Showing papers on "Methanogen published in 2016"
TL;DR: The discovery of divergent methyl-coenzyme M reductase genes in population genomes recovered from anoxic environments with high methane flux that belong to a new archaeal phylum, the Verstraetearchaeota, indicate that methanogen diversity is only beginning to understand and support an ancient origin for methane metabolism in the Archaea, which is changing the authors' understanding of the global carbon cycle.
Abstract: Methanogenesis is the primary biogenic source of methane in the atmosphere and a key contributor to climate change. The long-standing dogma that methanogenesis originated within the Euryarchaeota was recently challenged by the discovery of putative methane-metabolizing genes in members of the Bathyarchaeota, suggesting that methanogenesis may be more phylogenetically widespread than currently appreciated. Here, we present the discovery of divergent methyl-coenzyme M reductase genes in population genomes recovered from anoxic environments with high methane flux that belong to a new archaeal phylum, the Verstraetearchaeota. These archaea encode the genes required for methylotrophic methanogenesis, and may conserve energy using a mechanism similar to that proposed for the obligate H2-dependent methylotrophic Methanomassiliicoccales and recently described Candidatus ‘Methanofastidiosa’. Our findings indicate that we are only beginning to understand methanogen diversity and support an ancient origin for methane metabolism in the Archaea, which is changing our understanding of the global carbon cycle. The Verstraetearchaeota encode divergent methyl-coenzyme M reductase genes, which are required for methylotrophic methanogenesis, increasing methanogen diversity and the complexity of the global methane cycle.
375 citations
TL;DR: Findings reveal a remarkably unique methanogen ‘Candidatus Methanofastidiosum methylthiophilus’ as the first insight into the sixth class of methanogens ‘candidatus methanofastsidiosa’, which is a distinct class from other Euryarchaeota.
Abstract: The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota methanogenesis. To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol coenzyme M methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for carbon fixation, nitrogen fixation and biosynthesis of many amino acids. Acetate, malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially compete with CO2-reducing methanogens and even sulfate reducers. These findings reveal a remarkably unique methanogen 'Candidatus Methanofastidiosum methylthiophilus' as the first insight into the sixth class of methanogens 'Candidatus Methanofastidiosa'.
193 citations
TL;DR: It is shown that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production, consistent with the theory that a smaller rumen size with a higher turnover rate, results in lower hydrogen production and lower methane formation.
Abstract: Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep. Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings. Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep.
186 citations
TL;DR: Deep subsurface–derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs, and this mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl–coenzyme A metabolism.
Abstract: Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface–derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this “methoxydotrophic” mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl–coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.
174 citations
TL;DR: It is anticipated that this metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.
Abstract: Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones. Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using 13C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing. We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.
118 citations
TL;DR: A hybrid reactor, which coupled bioelectrolysis and AD, was developed to enhance methane recovery from WAS and revealed that hydrogenotrophic methanogen Methanobacterium dominated the cathode biofilm, which was the predominant contributor to accelerate the recovery of methane.
Abstract: Understanding the microbial community structure relative to enhancement of methane production from digestion of waste-activated sludge (WAS) coupled with a bioelectrochemical system is a key scientific question for the potential application of bioelectrochemistry in biogas production. Little has been known about the influence of electrode on the structure and function of microbial communities, especially methanogens in a bioelectrochemical anaerobic digestion (AD) reactor. Here, a hybrid reactor, which coupled bioelectrolysis and AD, was developed to enhance methane recovery from WAS. The methane production rate reached up to 0.0564 m3 methane/(m3 reactor*d) in the hybrid reactor at room temperature, which was nearly double than that of the control anaerobic reactor (0.0259 m3 methane/(m3reactor*d)) without bioelectrochemical device. Microbial community analysis revealed that hydrogenotrophic methanogen Methanobacterium dominated the cathode biofilm, which was the predominant contributor to accelerate the...
100 citations
TL;DR: In this paper, different voltages (0.3-1.5 V) were applied to investigate the influence of electrical stimulation on the anaerobic digestion of waste activated sludge (WAS).
Abstract: Electrical stimulation has been used conventionally for stimulation of microorganisms, and also be a promising technology to manage wastewater treatment by stimulating microbial metabolism. Previous studies on electrical stimulation were mainly focused on sewage treatment and groundwater purification, while little attention has been paid to its effect on anaerobic digestion of waste activated sludge (WAS). In this study, different voltages (0.3–1.5 V) were applied to investigate the influence of electrical stimulation on the anaerobic digestion of WAS. The results revealed that applied voltages could accelerate sludge hydrolysis and acidification process. The best performance in terms of methane production and sludge reduction was obtained with the applied voltage of 0.6 V. In this case, methane production increased by 76.2% with an enhanced VS removal rate (26.6%) compared to the control group. The energy consumption at 0.6 V could be neglected compared to the incremental energy generated from the methane. However, methane production decreased and hydrogen was produced when the applied voltage increased to 0.9 V. At higher voltages (1.2 V and 1.5 V), more soluble organic matters were released. In particular, the VFAs concentration peaked at 640 mg L−1 and 1001 mg L−1, respectively. Pyrosequencing revealed that hydrogenotrophic methanogens consisted majority of methanogen population when the applied voltage was over 0.6 V, while acetoclastic methanogens showed overwhelming dominance at 0.3 V. Moreover, 0.6 V enriched Pseudomonas for protein degradation and Methanoregula for methane generation with species richnesses of 19.1% and 53.3%, respectively.
93 citations
TL;DR: A novel method of two-phase anaerobic digestion where the acid reactor is operated at low pH 4.0 was proposed and investigated to examine the possibility of efficient degradation of lactate and to identify their optimal operating conditions.
87 citations
TL;DR: In this article, the effects of combined calcium peroxide (CaO2) and microwave pretreatment on anaerobic digestion of waste activated sludge, lab-scale experiments were conducted to measure the solubilization, biodegradation, and dewaterability of the waste activation sludge.
86 citations
TL;DR: This study proves for the first time that biohythane could be produced directly in biocathode MECs using waste sludge and reveals syntrophic interactions in microbial communities based on high-throughput sequencing and quantitative PCR targeting 16S rRNA gene.
Abstract: Biohythane is a new and high-value transportation fuel present as a mixture of biomethane and biohydrogen. It has been produced from different organic matters using anaerobic digestion. Bioenergy can be recovered from waste activated sludge through methane production during anaerobic digestion, but energy yield is often insufficient to sludge disposal. Microbial electrolysis cell (MEC) is also a promising approach for bioenergy recovery and waste sludge disposal as higher energy efficiency and biogas production. The systematic understanding of microbial interactions and biohythane production in MEC is still limited. Here, we report biohythane production from waste sludge in biocathode microbial electrolysis cells and reveal syntrophic interactions in microbial communities based on high-throughput sequencing and quantitative PCR targeting 16S rRNA gene. The alkali-pretreated sludge fed MECs (AS-MEC) showed the highest biohythane production rate of 0.148 L·L−1-reactor·day−1, which is 40 and 80 % higher than raw sludge fed MECs (RS-MEC) and anaerobic digestion (open circuit MEC, RS-OCMEC). Current density, metabolite profiles, and hydrogen-methane ratio results all confirm that alkali-pretreatment and microbial electrolysis greatly enhanced sludge hydrolysis and biohythane production. Illumina Miseq sequencing of 16S rRNA gene amplicons indicates that anode biofilm was dominated by exoelectrogenic Geobacter, fermentative bacteria and hydrogen-producing bacteria in the AS-MEC. The cathode biofilm was dominated by fermentative Clostridium. The dominant archaeal populations on the cathodes of AS-MEC and RS-MEC were affiliated with hydrogenotrophic Methanobacterium (98 %, relative abundance) and Methanocorpusculum (77 %), respectively. Multiple pathways of gas production were observed in the same MEC reactor, including fermentative and electrolytic H2 production, as well as hydrogenotrophic methanogenesis and electromethanogenesis. Real-time quantitative PCR analyses showed that higher amount of methanogens were enriched in AS-MEC than that in RS-MEC and RS-OCMEC, suggesting that alkali-pretreated sludge and MEC facilitated hydrogenotrophic methanogen enrichment. This study proves for the first time that biohythane could be produced directly in biocathode MECs using waste sludge. MEC and alkali-pretreatment accelerated enrichment of hydrogenotrophic methanogen and hydrolysis of waste sludge. The results indicate syntrophic interactions among fermentative bacteria, exoelectrogenic bacteria and methanogenic archaea in MECs are critical for highly efficient conversion of complex organics into biohythane, demonstrating that MECs can be more competitive than conventional anaerobic digestion for biohythane production using carbohydrate-deficient substrates. Biohythane production from waste sludge by MEC provides a promising new way for practical application of microbial electrochemical technology.
84 citations
TL;DR: In this paper, the authors combined biogeochemical and phylogenetic analyses with incubation experiments to unravel the origin of methane in the hypersaline sediments of Orca Basin in the northern Gulf of Mexico.
TL;DR: Strong population dynamics and the expression of taxonomically diverse GH families are observed, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.
Abstract: Summary
We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.
TL;DR: Investigation of microbial communities in a continuous anaerobic phenol-degrading system using a next generation sequencing tool found a progressive adaptation of the granules to phenol.
TL;DR: A novel crystalline form of the synthetic phenazine neutral red is described that harvests electrons from reduced inorganic and organic microbial sources in anaerobic environments and makes them available to methanogenic Archaea.
Abstract: With one billion tons of methane produced annually by microorganisms, biogas production can be appreciated both for its role in global organic matter turnover and as an energy source for humankind. The importance of electron transfer from electrically conductive surfaces or from bacteria to methanogenic Archaea has been implicated in widespread commercial anaerobic digestion processes, though a mechanism for reception of electrons from conductive surfaces or pili by methanogens has never been demonstrated. Here we describe a novel crystalline form of the synthetic phenazine neutral red that harvests electrons from reduced inorganic and organic microbial sources in anaerobic environments and makes them available to methanogenic Archaea. The novel crystalline form is so effective at harvesting reducing equivalents because it displays a potential for reduction 444 mV higher than the soluble form (E′ = 70 mV). Neutral red molecules solubilised in the reduced state by protonation at the point of methanogen cell contact with the crystal surface deliver electrons to methanogens at a negative midpoint potential (E′ = −375 mV). We demonstrate that soluble neutral red delivers reducing equivalents directly to the membrane bound HdrED heterodisulfide reductase of Methanosarcina, replenishing the CoM-SH and CoB-SH pool for methanogenesis and generating proton motive force. An order of magnitude increase in methane production is recorded in pure acetate fed Methanosarcina and coal and food waste fed mixed cultures in the laboratory. The phenomenon is also demonstrated at field scale in a sub-bituminous coal seam 80 m below ground level.
TL;DR: In this article, the authors apply measurements of multiply-substituted isotopologues, or clumped isotopes, of methane as a new tool to identify the origins of ebullitive fluxes in Alaska, Sweden and the Arctic Ocean.
TL;DR: The proposed method for development and maintenance of microorganism enrichment inoculum demonstrates consistent BMP data which is a requirement for dependable prediction of biogas production at field scale operations.
TL;DR: The results demonstrated the potential and operating condition of two-phase anaerobic digestion in treatment of oil refinery waste activated sludge and played important roles in hydrolytic-acidification and oil-organics degradation.
Abstract: Oil refinery waste activated sludge produced from oil wastewater biological treatment is a major industrial sludge. Two-phase anaerobic digestion of oil refinery waste activated sludge was studied for the first time. Thermal pretreatment under 170 °C is effective on sludge solubilization. At the optimum hydrolytic-acidogenic condition which was pH of 6.5, temperature of 55 °C and HRT of 2 days, 2754 mg/L volatile fatty acids (VFAs) were produced and acetic acid and butyric acid were the key components. Comparative studies of single-phase and two-phase anaerobic digestion in terms of organic removal, biogas production and methane concentration were conducted. The cumulative methane production and soluble COD (SCOD) removal efficiency in the two-phase system were 228 mL/g COD added and 77.8%, respectively, which were 1.6 and 2.1 times higher than those in single-phase anaerobic digestion. Such improved performance is attributed to intensification of dominant microbial population in separated reactors. Caloramator, Ureibacillus, Dechloromonas, Petrobacter, and T78 played important roles in hydrolytic-acidification and oil-organics degradation. Syntrophic bacteria in the family Porphyromonadaceae and the genus Anaerobranca provide acetate for methanogen. The results demonstrated the potential and operating condition of two-phase anaerobic digestion in treatment of oil refinery waste activated sludge.
TL;DR: Hydrogenotrophic methanogens seem to be favored under nickel- and cobalt-deficient conditions as their metabolism requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways, and TE supply is critical to sustain the activity of the versatile high-performance methanogen Methanosarcina.
Abstract: Trace elements play an essential role in all organisms due to their functions in enzyme complexes. In anaerobic digesters, control and supplementation of trace elements lead to stable and more efficient methane production processes while trace element deficits cause process imbalances. However, the underlying metabolic mechanisms and the adaptation of the affected microbial communities to such deficits are not yet fully understood. Here, we investigated the microbial community dynamics and resulting process changes induced by trace element deprivation. Two identical lab-scale continuous stirred tank reactors fed with distiller’s grains and supplemented with trace elements (cobalt, molybdenum, nickel, tungsten) and a commercial iron additive were operated in parallel. After 72 weeks of identical operation, the feeding regime of one reactor was changed by omitting trace element supplements and reducing the amount of iron additive. Both reactors were operated for further 21 weeks. Various process parameters (biogas production and composition, total solids and volatile solids, trace element concentration, volatile fatty acids, total ammonium nitrogen, total organic acids/alkalinity ratio, and pH) and the composition and activity of the microbial communities were monitored over the total experimental time. While the methane yield remained stable, the concentrations of hydrogen sulfide, total ammonia nitrogen, and acetate increased in the trace element-depleted reactor compared to the well-supplied control reactor. Methanosarcina and Methanoculleus dominated the methanogenic communities in both reactors. However, the activity ratio of these two genera was shown to depend on trace element supplementation explainable by different trace element requirements of their energy conservation systems. Methanosarcina dominated the well-supplied anaerobic digester, pointing to acetoclastic methanogenesis as the dominant methanogenic pathway. Under trace element deprivation, Methanoculleus and thus hydrogenotrophic methanogenesis was favored although Methanosarcina was not overgrown by Methanoculleus. Multivariate statistics revealed that the decline of nickel, cobalt, molybdenum, tungsten, and manganese most strongly influenced the balance of mcrA transcripts from both genera. Hydrogenotrophic methanogens seem to be favored under nickel- and cobalt-deficient conditions as their metabolism requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways. Thus, trace element supply is critical to sustain the activity of the versatile high-performance methanogen Methanosarcina.
TL;DR: This review highlights and discusses the interplay between ammonia and methane oxidizing bacteria, the potential interactions of microbial communities with microbially-enriched organic amendments and the possible role of these biofertilizers in augmenting the methane sink potential of soils.
TL;DR: Results indicate that the liquid and solid-adherent environments are distinguished mainly by the differential abundance of specific taxonomic groups, while dominant species in the fluid samples are primarily identified as consumers of soluble nutrients.
TL;DR: It is proposed that strain TC1T represents a novel species of a new genus, for which the name Flexilinea flocculi gen. nov., sp.
Abstract: A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3–0.4 μm wide. Growth of the isolate was observed at 25–43 °C (optimum 37 °C) and pH 6.0–8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).
TL;DR: A genome scale metabolic network of Methanococcus maripaludis is constructed that explicitly accounts for all major reactions involved in hydrogenotrophic methanogenesis, demonstrating the importance of electron bifurcation in central metabolism.
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. IMPORTANCE 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.
TL;DR: In this article, the hydrogen isotope ratio of CH4 produced during rapid growth of the thermophilic methanogen Methanothermococcus okinawensis under high pH2 conditions was affected by the isotopic composition of H2, as concluded in a previous study of Methanothermobacter thermautotrophicus.
Abstract: Hydrogen and carbon isotope systematics of H2O–H2–CO2–CH4 in hydrogenotrophic methanogenesis and their relation to H2 availability were investigated. Two H2-syntrophic cocultures of fermentatively hydrogenogenic bacteria and hydrogenotrophic methanogens under conditions of <102 Pa-H2 and two pure cultures of hydrogenotrophic methanogens under conditions of ~105 Pa-H2 were tested. Carbon isotope fractionation between CH4 and CO2 during hydrogenotrophic methanogenesis was correlated with pH2, as indicated in previous studies. The hydrogen isotope ratio of CH4 produced during rapid growth of the thermophilic methanogen Methanothermococcus okinawensis under high pH2 conditions (~105 Pa) was affected by the isotopic composition of H2, as concluded in a previous study of Methanothermobacter thermautotrophicus. This “
$$ {\updelta \mathrm{D}}_{{\mathrm{H}}_2} $$
effect” is a possible cause of the diversity of previously reported values for hydrogen isotope fractionation between CH4 and H2O examined in H2-enriched culture experiments. Hydrogen isotope fractionation between CH4 and H2O, defined by (1000 +
$$ {\updelta \mathrm{D}}_{{\mathrm{CH}}_4} $$
)/(1000 +
$$ {\updelta \mathrm{D}}_{{\mathrm{H}}_2\mathrm{O}} $$
), during hydrogenotrophic methanogenesis of the H2-syntrophic cocultures was in the range 0.67–0.69. The hydrogen isotope fractionation of our H2-syntrophic dataset overlaps with those obtained not only from low-pH2 experiments reported so far but also from natural samples of “young” methane reservoirs (0.66–0.74). Conversely, such hydrogen isotope fractionation is not consistent with that of “aged” methane in geological samples (≥0.79), which has been regarded as methane produced via hydrogenotrophic methanogenesis from the carbon isotope fractionation. As a possible process inducing the inconsistency in hydrogen isotope signatures between experiments and geological samples, we hypothesize that the hydrogen isotope signature of CH4 imprinted at the time of methanogenesis, as in the experiments and natural young methane, may be altered by diagenetic hydrogen isotope exchange between extracellular CH4 and H2O through reversible reactions of the microbial methanogenic pathway in methanogenic region and/or geological methane reservoirs.
TL;DR: This work hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts, and identified a protein from Methanobrevibacter ruminantium M1 as anAdhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium).
Abstract: Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein.
TL;DR: While the abundance of hydrogenotrophic microorganisms remained stable during the recovery period, an acetoclastic methanogen phylogenetically similar to Methanosarcina sp.
Abstract: An imbalance between acidogenic and methanogenic organisms during anaerobic digestion can result in increased accumulation of volatile fatty acids, decreased reactor pH, and inhibition of methane-producing Archaea. Most commonly the result of organic input overload or poor inoculum selection, these microbiological and biochemical changes severely hamper reactor performance, and there are a few tools available to facilitate reactor recovery. A small, stable consortium capable of catabolizing acetate and producing methane was propagated in vitro and evaluated as a potential bioaugmentation tool for stimulating methanogenesis in acidified reactors. Replicate laboratory-scale batch digesters were seeded with a combination of bioethanol stillage waste and a dairy manure inoculum previously observed to result in high volatile fatty acid accumulation and reactor failure. Experimental reactors were then amended with the acetoclastic consortium, and control reactors were amended with sterile culture media. Within 7 days, bioaugmented reactors had significantly reduced acetate accumulation and the proportion of methane in the biogas increased from 0.2 ± 0 to 74.4 ± 9.9 % while control reactors showed no significant reduction in acetate accumulation or increase in methane production. Organisms from the consortium were enumerated using specific quantitative PCR assays to evaluate their growth in the experimental reactors. While the abundance of hydrogenotrophic microorganisms remained stable during the recovery period, an acetoclastic methanogen phylogenetically similar to Methanosarcina sp. increased more than 100-fold and is hypothesized to be the primary contributor to reactor recovery. Genomic sequencing of this organism revealed genes related to the production of methane from acetate, hydrogen, and methanol.
TL;DR: Results demonstrated that archaeal and bacterial community structure was affected by changes in the acid/alkalinity ratio in the bioprocess, which could be useful for future implementations of two-stage anaerobic digestion processes at both bench- and full-scale.
Abstract: Two-stage technologies have been developed for anaerobic digestion of waste-activated sludge. In this study, the archaeal and bacterial community structure dynamics and bioprocess performance of a bench-scale two-stage anaerobic digester treating urban sewage sludge have been studied by the means of high-throughput sequencing techniques and physicochemical parameters such as pH, dried sludge, volatile dried sludge, acid concentration, alkalinity, and biogas generation. The coupled analyses of archaeal and bacterial communities and physicochemical parameters showed a direct relationship between archaeal and bacterial populations and bioprocess performance during start-up and working operation of a two-stage anaerobic digester. Moreover, results demonstrated that archaeal and bacterial community structure was affected by changes in the acid/alkalinity ratio in the bioprocess. Thus, a predominance of the acetoclastic methanogen Methanosaeta was observed in the methanogenic bioreactor at high-value acid/alkaline ratio, while a predominance of Methanomassilicoccaeceae archaea and Methanoculleus genus was observed in the methanogenic bioreactor at low-value acid/alkaline ratio. Biodiversity tag-iTag sequencing studies showed that methanogenic archaea can be also detected in the acidogenic bioreactor, although its biological activity was decreased after 4 months of operation as supported by physicochemical analyses. Also, studies of the VFA producers and VFA consumers microbial populations showed as these microbiota were directly affected by the physicochemical parameters generated in the bioreactors. We suggest that the results obtained in our study could be useful for future implementations of two-stage anaerobic digestion processes at both bench- and full-scale.
TL;DR: The biodegrading capacity of the Neocallimastix with methanogen co-cultures and the N. frontalis Yaktz1 mono-culture both have great potentials for different industrial use.
14 Mar 2016
TL;DR: Overall, methanogenic activity was lower when mesocosms were exposed to higher concentrations of both inhibitors, and aceticlastic methanogens were inhibited to a greater extent than hydrogenotrophic meethanogens.
Abstract: Methanogenic inhibitors are often used to study methanogenesis in complex microbial communities or inhibit methanogens in the gastrointestinal tract of livestock. However, the resulting structural and functional changes in archaeal and bacterial communities are poorly understood. We characterized microbial community structure and activity in mesocosms seeded with cow dung and municipal wastewater treatment plant anaerobic digester sludge after exposure to two methanogenic inhibitors, 2-bromoethanesulfonate (BES) and propynoic acid (PA). Methane production was reduced by 89% (0.5 mmol/L BES), 100% (10 mmol/LBES), 24% (0.1 mmol/LPA), and 95% (10 mmol/LPA). Using modified primers targeting the methyl-coenzyme M reductase (mcrA) gene, changes in mcrA gene expression were found to correspond with changes in methane production and the relative activity of methanogens. Methanogenic activity was determined by the relative abundance of methanogen 16S rRNA cDNA as a percentage of the total community 16S rRNA cDNA. Overall, methanogenic activity was lower when mesocosms were exposed to higher concentrations of both inhibitors, and aceticlastic methanogens were inhibited to a greater extent than hydrogenotrophic methanogens. Syntrophic bacterial activity, measured by 16S rRNA cDNA, was also reduced following exposure to both inhibitors, but the overall structure of the active bacterial community was not significantly affected.
TL;DR: The quantitative real time PCR and the optimized DNA extraction were implemented and validated to characterize and quantify the methanogen dynamic in dry batch AD process and methanogens populations were stabilized in liquid phase, except Methanosaetaceae.
TL;DR: Ordination analyses suggested that trimethylamine was the primary factor regulating shift in methanogen community structure and increased CH4 production potential in S. alterniflora marsh, which together facilitated increased CH 4 production potential.
Abstract: Invasion of Spartina alterniflora in coastal areas of China increased methane (CH4) emissions. To elucidate the underlying mechanisms, we measured CH4 production potential, methanogen community structure and biogeochemical factors along a coastal wetland transect comprised of five habitat regions: open water, bare tidal flat, invasive S. alterniflora marsh and native Suaeda salsa and Phragmites australis marshes. CH4 production potential in S. alterniflora marsh was 10 times higher than that in other regions, and it was significantly correlated with soil organic carbon, dissolved organic carbon and trimethylamine concentrations, but was not correlated with acetate or formate concentrations. Although the diversity of methanogens was lowest in S. alterniflora marsh, invasion increased methanogen abundance by 3.48-fold, compared with native S. salsa and P. australis marshes due to increase of facultative Methanosarcinaceae rather than acetotrophic and hydrogenotrophic methanogens. Ordination analyses suggested that trimethylamine was the primary factor regulating shift in methanogen community structure. Addition of trimethylamine increased CH4 production rates by 1255-fold but only by 5.61- and 11.4-fold for acetate and H2/CO2, respectively. S. alterniflora invasion elevated concentration of non-competitive trimethylamine, and shifted methanogen community from acetotrophic to facultative methanogens, which together facilitated increased CH4 production potential.