Engineering natural microbial communities: harnessing synthetic communities for bioremediation.

Microorganisms are ubiquitous in our world, and they form different communities between organisms and the surrounding environment. It is estimated that there are more than 108 bacteria in a small gram of soil, and there are more than 1013 bacteria in the human intestine. It is a complex ecosystem formed under the interaction mechanisms of resource competition, nutrient symbiosis, quorum sensing, and horizontal gene transfer. Among them, the microbial communities in the soil and hydrosphere environment play a key driving role in the global chemical cycle of carbon, nitrogen and other elements. In industrial production, the microbial communities can treat our waste and purify the environment. In the field of human diseases, the microbial community in the human intestine is closely related to the development, metabolism and immunity of the host. Therefore, studying the stability of the composition of the microbial community is of great significance for solving the problems of ecological environment, agricultural production, and human health. With the development of high-throughput sequencing technology, the diversity and stability of microbial communities in different environments have been deeply studied. Although the scientific community has paid more attention to microbial communities in recent years, there are still many difficulties before us effectively use these communities to meet social requirements and environmental challenges. Therefore, it is critical to use new scientific concepts such as synthetic biology to interpret existing scientific problems. Among them, the core cycle of synthetic biology, Design-Build-Test-Learn is expected to promote the perfection and discovery of the basic theory of microbial ecological communities, and promote the research and development of biotechnology lays a theoretical foundation for improving the application of human health, agricultural production and environmental pollution. The ecology of microbial communities in nature is highly complex, making it challenging to carry out reproducible, controllable perturbation experiments in natural ecosystems. In contrast, synthetic communities constructed in the laboratory using a “bottom-up” approach offer moderate complexity and high controllability, and can serve as a bridge between complex ecosystems and mathematical modeling. By combining synthetic microbial communities, quantitative models and sequencing techniques, we can systematically study interactions between microbial populations and predict the dynamics of microbial communities. In terms of application, research on synthetic microbial communities will provide critical insights for how to control and engineer complex microbial ecosystems, and help solve important problems in human health and agricultural production via the design of stable microbial communities with controllable functions. In this review article, we mainly focus on the construction methods and theoretical foundations of existing synthetic microbial communities and the potential applications of these communities from two aspects, namely the knowledge of creation and the application of creation. And we also discuss the potential application of synthetic biology methods in this field.

While natural microbial communities are composed of a mix of microbes with often unknown functions, the construction of synthetic microbial communities allows for the generation of defined systems with reduced complexity. Used in a top-down approach, synthetic communities serve as model systems to ask questions about the performance and stability of microbial communities. In a second, bottom-up approach, synthetic microbial communities are used to study which conditions are necessary to generate interaction patterns like symbiosis or competition, and how higher order community structure can emerge from these. Besides their obvious value as model systems to understand the structure, function and evolution of microbial communities as complex dynamical systems, synthetic communities can also open up new avenues for biotechnological applications.

Natural microbial communities generally have an unknown structure and composition because of their still not yet cultivable members. Therefore, understanding the relationships among the bacterial members, prediction of their behaviour, and controlling their functions are difficult and often only partly successful endeavours to date. This study aims to test a new idea that allows to follow community dynamics on the basis of a simple concept. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S ribosomal RNA genes was used to describe a community profile that we define as composition of a community. Flow cytometry and analysis of DNA contents and forward scatter characteristics of the single cells were used to describe a community profile, which we define as structure of a community. Both approaches were brought together by a non-metric multidimensional scaling (n-MDS) for trend interpretation of changes in the complex community data sets. This was done on the basis of a graphical evaluation of the cytometric data, leading to the newly developed Dalmatian plot tool, which gave an unexpected insight into the dynamics of the unknown bacterial members of the investigated natural microbial community. The approach presented here was compared with other techniques described in the literature. The microbial community investigated in this study was obtained from a BTEX contaminated anoxic aquifer. The indigenous bacteria were allowed to colonise in situ microcosms consisting of activated carbon. These microcosms were amended with benzene and one of the electron acceptors nitrate, sulphate or ferric iron to stimulate microbial growth. The data obtained in this study indicated that the composition (via T-RFLP) and structure (via flow cytometry) of the natural bacterial community were influenced by the hydro-geochemical conditions in the test site, but also by the supplied electron acceptors, which led to distinct shifts in relative abundances of specific community members. It was concluded that engineered environments can be successfully monitored by single cell analytics in combination with established molecular tools and sophisticated statistical analyses, a combination that holds great promise for studying and monitoring natural microbial community behaviour.

Natural (microbial) communities are complex ecosystems with many interactions and cross-dependencies. Among other factors, selection pressures from the environment are thought to drive the composition and functionality of microbial communities. Fermented foods, when processed using non-industrial methods, harbor such natural microbial communities. In non-alcoholic fermented foods the fermenting microbiota is commonly dominated by 4–10 species of bacteria, which make them suitable model systems to study ecosystem assembly and functioning. In this study, we assess the influence of the environment on the composition of microbial communities of traditional fermented products from Africa. We compare differences between microbial communities that are found in similar products but come from different countries, hypothesizing they experience different environmental selection pressures. We analyzed bacterial community composition in 36 samples of various cereal-based fermented foods from Benin, Tanzania and Zambia using 16S rDNA amplicon sequencing. The differential abundance analysis indicates that the bacterial communities of fermented foods from the three countries are dominated by mostly lactic acid bacteria belonging to the genera of Lactobacillus, Weisella and Curvibacter. The samples from Zambia contain the most dissimilar microbial communities in comparison with samples from Benin and Tanzania. We propose this is caused by the relatively low temperature in Zambia, suggesting that indeed environmental selection can shape community composition of fermenting microbes.

Common garden experiments that inoculate a standardised growth medium with synthetic microbial communities (i.e. constructed from individual isolates or using dilution cultures) suggest that the ability of the community to resist invasions by additional microbial taxa can be predicted by the overall community productivity (broadly defined as cumulative cell density and/or growth rate). However, to the best of our knowledge, no common garden study has yet investigated the relationship between microbial community composition and invasion resistance in microcosms whose compositional differences reflect natural, rather than laboratory-designed, variation. We conducted experimental invasions of two bacterial strains (Pseudomonas fluorescens and Pseudomonas putida) into laboratory microcosms inoculated with 680 different mixtures of bacteria derived from naturally occurring microbial communities collected in the field. Using 16S rRNA gene amplicon sequencing to characterise microcosm starting composition, and high-throughput assays of community phenotypes including productivity and invader survival, we determined that productivity is a key predictor of invasion resistance in natural microbial communities, substantially mediating the effect of composition on invasion resistance. The results suggest that similar general principles govern invasion in artificial and natural communities, and that factors affecting resident community productivity should be a focal point for future microbial invasion experiments.

Common garden experiments that inoculate a standardised growth medium with synthetic microbial communities (i.e. constructed from individual isolates or using dilution cultures) suggest that the ability of the community to resist invasions by additional microbial taxa can be predicted by the overall community productivity (broadly defined as cumulative cell density and/or growth rate). However, to the best of our knowledge, no common garden study has yet investigated the relationship between microbial community composition and invasion resistance in microcosms whose compositional differences reflect natural, rather than laboratory-designed, variation. We conducted experimental invasions of two bacterial strains (Pseudomonas fluorescens and Pseudomonas putida) into laboratory microcosms inoculated with 680 different mixtures of bacteria derived from naturally occurring microbial communities collected in the field. Using 16S rRNA gene amplicon sequencing to characterise microcosm starting composition, and high-throughput assays of community phenotypes including productivity and invader survival, we determined that productivity is a key predictor of invasion resistance in natural microbial communities, substantially mediating the effect of composition on invasion resistance. The results suggest that similar general principles govern invasion in artificial and natural communities, and that factors affecting resident community productivity should be a focal point for future microbial invasion experiments.

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

A BSTRACTThis study demonstrates microbial community changes over time in a nitroaromatic-contaminated groundwater upon amendment with hydrocarbons previously unknown to the microbial community (extrinsic) and hydrocarbons previously known to the microbial community (intrinsic). Sealed flasks, shaken and incubated at 25 degrees C, containing contaminated groundwater and salts were amended twice with extrinsic hydrocarbons including phenol, benzoic acid, and naphthalene, and intrinsic hydrocarbons including 2,4-dinitrotoluene (2,4-DNT) and para-nitrotoluene ( p-NT). Microbial growth, biodegradation, and community structure changes measured by random amplified polymorphic DNA (RAPD) and quantitative PCR (qPCR) targeting catechol-2,3-dioxygenase (C23O) genes were monitored over time. All amended substrates were biodegraded after both substrate amendments except for 2,4-DNT, which was only partially degraded after the second amendment. Unique microbial communities were developed in flasks amended with phenol, benzoic acid, and naphthalene. However, in the flasks amended with intrinsic hydrocarbons the microbial community remained similar to the unamended control flasks. The relative amount of C23O genes detected by qPCR correlated with the biodegradation of phenol and naphthalene but not with 2,4-DNT. The results showed that a selection for microorganisms capable of catabolizing extrinsic hydrocarbons naturally and initially present in the nitroaromatic-contaminated groundwater occurred. However, growth-linked biodegradation of added intrinsic hydrocarbons was not selective.

Synthetic and natural microbial communities in high-temperature Daqu production: Insights into metabolic pathways and volatile organic compounds.

Reprint of Design of synthetic microbial communities for biotechnological production processes

Design of synthetic microbial communities for biotechnological production processes

The use of novel high-throughput sequencing (HTS) technologies to examine the responses of natural multidomain microbial communities to scrubber effluent discharges to the marine environment is still limited. Thus, we applied metabarcoding sequencing targeting the planktonic unicellular eukaryotic and prokaryotic fraction (phytoplankton, bacterioplankton, and protozooplankton) in mesocosm experiments with natural microbial communities from a polluted and an unpolluted site. Furthermore, metagenomic analysis revealed changes in the taxonomic and functional dominance of multidomain marine microbial communities after scrubber effluent additions. The results indicated a clear shift in the microbial communities after such additions, which favored bacterial taxa with known oil and polycyclic aromatic hydrocarbons (PAHs) biodegradation capacities. These bacteria exhibited high connectedness with planktonic unicellular eukaryotes employing variable trophic strategies, suggesting that environmentally relevant bacteria can influence eukaryotic community structure. Furthermore, Clusters of Orthologous Genes associated with pathways of PAHs and monocyclic hydrocarbon degradation increased in numbers at treatments with high scrubber effluent additions acutely. These genes are known to express enzymes acting at various substrates including PAHs. These indications, in combination with the abrupt decrease in the most abundant PAHs in the scrubber effluent below the limit of detection-much faster than their known half-lives-could point toward a bacterioplankton-initiated rapid ultimate biodegradation of the most abundant toxic contaminants of the scrubber effluent. The implementation of HTS could be a valuable tool to develop multilevel biodiversity indicators of the scrubber effluent impacts on the marine environment, which could lead to improved impact assessment. Environ Toxicol Chem 2024;43:1012-1029. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

There is a growing concern about the simultaneous presence in the environment of different kinds of pollutants, because of the possible synergic or additive effects of chemical mixtures on ecosystems. Chlorpyrifos (CPF) is an organophosphate insecticide extensively used in agricultural practices. The anionic surfactant sodium lauryl ether sulphate (SLES) is the main component of several commercial products, including foaming agents used in underground mechanised excavation. Both compounds are produced and sold in high amounts worldwide and can be found in the environment as soil contaminants. The persistence of SLES and CPF in agricultural soils and their possible effects on the natural microbial community was evaluated in microcosms. The experimental set consisted of soil samples containing the autochthonous microbial community and treated with only SLES (70mg/kg), only CPF (2mg/kg) or with a mix of both compounds. Control microcosms (without the contaminants) were also performed. Soil samples were collected over the experimental period (0, 7, 14, 21 and 28days) and analysed for CPF, SLES and the main metabolite of CPF (3, 5, 6-trichloropyridinol, TCP). The half-life time (DT50) of each parent compound was estimated in all experimental conditions. At the same time, the abundance, activity and structure of the microbial community were also evaluated. The results showed that the co-presence of SLES and CPF did not substantially affect their persistence in soil (DT50 of 11 and 9days with co-presence and 13 and 10days, respectively, when alone); however, in the presence of SLES, a higher amount of the metabolite TCP was found. Interestingly, some differences were found in the bacterial community structure, abundance and activity among the various conditions.

Microbial degradation and utilization of proteins derived from bacterial detritus were investigated in a microcosm experiment using Pseudomonas aeruginosa detritus as a substrate. To assess the effects of natural marine microbial communities on degradation and utilization of protein derived from P. aeruginosa cells, four microcosms were prepared: natural seawater (containing the natural microbial community) with P. aeruginosa detritus (N+Pa), autoclaved seawater with P. aeruginosa detritus (A+Pa), natural seawater (N) and autoclaved seawater (A) without adding anything as a control. The numbers of total and growing bacterial cells, protease activity, and transition of P. aeruginosa proteins were monitored in the four microcosms. Changes in the numbers of total and growing bacterial cells and protease activities indicated that bacterial detritus significantly stimulated the microbial community in the microcosms. Both the surviving P. aeruginosa in A+Pa and natural microbial community in N+Pa microcosms were able to degrade and utilize P. aeruginosa detritus; however, the community in N+Pa including various microbes maintained high activity longer, indicating that diversity is an important factor in keeping the community active. Even under the very high protease activity in N+Pa, 39-kDa and 48-kDa proteins from P. aeruginosa remained in the microcosm during the entire experiment (150 days). Immunoblotting suggested the 48-kDa protein was an intact molecule of OprP, which had been detected from the dissolved fraction of natural seawater in previous studies. This result suggests that the protein molecules that had been detected from natural seawater actually had a high tolerance to microbial degradation.

Dispersal and environmental selection are two of the most important factors that govern the distributions of microbial communities in nature. While dispersal rates are often inferred by measuring the degree to which community similarity diminishes with increasing geographic distance, determining the extent to which environmental selection impacts the distribution of microbes is more complex. To address this knowledge gap, we performed a large reciprocal transplant experiment to simulate the dispersal of US East Coast salt marsh Spartina alterniflora rhizome-associated microbial sediment communities across a latitudinal gradient and determined if any shifts in microbial community composition occurred as a result of the transplantation. Using bacterial 16S rRNA gene sequencing, we did not observe large-scale changes in community composition over a five-month S. alterniflora summer growing season and found that transplanted communities more closely resembled their origin sites than their destination sites. Furthermore, transplanted communities grouped predominantly by region, with two sites from the north and three sites to the south hosting distinct bacterial taxa, suggesting that sediment communities transplanted from north to south tended to retain their northern microbial distributions, and south to north maintained a southern distribution. A small number of potential indicator 16S rRNA gene sequences had distributions that were strongly correlated to both temperature and nitrogen, indicating that some organisms are more sensitive to environmental factors than others. These results provide new insight into the microbial biogeography of salt marsh sediments and suggest that established bacterial communities in frequently-inundated environments may be both highly resistant to invasion and resilient to some environmental shifts. However, the extent to which environmental selection impacts these communities is taxon specific and variable, highlighting the complex interplay between dispersal and environmental selection for microbial communities in nature.