Rapid analysis of two food-borne microbial communities at the species level by Fourier-transform infrared microspectroscopy

The species composition of microbial communities in natural habitats may be extremely complex and therefore a quantitative analysis of the fraction each species contributes to the consortium has proven to be difficult. During recent years, the identification of bacterial pure cultures based on their infrared spectra has been established. Fourier-transform infrared microspectroscopy now proceeds a step further and allows identification of microorganisms directly plated from community dilutions. Infrared spectra of microcolonies of 70-250 microm in diameter can be recorded without producing a pure culture of the isolate. We have applied this novel technique for quantitative comparative analysis of two undefined, geographically separated food-borne smear cheese microbial consortia of limited complexity. Due to the high degree of automation, up to 200 microcolonies could be identified in 1 day and, in total, 3170 infrared spectra of microcolonies were recorded. The results obtained have been verified by Fourier-transform infrared macrospectroscopy and 16S rDNA sequencing. Interestingly, although the communities were unrelated, Staphylococcus equorum, Corynebacterium casei, Arthrobacter casei and Brevibacterium linens were found to be part of both consortia, however, with different incidence. In addition, Corynebacterium variabile, Microbacterium gubbeenense, Brachybacterium alimentarium, Enterococcus faecalis and an unknown species were detected in either one of the consortia.     

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set.     

The electric picnic: synergistic requirements for exoelectrogenic microbial communities

Characterization of the various microbial populations present in exoelectrogenic biofilms provides insight into the processes required to convert complex organic matter in wastewater streams into electrical current in bioelectrochemical systems (BESs). Analysis of the community profiles of exoelectrogenic microbial consortia in BESs fed different substrates gives a clearer picture of the different microbial populations present in these exoelectrogenic biofilms. Rapid utilization of fermentation end products by exoelectrogens (typically Geobacter species) relieves feedback inhibition for the fermentative consortia, allowing for rapid metabolism of organics. Identification of specific syntrophic processes and the communities characteristic of these anodic biofilms will be a valuable aid in improving the performance of BESs.     

Estimating ecotoxicological risk and impact using indigenous aquatic microbial communities

Emphasis has increased on accuracy in predicting the effect that anthropogenic stress has on natural ecosystems. Although toxicity tests low in environmental realism, such as standardized single species procedures, have been useful in providing a certain degree of protection to human health and the environment, the accuracy of such tests for predicting the effects of anthropogenic activities on complex ecosystems is questionable. The use of indigenous communities of microorganisms to assess the hazard of toxicants in aquatic ecosystems has many advantages. Theoretical and practical aspects of microbial community tests are discussed, particularly in related to widely cited problems in the use of multispecies test systems for predicting hazard. Further standardization of testing protocols using microbial colonization dynamics is advocated on the basis of previous studies, which have shown these parameters to be useful in assessing risk and impact of hazardous substances in aquatic ecosystems.     

Parasites, disease and the structure of ecological communities

Pathogens and parasites are fascinating to epidemiologists and ecologists alike; as well as causing disease in individual species, they can perturb the normal functioning of a community and thus give insights into the way that the community 'functions' Several recent studies on diseases in animal populations have confirmed the importance of pathogens and parasites as components of ecological systems, while also revealing the underlying structure of complex multispecies communities.     

How mutualisms arise in phytoplankton communities: building eco‐evolutionary principles for aquatic microbes

Extensive sampling and metagenomics analyses of plankton communities across all aquatic environments are beginning to provide insights into the ecology of microbial communities. In particular, the importance of metabolic exchanges that provide a foundation for ecological interactions between microorganisms has emerged as a key factor in forging such communities. Here we show how both studies of environmental samples and physiological experimentation in the laboratory with defined microbial co‐cultures are being used to decipher the metabolic and molecular underpinnings of such exchanges. In addition, we explain how metabolic modelling may be used to conduct investigations in reverse, deducing novel molecular exchanges from analysis of large‐scale data sets, which can identify persistently co‐occurring species. Finally, we consider how knowledge of microbial community ecology can be built into evolutionary theories tailored to these species’ unique lifestyles. We propose a novel model for the evolution of metabolic auxotrophy in microorganisms that arises as a result of symbiosis, termed the Foraging‐to‐Farming hypothesis. The model has testable predictions, fits several known examples of mutualism in the aquatic world, and sheds light on how interactions, which cement dependencies within communities of microorganisms, might be initiated.     

Links between soil microbial communities and plant traits in a species‐rich grassland under long‐term climate change

Climate change can influence soil microorganisms directly by altering their growth and activity but also indirectly via effects on the vegetation, which modifies the availability of resources. Direct impacts of climate change on soil microorganisms can occur rapidly, whereas indirect effects mediated by shifts in plant community composition are not immediately apparent and likely to increase over time. We used molecular fingerprinting of bacterial and fungal communities in the soil to investigate the effects of 17 years of temperature and rainfall manipulations in a species‐rich grassland near Buxton, UK. We compared shifts in microbial community structure to changes in plant species composition and key plant traits across 78 microsites within plots subjected to winter heating, rainfall supplementation, or summer drought. We observed marked shifts in soil fungal and bacterial community structure in response to chronic summer drought. Importantly, although dominant microbial taxa were largely unaffected by drought, there were substantial changes in the abundances of subordinate fungal and bacterial taxa. In contrast to short‐term studies that report high resistance of soil fungi to drought, we observed substantial losses of fungal taxa in the summer drought treatments. There was moderate concordance between soil microbial communities and plant species composition within microsites. Vector fitting of community‐weighted mean plant traits to ordinations of soil bacterial and fungal communities showed that shifts in soil microbial community structure were related to plant traits representing the quality of resources available to soil microorganisms: the construction cost of leaf material, foliar carbon‐to‐nitrogen ratios, and leaf dry matter content. Thus, our study provides evidence that climate change could affect soil microbial communities indirectly via changes in plant inputs and highlights the importance of considering long‐term climate change effects, especially in nutrient‐poor systems with slow‐growing vegetation.     

Bar-coded pyrosequencing reveals the responses of PBDE-degrading microbial communities to electron donor amendments

Polybrominated diphenyl ethers (PBDEs) can be reductively degraded by microorganisms under anaerobic conditions. However, little is known about the effect of electron donors on microbial communities involved in PBDEs degradation. Here we employed 454 Titanium pyrosequencing to examine the phylogenetic diversity, composition, structure and dynamics of microbial communities from microcosms under the conditions of different electron donor amendments. The community structures in each of the five alternate electron donor enrichments were significantly shifted in comparison with those of the control microcosm. Commonly existing OTUs between the treatment and control consortia increased from 5 to 17 and more than 50% of OTUs increased around 13.7 to 186 times at least in one of the microcosms after 90-days enrichment. Although the microbial communities at different taxonomic levels were significantly changed by different environmental variable groups in redundancy analysis, significant correlations were observed between the microbial communities and PBDE congener profiles. The lesser-brominated PBDE congeners, tri-BDE congener (BDE-32) and hexa-BDE, were identified as the key factors shaping the microbial community structures at OTU level. Some rare populations, including the known dechlorinating bacterium, Dehalobacter, showed significant positive-correlation with the amounts of PBDE congeners in the consortia. The same results were also observed on some unclassified bacteria. These results suggest that PBDEs-degrading microbial communities can be successfully enriched, and their structures and compositions can be manipulated through adjusting the environmental parameters.     

微生物生态学理论框架

微生物是生态系统的重要组成部分,直接或间接地参与所有的生态过程。微生物生态学是基于微生物群体的科学,利用微生物群体DNA/RNA等标志物,重点研究微生物群落构建、组成演变、多样性及其与环境的关系,在生态学理论的指导和反复模型拟合下由统计分析得出具有普遍意义的结论。其研究范围从基因尺度到全球尺度。分子生物学技术的发展,使人们可以直接从基因水平上考查其多样性,从而使得对微生物空间分布格局及其成因的深入研究成为可能。进而可以从方法学探讨微生物生物多样性、分布格局、影响机制及其对全球变化的响应等。在微生物生态学研究中,群落构建与演化、分布特征(含植物-微生物相互关系)、执行群体功能的机理(生物地球化学循环等)、对环境变化的响应与反馈机理是今后需要关注的重点领域。概述了微生物生态学的概念,并初步提出其理论框架,在对比宏观生态学基础理论和模型的基础上,分析微生物多样性的研究内容、研究方法和群落构建的理论机制,展望了今后研究的重点领域。     

Microbial Astronauts: Assembling Microbial Communities for Advanced Life Support Systems

Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth. The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space. Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments everything is everywhere given enough time). This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch. Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time. Research at the John F. Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support. Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed. In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity. Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment.     

Proteogenomic approaches for the molecular characterization of natural microbial communities

At the present time we know little about how microbial communities function in their natural habitats. For example, how do microorganisms interact with each other and their physical and chemical surroundings and respond to environmental perturbations? We might begin to answer these questions if we could monitor the ways in which metabolic roles are partitioned amongst members as microbial communities assemble, determine how resources such as carbon, nitrogen, and energy are allocated into metabolic pathways, and understand the mechanisms by which organisms and communities respond to changes in their surroundings. Because many organisms cannot be cultivated, and given that the metabolisms of those growing in monoculture are likely to differ from those of organisms growing as part of consortia, it is vital to develop methods to study microbial communities in situ. Chemoautotrophic biofilms growing in mine tunnels hundreds of meters underground drive pyrite (FeS(2)) dissolution and acid and metal release, creating habitats that select for a small number of organism types. The geochemical and microbial simplicity of these systems, the significant biomass, and clearly defined biological-inorganic feedbacks make these ecosystem microcosms ideal for development of methods for the study of uncultivated microbial consortia. Our approach begins with the acquisition of genomic data from biofilms that are sampled over time and in different growth conditions. We have demonstrated that it is possible to assemble shotgun sequence data to reveal the gene complement of the dominant community members and to use these data to confidently identify a significant fraction of proteins from the dominant organisms by mass spectrometry (MS)-based proteomics. However, there are technical obstacles currently restricting this type of "proteogenomic" analysis. Composite genomic sequences assembled from environmental data from natural microbial communities do not capture the full range of genetic potential of the associated populations. Thus, it is necessary to develop bioinformatics approaches to generate relatively comprehensive gene inventories for each organism type. These inventories are critical for expression and functional analyses. In proteomic studies, for example, peptides that differ from those predicted from gene sequences can be measured, but they generally cannot be identified by database matching, even if the difference is only a single amino acid residue. Furthermore, many of the identified proteins have no known function. We propose that these challenges can be addressed by development of proteogenomic, biochemical, and geochemical methods that will be initially deployed in a simple, natural model ecosystem. The resulting approach should be broadly applicable and will enhance the utility and significance of genomic data from isolates and consortia for study of organisms in many habitats. Solutions draining pyrite-rich deposits are referred to as acid mine drainage (AMD). AMD is a very prevalent, international environmental problem associated with energy and metal resources. The biological-mineralogical interactions that define these systems can be harnessed for energy-efficient metal recovery and removal of sulfur from coal. The detailed understanding of microbial ecology and ecosystem dynamics resulting from the proposed work will provide a scientific foundation for dealing with the environmental challenges and technological opportunities, and yield new methods for analysis of more complex natural communities.     

Emerging strategies for engineering microbial communities

From biosynthesis to bioremediation, microbes have been engineered to address a variety of biotechnological applications. A promising direction in these endeavors is harnessing the power of designer microbial consortia that consist of multiple populations with well-defined interactions. Consortia can accomplish tasks that are difficult or potentially impossible to achieve using monocultures. Despite their potential, the rules underlying microbial community maintenance and function (i.e. the task the consortium is engineered to carry out) are not well defined, though rapid progress is being made. This limited understanding is in part due to the greater challenges associated with increased complexity when dealing with multi-population interactions. Here, we review key features and design strategies that emerge from the analysis of both natural and engineered microbial communities. These strategies can provide new insights into natural consortia and expand the toolbox available to engineers working to develop novel synthetic consortia.     

Facilitation promotes invasions in plant‐associated microbial communities

While several studies have established a positive correlation between community diversity and invasion resistance, it is less clear how species interactions within resident communities shape this process. Here, we experimentally tested how antagonistic and facilitative pairwise interactions within resident model microbial communities predict invasion by the plant–pathogenic bacterium Ralstonia solanacearum. We found that facilitative resident community interactions promoted and antagonistic interactions suppressed invasions both in the lab and in the tomato plant rhizosphere. Crucially, pairwise interactions reliably explained observed invasion outcomes also in multispecies communities, and mechanistically, this was linked to direct inhibition of the invader by antagonistic communities (antibiosis), and to a lesser degree by resource competition between members of the resident community and the invader. Together, our findings suggest that the type and strength of pairwise interactions can reliably predict the outcome of invasions in more complex multispecies communities.     

Genomic and mechanistic insights into the biodegradation of organic pollutants

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.     

Deciphering microbial interactions in synthetic human gut microbiome communities

The ecological forces that govern the assembly and stability of the human gut microbiota remain unresolved. We developed a generalizable model‐guided framework to predict higher‐dimensional consortia from time‐resolved measurements of lower‐order assemblages. This method was employed to decipher microbial interactions in a diverse human gut microbiome synthetic community. We show that pairwise interactions are major drivers of multi‐species community dynamics, as opposed to higher‐order interactions. The inferred ecological network exhibits a high proportion of negative and frequent positive interactions. Ecological drivers and responsive recipient species were discovered in the network. Our model demonstrated that a prevalent positive and negative interaction topology enables robust coexistence by implementing a negative feedback loop that balances disparities in monospecies fitness levels. We show that negative interactions could generate history‐dependent responses of initial species proportions that frequently do not originate from bistability. Measurements of extracellular metabolites illuminated the metabolic capabilities of monospecies and potential molecular basis of microbial interactions. In sum, these methods defined the ecological roles of major human‐associated intestinal species and illuminated design principles of microbial communities.     

High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions,Useful for Exploring Interspecific Bacterial Interactions

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.     

New approaches in bioprocess‐control: Consortium guidance by synthetic cell‐cell communication based on fungal pheromones

Bioconversions in industrial processes are currently dominated by single‐strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single‐strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium‐based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell–cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone‐responsive gene expression, thereby creating pheromone‐dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone‐controlled sensor‐actor systems, exploiting their unique metabolic properties for microbial consortia approaches.     

Low root functional dispersion enhances functionality of plant growth by influencing bacterial activities in European forest soils

Current studies show that multispecies forests are beneficial regarding biodiversity and ecosystem functionality. However, there are only little efforts to understand the ecological mechanisms behind these advantages of multispecies forests. Bacteria are among the key plant growth-promoting microorganisms that support tree growth and fitness. Thus, we investigated links between bacterial communities, their functionality and root trait dispersion within four major European forest types comprising multispecies and monospecific plots. Bacterial diversity revealed no major changes across the root functional dispersion gradient. In contrast, predicted gene profiles linked to plant growth activities suggest an increasing bacterial functionality from monospecific to multispecies forest. In multispecies forest plots, the bacterial functionality linked to plant growth activities declined with the increasing functional dispersion of the roots. Our findings indicate that enriched abundant bacterial operational taxonomic units are decoupled from bacterial functionality. We also found direct effects of tree species identity on bacterial community composition but no significant relations with root functional dispersion. Additionally, bacterial network analyses indicated that multispecies forests have a higher complexity in their bacterial communities, which points towards more stable forest systems with greater functionality. We identified a potential of root dispersion to facilitate bacterial interactions and consequently, plant growth activities.     

Microbial community composition and dynamics in a semi-industrial-scale facility operating under the MixAlco™ bioconversion platform

Aims:  To monitor microbial community dynamics in a semi‐industrial‐scale lignocellulosic biofuel reactor system and to improve our understanding of the microbial communities involved in the MixAlco? biomass conversion process. Methods and Results:  Reactor microbial communities were characterized at six time points over the course of an 80‐day, mesophilic, semi‐industrial‐scale fermentation using community qPCR and 16S rRNA tag‐pyrosequencing. We found the communities to be dynamic, bacterially dominated consortia capable of changing quickly in response to reactor conditions. Clostridia‐ and Bacteroidetes‐like organisms dominated the reactor communities, but ultimately the communities established consortia containing complementary functional capacities for the degradation of lignocellulosic materials. Eighteen operational taxonomic units were found to share strong correlations with reactor acid concentration and may represent taxa integral to fermentor performance. Conclusions:  The results of this study indicate that the emergence of complementary functional classes within the fermentor consortia may be a trait that is consistent across scales, and they suggest that there may be flexibility with respect to the specific identities of the organisms involved in the fermentor’s degradation and fermentation processes. Significance and Impact of the Study:  This study provides new information regarding the composition, dynamics and potential flexibility of the microbial communities associated with the MixAlco? process and is likely to inform the improvement of this and other applications that employ mixed microbial communities.     

Phototrophic marine benthic microbiomes: the ecophysiology of these biological entities

Phototrophic biofilms are multispecies, self-sustaining and largely closed microbial ecosystems. They form macroscopic structures such as microbial mats and stromatolites. These sunlight-driven consortia consist of a number of functional groups of microorganisms that recycle the elements internally. Particularly, the sulfur cycle is discussed in more detail as this is fundamental to marine benthic microbial communities and because recently exciting new insights have been obtained. The cycling of elements demands a tight tuning of the various metabolic processes and require cooperation between the different groups of microorganisms. This is likely achieved through cell-to-cell communication and a biological clock. Biofilms may be considered as a macroscopic biological entity with its own physiology. We review the various components of some marine phototrophic biofilms and discuss their roles in the system. The importance of extracellular polymeric substances (EPS) as the matrix for biofilm metabolism and as substrate for biofilm microorganisms is discussed. We particularly assess the importance of extracellular DNA, horizontal gene transfer and viruses for the generation of genetic diversity and innovation, and for rendering resilience to external forcing to these biological entities.