Methane-producing Microbial Communities Research Articles

Evaluation of acidogenesis products\u2019 effect on biogas production performed with metagenomics and isotopic approaches

BackgroundDuring the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system.ResultsFour methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities.ConclusionsIn this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock.

Effective methane production from the Japanese weed Gyougi-shiba (Cynodon dactylon) is accomplished by colocalization of microbial communities that assimilate water-soluble and -insoluble fractions.

ABSTRACTWeed, an abundant biomass, is considered unsuitable as a raw material for methane production. There are few reports on the anaerobic digestion of weeds without the addition of other organic wastes. To solve this problem, a methane-producing microbial community with weed as a sole feedstock was established. This study mainly focused on the degree of contribution between water-soluble and -insoluble fractions of the weed to methane production; thus, methane production from both fractions was tested separately. Methane production after 80-day batch cultures with whole weed, water-soluble and water-insoluble fractions was 184.5, 96.8 and 26.5 NmL g−1 dry matter (DM), respectively. The results of 16S rRNA gene amplicon sequence analysis revealed that Proteiniphilum saccharofermentans and several Methanobacterium species commonly dominated all cultures, whereas the population dynamics of minor species differed in every culture. Moreover, the remixed culture of microbial communities adapted to water-soluble and -insoluble fractions recovered methane production (252.4 NmL g−1 DM). Based on these results, it can be strongly inferred that colocalizing the minor species in water-soluble and -insoluble fractions is important for effective methane production.

Microbial methane formation from different lithotypes of Miocene lignites from the Konin Basin, Poland: Geochemistry of the gases and composition of the microbial communities

The present research compares the yield of microbial methane production from different lithotypes of Miocene lignite from the Konin Basin in Poland and attempts to establish an understanding of the processes responsible for methanogenesis. A series of batch experiments were carried out with detritic and xylitic lignites inoculated with microorganisms from an external source (the anaerobic chamber of a wastewater treatment plant in a sugar factory). Biogas volume, concentration and stable carbon isotopes of microbial methane were measured. It was found that detritic lignites are a slightly more suitable raw material for microbial methane production than xylites. Methane yield for detritic lignites equalled 14.3 μmol CH4/g of total organic carbon (TOC); for xylites, 13.7 μmol CH4/g of TOC. The mean δ13C(CH4) value in experiments with detritic coal from Konin equalled −36.3‰; for fossil wood fragments, −47.3‰ and −42.7‰. We suppose that differences in mean δ13C(CH4) values from the biodegradation of different lithotypes of lignite from the Konin Basin most probably depend on cellulose and lignin content (%). The holocellulose content in xylites decreased over time, suggesting the important role of the enzymatic hydrolysis of cellulose to glucose. Methane from detritic lignite was formed due to lignin decomposition. Methane-producing microbial communities were dominated by Bacteria mainly from the phyla Proteobacteria (Alpha-, Beta- and Gamma- or Deltaproteobacteria), Firmicutes (Clostridia and Bacilli), Actinobacteria, and Bacteroidetes. Archaea constituted only 2–6% of the microbial community, including Methanosarcinales, Methanomicrobiales, and Methanobacteriales. The data presented here show no clear correlations between lignite type and specific bacterial or archaeal taxa. However, certain tendencies were observed. Exiguobacterium and Pseudomonas are more abundant in xylites, Rhizobacteriaceae in detritic lignites. This indicates the complexity and diversity of lignite material and processes leading to lignite degradation. In the presented research model, anaerobic oxidation of methane may occur.

Increasing sulfate levels show a differential impact on synthetic communities comprising different methanogens and a sulfate reducer.

Methane-producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulfate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulfate availability are not well understood. Here, we construct model synthetic communities using a sulfate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost twofold compared to co-cultures and are stable in the absence of sulfate. With increasing sulfate, system stability and productivity decreases and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in the metabolism of these methanogens towards co-utilization of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities.

Microbial Ecology: Community Coalescence Stirs Things Up

When methane-producing microbial communities are mixed experimentally, the resulting community is dominated by the community with the greatest resource-use efficiency. These results suggest a degree of community cohesion, or the maintenance of that initial community in the mix.

Metagenomic and Metatranscriptomic Analysis of an Electromethanogenic Microbial Community

Researchers have described the mechanisms of extracellular electron uptake in model electrogens like Shewanella oneidensis MR-11 and Geobacter sulfurreducens2 as well as providing evidence for direct electron uptake for some methanogenic strains in BESs3. However, practical applications of BESs will require the use of mixed microbial populations to ensure performance stability by taking advantage of microbial adaptation and symbioses that can reduce the impact of environmental or operational perturbations. However, little is known about the taxonomic composition of mixed microbial biofilms that successfully drive electromethanogenesis. Even less is known about the functional association of specific genes and pathways expressed by microbial community members that enable cooperation/competition in an electromethanogenic system. Here we utilized a novel stimulus-induced metatranscriptomic approach in combination with metagenomics to identify the microbial taxa in an electromethanogenic biofilm and quantify the dynamic responses of key genes associated with electron uptake, hydrogen production/utilization and carbon dioxide fixation. Two dual-compartment bioelectrochemical systems (BES1 and BES2) were inoculated with rice paddy soil, a minimal media without additional soluble carbon sources or chemical mediators, and a 20% CO2 headspace (balance N2). The systems were operated as described in Bretschger et al.4 using a subpassage technique to accelerate the enrichment of highly active electromethanogenic communities. The stimulus-induced metatranscriptomic experiments and metagenomic analyses were executed according to methods described in Ishii et al.5 , 6. Both systems were exposed to an open circuit condition for 45 minutes and biofilms were sampled before and after the operational change (Fig. 1a, main). At the time of sampling both systems were consuming electricity and producing methane (Fig. 1a, inset). However, BES2 was consuming more electrons per unit biomass than BES2, even though both systems had roughly equivalent methane production activity. The results from the metagenomic analyses show that the same strains of methanogens, sulfate reducers and fermenters were present in BES1 and BES2; however, they were present in different relative abundances, which may explain the performance differences (Fig. 1b and c). Further, the stimuli applied to both reactors induced different functional responses from the respective communities, which also indicates that the relative abundance of species impacts how they functionally respond in BESs. Future work will address the specific interactions that correlate with these performance differences. These data yield new perspectives on the functional and taxonomic dynamics of electromethanogenic microbial communities and how they may be controlled in BESs. 1 Ross, D. E., Flynn, J. M., Baron, D. B., Gralnick, J. A. & Bond, D. R. Towards electrosynthesis in Shewanella: energetics of reversing the Mtr pathway for reductive metabolism. PloS one 6, e16649 (2011). 2 Gregory, K. B., Bond, D. R. & Lovley, D. R. Graphite electrodes as electron donors for anaerobic respiration. Environmental Microbiology 6, 596-604 (2004). 3 Lohner, S. T., Deutzmann, J. S., Logan, B. E., Leigh, J. & Spormann, A. M. Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis. The ISME journal 8, 1673-1681 (2014). 4 Bretschger, O. et al. Functional and taxonomic dynamics of an electricity-consuming methane-producing microbial community. Bioresource technology 195, 254-264 (2015). 5 Ishii, S. i. et al. A novel metatranscriptomic approach to identify gene expression dynamics during extracellular electron transfer. Nature Communications, doi:doi:10.1038/ncomms2615 (2013). 6 Ishii, S. i. et al. Microbial metabolic networks in a complex electrogenic biofilm recovered from a stimulus-induced metatranscriptomics approach. Scientific reports 5 (2015). Figure 1

Diversity of methane-producing microbial community structure with 16S-V3 rDNA PCR-DGGE

The method of 16S-V3 rDNA PCR-DGGE was utilized to study the diversity of methane-producing microbial community structure under different methane-producing microbial community structure. Results showed that community structure of the methanogens under different environmental condition was significantly different. The number of bands about community under extreme condition was less than normal conditions, but the main bands were obvious. The sequence analysis showed that the predominant strains mainly were uncultured bacteria that could not be identified in the level of species .Z2 was Clostridia, Z8 was Clostridiales, D5 was Eubacteriaceae, and D8 was Flavobacteriaceae. This study laid some extent foundation for future research about establishing a methane-producing bacterial system with high gas yield in winter.

Functional and taxonomic dynamics of an electricity-consuming methane-producing microbial community

The functional and taxonomic microbial dynamics of duplicate electricity-consuming methanogenic communities were observed over a 6months period to characterize the reproducibility, stability and recovery of electromethanogenic consortia. The highest rate of methanogenesis was 0.72mg-CH4/L/day, which occurred during the third month of enrichment when multiple methanogenic phylotypes and associated Desulfovibrionaceae phylotypes were present in the electrode-associated microbial community. Results also suggest that electromethanogenic microbial communities are very sensitive to electron donor-limiting open-circuit conditions. A 45min exposure to open-circuit conditions induced an 87% drop in volumetric methane production rates. Methanogenic performance recovered after 4months to a maximum value of 0.30mg-CH4/L/day under set potential operation (−700mV vs Ag/AgCl); however, current consumption and biomass production was variable over time. Long-term functional and taxonomic analyses from experimental replicates provide new knowledge toward understanding how to enrich electromethanogenic communities and operate bioelectrochemical systems for stable and reproducible performance.

Plugging in or going wireless: strategies for interspecies electron transfer.

Interspecies exchange of electrons enables a diversity of microbial communities to gain energy from reactions that no one microbe can catalyze. The first recognized strategies for interspecies electron transfer were those that relied on chemical intermediates that are recycled through oxidized and reduced forms. Well-studied examples are interspecies H2 transfer and the cycling of sulfur intermediates in anaerobic photosynthetic communities. Direct interspecies electron transfer (DIET) in which two species establish electrical contact is an alternative. Electrical contacts documented to date include electrically conductive pili, as well as conductive iron minerals and conductive carbon moieties such as activated carbon and biochar. Interspecies electron transfer is central to the functioning of methane-producing microbial communities. The importance of interspecies H2 transfer in many methanogenic communities is clear, but under some circumstances DIET predominates. It is expected that further mechanistic studies and broadening investigations to a wider range of environments will help elucidate the factors that favor specific forms of interspecies electron exchange under different environmental conditions.

Co-digestion, biostimulation and bioaugmentation to enhance methanation of brewer’s spent grain

More than 300,000 tonnes of brewer's spent grain (BSG) is generated annually during beer production. This protein- and nutrient-rich by-product is mostly employed as an animal feedstuff. However, its marketability is compromised by its rapid deterioration owing to its high humidity and fermentable sugar content. Drying BSG can be achieved using the bio-energy generated from the anaerobic digestion of part of the BSG produced in the same brewery. We employed three types of strategies to enhance the biomethanation of BSG in mesophilic batch incubations. First, we co-digested BSG with peach flesh residues, juice residues, sewage sludge and pig slurry. Second, we supplemented BSG with chemical additives (carbon and energy sources) in order to biostimulate the methane-producing microbial communities. Finally, we used anaerobically acclimatised BSG to augment the initial microbial load in assays digesting BSG either alone or in co-digestion with sewage sludge. All co-substrates assayed were suitable to be fermented in combination with BSG, although methane production was highest for the mixtures with sewage sludge and pig slurry, with their high pH values and nutrient contents. Nine out of 14 combinations of stimulatory chemicals significantly enhanced BSG methanation compared with a non-supplemented control. Overall, bioaugmenting the anaerobic microbial consortia by using fermented BSG as an inoculum when co-digesting BSG with sewage sludge performed best in terms of methane yield.

A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor.

BackgroundA solid-state anaerobic digestion method is used to produce biogas from various solid wastes in China but the efficiency of methane production requires constant improvement. The diversity and abundance of relevant microorganisms play important roles in methanogenesis of biomass. The next-generation high-throughput pyrosequencing platform (Roche/454 GS FLX Titanium) provides a powerful tool for the discovery of novel microbes within the biogas-generating microbial communities.ResultsTo improve the power of our metagenomic analysis, we first evaluated five different protocols for extracting total DNA from biogas-producing mesophilic solid-state fermentation materials and then chose two high-quality protocols for a full-scale analysis. The characterization of both sequencing reads and assembled contigs revealed that the most prevalent microbes of the fermentation materials are derived from Clostridiales (Firmicutes), which contribute to degrading both protein and cellulose. Other important bacterial species for decomposing fat and carbohydrate are Bacilli, Gammaproteobacteria, and Bacteroidetes (belonging to Firmicutes, Proteobacteria, and Bacteroidetes, respectively). The dominant bacterial species are from six genera: Clostridium, Aminobacterium, Psychrobacter, Anaerococcus, Syntrophomonas, and Bacteroides. Among them, abundant Psychrobacter species, which produce low temperature-adaptive lipases, and Anaerococcus species, which have weak fermentation capabilities, were identified for the first time in biogas fermentation. Archaea, represented by genera Methanosarcina, Methanosaeta and Methanoculleus of Euryarchaeota, constitute only a small fraction of the entire microbial community. The most abundant archaeal species include Methanosarcina barkeri fusaro, Methanoculleus marisnigri JR1, and Methanosaeta theromphila, and all are involved in both acetotrophic and hydrogenotrophic methanogenesis.ConclusionsThe identification of new bacterial genera and species involved in biogas production provides insights into novel designs of solid-state fermentation under mesophilic or low-temperature conditions.

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO 2 as electron acceptor and H 2 as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.

Methane-Producing Microbial Community in a Coal Bed of the Illinois Basin

Methane-Producing Microbial Community in a Coal Bed of the Illinois Basin

Methane-Producing Microbial Community in a Coal Bed of the Illinois Basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.