": "1. Introduction Neuromorphic computing represents an exciting and promising approach to artificial intelligence (AI) that models its elements on the human brain and nervous system. Instead of relying on a central processing unit (CPU) like traditional von Neumann architectures, neuromorphic systems distribute computations across interconnected nodes, inspired by the remarkable capabilities of the human brain. This parallelism enables efficient and real-time processing of data, making it well suited to tasks such as image and speech recognition [ 1 , 2 ]. Recent research in the field of non-volatile memory technologies has enabled memory devices to tune their conductance and store multi-level states [ 3 , 4 ] such as resistive RAM (ReRAM), etc. On this basis, the utilization of memristor crossbar arrays to speed up matrix–vector multiplication (MVM) operations holds great promise for building energy-efficient and high-performance neural network architectures. However, the operations of a memristor crossbar array are indeed susceptible to various non-ideal effects due to its analog computing nature. These non-ideal effects can arise from several aspects, such as driver/sensing resistance, analog-to-digital converter (ADC) and digital-to-analog converter (DACs) non-linearity, process variations, and other physical phenomena. The above-mentioned faults can be further categorized to the device or circuit level [ 5 , 6 ]. This paper mainly focuses on device-level fault sources, including process variations [ 7 , 8 ], analog fluctuations, and noise during read and write operations [ 3 , 9 , 10 ]. The stochastic behavior exhibited by memristor devices can introduce deviations in its conductance, which then inevitably affect the reliability of the computations performed on the whole memristor crossbar array. Researchers are actively exploring multiple approaches to enhance the overall performance of memristor-based analog computing systems, such as redundancy and error correction [ 11 ], adaptive algorithms [ 12 ], statistical analysis and modeling [ 13 ], and even their combination. Process variations are inherent in nano-technology manufacturing processes, and can be decomposed into global and local variations. Global components are shared by all the memristors on a die. Local variation is related to the proximity effect and results in variation correlation of the devices across the array. Their combined impact makes neural network parameters heavily deviate from their expected values, thus making the hardware inference performance also significantly deviate from the software validation accuracy. As shown in Figure 1 , when mapping a neural network model onto one or more memristor crossbar arrays, the weights of the model are typically represented by the conductance values of the memristors. However, the programmed weights stored in the memristors may deviate from the expected values, i.e., the actual synapse weight may follow a distribution instead of a point value; thus, the inference accuracy cannot reach the same level as in training. From a system designer’s perspective, an as good as possible inference performance is always desired to maximize the benefit of the underlying system. Note that improving the fault tolerance of memristor crossbar arrays is an ongoing research area. Advancements in device fabrication, circuit design, and algorithmic techniques are necessary to develop effective solutions that mitigate the impact of conductance variations and enhance the fault tolerance of memristor-based neuromorphic computing systems. In this work, we propose a novel design methodology for fault-tolerant neuromorphic computing to address the weight disturbance problem without involving either redundancy design or adaptive retraining/remapping. In particular, by leveraging the probabilistic nature of Bayesian inference, we employ a Bayesian neural network (BNN) architecture to adjust the synapse weights of memristors, accounting for process variations. Specifically, we first model the synapse weights as probability distributions considering the effects of both process variations and noise, i.e., the programmed memristor conductances follow a log-normal distribution [ 12 ]. On this basis, we introduce a priori from the same distribution family to estimate the optimal posterior distributions over network parameters that can accommodate uncertain and stochastic behavior in memristor conductance."
} | 1,570 |
39050655 | PMC11268936 | pmc | 1,736 | {
"abstract": "Abstract Transcriptome data are frequently used to investigate coral bleaching; however, the factors controlling gene expression in natural populations of these species are poorly understood. We studied two corals, Montipora capitata and Pocillopora acuta , that inhabit the sheltered Kāne'ohe Bay, Hawai'i. M. capitata colonies in the bay are outbreeding diploids, whereas P. acuta is a mixture of clonal diploids and triploids. Populations were sampled from six reefs and subjected to either control (no stress), thermal stress, pH stress, or combined pH and thermal stress treatments. RNA‐seq data were generated to test two competing hypotheses: (1) gene expression is largely independent of genotype, reflecting a shared treatment‐driven response (TDE) or, (2) genotype dominates gene expression, regardless of treatment (GDE). Our results strongly support the GDE model, even under severe stress. We suggest that post‐transcriptional processes (e.g., control of translation, protein turnover) modify the signal from the transcriptome, and may underlie the observed differences in coral bleaching sensitivity via the downstream proteome and metabolome.",
"conclusion": "4 CONCLUSIONS Our findings broaden understanding of coral gene expression and demonstrate that two different coral species have divergent responses to stress vis‐à‐vis gene expression. P. acuta and M. capitata in Kāneʻohe Bay do not share a broadly conserved transcriptome response to stress across genotypes, which does not support the TDE model (Cziesielski et al., 2019 ) as defined here (Figure 2a ). Although we are unable to test for genotype‐by‐treatment effects, we find that genotype and ploidy drive genome‐wide gene expression variation much more than treatment (i.e., the GDE model, Figure 2b,c ) in these two species. These results provide a novel perspective, yet there are many coral genes whose expression has been shown to be stress‐responsive, including those that are “front‐loaded” and may confer physiological resilience under frequent stress exposure (Barshis et al., 2013 ; Fifer et al., 2021 ). Therefore, RNA‐seq data is undoubtedly informative about coral stress responses (Cleves, Shumaker, et al., 2020 ). The impact of genotype on gene expression is also not surprising, and likely reflects local adaptation of the parental lines, likely prior to transport to Kāneʻohe Bay (Dixon et al., 2015 ; Kenkel & Matz, 2016 ). Coral genotype in our model Hawaiian species clearly modulates the transcriptomic response to stress despite a convergence in observed stress response (Figure 1c,d ). Whereas the observed stress response could be due to genotype‐by‐treatment interactions, that is, every genotype has a different path to the same phenotype, an alternative but not mutually exclusive hypothesis is that post‐transcriptional processes may modify the signal from the transcriptome to stress‐related outcomes that we have observed. Proteome and metabolome data (e.g., Camp et al., 2022 ; Pei et al., 2022 ; Williams, Chiles, et al., 2021 ) may therefore be better proxies for coral physiology. This idea remains to be tested in future studies that target a range of different coral species from different geographic origins. Given these observations, our results underline the importance of characterizing genetic structure and reproductive behavior of corals to allow informed interpretation of “omics” data. The underpinnings of coral resistance and resilience involve not only algal symbiont and prokaryotic microbiome contributions but also fundamentally reflect host genotype(s) and their provenance (Dixon et al., 2015 ; Fuller et al., 2020 ). This is particularly pertinent with the acceleration of the search for transferrable mechanisms for use as human interventions to assist coral resistance and resilience and maintain these invaluable coral reef ecosystems under increasing global change.",
"introduction": "1 INTRODUCTION Coral reef ecosystems are built on the nutritional symbiosis between scleractinian coral hosts and their single‐celled dinoflagellate (algal) endosymbionts in the family Symbiodiniaceae (Davies et al., 2023 ; LaJeunesse et al., 2018 ). These ecosystems are increasingly at risk from impacts caused by climate change, including ocean acidification (Orr et al., 2005 ), rising sea surface temperatures, as well as more frequent and intense marine heatwaves (Cheng et al., 2023 ). Thermal stress is a leading cause of coral mass mortality worldwide, resulting in dysbiosis between algal endosymbionts and the coral host, which can lead to the expulsion of algae from coral tissues (i.e., “bleaching”; Hughes et al., 2017 ). Ocean acidification, alternatively, places a chronic energetic strain on the coral host by increasing the cost of maintaining homeostatic processes under altered acid–base balance (Chille et al., 2022 ). A recent meta‐analysis shows that the interactive effects of ocean acidification and thermal stress on corals are primarily additive, resulting in increasing bleaching severity and mortality (Klein et al., 2022 ). However, the range of responses to these stressors varies greatly both within and between coral species (Baird & Marshall, 2002 ; Burgess et al., 2021 ; Guest et al., 2012 , 2016 ; Mydlarz et al., 2010 ; Strand et al., 2024 ) and genotypes (Dixon et al., 2015 , 2018 ; Poquita‐Du et al., 2020 ). Understanding the range of coral responses to warming and acidifying oceans is of critical importance (Eakin et al., 2022 ). Yet, this remains a challenging area of research because of the genetically diverse collection of organisms (cnidarian animal host, algal symbionts, prokaryotic microbiome, fungi and other eukaryotes, and viruses) that comprise the coral holobiont and contribute to its health and resilience (Cumming et al., 2023 ; National Academies of Sciences, Engineering, and Medicine, 2019 ; Veron, 2011 ). Understanding the mechanisms underlying coral bleaching and the genomic basis for adaptive capacity has primarily been explored using transcriptome (RNA‐seq) data (Cziesielski et al., 2019 ; Kenkel & Wright, 2022 ; Young et al., 2023 ). However, these information‐rich datasets are often impacted by factors that can hinder straightforward interpretation, including host taxonomy and genotype, natural history, geographic origin, algal symbiont composition, and coral developmental stage (Ruggeri et al., 2022 ; Thomas et al., 2018 ). Understanding the factors that control gene expression, and therefore the bleaching response of corals in natural populations is crucial for inferring the correlation between these sources of information. To investigate the link between coral host natural history, genotype, and the abiotic stress response vis‐à‐vis gene expression, we compared RNA‐seq data from Pocillopora acuta , a species that spreads primarily clonally, to Montipora capitata , a species that is an obligate outbreeder (Stephens et al., 2023 ). We analyzed RNA‐seq data from Stephens et al. ( 2023 ) of 119 P. acuta corals from Kāne'ohe Bay, Hawai'i (Figure 1a,g ), which were randomly distributed in tanks that simulated thermal and/or low pH stress conditions. Post‐hoc analysis of single‐nucleotide polymorphism (SNP) data from the studied P. acuta colonies identified eight main genotypes (encompassing 113/119 [94.96%] of the samples) that comprise distinct diploid and triploid clonal lineages that have arisen in the bay via asexual larval propagation or fragmentation (Figure 1b,d ; Stephens et al., 2023 ). Therefore, we had the unique opportunity to assess intra‐ and inter‐genotype gene expression variation under abiotic stress, the effects of ploidy on this process, and to test the hypothesis that different genotypes of naturally derived clonal groups share gene regulation patterns for stress‐response genes. We also analyzed RNA‐seq data from 132 haphazardly sampled M. capitata colonies (Figure 1f,h ), collected from the same reefs as the P. acuta samples, that comprise 130 distinct, diploid genotypes (Caruso et al., 2022 ; Stephens et al., 2023 ). The M. capitata colonies were subjected to the same abiotic stressors as P. acuta , allowing us to determine if a population‐wide core set of gene expression patterns exist, while controlling for genotype. Our work is buttressed by the availability of a chromosome‐level reference assembly for Hawaiian M. capitata (Stephens et al., 2022 ) and a high‐quality draft assembly of P. acuta from a triploid individual collected in Kāne'ohe Bay (Stephens et al., 2022 ) that acted as the reference genomes for these analyses, and physiological data (e.g., color score, chlorophyll content, symbiont cell density, photosynthetic rates, total antioxidant capacity) that were collected in parallel (from different samples) during this experiment (Strand et al., 2024 ). These authors provided evidence that the M. capitata holobiont is more thermally resistant (Figure 1e ) and dominated by Durusdinium spp. algal endosymbionts, whereas P. acuta is relatively more thermally sensitive (Figure 1c ) and dominated by Cladocopium spp. endosymbionts. Both species showed a significant physiological response to temperature, but not pCO 2 treatment. Differences in host sensitivity to thermal stress appeared to be driven by elevated baseline photosynthetic rates in P. acuta and lower antioxidant capacity. Important for this study, because both species showed clear physiological responses to temperature, we were able to investigate the RNA‐seq data under two competing “strawman” models: (1) gene expression is largely independent of genotype, reflecting a shared response driven by physiological response to treatment (Treatment‐Driven Expression, TDE [e.g., Kenkel & Wright, 2022 ]) or, (2) genotype dominates gene expression, regardless of physiological response to treatment (Genotype‐Driven Expression, GDE). FIGURE 1 Experimental context for Pocillopora acuta and Montipora capitata RNA‐seq analyses. (a) Aerial image of Kāne'ohe Bay, O'ahu, Hawai'i with the six reefs where the coral samples were collected highlighted using yellow circles and labels (image modified from https://dlnr.hawaii.gov/ ). Proportional representation of the three studied clonal lineages of P. acuta (see key in bottom left) at each collection site is shown with the pie charts. These results do not provide evidence of genetic structure within and between the sampled reef areas. HIMB is the Hawai'i Institute of Marine Biology located on Moku o Lo'e. The legend for distance in feet is shown at the bottom left of the image. (b) Heatmap adapted from Stephens et al. ( 2023 ) showing P. acuta colony relatedness (based on Manichaikul et al., 2010 ) using all pair‐wise combinations of RNA‐seq data. The diploid and triploid groups are shown with green and red branches, respectively. The major clonal groups studied in the current paper are marked, with Clonal Groups 2 and 3 being triploid, and Clonal Group 6 being diploid. The eight major clonal groups that were identified by Stephens et al. ( 2023 ) are shown in different colors at the bottom of the heatmap. There is a single diploid P. acuta embedded among the triploids, which may be a case of reversion to the sexual state in this individual (for details, see Stephens et al., 2023 ). (c) Summary of color change results of P. acuta from Strand et al. ( 2024 ). Color change results indicate visual bleaching state and were quantified using color‐standardized photographs (Edmunds et al., 2003 ; Williams, Chiles, et al., 2021 ). The dashed vertical line indicates a return to ambient temperature between weeks 8 and 9. No results were collected from week 5. Significant effects from Strand et al. ( 2024 ) Type III ANOVA of linear mixed model outputs are listed in the bottom left of the panel. (d) PCA of P. acuta sample relatedness adapted from Stephens et al. ( 2023 ), whereby samples are colored by clonal group, showing the three analyzed in this study along with the other samples that were generated from Kāne'ohe Bay but were not analyzed here. A legend describing each color is shown in the bottom right of the panel. These plots are based on the covariance matrix produced by PCAngsd with estimated individual allele frequencies and show PC1 (17.54% variance explained) and PC2 (14.66% variance explained). (e) Summary of color change results of M. capitata from Strand et al. ( 2024 ). (f) PCA showing Stephens et al. ( 2023 ) results for M. capitata sample relatedness, with outliers removed, whereby samples are colored by clonal group (top) and reef (bottom). All samples in the clonal plot (top) are gray circles because no clonal groups were included in this analysis. (g) Two abutting colonies of the coral, P. acuta and (h) a large colony of M. capitata . Images taken by D. Bhattacharya.",
"discussion": "3 RESULTS AND DISCUSSION Transcriptome data are frequently used to investigate the genomic basis for coral bleaching. However, the factors controlling gene expression in natural populations of coral species are poorly understood. Here, we investigated the effect of ploidy and population genetic structure on gene expression in two sympatric coral species, the predominantly clonal P. acuta , and the predominantly outbreeding Montipora capitata (Stephens et al., 2023 ). Our study took advantage of an experimental system in which physiological (Strand et al., 2024 ) and RNA‐seq data (Stephens et al., 2023 ) were collected from both coral species during stress (high temperature, high pCO 2 [low pH], or both) and recovery. Given that both species exhibited a significant physiological response to thermal stress, but not to elevated pCO 2 (Strand et al., 2024 ), we investigated whether the gene expression response reflects physiological response to treatment, or whether it is more strongly influenced by genotype. Here, we show that even under severe stress, genotype, not treatment (i.e., the GDE model), drives genome‐wide gene expression variation in these two species. We hypothesize that post‐transcriptional processes (e.g., control of translation, protein turnover) may underlie the inter‐genotype and specific differences in coral bleaching sensitivity via the downstream proteome and metabolome. 3.1 Effects of treatment and genotype on global gene expression patterns in P. acuta \n For P. acuta , multiple genotypes representing extant Kāne'ohe Bay coral populations were exposed to abiotic stress (high temperature, high pCO 2 [low pH], or both). RNA‐seq (Stephens et al., 2023 ) and physiological data (Strand et al., 2024 ) were collected from 119 diploid and triploid colonies of P. acuta (the majority of which were clonally derived). Strand et al. ( 2024 ) showed that P. acuta exhibited a severe physiological response to the elevated temperature, marked by a decrease in color score (shown here in Figure 1c ), symbiont cell density, chlorophyll‐a, and respiration. Although physiological data were not taken from the same fragments as the RNA‐seq data, they were part of the same experiment, and because the fragments were randomly assigned (using R) to each dataset, the results from the physiology should be representative of the physiological state of the samples collected for RNA‐seq. Given the marked physiological response of the P. acuta exposed to elevated temperature, we asked whether a shared gene expression response to stress also exists within and between colonies from the three most abundant clonal genotypes (Groups 2, 3, and 6 [Figure 1d ]; which accounted for 85 of the 119 samples; Figure 1b ; Table 1 ). Stephens et al. ( 2023 ) showed that P. acuta clonal Groups 2 (34 colonies) and 3 (24 colonies) comprise distinct triploid lineages, whereas Group 6 (27 colonies) is a clonal diploid lineage (Figure 1d ), and that the triploid lineages are diverged both from each other and from the diploid lineage (Figure 1b ). We also expected limited confounding effects from collection site because these genotypes are distributed throughout Kāne'ohe Bay with no apparent fit to an isolation‐by‐distance model (Figure 1a , Stephens et al., 2023 ). Additionally, no significant gene expression response to mortality was observed (see Tables S10 and S11 , Figure S2 ). Therefore, these RNA‐seq data (comprising 21,048 expressed genes that passed low abundance filtering) allowed us to test the fit of the gene expression data to the TDE and GDE hypotheses, and to disentangle the effects of ploidy and genotype on gene expression. Given the marginal impact of high pCO 2 on P. acuta physiology when compared to thermal stress (Strand et al., 2024 ; Figure 1c ), we assessed the impact of this stressor on gene expression using PCA. Only 1.04% cumulative variation in gene expression across all significantly correlated principal components ( p ‐value < .05; Table 2 ) was found for pCO 2 , therefore our downstream analysis (see “Differential gene expression during heat stress”) targeted only the temperature treatment and not pCO 2 to test the competing TDE and GDE models. PCA was also used to test the fit of the competing TDE and GDE hypotheses. TABLE 1 Number of Pocillopora acuta colonies in each group per treatment. Treatment Group 2 (triploid) Group 3 (triploid) Group 6 (diploid) Grand total Ambient 16 11 14 41 Hot 18 13 13 44 Total 34 24 27 85 TABLE 2 Summary of Spearman's correlation coefficients and significance of sample attributes and principal components for Pocillopora acuta . \n \n \n Note : Only principal components significantly associated with sample attributes and with Spearman's correlation >|.2| are shown. Full results are available in Tables S2 and S4 . * p ‐value <.05; ** p ‐value <.01; *** p ‐value <.001. Under TDE (Figure 2a ), most of the variation that contributes to sample separation along theoretical PC1 would be attributable to treatment and most of the variation that contributes to sample separation along theoretical PC2, attributable to genotype. Under this scenario, P. acuta corals would share a conserved transcript‐based stress response that dominates the RNA‐seq data (see details below). Under GDE (Figure 2b ), most of the variation along theoretical PC1 would be attributable to genotype and most of the variation along theoretical PC2, attributable to treatment. Under both models, the amount of variation captured by PC1 is significantly higher than PC2, represented in Figure 2a,b by the relative font sizes of the axis labels. Given these scenarios, where do the P. acuta data fit? As is apparent in Figure 2c , the genome‐wide gene expression data overwhelmingly support the GDE model. The PCA shows that ploidy and genotype are highly correlated with PC1 (Genotype: r \n s = .9, p ‐value = 1.32E‐15, Ploidy: r \n s = −.81, p ‐value = 1.46E‐13, Table 2 ), which accounts for a significant proportion (24%) of the variation in the data, whereas PC7, which is the highest order component significantly correlated (albeit weakly) with treatment ( r \n s = .54, p ‐value = 16.43E‐07, Table 2 ), explains only 2.4% of data variation. The two triploid clonal genotypes (Groups 2 and 3) are positioned close together in the PCA, distinct from the diploids (Group 6). However, when considering only the two clonal triploid groups, Group 2 and Group 3 become greatly separated along PC1, which explains 28% of the variation in the data (Figure S1 ). This result demonstrates that coral colonies belonging to the same clonal group, despite originating from different reefs, have similar gene expression profiles, but the effect of ploidy (genome‐type) is stronger than genotype alone. Overall, these results show a weak genome‐wide shared transcriptomic stress response in P. acuta , which is consistent with the GDE model. That is, gene expression profiles observed in our experiment are primarily governed by genotype, as would be expected under GDE (Figure 2b ). FIGURE 2 Testing Treatment‐Driven (TDE) and Genotype‐Driven (GDE) models of coral gene expression. (a) Expected outcome of PCA under extreme cases of TDE and (b) GDE (see text for details). The size of the PC1 and PC2 legends in these cases reflect the amount of variation (i.e., larger text = greater variation) being explained by these components. A legend describing sample genotype and treatment for the TDE and GDE models is shown in (a). (c) The actual results of PCA using the Pocillopora acuta gene expression data from the three clonal lineages, which strongly supports the GDE model. Here we used PC1 and PC7, which were the principal components most highly correlated with genotype and treatment, respectively (Table 2 and Table S4 ). The colors used to denote the two triploid and one diploid lineage are consistent across images. Our results demonstrate that in P. acuta colonies, despite showing a significant physiological response to heat stress (i.e., bleaching [Figure 1c ], Strand et al., 2024 ), gene expression is primarily driven by genotype. These results are consistent with previous genotype‐controlled gene expression studies in Pocillopora acuta (Poquita‐Du et al., 2020 ), Acropora millepora (Dixon et al., 2015 , 2018 ), and Acropora cervicornis (Dilworth et al., 2024 ) that showed genotype explains a large proportion of variation in gene expression during thermal stress experiments (Dilworth et al., 2024 ; Dixon et al., 2015 ; Poquita‐Du et al., 2020 ) and reciprocal transplant experiments (Dixon et al., 2018 ). Therefore, we propose that the influence of genotype on gene expression is stronger than environment, that is, genotype‐by‐environment effects buffer the population‐wide transcriptomic response to stress. This leads to a central question: given that gene expression is presumed to respond to fluctuating environmental conditions, why does it not converge across genotypes under prolonged thermal stress, that in many cases leads to colony death in Pocillopora species (Baird & Marshall, 2002 ; Burgess et al., 2021 ; Guest et al., 2012 , 2016 ; Strand et al., 2024 )? One possible answer is that gene expression reflects past adaptive responses to local conditions that are not rapidly altered (e.g., “front‐loading”; Barshis et al., 2013 ). For this reason, downstream post‐transcriptional mechanisms such as differential access to the translation machinery, regulation of protein degradation, protein level buffering, and trans‐locus transcript abundance (Buccitelli & Selbach, 2020 ; Liu et al., 2016 ; Ponnala et al., 2014 ; Srivastava et al., 2022 ) may be critical to the coral stress response because they can be better tuned to local environmental conditions. These feedback systems (Arif et al., 2017 ; Kusnadi et al., 2022 ) will ultimately control enzyme abundance and activity (i.e., metabolite production that underlies the identified bleaching response). Our system is atypical in the respect that we have controlled for genetic background by exploiting the natural reproductive system of P. acuta in Kāneʻohe Bay, which relies on clonal propagation, allowing both diploids and triploids to spread throughout the region. Kāneʻohe Bay fits the “everything is everywhere” model (Caruso et al., 2022 ; Stephens et al., 2023 ) (Figure 1a ), resulting in a random distribution of coral larvae. Hence, we surmise that in this model system in which gene expression is controlled by genotype, post‐transcriptional regulatory mechanisms are likely critical to elicit the physiological response specific to different microhabitats. However, this pattern may not hold for other regions that show isolation‐by‐distance, whereby local adaptation is selectively advantageous for current and future generations. Understanding how these selective forces play out in different coral species and regions will likely be key to conservation efforts. 3.2 Effect of treatment on global gene expression patterns in M. capitata \n To test the GDE model in a second coral species, we analyzed RNA‐seq data from the sympatric M. capitata , which relies on a different reproductive strategy to ensure survival. This species (Figure 1h ) is relatively more resistant to thermal stress (Figure 1e ; Strand et al., 2024 , Williams, Pathmanathan, et al., 2021 , Williams, Chiles, et al., 2021 ) than P. acuta (see Figure 1c ) and is a hermaphroditic, mass‐spawning lineage. A strict outbreeder, M. capitata is essentially randomly distributed in Kāneʻohe Bay with respect to genotype (Figure 1f ; Caruso et al., 2022 ; Stephens et al., 2023 ). Under the same experimental conditions used for P. acuta , RNA‐seq data were generated from 132 colonies and the 22,587 genes that passed expression filtering were analyzed as described above. Support for the GDE model could not be explicitly tested because, except for four samples that likely originated via local fragmentation, each colony comprised a distinct genotype (Stephens et al., 2023 ). Therefore, we assessed fit to the TDE model, which these data do not support. Spearman correlations between the principal coordinates and sample attributes show very weak correlation of temperature with PC2 ( r \n s = .28, p ‐value = .0014) and PC8 ( r \n s = −.28, p ‐value = .0012), which explain 5% and 2% of variation in the gene expression data, respectively (Figure 3 ; Table S6 ), suggesting that stress treatment explains very little variation in these RNA‐seq data and does not fit the TDE model. FIGURE 3 Results of PCA (showing the two PCs with the highest correlation with treatment) using the Montipora capitata gene expression data. Although the GDE model could not be explicitly tested due to the low prevalence of clonal colonies, the results do not support the TDE model. A legend describing sample treatment is shown in the top right of the panel. 3.3 Differential gene expression during heat stress in P. acuta and M. capitata \n Under GDE, we expect a minor, albeit important treatment‐driven expression pattern. To determine which set of genes are driving the small difference between ambient and heat‐stressed corals, we performed differential gene expression analysis. M. capitata , which is comprised of distinct genotypes, provided a model in which we could search for the existence of a core set of population‐wide genes that are differentially regulated under stress in genetically heterogeneous colonies. Analysis conducted between heat‐stressed (HTAC) and non‐stressed (ATAC) M. capitata samples at 1‐week exposure revealed 31 differentially expressed (DE) genes out of the 18,225 that passed low‐abundance filtering (Table S8 ). Among the top over‐expressed genes under heat stress were paraspeckle component 1‐like isoform X2 (log 2 FoldChange 11.4, p \n adj < .01), which is associated with the formation of stress granules (An et al., 2019 ), and sacsin‐like (log 2 FoldChange 2.2; p \n adj < .01), which is a co‐chaperone of Hsp70 (Parfitt et al., 2009 ; Takahashi‐Kariyazono & Terai, 2021 ). These 31 genes likely represent a core set of genes that modulate the transcriptome‐based bleaching response in M. capitata . Given the number of clones identified from P. acuta , we were able to test whether genotypic effects abolish or reinforce the existence of a population‐wide set of stress‐responsive genes. In contrast to M. capitata , analysis conducted between HTAC and ATAC P. acuta samples at 1‐week exposure revealed no DE genes. These results are likely explained by the fact that genotypic variation in the expression of stress‐responsive genes exceeds treatment‐driven variation, because samples belonging to the same genotypes were present in both conditions. However, when testing for treatment effects between heat‐stressed (pooled HTAC+HTHC) and non‐heat stressed (pooled ATAC+ATHC) within a single genotype of P. acuta (Group 2, week 6), treatment‐driven expression was detected for 78 genes (out of the 17,981 that passed low‐abundance filtering; Table S9 ). Several of these DE genes in Group 2 have been identified in other coral heat‐stress studies, including gamma‐crystallin M1‐like (log 2 FoldChange 10.2, p \n adj < .01; Mayfield et al., 2018 ), 4‐hydroxyphenylpyruvate dioxygenase‐like isoform X2 (log 2 FoldChange 2.2, p \n adj < .01; Ip et al., 2022 ) and putative ammonium transporter 1 (log 2 FoldChange − 4.6, p \n adj < .01; Cleves, Krediet, et al., 2020 ; Williams, Pathmanathan, et al., 2021 ; Williams, Chiles, et al., 2021 ). Overall, these results show that whereas genome‐wide expression is driven primarily by genotype, treatment‐driven expression also occurs, but is dominated by genotypic variation. Therefore, the need to control and test for genotype and genotype‐by‐treatment interactions in future coral heat stress studies is paramount."
} | 7,222 |
34292745 | null | s2 | 1,737 | {
"abstract": "Gold is a critical resource in the jewelry and electronics industries and is facing increased consumer demand. Accordingly, methods for its extraction from waste effluents and environmental water sources have been sought to supplement existing mining infrastructure. Redox-mediated treatments, such as Fe(II)-based platforms, offer promise for precipitating soluble Au(III). We hypothesized that microbial generation of Fe(II) in the presence of sorbent metal-organic frameworks could capitalize on the advantages of both biological- and chemical-driven extraction approaches. Toward this aim, we tested Au(III) removal by "
} | 155 |
20463734 | PMC2872101 | pmc | 1,738 | {
"abstract": "Our ability to synthesize nanometer-scale particles with desired shapes and compositions offers the exciting prospect of generating new functional materials and devices by combining the particles in a controlled fashion into larger structures. Self-assembly can achieve this task efficiently, but may be subject to thermodynamic and kinetic limitations: Reactants, intermediates and products may collide with each other throughout the assembly timecourse to produce non-target instead of target species. An alternative approach to nanoscale assembly uses information-containing molecules such as DNA 1 to control interactions and thereby minimize unwanted crosstalk between different components. In principle, this method should allow the stepwise and programmed construction of target products by fastening individually selected nanoscale components – much as an automobile is built on an assembly line. Here, we demonstrate that a nanoscale assembly line can indeed be realized by the judicious combination of three known DNA-based modules: a DNA origami 2 tile that provides a framework and track for the assembly process, cassettes containing three distinct two-state DNA machines that serve as programmable cargo-donating devices 3 , 4 and are attached 4 , 5 in series to the tile, and a DNA walker that can move on the track from device to device and collect cargo. As the walker traverses the pathway prescribed by the origami tile track, it encounters sequentially the three DNA devices that can be independently switched between an ‘ON’ state allowing its cargo to be transferred to the walker, and an ‘OFF’ state where no transfer occurs. We use three different types of gold nanoparticles as cargo and show that the experimental system does indeed allow the controlled fabrication of the eight different products that can be obtained with three two-state devices."
} | 469 |
22549331 | null | s2 | 1,739 | {
"abstract": "The limitation of pH inside electrode-respiring biofilms is a well-known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode-respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm."
} | 301 |
39448370 | PMC11586630 | pmc | 1,740 | {
"abstract": "Abstract This publication highlights the latest advancements in the field of energy and nutrient recovery from organics rich municipal and industrial waste and wastewater. Energy and carbon rich waste streams are multifaceted, including municipal solid waste, industrial waste, agricultural by-products and residues, beached or residual seaweed biomass from post-harvest processing, and food waste, and are valuable resources to overcome current limitations with sustainable feedstock supply chains for biorefining approaches. The emphasis will be on the most recent scientific progress in the area, including the development of new and innovative technologies, such as microbial processes and the role of biofilms for the degradation of organic pollutants in wastewater, as well as the production of biofuels and value-added products from organic waste and wastewater streams. The carboxylate platform, which employs microbiomes to produce mixed carboxylic acids through methane-arrested anaerobic digestion, is the focus as a new conversion technology. Nutrient recycling from conventional waste streams such as wastewater and digestate, and the energetic valorization of such streams will also be discussed. The selected technologies significantly contribute to advanced waste and wastewater treatment and support the recovery and utilization of carboxylic acids as the basis to produce many useful and valuable products, including food and feed preservatives, human and animal health supplements, solvents, plasticizers, lubricants, and even biofuels such as sustainable aviation fuel. One-Sentence Summary Multifaceted waste streams as the basis for resource recovery are essential to achieve environmental sustainability in a circular economy, and require the development of next-generation waste treatment technologies leveraging a highly adaptive mixed microbial community approach to produce new biochemicals, biomaterials, and biofuels from carbon-rich organic waste streams.",
"conclusion": "Conclusion This paper clearly outlines the vast potential of organic rich waste utilization in bioenergy, biofuels, and biochemicals as a cornerstone for sustainable development. Organic waste from the food and beverage industry, high-strength wastewater, and marine seaweed biomass are valuable resources from a bioeconomy standpoint. Utilizing MMC as present in AD or wastewater treatment systems for bioenergy, biochemicals, and biofuels production from organic waste streams has enormous potential to reduce greenhouse gas emissions, decrease reliance on fossil fuels, promote sustainable waste management, and foster the transition to a circular economy. Pilot-scale results showed that an AM process has the potential for large-scale application to manage high-strength wastewater economically and environmentally friendly. AM is an exemplary waste-to-chemicals or waste-to-energy technology that transforms low- or negative-value waste streams into high-value bioproducts such as carboxylic acids, capitalizing on the carboxylate platform. This technology not only bypasses the generation of methane—a typical end product of AD—but also facilitates the production of valuable biochemicals and biofuels through metabolically versatile microbiomes. The strategic inhibition of methanogenesis to favour the production of SCCAs represents an innovative leap in optimizing the valorization of organic waste streams. The involved microbes demonstrate an impressive metabolic flexibility and, depending on their origin, can be adapted to a variety of waste streams for highly efficient MCCA production. Such developments not only promote the sustainability of industrial processes but also enhance economic outcomes by reducing operational costs and increasing the yield of marketable bioproducts. High purity carboxylic acids can be used ‘as is’ or as a platform chemical for producing chemicals and fuels, such as SAF, with a low carbon footprint. Carboxylate separation and purification cost can make up to 64% of total production cost since their separation and purification require energy- and chemical-intensive separation processes. A variety of low-cost and low carbon intensity separation technologies (resins, membrane, and electrochemistry-based technologies) instead of distillation reported promising results, effectively decreasing the separation and purification costs by 75%. However, more work is still needed to bring these technologies to an industrial scale. Biofilm technologies have introduced a robust platform for waste treatment, particularly in configurations such as dynamic MBRs. These systems exploit biofilms to enhance treatment efficiency and stability, facilitating the recovery of resources from wastewater and supporting the sustainability of water-intensive industries. The application of biofilms in waste treatment not only addresses the technical challenges of high-strength waste streams but also provides a scalable solution that can be integrated into existing infrastructure with minimal disruption. When it comes to understanding the complex structure, function, and regulation of biofilms and microbiomes involved, improved analytical methods are required to develop suitable targets and approaches for engineering the involved microbial consortia. Engineering multi-species biofilms like those present in wastewater systems or microbial conversion systems such as AM and wastewater treatment requires a detailed understanding of the pathways and regulatory system and the inter-species interactions, as well as associated biofilm formation. The energetic valorization of wastewater through advanced biogas technologies such as AnMBRs and the microbial upgrading of biogas to biomethane showcases the integration of waste treatment with energy recovery processes. These technologies enhance the efficiency of organic matter conversion and align with the objectives of reducing greenhouse gas emissions and utilizing renewable energy sources. In addition to improved product separation and purification techniques for carboxylates, this is another critical field for future research. A particularly innovative aspect covered in this paper is the treatment of recalcitrant waste streams, specifically through an AM process for the biosynthesis of SCCA from marine seaweed—a complex, and underutilized biomass resource. The anaerobic microbial pathways capable of breaking down the seaweed’s complex polysaccharide structure to produce valuable carboxylic acids highlight the potential for tapping into traditionally difficult-to-utilize waste streams. This approach not only enhances the economic viability of marine seaweed as low-cost biomass feedstock but also promotes sustainable waste management. In conclusion, this comprehensive review underlines a multi-dimensional strategy for waste management that harnesses technological innovations across several domains to transform waste into a resource, using MMC. This paradigm shift not only addresses the immediate challenges posed by increasing waste production and environmental degradation but also aligns with broader sustainability goals. The future of waste management, as suggested by this perspective, lies in the continued development and integration of these technologies into a coherent system that promotes environmental integrity, economic viability, and resource sustainability. As these technologies mature and scale, their integration into a global strategy for waste treatment and resource recovery will be pivotal in achieving a sustainable and circular bioeconomy.",
"introduction": "Introduction Reimagining our energy and carbon rich waste streams as a valuable resource can support overcoming current limitations with feedstock supply chains for biorefining approaches. Many current bio-based initiatives strongly rely on sugar-, lipid-, and starch-based input streams associated with land use change and the food/feed versus fuel debate. Extrapolations on how much carbon to build a future bioeconomy can be covered solely by sustainable biomass resources have their limitations. Although cascade utilization of the available biomass can counteract this limitation, the question remains where the necessary carbon should come from. A stepwise approach, where materials are the first priority, followed by chemicals production, and lastly fuel production, can enable a sustainable bioeconomy where the term ‘waste’ is redefined. Carbon waste streams are multifaceted and include municipal solid waste, industrial waste, agricultural by-products and residues, beached or residual seaweed biomass from post-harvest processing, and food waste. The food and beverage industry, including breweries, wineries, confectioners, and dairy producers, generates high-strength wastewater that requires treatment or costly disposal, varying by geography and quantity (Bochmann et al., 2020 ). Traditional treatment processes have low treatment efficiency or high operational costs and often ignore the economic potential of such carbon-rich waste streams. In a circular economy, resource recovery from waste streams is essential to achieve environmental sustainability. This requires the development of next-generation waste treatment technologies that produce new biochemicals, biomaterials, and biofuels from carbon-rich organic waste streams rather than simply disposing of them (Steinbusch et al., 2011 ; Tomás-Pejó et al., 2023 ). On top of solid waste streams, gaseous carbon sources have attracted scientific interest. Point source streams, including industrial emissions from the steel mill industry, CO 2 from biogas and bioethanol plants, as well as initiatives to concentrate CO 2 via Direct-Air Capture open up a new space for a gas fermentation platform. Gasification of various types of biomass allows for syngas production, introducing a new technology on its way to becoming a major contributor to the future bioeconomy. Companies like Lanzatech, its spinoff LanzaJet, Synata BIO—who incorporated Coskata, Inc.’s syngas conversion technology, INEOS Bio, and JUPENG BIO, have been developing gas fermentation technology to convert synthesis gas from low-cost feedstocks into high-value products (Heijstra et al., 2017 ; Köpke and Simpson, 2020 ; Benevenuti et al., 2021 ; Liew et al., 2022 ). These examples demonstrate the feasibility of reimagining waste streams as a highly valuable resource. Many current initiatives and future funding opportunities like the Sustainable Aviation Fuel (SAF) Grand Challenge Roadmap in the United States tackle challenges in sustainable fuel production and decarbonization efforts, particularly in hard-to-decarbonize sectors (Sustainable Aviation Fuel Grand Challenge Roadmap—Flight Plan for Sustainable Aviation Fuel, 2022 ). Developing a scalable, and robust bioconversion platform for carbon-rich waste streams contributes to advancing innovative energy technologies, facilitating the transition towards cleaner and more sustainable fuel production. Valorization of waste carbon streams into high-value and high-impact products as sustainable alternatives for the biomaterials, biochemicals, and biofuel sectors reduces environmental impact and promotes energy sustainability. In addition to reducing greenhouse gas emissions, bioconversion improves yield and energy efficiencies by using biogenic rather than fossil-derived inputs. This unique carbon benefit makes biomanufacturing at scale a more appealing alternative for producing most molecules, from the standpoint of CO 2 emissions. This perspective will highlight the latest advancements in the field of energy and nutrient recovery from municipal and industrial waste and wastewater. The emphasis will be on the most recent scientific progress in the area, the development of new and innovative technologies including microbial processes and the role of biofilms in the degradation of organic pollutants in wastewater, the production of biofuels and value-added products from wastewater and organic waste streams with a focus on carboxylic acids production via anaerobic digestion (AD). The authors acknowledge that other cutting-edge approaches such as gas fermentation strategies mentioned above will play a major role in the future circular economy that is based on waste carbon substrates. This highly important topic however, deserves in depth discussion elsewhere. Next-Generation Waste Treatment for Carbon-Rich Organic Waste Streams Industries such as breweries, wineries, confectioners, slaughterhouses, renderers, and dairies generate voluminous amounts of high-strength wastewater that often require a tipping fee for disposal, varying by geography and quantity. For example, the production of 1 kg of cheese can generate 9–10 l of wastewater (Pires et al., 2021 ), and the production of 1 l of beer can generate 3–10 l of wastewater (Chen et al., 2016 ). The high chemical oxygen demand (COD) (15–110 g COD/l) of high-strength wastewater necessitates adequate treatment prior to disposal, but current waste treatment processes have low treatment efficiency or high operation costs. Resource recovery from high strength organic wastewater not only allows the extraction of value-added products and offsets the operational costs of wastewater treatment, but it is also conducive to alleviating adverse environmental issues. The biodegradation of complex high-strength wastewaters requires a highly diverse microbial community structure. AD is an effective treatment process to convert large amounts of organic waste streams such as carbohydrate, protein, and lipid-rich wastewaters and food waste into high value renewable fuels (e.g., renewable methane) and products (e.g., volatile fatty acids) (Holtzapple et al., 2022 ). The mixed anaerobic microbial consortium or microbiome can have a significant biological diversity and syntrophic relationships, which enable the integration of multiple metabolic pathways from different kinds of microorganisms. Mixed microbial communities (MMC) are preferable to pure strains because the diversity of metabolic activities allows adaptation to varied operating conditions and complex feedstocks (Wu et al., 2021a ). Hydrolytic bacteria, acidogenic bacteria, acetogens, and methanogens are the main microbial communities responsible for AD of organic wastes. The first stage of AD is hydrolysis—complex and large organic molecules (proteins, carbohydrates, and lipids) are broken down into small compounds (amino acids, sugars, and long chain fatty acids) by microbes. In the second stage, acidogenesis, microorganisms convert the small molecules into volatile fatty acids, which are organic acids—also classified as short-chain (2–4 carbons) and medium-chain (5–8 carbons) carboxylic acids (acetic, propionic, butyric, valeric, caproic, heptanoic, and caprylic acids) (Holtzapple et al., 2022 ) along with other by-products. In the third stage (acetogenesis), the volatile fatty acids and other simple molecules created by the acidogenesis are converted into hydrogen, carbon dioxide, and acetic acid. In the final stage (methanogenesis), microorganisms convert the intermediate products of the preceding stages into methane, water, and carbon dioxide. AD technology can be modified for the valorization of organic waste streams to produce short chain- and medium chain- carboxylic acids (SCCAs and MCCAs) consistently (Wu et al., 2021a ). To this end, the AD process is rewired to produce carboxylic acids via arrested methanogenesis (AM), which is the basis for the carboxylate platform that can make a significant contribution to advanced waste and wastewater treatment (Holtzapple and Granda, 2009 ). To improve carboxylic acid production, the methanogens must be inhibited to avoid the consumption of SCCAs for methane production. The strategies inhibiting methanogens include inoculum pretreatment, a short sludge retention time and/or short hydraulic retention time, operation at low pH (pH <7.0), and the addition of chemical inhibitors. This modified AD process utilizes highly efficient, robust, and productive MMC structures for the conversion process. This MMC is an adaptable and stable ecosystem, which may not only adapt metabolically, but also microbially. The species present within the MMC fluctuate depending on the feedstocks, available nutrients, and operating conditions (e.g., temperature, pH, redox potential, etc). Such an MMC takes advantage of this diversity to efficiently convert any biodegradable material into carboxylic acids, ranging from acetic acid (C2) all the way to caprylic acid (C8). Figure 1a shows the different steps in the AM (or modified AD) conversion process, which starts with hydrolysis of complex molecules, such as carbohydrates, proteins and fats via enzymatic routes present in the MMC. The simpler molecules (sugars, amino acids, glycerol, and fatty acids) resulting from the hydrolysis step are then utilized by the same hydrolytic fermenters or syntrophically metabolized to SCCAs or short-chain fatty acids (formic, acetic, propionic, and butyric acid—C1, C2, C3, and C4), as well as hydrogen and CO 2 (and ammonia in the case of proteins) in primary fermentation. Certain other intermediates such as alcohols (e.g., ethanol), lactic acid and succinic acid, are also produced but immediately metabolized. The SCCAs from the primary fermentation may undergo secondary fermentation or chain elongation, where they are elongated into the MCCAs or medium-chain fatty acids (valeric, caproic, heptanoic, and caprylic acid—C5, C6, C7, and C8) using the metabolic pathway known as reverse β-oxidation, by organisms affiliated with the Clostridiaceae and Veillonellaceae families, mainly in the Clostridium and Megasphaera genus (De Groof et al., 2019 ; Candry and Ganigué, 2021 ). Several metabolic pathways are at play during these transformations as seen in Fig. 1b and c. In Fig. 1b , primary fermentation pathways are depicted, showing the conversion of glucose into pyruvate as the initial step. From pyruvate most other products form, including cellular biomass and acetic (C2) acid, with an acetyl-CoA intermediate, alongside other SCCAs, namely formate (C1), and propionate (C3). In addition, other intermediates such as lactate and ethanol may play a crucial role in secondary fermentations (Fig. 1c ) as electron donors for reverse β-oxidation. The oxidation of such electron donors to acetyl-CoA is coupled to the reductive elongation with SCCAs to form MCCAs, that is, butyric (C4), caproic (C6), and caprylic (C8) acids by adding two carbons in each step. Similar reverse β-oxidation can be observed with propionyl-CoA to form the odd-numbered MCCAs, valeric (C5) and heptanoic (C7) acids also by adding two carbons sequentially. In the anaerobic environment, these reactions occur under strongly reducing conditions, both primary and secondary fermentation are needed to provide sufficient free energy to generate ATP and restore NAD+/NADH balance in the cells. Fig. 1. (a) Overall conversion observed in AM acidogenic fermentation. (b) Metabolic pathways in primary fermentation in AM acidogenic fermentation. (c) Metabolic pathways in secondary fermentation in AM acidogenic fermentation showing chain-elongation (reverse β-oxidation) as described in (De Groof et al., 2019 ). SCCAs, short-chain carboxylic acids; MCCAs, medium-chain carboxylic acids. Figure 1a ( https://BioRender.com/l26u198 ) and c ( https://BioRender.com/q31o230 ) were created using Biorender. Although ethanol-consuming chain elongation occurs in a variety of natural environments (including animal faeces and anaerobic digesters), the metabolic differentiation of chain elongators is widely unexplored as the microbial diversity seems to differ significantly (Candry et al., 2020 ). In addition to previously described Clostridiaceae and Veillonellaceae involved in chain elongation via reverse β-oxidation, a metatranscriptomic study on MCCA formation from lignocellulosic ethanol fermentation conversion residue identified a number of other potential chain elongators (Scarborough et al., 2018 ). Not previously associated with reverse β-oxidation, Lachnospiraceae - and Eubacteriaceae -affiliated organisms were predicted to transform primary fermentation products (acetate and lactate) to MCCA. These were the only two families in the metagenome-assembled genomes that contained genes encoding homologues of enzymes known to catalyze chain elongation reactions in the reverse β-oxidation pathway, thus thought to be responsible for MCCA production in this specific microbiome. Using novel AM technology and associated MCCA producing MMCs, high concentrations of organic acids (35–78 g/l) from waste streams were produced at bench-scale digesters ranging from 0.5 to 14 l (Wu et al., 2021a , 2023 , 2024 ). These values were the highest acid titers reported for organic waste streams in the literature. The most promising conditions were tested to scale-up the process in a 100-gallon digester (Wu et al., 2023 ). The pilot-scale results showed that the newly developed AM process has the potential for large-scale application and is an exemplary waste-to-energy technology that transforms low- or negative-value waste streams into high-value bioproducts. Experimental data also showed that SCCAs separation can make up to 64% of total SCCA production cost since their separation and purification require energy- and chemical-intensive separation processes with high capital and operating cost. A variety of low-cost and low-carbon intensity separation technologies (resins, membrane, and electrochemistry-based technologies) instead of distillation were tested to increase the product titer by 2–10 times and decrease the separation/purification costs by 75% (Wu et al., 2021b , 2024 ). High purity SCCAs can be used ‘as is’ or as a platform chemical for producing chemicals and fuels, such as SAF, with a low-carbon footprint. This new waste treatment concept has the potential to disrupt the current waste treatment and management paradigm and can be readily integrated into current organic waste treatment and management practices. AM technology would eliminate the need for fossil-fuel derived feedstocks and introduce a responsible way to manage organic wastes and wastewater economically and environmentally friendly."
} | 5,588 |
30416644 | null | s2 | 1,742 | {
"abstract": "Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, "
} | 60 |
30271534 | PMC6157687 | pmc | 1,744 | {
"abstract": "Abstract The majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF) and rhizobia (i.e., nitrogen‐fixing bacteria). Understanding these associations has important implications for ecological theory and for restoration practice. Here, we tested whether the presence of AMF and rhizobia influences the performance of native woody plants invaded by a non‐native grass in experimental microcosms. We planted eight plant species (i.e., Acacia acuminata , A. microbotrya , Eucalyptus loxophleba subsp. loxophleba, E. astringens, Calothamnus quadrifidus , Callistemon phoeniceus , Hakea lissocarpha and H. prostrata ) in microcosms of field‐conditioned soil with and without addition of AMF and rhizobia in a fully factorial experimental design. After seedling establishment, we seeded half the microcosms with an invasive grass Bromus diandrus . We measured shoot and root biomass of native plants and Bromus , and on roots, the percentage colonization by AMF, number of rhizobia‐forming nodules and number of proteaceous root clusters. We found no effect of plant root symbionts or Bromus addition on performance of myrtaceous, and as predicted, proteaceous species as they rely little or not at all on AMF and rhizobia. Soil treatments with AMF and rhizobia had a strong positive effect (i.e., larger biomass) on native legumes ( A . microbotrya and A. acuminata ). However, the beneficial effect of root symbionts on legumes became negative (i.e., lower biomass and less nodules) if Bromus was present, especially for one legume, i.e., A. acuminata , suggesting a disruptive effect of the invader on the mutualism. We also found a stimulating effect of Bromus on root nodule production in A . microbotrya and AMF colonization in A. acuminata which could be indicative of legumes’ increased resource acquisition requirement, i.e., for nitrogen and phosphorus, respectively, in response to the Bromus addition. We have demonstrated the importance of measuring belowground effects because the aboveground effects gave limited indication of the effects occurring belowground.",
"introduction": "1 INTRODUCTION Mutualistic associations between soil organisms and plants are common in nature, particularly those involving mycorrhizal fungi and rhizobia (Schupp, Jordano, & Gómez, 2017 ). These plant symbionts can strongly influence dynamics of plant communities. For example, rhizobia have been reported to contribute to aboveground plant productivity and plant community evenness (Barrett, Bever, Bissett, & Thrall, 2015 ; van der Heijden et al., 2006 ). Additionally, arbuscular mycorrhizal fungi (AMF) have been shown to determine plant species diversity (Hiiesalu et al., 2014 ; Teste et al., 2017 ) and affect interspecific competition (Fonseca, Dias, Carolino, França, & Cruz, 2017 ; Lin, McCormack, & Guo, 2015 ) and plant productivity (Bauer, Blumenthal, Miller, Ferguson, & Reynolds, 2017 ). Indeed, Klironomos et al. ( 2011 ) have suggested that mycorrhizal associations could be as important as herbivory or competition for structuring plant communities. In recent years, soil microbial communities have widely been acknowledged to contribute to the success of invasive species (Callaway, Bedmar, Reinhart, Silvan, & Klironomos, 2011 ; Callaway, Thelen, Rodriguez, & Holben, 2004 ; Inderjit & van der Putten, 2010 ; Reinhart & Callaway, 2006 ; van der Putten et al., 2009 ) and there is some evidence for soil organisms being important for resistance to invasion. For example, soil organisms provided biotic resistance to native plants against invasive Potentilla (Callaway, Montesinos, Williams, & Maron, 2013 ). Additionally, biotic resistance conferred by soil pathogens was reported by Knevel, Lans, Menting, Hertling, and van der Putten ( 2004 ) for invasive dune grass Ammophila arenaria in South Africa. Thus, soil microbes may enhance biotic resistance of plant communities to weed invasion and in turn affect community structure. The role of plant root symbionts in invasion success has received increasing attention (Birnbaum, Bissett, Thrall, & Leishman, 2016 ; Klock, Barrett, Thrall, & Harms, 2015 ; Shelby et al., 2016 ; Stampe & Daehler, 2003 ; Wandrag, Sheppard, Duncan, & Hulme, 2013 ). An absence of ectomycorrhizal fungi has been reported to hinder the invasion success of exotic pines (Hayward, Horton, Pauchard, & Nuñez, 2015 ; Nuñez, Horton, & Simberloff, 2009 ). Other authors have suggested that invasive species have higher AMF colonization rates which may contribute to their higher total biomass compared with native species, and subsequently AMF may contribute to their invasion success (Paudel, Baer, & Battaglia, 2014 ). Rhizobia have also been suggested to facilitate legume establishment success in the introduced (invasive) ranges (Rodríguez‐Echeverria, 2010 ). Overall, these and other studies show that plant root symbionts play important roles as gate‐keepers to plant community membership. Understanding the contribution of plant root symbionts, their interactions, and their linkages to plants as determinants of plant community structure has important implications for ecological theory (Lambers et al., 2017 ). Beyond these theoretical implications, there are important practical outcomes too, i.e., this knowledge could help to refine frameworks for ecological restoration and could inform management practises more generally (Birnbaum, Bradshaw, Ruthrof, & Fontaine, 2017 ; Kardol & Wardle, 2010 ). For example, in old‐field restoration, often the aim is to overcome the resistance of the resident weedy community in order to establish a target community that is, in turn, resistant to reinvasion by the weedy species. Overcoming the resistance of the resident weedy community might be challenging if it is coupled with land‐use legacies in soil (Kulmatiski, Beard, Stevens, & Cobbold, 2008 ) or soil conditioning by invasive species (Hawkes, Wren, Herman, & Firestone, 2005 ; Vink et al., 2017 ). Emerging evidence suggests that better understanding of land‐use legacies on plants and their associated soil microbial communities could inform old‐field restoration (e.g., Hannula et al., 2017 ; Strickland et al., 2017 ). Here, the primary aim was to test the role of plant root symbionts in plant species coexistence and response to plant invasion using experimental microcosms. Our experimental design was informed by the Ridgefield Multiple Ecosystem Services Experiment (henceforth the Ridgefield Experiment) established on an old‐field in southwestern Australia (Perring et al., 2012 ). The Ridgefield Experiment was established to determine the relationship between the species diversity of woody plants and ecosystem functions in restoration, and additionally, the delivery of ecosystem services in the context of global change (e.g., N deposition, biological invasion; Perring et al., 2012 ). The microcosm experiment was designed to complement the questions being tested by the Ridgefield Experiment and uses soils, native plants from three different families (i.e., Fabaceae, Myrtaceae, and Proteacea), fungi and rhizobia from the vicinity of this field experiment. Specifically, we hypothesized that the presence of AMF and rhizobia would: a) positively influence the competitive outcomes among native woody plant species from Fabaceae that form associations with both symbionts and Myrtaceae that form association with AMF over Proteaceae that do not form associations with these symbionts and b) be beneficial to Fabaceae and Myrtaceae in resisting the Bromus diandrus invasion, whereas not affect response of Proteaceae to invasion in our experimental microcosms.",
"discussion": "4 DISCUSSION Plant species coexistence is mediated by negative feedbacks that promote cooccurrence of multiple species and ultimately contributes to species richness and ecosystem stability in plant communities (Bever, Platt, & Morton, 2012 ; Mack & Bever, 2014 ; Petermann, Fergus, Turnbull, & Schmid, 2008 ). Soil microorganisms, both beneficial plant root symbionts and plant pathogens, play an important role in mediating plant–soil feedbacks and contribute to ecosystem stability, species diversity as well as ecosystem invasibility (Bever, Mangan, & Alexander, 2015 ; Callaway et al., 2004 ; Dawson & Schrama, 2016 ; Klironomos et al., 2011 ; Pringle et al., 2009 ; van der Putten et al., 2013 ). In this study, our hypothesis was that the presence of arbuscular mycorrhizal fungi and rhizobia would influence the competitive outcomes among woody plant species and mediate the resistance of the native plant communities to weed invasion by Bromus . Our results suggest that AMF and rhizobia provided a competitive advantage (i.e., increased biomass) to the acacias (Fabaceae), had little to no effect on four species of Myrtaceae, and had a negative effect on the growth of Hakea lissocarpha (Proteaceae). It is well established that Fabaceae, especially acacias, benefit strongly from AMF and rhizobia (García‐Parisi, Lattanzi, Grimoldi, Druille, & Omacini, 2017 ; Ossler, Zielinski, & Heath, 2015 ; Simonsen, Dinnage, Barrett, Prober, & Thrall, 2017 ). Our results support these studies: the two acacias had significantly larger above‐ and belowground biomass in the presence of both plant root symbionts, but especially in the presence of AMF. Myrtaceae associate predominantly with ectomycorrhizal fungi (ECM; Lodge, 2000 ) as well as AMF, and some authors have suggested that eucalypts, in particular, receive more growth and nutritional benefits from ECM than AMF (Kariman, Barker, Finnegan, & Tibbett, 2012 ; Yuan, Huang, Li, & Christie, 2004 ). However, other studies have found that for some eucalypts, AMF associations can provide greater benefits during seedling establishment (but see Standish et al., 2007 ), whereas ECM are more prominent in adult trees (Adams, Reddell, Webb, & Shipton, 2006 ; Chen, Brundrett, & Dell, 2000 ). Here, we found, that +AMF+Rhiz treatment had a notable negative effect on AMF percent colonization in E. astringens roots. This result suggests that despite the AMF presence in soil inoculum, E. astringens had low AMF colonization. ECM tend to be ubiquitous in Eucalyptus tree roots (Kariman et al., 2012 ) and may have been present in our experimental plants; however, we did not quantify them. Taken together, the life‐stage‐dependent shifts in mycorrhizas for Myrtaceae and low interspecific competition for Proteaceae may explain the lack of observed soil treatment effect for these species. We did not expect the proteaceous species to benefit from access to plant root symbionts because they form cluster roots and thus are not reliant on mycorrhizas or rhizobia for nutrient uptake (Lamont, 2003 ). Rather, we predicted the proteaceous species would grow bigger in microcosms without plant root symbionts because of a competitive advantage. However, we did not observe this result, perhaps because interspecific competition for resources was weak. Overall, addition of Bromus to microcosms affected the native plant biomass and belowground root symbionts, suggesting a belowground effect of Bromus . Notably, for one species, A. acuminata , the plant biomass and the number of root nodules were significantly reduced when the microcosms were invaded with Bromus , while AMF colonization increased, suggesting an interaction between the invader and both AMF and rhizobia. It is plausible that A. acuminata increased its phosphorus acquisition, thus investing more into AMF to compensate for impeded growth in the presence of Bromus . In the same treatment, AMF percent colonization in Bromus was significantly reduced, suggesting a possible belowground competition between A. acuminata and Bromus for AMF and access to phosphorus. \n Acacia microbotrya had larger shoot and root biomass in the soil treatment with AMF but in the absence of rhizobia, irrespective of Bromus . Contrary to A. acuminata , Bromus addition had a strong positive effect on the number of root nodules in A. microbotrya . It is plausible that Bromus addition to microcosms (at 12 weeks) stimulated a belowground competitive response in Acacia that increased their investment in nodules or AMF to facilitate their own growth. It has been proposed that if soil fertility is high, grasses and legumes compete predominantly for light and little for soil nutrients (Eisenhauer & Scheu, 2008 ). However, if nitrogen (N) is limiting, grasses can benefit from N fixed by legumes, but this interaction may in turn reduce the competitive ability of legumes because grasses sequester a majority of the nitrogen (Munoz & Weaver, 1999 ; Schwinning & Parsons, 1996 ; Temperton, Mwangi, Scherer‐Lorenzen, Schmid, & Buchmann, 2007 ). In our study, Bromus did not appear to benefit from N fixed by legumes as its biomass was similar across all soil treatments. It is possible that native Acacia species were able to compete with Bromus because of extra N 2 from root nodules. In conclusion, our study highlights largely functional‐type specific responses of native plants to soil treatments and to Bromus addition in the microcosms. AMF and rhizobia influenced the competitive outcomes between Fabaceae, Myrtaceae and Proteacea by facilitating the Fabaceae. Fabaceae rely on these both mutualists for their establishment and growth, whereas Myrtaceae and Proteacea are less or not dependent on AMF and rhizobia for plant growth. Here, we showed that Bromus invasion disrupted the mutualisms and altered the belowground dynamics in Fabaceae by affecting nodulation and increasing mycorrhizal colonization (Hale, Lapointe, & Kalisz, 2016 ). Our results demonstrate that it is important to study plant competition from belowground as well as aboveground perspectives. In our case, the belowground data highlighted the role of plant root symbionts in mediating interactions among native and invasive plants to influence native plant performance, outcomes that were not apparent in the more easily measured aboveground data. This study provides a rare test of the role of belowground biota in structuring plant communities and supports the idea that soil biota are important in this role. From a restoration perspective, while it is often impractical to track belowground responses, our data suggest that it is important to remain cognisant of the likely interactions occurring belowground even if effects are not apparent aboveground. For example, there could be potentially negative effects of missing soil biota on plant interactions and ultimate restoration outcomes (Lin et al., 2015 ). A more surprising result was the stimulating effect of Bromus on root nodule production in Acacia microbotrya and AMF colonization in A. acuminata . This result adds yet another possible interaction to the gamut of interactions between native plants, their plant root symbionts and weeds in ecosystems. Our experiment has revealed some interesting and complex belowground dynamics that beg further research."
} | 3,772 |
31156716 | PMC6544526 | pmc | 1,746 | {
"abstract": "Surface-attached bacterial biofilms are self-replicating active liquid\ncrystals and the dominant form of bacterial life on earth ( 1 – 4 ). In\nconventional liquid crystals and solid-state materials, the interaction\npotentials between the molecules that comprise the system determine the material\nproperties. However, for growth-active biofilms it is unclear whether\npotential-based descriptions can account for the experimentally observed\nmorphologies, and which potentials would be relevant. Here, we overcame previous\nlimitations of single-cell imaging techniques ( 5 , 6 ) to reconstruct and track\nall individual cells inside growing three-dimensional (3D) biofilms with up to\n10,000 individuals. Based on these data, we identify, constrain, and provide a\nmicroscopic basis for an effective cell-cell interaction potential, which\ncaptures and predicts the growth dynamics, emergent architecture, and local\nliquid crystalline order of Vibrio cholerae biofilms.\nFurthermore, we show how external fluid flows control the microscopic structure\nand 3D morphology of biofilms. Our analysis implies that local cellular order\nand global biofilm architecture in these active bacterial communities can arise\nfrom mechanical cell-cell interactions, which cells can modulate by regulating\nthe production of particular matrix components. These results establish an\nexperimentally validated foundation for improved continuum theories of active\nmatter and thereby contribute to solving the important problem of controlling\nbiofilm growth."
} | 382 |
31297978 | null | s2 | 1,747 | {
"abstract": "Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations' involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next-generation biofuels."
} | 261 |
20188674 | null | s2 | 1,749 | {
"abstract": "Quorum-sensing (QS) bacteria assess population density through secretion and detection of molecules called autoinducers (AIs). We identify and characterize two Vibrio harveyi negative feedback loops that facilitate precise transitions between low-cell-density (LCD) and high-cell-density (HCD) states. The QS central regulator LuxO autorepresses its own transcription, and the Qrr small regulatory RNAs (sRNAs) posttranscriptionally repress luxO. Disrupting feedback increases the concentration of AIs required for cells to transit from LCD to HCD QS modes. Thus, the two cooperative negative feedback loops determine the point at which V. harveyi has reached a quorum and control the range of AIs over which the transition occurs. Negative feedback regulation also constrains the range of QS output by preventing sRNA levels from becoming too high and preventing luxO mRNA levels from reaching zero. We suggest that sRNA-mediated feedback regulation is a network design feature that permits fine-tuning of gene regulation and maintenance of homeostasis."
} | 263 |
35220660 | PMC9303955 | pmc | 1,752 | {
"abstract": "Abstract \n The presence of invasive alien plants (IAPs) alters the composition of soil arbuscular mycorrhizal (AM) fungal communities. Although fundamental for plant development, plant responses to AM from invaded soils have not been widely explored, especially under environmental stress. We compared plant growth, P accumulation, root colonization and the photosynthetic responses of the native AM‐dependent Plantago lanceolata growing in contact with AM fungi from communities invaded by Acacia dealbata Link (AM inv ) or non‐invaded communities (AM nat ) exposed to water and light restriction (shade). Under optimal growing conditions, plants in contact with AM nat produced higher leaf biomass and accumulated more P. However, plant responses to different AM inocula varied as the level of stress increased. Inoculation with AM inv promoted plant growth and root length under light restriction. When plants grew in contact with AM nat under drought, leaf P increased under severe water restriction, and leaf and root P increased under intermediate water irrigation. Growing in contact with the AM nat inoculum promoted root P content in both full light and light restriction. Colonization rates of P. lanceolata roots were comparable between treatments, and plants maintained photosynthetic activity within similar ranges, regardless of the level of stress applied. Our results suggest that origin of the inoculum (native soils versus invaded soils) did not affect the ability of AM species therein to establish effective mutualistic associations with P. lanceolata roots but did influence plant responses depending on the type and level of the abiotic stress.",
"conclusion": "CONCLUSIONS Habitat transformation induced by A . dealbata lead to water and light limitation for native plants, but the association with AM fungi can alleviate these effects. Growing in contact with mycorrhiza from native and invaded soils, P. lanceolata responded differently to the reduction in water and light availability. Nevertheless, the source of the AM inoculum had different effects on the photosynthetic and growth responses of P. lanceolata under abiotic stresses, without noticeable effects on root colonization. Similar infection levels and fungal structures were evidenced across treatments, regardless of the type and level of stress. Hence, our results separated plant performance from root colonization, also suggesting that potential changes in the fungal community induced by A. dealbata and the level of stress applied did not affect the ability of the AM community of invaded areas to associate with AM‐dependent plants, such as P. lanceolata . However, plant responses were affected to some extent by the origin of the AM inoculum used. Both AM inocula led to similar plant responses to drought stress, but the association with AM inv slightly improved plant growth under reduced light. Interestingly, inoculum origin influenced the capacity of the plants to maintain P supply, reducing plant P content when roots were associated with AM fungi from areas invaded by A. dealbata . With similar root colonization levels between treatments, the origin of the inoculum was the main factor influencing the ability of P. lanceolata to obtain soil P under water limitation.",
"introduction": "INTRODUCTION One of the main consequences and characteristics of the Anthropocene is the unprecedented increase in biological invasions in response to large‐scale phenomena, such as globalization, intensification of international trade and tourism and soil degradation. With consequences at the local, regional and global scale, the spread of invasive alien plants (IAPs) reduces and eliminates native species, limits plant biodiversity, modifies soil physicochemical parameters, nutrient cycling, water regimes and decreases the provision and quality of ecosystem services (Vilá et al . 2011 ; Pyšek et al . 2012 ; Simberloff et al . 2013 ; Banks et al . 2015 ). At the soil level, the presence of IAPs interferes with the structure and function of microbial communities, altering symbiotic relationships and the soil–plant exchange system (Van der Putten et al . 2007 ; Pringle et al . 2009 ; Vogelsang & Bever 2009 ; Tanner & Gange 2013 ; Inderjit & Cahill 2015 ). When IAPs arrive in new territories they create novel or selective associations with soil microbial species (Richardson et al . 2000a ; Moora et al . 2011 ; Nuñez & Dickie 2014 ; Kamutando et al . 2017 ; Le Roux et al . 2017 ) or bring their own mutualists (Correia et al . 2019 ; Kamutando et al . 2019 ). Soil microbiota and mycorrhiza in particular play a crucial role in determining the abundance and invasiveness of plant species (Levine et al . 2004 ). Arbuscular mycorrhizal (AM) fungi are located at the soil–root interface, expanding the range of plant influence and acting as intermediaries between plants and the soil matrix (Richardson et al . 2009 ). Through a symbiotic relationship established with more than 80% of terrestrial plants (Smith & Read 2010 ), AM fungi provide essential soil nutrients, mainly P and N, but also facilitate access and uptake of NH 4+ , NO 3 \n ‐ , Zn, Cu and K (Mathur et al . 2019 ) in exchange for C compounds derived from photosynthesis. Besides their contribution to plant nutrition, AM fungi also serve as a first defence, increasing plant resistance to biotic and abiotic stresses (Hajiboland et al . 2019 ; Diagne et al . 2020 ). Such AM communities are fundamental to maintaining forest soil balance as they can represent ecological barriers to limit the entry and spread of IAPs (Janos et al . 2013 ). Nevertheless, their role in the invasion process can vary, acting as facilitators (positive effect), inhibitors (negative effect) or having a neutral effect (Klironomos 2003 ; Levine et al . 2004 ; Shah et al . 2009 ). Reciprocally, once established in the novel community, IAPs may, in turn, affect the structure of AM fungal communities (Cantor et al . 2011 ; Meinhardt & Gehring 2012 ; Guisande‐Collazo et al . 2016 ), generally decreasing the abundance of native mycorrhiza and altering the structural composition of the soil fungal community (Grove et al . 2017 ). The AM fungi are ubiquitous and widely distributed among almost all terrestrial environments, but interactions with plants become especially relevant under stress conditions. Multiple studies indicate that AM fungi help plants to deal with drought stress (Augé 2001 ; Augé & Moore 2005 ; Boomsma & Vyn 2008 ; Zhu et al . 2012 ; Endresz et al . 2015 ; Begum et al . 2019 ; Mathur et al . 2019 ). Drought stress reduces plant growth, mainly by reducing photosynthetic capacity (Teskey et al . 2015 ; Mathur et al . 2019 ). In this sense, the association with AM fungi alleviates drought stress by regulating hormone balance, increasing water absorption through hyphae (Augé et al . 2007 ), contributing to osmotic adjustment (Porcel & Ruiz‐Lozano 2004 ), expanding antioxidant activity or increasing nutrient absorption (Fernández‐Lizarazo & Moreno‐Fonseca 2016 ). Plant association with AM fungi can also maintain photosynthetic capacity in shaded conditions (Shukla et al . 2009 ), improving responses to light deficiency (Liu et al . 2015 ; Koorem et al . 2017 ). On the other hand, light reduction limits C gain by the AM fungi due to a decrease in photosynthetic efficiency of host plants (Liu et al . 2015 ). Consequently, shady conditions can be further responsible for changes in AM community composition (Van Diepen et al . 2011 ; Shi et al . 2014 ; Liu et al . 2015 ). \n Acacia dealbata Link is a N 2 ‐fixing highly invasive species, native to Australia and Tasmania, that currently invades Mediterranean ecosystems in South Africa, South America and Europe (Fuentes‐Ramirez et al . 2011 ; Richardson & Rejmanek 2011 ; Souza‐Alonso et al . 2017 ). Here, A. dealbata severely impacts plant biodiversity, alters habitat conditions (including light intensity or water regime), soil physical‐chemical properties and nutrient cycling (Fuentes‐Ramírez et al . 2011 ; Lazzaro et al . 2014 ; Souza‐Alonso et al . 2014 ; Kamutando et al . 2017 ; Lorenzo et al . 2017 ). Besides reducing aboveground plant diversity (Fuentes‐Ramírez et al . 2011 ; Lorenzo et al . 2012 ; Lazzaro et al . 2014 ), the presence of A. dealbata also causes significant changes to the structure and function of the soil microbial community (Lorenzo et al . 2010 ; Souza‐Alonso et al . 2015 ; Kamutando et al . 2017 , 2019 ). Across geographical regions and nutritional levels, A. dealbata establishes relationships with different soil bacterial and fungal communities (Kamutando et al . 2017 , 2019 ). In general, the Acacia genus associates with AM and ectomycorrhizal (EM) fungi (Brundrett 2009 ), but although both types of mycorrhizae have been identified in its rhizosphere (Kamutando et al . 2017 ), A. dealbata does not obtain clear benefits from the association with AM fungi (Crisóstomo 2012 ). However, even when their mycorrhizal dependence is low, IAPs generally succeed in competition with obligate mycorrhizal plants by disturbing local AM communities (Mummey and Rillig 2006 ; Vogelsang et al . 2006 ; Pringle et al . 2009 ; Vogelsang & Bever 2009 ). The lower abundance of AM species (Kamutando et al . 2017 ) or changes in the community structure after A. dealbata invasion (Guisande‐Collazo et al . 2016 ) represent direct consequences of the invasion process. Novel AM communities in soils invaded by A. dealbata reduced plant growth, P acquisition and physiological activity of AM‐dependent plants (Guisande‐Collazo et al . 2016 ). However, how these structural changes affect plant establishment under different environmental conditions has not been explored to date. In this work, we hypothesize that the origin of the AM community influences the growth and development of plants exposed to different environmental stresses. Therefore, we compared growth of the mycorrhizal‐dependent Plantago lanceolata growing in contact with AM fungi obtained from areas invaded by A. dealbata with plants growing with inoculum from a local, non‐invaded plant community, when plants were exposed to different environmental stresses.",
"discussion": "DISCUSSION From a simplistic perspective, mycorrhizae are often perceived as mere appendages to obtain valuable resources: a symbiotic association to exchange part of the photosynthetic production in return for essential nutrients. However, the role of mycorrhizae and mycorrhizal networks on ecosystem configuration is central by regulating competition, colonization, interplant resource transfers and cross‐scale ecosystem interactions (Simard et al . 2012 ). The mycorrhizal contribution is particularly relevant for plant growth under environmental stress conditions (Brooker et al . 2008 ). Plant responses depend largely on their symbiotic relationships and, consequently, it would be expected that responses to inoculation with a novel AM consortium would vary depending on plant stress level (Bever 2002 ). Therefore, we aimed to address the mycorrhizal influence on a dependent plant under different water and light regimes, simulating limiting conditions of habitats transformed by A. dealbata . In general, we observed that both light and water restriction influenced plant growth and responses, but P. lanceolata was also affected, to some extent, by the origin of the AM inoculum. Photosynthesis and plant growth The establishment of mutualist relationships with different AM sources modulates plant physiological responses, e.g . by alleviating structural and functional damage to the PSII reaction centre and electron transport under drought stress (Mathur et al . 2019 ). However, it is important to note that different AM species/communities lead to variable plant photosynthetic responses during stress, such as water limitation (Augé 2001 ). In general, P. lanceolata maintained similar photosynthetic activity despite reduced water supply. Improving plant hydraulic conductivity through root expansion by AM fungi—the so‐called mycorrhizosphere —alleviates drought stress (Augé et al . 2007 ; Mathur et al . 2019 ), also minimizing energy losses in the form of heat while protecting the electron flow of the antenna complexes (LEF) (Boomsma & Vyn 2008 ). Commonly, plant photosynthetic activity decreases with drought stress; however, under mild or moderate water restriction it can remain temporarily stable or even increase (Morales et al . 2008 ), e.g . by increasing the rate of photorespiration (Massacci et al . 2008 ). Plants maintained similar photosynthetic activity across different water levels, regardless of the origin of the inoculum. In this sense, the different origin of the AM inoculum did not modify the photosynthetic response of P. lanceolata , since the photosynthetic efficiency (Ф II ) or F v '/F m ' —indicators of damage to the photosynthetic apparatus—were maintained in ranges considered adequate. Nevertheless, the Ф II value of plants inoculated with AM nat slightly decreased when plants were watered at 25% of field capacity. Nevertheless, plant growth and biomass production seemed to be more influenced by water availability (less availability, less growth) than by inoculum origin, since no differences were detected between plants treated with AM nat or AM inv . Sunlight reduction generally decreases plant photosynthetic activity and photosynthesis rates (Ojanguren & Goulden 2013 ). Low light leads to insufficient ATP produced to allow C fixation and carbohydrate biosynthesis (Shao et al . 2014 ), reducing photoassimilate production and, consequently, plant growth and biomass (Mathur et al . 2018 ). As a consequence, limited C products constrain the mutualistic investment (Fellbaum et al . 2014 ) due to the significant energy requirements necessary to maintain the mutualist relationship, since AM receive up to 20% of primary plant production (Hobbie & Hobbie 2008 ; Smith & Read 2010 ). On the other hand, mycorrhization contributes, to some extent, to ameliorate photosynthetic responses (Zhu et al . 2012 ; Liu et al . 2015 ; Koorem et al . 2017 ; Mathur et al . 2019 ), and plants colonized by AM often show higher stress tolerance (Jung et al . 2012 ; Augé et al . 2015 ). Contrary to previous studies (Graham et al . 1982 ; Gehring 2003 ), the attenuation of natural irradiance did not reduce fungal colonization of P. lanceolata roots. Similar colonization rates, including essential organs for lipid storage in vesicles (Smith & Read 2010 ), suggests that P. lanceolata maintained the energy investment to preserve AM structures, even under restrictive treatments (W 25 , shade). Nevertheless, although the mycorrhization levels were similar, our results indicated that growth responses of P. lanceolata were, to some extent, influenced by light conditions. The photosynthetic capacity (ф II ) and efficiency (ф NO ) of plants growing in contact with AM nat decreased compared to AM inv , which performed better under reduced irradiance. The adaptive response of plants to light restriction was reflected in the aerial length and biomass, suggesting a more beneficial role of symbiosis under limiting conditions (Zhang et al . 2015 ), in line with the stress‐gradient hypothesis (Brooker et al . 2008 ). Nevertheless, this effect also depends on the AM origin; plants in contact with AM inv could be more conservative in full light, prioritizing maintenance of the AM symbiosis over plant growth and the opposite under shade conditions. Growing in soils with AM inv allowed plants to maintain allocation patterns in shade, whereas plants in contact with AM nat relocated resources to the aerial parts, increasing LMF while reducing RMF. Although neither the quantum yield of PSII or the energy loss were affected, the allocation of plant resources to the aerial parts in AM nat plants, also the increase in SLA, would suggest an expansion of the available photosynthetic surface to compensate for the reduction in light. Plant phosphorus content From the plant point of view, the main advantage of investing in AM symbioses is the increased access to otherwise inaccessible soil nutrients, such as P or N, extending nutrient acquisition area beyond the limit of the root (Smith & Read 2010 ) and contributing up to 90% of the P obtained by the plant (Smith & Smith 2011 ). Phosphorus is taken up by extraradical hyphae, transported towards the root system and delivered to the plant via arbuscules (Smith & Read 2010 ), thus reducing plant dependence on soil environmental conditions for P uptake. In our study, water restriction caused a reduction in plant P content, which could be due to the decrease in P availability in water‐deficient soils (Gahoonia et al . 1994 ; García et al . 2008 ). However, plants in contact with AM nat tolerated water limitation better, maintaining root–stem transport, and therefore foliar P content. These plants had similar P levels under intermediate and severe water reduction ([P] at W 50 = W 25 ), unlike plants growing with AM inv ([P] at W 50 > W 25 ). Although root colonization generally decreases under drought (García et al . 2008 ; Mathur et al . 2019 ), under light restriction (Koorem et al . 2017 ) or due to the contact with AM from invaded soils (Tanner & Gange 2013 ), in our case, the origin of the inoculum did not affect colonization, with similar infection levels and comparable AM structures regardless of the level of stress applied. As stated above, mycorrhizal relationships are maintained at a high metabolic cost, and the stress severity, e.g . drought level, influences the investment in mycorrhizal symbiosis (Augé 2001 ). Phosphorus solubility and availability decreases under water limitation and, therefore, increasing the number of arbuscules (75%) in W 25 in plants growing with AM nat would suggest an additional effort to obtain P, probably at a higher metabolic cost (Roth & Paszkowski 2017 ). This increase contrasts with previous results that reported a reduction in the presence of arbuscules under drought (García et al . 2008 ). Despite the structural change in the AM community in soils invaded by A. dealbata (Guisande‐Collazo et al . 2016 ), the level of mycorrhizal colonization observed suggests that P. lanceolata associates effectively with the AM community provided from the invader. Therefore, it could be argued that it was the origin of the inoculum (and the species within), rather than the root colonization level, that influenced the ability of P. lanceolata to obtain soil P under water limitation. Although AM species vary in the capacity to acquire and provide P to P. lanceolata (Pel et al . 2018 ), how a specific set of AM species would influence P acquisition under different stresses seems difficult to predict. Noteworthy, the increased P availability (x2) in shrublands invaded by A. dealbata across the region (Lorenzo et al . 2010 ; Souza‐Alonso et al . 2014 ) could reduce the need to invest in specific mechanisms for P acquisition, and the mutualistic relationship could be focused on complementing other requirements (plant defence, abiotic stress, water uptake, etc.) (Jung et al . 2012 ; Hajiboland et al . 2019 ; Li et al . 2019 ; Diagne et al . 2020 ). Nevertheless, the costs and benefits of symbiotic exchanges are complex and depend on the relative resource availability and their balance between both symbiotic partners (Grman 2012 ). In Experiment 2 , plants growing in contact with AM nat did not show different responses to different light regimes, but in all cases accumulated more P in roots than plants inoculated with AM inv . In this case, plant response also varied between different irradiance conditions, showing different allocation patterns in the light (leaf P > root P) or shade (root P > leaf P). This trend was also observed for C resource allocation, where higher root length and biomass were observed under shade but not under full light. The interaction between light and soil nutrients can affect preferential bidirectional allocation patterns of C and P (Zheng et al . 2015 ). In this sense, leaf expansion and thus, increased photosynthetic surface, could be interpreted as a response of P. lanceolata when associated with AM inv to acquire more photosynthates under low‐light conditions that can be further used to increase or maintain the bidirectional exchange with the AM. The preservation of mycorrhizal structures in shade—and the associated energy cost—suggests that the stress level might not have been sufficiently intense (despite the 80% reduction in the natural irradiance) to affect plant growth or to produce noticeable changes. Considered globally, the benefit obtained in P acquisition was reduced when plants associated with the AM inv inoculum. Similar to observations in Experiment 1 , it could be argued that fungal communities in AM nat and AM inv affected P. lanceolata differently under shade. Considering similar root infection levels, the association with AM inv favoured P. lanceolata growth, whereas AM nat was more effective in obtaining soil P. Consequences of invasion and stress In our study, P. lanceolata showed different responses to changes in water or light regime, probably due to differences in the type and intensity of the stress applied. In this sense, it is important to note that unidirectional negative consequences produced by the association with AM from invaded communities (Guisande‐Collazo et al . 2016 ; Zubek et al . 2016 ) were not observed, at least in growing plants under different stress conditions. The presence of IAPs, such as A. dealbata , with low dependence on native mutualisms is expected to induce changes, decrease mutualist efficiency over time and affect mutualist‐dependent species after disturbances (Vogelsang & Bever 2009 ). To some extent, plant responses were altered under stress, but instead of decreasing plant performance, we observed what can be considered an adapted response . Under optimal growth conditions, plants in contact with AM nat were slightly favoured (leaf biomass, P content). However, the influence of AM nat and AM inv seemed to be related to the level of stress applied (water stress > light stress), providing slight advantages to plants growing in contact with AM nat under drought stress and to plants associated with AM inv under light reduction. Thus, as stated above, responses of P. lanceolata to cope with different environmental stresses would be conditioned by the origin of the AM inoculum. It is well established that AM fungi show interspecific functional diversity (Munkvold et al . 2004 ; Mensah et al . 2015 ), with differences, e.g . in soil exploration efficiency. The specific composition of inocula, and the intrinsic characteristics of species therein, might harbour different physiological attributes that produce different responses and benefits in mycorrhized plants according to the level and the type of stress applied (Augé 2001 ; Manoharan et al . 2017 ; Pel et al . 2018 ; Li et al . 2019 ) or also related to biotic factors (Kiers et al . 2011 ; Fellbaum et al . 2014 ). The presence of IAPs that do not depend on mycorrhiza disrupts the fungal community structure, negatively influencing native species that depend on AM symbioses (Tanner & Gange 2013 ). It is generally considered that IAPs take advantage of associations with soil microorganisms—in our case the low mycorrhizal dependence of A. dealbata —changing species composition and decreasing the effectiveness of the native mutualists over time (Vogelsang & Bever 2009 ). As a result, mutualistic‐dependent plants are adversely affected, especially under abiotic stress. In fact, the capacity of A. dealbata to modify its environment implies that changes occur on a much larger scale. It is not by chance that due to the ecosystem‐level changes produced, this and other Acacia species (Souza‐Alonso et al . 2017 ) are considered as transformer species (Richardson et al . 2000b ). Nevertheless, the interpretation of the results and the ecological implications assumed should be considered with caution because of the limitations to the experimental design (limited number of target species, inoculum or sampling sites). Our results indicated that the outcome of associating with AM nat or AM inv on plant performance is not unidirectional but is context dependent. Adapted responses of P. lanceolata could be related to its ability to associate with a wide range of AM species (Pel et al . 2018 ), mainly species of the genus Glomeromycota (Smith & Read 2010 ), and, at the same time, to the generalist character of AM species from different communities (invaded–native) to establish relationships with roots of different plants (Majewska et al . 2018 ). Here, the proportion, extent and number of AM structures, such as hyphae, vesicles or arbuscules, provide good insight into the plant–AM associative process. Considering that plant biomass and root mycorrhization are generally correlated (Zubek et al . 2016 ), our results separated plant performance from root colonization, suggesting that different sources of AM inocula and the level of stress applied do not limit the capacity of a generalist plant species such as P. lanceolata to associate with AM communities from areas invaded by A. dealbata ."
} | 6,412 |
22412902 | PMC3297609 | pmc | 1,753 | {
"abstract": "Plants and their pollinators form pollination networks integral to the evolution and persistence of species in communities. Previous studies suggest that pollination network structure remains nested while network composition is highly dynamic. However, little is known about temporal variation in the structure and function of plant-pollinator networks, especially in species-rich communities where the strength of pollinator competition is predicted to be high. Here we quantify temporal variation of pollination networks over four consecutive years in an alpine meadow in the Hengduan Mountains biodiversity hotspot in China. We found that ranked positions and idiosyncratic temperatures of both plants and pollinators were more conservative between consecutive years than in non-consecutive years. Although network compositions exhibited high turnover, generalized core groups – decomposed by a k -core algorithm – were much more stable than peripheral groups. Given the high rate of turnover observed, we suggest that identical plants and pollinators that persist for at least two successive years sustain pollination services at the community level. Our data do not support theoretical predictions of a high proportion of specialized links within species-rich communities. Plants were relatively specialized, exhibiting less variability in pollinator composition at pollinator functional group level than at the species level. Both specialized and generalized plants experienced narrow variation in functional pollinator groups. The dynamic nature of pollination networks in the alpine meadow demonstrates the potential for networks to mitigate the effects of fluctuations in species composition in a high biodiversity area.",
"introduction": "Introduction Community studies have shown that the network structure of plant-pollinator interactions is largely asymmetrical — the partners of specialists tend to also interact with generalists [1] – [5] . Within a community some plants are pollinated by a large proportion of the available flower visitors (generalization) while others are pollinated by a relatively smaller proportion (specialization) [6] , suggesting that ecological generalization predominates [7] – [10] . A relatively unexplored question is the extent to which ecological generalization is stable across multiple flowering seasons through the persistence of specific linkages. Documenting network stability is essential for an understanding of how specialized plants evolve in communities dominated by generalized pollination networks. If pollinator-mediated selection is one major force driving the evolution of flowers, the selective role of the pollinators could be diminished if plant-pollinator interactions are highly variable across years [11] , [12] . Recent multi-year studies, including four conducted in Europe [13] – [16] and two in North America [17] , [18] , show that large temporal differences in network structure and species interactions are largely attributable to species turnover across years and flexibility in interactions with new partners; but the basic topological properties of plant-pollinator networks remain unchanged [19] . For example, in a montane meadow community from southern California 36% of plant species and 18% of pollinator species were shown to have specialized links with at least one mutualistic partner across three summers [17] . In a scrub community in Greece, 53% of the plant species and 21% of pollinators, but only 5% of species interactions, were observed across four consecutive years [14] . However, our understanding of temporal variation in plant-pollinator interactions is currently limited, given that we know little about which kinds of species are likely to turn over, particularly in species-rich communities. A distinct property of pollination networks is that they are highly nested; that is, most species interact with hierarchical subsets of generalist partner species [6] . In a nested pattern, it has been suggested that generalists are key components to maintain network stability and to resist the susceptibility to extinction of specialists [20] . Although generalists contribute more to network stability than specialists, field investigations have shown that many plant or pollinator species link to only one partner per year and tended to be ecologically specialized, although they may be considered generalists in that they interact with different partner species in other years [14] . Furthermore, all community-level surveys must be considered to be samples of the true diversity and hence apparently specialist plants may be pollinated by other pollinators which were not detected in the surveys. To explore the dynamic nature of the plant-pollinator network, we investigated temporal stability in an alpine meadow over four years using a k -core analysis. The k -core algorithms can determine which plants and pollinators composed a “ k ”-core group ( k is an integer), depending on link number and partners' link quality. This k -core analysis is a bottom-up method to separate the network into subgroups [21] . It has been used to classify protein positions in protein networks to show the evolutionary trend in co-occurrence networks from single cells to multicellular eukaryotes [22] and also to identify subgroups of taxa in plant-pollinator networks [5] . We investigated plant-pollinator interactions in a species-rich alpine meadow of the Hengduan Mountains biodiversity hotspot in China [23] . Plants in species-rich communities may be pollinated by more specialized pollinators than those in species-poor communities. Although there was a positive relationship between plant species number and pollinator species number, studies suggested that high species diversity could reduce pollinator niche overlap, allowing pollinators to focus on specific plant species for nectar or pollen resources [24] , [25] . However, Ollerton et al. (2003) found significant pollinator overlap between asclepiad species in a species-rich grassland in South Africa [2] . Previous analysis suggested that plants in species-rich communities may be more prone to pollen limitation than those in less species-rich areas because of interspecific competition for pollinators [26] . Based on null models, a larger proportion of extreme specialists and generalists are both expected to appear in a species-rich community as the number of interacting species increases [9] . Therefore, plants in biodiversity hotspots are likely to experience a higher risk of extinction and/or to specialize on certain pollinators [26] . Plants may be pollinated by a taxonomically diverse group of pollinators that all function in a similar way [27] ; those pollinators sharing similar behavior or flower preference are categorized as being in the same functional group. For example, Pedicularis species in the study community were generalized – linked to several pollinator species – but they are specialized at the functional group level because they are only pollinated by bumble bees. It has been suggested that variation in pollinator composition can not be assumed to reflect coevolutionary relationships between plants and pollinators unless one considers the similar behavior and flower preference of members of the same pollinator functional group [28] , [29] . Thus, one would expect that temporal fluctuation in pollination networks could be cushioned if one lost pollinator species can be replaced by another from the same pollinator functional group. An estimate of the temporal stability of functional groups in pollination networks may provide insights into the difference between ecological and evolutionary specialization [28] , [30] . However, the temporal variation of functional pollinator groups has not been evaluated at a whole community level [19] , [28] , [31] . Studies of network stability have been conducted in Europe and North America, in relatively species-poor communities, ranging from 7 to 39 plant species and 23 to 597 pollinator species [13] , [15] – [18] . Only two investigations in large areas recorded over 100 plant species in Europe [14] , [15] . Community studies of the pollination network in the Hengduan Mountains biodiversity hotspot permit us to examine whether ecological specialization and/or generalization tends to be higher in species-rich communities through a comparison to previous studies in species-poor communities, as predicted by theoretical models [26] . Specifically, we addressed the following questions. (a) How great is inter-annual variation in the alpine meadow pollination network? Given that we have four years' data, we asked whether the network was more similar between consecutive years than non-consecutive years. (b) Which kinds of species are likely to turn over in pollination networks with relatively stable structures, the inner or periphery species? We examine whether there is a certain pattern in species turnover by k -core decomposition. (c) Is the variation in pollinator composition across years similar in ecologically generalized and specialized plants at species and functional group level?",
"discussion": "Discussion Our four-year investigation of pollination networks in a biodiversity hotspot indicated that the networks were relatively stable even though species assemblages changed significantly. Plants and pollinators were consistent in their positions and link qualities in networks between years. Furthermore, inner groups comprising relatively ecologically generalized species were more stable than peripheral groups comprising relatively ecologically specialized species. The same species linked to almost the entire partner spectrum each year. Theoretical modeling predicts a high proportion of specialized pollination links in species-rich communities [9] , a pattern not observed in this study of a biodiversity hotspot. Instead, both specialized and generalized plants experienced temporal stability of pollinator linkages. Our results are compatible with those of several previous studies. As in other networks, there were more pollinator species than plant species each year and plant-pollinator interactions were nested [8] , [17] , [39] . Plants and pollinators were in stable rank positions while their link qualities (IT) were more changeable. Species with high IT have a higher chance of forming specialist-to-specialist links. But the variation in IT across years rearranged the links and specialist-specialist links did not stabilize even though they may be formed in certain years. Examination of an Arctic pollination network in day-to-day resolution [13] also suggested that certain specialist-specialist links could continue for a certain period, but newly emerged species preferentially linked to generalists, and this rearranged links. More stable relationships in plant ranks may result from the steady links between specialized plants and bumblebee pollinators in the study community. Thus, even though species composition fluctuated across years, the remaining specialized plants could remain at their original position in the network. Morphological constraint (e.g., accessibility of nectar) has been considered an important factor governing pollination network structure [40] . Bumblebees, for example, had relatively long proboscises enabling them to probe nectar from various flowers with different tube lengths, and this may explain the positive relationship in IT of these species between years in our community. Our investigations indicate that plant-pollinator networks are relatively stable between years, consistent with previous studies [14] – [18] . A comparably high species turnover has also been observed in other ecosystems in the temperate zone [e.g. 41] , [42] . High turnover rate restricts the formation of specialist-specialist links. However, we did observe tight interactions between bumblebees and plant genera including Aconitum , Astragalus , Delphinium , Lotus , Pedicularis and Primula . In a nested pattern, generalized species are more important than specialists for their resilience to local extinction, because the extinction of specialized species removes fewer links in the network leaving a greater number of species linked to their generalist partners [20] . Our k -core analysis indicates that the stabilized inner groups, which consists of generalized plants and pollinators, links most partners across years, suggesting stability of the nested structure. The stability of the generalized core groups also supports Memmott's [20] simulation model of extinction by field observation, emphasizing that generalists are more important to network stability, especially for the most generalized pollinators, such as the bumblebee fauna which link to nearly 50% of plant species. Our investigation indicates that bumblebees are core species providing pollination service in the alpine meadow. The highly diverse pollination network studied here differs in several respects from networks studied in species-poor areas. First, the pollinator-plant ratio (average 1.48±0.09 for each year; 2.49 over all four years) in our community is much lower than that of other pollinator-plant networks [6] , which generally have ratios above 4. Species richness has been considered to influence structural properties of a network [6] , [43] . For example, as networks grow larger, there tend to be more asymmetric interactions in larger pollination networks compared to smaller ones [3] . We observed that some plant genera share the same pollinator functional group or even the same pollinator species [e.g. 44] . For example, Bombus richardsi was always the most generalized pollinator over the four years, and it was observed to link with over 50% of plant species in the community each year. These results support the prediction of strong pollinator competition in species-rich communities [26] . Second, the predicted high proportion of specialized pollinators in a species-rich community [9] , [26] was not observed in our study. Only a small proportion of plant species were pollinated by a single pollinator species (13.5%±7.7%) and single pollinator functional group (20.4%±6.9%). Similarly, there was on average 13.0±7.1% of plant species pollinated by a single pollinator based on four surveys in North America [see 7] ; in one community 54.0% of plant species were pollinated by one functional group [see 29] . Contrary to a null model that predicts a higher proportion of both generalized and specialized pollination links in species-rich than species-poor communities [9] , our four-year data in this community showed that nearly 80% of plant species were pollinated by at least two pollinator functional groups. The proportion of specialized pollination links was not high in this species-rich community, consistent with two recent studies [16] , [45] . The predicted increase of specialization of pollination systems during community succession was not observed along the chronosequence of a glacier foreland in southeastern Switzerland [16] . An investigation of flower supply and flower-visiting insects in 27 meadows in southern Germany indicated that the level of specialization did not significantly differ across the gradient of flower diversity [45] . Third, in our study Hymenoptera (bumblebees and other bees), rather than Diptera (flies), were highly generalized. Flies were observed to be more abundant in species and links than were bees in a sub-arctic alpine site in north Sweden [46] and in Andean meadows [47] . Dipteran species number (49.9%±5.2%) and link number (47.2%±5.1%) were predominant in our study, while Hymenopteran species contributed less, 29.5%±4.0% in species number and 38.1%±6.1% in link number. However, Hymenoptera linked to more plant species compared to Diptera (F = 2.36, p<0.01), indicating that Hymenoptera were more generalized than Diptera. Another investigation at the same site indicated that visits of bumblebees were more numerous than the total visits of all other pollinator groups [33] . High turnover rate in pollinator species does not necessarily imply high variation in pollination roles at a functional level. We found that plant species usually retain steady relationships to one or several functional groups over time, despite various levels of generalization. For example, the most generalized plant Pleurospermum davidii (Apiaceae) linked to 116 pollinator species across all 9 functional groups in four years. However, it had 4 functional groups consecutively, accounting for 88.4%±1.7% of the pollinators of the annual networks. These results suggest that generalized plants might experience stable evolutionary relationships with diverse functional groups. Different functional pollinator groups showed preferences for certain flower trait or traits combinations in the community [48] . Such stability and preferences of functional pollinator groups may contribute to the maintenance of diverse species in one community, although network compositions are highly changeable. Our finding of the same pattern of network structures in all four years suggests that we have captured the ecologically important interactions, given that a transect sample procedure was used in 2007 but timed observation procedures were used from 2008 to 2010. Our binary data prevent us from considering the visit frequency of each link. However, from the perspective of species duration and link partner turnover, a binary network could represent the dynamic nature of pollination at the community level. In summary, by comparing pollination networks over four years, we found the structure of the pollination network to be stable, and the fluctuation in species composition mostly represented in the periphery of the networks, without changing network shape. The pollination network in this highly diverse community is robust and plants at different generalization levels experience similar variation in pollinator functional groups, although there is a great fluctuation in community species composition as observed at other sites [6] , [19] . Our results also supported a recent 12-year butterfly plant network study, which suggested a separation between relatively stable species and sporadic species [49] . Our multi-year survey represents one of first studies on pollination networks in the biodiversity hotspot from China. Clearly, further study is needed if we are to understand how the generalized pollinators sustain diverse plant species in this alpine area. For example, more explicit data sets are needed to quantify the dynamics of pollination systems and explain how plants avoid reproductive interference in a highly generalized pollination system."
} | 4,695 |
29887307 | null | s2 | 1,755 | {
"abstract": "Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components, exopolysaccharides (EPSs) and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS producers, and generalists, which produce both EPSs and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPSs and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modeling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components."
} | 436 |
23879839 | PMC4231231 | pmc | 1,760 | {
"abstract": "Ecology, with a traditional focus on plants and animals, seeks to understand the mechanisms underlying structure and dynamics of communities. In microbial ecology, the focus is changing from planktonic communities to attached biofilms that dominate microbial life in numerous systems. Therefore, interest in the structure and function of biofilms is on the rise. Biofilms can form reproducible physical structures (i.e. architecture) at the millimetre-scale, which are central to their functioning. However, the spatial dynamics of the clusters conferring physical structure to biofilms remains often elusive. By experimenting with complex microbial communities forming biofilms in contrasting hydrodynamic microenvironments in stream mesocosms, we show that morphogenesis results in ‘ripple-like’ and ‘star-like’ architectures – as they have also been reported from monospecies bacterial biofilms, for instance. To explore the potential contribution of demographic processes to these architectures, we propose a size-structured population model to simulate the dynamics of biofilm growth and cluster size distribution. Our findings establish that basic physical and demographic processes are key forces that shape apparently universal biofilm architectures as they occur in diverse microbial but also in single-species bacterial biofilms.",
"introduction": "Introduction The realization of the extent to which microorganisms develop on surfaces, as matrix-enclosed communities has increasingly moved the interest of microbial ecology from planktonic to biofilm communities over the last decades (Costerton and Lewandowski, 1995 ; Hall-Stoodley et al ., 2004 ). Biofilms dominate microbial life in numerous aquatic ecosystems where they orchestrate key biogeochemical processes (Battin et al ., 2003 ; 2008 ). Biofilms are also important agents of biofouling and biocorrosion in technical systems (Bixler and Bushan, 2012 ) and account for numerous persistent and chronic infections (Costerton et al ., 1999 ; Hall-Stoodley et al ., 2004 ; Percival et al ., 2012 ). It is notably the recognition of the role biofilms play in medical and technical systems, which has boosted biofilm research over the last decades with a clear focus on monospecies bacterial cultures grown in vitro (Costerton and Lewandowski, 1995 ; Costerton et al ., 1999 ; Hall-Stoodley et al ., 2004 ; Percival et al ., 2012 ). This approach is now shifting towards multispecies bacterial biofilms if possible grown under more realistic in vivo conditions (Hibbing et al ., 2010 ; Elias and Banin, 2012 ; Rendueles and Ghigo, 2012 ). The ability of biofilms to form highly differentiated architectural structures is thought to be an ancient and integral characteristic of microorganisms, which over evolutionary time has lead to strategies of microorganisms to optimize growth even in adverse environments (Costerton and Lewandowski, 1995 ; Stoodley et al ., 2002 ; Hall-Stoodley et al ., 2004 ). Biofilms, whether monospecies or multispecies bacterial communities or even more complex communities, also including algae, protozoa and non-living particles as occurring in streams or tidal flats, can form reproducible architectures across scales. Mushroom-like caps, microcolonies with pores and channels (Parsek and Tolker-Nielsen, 2008 ), filamentous streamers (Stoodley et al ., 1999 ), and even quasipolygonal or reticulated geometries (Stoodley et al ., 1999 ; 2002 ; Battin et al ., 2003 ; Cogan and Wolgemuth, 2005 ; Parsek and Tolker-Nielsen, 2008 ; Baum et al ., 2009 ; Xavier et al ., 2009 ; Shepard and Sumner, 2010 ; Elias and Banin, 2012 ) figure among the most commonly observed architectures of microbial biofilms. An enduring question remains, what are the key forces driving biofilm structural differentiation (i.e. morphogenesis) and resulting architectures? Addressing this question is fundamental as morphogenesis can determine functional properties of biological systems (Bourgine and Lesne, 2011 ). Notably the spatial organization of biofilm architecture at the mesoscale (millimetre range) is recognized to affect biofilm functions and, well beyond, even ecosystem and engineering processes (Battin et al ., 2003 ; Morgenroth and Milferstedt, 2009 ; Wagner et al ., 2010 ). Understanding biofilm morphogenesis may also be helpful to unveil the success of the biofilm mode of life as biofilm function is tightly connected to architecture. Over the last decades, various conceptual and theoretical models were put forward to explain biofilm formation and structural differentiation (Wimpenny and Colasanti, 1997 ; Picioreanu et al ., 1998 ; 2007 ; Kreft et al ., 2001 ; Stoodley et al ., 2002 ; Parsek and Tolker-Nielsen, 2008 ; Monds and O'Toole, 2009 ). For instance, the developmental model (Monds and O'Toole, 2009 ) proposes genetic networks to guide phase transition in biofilm formation and emphasizes selection for the evolution of cooperation between microorganisms. Alternative models propose that stochastic interactions of microorganisms with the environment shape biofilm structure and function; here, biofilm morphogenesis is supposedly driven by selection in dynamic environments (Monds and O'Toole, 2009 ; Xavier et al ., 2009 ). Mathematical studies have related biofilm architecture to the availability of nutrients, carbon and oxygen, which ultimately results from the interplay between replenishment and uptake, processes that are linked to hydrodynamics (Wimpenny and Colasanti, 1997 ; Picioreanu et al ., 1998 ; 2007 ; Kreft et al ., 2001 ; Cogan and Keener, 2004 ; Klapper and Dockery, 2010 ). The hydrodynamics of the bulk liquid above the biofilms affects solute replenishment (Picioreanu et al ., 1998 ; Battin et al ., 2003 ) and, at the same time, imposes a physical control on architectural differentiation (Battin et al ., 2003; 2007 , ; Hall-Stoodley et al ., 2004 ). For instance, turbulent flow induces the formation of filamentous streamers oscillating in the water, whereas laminar flow seems to favour the formation of largely isotropic microcolonies (Stoodley et al ., 1999; 2002 , ; Hall-Stoodley et al ., 2004 ). Furthermore, as purported by the emerging field of sociomicrobiology, the balance between microbial growth and competition for nutrients, including cell motility, may also contribute to the emergence of higher order biofilm structures from individual clusters (Picioreanu et al ., 2007 ; Xavier et al ., 2009 ; Mabrouk et al ., 2010 ). By experimenting with complex biofilms under quasinatural flow and by applying a mathematical model, we study physical and demographic mechanisms that possibly underlie biofilm morphogenesis and mesoscale architectures in contrasting hydrodynamic microenvironments. Experiments were conducted in 40 m long streamside flumes where biofilm communities could assemble from the natural microbial communities suspended in the stream water (Besemer et al ., 2012 ). In these flumes, graded bedforms ( n = 40) induced reproducible flow landscapes typical of low-submergence headwater streams. Within these landscapes, we compared biofilm morphogenesis at the bedform crest and in the trough between consecutive bedforms as two contrasting and well-defined hydrodynamic microenvironments. We deliberately included into our study other biofilm components beside prokaryotes (i.e. bacteria and archaea), such as algae and non-living particles, as these are common in stream biofilms. Furthermore, we purposely focused on the dynamics of individual clusters such as microcolonies, but also single cells and non-living particles, as they are the fundamental building blocks of biofilms. Biomass clusters have traditionally received attention to study mass transfer phenomena and both chemical and microbial heterogeneity in biofilms (Stoodley et al ., 1998 ; Stewart, 2003 ; Stewart and Franklin, 2008 ).",
"discussion": "Discussion The study of the effects of hydrodynamics on biofilm formation and structural differentiation has been at the core of biofilm research (de Beer et al ., 1994 ; Lewandowski et al ., 1994 ; Stoodley et al ., 1998 ; Eberl et al ., 2000 ; Purevdorj et al ., 2002 ; Horn et al ., 2003 ). Our combined experimental and modelling findings expand on those suggesting that hydrodynamics imposes a major physical template on cluster dynamics and resulting biofilm morphogenesis. Biofilms were exposed to the same seeding material from untreated streamwater and had comparable biomass and even comparable bacterial community composition (Besemer et al ., 2009 ). Remarkably, however, biofilm morphogenesis resulted in diverging architectures in both hydrodynamic microenvironments. Despite quantitative differences of cluster size distributions in the two microenvironments, cluster size distributions were very similar from a qualitative perspective. This observation indicates that few basic processes linked to the different hydrodynamics at the crest and in the trough suffice to impose a major physical template on biofilm morphogenesis. Non-living particles, which contribute significantly to stream biofilms, but also cells with low motility are likely most susceptible to the physical constraints. More motile microorganisms may escape these constraints and contribute to biofilm morphogenesis and resulting structures via migration and coalescence, for instance. Mesoscale biofilm structures largely shaped by physical processes seem comparable with sand dune formation and other landform patterns (Werner, 1999 ; Bourgine and Lesne, 2011 ; Zhang et al ., 2012 ). Here, longitudinal dunes may elongate parallel to the prevailing wind, whereas star dunes may result from the combination of individual longitudinal dunes depending on the frequency of wind reorientation (Zhang et al ., 2012 ). These observations are in line with the concept of biofilms as microbial landscapes, where the interplay between hydrodynamics and substratum topography was postulated to shape biofilm architecture (Battin et al ., 2007 ). Multidirectional flow, as prevalent in the trough, allows higher degrees of freedom to the directionality of growing cluster to spread through cell migration and coalescence. This enhances the chance to interconnect with adjacent clusters and may ultimately result in the star-like architecture of larger clusters, which in turn causes the observed decline of cluster abundance in the trough. However, we are presently not able to provide a suitable mechanistic explanation for the onset of this process. In fact, the complexity of stream biofilms entails innumerable biotic interactions, internal elemental fluxes and even feedback loops between microbial heterotrophs and photoautotrophs (Lyon and Ziegler, 2009 ), which altogether would require highly sophisticated modelling approaches. Work on more simple systems such as on Pseudomonas aeruginosa biofilms has shown that the coalescence of adjacent cluster with similar pattern formation as observed in our biofilms involves the interplay between cell proliferation, surface-associated motility and the production of extracellular polymeric substances that form the biofilm matrix (Parsek and Tolker-Nielsen, 2008 ; Mabrouk et al ., 2010 ). Mabrouk and colleagues ( 2010 ) suggest that interconnected microcolonies in these P. aeruginosa biofilms appear when extracellular polymers are expressed at low rate and persist on the path generated by motile cells. We suggest that star-like structures as observed in the trough between bedforms optimize the exploitation of space, especially in an environment where turbulent wakes may impede solute replenishment. In fact, given that microbial biomass did not differ between crest and trough, it is reasonable to assume that diverging morphogenesis and concurrently different spatial coverage optimize space in contrasting flow environments. The connection of star-like clusters may ultimately result in quasipolygons as reported from laboratory-based bacterial (Xavier et al ., 2009 ; Mabrouk et al ., 2010 ) and stream biofilms (Battin et al ., 2003 ), and from cyanobacterial mats (Shepard and Sumner, 2010 ). General ecology relates such polygonal shapes to foraging optimality (Covich, 1976 ), which would support that such biofilm structures may be dynamically accessible optimal states frustrated by the physical constraints like the local turbulence structure. The unidirectional flow at the crest reduces the degrees of freedom to migrating cells and cluster coalescence to spread in space. This constrains cluster anisotropy with elongated shapes characterizing biofilm architecture. Shear stress is higher at the crest because of elevated flow velocity and may induce the higher fraction of cells migrating over short ranges (Table 1 ). Guided by the unidirectional flow, cells may preferentially settle downstream in the wake of the parental cluster. The reduced capacity of areal growth (as coverage) in this microenvironment is consistent with general ecological theory predicting physical disturbance to reduce growth efficiency (White and Pickett, 1985 ). Our study suggests that basic physical and demographic processes are sufficient to explain the morphogenesis and resulting higher order structures of biofilms containing high microbial diversity and even non-living particles. This may run counter the view of sociomicrobiology stating that multispecies bacterial biofilms with high cell density result from the balance between cooperation and competition, and that the understanding of this balance is essential to model biofilm formation (Kreft et al ., 2001 ; West et al ., 2006 ; Nadell et al ., 2009 ). The fact that hydrodynamics is a major control on cluster dynamics and resulting biofilm morphogenesis in our study may be attributable to the complex flow environment and flow velocities that reflect natural conditions in streams yet not the environment typically mimicked in flow chambers. However, our experimental results, emphasizing hydrodynamics as a physical forcing, are essentially consistent with mathematical models that predict biofilm architecture from hydrodynamics and related mass transfer phenomena (Cogan and Wolgemuth, 2005 ; Klapper and Dockery, 2010 ). We acknowledge that we have not attempted to study mass transfer in our biofilms and future studies will therefore focus on this aspect, also including metabolic capabilities of biofilms that diverge in architecture. Still, our study, combining physical forces and ecological processes, offers a fresh view on biofilm architectures, which appears universal independent of scale and community complexity."
} | 3,682 |
39710769 | PMC11663328 | pmc | 1,762 | {
"abstract": "Background Crustose Coralline Algae (CCA) play a crucial role in coral reef ecosystems, contributing significantly to reef formation and serving as substrates for coral recruitment. The microbiome associated with CCAs may promote coral recruitment, yet these microbial communities remain largely understudied. This study investigates the microbial communities associated with a large number of different CCA species across six different islands of French Polynesia, and assess their potential influence on the microbiome of coral recruits. Results Our findings reveal that CCA harbor a large diversity of bacteria that had not been reported until now. The composition of these microbial communities was influenced by geographic location, and was also closely linked to the host species, identified at a fine taxonomic unit using the 16S rRNA gene of the CCA chloroplast. We demonstrate the usefulness of these ecologically meaningful units that we call CCA chlorotypes. Additionally, we observed a correlation between host phylogeny and microbiome composition (phylosymbiosis) in two CCA species. Contrary to expectations, the CCA microbiome did not act as a microbial reservoir for coral recruits. However, the microbial community of coral recruits varied according to the substrate on which they grew. Conclusions The study significantly expands the number of characterized CCA microbiomes, and provides new insight into the extensive diversity of these microbial communities. We show distinct microbiomes between and within CCA species, characterized by specific chloroplast 16S rRNA gene sequences. We term these distinct groups “chlorotypes”, and demonstrate their utility to differentiate CCA. We also show that only few bacterial taxa were shared between CCA and coral recruits growing in contact with them. Nevertheless, we observed that the microbial community of coral recruits varied depending on the substrate they grew on. We conclude that CCA and their associated bacteria influence the microbiome composition of the coral recruits. Supplementary Information The online version contains supplementary material available at 10.1186/s40793-024-00640-y.",
"conclusion": "Conclusion Our results provide a new insight into the large diversity of the CCA microbial communities by considerably extending the number of CCA microbiomes studied to date. We show distinct microbiomes, between and within CCA species characterized with distinct chloroplast 16S rRNA gene sequences. We call these distinct groups chlorotypes and demonstrate their utility to differentiate groups of CCAs. It illustrates the importance of taking into account the host genetics for a better understanding of the microbiome composition. We also show that only few bacterial taxa were shared between the CCA and the coral growing in contact with it. These results suggest that the CCA-algal substrata did not act as the main source for the coral’s microbiome. Nevertheless, we observed that the microbial community of coral recruits varied depending on the substrate they grew on. We conclude that CCAs and/or their associated bacteria influence the composition of the coral recruits’ microbiome.",
"discussion": "Discussion The diversity of CCA microbiomes This study considerably increased the number of CCA analysed to date with up to 55 different chlorotypes detected. However, we could not saturate the discovery curve of the microbial community diversity since each addition of a new CCA species, or chlorotype, to the study increased the microbial diversity. It shows that there is still a large potential of undescribed microbial communities among the > 1,600 CCA species known globally [ 55 ]. This newly described diversity adds to the diverse communities of microorganisms recently reported in corals, tropical fish, and reef plankton [ 17 , 26 ], and demonstrates that reef ecosystems harbor a tremendous undescribed microbial diversity. Our study shows that chlorotypes (based on chloroplast 16S rRNA) could be used as a useful barcoding tool to differentiate genus of CCA, and even intraspecific variants. Classical CCA identification requires examination of different vegetative and reproductive features [ 20 ]. However, due to the need of expert eyes, and laboratory microscopy processing, the identification and differentiation of taxa based on morphological and development features remain challenging [ 20 ], especially since geographic, seasonal and environmental conditions can influence morphology [ 45 , 76 , 91 ]. To elucidate the phylogeny of CCA, molecular approaches have been increasingly applied, mainly using specific markers such as the 18 S rRNA gene, the chloroplast PsbA gene, and the mitochondrial cox1 gene [ 5 – 7 , 83 ]. We were not able to directly compare the 16S chlorotype from this study to the ones from other known CCA species due to lack of reference sequences for this gene. Our chloroplast 16S rRNA distance tree was nevertheless precise enough to separate groups of CCA, to explain that variations in microbial composition within a same CCA species followed chlorotype delineations, and that the addition of chlorotypes to the rarefaction curve added microbial diversity. It suggests that chlorotypes could in some cases represent taxonomic units that are not distinguishable by classical identification techniques. The potential for chlorotypes to resolve finer clades varied between species giving for instance better results for P. onkodes than for N. frutescens . However, the chlorotypes did not allow a precise phylogenetic separation of monophyletic groups, and did not directly support the known phylogenetic separation of CCA in the tree. For instance, the two Porolithon species did not form a monophyletic clade. The CCA microbiome communities were significantly different between CCA genera, but there were also significant differences between species from the same genus. It had been observed earlier for Porolithon [ 70 ], and our finding thus extends these observation to Neogoniolithon. Previous studies have also shown CCA species specific microbiomes [ 37 , 73 ], but our results go beyond current knowledge by showing that there were often differences in microbial community composition between CCA chlorotypes. We showed that even at the fine chlorotype or sub-species level, microbial communities were specific and strongly linked to the host genetics. It provides solid evidence of the specificity of the CCA – microbiome association. We originally hypothesized that CCA would show patterns of phylosymbiosis as previously observed in corals [ 60 ]. Our results partly confirmed this hypothesis, as we observed phylosymbiosis in P. onkodes and N. fosliei . In these cases, the host chlorotype was the main determinant of microbiome composition before the environment, and the geographic location. Phylosymbiosis patterns may emerge as a consequence of different factors like co-diversification, host and microbial biogeography interaction, or host trait preference [ 12 , 53 ]. Further work should be conducted by integrating sets of phylogenetic markers (SSU, LSU, psbA, COI, 23 S) [ 66 ], as well morphological taxonomy, to confirm our findings. In our study, CCA were overall dominated by Flavobacteriia (phylum: Bacteroidota ) followed by Alphaproteobactaria (phylum: Proteobacteria ). These two phyla were also identified as co-dominants in several other CCA microbiomes studies: in P. onkodes collected from the China Sea [ 92 ], Neogoniolithon sp. from the Mediterranean sea [ 27 ], and in N. mamillare from the Caribbean sea [ 46 ]. However, this is not always the case as seen with the absence or rarity of Bacteroidota in N. brassica-florida from Mediterranean sea [ 62 ]d fosliei and P. onkodes from Moorea, French Polynesia [ 37 ]. The differences may be due to different sampling design as some analyses were conducted on CCA from aquarium a day after the collection from the back reef [ 37 ], and aquaria and field microbial communities of Neogoniolithon sp. have been shown to differ significantly [ 27 ]. Additionally, they may be spatial or seasonal variations in communities as seen for several other benthic groups (e.g. corals, sponges)(White et al., [ 21 , 88 ]. For instance, N. fosliei microbial community changed from Alphaproteobacteria to Bacteroidota (formerly Bacteroidetes ) under rising sea surface temperature [ 86 ]. This change in the microbiome composition was accompanied by a reduction in the ability of N. fosliei to induce coral larval metamorphosis of Acropora millepora by half [ 86 ]. Similarly, our CCA were sampled following the 2016 bleaching event which increased heat stress up to 9.2 °C weeks and caused large-scale coral mortality in the Tuamotu Archipelago [ 30 ]. In our study, the most frequent CCA ASV (asv0000053) was identified as Ruegeria profundi [ 93 ] (100% identity). Interestingly, bacteria belonging to the Ruegeria genus could have antimicrobial activities, and notably against the diverse marine host-pathogen genus Vibrio (family Vibrionaceae ) [ 48 ]. Ruegeria are also known for their ability to form biofilms and their inhibition capabilities [ 19 , 77 ]. Additionally, a strain similar to the one we found ( Ruegeria mobilis , 99.3% identity), isolated from seawater in Eilat (northern Red Sea, Israel), was beneficial for larval development in the octocoral Rhytisma fulvum [ 25 ]. However, members of this bacterial genus were also found associated with diseased corals [ 3 , 78 ]é et al., [ 68 ]), so its role remains to be precisely determined. We also noted that asv0003145 represented a bacterium assigned to another genus from the Roseobacter family, Roseovarius , which contains the known pathogen Roseovarius crassostreae , the causative agent associated with juvenile oyster disease [ 11 ]. Microbial communities shared between CCA and the coral Pocillopora In our study, some bacterial taxa were shared between CCA and corals. This observation is interesting as microbial communities have been hypothesized to play a role in coral larvae recruitment and recruit health, but also as reservoir for the development of the coral’s microbiome [ 73 ]. However, here, all the bacterial taxa shared between the CCA and the associated coral recruits were found in very low abundance. Although limited to Pocillopora corals, these results suggest, contrary to an earlier hypothesis [ 73 ], that only few bacteria could have been transferred from the algal substrata to the coral colony. Furthermore, we observed that there were fewer shared taxa in adult corals compared to recruits. It implies that although bacteria taxa from the environment, here the algal substrate, may settle on coral recruits, further coral-bacteria, and bacteria-bacteria interactions select a stable and less diverse bacteria community in adult corals. Further studies on younger coral recruits are needed to explore the potential interaction between the algal microbiome and that of coral recruits. Although there was no clear transfer of bacteria from CCA to corals, we noted that the composition of the coral recruit microbiome differed according to their surrounding substrate. The coral microbial community may thus be influenced by the substrate. One explanation maybe the release of algal-specific metabolites. CCA and turf algae exude specific exometabolites to their surrounding environment, and these differentially influence reef microbial dynamics, and biogeochemical parameters, including labile DOC, oxygen availability, bacterial abundance and metabolism [ 28 , 87 ]. In turn, these could impact the bacterial communities recruited by the coral. CCA-associated bacteria may also produce secondary compounds which trigger variable degrees of coral larval settlement and metamorphosis [ 75 , 79 ]. These could attract specific bacteria. Microbiomes of Pocillopora recruits and adult colonies We observed significant differences in bacterial diversity and composition between Pocillopora young stages (recruits and juveniles: mean diameter of the colony 13.9 ± 4.82 mm and less than 1 year old) and adult corals. The highest bacterial species richness was associated with the coral recruits and the lowest richness with the adult, which has been observed earlier in Acropora tenuis and A. millepora [ 42 ]. We also observed that the composition of the recruit bacterial community was more variable than the one of the adult. Additionally, the bacterial communities associated with adult corals were dominated by bacterial strains also found in recruits, but in lower proportions, notably the well-known coral endosymbiont Endozoicomonas [ 33 , 59 ]. We further observed an increase in the proportion of recruits-adult common taxa with increasing coral colony size. These results suggest that the establishment of the adult bacterial community may have involved different selection processes from the one of the coral recruit. The highly diverse and heterogeneous bacterial community of recruits may progressively change in the well-established and conserved adult bacterial community comparable to a winnowing process [ 52 ], which has been described for the establishment of the Symbiodinium symbiosis [ 1 , 41 ], and with emerging evidence for microbes [ 4 , 40 , 42 , 69 ]."
} | 3,335 |
34721322 | PMC8551758 | pmc | 1,764 | {
"abstract": "Bioenergy crops are a promising energy alternative to fossil fuels. During bioenergy feedstock production, crop inputs shape the composition of soil microbial communities, which in turn influences nutrient cycling and plant productivity. In addition to cropping inputs, site characteristics (e.g., soil texture, climate) influence bacterial and fungal communities. We explored the response of soil microorganisms to bioenergy cropping system (switchgrass vs. maize) and site (sandy loam vs. silty loam) within two long-term experimental research stations. The live and total microbial community membership was investigated using 16S and ITS amplicon sequencing of soil RNA and DNA. For both nucleic acid types, we expected fungi and prokaryotes to be differentially impacted by crop and site due their dissimilar life strategies. We also expected live communities to be more strongly affected by site and crop than the total communities due to a sensitivity to recent stimuli. Instead, we found that prokaryotic and fungal community composition was primarily driven by site with a secondary crop effect, highlighting the importance of soil texture and fertility in shaping both communities. Specific highly abundant prokaryotic and fungal taxa within live communities were indicative of site and cropping systems, providing insight into treatment-specific, agriculturally relevant microbial taxa that were obscured within total community profiles. Within live prokaryote communities, predatory Myxobacteria spp. were largely indicative of silty and switchgrass communities. Within live fungal communities, Glomeromycota spp. were solely indicative of switchgrass soils, while a few very abundant Mortierellomycota spp. were indicative of silty soils. Site and cropping system had distinct effects on the live and total communities reflecting selection forces of plant inputs and environmental conditions over time. Comparisons between RNA and DNA communities uncovered live members obscured within the total community as well as members of the relic DNA pool. The associations between live communities and relic DNA are a product of the intimate relationship between the ephemeral responses of the live community and the accumulation of DNA within necromass that contributes to soil organic matter, and in turn shapes soil microbial dynamics.",
"conclusion": "Conclusion Our study highlights the dynamic nature of live and total microbial communities under major bioenergy cropping systems (switchgrass and maize) at two sites with differing soil texture and fertility. Our indicator species analyses demonstrate distinct live and total communities within early growing season soil. Live and total bacterial and fungal communities were significantly influenced by site and crop with all communities showing a slightly higher site effect. Although the live community represented a snapshot of the total community, site and crop-specific species were identified predominately within the live community. Bacterial indicator species were highly diverse with Myxobacteria generally associating with silty switchgrass communities. Mortierellomycota species, which scavenge for phosphorous and associate with mycorrhizal fungi, were indicators of silty soil. Glomeromycota species, which form beneficial associations with rhizomes, were indicative of switchgrass. Our comparison of live and total bacterial and fungal communities residing under switchgrass and maize revealed distinct live communities driven by similar environmental variables. Understanding the effect of agricultural management on live and total communities will further our understanding of how agricultural management and environmental change alter the soil microbiome.",
"introduction": "Introduction Bioenergy crop production provides a promising opportunity to decrease energy dependence on fossil fuels and limit increases in atmospheric carbon dioxide (CO 2 ) concentrations ( Gelfand et al., 2013 ). Low-nitrogen, marginal lands are targeted for bioenergy feedstock production to reserve productive land for food crops while increasing soil organic matter (SOM) stocks ( Paustian et al., 2016 ). SOM formation and nutrient cycling are mediated via decomposition of plant inputs by soil biota, including microorganisms. Microbial community composition influences nitrogen (N) availability and carbon (C) mineralization, while their biomass and biochemistry influence the production of microbial necromass and the formation of persistent SOM ( Simpson et al., 2007 ; Kallenbach et al., 2016 ; Crowther et al., 2019 ; Liang et al., 2019 ). Therefore, understanding how bioenergy cropping systems influence microbial community composition and necromass production is fundamental to sustainable bioenergy crop production ( Jansson and Hofmockel, 2020 ; Zhu et al., 2020 ; Kasanke et al., 2021 ). Annual (e.g., maize, Zea mays L.) and perennial (e.g., switchgrass, Panicum virgatum L.) bioenergy crops recruit diverse microbial communities with varied agronomic benefits ( Liang et al., 2012a ; Hargreaves et al., 2015 ; Jesus et al., 2016 ). Microbial recruitment is influenced by crop inputs, such as plant litter and root exudation, with annuals and perennials differing in their spatial and temporal delivery of these inputs ( Hargreaves and Hofmockel, 2014 ). After the majority of aboveground biomass is harvested for bioenergy production, live and dead roots are the dominant source of new substrates supporting soil microbial decomposition ( Sanford et al., 2016 ). In addition, perennial crops, like switchgrass, have year-round live root systems which can recruit symbiotic, nutrient-acquiring microbes such as arbuscular mycorrhizal fungi (AMF; Mafa-Attoye et al., 2020 ). The extent of AMF recruitment varies with sampling time and plant type with higher AMF abundance seen early in the growing season ( Hijri et al., 2006 ) and after long-term (>10year) perennial management ( Jesus et al., 2016 ). Perennials also have more extensive root systems than annuals, which have been shown to sustain a more diverse, but not necessarily larger (μg biomass C g −1 soil) microbial community ( Liang et al., 2012a ; Dou et al., 2013 ; Hargreaves and Hofmockel, 2014 ). Cropping system also incorporates the different fertilization requirements of each plant type, which is generally higher for annual plants than for perennials. A higher fertilization rate can impact microbial recruitment by decreasing the necessity for plants to support nutrient-acquiring symbionts such as AMF, and thereby lowering AMF root colonization and/or diversity ( Oates et al., 2016 ; Emery et al., 2017 ; Jach-Smith and Jackson, 2018 ). However, other studies show no or a positive AMF response to fertilization ( Treseder and Allen, 2002 ; Egerton-Warburton et al., 2007 ; Cheng et al., 2013 ). It is essential to understand the plant-microbe interactions within bioenergy cropping systems, to optimize microbial contributions to C and nutrient cycling, plant productivity, and long-term soil C storage. In addition to the influences of cropping system on microbial community composition, variability in soil characteristics between experimental sites is often the greatest determinant in explaining soil microbial community differences ( Mao et al., 2013 ; He et al., 2017 ; Zhou et al., 2017 ; Xue et al., 2018 ). Soil properties that influence soil moisture and nutrient status, such as texture, fertility, and pH, have been correlated to changes in bacterial and fungal community composition within unmanaged systems ( Lauber et al., 2008 ; Vieira et al., 2019 ). Specifically, bacterial communities are shown to be structured by soil type and pH due to their sensitivity to microhabitat conditions as well as nutrient status ( Ranjard and Richaume, 2001 ; Girvan et al., 2003 ; Fierer and Jackson, 2006 ; Fierer et al., 2009 ; Ramirez et al., 2012 ). Fungal communities are shown to be influenced by differences in soil moisture and nutrient status ( Lauber et al., 2008 ; Talbot et al., 2014 ; Peay et al., 2016 ). Studies on agroecosystem soil microbiomes have largely focused on the effect of management type (e.g., land-use change, tillage, and biochar; Zhang et al., 2016 ; Sheng and Zhu, 2018 ; Gu et al., 2019 ); yet the contemporary and cumulative effects of bioenergy crops on the soil microbiome is still lacking, especially in a long-term (decadal), cross-site context ( Liang et al., 2012b ; Jesus et al., 2016 ). Although the importance of cropping system and site characteristics in defining the assembly and function of microbial communities is generally recognized, there are still gaps in our knowledge of how these factors differentially impact microbial community membership to enhance necromass production and SOM formation. This is because the soil microbiome is extremely diverse and the community structure varies over time and space, making generalizable patterns difficult to identify. The influence of contemporary environmental conditions on microbial community structure may be best characterized with RNA-based measures, while DNA provides an integrated signature of both past and present microbiomes ( Girvan et al., 2003 ; Orellana et al., 2019 ). Though there remains uncertainty surrounding the interpretation of RNA and DNA measurements, current consensus suggests that RNA-inferred communities include both metabolically active and dormant cells; therefore, RNA-based analyses are thought to represent organisms that may quickly respond to changes in environmental conditions ( Anderson and Parkin, 2007 ; Blazewicz et al., 2013 ; Emerson et al., 2017 ). DNA is more stable in the environment; therefore, it is a less sensitive measure of temporal shifts in environmental microbiomes. Like RNA communities, DNA communities include active and inactive live cells. In addition, DNA communities include the roughly 40% of bacterial and fungal DNA that originates from non-living cells (“relic” DNA), whose persistence is a result of factors such as pH and soil mineralogy ( Carini et al., 2016 ; Lennon et al., 2018 ). This relic DNA can persist for years and obscure measures of the dominant, live members, potentially skewing interpretations of microbial contributions to ecosystem function ( Nagler et al., 2018 ). At the same time, this persistent relic DNA can potentially help identify organisms that produce necromass and contribute to SOM formation. While we recognize that RNA and DNA-inferred community interpretation is a dynamic field, we will refer to RNA and DNA communities from this point on as “live” and “total,” respectively. Past literature has found markedly different dominant members in live and total communities during SOM decomposition ( Baldrian et al., 2012 ), after long-term N deposition ( Freedman et al., 2015 ), following forest to plantation land conversion ( Meyer et al., 2019 ), and after pulses of OM and metalloids ( Birrer et al., 2018 ). Live bacterial communities have also been shown to be less diverse than total bacterial communities in bulk and rhizosphere rice paddy soils ( Li et al., 2019 ). Assessing differences in live and total community diversity is a powerful approach for characterizing the response of microbial communities to contemporary disturbances as well as long-term environmental change. The contributions of both the live and total communities are critical to developing cropping systems that manage microbiomes to promote plant production and enhance SOM formation. To date, live and total microbial community analyses have not been compared within bioenergy cropping systems which is important for understanding how promising bioenergy crops, like switchgrass, alter microbial community composition and microbially-driven processes. By characterizing live and total microbial communities at long-term maize and switchgrass sites with differing soil types, we have the unique opportunity to address this knowledge gap. This brings us to our main question: How do crop and site differentially influence the live and total prokaryotic and fungal communities within bioenergy cropping systems? H1: Fungal community composition is primarily influenced by crop type (perennial vs. annual) due to the importance of symbiotic relationships between plants and fungi for fungal metabolism. H2: Prokaryotic community composition is primarily influenced by soil type (sandy vs. silty) due to the importance of microhabitats created by soil texture. H3: Live prokaryotic and fungal community compositions are more sensitive to crop and site than their total community counterparts because they represent the most recent field stimuli without the influence of relic DNA.",
"discussion": "Discussion Despite Compositional Differences, Live and Total Communities Have Similar Environmental Drivers We performed the first analysis of the influence of bioenergy cropping systems on live and total soil microbial communities to better understand the contemporary and lasting selective forces of cropping system and site selection on bacterial and fungal communities. We hypothesized that live communities would be more sensitive to contemporary conditions because they represent the most recent field stimuli without the influence of relic DNA (H3). Instead, we found that site and cropping system contributed similarly to selecting live and total community structure, although their community membership varied strongly ( Tables 1 and 2 ). Our results differ from other studies that found a stronger treatment response in the live compared to the total community after addition of biochar to rice paddies ( Chen et al., 2016 ) and influx of methane into a landfill biocover ( Kim et al., 2013 ). A distinguishing factor of our study is the consistent field management during the previous 9years, while the cited studies measured the effect of acute treatments on the live community. Our results suggest that our consistent cropping system produced a strong present-day and legacy effect on microbial communities, validating the importance of DNA-based analyses for detecting lasting environmental impacts while highlighting the importance of RNA for identifying community members that are responsive to current environmental conditions ( Zhang et al., 2014 ; Nawaz et al., 2019 ; Orellana et al., 2019 ; Wutkowska et al., 2019 ). The cumulative effects of site and crop on community structure explained 18% more variation within live bacterial communities than for live fungal communities (0.49 vs. 0.31; Table 2 ). These findings indicate that bacteria and fungi react differently to biotic and abiotic factors, and we are missing a larger portion of the variation behind fungal community composition when only accounting for broad effects such as cropping system and site. Site and cropping system explained a similar amount of variance to past studies at our experimental sites ( Jesus et al., 2016 ), yet a large portion was left unexplained likely due to several unaccounted drivers such as phosphate concentration and soil moisture ( Fierer et al., 2009 ; Kuramae et al., 2012 ; Deepika and Kothamasi, 2015 ). Stochastic processes, such as random birth/death events, are inherently unpredictable and are likely playing a role in the unexplained variance as well ( Evans et al., 2017 ). Site Was the Primary Influence on Bacterial and Fungal Communities While we were supported in hypothesizing that site would be the primary driver of prokaryotic community composition (H2), we did not anticipate a primary site effect within the fungal community as well. Even though live and total communities differed in composition, they were similarly influenced by environmental factors ( Table 2 ), which has been shown within forested and aquatic systems ( Romanowicz et al., 2016 ; Nawaz et al., 2019 ). Site can encompass many attributes; because pH and climate are similar between our two study sites, soil type and nutrient status are likely the main drivers of community structure. The importance of texture and nutrient status indicates a reliance on habitat niche, a pattern consistent with previous studies ( Girvan et al., 2003 ; Oehl et al., 2010 ; Jesus et al., 2016 ; Jach-Smith and Jackson, 2018 ; Kasanke et al., 2021 ). It is interesting to note that Jesus et al. (2016) studied the same field sites and found a strong site effect on DNA communities after 2years of establishment ( Kasanke et al., 2021 ), and after 8years of establishment also found a strong long-term site effect. The novelty of our results come from the inclusion of RNA-based data as well as sampling pre-growing season, which reveal that the strong site effect on microbial community characteristics is consistent for both the time-integrated total community and the contemporary live community. DNA is appropriate for some questions, particularly when comparing communities across time or space, but is less ideal for deciphering relationships between environmental effects and microbial members due to differences in dominant membership between the live and total community. Crop Had a Secondary Influence on Bacterial and Fungal Communities We hypothesized that crop type would have a stronger impact than site in shaping fungal communities due to the importance of plant inputs as a carbon/energy source for fungal metabolism (H1), but live and total communities sampled pre-growing season did not support this hypothesis. Crop management explained a smaller but significant amount of variance in both bacterial and fungal communities, yet the live and total communities were composed of different proportions of members ( Tables 1 and 2 ). Both communities uncovered the prevalence of Basidiomycota in maize plots and Glomeromycota under switchgrass. Live and total communities revealed different dominant crop-specific members with a higher proportion of Mortierellomycota within live maize communities and more Olpidiomycota found within total switchgrass communities ( Table 1 ). Mortierellomycota contains several genera of saprotrophic fungi which points to a possible prevalence of fungi decomposing recalcitrant maize residue left over from the previous season ( Webster and Weber, 2007 ). Olpidiomycota is a new phylum composed primarily of plant-pathogenic genera which indicates a potential relationship between pathogens and perennial crops ( Naranjo-Ortiz and Gabaldón, 2019 ). Even in the absence of actively growing plants, cropping system had a lasting impact on both the live and total community membership. Although our conventional cropping system did not allow for a true test of crop type vs. fertilizer rate (i.e., lower N rates on maize and vice versa), it is a powerful systems approach which provides insight into long-term impacts of cropping management on the soil microbial community. Past studies have highlighted bioenergy crop type as an important secondary driver of bacterial community structure ( Zhang et al., 2017 ), yet our results uncovered the universality of this trend (i.e., for fungi and within both live and total communities). Seasonality can affect the delivery of plant-derived substrates (i.e., aboveground litter production and root exudate production) as well as delivery of inorganic nutrients (i.e., fertilization). By sampling early in the growing season, rhizodeposition was minimized, thereby focusing the attention on the recent and long-term stimuli of fertilization and perennial/annual cropping, respectively. Other studies have documented the effect of rhizodeposition during the growing season ( Upton et al., 2019 ; Wattenburger et al., 2019 ); we captured the persisting effects of cropping system and site by sampling pre-growing season. Jesus et al. (2016) found crop to be a greater driver than site within long-term (10years) bioenergy cropping systems which contradicts our primary site effect within both contemporary (RNA) and long-term (DNA) communities. This difference could be explained by differences in sampling time, wherein our pre-growing season soil conditions determined community composition instead of growing season crop inputs. While we expected recent N fertilization to heavily impact live communities, soil properties such as C/N are important for satisfying microbial metabolic needs ( Zhao et al., 2019 ) and differed between sites ( Supplementary Table 10 ) Although the crop effect was secondary, it was still significant within live communities, which indicates the influence of recent field management such as fertilization and crop types (perennial/annual). Myxobacteria Were Indicative of Silty Switchgrass Communities Assessing which microbes are indicative of different soil niches and crops can assist in connecting the micron-scale functions of microorganisms to the broader ecological context ( Bach et al., 2018 ; Biesgen et al., 2020 ). After identifying unique membership, relationships between membership and ecosystem processes were explored. Indicator species analyses on the bacterial community revealed a highly diverse suite of prokaryotic indicator species (59 phyla) within both the live and total communities ( Supplementary Files 4 and 5 ). Unlike the fungal indicator species, specific phyla or orders of prokaryotes were not solely indicative of a site or cropping system. While the proteobacterial order, Myxococcales , was found within all sites, crops, and combinations, live silty switchgrass communities had the highest proportion of Myxococcales as well as highly abundant members ( Supplementary Figure 2 ; Supplementary File 4 ). Members of Myxococcales represent facultative predatory bacteria which hunt in packs to lyse cells and consume necromass ( Muñoz-Dorado et al., 2016 ; Hungate et al., 2021 ). They have been shown to prey on gram-negative bacteria and assimilate necromass carbon ( Hanajima et al., 2019 ). It is intriguing to consider what characteristics of the spring pre-growing season conditions within silty and switchgrass communities were ideal for Myxococcales to bloom. The pre-growing season is characterized by low plant-derived inputs which might be an indication of the ability of Myxobacteria to survive and thrive when food is scarce. Mortierellomycota Were Strong Indicator Species for Silty Soil Mortierellomycota species were top indicators for both live and total fungi within silty soil. Compared to sandy soils, the silty soil had higher total dissolved nitrogen (TDN) levels which were positively correlated with the abundance of genus Mortierella fungi at our sites ( R 2 =0.22, p =0.001; Supplementary Figure 4 ) and reported within past studies ( Detheridge et al., 2016 ). In addition to Mortierellomycota, both live and total silty switchgrass indicator species were dominated by AMF. The prevalence of Mortierella species might be beneficial to crop growth, since these species have been found to increase plant phosphorus uptake when in the presence of AMF ( Osorio and Habte, 2001 ). Thus, silty switchgrass communities may benefit from microbe-microbe interactions between AMF and Mortierellomycota. Additionally, Mortierellomycota are generally fast-growing fungal species that thrive on organic substrate additions in arable soils ( Schlatter et al., 2017 ). Thus, the higher C and N status of silty soil could have stimulated the unique occurrence of highly abundant Mortierella species. The abundant Mortierellomycota indicator species were shared between the live and total fungal communities, suggesting they are persistent and may be all-time ecologically important members of the fungal community. These results support the notion that Mortierella are potentially important contributors to microbial necromass production and SOM formation ( Fernandez and Kennedy, 2018 ; Li et al., 2018 ; Kasanke et al., 2021 ). Glomeromycota (AMF) Were Strong Indicator Species for Switchgrass Switchgrass supported unique fungal members, as demonstrated by higher counts of indicator species compared to maize within both the live and total fungal community. Switchgrass indicator species were predominantly Glomeromycota, especially within the live community, alluding to the importance of perennial rhizomes for early season switchgrass growth symbionts ( Somenahally et al., 2018 ). AMF form associations with ~75% of plant species and acquire nutrients which could be especially important on marginal lands with limited nutrient pools ( Emery et al., 2018 ). In native prairies, switchgrass is known to be strongly reliant on AMF ( Bingham and Biondini, 2009 ), but this is the first RNA-based documentation of AMF activity in early season switchgrass bioenergy cropping systems. Switchgrass receives a much lower fertilization rate compared to maize which might also promote higher AMF colonization, since mycorrhizal abundance has been shown to negatively correlate with N fertilization within agriculture and across biomes ( Treseder, 2004 ; Emery et al., 2017 ; Jach-Smith and Jackson, 2018 ). Sandy switchgrass had more indicator species than silty switchgrass and a higher proportion of Glomeromycota species. A higher proportion of Glomeromycota indicators in sandy soil coincides with past findings that sand content positively correlated with higher colonization of AMF fungi ( Zaller et al., 2011 ). Members of the AMF-genus Rhizophagus were also marginally negatively correlated with salt-extractable N concentrations ( R 2 =0.13, p =0.065; Supplementary Figure 5 ); thus, a lower N level could have encouraged Glomeromycota colonization in sandy soils like past studies ( Detheridge et al., 2016 ). The prevalence of AMF is exciting because of their role in supporting plant health by scavenging nutrients from outside the rhizosphere ( Chen et al., 2018 ). Although cropping system was a secondary microbial filter compared to soil type, crop-specific colonization of beneficial fungi under switchgrass demonstrates the lasting influence of cropping system on the soil microbiome even before the growing season. Live and Total Communities Highlight Different Members Live and total bacterial communities contained different dominant members, making it difficult to link relatively abundant bacterial taxa to their environmental roles or preferred habitats using only an amplicon DNA-based approach ( Table 1 ). DNA-based investigations into soil bacterial community structure are less technically involved and therefore much more common than RNA-based studies ( Knight et al., 2018 ). While temporally sampling the DNA community can help distinguish between seasonal or event-driven blooms, RNA is important for highlighting live members continually concealed in the DNA community by a potentially large relic DNA pool ( Carini et al., 2020 ). However, it is not possible to determine if members of the live community are active, perhaps except for ITS RNA sequencing due to the presence of ribosomes in dormant cells ( Blazewicz et al., 2013 ). In addition, slow growing microorganisms in this environment (e.g., oligotrophic) may be less represented in the live community compared to the total community due to lower ribosome cell content. Alternatively, differences in fungal and bacterial live and total communities may be driven by a difference in residence time between bacterial and fungal DNA. Fungal cell walls are composed of recalcitrant materials which decay slowly, favoring their accumulation in soil ( Li et al., 2015 ; Starke et al., 2019 ). This suggests that differences between total and live communities could be used to identify relic DNA from recalcitrant fungal biomass and provide a signature for taxa contributing to microbially-derived SOM (“necromass”; Liang et al., 2019 ). The communities represented by RNA and DNA sequencing is still a topic of debate and differentiating the in-situ diversity of living microbial biomass from long-term persistent signatures remains an important frontier. By uncovering unique, dominant members within the RNA-based community, our results emphasize the importance of recognizing that the DNA-based community is an integrative measure of past and present communities. To avoid the significant biases imposed by relic environmental DNA, other approaches (e.g., RNA, qSIP, and culturing) should be used in tandem for linking microbial taxonomy to environmental factors."
} | 7,091 |
21625650 | PMC3078317 | pmc | 1,765 | {
"abstract": "Social insects rank among the most abundant and influential terrestrial organisms. The key to their success is their ability to form tightly knit social groups that perform work cooperatively, and effectively exclude non-members from the colony. An extensive body of research, both empirical and theoretical, has explored how optimal acceptance thresholds could evolve in individuals, driven by the twin costs of inappropriately rejecting true nestmates and erroneously accepting individuals from foreign colonies. Here, in contrast, we use agent-based modeling to show that strong nestmate recognition by individuals is often unnecessary. Instead, highly effective nestmate recognition can arise as a colony-level property from a collective of individually poor recognizers. Essentially, although an intruder can get by one defender when their odor cues are similar, it is nearly impossible to get past many defenders if there is the slightest difference in cues. The results of our models match observed rejection rates in studies of ants, wasps, and bees. We also show that previous research in support of the optimal threshold theory approach to the problem of nestmate recognition can be alternatively viewed as evidence in favor of the collective formation of a selectively permeable barrier that allows in nestmates (at a significant cost) while rejecting non-nestmates. Finally, this work shows that nestmate recognition has a stronger task allocation component than previously thought, as colonies can nearly always achieve perfect nestmate recognition if it is cost effective for them to do so at the colony level. Electronic supplementary material The online version of this article (doi:10.1007/s00265-010-1094-x) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction The ability to discriminate self from non-self is fundamental to the evolution and function of biological systems ranging from multicellular organisms to colonies of social insects (Grosberg 1988 ; Hölldobler and Wilson 1990 ; Bourke and Franks 1995 ; Crozier and Pamilo 1996 ; Tsutsui 2004 ). In the social insects, it is believed that a worker discriminates nestmates from non-nestmates by comparing the olfactory cues on the surface of individuals they encounter with a cognitive representation of the suite of acceptable odors, known as a template (Getz 1982 ; Lacy and Sherman 1983 ; Stuart 1988 ; Breed et al. 1988 ; Reeve 1989 ; Tsutsui 2004 ). In many cases, young workers form their template by imprinting on the odors of nestmates (Lacy and Sherman 1983 ). The odor profile is a composite of various chemicals, which may be of genetic or environmental origin, with cuticular hydrocarbons, which have a genetic basis, playing a prominent role (Vander Meer and Morel 1998 ; Soroker et al. 1998 ; Lah av et al. 1999 ; Howard and Blomquist 2005 ; Foitzik et al. 2007 ; Martin et al. 2008 , 2009 , D’Ettorre and Lenoir 2010 ; Guerrieri et al. 2009 ; D’Ettore and Lenoir 2009). A widely accepted and influential model for nestmate recognition has explored the forces that drive the evolution of optimal acceptance thresholds in individual workers (Lacy and Sherman 1983 ; Reeve 1989 ; Downs and Ratnieks 2000 ; Couvillon et al. 2008 ). In this approach, individuals are unable to distinguish nestmates from non-nestmates with perfect precision because there may be overlap in the labels (odor cues) possessed by individuals in separate colonies and because sensory systems may be insufficiently sensitive to detect small differences in labels. The resulting recognition errors can take two forms. On one hand, extremely stringent recognition systems will effectively exclude non-colony members, but may also inadvertently lead to the inappropriate rejection of nestmates (referred to as false rejections). On the other hand, extremely permissive recognition systems should minimize false rejections, but open the door to the inappropriate acceptance of non-nestmates (acceptance errors). Although the costs of recognition errors vary among species and through time, the trade-off between them is thought to lead to an optimal acceptance threshold that dictates whether an individual accepts or rejects in a given encounter (Reeve 1989 ). In contrast to theory, which assumes an overlap between the cues of different colonies such that members of different colonies can have identical cues, work on the chemistry of nestmate recognition shows that olfactory cues are variable between colonies and uniform within colonies (reviewed in Howard and Blomquist 2005 ; Martin et al. 2009 , in press ). The diversity of recognition cues allows an experimenter, for example, to reliably distinguish one colony’s members from another’s (Vander Meer et al. 1989; Martin et al. 2008 ). Research has also shown that even in polygynous colonies, which have the greatest genetic diversity underlying cues, mechanisms for sharing chemical cues ensure that each member of a colony has the same overall cue profile (Martin et al. 2009 ). In spite of these results, however, there is still likely a functional overlap between the cue distributions of different colonies because workers may not be able to perceive relatively small differences in composite odor profiles. Further, the complex environment in which nestmate recognition occurs ensures numerous errors. For one thing, the nest can be crowded and workers may not get a good chance to inspect another individual as it pushes its way through the bustle of workers. Returning workers may also have been exposed to food stuffs with strong and/or similar odors to their own, for example (Liang and Silverman 2000 ; Chen and Nonacs 2000 ). Likewise, a honey bee that has partially acquired the odor cues of a foreign colony while robbing must still be accepted by its own colony (Breed et al. 1988 ; Couvillon and Ratnieks 2008 ). Hence, there is likely some limitation on the part of a worker to quickly distinguish between similar odors that create an error window around the threshold for acceptance, which itself is modified by the current environment. This context is illustrated in Fig. 1 . If the acceptance window is moved to the left to exclude all non-nestmates, it would result in the rejection of nestmates; thus, illustrating the basic trade-off between true and false rejections.\n Fig. 1 Conceptual model for the trade-off between the prevalence of true and false rejections based on the limited olfactory acuity of individual workers. Any perceived worker with a template cue dissimilarity value to the right of the acceptance threshed is rejected, while those to the left are accepted. Imperfect discrimination ability on the part of the worker is proposed to generate an error window (within which odor differences cannot be distinguished). Depending on the width of this window, and the placement of the acceptance threshold, this window can generate a trade-off between acceptance and rejection errors. In order to account for studies showing no false rejections, the acceptance level must be set far to the right of the nestmate distribution, necessitating a large number of false acceptances of non-nestmates \n Here, we explore nestmate recognition as a collective process. We test the hypothesis that colonies, unlike individuals, can achieve near-perfect nestmate discrimination (in spite of the above-mentioned physiological trade-offs) because a colony, unlike a single worker, gets multiple chances to make the correct decision. We first review the literature to determine whether data on recognition errors supports the traditional individual-level model or a collective one. We then develop two agent-based models to explore nestmate discrimination when it is treated as a collective process. The first focuses on the context of when a colony posts no dedicated guards. The second model explores the more complex case of active nest defense, in which the colony posts guards specialized for the detection of intruders.",
"discussion": "Discussion This study advances our understanding in two ways. First, it shows that physiological trade-offs, which can create severe barriers to adaptation at the individual level (Reeve 1989 ), can be easily overcome by collective decision making (Marshall and Franks 2009 ). Although individual workers cannot exclude all non-nestmates without also excluding nestmates (as suggested by previous studies), we show that colonies can because they have multiple opportunities to make the assessment. Thus, a colony can accomplish a task that an individual cannot, a central tenet of collective decision making (Seeley et al. 2006 ; Marshall and Franks 2009 ). Further, although much of this study deals with honey bees, the factors modeled are common to all social insects, so the results should be broadly applicable. In addition, Table 1 reviews mainly work on ants, showing that this hypothesis may be equally applicable to ants, as well as bees and wasps. Finally, this collective decision-making result has consequences for the physiological study of nestmate recognition because it suggests that for many species there may only be weak selection pressure for sophisticated individual-level recognition ability. Although our study is primarily focused on the general theoretical underpinnings of nestmate recognition, we found theoretical support for some interesting hypotheses related to the natural history of nestmate recognition in ants and bees. First, we found that a worker trying to invade another colony faces a daunting task, as it must evade detection almost continuously as it moves through the nest (Fig. 3 ). This is perhaps why some species do not post guards. They are unnecessary, as penetrating a nest involves numerous encounters between an invader and the host workers. Second, posting dedicated guards at the nest entrance is likely a costly trait. This is because, although guarding allows for stringent nest defense, it carries the cost of disrupting the colony’s own foraging, as guards inspect and falsely reject nestmates (Fig. 5 ). Therefore, it might not be cost effective to post guards in many contexts in which the cost in lost foraging outweighs the benefits of preventing robbing. This may be why colonies, such as honey bees, only intermittently post guards (Downs and Ratnieks 2000 ). Previous research predicts a correlation between true and false rejections due to the similarity of the odor profiles of nestmates and non-nestmates (Reeve 1989 ). That work assumed each individual-level error carried with it a cost, however, and as we show here, when this assumption is relaxed, as the biological context suggests it must, the prediction changes. When a colony gets many chances to make the correct decision, it does better than an individual who gets only one chance. Table 1 supports this by showing the permissive individual-level acceptance thresholds of species of ants, wasps, and bees. Although the basic prediction of the individual-level and collective approach differ, there is some overlap, however. Studies of bees and wasps have shown significant nestmate rejection in some contexts (guarding) predicted by the individual-level approach (Downs and Ratnieks 2000 ; Couvillon et al. 2008 ). When we modeled this guarding behavior as a collective process, we found that colonies are nevertheless still making near-perfect decisions. Thus, when individuals lower their acceptance thresholds (causing the false rejection of nestmates at the per-encounter level), it is best interpreted as a colony-level adaptation to create a selectively permeable barrier at the nest entrance, rather than as an individual-level adaptation focused on optimizing the costs and benefits of accepting nestmates versus non-nestmates. In general, the present work suggests that colonies should typically be capable of near-perfect nestmate recognition, in spite of the physiological constraints at the individual level. However, strong nestmate recognition may not always be cost effective, as it slows the colony’s foraging rate. Thus, nestmate recognition, like most social insect problems, is a task allocation problem (Seeley 1995 ). As for every task, nestmate recognition has a rich proximate basis, but the individual-level characteristics of the workers must always be considered from a colony-level perspective when questions of adaptation are considered. It has previously been argued that context-dependent nestmate recognition is a solution to the problem modeled by our within-nest nestmate recognition model (Errard et al. 2006 ; Lenoir et al. 2009 ; Ozaki et al. 2005 ). From this perspective, they argue that the problem of multiple interactions potentially leading to large numbers of false rejections in the nest, and hence within-nest fighting, is solved by only performing nestmate recognition outside the nest and never within it. This is an intriguing alternative to our solution to this problem, which is nevertheless not mutually exclusive with the main point of this study. This is because the studies reviewed in Table 1 were not conducted within nests. Thus, low rejection of nestmates is something that occurs both outside and inside the nest. Hence, our result that multiple interactions can lead to near-perfect nestmate recognition at the colony entrance is independent of the notion of context-dependent nestmate recognition. Because a colony of weak discriminators is more effective than an individual with even maximal recognition ability (given physiological constraints), there may be less selective pressure at the individual level for increased recognition ability than previously thought. However, a greater ability, at the individual level, for accurate nestmate discrimination is adaptive, as it decreases the time (number of encounters) to rejection for non-nestmates (Fig. 2 ). Hence, we might expect a range of individual-level recognition abilities resulting from the need of each species at the colony level to solve their unique nestmate and parasite recognition problems (Martin et al. 2010 ). Studies show a broad range of recognition abilities (reviewed in Table 1 ) which, although known to correlate with the complexity of the genetic and environmental cues being used, also supports this prediction. This range of individual-level discrimination abilities between species may also magnify the difficulty of determining the neural mechanisms by which nestmate recognition occurs, as mechanisms sufficient for producing weak recognition ability could be different from those that are considerably stronger. It is also likely that neuronal mechanisms may strongly vary between species, as there are likely many ways to generate the weak recognition abilities social insects possess and because nestmate recognition systems are not likely to have been inherited intact from a common ancestor."
} | 3,731 |
24688666 | PMC3962098 | pmc | 1,766 | {
"abstract": "Technological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with –omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds.",
"introduction": "Introduction Since antiquity, microbial fermentation processes have been extensively used for the processing of foods and the production of beverages, while technological developments over the past century made microbes the work-horses for large scale industrial production processes. Since the 1980s in particular, significant advances in genetic engineering have converted microbes to “cell factories” for the production of a diverse range of important chemical compounds. Manipulation of microbial metabolism holds major advantages, since microbes offer an environmentally friendly means to efficiently convert cheap raw materials like glucose, sucrose, and biomass derived materials into high value chemicals and fuels. Saccharomyces cerevisiae is an organism highly preferred by the industry, as it can withstand high osmotic pressure and reduced pH compared to bacteria [ 1 ]. Currently, there is continuous development and improvement of yeast strains for the production of high levels of desirable end products. The pace of strain development has accelerated as new tools for metabolic engineering manipulations are introduced. The overall approach for generating high production strains is currently based on a number of complementary approaches that include: a) the upregulation of desirable biosynthetic pathways, b) the suppression of pathways that drain resources or precursors (competing pathways), c) the introduction of exogenous genes or biosynthetic pathways, and d) the development of methodologies to alleviate stress and/or toxicity caused by the production of high levels of the product or of an undesirable intermediate. The arsenal of the contemporary metabolic engineer contains tools that allow for either targeted rational interventions that introduce changes in the strain's genotype based on past knowledge of the biosynthetic machinery and its regulation, or global screens that combine classical approaches of strain evolution through adaptation and selection with –omics approaches that can globally assess changes leading to desirable outputs. Efforts to produce terpenoids in S. cerevisiae are characteristic of the variety of novel approaches used for strain improvement. Terpenoids and isoprenoids are an important class of secondary metabolites contributing more than 50,000 compounds to the rich chemical diversity of natural product structures [ 2 ]. Members of this group have attracted commercial interest as flavour and fragrance additives in the food and cosmetic industry. One such example is sclareol, an industrially important diterpene used by the fragrance industry [ 3 , 4 ]. Many terpenoids also possess pharmaceutical properties and are currently used in clinical practice. Among them taxol, a diterpene from yew, which has successfully been established as a major antineoplastic agent, and artemisinin, a sesquiterpene lactone, which is an effective antimalarial agent [ 5 – 10 ]. Recently, attention has also focused on microbially produced terpenes as biodiesel [ 11 – 13 ]. Terpenoids are biosynthesized from two C 5 precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [ 14 ]. In yeast and mammals, IPP originates from acetyl-CoA through the intermediate mevalonic acid (MVA). IPP then gives rise through the action of prenyltransferase enzymes to the higher order building blocks, geranyl pyrophosphate (GPP; C 10 ), farnesyl pyrophosphate (FPP; C 15 ) and geranylgeranyl pyrophosphate (GGPP; C 20 ) [ 14 ]. In yeast, most of the pathway output in the form of FPP is utilized for the biosynthesis of sterols. Sterols are essential structural and regulatory components of eukaryotic cell membranes. Ergosterol, the main sterol in yeast, is responsible for structural membrane features such as fluidity and permeability, in a similar way as cholesterol is in human cells [ 15 ]. The pathway has been extensively studied for several years, since it functioned as a model for understanding human disease caused by high cholesterol levels [ 16 ] and is target of an important class of antifungal compounds [ 16 ]. Extensive knowledge on the biosynthesis and regulation of the pathway provided the first set of targeted interventions to increase the pool of the intermediates GPP, FPP, and GGPP, which are the substrates of terpene synthases. The terpene hydrocarbon scaffolds are generated by the action of mono-, sesqui-, and diterpene synthases that catalyze multi-step reactions with diphosphorylated substrates of 10 (GPP), 15 (FPP) or 20 (GGPP) carbon atoms. The reactions catalyzed by terpene synthases are unparalleled relative to other classes of enzymes because they often consist of a series of stereochemically complex steps. These reactions initiate by the ionization of the diphosphate substrate to create an acyclic and reactive carbocationic intermediate. Subsequent regio- and stereo-specific formation of single or multiple rings, proton eliminations to form double bonds, water quenching of carbocations to create terpene alcohols, and stereospecific hydride, methyl, and methylene migrations, give rise to a vast complexity of structures [ 17 , 18 ]. All this chemical complexity is catalyzed by enzymes whose three dimensional structure is highly conserved from fungi to plants, characterized by an active site lined mainly by inert amino acids [ 18 ]. Yeast does not normally produce terpenoids. However, expression of plant derived terpene synthases in yeast cells showed that it was possible for the enzymes to utilize the endogenous substrates (GPP, FPP, GGPP) and produce a range of terpenoid compounds [ 9 , 19 , 20 ]. The number of terpenoids produced is continuously growing as new genes from plant sources are cloned and characterized [ 21 – 26 ]. Following the formation of the olefin structure of terpenes, the metabolites can be further modified in nature by various cytochrome P450-dependent mono-oxygenases (P450), reductases, dehydrogenases or various classes of transferases, which expands immensely the variety of chemical structures synthesized [ 27 ]. Yeast, is the preferred host for P450 expression as it can express functionally active plant derived P450 enzymes [ 9 , 28 – 30 ]."
} | 1,801 |
24695678 | PMC3973573 | pmc | 1,767 | {
"abstract": "Even as demand for their services increases, honey bees ( Apis mellifera ) and other pollinating insects continue to decline in Europe and North America. Honey bees face many challenges, including an issue generally affecting wildlife: landscape changes have reduced flower-rich areas. One way to help is therefore to supplement with flowers, but when would this be most beneficial? We use the waggle dance, a unique behaviour in which a successful forager communicates to nestmates the location of visited flowers, to make a 2-year survey of food availability. We “eavesdropped” on 5097 dances to track seasonal changes in foraging, as indicated by the distance to which the bees as economic foragers will recruit, over a representative rural-urban landscape. In year 3, we determined nectar sugar concentration. We found that mean foraging distance/area significantly increase from springs (493 m, 0.8 km 2 ) to summers (2156 m, 15.2 km 2 ), even though nectar is not better quality, before decreasing in autumns (1275 m, 5.1 km 2 ). As bees will not forage at long distances unnecessarily, this suggests summer is the most challenging season, with bees utilizing an area 22 and 6 times greater than spring or autumn. Our study demonstrates that dancing bees as indicators can provide information relevant to helping them, and, in particular, can show the months when additional forage would be most valuable.",
"introduction": "Introduction Pollinating insects, including honey bees ( Apis mellifera ), continue to decline in Europe and North America [1] – [5] , even though the demand for their services is increasing [6] – [8] . The number of managed hives in Great Britain has decreased 75% in the past century; in the United States, the 62% decline from 6 million in the 1940s to 2.3 million in 2008 is even more rapid [5] , [9] . Honey bees face many challenges including pests [10] , pathogens [11] and pesticides [12] . However, independent of these is another major issue affecting wildlife in general: landscape changes in the last century such as agricultural intensification have reduced flowers and flower-rich habitats that provide nectar and pollen for honey bees and other insects [13] – [18] . These changes are predicted to continue [19] . One suggestion on how to help bees is to provide more flowers when they are lacking [4] , [9] . Although simple in principle, this is less easy in practice: when do bees most need additional flowers? To obtain directly data on the amount of forage available in a landscape-wide area, one could, with great effort, count competing flower-visiting insects and flowers and determine nectar and pollen availability. Perhaps this difficulty may explain why lack of forage is an often-mentioned reason behind bee declines [4] , [9] , but is relatively under-studied (although see Carvell et al. 2006 for bumble bees [15] ). The honey bee possesses a unique and fascinating behaviour in which a successful forager, upon returning to the hive, communicates to unemployed nestmate foragers the location of where she has collected food [20] , [21] . The vector information is therefore available for eavesdropping researchers as a tool for ecology. Honey bees, as economically savvy foragers, weigh the relevant costs and benefits for that forage in their decision to recruit to the location [22] – [24] , which makes the dance an integrative message that evaluates landscape profitability. Because honey bees are adept at scouting the landscape for food [25] and because flight is costly [23] , foragers will not collect at long distances unnecessarily [20] , [24] . Communicated distance therefore is a simple and powerful proxy for forage availability. Previous work investigating recruitment distances using the waggle dance has focused on a few weeks or months of the much longer foraging year [26] – [29] , most likely because dance decoding is time-costly and must be done by hand. However, an increased understanding of intra-dance variation has greatly streamlined the process [30] , making it easier to decode high numbers of dances. Decoded dances provide a unique data set that is integrated and not confounded by weather and competition from other insects (see Discussion ). Here, for the first time, we investigate month by month and season by season variation in honey bee foraging distance over a representative rural-urban landscape. In year 3, we determined the nectar sugar concentration, which correlates with quality, returned by foragers. We found significant and consistent variation in mean foraging distance and area, where summer is the season in which bees must roam further and utilize a foraging area 22 and 6 times greater than spring or autumn respectively, even though they do not necessarily bring back better quality forage. More generally, our study demonstrates that honey bees may be used as indicators and can show through their dance the seasons in which forage is relatively less available and, by extension, when additional forage would be most beneficial.",
"discussion": "Discussion Here we have shown that honey bees, foraging over a landscape that is typical of most of the Western world, must travel further, covering a significantly larger area, in the summers (2156 m, 15.2 km 2 ) compared to springs (493 m, 0.8 km 2 ) or even autumns (1275 m, 5.1 km 2 ) to collect forage that is not of better quality. Our study is necessarily set in one location to investigate the spatio-temporal changes in foraging patterns; however, these data also demonstrate that dancing bees may act as indicators, pinpointing in particular what months are representing relative dearth in forage availability. Because not all foragers make waggle dances, dance decoding does not give information about all the foraging sites currently being used by a honey bee colony; rather, waggle dances are filtered information that communicate the most profitable feeding locations known to a colony at that time [42] . Foraging honey bees are very sensitive to relative energetic reward [23] , [24] , which heavily weighs flight cost. Patches of better quality, because they are closer or possess higher quality nectar, will be valued higher by honey bees and generate more dancing and more repeated waggle runs within each dance [24] , [41] , all of which cause increased recruitment. Each dance represents economically savvy advice for a colony's unemployed foragers as to where to collect food. Dance decoding, therefore, provides an integrated picture of the best feeding locations. The fact that waggle dances in August and July advertise patches at the greatest distances indicates that summer is the most challenging season to find food in the study landscape. In contrast to summer, during spring the bees danced for much closer locations, mostly within 500 m from the hives ( Fig. 4C, 4F ). In many temperate habitats, spring is a season of great flower abundance, with woodland flowering species that bloom before the tree canopy matures, including trees, shrubs, perennial herbs and annuals [43] . Abundant flowers mean that bees are able to forage and to recruit locally. Additionally, even though the weather in autumn is less favourable than in summer, the dance decoding indicates that foraging conditions actually improve from summer to autumn. This is due to ivy ( Hedera spp.), a common European flowering plant that is very abundant in both urban and rural settings. In the study area, ivy begins to bloom in August, with the first flowers seen on 29 August, 2009 and 14 August, 2010, and peaks in September and October. Honey bees feed almost exclusively on ivy for both nectar and pollen in the autumn [44] , and its ubiquity means that they can forage closer than in summer ( Fig. 4B, 4E ). Ivy nectar is also high in sugar, c. 45%, which most likely accounts for the improved quality of autumn nectar compared to summer [44] . What general lessons can be learned from our study, which was necessarily set in a particular location? Historically, the landscape contained more habitats, such as hay meadows with abundant summer-flowering plants. Since World War II, these have been much reduced due to agricultural intensification [9] , [13] – [16] , [45] . Concurrent with these reductions, the number of managed honey bee hives has decreased 75% in Great Britain in the past century [5] , which mirrors the drop in other flower-visiting insects, including bumble bees, solitary bees, butterflies, and hoverflies [1] – [4] . These declines and their link to landscape changes have generated much attention, including initiatives by governments and commercial organizations, such as seed companies, to increase forage [46] . However, the information on how to help bees appears not always to be soundly based on scientific data, such as recommendations to grow winter-blooming garden plants, when most bee species (including honey bees) are dormant, or the description, without supportive data, of a June “hungry gap” [47] . The UK Royal Horticultural Society, in its “Perfect for Pollinators” campaign, recently advised the planting of garden flowers to bloom during “the entire period of bee activity”. While it is certainly correct that bees require flowers throughout the entire foraging season, the herculean task of increasing the availability of forage would be more manageable and cost-effective if aid could be better targeted. Our study suggests that in a particular location, the greatest challenge for finding food will be concentrated in a portion of the much longer foraging season. The question then becomes how do we identify these periods of relative food dearth? Beekeepers sometimes point to changes in hive weight as identifying periods of forage dearth and abundance; however, this practice is confounded by hive size and by weather. A second way in which to obtain directly the data on the amount of forage available in a landscape-wide area would be, with great effort, to count competing flower-visiting insects and flowers and determine nectar and pollen availability. In contrast, the alternative, as we have done in this study, is to use honey bee dances to obtain a picture that already integrates all these factors. Our study region is temperate and similar to most of Great Britain and parts of Europe and North America possessing strong spring flowering, some autumn flowering (e.g., ivy in Europe; golden rod, asters in the USA), and a mixed landscape of urban and rural habitats with large agricultural areas of monocrops. Therefore, our particular results of summer foraging challenges could be generally applicable. However, of more widespread practical importance is our general result: we show that honey bees can act as indicators, and dance decoding can be implemented to survey landscapes to determine when forage is hardest to locate. Such information will help place recommendations to help bees and flower-visiting insects onto a more solid foundation based on empirical evidence. Determining where foraging animals collect food is valuable in conservation work, and recent years have witnessed an explosion in the use of GPS trackers for this purpose [48] . Although insects are too small for these technologies, trackers are actually unnecessary with the honey bee, which is the only animal that directly tells eavesdropping researchers where it has collected food. Additionally, although the honey bee is only one of many flower-visiting insects, it is a generalist forager, and flower-rich locations visited by honey bees will be visited by other flower-visiting insects as well [44] , [49] . This makes the evidence for seasonal forage scarcity widely relevant for insect pollinators, especially as there is a valuable pollination synergy between honey bees and other bees [50] . The honey bee is the only animal who tells you where it has collected food. Here we have shown that listening to the bees will allow us to better direct efforts to make our landscape more insect-friendly."
} | 3,005 |
29321811 | PMC5759196 | pmc | 1,769 | {
"abstract": "Background Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Results We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. Conclusion The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants. Electronic supplementary material The online version of this article (10.1186/s13068-017-1007-6) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion Here, we have engineered plants with up to a 3.3-fold increase of the C6/C5 sugar ratio in the TFA-hydrolyzable biomass fraction and with reduced lignin. No morphological differences were observed in these plants, except for a slight decrease in stem stiffness and change in height in some of the lines. No significant changes in stem biomass accumulation were observed. The approach demonstrated here can be transferred to bioenergy crops such as poplar ( Populus sp.) and possibly also to bioenergy grasses in which galactan is not an abundant polymer. In the absence of mutants in xylan biosynthesis, an alternative method to obtain a similar reduction in xylan specifically in fibers could be achieved by targeted CRISPR/Cas9 mutagenesis directed against a xylan biosynthesis gene in fiber cells [ 30 ]. The effect of changing the biomass in bioenergy crops on agronomical performance is a key question to be resolved and ultimately will require field tests. The galactan-engineered lines described in this study are a valuable tool for further investigation of the potential relationship between galactan content in the secondary cell wall, mechanical properties, and stress responses.",
"discussion": "Discussion Overexpression of GALS1 , UGE2 , URGT1, and NST1 and additional expression of QsuB in W- and X-Arabidopsis genetic backgrounds In this report, multiple traits beneficial to biofuel production were combined in Arabidopsis using the jStack cloning method. Up to three individual traits were simultaneously introduced via a gene stacking approaches into wild-type (W-) and xylan-engineered (X-) backgrounds (Figs. 1 , 2 ). Plants were generated to overexpress three key genes ( GALS1 , UGE2, and URGT1 ) involved in galactan biosynthesis, and showed increased expression under both constitutive and fiber-specific promoters in T3 generation (Fig. 3 ). These W1 and W2 lines did not show a morphological phenotype compared to Col-0, and exhibited similar development during their life cycles. Lines expressing the C4 construct displayed opposite phenotypes in the W- and in X-genetic backgrounds (Fig. 2 ). While the inflorescence stems of engineered lines tended to be shorter in the W-background, they had the same height or were taller in the X-genetic background. Lines in the W- or X-genetic backgrounds transformed with all the traits in combination (construct C5) show poor growth and weak stems. Thus, this may be indicative that brute-force overexpression strategies may have been exhausted and, in this specific combination, it is quite possible that we have reached the limits of genetic engineering. Alternatively, using different promoters or expressing the NST1 APFL independently from UGE2 could show different outcomes, as the increased expression of NST1 results in higher than optimal level of expression of QsuB when the promoter ( pCesA7 ) used to drive its expression is induced by the NST1 transcription factor. Testing different promoter combinations could likely overcome the detrimental phenotype. Using the jStack cloning method, we generated engineered lines carrying all the transgenes at the same locus and the use of a tissue-specific promoter allowed the coordinating spatio-temporal expression of the desired transgenes. Because we had experience with a limited number of strong fiber-specific promoters, we decided to use the 2A peptide to stack genes in addition to the jStack approach. This technique has previously been used successfully by others and in our laboratory. However, in W3-1 and W3-5 stem sections, the presence of the APFL driven by NST1 did not appear to result in a substantial increase of cell wall thickness in fibers compared to what we had observed in previous studies [ 19 , 24 ]. As mentioned before, a better comprehension of the network regulation of cell wall-related genes and a different sequence arrangement could possibly lead to the desired increase in fiber cell wall density. Alternatively, it is possible that the enhancement of galactan biosynthesis in fibers with promoters that are responsive to NST1 may cause a drastic substrate competition for UDP-glucose between the cellulose and galactan biosynthesis pathways. That could perhaps lead to reduced ability to also increase cellulose and hemicellulose biosynthesis by overexpression of NST1. Engineered plants showed an increase of galactan in fibers Previously, we showed that the overexpression of UGE2 and GALS1 driven by 35S promoter in Col-0 plant leads to 80% more galactose in the cell wall of stems [ 19 ]. Here, we engineered plants able to accumulate up to 150% more galactose in stem cell walls compared to the wild type. However, our qPCR data did not confirm increased URGT1 expression in all of our engineered lines relative to the control plants (Fig. 3 a, b). Some lines did show a three- to 11-fold increase in URGT1 expression (i.e., W4-1 and X4-12) and these showed the highest increase in cell wall galactose accumulation, indicating the importance of boosting the expression of this gene. Expressing URGT1 under a stronger fiber-specific promoter or inserting its native introns could perhaps result in producing even more galactan in cell walls due to increased transport of UDP-galactose into the Golgi lumen. LM5 immunodetection showed that the additional galactose is located in fibers and assembled into galactan polymers (Fig. 4 ). Moreover, by introducing more galactan in fibers of XE, we demonstrated that it is conceivable to produce dicot plants designed to have reduced xylan content and a high amount of C6 sugars in the secondary cell wall of fibers (Figs. 4 , 5 , Additional file 2 ). The C6/C5 sugar ratio determined by TFA hydrolysis of cell wall preparations of the X4-12 plant line is 2.66 as compared to 0.80 in Col-0, corresponding to a 3.3-fold increase (Additional file 2 ). Assuming that TFA hydrolysis-resistant cellulose levels are the same in all the plants, we estimated a C6/C5 ratio of 6.3 in the best X4 line versus 2.6 in Col-0, corresponding to a 2.4-fold increase. We also demonstrated that the low lignin trait conferred by QsuB engineering is compatible with our hexose-enrichment strategies. Indeed, QsuB expression does not interfere with the accumulation of galactose (Fig. 5 ) in contrast to the biomass densification trait controlled by APFL. In lines W4-8 and W4-1, QsuB and galactan biosynthesis gene expression seems to have a synergistic effect (Fig. 5 a). Galactan content in fibers can impact stem stiffness Modifying the composition of cell walls in stem fibers could affect the mechanical properties of the entire organ. Such modifications could be advantageous to prevent lodging or, on the contrary, enhance lodging susceptibility. To address this question beyond the macroscopic phenotype, we tested stem stiffness with a three-point bending test. Our results showed no substantial effect on stem stiffness in most of the lines (Fig. 7 ). However, some lines carrying the C4 construct, in both the W- and X-genetic background, showed a decrease in stiffness, which was not observed in the QsuB control line. This decrease in stiffness was observed in the same lines where the elevated contents of galactose were found (Figs. 5 , 7 ). In these plants, we may have reached the critical point where accumulation of galactan impacts tissue properties and consequently stem mechanical properties. Indeed, galactose content in the cell wall has been demonstrated to impact mechanical properties of Arabidopsis leaves [ 29 ]. However, we cannot conclude from the data whether the decreased stiffness in the W4 and X4 lines is due only to the high galactan content or due to the combination of high galactan with low lignin mediated by QsuB expression. Optimized C6/C5 sugar ratios are compatible with saccharification improvement traits Because most microbes used for conversion into biofuels and bioproducts are more efficient in metabolizing C6 than C5 sugars, we aimed to increase the C6/C5 sugar ratio to optimize plant biomass for biofuels production. The saccharification assays conducted in this study showed that our engineered plants released more C6 sugars in both the wild-type and in a low C5 sugar (XE) background (Fig. 8 ). The best engineered line resulted from fiber-specific overexpression of galactan biosynthesis-related genes and the bacterial gene QsuB . Previously, we have shown that the expression of QsuB itself doubled the saccharification efficiency of Arabidopsis biomass [ 12 ]. QsuB expression leads to a decrease of G/S ratio and an increase of H-units in lignin, and biomass of QsuB-expressing plants is more easily hydrolyzed by the Cellic CTec2 enzymatic cocktail than control biomass [ 12 ]. In our experiments, the same phenomenon was observed with the QsuB control line. In W4 and X4 lines, the improvement in saccharification due to QsuB combined with the galactan increase in fibers could have an additive effect, resulting in an even better sugar release."
} | 2,776 |
21438624 | null | s2 | 1,771 | {
"abstract": "Despite widespread use of silk, it remains a significant challenge to fabricate fibers with properties similar to native silk. It has recently been recognized that the key to tuning silk fiber properties lies in controlling internal structure of assembled β-sheets. We report an advance in the precise control of silk fiber formation with control of properties via microfluidic solution spinning. We use an experimental approach combined with modeling to accurately predict and independently tune fiber properties including Young's modulus and diameter to customize fibers. This is the first reported microfluidic approach capable of fabricating functional fibers with predictable properties and provides new insight into the structural transformations responsible for the unique properties of silk. Unlike bulk processes, our method facilitates the rapid and inexpensive fabrication of fibers from small volumes (50 μL) that can be characterized to investigate sequence-structure-property relationships to optimize recombinant silk technology to match and exceed natural silk properties."
} | 272 |
32200735 | PMC7133524 | pmc | 1,772 | {
"abstract": "The challenge of moving beyond descriptions of microbial community composition to the point where understanding underlying eco-evolutionary dynamics emerges is daunting. While it is tempting to simplify through use of model communities composed of a small number of types, there is a risk that such strategies fail to capture processes that might be specific and intrinsic to complexity of the community itself. Here, we describe approaches that embrace this complexity and show that, in combination with metagenomic strategies, dynamical insight is increasingly possible. Arising from these studies is mounting evidence of rapid eco-evolutionary change among lineages and a sense that processes, particularly those mediated by horizontal gene transfer, not only are integral to system function, but are central to long-term persistence. That such dynamic, systems-level insight is now possible, means that the study and manipulation of microbial communities can move to new levels of inquiry. This article is part of the theme issue ‘Conceptual challenges in microbial community ecology’.",
"conclusion": "6. Conclusion Mounting evidence supports the view that microbial communities are complex dynamic systems. Interactions among an immense diversity of types shape the relationship between cells and their biotic and abiotic environment. These myriad of interactions—which are not hardwired—establish feedbacks between the environment each cell experiences and determines the future eco-evolutionary response of those cells and descendant lineages. This acts to further change the nature of interactions, provoking continual change—and change that maybe further influenced by the migration of communities between environments [ 34 ]. The challenge of studying such dynamic systems is considerable. Here, we have advocated approaches that embrace this complexity. The reason for doing so stems from our sense that microbial community function depends on processes that are intrinsic to the system itself: simplify, via, for example, analysis of the interactions among a small number of focal genotypes maintained under laboratory conditions, and the opportunity to detect and quantify processes that define the system may well be lost. On one level this is trivial: if the nitrogen cycle requires 10 species to operate, then a two-fold reduction of diversity will clearly eliminate a given community's capacity to cycle nitrogen. But there is arguably a further and analogous layer of functional significance that embraces ecologically significant genes moved by HGT. There are parallels between this view and that recently advocated by Doolittle and colleagues [ 35 , 74 ], the so named ‘it's the song not the singer’ (ITSNTS) theory. ITSNTS argues the importance of recognizing that selection can act on processes leading to persistence of, for example, microbial communities, even though such communities are for the most part not participating directly in the process of evolution by natural selection—at least not as units in the sense of Lewontin [ 70 ]. By encompassing a process-focused view, attention shifts to the nature of the process that sustains function. In the context of microbial communities, a process with the capacity to ensure persistence of function, and that is arguably shaped by selection, is HGT. The movement of genes between individuals in diverse communities serves to promote convergence upon shared function provided by diverse components [ 45 ]. Taking this a further step, there exists the intriguing possibility that the genetic information moved between entities defines a community-level interaction network that achieves a dynamic and functional effect that vastly exceeds the sum of the component HGT events. There are important implications that arise from this way of thinking, but first the hypothesis needs testing. A testable prediction is that any emergent community-level effect of HGT will depend on the diversity of interacting components. For example, a community-level process dependent on a network of genetic interactions is likely to require the number of interacting partners—and thus combinatorial possibilities—to exceed a threshold level. If so, then one way to explore its existence would be to manipulate resource availability and measure the functional impact of ensuing changes in diversity on the presumed community-level process.",
"introduction": "1. Introduction No matter one's perspective, microbial communities are mind-bogglingly complex [ 1 – 3 ]. The sense of complexity comes not just from the vast number of often densely packed cells [ 4 , 5 ], or the genetic, physiological and functional diversity of types [ 6 – 8 ], but particularly from the recognition that cells in close proximity compete and interact [ 9 , 10 ]. The range, scale and dynamic of these interactions are largely unknown. Attempts to comprehend the multiplicity of effects, including those on the patterning of diversity, addle the mind. But the challenge is even more acute: ecology is only part of the picture. Ecological processes determine the selective conditions that drive evolutionary change, which in turn feed back to affect patterns of diversity, which prompt further evolutionary change in iterative and continual cycles [ 11 , 12 ]. But what is the dynamic of this process? Over what spatial and temporal scales should the effects be measured? How can the effects even be measured? Does evolution matter, or is it all just ecology? How does the field move beyond description of types to the point where insight emerges into the feedback between ecological and evolutionary process—ideally with links to function? How do we even decide what matters? Is it conceivable that community function is shaped by processes that are intrinsic to community complexity? At one level, answers to most of these questions are apparent and we do not wish to downplay the enormous advances that have come from the ability to describe community composition [ 13 , 14 ], or from the use of simplified model communities composed of representative types [ 12 , 15 , 16 ], including interactions [ 17 , 18 ], but here we aim to advocate thinking about communities as dynamic systems. Such a view places emphasis on process, on the connection between process and function, and on the connection between process and ecological opportunity. It brings to the fore eco-evolutionary feedbacks between genes, the phenotypes they determine, and the effects wrought by selection acting on phenotypic differences among the populations that compose communities and even ecosystems [ 11 ]. Ultimately, it raises the possibility that microbial communities might be shaped by processes, such as that mediated by horizontal gene transfer (HGT), that while recognized at the population level, assume, in the context of communities, a dynamic and impact that remain under-appreciated. If true, then simplification, for example through use of synthetic or model communities—typically such a powerful way of doing science—may fail to capture community-level processes."
} | 1,762 |
22983030 | null | s2 | 1,773 | {
"abstract": "Omics, including genomics, proteomics, and metabolomics, enable us to explain symbioses in terms of the underlying molecules and their interactions. The central task is to transform molecular catalogs of genes, metabolites, etc., into a dynamic understanding of symbiosis function. We review four exemplars of omics studies that achieve this goal, through defined biological questions relating to metabolic integration and regulation of animal-microbial symbioses, the genetic autonomy of bacterial symbionts, and symbiotic protection of animal hosts from pathogens. As omic datasets become increasingly complex, computationally sophisticated downstream analyses are essential to reveal interactions not evident from visual inspection of the data. We discuss two approaches, phylogenomics and transcriptional clustering, that can divide the primary output of omics studies-long lists of factors-into manageable subsets, and we describe how they have been applied to analyze large datasets and generate testable hypotheses."
} | 255 |
34835373 | PMC8621161 | pmc | 1,775 | {
"abstract": "Methanogens are archaea that grow by producing methane as a catabolic end product and thrive in diverse anaerobic habitats, including soil, sediments, oil reservoirs, digestive tracts, and anaerobic digesters. Methanogens have typically been classified into three types—namely, hydrogenotrophic, acetoclastic, and methylotrophic methanogens. In addition, studies have found methanogens that require both hydrogen/CO 2 and organics, such as acetate, for growth. Genomic analyses have shown that these methanogens lack genes for carbon monoxide dehydrogenase/acetyl-CoA synthase (Codh/Acs), one of the oldest enzymes that catalyzes the central step in the Wood–Ljungdahl pathway. Since these methanogens have been found dominant in such habitats as digestive tracts and anaerobic digesters, it is suggested that the loss of Codh/Acs confers ecological advantages on methanogens in these habitats. Comparisons in genomes of methanogens suggest the possibility that these methanogens have emerged recently in anaerobic digesters and are currently under the process of prevalence. We propose that an understanding of the genetic and ecological processes associated with the emergence and prevalence of these methanogens in anaerobic digesters would offer novel evolutionary insights into microbial ecology.",
"conclusion": "4. Conclusions The analyses of genomes of methanogens suggest that Codh/Acs-deficient methanogens have arisen in different lineages and that these methanogens have gained ecological advantages in such habitats as animal digestive tracts and anaerobic digesters, where organics and fermentative bacteria are abundantly present. It is however still enigmatic how the loss of Codh/Acs confers ecological advantages on these methanogens, and future studies will address underlying molecular and ecological mechanisms. While molecular biology tools for methanogens are still limited, a recent work has developed a genome-editing technique for methanogens [ 27 ], allowing us to expect that molecular studies using such techniques would deepen our understanding of the ecology and evolution of methanogens. We propose that such studies on Codh/Acs-deficient methanogens in anaerobic digesters will provide us with novel insights into evolutionary ecology.",
"introduction": "1. Introduction Diverse organisms thrive on our planet and interact with each other to establish biomes. Ecology is a subdiscipline of biology that investigates interactions among organisms and between organisms and their habitats. Ecological interactions are formed as a result of evolution of individual organisms, while they also serve as driving forces for the evolution. It is therefore important to study ecology in relation to evolution, and the advent of genomics/metagenomics has provided microbial ecology with promising approaches to incorporate evolutionary insights. In the genome of an organism, we find genetic signatures that assist us in deducing how the organism has evolved [ 1 ]. In addition, one may be able to predict how organisms will evolve, if genomic signatures can be linked to changes in ecological settings [ 2 ]. Such an attempt of evolutionary ecology would deepen our understanding of the emergence and transition of niches, and we expect that the examination of specialist organisms that thrive in diverse habitats would offer novel concepts in evolutionary ecology. Methanogens are archaea that grow by producing methane as the catabolic end product (methanogenesis) and thrive in diverse anaerobic habitats, including soil, sediments, oil reservoirs, digestive tracts, and anaerobic digesters [ 3 ]. They play pivotal roles in the global carbon cycle, and it has been estimated that methane produced by methanogens shares over one half of all methane produced on the planet [ 4 ]. We are interested in studying the evolutionary ecology of methanogens since they share distinct but indispensable niches in various anaerobic habitats, and genomic information has so far been gained for a substantial number of methanogens [ 5 ]. Based on growth substrates, methanogens have been classified into three types—namely, hydrogenotrophic methanogens (HMs, producing methane from carbon dioxide with hydrogen and/or formate as reducing agents), acetoclastic methanogens (AMs, using acetate as the sole substrate), and methylotrophic methanogens (MMs, producing methane from the methyl group in organic compounds) [ 3 ]. Among these, HMs utilize carbon dioxide as a carbon source and are therefore considered autotrophs. In addition to these typical methanogens, studies have also found methanogens that require both hydrogen/CO 2 and acetate for growth. Genomic analyses have shown that these methanogens lack genes for carbon monoxide dehydrogenase/acetyl-CoA synthase (Codh/Acs), one of the oldest enzymes that catalyzes the central step in the Wood–Ljungdahl (WL) pathway. Since these methanogens have been found dominant in such habitats as digestive tracts [ 6 ] and anaerobic digesters [ 7 , 8 ], it is considered that the loss of Codh/Acs confers ecological advantages that facilitate the ability of methanogens to overgrow in these habitats. In the present work, we performed comparative genome analyses of diverse methanogens in order to gain insights into the ecology and evolution of Codh/Acs-deficient methanogens. We suggest that Codh/Acs-deficient methanogens are on the verge of prevalence in anaerobic digesters and that more attention should be paid to these methanogens for the successful operation of anaerobic digesters.",
"discussion": "3. Results and Discussion Catabolic pathways for typical methanogens (HMs, AMs, and MMs) are collectively illustrated in Figure 1 . A catabolic step all methanogens share is the final step of the methanogenesis pathway, where methane is released from methyl coenzyme M by methyl coenzyme M reductase (Mcr). Since this enzyme is present only in methanogens and anaerobic methanotrophs, metagenomic studies use mcr genes as indices for identifying whether metagenome-assembled genomes (MAGs) encode these organisms [ 5 ]. Many methanogens also possess the WL pathway [ 11 ]. HMs utilize this pathway for carbon fixation, while AMs use it in reverse for converting acetyl-CoA into the methyl group and carbon dioxide ( Figure 1 ). Many methanogens that solely perform methylotrophic methanogenesis are known not to have the WL pathway. All methanogens so far isolated from natural and engineered habitats are affiliated with the phylum Euryarchaeota , while metagenomic studies have suggested that methanogens are more diverse than previously thought [ 5 ]. In the present study, in order to summarize the phylogenetic distribution of the three types of methanogens (isolates and MAGs) in the domain Archaea , a phylogenetic tree was constructed for class-level phylogenetic groups using amino acid sequences of concatenated ribosomal proteins ( Figure 2 ), and lineages that include methanogens are marked. As indicated in this figure, diverse archaeal lineages are now thought to include methanogens. For instance, Mcr-encoding MAGs that are affiliated with the phylum Bathyarchaeota (included in the TACK group) were recovered from a deep aquifer and are considered to represent MMs [ 12 ]. In addition, MAGs that encode MMs in the phylum Verstraetearchaeota (the TACK group) were discovered from a cellulose-degrading methanogenic bioreactor [ 13 ]. These findings, along with the knowledge on MMs in the Euryarchaeota , suggest that the methanogenesis and WL pathways were not necessarily linked at early stages in the evolution of methanogens [ 11 ]. On the other hand, a recent study of microbiomes in the hot springs of Yellowstone National Park has shown that MAGs affiliated with the Verstraetearchaeota encode HMs that have the WL pathways [ 14 ]. This finding has challenged the above-mentioned view on the early evolution of methanogens, while it is in line with the idea that ancestral methanogens had the WL pathway [ 15 ]. This idea is based on the fact that the WL pathway is present in organisms affiliated both with the domains Bacteria (e.g., acetogens) and Archaea (e.g., methanogens) and is related to the view that the last universal common ancestor had the WL pathway [ 15 , 16 ]. Although debates still exist concerning the early evolution of methanogens, it is possible to conclude that methanogens have diverged as a result of dynamic evolutionary events that have been associated with the loss of catabolic genes. Genomic analyses of methanogens also suggest the possibility that catabolic pathways in methanogens are currently subjected to dynamic evolution for adapting to emerging habitats. This idea was to be apparent when we comparatively analyzed genomes of methanogens to know the presence and absence of genes for Codh/Acs, an enzyme that constitutes the carbonyl branch in the WL pathway [ 15 ]. This enzyme is known to be essential for carbon fixation in HMs and methanogenesis in AMs ( Figure 1 ). Studies of the genomes of methanogens have however found that some methanogens considered to perform hydrogenotrophic methanogenesis are deficient in Codh/Acs genes. These include Methanocella paludicola [ 17 ], Methanobrevibacter spp. represented by Methanobrevibacter smithii [ 18 ], Methanoculleus spp. represented by Methanoculleus sp. MAB1 [ 19 ], and Methanothermobacter sp. Met2 [ 7 ]. Among these, M. paludicola is the type genus and species of the order Methanocellales that corresponds to Rice Cluster I, an archaeal group that has been abundantly detected in rice paddy fields [ 20 ]. A study has shown that this methanogen requires acetate, in addition to hydrogen and carbon dioxide, for growth, and its genome does not encode Codh/Acs [ 18 ]. Methanogens affiliated with the genus Methanobrevibacter have been isolated from digestive tracts of animals, such as guts and rumens [ 21 ], and a representative archaeon M. smithii has been found to be the most abundant methanogen in the human gut (~10% of the total anaerobes) [ 6 ]. It has been shown that its genome encodes a number of traits beneficial to growth in the gut of animals, but not Codh/Acs, and this HM requires acetate for growth [ 19 ]. Methanogens affiliated with the genus Methanoculleus are present in diverse anaerobic habitats, and studies have frequently detected these methanogens as one of the major populations in anaerobic digesters [ 22 ]. A representative strain, Methanoculleus bourgensis BA1 isolated from a laboratory biogas reactor, is an HM also requiring acetate for growth [ 23 ], and its genome does not contain the complete set of genes for Codh/Acs [ 24 ]. Another HM that does not possess Codh/Acs is Methanothermobacter sp. Met2 (its complete genome is deposited in the databases as Methanothermobacter MT-2) that was abundantly detected from biofilms in thermophilic fixed-bed anaerobic digesters (over 20% of the total biofilm microbes) [ 7 ]. In that study, a closely related archaeon ( Methanothermobacter Met20) was also detected, albeit as a minor population (approx. 0.2%), from the same biofilm, and genomic analyses have revealed that this methanogen has Codh/Acs [ 7 ]. In a subsequent study, archaeal strains that represent Met2 and Met20 were isolated, and growth tests have demonstrated that Met20 is able to grow autotrophically on hydrogen and carbon dioxide, while Met2 requires acetate in addition to hydrogen and carbon dioxide [ 8 ]. According to the catabolic pathways illustrated in Figure 1 , it is likely that Codh/Acs-deficient methanogens utilize hydrogen and carbon dioxide only for conserving energy by methanogenesis, while acetate is activated by acetyl-CoA synthase and/or acetate kinase plus phosphotransacetylase and solely used as a carbon source. Since previous studies have shown that Codh/Acs-deficient methanogens require acetate for growth, these methanogens may have emerged in acetate-rich habitats. It is however also conceivable that other organic compounds may support the growth of some Codh/Acs-deficient methanogens, and this should be addressed in future studies. It is also noteworthy that the growth of Met2 was slower than Met20 even in the presence of acetate [ 8 ], suggesting that Met2 may have some advantages other than growth rate over Met20, which facilitate Met2 to constitute dominant populations in anaerobic digesters. In order to deepen our understanding of the diversity of Codh/Acs-deficient methanogens, we extensively analyzed genomes of methanogens deposited in the public databases ( Figure 3 ). In this analysis, we only analyzed complete genomes since solid conclusions on the loss of genes from a genome cannot be obtained from an incomplete draft genome. Figure 2 shows a phylogenetic tree based on 16S rRNA genes of genome-completed methanogens, with accompanying information on the presence and absence of the Codh/Acs genes (genes for 5 subunits in the enzyme). It was found that, in addition to the above-mentioned methanogens, the genes were also lost from some other methanogens, including Methanosphaera spp. and Methanococcus voltae . Methanogens affiliated with the genus Methanosphaera are hydrogen-utilizing MMs that are abundantly present in animal guts [ 6 , 21 ]. In contrast, Methanococcus voltae are known to be an HM, while a study has indicated that this archaeon requires acetate for growth [ 25 ]. The phylogenetic tree in Figure 3 shows that unexpectedly diverse methanogens do not possess the complete set of genes for Codh/Acs. In addition, since the loss of Codh/Acs is multi-phyletic, it is suggested that this evolutionary event occurred independently and was fixed in different lineages, probably conferring ecological advantages that facilitate methanogens to overgrow in respective habitats. Figure 3 also shows that all strains in the genera Methanobrevibacter and Methanosphaera are completely deficient in the Codh/Acs genes, while all the 12 strains in the closely related Methanobacterium have the complete set. Given that a large portion of methanogens affiliated with the class Methanobacteria have the genes, it is likely that ancestral Methanobacteria methanogens had the genes. We also deduce that these genes were lost from the genomes of Methanobrevibacter and Methanosphaera immediately after they were diverged from other genera, since all the members do not have the genes. It is likely that this genotype has been settled in these methanogens in association with their prevalence in the digestive tracts of animals. In contrast, most strains in the genus Methanothermobacter possess Codh/Acs, while Met2 found in thermophilic digesters [ 7 ] and EMTCatA1 detected from an electromethanogenic reactor [ 26 ] do not, suggesting that this genotype (the lack of genes for Codh/Acs) is not well fixed in the genus Methanothermobacter . Given that Methanothermobacter methanogens, including those shown in Figure 3 , have been found in and isolated from anaerobic digesters, it is conceivable that Codh/Acs-deficient Methanothermobacter methanogens have emerged relatively recently in some anaerobic digesters and that they are subjected to the process of prevalence. This idea is related to the fact that, compared with animal digestive tracts, anaerobic digesters are the latest habitats for methanogens, while characteristics of these habitats, including, abundant organics, rich fermentative bacteria, and stable environmental parameters (e.g., temperature), are similar to each other. Partial deletion of genes for Codh/Acs from genomes of Methanoculleus methanogens in anaerobic digesters supports this idea ( Figure 3 ). It is suggested that anaerobic digesters are emerging habitats for methanogens, in which Codh/Acs-deficient methanogens have evolved and have become prevalent relatively recently."
} | 3,963 |
34937895 | PMC8695373 | pmc | 1,776 | {
"abstract": "Carbon capture and storage (CCS) is a key technology to mitigate the environmental impact of carbon dioxide (CO 2 ) emissions. An understanding of the potential trapping and storage mechanisms is required to provide confidence in safe and secure CO 2 geological sequestration 1 , 2 . Depleted hydrocarbon reservoirs have substantial CO 2 storage potential 1 , 3 , and numerous hydrocarbon reservoirs have undergone CO 2 injection as a means of enhanced oil recovery (CO 2 -EOR), providing an opportunity to evaluate the (bio)geochemical behaviour of injected carbon. Here we present noble gas, stable isotope, clumped isotope and gene-sequencing analyses from a CO 2 -EOR project in the Olla Field (Louisiana, USA). We show that microbial methanogenesis converted as much as 13–19% of the injected CO 2 to methane (CH 4 ) and up to an additional 74% of CO 2 was dissolved in the groundwater. We calculate an in situ microbial methanogenesis rate from within a natural system of 73–109 millimoles of CH 4 per cubic metre (standard temperature and pressure) per year for the Olla Field. Similar geochemical trends in both injected and natural CO 2 fields suggest that microbial methanogenesis may be an important subsurface sink of CO 2 globally. For CO 2 sequestration sites within the environmental window for microbial methanogenesis, conversion to CH 4 should be considered in site selection."
} | 351 |
27917379 | PMC5114238 | pmc | 1,778 | {
"abstract": "Lignin, a complex aromatic polymer in terrestrial plants, contributes significantly to biomass recalcitrance to microbial and/or enzymatic deconstruction. To reduce biomass recalcitrance, substantial endeavors have been exerted on pretreatment and lignin engineering in the past few decades. Lignin removal and/or alteration of lignin structure have been shown to result in reduced biomass recalcitrance with improved cell wall digestibility. While high lignin content is usually a barrier to a cost-efficient application of bioresources to biofuels, the direct correlation of lignin structure and its concomitant properties with biomass remains unclear due to the complexity of cell wall and lignin structure. Advancement in application of biorefinery to production of biofuels, chemicals, and bio-derived materials necessitates a fundamental understanding of the relationship of lignin structure and biomass recalcitrance. In this mini-review, we focus on recent investigations on the influence of lignin chemical properties on bioprocessability—pretreatment and enzymatic hydrolysis of biomass. Specifically, lignin-enzyme interactions and the effects of lignin compositional units, hydroxycinnamates, and lignin functional groups on biomass recalcitrance have been highlighted, which will be useful not only in addressing biomass recalcitrance but also in deploying renewable lignocelluloses efficiently.",
"introduction": "Introduction The conversion of renewable lignocellulosic biomass to fuels and valuable co-products, usually referred as biorefining, has been advanced recently (Ragauskas et al., 2006 ). However, transition from fossil-based to biomass-based products using current feedstocks and technologies has been challenged by the inherent resistance of plant cell walls to microbial and enzymatic deconstruction, namely, recalcitrance (Himmel et al., 2007 ). While other factors could not be neglected, the presence of lignin (ca. 15–35% by weight) is one of the most significant recalcitrance contributors, which escalates the processing costs (i.e., necessitated pretreatment and enlarged enzyme amount; Mosier et al., 2005 ; Pu et al., 2011 , 2013 ). Lignin is an amorphous and complex aromatic polymer providing terrestrial plants mechanical support, stress response, pathogen resistance, and water transport (Boerjan et al., 2003 ). By bonding or embedding with other biopolymers (cellulose and hemicellulose), lignin strengthens the integrity and rigidity of the plant cell wall yielding a complex macro-molecular assembly (Figure 1 ). This lignin-polysaccharides matrix renders cell walls recalcitrance for biorefining. Depending on biological species, lignin in plants derives primarily from three phenylpropanoid monolignols— p -coumaryl, coniferyl, and sinapyl alcohols (Li M. et al., 2016 ; Yoo et al., 2016b ), which give rise to p -hydroxyphenyl (H), guaicyl (G), and syringyl (S) units, respectively (Figure 1 ). The inter-linkages between these subunits are β- O -4′, β-5′, α- O -4′, 4- O -5′, β-β′ in common, and β-1′, and 5-5′ in minor amount. Figure 1 Simplified structure of plant cell walls (A) (Phitsuwan et al., 2013 ), lignin isolated from poplar (B) , and schematic structure of poplar lignin (C) (Vanholme et al., 2010 ). In biomass, lignin content, structure, subunits, and linkages with polysaccharides own their remarkable importance to cell wall recalcitrance, usually gauged by biomass enzymatic hydrolyzability (Zeng et al., 2014 ; McCann and Carpita, 2015 ). Lignin limits the enzymatic hydrolysis of biomass through two primary mechanisms: restricting polysaccharides accessibility (physical barrier and/or lignin-carbohydrate complexes) and non-productive binding with enzymes (inhibition). Current strategies to reduce lignin involved biomass recalcitrance mainly include (i) pretreatment technologies (Mood et al., 2013 ; Pu et al., 2013 ), (ii) perturbing lignin biosynthesis (Bonawitz and Chapple, 2010 ; Simmons et al., 2010 ), and (iii) enzyme engineering and modification (Güven et al., 2010 ). Whichever strategy is used, fundamental understanding of the influence of lignin's physicochemical properties on biomass recalcitrance is crucial to advance the biologically-based biorefinery. This mini-review aims at recent findings on the relationship between lignin structure and biomass recalcitrance."
} | 1,090 |
34521754 | PMC8463893 | pmc | 1,779 | {
"abstract": "Significance Stable endosymbiosis between eukaryotic microbes has driven the evolution of further cellular complexity. Yet the mechanisms that can act to stabilize an emergent eukaryote–eukaryote endosymbiosis are unclear. Using the model facultative endosymbiotic system, Paramecium bursaria , we demonstrate that endosymbiont–host RNA–RNA interactions can drive a cost to host growth upon endosymbiont digestion. These RNA–RNA interactions are facilitated by the host RNA-interference system. For endosymbiont messenger RNA sharing a high level of sequence identity with host transcripts, this process can result in host gene knockdown. We propose that these endosymbiont–host RNA–RNA interactions—“RNA-interference collisions”—represent an emergent mechanism to sanction the host for breakdown of the endosymbiosis, promoting the stability of the facultative endosymbiotic interaction.",
"discussion": "Discussion Through manipulation of the P. bursaria endosymbiotic system, we have demonstrated that RNA released upon digestion of the algal endosymbiont is processed by the host RNAi system. For endosymbiont-derived mRNA sharing a high level of sequence identity with host transcripts, this processing can result in knockdown of endogenous host gene expression, resulting in a cost to host growth ( SI Appendix , Fig. S1 ). We therefore postulate that these RNA–RNA interactions are of importance in eukaryote–eukaryote endosymbioses where partners are likely to share greater sequence similarity, especially among conserved transcripts which tend to be more highly represented among lethal or conditionally essential genes ( 61 ). Due to the inherent difficulty in characterizing such mechanisms directly, we have relied on multiple-experimental approaches to demonstrate the viability of putative RNA–RNA interactions at each stage of the process. We have tracked the interaction through sRNA sequencing, recapitulated the effect through exposure to synthetic endosymbiont-derived RNA—including sense ssRNA analogous to endosymbiont mRNA—and demonstrated that this mechanism is mediated by host Dicer, AGO-Piwi, Pds1, and RdRP proteins. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term “RNAi collisions,” represents a mechanism to sanction the host for breakdown of the interaction, a factor that would promote stability in a facultative eukaryote–eukaryote endosymbiosis. The long-term maintenance of symbiotic interactions represents a quandary for evolutionary theory ( 31 – 36 ). How do such relationships avoid overexploitation by one partner that would ultimately lead the interaction to collapse? Partner switching is one option; however, the result is typically a reduced pattern of coevolution between symbiont and host that can inhibit the process of metabolic and genetic integration, stalling the evolution of stable interactions which are needed if a system is to move toward evolution of an organelle ( 13 , 62 – 65 ). Previous studies have suggested that when selfish behaviors arise, the evolution of enforcement to punish or suppress the exploitative partner can act to restore cooperation ( 9 , 10 ). Enforcement mechanisms have been identified in diverse biological systems ( 66 , 67 ), and are argued to be one of the most effective drivers of cooperation between individuals from different species. The results presented here suggest that “RNAi collisions” between endosymbiont and host, which are capable of imposing a cost to host growth for breakdown of the symbiosis, could provide an emergent mechanism to discourage overexploitation of the endosymbiont population by the host. Under such a scenario the host would gain greater benefit from sequestering the endosymbiont within a stable perialgal vacuole, protecting them from digestion and harvesting the nutrients which leak from them ( 26 , 62 ), rather than simply digesting the would-be food through lysosomal fusion ( 14 ). Interestingly, the emergence of this mechanism appears to be a by-product of preexisting biological features that are already likely to be under strong selective pressure. For instance, the essential RNAi system of the host, which in some ciliates can function in exogenous RNA processing, transposon elimination, nuclear rearrangement and transcriptional regulation ( 43 – 46 , 48 , 53 , 68 – 71 ). Or, furthermore, the conserved gene repertoire and sequence composition of the host and endosymbiont transcriptomes from which some “RNAi collisions” have here been identified [including many transcripts which are conditionally essential in other systems ( 61 )]. Unlike comparable mechanisms of RNA–RNA interactions that have been studied in host–pathogen symbioses ( 37 – 41 ), these “RNAi collisions” appear to be untargeted and, hence, emergent. In the aforementioned host–pathogen systems, targeted RNA is passed from one partner to the other in order to modulate expression of transcripts involved in virulence or resistance ( 37 , 40 , 41 ). However, in order for such systems to evolve, they must first exist in an untargeted form upon which selection is able to act, allowing the emergence of specific RNA factors ( 38 , 39 ). Identification of undirected “RNAi collisions” in the P. bursaria system represents one such intermediary state, emergent in nature and untargeted, upon which sustained cellular interaction coupled with the potential for host-symbiont conflict could drive the selection of targeted RNA–RNA interactions. We therefore propose that “RNAi collisions” represent a putative mechanism to discourage overexploitation of the endosymbiont population by the host. Here we have used the example of mass endosymbiont digestion in response to drug treatment to simulate this effect in the extreme. In natural interactions between P. bursaria and its algal endosymbiont, such a cost would only need to occur in the drastic occurrence of mass endosymbiont digestion in order to drive stability of the interaction. Importantly, the endosymbiotic algal population within P. bursaria is largely composed of closely related or clonal lineages ( 11 – 13 ), and as such, the fate of the algal population should be considered as a collective unit. This cost therefore need only act to suppress large-scale, rapid destruction by the host in order to drive the maintenance of a surviving subsection of the endosymbiont population. Previous studies have demonstrated how P. bursaria is capable of manipulating endosymbiont load in response to varying light conditions to better suit its own ends ( 28 , 29 ), however, in these examples, reduction of endosymbiont number through digestion is slow and partial. By providing a system that selects against rapid and near-complete digestion of the endosymbiont population, “RNAi collisions” effectively buffer the endosymbiotic interaction against total breakdown. We suggest that this has allowed the relationship to be maintained across time and varying ecological conditions, even in the event of host-symbiont conflict ( 28 , 29 ) and fluctuating endosymbiont numbers ( 14 , 27 , 28 ). As an alternate route to conflict resolution that avoids partner switching, we propose that such a mechanism would facilitate greater coevolution between endosymbiont and host. Over time, this would allow the metabolic and genetic integration that drives the formation of obligate symbioses to become manifest. We therefore present “RNAi collisions” as a mechanism in this endosymbiotic system, a factor that can promote stability in the face of conflict in an emergent endosymbiotic eukaryote–eukaryote cell–cell interaction."
} | 1,916 |
22232619 | PMC3252642 | pmc | 1,780 | {
"abstract": "Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H 2 ). In order to assess the potential for microbial H 2 utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H 2 -oxidizers. Both sites also yielded metagenomic evidence for microbial H 2 production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood–Ljungdahl pathway. In general, our results point to H 2 -oxidizing Betaproteobacteria thriving in shallow, oxic–anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H 2 -powered primary production in serpentinite-hosted subsurface habitats.",
"conclusion": "Conclusion Both the marine and continental serpentinite springs investigated in this study show evidence of aerobic organisms capable of H 2 -fueled (or at least H 2 -regulated) primary production (i.e., Burkholderiales) and anaerobic organisms capable of H 2 production from fermentation of organic carbon (i.e., Clostridia). This community structure resembles that of the deep subsurface habitat sampled by a ∼3 km deep borehole in South Africa (Moser et al., 2005 ), indicating that the surface-exposed springs described in this study provide access to organisms flushed from the subsurface. Furthermore, the remarkably high density of hydrogenases in both the marine and continental springs (Lost City and WHC2b) in this study and their complete absence in a spring showing evidence of extensive mixture with surface runoff (TLE) indicate that the H 2 -associated metabolic activities discussed here are specific to subsurface processes. The predicted metabolic characteristics of the dominant organisms in the Tablelands springs are consistent with the known abiogenic products of subsurface serpentinization-associated processes: H 2 and low molecular-weight organic compounds. A major unanswered question, however, is whether the H 2 -oxidizing Burkholderiales subsist on abiogenic H 2 generated by serpentinization in the subsurface or if they depend on biogenic H 2 produced by Clostridia. In either case, it seems likely that the Burkholderiales in the Tablelands springs and Lost City chimneys inhabit oxic–anoxic interfaces where they have access to both H 2 and oxygen. Our metagenomic evidence also suggests that these organisms may be able to survive on carbon monoxide if H 2 is unavailable. The Clostridia are likely inhabitants of anoxic, subsurface habitats where they ferment organic compounds into H 2 , but the source of these organic compounds is unknown. If they are ultimately derived from reduction of carbon by serpentinization-associated reactions (as evidenced in (Proskurowski et al., 2008 ) and predicted by experiments reviewed in (McCollom and Seewald, 2007 ), then fermentation of these compounds could be considered a kind of primary production as it would be the generation of new biomass from non-biological carbon and energy. Therefore, both the H 2 -oxidizing Burkholderiales and H 2 -producing Clostridia may be important mediators of carbon and energy exchange between the deep Earth and the surface biosphere. Further research should investigate whether these organisms are bona fide denizens of the anoxic subsurface by probing deeper to obtain more representative samples of deep subsurface habitats. In particular, the Clostridia and methanogens should be better represented in deeper samples. Nevertheless, the datasets presented here represent a proof-of-concept metagenomic study that demonstrates the potential of surface-exposed springs to yield insights into the microbial diversity of the subsurface biosphere.",
"introduction": "Introduction The potentially vast microbial diversity and biomass of the subsurface biosphere (Whitman et al., 1998 ) has been frequently noted (Biddle et al., 2006 ; Huber et al., 2007 ; Santelli et al., 2008 ; Schrenk et al., 2010 ), but there is very little evidence to indicate how much of it is supported by new primary production or by recycling of buried organic carbon. Earth’s mantle is primarily composed of ultramafic rocks that undergo a geochemical process known as serpentinization when they are tectonically uplifted into the crust and exposed to water. Serpentinization is highly exothermic and can release large quantities of hydrogen gas (H 2 ) and variable amounts of methane and low-molecular weight organic compounds (McCollom and Seewald, 2007 ; Proskurowski et al., 2008 ). Therefore, serpentinization is a potential source of reducing power and organic carbon for organisms inhabiting the ultramafic subsurface. Actively serpentinizing rocks are present on all of the world’s continents and comprise significant portions of the deep seafloor, and yet they are some of the most poorly understood portions of the biosphere. The most dramatic example of an ecosystem supported by serpentinization is the Lost City hydrothermal field, which is situated on a serpentinite-rich massif 15 km from the Mid-Atlantic Ridge. Carbonate chimneys at Lost City vent warm (up to 90°C), pH 9–11 fluids rich in calcium, H 2 (up to 14 mmol/kg), and methane (1–2 mmol/kg; Kelley et al., 2005 ). Methane and larger hydrocarbon chains with up to four carbon atoms in Lost City fluids show evidence of an abiogenic origin in the deep subsurface (Proskurowski et al., 2008 ), but the amount of microbial activity supported by this abiotic source of organics has not been quantified. The anoxic interiors of Lost City carbonate chimneys are dominated by Methanosarcinales-related archaea potentially involved in methane production and oxidation (Schrenk et al., 2004 ; Brazelton et al., 2011 ). The oxic chimney exteriors are dominated by aerobic methane- and sulfur-oxidizing bacteria (Brazelton et al., 2006 , 2010 ). The role of H 2 -metabolizing bacteria in Lost City chimneys, though, has not been explicitly investigated. In addition to marine hydrothermal systems such as Lost City, Ophiolites abducted onto continents also provide a potential window into subsurface habitats supported by serpentinization. The Tablelands Ophiolite in western Newfoundland, Canada features extensive ultramafic exposures including serpentinites associated with the seepage of highly reducing, pH 12 fluids, and extensive calcium carbonate (travertine) deposits. The Tablelands fluids are enriched in calcium, H 2 (up to ∼500 μM), and methane (Szponar et al., submitted). The geochemical similarities between the alkaline springs at the Tablelands and the vent fluids at Lost City strongly suggest that they are the surface expressions of ongoing serpentinization-associated reactions occurring in the underlying ultramafic subsurface. In order to identify potential inhabitants of the ultramafic subsurface in both marine and continental settings, we conducted a metagenomic survey of two Tablelands alkaline springs as a comparison to a previously published metagenome from a Lost City chimney (Brazelton and Baross, 2009 , 2010 ). In this report, we focus on the potential for H 2 -fueled microbial activity by investigating the incidence and diversity of sequences encoding hydrogenase enzymes. Hydrogenases catalyze the reversible conversion between molecular hydrogen and its component protons and electrons: H 2 ↔2H + + 2e − . This reaction is catalyzed by two main classes of hydrogenase: [NiFe]-hydrogenases are required for uptake and oxidation of H 2 , while [FeFe]-hydrogenases are typically involved in microbial H 2 production. Although the two classes share some sequence similarity, they do not appear to be monophyletic (Vignais et al., 2001 ; Vignais and Billoud, 2007 ). Therefore, phylogenetic analyses of possible hydrogenase-encoding sequences should reliably indicate a genetic potential for H 2 oxidation or H 2 production. The results presented here indicate that both types of hydrogenase are abundant in the Tablelands and Lost City metagenomes and that the identity of the H 2 -metabolizing organisms at both sites are intriguingly similar. Additional metagenomic evidence also indicates the potential for carbon fixation pathways involving carbon monoxide utilization or acetogenesis by H 2 -metabolizing organisms. In general, this initial metagenomic survey highlights the potential for H 2 -fueled primary production in the ultramafic subsurface.",
"discussion": "Discussion Potential for H 2 -fueled carbon fixation by Burkholderiales The metagenomic and phylogenetic data presented above indicate that Betaproteobacteria belonging to order Burkholderiales are potentially important primary producers adapted to the extreme conditions of the Tablelands springs. Their potential for H 2 oxidation is indicated by the diversity of uptake [NiFe]-hydrogenase sequences (Figure 5 ) in the WHC2b spring, which are absent in the TLE spring. Their potential for carbon fixation is indicated by the presence of gene clusters encoding carbon monoxide dehydrogenase (CODH) and Rubisco in the largest WHC2b contig (WHC2b.C1; Figure 4 ). The phylogenies of the [NiFe]-hydrogenases, CODH, and 16S rRNA sequences in WHC2b contigs indicate close relationships with Hydrogenophaga species and Ralstonia eutropha (now Cupriavidus necator ). These organisms are facultatively autotrophic; i.e., they only utilize H 2 or fix carbon when organic carbon is unavailable (Willems et al., 1989 ; Schwartz et al., 2009 ). Therefore, further characterization of the physiology of these organisms and their access to organic matter in the Tablelands springs is required to estimate their contribution to primary production. Furthermore, all Hydrogenophaga and Ralstonia species are aerobic or facultatively anaerobic, so the corresponding organisms at the Tablelands and Lost City are likely to inhabit oxic–anoxic transition zones where they have access to both H 2 and oxygen. Both of these systems feature strong oxygen gradients between the atmosphere and spring water (at the Tablelands) and between oxygenated seawater and hydrothermal fluid (at Lost City), so there is potential in each system for organisms to utilize both H 2 and oxygen. It is unclear whether the TLE spring also hosts H 2 -fueled carbon fixation. Although a Burkholderiales 16S rRNA sequence was identified in a metagenomic contig from TLE and automated taxonomy classifiers identify many Burkholderiales-related sequences in the TLE metagenome (MG-RAST and TaxSOM, data not shown), no hydrogenases were detected in TLE. The absence of hydrogenases could be due to a combination of lower abundance of Burkholderiales in TLE and lower sequencing depth of the TLE metagenome compared to WHC2b. It is also possible that the Burkholderiales species in TLE have lost their hydrogenase genes, which may have resulted from the loss of the plasmid potentially represented by contig WHC2b.C1, as discussed above in the description of Figure 4 . Most H 2 -oxidizing autotrophs utilize both membrane-bound (Group 1) and cytoplasmic (Group 3) [NiFe]-hydrogenases. The [NiFe]-hydrogenase in the WHC2b.C1 contig belongs to the Group 2 H 2 sensor proteins, which are involved in the regulation of carbon fixation by H 2 but do not directly couple H 2 oxidation with energy conservation (Vignais et al., 2001 ). Therefore, the evidence from this one contig indicates only that carbon fixation in the corresponding organism is regulated by the presence of H 2 and not necessarily fueled by H 2 oxidation. The phylogeny of the Group 2 [NiFe]-hydrogenase in WHC2b.C1, however, is congruent with the phylogeny of the Group 3 [NiFe]-hydrogenase in a 21 kb contig (WHC2b.C15; Figure 5 ). It seems highly likely that both contigs are derived from the same species, and both hydrogenases are highly similar to putative homologs in Ralstonia eutropha . The Group 1, 2, and 3 hydrogenases in R. eutropha are all encoded in a 452 kb megaplasmid, and it is possible that contigs WHC2b.C1 and WHC2b.C15 are partial sequences of the same plasmid (as described above). The lack of a Ralstonia -related Group 1 [NiFe]-hydrogenase in the WHC2b metagenome is puzzling, however (Figure 5 ). Nitrosospira multiformis is one of the few examples listed in the exhaustive survey by Vignais and Billoud ( 2007 ) of an organism that has only a Group 3d [NiFe]-hydrogenase and no representative from Group 1. The function of the N. multiformis Group 3d [NiFe]-hydrogenase is unknown but suspected to be the catalysis of NAD reduction by H 2 in order to “increase the overall energetic yield from ammonia oxidation” (Norton et al., 2008 ). Therefore, it is possible that the Ralstonia -like organisms in the Tablelands only utilize H 2 to supplementary their primary electron donor (e.g., organic carbon). The current metagenomic dataset from WHC2b is relatively low coverage, however, and additional sequencing at higher coverage may eventually recover a Group 1 homolog. Both Group 1 and Group 3 [NiFe]-hydrogenases related to R. eutropha are present in several Lost City contigs (Figure 5 ), indicating that a Ralstonia -related organism with the genetic potential for H 2 oxidation also inhabits Lost City chimneys. The Lost City metagenomic dataset is dominated by sequences with high similarity to that of Thiomicrospira crunogena , a cosmopolitan sulfur-oxidizing autotroph in marine hydrothermal vents. Previous studies have noted the inability of T. crunogena to utilize H 2 as a sole electron donor despite the presence of a Group 1 [NiFe]-hydrogenase in its genome (Scott et al., 2006 ). None of the hydrogenases detected in this study have high sequence similarity to the T. crunogena hydrogenase, nor do any of the large Lost City contigs expected to correspond to Thiomicrospira -like organisms contain predicted hydrogenases. Therefore, H 2 -oxidizing organisms in young, hot Lost City chimneys are most likely aerobic or facultatively anaerobic Betaproteobacteria belonging to order Burkholderiales and appear to be less abundant than the dominant sulfur-oxidizing Thiomicrospira -like population. Potential for CO utilization by Burkholderiales Carbon dioxide is extremely scarce in the highly reducing, high pH fluids of the Tablelands and Lost City, so alternative carbon species may be more favorable substrates for carbon fixation. The largest Tablelands contig (WHC2b.C1; Figure 4 ) includes the CoxMSL gene cluster which encodes all three subunits of the carbon monoxide dehydrogenase (CODH) used by aerobic carboxydotrophs (Ragsdale, 2004 ; King and Weber, 2007 ). This enzyme is frequently plasmid-encoded (Hugendieek and Meyer, 1992 ), providing additional but not conclusive evidence that the WHC2b.C1 contig represents a plasmid. The phylogeny of the large subunit of CODH from WHC2b.C1 indicates a close phylogenetic relationship with Hydrogenophaga pseudoflava , an aerobic autotrophic member of Burkholderiales that can grow on either H 2 or CO (Willems et al., 1989 ; Kang and Kim, 1999 ). Therefore, the phylogeny of the CODH in the WHC2b.C1 contig is consistent with that of the [NiFe]-hydrogenases discussed above. CODH is typically involved in aerobic oxidation of CO, but some studies indicate that oxidation of low levels of CO can be coupled to nitrate rather than oxygen (King, 2006 ). Therefore, CO utilization could be advantageous in Tablelands springs when concentrations of H 2 , oxygen, and organic compounds are too low to support growth, but any conclusions about the importance of CO in these systems will require further investigations. At Lost City, CO utilization seems unlikely because of the abundance and ubiquity of H 2 and because CODH appears to be very rare (identified in only a single shotgun sequencing read). Potential for H 2 production by Clostridia Nearly all of the [FeFe]-hydrogenases detected in the WHC2b spring at the Tablelands and in the Lost City chimney have close phylogenetic relationships with putative homologs in Clostridia. [FeFe]-hydrogenases catalyze H 2 production by anaerobic bacteria, typically during fermentation, so one would expect them to be prevalent in anoxic environments where H 2 production is favorable. Potential subsurface sources of fermentable organic material are indicated by elevated levels of dissolved organic carbon in Lost City fluids (Lang et al., 2010 ) and the presence of low molecular weight hydrocarbons with potentially abiogenic origins in both Lost City and Tablelands fluids (Proskurowski et al., 2008 ; Szponar et al., submitted). No hydrogenases were detected in the Tablelands spring (TLE) that was collected from a more dilute and oxidizing seep (pH 10.5, E h + 25 mV) only ∼2 km from WHC2b (pH 12.06, E h − 733 mV). Therefore, the presence of [FeFe]-hydrogenases in the WHC2b metagenome supports the notion that the spring is supplied by fluid from an anoxic environment. Their presence in the Lost City chimney could be indicative of anoxic niches within chimney biofilms and/or the contribution of subsurface fluid to the chimney sample. The greater abundance of Clostridia-related 16S rRNA gene sequences in younger, hotter Lost City chimneys is consistent with both of these possibilities (Brazelton et al., 2010 ). The community structure of Tablelands springs and Lost City chimneys, as described here, resembles that of deep boreholes in South Africa (Moser et al., 2005 ; Lin et al., 2006 ). The subsurface fluids sampled by these boreholes are also basic (pH ∼9) and enriched in H 2 (up to 3.7 mM). The shallow fluids described by Moser et al. ( 2005 ) are dominated by Betaproteobacteria belonging to the Comamonadaceae family, and deeper fluids are comprised almost exclusively of Clostridia affiliated with genus Desulfotomaculum . The deep subsurface Desulfotomaculum -related organisms are predicted to be sulfate-reducers in these environments, which is consistent with metagenomic data representing the dominant organism, Candidatus “ Desulforudis audaxviator ” (Chivian et al., 2008 ). Some closely related species, however, are known to lack the genes required for sulfate reduction and instead subsist on fermentation, producing H 2 as part of a syntrophic relationship with methanogens (Imachi et al., 2006 ). Indeed, no sequences encoding dissimilatory sulfite reductase were identified in the Tablelands metagenomes, a striking result compared to the abundance of hydrogenases. The Lost City metagenome encodes a dissimilatory sulfite reductase with high sequence similarity to multiple Desulfotomaculum species (Brazelton, 2010 ), but it was detected in only a single sequencing read, indicating that it is far less abundant than the hydrogenases. Therefore, the Clostridia in Tablelands springs and Lost City chimneys are potential sulfate-reducers, but the abundance of [FeFe]-hydrogenases in metagenomic data from both environments indicates that they are more likely to be involved in H 2 -generating fermentation. It is unclear whether this putative fermentation is syntrophic with H 2 -utilizing methanogens. Automated annotation predicted very few methanogen sequences in the Tablelands and Lost City metagenomes (data available on the MG-RAST server), but they were present and may be more abundant in deeper habitats that were not well-represented in the samples described in this study. It is also possible that the Clostridia detected in this study are acetogens that are adapted to the elevated H 2 concentrations in the Tablelands and Lost City fluids. No sequences encoding acetyl-CoA synthase were detected at Lost City, but the phylogeny of ACS sequences from WHC2b is consistent with the presence of clostridial acetogens in very low abundance. Acetogens are known to be capable of producing H 2 and harboring a wide diversity of [FeFe]-hydrogenases (Kellum and Drake, 1984 ; Schmidt et al., 2010 , 2011 ), so determining the role of these Clostridia in the H 2 budget of these systems will require physiological and biogeochemical investigations. Only three ACS sequences were recovered from WHC2b, however, and none of these were assembled into contigs. Therefore, the current dataset indicates that acetogenesis may occur but does not appear to be prevalent in the Tablelands springs. It is possible that a more representative sample of the subsurface habitat underlying the spring could reveal more abundant evidence of acetogenesis, as well as other anaerobic metabolic pathways."
} | 5,345 |
23516610 | PMC3596300 | pmc | 1,781 | {
"abstract": "Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba ) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana . The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R 2 = 97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations.",
"conclusion": "Conclusion Genetically modified poplars are a potential alternative for the production of renewable energy since their properties can be optimized to facilitate saccharification. The release of transgenic organisms into the field needs to be carefully controlled to avoid negative effects on environmental interactions, especially with potentially beneficial soil microbes. In this study we demonstrated that transgenic poplar lines modified in the lignin biosynthesis pathway show normal abilities to form ectomycorrhizas. Gene-specific effects of the transformed poplars on mycorrhizal community structure were not found. Variations in EM community structures found between different GM poplar genotypes were in a range similar to the intra-specific variation of commercial poplar clones. The transgenic lines displayed strong differences in stem biomass production. Wood production in the initial phase of plantation establishment was positively correlated with EM colonization rates and negatively with stem N concentrations. Growth advantages realized in the establishment phase were pertained in the following year. Our results suggest that initial differences in EM colonization may have consequences for long term biomass production.",
"introduction": "Introduction The growing world population inevitably entails an increasing energy demand along with diminishing fossil fuel resources [1] . Renewable energies from biomass can be used as an alternative to partially replace conventional energy supplies. Trees, especially fast-growing species such as poplars, are an appealing feedstock for this purpose because they can be grown in dense short rotation plantations allowing several harvests without the need to re-plant [2] . Furthermore, poplars have a low nitrogen demand compared with other potential bioenergy crops [3] . Thus, their cultivation may contribute to the mitigation of nitrogen emissions from intensely used agricultural areas [4] . The conversion process of biomass to biofuels requires the breakdown of plant cell walls, which mainly consist of cellulose, hemicelluloses, and lignin [5] . Lignin is a recalcitrant polymer composed of phenylpropanoid units that hinder chemical and enzymatic cellulose degradation necessary for bioethanol production [6] . To amend wood utilization cell wall properties have been changed by targeted genetic approaches [7] . Genes of the biosynthetic pathway of lignin and cellulose have been isolated and characterized [8] – [10] . Suppression of cinnamyl alcohol dehydrogenase (CAD), an enzyme which converts cinnamyl aldehydes to the respective alcohols [5] and caffeate/5-hydroxyferulate O-methyltransferase (COMT), an enzyme involved in biosynthesis of syringyl lignin [5] result in altered lignin composition compared to wildtype (WT) poplars [11] – [13] . Overexpression of ferulate 5-hydroxylase (F5H), an enzyme that catalyzes an intermediate step in lignin biosynthesis, also results in compositional changes and less polymerization of monolignol units compared to the WT [14] . Suppression of cinnamoyl-CoA reductase (CCR) causes reduced lignin contents [15] . Transgenic poplars with alterations in lignin content and composition have been tested for industrial usage and display improved Kraft pulping [16] . The saccharification efficiency is also increased by genetic engineering of the lignin biosynthetic pathway [17] . If the use of genetically modified (GM) poplars with improved wood properties for bioenergy production was expanded, it will be necessary to know whether nutrient status and ecological interactions of GM poplars are changed compared with the WT. In a preceding study we compared whole fungal communities in soil and roots of poplars with suppressed CAD activities and of the WT by pyrosequencing and found a strong dominance of ectomycorrhizal (EM) in roots, whereas saprophytes were prevalent in soil [18] ; significant differences of these traits between the CAD lines and WT were not found [18] . The interaction of poplar roots with EM fungi is of particular importance for nutrient acquisition [19] . But other benefits have also been reported such as higher survival rates of EM-inoculated young poplar saplings [20] – [23] and increased resistance to drought stress [24] – [26] , issues gaining importance with increasing poplar cultivation in a warming climate. Currently it is still unclear if changes in the lignification pathway have significant ecological implication for interacting organisms. Lignin is the end product of the phenylpropanoid pathway, whose modification generally has consequences for the biosynthesis of other phenol-bearing compounds. For example, the suppression of CCR results in decreased lignin, but increased concentrations of phenolic compounds [15] . Phenolic compounds have been implicated in a wide range of ecological interactions. Greenhouse studies have shown that enzymatic activities of microbial communities are altered in soil of poplars with reduced lignin concentrations [27] . Field studies on the EM communities in relation to the performance of poplars with changes in the lignin composition and reduction of the lignin concentrations are lacking. The aim of this study was to characterize the EM community composition and dynamics in the first cycle of a short rotation plantation with poplars modified in the lignification pathway. To assess the relationship between EM diversity, plant nutrient status and dendromass we analyzed height growth, biomass, and nutrient element composition in leaves, stem and roots of transgenic Populus × canescens with suppressed activities of COMT (L9 and L11), CCR (L5 and L7), or CAD (L18, L21 and L22) and the wildtype (WT). We further compared the EM assemblages in the GM plantation with those of commercial poplar clones ( P . x euramericana , syn, Populus deltoides × Populus nigra c.v. Ghoy, I-214, and Soligo). Our study shows that in the first year after plantation establishment, EM fungal colonization and diversity were linked with tree productivity and low stem nitrogen concentrations. The variation of the EM fungal community composition found on roots of different transgenic poplar genotypes was similar to that found on different commercial poplar genotypes.",
"discussion": "Discussion Influence of gene modification on mycorrhizal colonization and community structure Poplars can form mutualistic associations with both arbuscular mycorrhizal and EM fungi [19] . However, in poplar plantations associations with EM fungi are the dominant symbiotic form [18] , [21] . Age-related increases in root tip colonization and EM species diversity as observed here for GM and WT poplars are well known for non-transgenic as well as transgenic poplars (e.g., suppression of the rolC gene in P. x canescens \n [43] , wildtype P. tremuloides \n [44] ). Besides the dynamic fungal succession, we observed initially differences in root tip colonization, which vanished in the second year and a differentiation of distinct EM communities on different poplar genotypes. A main question of the current study, therefore, was if the changes in EM colonization and fungal species composition were caused by the suppression of genes of the lignification pathway. Decreases in lignin as caused by CCR suppression or changes in the lignin composition as caused by CAD and COMT suppression interfere with secondary metabolism and entail changes in the profiles of phenolic compounds [45] . Since phenolic compounds belong to the defense arsenal of poplars [46] – [49] , negative effects on biotic interactions with EM fungi may be anticipated in transgenic trees with changed lignin biosynthesis. Although we found differences in the EM community composition in the second year after planting, these differences could not be related to the suppression of CCR, CAD or COMT. The composition of EM communities can be influenced by abiotic and biotic environmental factors such as fungal competition [50] , soil nutrient and water availability [51] – [53] and the physiology and genetic constitution of the host [34] , [54] , [55] . Variations of abiotic factors and patchiness of soil fungi were not detected in our study plantation. Therefore, EM species composition and abundance might have been influenced by host factors. During transformation the positioning of the introduced DNA in the genome cannot be controlled. Thus, the insertion may have side-effects when the introduced DNA fragment unintentionally hits a functional plant gene locus. Therefore, each transformation event may cause intra-specific variation of traits, in addition to the target gene. Controlled experiments testing the colonization efficiency of the EM fungus Laccaria bicolor with the F1 progeny of an inter-specific poplar hybrid revealed that the ability to form mycorrhizas underlies natural intra-specific variation [55] – [57] . Different EM assemblages were also observed in the present study for different varieties of P . x euramericana , a poplar hybrid bred for biomass plantations [58] , [59] . The intra-specific and inter-specific variation in EM assemblages on the WT hybrids of P . x euramericana and P . x canescens was similar to that between CCR line L7 and CAD line 22, which exhibited the largest difference of EM species composition. Our study, therefore, supports that the host genotype can affect the colonization ability of distinct mycorrhizal fugal species. However, the intra-specific variation introduced by the transformation of poplars with the antisense constructs to suppress CCR, COMT or CAD activities did not result in larger differences in the EM community composition than those observed for different varieties of conventionally bred high-yielding poplar clones. The link between EM colonization and diversity and poplar dendromass and nutrient status The GM poplars with suppressed activities of enzymes of lignin biosynthesis showed strong (ca. 5-fold) differences in growth and biomass in the plantation. This was not surprising since similar results had been obtained by others studying the performance of lignin-modified plants. For example, Leplé et al . [15] found reduced growth in two of five investigated CCR-suppressed poplar lines under field conditions. Voelker et al . [60] observed extensive variations in aboveground biomass of 14 different lines of P. × canescens down-regulated in 4-coumarate:coenzyme A ligase (4CL). Furthermore, greenhouse-grown transgenic poplars with suppressed coumaroyl 3′-hydrolase (C3′H) activity showed drastic growth reductions [61] . The suppression of C3′H activity also reduced the water use efficiency resulting in lower δ 13 C signatures in the transgenic compared to WT poplars [61] . If the growth reductions found here were due to impairment of photosynthesis such as reduced stomatal conductance, we would have expected a shift in the δ 13 C signature to higher values because of decreased carbon discrimination. However, this was not observed and, therefore, effects on water use and carbon allocation to wood are unlikely reasons for growth reductions in the GM poplars of our study. Another possibility is that changes in EM colonization and changes in the EM communities had negative impact on tree nutrition leading to reduced growth. This option is not unlikely since the interactions of mycorrhizas with their hosts cover the whole range from beneficial to parasitic effects [62] , [63] . For example, colonization of P . x euramericana (cv Ghoy) with different arbuscular mycorrhizal fungal species caused reductions in plant biomass [64] . Although the P concentrations of the aboveground tissues increased, P content of the shoot was diminished because of overall biomass loss [64] . In our study, the abundance of the EM fungi Peziza ostracoderma and the ascomycete JQ JQ409294 on root tips of the transgenic poplar genotypes showed negative and positive correlations with foliar P concentrations, respectively. Paxillus involutus , which was present in our plantation, has been shown to increase K and P nutrition of poplars [20] – [23] , [65] . These observations might imply that distinct EM-poplar genotype associations contributed to facilitating or suppressing P or K transfer to their host trees. However, this suggestion is currently speculative since a full nutrient budget of the trees was not possible and the regulation of tree-fungal-environmental interaction is barely understood. Further functional analyses of EM fungi are, therefore, required. N is one of the most important nutrient elements for plant growth [66] . In young strongly growing poplars N is mainly present in leaves, but a significant fraction is resorbed in fall, present in woody tissues during the dormant season and re-utilized for sprouting in spring [67] , [68] . Here, we observed a negative relationship between stem N concentrations and stem biomass indicating higher storage in the wood of smaller poplars than in those of taller plants. The biomass differences of stems were maintained in the following season, and could obviously not be compensated by increased internal N utilization of smaller trees for stem growth. Thus, poplars with low growth have the additional disadvantage of wasting N when utilizing woody biomass. There is evidence that N allocation differs between fast and slow growing poplar species since trees with inherently higher biomass production exhibit lower N concentrations in the wood and higher nitrogen productivity [69] – [71] . Poplars grown on a previous agricultural field also showed increased biomass production, decreased N concentrations, and increased nitrogen use efficiency in response to long-term free air CO 2 enrichment [72] , [73] . Our present data support that, at least in the initial phase, EM colonization is linked with these traits. Positive relationships for growth, nitrogen utilization and EM colonization rates have also been found in Douglas fir [74] . Based on the current data it is not possible to distinguish if poplar growth was stimulated because of higher rates of EM colonization or if trees with higher growth were more amenable to EM colonization. However, the latter possibility is more likely since other studies have already shown that EM colonization and diversity were driven by carbon availability and productivity of the host tree and not vice versa [34] , [54] , [74] . Since the root tips of the GM poplars were almost completely colonized with EM at the end of the second growing season, it is clear that the GLM model developed for biomass, nitrogen and root colonization will not be applicable in older plantations. The establishment phase is, however, very important and biomass increments realized during this crucial period will result in further gains because of the exponential nature of growth."
} | 4,243 |
33575251 | PMC7870715 | pmc | 1,782 | {
"abstract": "Global warming and uneven distribution of fossil fuels worldwide concerns have spurred the development of alternative, renewable, sustainable, and environmentally friendly resources. From an engineering perspective, biosynthesis of fatty acid-derived chemicals (FACs) is an attractive and promising solution to produce chemicals from abundant renewable feedstocks and carbon dioxide in microbial chassis. However, several factors limit the viability of this process. This review first summarizes the types of FACs and their widely applications. Next, we take a deep look into the microbial platform to produce FACs, give an outlook for the platform development. Then we discuss the bottlenecks in metabolic pathways and supply possible solutions correspondingly. Finally, we highlight the most recent advances in the fast-growing model-based strain design for FACs biosynthesis.",
"conclusion": "Conclusion The ongoing reliance on fossil fuels of human society is driving elevated atmospheric CO 2 and increasing global temperatures, thereby escalating the risk of widespread environmental disasters in the near future. We anticipate that microbial synthesis of products from CO 2 , which can provide chemicals with near-zero net greenhouse gas emissions, will play as a game-changer in the future ( Ediger, 2019 ). Great progress has been made in the areas of enzyme engineering, metabolic engineering, and model-assisted engineering to assist microbial production of FACs ( Cao et al., 2016 ; Herman and Zhang, 2016 ; Kim et al., 2016 ; Zhou et al., 2016 ; Fatma et al., 2018 ; Marella et al., 2018 ; Kim and Park, 2019 ; Liu and Nielsen, 2019 ; Lynch et al., 2019 ). However, the present-day microbial cell factories still have major challenges to overcome, such as controlling the length and types of released FACs and improving the conversion efficiency via RBO. We expect that directed enzyme evolution and rational enzyme engineering will contribute to the production of target FACs through the RBO pathway. Recently, there are some machine learning-based algorithms developed for computational protein design, which can also be used in enzyme engineering ( Masso and Vaisman, 2008 ; Fang, 2019 ; Zu Belzen et al., 2019 ). In addition, new methods for design and build of synthetic microorganism communities can contribute to the construction of novel microbial platforms, which combine carbon-fixing autotrophs with heterotrophs for efficient FACs biosynthesis with net-zero greenhouse gas emissions.",
"introduction": "Introduction Increasing consumption of petroleum-derived products leads to increasing atmospheric carbon dioxide (CO 2 ) levels and global warming ( Sperry et al., 2019 ). Furthermore, the uneven distribution and unsustainability of fossil resources have motivated engineers to seek alternative sustainable solutions ( Raslavičius et al., 2014 ; Chen et al., 2020 ). Compared with the traditional strategies to convert plant oils and animal fats into biodiesel, microbial synthesis of fuels, and chemicals presents several advantages. Firstly, feedstocks can be shifted from edible plant oils and animal fats to non-edible biomass feedstocks, especially CO 2 . Secondly, due to the flexibility of pathways in microbial chassis, a large diversity of bioproducts can be produced in microbial cell factories. Among these bioproducts, fatty acid-derived chemicals (FACs) have attracted significant attention, because fatty acids (FAs) are essential metabolites in all organisms. FAs and their biosynthetic/catabolic intermediates can be used as precursors for a large diversity of FACs, which have an unprecedented wide application range: biofuels, pharmaceuticals, feed additives, and others. Thirdly, bioproducts are green alternatives to petroleum-based fuels, given the capacity of net-zero greenhouse gas emissions. Microbial chassis must be extensively designed and engineered to produce FACs at high titer, rate and yield from various substrates. Recent successes in model-based strain design have speed-up the Design-Build-Test-Learn (DBTL) cycle in metabolic engineering ( Carbonell et al., 2018 ; Hamedirad et al., 2019 ; Opgenorth et al., 2019 ). Although FACs biosynthesis has been reviewed from different angles ( Marella et al., 2018 ; Liu and Li, 2020 ), the purpose of this review is to update the most recent advances in this fast-developing field, with an emphasis on possible synthetic microbial chassis and computational modeling for biosynthesis of FACs."
} | 1,117 |
36479317 | PMC9720408 | pmc | 1,784 | {
"abstract": "A central endeavor in bioengineering concerns the construction of multistrain microbial consortia with desired properties. Typically, a gene network is partitioned between strains, and strains communicate via quorum sensing, allowing for complex behaviors. Yet a fundamental question of how emergent spatiotemporal patterning in multistrain microbial consortia affects consortial dynamics is not understood well. Here, we propose a computationally tractable and straightforward modeling framework that explicitly allows linking spatiotemporal patterning to consortial dynamics. We validate our model against previously published results and make predictions of how spatial heterogeneity impacts interstrain communication. By enabling the investigation of spatial patterns effects on microbial dynamics, our modeling framework informs experimentalists, helps advance the understanding of complex microbial systems, and supports the development of applications involving them.",
"introduction": "Introduction Since the discovery of quorum sensing in the early 1970s ( 1 ), understanding of microbial communication has grown tremendously. Multiple communication mechanisms such as acyl-homoserine lactones (AHLs) ( 2 , 3 ) and autoinducing polypeptides (AIPs) ( 4 , 5 ) have been uncovered, and their role in the formation of complex social behaviors such as biofilms and swarming motility have been deciphered ( 6 , 7 , 8 ). Recognizing that consortia of interacting microbial populations can perform more complicated behaviors than any one species individually, researchers have begun to leverage these systems for various biotechnology applications ( 9 ). For example, microbial consortia have been designed using natural or genetically engineered bacterial strains for use in biosensors ( 10 ), bioremediation ( 11 , 12 ), and more efficient bioproduction ( 13 , 14 , 15 ). With this wide level of utility, the impact of microbial consortia on industry and human health are likely to grow as more efficient tools are developed for their design and application. Substantial tools exist for controlling a particular consortium’s behavior genetically using synthetic regulatory circuits ( 16 , 17 , 18 , 19 ), and tools for engineering a consortium’s spatial organization have also begun to be developed ( 20 ). For example, Romano et al. recently developed a novel optogenic method for regulating gene expression that allowed them to re-create highly complex images using bacterial lawns ( 21 ). Similarly, Alnahhas et al. demonstrated that the number of subpopulation bands in an extended microfluidic device can be regulated through the seeding of the trap. Other methods for regulating the spatial organization of consortia including adhesion, cell-communication, and motility are also being developed ( 20 , 22 , 23 , 24 , 25 , 26 ). With previous studies demonstrating that the spatial organization of a consortium can significantly alter its dynamics ( 18 , 27 ), these experimental methods are enabling an additional layer of control for engineering microbial consortia. What is lacking from the research are sufficiently flexible modeling frameworks for investigating the impact of complex spatiotemporal structure on microbial consortium dynamics. These tools have the potential to streamline the rational engineering of spatial patterns to control consortium behavior. Previous approaches to modeling the spatiotemporal dynamics of microbial consortia have traditionally relied on well-mixed compartment models ( 16 , 17 ) or agent-based frameworks ( 28 , 29 , 30 ). However, the well-mixed compartment models do not allow for the explicit incorporation of strain patterns, and agent-based modeling frameworks are computationally expensive for larger systems ( 31 ). To address this gap in the literature, we borrow from previous research focused on modeling diffusion in heterogeneous media ( 32 , 33 , 34 ) and develop a piecewise-defined reaction-diffusion equation framework for modeling spatiotemporal consortium dynamics. Here, we show that this modeling framework allows for the explicit incorporation of consortium spatial organization while remaining computationally tractable for larger systems. We then use this framework to investigate the impact of spatial heterogeneity on consortium dynamics and, using our results, demonstrate the rational design of consortium dynamics through spatial organization. Importantly, our framework provides an explicit link between interstrain communication and spatiotemporal patterning. Modeling framework For diffusion in heterogeneous media, variations in the composition of the medium results in corresponding discontinuities in its physical properties that need to be accounted for when modeling molecular transport. Researchers traditionally model this phenomenon using a reaction-diffusion equation ( 32 , 33 , 34 ): (Reaction-Diffusion Equation) ∂ c ∂ t − ∇ · ∇ D x c x , t = f c , x , t , t ≥ 0 , x = x , y , z ∈ Ω , where c = c ( x , t ) is the local concentration of the diffusing molecule, D is the diffusion coefficient, t is time, Ω is the domain, and f ( c , x , t ) describes the production and degradation of c . To account for variations in transport properties in heterogeneous media, D is piecewise-defined according to the medium composition to have the appropriate value in the appropriate (user-controlled) location. Recognizing a natural correspondence between media heterogeneity and cell heterogeneity, we modify this modeling approach so that it can be used to study the spatiotemporal dynamics of microbial consortia. In our modified approach, the concentration of each signaling molecule in the consortium is modeled using a reaction-diffusion equation with components representing the local production and degradation described by a source term f ( c , x , t ) . We incorporate the spatial organization of the consortium strains into the resulting partial differential equation (PDE) model by structuring the source term for each signaling molecule on Ω as a piecewise function. This ensures that each signal is only generated in areas that contain a strain capable of producing it. Since each strain is uniquely defined by the signaling molecules that it produces, piecewise defining our system creates areas of production that mimic the spatial organization of the strains (see Fig. 1 \n A ). This allows the resulting system to incorporate the influence of spatial organization on the dynamics of the microbial consortium and allows for the investigation of any desired organization. Figure 1 Consortial organization. ( A ) Micro and macro illustration of our spatial discretization that allows for the incorporation of columnar spatial patterns in the P 2 N 1 and P 1 N 1 consortia. For spatial points corresponding to activator segments (blue), the production terms for the repressor, η r 0 and η r 1 , are set to zero. The opposite holds in the repressor segments (orange) where the the production terms for the activator, η a 0 and η a 1 , are set to zero. Diagram of the P 2 N 1 ( B ) and P 1 N 1 ( C ) gene circuit topology. Evaluation of framework and results As a first application of this framework, we used it to model a recent experimental study of the spatiotemporal dynamics of a well-known microbial consortium ( 16 ). There, Kim et al. compared the dynamics of four microbial consortia in a spatially extended microfluidic trap consisting of different types of feedback networks. Each consortium consisted of an activator and repressor microbial strain in an alternating columnar spatial arrangement (diagrammed in Fig. 1 \n A ). The two strains regulated each other’s gene expression in a dual-feedback mechanism to produce an oscillating fluorescent pattern ( 17 ). Through experimentation and modeling, Kim et al. found that the addition of a positive feedback loop resulted in globally coordinated oscillations. This was in spite of the fact that the length of the trap was significantly larger than the diffusion correlation length of the signaling molecules, thereby preventing direct communication between strains located far apart from each other ( 16 ). However, their model omitted the organization of the stripe spatial patterns. Stripe thickness could affect the ability of the strains to couple their behavior through diffusion. Thus, the influence of strain spatial organization on consortium dynamics warrants investigation. For simplicity, we only model two of the consortia studied in Kim et al., P 2 N 1 and P 1 N 1 (diagrammed in Fig. 1 \n B and C ), since they behave very similarly to the other two consortia. The P 2 N 1 and P 1 N 1 consortia also exhibited more robust columnar stripe patterns making them better suited to study in 1D. Model derivation To model the spatiotemporal dynamics of the P 2 N 1 and P 1 N 1 consortia using our framework, we constructed reaction-diffusion equations describing the dynamics of the consortium’s extracellular signaling molecules. Like previous research into microbial consortia ( 35 ), we developed a simplified model of the dynamics since a thorough description of the underlying gene circuit interactions has been previously shown to require 16 delay-differential equations (DDEs) ( 17 ). To do so, we suppressed the dynamics of the fluorescent reporters and considered only the dynamics of the extracellular signaling molecules. This simplification is valid because the fluorescent reporters are produced in proportion to the local concentration of the lactone-based signaling molecules ( 17 ). We also ignored the role of intermediaries in producing these signaling molecules by modeling their production using Langmuir-functions, which take into account the overall activator-repressor network interactions. These functions mirror the form of previously developed equations for modeling production in consortia ( 16 , 17 ) and are provided below, where a represents the local concentration of the activator N-butanoyl-L-homoserine lactone (C4-HSL, C4) and r represents the local concentration of the repressor N-(3-hydroxytetradecanoyl)-DL-homoserine lactone (3-OHC14-HSL, C14). We chose a similar strategy to model their enzymatic degradation by AHL-lactonase, AiiA, since it is produced in response to the local concentration of the repressor. We also borrowed from previous research by assuming linear degradation of the signaling lactones and a 7.5-min transcriptional delay, τ ( 16 , 17 ). With these interactions characterized, the source terms f a ( x , t ) and f r ( x , t ) are given by (1) f a ( x , t ) = η a 0 + η a 1 ( a τ K a ) n a 1 + ( a τ K a ) n a + ( r τ K r ) n r ︸ production − d e a ( r τ K e ) n e 1 + ( r τ K e ) n e ︸ AiiA degradation − γ a ︸ dilution (2) f r ( x , t ) = η r 0 + η r 1 ( a τ K a ) n a 1 + ( a τ K a ) n a ︸ production − d e r ( r τ K e ) n e 1 + ( r τ K e ) n e ︸ AiiA degradation − γ r ︸ dilution , where a τ ≡ a ( x , t − τ ) and r τ ≡ r ( x , t − τ ) . A complete description of all the parameters and their corresponding values can be found below in Table 1 , and a description of how the parameters values were determined can be found in the Methods section of the Supporting material . To differentiate between the P 2 N 1 and P 1 N 1 consortia, η a 1 is set to zero for the P 1 N 1 consortium to account for the absence of the auto-activation, and the ( a τ K a ) n a in the denominator of the production term is removed. We thus arrived at the following two-equation system, partial delay-differential equation model of the P 2 N 1 and P 1 N 1 consortium spatiotemporal dynamics: (3) { ∂ a ∂ t − D ∇ 2 a = f a ( x , t ) ∂ r ∂ t − D ∇ 2 r = f r ( x , t ) Table 1 Parameter Values and Descriptions Parameter Description Value η a 0 Basal production rate of activator signaling molecule from promoter. 985.56 nM min − 1 η a 1 Maximal production rate of activator signaling molecule from promoter in response to activator signal. 17,991.82 / 0 nM min − 1 η r 0 Basal production rate of repressor signaling molecule from promoter. 74.46 nM min − 1 η r 1 Maximal production rate of repressor signaling molecule from promoter in response to activator signal. 46,495.80 nM min − 1 K a EC 50 of activator signaling molecule for production. 5937 nM K r IC 50 of repressor signaling molecule for repression. 10 nM n a Langmuir coefficient of production for activator signaling molecule. 4 n r Langmuir coefficient of repression for repressor signaling molecule. 2 D Diffusion coefficient signaling molecules. 4080 μ m 2 min − 1 d e Maximal rate of internal enzymatic degradation of signaling molecules. 2257 min − 1 K e EC 50 of repressor signaling molecule for enzymatic degradation. 1000 nM n e Langmuir coefficient of internal enzymatic degradation for repressor signaling molecule. 4 γ Inherent rate of degradation of signaling molecules caused by dilution from both media flow and cell growth. 0.128 min − 1 τ Time delay for transcription and translation. 7.5 min The strain spatial organizations are included in this model in a user-defined fashion through the piecewise definition of f a ( x , t ) and f r ( x , t ) . Specifically, we set the production rate terms in f a ( x , t ) and f r ( x , t ) to be zero in areas that did not include an activator or repressor strain, respectively. Model validation To validate our simplified model of the P 2 N 1 and P 1 N 1 consortia, we investigated its ability to accurately capture the spatiotemporal dynamics recently observed in Kim et al. The main result of this paper was that strains containing a second positive feedback loop synchronized their dynamics across a spatially extended microfluidic trap. Thus, we validated our model by testing whether it could reproduce this key behavior. For this test, we simulated the P 2 N 1 and P 1 N 1 consortia in one dimension. A 1D model was chosen since diffusion and variations in strain composition in the vertical direction are assumed to be negligible in the spatially extended trap ( 16 ). We therefore modeled the L = 2 mm long trap (e.g., device size in Kim et al. ( 16 )) using the 1D spatial domain, Ω ≡ [ 0 , L ] . The consortia were implemented by dividing the spatial domain into 100 stripes of equal width alternating between activator and repressor strains. The consortium was then divided into two equally spaced halves oscillating with an initial phase difference of | Δ φ 0 | = 25 % of the oscillation period, φ . We chose this value because it resulted in synchrony for P 2 N 1 and asynchrony for P 1 N 1 in Kim et al. Both the P 2 N 1 and P 1 N 1 consortia were then analyzed for whether synchronization between the two halves occurred. To determine the presence of synchronization, we calculated the phase difference of the activator concentration oscillations for the middle spatial points of the left and right subpopulations, Δ t φ (detailed explanation provided in the Methods section of the Supporting material ). A percent phase difference, | Δ φ | was then calculated as follows: (4) | Δ φ | = Δ t φ φ ∗ 100 We assumed a phase difference of | Δ φ | < 2.5 % between the two halves corresponded to synchronization across the trap. Since φ depends on the basal production rate, η a 0 , we chose to measure the phase difference after the 10th oscillation instead of after a specified period of time had passed. The presence of the autoinducing positive feedback loop in the P 2 N 1 consortium was cited in Kim et al. as the reason P 2 N 1 oscillations became coordinated, whereas P 1 N 1 oscillations did not. Thus, we originally only varied the value of η a 1 between our P 2 N 1 and P 1 N 1 simulations, setting it to zero in the P 1 N 1 simulation to account for an absence of the feedback loop. We failed to see any significant difference in coordination between the P 2 N 1 and P 1 N 1 consortia ( Fig. 2 \n C ). With this finding, it was clear that the difference in the activator basal production rate between the P 2 N 1 and P 1 N 1 consortia needed to be incorporated into our model. In Chen et al., it was reported that the activator basal production rate in the P 1 N 1 consortium is approximately nine times greater than in the P 2 N 1 consortium, and this difference was even included in the model used in Kim et al. Thus, to investigate the role changing this basal production rate had on P 1 N 1 behavior, we re-ran the P 1 N 1 synchronization tests for different values of η a 0 . Specifically, we scaled η a 0 by a basal multiplier, b m , that varied from 0.1 to 10. The results of these tests are provided in Fig. 2 \n C and show that the coordination of oscillations is strongly influenced by the value of the activator basal production rate, η a 0 . Kymographs illustrating the level of coordination in the P 2 N 1 and P 1 N 1 consortia are provided in Fig. 2 \n A and B , respectively. Figure 2 Basal production’s influence on synchrony. Results of our synchronization simulations including kymograph of activator concentration during the 8th to 11th oscillations of the P 2 N 1 ( A ) and P 1 N 1 ( B ) consortia (basal multiplier of 0.7). ( C ) Graph showing the relationship between phase difference and the value of the basal multiplier for the P 1 N 1 consortium at two different initial phase differences, | Δ φ 0 | = 25 % and 20 % . Investigating the role of spatial heterogeneity Having validated our model against the experimentally observed difference between the P 2 N 1 and P 1 N 1 consortia, provided the difference in activator basal production rates is incorporated, we proceeded to test our model’s ability to capture the influence of spatial heterogeneity on consortium dynamics. This is the primary focus of this work and will provide new insight into heterogeneous systems. First, we investigated the impact changing stripe width had on the coordination of oscillations in the P 2 N 1 consortium. This was done by repeating the aforementioned synchronization test for the P 2 N 1 consortium with stripe widths varying from 10 to 200 μ m. The results of these simulations are provided in Fig. 3 \n B and show that coordination breaks down as stripe width increases. In fact, we observe a significant breakdown in synchrony for stripe widths larger than 90 μ m. This shows quantitative agreement with previous experimental studies of the same consortia in Gupta et al. There, they found that separation distances larger than 100 μ m between activator and repressor populations led to a loss of oscillatory behavior. This demonstrates that our modeling framework results are consistent with experimental results from different studies ( 16 , 18 ). Figure 3 Spatial organization’s influence on synchrony. ( A ) Kymograph of activator concentration in the P 2 N 1 consortium consisting of heterogeneous stripes. The left side of the consortium consists of small-width stripes (0.56–47.06 μ m), and the right side of the consortium consists of large-width stripes (19.05–266.67 μ m). ( B ) Graph showing the relationship between phase difference and stripe width for the P 2 N 1 consortium. Interestingly, a stripe width of 70 μ m is optimal for synchronization. We intuit this result as follows. For stripe widths larger than 70 μ m, the diffusion correlation length of the quorum sensing molecules is not great enough to allow for interstrain communication. That is, the coupling is too weak. On the other hand, for stripe widths less than 70 μ m, chemical signals can diffuse across several stripes, flooding the trap with chemical signal. When active, the activator promoters are maximally productive, leading to an effective basal production rate that corresponds to the right-hand side of Fig. 2 \n C . In effect, the spatial heterogeneity provides an engineering methodology to control the effective basal production of the strains. With this result in mind, we hypothesized that the coordination of the consortium could be controlled by only varying its spatial organization. To test this hypothesis, we simulated two P 2 N 1 consortia side by side in a 4-mm-long trap. Both consortia contained alternating activator and repressor stripes whose widths increased linearly. However, the first consortium contained small-width stripes (0.56–47.06 μ m), and the second consortium contained large-width stripes (19.05–266.67 μ m). The results of this test are provided in a kymograph showing the concentration of the activator across the consortium and time. As anticipated by the results provided in Fig. 3 \n B , the left half of the consortium oscillated in a coordinated manner, and the right half of the consortium, containing larger stripes, had its oscillations become decoupled.",
"discussion": "Discussion We developed a new modeling framework to investigate the impact of spatial heterogeneity on consortial dynamics. We verified our framework results against the modeling and experimental results found in Kim et al. ( 16 ). We also observed quantitative agreement between our stripe width investigation study and experimental findings from Gupta et al. ( 18 ). Although our model is much simpler than what is used by Kim et al. and coarse-grains over many subcellular processes, the qualitative behaviors of the two models coincide. Our model gains tractability at the expense of some fidelity to reality. But it is exactly in these scenarios that mathematical modeling can provide unexpected qualitative predictions regarding the biological system. The results demonstrate that our new modeling framework is able to effectively incorporate the effect of spatial organization on microbial consortium dynamics. By allowing for the explicit incorporation of stripe patterns, our work complements and extends previous research into modeling spatiotemporal dynamics of microbial consortia ( 16 , 18 , 22 , 28 , 29 , 30 , 36 , 37 ). For example, Kim et al. modeled spatiotemporal dynamics using well-mixed compartments containing both activator and repressor strains. However, this approach prevents the incorporation of well-defined spatial patterns into the consortia, which, in turn, limits any investigation into their effect on consortium dynamics. Our piecewise-defined diffusion equation framework allowed us to investigate this impact in the present study. Our modeling framework is thus more faithful to the robust single-strain stripes observed in Alnahhas et al. Compartment models force all cells in a given compartment to experience the same level of chemical signal. Our piecewise framework allows for spatial variations within and between single-strain stripes. As expected, we found that small-width stripes allowed for more coordination of consortium dynamics due to better communication between stripes over shorter distances ( 18 , 27 ). Interestingly, our heterogeneous stripe test demonstrates consortium behavior can be controlled through the consortium’s spatial organization alone. Combined with a rise in experimental methods for controlling spatial organizations in consortia ( 20 , 23 , 24 , 38 ), this opens an interesting avenue for regulating consortium dynamics in the future ( 36 ). An unexpected finding from our work was the strong influence of the basal multiplier on consortium coordination. From the results of Kim et al., it was expected that the presence or absence of the positive feedback loop would be sufficient to control coordination. Our simulation results indicate that the basal production rate had a stronger impact. Since the results of the basal multiplier test mirror that of the stripe width investigation (see Figs. 2 \n C and 3 \n B ), we hypothesize that the observed behavior was due to variations in communication strength at various multiplier values. For lower values, the production rate of signaling molecule is lower, which results in weaker signaling between stripes. The opposite holds for higher production rates. The fact that Kim et al. included differences in basal production rates into their model may have obfuscated the actual impact of the positive feedback loop. However, since our model substantially simplifies the underlying gene circuits in exchange for computational efficiency and understandability, the basal multiplier results could be an artifact of this simplification. Separate from this simplified model is the underlying framework of using piecewise-defined reaction-diffusion equations to incorporate spatial patterns into studies on consortium dynamics. Our ability to study various spatial patterns using this framework demonstrates its usefulness for modeling the spatiotemporal dynamics of microbial consortia. One strength of this framework is its flexibility. By varying the spatial points where the production terms are nonzero, a researcher can precisely control the spatial organization they are studying, including heterogeneous arrangements. Besides flexibility, another benefit of our model is its computational tractability. One strategy that previous studies have used to explicitly incorporate consortium spatial patterns is agent-based modeling ( 28 , 29 , 30 ). Although the fine-grained nature of these frameworks provides the greatest level of control when investigating the impact of spatial patterns, the complexity of these systems makes the study of larger consortia computationally infeasible ( 29 , 31 ). Our continuum-based framework complements these studies and fills a gap in the literature by allowing for incorporation of spatial organizations into studies on the dynamics of large, spatially extended consortia. Although we did not investigate other geometries or consortia in this study, we are confident the flexibility and computational tractability of our modeling framework will enable the study of more complicated systems. For example, our model could be used to investigate the effects of square size on consortium dynamics for the checkerboard pattern recently developed by Perkins et al. Besides allowing for a better understanding of these systems, our heterogeneous stripe results show that our modeling framework could potentially be used to engineer consortium behavior by finding spatial organizations that modulate the communication between consortium subpopulations in a desired fashion. Specifically, this can be done by making the distance between subpopulations either smaller or larger to modify the signaling strength between the subpopulations. We used this principle here to design a consortium that oscillated synchronously on the half that contained small stripes and asynchronously on the half that contained large stripes. Combined with a growing number of experimental approaches for designing consortia with specific spatial arrangements, including optogenic, seeding, and other approaches ( 20 , 22 , 23 , 24 ), modulating consortium dynamics through spatial organization is becoming more practical. Since the underlying numerical scheme used to solve our reaction-diffusion equation model has routinely been applied in higher dimensions ( 39 , 40 , 41 , 42 ), our framework is well suited to interrogate the effects of the more complex spatial organizations implemented by these experimental methods ( 23 , 43 , 44 , 45 ). Through enabling these investigations into the impact of spatial organization on microbial consortium dynamics, we hope that our modeling framework helps advance the understanding of these systems and supports the development of new applications involving them."
} | 6,896 |
28362723 | PMC5584475 | pmc | 1,785 | {
"abstract": "From microbial biofilms to human migrations, spatial competition is central to the evolutionary history of many species. The boundary between expanding populations is the focal point of competition for space and resources and is of particular interest in ecology. For all Escherichia coli strains studied here, these boundaries move in a counterclockwise direction even when the competing strains have the same fitness. We find that chiral growth of bacterial colonies is strongly suppressed by the expression of extracellular features such as adhesive structures and pili. Experiments with other microbial species show that chiral growth is found in other bacteria and exclude cell wall biosynthesis and anisotropic shape as the primary causes of chirality. Instead, intimate contact with the substratum is necessary for chirality. Our results demonstrate that through a handful of surface molecules cells can fundamentally reorganize their migration patterns, which might affect intra- and interspecific competitions through colony morphology or other mechanisms.",
"conclusion": "Conclusions Chirality is a general property of biological systems which is shared by very different species as the Gram-positive B. subtilis and the Gram-negative E. coli . Efficient adaptation of bacterial colonies to changing environmental conditions requires cooperative behavior and self-organization, which is based on the exchange of information. The different communication channels range from direct physical and chemical interaction to indirect interactions through chemotaxis and trails on the agar. Pattern formation in microbial colonies is a way to exchange information between the microscopic level and the macroscopic level. In other words, an exchange between the individual cells and the colony. Therefore, the full understanding of chiral growth, its origins and implications must focus on the relation between the individual cell and the colony organization. For E. coli colonies expanding on a substratum, the border between competing strains often exhibits chirality and always in a left-handed manner (when observed from the agar side). We find a correlation between the expression of surface structures involved in cell–cell and cell–surface adhesion or biofilm formation and the macroscopic degree of chirality; the more surface structures expressed, the less chiral the border between competing strains. This result was obtained by comparing pattern formation of MG1655 to pattern formation of MG1655 mutants with deletion of pili, curli fimbriae, colanic acid and antigen 43. Furthermore, we found chirality to be independent of flagella-mediated motility. The DH5α strain had more chiral borders than any of the MG1655 mutants, possibly because DH5α is a poorer biofilm former. We also find that substituting agar with agarose reduces chirality, suggesting that chirality largely depend on substratum composition. The exact mechanism by which the substratum mediates the chiral growth will be a subject of future investigation. Overall, intimate cell–substratum adhesion is necessary for chirality and extracellular features, such as fimbriae or biofilm, weakens this contact. Our results link macroscopic chiral colony formation to the microscopic biological features of E. coli ’s cell wall and the substratum and suggest new ecological roles for several membrane-associated proteins during colony growth.",
"introduction": "Introduction From the structure of amino acids to the shape of our galaxy, chirality is ubiquitous in nature and has fascinated scientists for centuries. The accepted mechanism for the emergence of left–right asymmetry is a fluctuation producing a chiral state followed by positive feedback favoring homochirality ( Frank, 1953 ; Gayathri and Rao, 2005 ; Saito et al. , 2007 ). More recent work further demonstrates that intrinsic noise in autocatalytic reactions is also sufficient to produce and stabilize left–right asymmetry ( Jafarpour et al. , 2015 ). While these physical mechanisms might explain chirality in the inanimate world, chirality in living systems could be a product of natural selection. Although the ecological and evolutionary role of chirality is largely unexplored, there are several examples that point to the possibility that chirality could be advantageous. Extensive work with Paenibacillus showed that this microbe switches from chiral to non-chiral phenotypes in response to changing environmental conditions ( Ben-Jacob et al. , 2000 ). More recently, Wan et al. (2011) found that cancer cells are chiral and have the opposite chirality of the normal tissue from the same patient. In both cases, molecular changes propagate to population-level scales and alter the ecology and evolution of the species. The details of these processes are poorly understood. These striking examples motivated us to develop a molecular understanding of chirality in a simple model system of an Escherichia coli colony. In the lab, E. coli typically grows as a compact circular colony when inoculated on an agar plate; yet internal dynamics of those colonies are often chiral. These dynamics can be revealed by fluorescently labeling subpopulations of growing cells. Strong genetic drift at the growing edge of the colony promotes local loss of genetic diversity and results in spatial de-mixing of the subpopulations producing a characteristic pattern of flaring sectors on long timescales ( > 30 generations) ( Kreft, 2004 ; Hallatschek et al. , 2007 ; Ali and Grosskinsky, 2010 ; Hallatschek and Nelson, 2010 ; Korolev et al. , 2010 ). Without chirality, the boundaries between the sectors extend radially, but, in chiral species, boundaries appear as spirals emerging from the center of the colony ( Korolev et al. , 2011 ). While the existence of chirality in bacterial colony growth has been established, there are still open and interesting questions regarding the origin and evolutionary importance of sector boundary chirality: (i) why and when is chirality beneficial, (ii) how does microscopic chirality translate into a macroscopic chirality and (iii) what are the molecular origins of the chirality? The first question is of great interest and, while our work does not address this question directly, it provides important foundation for future studies. The second question has been largely resolved for bacteria via thorough mathematical modeling and experimentation ( Ben-Jacob et al. , 2000 ; Xue et al. , 2011 ). Here, we primarily focus on the last question and uncover the key molecular players shaping chirality of bacterial growth. Already in 1848, Louis Pasteur related macroscopic chirality of a crystal to a microscopic symmetry breaking of constitutive molecules ( Flack, 2009 ). Similar mechanisms operate in biological systems; for example, Lymnaea stagnalis snail shell chirality could be reversed by altering asymmetry in the blastomere ( Kuroda et al. , 2009 ). Although the general principle that macroscopic chirality originates at cellular or subcellular level is well established, a detailed molecular and biophysical understanding of these processes is still lacking for all but a handful of systems. Perhaps the best-studied example of chiral behavior in bacteria is that of E. coli , which turns clockwise while swimming near surfaces due to the chiral motion of its flagella ( DiLuzio et al. , 2005 ). To uncover the molecular mechanisms of chiral growth, we studied several microbial species and E. coli mutants lacking certain surface structures under varying environmental conditions. Our results ruled out shape and the direction of cell wall biosynthesis as the primary drivers of chiral growth. Instead, the data suggest that chirality relies on intimate contact between the substratum and the E. coli cell surface. This contact can be masked by surface structures; thus, the deletion of surface factors progressively increases E. coli chirality compared with wild type. Not only the cell surface, but also the substratum itself is important for the interaction mediating chirality. We found maximal chiral behavior at intermediate agar concentration (1–1.5%) and a significant dependence on substrate composition. Collectively, our results reveal how molecular mechanisms control spatial patterning during colony growth and suggest new ecological roles for several proteins expressed on cell surfaces.",
"discussion": "Discussion Some E. coli strains display chiral borders between competing subpopulations during colony expansion. We found that a systematic removal of extracellular constituents involved in biofilm formation, cell–cell and cell–surface adhesion increased chirality. Deletion of pili, curli fimbriae and colanic acid in E.coli MG1655 produced sector boundaries that were more chiral. Loss of two extracellular constituents (antigen 43 and pili) increased chirality further. Interestingly, E.coli MG1655 colony formation displayed relatively low chirality compared with the other K-12 strain, DH5α. This could be due to inadvertent selection in the construction of E. coli DH5α, which is known to be a poor biofilm former and likely lacks certain extracellular structures present in MG1655. These results support a view that intimate contact with the substratum is responsible for the chirality in E. coli , and the adhesive surface structures are masking it by physical shielding or by reducing cell movement during colony growth. Some of the largest surface structures on E. coli are the pili, which are thin, rigid and adhesive organelles. They are found on many E. coli strains and other members of the Enterobacteriaceae ( Klemm and Krogfelt, 1994 ) and are important for adherence to mammalian host tissues ( Krogfelt et al. , 1990 ; Connell et al. , 1996 ) and for biofilm formation ( Pratt and Kolter, 1998 ; Schembri and Klemm, 2001 ). On a piliated cell, these peritrichous surface structures can radiate to a distance of up to 2 μm from the cell surface ( Figure 3a ), making them prime candidates for a physical masking of an underlying cell envelope structure. Accordingly, we found that the deletion of piliating genes in MG1655 (MG1655 fim ) significantly increased colony chirality. Interestingly, pili expression, which is subject of phase variation, that is, individual cells can switch between a piliated and non-piliated state, is normally considered low on solid media. Nevertheless, the presence of pili clearly decreases chirality. Antigen 43, the product of the flu gene, is a self-recognizing auto-transporter protein. It confers auto-aggregation giving rise to a characteristic frizzy colony morphology on solid media as well as flocculation of cells in static liquid cultures ( Hasman et al. , 1999 ). Antigen 43 is known to be important for bacterial biofilm formation ( Danese et al. , 2000 ; Kjærgaard et al. , 2000 ; O’Toole et al. , 2000 ) and for cellular chain formation ( Vejborg and Klemm, 2009 ). Interestingly, antigen 43 mediated auto-aggregation is inhibited by the presence of pili on the cells ( Hasman et al. , 1999 ). Consistent with this finding, we observed little change of chirality in the MG1655 flu mutant, but a significant enhancement in θ of the double flu , fim mutant (MG1655 flufim ). Overexpression of pili in the MG1655 flufim also could not fully reverse the phenotype, clearly suggesting that antigen 43 also plays an important role, and both surface structures mask the underlying chirality of the cell. The deletion of curli fimbriae (MG1655 csgAB ) or colanic acid (MG1655 cps ) also made sector boundaries significantly more chiral. This increase in chirality was somewhat surprising, given that curli and colanic acid are not typically expressed in K-12 strains at 37 °C ( Olsén et al. , 1989 ). Given that the expression of many surface structures is intricately connected, we cannot exclude the possibility that the deletion of these genes could affect the expression of other surface structures. It should also be noted that MG1655 cps was independently derived and could have acquired additional genetic and phenotypic variations, which might obscure direct comparison with our version of E.coli MG1655. Flagella-dependent motility is known to play a major role in chiral spreading in other bacteria. For example, Proteus mirabilis produces chiral swarming patterns via this mechanism ( Xue et al. , 2011 ). E. coli also use flagella for active swimming, which can produce chiral motion due to hydrodynamic interactions ( DiLuzio et al. , 2005 ). In addition, flagella are involved in biofilm formation ( Pratt and Kolter, 1998 ), and they may play a structural role in colony biofilms ( Serra et al. , 2013 ). Not surprisingly, flagella have been put forward as the primary suspect for chiral behavior in bacteria. For instance, for Paenibacillus dendritiformis, it is proposed in Ben-Jacob et al. (1995) that chirality is caused by flagella and is mediated by strong cell–cell interaction. We found that chirality decreases slightly, and not statistically significantly, upon flagella removal. However, there was no link between the magnitude of chirality and flagella-mediated motility (as determined in swimming motility assays); in particular, the most chiral strains (DH5α and MG1655 flufim ) were poorly motile. Taken together, our data suggest that flagella, for these types of assays, contributes little to chirality in E. coli colonies under the studied growth conditions. The macroscopic handedness of the chiral borders between competing E. coli strains is opposite of the microscopic handedness of the growth of the peptidoglycan layer ( Wang et al. , 2012 ). Hence, although chirality is increased upon the removal of surface structures that possibly mask underlying cell wall structures, there is no direct link between the spiral growth direction of the peptidoglycan and the chirality of colony borders. This was confirmed by the same chirality direction of E. coli and B. subtilis despite the opposite directions of cell wall biosynthesis in these two species. However, spiral growth has in Myxococcus xanthus been linked to another kind of motility (gliding) than employed by E. coli . Similar to chirality in regular matter ( de Gennes and Prost, 1995 ), chirality in bacterial colonies may require parallel geometric ordering of cells during cell division and colony expansion ( Shapiro and Hsut, 1989 ; Su et al. , 2010 ), which is clearly visible at the border between competing strains ( Figure 4b ). The composition of the substratum has been shown to control cell morphogenesis ( Su et al. , 2010 ). Hence, the alignment of mother–daughter cells is probably affected by the cells’ ability to attach to the substratum and possibly to each other. Supporting the importance of the substratum for chiral growth, we also found that interchanging agar with agarose in the substratum reduced chirality. This suggests that the agar polymer network itself could be chiral or that it includes chiral entities. However, it could also be that cell–surface adhesion were modulated or that gene expression was changed. Active chiral processes play an important role in pattern formation as well as during embryonic development ( Coutelis et al. , 2014 ). While the ecological role of chirality in the microbial world is still poorly understood, chiral phenotypes could be functional or even advantageous to microbial communities as they are during embryogenisis. Indeed, one can view microbial colonies as a stepping-stone towards multi-cellularity ( Shapiro and Dworkin, 1988 ), and, therefore, related pattern forming mechanisms could be required. Consistent with these ideas, a switch of chirality has been reported in bacteria as a response to changes in the environmental conditions ( Ben-Jacob et al. , 2000 ) and during tumorigenesis ( Wan et al. , 2011 ). We found that E. coli employs a mechanisms to generate chirality distinct from higher organisms, which often rely on the asymmetric orientation of the mitotic spindle during cell division ( Grande and Patel, 2009 ) or, in the case of Caenorhabditis elegans , on chiral torque generation in the cortex ( Naganathan et al. , 2014 ). Although the microscopic mechanisms for chirality in bacteria are distinct, some conclusions of our work could generalize to other settings. In particular, we established an experimental approach to examine the role of surface structure and adhesion in chiral pattern formation."
} | 4,130 |
32069978 | PMC7074724 | pmc | 1,786 | {
"abstract": "Aerobic moderately thermophilic and thermophilic methane-oxidizing bacteria make a substantial contribution in the control of global warming through biological reduction of methane emissions and have a unique capability of utilizing methane as their sole carbon and energy source. Here, we report a novel moderately thermophilic Methylococcus -like Type Ib methanotroph recovered from an alkaline thermal spring (55.4 °C and pH 8.82) in the Ethiopian Rift Valley. The isolate, designated LS7-MC, most probably represents a novel species of a new genus in the family Methylococcaceae of the class Gammaproteobacteria . The 16S rRNA gene phylogeny indicated that strain LS7-MC is distantly related to the closest described relative, Methylococcus capsulatus (92.7% sequence identity). Growth was observed at temperatures of 30–60 °C (optimal, 51–55 °C), and the cells possessed Type I intracellular membrane (ICM). The comparison of the pmoA gene sequences showed that the strain was most closely related to M. \n capsulatus (87.8%). Soluble methane monooxygenase (sMMO) was not detected, signifying the biological oxidation process from methane to methanol by the particulate methane monooxygenase (pMMO). The other functional genes mxaF , cbbL and nifH were detected by PCR. To our knowledge, the new strain is the first isolated moderately thermophilic methanotroph from an alkaline thermal spring of the family Methylococcaceae . Furthermore, LS7-MC represents a previously unrecognized biological methane sink in thermal habitats, expanding our knowledge of its ecological role in methane cycling and aerobic methanotrophy.",
"conclusion": "5. Conclusions We have retrieved an obligate moderately thermophilic Type Ib methanotroph that belongs to the family Methylococcaceae of the class Gammaproteobacteria . This new isolate is a Methylococcus -like bacterium that contains the particulate methane monooxygenase (pMMO) but does not contain soluble methane monooxygenase (sMMO) in the methane oxidation process. Based on the physiological, biochemical and genotypic properties, strain LS7-MC most probably represents a novel genus within the family Methylococcaceae . This strain also denotes a previously unrecognized biological methane sink, diversity of methane oxidation and on the adaptation of this process to alkaline thermal habitats. Furthermore, this finding will increase our knowledge of methanotroph ecology and its involvement to global cycles of carbon and nitrogen, and the thermophilic nature of this strain possibly makes a considerable candidate for potential biotechnological applications [ 56 ]. Additional studies regarding the molecular biology, biochemistry and whole genome of LS7-MC are needed to provide insight into how biological methane oxidation processes and mechanisms are regulated in alkaline thermal ecosystems.",
"introduction": "1. Introduction Methane plays a key role in the global carbon cycle, being 34 times more powerful as a greenhouse gas than CO 2 , and is the most substantial contributor to climate effect [ 1 ]. Abiogenic methane from underground reservoirs is produced in catalytic reactions at high pressure and temperature. Especially in geothermal habitats, a mixture of methane and other gases (known as natural gas) enter the Earth’s atmosphere as a part of volcanic gases and hydrothermal solutions through seeps, degassing of spring water and gas venting. Moreover, anaerobic microbials (the presence of methanogenic archaea) in hyperthermal hot springs also contribute formation and releasing of biogenic methane into the atmosphere [ 2 , 3 , 4 ]. In some parts of Ethiopia (the Great Rift Valley regions), natural methane is released through thermal springs nearby the Rift Valley lakes. Such lakes and thermally heated water sediments from hot springs may affect the community structure and diversity of microorganisms and may have a major influence in the global carbon cycle. The study of aerobic methane-oxidizing bacteria (MOB) or methanotrophs is of special interest because of their significant ecological role in the global carbon cycle and natural reduction of methane emission to the atmosphere from many different ecosystems. Moreover, these microorganisms have the ability of utilizing methane as their sole energy and have a unique multicomponent enzyme system called methane monooxygenase (MMO), of which two distinct types, a particulate membrane-bound enzyme (pMMO) and a cytoplasmic soluble, membrane-free form (sMMO) have been described. These bacteria are found worldwide in nature and have been detected and isolated from a variety of thermal and non-thermal habitats [ 5 , 6 , 7 , 8 ]. Until now, taxonomical and molecular diversity studies of aerobic methanotrophs comprise the three phyla of Proteobacteria, Verrucomicrobia and “Methylomirabilaeota” (candidate phylum NC10). In the phylum Proteobacteria , methanotrophs are currently reclassified and defined into five distinct families: the gammaproteobacterial Methylomonadaceae (referred to as Type Ia), Methylococcaceae (Type Ib, formerly named as Type X), Methylothermaceae (Type Ic) and the alphaproteobacterial Methylocystaceae (Type IIa) and Beijerinckiaceae (Type IIb), based on their genomic comparisons (digital DNA-DNA hybridization (dDDH)), reconstruction of genome phylogeny, average nucleotide identity (ANI) and average amino acid identity [ 9 , 10 , 11 , 12 , 13 ]. Within the phylum Verrucomicrobia (sometimes referred also to Type III methanotrophs), only one family is defined as Methylacidiphilaceae that consists of, up to now, two genera: Methylacidiphilum and Methylacidimicrobium [ 14 , 15 , 16 ]. Although the majority of reported aerobic methanotrophs are mesophilic (optimal between 10 and 35 °C) and neutrophilic, their actual physiological tolerance ranges from 0 to 72 °C, pH from 1 to 11 and salinities up to 30%. In fact, several thermotolerant (growth up to 50 °C) and moderately thermophilic methanotrophs have also been described [ 9 , 13 , 17 ]. Our knowledge of truly thermophilic or moderately thermophilic proteobacterial methanotrophs (T opt > 40 °C and T max < 67 °C) is still limited compared to their thermotolerant (T max < 50 °C), mesophilic (T max < 42 °C) or psychrotolerant (T max < 36 °C) counterparts. Only a few validly described species of thermophilic methanotrophs like Methylothermus thermalis (growth at 37−67 °C), Methylothermus subterraneus (growth at 37−65 °C) within the family Methylothermaceae and Methylocaldum szegediense (growth at 37−62 °C) within the family Methylococcaceae , could grow optimally above 55 °C [ 13 , 18 , 19 ]. The family Methylococcaceae presently comprises only seven phylogenetically associated genera: Methylococcus, Methylocaldum, Methyloparacoccus, Methylogaea, Methylomagnum, Methyloterricola and Methylotetracoccus [ 13 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. In addition to these genera, the first isolated acid-tolerant moderately thermophilic (at a temperature range of 30−60 °C and at pH range 4.2−7.5) gammaproteobacterial methane oxidizers (methanotrophic isolates BFH1 and BFH2) were retrieved from a tropical topsoil with methane seeps habitat in Bangladesh, and possibly represent a novel new genus within the Type Ib methanotrophs. 16S rRNA gene phylogeny of both strains formed a cluster with the genus Methylocaldum as the closest described relative [ 26 ]. Furthermore, three novel isolates (strains GFS-K6, BRS-K6 and AK-K6) were also recovered from three different geographical regions and habitats: rice field soil, a methane seeps pond sediment from Bangladesh and a warm spring sediment from Armenia. But these microorganisms are mesophilic rather than thermotolerant and represent the members of Type Ib methanotrophs, and make, phylogenetically, a cluster together of the mesophilic genera Methylomagnum and Methyloparacoccus [ 27 ]. Methylococcus capsulatus strains, Texas and Bath, were the first reported thermotolerant methane oxidizers, growing up to 50 °C and at pH range 5.5−8.5, and were isolated from sewage sludge and geothermally heated water, respectively [ 21 , 28 ]. So far, strain M. capsulatus Bath is the most studied methanotroph and expanded knowledge of its ecophysiology, genetic and biochemistry. Bodrossy and colleagues, reported a bona fide novel thermophilic gammaproteobacterial methane-oxidizing bacterium (informally named as “Methylothermus” strain HB), which was recovered from underground hot springs in Hungary, and this bacterium represented the highest recorded growth temperature range at 40−72 °C, with an optimum at 62−65 °C, until now [ 29 ]. Sequence comparisons of both 16S rRNA and pmoA genes revealed that strain HB represents a novel genus of the Type Ic methanotrophs in the family Methylothermaceae , but unfortunately, this strain does not exist any longer [ 13 ]. Recently, the detection of a new gammaproteobacterial group of methanotrophs, distantly related to Methylococcus and Methylocaldum , in a Russian Far East thermal spring, provides new insights into the diversity and distribution of thermophilic methanotrophs [ 30 ]. Moreover, three thermoacidophilic strains, M. infernorum, M. fumiolicum and M. kamchatkense , were able to grow at temperatures of 37 to 65 °C and pH at up to 6.0. These were isolated from acidic geothermally heated soils and a hot spring, but they are members in the genus Methylacidiphilum of the phylum Verrucomicrobia . Verrucomicrobial methane oxidizers appear to be found only in acidic geothermal environments [ 14 , 15 , 16 ]. Several key functional molecular gene markers like pmoA (encoding a subunit of the particulate methane monooxygenase, pMMO: a copper-dependent enzyme), mmoX (encoding a subunit of the soluble methane monooxygenase, sMMO: an iron-dependent enzyme) and mxaF (encoding the large subunit of PQQ-dependent methanol dehydrogenase, MDH: a calcium-containing enzyme) were frequently applied for detecting and diversity analysis of C 1 -utilizing bacteria. Especially, the pmoA gene is often applied as a phylogenetic marker for identifying aerobic methanotrophs in various habitats [ 6 , 31 ]. Hitherto, no thermophilic methane oxidizers have been reported to exhibit both enzymes systems, indicating that methanotrophic cytoplasmic sMMO might not be existing in cells living above 55 °C [ 13 , 18 , 29 ]. The gene cbbL , encodes the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo: an essential enzyme in the Calvin-Benson-Bossham cycle), which is responsible for autotrophic growth. This gene has been commonly utilized for analyzing marine and hypersaline microbial communities as well as studies of Type Ib methanotrophs [ 26 , 32 , 33 , 34 ]. Recovering of moderately thermophilic methane-oxidizing bacteria from alkaline thermal springs (pH 8.0 to 9.0) is still a challenging process. Little is known about the identity, community structure and distribution of these bacteria from such ecosystems. In this study, we have isolated and characterized the first moderately thermophilic Type Ib methanotroph, recovered from an alkaline thermal spring sample from the Ethiopian Rift Valley. The new isolate likely represents a new genus within the family Methylococcaceae of the class Gammaproteobacteria and extends our knowledge of this group of microorganisms in these environments.",
"discussion": "4. Discussion Research on methane-oxidizing bacteria has mainly focused on low-temperature ecosystems. Most of the more than 50 validates species are either mesophilic or psycrophilic. Only a few thermophilic or moderately thermophilic species (8 species of proteobacterial and 3 species of verrucomicrobial methanotrophs) with optimal growth temperature between 50 and 55 °C have so far been reported [ 13 , 14 , 26 ]. In this study, enrichments for aerobic methanotrophs were established by inoculating sediment slurry from a thermal spring close to an alkaline lake (Lake Shalla) in the Ethiopian Rift Valley. From these enrichments, a novel moderately thermophilic Type Ib methanotroph, termed LS7-MC, was retrieved. This novel isolate shows an obligate aerobic methylotrophic growth with methane and methanol as the sole carbon and energy sources. According to 16S rRNA, PmoA, MxaF, CbbL and NifH sequence analyses, strain LS7-MC is most closely related to the Type Ib thermotolerant and moderately thermophilic methanotrophic bacteria within the family Methylococcaceae . Both the particulate methane monooxygenase gene pmoA and the methanol dehydrogenase gene mxaF, routinely used as functional and phylogenetic biomarkers for methanotrophic proteobacteria in natural environments [ 31 , 47 ], were detected in strain LS7-MC, indicating that methanotrophic proteobacteria are more widespread in thermal environments than previously thought. Furthermore, the difference in 16S rRNA gene sequences between strain LS7-MC and other related validated Type Ib methanotrophic genera ranges between 7% and 10%. The lack of soluble methane monooxygenase gene mmoX implies another significant difference between the thermotolerant genus Methylococcus and the strain LS7-MC presented here. These results ascertain that strain LS7-MC is, most probably, not a new species or subspecies of the genus Methylococcus or other genera of the families Candidate Methylomonadaceae , Methylococcaceae or Methylothermaceae . The 16S rRNA gene analyses suggested that this strain most probably represents a new genus in the methane-oxidizing bacterial family Methylococcaceae of the class Gammaproteobacteria. Thermotolerant and moderately thermophilic proteobacterial methanotrophs have been found in various ecosystems from thermal spring sediments, subsurface hot aquifer, tropical landfill wetlands, methane seeps topsoil, compost and marine sediments [ 17 , 18 , 26 , 42 ]. The existence of these bacteria was supported by phylogenetic analyses of genes (like 16S rRNA, pmoA, mxaF and mmoX ), through cultivation efforts and bacterial community analyses (using DNA-SIP, metagenomics, metatranscriptomics and next-generation sequencing) [ 48 , 49 , 50 , 51 ]. The comparative analysis of the 16S rRNA gene showed a relatively high sequence identity (>98%) to a clone of subsurface water sample (average pH 7.88) from an Austrian radioactive thermal spring [ 44 ]. The high sequence similarity of this clone to strain LS7-MC may indicate common physiology and metabolism properties between the strain LS7-MC and the uncultured thermal spring bacterium. Moderately thermophilic methanotrophs related to strain LS7-MC could be present in the central Austrian radioactive thermal spring and possibly in other related habitations of radioactive geothermal, and pmoA gene sequences obtained from the same thermal spring showed 93.8% sequence identity to pmoA of the strain LS7-MC. This is an indication that moderately thermophilic gammaproteobacterial methanotrophs may play a significant role in the carbon cycle in such subsurface thermal spring ecosystems. A relatively lower percentage identity of 16S rRNA, pmoA and mxaF sequences have also been detected in coal mine, diverse soils, wetland and landfill cover soils as well as hot spring water [ 19 , 52 , 53 , 54 ], suggesting that Methylococcus -like mesophilic or moderately thermophilic methanotrophs might be found in these various environments. Strain LS7-MC required vitamins and copper for consistent growth and was negative with the naphthalene-oxidation assay. The lack of sMMO was also verified using the Southern blotting technique and PCR amplification. These central observations have also been described in other thermophilic proteobacterial isolates such as ‘ Methylothermus’ strain HB [ 29 ], Methylothermus spp. [ 18 , 48 ] and thermophilic Methylocaldum spp. [ 17 ], as well as the verrucomicrobial M. kamchatkense Kam1 [ 36 ], which suggests that genes encoding soluble methane monooxygenase most probably do not exist in moderately thermophilic or thermophilic proteobacterial methanotrophs or verrucomicrobial thermoacidophilic methanotrophs [ 14 ]. On the other hand, within Type Ib methane-oxidizers, thermotolerants such as M. capsulatus and M. marinum, and mesophilic M. ishizawai (growth range 4–37 °C) possess both sMMO and pMMO enzyme systems [ 13 , 42 , 55 ]."
} | 4,098 |
38444935 | PMC10911227 | pmc | 1,788 | {
"abstract": "Artificial synapses based on resistive switching have emerged as a promising avenue for brain-inspired computing. Hybrid metal halide perovskites have provided the opportunity to simplify resistive switching device architectures due to their mixed electronic–ionic conduction, yet the instabilities under operating conditions compromise their reliability. We demonstrate reliable resistive switching and synaptic behaviour in layered benzylammonium (BzA) based halide perovskites of (BzA) 2 PbX 4 composition (X = Br, I), showing a transformation of the resistive switching from digital to analog with the change of the halide anion. While (BzA) 2 PbI 4 devices demonstrate gradual set and reset processes with reduced power consumption, the (BzA) 2 PbBr 4 system features a more abrupt switching behaviour. Moreover, the iodide-based system displays excellent retention and endurance, whereas bromide-based devices achieve a superior on/off ratio. The underlying mechanism is attributed to the migration of halide ions and the formation of halide vacancy conductive filaments. As a result, the corresponding devices emulate synaptic characteristics, demonstrating the potential for neuromorphic computing. Such resistive switching and synaptic behaviour highlight (BzA) 2 PbX 4 perovskites as promising candidates for non-volatile memory and neuromorphic computing.",
"conclusion": "Conclusions We investigated the resistive switching of benzylammonium (BzA) based (BzA) 2 PbX 4 layered RP lead halide perovskites, showcasing their potential for non-volatile memory and neuromorphic computing applications. The study revealed a shift from digital to analog resistive switching through halide substitution, offering versatile functionality and reduced power consumption. The underlying mechanism was primarily attributed to halide ion migration and halide vacancy conductive filament formation. Both the bromide- and iodide-based systems exhibited high performances, with the former excelling in achieving a superior on/off ratio and the latter displaying excellent retention and endurance. The (BzA) 2 PbBr 4 established reliable random-access data storage and retrieval capabilities through write-read-erase-read (WRER) pulses, complemented by consistent switching voltages for uniform performance in practical random-access memory applications. In addition, (BzA) 2 PbI 4 demonstrated gradual (analog) set and reset processes, enhancing energy efficiency and emulating critical synaptic characteristics, such as potentiation, depression, and paired-pulse facilitation, showcasing its potential for neuromorphic computing. These resistive switching properties and synaptic behaviour position (BzA) 2 PbX 4 layered hybrid halide perovskites as compelling candidates for advanced non-volatile memory and neuromorphic computing. This further contributes to the development and understanding of hybrid halide perovskites in the realm of emerging memory technologies.",
"introduction": "Introduction Resistive switching has garnered significant attention in non-volatile memory technology due to its rapid switching speed, remarkable endurance, exceptional density, and minimal power consumption. 1 The principle of resistive switching is associated with resistive random-access memory (RRAM) operation, characterized by an abrupt variation in resistance. 2 These systems can be classified into digital or analog, depending on whether the resistive switching effect is abrupt or gradual. While digital memristors are primarily studied for non-volatile memory applications, analog ones hold significant potential in the field of neuromorphic computing. 2 To this end, two-terminal devices with a vertical metal–insulator–metal structure have provided a simplified design for artificial synapses owing to their capability to mimic the transmission properties of biological synapses. 3 Such devices play a pivotal role as fundamental functional units in neuromorphic computing, 4 where electrical stimuli must transfer from a pre-synaptic to a post-synaptic neuron for an artificial synapse to operate. In a ‘memristor’, a resistive switching memory element that can mimic the operation of a biological system, one metal electrode serves as a pre-neuron, another as a post-neuron, and the insulating layer acts as the synapse. 5,6 A wide range of materials have demonstrated the ability to exhibit resistive switching, including polymers, 7 organic materials, 8 transition metal dichalcogenides, 9 binary oxides, 10 and perovskites. 11 While oxides are the most extensively studied materials for resistive memories, their efficacy is impeded by the difficult fabrication, high-power consumption, and lack of precise control over filament formation that is associated with memristive functions, which compromises their performance. 12,13 In particular, the necessity of high-temperature processing and vacuum conditions limits the application of perovskite oxide materials in RRAM. 14 Alternatively, hybrid halide perovskite materials have emerged as promising contenders in the realm of optoelectronic devices, notably in applications such as photovoltaics, 15 light-emitting diodes, 16 lasing, 17 and photodetectors. 18,19 Due to their mixed ionic–electronic conduction, 20,21 low leakage currents, and tuneable band gap, hybrid metal halide perovskites have extended their utility to memory devices, 13,22,23 offering the possibility of integrating the benefits of both inorganic and organic resistive switching. However, the instability of halide perovskites under operating conditions poses a challenge for their application. 21,24 Toward overcoming the instability of metal halide perovskites, layered (2D) hybrid perovskites incorporating larger organic moieties have shown potential for stabilizing perovskite materials and their devices toward more reliable memory elements. 25,26 These materials are often defined by the general formulation of S 2 A n +1 MX 4 , where S and A are ( i.e. , alkyl or arylammonium) spacers and central ( e.g. , methylammonium (MA), formamidinium (FA), etc. ) organic cations, respectively, M is a divalent metal ion (mostly Pb 2+ ) and X halide anion (I − , Br − , or Cl − ). 22,25,26 The value of n represents the number of {MX 6 } corner-sharing octahedra between organic spacer (S) layers. While these systems present a diverse class of materials defined by different compositional elements and number of layers ( n ), they are mostly associated with Ruddlesden–Popper (RP) phases formed by van der Waals interactions between spacer cations with a half-a-unit-cell displacement between the adjacent slabs ( Fig. 1 ). 26,27 As a result, the 2D perovskite structure exhibits anisotropic charge transport, particularly in the out-of-plane direction, and this unique characteristic holds promise for designing resistive memories with low programming currents. 28 However, the application of 2D perovskites in resistive switching memories has so far been limited, primarily relying on archetypical n -butylammonium (BA) and 2-phenylethylammonium (PEA) based materials. 22,28 For instance, it was shown that the reduction of dimensionality from 3D to 2D induced the resistive switching properties of BA/MA-based RP phases with an improvement in the endurance and on/off ratios. The 2D structure was also found to be advantageous for the formation of a conductive filament, primarily due to the anisotropic migration of defects. 28 Similarly, 2D RP (PEA) 2 PbBr 4 perovskite single crystals exhibited resistive switching behaviour with significantly reduced currents of 10 pA, attributed to the mixed transport. 22 A recent study also reported an exceptionally high on/off ratio of 10 9 using a quasi-2D perovskite based on (PEA) 2 Cs 3 Pb 4 I 13 composition, higher than that of the 3D CsPbI 3 , which was attributed to the wider bandgap and thus an increased Schottky barrier. 29 The composition of halide perovskite has also been found to impact its switching properties in 3D CH 3 NH 3 PbX 3 (X = I, Br), where the set voltage was found to decrease when the iodide is replaced by bromide due to the lower barrier for Br − migration compared to I − ions. 30 This is unexploited in 2D perovskites, which still remain underrepresented in resistive switching devices despite their potential. Fig. 1 Layered hybrid perovskite resistive switching device. Schematic of a resistive switching device architecture (left) based on Ruddlesden–Popper (RP) layered perovskite phase as active material (right) comprising benzylammonium (BzA) cations between aluminium (Al) contacts and fluorine-doped tin oxide (FTO) conductive glass used in this study. Here, we report the resistive switching behaviour of layered benzylammonium (BzA) based hybrid halide perovskite of (BzA) 2 PbX 4 (X = Br, I) composition. The memory devices based on a simple Al/(BzA) 2 PbX 4 /fluorine-doped tin oxide (FTO) architecture exhibited bipolar resistive switching behaviour with low switching voltage, good data retention, and excellent endurance. Moreover, we observed an unusual transformation from digital to analog resistive switching upon the substitution of Br with I ions. A model is proposed in which a conductive filament is formed by halide vacancies to explain the underlying mechanism. 31 Finally, voltage pulses were applied to the (BzA) 2 PbI 4 device to validate its capability to emulate a synapse and function as an artificial synapse. 32 The observed gradual set and reset behaviour of the device signifies its potential for neuromorphic applications, where the ability to tune conductance dynamically is highly desirable. The artificial synapse effectively emulated key synaptic characteristics, such as potentiation, depression, and paired-pulse facilitation. 5 This study thereby reveals unique resistive switching characteristics for layered BzA-based hybrid halide perovskites, providing a better understanding of their memristive effects and revealing the potential for neuromorphic computing in the future.",
"discussion": "Results and discussion The resistive switching properties of (BzA) 2 PbX 4 (X = I, Br) perovskites were studied in a metal–insulator–metal device based on a thin film spin-coated on the FTO substrate and top circular Al electrode contacts ( Fig. 1 ). The experimental details are provided in the Experimental Section of the Electronic Supporting Information (ESI, † Fig. S1–S5). Layered (2D) perovskite materials were prepared mechanosynthetically by ball-milling, followed by solution-based spin-coating and subsequent annealing. The formation of a 2D perovskite was evidenced by X-ray diffraction, UV-vis absorption, and photoluminescence spectroscopy (Fig. S1, ESI † ). 27 Resistive switching characteristics of the Al/(BzA) 2 PbX 4 /FTO devices reveal a distinct bipolar switching ( Fig. 2a and b ). Digital switching is observed in (BzA) 2 PbBr 4 upon application of voltage in the range between +2 V and −4 V ( Fig. 2a ). 33 Upon sweeping the voltage from 0 to 2 V, an abrupt transition from a high (HRS) to a low resistance state (LRS) is observed at +1.04 V, indicating the set process. The LRS persisted until a negative bias voltage was applied, after which the state reverted back to HRS at −1.33 V, signifying the reset process. In contrast, Al/(BzA) 2 PbI 4 /FTO showed a more gradual set and reset ( Fig. 2b ). The set and reset voltages of +0.5 V and −1 V for (BzA) 2 PbI 4 were also smaller, and the device featured lower power consumption, with the estimated set and reset powers of 0.63 mW (at 0.5 V and 1.26 mA) and 12.25 mW (at −1 V and 12.2 mA), respectively. In addition, current–voltage characteristics exhibited a negative differential resistance (NDR), where the current decreased as the voltage increased after reset, which is relevant for achieving diverse resistance states in neuromorphic computing applications. 34 Fig. 2 Resistive switching in bromide (left) and iodide (right) based layered hybrid perovskites. Current–voltage characteristics for bipolar resistive (a) digital switching in (BzA) 2 PbBr 4 and (b) analog switching in (BzA) 2 PbI 4 devices at the voltage scan rate of 20 mV and 8 ms time sweep. More information about the frequency and contact dependence is detailed in ESI † (Fig. S3 and S4). To calculate the set voltage for the I-based system, the current–voltage curve was replotted on a linear scale to visualise the point where the current starts increasing (Fig. S5, ESI † ). (c) and (d) Retention time and (e) and (f) endurance over repeated cycles for resistive switching devices at 0.2 V bias over (e) 125 cycles in (BzA) 2 PbBr 4 -based system and (f) 500 cycles in (BzA) 2 PbI 4 -based system. Current in HRS (black) and LRS (red) was read at +0.2 V for the endurance and retention tests. To assess the nonvolatility and reliability of each state ( i.e. , resistance level) in Al/(BzA) 2 PbX 4 /FTO devices, retention time and endurance characteristics were monitored ( Fig. 2c–f ). The iodide-based (X = I) system exhibited better retention and endurance than the bromide-based (X = Br) system by showing a stable switching for more than 500 cycles with a retention time of 1.5 × 10 4 s ( Fig. 2d and f ), whereas the bromide-based system showed a superior on/off ratio of >10 2 as compared to iodide system ( Fig. 2c and e ). These characteristics highlight the non-volatile potential of the devices that can be further optimised in terms of morphology, thickness, and device architecture beyond the scope of this study, such as by tailoring contact layers and using interlayers to reach longer retention time and better on/off ratios for practical applications. To ensure that the device can reliably store and retrieve data, write-read-erase-read (WRER) pulses were applied ( Fig. 3a and Fig. S6, ESI † ). A pulse voltage of 2 V was first applied for a duration of 4 s to write data into the (BzA) 2 PbBr 4 device ( Fig. 3a ). This voltage pulse causes the resistive state of the device to change and store the data. After the writing process, a read voltage of 0.2 V was applied for 6 s to verify the data stored. Finally, an erase voltage of −4 V was applied for 4 s, causing the resistive state to change and erase the data. Once the erase process was complete, a pulse voltage of 0.2 V was used for 6 s to read the erased state ( Fig. 3b ). A reliable switching between these states was observed for 100 WRER cycles. Moreover, a comparable procedure was applied to the (BzA) 2 PbI 4 system, with an erase pulse of −3 V instead of −4 V (Fig. S6, ESI † ), evidencing the utility of the system for storing and retrieving data. Fig. 3 Bromide-based perovskite memory data storage and retrieval. (a) Write-read-erase-read (WRER) cycles. (b) Enlarged view of 1st WRER cycle indicating write (W), read (R), and erase (E) states with the corresponding voltage bias. (c) Cumulative probability for set and reset voltages based on 125 cycles. (d) Statistical distribution of set and reset voltages with Gaussian fitted curve. WRER cycles for the iodide-based system are detailed in Fig. S6 of the ESI. † The practicality of WRER cycles for RRAM applications was further assessed for their consistency and reliability by analysing the uniformity in switching voltages. For this purpose, cumulative probabilities of the set and reset voltages of the (BzA) 2 PbBr 4 device were calculated using data obtained from 125 switching cycles ( Fig. 3c and d ). The statistical distribution of set and reset voltages indicated the voltage range where the switching occurs ( Fig. 3c ). The set voltage range was found to be between 0.69 V to 1.98 V with an average of 1.04 V, while the reset voltage was distributed between −0.81 V and −2.38 V with an average of −1.33 V in (BzA) 2 PbBr 4 ( Fig. 3d ). This voltage profile permits reliable and consistent switching in RRAM applications. The gradual switching behaviour in (BzA) 2 PbI 4 devices, however, suggests a distinct operation mechanism that is more relevant in the design of artificial synapses ( Fig. 4 ). Fig. 4 Resistive switching mechanism in layered hybrid perovskites. Schematic of (a) set (LRS) and (b) reset (HRS) switching governed by ion migration. Illustration of a (c) biological snapse and (d) (BzA) 2 PbI 4 -based artificial synapse mimicking the biological function. The differences in the resistance switching mechanism of (BzA) 2 PbX 4 devices can be governed by the halide ion (X − ) migration dynamics and the subsequent halide vacancy (V X ) conductive filament formation under the influence of the applied electric field ( Fig. 4a and b ). 22,23 Specifically, when a positive voltage is applied, negatively charged halide ions migrate toward the top electrode. Such a migration of ions leads to the formation of halide vacancies, which are gradually accumulating and contributing to the formation of a halide vacancy conductive filament in the LRS ( Fig. 4a ). When a negative voltage is applied to the top electrode, halide ions migrate in the opposite direction, causing the ‘rupture’ of the filament, switching the device to the HRS ( Fig. 4b ). This halide ion dynamics can be related to the distinct digital and analog switching mechanisms, as analog switching can be associated with the gradual migration of iodide ions, whereas digital switching corresponds to the faster migration of bromide ions. 30 With lower activation energy, bromide ions exhibit faster migration, whereas higher activation energy of iodide vacancies hinders rapid migration, 30 leading to digital switching in (BzA) 2 PbBr 4 and a more gradual movement of iodide ions and, consequently, analog switching behaviour in (BzA) 2 PbI 4 . To gain a better understanding of the conduction mechanism, current–voltage characteristics were assessed for both bromide- and iodide-based devices ( Fig. 5 ). In their HRS, the analysis of the double logarithmic scale discerns the presence of three distinctive conductive regimes (I–III). First, at low voltages, the curve exhibits a linear region with a slope close to unity, suggesting Ohmic conduction, which is dominated by thermally generated free charge carriers (I). Next, as the voltage increases, the deviations from linearity become apparent, marking the onset of space charge-limited conduction (SCLC). As the applied bias voltage is raised from 0.15 V to 0.46 V, the injected electrons dominate the conduction mechanism, resulting in a trap-unfilled SCLC where the current is closely proportional to the square of the bias voltage (II). In this case, the conduction may be expressed by the eqn (1) : 35 1 where ε , μ eff , and d are dielectric constant, effective drift mobility, and thickness of the film, respectively. Finally, in the high voltage regime with a slope above 4, the presence of inherent defects leads to the trapping of injected electrons, resulting in a trap-filled SCLC (III). Upon complete trap filling by injected electrons, a transition to a low resistance state (LRS) occurred, accompanied by the emergence of ohmic behaviour. These changes in the conduction regimes suggest that the switching mechanism is likely to be associated with the halide ion and vacancy migration. The absence of switching upon the use of an alternative, more reactive (Ag) contact electrode corroborates this (Fig. S4, ESI † ), excluding metallic filament formation. The use of ion-blocking interlayers (such as polymers) could affect this response. 36 The dynamics of these processes could also determine prospective synaptic switching. Fig. 5 Conduction regimes in layered hybrid perovskite devices. (a) and (b) Linearly fitted current–voltage curves for LRS (top) and HRS (bottom) on a double logarithmic scale for a better understanding of the conduction mechanism related to the resistive switching in (a) bromide- and (b) iodide-based perovskite devices. The changes in slopes correspond to different conduction regimes. The synaptic behaviour is often described by potentiation ( i.e. , increase in synaptic weight through a gradual current increase upon biasing) and depression ( i.e. , decrease in synaptic weight through gradual current decrease upon biasing), which represent the changes in the strength of the artificial synapse that mimics biological systems. 5 Analog resistive switching behaviour observed in the current–voltage sweeps of (BzA) 2 PbI 4 system suggested that it could exhibit potentiation and depression, which was assessed through pulse biases of +0.5 V and −1 V, respectively. Upon application of 20 positive pulses of +0.5 V, the current level increased gradually from around 0.74 to 1.1 mA, resembling synaptic potentiation ( Fig. 6a and c ). Subsequently, the application of 20 negative pulses of −1 V caused a gradual decrease in the current level from about 10.2 to 4.9 mA, mimicking synaptic depression ( Fig. 6b and d ). Accordingly, positive and negative pulses induce a weight change that leads to potentiation and depression. With positive pulses, iodide ions are expected to move towards the top electrode ( Fig. 4 ), initiating the formation of filaments due to iodide vacancies, which would gradually enhance the conductance associated with potentiation. Similarly, negative pulses would induce the retrograde movement of ions towards the iodide vacancies, restoring the conductance to the original (lower) current state in a depression. 22 Fig. 6 Synaptic behaviour in the iodide-based layered hybrid perovskite. (a) Gradual increase in current on applying consecutive 20 positive pulses of 379 ms width and 0.5 V height. Additional scans confirming the reliability are shown in the ESI † (Fig. S7). (b) A gradual decrease in current on the application of 20 negative pulses of 379 ms width and −1 V height. (c) Potentiation and (d) depression, mimicking the increase and decrease in synaptic weight, respectively. Additional information about the PPF effect is provided in Fig. S8 of the ESI. † The synaptic behaviour is relevant to neural signalling, which is commonly described by the paired-pulse facilitation (PPF), a synaptic plasticity phenomenon where the response of a neuron to a pair of stimuli is enhanced when the second stimulus is delivered shortly after the first one. 37 This is an important mechanism for information processing and memory in the brain. Additional scans provide evidence of the PPF effect in (BzA) 2 PbI 4 devices (Fig. S7 and S8, ESI † ). The enhancement in the synaptic response can be related to the migration of iodide ions after the first stimulus, facilitating conduction in response to the second stimulus. Such emulation of key synaptic characteristics in the iodide-containing perovskite artificial synapse provides a proof-of-concept for its viability in neuromorphic systems that can be further explored in the future."
} | 5,709 |
39471225 | PMC11551444 | pmc | 1,789 | {
"abstract": "Significance Coral reefs are exceptional ecosystems and support hundreds of millions of people around the world, yet they are under severe threat due to ocean warming and acidification. Reefs are predicted to collapse over the next few decades under these climate change stressors, with grave consequences for society. Contrary to predictions of near total destruction, this study shows that with effective climate change mitigation, coral reefs will continue to change, but global reef collapse may still be avoidable.",
"conclusion": "Conclusions Perhaps the most important question that emerges from this work is why these results contrast with many earlier projections about reef futures under global change. We believe that there are three especially important factors which help to explain these discrepancies. First, previous studies have focused on a small subset of the natural diversity of species-specific and genotype-specific coral responses to global change with half of published research focused on just three coral species ( Acropora millepora , Pocillopora damicornis , and Stylophora pistillata ) ( 50 ). In line with many projections, the Pocillopora species in this study ( P. meandrina and P. acuta ) suffered greatest mortality under heating. In contrast, many other coral species, and particularly some individual genotypes within these species, showed less severe responses under warmer and more acidic conditions than has been projected. Second, these communities exhibited reduced calcification due to both warming and acidification, but did not transition to net carbonate dissolution. Projections of future reef decalcification rely on both the chemical effects of acidification (which we can confirm) as well as the predicted near total loss of corals, which did not occur here. Instead, corals and some other calcifiers exhibited greater persistence under future ocean conditions than has often been assumed, reflecting the natural diversity in their responses. In particular, CCA have have been shown to acclimatize to ocean acidification over the course of months ( 56 , 57 ) and these taxa exhibited their highest performance under the combined future ocean treatment in this experiment. Persistence of these calcifying taxa in the mesocosms allowed these communities to maintain net calcification. Third, many projections are based on single factor studies of warming or acidification showing negative impacts on corals. While combinatorial experiments remain relatively less common, it has often been assumed that most reef organisms will exhibit similarly negative outcomes under warming and acidification as compared to corals. However, very few other reef organisms have ever been investigated for their responses under future ocean conditions. Indeed, many reef organisms are yet to be formally described or named, much less investigated for their responses to global change ( 32 ). While coral species richness did decline under ocean warming and was unresponsive to acidification, most other functional groups exhibited fundamentally different responses to each factor as compared to the corals. These discrepancies may reflect our lack of knowledge about most of these species, but also likely reflects a failure to account for the complexity of species interactions within biologically diverse communities. These reef communities persisted when exposed to chronic warming and acidification for two years, yet they were fundamentally transformed and, in several ways, they were diminished. Our results emphasize the critical importance of mitigating both climate change and the intensity of local stressors. Without effective climate change mitigation, reefs will become increasingly degraded, yet it seems likely that coral reefs will face severe environmental stress, even under the best-case climate change scenario. Without effective local management, corals will be unable to recolonize damaged reefs ( 18 , 58 ). The treatments we imposed here are on par with a business-as-usual climate change scenario of +2 °C warming above present-day (about +3 °C above the preindustrial) and 0.2 pH units below present-day (about 0.3 below the preindustrial). If the world achieves Paris Climate Agreement targets of limiting climate change to no more than 2 °C above the preindustrial, then most reefs will rarely. if ever, experience the intensity of heat and acidification stress that we imposed here ( 59 ). Overall, our results imply that with effective climate change and local stressor mitigation, reef communities will continue to change, but global collapse of coral reefs may still be avoidable.",
"discussion": "Discussion We created biologically diverse mesocosms with predicted ocean warming (+2 °C) and acidification (−0.2 pH units) supplied with unfiltered seawater from the adjacent reef to examine the coral reef communities that developed over the course of two years. The IPCC Climate Change 2023 Report concludes with very high confidence that coral reefs will decline by >99% under these future ocean conditions ( 27 ), but we observed comparatively less severe responses than have been projected. Rather than collapsing into extremely low coral cover, net carbonate dissolution, and markedly reduced biodiversity under future ocean conditions, these communities instead transitioned into novel calcifying reef systems with diminished yet substantial coral cover and maintained high biodiversity. Reef communities that developed in each of warming, acidification, and the future ocean combination of both factors, showed substantial changes in community structure and composition relative to the community that developed under present-day conditions. Reefs of the future will certainly be different from those of today ( 34 , 35 ) Coral-specific Responses. Corals in the treatments with elevated temperature (both ocean warming and combined future ocean treatments) were exposed to severe ( 3 ) heat stress in successive years. These corals experienced temperatures at or above the nominal bleaching threshold for the Main Hawaiian Islands for 3.5 mo per year, during which they accumulated 24 degree heating weeks (DHW) annually ( Figs. 1 and 2 and SI Appendix , Figs. S1, S4, and S5 ). Many studies predict that this level of repeated annual bleaching stress should have been sufficient to nearly extirpate corals in our elevated temperature mesocosms ( 1 , 3 , 7 , 8 ) and that acidification should have exacerbated the heat stress ( 3 ). The accumulation of 15 to 22 DHW on the Great Barrier Reef, in the Florida reef tract, and in many other locations worldwide during the unprecedented 2023–2024 marine heatwave has resulted in devastating consequences for some corals and other organisms. Likewise, many of the corals that bleached severely in this study subsequently died ( Fig. 1 and SI Appendix , Fig. S4 and Table S1 ). However, contrary to projections of near total mortality ( 7 , 8 ), coral survivorship was reduced by an average of only 35% in the heated treatments compared to the present-day temperature treatments, and with no evidence that acidification affected survivorship ( Fig. 1 and SI Appendix , Fig. S4 and Table S1 ). The effects of heating on survivorship, however, differed dramatically among coral species. While P. meandrina suffered catastrophic mortality (97 to 100%) in the heated treatments, P. evermanni exhibited high survivorship (92 to 95%) regardless of heat stress. The remaining six species exhibited intermediate levels of survivorship with a general tendency toward higher survivorship among Porites spp., intermediate survivorship among Montipora spp., and lower survivorship among Pocillopora spp. Indeed, while some individual corals in this study bleached and died following the first heat stress event, others bleached annually yet survived to the end, and still others never bleached at all. Despite severe annual heat stress in the heated treatments, live coral cover increased substantially over the course of the experiment in all four treatments, from about 3% at the beginning to about 40% in the control and ocean acidification treatments, and about 21% in the ocean warming and combined future ocean treatments by the end of the study ( Fig. 1 and SI Appendix , Fig. S1 and Table S2 ). Hence, the rate of increase was reduced by about half in the heated treatments (ocean warming and combined future ocean) as compared to the nonheated treatments (control and ocean acidification). This decrease resulted from lower coral survivorship, partial mortality, and reduced growth rates among many of the survivors under heating. Similar to the survivorship responses, acidification had no significant effect on live coral cover. After nearly two years of exposure under treatment conditions, we examined the physiological performance of a subset of the corals. This subset was chosen based on logistical constraints about how many coral ramets could possibly be measured within a reasonable timeframe and we focused on the three most common species ( M. capitata , P. compressa , and P. lobata ). Among the survivors, M. capitata exhibited reduced carbon budgets under heating, indicative of stress, whereas P. compressa experienced enhanced photosynthesis under acidification, while P. lobata did not differ physiologically among treatments ( 36 ). Part of these physiological responses appear to be related to the coral-associated microbiome ( 33 ). Surviving M. capitata genets had a distinct, seemingly advantageous, microbial community composition compared to genets that died. In contrast, the microbial community composition associated with each of P. compressa and P. lobata shifted in response to the treatments. These results suggest that some of the corals (but not necessarily others) experienced a degree of physiological and microbial acclimatization to the treatments over the course of the study. Indeed, coral species exhibit substantial variability in their capacity to acclimatize to heat stress ( 37 ). After the conclusion of the mesocosm experiment, we tested for the possible effects of temperature preconditioning on future performance within two of the coral species included in this study ( M. capitata and M. flabellata ) ( 38 ). Replicate coral ramets that were maintained under ocean warming conditions for more than two years were compared to those maintained under present-day conditions throughout, and their performance was assessed before and during a natural coral bleaching event in an open ocean nursery. Over four years of consecutive heat stress events, we found no evidence that preconditioning provided either long-term temperature acclimatization and resistance or sensitization to future bleaching for either species, because responses were indistinguishable based on temperature history during subsequent transplantation to a common garden. Differences in growth rate and survival in each species were driven by individual genotype of the corals, rather than preconditioning to thermal stress ( 38 ). These results were confirmed by a recent field survey wherein individually tagged colonies of M. capitata exhibited no long-term change in thermal tolerance over multiple, natural bleaching events ( 39 ). In contrast, P. compressa , which we included in the mesocosm study but were unable to include in the field trial, does exhibit increased thermal tolerance over the course of years under repeated marine heatwaves ( 39 ). Thus, acclimatization is unlikely to increase long-term thermal tolerances for all corals and adaptation via natural selection must play an important role in future responses for some ( 30 ). Large numbers of one coral species ( P. acuta ) recruited into the mesocosms due to spawning of the adult corals housed within them ( 20 , 40 ), but recruitment rate was unaffected by any of the treatments ( 40 ). Recruitment only occurred in mesocosms which contained live adults of the species and the lack of treatment effects on recruitment might be related to the weedy life history strategy exhibited by this coral. Indeed, P. acuta is effective at rapid colonization and can often persist in marginal habitats, despite the fact that it was one of the most thermally sensitive species examined here. We also observed widespread spawning of one of the competitively dominant coral species ( M. capitata ) across all four treatments (CPJ pers. obs. in June 2018). Hence, at least some of these corals were reproductive under future ocean levels of warming and acidification. Unfortunately, it was infeasible to assess reproductive output or gamete quality at the time of release. Early life stages are often especially vulnerable to environmental stressors, such as heating and acidification. If the larvae and recruits of these coral species exhibit more severe responses to warming and acidification than do the adults, then our results may underestimate the coral decline which should be expected later this century. While our observations of recruitment are limited to one species in this study, if coral individuals that are resistant to these conditions (like some of those in this study) proliferate in the future, then they could help to reduce the decline in coral abundance predicted for coral reefs under future ocean warming and acidification. Indeed, coral communities in Hawai’i already appear to be mounting adaptive responses to climate change with bleaching and mortality occurring at higher temperatures and after longer exposures than reported 50 y ago ( 18 , 41 ), and all eight of the coral species examined here exhibit clear scope to adapt to ocean warming, ocean acidification, and the combination of both factors ( 30 , 42 ). Community Calcification. The calcification rates measured in the control mesocosm communities were very similar to those measured on the nearby reefs ( 43 ), suggesting that the mesocosms adequately replicated the processes involved in community calcification. Net calcification of the mesocosm communities (sometimes referred to as net community calcification, NCC, or net ecosystem calcification, NEC, in other studies) declined in all treatments relative to the control, with the largest decline under the combined future ocean scenario ( Fig. 2 and SI Appendix , Table S2 ). The 19 to 24% reduction in mesocosm calcification attributable to acidification is lower but roughly similar to the 30% reduction measured on an experimentally acidified reef flat ( 44 , 45 ), further illustrating the efficacy of our approach to simulate the natural system. Nevertheless, all communities continued calcifying. Even under the combined future ocean treatment, reef community calcification was positive, albeit at only 56% the rate of control reef communities. At present-day rates of calcification, however, few reefs are expected to accrete fast enough to be able to keep up with sea level change and many future reefs may become submerged as the oceans rise ( 46 ). Indeed, even under the scenario we present here, many reefs are likely to be drowned or move shoreward given the rising ocean ( 34 , 35 , 46 ) Acidification does not by itself kill corals but rather tends to inhibit their skeletal growth by an average of 15 to 20% ( 11 ), which may compromise their competitive abilities in nature ( 12 , 13 , 16 ). Acidification, however, had no effect on net coral community calcification within this study ( Fig. 2 and SI Appendix , Table S2 ). While these results differ from many prior laboratory experiments, both ex situ and in situ studies have found that some corals can maintain normal calcification rates under lower pH ( 13 , 14 , 18 , 47 – 49 ). Further, irradiance and water flow are both known to affect coral responses to acidification ( 21 ). We conducted this experiment using natural sunlight (attenuated by shade cloth to ambient levels at mean collection depth of 2 m), rapid turnover (1 h) with unfiltered natural seawater, and additional water circulation provided by seawater pumps (10 to 15 cm s −1 ) to replicate light and flow conditions on the natural reefs as closely as possible ( SI Appendix , Figs. S1 and S2 ). These more natural conditions may help to explain the observed insensitivity of coral calcification to low pH relative to many previous laboratory studies, which rarely replicate natural reef conditions ( 50 ). In addition, corals may show threshold responses to acidification such that they are able to maintain calcification rates under a 0.2 pH unit reduction yet experience reduced calcification rates at higher levels of acidification ( 18 , 20 , 30 ). Unlike acidification, elevated temperature reduced coral community calcification by nearly half due to bleaching, mortality, and reduced growth among the survivors ( Fig. 2 and SI Appendix , Table S2 ). In contrast, net calcification by rubble-associated communities declined under ocean acidification conditions yet was insensitive to warming ( Fig. 2 and SI Appendix , Table S2 ). These results suggest that the measured reductions in calcification for mesocosm communities ( 24 , 25 , 51 ) and natural communities ( 9 , 44 , 52 ) due to acidification are driven largely by processes occurring within the reef framework rather than by the corals themselves. This may result in the reef being more brittle and more vulnerable to storm damage. The calcification budget of the mesocosms exceeded that explained by the corals and rubble, and this additional carbonate production was likely from the growth of crustose coralline algae (CCA) and other organisms which formed thick, calcified crusts on the mesocosm walls. These crusts were slowly eroded and regrown over time in a continuous cycle over the course of the experiment. Future reefs will undoubtedly experience a major decline in growth due to the loss of corals from heat stress, and reduced calcification of the reef framework under acidification. Nonetheless, these communities continued calcifying at about half of present-day rates under potential future ocean conditions. The maintenance of live coral cover along with the growth of CCA and other calcifiers within all mesocosms helped to support net calcification by these communities. Community Structure and Species Richness. Corals are ecosystem engineers, yet coral reef biodiversity is derived largely from the array of algae, invertebrates, and microbes which live within, among, and upon the reefs. Coral reefs occupy less than 0.2% of the seafloor but are home to an estimated 32 to 38% of all marine species ( 53 ). Yet the vast majority of research is focused on a handful of reef-building coral species ( 50 ), and almost nothing is known about how this biodiversity will respond to ocean warming, acidification, or the combination of both factors. To determine how algal, microbial, and noncoral invertebrate composition varied within each treatment, at the end of the experiment we 1) retrieved 3-tiered settlement tile arrays [modified Autonomous Reef Monitoring Structures ( 54 )] which had recruited diverse benthic assemblages while soaking in the mesocosms for the duration of the experiment; 2) sampled the coral-associated algal endosymbionts and coral-associated microbes, as well as the water column-associated microbes; and 3) sampled the mesocosms for both CCA and benthic, fleshy algae. The settlement tile arrays mimicked the three-dimensional structure of the reef framework (albeit not the microstructural complexity of the reef) and provided a standardized tool with which to examine this often-overlooked yet highly diverse cryptobenthic community. Given the relatively short reproductive cycles of many algae and invertebrate species (weeks to months), they experienced multiple generations over the course of the experiment, providing a time-integrated measure of the treatment effects on community composition and abundances. Throughout the course of the experiment the benthic community transitioned from early colonizing species to a mature and diverse community that underwent seasonal variation in abundance similar to adjacent reef communities ( 55 ). Benthic cover analyses of the settlement tiles by functional group revealed that community structure differed only by temperature, and this effect was driven largely by separation of the control and ocean warming treatments in a community ordination ( Fig. 3 and SI Appendix , Table S3 ). Indeed, only a subset of the functional groups responded significantly to the treatments. Calcifying vermetid gastropods declined under acidification but increased under warming ( Fig. 4 and SI Appendix , Table S2 ). Calcifying CCA cover also increased with warming ( Fig. 4 and SI Appendix , Table S2 ). The CCA crusts in the ocean acidification treatment were noticeably thin as compared to the other treatments but the trend toward lower cover was nonsignificant. Metabarcoding revealed that the biomass of CCA and other red algae was dramatically reduced under acidification alone, yet these algae reached maximum biomass under the combined future ocean treatment, suggesting a compensatory interaction between warming and acidification ( 32 ). In contrast, the cover of noncalcifying biofilm/turf algae decreased with warming, but was likewise unaffected by acidification ( Fig. 4 and SI Appendix , Table S2 ). This response may be an indirect effect of reduced space availability as some other groups increased their abundances under warming. Encrusting green algal cover was below detection limits in most of the treatments but achieved modest abundance in the combined future ocean scenario ( Fig. 4 and SI Appendix , Table S4 ). Finally, the abundance of motile fauna (consisting primarily of calcifying amphipods and brittle stars) increased under warming and exhibited a nonsignificant trend toward higher abundance due to acidification ( Fig. 4 and SI Appendix , Table S2 ). Results from visual surveys were confirmed through metabarcoding wherein the abundance of most members of these motile groups increased substantially due to both warming and acidification ( 32 ). The higher abundance of these organisms under warming and acidification might result from differences in detritus production ( 55 ), which serves as a primary food source for these animals. The benthic cover of the other functional groups on the settlement tiles (anemones, bivalves, macroalgae, sediment, serpulid worms, sponges, tunicates, and uncolonized space) did not respond to the treatments ( Fig. 4 and SI Appendix , Tables S2, S4 ). However, metabarcoding revealed that all four treatments exhibited distinct community structure ( 32 ). These data show that community composition shuffles, with some species losses offset by others’ gains, depending on treatment, and these differences were not detected at the coarser level of functional group. Taxonomic and functional groups showed highly variable responses to the experimental treatments ( Fig. 4 ), complicating predictions about exactly how reefs will respond to future ocean conditions. However, when all taxonomic datasets (corals, sponges, metabarcoding of metazoans from settlement tiles, CCA, fleshy algae, coral-associated microbes, water column-associated microbes, and coral-associated algal endosymbionts) were pooled to examine the effects of elevated temperature and reduced pH on proportional changes in overall biodiversity, the number of species was not significantly affected by any of the treatments ( Fig. 5 and SI Appendix , Table S1 )."
} | 5,894 |
36132471 | PMC9473270 | pmc | 1,790 | {
"abstract": "Both superwettability and structural colours have attracted considerable attention in recent years. In addition, the combination of structural colours and superwettability could endow materials with broader application prospects. The combination provides a new strategy to design novel functional materials, and there are many studies pertaining to these materials that have been reported in recent years. Herein, a polysulfide (PSF) superhydrophobic coating was synthesized successfully. The PSF superhydrophobic coating possesses excellent superhydrophobicity, oleophobicity for diesel and macroscopic structural colour variation when wetted. The colour is changed when the coating is wetted and it returns to its original colour after drying. In addition, the surface presents better reusability and thermostability which satisfies various daily needs. The PSF superhydrophobic coating can be considered as an excellent candidate for designing wetting responsive materials, and it has enormous application potential in the fields of detection, sensing, anti-counterfeiting and security. For the first time, we present a novel and low-cost strategy to fabricate materials with both superhydrophobicity and structural colour, offering significant insights into the practical application of these functional materials.",
"conclusion": "Conclusions For the first time, a polysulfide coating with superhydrophobicity and structural colour was successfully prepared by a facile process. The PSF superhydrophobic coating possesses excellent superhydrophobicity, oleophobicity for diesel and macroscopic structural colour variation when wetted. The colour is changed when the coating is wetted and it returns to its original colour after drying. In addition, the surface presents better reusability and thermostability which satisfies various daily needs, exhibits better survivability under rainy, windy and hot natural environments. Hence, it has enormous application potential in the fields of detection, sensing, anti-counterfeiting and security, and the PSF superhydrophobic coating can be considered as an excellent candidate for designing liquid responsive materials. For the first time, we present a novel and low-cost strategy to fabricate a PSF surface with both superhydrophobicity and structural colour, offering significant insights into the practical application of this functional material.",
"introduction": "Introduction As one of the most important surface characteristics, superhydrophobicity has been attracting increasing attention in recent years. And this characteristic endows surfaces with many special properties such as self-cleaning, 1 anti-fouling, 2 anti-fogging 3,4 and anti-frosting. 5,6 Generally speaking, superhydrophobic surfaces possess properties of contact angle (CA) more than 150° and slide angle (SA) less than 5°. 7,8 There are many classical theories for explaining this magical phenomenon, including the Wenzel state, 9 Cassie–Baxter state 10 and Marmur state. 11 All of the theories emphasize the necessity of synergy between surface roughness and chemical composition for acquiring excellent superhydrophobic surfaces. Therefore, constructing a surface which possesses special surface topography with a certain amount of interface energy is the mainstream approach for obtaining superhydrophobic surfaces. 12–14 Another important surface characteristic, structural colour, has also attracted considerable attention in recent years. The phenomenon is attributed to the interference, diffraction and dispersion of light with facile periodic morphologies. 15–17 Materials with this characteristic have been widely used in many fields such as sensing, 18,19 bioassay, 20,21 anti-counterfeiting, 22 and optical components. 23 There are many materials with improved optical properties that have been developed and reported to obtain materials with structural colour, including silicon dioxide, polymethyl methacrylate and polystyrene, 24–35 since the fabrication of structural materials from colloidal spheres is low-cost and facile. And we can adopt a colloidal self-assembly method to obtain a surface with periodic morphology. Therefore, this method has been considered as the mainstream approach to fabricate materials with structural colour. 36–39 In contrast, the occurrence of structural defects on the surface during the fabrication process limits the application of structural colour. Furthermore, with most polymers, even some traditional optical materials, it is hard to obtain bright and obvious structural colour due to their low refractive index which hinders the generation of a wider (even complete) photonic band gap (PBG). 17,40–42 And to improve the structural color visibility, black materials are usually added into the photonic ordered arrays to absorb the scattered light on a broadband. 43 Gianneschi et al. and Kohri et al. adopted this strategy and successfully acquired non-iridescent structural color using black particles. 44,45 However, the original black color of particles limited the application of the responsive material. Although it can present bright color, it cannot generate an obvious color change for darker colors. The addition of melanin nanoparticles also complicates the preparation technology. But when particles themselves possess a high refractive index, high surface charge, low mass density, and the ability for visible light absorption, this problem is solved perfectly, and the color visibility can be greatly improved. In order to improve this situation, Zhang et al. made use of persulfide (PSF) polymeric microspheres which possess higher refractive index (as high as 1.858) to construct a surface with structural colour. 37 The report introduced the production of polysulfide rubber in highly monodisperse spheres with bright colour. As is known to us, micro–nano-structured morphologies could be created to obtain a superhydrophobic surface. Structural colour can also be obtained through the same morphology. 16 Additionally, the combination of these characteristics improves the surface properties, such as self-cleaning, selective wetting and colour variation via angle change. The combination provides a new strategy to design novel functional materials, and there are many studies pertaining to these materials that have been reported in recent years. 46–52 So, it is necessary to explore the possibility of combining superhydrophobicity and structural colour. Due to the special wettability, materials can only be wetted by specific liquids, which provides the possibility of fabricating materials with selective responsive structural colour, as shown in Fig. 1 . And the special colour variation by wetting provides a novel strategy to design anti-counterfeit, responsive and even security materials. Because PSF microspheres can greatly enhance colour visibility, due to their higher refractive index, PSF was chosen in this work to fabricate a material with both superhydrophobicity and structural colour. Up until now, this is the first successful synthesis of a superhydrophobic PSF surface with selective responsive structural colour. In this work, nanoscale PSF microspheres were self-assembled to form blocks with a micro–nano-structure. After that, the powders were modified with triethoxy-1 H ,1 H ,2 H ,2 H -tridecafluoro- n -octylsilane (TTO), and superhydrophobic PSF was synthesized successfully. After concentration in ethyl alcohol, superhydrophobic PSF solution was coated on the glass slide, acquiring a superhydrophobic PSF surface. The PSF superhydrophobic coating possesses excellent superhydrophobicity, oleophobicity for diesel and macroscopic structural colour variation when wetted. The colour is changed when the coating is wetted and it returns to its original colour after drying. In addition, the coating can maintain its superhydrophobicity after more than 130 cycles of wetting. The coating is thermally stable below 150 °C, which satisfies daily needs. The PSF superhydrophobic coating can be considered as an excellent candidate for designing wetting responsive materials, and it has enormous application potential in the fields of detection, sensing, anti-counterfeiting and security. For the first time, we present a novel and low-cost strategy to fabricate materials with both superhydrophobicity and structural colour, offering significant insights into the practical application of these functional materials. Fig. 1 A schematic diagram of the material changing its structural colour by swelling or shrinking of microspheres.",
"discussion": "Results and discussion Polysulfide microspheres There are many studies that have reported the theory of structural colours, and they have been reviewed by our previous studies. 15,16 Light with a specific wavelength can be captured and located in the structure of the photonic band-gap in a photonic crystal material, and other wavelength lights will be reflected. Hence, the material can exhibit various structural colours such as metal colours, iridescence and other single colours. The wavelength of reflected light on the surface can be quantitatively calculated by Bragg's law and Snell's law ( eqn (1) ): 52 1 where λ is the position of the PBG, n is the refractive index of the material, and n PSF = 1.65. θ is the included angle between the incident light and the surface, m is the diffraction series, “ d hkl ” is the spacing of diffraction layers for the FCC packing structure shown in the material which can be calculated using eqn (2) : 2 where D is the space between adjacent pore layers and can also be seen as the diameter of the particle and h , k , and l are parameters which accord with the Miller indices of a given diffraction plane. Structural color can be attributed to the interaction between light and periodically organized architectures. Colloidal microspheres distribute uniformly on the surface to generate a photonic band gap structure. When the structure is exposed to light, a specific wavelength of incident light will be located in the structure, and others will be reflected. Therefore, the surface can present special structural color. Judging from Bragg's law and Snell's laws, the color is affected by the refractive index of the material, angle of incidence and particle spacing. We can modulate these specific parameters to acquire special structural colors. Hence, it is possible to select structural colours by controlling the size of PSF spheres and the distance between each of the particles. The size of PSF spheres can be controlled by adjusting the ratio of ethyl alcohol and deionized water. 37 And the distance between each of the particles can also be controlled by liquid absorption or dehydration of the spheres. As shown in Fig. 1(a) and (b) , the distance between each of the particles will diminish by inflation when the spheres absorb liquid. Due to the infiltration of liquid, each microsphere is full of ethanol. At this time, the reflectance of this material is changed and can be calculated using eqn (1) . And the surface can exhibit a single structural colour. But as the liquid is drying, the spheres will shrink and the distance between each of the spheres will increase. Hence, the surface cannot exhibit structural colour. Hence, PSF sphere surfaces, which can present a single structural colour, were fabricated in this work ( Table 1 ). The as-prepared emulsion was filtered by vacuum filtration, all liquid was removed from the system, and PSF spheres remained and distributed on the filter paper uniformly, as shown in Fig. 2(a) and (c) . The single structural colour exhibited on the filter papers is also present on the bottom of the centrifuge tube after centrifugation ( Fig. 2(b) ). The filter papers present green (I), red (II) and violet (III) colors when the three kinds of PSF spheres with different sizes are arranged on the surface, respectively. Sizes of PSF spheres were measured with a Zetasizer Nano ZS ( Fig. 2(d) ), and with increasing PSF sphere size, the structural colour was transformed from green to red to violet. Different ratios of deionized water and ethyl alcohol to fabricate PSF microspheres No. The Na 2 S 2 precursor solution (mL) Deionized water (mL) Ethyl alcohol (mL) F-127 (g) TCP (g) I 30 150 30 0.3 0.75 II 30 108 42 0.3 0.75 III 30 96 54 0.3 0.75 Fig. 2 (a) A schematic diagram of the preparation process to fabricate the PSF coating on filter paper. (b) The sediment of PSF microspheres on the bottom of centrifuge tubes. And I, II and III present different structural colours (I green, II red and III violet). (c) Different size PSF microspheres deposited on filter paper (I green, II red and III violet). (d) Size distribution of I, II and III PSF microspheres. (e) SEM images of II red PSF microspheres deposited on filter paper. In addition, the microspheres of 180 nm (green), 240 nm (red) and 310 nm (violet) diameters mainly distribute on the filter paper, which almost accord with Bragg's law and Snell's laws. Judging from the SEM images of the filter paper ( Fig. 2(e) ), countless PSF spheres were distributed on the filter paper, almost covering the paper structure completely. Spheres were uniformly distributed on the paper surface with a hexagonal closest packed structure. On the other hand, some defects were created during the packing process, but the structure retained periodic permutations which is the required condition for the generation of structural colour. Superhydrophobic polysulfide powders with non-iridescent colour were prepared. Based on the classical model of superhydrophobicity, surface roughness and chemical composition are essential factors which together determine the wettability properties. 9–11 Hence, it is necessary to increase surface roughness and decrease surface energy for the surface wettability transformation of hydrophilic to superhydrophobic. Considering that structural colours require an orderly and regular distribution of particles, surface roughness requires an appropriate increase to guarantee that the surface presents not only super-hydrophobicity but also structural colours. An infinitesimally small amount of triethoxy-1 H ,1 H ,2 H ,2 H -tridecafluoro- n -octylsilane (TTO) was used to modify PSF powders. As shown in Fig. 3(a) , PSF powders exhibited a single structural colour and the colour was disparate due to the difference in PSF microsphere sizes. The powders were added into 0.25% TTO solution and kept stirring for 24 h. The solutions after the reaction and concentration were stored in three small transparent bottles, respectively, as shown in Fig. 3(b) . And each bottle also exhibits a specific single structural colour which is similar to the PSF powders' colour before the reaction. That can be attributed to the low dispersibility of the modified particles. Hence, the particles can retain their aggregation structure in ethyl alcohol without any destruction of color. Micro-sized aggregates usually precipitate; after a while, the particles will precipitate at the bottom of the bottle, as shown in the bottom right corner of each of the figures in Fig. 3(b) . Hence, when using the solution, it needs to be stirred. Fig. 3 (a) The images of I green, II red and III violet PSF particles. (b) The solution of the modified particles. The solution also exhibits the same structural colour as that of the particles after stirring. And the bottom right corner of each of the images shows the status of precipitation of the particles after being left to stand for a while. XPS spectra ( Fig. 4(a) ) of the particles demonstrated that many elements were introduced into the particles after modification with TTO. Intense peaks of F, C, Cl and Si elements were observed for the modified particles compared with the original ones. Furthermore, the modified particles generate a more complicated curve in the FT-IR spectra of PSF particles ( Fig. 4(b) ). The spectra of both modified and unmodified particles present an intense absorption peak of C–H at 2800 cm −1 . Especially, there is a weak absorption peak of S–H stretching at 2600 cm −1 . The C–S bond generated an absorption peak at 1200 cm −1 . Moreover, the C–F bond produced an absorption peak at 1050 cm −1 , and the C–F bond in the modified particles generated a stronger absorption peak than that in the original particles. After modification, there was an absorption peak at 750 cm −1 due to C–Cl. And the absorption peak at 1050 cm −1 was generated by C–F. C–Si also generated an absorption peak at 1642 cm −1 . And the difference in intensity of the –OH absorption peak at 3400 cm −1 also demonstrated that TTO modified the PSF powders by reaction with –OH. In conclusion, the FT-IR and XPS spectra demonstrated that TTO modified the PSF powders successfully in the reaction process. And the modified particles possess a massive amount of –Si(CF 3 ) 3 which endows the surface with superhydrophobicity and superoleophobicity of diesel compared to the unmodified particles. Fig. 4 (a) XPS spectra, (b) FT-IR spectra, (c) TG curves and (d) XRD patterns of the original PSF particles and modified particles. (e) The reflection spectra of the modified PSF particles when generating structural color. (f) CIE chromaticity diagram for the green, red, and violet color changes of the modified PSF coatings. The TG curves ( Fig. 4(c) ) demonstrated that the modification with TTO did not have any influence on the thermodynamic properties of the PSF powders. An infinitesimally small amount of TTO was used during the reaction. Therefore, the thermodynamic properties were not affected by modification. And the XRD patterns ( Fig. 4(d) ) also confirmed that the crystal form did not transfer after modification. Both original particles and modified particles were amorphous and exhibited a smaller percentage of microsphere contraction and little warping on the substrate. However, the smaller percentage of contraction can suitably transform the structural colour of the surface when drying or wetting. Therefore, it is an excellent candidate for designing a responsive surface. For standardization of the structural colour expression, the reflection spectra of the modified PSF particles when generating structural color ( Fig. 4(e) ) were measured. And data of the modified coating were converted into Commission Internationale deL'Eclairage (CIE) chromaticity, which is shown in Fig. 4(f) . The CIE chromaticity diagram was used to visualize the variation of structural colour, and it is important for the study of the structural discolouration of surfaces. Particles with a single structural colour possess broad prospects in painting and decoration. And superhydrophobicity also extends the application fields of particles and endows the coating with responsiveness for the liquid. Superhydrophobic surface with non-iridescent colour The superhydrophobic PSF coating solution was prepared and stored in a glass bottle ( Fig. 3(b) ). The solution also exhibited a single structural colour which is same as the colour that the PSF powders presented. 5 mL of the solution was dropped and spread out evenly on cleaned glass substrates. After drying for 6 h at 60 °C, the ethanol evaporated completely and the superhydrophobic coating was formed on the substrates, which are displayed in Fig. 5(a) . The coating did not exhibit the same single structural colour as that of the particles ( Fig. 3(a) ). However, the coating exhibited excellent super-hydrophobicity (CA ≥ 150° and sliding angle ≤ 5°), as shown in Fig. 5(b) and (c) . The surface presented strong repellence against water. The droplet can easily be removed and rolled without any resistance, which is demonstrated in Fig. 5(d) , where a droplet was lowered down and brought into contact with the surface. But the droplet still maintained its globular shape throughout the process. When the syringe with the droplet at the tip was lift up, the droplet remained at the tip and left the surface in a whole without any shape change. Fig. 5 (a) Optical photographs of no. I, II and III solutions coated on a glass slide. (b) Optical photographs of a liquid droplet distributed on the coating and CA images of I, II and III coatings (in the top right corner). (c) Images of a liquid droplet rolling on the surface (sliding angle ≤ 5°). (d) Images of the surface with low adhesion for the droplet. As shown in the optical photographs and SEM images of the coating prepared from the original green particles and modified particles ( Fig. 6(a) ), the superhydrophobic surface did not exhibit the green structural colour that was presented by the original particles. The SEM images of the original coating ( Fig. 6(b) ) revealed that the original PSF spheres were distributed uniformly and were well-structured due to the capillary force of liquid evaporation. Countless homogeneous PSF spheres were arranged on the slide substrate uniformly and distributed on the substrate by hexagonal closest packing which led to a PBG structure. Because of this tight arrangement, the surface exhibited a single macroscopic green structural colour. Moreover, it was hydrophilic, and could be wetted by a liquid ( Fig. 6(a) ). The coating composed of modified PSF particles presented excellent superhydrophobicity, which is revealed by Fig. 5(b) and 6(c) . The SEM images ( Fig. 6(d) ) were different from those in Fig. 6(b) . This can be attributed to particle formation. Countless microspheres aggregated and generated particles with a micro–nano-structure before being modified by TTO. Fig. 6 (a) The optical photograph and CA image of the coating composed of the original PSF particles. (b) SEM images of the coating composed of the original PSF particles. (c) The optical photograph and CA image of the coating (I green) composed of the modified PSF particles. (d) SEM images of the coating composed of the modified PSF particles. (e) AFM images of the coatings composed of the original PSF particles and modified PSF particles. (f) Schematic diagram of the Marmur state. (g) Self-cleaning experiment of the coating composed of the modified PSF particles. (h–j) Cross section of the coating (II red) with different thicknesses: (h) 8.875 μm, (i) 10.066 μm, and (j) 15.657 μm. The top right corner presents the CA image of the coating. (k) The reflection spectra of the II red sample with different thicknesses: (h) 8.875 μm, (i) 10.066 μm, and (j) 15.657 μm. Therefore, the particles can maintain their structural colour in solution, as is displayed in Fig. 3(b) ; nonetheless, they cannot generate enough roughness, as shown in Fig. 6(e) . Fig. 6(d) clearly demonstrates that the coating is composed of polymeric microspheres and generates micro–nano-roughness on the substrate. Comparing the AFM images of the original and modified surfaces ( Fig. 6(e) ), the modified surface is found to be rougher than the original one. And the value of roughness of the modified surface is also higher. Considering that the Marmur state combines the Wenzel state and Cassie–Baxter state, 16 some air can be trapped in grooves of the rough surface when a droplet comes into contact with the surface, as simulated in Fig. 6(f) . These grooves can also be discovered in the AFM images ( Fig. 6(e) ). Also, in the surface composed of the original PSF powders, these structures can be obtained. However, the surface presented hydrophilia. That is because it exhibits nanoscale roughness and there are no hydrophobic groups in the molecule. Due to the particle aggregation and modification by reaction with TTO, the surface composed of modified PSF powders generates greater roughness, and numerous hydrophobic groups were introduced into the molecular chain. Hence, the surface can obtain excellent superhydrophobicity. As one of the applications of a superhydrophobic surface, self-cleaning is necessary for the coating to survive in natural environments. Dust is one of the major contaminants that can pollute and damage the functional surface. And self-cleaning is important for the surface to protect its functionality. Therefore, dust was used in this study to act as a contaminant. The whole process is presented in Fig. 6(g) . A certain amount of dust was placed on the surface. After that, a water column was injected onto the surface. When a droplet made contact with the dust, the dust adhered to the droplet surface and rolled down with the droplet. Then the surface cleanness was restored without any dust on it. And in the whole process, the surface was neither wetted nor cracked by the injection of the water column, which demonstrates that the coating possesses a certain degree of mechanical strength. In addition, the thickness of the coating (II red) was measured, and the cross-sectional SEM images of the coating on the glass substrate are shown in Fig. 6(h)–(j) . The thickness can be modulated by adjusting the amount of solution. The coated area was limited to 2 cm × 2 cm. And 1.5, 2 and 2.5 mL of the as-prepared solution were dropped and spread out evenly onto cleaned glass substrates, respectively. And the thickness of the as-prepared coating can be clearly measured from the SEM images. The coating thicknesses when using 1.5, 2 and 2.5 mL solution are 8.875 μm, 10.066 μm, and 15.657 μm, respectively. With increasing solution volume, the thickness of the coating increases. As shown in the top right corner of Fig. 6(h)–(j) , the CA is almost 150°, and it cannot be affected by the thickness. The reflection spectra are shown in Fig. 6(k) . They demonstrate that the structural color cannot be affected by the thickness either. Responsive and repeatable structural colour conversion by liquids The as-prepared coating possesses a macroscopic colour response for liquids which can wet the surface. That is attributed to the swelling or shrinkage of microspheres which has been mentioned in the previous section ( Fig. 1 ). And the coating can repeatedly respond to the stimulation of liquids, even different kinds of liquids. The response process is presented in Fig. 7(a) . A droplet wetted the surface and generated a localized wet spot on the surface. The area exhibited a special single structural colour under visible light. (In Fig. 7(a) , green colour was chosen as an illustration.) After the evaporation of the liquid, the colour faded away, and the coating returned to its original colour. As shown in Fig. 7(b)–(d) , all the coating samples presented a macroscopic colour response for ethyl alcohol. When a drop of ethyl alcohol was dropped on the surface, the coating generated high-contrast colour variation and exhibited structural colours which were similar to the colours of the original particles ( Fig. 3 ). After the evaporation of the liquid, the coatings returned to their original colour and also possessed excellent superhydrophobicity and water repellency (CA ≥ 150°), as shown in the top right corner of Fig. 7(b)–(d) . Moreover, the reflectance at this time can also be calculated. Judging from eqn (1) and the parameters of Fig. 2(d) and 4(e) , the reflectance at this time can be calculated, and the valve is 1.513. Fig. 7 (a) Schematic diagram of the colour variation of the coating wetted by a droplet. (b–d) The optical photographs of colour variation after wetting of no. I, II and III superhydrophobic PSF coatings. From left to right, the images present the original coating, the coating wetted by ethyl alcohol, and the coating after drying. The rightmost images are the CA images and optical photographs of the droplets distributed on the surface. Considering the degree of colour variation, the no. I sample was chosen to perform various performance tests because of the bright colour variation. Firstly, the thermostability of the coating was tested. The no. I sample was put into a muffle furnace and heated for 2 h at 20, 30, 40, …, 150 °C, respectively. After cooling, its superhydrophobicity and colour variation were tested. And the whole test process is demonstrated in Fig. 8(b) . The thermostability of the coating is shown in Fig. 8(a) . Judging from the TG curves of the particles ( Fig. 4(c) ), the test temperature range was maintained between 20 °C and 150 °C. Fig. 8(a) demonstrates that the coating possessed a certain degree of thermostability, and can also retain its superhydrophobicity and responsive structural colour below 150 °C. However, persulfide is unstable at high temperature and can easily be oxidized to sulfur dioxide. However, it can still retain its functionality below 150 °C, which means that the coating is suitable for use in daily life. In addition, the responsive performance can aid the detection of the liquid by the naked eye. Moreover, we tested the responses of the coating for several other liquids. To our astonishment, all three coatings (I, II and III) presented not only excellent superhydrophobicity, but also a certain degree of oleophobicity. As shown in Fig. 8(c) and (d) , five kinds of liquid were chosen to test the performance. And only diesel and water could maintain a globular shape on the surface and presented a large CA, almost ∼150°. Other liquids such as ethyl alcohol, 1,2-dichloroethane, normal octane and hexamethylene wetted and spread on the surface, which means that the coating can detect liquids whether they are pure water or diesel. And when other liquids made contact with the surface, the coating exhibited a specific single structural colour ( Fig. 8(f) ). Cycling durability was also tested for this paper. As shown in Fig. 8(b) , one cycle included three steps. First of all, the coating was wetted by a liquid and then it exhibited a specific structural colour. After that, the coating was dried at 60 °C. After drying completely, the superhydrophobicity of the coating was tested. Next, these steps were repeated several times until the coating began to lose its functions. Four liquids were chosen to test the cycling durability of the coating. The results are presented in Fig. 8(e) . The coating can maintain its functions for 148 cycles when wetted by ethyl alcohol. With increasing number of cycles, the surface began to be damaged and lost both its superhydrophobicity and structural colour. This can be attributed to the weak dissolution of PSF particles in ethyl alcohol. And in other oil liquids, the coating could withstand approximately 130 cycles. In this test, the coating presented better cycling durability and could respond to various liquids. Fig. 8 (a) The thermostability of the no. I superhydrophobic coating. (b) The images of colour variation by ethyl alcohol wetting and optical photograph of a droplet distributed on the superhydrophobic surface. (c) The statistics of the water and diesel CA on the no. I, II and III superhydrophobic surfaces. (d) CA images of water, diesel, 1,2-dichloroethane and ethyl alcohol on the no. I surface. (e) The statistical data of cycle testing of the no. I coating wetted by different liquids. (f) The optical photographs of the no. I coating wetted by different liquids. The coating is adequate for use in daily life and its macroscopic colour response behavior gives it enormous application potential in military, medicine, mechanical and other fields."
} | 7,813 |
24909922 | null | s2 | 1,793 | {
"abstract": "Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil-dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programmes of cellular specialization and cell-cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis and thereby placing a special emphasis on summarizing the most recent discoveries in the field."
} | 264 |
25475855 | PMC4256709 | pmc | 1,796 | {
"abstract": "Investigation of the response of coral microbial communities to seasonal ecological environment at the microscale will advance our understanding of the relationship between coral-associated bacteria community and coral health. In this study, we examined bacteria community composition from mucus, tissue and skeleton of Porites lutea and surrounding seawater every three months for 1 year on Luhuitou fringing reef. The bacterial communities were analyzed using pyrosequencing of the V1-V2 region of the 16S rRNA gene, which demonstrated diverse bacterial consortium profiles in corals. The bacterial communities in all three coral compartments studied were significantly different from the surrounding seawater. Moreover, they had a much more dynamic seasonal response compared to the seawater communities. The bacterial communities in all three coral compartments collected in each seasonal sample tended to cluster together. Analysis of the relationship between bacterial assemblages and the environmental parameters showed that the bacterial community correlated to dissolved oxygen and rainfall significantly at our study site. This study highlights a dynamic relationship between the high complexity of coral associated bacterial community and seasonally varying ecosystem parameters.",
"discussion": "Discussion Seasonal influences on the P. lutea–associated bacterial community Seasonal factors influencing coral-associated bacteria communities have been reported for several coral species distributed throughout different regions 9 10 18 . This temporal variation was also present within the bacterial communities associated with other invertebrate 19 , but not in sponges 20 . In this study, we suggest that bacterial populations in the coral P. lutea on Luhuitou fringing reef showed variation with seasons ( Fig. 2 and 3 ). Similar to previous observations on the coral Acropora in the Great Barrier Reef 4 and Isopora palifera at Ken-Ting 9 , Alphaproteobacteria and Gammaproteobacteria were the two constant predominant groups in all three compartments of P. lutea . However, when analyzed based on lower taxonomic levels, the compositions of these two constant major groups in three coral compartments in different time were distinct ( Table S1 and Fig. S3, S4 and S5 ). Such as Rhodobacteraceae was the most abundant alphaproteobacterial group in both of mucus and skeleton in February and in coral tissue and skeleton collected in May, while Sphingobium was most abundant in all of mucus, tissue and skeleton in August and November. In addition, Rhizobium and unclassified Rhiziobales were the following abundant groups in the coral skeleton collected in November. Mutualistic benefits of coral-associated bacterial communities have been suggested 3 21 22 23 , it makes sense that different bacterial combinations fit the requirements of their host exposed to dynamic environmental factors. Consistent with previous observations, distinct partitioning has been observed in the composition of bacterial communities inhabiting the coral and overlying seawater 9 17 24 . Furthermore, the bacterial communities were much more stable in surrounding seawater in contrast to bacteria associated with corals on Luhuitou fringing reef. Flavobacteria , Alphaproteobacteria and Cyanobacteria were dominant in seawater in all four sampling months. Even at the lower taxonomic level, the major groups, such as Flavobacteriaceae , Ruegeria , unclassified Rhodobacteraceae and GpIIa, showed stability ( Table S2 ). nMDS and ANOSIM analysis further determined the similar composition of bacterial communities in seawater at different times. The result that coral-associated bacteria community was more susceptible compared with water column-associated bacteria suggested that the relationship between corals and their associated microbes might more directly linked to coral holobiont health. However, recent reports showed that the mucus community from Brazilian coral species was more stable and resistant to seasonal variations compared to the water and sediment communities 7 . Whether the contradictions are due to environmental conditions in different coral reef ecosystems, or due to differing coral species, might be resolved when we investigate a larger number of coral species distributed in various geographic positions. Bacterial communities correlated to the P. lutea compartments Investigation of the spatial organization of bacterial communities within the coral holobiont is crucial for understanding the relationship between coral and bacterial assemblages. We investigated the spatial structures of bacterial communities in different microhabitats of coral including the mucus layer, tissue and skeleton at every three months throughout a year. It should be noticed that the composition of bacteria showed dramatically low similarities (< 30%) among different compartments of coral sampled at the same time ( Fig. 3 ). This is similar to the results presented by Sweet and colleagues 13 that bacterial communities within separated coral compartments were significantly different, with average similarity between each other ranging from 24%–46%. In this study, the bacterial communities of all three coral fractions, mucus, tissue and skeleton, tended to cluster together according to the sampling time ( Fig. 2 and 3 ). In the previously published research, the bacterial communities in all Acropora eurystoma fractions were influenced by the pH and coral fractions were distributed within the clusters divided according to the pH level to which the coral was exposed 25 . These results suggested that the bacterial assemblages in the microhabitats of corals including the surface mucus layer, tissue and the skeletal matrix, may synchronously respond to environmental shift. Specialists associated with coral compartments INDVAL analysis showed that several species (OTUs) were associated with a specific compartment. Bacteria inhabiting a specific compartment may reflect the properties of contemporaneous microhabits provided by the coral as well as the coral physiological and adaptive requirements. In this study, we noticed that OTUs belonging to Prosthecochloris and Clostridium strongly associated with coral tissue, both of which were previously reported potential nitrogen fixers 26 27 . It seems that these potential nitrogen fixers specifically live in the tissue of P. lutea . Several bacterial groups such as Acinetobacter 28 and Pseudomonadaceae 29 , considered to be related to coral bleaching and/or disease, were representative specialists in P. lutea tissue. In addition, Vibrio species have been previously reported as an implication for bleaching of some coral species 30 31 32 33 , the coral samples collected in May, in which mucus bacterial community was dominated by Vibrio , did not show any visible signs of disease or bleaching. These bacteria may form a natural part of the healthy coral microbiota 34 , though when the balance of bacterial consortium is disrupted, they may become enriched and switch on the virulence factors 35 36 37 . Whether these small specialist assemblages play role in restoring the bacterial community balance as a functional conserved community 38 39 remains to be investigated. Relationship of environmental factors and the P. lutea bacterial community Six environmental factors ( Table S4 ) and three separate coral compartments were included in the analysis with the bacterial groups. Dissolved oxygen (DO) and rainfall appeared as the most influential environmental parameters out of all those measured that significantly contributed to the variation in bacterial community of P. lutea on Luhuitou fringing reef. Chen et al 9 suggested that the rainfall was a factor with greatest effect on the bacterial community of I. palifera at Ken-Ting, and the changes in the pattern of Bacilli were associated with rainfall. The significant influence of rainfall observed in this study further supports the previous viewpoint. In our study, the Bacilli were the most abundant group in mucus and skeleton during August; the highest rainfall occurred from June to August ( Fig. S6 ). The predominant Bacilli , Exiguobacterium and Planococcus in mucus and Paenibacillus in the skeleton had a high positive correlation to rainfall in RDA analysis ( Fig. 4 ). This supported the speculation that these bacteria might be derived from terrestrial soil and might occur transiently in coral 8 9 . Moreover, Exiguobacterium and Paenibacillus are facultative anaerobes, and the oxygen depleted microhabitats within the coral holobiont may favor their existence. In this study, 60.9% of variance in P. lutea –associated bacterial community composition could be explained both by the measured environmental parameters and the three coral compartments. It implies there are other undetected drivers of the shift of bacterial assemblages. We noticed that Alteromonas was dominant in seawater during May, but it was rarely observed in the other three months. At this sampling time, Vibrio was the absolute dominant group in coral mucus ( Table S2 ). This synchronous phenomenon might reflect the potential correlations between certain bacterial groups in water column and corals. Comprehensively investigating the ecological network between the coral associated microbiota and water column-derived microbes may shed light on this hypothesis. Differential bacterial communities detected from separate coral compartments have been seen in this study as well as previous studies 3 13 34 , reflecting the significant influence of coral microhabitats. These findings highlight the internal drivers of the structure of coral associated bacterial communities 3 13 34 . Therefore, including the physiochemical properties of coral microhabitats into the environmental parameters might better explain the variance in the coral-associated bacterial communities. In conclusion, our results suggest that bacterial populations in all three compartments of the coral P. lutea showed seasonal variations. Dissolved oxygen and rainfall were the most influential environmental factors, out of all those measured, that significantly contributed to the variation in bacterial community of P. lutea on Luhuitou fringing reef. Results of analysis of relationship between bacterial assemblages and the environment showed that the P. lutea –associated bacterial community composition variance could not be completely explained by both the environmental parameters measured and the coral structure compartments, which suggested that differences in microbial populations could be a result of factors other than those included in our analysis. Such factors could include the interactions with water column bacteria communities and the properties of physiochemical environments of coral microhabitats. The probability of bacterial adaptation to a specific compartment was extremely small; however, the study of the ecological role of the small number of specialists is highly desirable. In order to illuminate the relationship between coral and the associated microbes and further understand the roles coral-associated microbes have in maintaining coral holobiont health, investigation of coral-associated microbial consortium at different time scales, their restoring capability and mechanism, and measurement of the microenvironmental conditions of coral compartments is definitely needed in future studies."
} | 2,874 |
33143336 | PMC7693878 | pmc | 1,797 | {
"abstract": "Ultrafine fibers are widely employed because of their lightness, softness, and warmth retention. Although silkworm silk is one of the most applied natural silks, it is coarse and difficult to transform into ultrafine fibers. Thus, to obtain ultrafine high-performance silk fibers, we employed anti-juvenile hormones in this study to induce bimolter silkworms. We found that the bimolter cocoons were composed of densely packed thin fibers and small apertures, wherein the silk diameter was 54.9% less than that of trimolter silk. Further analysis revealed that the bimolter silk was cleaner and lighter than the control silk. In addition, it was stronger (739 MPa versus 497 MPa) and more stiffness (i.e., a higher Young’s modulus) than the trimolter silk. FTIR and X-ray diffraction results revealed that the excellent mechanical properties of bimolter silk can be attributed to the higher β-sheet content and crystallinity. Chitin staining of the anterior silk gland suggested that the lumen is narrower in bimolters, which may lead to the formation of greater numbers of β-sheet structures in the silk. Therefore, this study reveals the relationship between the structures and mechanical properties of bimolter silk and provides a valuable reference for producing high-strength and ultrafine silk fibers.",
"conclusion": "5. Conclusions We reported on the treatment of a trimolter mutant silkworm strain with the anti-juvenile hormone (AJH) KK-42 to induce bimolters, which produced more closely packed cocoons and ultrafine silk fibers. In addition, attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR) showed that the bimolter silk fibroin possessed a higher content of β-sheet structures and fewer random coil and helical structures. The X-ray diffraction measurements revealed that the bimolter silk exhibited a more crystalline structure than the control trimolter silk, whereas mechanical testing indicated that the bimolter silk fibers were stronger and stiffer. We also stained the silk glands with chitin to investigate the origin of the enhanced mechanical properties of the silk sample, and it was found that the anterior silk gland lumen in bimolters is significantly smaller than that in trimolters. In summary, this study reveals the relationship between the morphological changes of bimolter silkworms and the structures and mechanical properties of the resulting silk fibers, thereby providing a valuable fundamental reference for the production of ultrafine and high-strength silk fibers.",
"introduction": "1. Introduction Bombyx mori silk fiber has a long history of application in the textile industry since it was discovered; the fibers were unwound from cocoons and used to manufacture strong and unique fabrics. However, the rapid development of modern fiber technologies has led to stricter requirements for the fineness of such silk fibers. In recent years, ultrafine fiber has been widely used in many fields because of its excellent characteristics, including its high detergency, heat preservation, softness, and lightness [ 1 , 2 , 3 ]. Ultrafine fiber can be converted into ultrafine fabric, where the fabric surface is extremely smooth, and it exhibits minimal air resistance; its optical properties (e.g., the refractive index, transmittance, and reflectance) differ significantly than traditional fabric. Indeed, the light weight fabrics and clean cloths composed of ultrafine fibers are popular in Japan and Europe. In sericulture, thin silk fibers are typically obtained by breeding trimolter strains or inducing normal tetramolters into trimolters. This process is followed since trimolter silk is thinner than typical tetramolter silk, which suggests that further reducing the number of molts could produce thinner silk. To date, numerous studies have focused on trimolters induced by hormone-like chemicals [ 4 , 5 , 6 ]. For example, Asano et al. first introduced and successfully applied anti-juvenile hormones (AJHs) that induce tetramolter silkworms into trimolters [ 4 ], whereas Zhuang et al. induced two Chinese tetramolter silkworm species into trimolters using the imidazole compound YA 20 [ 5 ]. In addition, Niu et al. obtained trimolter silkworms and found that the obtained trimolter silk exhibited thin filaments, high reliability, excellent neatness, and little filament deviation [ 6 ]. In the textile industry, linear density is generally used as a specification corresponding to silk fineness. More specifically, ultrafine fiber is defined as a filament with a fineness of less than 1 dtex [ 7 ]; thus, the fineness of trimolter silk (i.e., >1.5 dtex) does not meet modern expectations. Therefore, the induction of bimolters to produce finer silk fibers has been considered. Unfortunately, normal tetramolter silkworms cannot be induced into bimolters, which have been previously induced from trimolter strains [ 8 ]. Under this previously reported treatment, trimolter larvae could be induced into bimolter which spun cocoons comprising ultrafine silk. However, the study of bimolter silk is an essentially undeveloped field, and the structure and properties of bimolter silk remain unknown. In our previous study, we induced trimolters to obtain thinner silk fibers compared to those of the corresponding tetramolters. Interestingly, we found that the conformation of trimolter silk fibroin possessed a higher content of β-sheet structures and superior mechanical properties compared to the normal tetramolter silk fibroin [ 9 ]. A number of studies have also reported that the mechanical properties of silk fibers are closely related to their diameter [ 10 , 11 , 12 ], where thin silk fiber has a higher breaking strength than that coarse fiber [ 10 , 11 ]. Evidently, thin fibers with small filaments are strong because of their high hydrogen bond energy density [ 12 ], and this can result in the formation of thinner silk fibers and improved silk performances. In general, silkworm silk is considered significantly weaker than spider dragline silk [ 13 ]. To date, various physical, chemical, and biological approaches have been proposed and implemented to enhance the mechanical properties of silkworm silk [ 14 , 15 , 16 , 17 , 18 ]. In this context, post-functionalization approaches have most often been applied to produce high-strength silk fibers from regenerated silk fibroin solutions containing additives through dry or wet spinning [ 19 , 20 ]. However, these approaches inevitably require the use of toxic solvents and complex multistep procedures. Thus, we report on the treatment of a trimolter silkworm strain with the AJH KK-42 to induce bimolters and obtain ultrafine silk fibers. In addition, the strength and Young’s modulus of the filament silk of the bimolter are determined. Furthermore, the morphologies of the bimolter cocoon and silk are observed by scanning electron microscopy (SEM), and the crystalline structure of the silk is characterized and analyzed via attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray diffraction (XRD) measurements. Finally, the silk gland structure of the bimolter is also used to explain the improved mechanical properties of the obtained silk.",
"discussion": "4. Discussion Ultrafine silk has particular economic value and significant potential for application in a range of fields, and as a result, methods of inducing silkworm larvae to spin thin silk have attracted significant research interest. Silkworms spin silk throughout their larval stages; the diameters of the spun fibers vary among larval instars. In general, the smaller the body of the silkworm, the thinner the silk. In this study, an appropriate dose of KK-42 was used topically to the third instar trimolter larvae to induce precocious metamorphosis. This change during growth also affected tissue and organ development, with the body sizes and silk glands of the bimolters differing significantly, relative to those of the control trimolters. Typically, silkworms undergo four molts (i.e., tetramolters) and spin cocoons during the fifth larval instar. The growth and molting of silkworms are tightly regulated by two hormones: Ecdysone, which causes molting, and juvenile hormone (JH), which prevents metamorphosis [ 49 , 50 ]. Breaking this hormone balance alters the growth and molting processes, which can convert normal silkworms into trimolters or pentamolters, which exhibit one fewer or one more instar stage, respectively [ 51 , 52 ]. The compounds that induce precocious metamorphosis or other symptoms of JH deficiency in insects are collectively called AJH agents, and these function by interfering with the biosynthesis, transport, or secretion of hormones [ 53 , 54 ]. Several AJH agents have been used to induce trimolters for producing thin silk fibers such as the JH inhibitors SD-III, SM-1, and YA 20 , [ 5 , 55 , 56 , 57 ]. Although these methods cause the silk spun by trimolters to become thinner, the diameter remains too wide to be considered ultrafine fiber. Nonetheless, this tendency suggests that silkworms can be further treated to spin even thinner silk. Additionally, our previous studies have shown that the mechanical properties of the silk of trimolters induced by KK-42 are better than those of the tetramolter silk. Thus, to obtain thinner and stronger silk, we attempted to induce normal tetramolter silkworms into bimolters; however, this was unsuccessful. Therefore, in this work, we applied KK-42 to a trimolter mutant strain and successfully induced bimolters. The average diameter of the resulting bimolter silk was less than 10 μm. Compared with that of the trimolter mutant, the shortened period of larval development resulted in a notably smaller body ( Figure 1 B), which may affect the silk gland. Previous studies have shown that the liquid silk fibroin flows from the posterior silk gland to the anterior silk gland, during which the conformation of the silk fibroin changes gradually from a helical structure to a β-sheet structure [ 35 , 58 ]. Analysis of chitin-stained silk glands also indicated that the anterior silk gland was significantly smaller in bimolter larvae ( Figure 5 ). These small anterior silk glands lead to the production of silk fibers with smaller diameters as well as different structures. Furthermore, they induce a greater compressive stress and shearing force on the silk protein solution in the anterior silk gland, which may result in a more compact arrangement of fibroin molecules within the silk [ 59 , 60 ]. Recent studies have indicated that the shearing force plays a key role in the transition of the silk protein conformation from a random coil and/or helical conformations to β-sheets in the anterior silk gland [ 60 , 61 , 62 ]. The structural and size changes of the anterior silk glands observed in this study were therefore expected to provide greater shear and compressive stresses in the anterior silk gland to result in the formation of additional β-sheet structures and give a higher crystallinity in the silk protein. In general, increases in the number of β-sheets and crystallinity are associated with an increase in rigid connections between molecules in the silk fiber. This increases the strength and elastic modulus of the silk fiber because β-sheets are responsible for the physical properties and highly dense crystal structure [ 31 , 63 , 64 , 65 , 66 , 67 ]. We also found that the extensibility of the silk fibers decreased after KK-42 treatment, which may be owing to a relatively low content of random coil and β-turn structures in the bimolter silk. In addition, it has been reported that thinner silk has higher strength and stiffness because of its high hydrogen bond energy density [ 12 ]; thinner silk also exhibited a higher “draw ratio” when a polymer jet was applied to a stenotic lumen [ 11 ]. Notably, it was reported that the improved strength was not only originated from the increase in crystallinity but may due to the improved degree of orientation or alignment of the silk molecular chains [ 68 ]. We considered it was possible. The work will be investigated in the following study. The demand for silk products is developing towards diversification and functionalization. Ultrafine silk fiber has the advantages of lightness, strength, and cleanliness, and it has been used to produce high-grade and special-purpose silk products for textiles [ 6 , 69 ]. The ultrafine fibers reported previously were mainly prepared via a composite spinning technique, which requires complex technologies and incurs high costs [ 7 ]. In contrast, the method employed in this study is a safe, simple and an efficient approach to obtain high-strength and ultrafine silk fibers directly from silkworms. In addition, the growth cycle of bimolters in sericulture was shortened, which will not only save labor time and decrease costs but also reduce the occurrence of silkworm diseases. Moreover, the greater economic value and potential advantages of bimolter silk indicate that it will have a bright future."
} | 3,256 |
24794435 | null | s2 | 1,798 | {
"abstract": "The interspecies exchange of metabolites plays a key role in the spatiotemporal dynamics of microbial communities. This raises the question of whether ecosystem-level behavior of structured communities can be predicted using genome-scale metabolic models for multiple organisms. We developed a modeling framework that integrates dynamic flux balance analysis with diffusion on a lattice and applied it to engineered communities. First, we predicted and experimentally confirmed the species ratio to which a two-species mutualistic consortium converges and the equilibrium composition of a newly engineered three-member community. We next identified a specific spatial arrangement of colonies, which gives rise to what we term the \"eclipse dilemma\": does a competitor placed between a colony and its cross-feeding partner benefit or hurt growth of the original colony? Our experimentally validated finding that the net outcome is beneficial highlights the complex nature of metabolic interactions in microbial communities while at the same time demonstrating their predictability."
} | 269 |
21966516 | PMC3179512 | pmc | 1,799 | {
"abstract": "Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO 2 , carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria .",
"conclusion": "Conclusions Genome analysis of M. ferrooxydans PV-1 revealed first insights into the Zetaproteobacteria and disclosed candidate genes involved in inorganic and organic carbon acquisition, oxygen scavenging and defense, energy acquisition in the form of poly P, chemotaxis, and neutrophilic Fe(II) oxidation. The relative abundance of regulatory and signaling protein-encoding genes in PV-1 may be a reflection of the temporal and spatial heterogeneity of its hydrothermal vent habitat as previously described for the genome of T. crunogena XLC-2 [64] . The genomic potential predicting ability and tight regulation of mixotrophic growth, CO 2 fixation under a variety of CO 2 /O 2 concentration ratios and energy storage in phosphates as predicted from genomic potential show previously unknown degrees of flexibility that PV-1 may use to adapt to rapid redox chemistry changes at Loihi. Genes that have a potential role in Fe(II) oxidation show closest resemblance in gene content and synteny to organisms known to perform metal redox processes. M. ferrooxydans may be thus used as a model organism for future studies on neutrophilic, microaerophilic Fe(II) oxidation, which should address experimental verification of the suite of genes required for the enzymatically catalyzed conversion of Fe(II) to Fe(III). Despite apparent genomic parallels to other FeOB from various Proteobacteria classes, relatively low amino acid sequence similarities between PV-1 and other Proteobacteria limit the ability to evaluate the evolutionary history of this organism's genome. The completion of this genome would allow more meaningful comparative genomics, verify or disprove speculations about missing functional genes, and provide insights into events in genome evolution, e.g. gene duplication and loss. Sequencing of additional Zetaproteobacteria strains will be useful to understand the metabolic and phylogenetic diversity within this recently discovered class and to examine the degree, to which the genomic potential is responsible for its dominance at the Loihi Seamount and possibly in other environments.",
"introduction": "Introduction \n Zetaproteobacteria \n \n Zetaproteobacteria are proposed as a novel class of Proteobacteria that were first discovered at iron-rich low temperature hydrothermal vents of the Loihi Seamount, Hawaii [1] . Biogenically formed iron oxide mats that cover the seafloor around the seamount are dominated by Zetaproteobacteria \n [2] . M. ferrooxydans PV-1 is a representative of a cluster of related isolates that share in common the production of an Fe-oxyhydroxide encrusted helical stalk, and an apparent obligate requirement for ferrous iron (Fe(II)) as an energy source. 16S diversity of the Zetaproteobacteria class appears to be high [3] , however, all known strains have the ecological and biogeochemically important trait of Fe(II) oxidation, biomineral and iron mat formation in common ( e.g. \n [4] ). Other related iron-oxidizing Zetaproteobacteria have been identified using cultivation-independent techniques at widely distributed sites in deep-sea environments: these include the Red Sea, the Guaymas basin, the Cleft segment hydrothermal system off the coast of Oregon, the Mariana Trench in the Western Pacific, microbial mats from NW Eifuku Volcano along the Marian Island Arc, the South Tonga Arc, and the Cleft Segment of the Juan de Fuca Ridge [1] , [5] , [6] , [7] , [8] , [9] . Recently, Zetaproteobacteria have also been found associated with deep oceanic crustal boreholes in the western Pacific [10] and coastal environments in the eastern United States [11] . Despite the apparent global distribution and biogeochemical importance of the Zetaproteobacteria , there has been no genetic or biochemical data on this class; the PV-1 genome thus provides the first molecular insights into potential mechanisms employed by this group to succeed in the deep ocean. \n Mariprofundus ferrooxydans \n Cells of M. ferrooxydans are gram-negative, motile curved rods [1] . During its cell cycle, M. ferrooxydans alternates between a free-living, often motile stage, and a stage where cells excrete highly structured stalks, primarily composed of iron oxyhydroxides and an organic matrix ( Fig. 1 ) [12] . In the model proposed by [12] , stalks direct iron oxide formation, preventing engulfment of the cell by solid phase iron minerals by positioning cells in the dynamic gradients of Fe(II) and O 2 . As noted above, Fe oxide filaments similar to those made by PV-1 have been found broadly in the deep ocean ( e.g. Axial Volcano, Juan de Fuca Ridge, Vailul'u Volcano, and Loihi) [12] , [13] , [14] . 10.1371/journal.pone.0025386.g001 Figure 1 PV-1 cultures. Left: the bottle on the right contains a PV-1 culture in log phase showing orange biological iron oxide precipitates, the left bottle contains the uninoculated control; right: Transmission Electron Microscopy (TEM) picture of a PV-1 cell with twisted stalk made of iron oxides and organic matrix. PV-1 is an obligate chemolithoautotroph that oxidizes reduced Fe from a variety of substrates at pH 5.5–7.2 ( e.g. FeS, FeCO 3 , FeCl 2 , Fe(NH 4 ) 2 (SO 4 ) 2 , FeSO 4 , Fe 0 ). Oxygen serves as the only electron acceptor and cells are aerotactic [1] . Though Fe(II) oxidizing bacteria (FeOB) have also been isolated from freshwater environments, e.g. \n [2] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , little is known about the molecular basis of Fe(II) oxidation: to date most genetic and biochemical studies have been conducted on the acidophilic bacterium Acidithiobacillus ferrooxidans \n [25] , and the anoxygenic photosynthetic organisms Rhodobacter sp. strain SW2002 [26] , and Rhodopseudomonas palustris \n [27] . These studies have led to the discovery of various proteins that are implicated in the enzymatic oxidation of Fe(II), however, proteins with an active role in microaerophilic Fe(II) oxidation by chemolithoautrophic bacteria at circumneutral pH have not been identified to date. We have conducted a functional annotation of the genome of Mariprofundus ferrooxydans PV-1 with the aim of gaining insights into its phylogeny, physiology, and biochemistry. Comparative genomic analyses including genomes from other FeOB were used to define genomic commonalities between these phylogenetically and ecologically distinct neutrophilic Fe(II) oxidizing bacteria.",
"discussion": "Results and Discussion Phylogenetic context A previous phylogenetic analysis based on comparisons of the 16S rRNA gene, as well as GyrB and RecA proteins indicated that M. ferrooxydans did not belong to any of the recognized classes of Proteobacteria ( Fig. S1 ) [1] . Analysis based on an amino acid sequence tree of ten concatenated conserved proteins ( Fig. 2 ) supports these previous analyses and further demonstrates that PV-1 belongs to a new class within the Proteobacteria . 10.1371/journal.pone.0025386.g002 Figure 2 Phylogenetic placement of PV-1. Maximum-likelihood tree of ten proteins considered evolutionarily conserved: FusA, GyrB, IleS, LepA, LeuS, PyrG, RecA, RecG, RplB, RpoB [72] . Mariprofundus ferrooxydans PV-1 branches out as a distinct class within the Proteobacteria and appears most closely related to the Magnetococci subdivision, which only comprises one sequenced genome, Magnetococcus sp. MC-1, to date. General genome organization and content The Mariprofundus ferrooxydans PV-1 draft genome sequence consists of 32 scaffolds. It comprises 2,867,087 bp with an average G+C content of 54% and has 2,866 protein coding sequences (CDSs). A mathematical model by [28] of the draft predicts the genome to include ∼98.5% of all CDSs, thus implying only ∼44 genes are missing. PV-1 carries 6 phage integrases and 21 transposases. The transposases are distributed relatively evenly across the genome scaffolds and are typically located next to genes with higher or lower G+C content compared to the genome average, required for signal transduction mechanisms, posttranslational modification, and cell motility, suggesting that some of the genes encoding these functions were obtained via lateral gene transfer (LGT). One phage gene cluster consists of 32 CDSs and is flanked by a transposase (SPV1_02953) and three hypothetical proteins located upstream ( Fig. S2 ). The G+C content varies between 48%–60% across the gene cluster, with 19 genes at 2–10 higher G+C% and 7 genes at 2–10 lower G+C%. The phage gene clusters with most significant nucleotide sequence alignment scores across the entire cluster of all 32 genes are found in Pseudomonas phage MP29 (30% NAID), Bacteriophage D3112 (29% NAID), and Sideroxydans lithotrophicus ES-1 (24% NAID) indicating potential LGT events between these organisms. Considering the similarity of prophage sequences between PV-1 and ES-1, this prophage region may have provided a selective advantage to neutrophilic FeOB. Metabolic processes Carbon acquisition and storage \n M. ferrooxydans is capable of growth in a mineral salts medium with Fe(II) as an energy source and CO 2 as a carbon source [1] . The genome contains two sets of ribulose bisphosphate carboxylase (RuBisCo) genes, including the large and small subunit Form IAq RuBisCo (SPV1_12797, SPV1_12802) and a Form II RuBisCo (SPV1_04963). Both, Form I and Form II RuBisCo genes are located in typical gene clusters containing the two RuBisCo activation proteins CbbQ (SPV1_12807, SPV1_04958) and CbbO (SPV1_12812, SPV1_04953). Form IAq RuBisCo appears predominantly in obligate chemolithotrophs and functions best in niches with medium to low CO 2 concentrations (0.1–1%) and O 2 present [29] . Form IAq RuBisCo is not associated with carboxysomes and carbon concentrating mechanisms [29] , however, it is not clear if that necessarily implies that organisms, which solely contain form IAq RuBisCo, are not capable of building carboxysomes. Form II RuBisCo proteins have a low discrimination threshold against O 2 as an alternative substrate, poor affinity for CO 2 , and therefore potentially take over when the organism moves to a high-CO 2 (>1.5%) and low-O 2 environment [29] . It has been suggested that Form II RuBisCo may be a more ancient type of enzyme and Form I RuBisCo therefore an aerotolerant descendant [30] . Proteobacteria that encode both Form I and II RubBisCo proteins include purple non-sulfur bacteria and certain chemoautotrophic bacteria; most of these organisms appear to be predominantly facultative anaerobes that are metabolically versatile and globally distributed [31] , [32] , [33] . At the Loihi Seamount, temperature differences between bottom water (4°C) and hydrothermal efflux (55°C) may create turbulent eddies in the water column, which would expose cells to oscillating anaerobic and microaerobic conditions, where CO 2 levels are variable (ranging from 2 µM to 20 µM) and dependent on positioning within the chemocline interface [29] , [34] . Utilization of Form I and II RuBisCo proteins could thus enable PV-1 to optimize the acquisition of carbon under a wider range of CO 2 and O 2 concentrations in this dynamic system. PV-1 also has three carbonic anhydrase-encoding genes (SPV1_01467, SPV1_09083, SPV1_07931) predicted to function in the rapid conversion of CO 2 to bicarbonate (typically ∼10 6 reactions per second) [35] . Two gene homologs to cmpB (SPV1_06134) and cmp C (SPV1_06129), which were shown to function in an operon ( cmpABCD ) encoding for bicarbonate uptake in Synechococcus sp. strain PCC 7942 [36] , are located on a large gene cluster (20 genes), which includes a predicted urea carboxylase (SPV1_06124). Urea carboxylase is known to catalyze the conversion of ATP, urea, and bicarbonate to ADP, phosphate, and urea-1-carboxylate. CmpB, cmpC, and urea carboxylase could be part of a carbon-concentrating mechanism (CCM), although neither ccmKLMNOP , chpXY nor cmpABCD operons are observed and no carboxysomes have ever been observed by TEM (Chan, unpublished data). The range of inorganic and organic carbon substrates appears to be rather narrow for M. ferrooxydans \n [1] , however PV-1 possesses a predicted operon (SPV1_t10271, SPV1_10194, SPV1_10199, SPV1_10204, SPV1_10209, SPV1_10214, SPV1_10219, SPV1_10224, SPV1_10229, SPV1_10234, SPV1_, SPV1_10239) encoding for a phosphoenolypyruvate-dependent sugar phosphotransferase system (PTS), which is the major carbohydrate transport system in bacteria [37] . The PTS enzyme II is a fructose/mannose-specific transporter in PV-1 (SPV1_10229). Fructose metabolism requires 1-P-phosphofructokinase [38] , which appears to be missing in the PV-1 genome, however, imported mannose-6-phosphate could be converted by manA (SPV1_07961) to fructose-6-phosphate, which may then enter glycolysis I. This raises the possibility that carbon could be acquired in the form of carbohydrates from the environment, which would allow PV-1 to grow mixotrophically, although such behavior has not yet been observed in previous experiments, but was also not tested for fructose or mannose [1] , [37] . Mannose may otherwise be used in glycoproteins and glycolipids, e.g. proteins that extend into the extracellular space, such as are required during stalk formation, and integral membrane proteins. The genome of M. ferrooxydans shows the organism's potential ability to acquire and potentially store carbon from various sources as well as genomic evolution to the highly dynamic hydrothermal vent environment at Loihi. It remains to be experimentally tested if PV-1 solely utilizes imported carbohydrates for membrane and/or stalk synthesis or if this organism is in fact a mixotroph. The latter would imply that the organism could acquire carbon even when CO 2 fixation is not possible in the niche it resides, and thereby enhance its chance of survival although carbon storage does not appear to be encoded in the genome. Energy acquisition: Aerobic Fe(II) oxidation at neutral pH Microaerophily The aerobic oxidation of Fe(II) requires M. ferrooxydans to live at the anoxic-oxic interface where it can outcompete the abiotic oxidation of Fe(II) [39] , thus PV-1 should be adapted to capture oxygen at very low concentrations. Additionally, in oxic environments Fe(II) may react with hydrogen peroxide that is generated through oxidative processes, to form highly reactive oxygen species (ROS) via Fenton chemistry [40] . Since ROS have the potential to cause oxidative damage to DNA, RNA, and proteins, bacteria require defense mechanisms to convert these compounds into oxygen and water. The PV-1 genome contains a cytochrome cbb 3 oxidase regulon ( ccoNOP ) (SPV1_10291, SPV1_10301, SPV1_10306). CcoQ does not appear to be encoded, however, lack of this gene was shown to have no apparent effect upon the assembly or activity of cytochrome cbb 3 oxidase [41] . Cbb 3 -type cytochromes are members of the heme-copper oxidase superfamily that have the highest affinity for O 2 among all cytochrome oxidases involved in microaerobic respiration [42] , [43] . Substrate affinities have been measured in very few organisms so far, however, the high degree of sequence conservation of the catalytic subunit CcoN in cbb 3 -type cytochrome oxidases and the exclusive bacterial gene expression patterns under microaerophilic conditions suggests that cytochrome cbb 3 -type share oxygen affinities in M. ferrooxydans that are likely to be similar to such measured in other microaerophilic microorganisms [43] . In addition, there are two distinct cytochrome bd quinol oxidases (SPV1_03663, SPV1_03668) in the genome. These enzymes are distinct from heme-copper terminal oxidases and can function as oxidases and O 2 -scavengers [44] with K m values for O 2 in the range of 3–8 nM reported for E. coli \n [45] . Cytochrome bd may also help to mitigate oxidative stress by protecting cells from reactive oxygen species [44] . The expression of these genes could allow growth in oxygen limited habitats, such as is required in the reducing environment of the Loihi hydrothermal vents. Protection against free oxygen radicals inside the cell is provided in part by a superoxide dismutase (SPV1_10466), several peroxidases (SPV1_03628, SPV1_11291, SPV1_13092), and alkyl hydroperoxide reductases (SPV1_06464, SPV1_08671), which also encode for predicted antioxidant response. Interestingly, genes encoding catalase and glutathione reductase that are present in nearly all organisms that are exposed to oxygen, including microaerophiles, such as S. lithotrophicus , were not found in the PV-1 genome [46] . While catalase produces H 2 O and O 2 during the breakdown of H 2 O 2 , peroxidase requires NADH, but only produces H 2 O. The use of several peroxidases may therefore also be favored over that of catalase and superoxide dismutase because peroxidase reactions do not yield O 2 , which - when released to the environment - could affect the sensitive redox balance of iron and exacerbate microaerophilic Fe(II) oxidation. The suite of genes involved in respiration under microaerobic conditions as well as oxygen radical defense display how performance in a low oxygen environment is supported in the genome. Fe(II) oxidation model All current models for microbial Fe(II) oxidation and reduction involve the coupling of electron transfer to iron in the cytoplasmic membrane, so that insoluble minerals precipitate outside the cell. In the case of Fe(III) reduction, this concept is referred to as extracellular electron transfer [47] and several key genes have been identified in Shewanella oneidensis and Geobacter sulfurreducens \n [48] , [49] . Similarly, there have been various key genes identified as relevant for Fe(II) oxidation. These include the pio and fox operon in the phototrophic organisms Rhodobacter sp. strain SW2 and R. palustris , respectively [27] , and iro , cyc1 , cyc2 , cox genes and rus in the acidophilic A. ferrooxidans \n [25] , [50] . The diversity of environmental conditions, under which microbial Fe(II) oxidation and ferric iron (Fe(III)) reduction may be performed, gives rise to diverse physiological mechanisms, biochemical pathways, and gene families involved in this process. Conservation of gene families between different microbial groups involved in Fe(II) oxidation and Fe(III) reduction is absent in most cases, however, few homologs with variable - generally low - sequence identities among key genes are observed [51] . The PV-1 genome harbors more than 70 genes required for electron transport (identified with Pfam domains). Most redox carriers belong to the cytochrome family, however, there are no gene homologs to the above mentioned iron redox genes in the PV-1 genome. Heme-containing cytochromes with peroxidase activity were shown to be specifically expressed during Fe(II) oxidation in various organisms [26] , [27] , [50] . A protein significantly expressed in PV-1 cells oxidizing Fe(II) was extracted and identified as molybdopterin oxidoreductase Fe 4 S 4 region (SPV1_03948). Protein topology prediction indicates a location of the encoded protein outside either membrane, possibly within the periplasm ( Fig. S3 ). The gene neighborhood includes a cluster of 17 CDSs together with other cytochrome, succinate dehydrogenase, and ferredoxin encoding genes ( Fig. 3A ). Orthologous gene neighborhood comparison suggest most conserved gene content and synteny occurs with G. capsiferriformans and S. lithotrophicus , and to a lesser extent in Geobacter uraniumreducens , Geobacter metallireducens , and Geobacter sp. ( Fig. 3B ). 10.1371/journal.pone.0025386.g003 Figure 3 Iron oxidation candidate genes. A) Gene neighborhood of the extracted molybdopterin oxidoreductase protein (scaffold 1). Unlabeled genes are annotated as “hypothetical protein”. Putative functions of orange labeled genes were acquired via BLASTP search. B) Most similar orthologous neighborhoods were found in genomes from other metal oxidizing and reducing organisms in various Proteobacteria subdivisions. Most PV-1 genes within the red box (underlined) contain transmembrane helices indicating a location in either the inner or outer membrane and a potential role in electron transfer across membranes during Fe(II) oxidation. Coloring in A) and B) follows COG classification: blue = energy production and conversion; red: carbohydrate transport and metabolism; purple = general function prediction only; light green = posttranslational modification; rose = cell motility; grey = signal transduction mechanisms; orange = cell wall/membrane/envelope biogenesis; pink = inorganic ion transport and metabolism; dark green = coenzyme transport and metabolism. Source: IMG. Molybdenum functions as a redox-active center, constituting a pterin cofactor in various enzymes involved in catalyzing oxygen atom transfer reactions to or from an electron donating/accepting substrate. Some of these enzymes facilitate the first step in redox reactions, ( e.g. sulfite oxidase and assimilatory nitrate reductase), whereas other enzymes function as terminal respiratory oxidases, ( e.g. DMSO reductase and biotin- S -oxide reductase) [52] . Electron transfer pathways proposed to specifically involve a molybdopterin oxidoreductase, include H 2 oxidation during sulfate reduction [53] and the alternative complex III respiratory system [54] . Considering these examples and the protein isolation results (Barco et al. , in prep.), molybdopterin oxidoreductase and genes located in the same potential operon may play a significant role in the electron transport during Fe(II) oxidation. We propose an Fe(II) oxidation model as shown in Fig. 4A . The conversion of Fe(II) to Fe(III) may be catalyzed by an iron oxidase located in the outer membrane that is closely associated with a molybdopterin oxidoreductase Fe-S region located in the periplasm. The enzyme accepts electrons from ferrous iron and passes them on to an electron transport chain consisting of several oxygen sensitive cytochromes, which are predicted to be essential in the microaerobic environment PV-1 inhabits. Since the electrons obtained from the oxidation of Fe(II) with O 2 are low potential electrons, reverse electron transport and the concurrent consumption of proton motive force are required for NADH synthesis. 10.1371/journal.pone.0025386.g004 Figure 4 Conceptual iron oxidation model in relation to the life cycle in M. ferrooxydans PV-1. A: Proteins potentially involved in the energy acquisition via Fe(II) oxidation through the outer and inner membrane as predicted from genomic analysis. The “Mob gene”, possibly located in the periplasm, represents the experimentally identified molybdopterin oxidoreductase Fe 4 -S 4 region (SPV1_03948), which was extracted under iron oxidizing conditions as mentioned earlier. Its function may include the shuttling of electrons between outer and inner membrane. B: Biologically formed iron oxides are stored in the stalk of PV-1 as edge-sharing Fe-O 6 octahedral linkages as previously described in [76] . As the cell performs Fe(II) oxidation, it rotates, which results in a twisted, coiled stalk. C: Schematic of the life cycle in PV-1. The cell moves in the environment until it identifies conditions suitable for Fe(II) oxidation. The flagella are discarded and stalk growth initiated. As the cell divides, the stalk becomes bifurcated, and each cell continues to form a stalk that is initially half the width as observed by [12] . When O 2 concentrations exceed the maximum tolerable by PV-1, the cell detaches from the stalk and forms flagella to move to a better-suited niche, where the life cycle starts over. Energy storage and life cycle PV-1 exhibits a cell cycle from free-living motile cells to attached, stalk producing cells, which attach to substrates (glass surfaces, other Fe oxides, etc.), and produce Fe oxyhydroxides ( Fig. 4C ; also see [12] ). Cells often undergo division and stalk bifurcation prior to detaching from attached substrate, when single cells enter a free living, motile stage. Motile cells are unattached to stalks and do not appear to oxidize Fe(II). During this motile phase cells are presumably using stored energy, like other obligate chemolithoautotrophic and photolithoautotrophic bacteria [55] , [56] , and may ferment stored organic compounds under anaerobic conditions to obtain ATP [56] , [57] . Neither carboxysomes, nor poly-β-hydroxybutyric acid subcellular bodies have been identified in cells [58] , and no genes ( ccmKLMNOP , chpXY , cmpABCD ) encoding carbon-concentrating mechanisms were identified in the genome. However, two genes encode glycogen/starch synthesis proteins (SPV1_03773, SPV1_01897) and glycogen and starch hydrolysis, i.e. usage of stored polysaccharides, are encoded by several amylases (SPV1_09118, SPV1_09123, SPV1_05592). Polyphosphate (poly P) has previously been proven to serve as energy and/or phosphate reservoir in Thiobacillus strain Q and Accumulibacter phosphatis \n [55] , [59] , [60] . Candidate genes involved in poly P synthesis were identified and poly P bodies were observed in PV-1 ( Fig. 5 ). Several metabolic models for the use of poly P have been proposed, the consensus of which describes the uptake of inorganic phosphate (P i ) via either low or high affinity P i transporters ( e.g. SPV1_07119, SPV1_07314, SPV1_07139) and conversion into poly P via ATP during conditions of carbon and energy excess in an aerobic environment [59] . Under anaerobic conditions, when the organism is in need of energy for the uptake of volatile fatty acids (VFAs), such as acetate and propionate that are stored as polyhydroxyalkanoates (PHAs), the phosphodiester bonds of the stored poly P are broken [59] . Enzymes shown to be involved in the degradation of poly P are polyphosphate:AMP phosphotransferase (SPV1_08276), which catalyzes the phosphorylation of AMP to ADP, and polyphosphate kinase (PPK) (SPV1_07169), which catalyzes ATP formation from ADP thereby enabling the use of poly P as energy source. Poly P may also be degraded into P i for ATP production via V- and F-type ATPases ( e.g. SPV1_13804, SPV1_13814, SPV1_13824) [59] . The source of the reducing power (NAD(P)H) required for PHA production may originate in the reverse electron transport chain through a bc 1 and NADH-Q oxidoreductase complex ( e.g. SPV1_03858, SPV1_03863, SPV1_13739, SPV1_13744), as shown in Thiobacillus ferrooxidans \n [61] . Since the genome of PV-1 appears to encode for a complete set of genes required for the uptake and conversion of poly P to ATP, there is strong indication that the organism may use poly P as energy source as well as phosphate reserve during anaerobic conditions. 10.1371/journal.pone.0025386.g005 Figure 5 Cryo-TEM image of a M. ferrooxydans cell showing two polyphosphate bodies. Identification of polyphosphate bodies is based on electron density, electron dose tolerance, and shape as previously characterized by [58] , who correlated these features to P electron spectroscopic imaging. Small dots on lacey carbon are 10 nm gold particles added to the sample. Regulation and Signaling \n M. ferrooxydans thrives best at low oxygen and high Fe(II) concentrations, however, the hydrothermal vent environment at Loihi is chemically heterogeneous and highly dynamic [34] . The organism therefore requires a chemotactic system that allows rapid sensation, signal transduction, and cell response in order to ensure flexibility and survival under suboptimal conditions. 9.35% of all CDSs in PV-1 are predicted to encode regulatory and signaling proteins, dominated by histidine kinases (43 CDSs) with various function domains, including PAS/PAC sensors, GGDEF/EAL, and multisensors. Other abundant functional genes include diguanylate cyclases (15), sensory box proteins (9), and (two-component) transcriptional regulators (19). In comparison, among other known neutrophilic Fe(II) oxidizers, regulatory and signaling genes comprise 10.61% ( G. capsiferriformans ) to 12.61% ( S. lithotrophicus ) of all CDSs, very similar to PV-1. In Thiomicrospira crunogena XCL-2, a sulfide oxidizer known to inhabit hydrothermal vents, 9.5% of all CDSs fulfill these functions, similar to strain PV-1. The primary role of PAS/PAC domains is the sensing of oxygen, redox, small ligand and overall cell energy level by binding redox or oxygen-sensitive ligands, such as heme and FAD in the cytosol [62] . PAS domains are understood to provide enhanced flexibility in adapting to complex redox environments [62] . EAL/GGDEF domain proteins catalyze the hydrolysis and the synthesis of cyclic diguanylate, an important intracellular signaling molecule, which in some species dictates the switch between attached and planktonic lifestyle via initiation of flagellar degradation and stalk formation [63] . All of these protein domains may provide an advantage when PV-1 detaches from its stalk and enters a stalk-free phase until it initiates Fe(II) oxidation and stalk formation in a better suited redox environment. Interestingly, the PV-1 genome draft harbors very few methyl-accepting chemotaxis protein-encoding (MCP) genes compared to other Fe(II) oxidizers as well as hydrothermal vent inhabiting organisms. There are only three CheY-like receiver proteins and one CheW protein. The family of MCP genes mediates chemotaxis to diverse signals, responding to changes in the concentration of attractants and repellents in the environment by altering swimming behavior. Each MCP is specific to a particular nutrient or toxin [64] , therefore PV-1 may not require a large suite of MCPs if it follows a simple autotrophic lifestyle. There is also a full complement of flagellar genes (SPV1_01957-1967, SPV1_05769, SPV1_05579-05784, SPV1_07696-07701, SPV1_13924, SPV1_13954-13979) in the genome, which is consistent with the observation that PV-1 has a motile cell cycle stage ( Fig. 4C ). The mechanism by which it coordinates motility in response to chemical gradients remains to be biochemically established. Conclusions Genome analysis of M. ferrooxydans PV-1 revealed first insights into the Zetaproteobacteria and disclosed candidate genes involved in inorganic and organic carbon acquisition, oxygen scavenging and defense, energy acquisition in the form of poly P, chemotaxis, and neutrophilic Fe(II) oxidation. The relative abundance of regulatory and signaling protein-encoding genes in PV-1 may be a reflection of the temporal and spatial heterogeneity of its hydrothermal vent habitat as previously described for the genome of T. crunogena XLC-2 [64] . The genomic potential predicting ability and tight regulation of mixotrophic growth, CO 2 fixation under a variety of CO 2 /O 2 concentration ratios and energy storage in phosphates as predicted from genomic potential show previously unknown degrees of flexibility that PV-1 may use to adapt to rapid redox chemistry changes at Loihi. Genes that have a potential role in Fe(II) oxidation show closest resemblance in gene content and synteny to organisms known to perform metal redox processes. M. ferrooxydans may be thus used as a model organism for future studies on neutrophilic, microaerophilic Fe(II) oxidation, which should address experimental verification of the suite of genes required for the enzymatically catalyzed conversion of Fe(II) to Fe(III). Despite apparent genomic parallels to other FeOB from various Proteobacteria classes, relatively low amino acid sequence similarities between PV-1 and other Proteobacteria limit the ability to evaluate the evolutionary history of this organism's genome. The completion of this genome would allow more meaningful comparative genomics, verify or disprove speculations about missing functional genes, and provide insights into events in genome evolution, e.g. gene duplication and loss. Sequencing of additional Zetaproteobacteria strains will be useful to understand the metabolic and phylogenetic diversity within this recently discovered class and to examine the degree, to which the genomic potential is responsible for its dominance at the Loihi Seamount and possibly in other environments."
} | 8,511 |
37181279 | PMC10167139 | pmc | 1,800 | {
"abstract": "Plant-soil feedbacks have been recognised as playing a key role in a range of ecological processes, including succession, invasion, species coexistence and population dynamics. However, there is substantial variation between species in the strength of plant-soil feedbacks and predicting this variation remains challenging. Here, we propose an original concept to predict the outcome of plant-soil feedbacks. We hypothesize that plants with different combinations of root traits culture different proportions of pathogens and mutualists in their soils and that this contributes to differences in performance between home soils (cultured by conspecifics) versus away soils (cultured by heterospecifics). We use the recently described root economics space, which identifies two gradients in root traits. A conservation gradient distinguishes fast vs. slow species, and from growth defence theory we predict that these species culture different amounts of pathogens in their soils. A collaboration gradient distinguishes species that associate with mycorrhizae to outsource soil nutrient acquisition vs. those which use a “do it yourself” strategy and capture nutrients without relying strongly on mycorrhizae. We provide a framework, which predicts that the strength and direction of the biotic feedback between a pair of species is determined by the dissimilarity between them along each axis of the root economics space. We then use data from two case studies to show how to apply the framework, by analysing the response of plant-soil feedbacks to measures of distance and position along each axis and find some support for our predictions. Finally, we highlight further areas where our framework could be developed and propose study designs that would help to fill current research gaps. Supplementary Information The online version contains supplementary material available at 10.1007/s11104-023-05948-1.",
"conclusion": "Conclusion Plant soil feedbacks are the result of a complex interplay between plants, mutualists and antagonists and it remains difficult to predict when PSF should be strong. Here, we propose and test a novel concept that can help to predict the outcome of plant-soil feedbacks by linking plant belowground strategies to soil community components. We predict that pathogens and mutualists scale on independent, orthogonal axes of the recently described root economics space, leading to different soil communities for plants with different combinations of belowground strategies. As the variation along these gradients is continuous, we suggest that continuous measures of functional distance are needed to predict variation in plant-soil feedbacks, and we predict that there should be an interaction between the two distances. Our predictions would also suggest that soil communities could mediate competitive exclusion or coexistence between certain strategies and incorporating root traits into plant competition theory could be a promising future direction. The different effects of the various root strategies on soil microbes would also have consequences for understanding biodiversity functioning relations and might lead to different relationships when different combinations of root strategies are combined. Our first application of the frame work to data from two case studies provides some support for our predictions and the variables are able to explain almost half of the variation in plant-soil feedbacks between the species pairs. Our approach of predicting plant soil feedbacks using the functional similarity between the focal and the soil conditioning species, together with the position of the species pair along each gradient, could therefore be widely applied to analyse plant-soil feedbacks in multispecies experiments. We argue that this framework could lead to a more mechanistic understanding of the outcome of plant-soil feedbacks and that differences in root traits and the root strategies resulting from the root economics space may be key predictors of the strength and direction of plant-soil feedbacks.",
"introduction": "Introduction Interactions between plants and soil biota that affect the subsequent growth of conspecific or heterospecific plants are referred to as plant soil feedbacks (Bever 2003 ; Van der Putten et al. 2013 ). Such biotic plant soil feedbacks (PSFs) are ubiquitous and have wide-ranging ecological effects (Van der Putten et al. 2013 ). For example, PSFs can affect plant population dynamics (Bennett et al. 2017 ; Crawford et al. 2019 ), plant abundance (Mangan et al. 2010 ; Rutten et al. 2016 ; Reinhart et al. 2021 ), diversity (Teste et al. 2017 ) and biodiversity-ecosystem functioning relationships (van der Heijden et al. 2008 ; Maron et al. 2011 ; Schnitzer et al. 2011 ; Mommer et al. 2018 ; Forero et al. 2021 ). However, it remains hard to forecast the outcome of plant soil feedbacks, in part due to an incomplete understanding of the mechanisms underlying PSFs. In the last years, several quantitative reviews have improved our understanding of PSFs. They show that negative plant soil feedbacks dominate, which suggests an important role for pathogens in determining PSFs (Kulmatiski et al. 2008 ; Lekberg et al. 2018 ; Crawford et al. 2019 ; Reinhart et al. 2021 ). However, the strength and direction of PSFs can vary substantially and factors such as plant functional group and growth form, plant native status, evolutionary relatedness, plant abundance and local environmental conditions can explain the outcome of PSFs (Kulmatiski et al. 2008 ; Lekberg et al. 2018 ; Crawford et al. 2019 ; Reinhart et al. 2021 ). Several reviews have evaluated different approaches to testing plant-soil feedbacks and have pinpointed knowledge gaps in the field. However, large parts of the variation in PSFs remained unexplained in previous meta-analyses (Lekberg et al. 2018 ; Crawford et al. 2019 ), suggesting that we need to consider additional predictors of plant-soil feedback strength. Box 1. Measuring PSFs\n Multiple experimental approaches have been proposed to quantify PSFs, each with their own metric (Fig. 1 ) . The most commonly-used approach is to calculate the PSF home/away , which compares plant performance on soil trained by own vs other plant species (Kulmatiski et al. 2008 ; Lekberg et al. \n 2018 ). The advantage of this metric is that it assesses the net effects of the species-specific soil communities. However, the home/away approach cannot assess which soil community component is responsible for the PSF, nor does it directly predict the outcome of competitive interactions. The pairwise feedback approach, PSF pairwise , compares the performance of two plant species growing separately in their respective soils, which corrects for overall differences between soils and relates more closely to the capacity of the pair of plant species to coexist or not (Bever et al. \n 1997 ; Crawford et al. \n 2019 ). The last approach, PSF live/control , compares plant performance on soil trained by a particular plant species vs an unconditioned control soil. Variations of this approach include comparing home/sterile, away/sterile, home/unconditioned and home/fungicide, where the effects of specific agents can be isolated (Petermann et al. \n 2008 ; MacDougall et al. \n 2011 ; Bagchi et al. \n 2014 ; Rutten et al. \n 2016 ; Lekberg et al. \n 2018 ). This metric isolates the effects of one soil community, where negative feedbacks suggest an accumulation of pathogens and positive PSF an accumulation of mutualists. All these three approaches mostly use small amounts of inoculum added to a common background soil, to isolate the biotic feedback and reduce the effects of other drivers, such as differences in nutrient availability or nutrient flush after sterilization (Brinkman et al. \n 2010 ). The proportion of inoculum added, ranges from 0.8–100% across studies, but this did not consistently affect strength of PSFs in a recent meta-analysis (Crawford et al. \n 2019 ). With this, each of the three commonly-used plant soil feedback metrics answers a slightly different question. PSF home/away evaluates the effects of the specialized soil community cultured by a particular plant species by comparing its effect with a soil community cultured by one or more other plant species, where only generalist taxa should affect plant growth. PSF pairwise evaluates how changes in soil communities conditioned by different plant species affect interactions between them. Finally, PSF live/control evaluates the net effects of a soil community including both its specialized and generalist components, where the \"control\" soils may be unconditioned, sterilized, fungicide or AMF soils. However, whilst almost all PSF studies test the growth of species on their home soils (α) the control soils that are used for comparison vary substantially, e.g., they can be unconditioned or sterilised or treated soil; γ or soil conditioned by one or more other species; β. Therefore, it is important to carefully consider the control soils used as these determine which conclusion can be drawn. A combination of approaches likely leads to the best understanding but is also most labour intensive. Fig. 1 PSF metrics have in common that they compare the growth of a focal species ( A ) on soil conditioned by the same species (α) versus on a control soil. The control can be unconditioned soil (γ) or soil conditioned by another species (β). Pairwise feedbacks reciprocally compare the performance of focal species ( A ) and soil conditioning species ( B ) on their respective soils α and β, where α A is A’s performance in conspecific soil, α B is B’s performance in heterospecific soil, β A is A’s performance in heterospecific soil, β B is B’s performance in conspecific soil. Consequently, feedback can be measured as PSF home/away (α A – β A ), PSF pairwise (α A —α B —β A + β B ) or PSF live/control (α A —γ A ). Note that ‘away’ soils are often pooled across species in a community and ‘control’ soils can be unconditioned, sterilized, fungicide or AMF additions"
} | 2,515 |
36212906 | PMC9535326 | pmc | 1,801 | {
"abstract": "Lignocellulose utilization has been gaining great attention worldwide due to its abundance, accessibility, renewability and recyclability. Destruction and dissociation of the cross-linked, hierarchical structure within cellulose hemicellulose and lignin is the key procedure during chemical utilization of lignocellulose. Of the pretreatments, biological treatment, which can effectively target the complex structures, is attractive due to its mild reaction conditions and environmentally friendly characteristics. Herein, we report a comprehensive review of the current biological pretreatments for lignocellulose dissociation and their corresponding degradation mechanisms. Firstly, we analyze the layered, hierarchical structure of cell wall, and the cross-linked network between cellulose, hemicellulose and lignin, then highlight that the cracking of β-aryl ether is considered the key to lignin degradation because of its dominant position. Secondly, we explore the effect of biological pretreatments, such as fungi, bacteria, microbial consortium, and enzymes, on substrate structure and degradation efficiency. Additionally, combining biological pretreatment with other methods (chemical methods and catalytic materials) may reduce the time necessary for the whole process, which also help to strengthen the lignocellulose dissociation efficiency. Thirdly, we summarize the related applications of lignocellulose, such as fuel production, chemicals platform, and bio-pulping, which could effectively alleviate the energy pressure through bioconversion into high value-added products. Based on reviewing of current progress of lignocellulose pretreatment, the challenges and future prospects are emphasized. Genetic engineering and other technologies to modify strains or enzymes for improved biotransformation efficiency will be the focus of future research.",
"conclusion": "8 Conclusion This review mainly focuses on lignocellulose degradation technologies related to biological methods. In this regard, enzyme treatment has gained increasing attention due to the advantages of mild and simple biological treatment conditions. The current research directions mainly focus on the following aspects: (1) searching for new efficient functional strains or enzymes; (2) deeply understanding the degradation mechanisms; (3) determining the best pretreatment conditions; (4) improving the function of strains through genetic engineering; and (5) adding auxiliary materials to strengthen the process. Additionally, this article summarizes the potential value of lignocellulose in high-value utilization. As a renewable resource, lignocellulose could be a potential alternative to fossil fuels. In the process of biotransformation, high-yield bacteria screening, designing reasonable transformation process, improving strain stability, and avoiding feedback inhibition could meet the needs of industrial mass production. However, finding an efficient and universal solution is highly necessary for achieving sustainable development and improving economic benefits. Overall, the biological refining of lignocellulose is still a complex process. When strengthening a single step, we should also pay attention to the relationship between different processes, and improve the process performance on the basis of biotechnology combined with other methods, so as to realize the efficient utilization of lignocellulose.",
"introduction": "1 Introduction According to reports, fossil fuels and other nonrenewable resources are in short supply. In this context, the utilization of renewable energy and its derivatives as substitutes can effectively reduce the dependence on nonrenewable energy, thereby achieving sustainable development and economic benefits [ 1 ]. Lignocellulose is an abundant renewable biomass resource on earth, with an annual output of more than 20 billion tons [ 2 ], including wood, plants, agricultural waste, forest residues and municipal solid waste, which can be converted into eco-friendly high value-added products [ 3 ]. It was reported that if all straw waste in China could be converted into hydrogen, the energy generated would be equivalent to a total of 170 million tons of standard coal [ 4 ]. Moreover, the production of biofuels from lignocellulose could alleviate the pressure caused by rapid energy consumption. Biofuel conversion technologies, such as those producing bioethanol, biohydrogen and biomethane, are mature. Moreover, high-value chemicals produced from lignocellulose increase the profitability. However, the large-scale utilization of lignocellulose is still challenging. Enzymatic hydrolysis of cellulose and hemicellulose is a key step in the transformation process, but the complex cross-linked structure of lignocellulose hinders the process by inhibiting enzymatic interactions ( Fig. 1 ) [ 5 ], and the unproductive binding between enzymes and lignin also inhibits the enzymatic hydrolysis process [ 6 ]. In recent years, pretreatment has emerged as an effective approach to overcome these bottlenecks, thereby eliminating the compact and complex structure of biomass components and improving the accessibility of enzymes to cellulose. Pretreatment can accelerate the dissociation of fibers, promote biomass conversion, and hydrolyze polysaccharides into monosaccharides. Pretreatment also decreases the lignin contents, cellulose crystallinity and energy consumption [ 7 ]. Fig. 1 Function of pretreatment [ 5 ]. Fig. 1 In the lignocellulose process, effective pretreatment should meet the following requirements: (1) conducive to subsequent hydrolysis; (2) avoid producing by-products that inhibit enzyme saccharification; (3) be economically feasible; and (4) reduce pollution and waste of resources. At present, physical, chemical and biological treatments have been widely used for lignocellulose pretreatment. Physical treatments include mechanical crushing, ultrasonic treatment, microwave treatment, but these consume a lot of energy and are usually used as an auxiliary method. Chemical treatments have the advantage of high efficiency. For instance, rice straw treated with KOH and urea could obtain 92.38% sugar yield after 32.47% of lignin was removed under the optimal conditions [ 8 ]. However, the traditional chemical reagents have some disadvantages, such as the incomplete recovery of polysaccharides, possible generation of inhibitors and the risk of environmental pollution, thereby affecting the production efficiency [ 9 ]. Recently, ionic liquids have been used in pretreatment due to their non-flammable, non-volatile and recyclable characteristics [ 10 ]. When rice straw was treated with 1-H-3-methylmorpholinium chloride ionic liquid with water as co-solvent for 5 h, the ethanol production yield increased from 21.9% to 64% [ 11 ]. However, the high cost of ionic liquids limits large-scale implementation. Therefore, other types of green solvents have been developed as good substitutes for traditional reagents. It was reported that a high lignin removal rate of 73.17% was obtained after pretreatment with 10% ethylenediamine (EDA) and 200 °C. Compared with traditional alkali treatment, the residual EDA in wastewater can be separated by simple evaporation [ 12 ]. In addition, deep eutectic solvent (DES) has been shown to be a highly cost-effective and promising solvent [ 13 ]. Despite the fact that the lignin removal rate of DES might not be as high as that of alkali reagents, lignin pretreated with DES was substantially depolymerized and no condensation structure was observed [ 14 ]. The lignin from the corn stover was effectively removed after pretreatment with DES consisting of quaternary ammonium salts and hydrogen donors. After hydrolysis, no unfavorable inhibitors to the subsequent fermentation process were produced, indicating that DES possesses good biocompatibility [ 15 ]. In addition, it was found that the energy required for DES pretreatment was 28% and 72% lower than that of NaOH pretreatment and the steam explosion process, respectively [ 16 ]. These results all reflect the potential value of DES. Nevertheless, there are still challenges in the application of DES, whose inherent properties such as high viscosity and hygroscopicity may limit its application in biomass pretreatment. At the same time, further research is needed to identify ways in which to efficiently recover DES [ 10 ]. In short, traditional physical and chemical pretreatments require special equipment, and inhibitors can be produced in the process, which is inconducive to subsequent fermentation. Due to the great influence of pretreatment on downstream technologies, from the overall economic perspective, the eco-friendly characteristics make biological pretreatment stand out. Relatively speaking, biological methods consume less energy and do not produce inhibitors during the treatment process because it is carried out under mild conditions. There are a large number of microorganisms in nature that can be applied in biological pretreatment. The degradation capability of microorganisms mainly originates from their unique enzymatic systems. Lignolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase have been applied to open the recalcitrant structure of lignocellulose. Numerous studies have shown that white rot fungi are highly selective for lignin, which can improve the hydrolysis efficiency and reduce energy consumption. At present, several studies are seeking to understand the degradation mechanism of different strains. However, fungal pretreatment still has some challenges, such as improving the stability and tolerance of strains, selecting appropriate strains and improving degradation efficiency. In biological pretreatment bacteria with strong viability, which could adapt well to variation in the microenvironment, have also been isolated and applied. Effective biodegradation can be accomplished with microbial consortium comprising different strains. Synergism of consortium is the key to degradation. Due to higher substrate utilization rates, microbial consortium has gradually replaced the single strain and has become the first choice for this process. Furthermore, combining biological pretreatment with other methods has been a research hotspot. Various technologies have also been developed to improve the biological pretreatment process, with a focus on gene editing technology. In addition to being widely accepted in new fields, lignocellulose has been utilized in traditional pulping and papermaking for thousands of years. With technological advancements, lignocellulosic derivatives have been used in agriculture, industry and other fields. In line with the requirements of green technology, the introduction of microorganisms or enzymes for biological modification is conducive to saving energy consumption. Relevant studies have indicated that cellulases could improve the performance of pulp dissolution [ 17 ]. Xylanase is mainly used to assist in pulp bleaching to reduce the formation of organic halogens and consumption of chemicals [ 18 ]. The reduction in total production costs ensures sustainable development of the pulp and paper industry. This review specifically summarizes and discusses the research progress of biological pretreatments to date, providing a theoretical basis for the selection of strains. Additionally, the insightful degradation mechanisms are further clarified, and the methods for improving the process are discussed. Finally, the application of lignocellulose in fuel, chemical and pulping industries is introduced, and the influence of pretreatment on resource utilization is highlighted."
} | 2,909 |
26840490 | PMC4780264 | pmc | 1,802 | {
"abstract": "The origin of eukaryotes stands as a major conundrum in biology 1 . Current evidence indicates that the Last Eukaryotic Common Ancestor (LECA) already possessed many eukaryotic hallmarks, including a complex subcellular organization 1 – 3 . In addition, the lack of evolutionary intermediates challenges the elucidation of the relative order of emergence of eukaryotic traits. Mitochondria are ubiquitous organelles derived from an alpha-proteobacterial endosymbiont 4 . Different hypotheses disagree on whether mitochondria were acquired early or late during eukaryogenesis 5 . Similarly, the nature and complexity of the receiving host are debated, with models ranging from a simple prokaryotic host to an already complex proto-eukaryote 1 , 3 , 6 , 7 . Most competing scenarios can be roughly grouped into either mito-early , which consider the driving force of eukaryogenesis to be mitochondrial endosymbiosis into a simple host, or mito-late , which postulate that a significant complexity predated mitochondrial endosymbiosis 3 . Here we provide evidence for late mitochondrial endosymbiosis. We used phylogenomics to directly test whether proto-mitochondrial proteins were acquired earlier or later than other LECA proteins. We found that LECA protein families of alpha-proteobacterial ancestry and of mitochondrial localization show the shortest phylogenetic distances to their closest prokaryotic relatives, when compared to proteins of different prokaryotic origin or cellular localization. Altogether, our results shed new light on a long-standing question and provide compelling support for the late acquisition of mitochondria into a host that already had a proteome of chimeric phylogenetic origin. We argue that mitochondrial endosymbiosis was one of the ultimate steps in eukaryogenesis and that it provided the definitive selective advantage to mitochondria-bearing eukaryotes over less complex forms."
} | 480 |
31349557 | PMC6722850 | pmc | 1,803 | {
"abstract": "Autothermal thermophilic aerobic digestion (ATAD) is a microbial fermentation process characterized as a tertiary treatment of waste material carried out in jacketed reactors. The process can be carried out on a variety of waste sludge ranging from human, animal, food, or pharmaceutical waste where the addition of air initiates aerobic digestion of the secondary treated sludge material. Digestion of the sludge substrates generates heat, which is retained within the reactor resulting in elevation of the reactor temperature to 70–75 °C. During the process, deamination of proteinaceous materials also occurs resulting in liberation of ammonia and elevation of pH to typically pH 8.4. These conditions result in a unique microbial consortium, which undergoes considerable dynamic change during the heat-up and holding phases. The change in pH and substrate as digestion occurs also contributes to this dynamic change. Because the large reactors are not optimized for aeration, and because low oxygen solubility at elevated temperatures occurs, there are considerable numbers of anaerobes recovered which also contributes to the overall digestion. As the reactors are operated in a semi-continuous mode, the reactors are rarely washed, resulting in considerable biofilm formation. Equally, because of the fibrous nature of the sludge, fiber adhering organisms are frequently found which play a major role in the overall digestion process. Here, we review molecular tools needed to examine the ATAD sludge consortia, what has been determined through phylogenetic analysis of the consortia and the nature of the dynamics occurring within this unique fermentation environment.",
"conclusion": "8. Conclusions ATAD is a tertiary treatment process to stabilize waste sludge from a variety of sources. The process carried out in jacketed reactors with aeration results in a unique thermophilic microbial community capable of generating heat, reducing the total solids of the sludge and producing Class A Biosolids with some fertilizer potential. Tools for molecular analysis of the microbial community have demonstrated it to be dynamic and responsive to the changes in temperature and pH with the community adapting to the ATAD niche. The nature of the biodegradation processes may mean that the unique thermophilic population possesses a unique arsenal of enzymes and biodegradative capabilities, which have yet not been exploited. One of the limitations of widespread use of ATAD is the energy cost of aeration and mixing [ 3 , 8 ]. However, this cost could be reduced significantly by utilizing heat recovery processes [ 64 ] from the heated ATAD sludge and by decreasing the treatment time, perhaps by using the heat generated to decrease the autothermal phase.",
"introduction": "1. Introduction ATAD (autothermal aerobic thermophilic digestion) is a liquid composting, tertiary sludge treatment process which utilizes the metabolic heat generated by way of microbial digestion to stabilize the treated sludge and effectively pasteurize the digestate [ 1 , 2 , 3 ]. This produces class A biosolids suitable for land spread. The ATAD process has been utilized to treat a variety of waste streams ( Table 1 ) including animal waste, food, brewery slaughterhouse wastes and domestic wastes. It has been demonstrated that ATAD can be used to process a variety of high strength waste streams at elevated temperatures with biological oxygen demand (BOD) reduction of 95% and chemical oxygen demand (COD) reductions up to 99% [ 4 , 5 ]. The process can operate as a one-stage system [ 6 ] or as a two-stage system [ 7 ] that incorporates a mesophilic stage and then follow on thermophilic stage. Generally, the sludge itself is thickened via polymer addition to 7–8% total solids followed by aeration in jacketed bioreactors, where the addition of air and mixing causes biodegradation to proceed with the energy converted to biomass and heat. Because of the jacketed nature of the bioreactors, the trapped heat results in a rise in temperature to above 60 °C, often reaching up to 75 °C [ 8 ]. The thermal processing results in a sanitized digestate [ 9 ] where pathogens, carried over from the initial secondary sludge, are inactivated. This results in a Class A Biosolids, which has the potential for land spread as a liquid fertilizer. During the process, stages deamination of proteinaceous material results in ammonia release [ 7 ]. This results in the case of domestic wastes, in a rise in pH to above 8. This increased pH has been proposed to allow better reactor performance, higher enzyme activity, facilitates oxidation, accelerates humification but limits microbial diversity by the unique combination of pH and temperature [ 10 ]. These conditions provide a unique environmental niche with a distinctive nutrient profile of often somewhat recalcitrant biological material that results from primary and secondary treatment waste processes the nature of which varies according to waste type ( Table 1 ). In addition, the elevated slightly alkaline pH coupled to the elevated temperature also selects a unique microbial population that will be discussed below. Many of the large-scale reactors and their operating parameters (mixing, air addition, sludge additions and removal in semi continuous mode) are imperfect in terms of providing optimal aerobic conditions. This is due to low oxygen solubility at elevated temperatures, low dissolved oxygen concentration because of the high level of microbial degradation at the thermophilic stage, imperfect mixing and aeration to account for these changes, biofilm formation on the reactor surfaces, and the sludge viscosity [ 4 , 7 ]. Due to a combination of these conditions, although termed aerobic digestion, there are many reports of the recovery and activity of anaerobes, which undoubtedly play a major part in the ATAD process [ 2 , 3 , 14 , 19 ]. The process is also dynamic from a microbial perspective with various transitions occurring at various stages during the digestion. Initially, mesophiles dominate and degrade the more readily digestible substrates. Then, as the pH and temperature rises, the mesophilic population is replaced by a more thermoduric and thermophilic population adapted for growth at slightly alkaline thermophilic temperatures [ 20 ]. The microbial diversity operating within ATAD reactors is key to the degradation and stabilization of the sludge substrate and the maintenance of thermophilic conditions [ 2 , 20 ]. This diversity has been examined for several different ATAD processes such as swine waste [ 21 ], pharmaceutical waste [ 17 ], and domestic sludge [ 2 , 3 , 19 , 22 , 23 ]. Previous studies attempted to utilize culture-based methods [ 24 ] to identify the nature of the microbial diversity, but these proved to be limiting [ 25 ], thereby necessitating the use of molecular tools to definitively address the diversity present in ATAD reactors."
} | 1,726 |
33986540 | PMC8263491 | pmc | 1,804 | {
"abstract": "Directed evolution has been used for decades to engineer biological systems at or below the organismal level. Above the organismal level, a small number of studies have attempted to artificially select microbial ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. Here, we have developed a flexible modeling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions. By artificially selecting hundreds of in-silico microbial metacommunities under identical conditions, we first show that the main breeding methods used to date, which do not necessarily let communities to reach their ecological equilibrium, are outperformed by a simple screen of sufficiently mature communities. We then identify a range of alternative directed evolution strategies that, particularly when applied in combination, are well suited for the top-down engineering of large, diverse, and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure-function landscape to be navigated in search for dynamically stable and ecologically resilient communities with desired quantitative attributes.",
"introduction": "INTRODUCTION Harnessing microbial communities is a major aspiration of modern biology, with implications in fields as diverse as medicine, biotechnology, and agriculture 1 . Several groups have demonstrated that small synthetic communities can be engineered to carry out functions such as biodegrading environmental contaminants 2 – 4 , manipulating plant phenotypes 5 , or producing biofuels 6 , 7 , among others 8 , 9 . Despite these success stories, engineering consortia from the bottom-up (i.e. “rational design”) remains challenging. The function of a consortium is generally affected by species interactions, which are difficult to predict from first principles and expand rapidly with species richness 10 – 16 . Perhaps more importantly, microbial communities are rapidly evolving ecological systems, and their engineered functions can be disrupted by environmental fluctuations, invasive species, species extinctions, or the fixation of mutant genotypes 17 – 20 . Rather than fighting these eco-evolutionary forces, an alternative “top-down engineering” approach seeks to leverage ecology and evolution to find microbial consortia with desirable attributes 20 – 26 . Most work has focused on enrichment approaches 22 , 25 – 28 , but a small number of studies have gone further and empirically demonstrated that ecological communities can respond to artificial selection applied at the level of the community itself 29 , 30 . This strategy has been deployed to iteratively optimize complex microbial communities that modulate plant phenotypes 1 , 30 – 34 , animal development 35 , or the physico-chemical composition of the environment 36 – 40 . Despite its conceptual elegance, the success of artificial selection at the microbiome level has been mixed and generally modest, and artificial selection has not yet been widely adopted in microbiome engineering 1 , 41 . A limiting factor is that we do not know how to design efficient artificial selection protocols at the microbial community level. The selection methods used in early studies (e.g. 30 , 42 ) were inspired by even earlier work on artificial group selection of either single-species populations 43 – 45 , or two-species communities of sexually reproducing animals 29 , 46 . In these studies, new generations of communities were created through either: (i) a sexual reproduction-like “migrant-pool” strategy, where the communities with the highest function were mixed together and then used to inoculate a new generation, or (ii): an asexual-like “propagule” reproduction strategy, where the best communities were selected and then propagated without mixing 29 , 30 , 36 . All subsequent microbial ecosystem-selection studies followed suit and employed variations of those two methods. But are selection strategies originally developed for small populations of sexually reproducing organisms well suited to efficiently direct the evolution of much larger and diverse communities of generally asexual microbes? Are there other alternatives? To address these questions, we set out to explore the effectiveness of all previous selection strategies we could find in the literature. To do this, we evaluated them in parallel on the same set of in-silico microbial communities and for a number of different functions. We show that all of these protocols do worse than a simple screen, a no-selection control that has been largely missing from previous microbiome selection experiments. The limitations of past protocols led us to propose an alternative framework for top-down microbial community engineering that is based on the directed exploration of the ecological structure-function landscape (i.e. the map between community composition and community function), through iterated rounds of randomization and selection 10 – 12 , 15 , 47 , 48 . This approach is inspired by the directed evolution field, where proteins and RNA molecules are evolved in the laboratory through a guided random exploration of their genotype-phenotype maps 49 , 50 . In the second part of this paper, we address how these structure-function landscapes can be systematically navigated in search for stable communities of high function.",
"discussion": "DISCUSSION Directed evolution can be used to iteratively optimize the function of microbial communities, through sequential rounds of exploration and selection. Previous approaches to engineer communities from the top-down include enrichment (which is often followed by a perturbation such as a bottleneck, to reduce community complexity) 20 , 22 – 24 , 28 , 81 , 82 , and selective breeding by artificial selection 1 , 30 – 33 , 35 – 41 . The directed evolution approach we have studied here combines components of both approaches: the iterative search that is inherent of the latter, with the idea of building stable consortia and exploring compositional variants of the former. In addition to inducing evolutionary changes in the resident species, the methods to generate compositional variants and explore the ecological structure-function landscape include many ecological perturbations that randomly sample new species in and out of the community. For instance, bottlenecking (also known as dilution-to-extinction 21 , 22 , 27 , 70 , 81 , 83 ) is a blunt method for randomly removing “deleterious” taxa, which has the cost of also eliminating potentially beneficial species. Horizontal immigration from the regional pool may create variants that contain new and potentially “beneficial” species, but it has the cost of potentially adding species with deleterious effects. A selection method that combines the two with strong selection is able to compensate for the specific weaknesses of each, leading us to high-function regions of the ecological structure-function landscape that were not reached by any of the two individual strategies alone ( Fig. 4 ). These communities are also ecologically resistant to invasions compared to both enrichment and synthetic communities assembled by artificially mixing together species with high per-capita function. As is the case for any computational model, ours has simplifying assumptions and, therefore, limitations. We discuss them at length in the Supplementary Discussion . Perhaps the most notable one is that, for simplicity, we have focused on a function that is additive on the species contributions and which carries no cost at the individual level. We relax both these assumptions in the Supplementary Methods . We show that our main findings also hold when we work with non-additive functions, including those modeling realistic community objectives such as resisting invasion from an undesired organism or the elimination of a specific metabolite ( Extended Data Fig. 1 ). In addition, our main results were found to hold true under alternative ecological scenarios, which include growth media of different richness and interactions ranging from pure nutrient competition to cross-feeding ( Extended Data Fig. 2 ); alternative functional responses by the species in our communities ( Extended Data Fig. 3 ); different methods of sampling taxa from the environment ( Extended Data Fig. 4 ); and various distributions of per-capita species contributions to the community function: from highly redundant to rarefied ( Extended Data Fig. 5 ). Finally, we also show that our results hold true when species contributions to the function under selection are not fitness neutral ( Extended Data Fig. 1 ). Although many microbial functions, such as the secretion of metabolic byproducts and overflow metabolites do not incur any cost to their producer 84 , many other functions are costly for the contributing cell 85 . On this note, it is important to note that our simulations do not include within-species evolution. It is thus possible that, on an evolutionary timescale, the directly evolved communities would be vulnerable to “cheater” mutants which forgo the cost of functional contributions in favour of faster growth, outcompeting their direct ancestors as would be predicated by social evolution theory 85 . The timescale over which evolution would degrade community function is unknown, through recent community evolution experiments suggest that evolution is heavily constrained when species are embedded within a complex community 86 . Furthermore, recent artificial community-level selection experiments suggest that one may be able to preserve a costly community function that may be prone to exploitation by cheaters (the expression of an extracellular enzyme) by continuously purging those communities where cheating phenotypes arise (i.e. purifying selection at the community-level) 40 . It is important to highlight that the mechanisms we have considered here to generate variation between communities are all purely ecological, as we do not allow evolution within species. Taken as a unit, one can consider communities to be evolving: we are introducing heritable variation between them and then selecting upon that variation, and this results in changes in the genetic makeup of the communities (i.e. their metagenomes) as well as in their attributes 55 , 87 . Explicitly incorporating within-species evolution into our framework (for example by allowing new mutants to arise within each growth cycle) represents an exciting future direction for this work and would allow us to explicitly explore the complex trade-offs between community function, ecological stability and evolutionary resilience. We hope that our results will not only clarify the limitations of previous approaches to artificially selecting communities, but also motivate the development of new empirical methods for the directed evolution of microbial communities."
} | 2,782 |
38584951 | PMC10995308 | pmc | 1,805 | {
"abstract": "The world has undergone a remarkable transformation from the era of famines to an age of global food production that caters to an exponentially growing population. This transformation has been made possible by significant agricultural revolutions, marked by the intensification of agriculture through the infusion of mechanical, industrial, and economic inputs. However, this rapid advancement in agriculture has also brought about the proliferation of agricultural inputs such as pesticides, fertilizers, and irrigation, which have given rise to long-term environmental crises. Over the past two decades, we have witnessed a concerning plateau in crop production, the loss of arable land, and dramatic shifts in climatic conditions. These challenges have underscored the urgent need to protect our global commons, particularly the environment, through a participatory approach that involves countries worldwide, regardless of their developmental status. To achieve the goal of sustainability in agriculture, it is imperative to adopt multidisciplinary approaches that integrate fields such as biology, engineering, chemistry, economics, and community development. One noteworthy initiative in this regard is Zero Budget Natural Farming, which highlights the significance of leveraging the synergistic effects of both plant and animal products to enhance crop establishment, build soil fertility, and promote the proliferation of beneficial microorganisms. The ultimate aim is to create self-sustainable agro-ecosystems. This review advocates for the incorporation of biotechnological tools in natural farming to expedite the dynamism of such systems in an eco-friendly manner. By harnessing the power of biotechnology, we can increase the productivity of agro-ecology and generate abundant supplies of food, feed, fiber, and nutraceuticals to meet the needs of our ever-expanding global population.",
"conclusion": "6 Conclusion In conclusion, biotechnology in agriculture has emerged as a multifaceted tool that encompasses a diverse range of techniques, ranging from traditional breeding methods to advanced genetic engineering. This comprehensive approach has played a pivotal role in the 21st-century agricultural revolutions, contributing significantly to enhanced productivity and the socio-economic development of countries, with agricultural biotechnology standing as a key segment within the Indian biotech sector. The association of biotechnology with industrial farming practices has led to misconceptions and a stringent regulatory framework in many countries. It is crucial to distinguish between the biotechnological production process and the safety of the end product, addressing the misperception that underlies regulatory challenges. Biotechnology, when applied judiciously, addresses various aspects of agriculture, promoting sustainability in three major criteria: improving plants, modifying soil, and developing alternatives to fuel inputs for agricultural equipment. The integration of functional omics, computational biology, and advanced techniques like RNA-Seq and GWAS to modify critical agro-morphological traits in plants besides altering host–pathogen interactions, signaling mechanisms, and associated proteins holds promise for disease-resistant high-yielding varieties. These advancements are crucial for addressing contemporary challenges, including climate change and resource constraints, in the pursuit of sustainable agriculture. As we anticipate a new biotechnological revolution focused on deciphering gene codes and the “gene revolution,” it is imperative to foster a balanced understanding of biotechnology’s potential in synergy with natural farming practices. This synergy holds the key to pioneering agricultural sustainability through innovative interventions, encompassing microbe-mediated bio-fortification, bioremediation, restructuring soil through composting, and developing alternatives to petroleum-based fuels for agricultural equipment. By embracing these innovative approaches, we can pave the way for a sustainable future in agriculture that maximizes productivity while minimizing environmental impact and ensuring food security for generations to come. In terms of environmental sustainability, genetically engineered crops have proven to be advantageous over conventional insecticides, conserving non-target species, enhancing arthropod abundance and diversity, and promoting more effective biological control of pests. The incorporation of insect-resistant crops not only reduces the need for expensive chemical inputs but also contributes to soil modification for water conservation, decreasing erosion-induced damage and lowering irrigation costs. The implementation of refuges alongside insect-resistant crops serves as a strategic measure to delay the evolution of resistant pest populations, emphasizing the importance of maintaining a balanced ecosystem. The economic sustainability of natural farming is underscored by its inherent link to financial viability and income generation for farmers. Biotechnology-assisted natural farming facilitates precision agriculture, reducing costs and offering a diversified approach to mitigate risks associated with weather, market fluctuations, and disease/pest outbreaks. On a societal level, the social system surrounding agriculture is positively influenced by the adoption of natural farming practices. The alignment of natural farming with cultural traditions fosters a sense of identity and community resilience. It serves as a source of job creation, wealth generation, and economic growth within the community, reinforcing the interdependence of agriculture with social wellbeing. In conclusion, the impact of biotechnology-assisted natural farming on environmental health, economic status, and social systems demonstrates the potential for a harmonious integration of technological advancements with sustainable agricultural practices.",
"introduction": "1 Introduction The term “sustainability” finds its origin from the Latin word “Sustinere”, which denotes the enhancement of environmental quality and the resource base that can uphold and endure future societal development. The term “sustainable” was used for the first time at the United Nations Conference on Human Environment, Stockholm in 1972 focusing on the preservation of environment for the benefit of human beings across the globe. The major outcome of the Stockholm Conference (1972) was the establishment of the United Nations Environment Programme (UNEP), which became the leading global environmental authority for setting the global environmental agenda. Later on in 1992 in Rio de Janeiro, Brazil, the UN General Assembly called for the United Nations Conference on Environment Development (UNCED) commonly known as the Rio Summit or Earth Summit, 1992 with primary goals of socio-economic development while preventing environmental deterioration ( Grubb et al., 2019 ). A number of multilateral environmental agreements have taken place since 1992. However, the global environment has continued to suffer in terms of loss of biodiversity, desertification, and increasing natural disasters. Over the past two decades, there has been a growing concern about the need for sustainable agriculture to address the food and fiber requirements of society while also providing enduring solutions for both present and future generations. A fundamental prerequisite for sustainable agriculture is to guarantee social equity and economic viability for farmers and all individuals engaged in agriculture and its associated enterprises. This will encourage them to maintain a healthy environment and support the development of climate-resilient agriculture. One of the popular approaches toward sustainable agriculture is natural farming, popularly known as Zero Budget Natural Farming (ZBNF). The Indian civilization thrived on natural farming for ages and India was one of the most prosperous countries in the world. Traditionally, the entire agriculture was practiced using natural inputs where the fertilizers, pesticides, etc. were obtained from plant and animal products. This continued till the advent of colonial rule in India, which introduced plantation agriculture and turned the focus of farmers from self-sufficient crops to cash crops like indigo, jute, tea, and tobacco. Furthermore, the burgeoning population, the pressure to grow cash crops, and drastic climatic calamities led to the shift of the farming sector toward high-input agriculture. The concept of natural farming was regained by the Japanese scientist Fukuoka in the 1970s through his book The One Straw Revolution: An Introduction to Natural Farming , in which he mentioned it as a do-nothing technique. The concept of natural farming revolves around the idea of self-sufficiency of the natural ecosystem without much human intervention. In India, Padma Shri recipient Mr. Subhash Palekar became the first to adopt the ZBNF system in the 1990s. His concern with the increasing indebtedness and suicide among farmers in India due to the increasing costs of fertilizers and pesticides and their long-term devastating effects on the environment compelled him to advocate the use of low-input technologies in agriculture that should be available within farmlands. He started the natural farming concept in Karnataka and subsequently converted over 50 lakh farmers into practicing ZBNF in various states of India. This method promotes soil aeration, minimal irrigation, intercropping, bunds, and topsoil mulching with crop residue and strictly prohibited intensive irrigation like flooding and deep ploughing tillage practices. However, these traditional practices will not be sufficient to provide food to the estimated 9.7 billion population in 2050. Recently, the Indian Council of Medical Research (ICMR) has set guidelines for per person per day calorie intake to achieve nutritional sufficiency ( Chellamuthu et al., 2021 ). Incorporating modern biotechnological techniques into agriculture is the prerequisite to attaining this goal and mitigating the climate crisis ( \n Figure 1 \n ). Figure 1 Catalyzing sustainable growth through Zero Budget Natural Farming for India’s burgeoning population. However, adopting biotechnology in natural farming system is not that easy. There exists an ideological war between natural farming and biotechnology-assisted farming, leading to complete incompatibility among these two systems ( Purnhagen and Wesseler, 2021 ). Biotechnology in agriculture encompasses a diverse range of techniques, which may include traditional breeding methods that modify living organisms or their components to create or enhance products, improve plants or animals, or engineer microorganisms for particular agricultural applications. It is not exclusive but includes the tools of genetic engineering. It has emerged as a promising tool for crop improvement and led to significant enhancement in agricultural productivity in the 21st century through agricultural revolutions. Within the Indian biotech sector, agricultural biotechnology stands as the third largest segment (as reported by Business Standard in 2013). It is widely recognized as a pivotal sector that plays a significant role in driving the socio-economic development of the country ( ABLE INDIA, 2013 ; Shukla et al., 2018 ; Lima, 2022 ). A new biotechnological revolution is estimated to revolve around deciphering the gene codes of living beings leading to “gene revolution”. Biotechnology often carries a perplexing association with industrial, commodity-based farming, monoculture practices, the extensive use of pesticides, and patented seeds. However, the most significant misinterpretation lies in conflating biotechnology—a production process—with an inherently unsafe and perilous product. This misperception forms the foundation of the stringent regulatory framework that many countries apply to biotech crops. The current review seeks to advocate the idea that integrating biotechnology with natural farming can offer a promising solution to address key challenges in achieving sustainable agriculture. These challenges include the need to produce sufficient food within the constraints of limited arable land and finite resources, particularly in the face of stresses like drought, salinity, high temperature, and diseases. The aim is to achieve these goals while reducing reliance on synthetic fertilizers and pesticides."
} | 3,108 |
36685258 | PMC9811514 | pmc | 1,806 | {
"abstract": "Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing 13 C at the sidechain β-carbon. Feeding of [β- 13 C]-labelled DHP lignin to Rhodococcus jostii RHA1 has led to the incorporation of 13 C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, confirming that they are derived from lignin breakdown. We have identified a glycolate oxidase enzyme in Rhodococcus jostii RHA1 which is able to oxidise glycolaldehyde via glycolic acid to oxalic acid, thereby identifying a pathway for the formation of oxalic acid. R. jostii glycolate oxidase also catalyses the conversion of 4-hydroxyphenylacetic acid to 4-hydroxybenzoylformic acid, identifying another possible pathway to 4-hydroxybenzoylformic acid. Formation of labelled oxalic acid was also observed from [β- 13 C]-polyferulic acid, which provides experimental evidence in favour of a radical mechanism for α,β-bond cleavage of β-aryl ether units.",
"introduction": "Introduction Lignin is a high molecular weight heteropolymer found as a major component of plant cell wall lignocellulose, formed by oxidative polymerisation of hydroxycinnamyl alcohol precursors. Although recalcitrant, lignin can be degraded by Basidiomycete white-rot fungi and some soil bacteria, via production of lignin-oxidising peroxidase and laccase enzymes. 1,2 There is current interest in metabolic engineering of lignin-degrading bacteria such as Rhodococcus jostii RHA1 and Pseudomonas putida KT2440 to generate high-value bioproducts such as vanillin, 3 muconic acid, 4 and aromatic dicarboxylic acids. 5 However, using polymeric lignin feedstock, conversion yields are at best in the range 3–10%. A major unsolved problem is the understanding of pathways by which the lignin heteropolymer is converted into low molecular weight products. 6 While there is general agreement that vanillic acid and protocatechuic acid are important intermediates, which are usually degraded via the β-ketoadipate pathway, the possible routes to these intermediates from substructures found in polymeric lignin are less certain. 6 Oxidative C α –C β bond cleavage of β-aryl ether units is known to generate vanillin, which can be converted to vanillic acid and protocatechuic acid. 7 Phenylcoumaran (β-5) model compounds can be converted in Sphingobium SYK-6 to lignostilbene intermediates, followed by oxidative cleavage to generate vanillin. 8 Lignostilbene intermediates can also be formed from β-arylpropane (β-1) model compounds in Novosphingobium aromaticivorans , followed by oxidative cleavage to generate vanillin. 9 Pinoresinol (β–β) units can be cleaved reductively in Sphingobium SYK-6, 10 but are cleaved oxidatively in Pseudomonas sp. SG-MS2. 11 However, homologues for the genes involved in these pathways are not found in other lignin-degrading microbes, so it is not clear whether these pathways are used widely. Understanding these pathways is therefore an important task in achieving high yields from lignin bioconversions, and such studies may reveal new opportunities for generation of renewable bioproducts from lignin breakdown. While many metabolites have been observed by GC–MS or LC–MS in bioconversions of lignin or lignocellulose, 6 in some cases it is uncertain whether they are derived from polymeric lignin or not. A further complication is that the method used to isolate lignin from the plant also generates some low molecular weight by-products present in the lignin preparation, and this is especially true of industrially derived lignins such as Kraft lignin. We wished to investigate two particular metabolites. Firstly, we have previously observed oxalic acid as a bioproduct from lignin bioconversion in Rhodococcus jostii RHA1 and Pseudomonas putida , 12 which we suspected is derived from the two-carbon fragment released after C α –C β cleavage of polymeric lignin, 7 however, oxalic acid in fungi is thought to be derived from primary metabolism. 13 Secondly, we have identified a gene cluster in Rhodococcus jostii RHA1 for degradation of aryl-C 2 lignin fragments to vanillin via 4-hydroxy-3-methoxybenzoylformate, but the route from polymeric lignin to this intermediate is uncertain. 14 In order to provide more definitive evidence for the conversion of polymeric lignin to these metabolites and other low molecular weight metabolites, we have prepared synthetic isotope-labelled lignins, which we have used for bioconversion by wild-type and engineered lignin-degrading bacteria. Dehydrogenatively polymerised (DHP) lignin can be prepared in the laboratory by peroxidase-catalysed oxidative polymerisation of coniferyl alcohol. 15 14 C-labelled DHP lignins have been used to study lignin degradation by fungi 16,17 and soil bacteria, 18,19 and in compost. 20 DHP lignins have also been used to study the action of laccase enzymes on polymeric lignin, 21 and the chemical demethylation of lignin. 22 Here we describe the verification of the conversion of polymeric lignin to oxalic acid and 4-hydroxy-3-methoxyphenylacetic acid, and the identification of a flavin-dependent enzyme in Rhodococcus jostii RHA1 involved in their metabolism.",
"discussion": "Discussion The use of isotope-labelled DHP lignin for isotope incorporation into metabolites of lignin degradation described herein is a useful and sensitive method for investigating or confirming whether observed metabolites are derived from lignin breakdown. We show that DHP lignin does serve as a substrate for microbial conversion by Rhodococcus jostii RHA1, and therefore it is likely that it could be applied to other lignin-degrading microbes. The synthetic route used to make the 13 C-labelled DHP lignin in this work could be used to prepare DHP lignins containing 13 C in other positions, 24 or isotopes such as 2 H. Although the intensities for the observed metabolites are only 10–40 fold above background ( Fig. 4 and 6 ), the observation of lignin degradation intermediates is challenging, due to the presence of multiple microbial lignin degradation pathways, 6,8–11,39 and further microbial degradation of these intermediates. 14,39 The results obtained here confirm that a pathway exists from lignin breakdown to oxalic acid in Rhodococcus jostii RHA1, which can be mediated by glycolate oxidase, although we note that there are other pathways to oxalic acid in fungi. 8,30,31 The formation of 13 C-labelled oxalic acid also observed from poly-ferulic acid, which implies that C α –C β oxidative cleavage of lignin units can occur in the presence of a γ-carboxylic acid. This observation has implications for the mechanism of α,β-bond cleavage, which could take place via a radical mechanism, as proposed for R. jostii DypB, 7 or via a carbocation intermediate, as illustrated in Fig. 7 . The formation of a radical upon bond cleavage would be stabilised by an adjacent carbonyl group, but a carbocation intermediate would be considerably destabilised by an adjacent carbonyl group. The observation that oxalic acid is formed from poly-ferulic acid is therefore evidence in favour of a radical mechanism for α,β-bond cleavage. Fig. 7 Mechanisms for α,β-bond cleavage of β-aryl ether lignin units in DHP lignin (R = CH 2 OH) or poly-ferulic acid (R = CO 2 H). Path A, radical mechanism proposed for R. jostii RHA1 peroxidase DypB; 7 path B, possible 2-electron mechanism for oxidative cleavage. The observed formation of homovanillic acid from DHP lignin provides evidence for the conversion of lignin G units to homovanillic acid, and the likely conversion of lignin H units to 4-hydroxyphenylacetic acid. The conversion of substituted phenylacetic acids to substituted benzoylformates, catalysed by glycolate oxidase, provides a further route to aryl C 2 metabolites from lignin, which could be processed in R. jostii RHA1 via the 4-hydroxybenzoylformate degradation pathway. 14 Although the latter pathway is not present in other lignin-degrading bacteria, other bacteria contain the hpc gene cluster for 4-hydroxyphenylacetate degradation. 39 The mechanism of formation of substituted phenylacetic acids from lignin is an interesting question, since 4-hydroxyphenylacetaldehyde can be formed via chemocatalytic breakdown of β-O-4 units in lignin under acidic 40 or solvothermolytic conditions, 41 via protonation of the α-hydroxyl group, followed by a C–C fragmentation reaction releasing formaldehyde (see Fig. 8 , path A). Although such a process is not known biologically, a related reaction generating formaldehyde from β-arylpropane lignin fragments has been recently identified in Novosphingobium aromaticivorans , 9 which provides a possible precedent that such a mechanism might occur biologically. Alternatively, sidechain oxidation to a γ-carboxylic acid intermediate in a lignin fragment, followed by decarboxylation (see Fig. 8 , path B), would also be a possible mechanism. The results of isotope incorporation studies therefore give new insight into microbial lignin degradation pathways. Fig. 8 Possible mechanisms for the formation of substituted phenylacetic acids from polymeric lignin or oxidised lignin fragments, either (A) via C–C fragmentation, or (B) via γ-oxidation, followed by decarboxylation. Possible routes for catabolism of substituted phenylacetic acids are illustrated. Position of 13 C label is indicated."
} | 2,403 |
35993149 | PMC10086978 | pmc | 1,807 | {
"abstract": "The light environment in a mixing water column is arguably the most erratic condition under which photosynthesis functions. Shifts in light intensity, by an order of magnitude, can occur over the time scale of hours. In marine Synechococcus , light is harvested by massive, membrane attached, phycobilisome chromophore‐protein complexes (PBS). We examined the ability of a phycobilisome‐containing marine Synechococcus strain (WH8102) to acclimate to illumination perturbations on this scale. Although changes in pigment composition occurred gradually over the course of days, we did observe significant and reversible changes in the pigment's fluorescence emission spectra on a time scale of hours. Upon transition to ten‐fold higher intensities, we observed a decrease in the energy transferred to Photosystem II. At the same time, the spectral composition of PBS fluorescence emission shifted. Unlike fluorescence quenching mechanisms, this phenomenon resulted in increased fluorescence intensities. These data suggest a mechanism by which marine Synechococcus WH8102 detaches hexamers from the phycobilisome structure. The fluorescence yield of these uncoupled hexamers is high. The detachment process does not require protein synthesis as opposed to reattachment. Hence, the most likely process would be the degradation and resynthesis of labile PBS linker proteins. Experiments with additional species yielded similar results, suggesting that this novel mechanism might be broadly used among PBS‐containing organisms.",
"introduction": "Introduction Marine photosynthetic organisms are responsible for approximately 50% of the world's primary production [ 1 ] and play a pivotal role as the energetic drivers of the ocean's biogeochemical cycles [ 2 ]. They are responsible for absorbing an estimated 25% of annual anthropogenic carbon emissions from the atmosphere [ 2 ]. In much of the global ocean, the water column is susceptible to seasonal changes. During summer months, the water column is stratified. As winter commences, warm water bodies are exposed to colder air. Denser upper layer water forces itself downwards causing vertical mixing as a result of the temperature change [ 3 ]. These dynamics create the conditions for spring blooms in oligotrophic oceans [ 4 ]. Fluctuations in nutrient availability, salinity, pressure and temperature require strategies for adjustment to the changing conditions, within the relevant time scales for mixing. Light harvesting systems of photosynthetic organisms in these waters face substantial changes in light intensity and spectrum and must re‐acclimate accordingly and continuously. These dynamics are suggested to be on the scale of hours to days [ 3 ]. The Synechococcus group of cyanobacteria are considered to be ubiquitous in the world open oceans [ 5 ]. The distribution of major Synechococcus clades coincides with the mixing conditions that promote spring blooms [ 6 ]. This makes Synechococcus prime candidates for an investigation of the adaptations to such fluctuating conditions [ 7 ]. Synechococcus use phycobilisomes (PBS) for light harvesting. To efficiently react in a changing environment, the PBS has to be able to tune its light harvesting to fit the erratic dynamics of the light intensity and spectrum changes in a mixing water column. The PBS is a soluble membrane attached pigment–protein complex composed of two subcomplexes: (a) a core attached to the thylakoid membrane surface and (b) rods projecting outwards from the core in a fan‐like layout [ 8 , 9 , 10 ]. The Synechococcus PBS system is composed of four major subgroups of light harvesting chromophore‐protein complexes called phycobiliproteins (PBPs): allophycocyanin (APC) in the cores, phycocyanin (PC) in rods closest to the core, and two types of phycoerythrin I (PEI) then phycoerythrin II (PEII) in the rods, farthest from the cores. PEII is the most distal in the rod structure. PBPs covalently bind chromophores as shown in Fig. 1 [ 7 , 11 ]. Fig. 1 PBPs and their pigments. The nomenclature of PBS proteins and pigments is complex. Phycocyanobilins (PCB) are pigments bound to phycocyanin (PC) and allophycocyanin (APC) PBPs. PC PBSs in Synechococcus can also bind phycoerythrobilin (PEB) pigments [ 11 ]. The difference in the emission maxima between PC and APC is a result of the different interaction of PC pigments with the PBP protein environment. The phycoerythrin PBPs (PEI and PEII) bind PEB and phycourobilin (PUB). PEII binds a higher proportion of PUB than PEI. The difference in PUB/PEB ratio results in a difference in the wavelength of the emission maxima. An additional fluorescence band observed in emission spectra is that of the chlorophylls associated with PSII. PSI associated chlorophylls do not fluoresce strongly at room temperature. The structure of the PBS in respect to the thylakoid membrane plane is presented in the cartoon on the left‐hand side. The number of rods in a PBS, the number of PBP units in each rod and their ratio is variable [ 7 , 12 , 13 ]. Phycobilisomes also play a role in immediate (seconds to minutes) reaction processes. These include state transitions [ 14 , 15 , 16 ] and non‐photochemical energy quenching mechanisms driven by the function of the orange carotenoid protein (OCP) [ 17 ]. These mechanisms come into play immediately, within seconds to minutes of exposure to high, potentially photo‐inhibitory, light intensities [ 15 ]. The issue of PBS acclimation to an erratically changing light intensity within the time scale of hours relevant to a mixed water column warrants further exploration. In the present study, we used Synechococcus WH8102 as a model organism. It is a clade II marine Synechococcus comprising a group that is more abundant during winter [ 6 ] and has a wide growth irradiance range (from 15 to 650 μmol photons·m −2 ·s −1 continuous white light [ 7 ]).",
"discussion": "Discussion In the present study, we tracked the changes in pigment content and fluorescence properties during the transition between two physiologically relevant light intensities for marine Synechococcus species. The results indicate that an extensive change in fluorescence properties occurs within hours following the transition. These fluorescence changes were faster than the changes observed for the absorption of light harvesting and photosystem pigments, which take place over time scales of days. Modulations of energy transfer efficiency in photosynthetic antennas are usually discussed in the framework of excitation quenching. However, in the Synechococcus PBS under the conditions examined here, we observed an increase in fluorescence rather than quenching. Our analysis suggests a model in which PBS structures are disassembled during the first few hours following the transition from LL to ML. The most likely mechanism is that the disassembly is driven by the degradation of linker proteins. Linker proteins control the PBS structure and are known to be very susceptible to degradation [ 10 , 25 , 26 ]. The process does not require protein synthesis and therefore we can assume that the mechanism for PBS disassembly is present and ready to be activated. Recovery upon return to LL requires protein synthesis, which fits with the hypothesis that linkers are degraded in the disassembly process. Inhibiting protein synthesis provided information for an additional acclimation strategy. PSII core D1 proteins are degraded to a certain extent. The rate of D1 synthesis is sufficient to compensate for this loss and therefore the shift from LL to ML cannot be considered as truly photo‐inhibitory [ 27 ]. However, an increase in the ratio of high light to low light psbA transcripts would ensure a switch to the high light form of the D1 protein. As a rule, adjusting photosynthetic energy flow to fit environmental conditions is achieved by multiple mechanisms to ensure the robustness of the process. In marine Synechococcus , short‐term acclimation is achieved by quenching mechanisms [ 15 ]. Long‐term acclimation is achieved by changes in pigment content and their associated proteins [ 18 ]. In the present study, we describe an intermediate mechanism, over the scale of hours, that appears to be uniquely fit for addressing the erratic light environment of the mixed water column in which these organisms thrive."
} | 2,084 |
32194877 | PMC7067176 | pmc | 1,809 | {
"abstract": "ABSTRACT The triboelectric nanogenerator (TENG) is a recent mechanical energy harvesting technology that has been attracting significant attention. Its working principle involves the combination of triboelectrification and electrostatic induction. The TENG can harvest electrical energy from both solid–solid and liquid–solid contact TENGs. Due to their physical difference, triboelectric materials in the solid–solid TENG need to have high mechanical properties and the surface of the liquid–solid contact TENG should repel water. Therefore, the surface of the TENG must be versatile for applications in both solid–solid and liquid–solid contact environments. In this work, we develop a solid–solid/liquid–solid convertible TENG that has a slippery liquid-infused porous surface (SLIPS) at the top of the electrode. The SLIPS consists of a HDFS coated hierarchical Al(OH) 3 structure and fluorocarbon liquid. The convertible TENG developed in this study is capable of harvesting electricity from both solid–solid and liquid–solid contacts due to the high mechanical property of Al(OH) 3 and the water-based liquid repelling nature of the SLIPS. When the contact occurs in freestanding mode, electrical output was generated through solid–solid/liquid–solid sliding motions. The convertible TENG can harvest electricity from both solid–solid and liquid–solid contacts; thus, it can be a unified solution for TENG surface fabrication.",
"conclusion": "4. Conclusions In summary, we developed a solid–solid/liquid–solid convertible TENG using a PFPE infused surface. Using fluorine abundant materials, the SLIPS could be charged negatively when it came in contact with a counter-charged triboelectric material and utilized in both solid–solid and liquid–solid contact environments. Due to the negatively charged surface, the convertible TENG produced the highest peak V OC output of 122 V and peak I CC output of 6.4 μA when the contact material was solid nylon. In addition, the SLIPS on the convertible TENG was capable of repelling water-based liquids. The convertible TENG could produce 15–20 V peak voltages on average using various common used liquids. To demonstrate the applicability of the solid–solid/liquid–solid convertible TENG, a freestanding mode TENG was developed that could harvest electricity from the sliding mechanical motions of both solid and liquid materials. Therefore, the convertible TENG that can harvest electricity from both solid–solid and liquid–solid contacts can be unified solution for TENG surface fabrication.",
"introduction": "1. Introduction Owing to the rising demand for portable electronics, an increasing number of studies have focused on harvesting electrical energy from ambient sources, including solar [ 1 – 3 ], thermal [ 4 – 6 ], and salinity difference [ 7 – 9 ]. Among these, mechanical energy sources are suitable for harvesting electrical energy since they are less affected by external conditions, such as weather, temperature, and location. Several technologies have been developed for the effective conversion of mechanical energy into electricity, including piezoelectric transducers [ 10 – 12 ], and electromagnetic induction [ 13 – 15 ]. Among these technologies, the triboelectric nanogenerator (TENG), a recently developed mechanical energy harvesting technology, has been attracting significant attention; its working principle is based on the combination of triboelectrification and electrostatic induction [ 16 – 19 ]. In typical TENGs, the electrode is covered with a polymer material to maximize the surface charge, after which it is placed in contact–separation with a counter-charged triboelectric material to generate electricity [ 20 – 23 ]. This counter-charged triboelectric material can be either solid or liquid depending on the working condition [ 24 – 27 ]. Both solid–solid and liquid–solid contact TENGs have distinct characteristics due to their different physical phases. Due to this difference, these two TENGs require different material properties; triboelectric materials in solid–solid TENGs require high mechanical properties for a long lifespan, and the surface of liquid–solid contact TENGs needs to be water repellent for constant liquid separation [ 28 – 31 ]. Previous studies have presented these TENGs as separate devices; therefore, the triboelectric surfaces of TENGs were developed separately as well. However, for a TENG to harvest electricity from ambient mechanical energy sources such as wind and raindrops, it must be able to adapt to both solid–solid and liquid–solid contact environments. Therefore, a unified TENG surface that is capable of effectively harvesting electrical energy from both solid–solid and liquid–solid contacts is required. In this study, we develop a solid–solid/liquid–solid convertible TENG that has a slippery liquid-infused porous surface (SLIPS) at the top of its electrode. On this device, a low-surface tension fluorocarbon liquid (perfluoropolyether, PFPE, Krytox) was placed over a trichloro(1H,1H,2H,2H-perfluorooctyl) silane (HDFS)-coated Al(OH) 3 micro-/nanostructure on the aluminum surface. Due to the large number of fluorine atoms on the surface, both the PFPE liquid and HDFS-coated surface can be negatively charged during the contact–separation process, which can lead to the generation of electrical energy from the solid–solid contact. In addition, the surface can effectively repel water-based liquids; thus, it can induce constant contact and separation between the liquid and the TENG surface. The convertible TENG developed in this study could generate electrical energy when in contact with various water-based liquids, such as tap water, carbonated water, liquor, vinegar, and sports drink. When the contact occurred in the freestanding mode, electrical energy was generated from the solid–solid and liquid–solid sliding motions. Thus, this paper presents a unified TENG surface that can effectively harvest electrical energy from both solid–solid and liquid–solid mechanical input.",
"discussion": "3. Results and Discussion Figure 1 shows the schematic illustration and magnified images of the micro-/nanostructures on the aluminum surface. As shown in Figure 1(a ), the hierarchical structure of Al(OH) 3 is constructed on the aluminum surface. On the outer side of the Al(OH) 3 layer, a self-assembled monolayer coating of HDFS is fabricated. The PFPE liquid is applied to the hydrophobic hierarchical structure to form a SLIPS. The SLIPS is extremely liquid-repellent, and it can effectively repel water-based liquids [ 32 ]. In this study, 1 mL of liquid PFPE is applied to the hierarchical structure and spin-coated for 1 min at 500 rpm to form an evenly distributed thin liquid layer. Figures 1(b,c ) and S1 are the magnified images of the hierarchical structure taken by FE-SEM. As shown in the image, both a micro-sized stair-like structure ( Figure 1(b )) and a nano-sized wall structure ( Figure 1(c )) are formed on the aluminum surface. Figure 1. (a) Schematic illustration of the solid–solid/liquid–solid convertible TENG. SAM stands for self-assembled monolayer. Field emission scanning electron microscopy (FE-SEM) image of the (b) microstructures and (c) nanostructures on the aluminum surface In TENGs, selecting a material with a high surface charge is important. A high surface charge will facilitate the flow of electrons, which would, in turn, generate a relatively high electrical output. Generally, materials with a high electron affinity have a corresponding high surface charge. This accounts for the high usage frequency of fluoropolymers, such as polytetrafluoroethylene (PTFE), in TENGs. For comparison with the material used in this device, the PFPE liquid can be expressed as F-(CF(CF 3 )-CF2-O) n -CF 2 CF 3 , where n lies within the range of 10–60, and HDFS can be expressed as CF 3 (CF 2 ) 5 CH 2 CH 2 SiCl 3 . Both the PFPE liquid and HDFS contain a large number of fluorine atoms that has a high electron affinity. Therefore, the PFPE liquid-applied HDFS surface, which has a high negative surface charge, can be suitable for the solid–solid contact. In addition, the hierarchical structure on top of aluminum is that of Al(OH) 3 , which has more mechanical properties than PTFE [ 33 , 34 ]. The TENG generates electrical output through mechanical contact and friction; consequently, a long lifespan can be expected with high mechanical properties. Figure 2(a ) is a schematic illustration of the solid–solid contact TENG working principle, which is the same as that of the single electrode TENG [ 35 , 36 ]. As shown in the figure, the triboelectric material at the top is positively charged and the PFPE liquid-applied HDFS surface is negatively charged due to repeated contact and separation processes. The aluminum electrode at the bottom is affected by the electric field of the SLIPS. As external pressure is applied to the triboelectric material, its surface approaches the single electrode TENG surface. The electrical equilibrium of the aluminum electrode is disrupted by the electric field on the surface of the triboelectric material, and electrons flow into the aluminum electrode. When the triboelectric material contacts the SLIPS, the aluminum electrode attains electrical equilibrium once more. Once the external pressure is eliminated, the triboelectric material detaches from the SLIPS and electrons flow back to the electrical ground owing to the electric field of the SLIPS. By repeating this process, the TENG can produce alternating current (AC) by the contact–separation process between two solid materials. Figure 2. (a) Schematic illustration and working mechanism of the solid–solid contact TENG. (b) V OC and (c) I CC outputs of the convertible TENG depending on various materials. PVC, PI and PU stand for polyvinyl chloride, polyimide, and polyurethane, respectively Figure 2(b,c ) show the open-circuit voltage ( V OC ) and closed-circuit current ( I CC ) outputs of the device, respectively. The TENG was supplied with 6 Hz input using a mechanical vibration tester. As shown in the plot, the TENG generated the highest output when nylon came in contact with the SLIPS. The nylon contact produced high positive peaks, while the PTFE and PVC contacts produced high negative peaks. This is because the PTFE and PVC surfaces became negatively charged when they came in contact with the SLIPS, whereas the nylon surface became positively charged. The SLIPS was formed with the PFPE liquid and HDFS, and for it to produce the highest output when in contact with nylon, it should be negatively charged. When the contact material is nylon, the TENG produces a maximum V OC of 122 V and a maximum I CC of 6.4 μA. The TENG can generate electrical energy from liquid–solid contact as well because the SLIPS has an excellent liquid repellant property. Figure 3(a ) shows the working mechanism of the liquid–solid contact TENG [ 37 , 38 ]. In Figure 3(a ), the waterdrop becomes positively charged when it moves through the air and water pipe, and the SLIPS is negatively charged due to the constant contact and separation of the waterdrop. Due to the negatively charged SLIPS, the aluminum electrode will have a positive net charge. When the waterdrop approaches the SLIPS, the positively-charged waterdrop neutralizes the negatively charged SLIPS; therefore, the electrons will flow from the electrical ground to the aluminum electrode. After the waterdrop attaches completely, there will be a minimal surface area difference as the waterdrop moves toward the edge of the TENG. When the waterdrop separates from the TENG, the electrons will flow back to the ground. A repetition of the waterdrop contact and separation processes produces AC. Figure 3. (a) Working mechanism of the liquid–solid contact TENG. (b) Thickness difference of the PFPE liquid depending on the spin-coating process. (c) V OC output depending on the spin-coating process. (d) Photographs of the vinegar droplet on a 10°-slope SLIPS. (e) Average maximum peak voltage of the liquid–solid contact TENG depending on the liquid In a typical TENG, a thin layer of dielectric material is preferred to effectively induce charges on the electrode [ 39 , 40 ]. In this device, the thickness of the dielectric material is equal to the amount of the PFPE liquid remaining on the hierarchical structure. As shown in Figure 3(b–i ), the PFPE liquid forms a flat liquid film when initially applied. However, when spin-coated, it forms a thin liquid film along the hierarchical structure on the aluminum electrode ( Figure 3(b–ii) ). This thickness difference of the PFPE liquid affects the power generation of the device. For comparison, two devices with identical surfaces that have equal amounts of the PFPE liquid were prepared. Subsequently, one sample was spin-coated for 1 min at 500 rpm. Afterward, 2 mL of tap water was dropped on each sample from a height of 20 cm for electrical measurement. As shown in the plot of Figure 3(c ), the spin-coated devices produced a peak voltage approximately 5 times higher than that produced by the non-spin-coated device. This indicates that the PFPE liquid is able to properly charge the aluminum electrode when the PFPE liquid film is thin. In addition, the peak-like shape of Al(OH) 3 hierarchical structure accumulates the electrical charge and enhances the output accordingly. The SLIPS at the top of the aluminum electrode can repel water-based liquids effectively, including various liquids that are frequently used in everyday life. Figures 3(d ) and S2 are photographs of 30 μL-drops of various liquids on a SLIPS, which were taken at 2 s intervals. The surface was tilted 10° for the liquid drop to gravitate toward the edge due to gravitational force. The tested liquids are tap water, carbonated water, liquor (17.8% alcohol), vinegar, and sports drink. As shown in the images, all these liquids slipped to the ground without leaving liquid residues on the surface. Photograph of hierarchical structure without PFPE liquid is shown in Figure S3, after 100 mL of vinegar was poured. As shown in Figure S3, there are many liquid drops pinned on the surface after pouring. These liquid drops left from on the surface would lower the electrical potential difference between liquid and electrode resulting lower output [ 37 ]. When 2 mL-liquid drops were dropped from a height of 20 cm, each liquid produced electrical output, as shown in Figure 3(e ). The plot represents the maximum peak voltage when each liquid drop was dropped. Although the standard deviations of the voltage peaks are quite large due to the unconstrained nature of the drops, each liquid drop produced 15–20 V on average. This shows the possibility of producing electricity from common used water-based liquids using a SLIPS. A single-electrode-mode TENG discussed in previous paragraphs required an electrical ground for electrons to flow in between. For portable applications, having extra components, such as an electrical ground, can be a critical factor. Therefore, in Figure 4(a ), two aluminum electrodes with SLIPSs were attached to an acrylic substrate to generate electrical output in freestanding mode. In the freestanding mode, the TENG can effectively convert sliding mechanical input into electricity. Figure 4(a–i ) shows the solid–solid contact freestanding TENG, and Figure 4(a–ii ) shows the liquid–solid contact freestanding TENG. For the solid–solid contact TENG, nylon was used as the triboelectric material, and the sliding input was supplied manually (by hand). For the liquid–solid contact TENG, water was sprayed using a commercial shower head. The produced V OC output is shown in Figure 4(b,c ), and the current output is shown in Figure S4. As shown in Figures 4(b ) and S4(a), both the V OC and I CC show periodic outputs as the solid triboelectric material slides in between two electrodes. In contrast, Figures 4(c ) and S4(b) show rather random peak outputs due to the combination of waterdrops falling on to the surface randomly and waterdrops slipping to the ground. The electrical outputs by the solid triboelectric material and liquid drop show their possible application in unified-surface convertible TENGs. Figure 4. (a) Schematic illustration of the solid–solid/liquid–solid freestanding TENG. V OC output of the convertible TENG from (b) solid–solid sliding and (c) tap water spraying"
} | 4,099 |
33857631 | null | s2 | 1,812 | {
"abstract": "The recent development of synthetic biology has expanded the capability to design and construct protein networks outside of living cells from the bottom-up. The new capability has enabled us to assemble protein networks for the basic study of cellular pathways, expression of proteins outside cells, and building tissue materials. Furthermore, the integration of natural and synthetic protein networks has enabled new functions of synthetic or artificial cells. Here, we review the underlying technologies for assembling protein networks in liposomes, water-in-oil droplets, and biomaterials from the bottom-up. We cover the recent applications of protein networks in biological transduction pathways, energy self-supplying systems, cellular environmental sensors, and cell-free protein scaffolds. We also review new technologies for assembling protein networks, including multiprotein purification methods, high-throughput assay screen platforms, and controllable fusion of liposomes. Finally, we present existing challenges towards building protein networks that rival the complexity and dynamic response akin to natural systems. This review addresses the gap in our understanding of synthetic and natural protein networks. It presents a vision towards developing smart and resilient protein networks for various biomedical applications."
} | 334 |
33857631 | null | s2 | 1,813 | {
"abstract": "The recent development of synthetic biology has expanded the capability to design and construct protein networks outside of living cells from the bottom-up. The new capability has enabled us to assemble protein networks for the basic study of cellular pathways, expression of proteins outside cells, and building tissue materials. Furthermore, the integration of natural and synthetic protein networks has enabled new functions of synthetic or artificial cells. Here, we review the underlying technologies for assembling protein networks in liposomes, water-in-oil droplets, and biomaterials from the bottom-up. We cover the recent applications of protein networks in biological transduction pathways, energy self-supplying systems, cellular environmental sensors, and cell-free protein scaffolds. We also review new technologies for assembling protein networks, including multiprotein purification methods, high-throughput assay screen platforms, and controllable fusion of liposomes. Finally, we present existing challenges towards building protein networks that rival the complexity and dynamic response akin to natural systems. This review addresses the gap in our understanding of synthetic and natural protein networks. It presents a vision towards developing smart and resilient protein networks for various biomedical applications."
} | 334 |
35243332 | PMC8861577 | pmc | 1,816 | {
"abstract": "Biofilm formation is a ubiquitous process of bacterial communities that enables them to survive and persist in various environmental niches. The Bacillus cereus group includes phenotypically diversified species that are widely distributed in the environment. Often, B. cereus is considered a soil inhabitant, but it is also commonly isolated from plant roots, nematodes, and food products. Biofilms differ in their architecture and developmental processes, reflecting adaptations to specific niches. Importantly, some B. cereus strains are foodborne pathogens responsible for two types of gastrointestinal diseases, diarrhea and emesis, caused by distinct toxins. Thus, the persistency of biofilms is of particular concern for the food industry, and understanding the underlying mechanisms of biofilm formation contributes to cleaning procedures. This review focuses on the genetic background underpinning the regulation of biofilm development, as well as the matrix components associated with biofilms. We also reflect on the correlation between biofilm formation and the development of highly resistant spores. Finally, advances in our understanding of the ecological importance and evolution of biofilm formation in the B. cereus group are discussed.",
"conclusion": "6 Conclusions and future perspectives While extensive studies have been conducted addressing the mechanisms and applications of B. subtilis biofilms, much less attention has been paid to B. cereus group bacteria. This could be due to the complex phylogenetic relationship that creates diverse genotypes and phenotypes among B. cereus group bacteria. For example, the basic building blocks of biofilm matrix are still being debated, as well as the regulatory networks influencing biofilm production. For example, the role of epsA-O , the major polysaccharide locus in B. subtilis , seems to be unrelated to biofilm formation in B. cereus . Furthermore, the lack of genetic accessibility for most B. cereus isolates has delayed progress in this field. Nevertheless, some studies have focused on how to control biofilm contamination, while few studies have investigated the underlying mechanism underpinning biofilm formation. To develop highly efficient cleaning procedures, a deeper understanding of how B. cereus biofilms are regulated may be needed, especially during the dispersal period. As phage-mediated competition can influence biofilm structures, further understanding of how phages influence biofilm formation and evolution in the B. cereus group may be critical given the potential of phage treatment as an alternative antibacterial method. Regarding the pathogenic traits of B. cereus , one of the most critical issues is uncovering the relationship between biofilms, spores, and toxins. Although sporulation of the B. cereus group within biofilms has been documented, further study is needed to explore whether biofilms directly influence sporulation. Additionally, in B. cereus there is limited evidence of a relationship between toxin synthesis and biofilm development. Furthermore, the existence and evolution of biofilms in vivo , as well as their precise contribution to bacterial pathogenicity, have yet to be determined. Toxin production is vital for a foodborne pathogen, and sporulation or biofilm formation are likely to increase the risk of food poisoning. Investigations on the correlations between biofilms, spores, and toxins are needed, with a focus on biofilm evolution and gene expression. In terms of plant-associated biofilms, although it has been demonstrated that pre-engineered bacteria may rapidly turn into a plant endosymbiont, laboratory-based guided evolution of root colonization has been established only recently. Our understanding of plant-microbe interactions will be further facilitated by future studies on multispecies setups in these evolution experiments. Importantly, evolution experiments based on field trails should be conducted to gather data from natural settings, providing huge potential for optimizing biofertilizers based on B. cereus group isolates. In summary, the B. cereus group is a large group of bacteria with diverse phenotypes that form biofilms. Further knowledge in this area will help resolve problems in food contamination, and facilitate bioresource optimization in a strain-specific manner.",
"introduction": "1 Introduction Biofilms are bacterial communities living in a collective form that confers various advantages on the inhabitants, and cells in biofilms represent a higher level of organization than solitary cells [ 1 ]. Bacterial biofilms are ubiquitous and widespread in both natural and artificial environments. Cells in biofilms are encased in a self-produced matrix typically comprising exopolysaccharides (EPS), fiber proteins, and frequently also extracellular DNA (eDNA) [ 2 , 3 ]. The driving forces of the transition from a unicellular to a multicellular lifestyle are a rapidly-growing field of research, especially the evolutionary and ecological factors. Bacillus cereus sensu lato (s.l.) includes three main species; the foodborne pathogen Bacillus cereus , the biopesticide control agent Bacillus thuringiensis , and the anthrax-causing pathogen Bacillus anthracis [ 4 , 5 ]. High levels of genome similarity between these three species of B. cereus sensu lato makes their taxonomical classification difficult to discern [ 6 ]. Importantly, the ecological niches of B. cereus s.l. are widely distributed among soil, plant rhizosphere, and arthropod and nematode guts [ [7] , [8] , [9] , [10] , [11] ]. The highly diversified ecology of B. cereus s.l. is also reflected by the fact that both probiotic and pathogenic traits have been identified in the group [ 12 ]. Furthermore, besides being widely commercialized as pesticides, strains of the B. cereus s.l. group have also been exploited as plant growth-promoting bacteria (PGPB), suggesting an intrinsic ability to colonize plants [ 13 , 14 ]. B. cereus isolates vary in their physiological properties and survival abilities under different stress conditions. Nevertheless, the formation of biofilms by B. cereus strains is a universal trait that facilitates survival and persistence in harsh environmental conditions [ 5 , 15 ]. Most scenarios, such as colonization of plant rhizosphere and soil, are related to the sessile state of bacterial biofilms. For instance, B. cereus colonizes plant roots by forming biofilms. The tasA gene is an essential gene for Bacillus subtilis biofilms, and its paralog is needed for root colonization in B. cereus [ 16 , 17 ] . B. cereus biofilms are known to be the source of device contamination in clinical settings and in food industries [ 18 ]. Furthermore, the production of endospores during the late developmental stage complicates the removal of biofilms during the cleaning process due to the ability of spores to survive heating and irradiation processes [ 19 ]. Owing to the persistence of biofilms and the secretion of potential enterotoxins such as nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), and cytotoxin K (CytK), a considerable amount of research has focused on strategies to prevent biofilm formation or remove mature biofilms, which has been systematically reviewed in other studies [ 20 , 21 ]. The bacterial biofilm lifestyle is a cyclic process for most if not all species, involving at least five phenotypically distinct stages [ 22 ]; a complete biofilm cycle typically includes initial attachment, irreversible attachment, biofilm maturation, initiation of biofilm dispersion, and dispersal. Among these stages, studies on B. cereus biofilms have mostly focused on the first three stages, especially the involvement of biofilm matrix components, the role of flagella, and regulatory networks. Similarly, these developmental stages have been extensively explored for B. subtilis biofilms, and comparative studies have uncovered both shared and distinct molecular mechanisms between these two species [ 23 ]. For instance, EPS synthesized by the coded enzymes of the epsA–O operon in B. subtilis is one of the main extracellular matrix components, while its homolog has a minor role in B. cereus biofilms. The genomes of B. cereus lack paralogs of bslA and tapA genes in B. subtilis , whereas there are two paralogs of B. subtilis tasA [ 17 , 24 ]. Various in-depth studies into the B. cereus biofilm lifestyle are being driven by these variations in biofilm formation between the two species. In the previous decade, a substantial amount of knowledge about biofilm formation in the. B. cereus group has been acquired through a wide field of research topics. This review summarizes recent advances in our knowledge of both the mechanisms and applications governing biofilm formation in B. cereus s.l. We explore advances in B. cereus biofilm formation within the context of global regulation and the components of the biofilm matrix, and expand on the heterogeneity within biofilm structures. Finally, we address advances in terms of ecological importance of several aspects including plant-associated biofilms and food industry contamination."
} | 2,295 |
38264162 | PMC10804224 | pmc | 1,818 | {
"abstract": "Abstract Multicellular eukaryotic organisms are hosts to communities of bacteria that reside on or inside their tissues. Often the eukaryotic members of the system contribute to high proportions of metagenomic sequencing reads, making it challenging to achieve sufficient sequencing depth to evaluate bacterial ecology. Stony corals are one such complex community; however, separation of bacterial from eukaryotic (primarily coral and algal symbiont) cells has so far not been successful. Using a combination of hybridization chain reaction fluorescence in situ hybridization and fluorescence activated cell sorting (HCR-FISH + FACS), we sorted two populations of bacteria from five genotypes of the coral Acropora loripes , targeting (i) Endozoicomonas spp, and (ii) all other bacteria. NovaSeq sequencing resulted in 67–91 M reads per sample, 55%–90% of which were identified as bacterial. Most reads were taxonomically assigned to the key coral-associated family, Endozoicomonadaceae, with Vibrionaceae also abundant. Endozoicomonadaceae were 5x more abundant in the ‘ Endozoicomonas ’ population, highlighting the success of the dual-labelling approach. This method effectively enriched coral samples for bacteria with <1% contamination from host and algal symbionts. The application of this method will allow researchers to decipher the functional potential of coral-associated bacteria. This method can also be adapted to accommodate other host-associated communities.",
"conclusion": "Conclusion Our findings show that HCR-FISH + FACS is a substantially improved method to obtain host-associated bacteria for metagenome sequencing, where standard metagenomic techniques do not work for low-abundant bacteria due to noise from more common species (typically the host species).Our method makes the analysis of uncharacterized microbes simpler and more accessible, and provides researchers with an enhanced platform to address the grand challenge of deciphering the functions of host-associated bacteria in symbiosis. As our method can be implemented in holobionts other than coral, we believe that this innovative approach holds promise for advancing the field of microbial ecology.",
"introduction": "Introduction It is now widely accepted that all animals and plants depend on bacteria and other microbes for their health and functioning (McFall-Ngai et al. 2013 , Mueller and Sachs 2015 , Sessitsch et al. 2023 ). In eukaryotic hosts, bacteria have been shown to play roles in processes and traits as diverse as immunity, development, digestion of food, adaptation to different environmental conditions, mate choice and other behaviours (McFall-Ngai et al. 2013 , McCutcheon 2021 ). Corals are a notable example, as these marine cnidarians associate with several groups of microbes critical to their health and survival, including bacteria (Blackall et al. 2015 , Bourne et al. 2016 , Maire et al. 2022 , Mohamed et al. 2023 ). While genomic approaches have provided an in-depth understanding of the composition of bacterial communities associated with reef-building corals (van Oppen and Blackall 2019 ), the functions of the bacteria within the coral holobiont (i.e. the coral animal and its associated microbiota) are still poorly understood (Sweet and Bulling 2017 ). Because not all coral-associated bacteria can be isolated with conventional methods, high-quality assembled genomes derived from metagenomic analyses can be used in their absence to study whole communities. The close symbiotic relationships bacteria have with their coral hosts, in combination with the lack of host reference genomes, make it difficult to eliminate contaminating coral DNA for subsequent analyses. One approach to achieve sufficient bacterial read depth is to physically isolate them from other microorganisms and eukaryotic cells prior to DNA extraction and sequencing (Grieb et al. 2020 ). Fluorescence in situ hybridization (FISH) can assist with this enrichment process. FISH was introduced >30 years ago as a valuable molecular tool to detect specific DNA or RNA sequences using complementary DNA- or RNA-probes labelled with fluorescent dyes (DeLong et al. 1989 ). Host-associated bacterial identification by standard FISH methods (i.e. the use of oligonucleotide probes labelled at either the 5′ or 3′ end with a single fluorophore) targeting ribosomal RNA (rRNA) has been explored in corals (Ainsworth et al. 2006 , Apprill et al. 2012 , Ainsworth et al. 2015 , Damjanovic et al. 2019 , Maire et al. 2023 ), but suffers from several limitations, such as host autofluorescence and non-specific probe binding to certain host cells and structures, that may prevent the successful detection of target organisms (Wada et al. 2016 ). Autofluorescence associated with corals is the direct result of high densities of chlorophyll-containing dinoflagellates within the coral tissue and an abundance of host-derived fluorescent proteins, including green, red, cyan, and orange fluorescent protein-like molecules (reviewed in Alieva et al. 2008 , Wada et al. 2016 ). Further, target bacterial cells could be in low abundance in some compartments of the coral animal (Maire et al. 2021 ) or might not be detected due to low ribosome content (Poulsen et al. 1993 ), or lack of permeabilization, with additional unsatisfactory signal-to-noise ratio. While some variations of FISH can amplify signal to address these challenges, such as catalysed reporter deposition (CARD)-FISH, they often require reagents that damage DNA (Keller and Pollard 1977 ) making downstream genomic analyses difficult. Instead, hybridization chain reaction (HCR)-FISH (Choi et al. 2010 , Yamaguchi et al. 2015a ) can boost the probe signal and transcend specific limitations ranging from low signal detection to the interference of host autofluorescence. In this approach, a specific oligonucleotide probe complementary to the rRNA target, carrying an initiator sequence, is hybridized to the cells. Next, two fluorescently labelled hairpin oligos (X1 and X2) bind subsequently in a chain reaction to the initiator sequence, thus multiplying the fluorescent signal. HCR-FISH has previously been paired with fluorescence activated cell sorting (FACS) to sort bacteria from environmental samples for single cell genomics (Grieb et al. 2020 ) but has yet to be applied to animals hosting complex communities of microorganisms. In this study, we developed a combined HCR-FISH + FACS pipeline for the targeted retrieval of bacteria from coral tissues for metagenomic sequencing using the coral Acropora loripes as a model. Coral 16S rRNA gene metabarcoding studies have revealed that while many corals associate with several hundred and sometimes even more than a thousand different bacterial taxa (amplicon sequence variants [ASVs]) (Blackall et al. 2015 ), adult colonies of A. loripes partner with as few as 20–30 ASVs with only one or a few Endozoicomonas ASVs dominating the communities (Damjanovic et al. 2020 ). We developed our protocol using mock bacterial communities and A. loripes tissue to select appropriate fluorophores and FACS gates to sort two populations: 1) ‘ Endozoicomonas ’ and 2) ‘all-bacteria’.",
"discussion": "Results and Discussion HCR-FISH + FACS Traditional in-solution FISH (Hugenholtz et al. 2001 ) was used in preliminary trials during the development of this method. However, we were unable to sort pure labelled bacteria from some autofluorescent coral cells as evidenced by the presence of mixed populations, observed by confocal laser scanning microscopy post FACS. To combat this issue, we successfully employed HCR-FISH (Yamaguchi et al. 2015a , b , Grieb et al. 2020 ) to label and sort two populations of bacteria in coral tissue homogenates from A. loripes . This study represents the first application of HCR-FISH in-solution for the enrichment of bacteria from holobiont samples, in this case corals. To date, HCR-FISH has been used to overcome high autofluorescence to visualize bacteria in tissue sections in one coral study (Wada et al. 2022 ), but it has also been applied to anemones (Goffredi et al. 2021 ) and the bobtail squid (Nikolakakis et al. 2015 , Moriano-Gutierrez et al. 2019 ). CARD-FISH has been used on histology sections to better resolve bacteria within autofluorescent shallow (Chiu et al. 2012 , Neave et al. 2016 ) and deep-water (Thompson and Gutierrez 2021 ) coral tissues, but this approach requires reagents that damage DNA (Keller and Pollard 1977 ). CARD-FISH would therefore not be suitable for post-labelling metagenomic applications. Previous FACS work in corals have prepared cell suspensions by mechanical disruption after incubation in calcium free media (Rosental et al. 2017 , Levy et al. 2021 ). Here, the dissociation of cells was accomplished using enzymatic tools rather than mechanical approaches to fully dissociate cells and reduce bacterial contamination. Future applications of this method should consider quantifying bacterial load via quantitative or digital PCR to compare the efficiency of a mechanical versus enzymatic approach. This step could be improved further by visualizing cells after dissociation to compare methods. Sorting of FISH-labelled bacterial cells has previously been done using standard FISH on mixed bacterial cultures (Wallner et al. 1997 ), sludge from a bioreactor (Miyauchi et al. 2007 ), or marine sediment (Kalyuzhnaya et al. 2006 ), CARD-FISH on seawater samples (Sekar et al. 2004 ), or HCR-FISH on marine phytoplankton samples (Grieb et al. 2020 ). These studies have sequenced PCR products of specific genes like the 16S rRNA gene from sorted cells. Whole genome sequencing has been attempted from FISH labelled and sorted cells, but the recovered genomes suffered from low completeness (Podar et al. 2007 , Yilmaz et al. 2010 ). Using physical parameter properties FSC and SSC, and fluorescent probes, we established conditions to differentiate bacteria from non-bacterial particles and to distinguish and separate ‘ Endozoicomonas ’ (dual labelled) and ‘all-bacteria’ (labelled with EUBMix338 only) populations (Fig. 2 ; Fig. S1 ). FACS is a highly sensitive technique for detecting and measuring fluorescence signals from individual cells or particles with high precision and resolution. So, while the sorted populations were visually free of host cells and debris (Fig. 3 ), the sensitivity of confocal microscopy may be limited compared to FACS due to factors such as background noise and detection efficiency. Because coral tissue is known to exhibit non-specific binding of the EUBMix338 probe (Wada et al. 2016 ), we also trialled HCR-FISH+FACS with a nonsense probe, NONEUB338, which has a nucleotide sequence complementary to the nucleotide sequence of probe EUBMix338 (Christensen et al. 1999 ). These trials were inconclusive because the NONEUB338 initiator sequence can bind to the 16S rRNA gene in the bacterial DNA and, with HCR-FISH amplification, the signal is sufficient for FACS detection. Future studies should consider ways to address non-specific binding with HCR-FISH+FACS, such with the addition of a blocking reagent (Yamaguchi et al. 2015a ). Figure 3. A labelled A. loripes sample (colony Al13) prior to sorting with FACS (A–C) or sorted cell populations that were single (‘all-bacteria’, D–F), or dual (‘Endozoicomonas’, G–I) labelled. Each sample was visualized on a Nikon A1R confocal laser scanning microscope with channels for brightfield (A, D, G), 561 nm excitation (B, E, H), and 405 nm excitation (C, F, I). S=Symbiodiniaceae, Cn=host cnidocyst, H=uncharacterised host cell, B=bacteria. All scale bars are 25 µm. Metagenomics Merging all data for the ‘ Endozoicomonas ’ and ‘all-bacteria’ populations resulted in 326 M and 307 M read pairs, with 313 M and 297 M read pairs remaining after quality filter and trimming by trimmomatic, respectively. Of these, only 0.68% of reads in the ‘ Endozoicomonas ’ and 0.01% in the single-labelled ‘all-bacteria’ population aligned to the reference host A. loripes genome (Salazar et al. 2022 ). Of the total reads, 169.3 M and 181.5 M from the ‘ Endozoicomonas ’ and ‘all-bacteria’ populations, respectively, were used to assemble contigs. A total of 4785 and 4594 contigs were assembled from the ‘all-bacteria’ and ‘ Endozoicomonas ’ populations, respectively (Table 3 ). For the ‘all-bacteria’ contigs, 822 were taxonomically identified as eukaryotes (12.0% reads), 2033 contigs were unidentified (18.1% reads), and 1930 contigs were identified as bacteria (69.8% reads). Of those bacterial contigs, the most abundant families were Endozoicomonadaceae (584 contigs, 9.1% reads), Sporolactobacillaceae (160 contigs, 1.1% reads), Peptostreptococcaceae (74 contigs, 1.9% reads), and Vibrionaceae (36 contigs, 0.2% reads). However, the contigs were generally short (Endozoicomonadaceae mean±SD 5 459±3790 bp), with highly variable fold coverage (Endozoicomonadaceae ranges 5–37 744x coverage, mean±SD 1188±3658x). There were only nine symbiodiniacean and four cnidarian contigs, highlighting the success of the sorting in enriching samples for bacteria. For the ‘ Endozoicomonas ’ contigs, 196 were identified as cnidarian (0.4% reads), three were apicomplexan (0.007% reads), and 18 were Symbiodiniaceae (0.1% reads). Of the remaining contigs, 890 were unidentified (12.0% reads), one contig (0.1% reads) was identified as a virus, and 3 055 contigs were identified as bacteria (75.4% reads; Table 3 ). Of the bacterial contigs, by far the most abundant family was Endozoicomonadaceae with 1298 contigs (47.8% reads). These contigs averaged 7705±7346 bp (mean±SD), had 75 081–5x fold coverage (mean±SD 2538 ± 5429x) and total length of 10 Mbp. Table 3. Stats from metagenomic data analysis for the ‘Endozoicomonas’ and ‘all-bacteria’ populations. Sorted population Raw reads (M) Reads after trimmomatic (M) Read pairs after removing A. loripes (M) Reads used to assemble contigs (M) Contigs Total contig length Mbp Max/Avg contig length (bp) N50 Avg. fold coverage #contigs no hit—read pairs (M) #contigs euk—read pairs (M) #contigs bacteria—read pairs (M) # of Endozoicomonadaceae contigs—read pairs (M) \n ‘Endozoicomonas’ (P5) 326 313 311 169.3 4 594 27.4 165 029/5 972 7 643 1 434 890–20.3 648–21.1 (18 Symbiodiniaceae; 196 Cnidarian) 3 055–127.6 1 298–81.0 ‘All-bacteria’ (P3) 307 297 297 181.5 4 785 24.9 102 783/5 207 6 059 1 100 2 033–32.9 822–21.9 (9 Symbiodiniaceae; 4 Cnidarian) 1 930–126.7 584–16.6 While Endozoicomonadaceae reads were still present in the ‘all-bacteria’ population, they were ∼5-fold less abundant than in the ‘ Endozoicomonas ’ population (9.1% reads in ‘all-bacteria’ versus 47.85% reads in ‘ Endozoicomonas ’; Supp. File 2 ), highlighting the success of the dual-labelling approach. Because Endozoicomonas spp. are highly abundant in A. loripes (Damjanovic et al. 2020 ), by concentrating this taxon in one population of sorted cells we hoped to provide greater read depth of rare or novel taxa. This is apparent in the ‘all-bacteria’ population, where 52% of the bacterial reads ( Supp. File 2 ) were unidentified, suggesting that they represent novel environmental microbes not yet present in the GTDB reference database. This is compared to only 22% of bacterial reads assigned as no support in the ’ Endozoicomonas ’ population ( Supp. File 2 ). The low level (<1%) of coral and algal symbiont contamination in the metagenomic sequence data for our coral tissue samples is unique. Previous work to enrich prokaryotes from coral samples prior to metagenomic sequencing (Table 4 ) have applied percoll gradient fractionation (Wegley et al. 2007 , Dinsdale et al. 2008 , Vega Thurber et al. 2009 , Littman et al. 2011 ), differential centrifugation (Keller-Costa et al. 2021 , Keller-Costa et al. 2022 ), or sequential filtration (Robbins et al. 2019 ). These approaches however have been unsuccessful in providing researchers with metagenomic data dominated by bacterial reads, in part because percoll fractionation to enrich for bacteria will also capture host mitochondria (Wegley et al. 2007 ), and residual host DNA can contaminate centrifugation and filtration strategies. When coral tissue has been sequenced more recently without enrichment for prokaryotes, reads from the eukaryotic populations accounted for over 90% of the reads in most cases (Roach et al. 2020 , Rosales et al. 2022 ). Our enrichment method implementing HCR-FISH prior to FACS is the first to average >70% bacterial reads from coral tissue samples. Table 4. Details from existing stony coral metagenomics work where the primary sequencing target was coral-associated microbes. *The % of total reads that were classified as bacterial from the authors chosen reference database†. Where studies collected metagenomes from corals and other substrates (i.e. seawater, algae), only the data from the corals is included. Coral Species Geographic location Target Enrichment method Classified bacterial sequences* (% of total reads) Classification database† Sequencing Platform Reference \n Porites asteroides \n Caribbean All microbes Percoll fractionation 1.5% SEED 454 Pyrosequencing (Wegley et al. 2007 ) \n Porites compressa \n Hawaii All microbes Percoll fractionation 0.6–1.0% SEED 454 Pyrosequencing (Dinsdale et al. 2008 , Vega Thurber et al. 2009 ) \n Acropora millepora \n Magnetic Island, GBR All microbes Percoll fractionation 0.2–0.9% SEED 454 Pyrosequencing (Littman et al. 2011 ) \n Platygyra carnosa \n Lamma Island and Crescent Bay, South China Sea Bacteria Percoll fractionation 0–40% NCBI NR Illumina HiSeq (Cai et al. 2017 ) \n Porites lutea \n Orpheus Island, GBR Bacteria and archaea Sequential filtration 0.2–15% Greengenes/GTDB Illumina HiSeq2500 (2×150 bp) (Robbins et al. 2019 ) \n Pseudodiploria strigosa Orbicella faveolata \n Curaçao, Caribbean All microbes NA 0.93% SEED Illumina MiSeq (2×300 bp) (Roach et al. 2020 ) \n Acropora tenuis Goniastrea minuta Pocillopora verrucosa Pocillopora meandrina \n Xiane Reef, South China Sea All microbes NA <20% in healthy corals; 33–79% in bleached corals NCBI NR Illumina HiSeq (2×150 bp) (Sun et al. 2020 ) \n Pseudodiploria strigosa \n Bermuda Bacteria in coral surface mucus layer (SML) Modified syringe used to collect SML only 50.2% average MG-RAST (Refseq/SEED) Illumina MiSeq (Lima et al. 2022 ) \n Stephanocoenia intersepta, Diploria labyrinthiformis, Dichocoenia stokesii , and Meandrina meandrites Looe Key and East Washerwoman Reef, Florida Keys All microbes in tissue and mucus NA 19.2% average (skewed by one sample); ranged 0.9–67.3% GTDB Illumina HiSeq 4000 (Rosales et al. 2022 ) \n Porites lutea Goniastrea edwardsi \n Abu Shoosha Reef, Red Sea Skeleton bacteria Fragmentation to remove tissue Not provided GTDB Illumina HiSeq 4000 (2×151 bp) (Cardenas et al. 2022 ) \n Porites lutea Isopora palifera \n Heron Island, GBR Skeleton bacteria and archaea Coral tissue was removed from the fragments using a Waterpik and sterile seawater; the remaining skeletons were snap frozen. Not provided GTDB DNBSeq (Tandon et al. 2023 ) 270 samples from Pocillopora, Porites , and Millepora Tara Pacific expedition All microbes NA Not provided Centrifuge v1.0.3, NCBI NR Illumina NovaSeq6000 or HiSeq4000 (Belser et al. 2023 , Hochart et al. 2023 , Lombard et al. 2023 ) \n Acropora loripes \n Davies and Backnumbers Reef, GBR Tissue Bacteria HCR-FISH + FACS 72.6% average GTDB Illumina NovaSeq6000 SP (2×150 bp) This study When completing metagenomic sequencing on compartments of the coral holobiont that do not include tissue, contamination by host or other eukaryotic reads is less prominent. Tandon et al. ( 2023 : effectively assembled 393 high-quality metagenome assembled genomes (MAGs) from coral skeleton fragments by sequencing samples on individual lanes; in only two samples were there >45% host reads. Cardenas et al. ( 2022 ) were able to achieve metagenome sequences that were dominated (∼75%) by bacterial reads when working with skeletal material. However, to compare bacterial communities between skeleton and tissue they had to use 16S rRNA gene metabarcoding. Metagenomics of the surface mucus layer (SML) of a Caribbean coral species contained ∼50% of reads that were identified as bacteria with no additional enrichment required (Lima et al. 2022 ). MDA (Dean et al. 2002 ) was used in this study to obtain sufficient DNA from sorted bacteria. However, it has drawbacks such as amplification bias (Ahsanuddin et al. 2017 ), poor uniformity, errors and artifacts, low genome coverage, inability to address all variant classes, low accuracy, poor reproducibility, and/or complex protocols that are difficult to automate or scale. Reads generated in this study were heavily skewed toward some regions of bacterial genomes resulting in orders of magnitude differences in coverage and an inability to generate MAGs, which is likely a result of uneven amplification during the 8 hrs of MDA. Ideally, researchers should aim to collect enough cells so that amplification is not necessary. In these cases, DNA can be extracted with low biomass-input methods (Bramucci et al. 2021 ). When this is not possible, a potential alternative to MDA is primary template-directed amplification (PTA) (Gonzalez-Pena et al. 2021 ). PTA is an isothermal whole genome amplification method that reproducibly captures near-complete genomes of single cells while suppressing the formation of experimental artifacts such as chimeric molecules and non-specific priming (Telenius et al. 1992 ). PTA may be performed directly on DNA from single cells (collected by FACS, microfluidic or other methods), multiple cells, or ultra-low inputs of DNA (>4 pg– 10 ng). Future applications of this method to enrich complex communities for bacteria prior to metagenomics should use caution with MDA and amplify for the shortest duration of time required to get sufficient DNA for sequencing."
} | 5,488 |
31160678 | PMC6547642 | pmc | 1,819 | {
"abstract": "Ambient vibration energy is highly irregular in force and frequency. Triboelectric nanogenerators (TENG) can convert ambient mechanical energy into useable electricity. In order to effectively convert irregular ambient vibrations into electricity, the TENG should be capable of reliably continuous operation despite variability in input forces and frequencies. In this study, we propose a tandem triboelectric nanogenerator with cascade impact structure (CIT-TENG) for continuously scavenging input vibrations with broadband frequencies. Based on resonance theory, four TENGs were explicitly designed to operate in tandem and cover a targeted frequency range of 0–40 Hz. However, due to the cascade impact structure of CIT-TENG, each TENG could produce output even under non-resonant conditions. We systematically studied the cascade impact dynamics of the CIT-TENG using finite element simulations and experiments to show how it enables continuous scavenging from 0–40 Hz even under low input accelerations of 0.2 G–0.5 G m/s 2 . Finally, we demonstrated that the CIT-TENG could not only scavenge broadband vibrations from a single source such as a car dashboard, but it could also scavenge very low frequency vibrations from water waves and very high frequency vibrations from air compressor machines. Thus, we showed that the CIT-TENG can be used in multiple applications without any need for redesign validating its use as an omnipotent vibration energy scavenger.",
"introduction": "Introduction Triboelectrification is a ubiquitous and naturally occurring phenomena that occurs when two different materials come into contact and rub against each other. The effect can be constructively used for various applications such as nano-patterning 1 , self-assembly 2 , recycling 3 , smoke filtration 4 , and mechanical energy harvesting using triboelectric nanogenerators (TENGs) 5 . Due to the increasing demand for renewable energy, there has been considerable interest shown in TENG based energy harvesting systems by the research community, and several approaches ranging from material processing to structural design have been proposed to improve and regulate its output 6 – 8 . Vibration energy harvesters based on TENGs rely on resonance design, that is, the natural frequency of the TENG vibration system should approximately equal the input source vibration frequency. The natural frequency of the TENG in turn depends on parameters such as mass and stiffness which can be selected based on target frequency 8 . Furthermore, under non-linear impact the output frequency response of a vibration TENG stiffens near the resonance region resulting in a broadband output which depends on level of input forces and gap distance between the moving and fixed layers of the TENG 9 , 10 . It is important for a vibration energy harvester to have broadband operation since ambient vibration energy is highly irregular in force and frequency. In order to increase the operation bandwidth, the idea of tandem TENGs was proposed by the authors where helical coil spring based vibration TENGs were explicitly designed to operate at specific target frequencies, and then stacked together as one system to achieve the desired bandwidth coverage 10 . Similar tandem systems have been proposed for cantilever beam spring based vibration TENGs 11 and fixed-fixed beam spring based vibration TENGs 12 . However, under low input vibration forces the typical tandem TENGs have gaps in their output frequency response resulting in non-continuous scavenging. Previously some research works have proposed frequency up-conversion structures for piezoelectric 13 and electromagnetic 14 vibration energy harvesters. In these frequency up-conversion structures, two vibration systems are employed, one designed to oscillate at low frequency and the other at high frequency. When the low frequency vibration system is in resonance, it impacts with the high frequency vibration system resulting in the translation of low frequency oscillations into high frequency oscillations. Similarly, high frequency oscillations can be translated into low frequency in frequency down-conversion structures. In this work we show that by adopting ideas from frequency up-conversion structures we can achieve continuous scavenging even under low input vibration forces. Furthermore, not only do different ambient vibration sources have different frequencies, but even a single vibration source can have ever changing attributes. For example, sea waves typically have very low frequencies, but they can change their features as they approach the shore 15 ; car vibrations, such as on the front dashboard or the rear deck can depend on suspension design, road conditions and the car speed 16 ; other machine vibrations such as from engines or motors can have several frequency components at low as well as high frequencies 17 . Thus, in this work we propose an all-purpose and omnipotent vibration energy harvester that can operate continuously under any variations in the properties of the vibration input source. We investigate a tandem TENG with cascade impact structure (CIT-TENG) that shows continuous operation within target frequency range even under low input vibration forces. The CIT-TENG uses four layers of TENGs such that the resonating TENG impacts with the next TENGs thereby producing a domino impact effect. Due to cascade impact, the TENGs that are non-resonating can also produce output thereby contributing the bandwidth broadening. The non-linear dynamics of the CIT-TENG were analyzed using finite element simulation as well as experimentally. The continuous scavenging of CIT-TENG under different levels of input forces was verified and compared to the non-continuous scavenging of a typical tandem TENG. Finally, omnipotence of the CIT-TENG was demonstrated by using the same device for harvesting low frequency vibrations from water waves, high frequency vibrations form an air compressor machine, and broadband frequency vibrations from a car dashboard.",
"discussion": "Results and Discussion Figure 1a conceptually illustrates that the CIT-TENG can continuously scavenge broadband frequency vibrations from multiple ambient sources such as water waves, car dashboard, and air compressor machines. The reader is pointed to Supporting Fig. S1 for construction and operation of a basic vertical contact separation mode TENG. The CIT-TENG employs four such vertical contact separation mode TENGs as shown in the photograph Fig. 1b and cross-section schematic in Fig. 1c . The TENGs were each explicitly designed to resonate at natural frequencies of 8 Hz, 24 Hz, 32 Hz, and 40 Hz, respectively. Figure 1c also illustrates the cascade impact mechanism, where the resonating motion of activated TENG-1 causes impact with TENG-2 which propagates through the rest of the structure resulting in impact between TENG-2 and TENG-3, and between TENG-3 and TENG-4. Figure 1d shows the limitation of a typical tandem TENG structure for scavenging irregular frequency vibrations. The voltage output frequency response shows non-continuous scavenging under low input acceleration of 0.2 G m/s 2 . Supporting Note 1 describes the fabrication and assembly of the simply stacked structure type typical tandem TENG, Supporting Fig. S2 shows its photograph and Supporting Fig. S3 shows its frequency response under input accelerations of 0.2G-0.5 G m/s 2 . It was observed that under the higher input accelerations of 0.4 G m/s 2 and beyond, the output frequency response could be adequately continuous, however under low input accelerations of 0.2 G–0.3 G m/s 2 , there were significant gaps in the response where the output was zero. This is because in this simply stacked structure of the typical tandem TENG, each TENG vibrates in solitary fashion with no interaction with the other TENGs. The CIT-TENG, on the contrary, has considerable interactions between the individual TENGs, and thus shows continuous energy scavenging within the targeted frequency range of 40 Hz even under low input acceleration of 0.2 m/s 2 as shown in Fig. 1e . Detailed photograph of the CIT-TENG is shown in Supporting Fig. S4 and its frequency response under input accelerations of 0.2G–0.5 G m/s 2 is shown in Supporting Fig. S5 . As the input acceleration level of the vibration shaker was increased from 0.2 G m/s 2 to 0.5 G m/s 2 , the CIT-TENG output levels also increased, and no gaps were observed in the response. Thus, the proposed CIT-TENG structure is superior to the previously proposed tandem TENG structure since it can operate continuously within the targeted frequency range even under low input accelerations. Figure 1 Basic idea behind CIT-TENG. ( a ) Conceptual illustration showing that CIT-TENG can continuously scavenge broadband frequency vibrations from multiple ambient sources. ( b ) Photograph of actual CIT-TENG developed in this work. ( c ) Cross-section schematic of CIT-TENG design and the cascade impact behavior initiated by activated TENG-1. ( d ) Voltage output frequency response of typical tandem TENG showing non-continuous scavenging at input acceleration of 0.2 G m/s 2 . ( d ) Voltage output frequency response of CIT-TENG showing continuous scavenging within 40 Hz bandwidth at input acceleration of 0.2 G m/s 2 . The design of each vibration TENG involved the consideration of its natural resonance frequency as well as the bandwidth broadening due to impact stiffening. If the vibration TENG mass and stiffness are represented as m and k , respectively, then based on the resonance equation, the TENG natural frequency is given as, \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$2\\pi {f}_{n,sys}=\\sqrt{k/m}$$\\end{document} 2 π f n , s y s = k / m . Accordingly, in order to achieve the target natural resonance frequencies of 8 Hz, 24 Hz, 32 Hz, and 40 Hz for each of the TENGs, their mass and stiffness values were determined as 40 grams and 25*4 N/m for TENG-1, 30 grams and 160*4 N/m for TENG-2, 38 grams and 400*4 N/m for TENG-3, and 26 grams and 400*4 N/m for TENG-4, respectively. The reader is pointed to our previous work 10 for details on how to select the mass and stiffness values. In this study, we employed finite element simulation using ABAQUS/CAE software to determine the vibration dynamics of each TENG. However, for completeness, the vibration equations of motion for the individual TENGs during no-impact and impact conditions is described in Supporting Note 2. Supporting Movie S1 shows the simulated vibration dynamics of the CIT-TENG structure at sinusoidal input frequencies of 10 Hz, 20 Hz, 30 Hz, and 40 Hz, under an input acceleration on 0.5 G m/s 2 . The first point of observation from the movie is that different TENGs are actively resonating during each of these input frequencies, related to the natural resonance frequency of the respective TENG. The second point of observation is that the motion of a single TENG initiates cascade impact behavior amongst adjacent TENGs making them vibrate at their non-resonant frequencies. The simulated displacement time response of each TENG at 10 Hz, 20 Hz, 30 Hz, and 40 Hz input frequencies is provided in Supporting Fig. S6 . It can be observed from the simulation results that not only do the resonating TENG (corresponding to the input frequency) show high amplitude motion, but also the non-resonating TENGs show significantly large amplitudes due to cascade impact initiation. In order to better understand the dynamic behavior each TENG at their resonating and non-resonating frequencies, their frequency responses were simulated and validated experimentally. Figure 2a shows simulation image from Supporting Movie S1 indicating the actively resonating vibration of TENG-1. Figure 2b shows the simulated amplitude frequency response results for TENG-1 under input accelerations of 0.1 G m/s 2 , 0.3 G m/s 2 , and 0.5 G m/s 2 . Figure 2c shows the corresponding voltage output frequency response results for TENG-1 under input acceleration of 0.5 G m/s 2 . It can be seen from the simulation and experimental results that even though the natural resonance frequency of TENG-1 is 8 Hz, due to cascade impact the TENG-1 output can be high even at non-resonant frequencies such as when TENG-2 and TENG-3 are in resonance, due to frequency down-conversion behavior. Furthermore, the simulated amplitude response and experimental voltage response results are reasonably well matched, validating the finite element simulation method. Figure 2d shows simulation image indicating the actively resonating vibration of TENG-2, which can be expected to directly impact both TENG-1 and TENG-3. Thus even though the natural resonance frequency of TENG-2 is 24 Hz, the TENG-2 also shows high amplitude and voltage at the non-resonant frequencies when TENG-1 and TENG-3 are in resonance, due to frequency up-conversion and frequency down-conversion, respectively, as shown in Fig. 2e,f . Next, the simulation image Fig. 2g indicates the actively resonating vibration of TENG-3, which can be expected to directly impact TENG-2 and TENG-4. Here TENG-3 not only shows high amplitude and voltage output at its own resonant frequency (32 Hz) and the resonating frequencies of TENG-2 (due to frequency up-conversion) and TENG-4 (due to frequency down-conversion), but also the resonating vibration of TENG-1 contributes to the output of TENG-3 due to cascade impact and frequency up-conversion, as shown in Fig. 2h,i . Finally, the simulation image Fig. 2j indicates the actively resonating vibration of TENG-4, which is expected to directly impact only TENG-3. Thus apart from high amplitude and voltage response at its own natural frequency (40 Hz), its output is also influenced by TENG-3 vibration due to frequency up-conversion, as shown in Fig. 2k,l . Based on these results we conclude that the cascade impact structure induced each TENG to show significant vibration not only at their respective natural resonance frequencies, but even at non-resonant frequencies which become more prominent as the input acceleration levels were increased. This behavior ultimately helps obtain continuous scavenging within targeted frequency range from CIT-TENG. Figure 2 TENG dynamics in the cascade impact structure. Simulation image indicating resonance, simulated amplitude frequency response results, and voltage output frequency response results of TENG-1 ( a–c ) respectively, TENG-2 ( d–f ) respectively, TENG-3 ( g–i ) respectively, and TENG-4 ( j–l ) respectively. The simulated amplitude frequency response results are shown for input accelerations of 0.1 G m/s 2 , 0.3 G m/s 2 , 0.5 G m/s 2 . The voltage output frequency response results are shown for input acceleration of 0.5 G m/s 2 . Further analysis of the CIT-TENG system was performed by studying the effect of layer by layer installing of TENGs. Figure 3 shows that as each additional TENG is installed above the previous TENG, the output gradually broadens at the expense of the output level. Figure 3a shows that only TENG-4 will produce resonating output at 40 Hz. When TENG-3 is added above TENG-4 as in Fig. 3b , they not only vibrate at their resonating frequencies but also interact with one another. The motion of TENG-4 is restricted by TENG-3 which results in the reduction of its output level. However it now also experiences double impact which has been shown to broaden the TENG bandwidth 18 . When TENG-2 is added above TENG-3 as in Fig. 3c , cascade impact initiation begins to take its effect. Now the TENGs not only operate at their resonating frequencies but also at the resonating frequencies of the other TENGs as discussed previously. This further contributes to smoothly broadening out the output frequency response. Finally when TENG-1 is added above TENG-2 as in Fig. 3d , continuous operation from 3–40 Hz could be observed. Supporting Fig. S7 tracks the decrease in TENG-4 output voltage at 40 Hz input frequency, as TENGs are installed layer by layer above it. The rate of output voltage decrease from TENG-4 was determined as 14 V per TENG installed. Supporting Movie S2 shows the actual vibration dynamics of the individual TENGs during CIT-TENG frequency response. As the input frequency was increased from 3 Hz to 50 Hz, the resonating TENG can be observed to show the maximum amplitude motion which in turn effects the motion of adjacent TENGs through cascade impact. Apart from up-down motion, other vibration mode motion such as the sideways rocking motion can be observed for TENG-1 and TENG-2 at frequencies higher than their resonant frequencies. These additional vibration modes have lower amplitude vibrations so they can only significantly contribute to the output when the gap distance between the TENGs is reduced. Thus for the CIT-TENG used in application demonstration, the gap distances between the TENGs were slightly reduced to make use of these additional vibration modes which positively contributed to the continuous scavenging behavior of CIT-TENG, as previously shown in the voltage output frequency response results in Supporting Fig. S5 . Figure 3 Layer by layer installing of TENGs. Voltage output frequency response results from CIT-TENG with ( a ) only TENG-4, ( b ) TENG-3 added above TENG-4, ( c ) TENG-2 added above TENG-3, and ( d ) TENG-1 added above TENG-2. In order to demonstrate application of CIT-TENG as broadband frequency vibration energy scavenger, it was placed on a car dashboard and experimental measurements were taken at driving speeds of 20 km/hr and 40 km/hr. Figure 4a shows photograph of the car speedometer at 20 km/hr driving speed. At this speed, the input vibration acceleration was measured to be around 0.22 G m/s 2 (rms) as shown in Fig. 4b . The input frequency spectrum was obtained by taking fast fourier transform (FFT) of the input acceleration data, following which smoothing function was used to make the input vibration frequencies easier to identify. Figure 4c shows that the input frequency spectrum of the car dashboard vibrations was broadband with resonance peaks observed at 2 Hz, 12 Hz and 30–50 Hz. The voltage and current of the CIT-TENG under these input conditions were 9.55 V (rms) and 0.1 μA (rms) as shown in Fig. 4d,e , respectively. FFT of the voltage output in Supporting Fig. S8a showed that the CIT-TENG scavenged the input vibrations of 12 Hz and 30–40 Hz frequencies. Figure 4f shows photograph of the car speedometer at 40 km/hr driving speed. At this speed, the input vibration acceleration was measured to be around 0.56 G m/s 2 (rms) as shown in Fig. 4g . The corresponding input frequency spectrum had resonance peaks at the same frequencies as 20 km/hr driving speed. However, compared to the frequency spectrum at 20 km/hr, the higher frequencies of 30–50 Hz were more dominant at 40 km/hr as shown in Fig. 4h . The voltage and current of the CIT-TENG at 40 km/hr were 13.33 V (rms) and 0.22 μA (rms) as shown in Fig. 4i,j , respectively. The increase in the CIT-TENG output levels at 40 km/hr compared to the output at 20 km/hr was attributed to the increase in the input acceleration level. FFT of the voltage output at 40 km/hr in Supporting Fig. S8b showed that the CIT-TENG could scavenge the more dominant higher frequency components observed in the input frequency spectrum. Thus we showed that input vibrations from even a single vibration source can be broadband in nature, and that in order to effectively scavenge broadband frequency vibrations from such ambient sources we require continuous scavenging capability as shown by the CIT-TENG. Figure 4 CIT-TENG as broadband frequency vibration energy scavenger. Car driving speed of 20 km/hr: ( a ) speedometer photograph, ( b ) input acceleration measurement, ( c ) input frequency spectrum with raw data (grey) and smoothing curve (magenta), ( d ) CIT-TENG output voltage results, and ( e ) CIT-TENG output current results. Car driving speed of 40 km/hr: ( f ) speedometer photograph, ( g ) input acceleration measurement, ( h ) input frequency spectrum with raw data (grey) and smoothing curve (magenta), ( i ) CIT-TENG output voltage results, and ( j ) CIT-TENG output current results. In order to demonstrate CIT-TENG as an omnipotent or multi-purpose vibration energy scavenger, it was used for scavenging low frequency water wave vibrations and high frequency air compressor machine vibrations. Figure 5a shows photograph of the experimental setup for water waves vibration energy scavenging. The input vibration acceleration from the waves was measured to be around 0.2 G m/s 2 as shown in Fig. 5b . The corresponding input frequency spectrum had resonance peak at about 1 Hz as shown in Fig. 5c . The voltage and current output of the CIT-TENG from the water waves was found to be about 0.75 V and 0.1 μA as shown in Fig. 5d,e , respectively. FFT of the voltage output in Supporting Fig. S9a showed prominent peak at 1 Hz, similar to the input frequency spectrum. Figure 5f shows photograph of the experimental setup for air compressor vibration energy scavenging, where the CIT-TENG was affixed on top of the motor housing. The input vibration acceleration was measured to be around 1.5 G m/s 2 as shown in Fig. 5g . The corresponding input frequency spectrum had multiple resonance peaks with the highest peak at 100 Hz as shown in Fig. 5h . However, since the CIT-TENG operating bandwidth was up to 40 Hz as indicated in the figure, only the resonance peaks at 20 Hz, 30 Hz and 40 Hz could be expected to activate the TENGs. The voltage and current output of the CIT-TENG from the air compressor was found to be about 5 V and 0.75 μA as shown in Fig. 5i,j , respectively. FFT of the voltage output in Supporting Fig. S9b showed prominent peak at 40 Hz, similar to the input frequency spectrum. The reader is pointed to Supporting Movie S3 which shows the CIT-TENG dynamics during car dashboard, water wave and air compressor application experiments. Through these three applications we demonstrated the effectivity of CIT-TENG as an omnipotent vibration energy scavenger. Figure 5 CIT-TENG as omnipotent vibration energy scavenger. Water wave vibration energy scavenging: ( a ) photograph of experimental setup, ( b ) input acceleration measurement, ( c ) input frequency spectrum, ( d ) CIT-TENG output voltage results, and ( e ) CIT-TENG output current results. Air compressor vibration energy scavenging: ( f ) photograph of experimental setup, ( g ) input acceleration measurement, ( h ) input frequency spectrum with CIT-TENG operation range indicated, ( i ) CIT-TENG output voltage results, and ( j ) CIT-TENG output current results. The usage of CIT-TENG in powering practical applications requires an analysis of the output voltage, output current, and output power under electrical loading conditions. Supporting Fig. S10 provides these results at 10 Hz, 20 Hz, 30 Hz, and 40 Hz input frequencies for an input acceleration of 0.5 G m/s 2 . The output voltage ( V ) and output current were experimentally measured at different loading resistances ( R ), whereas output power ( P ) was determined using the formula, P = V 2 / R . The maximum power point was found to be at 5 MΩ impedance for 10 Hz input frequency, and at 10 MΩ impedance for 20 Hz, 30 Hz, and 40 Hz input frequencies. Supporting Movie S4 shows feasibility of using the CIT-TENG in powering practical applications. Indicator LEDs could be directly powered by the CIT-TENG when driven by an air compressor. Since the LEDs would only light-up when the air compressor was running, they could be used to indicate that the machine is in ON state. In summary, we studied a tandem triboelectric nanogenerator with cascade impact structural design that enabled continuous scavenging of ambient vibrations with broadband frequencies within a targeted frequency range even at low input accelerations. We demonstrated that our CIT-TENG could scavenge broadband vibrations from a single vibration source in a car dashboard under variable driving speeds. Finally, we demonstrated that the CIT-TENG can be reliably used for different vibration energy scavenging applications such as scavenging low frequency water wave vibrations and high frequency air compressor machine vibrations without any need for redesign. The CIT-TENG is thus an omnipotent solution for all vibration energy scavenging applications."
} | 6,166 |
26441847 | PMC4562303 | pmc | 1,820 | {
"abstract": "Microbial biomineralization sometimes leads to periplasmic encrustation, which is predicted to enhance microorganism preservation in the fossil record. Mineral precipitation within the periplasm is, however, thought to induce death, as a result of permeability loss preventing nutrient and waste transit across the cell wall. This hypothesis had, however, never been investigated down to the single cell level. Here, we cultured the nitrate reducing Fe(II) oxidizing bacteria Acidovorax sp. strain BoFeN1 that have been previously shown to promote the precipitation of a diversity of Fe minerals (lepidocrocite, goethite, Fe phosphate) encrusting the periplasm. We investigated the connection of Fe biomineralization with carbon assimilation at the single cell level, using a combination of electron microscopy and Nano-Secondary Ion Mass Spectrometry. Our analyses revealed strong individual heterogeneities of Fe biomineralization. Noteworthy, a small proportion of cells remaining free of any precipitate persisted even at advanced stages of biomineralization. Using pulse chase experiments with 13 C-acetate, we provide evidence of individual phenotypic heterogeneities of carbon assimilation, correlated with the level of Fe biomineralization. Whereas non- and moderately encrusted cells were able to assimilate acetate, higher levels of periplasmic encrustation prevented any carbon incorporation. Carbon assimilation only depended on the level of Fe encrustation and not on the nature of Fe minerals precipitated in the cell wall. Carbon assimilation decreased exponentially with increasing cell-associated Fe content. Persistence of a small proportion of non-mineralized and metabolically active cells might constitute a survival strategy in highly ferruginous environments. Eventually, our results suggest that periplasmic Fe biomineralization may provide a signature of individual metabolic status, which could be looked for in the fossil record and in modern environmental samples.",
"introduction": "Introduction Microbial biomineralization is a widespread process, responsible for the formation of a diversity of minerals (e.g., Ca and Mg-carbonates, iron and manganese oxides, silica, calcium, and iron phosphates…) that can be found in both modern environments and ancient geological formations. This process leads to the close association of microbial cells and minerals that precipitate at the contact of cells or even intracellularly. Highly mineralized conditions are commonly thought to be deleterious and to prevent microorganism growth or even survival. However, bacterial viability is maintained under some mineralized conditions in the environment, as exemplified by the diversity of bacteria thriving in stromatolitic formations ( Dupraz and Visscher, 2005 ; Goh et al., 2009 ; Gérard et al., 2013 ). Bacterial survival has also been observed and monitored under experimental conditions, e.g., in encapsulation silica matrices ( Nassif et al., 2004 ; Blondeau and Coradin, 2012 ; Eleftheriou et al., 2013 ; Le Ouay et al., 2013 ). Microorganisms have developed multiple strategies to deal with minerals that are sometimes products of their own metabolism: (1) Magnetotactic bacteria and some cyanobacteria localize biomineralization intracellularly ( Couradeau et al., 2012 ; Lefevre and Bazylinski, 2013 ; Benzerara et al., 2014 ). This provides a fine control of metal transit, mineral composition and/or crystallinity and offers the possibility to take advantage of specific properties of these biominerals (e.g., magnetic properties). (2) Biomineralization conditions can promote the production of extracellular polymeric substances (EPS) acting as templates for the precipitation of minerals. This has been shown in stromatolites ( Decho et al., 2005 ) and in other systems, among which Fe cycling bacteria. For instance, EPS are produced by the Fe(III)-reducer Shewanella oneidensis promoting the precipitation of uraninite ( Shao et al., 2014 ) and by nitrate-dependent iron(II)-oxidizing bacteria precipitating Fe-oxyhydroxides or phosphates ( Miot et al., 2009b ; Klueglein et al., 2014 ). (3) Micro-aerobic iron oxidizing bacteria also produce extracellular organics in the form of stalks ( Gallionella and Mariprofundus genus) or sheaths (e.g., Leptothrix or Sphaerotilus genus) templating Fe-(oxyhydr)oxide precipitation ( Banfield, 2000 ; Chan et al., 2011 , 2009 ; Seder-Colomina et al., 2014 ). (4) In addition, the microaerobic iron-oxidizer Mariprofundus ferroxydans PV-1 was shown to exhibit specific cell surface properties mitigating interaction with Fe minerals ( Saini and Chan, 2013 ). (5) Recently, S. oneidensis was shown to form membrane vesicles that get mineralized when exposed to uranium, hence lessening cell surface mineralization ( Shao et al., 2014 ). (6) Finally, phototrophic iron oxidizing bacteria produce a locally slightly acidic microenvironment around them that could increase Fe(III) solubility in the immediate vicinity of the cells and thus inhibit its precipitation at direct contact of the cells ( Hegler et al., 2010 ). Nonetheless, there have been numerous reports of microorganisms becoming encrusted within their cell wall in laboratory experiments, modern environments as well as the fossil record. Such periplasmic encrustation has been observed with bacteria mineralized by calcium phosphate ( Benzerara et al., 2004 ; Cosmidis et al., 2013 ), chromium phosphate ( Goulhen et al., 2006 ), uranyl phosphate ( Dunham-Cheatham et al., 2011 ), As-Fe-hydroxysulfate ( Benzerara et al., 2008 ), Fe sulfide ( Donald and Southam, 1999 ), and a diversity of Fe-minerals ( Miot et al., 2011 , 2014a , b ; Cosmidis et al., 2014 ; Klueglein et al., 2014 ). Periplasmic mineralization is a potential consequence of the activity of periplasmic enzymes, promoting (most plausibly indirectly) the precipitation of insoluble elements. For instance, activity of the periplasmic Nar enzyme involved in nitrate reduction was proposed to be responsible for Fe(III) precipitation in the periplasm of some anaerobic iron oxidizing bacteria ( Miot et al., 2011 ; Picardal, 2012 ; Carlson et al., 2013 ; Etique et al., 2014 ; Klueglein et al., 2014 ). Another example is the precipitation of periplasmic and/or cell surface meta-autunite (uranium phosphate) linked to the expression of a periplasmic alkaline phosphatase and subsequent export of inorganic phosphate in cultures of Caulobacter crescentus exposed to uranium ( Yung and Jiao, 2014 ). However, the periplasm is a key transit site, across which nutrients and wastes circulate. Its encrustation is thus thought to be lethal and/or at least to strongly limit cell – medium exchanges and hence microbial growth. Such hypotheses related to the viability of mineralized bacteria have, however, never been explored down to the single cell level. In the present study, we evaluated the link between Fe biomineralization and viability of the anaerobic nitrate-dependent Fe(II)-oxidizer Acidovorax sp. strain BoFeN1 under biomineralization conditions in batch cultures. We followed these processes down to the single cell level using a combination of electron microscopy and Nano-Secondary Ion Mass Spectrometry (NanoSIMS). This strain has been shown to promote the biomineralization of a diversity of Fe-minerals depending on biomineralization conditions: Fe-phosphates ( Miot et al., 2009b ), green rust ( Pantke et al., 2012 ), goethite ( Pantke et al., 2012 ), and lepidocrocite ( Miot et al., 2014b ) sometimes in association with extracellular magnetite ( Miot et al., 2014a ). In each case, Fe minerals (except extracellular magnetite) were shown to precipitate at least partly within the periplasm and at the cell surface at more advanced stages. We explored how cells assimilated a labeled organic carbon substrate ( 13 C-acetate) as a function of the stage of biomineralization and of the nature of the biominerals precipitated in the periplasm. NanoSIMS has been widely used in the last years to investigate single cell metabolism in laboratory and environmental samples, shedding light for instance on strong individual heterogeneities of carbon or nitrogen assimilation (e.g., Musat et al., 2008 ; Remusat et al., 2012 ; Zimmermann et al., 2015 ). In the present study, NanoSIMS analyses provided a way to analyze single cells and discriminate the C assimilation behavior of mineralized vs. non-mineralized bacteria. We show that BoFeN1 community remains viable under biomineralization conditions as a consequence of heterogeneous biomineralization of the cells. In addition, we show that carbon assimilation is correlated with the amount of Fe precipitated at the cell contact.",
"discussion": "Discussion Periplasmic Encrustation Control over Carbon Assimilation and Viability A diversity of nitrate reducing bacteria have been shown to promote Fe(II) oxidation and precipitation of Fe minerals ( Kappler et al., 2005 ; Chakraborty et al., 2011 ; Carlson et al., 2012 ; Chakraborty and Picardal, 2013 ; Etique et al., 2014 ; Klueglein et al., 2014 ). Under biomineralization conditions, all these strains become progressively encrusted with Fe phases. In Acidovorax sp. strain BoFeN1, mineralization starts in the periplasm at the contact of the inner membrane then progressively encrusts the periplasmic space. Further, minerals deposit at the cell surface ( Miot et al., 2011 ). At ultimate stages, minerals fill in the cytoplasm ( Li et al., 2013a ; Klueglein et al., 2014 ). In addition, existence of mineralized cells in division has been reported ( Miot et al., 2011 ). However, it was unclear whether mineralized cells were still able to divide or whether mineralization occurred very rapidly during division hence freezing the cell division process. This raises the question of the chronology of Fe mineralization vs. cell death. Here, we observed that carbon incorporation depended on the level of cell mineralization ( Figures 4 and 6 ). At low cell encrustation levels, carbon assimilation by mineralized cells was comparable to that of the non-mineralized cells ( Figure 7 ). However, carbon assimilation decreased exponentially with increasing cell mineralization by Fe minerals ( Figure 4E ). Interestingly, carbon assimilation by mineralized cells was not dependent on the nature of Fe minerals precipitated in the cell wall, but only on the amount of Fe precipitated in the periplasm and at the direct cell contact. There is thus a biomineralization threshold above which carbon assimilation ceases, but below which carbon assimilation is not or only partially hampered. Whereas carbon assimilation by mineralized cells dropped with increasing the level of periplasmic encrustation, carbon assimilation by non-mineralized cells remained constant ( Figure 7B ). Present results therefore strongly support that periplasmic encrustation and cell death are temporarily decoupled, these two processes occurring successively through the following scenario: as the periplasm becomes slightly mineralized, carbon assimilation, and cell division are still possible ( Miot et al., 2011 ). Then, further cell wall mineralization progressively limits carbon assimilation, until reaching high Fe mineralization levels that lead to cessation of metabolic activity, and potentially cell lysis. Hence, these results suggest that first stages of periplasmic Fe biomineralization occur while bacteria are alive and not post mortem . Ultimate stages of mineralization leading to cytoplasm encrustation ( Li et al., 2013a ; Klueglein et al., 2014 ) most probably induce membrane perforation and cell death. Carlson et al. (2013) reported faster rates of Fe(II) oxidation by lysates compared with intact cells. Abiotic oxidation reactions may thus promote cytoplasmic precipitation of Fe minerals. Our present results shed light on the impact of Fe biomineralization on carbon assimilation. Although we did not investigate more specifically the link with cell death, we can propose that periplasmic encrustation may induce multiple processes ultimately leading to cell death. Carlson et al. (2013) demonstrated that the main response to Fe biomineralization conditions was a stress response with an up-regulation of metal efflux pumps and of proteins involved in the response to nitrosative/redox stress. Inactivation of proteins from respiratory complexes (e.g., nitrite reductase, NO reductase) might occur in biomineralized cells, consistently with the accumulation of nitrate reduction intermediates in cultures of Acidovorax ebreus under nitrate dependent Fe(II) oxidation conditions ( Carlson et al., 2013 ). In addition, although there is no proteomic evidence for a major role played by these processes, we cannot exclude that Fe biomineralization would directly inactivate proteins involved in C assimilation pathways. Biomineralization conditions may also lead to overexpression of membrane proteins such as porins ( Carlson et al., 2013 ), which would promote pore formation as a step toward cell death (e.g., Bednarska et al., 2013 ). Eventually, periplasmic biomineralization would promote protein aggregation. Indeed, protein aggregates entrapped within the mineralized cell wall of BoFeN1 cells have been previously identified ( Miot et al., 2011 ), and such protein aggregation has been associated with loss of function ( Bednarska et al., 2013 ). Segregation of protein aggregates toward the old poles of bacterial cells is a common process limiting toxicity of protein aggregates and preserving cell integrity (e.g., Dougan et al., 2002 ). This process is consistent with previous observations of periplasmic biomineralization occurring preferentially at the old poles of the cells, while new septae remain free of Fe precipitates in BoFeN1 ( Miot et al., 2011 ). At advanced stages of biomineralization, protein aggregation combined with mineral accumulation within the periplasm may induce stress response including reduction of membrane permeability ( Ami et al., 2009 ). In the end, all the aforementioned processes would contribute to loss of viability. Link between Individual Phenotypic Variability of Carbon Assimilation and Fe Biomineralization Our study evidences the co-existence of mineralized and non-mineralized cells at each stage of biomineralization and whatever the nature of Fe biominerals formed. Whereas mineralized cells did not incorporate carbon, moderately mineralized and non-mineralized cells significantly incorporated acetate. Previous studies evidenced persistence of acetate incorporation by some nitrate-reducing Fe(II) oxidizing strains even at advanced stages of Fe biomineralization ( Klueglein et al., 2014 ). This has been observed not only for Acidovorax sp. strain BoFeN1 but also for Pseudogulbenkania sp. strain 2002 and for heterotrophic nitrate reducers such as Paracoccus denitrificans ATCC 19367 and P. denitrificans Pd 1222 ( Klueglein et al., 2014 ). Our results are consistent with these previous observations, as acetate incorporation at advanced stages of Fe biomineralization can be attributed to metabolic activity of non-mineralized and moderately mineralized cells. Interestingly, low proportions of metabolically active cells (∼10%) were enough for the cultures to recover ( Figure 5 ). This is consistent with previous estimations and suggestions that low proportions of non-mineralized cells could account for acetate consumption at advanced biomineralization stages ( Klueglein et al., 2014 ). Phenotypic variations within a monospecific bacterial culture under controlled conditions have been reported in multiple systems (e.g., Davidson and Surette, 2008 ; Raj and van Oudenaarden, 2008 ; Ackermann, 2013 ), shedding light on the fact that a microbial population is a heterogeneous group of physiologically distinct individuals. Individual variations are increasingly studied with the advent of powerful tools such as NanoSIMS allowing the exploration of metabolic processes down to the single cell level in microbial cultures and environmental samples ( Musat et al., 2008 , 2012 ; Dekas et al., 2009 ; Morono et al., 2011 ; Milucka et al., 2012 ; Remusat et al., 2012 ; Kopp et al., 2013 ; Zimmermann et al., 2015 ). Individual non-genetic phenotypic heterogeneity has been investigated for multiple traits including metabolism (e.g., Ackermann et al., 2008 ; Kiviet et al., 2014 ) and stress response ( Balaban et al., 2004 ; Wakamoto et al., 2013 ; Holland et al., 2014 ). In particular, a subpopulation of bacteria known as “persisters” usually overcomes lethal stress induced by antibiotics, being responsible for antibiotic resistance (e.g., Lewis, 2010 ). It has been recently shown that stochastic expression of genes (non-responsive, selection-mediated adaptation) is a robust (in terms of fitness) alternative adaptation to stresses compared to the expression of dedicated repair mechanisms (sense-and-respond adaptative strategy; Wakamoto et al., 2013 ). In the present study phenotypic cell-to-cell variability of carbon assimilation associated with individual variability of periplasmic Fe biomineralization may mirror non-genetic heterogeneity caused by different gene expression by individual bacteria. Although such a hypothesis would deserve a dedicated study, we may tentatively attribute these observed phenotypic differences to several individual differences in gene expression along the course of the cultures. Heterogeneities of Fe biomineralization may mirror (1) individual differences in response to the toxicity of Fe 2+ and reactive nitrogen species, via the differential expression (up/down regulation) of metal efflux pumps and proteins involved in the cytoplasmic response to nitrosative stress ( Carlson et al., 2013 ) and (2) variations in the individual rates of enzymatic nitrogen species reduction (NO 3 - , NO 2 - , NO, N 2 O; Carlson et al., 2013 ), themselves dependent on concentrations of organic co-substrates and access to nutrients (acetate, nitrate). (3) Finally, Acidovorax sp. strain BoFeN1 has been shown to produce EPS that get progressively mineralized with Fe minerals ( Miot et al., 2009b ). This has been observed in other nitrate-dependent Fe(II)-oxidizers as well, though with notable interspecific differences ( Klueglein et al., 2014 ). Different individual rates of EPS production might partly account for different individual capabilities to overcome encrustation. This assumption might be tested in the future by correlating individual rates of carbon assimilation and Fe encrustation with local amounts of EPS produced, e.g., through the use of correlative NanoSIMS and carbon K-edge STXM analyses ( Remusat et al., 2012 ). Phenotypic Heterogeneity as a Strategy to Cope with Fe Biomineralization? In the present study, we analyzed bacteria from batch cultures exposed to relatively high (millimolar) Fe(II) concentrations. In contrast, in continuous flow systems at much lower Fe 2+ concentrations, nitrate reducing Fe(II)-oxidizing bacteria were shown to exhibit minimized cell encrustation by Fe minerals ( Chakraborty et al., 2011 ). Under these conditions, it was shown that nutrient uptake by Acidovorax sp. strain 2AN continued, whereas it ceased after 3–4 days of exposure to millimolar Fe 2+ concentrations under batch conditions. Our results are consistent with these previous observations, showing that low levels of periplasmic encrustation do not significantly hamper carbon assimilation, hence suggesting that under low Fe 2+ concentrations, Fe biomineralization would not significantly interfere with cell growth. Besides, some modern and past environmental conditions might offer higher Fe(II) concentrations, comparable to those set in the present study. Indeed, high dissolved Fe 2+ concentrations might have been predominant in the Precambrian ocean ( Poulton and Canfield, 2011 ; Li et al., 2013b ). Some modern environments exhibit millimolar Fe 2+ concentrations as well. For instance, the deep anoxic layer of the meromictic lake Pavin displays Fe 2+ concentrations at the saturation with the Fe phosphate vivianite ( Viollier et al., 1997 ), comparable to the conditions reached in the Gt and FeP media used in the present study. Bacteria encrusted with Fe phosphate have been evidenced in this lake ( Cosmidis et al., 2014 ), showing morphological and compositional similarities with encrusted nitrate reducing Fe(II)-oxidizers. It is worth understanding why nitrate reducing Fe(II) oxidizing bacteria did not evolve pathways to avoid cell encrustation. Indeed, many bacterial groups have developed strategies to cope with metals, in particular Fe. On the one hand, some bacteria produce organics that template Fe mineral precipitation, thus avoiding cell encrustation. For instance, microaerobic Fe(II)-oxidizers such as Gallionella sp. and Mariprofundus sp., or Leptothrix sp. produce stalks or microtubes composed of organic fibers at the surface of which Fe minerals nucleate ( Chan et al., 2011 ; Comolli et al., 2011 ; Saini and Chan, 2013 ). Extrusion of these organo-mineral structures leaves cells free of precipitates. On the other hand, phototrophic Fe(II)-oxidizers have been proposed to avoid encrustation by maintaining a local acidic pH in their surroundings ( Hegler et al., 2010 ). Our results suggest that the periplasmic encrustation trait could have been maintained through (1) the limitation of periplasmic encrustation to low levels in environments exhibiting low Fe 2+ concentrations, and (2) the persistence of a low proportion of cells remaining free of precipitates, while the majority of the cells do not avoid cell encrustation in more ferruginous environments. Further investigations would be needed to explore the underlying mechanisms and clarify how such bacteria may have persisted in natural ferruginous habitats despite the deleterious effects of periplasmic encrustation. Noteworthy, depletion of C assimilation concomitantly with Fe biomineralization was observed whatever the composition of Fe biomineralization media (e.g., with or without phosphate) and the nature of Fe biominerals precipitated. As a consequence, we would expect that this link between individual phenotypic heterogeneities of Fe biomineralization and C assimilation would be ubiquitous in natural ferruginous environments. Using a combination of NanoSIMS and flow cell sorting, phenotypic heterogeneities of carbon and nitrogen assimilation have been recently evidenced among different bacterial populations in the meromictic lake Cadagno ( Musat et al., 2008 ; Zimmermann et al., 2015 ), suggesting potential non-genetic phenotypic diversity in this natural habitat. Here, we propose that Fe biomineralization would influence such non-genetic phenotypic variability under ferruginous conditions. In addition, our study shows that Fe biomineralization would provide a signature of the metabolic status of the cells. Exploring the relationship between phenotypic heterogeneity and biomineralization processes in natural habitats would provide a step toward understanding ecosystem functioning and evolutionary adaptation to metal-rich niches."
} | 5,799 |
25565556 | null | s2 | 1,821 | {
"abstract": "Bombyx mori (BM) silk fibroin is composed of two different subunits: heavy chain and light chain fibroin linked by a covalent disulfide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fiber mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibers formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fiber formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications, and potential applications of these materials in biomedical and industrial applications."
} | 440 |
38260628 | PMC10802371 | pmc | 1,823 | {
"abstract": "DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.",
"introduction": "INTRODUCTION Recent advances in DNA nanotechnology have presented promising opportunities for applications in areas like drug delivery, biosensing, and biomanufacturing [ 1 ]–[ 3 ]. In particular, DNA origami (DO),[ 4 ] where a long template strand is folded into a compact shape by base-pairing with many shorter strands, enables fabrication of nanostructures with complex and precise shape, custom functionalization, and tunable mechanical properties[ 5 ], [ 6 ]. These features make DO devices attractive as platforms for targeted therapies,[ 7 ] biophysical measurements[ 8 ], or controlling molecular interactions[ 9 ], [ 10 ]. Many of these applications either require or can be enhanced by effective methods to deliver DO into intracellular environments. Prior studies have demonstrated uptake of DO into cells[ 11 ]–[ 13 ], but the trafficking of DOs upon entry into live cells and specifically to nuclei is less well-understood and/or developed. Methods for the efficient delivery of DOs into live cell nuclei could greatly enhance existing applications in therapeutic delivery, for example gene delivery,[ 14 ]–[ 16 ] and could enable translation of other functions of DO like biophysical measurement or imaging into cell nuclei. The nucleus houses the cell’s genetic material and the machinery essential for transcription and other processes vital to gene expression and regulation[ 17 ], [ 18 ]. Consequently, targeting molecular structures and devices to the nucleus is an attractive approach for many therapies and may present opportunities for nanoscale tools to probe or control the genetic or epigenetic processes that regulate cell function. For example, recent in vitro work has demonstrated nanodevices as tools for sequestering or organizing biomolecules or larger complexes,[ 19 ]–[ 21 ] imaging biomolecules at high resolution,[ 22 ], [ 23 ] and manipulating enzymatic reactions,[ 24 ], [ 25 ] all of which could be useful inside cells and cellular compartments. Delivering DO nanodevices to cell nuclei is attractive for applications like nucleic acid detection[ 26 ], [ 27 ], biophysical probing of chromatin sub-structures (previously demonstrated in vitro [ 28 ], [ 29 ]), and gene delivery.[ 14 ]–[ 16 ] While significant efforts have studied the delivery and uptake of DO nanostructures into live cells,[ 11 ], [ 13 ], [ 30 ], [ 31 ] only recently has the specific delivery of DO structures to the nucleus been explored, focused in the context of gene delivery.[ 14 ]–[ 16 ], [ 32 ] These studies have established DO as useful tool for the delivery of genetic information into live cells. Even though these prior studies focused on gene expression, key questions remain unclear: i) are these DO structures stable inside the cell?, ii) how many of the DO structures reach the nuclei, and iii) can intact DOs can be delivered into the nucleus? Hence, there remains a critical need for robust methods to deliver DO nanostructures to live cell nuclei, which would be an essential step to enabling intranuclear functions that rely on the structure and not just the encoded sequence. Here, we present a novel approach for the delivery of intact DO nanstructures into live cells and specifically to the nucleus ( Figure 1 ). Inspired by recent work focused on the delivery of antibodies into live cell nuclei,[ 33 ]–[ 35 ] our method involves the conjugation of DO nanostructures to antibodies that bind to neosynthetized proteins in the cytoplasm, which function in the nucleus and thus naturally cycle to the nucleus, thereby carrying, or “piggybacking,” the DOs along with them. We chose the large subunit of RNA polymerase Pol II, a pivotal enzyme involved in gene transcription, as a molecule to target the neosynthetized subunit in the cytoplasm. Our prior work demonstrated that the piggybacking approach is effective for the delivery of antibodies with high affinity towards Pol II into live cell nuclei.[ 33 ] Here we show that, after electroporation into the ctyoplasm, Pol II antibody-conjugated 30 nm nanorod DO structures can enter the nuclei of U2OS cells, as confirmed by fluorescence microscopy, and exhibit sub-diffusive motion within live cell nuclei. We also studied the stability of DO in cell culture media and different cell lysates using gel electrophoresis and transmission electron microscopy (TEM), and inside live cells using fluorescence imaging. These analyses reveal the structural integrity of the DO over extended periods in cell media and extracts, and confirm that DOs remain structurally stable 24 hr after electroporation both in the cytoplasm and after piggybacking into the nucleus. Combined, our results establish a basis to implement DO nanodevices as tools for imaging, detection, biophysical measurements, or other applications inside cell nuclei.",
"discussion": "DISCUSSION DO nanostructures have been demonstrated for applications like biophysical measurements[ 8 ], [ 67 ], manipulating molecular interactions[ 9 ], [ 10 ], and delivery of therapeutic agents[ 2 ], [ 7 ], [ 68 ], which could all be useful intracellular functions; and other applications like high resolution imaging[ 22 ], nucleic acid and protein detection[ 2 ], [ 69 ], probing of chromatin sub-structures[ 28 ], [ 29 ], and gene delivery[ 14 ]–[ 16 ] could particularly benefit from mechanisms to specifically deliver DO to live cell nuclei. As a critical step for intracellular delivery and applications, we evaluated the stability of DOs in relevant conditions including cell culture media, cell ctyoplasmic and nuclear extracts, upon electroporation, and inside cells. Our results show that the DO designs used here are stable in cell media and in nuclear and cytoplasmic extracts for 24 hr, which is consistent with prior work showing DO can exhibit extended stability in cell culture or in cell lysates[ 36 ], [ 70 ], [ 71 ]. It is worth noting the stability is design dependent and DO can degrade more rapidly at higher serum levels[ 72 ], [ 73 ], which is an important consideration especially for translational applications. However, multiple strategies exist such as UV cross-linking or polymer coating and brushes that can extend the stability of DOs [ 74 ]–[ 76 ]. Prior work has shown that the process of electroporation can impact structural integrity of DO [ 77 ], while others studies have demonstrated some DO designs can remain stable through electroporation [ 14 ], [ 15 ], suggesting the electroporation stability is dependent on the design and electroporation parameters. Our results show that the 8HB DO structure and antibody attachment is stable after electroporation. We also demonstrate that the 8HB DO can remain stable for 24 hr after electroporation into cells in the cytoplasm or after entering the nucleus. While prior work have not evaluated DO inside nuclei, our results are in agreement with prior studies showing some DNA nanostructure designs can exhibit extended stability inside cells [ 78 ]–[ 80 ]. Several prior efforts have studied interactions between DO and cells (e.g. see recent reviews[ 12 ], [ 81 ]), and a few recent studies have demonstrated effective delivery of gene sequences folded into DO structures where genes can be expressed[ 14 ]–[ 16 ], [ 32 ]. Two of these studies leveraged either Cas9[ 14 ] or an SV40 derived DNA sequence[ 32 ] to promote delivery to the nucleus. However, these studies were focused on delivering information through the DNA sequence to the nucleus, rather than intact DO structures. Unlocking potential device functions of DO inside cell nuclei requires methods that allow for the delivery and tracking of intact DO into live cells and targeted delivery to nuclei. Here we targeted DOs to the nucleus by functionalizing them to bind neosynthetized nuclear factors in the cytoplasm, in this case the largest subunit of the RNA polymerase Pol II. As the nuclear factor is imported to the nucleus, the DO can be carried, or “piggybacked,” along with them. We found this piggybacking approach is size dependent, with no clear nuclear delivery observed using a larger size DNA origami (~4.8 MDa, ~90 nm long nanorod), while the piggybacking approach worked effectively to deliver smaller structures (~0.5 MDa, ~30 nm long nanorod) to the nucleus. We confirmed that these DO remain intact inside cells for 24 hr using two-color fluorescence co-localization (DO dual-labeled with Cy3 and Cy5), including comparison to co-delivery of a single-labeled structures (Cy3-labeled DO plus Cy5-labeled DO) to verify that co-localization is the result of intact structures[ 65 ]. iSIM imaging further revealed DO can remain intact in live cell nuclei 24 hr after electroporation, hence opening a door to leverage the diverse functions of DO inside the nucleus. These ~30 nm nanorod DO already provides a useful basis for functions like imaging, or detection with the simple inclusion of fluorophores or aptamers[ 82 ]. Our results further showed these DO are mobile inside the nucleus. They exhibit sub-diffusive motion similar to what has previously been measured for other nuclear factors[ 66 ], which is likely due to the highly constrained environment inside the nucleus. Nevertheless, our results suggest the piggybacked DO can explore the nuclear volume. Some functions of DO would likely be enhanced through the use of larger structures. Here a key factor limiting our use of the larger 26HB DO was aggregation in the cytoplasm. The large design space of DO in terms of size, shape, surface coating, and functionalization can likely enable engineering of intracellular behaviors like aggregation, passive or active transport, and entry to the nucleus or other cell compartments. Our results and other recent efforts[ 12 ], [ 14 ], [ 32 ], [ 65 ], [ 78 ], [ 80 ] provide a framework to guide these studies. In the future, a better understanding of these intracellullar behaviors of DO will be important to for enabling additional applications, for example those that leverage multi-component devices like biophysical measurements[ 28 ]. The piggybacking approach we presented here relies on binding neosynthetized nuclear factors in the cytoplasm that will be imported to the nucleus. Here we targeted the RNA polymerase II building on prior studies that established the piggybacking approach for delivering antibodies to the nucleus.[ 33 ]–[ 35 ] These studies used the same approach to target multiple transcription factors, including TATA binding protein (TBP), TBP-associated factor 10 (TAF10), suggesting these, and likely a variety of other nuclear factors, could be used to piggyback DO structures to the nucleus. These proteins have specific mechanisms that drive localization to the nucleus, such as interactions with other proteins (e.g. RNA Pol II associated protein, RPAP2[ 83 ]) that mediate trafficking or direct interactions with importins via nuclear localization signal (NLS) sequences or other domains[ 84 ]. Indeed, prior studies showed the expression of gene sequences delivered via DO is increased with inclusion of either amino acid NLS or DNA nuclear targeting sequences (DTS)[ 14 ], [ 32 ]. Combined with our results, these studies suggest a variety of proteins or motifs or direct inclusion of NLS or DTS sequences onto DO could be alternative routes to specifically deliver intact DO devices to the nucleus."
} | 3,143 |
37348845 | PMC10327651 | pmc | 1,824 | {
"abstract": "Wetting of solid surfaces is crucial for biological and\nindustrial\nprocesses but is also associated with several harmful phenomena such\nas biofouling and corrosion that limit the effectiveness of various\ntechnologies in aquatic environments. Despite extensive research,\nthese challenges remain critical today. Recently, we have developed\na facile UV-grafting technique to covalently attach silicone-based\ncoatings to solid substrates. In this study, the grafting process\nwas evaluated as a function of UV exposure time on aluminum substrates.\nWhile short-time exposure to UV light results in the formation of\nlubricant-infused slippery surfaces (LISS), a flat, nonporous variant\nof slippery liquid-infused porous surfaces, longer exposure leads\nto the formation of semi-rigid cross-linked polydimethylsiloxane (PDMS)\ncoatings, both covalently bound to the substrate. These coatings were\nexposed to aquatic media to evaluate their resistance to corrosion\nand biofouling. While the UV-grafted cross-linked PDMS coating effectively\ninhibits aluminum corrosion in aquatic environments and allows organisms\nto grow on the surface, the LISS coating demonstrates improved corrosion\nresistance but inhibits biofilm adhesion. The synergy between facile\nand low-cost fabrication, rapid binding kinetics, eco-friendliness,\nand nontoxicity of the applied materials to aquatic life combined\nwith excellent wetting-repellent characteristics make this technology\napplicable for implementation in aquatic environments.",
"conclusion": "4 Conclusions In this study, we further\nexplored a one-pot approach of UV-grafting\nof PDMS to convert a surface of Al into LISS. Here, PDMS was covalently\ngrafted to Al by UV irradiation at a specific wavelength and served\nas a surface energy-reducing agent, while the remaining unbound oil\nwas simultaneously utilized as an infusing lubricant. Our approach\ndoes not require harsh pre/post-grafting harsh treatments or micro-\nor hierarchical micro/nano-surface structuring to be suitable for\nindustrial applications, making it advantageous over superhydrophobic\nand SLIPS surfaces as long as the substrate is UV-stable, i.e., metals,\nmetal oxides, ceramics, and glasses. Since Al is used in highly corrosive\nenvironments and at the same time is susceptible to biofouling, the\nUV-grafted Al samples were investigated for their corrosion resistance\nin aqueous environments with and without aquatic organisms such as\nfreshwater algae and seawater diatoms. We investigated two types of\ncoatings: (i) semi-rigid, CL-PDMS and (ii) liquid-like LISS, which\nwere obtained by different exposure times to UV irradiation. While\nbare Al corroded severely in seawater media within a short period\nof exposure, both coatings exhibited superior corrosion resistance,\nshowing a corrosion-free surface after 143 days of immersion. The\nlatter was also confirmed by potentiodynamic polarization measurements,\nespecially for the CL-PDMS coating. This superior corrosion resistance\nwas attributed to the inability of the corrosive electrolyte to penetrate\nthe CL-PDMS layer, which was also firmly attached to the Al substrate.\nTo test the biofouling resistance of the UV-grafted coatings, the\nbare and coated Al surfaces were exposed for 8 days to a medium containing\naquatic species such as freshwater C. reinhardtii algae and seawater O. aurita diatoms.\nWhile both coatings exhibited superior corrosion resistance, LISS\nprevented almost complete biofilm attachment of both aquatic species\nin short-term laboratory studies. In contrast to CL-PDMS, the presence\nof residual, unbound silicone oil in LISS enhances the anti-biofouling\nperformance of PDMS by forming an additional stable liquid–liquid\ninterface that separates the solid surface from the fouling liquid\nmedia. 69 Despite the superior corrosion\nresistance, the CL-PDMS coating should be further investigated for\nits damage tolerance, while the self-healing properties imparted to\nCL-PDMS by the infusions of silicone oil could further improve the\noverall corrosion resistance. Given the above advantages and considering\nthat the proposed approach is easy to implement, fast, nontoxic, environmentally\nfriendly, scalable, and inexpensive, we envision that the UV-grafting\napproach of PDMS will advance the development of a nontoxic omni-repellent\nand corrosion-resistant coating technology for challenging but highly\ndesirable aquatic applications.",
"introduction": "1 Introduction The oceans that cover\n∼70% of the surface of our planet\nare in constant motion due to wind, the moon, and the planet’s\nrotation, temperature, and salinity differences. 1 While such endless movements provide food and oxygen to\naquatic life and move ships across the ocean, 2 they are also regarded as a green, renewable, and reliable source\nof energy around the world. However, the potential of the ocean as\none of the greatest sources of energy is under-explored compared to\nother green alternatives. 3 This is because\nthe aquatic environment is extremely challenging due to (i) the high\nconcentration of ions such as chlorine, which leads to increased corrosion\nof metals, and (ii) the accumulation of aquatic organisms on solid\nsurfaces, i.e., biofouling, which degrades the desired functionality\nof devices. Among the available engineering materials, aluminum\n(Al) is one\nof the most widely used structural metals in marine applications due\nto its lightweight, high strength, good corrosion resistance, easy\nrecyclability, extrudability, and weldability for design flexibility.\nToday, it is used in fast ferries, wave-piercing catamarans, cruise\nships, as well as offshore oil drilling rigs, deep submersible vessels,\nand helicopter landing pads, to name but a few, 4 in its pure form or as an alloy to adjust the mechanical\nand corrosion properties. 5 , 6 It is common knowledge\nthat Al is susceptible to corrosion. Under mild conditions (e.g.,\nthe near-neutral pH range of many aqueous solutions), Al exhibits\nstable passivity and, hence, very low corrosion rates. 7 , 8 However, under more aggressive conditions, Al can corrode severely. 9 The most common type of corrosion is pitting,\nwhich occurs in the presence of chloride ions. 10 In addition to direct corrosion-related structural failures\nof constructions, this results in the release of Al ions into the\nmarine environment, which can be toxic to aquatic life. 11 However, it is well known that Al is neither\nrequired in biological\nsystems nor involved in any essential biological processes. 12 Nevertheless, all living organisms today contain\nsome aluminum due to its abundance in the Earth’s crust. On\nthe contrary, there are known biological effects of Al, most of which\nare negative. 13 The toxicity of Al is closely\nrelated to pH, as the metal is soluble and biologically available\nin acidic (pH < 5.5) soils and waters but relatively harmless in\nneutral conditions (pH = 5.5–7.5). Forest dieback and impaired\nreproduction of aquatic invertebrates, fish, and amphibians have been\ndirectly linked to Al toxicity. 11 Another issue to consider in aquatic applications is biofouling,\nwhich is the undesired colonization and accumulation of aquatic organisms\non submerged surfaces. 14 Biofouling is\na multistep process initiated by the formation of a “conditioning\nlayer” of microfoulers, which are bacteria, diatoms, and/or\nmicroalgae that promote the formation of biofilm matrix upon which\nmulticellular micro- and macrofoulers develop. 15 For centuries, it has been common practice to apply anti-biofouling\ncoatings containing toxic biocidal chemicals to prevent biofouling\nby locally killing or slowing down their growth. 16 , 17 Such anti-biofouling coatings typically contained active biocidal\nchemicals based on As, Sn, Cu, and Zn, among others. 17 − 19 Over time, the environmental impact of these compounds became apparent\non aquatic life, and in particular on nontarget fouling organisms,\nwhich finally led to their banning in 2008, opening up a new era in\nthe development of nontoxic anti-biofouling coatings. 20 In particular, the focus is on reducing the attachment\nor increasing the release of biological foulants from the surface\nby tailoring the surface properties of the material, such as energy,\nroughness, wettability, and chemistry. In this regard, superhydrophobic\nsurfaces have been studied for many years as a vital solution for\nanti-biofouling surfaces. 21 The trapped\nair layer, so-called plastron, formed on a superhydrophobic surface\nreduces the contact between the biomaterial-containing fluid and the\nsubstrate. However, to date, the plastron on engineered superhydrophobic\nsurfaces has been shown to be metastable, making such surfaces unsuitable\nfor long-term immersion. 22 Furthermore,\nthe increased surface roughness after plastron loss triggers surface\ncoverage by fouling organisms and also provides a larger surface area\nto accelerate corrosion. Therefore, in 2011, a new type of coating\ncalled slippery liquid-infused porous surfaces (SLIPS) was introduced. 23 Inspired by the Nepenthes pitcher\nplant, the surface chemistry and roughness were tailored to immobilize\na liquid layer on the rough solid surface, thereby introducing an\nadditional liquid–liquid interface that separates the solid\nsubstrate from direct contact with an aggressive and fouling liquid. 24 By controlling the composition and viscosity\nof the lubricant, a wide variety of liquids can be repelled. 25 − 34 However, there are several drawbacks associated with the SLIPS technology:\n(i) the requirement for rough, porous structuring of the substrate\nsurface, which weakens the mechanical robustness, and (ii) the multistep\npreparation processes. 23 Recently,\nwe developed a one-pot process to form a nontoxic lubricant-infused\nslippery surface (LISS) coating on smooth surfaces based on the UV-grafting\nof polydimethylsiloxane (PDMS or silicone oil). 35 A layer of PDMS molecules is covalently grafted to the\nsolid surface by selective dissociation of the Si–CH 3 terminal bond, lowering the surface energy of the substrate and\nmatching it to the surface tension of the nongrafted, i.e., residual\nPDMS used as the infusing lubricant. PDMS is commercially available,\ninexpensive, and nontoxic to aquatic environments, providing an optimal\nsolution compared to fluorinated compounds. Previously, we demonstrated\nthat the UV-grafted LISS coating prepared on austenitic stainless\nsteel prevented the adhesion of aquatic organisms even when severely\ndamaged. 36 In this study, we fabricated\na UV-grafted PDMS coating on aluminum\nto simultaneously investigate the resistance of the UV-grafted PDMS\ncoatings to corrosion and biofouling in aquatic environments. Corrosion\nof metallic substrates can deteriorate the stability of the infused\nlubricant, increase its depletion, and ultimately lead to coating\nfailure. This is because metal oxides have a high surface energy, 37 which makes them significantly less attractive\nfor wetting by low surface tension liquids such as lubricants, which\nis contrary to the basic requirement for liquid-infused surfaces. 23 First, the LISS coating was fabricated according\nto our previously reported process. 35 , 36 Then, the\nUV exposure time was extended to achieve a cross-linked PDMS (CL-PDMS)\ncoating. In both cases, PDMS is covalently grafted onto the substrate.\nThe corrosion protection of the UV-grafted coating was investigated\nby potentiodynamic polarization curves measured in an aqueous electrolyte\ncontaining 3.5 wt % NaCl and by long-term immersion in freshwater\nand seawater media. Finally, the UV-grafted and bare aluminum samples\nwere exposed to aquatic organisms such as freshwater algae and seawater\ndiatoms, the latter known as early colonizers. 38 , 39 We show that the LISS coating is effective in preventing biofouling\nadhesion, while the CL-PDMS is superior in corrosion protection of\nAl in aquatic environments.",
"discussion": "3 Results and Discussion Figure 1 a shows\na schematic representation of the UV-grafting process of PDMS (i.e.,\nsilicone oil) used to convert 1000-grade Al surfaces into either LISS 35 or CL-PDMS coatings. Prior to UV-grafting, the\nsubstrates were ground to ensure the same surface roughness for all\nsamples, and particularly for corrosion characterization, although\nthe coating can be applied equally well to polished or rough surfaces\nregardless of their roughness characteristics. The formation process\nis simple, inexpensive, and easily scalable. The samples were placed\nhorizontally in a glass Petri dish, and then, droplets of trimethylsiloxy-terminated\nsilicone oil (PDMS) were added to spread over the entire substrate\nsurface. In this study, PDMS with a kinematic viscosity of 500 cSt\n( M w = 17.3 kDa) was used. The setup was\nthen illuminated with a medium-pressure mercury UV lamp at different\ntimes. Figure 1 (a) Schematic representation of the silicone oil UV-grafting process\nto convert Al surfaces into either LISS or CL-PDMS coatings, both\ncovalently bound to the substrate. (b–e) High-resolution XPS\nspectra of (b) Si 2p, (c) O 1s, (d) Al 2p, and (e) C 1s. (f) XPS depth\nprofile of Si, C, O, and Al, UV-grafted PDMS on polished Al substrates.\n(g) Cross-sectional SEM image of the CL-PDMS layer prepared on 200\nμm thick Al foil. Air–PDMS and PDMS–aluminum foil\ninterfaces are highlighted by orange dashed lines. (h) EDX analysis\nof bare and CL-PDMS (measured at the top and near the Al substrate)\nUV-grafted on polished Al substrates. The typical spectrum of a light source is presented\nin Figure S1 , Supporting Information. As\nshown,\nthe most intense peaks in the UVA region correspond to λ = 320\nand 365 nm. As we have previously shown, the 321 nm wavelength is\nthe minimum required to dissociate the trimethylsilyl bonds of the\nPDMS terminal groups. 35 Other bonds, such\nas Si–O or even the backbone Si–CH 3 , require\nhigher energy photons, i.e., a lower wavelength, to ensure that there\nis a higher probability of preferentially dissociating the Si–CH 3 terminal bonds when using this source of UV irradiation.\nThe UV-grafting process was previously tested on various metals and\noxides, while the LISS samples were thoroughly washed in toluene to\ndissolve the remaining, nongrafted silicone oil, leaving only covalently\nbound PDMS molecules. It was shown that the surface was completely\nand homogeneously covered with covalently bound PDMS molecules. 35 To verify covalent bonding of PDMS molecules\nto Al substrates,\na water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS)\nwere applied. Bare Al substrates are hydrophilic and exhibit a WCA\nof 74.0° ± 0.2°. Note that Al is considered a high\nsurface energy material with a low WCA due to the formation of a native\noxide layer. 41 Here, such a relatively\nhigh WCA is due to hydrophobic contaminants, well in agreement with\nthe literature, 41 , 42 while the high-resolution XPS\nmeasurements support the adventitious carbon signature typical of\nairborne contamination ( Figure S2 , Supporting\nInformation). 43 To form the LISS coating,\nAl samples were exposed to UV light for 30 min. The UV light partially\ndissociates silicone oil and covalently grafts it to the substrate,\nwhile the remaining nondissociated PDMS molecules are considered to\nbe an infusing lubricant. The stability of LISS is ensured by matching\nthe surface tension of the residual silicone oil with the surface\nenergy of Al, which is composed of the same but covalently grafted\nPDMS molecules. 23 The LISS Al samples show\na WCA and CA hysteresis (CAH) of 100.9° ± 3.9° and\n0.9° ± 0.5°, respectively ( Figure 1 a, top inset images), due to the liquid nature\nof the infused lubricant, i.e., the formation of the atomically smooth\nliquid surface typical for SLIPS/LISS. 23 , 24 , 44 To investigate the chemical composition and\nsurface coverage of\nthe UV-grafted PDMS layer on Al substrates, angle-resolved XPS analysis\nwas performed at incident angles of 15°, 45°, and 75°,\ni.e., the detection depth changes due to the sample tilt angle. Silicone\noil was UV-grafted to polished Al substrates, and the remaining oil\nwas dissolved in toluene. The typical XPS survey spectra of these\nsamples are shown in Figure S3 , Supporting\nInformation, and the high-resolution XPS spectra are shown in Figure 1 b–e. Bare\nAl was measured as a reference, showing Al, O, and C peaks ( Figure S3a , Supporting Information). When UV-grafted,\nthe survey XPS spectra consist mainly of Si, O, and C peaks associated\nwith PDMS, while the Al 2p peak originating from the substrate is\nbarely noticeable ( Figure S3b , Supporting\nInformation). The high-resolution Si 2p XPS spectrum consists of a\nsingle peak with a binding energy of 102.64 eV, which was deconvoluted\ninto two components at 102.61 and 103.51 eV corresponding to Si–C\nand Si–O bonds, respectively ( Figure 1 b). 45 The O 1s\nspectra with the peak centered at 532.80 eV consist of three components:\nat 532.73 eV corresponding to the Si–O–Si bond (89.8%) 46 and at 531.53 and 534.13 eV corresponding to\nthe Al–O–Si (5.3%) and Al–OH (4.9%) bonds, respectively\n( Figure 1 c). 47 , 48 When measured at an angle of 75°, the components corresponding\nto the substrate, Al–O–Si and Al–OH, increase\nslightly to 13.3 and 7.0%, respectively ( Figure S4 , Supporting Information). The high-resolution XPS peak of\nAl 2p was clearly observed at 45° and 75° but almost disappears\nat 15°, confirming that the PDMS-grafted layer covers the substrate\nentirely ( Figure S5 , Supporting Information).\nWhen measured at 45° and 75°, two peaks were observed at\n72.70 and 75.55 eV, corresponding to metallic Al and its oxide (in\nthe case of metallic Al, the peaks are asymmetric and the splitting\nof the 2p peak is evident with a spin separation of 0.44 eV, while\nthe oxide has a symmetrical peak and the splitting can typically be\nignored), 49 , 50 respectively ( Figures 1 d and S5 , Supporting\nInformation). 48 The C 1s spectra show a\nsingle peak at 285.10 eV, which is associated with Si–C bonds\n(284.49 eV, 16.5%) and C–H bonds (285.19 eV, 82.5%) in PDMS,\nas well as small amounts of CO bonds (286.74 eV, 0.9%) ( Figure 1 e). 51 The XPS sputter profiles of the UV-grafted PDMS on Al substrates\ndemonstrate the thickness of the grafted layer to be ∼10 nm,\nwhich is in good agreement with our previous results obtained on Si\nwafers ( Figure 1 f). 35 However, when the Al samples with silicone\noil were exposed to\nUV irradiation for a longer time, i.e., 180 min, cross-linkage of\nPDMS was obtained. In this case, the coating appears as a thin semi-rigid\nlayer in contrast to its liquid-like LISS counterparts ( Figure 1 g). The thickness of the CL-PDMS\nlayer varies from tens to hundreds of microns as a function of the\ninitial silicone oil volume dispersed ( Figure S6 , Supporting Information). The UV-grafted CL-PDMS layer has\na CA and CAH of 98.7° ± 4.2° and 8.6° ± 4.8°,\nrespectively ( Figure 1 a, bottom inset images). Attenuated total reflectance Fourier-transform\ninfrared (ATR-FTIR)\nand Raman spectroscopy analyses were performed on polished Al, plain\nPDMS, LISS, and CL-PDMS to study the time dependence of the UV-grafting\nprocess on Al. The FTIR spectra in the wave number range of 250–4000\ncm –1 are shown in Figure 2 a. The peaks at 1258, 1065, 1011, and 788\ncm –1 are the fingerprint for silicone oil. 52 The two PDMS peaks at 1258 and 788 cm –1 are due to CH 3 deformation and CH 3 rocking\nin Si–CH 3 , respectively, and the two adjacent peaks\nat 1065 and 1011 cm –1 are due to Si–O–Si\nasymmetric deformation ( Figure 2 b). The peaks at 856 and 900 cm –1 in the\nCL-PDMS spectrum can be assigned to Si–O–Al bonds ( Figure 2 b, blue spectrum). 53 Figure 2 c shows the symmetric and asymmetric CH 3 stretching\npeaks at 2962 and 2906 cm –1 , respectively. 54 A reduction in the peak intensity at 2962 cm –1 and a shift to 2963 cm –1 were observed\nin CL-PDMS. Such a shift of the methylene vibrations is generally\nbelieved to reflect an increase in the intermolecular interactions,\nimproved long-range order, and, consequently, crystallinity of the\nPDMS layer. 55 , 56 Figure 2 ATR-FTIR spectra in the wave number range\nof (a) 4500–250\ncm –1 , (b) 1400–250 cm –1 , and (c) 3000–2880 cm –1 of the polished\nAl (black lines), plain PDMS (red lines), LISS (green lines), and\nCL-PDMS (blue lines) samples. (d) Mean Raman spectra and corresponding\nimages of (e) plain and (f) UV-grafted CL-PDMS. The color bar in (e)\nand (f) indicates the percentage area change under the Si–O–Si\npeak. Raman spectroscopy shows a typical spectrum of\nplain silicone oil\nwith the repeating unit of (Si(CH 3 ) 2 –O)\n( Figure 2 d). The intense\npeaks at 2965 and 2906 cm –1 in plain silicone oil\ncorrespond to the stretching modes of the CH 3 group, 1411\ncm –1 (CH 3 asymmetric bending), 1260 cm –1 (CH 3 symmetric bending), and 862 (CH 3 symmetric rocking), 788 (CH 3 asymmetric rocking\nand Si–C asymmetric stretching), and 708 cm –1 (Si–C symmetric stretching) modes. 57 The peak at 688 cm –1 is assigned to the Si–CH 3 symmetric rocking mode, and the peak at 489 cm –1 corresponds to the Si–O–Si stretching mode. 58 Upon cross-linking, the Si–O–Si\npeak shifts to 491 cm –1 , and the faint peak at 742\ncm –1 appears ( Figure 2 d). While the Si–O–Si peak shift to higher\nwavenumbers indicates the increase in hardening/crystallinity of the\nCL-PDMS, the peak at 742 cm –1 was typically observed\nin the thermally cured CL-PDMS elastomers. 59 The color maps in Figure 2 e,f, superimposed on the optical images, show the area under\nthe Si–O–Si peak, indicating only slight spatial differences\nin the thickness of the films. As can be seen, the percentage change\ncalculated from the average of the entire map is smaller for the CL-PDMS\nas compared to the unlinked one. The latter can be attributed to the\ndifferent sample preparation, i.e., the plain PDMS was dropped into\nan Al well, resulting in a drop-like thickness pattern after drying,\nwhile CL-PDMS forms a more uniform thick film under UV illumination\non a flat Al substrate. Finally, the standard ASTM D2765-16 procedure\nwas used to estimate the degree of cross-linking. According to the\ncalculations, the degree of cross-linking of UV-grafted samples exposed\nto UV irradiation for 180 min was ∼98%, while the CL-PDMS layer\nwas barely swollen in toluene. The ATR-FTIR, Raman, and percentage\ncross-linking measurements indicate that with prolonged UV irradiation,\nthere is a probability of dissociation of more than one trimethylsilyl\nbond to form a gel-like CL-PDMS coating. As a next step, LISS\nand CL-PDMS coatings on Al were investigated\nfor their resistance to wetting-related phenomena such as corrosion\nand biofouling in aqueous environments. As previously described, Al\nis a non-biocompatible material that is toxic to aquatic life. 11 Therefore, in addition to direct corrosion-related\nstructural failure of metallic components, it is essential to protect\nAl from contact with a corrosive environment. Figure 3 a shows potentiodynamic polarization curves\nmeasured in an aqueous electrolyte containing 3.5 wt % NaCl, the standard\nconcentration used to simulate the seawater chloride concentration.\nOptical microscopy images show the original surface after the grinding\nand cleaning steps with micron-scale roughness features caused by\nthe SiC abrasive paper ( Figure 3 b). The bare Al curve shows active corrosion upon anodic polarization;\nthis is due to pitting corrosion with pits uniformly distributed over\nthe entire surface ( Figure 3 c). 60 The substrate covered with\nthe LISS coating shows a lower current density in the cathodic branch\ndue to the lower conductivity of the PDMS-covered surface. However,\nduring anodic polarization, active dissolution is still observed,\nwith only slightly reduced anodic current densities compared to the\nbare Al surface. However, no localized corrosion was observed on the\nsurface of the LISS samples ( Figure 3 d). This is explained by a corrosion attack at the\ninterface between the protective lacquer and the LISS coating, where\ncrevice corrosion occurred ( Figure S7 ,\nSupporting Information). When the potentiodynamic polarization was\napplied to the CL-PDMS coating, only noise was obtained and no current\nsignal in response to polarization could be measured ( Figure 3 a, red spectrum, and 3 e). This is due to the inability\nof the corrosive electrolyte to penetrate the cross-linked layer,\nas well as the covalent bonding of the PDMS to the Al substrate, making\nelectrochemical measurements impossible. Such complete isolation of\nthe material surface from an aggressive environment gains the system\nsuperior corrosion resistance. Figure 3 (a) Potentiodynamic polarization curves\nand (b–e) bright-field\noptical microscopy images of the ground bare aluminum surface before\npotentiodynamic polarization (b) and after potentiodynamic polarization\nof bare (c), LISS (d), and covalently bound CL-PDMS (e), measured\nin a 3.5 wt % NaCl aqueous electrolyte. (f) Digital images of bare,\nCL, and LISS covalently bound PDMS prepared on aluminum before (day\n0) and after 85 and 143 days of submersion in artificial seawater. Nevertheless, it has been shown previously that\nwater can infiltrate\nPDMS. 61 To investigate the long-term stability\nand corrosion protection of PDMS-based coatings, bare, LISS, and CL-PDMS\nwere immersed in freshwater and seawater media for several months\n( Figures 3 f and S8 , Supporting Information). As shown, bare Al\nsamples corrode severely in both freshwater and seawater media used\nto grow aquatic organisms (mimicking the freshwater and seawater composition)\n(see Tables S1 and S2 for chemical composition,\nSupporting Information). In the case of LISS and CL-PDMS, no corrosion\nwas observed after 85 and 143 days. Note that this set of samples\nis still immersed in aquatic media, while the full period of corrosion\nprotection will be published elsewhere. One of the most challenging\nphenomena associated with the wetting\nof solid surfaces in aquatic environments is the formation of biofilms\nor biofouling. Biofilm formation is a complex, multistep process involving\na wide variety of microorganisms. Once a solid is immersed in water,\nit adsorbs glycoprotein organic materials, forming a “conditioning\nfilm” within minutes. Bacteria, unicellular algae, and cyanobacteria\n(blue-green algae) are typically the next species to appear on the\nmaterial, colonizing the material surface within hours. 62 Species such as freshwater algae are critical\nto maintaining a healthy aquatic ecosystem as they increase the availability\nof dissolved oxygen for the organisms below, while the negative effects\nof Al on algae have been demonstrated. 13 Here, green algae of the species Chlamydomonas reinhardtii , a motile alga that moves freely in the aquatic environment, were\nused as a freshwater model organism to study the growth and adhesion\nof algal biofilms on bare, CL-PDMS, and LISS-coated Al ( Figure 4 ). Such green algal biofilms,\ngrown on slippery lubricant-infused surfaces, have been shown to be\nremarkable indicators of the effectiveness of the liquid-infused layer. 24 , 35 , 36 , 63 , 64 Here, treated and control samples were immersed\nin freshwater growth media with green algae for 8 days under a 16/8\nh on/off illumination cycle, and the results are summarized in Figure 4 a–d. Over\nthe growth period, there was a significant increase in the algal density\nof LISS and CL-PDMS samples with no evidence of substrate-related\ngrowth reduction or mortality, indicating the nontoxic nature of the\ncoating. Figure 4 Digital images of aluminum samples immersed for 8 days in freshwater\nmedium containing C. reinhardtii green\nalgae just before (top-left) and immediately after (bottom-left) harvesting\nby passing through the water–air interface and corresponding\nbright-field reflectance (top-right) and fluorescence (bottom-right)\nimages obtained immediately after harvesting. (a) Bare, (b) CL-PDMS,\nand (c) LISS. (d) Calculated surface coverage as obtained from digital\n(red columns) and fluorescence (blue columns) images. Note that fluorescence\ncoverage is plotted on a logarithmic scale. The surface coverage of green algae was obtained\nfrom digital and\nconfocal fluorescence microscopy images. As shown in Figure 4 a–c, freshwater green\nalgae grow uniformly in Petri dishes. However, the biofilms formed\non the treated and control samples show significantly different affinity\nto the substrates. When the bare samples were pulled through a water–air\ninterface, algal biofilms were firmly attached to Al surfaces covering\n97.4 ± 0.5% of the total sample area ( Figure 4 a,d and Movie S1 , Supporting Information). Furthermore, all bare Al samples showed\nsevere corrosion, resulting in the appearance of dark green-black\ncolor ( Figure 4 a).\nThe biofilms formed on the CL-PDMS samples covered 63.8 ± 18.4%\nof the total sample area without showing any signs of corrosion ( Figure 4 b,d and Movie S2 , Supporting Information). When the Petri\ndish containing LISS Al samples was immersed in a larger reservoir\nof fresh water prior to harvesting, the algal biofilm spontaneously\ndelaminated from the LISS substrates. When pulled through the water–air\ninterface, 99.6 ± 0.3% of the Al surface was both biofilm-free\nand corrosion-free, showing a highly reflective metallic luster ( Figure 4 c,d and Movie S3 , Supporting Information). Statistical\nanalysis was performed to determine the significance of the resistance\nof the coated samples to freshwater green algae and showed a p -value of 0.045 and ≪0.005 for CL-PDMS and LISS\naluminum, respectively ( Figure 4 d). Although bacteria are usually associated with primary\nbiofilm communities,\nother microscopic organisms such as diatoms are also known to be early\ncolonizers. 38 , 39 Diatoms are unicellular algae,\nin which the protoplast is enclosed in an elaborately decorated silica\nshell (the frustule) composed of overlapping halves or “valves”. 65 Diatoms adhere to surfaces through the production\nof sticky extracellular polymeric substances, which are secreted through\nan elongated slit in one or both valves, while the division of attached\ncells rapidly gives rise to colonies that eventually coalesce to form\na compact biofilm. 36 , 66 While silicone elastomers have\nbeen investigated and commercially tested with anti-biofouling paints\nto “release” fouling organisms under hydrodynamic conditions, 38 it has been demonstrated that diatoms can barely\nbe released from PDMS elastomer coatings even under high-speed operating\nconditions (>30 knots). 38 , 66 Therefore, it is critical\nto evaluate the developed coatings for their fouling release characteristics\nagainst early colonizers. Odontella aurita is the benthic diatom that belongs to the cosmopolitan taxa category\nand can be found in the Arctic, sub-Arctic, and even in tropical marine\nwaters. 67 It is an unicellular organism,\na cylindrical, chain-forming diatom with a yellow to brown color.\nThe cells are connected to each other by protrusions that extend from\nthe end of each cell. O. aurita can\neither swim freely in the water or be firmly attached to rocks, plants,\nor animals. It has also been shown that O. aurita can act as a substrate for other diatoms in freshwater, brackish,\nand marine environments. 68 Figure 5 Digital images of aluminum\nsamples immersed for 8 days in seawater\nmedium with O. aurita diatoms just\nbefore (upper-left) and immediately after (lower-left) harvesting\nby passing through the water–air interface and corresponding\nbright-field reflectance (upper-right) and fluorescence (lower-right)\nimages obtained immediately after the harvesting of (a) bare, (b)\nCL-PDMS, and (c) LISS. (d) Calculated surface coverage from digital\n(red columns) and fluorescence (blue columns) images. Note that fluorescence\ncoverage is plotted on a logarithmic scale. The treated and control samples were immersed in\nseawater growth\nmedia containing O. aurita diatoms\nfor 8 days under a 16/8 h on/off lighting cycle, and the results are\nsummarized in Figure 5 . Again, there was a significant increase in diatom density in all\nsamples over the growth period. Surface coverage calculations were\nperformed using digital and confocal fluorescence microscopy images.\nBare and CL-PDMS samples were pulled through a water–air interface\nand showed surface coverage of 99.9 ± 0.1 and 91.2 ± 2.6%,\nrespectively ( Figure 5 a,b,d and Movies S4 and S5 , Supporting Information). The surface coverage of the LISS\nsamples was 4.6 ± 2.7%, a significant difference ( p ≪ 0.005) compared to the bare and CL-PDMS samples ( Figure 5 c,d and Movie S6 , Supporting Information). Such passive\nshedding, i.e., no external stimuli applied to remove the biofilm\nrather than pulling it through a water–air interface, indicates\npoor adhesion of the diatom biofilms to the LISS-coated Al substrates.\nFurthermore, all treated substrates, i.e., CL-PDMS and LISS, exhibit\na well-reflective metallic luster after 8 days of immersion in seawater\nmedia, indicating superior resistance to a highly corrosive environment\n( Figure 5 b,c)."
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