Project description:Inhibition of the anaerobic digestion process through accumulation of volatile fatty acids (VFA) is a recurring problem which is the result of unbalanced growth between acidogenic bacteria and methanogens. A speedy recovery is essential for an establishment of a feasible economical biogas productions. Yet, little is known regarding the organisms participating in the recovery. In this study the organisms involved in the recovery were studied using protein-stable isotope probing (Protein-SIP) and mapping this data onto a binned metagenome. Under acetate-accumulated simulating conditions a formation of 13C-labeled CO2 and CH4 was detected immediately after the addition of [U-13C]acetate, indicative of a high turnover rate of acetate. Several labeled peptides were detected in protein-SIP analysis. These 13C-labeled peptides were mapped onto a binned metagenome for improved taxanomical classification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia and one Bacteroidetes. The organisms affiliating with Clostridia and Bacteroidetes all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis; indicating that these organisms are possible syntrophic acetate-oxidizing bacteria (SAOB) that can facilitate acetate consumption via syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms involved in specific pathways.
Project description:Purpose: To understand the adaptive mechanisms of Methanocellales to low H2 and syntrophic growth. Methods: We analyzed the transcriptomes of M. conradii and P. thermopropionicum under monoculture and syntrophic coculture conditions by strand specific mRNA sequencing using Illumina Hiseq 2000. Four biological replicates were sequenced. The sequence reads that passed quality filters were analyzed by Burrows–Wheeler Aligner (BWA) followed by HTSeq and DESeq2. qRT–PCR validation was performed using SYBR Green assays Results: The results showed that M. conradii and P. thermopropionicum interacted closely and synchronized their gene transcription during the syntrophic growth. In coculture, M. conradii and P. thermopropionicum significantly enhanced the transcription of genes related to energy conservation processes, including methanogenesis, propionate degradation and electron bifurcation. By contrast, the genes coding for biosynthesis steps were downregulated in both M. conradii and P. thermopropionicum during the syntrophic growth. The physiology experiment showed that formate but not H2 inhibited syntrophic oxidation of propionate. Accordingly, formate dehydrogenase-encoding genes in both M. conradii and P. thermopropionicum were markedly upregulated, indicating that formate plays an important role in the interspecies electron transfer between M. conradii and P. thermopropionicum in coculture. Conclusions: our study provides abundant transcriptome data indicating the adaptations of Methanocella spp. to H2 limitation and suggests that flavin based electron bifurcations are critical to the syntrophic growth in both M. conradii and P. thermopropionicum.
Project description:Biogas plants (BGPs) produce methane and carbon dioxide through the anaerobic digestion of agricultural waste. Identification of strategies for more stable biogas plant operation and increased biogas yields require better knowledge about the individual degradation steps and the interactions within the microbial communities. The metaprotein profiles of ten agricultural BGPs and one laboratory reactor were investigated using a metaproteomics pipeline. Fractionation of samples using SDS-PAGE was combined with a high resolution Orbitrap mass spectrometer, metagenome sequences specific for BGPs, and the MetaProteomeAnalyzer software. This enabled us to achieve a high coverage of the metaproteome of the BGP microbial communities. The investigation revealed approx. 17,000 protein groups (metaproteins), covering the majority of the expected metabolic networks of the biogas process such as hydrolysis, transport, fermentation processes, amino acid metabolism, methanogenesis and bacterial C1-metabolism. Biological functions could be linked with the taxonomic composition. Two different types of BGPs were classified by the abundance of the acetoclastic methanogenesis and by abundance of enzymes implicating syntrophic acetate oxidation. Linking of the identified metaproteins with the process steps of the Anaerobic Digestion Model 1 proved the main model assumptions but indicated also some improvements such as considering syntrophic acetate oxidation. Beside the syntrophic interactions, the microbial communities in BGPs are also shaped by competition for substrates and host-phage interactions causing cell lysis. In particular, larger amounts of Bacteriophages for the bacterial families Bacillaceae, Enterobacteriaceae and Clostridiaceae, exceeding the cell number of the Bacteria by approximately four-fold. In contrast, less Bacteriophages were found for Archaea, but more CRISPR proteins were detected. On the one hand, the virus induced turnover of biomass might cause slow degradation of complex biomass in BGP. On the other hand, the lysis of bacterial cells allows cycling of essential nutrients.
Project description:In the syntrophic interaction between fermentative bacteria (Pelotomaculum thermopropionicum) and methanogenic archaea (methanogens: Methanothemobacter thermautotrophicus), reducing equivalents (e.g., H2) produced by fermentative bacteria should efficiently be consumed by methanogens in order for the fermentation of volatile fatty acids (VFA, e.g., butyrate, propionate, and acetate) to be thermodynamically feasible. It has been known that physical approximation (e.g., coaggregation) between VFA-fermenting syntrophic bacteria (syntrophs) and hydrogenotrophic methanogens is necessary for efficient H2 transfer between them. Our previous study has shown that, at an early exponential growth phase of syntrophic coculture, cells of Pelotomaculum thermopropionicum (syntroph) were connected to cells of Methanothermobacter thermautotrophicus (methanogen) via unidentified extracellular filamentous appendages, after which they started to coaggregate, suggesting that the filamentous appendages may have been important for their syntrophic interaction. The filamentous appendages seemed to specifically connect these syntrophic partners, since such pairwise connection has been observed neither in single-species cultures (monocultures) nor in mixtures with other microbes.<br>We found that P. thermopropionicum has putative gene clusters for flagellum and pilus, while no extracellular filament gene was identified in the M. thermautotrophicus genome. So we examined transcriptome responses of M. thermautotrophicus to the contact with flagellar filament protein (FliC) and flagellar cap protein (FliD) of P. thermopropionicum.
Project description:In the syntrophic interaction between fermentative bacteria (Pelotomaculum thermopropionicum) and methanogenic archaea (methanogens: Methanothemobacter thermautotrophicus), reducing equivalents (e.g., H2) produced by fermentative bacteria should efficiently be consumed by methanogens in order for the fermentation of volatile fatty acids (VFA, e.g., butyrate, propionate, and acetate) to be thermodynamically feasible. It has been known that physical approximation (e.g., coaggregation) between VFA-fermenting syntrophic bacteria (syntrophs) and hydrogenotrophic methanogens is necessary for efficient H2 transfer between them. Our previous study has shown that, at an early exponential growth phase of syntrophic coculture, cells of Pelotomaculum thermopropionicum (syntroph) were connected to cells of Methanothermobacter thermautotrophicus (methanogen) via unidentified extracellular filamentous appendages, after which they started to coaggregate, suggesting that the filamentous appendages may have been important for their syntrophic interaction. The filamentous appendages seemed to specifically connect these syntrophic partners, since such pairwise connection has been observed neither in single-species cultures (monocultures) nor in mixtures with other microbes. <br> We found that P. thermopropionicum has putative gene clusters for flagellum and pilus, while no extracellular filament gene was identified in the M. thermautotrophicus genome. So we examined transcriptome responses of M. thermautotrophicus to the contact with flagellar filament protein (FliC) and flagellar cap protein (FliD) of P. thermopropionicum.