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: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.
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:Richards2016 - Genome-scale metabolic
reconstruction of Methanococcus maripaludis (iMR539)
This model is described in the article:
Exploring Hydrogenotrophic
Methanogenesis: a Genome Scale Metabolic Reconstruction of
Methanococcus maripaludis.
Richards MA, Lie TJ, Zhang J,
Ragsdale SW, Leigh JA, Price ND.
J. Bacteriol. 2016 Dec; 198(24):
3379-3390
Abstract:
Hydrogenotrophic methanogenesis occurs in multiple
environments, ranging from the intestinal tracts of animals to
anaerobic sediments and hot springs. Energy conservation in
hydrogenotrophic methanogens was long a mystery; only within
the last decade was it reported that net energy conservation
for growth depends on electron bifurcation. In this work, we
focus on Methanococcus maripaludis, a well-studied
hydrogenotrophic marine methanogen. To better understand
hydrogenotrophic methanogenesis and compare it with
methylotrophic methanogenesis that utilizes oxidative
phosphorylation rather than electron bifurcation, we have built
iMR539, a genome scale metabolic reconstruction that accounts
for 539 of the 1,722 protein-coding genes of M. maripaludis
strain S2. Our reconstructed metabolic network uses recent
literature to not only represent the central electron
bifurcation reaction but also incorporate vital biosynthesis
and assimilation pathways, including unique cofactor and
coenzyme syntheses. We show that our model accurately predicts
experimental growth and gene knockout data, with 93% accuracy
and a Matthews correlation coefficient of 0.78. Furthermore, we
use our metabolic network reconstruction to probe the
implications of electron bifurcation by showing its
essentiality, as well as investigating the infeasibility of
aceticlastic methanogenesis in the network. Additionally, we
demonstrate a method of applying thermodynamic constraints to a
metabolic model to quickly estimate overall free-energy changes
between what comes in and out of the cell. Finally, we describe
a novel reconstruction-specific computational toolbox we
created to improve usability. Together, our results provide a
computational network for exploring hydrogenotrophic
methanogenesis and confirm the importance of electron
bifurcation in this process.Understanding and applying
hydrogenotrophic methanogenesis is a promising avenue for
developing new bioenergy technologies around methane gas.
Although a significant portion of biological methane is
generated through this environmentally ubiquitous pathway,
existing methanogen models portray the more traditional energy
conservation mechanisms that are found in other methanogens. We
have constructed a genome scale metabolic network of
Methanococcus maripaludis that explicitly accounts for all
major reactions involved in hydrogenotrophic methanogenesis.
Our reconstruction demonstrates the importance of electron
bifurcation in central metabolism, providing both a window into
hydrogenotrophic methanogenesis and a hypothesis-generating
platform to fuel metabolic engineering efforts.
This model is hosted on
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and identified by:
MODEL1607200000.
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Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase a key enzyme in the methanogenesis pathway.
Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase -- a key enzyme in the methanogenesis pathway.