Formylmethanofuran Dehydrogenase Research Articles

Isolation of an H2 -dependent electron-bifurcating CO2 -reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis.

In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2 . Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420 , respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13 C]-CO2 to form [13 C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2 , which likely involves MvhB polyferredoxin as an electron relay.

Comparing the mechanisms of syntrophic volatile fatty acids oxidation and methanogenesis recovery from ammonia stress in regular and biochar-assisted anaerobic digestion: Different roads lead to the same goal

Biochar has been recognized as a promising additive to mitigate ammonia inhibition during syntrophic methanogenesis, while the key function of biochar in this process is still in debates. This study clarified the distinct mechanisms of syntrophic volatile fatty acids -oxidizing and methanogenesis recovery from ammonia inhibition in regular and biochar-assisted anaerobic digestion. Under 5 g/L ammonia stress, adding biochar shortened the methanogenic lag time by 10.9% and dramatically accelerated the maximum methane production rate from 60.3 to 94.7 mLCH4/gVSsludge/d. A photometric analysis with a nano-WO3 probe revealed that biochar enhanced the extracellular electron transfer (EET) capacity of suspended microbes (Pearson's r = −0.98), confirming that biochar facilitated methanogenesis by boosting EET between syntrophic butyrate oxidizer and methanogens. Same linear relationship between EET capacity and methanogenic rate was not observed in the control group. Microbial community integrating functional genes prediction analysis uncovered that biochar re-shaped syntrophic partners by enriching Constridium_sensu_stricto/Syntrophomonas and Methanosarcina. The functional genes encoding Co-enzyme F420 hydrogenase and formylmethanofuran dehydrogenase were upregulated by 1.4–2.3 times, consequently enhanced the CO2-reduction methanogenesis pathway. Meanwhile, the abundances of gene encoding methylene-tetrahydrofolate transformation, a series of intermediate processes involved in acetate oxidation, in the biochar-assisted group were 28.2–63.7% higher than these in control group. Comparatively, Methanosaeta played a pivotal role driving aceticlastic methanogenesis in the control group because the abundance of gene encoding acetyl-CoA decarbonylase/synthase complex increased by 1.9 times, suggesting an aceticlastic combining H2-based syntrophic methanogenesis pathway was established in control group to resist ammonia stress. A 2nd period experiment elucidated that although depending on distinct mechanisms, the volatile fatty acid oxidizers and methanogens in both groups developed sustained and stable strategies to resist ammonia stress. These findings provided new insights to understand the distinct methanogenic recovery strategy to resist toxic stress under varied environmental conditions.

Anaerobic digestion of fat, oil, and grease (FOG) under combined additives: Enhanced digestibility, biogas production, and microbiome

Addition of a single additive (such as biochar or calcium) in anaerobic digestion has been studied previously. However, the effect of combined additives in anaerobic digestion (including fat, oil, and grease) has not been well-investigated yet. In this study, the biogas/biomethane, lipid degradation, and volatile fatty acids were investigated under various concentrations of combined additives. The effect on principal reactions, microbial community, and functional enzymes was also analyzed under different concentrations of calcium-modified biochar. The fat, oil, and grease was degraded up to 81.91 % resulting in 9.03-fold higher biomethane production. Acidogenesis and methanogenesis efficiencies were induced up to 53.31 % and 67.91 %, respectively, due to the enrichment of bacteria (such as Clostridium >50 %, Sporosarcina >18 %, Cellulosilyticum >8 %) and archaea (such as Methanosphaera >49 %). Increased abundance of Co-A ligase, short-chain acyl-CoA dehydrogenase, acetate kinase, and formylmethanofuran dehydrogenase, represented mixed hydrogenotrophic and acetoclastic methanogenesis. The current study demonstrated that co-additives can be added to overcome microbial inhibition and facilitate biomethanation at high fat, oil, and grease concentrations.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Bioelectrocatalytic CO2 Reduction by Mo‐Dependent Formylmethanofuran Dehydrogenase

AbstractMassive efforts are invested in developing innovative CO2‐sequestration strategies to counter climate change and transform CO2 into higher‐value products. CO2‐capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2, it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2‐reducing‐and‐fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2‐fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Biochar facilitates methanogens evolution by enhancing extracellular electron transfer to boost anaerobic digestion of swine manure under ammonia stress

This study explored the mechanisms by which biochar mitigates ammonia inhibition in anaerobic digestion (AD) of swine manure. Findings show 2–8 g/L exogenous ammonia dosages gradually inhibited AD, leading to decreases in the efficiencies of hydrolysis, acidogenesis and methanogenesis by 3.4–70.8%, 6.0–82.0%, and 4.9–93.8%, respectively. However, biochar addition mitigated this inhibition and facilitated methane production. Biochar enhanced microbial activities related to electron transport and extracellular electron transfer. Moreover, biochar primarily enriched Methanosarcina, which, consequently, upregulated the genes encoding formylmethanofuran dehydrogenase and methenyltetrahydromethanopterin cyclohydrolase for the CO2-reducing methanogenesis pathway by 26.9–40.8%. It is believed that biochar mediated direct interspecies electron transfer between syntrophic partners, thereby enhancing methane production under ammonia stress. Interestingly, biochar removal did not significantly impact the AD performance of the acclimated microbial community. This indicated the pivotal role of biochar in triggering methanogen evolution to mitigate ammonia stress rather than the indispensable function after the enrichment of ammonia-resistance methanogen.

Analysis on methane production from various coal slime fermentations based on metagenomics

Metagenomic sequencing technology was applied to evaluate differences in the anaerobic fermentation process of coal slimes by analyzing microbial diversity, functional activity structure, and cooperative relationship during the anaerobic fermentation of coal slimes with different coal ranks. The obtained results showed that the production of biomethane from coal slime was decreased by increasing metamorphism degree. Internal reason was higher abundance of microbial community in low rank coal slimes compared to that in high rank coal which had higher activity in the gene expression of key steps such as hydrolysis and acidification, methanation and the production of hydrogen and acetic acid. Acetic acid decarboxylation and CO2 reduction are two key pathways of methanation process. At the same time, K11261 (formylmethanofuran dehydrogenase subunit) and K01499 (methenyltetrahydromethanopterin cyclohydrolase) genes were further enriched in low rank slime systems, which enhanced the proportion of CO2 reduction in methanation pathway and was beneficial to biomethane production. Research revealed the roles of different coal slime ranks in biomethane production process and is considered as an important reference significance for further exploration of coal slime resource utilization.

Effect of extreme pH conditions on methanogenesis: Methanogen metabolism and community structure

The control of pH is effective for inhibiting methanogenesis in the chain elongation fermentation (CEF) system. However, obscure conclusions exist especially with regard to the underlying mechanism. This study comprehensively explored the responses of methanogenesis in granular sludge at various pH levels, ranging from 4.0 to 10.0, from multiple aspects including methane production, methanogenesis pathway, microbial community structure, energy metabolism and electron transport. Results demonstrated that compared with that at pH 7.0, pH at 4.0, 5.5, 8.5 and 10.0 triggered a 100%, 71.7%, 23.8% and 92.1% suppression on methanogenesis by the end of 3 cycles lasting 21 days. This might be explained by the remarkably inhibited metabolic pathways and intracellular regulations. To be more specific, extreme pH conditions decreased the abundance of the acetoclastic methanogens. However, obligate hydrogenotrophic and facultative acetolactic/hydrogenotrophic methanogens were significantly enriched by 16.9%-19.5 fold. pH stress reduced the gene abundance and/or activity of most enzymes involved in methanogenesis such as acetate kinase (by 81.1%–93.1%), formylmethanofuran dehydrogenase (by 10.9%–54.0%) and tetrahydromethanopterin S-methyltransferase (by 9.3%–41.5%). Additionally, pH stress suppressed electron transport via improper electron carriers and decreased electron amount as evidenced by 46.3%–70.4% reduced coenzyme F420 content and diminished abundance of CO dehydrogenase (by 15.5%–70.5%) and NADH:ubiquinone reductase (by 20.2%–94.5%). pH stress also regulated energy metabolism with inhibited ATP synthesis (e.g., ATP citrate synthase level reduced by 20.1%–95.3%). Interestingly, the protein and carbohydrate content secreted in EPS failed to show consistent responses to acidic and alkaline conditions. Specifically, when compared with pH 7.0, the acidic condition remarkably reduced the levels of total EPS and EPS protein while both levels were enhanced in the alkaline condition. However, the EPS carbohydrate content at pH 4.0 and 10.0 both decreased. This study is expected to promote the understanding of the pH control-induced methanogenesis inhibition in the CEF system.

Enrichment culture combined with microbial electrochemical enhanced low-temperature anaerobic digestion of cow dung

Enrichment culture combined with the microbial electrochemical system was used to co-enhance the low-temperature (20 °C) anaerobic digestion. The results showed that enrichment culture combined with microbial electrochemical system increased the cumulative methane production in low-temperature anaerobic digestion system by 39.64 % and 133.29 % compared to single and no enrichment culture, respectively. Enrichment culture combined with microbial electrochemical system increased the relative abundance of methanogenic archaea (Methanomassiliicoccus, Methanocorpusculum, unclassified Methanomicrobiaceae, Methanobacterium, Methanoculleus, Methanocalculus) and the relative abundance of cold-tolerant hydrolytic acidifying bacteria (unclassified Bacteroidetes, Treponema). The expressions of specific enzyme genes in the methanogenesis pathway were enhanced, including acetyl-CoA synthetase, formylmethanofuran dehydrogenase, methanol cobalamin methyltransferase, etc. These results indicated that enrichment culture combined with microbial electrochemical system enhanced low-temperature anaerobic digestion methanogenesis by altering microbial communities and stimulating enzyme gene expression to affect volatile fatty acids, pH, redox potential, and reducing sugar parameters.

Insights into the impact of polyethylene microplastics on methane recovery from wastewater via bioelectrochemical anaerobic digestion

Bioelectrochemical anaerobic digestion (BEAD) is a promising next-generation technology for simultaneous wastewater treatment and bioenergy recovery. While knowledge on the inhibitory effect of emerging pollutants, such as microplastics, on the conventional wastewater anaerobic digestion processes is increasing, the impact of microplastics on the BEAD process remains unknown. This study shows that methane production decreased by 30.71% when adding 10 mg/L polyethylene microplastics (PE-MP) to the BEAD systems. The morphology of anaerobic granular sludge, which was the biocatalysts in the BEAD, changed with microbes shedding and granule crack when PE-MP existed. Additionally, the presence of PE-MP shifted the microbial communities, leading to a lower diversity but higher richness and tight clustering. Moreover, fewer fermentative bacteria, acetogens, and hydrogenotrophic methanogens (BEAD enhanced) grew under PE-MP stress, suggesting that PE-MP had an inhibitory effect on the methanogenic pathways. Furthermore, the abundance of genes relevant to extracellular electron transfer (omcB and mtrC) and methanogens (hupL and mcrA) decreased. The electron transfer efficiency reduced with extracellular cytochrome c down and a lower electron transfer system activity. Finally, phylogenetic investigation of communities by reconstruction of unobserved states analysis predicted the decrease of key methanogenic enzymes, including EC 1.1.1.1 (Alcohol dehydrogenase), EC 1.2.99.5 (Formylmethanofuran dehydrogenase), and EC 2.8.4.1 (Coenzyme-B sulfoethylthiotransferase). Altogether, these results provide insight into the inhibition mechanism of microplastics in wastewater methane recovery and further optimisation of the BEAD process.

Effect of bioaugmentation combined with activated charcoal on the mitigation of volatile fatty acids inhibition during anaerobic digestion

Volatile fatty acids (VFAs) accumulation is a common problem in anaerobic digestion, which is constraining the process. In this study, bioaugmentation was initially adopted as a measure to tackle the acidification problem in a continuous anaerobic digester. However, after bioaugmentation stopped, the promotive effect of bioaugmentation was hard to sustain. In comparison, the combination of bioaugmentation and Activated charcoal (AC) enabled a better reactor performance. The positive impact of AC on recovery from acidification in sequential batch reactors was thus investigated by feeding either propionate or acetate. The results implied that the addition of AC significantly increased the degradation rate of propionate, thus accelerating the biogas production rate (104 ~ 371%). Moreover, the role of AC is mainly in the immobilization of microorganisms to form bio-AC owing to AC’s physical characteristics (high-surface-area and large number of pores enables adsorption of microorganisms). The biofilms on the AC decreased the distance between the microbes and delayed the washout effect of microorganisms, enabling a longer-lasting effect of bioaugmentation. The 16S-rRNA analysis showed that the addition of AC led to the enrichment of syntrophic microbial guilds (syntrophic VFAs oxidizing bacteria and hydrogenotrophic methanogens). The potential function analysis (PICRUSt2) indicated the significant enhancement of formylmethanofuran dehydrogenase (EC 1.2.7.12) and coenzyme F420 hydrogenase (EC 1.12.98.1) by AC. The results suggested that the addition of AC promoted interspecies electron transfer.

Unveiling the Biodiversity of Hyperthermophilic Archaea in Jharia Coal Mines: Potential Threat to Methanogenesis?

Aim To examine the biodiversity of archaeal sulfate reducers and methanogens present in the underground coal mines of Jharia using metagenomics and pyrosequencing.Objectives 1) Bioinformatical analysis of the metagenomic data related to a taxonomic analysis obtained from the coal to investigate complete archaeal taxonomic features of the coal bed methane (CBM) microbiome. 2) Bioinformatical analysis of the metagenomic data related to a functional analysis obtained from the coal to investigate functional features relating to taxonomic diversity of the CBM microbiome. 3) The functional attributes have been examined specifically for ORFs related to sulfite reduction and methanogenesis.The taxonomic and functional biodiversity related to euryarchaeota will help in a better understanding of the obstacles associated with methane production imposed by the sulfate reducers.Background The microbial methanogenesis in the coal microbiome is a resultant of substrate utilization by primarily fermentative bacteria and methanogens. The present work reveals the biodiversity of archaeal sulfate reducers and methanogens present in the underground coal mines of Jharia using metagenomics and pyrosequencing.Methodology Bioinformatical analysis for structural and functional attributes was accomplished using MG-RAST. The structural analysis was accomplished using RefSeq database, whereas the functional analysis was done via CoG database with a cut off value, a sequence percent identity, and sequence alignment length cut off of 1e−5, 60% and 45, respectively.Results Attained communities revealed the dominance of hyperthermophilic archaea Pyrococcus furiosus along with Thermococcus kodakarensis in the coal metagenome.The obtained results also suggest the presence of dissimilatory sulfite reductase and formylmethanofuran dehydrogenase, formylmethanofuran: tetrahydromethanopterin formyltransferase involved in sulfite reduction and methanogenesis, respectively, in the microbiome.Conclusion This report is the first attempt to showcase the existence of specific euryarchaeal diversity and their related functional attributes from Jharia coal mines through high throughput sequencing. The study helps in developing a better understanding of the presence of indigenous microbes (archaea) and their functions in the coal microbiome, which can be utilized further to resolve the energy crisis.

Tungstoenzymes: Occurrence, Catalytic Diversity and Cofactor Synthesis

Tungsten is the heaviest element used in biological systems. It occurs in the active sites of several bacterial or archaeal enzymes and is ligated to an organic cofactor (metallopterin or metal binding pterin; MPT) which is referred to as tungsten cofactor (Wco). Wco-containing enzymes are found in the dimethyl sulfoxide reductase (DMSOR) and the aldehyde:ferredoxin oxidoreductase (AOR) families of MPT-containing enzymes. Some depend on Wco, such as aldehyde oxidoreductases (AORs), class II benzoyl-CoA reductases (BCRs) and acetylene hydratases (AHs), whereas others may incorporate either Wco or molybdenum cofactor (Moco), such as formate dehydrogenases, formylmethanofuran dehydrogenases or nitrate reductases. The obligately tungsten-dependent enzymes catalyze rather unusual reactions such as ones with extremely low-potential electron transfers (AOR, BCR) or an unusual hydration reaction (AH). In recent years, insights into the structure and function of many tungstoenzymes have been obtained. Though specific and unspecific ABC transporter uptake systems have been described for tungstate and molybdate, only little is known about further discriminative steps in Moco and Wco biosynthesis. In bacteria producing Moco- and Wco-containing enzymes simultaneously, paralogous isoforms of the metal insertase MoeA may be specifically involved in the molybdenum- and tungsten-insertion into MPT, and in targeting Moco or Wco to their respective apo-enzymes. Wco-containing enzymes are of emerging biotechnological interest for a number of applications such as the biocatalytic reduction of CO2, carboxylic acids and aromatic compounds, or the conversion of acetylene to acetaldehyde.

Methane, arsenic, selenium and the origins of the DMSO reductase family

Mononuclear molybdoenzymes of the dimethyl sulfoxide reductase (DMSOR) family catalyze a number of reactions essential to the carbon, nitrogen, sulfur, arsenic, and selenium biogeochemical cycles. These enzymes are also ancient, with many lineages likely predating the divergence of the last universal common ancestor into the Bacteria and Archaea domains. We have constructed rooted phylogenies for over 1,550 representatives of the DMSOR family using maximum likelihood methods to investigate the evolution of the arsenic biogeochemical cycle. The phylogenetic analysis provides compelling evidence that formylmethanofuran dehydrogenase B subunits, which catalyze the reduction of CO2 to formate during hydrogenotrophic methanogenesis, constitutes the most ancient lineage. Our analysis also provides robust support for selenocysteine as the ancestral ligand for the Mo/W atom. Finally, we demonstrate that anaerobic arsenite oxidase and respiratory arsenate reductase catalytic subunits represent a more ancient lineage of DMSORs compared to aerobic arsenite oxidase catalytic subunits, which evolved from the assimilatory nitrate reductase lineage. This provides substantial support for an active arsenic biogeochemical cycle on the anoxic Archean Earth. Our work emphasizes that the use of chalcophilic elements as substrates as well as the Mo/W ligand in DMSORs has indelibly shaped the diversification of these enzymes through deep time.

Long-term evaluation of bioaugmentation to alleviate ammonia inhibition during anaerobic digestion: Process monitoring, microbial community response, and methanogenic pathway modeling

The effect of different bioaugmentation strategies on anaerobic digestion related to the alleviation of ammonia inhibition was investigated in a long-term operation. The long-term operation confirmed that bioaugmentation is a stable method. A 35% increase in methane production (MP) was observed in bottles bioaugmented with Methanosarcina barkeri (MSB) or Syntrophaceticu schinkii (SS) + Methanobrevibacter smithii (MBS), and a 49% increment was obtained from the bottles bioaugmented with Methanosaeta harundinacea (MSH) + SS + MBS. Results suggest that the enhancement in both aceticlastic and hydrogenotrophic methanogenic pathways should be considered, and bioaugmentation strain should be properly selected to achieve a synergistic effect. The microbial community analysis indicated Methanosarcina spp. was the dominant archaea. Combined with specific methanogenic activity and carbon isotope fractionation analysis, it was suggested that Methanosarcina spp. performed differently in methanogenic pathways in different bottles. The abundance of COG and total enzymes in the bottles with high MP (MSB and MSH + SS + MBS) was higher than that in the other bottles. The ratio of the functional enzyme tetrahydromethanopterin S-methyltransferase subunit H (EC 2.1.1.86) to formylmethanofuran dehydrogenase subunit E (EC 1.2.99.5) and the relative abundance of enolase (EC 1.2.1.2) confirmed the aceticlastic methanogenic pathway of MSB in Group 1 and the pathway enhancement balance in MSH + SS + MBS. The modified Anaerobic Digestion Model No.1 including syntrophic acetate oxidation was used for simulation with R2 > 0.96. Simulated contribution rate data indicated that the hydrogenotrophic methanogenic pathway was dominant while both two pathways got strengthened, which is consistent with the findings of the microbial analysis.

Molybdenum- and tungsten-containing formate dehydrogenases and formylmethanofuran dehydrogenases: Structure, mechanism, and cofactor insertion.

An overview is provided of the molybdenum- and tungsten-containing enzymes that catalyze the interconversion of formate and CO2 , focusing on common structural and mechanistic themes, as well as a consideration of the manner in which the mature Mo- or W-containing cofactor is inserted into apoprotein.

How does binuclear zinc amidohydrolase FwdA work in the initial step of methanogenesis: From formate to formyl-methanofuran

The initial step of methanogenesis is the fixation of CO2 to formyl-methanofuran (formyl-MFR) catalyzed by formyl-MFR dehydrogenase, which can be divided into two half reactions. Herein, the second half reaction catalyzed by FwdA (formyl-methanofuran dehydrogenase subunit A), i.e., from formate to formyl-methanofuran, has been investigated using density functional theory and a chemical model based on the X-ray crystal structure. The calculations indicate that, compared with other well-known di-zinc hydrolases, the FwdA reaction employs a reverse mechanism, including the nucleophilic attack of MFR amine on formate carbon leading to a tetrahedral gem-diolate intermediate, two steps of proton transfer from amine to formate moieties assisted by the Asp385, and the CO bond dissociation to form the formyl-MFR product. The second step of proton transfer from the amine moiety to the Asp385 is rate-limiting with an overall barrier of 21.2 kcal/mol. The two zinc ions play an important role in stabilizing the transition states and intermediates, in particular the negative charge at the formate moiety originated from the nucleophilic attack of the MFR amine. The work here appends a crucial piece in the methanogenic mechanistics and advances the understanding of the global carbon cycle.

Metabolic Adaptation of Methanogens in Anaerobic Digesters Upon Trace Element Limitation.

Anaerobic digestion (AD) is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The lower diversity of methanogenic archaea compared to the bacterial groups involved in AD and the corresponding lack of functional redundancy cause a stronger susceptibility of methanogenesis to unfavorable process conditions such as trace element (TE) deprivation, thus controlling the stability of the overall process. Here, we investigated the effects of a slowly increasing TE deficit on the methanogenic community function in a semi-continuous biogas process. The aim of the study was to understand how methanogens in digester communities cope with TE limitation and sustain their growth and metabolic activity. Two lab-scale biogas reactors fed with distillers grains and supplemented with TEs were operated in parallel for 76 weeks before one of the reactors was subjected to TE deprivation, leading to a decline of cobalt and molybdenum concentrations from 0.9 to 0.2 mg/L, nickel concentrations from 2.9 to 0.8 mg/L, manganese concentrations from 38 to 18 mg/L, and tungsten concentrations from 1.4 to 0.2 mg/L. Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing TE limitation, the relative abundance of Methanosarcina dropped to 67% and a slight decrease of acetoclastic methanogenic activity was observed in batch tests with 13C-methyl-labeled acetate, suggesting a shift toward syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways. Proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase from Methanosarcinaceae increased, confirming our hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. Both Methanosarcina and Methanoculleus increased the abundance of N5-methyltetrahydromethanopterin-coenzyme M methyltransferase and methyl-coenzyme M reductase. However, these efforts to preserve the ion motive force for energy conservation were seemingly more successful in Methanoculleus. We conclude that both methanogenic genera use different strategies to stabilize their energy balance under TE limitation. Methanosarcina switched from TE expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis. Methanoculleus showed a higher robustness and was favored over the more fastidious Methanosarcina, thus stabilizing reactor performance under TE limitation.

Theoretical exploration of the mechanism of formylmethanofuran dehydrogenase: the first reductive step in CO2 fixation by methanogens.

A theoretical exploration of the possible active site models of methanofuran dehydrogenase reveals that the free energy of the reduction of the carbamate group is substantially negative and is driven by the electron withdrawing amide group next to the carbonyl carbon. Comparison of the computed transition state energies with the experimental energy barrier indicates that the active site is likely to have an axial oxo and equatorial hydrosulfide ligand, the substrate is likely to be protonated and a second-sphere hydrogen-bonding interaction with the axial ligand can, substantially, lower the barrier of this reaction which involves reduction of the carbonyl center of the a carbamate to form an N-formyl group via a hydride shift from a Mo(IV) center.

Metagenome changes in the biogas producing community during anaerobic digestion of rice straw

The present investigation was undertaken to study the microbial community succession in a sour and healthy digester. Ion torrent next-generation sequencing (NGS)-based metagenomic approach indicated abundance of hydrolytic bacteria and exclusion of methanogens and syntrophic bacteria in sour digester. Functional gene analysis revealed higher abundance of enzymes involved in acidogenesis and lower abundance of enzymes associated with methanogenesis like Methyl coenzyme M-reductase, F420 dependent reductase and Formylmethanofuran dehydrogenase in sour digester. Increased abundance of methanogens (Methanomicrobia) and genes involved in methanogenesis was observed in the restored/healthy digester highlighting revival of pH sensitive methanogenic community.