Artificial Microbial Community Research Articles

A novel exoelectrogen from microbial fuel cell: Bioremediation of marine petroleum hydrocarbon pollutants

In the past decades, the microbial fuel cell (MFC) technology has caught the attention of the scientific community for its potential in transforming petroleum hydrocarbon (PHC) pollutants directly into electricity through microbial catalyzed anodic. The microbe was one of the most important factors that both influence MFCs and PHC degradation. Here we aimed to identify new microbes to expand the list of microbial species which are both electrogenic and diesel hydrocarbon degrading. In this text, we depicted a strain of microbe named E2, isolated from on the anode surface of MFC, and using diesel as sole carbon source. E2 exhibited electrochemical activity in cyclic voltammetry curve, implicating that it had electrogenic ability. E2 degraded about 50% diesel (3.26 g/L) in maximum during 8 days. Pyrosequencing of 16S rRNA gene of E2 revealed E2 was a sub-strain of Vibrio. Corresponding to salt and alkali tolerant properties of vibrio, the optimal condition for E2 in degrading diesel was 3%–4% in salinity, and pH 8–9 in mineral medium. Collectively, as a member of Gammaproteobacteria class, E2 was novel marine microbe both electricity generation and diesel degradation, which may attract its future application toward artificial microbial community construction in MFC in promoting the PHC pollution removal.

Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium.

In order for human microbiome studies to translate into actionable outcomes for health, meta-analysis of reproducible data from population-scale cohorts is needed. Achieving sufficient reproducibility in microbiome research has proven challenging. We report a baseline investigation of variability in taxonomic profiling for the Microbiome Quality Control (MBQC) project baseline study (MBQC-base). Blinded specimen sets from human stool, chemostats, and artificial microbial communities were sequenced by 15 laboratories and analyzed using nine bioinformatics protocols. Variability depended most on biospecimen type and origin, followed by DNA extraction, sample handling environment, and bioinformatics. Analysis of artificial community specimens revealed differences in extraction efficiency and bioinformatic classification. These results may guide researchers in experimental design choices for gut microbiome studies.

Modeling and Visualizing Bacterial Colony Purification Without the Use of Bacteria or Laboratory Equipment.

Microorganisms typically exist in diverse and heterogeneous communities within their various environmental niches. The isolation of an individual species from these communities is an essential laboratory skill to study of the properties and behaviors of that organism. To achieve this separation, the “quadrant streak” for single colony purification is often included in undergraduate microbiology laboratory curricula. To aid student mastery of this technique, I have developed a simulated culture purification activity that allows students to immediately visualize the dilution and separation of an artificial microbial community with the goal of isolating purified colonies. This tool uses readily available, inexpensive, art supplies to simulate a mixed bacterial culture. The “mixed culture” consists of craft glitter of at least two distinct colors, held together with water-soluble, highly pigmented watercolor or gouache paint. Students practice aseptic technique by using a paintbrush to mimic an inoculating loop to streak and dilute the culture on a piece of cardstock. Sterilization of the “loop” is simulated by rinsing the brush. Students will immediately self-assess whether they are correctly performing the quadrant streak, rather than having to wait until the next laboratory session for bacteria to grow, which may allow them to master the technique sooner.

Effects of pyrite and sphalerite on population compositions, dynamics and copper extraction efficiency in chalcopyrite bioleaching process.

This study used an artificial microbial community with four known moderately thermophilic acidophiles (three bacteria including Acidithiobacillus caldus S1, Sulfobacillus thermosulfidooxidans ST and Leptospirillum ferriphilum YSK, and one archaea, Ferroplasma thermophilum L1) to explore the variation of microbial community structure, composition, dynamics and function (e.g., copper extraction efficiency) in chalcopyrite bioleaching (C) systems with additions of pyrite (CP) or sphalerite (CS). The community compositions and dynamics in the solution and on the ore surface were investigated by real-time quantitative PCR (qPCR). The results showed that the addition of pyrite or sphalerite changed the microbial community composition and dynamics dramatically during the chalcopyrite bioleaching process. For example, A. caldus (above 60%) was the dominant species at the initial stage in three groups, and at the middle stage, still dominated C group (above 70%), but it was replaced by L. ferriphilum (above 60%) in CP and CS groups; at the final stage, L. ferriphilum dominated C group, while F. thermophilum dominated CP group on the ore surface. Furthermore, the additions of pyrite or sphalerite both made the increase of redox potential (ORP) and the concentrations of Fe3+ and H+, which would affect the microbial community compositions and copper extraction efficiency. Additionally, pyrite could enhance copper extraction efficiency (e.g., improving around 13.2% on day 6) during chalcopyrite bioleaching; on the contrary, sphalerite restrained it.

Additions of Pyrite or Chalcopyrite Alters the Microbial Community Diversity, Composition and Function in Sphalerite Bioleaching Systems

Extraction of zinc from sphalerite using bio-hydrometallurgical technologies has become more and more popular. This study used an artificial microbial community with five known microorganisms to examine the relationship among microbial diversity, composition, and function (e.g., zinc extraction rates) in sphalerite bioleaching systems with additional pyrite (SP), chalcopyrite (SC), or both (SPC). Real-time quantitative PCR (qPCR) analysis showed that additional pyrite or chalcopyrite changed the microbial community composition dramatically during the sphalerite bioleaching process. Shannon diversity index, compared with Sphalerite (0.109), showed an increase in SP (0.508), SC (0.536) and SPC (0.289) on day 30, and zinc extraction rates were enhanced by about 12.1%, 4.3% and 9.47%, respectively. Also, additional pyrite or chalcopyrite made ORP and the concentrations of Fe3+, Zn2+ and H+ increased, which were the main factors on shaping the microbial community composition by Mantel test analysis. We developed a unitary model, showing that additional pyrite or chalcopyrite increased the microbial community diversity.

Evolution of species interactions determines microbial community productivity in new environments.

Diversity generally increases ecosystem productivity over short timescales. Over longer timescales, both ecological and evolutionary responses to new environments could alter productivity and diversity–productivity relationships. In turn, diversity might affect how component species adapt to new conditions. We tested these ideas by culturing artificial microbial communities containing between 1 and 12 species in three different environments for ∼60 generations. The relationship between community yields and diversity became steeper over time in one environment. This occurred despite a general tendency for the separate yields of isolates of constituent species to be lower at the end if they had evolved in a more diverse community. Statistical comparisons of community and species yields showed that species interactions had evolved to be less negative over time, especially in more diverse communities. Diversity and evolution therefore interacted to enhance community productivity in a new environment.

UPARSE: highly accurate OTU sequences from microbial amplicon reads

Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

Bar-Coded Enterobacteria: An Undergraduate Microbial Ecology Laboratory Module

Microbial community ecology is an area of rapid growth centered within the larger discipline of microbiology. Newly developed research methods using molecular strategies have transformed this area into an accessible research topic. Despite such advances, transmission of this topic into pedagogical form has lagged behind. To improve this situation, an undergraduate research team created an artificial microbial community for class room use. They used color-coded enterobacterial taxa transformed with broad-host range plasmids that encoded green fluorescent protein color variants. Using this instructional tool, a class room teaching module was developed about microbial fitness. Over a multi-semester period, the module was introduced into a conventional microbiology curriculum and refined. The learning outcomes for this module include; understanding community composition, that the members of a community can respond in different ways to external events and, that these responses can be used to measure fitness. Learning outcomes were measured through pre and post testing and indicated a gain in understanding about microbial communities.

Proteomic Analysis of Ketogulonicigenium vulgare under Glutathione Reveals High Demand for Thiamin Transport and Antioxidant Protection

Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4–6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.

Integrated Proteomic and Metabolomic Analysis of an Artificial Microbial Community for Two-Step Production of Vitamin C

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.

Development and application of functional gene arrays for microbial community analysis

Functional gene markers can provide important information about functional gene diversity and potential activity of microbial communities. Although microarray technology has been successfully applied to study gene expression for pure cultures, simple, and artificial microbial communities, adapting such a technology to analyze complex microbial communities still presents a lot of challenges in terms of design, sample preparation, and data analysis. This work is focused on the development and application of functional gene arrays (FGAs) to target key functional gene markers for microbial community studies. A few key issues specifically related to FGAs, such as oligonucleotide probe design, nucleic acid extraction and purification, data analysis, specificity, sensitivity, and quantitative capability are discussed in detail. Recent studies have demonstrated that FGAs can provide specific, sensitive, and potentially quantitative information about microbial communities from a variety of natural environments and controlled ecosystems. This technology is expected to revolutionize the analysis of microbial communities, and link microbial structure to ecosystem functioning.

Defined spatial structure stabilizes a synthetic multispecies bacterial community.

This paper shows that for microbial communities, "fences make good neighbors." Communities of soil microorganisms perform critical functions: controlling climate, enhancing crop production, and remediation of environmental contamination. Microbial communities in the oral cavity and the gut are of high biomedical interest. Understanding and harnessing the function of these communities is difficult: artificial microbial communities in the laboratory become unstable because of "winner-takes-all" competition among species. We constructed a community of three different species of wild-type soil bacteria with syntrophic interactions using a microfluidic device to control spatial structure and chemical communication. We found that defined microscale spatial structure is both necessary and sufficient for the stable coexistence of interacting bacterial species in the synthetic community. A mathematical model describes how spatial structure can balance the competition and positive interactions within the community, even when the rates of production and consumption of nutrients by species are mismatched, by exploiting nonlinearities of these processes. These findings provide experimental and modeling evidence for a class of communities that require microscale spatial structure for stability, and these results predict that controlling spatial structure may enable harnessing the function of natural and synthetic multispecies communities in the laboratory.

A Quantitative Assay for Linking Microbial Community Function and Structure of a Naphthalene-Degrading Microbial Consortium

A comprehensive culture-independent assay, called Q-FAST, was developed for concurrent identification and quantification of active microorganisms involved a specific function in a given microbial community. The development of Q-FAST was achieved by integrating the concept of stable isotope probing technique into a new quantitative fingerprinting assay called real-time-t-RFLP for microbial community structure analysis. The Q-FAST was successfully validated by using a three-member artificial microbial community containing a known naphthalene-utilizing bacterium (Pseudomonas putida G7) and two nonnaphthalene-degrading bacteria (Escherichia coli and Bacillus thuringiensis). The application of Q-FAST to identify and quantify a guild of naphthalene-utilizing microorganisms in soils revealed the involvement of eight members, with six members relating to several phylogenetic groups of eubacteria (three in beta-proteobacteria, two in gamma-proteobacteria, and one in genera Intrasporangium of Gram-positive bacteria) and two members showing no close phylogenetic affiliation to any known bacterial sequences deposited in GenBank. The quantity of three members belonging to beta-proteobacteria accounted for 34% of total 16S rDNA copies measured from the "heavier" fraction of DNA that was contributed from the DNA of microorganisms capable of incorporating 13C-labeled naphthalene into their genetic biomarkers. The other five members composed 66% of total 16S rDNA copies of active naphthalene-utilizing populations measured. Offering a powerful tool for studying microbial ecology, Q-FAST thus opens a new avenue for deeper exploration of microbial-mediated processes, mainly the quantitative relationship between microbial diversity and microbial activity in a given environment.

Interspecies Protection against Freezing Stress within a Food Microbial Community

This study was completed on an artificial microbial community (consortium) including three microorganisms of dairy interest, two mesophilic (Lactococcus lactis subsp. cremoris and Geotrichum candidum) and one thermophilic (Lactobacillus delbrueckii subsp. bulgaricus). The aim of this work was to study the stress responses of the food microbial consortium subjected to a freeze–thaw challenge (–20°C, 10 min / +25°C, 3 min) combined with physiological cryoadaptation (cryotolerance). Our results show that induction of cryotolerance by near-freezing temperature pretreatment of G. candidum and L. cremoris within the microbial community is similar to the one obtained with pure cultures. However, cryotolerance of L. bulgaricus was only induced within the consortium in milk. An interspecies cryoprotection was thus identified. This acquisition is correlated with the cell envelope extracts of G. candidum and/or the extracellular fractions of the two adapted mesophilic microorganisms. Therefore, in our experimental c...