Artificial Microbial Community Research Articles

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Metabolomics-Guided Discovery of New Dimeric Xanthones from Co-Cultures of Mangrove Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11.

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool for the activation of biosynthetic gene clusters (BGCs) to generate new metabolites, as well as to increase the yield of respective target metabolites. As part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, we selected the co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophyted in mangrove Rhizophora mangle considering the impart of the taxonomic criteria and ecological data. The competition interaction of the two strains was investigated through morphology observation and scanning electron microscopy (SEM), and it was found that the mycelia of the DHS-48 and DHS-11 compacted and tangled with each other with an interwoven pattern in the co-culture system. A new approach that integrates HPLC chromatogram, 1HNMR spectroscopy, UPLC-MS-PCA, and molecular networking enabled the targeted isolation of the induced metabolites, including three new dimeric xanthones phomoxanthones L-N (1-3), along with six known analogs (4-9). Their planar structures were elucidated by an analysis of their HRMS, MS/MS, and NMR spectroscopic data and the absolute configurations based on ECD calculations. These metabolites showed broad cytotoxic activity against the cancer cells assessed, of which compounds 7-9 displayed significant cytotoxicity towards human liver cells HepG-2 with IC50 values ranging from 4.83 μM to 12.06 μM. Compounds 1-6 exhibited weak immunosuppressive activity against the proliferation of ConA-induced (T-cell) and LPS-induced (B-cell) murine splenic lymphocytes. Therefore, combining co-cultivation with a metabolomics-guided strategy as a discovery tool will be implemented as a systematic strategy for the quick discovery of target bioactive compounds.

The effectiveness of artificial microbial community selection: a conceptual framework and a meta-analysis.

The potential for artificial selection at the community level to improve ecosystem functions has received much attention in applied microbiology. However, we do not yet understand what conditions in general allow for successful artificial community selection. Here we propose six hypotheses about factors that determine the effectiveness of artificial microbial community selection, based on previous studies in this field and those on multilevel selection. In particular, we emphasize selection strategies that increase the variance among communities. We then report a meta-analysis of published artificial microbial community selection experiments. The reported responses to community selection were highly variable among experiments; and the overall effect size was not significantly different from zero. The effectiveness of artificial community selection was greater when there was no migration among communities, and when the number of replicated communities subjected to selection was larger. The meta-analysis also suggests that the success of artificial community selection may be contingent on multiple necessary conditions. We argue that artificial community selection can be a promising approach, and suggest some strategies for improving the performance of artificial community selection programs.

Customized spatial niches for synthetic microbial consortia

The construction of synthetic microbial consortia has been considered a new frontier. However, maintaining artificial microbial communities remains challenging because the dominant strain eventually outcompetes the others. Inspired by natural ecosystems, one promising approach to assemble stable consortia is to construct spatial niches partitioning subpopulations and overlapping abiotic requirements.

High-resolution phylogenetic and population genetic analysis of microbial communities with RoC-ITS

Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities at the subspecies designation. Using rolling-circle amplification, this methodology produces long-read sequences from a circular construct containing the complete 16S ribosomal gene and the neighboring internally transcribed spacer (ITS). These long reads can be used to generate a high-fidelity circular consensus sequence. Generally, the ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism and the assessment of their relative stoichiometry, as well as the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to an artificial microbial community that was also sequenced using the Illumina platform, to assess its accuracy in quantifying the relative abundance and identity of each species.

Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level.

Synthetic microbial consortia with programmable ecological interactions

Abstract Central to the composition, structure and function of any microbial community is the complex species interaction web. But understanding the overwhelming complexity of ecological interaction webs has been challenging, owing at least partly to the lack of efficient tools for disentangling species interactions in natural or artificial microbial communities. In this study, we developed a microbial experimental system which allows for rapidly generating microbial consortia with programmable ecological interactions. We engineered the model organism Escherichia coli to construct metabolism‐ and quorum sensing‐based modules. The two engineered modules were used to create synthetic microbial consortia of synergy, competition and exploitation. We showed that each of synthetic microbial consortia displayed the unique mode of population dynamics under certain initial inoculation conditions. We also demonstrated that the transitions between exploitation and the types of competition or synergy based on the same paired strains were plausible by tuning the two engineered modules. We lastly derived mathematical models to quantitatively capture the experimentally observed population dynamics of these synthetic microbial consortia. This approach offers a fresh angle to engineering microbial systems for experimentally testing ecological questions with a much greater control and manipulation.

Co-culture of Aspergillus sydowii and Bacillus subtilis induces the production of antibacterial metabolites

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool to activate the biosynthetic gene clusters to generate new compounds. However, without optimization of fermentation conditions, the antagonism between the microbes often interferes with the production of secondary metabolites. In this study, the fermentation conditions of co-culture of Aspergillus sydowii and Bacillus subtilis were optimized by response surface methodology to increase the production of active metabolites against Staphylococcus aureus. After optimization, the inhibitory rate of the co-culture extract was 74.62%, which was 29.20% higher than that of the initial conditions. Meanwhile, a total of 15 newly biosynthesized metabolites were detected only in optimized co-culture, occupying 13.2% of all detected metabolites. The structures of the 12 metabolites with high variable importance in projection score were elucidated by the established LC-MS/MS approach integrated with various metabonomic tools. Among them, 7 metabolites were newly induced and the content of other 5 metabolites increased by 1.1–2.4 folds in optimized co-culture. The bioassay of metabolites in co-culture against S. aureus indicated that compounds (−)- (7S)- 10-hydroxysydonic acid, serine sydonate and macrolactin U’ contributed much to the increment of antibacterial activity. This study demonstrated that optimizing the fermentation conditions of co-culture was beneficial to changing the metabolite profile and effective to induce the biosynthesis of active metabolites.

Generation of Stable, Light-Driven Co-cultures of Cyanobacteria with Heterotrophic Microbes.

Co-cultivation of an autotrophic species with one or more heterotrophic microbes is a strategy for photobiological production of high-value compounds and is relatively underexplored in comparison to cyanobacterial or microalgal monocultures. Long-term stability of such consortia is required for useful collaboration between the partners, and this property can be increased by encapsulation of phototrophic partners within a hydrogel. Encapsulated cyanobacteria have advantages relative to planktonic cultures that may be useful to explore the potential for artificial microbial communities for targeted biomolecule synthesis, such as increased control over population sizes and reduced liquid handling requirements. In this chapter, we describe a method for encapsulation of genetically modified cyanobacterial strain (Synechococcus elongatus PCC 7942, CscB+) into a sodium alginate matrix, and the utilization of these encapsulated cells to construct stable, artificial autotroph/heterotroph co-cultures. This method has applications for the study of phototroph-based synthetic microbial consortia, and multi-species photobiological production.

Copper and Zinc Recovery from Sulfide Concentrate by Novel Artificial Microbial Community

Exploring efficient methods to enhance leaching efficiency is critical for bioleaching technology to deal with sulfide concentrate. In our study, a novel artificial microbial community was established to augment the bioleaching efficiency and recovery of copper (Cu) and zinc (Zn). The optimum parameters in bioleaching experiments were explored according to compare a series of conditions from gradient experiments: the pH value was 1.2, temperature was 45 °C, and rotation speed was 160 r/min, which were different with pure microorganism growth conditions. Under optimal conditions, the result of recovery for Cu and Zn indicated that the average leaching rate reached to 80% and 100% respectively, which almost increased 1.8 times and 1.2 times more than control (aseptic condition) group. Therefore, this method of Cu and Zn recovery using a new-type artificial microbial community is expected to be an environmentally-friendly and efficient bioleaching technology solution, which has the potential of large-field engineering application in the future.

Metabolomic profiles of the liquid state fermentation in co-culture of A. oryzae and Z. rouxii

Aspergillus oryzae and Zygosaccharomyces rouxii are the perquisites microorganisms in food fermentation due to the broad application prospects of their secondary metabolites. The co-culture strategy simulates the naturally occurring ecology by developing artificial microbial communities. This strategy has also been widely adopted to investigate the novel secondary metabolites. In the present study, the effects of co-culture on extracellular and intracellular secondary metabolites of fungi in liquid culture were investigated through UPLC-QTOF-MS. Notably, A. oryzae could significantly inhibit the growth of Z. rouxii when A. oryzae and Z. rouxii were co-cultured. The results further indicated that the co-culture of fungi could affect the secondary metabolites and produce various metabolic pathways. Overall, this study will provide insights into fungal interactions and cell metabolism mechanisms, contributing to supplementing and enriching the fermentation potential of fungi.

Effect of diurnal temperature range on bioleaching of sulfide ore by an artificial microbial consortium

Temperature is considered to be one of the main factors affecting bioleaching, but few studies have assessed the effects of diurnal temperature range (DTR) on the bioleaching process. This study investigates the effects of different bioleaching temperatures (30 and 40 °C) and DTR on the bioleaching of metal sulfide ores by microbial communities. The results showed that DTR had an obvious inhibitory effect on the bioleaching efficiency of the artificial microbial community, although this effect was mainly concentrated in the early and middle stages (0–18 days) of exposure, gradually decreasing until almost disappearing in the late stage (18–24 days). Extracellular polymeric substance (EPS) analysis showed that DTR did not change the composition of the EPS matrix (humic acid-like substances, polysaccharides and protein-like substances), but had a significant effect on the generative behavior of EPS, inhibiting the secretion of EPS during the early and middle stages of the bioleaching process. However, the continual increase in EPS secretion in the bioleaching system gradually reduced the adverse effects of DTR on mineral dissolution. X-ray diffraction (XRD), Fourier transform infrared (FTIR) and Scanning electron microscopy- energy dispersive spectrometry (SEM-EDS) analysis of the bioleached residue showed that DTR had no obvious effect on the mineralogical characteristics of sulfide ore. Therefore, in industrial sulfide ore bioleaching applications, in order to accelerate the artificial microbial community start-up process, temperature control measures should be increased in the bioleaching process to reduce the adverse effects of DTR on mineral dissolution.

Microbial diversity formation and maintenance due to temporal niche differentiation caused by low-dose ionizing radiation in oligotrophic environments

The planetary protection strives to minimize the contamination of microorganisms in spacecrafts. However, it is reported that microbial diversity is abnormally high in the International Space Station (ISS) after long-term exposure to low-dose ionizing radiation (LDIR). It remains a mystery why LDIR leads to the formation and maintenance of high microbial diversity in oligotrophic environments like the ISS. In this study, an artificial microbial community has been cultivated without and with LDIR, respectively. The microbial community was composed of three common microbial species, i.e., Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa in the ISS. After analyzing the differences in microbial physiological and behavioral response characteristics in the two scenarios, a reasonable hypothesis was proposed to elucidate the formation and maintenance mechanisms of high microbial diversity in oligotrophic environments with the LDIR. Then a set of kinetic models with time-lag were developed based on this hypothesis, observed phenomena, and experimental data. Finally, these kinetic models were sufficiently validated, and the hypothesis was fully confirmed through large-scale digital simulations. Briefly, as a decisive succession mechanism in oligotrophic environments with LDIR, temporal niche differentiation (TND) caused by microbial delayed responses to LDIR can give rise to asynchronously convergent fluctuations of microbial populations and significantly alleviate the intra- and interspecific competitions. Such a mechanism can drive the microbial communities in oligotrophic environments with LDIR to form and maintain high species diversity.

Microbial diversity formed and maintained through substrate feedback regulation and delayed responses induced by Low-Dose Ionizing Radiation

Microbial diversity is essential for the maintenance of the normal structure and function of bioregenerative life support systems (BLSS). As a typical nutrient-deficient environment (NDE), the BLSS does not provide sufficient types of available substrates for microbial communities, and its internal microbial diversity is usually not high due to interspecific competitive exclusion. However, it is reported that microbial diversity is abnormally high in the International Space Station (ISS) after long-term exposure to low-dose ionizing radiation (LDIR). It remains a mystery why LDIR leads to the formation and maintenance of high microbial diversity. In this study, a series of artificial microbial communities have been cultivated in NDE without and with LDIR, respectively. These communities are composed of three common microbial species (Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa) in the ISS. By comparing and analyzing the differences in the microbial physiological and behavioral response characteristics in the two scenarios, a reasonable hypothesis was put forward to elucidate the formation and maintenance mechanisms of high microbial diversity in NDE with LDIR. Then a set of kinetic models were developed based on this hypothesis, observed phenomena, and experimental data. Finally, these kinetic models were sufficiently validated and the hypothesis was fully confirmed through large-scale digital simulations. Briefly, two fundamental succession mechanisms of the microbial communities are supposed to exist in NDE with LDIR: substrate-based negative feedback regulation (SNFR) and microbial delayed responses. These two decisive succession mechanisms can give rise to asynchronously convergent fluctuations of microbial populations and significantly alleviate the interspecific competitions. Such a species-for-quantity strategy drives the microbial communities to form and maintain species diversity with higher richness and evenness. This study can lay the theoretical foundation and provide new ideas for the construction of advanced BLSS featured with more robust structures and stronger function.

De Novo Biosynthesis of Chlorogenic Acid Using an Artificial Microbial Community.

Engineering an artificial microbial community for natural product production is a promising strategy. As mono- and dual-culture systems only gave non-detectable or minimal chlorogenic acid (CGA) biosynthesis, here, a polyculture of three recombinant Escherichia coli strains, acting as biosynthetic modules of caffeic acid (CA), quinic acid (QA), and CGA, was designed and used for de novo CGA biosynthesis. An influx transporter of 3-dehydroshikimic acid (DHS)/shikimic acid (SA), ShiA, was introduced into the QA module-a DHS auxotroph. The QA module proportion in the polyculture and CGA production were found to be dependent on ShiA expression, providing an alternative approach for controlling microbial community composition. The polyculture strategy avoids metabolic flux competition in the biosynthesis of two CGA precursors, CA and QA, and allows production improvement by balancing module proportions. The performance of this polyculture approach was superior to that of previously reported approaches of de novo CGA production.

Inducing secondary metabolite production of Aspergillus sydowii through microbial co-culture with Bacillus subtilis

BackgroundThe co-culture strategy which mimics natural ecology by constructing an artificial microbial community is a useful tool to activate the biosynthetic gene clusters to generate new metabolites. However, the conventional method to study the co-culture is to isolate and purify compounds separated by HPLC, which is inefficient and time-consuming. Furthermore, the overall changes in the metabolite profile cannot be well characterized.ResultsA new approach which integrates computational programs, MS-DIAL, MS-FINDER and web-based tools including GNPS and MetaboAnalyst, was developed to analyze and identify the metabolites of the co-culture of Aspergillus sydowii and Bacillus subtilis. A total of 25 newly biosynthesized metabolites were detected only in co-culture. The structures of the newly synthesized metabolites were elucidated, four of which were identified as novel compounds by the new approach. The accuracy of the new approach was confirmed by purification and NMR data analysis of 7 newly biosynthesized metabolites. The bioassay of newly synthesized metabolites showed that four of the compounds exhibited different degrees of PTP1b inhibitory activity, and compound N2 had the strongest inhibition activity with an IC50 value of 7.967 μM.ConclusionsCo-culture led to global changes of the metabolite profile and is an effective way to induce the biosynthesis of novel natural products. The new approach in this study is one of the effective and relatively accurate methods to characterize the changes of metabolite profiles and to identify novel compounds in co-culture systems.

Succession mechanism of microbial community with high species diversity in nutrient-deficient environments with low-dose ionizing radiation

It remains a mystery why microbial community succession can violate the competitive exclusion principle to form high species diversity in nutrient-deficient environments (NDE) with low-dose ionizing radiation (LDIR). In this research, artificial microbial communities containing three common strains downloaded from China Tiangong-1 space station were cultivated in NDE with and without LDIR, respectively. By comparing their identities and differences in cellular phenotype, extracellular pH, intracellular triglycerides and population dynamics under the two circumstances, a new hypothesis was put forward from observed phenomena and experimental data to expound new interspecific relationships and interactions induced by LDIR to produce new dynamic mechanisms to drive microbial community succession with high species diversity. A set of highly valid kinetic models was then developed from this hypothesis, and finally, the hypothesis proposed was verified through a large number of digital simulations. The results show that induced by LDIR, specific feedback regulation of microbial populations by mutual promotion and restraint mechanisms might exist in microbial communities, which can cause asynchronous convergent fluctuations of microbial populations. These fluctuations remarkably alleviate interspecific competition and inhibit the emergence of dominant species, leading to the formation and maintenance of higher species diversity. The results can provide a theoretical basis for understanding the ecological effect of LDIR on microbial communities in NDE as well as its application for ecological engineering purposes.

FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties

Fluorescence-activated cell sorting (FACS) has rarely been applied to screening of microorganisms because of poor detection resolution, which is compromised by poor stability, toxicity, or interference from background fluorescence of the fluorescence sensors used. Here, a fluorescence-based rapid high-throughput cell sorting method was first developed using a fluorescence resonance energy transfer (FRET) fluorescent nanoprobe NP-RA, which was constructed by coating a silica nanoparticle with Rhodamine B and methyl-red (an azo dye). Rhodamine B (inner layer) is the FRET donor and methyl-red (outer layer) is the acceptor. This ready-to-use NP-RA is non-fluorescent, but fluoresces once the outer layer is degraded by microorganisms. In our experiment, NP-RA was ultrasensitive to model strain Shewanella decolorationis S12, showing a broad detection range from 8.0 cfu/mL to 8.7 × 108 cfu/mL under confocal laser scanning microscopy, and from 1.1 × 107 to 9.36 × 108 cfu/mL under a fluorometer. In addition, NP-RA bioimaging can clearly identify other azo-respiring cells in the microbial community, including Bosea thiooxidans DSM 9653 and Lysinibacillus pakistanensis NCCP-54. Furthermore, the fluorescent probe NP-RA is compatible with downstream FACS so that azo-respiring cells can be rapidly sorted out directly from an artificial microbial community. To our knowledge, no fluorescent nanoprobe has yet been designed for tracking and sorting azo-respiration functional microorganisms.

Co-Culture Systems for the Production of Secondary Metabolites: Current and Future Prospects

Microorganisms are the great sources of Natural Products (NPs); these are imperative to their survival apart from conferring competitiveness amongst each other within their environmental niches. Primary and secondary metabolites are the two major classes of NPs that help in cell development, where antimicrobial activity is closely linked with secondary metabolites. To capitalize on the effects of secondary metabolites, co-culture methods have been often used to develop an artificial microbial community that promotes the action of these metabolites. Different analytical techniques will subsequently be employed based on the metabolite specificity and sensitivity to further enhance the metabolite induction. Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS are commonly used for metabolite separation while Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) have been used as tools to elucidate the structure of compounds. This review intends to discuss current systems in use for co-culture in addition to its advantages, with discourse into the investigation of specific techniques in use for the detailed study of secondary metabolites. Further advancements and focus on co-culture technologies are required to fully realize the massive potential in synthetic biological systems.

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.