Microfluidic approaches in microbial ecology.

Microbial life is at the heart of many diverse environments and regulates most natural processes, from the functioning of animal organs to the cycling of global carbon. Yet, the study of microbial ecology is often limited by challenges in visualizing microbial processes and replicating the environmental conditions under which they unfold. Microfluidics operates at the characteristic scale at which microorganisms live and perform their functions, thus allowing for the observation and quantification of behaviors such as growth, motility, and responses to external cues, often with greater detail than classical techniques. By enabling a high degree of control in space and time of environmental conditions such as nutrient gradients, pH levels, and fluid flow patterns, microfluidics further provides the opportunity to study microbial processes in conditions that mimic the natural settings harboring microbial life. In this review, we describe how recent applications of microfluidic systems to microbial ecology have enriched our understanding of microbial life and microbial communities. We highlight discoveries enabled by microfluidic approaches ranging from single-cell behaviors to the functioning of multi-cellular communities, and we indicate potential future opportunities to use microfluidics to further advance our understanding of microbial processes and their implications.

High immigration rates critical for establishing emigration-driven diversity in microbial communities.

Unraveling the mechanisms governing the diversity of ecological communities is a central goal in ecology. Although microbial dispersal constitutes an important ecological process, the effect of dispersal on microbial diversity is poorly understood. Here, we sought to fill this gap by combining a generalized Lotka-Volterra model with experimental investigations. Our model showed that emigration increases the diversity of the community when the immigration rate crosses a defined threshold, which we identified as Ineutral. We also found that at high immigration rates, emigration weakens the relative abundance of fast-growing species and thus enhances the mass effect and increases the diversity. We experimentally confirmed this finding using co-cultures of 20 bacterial strains isolated from the soil. Our model further showed that Ineutral decreases with the increase of species pool size, growth rate, and interspecies interaction. Our work deepens the understanding of the effects of dispersal on the diversity of natural communities.

Metabolism of novel potential syntrophic acetate-oxidizing bacteria in thermophilic methanogenic chemostats.

Acetate is a major intermediate in the anaerobic digestion of organic waste to produce CH4. In methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or syntrophic degradation by acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in the isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB and the mechanisms of their interactions with methanogenic partners are not fully characterized. In this study, the in situ activity and metabolic characteristics of potential SAOB and their interactions with methanogens were elucidated through metagenomics and metatranscriptomics. In addition to the reported SAOB classified in the genera Tepidanaerobacter, Desulfotomaculum, and Thermodesulfovibrio, we identified a number of potential SAOB that are affiliated with Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. The potential SAOB possessing the glycine-mediated acetate oxidation pathway dominates SAOB communities. Moreover, formate appeared to be the main product of the acetate degradation by the most active potential SAOB. We identified the methanogen partner of these potential SAOB in the acetate-fed chemostat as Methanosarcina thermophila. The dominated potential SAOB in each chemostat had similar metabolic characteristics, even though they were in different fatty-acid-fed chemostats. These novel syntrophic lineages are prevalent and may play critical roles in thermophilic methanogenic reactors. This study expands our understanding of the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders and presents novel insights into how they interact with methanogens.IMPORTANCECombining reactor operation with omics provides insights into novel uncultured syntrophic acetate degraders and how they perform in thermophilic anaerobic digesters. This improves our understanding of syntrophic acetate degradation and contributes to the background knowledge necessary to better control and optimize anaerobic digestion processes.

The trade-off between individual metabolic specialization and versatility determines the metabolic efficiency of microbial communities.

In microbial systems, a metabolic pathway can be either completed by one autonomous population or distributed among a consortium performing metabolic division of labor (MDOL). MDOL facilitates the system's function by reducing the metabolic burden; however, it may hinder the function by reducing the exchange efficiency of metabolic intermediates among individuals. As a result, the function of a community is influenced by the trade-offs between the metabolic specialization and versatility of individuals. To experimentally test this hypothesis, we deconstructed the naphthalene degradation pathway into four steps and introduced them individually or combinatorically into different strains with varying levels of metabolic specialization. Using these strains, we engineered 1,456 synthetic consortia and found that 74 consortia exhibited higher degradation function than both the autonomous population and rigorous MDOL consortium. Quantitative modeling provides general strategies for identifying the most effective MDOL configuration. Our study provides critical insights into the engineering of high-performance microbial systems.

Assembly of Microbial Communities Engaging in Metabolic Division of Labor in a Diffusion-Limited Environment Is Governed by Metabolic Flux.

Metabolic division of labor (MDOL) represents one of the most commonly occurring interactions within natural microbial communities. Specifically, in a variety of MDOL systems engaged in hydrocarbon degradation, a sequential degradation is performed by several members with final products that are necessary for the growth of each member. In these MDOL systems, each strain catalyzes one or more specific reactions of a multistep metabolic pathway, whose end products are then allocated among the participants. While the benefit allocation is independent of metabolic flux in well-mixed environments, it remains unclear how the benefits are allocated when diffusion is limited. Here, we investigated how MDOL communities assemble in a diffusion-limited environment, by combining mathematical modeling with experimental inquiry using a synthetic consortium engaged in MDOL. Our model analysis in a diffusion-limited environment showed that, when the growth of all populations in the community relies on the final product that can only be produced by the last population, a diffusion gradient of the final products may create a bias favoring the member producing the final products, resulting in a higher relative abundance of the final product producer. Moreover, such asymmetric allocation of the final products is enhanced by both the lower diffusion rate and the higher metabolic flux (i.e., the higher yields of the final products) in the MDOL. Our results show that in a diffusively confined environment, metabolic flux constitutes a determining factor in the assembly of the MDOL community. Together, our findings are critical for a better understanding of how resource-sharing microbial communities are established and should assist in designing such communities for improved biomanufacturing and bioremediation.

Volatile compounds-induced environmental pH shifts mediate interspecific bacterial interactions over long-distance.

Microorganisms interact with each other through exchanging metabolites as well as signals molecules. This exchange typically depends on metabolites or signaling molecules diffusing in aqueous media within spatially connected habitats, and it only occurs within a short range. However, different microorganisms frequently live in spatially discontinuous micro-habitats without the connections of aqueous media. How microorganisms populating such spatially discontinuous micro-habitats interact remains poorly understood. Here, we show that a bacterial strain, Corynebacterium glutamicum ATCC13032T, produces high amounts of ammonia in its local habitat, which disperses over long distances (up to 12 cm) through the air. As a result, the pH of another spatially disconnected habitat increases, thus inducing the growth of an acid-sensitive strain (Glycocaulis alkaliphilus 6B-8T). We also show that ammonia-mediated long-distance interactions can be commonly found in various bacterial communities. In conclusion, our work demonstrates that bacteria growing in spatially discontinuous micro-habitats can interact with each other through gaseous diffusion of volatile compounds. This finding expands our understanding of the spatial scale at which bacteria can interact and provides new insights into how spatially separated species are connected to maximizing community-level commensalism.

Even allocation of benefits stabilizes microbial community engaged in metabolic division of labor.

Microbial communities execute metabolic pathways to drive global nutrient cycles. Within a community, functionally specialized strains can perform different yet complementary steps within a linear pathway, a phenomenon termed metabolic division of labor (MDOL). However, little is known about how such metabolic behaviors shape microbial communities. Here, we derive a theoretical framework to define the assembly of a community that degrades an organic compound through MDOL. The framework indicates that to ensure community stability, the strains performing the initial steps should hold a growth advantage (m) over the "private benefit" (n) of the strain performing the last step. The steady-state frequency of the last strain is then determined by the quotient of n and m. Our experiments show that the framework accurately predicts the assembly of our synthetic consortia that degrade naphthalene through MDOL. Our results provide insights for designing and managing stable microbial systems for metabolic pathway optimization.

Co-inoculation with beneficial microorganisms enhances tannery sludge bioleaching with Acidithiobacillus thiooxidans.

Bioleaching of tannery sludge is an efficient and environmentally friendly way for chromium (Cr) removal, which supports the sustainable development of the leather industry. Acidithiobacillus thiooxidans has been reported effective in Cr bioleaching of tannery sludge. However, little is known about whether the presence of other benefiting species could further improve the Cr leaching efficiency of A. thiooxidans. Here, we studied the enhancing roles of four species namely Acidiphilium cryptum, Sulfobacillus acidophilus, Alicyclobacillus cycloheptanicus, and Rhodotorula mucilaginosa in chromium bioleaching of tannery sludge with A. thiooxidans by batch bioleaching experiments. We found that each of the four species facilitated the quick dominance of A. thiooxidans in the bioleaching process and significantly improved the bioleaching performance including bioleaching rate and efficiency. The bioleaching efficiency of Cr in the tannery sludge could reach 100% on the sixth day by co-inoculating A. thiooxidans and four auxiliary species. The achievements shed a light on the role of the community-level interactions on bioleaching and may also serve as guidance for managing bioleaching consortiums for better outcomes.

Type IV Pilus Shapes a 'Bubble-Burst' Pattern Opposing Spatial Intermixing of Two Interacting Bacterial Populations.

Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be important determinants of community-level properties. Spatial intermixing emerging from microbial interaction is one of the best-studied characteristics of spatial patterns. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two mutualistic Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (here referred to as a 'bubble-burst' pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments. IMPORTANCE When growing on surfaces, multispecies microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, enabling otherwise impermissible metabolic functions to occur as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells, and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganisms, but its role on pattern formation within microbial communities is unclear. Here, we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy for engineering high-performance synthetic microbial communities.

Substrate availability and toxicity shape the structure of microbial communities engaged in metabolic division of labor.

Metabolic division of labor (MDOL) represents a widespread natural phenomenon, whereby a complex metabolic pathway is shared between different strains within a community in a mutually beneficial manner. However, little is known about how the composition of such a microbial community is regulated. We hypothesized that when degradation of an organic compound is carried out via MDOL, the concentration and toxicity of the substrate modulate the benefit allocation between the two microbial populations, thus affecting the structure of this community. We tested this hypothesis by combining modeling with experiments using a synthetic consortium. Our modeling analysis suggests that the proportion of the population executing the first metabolic step can be simply estimated by Monod-like formulas governed by substrate concentration and toxicity. Our model and the proposed formula were able to quantitatively predict the structure of our synthetic consortium. Further analysis demonstrates that our rule is also applicable in estimating community structures in spatially structured environments. Together, our work clearly demonstrates that the structure of MDOL communities can be quantitatively predicted using available information on environmental factors, thus providing novel insights into how to manage artificial microbial systems for the wide application of the bioindustry.

Guided by the principles of microbiome engineering: Accomplishments and perspectives for environmental use.

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities, knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology, especially for environmental use. Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering. Here, we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation. Moreover, a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications. A strategy to engineer microbiomes for environmental use, which avoids the build-up of toxic intermediates that pose a risk to human health, is suggested. We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.

Energy Availability Determines Strategy of Microbial Amino Acid Synthesis in Volatile Fatty Acid-Fed Anaerobic Methanogenic Chemostats.

In natural communities, microbes exchange a variety of metabolites (public goods) with each other, which drives the evolution of auxotroph and shapes interdependent patterns at community-level. However, factors that determine the strategy of public goods synthesis for a given community member still remains to be elucidated. In anaerobic methanogenic communities, energy availability of different community members is largely varied. We hypothesized that this uneven energy availability contributed to the heterogeneity of public goods synthesis ability among the members in these communities. We tested this hypothesis by analyzing the synthetic strategy of amino acids of the bacterial and archaeal members involved in four previously enriched anaerobic methanogenic communities residing in thermophilic chemostats. Our analyses indicate that most of the members in the communities did not possess ability to synthesize all the essential amino acids, suggesting they exchanged these essential public goods to establish interdependent patterns for survival. Importantly, we found that the amino acid synthesis ability of a functional group was largely determined by how much energy it could obtain from its metabolism in the given environmental condition. Moreover, members within a functional group also possessed different amino acid synthesis abilities, which are related to their features of energy metabolism. Our study reveals that energy availability is a key driver of microbial evolution in presence of metabolic specialization at community level and suggests the feasibility of managing anaerobic methanogenic communities for better performance through controlling the metabolic interactions involved.

A novel temperate phage, vB_PstS-pAN, induced from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10.

A novel temperate phage named vB_PstS-pAN was induced by mitomycin C treatment from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10. The phage particles have icosahedral heads and long non-contractile tails, and vB_PstS-pAN can therefore be morphologically classified as a member of the family Siphoviridae. The whole genome of vB_PstS-pAN is 39,466 bp in length, with an 11-nt 3' overhang cohesive end. There are 53 genes in the vB_PstS-pAN genome, including genes responsible for phage integration, replication, morphogenesis, and bacterial lysis. The vB_PstS-pAN genome has low similarity to other phage genomes in the GenBank database, suggesting that vB_PstS-pAN is a novel member of the family Siphoviridae.

Isolation Chip Increases Culturable Bacterial Diversity and Reduces Cultivation Bias.

Conventional cultivation methods, including petri dish plating, are selective and biased to enrich specific microorganisms, such as big population and fast-growing bacteria. In this study, we evaluated the ability of isolation chip (ichip) to reduce cultivation bias. We used the ichip and petri dish plating methods to cultivate bacteria from soil contaminated with (contaminated soil) or without (natural soil) crude oil. Ichip improved the richness and evenness of bacterial isolates in both the natural and contaminated soil samples. Using the petri dish plating method, Pseudomonas and Lysinibacillus isolates were found to be the most abundant, with over 50% of the relative abundance in the natural and oil-polluted soil-cultured communities, respectively. In comparison, using the ichip method, the isolates with the highest relative abundances were from Bacillus and Aeromonas in natural and contaminated soil-cultured communities, which only accounted for 20% and 28% of the total isolates, respectively. Interestingly, the evenness and richness of the bacteria varied slightly between the natural and oil-polluted soil samples, indicating that ichip had the ability to reduce the cultivation bias. In addition, oil selective pressure enriched the functional bacteria isolated using the petri dish plating method. In summary, ichip allows bacteria to grow evenly, as well as allowing for substance exchange between the environment and single cells. As such, it is a very good method for increasing culturable bacterial diversity and reducing cultivation bias.

Selfishness driving reductive evolution shapes interdependent patterns in spatially structured microbial communities.

Microbes release a wide variety of metabolites to the environment that benefit the whole population, called public goods. Public goods sharing drives adaptive function loss, and allows the rise of metabolic cross-feeding. However, how public goods sharing governs the succession of communities over evolutionary time scales remains unclear. To resolve this issue, we constructed an individual-based model, where an autonomous population that possessed functions to produce three essential public goods, was allowed to randomly lose functions. Simulations revealed that function loss genotypes could evolve from the autonomous ancestor, driven by the selfish public production trade-off at the individual level. These genotypes could then automatically develop to three possible types of interdependent patterns: complete functional division, one-way dependency, and asymmetric functional complementation, which were influenced by function cost and function redundancy. In addition, we found random evolutionary events, i.e., the priority and the relative spatial positioning of genotype emergence, are also important in governing community assembly. Moreover, communities occupied by interdependent patterns exhibited better resistance to environmental perturbation, suggesting such patterns are selectively favored. Our work integrates ecological interactions with evolution dynamics, providing a new perspective to explain how reductive evolution shapes microbial interdependencies and governs the succession of communities.

Successful microbial colonization of space in a more dispersed manner.

Many organisms live in habitats with limited nutrients or space, competition for these resources is ubiquitous. Although spatial factors related to the population's manner of colonizing space influences its success in spatial competition, what these factors are and to what extent they influence the outcome remains underexplored. Here, we applied a simulated competitive model to explore the spatial factors affecting outcomes of competition for space. By quantifying spatial factors, we show that colonizing space in a more dispersed manner contributes to microbial competitive success. We also find that the competitive edge deriving from a more dispersed manner in colonization can compensate for the disadvantage arising from either a lower growth rate or lower initial abundance. These findings shed light on the role of space colonization manners on maintaining biodiversity within ecosystems and provide novel insights critical for understanding how competition for space drives evolutionary innovation.

Sessile bacterium unlocks ability of surface motility through mutualistic interspecies interaction.

In addition to their common planktonic lifestyle, bacteria frequently live in surface-associated habitats. Surface motility is essential for exploring these habitats for food sources. However, many bacteria are found on surfaces, even though they lack features required for migrating along surfaces. How these canonical non-motile bacteria adapt to the environmental fluctuations on surfaces remains unknown. Here, we report a previously unknown surface motility mode of the canonical non-motile bacterium, Dietzia sp. DQ12-45-1b, which is triggered by interaction with a dimorphic prosthecate bacterium, Glycocaulis alkaliphilus 6B-8T. Dietzia cells exhibits 'sliding'-like motility in an area where the strain Glycocaulis cells was pre-colonized with a sufficient density. Our analysis also demonstrates that Dietzia degrade n-alkanes and provide Glycocaulis with the resulting metabolites for survival, which in turn induced directional migration of Dietzia towards nutrient-rich environments. Such interaction-triggered migration was also found between Dietzia and Glycocaulis strains isolated from other habitats, suggesting that this mutualistic relationship ubiquitously occurs in natural environments. In conclusion, we propose a novel model for such a 'win-win' strategy, whereby non-motile bacteria pay metabolites to dimorphic prosthecate bacteria in return for migrating to seek for nutrients, which may represent a common strategy for canonically non-motile bacteria living on a surface.

Metabolic Exchange with Non-Alkane-Consuming Pseudomonas stutzeri SLG510A3-8 Improves n-Alkane Biodegradation by the Alkane Degrader Dietzia sp. Strain DQ12-45-1b.

Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are sometimes composed of not only hydrocarbon degraders but also nonconsumers, but the synergistic mechanisms remain unknown. Here, we found that two bacterial strains isolated from Chinese oil fields, Dietzia sp. strain DQ12-45-1b and Pseudomonas stutzeri SLG510A3-8, had a synergistic effect on hexadecane (C16 compound) biodegradation, even though P. stutzeri could not utilize C16 individually. To gain a better understanding of the roles of the alkane nonconsumer P. stutzeri in the C16-degrading consortium, we reconstructed a two-species stoichiometric metabolic model, iBH1908, and integrated in silico prediction with the following in vitro validation, a comparative proteomics analysis, and extracellular metabolomic detection. Metabolic interactions between P. stutzeri and Dietzia sp. were successfully revealed to have importance in efficient C16 degradation. In the process, P. stutzeri survived on C16 metabolic intermediates from Dietzia sp., including hexadecanoate, 3-hydroxybutanoate, and α-ketoglutarate. In return, P. stutzeri reorganized its metabolic flux distribution to fed back acetate and glutamate to Dietzia sp. to enhance its C16 degradation efficiency by improving Dietzia cell accumulation and by regulating the expression of Dietzia succinate dehydrogenase. By using the synergistic microbial consortium of Dietzia sp. and P. stutzeri with the addition of the in silico-predicted key exchanged metabolites, diesel oil was effectively disposed of in 15 days with a removal fraction of 85.54% ± 6.42%, leaving small amounts of C15 to C20 isomers. Our finding provides a novel microbial assembling mode for efficient bioremediation or chemical production in the future.IMPORTANCE Many natural and synthetic microbial communities are composed of not only species whose biological properties are consistent with their corresponding communities but also ones whose chemophysical characteristics do not directly contribute to the performance of their communities. Even though the latter species are often essential to the microbial communities, their roles are unclear. Here, by investigation of an artificial two-member microbial consortium in n-alkane biodegradation, we showed that the microbial member without the n-alkane-degrading capability had a cross-feeding interaction with and metabolic regulation to the leading member for the synergistic n-alkane biodegradation. Our study improves the current understanding of microbial interactions. Because "assistant" microbes showed importance in communities in addition to the functional microbes, our findings also suggest a useful "assistant-microbe" principle in the design of microbial communities for either bioremediation or chemical production.

Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium.

n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48 bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (± 0.03) [k(D) = 1.06 (± 0.16) µM, k(D) ' = 0.05 (± 0.01) µM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria.