Methylotrophic Methanogens Research Articles

Polyphenol rewiring of the microbiome reduces methane emissions.

Methane mitigation is regarded as a critical strategy to combat the scale of global warming. Currently, about 40% of methane emissions originate from microbial sources, which is causing strategies to suppress methanogens, either through direct toxic effects or by diverting their substrates and energy, to gain traction. Problematically, current microbial methane mitigation knowledge derives from rumen studies and lacks detailed microbiome-centered insights, limiting translation across ecosystems. Here we utilize genome-resolved metatranscriptomes and metabolomes to assess the impact of a proposed methane inhibitor, catechin, on greenhouse gas emissions for high-methane-emitting peatlands. In microcosms, catechin drastically reduced methane emissions by 72-84% compared to controls. Longitudinal sampling allowed for reconstruction of a novel catechin degradation pathway involving Actinomycetota and Clostridium, which break down catechin into smaller phenolic compounds within the first 21 days, followed by degradation of phenolic compounds by Pseudomonas_E from days 21 to 35. These genomes also co-expressed hydrogen-uptake genes, suggesting that hydrogenases may act as a hydrogen sink during catechin degradation, depriving methanogens of substrates. This was supported by decreased gene expression in hydrogenotrophic and hydrogen-dependent methylotrophic methanogens under catechin treatment. We also saw reduced gene expression from genomes inferred to be functioning syntrophically with hydrogen-utilizing methanogens. We propose that catechin metabolic redirection effectively starves hydrogen-utilizing methanogens, offering a potent avenue for curbing methane emissions across diverse environments including ruminants, landfills, and constructed or managed wetlands.

Hydroxylated GDGTs-0 in marine methane seep environments: A putative indicator for archaeal methanogenesis

Marine methane seeps are environments with a high microbial diversity, but are known for one biogeochemical key process, the sulfate-driven anaerobic oxidation of methane (SD-AOM) performed by anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). SD-AOM is also the dominant process at methane seeps in the South China Sea based on the lipid biomarker inventory of authigenic seep carbonates characterized by crocetane, a high sn2-hydroxyarchaeol over archaeol ratio, low contents of glycerol dibiphytanyl glycerol tetraethers (GDGTs), and δ13Clipid-methane offsets of −52 ± 4 ‰. Combined with the dominance of aragonite over other carbonate minerals, such pattern suggests high seepage intensity with a predominance of ANME-2/SRB consortia. Interestingly, the studied seep carbonates also yielded some uncommon biomarkers, which may derive from methanogenic archaea. Methylotrophic methanogenesis has been shown to be the dominant mode of methanogenesis in seep environments where non-competitive substrates like methanol or trimethylamine are abundant. The presence of methylotrophic methanogens is possibly indicated by high contents (more than 50 % of all GDGTs) of hydroxylated GDGTs-0 (OH-GDGT-0 and 2OH-GDGT-0) with extreme 13C-depletion (−128 ‰ to −116 ‰); this unique pattern is recognized in only some of the studied seep carbonates, while other carbonates are dominated by typical distributions of ANME-2 lipids, also comprising GDGTs with 0 to 4 rings, but lacking high contents of OH-GDGTs. The overall lack of crenarchaeol, the specific biomarker of planktonic Thaumarchaeota, agrees with the tentative assignment of the highly abundant OH-GDGTs-0 to methanogenic archaea. Such interpretation is necessarily circumstantial considering that the compound and carbon isotope composition of the membranes of ANMEs and methanogenic archaea is similar. Although production of OH-GDGTs has been previously reported for planktonic Thaumarchaeota and ANMEs, OH-GDGT-0 as sole and highly abundant OH-GDGT has only been recognized in one culture of methanogenic archaea. Therefore, the high abundance of 13C-depleted hydroxylated GDGTs-0 compared to only minor contents of regular, ANME-derived GDGTs 1–4 with similar 13C-depletions can possibly be used as an indicator for methylotrophic methanogenesis in seep environments. Future experimental work is needed and should test if 13C-depleted hydroxylated GDGTs-0 are indeed biomarkers of methylotrophic methanogenesis at seeps.

Isolation of a methyl-reducing methanogen outside the Euryarchaeota.

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades1-4, thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle.

Methanomethylovorans are the dominant dimethylsulfide-degrading methanogens in gravel and sandy river sediment microcosms

BackgroundRivers and streams are important components of the global carbon cycle and methane budget. However, our understanding of the microbial diversity and the metabolic pathways underpinning methylotrophic methane production in river sediments is limited. Dimethylsulfide is an important methylated compound, found in freshwater sediments. Yet, the magnitude of DMS-dependent methanogenesis nor the methanogens carrying out this process in river sediments have been explored before. This study addressed this knowledge gap in DMS-dependent methanogenesis in gravel and sandy river sediments.ResultsSignificant methane production via DMS degradation was found in all sediment microcosms. Sandy, less permeable river sediments had higher methane yields (83 and 92%) than gravel, permeable sediments (40 and 48%). There was no significant difference between the methanogen diversity in DMS-amended gravel and sandy sediment microcosms, which Methanomethylovorans dominated. Metagenomics data analysis also showed the dominance of Methanomethylovorans and Methanosarcina. DMS-specific methyltransferase genes (mts) were found in very low relative abundances whilst the methanol-, trimethylamine- and dimethylamine-specific methyltransferase genes (mtaA, mttB and mtbB) had the highest relative abundances, suggesting their involvement in DMS-dependent methanogenesis.ConclusionsThis is the first study demonstrating a significant potential for DMS-dependent methanogenesis in river sediments with contrasting geologies. Methanomethylovorans were the dominant methylotrophic methanogen in all river sediment microcosms. Methyltransferases specific to methylotrophic substrates other than DMS are likely key enzymes in DMS-dependent methanogenesis, highlighting their versatility and importance in the methane cycle in freshwater sediments, which would warrant further study.

Organic matter accumulation drives methylotrophic methanogenesis and microbial ecology in a hypersaline coastal lagoon

AbstractHypersalinity is common in coastal wetlands throughout warm, tropical, and arid regions. Climate‐induced changes in rainfall, sea level, and anthropogenic modification to basins and coastlines are likely to further increase salinization in these ecosystems. Yet, carbon cycling in hypersaline coastal wetlands is not well understood, and poorly constrained in climate models. In the Coorong, a eutrophic, hypersaline coastal lagoon, recognized as internationally important under the Ramsar convention, organic matter rapidly accumulates in deeper areas of the lagoon, through the settling of fine detrital particles, phytoplankton and suspended sediments. During initial surveys, elevated surface water methane (CH4) concentrations were observed above these fine depositional sediments. To identify the drivers of CH4 production, organic matter and sediment characteristics were assessed in surface sediments. Genetic markers (i.e., 16rDNA and the mcrA functional gene) were used to characterize microbial communities. With multiple lines of evidence, this study identifies organic matter, methanogen abundance, and salinity as important drivers of CH4 production, which is concentrated in depositional zones. Archaea were also more abundant in depositional zones, including methylotrophic methanogens: Methanofastidiosales, Methanomasiliicoccales, Methermicoccaceae, and Methanococcoides. These methanogens were highly correlated to CH4 in porewater, suggesting an influence of methylotrophic methanogenesis. To investigate further, metabolic genes were predicted from 16S rRNA with PICRUSt2. This represents the first effort to analyze CH4 dynamics in the Coorong, underscoring the need to integrate these unique ecosystems into global climate models to enhance our understanding of greenhouse gas dynamics and emissions in a changing climate.

Potential coupling of microbial methane, nitrogen, and sulphur cycling in the Okinawa Trough cold seep sediments.

The Okinawa Trough (OT) is a back-arc basin with a wide distribution of active cold seep systems. However, our understanding of the metabolic function of microbial communities in the cold seep sediments of the OT remains limited. In this study, we investigated the vertical profiles of functional genes involved in methane, nitrogen, and sulphur cycling in the cold seep sediments of the OT. Furthermore, we explored the possible coupling mechanisms between these biogeochemical cycles. The study revealed that the majority of genes associated with the nitrogen and sulphur cycles were most abundant in the surface sediment layers. However, only the key genes responsible for sulphur disproportionation (sor), nitrogen fixation (nifDKH), and methane metabolism (mcrABG) were more prevalent within sulfate-methane transition zone (SMTZ). Significant positive correlations (P < 0.05) were observed between functional genes involved in sulphur oxidation, thiosulphate disproportionation with denitrification, and dissimilatory nitrate reduction to ammonium (DNRA), as well as between AOM/methanogenesis and nitrogen fixation, and between sulphur disproportionation and AOM. A genome of Filomicrobium (class Alphaproteobacteria) has demonstrated potential in chemoautotrophic activities, particularly in coupling DNRA and denitrification with sulphur oxidation. Additionally, the characterized sulfate reducers such as Syntrophobacterales have been found to be capable of utilizing nitrate as an electron acceptor. The predominant methanogenic/methanotrophic groups in the OT sediments were identified as H2-dependent methylotrophic methanogens (Methanomassiliicoccales and Methanofastidiosales) and ANME-1a. This study offered a thorough understanding of microbial ecosystems in the OT cold seep sediments, emphasizing their contribution to nutrient cycling.IMPORTANCEThe Okinawa Trough (OT) is a back-arc basin formed by extension within the continental lithosphere behind the Ryukyu Trench arc system. Cold seeps are widespread in the OT. While some studies have explored microbial communities in OT cold seep sediments, their metabolic potential remains largely unknown. In this study, we used metagenomic analysis to enhance comprehension of the microbial community's role in nutrient cycling and proposed hypotheses on the coupling process and mechanisms involved in biogeochemical cycles. It was revealed that multiple metabolic pathways can be performed by a single organism or microbes that interact with each other to carry out various biogeochemical cycling. This data set provided a genomic road map on microbial nutrient cycling in OT sediment microbial communities.

Anaerobic co-digestion of corn straw, sewage sludge and fresh leachate: Focusing on synergistic/antagonistic effects and microbial mechanisms

Effects of sewage sludge (SS) and fresh leachate (FL) addition on corn straw (CS) digestion and underlying mechanisms were investigated. Co-digestion of CS, SS and FL significantly increased cumulative methane production by 7.2–61.1%. Further analysis revealed that co-digestion acted mainly on slowly degradable substrates and exerted dual effects on methane production potential, which was closely related to the volatile solids (VS) content. Antagonistic effects of co-digestion resulted from the dominance of norank_c_Bathyarchaeia, a mixotrophic methanogen that may generate methane inefficiently and consume existing methane. The synergistic enhancement of methane production (0.7–12.7%) was achieved in co-digestion with 33.5–45.5% of total VS added as SS and FL. Co-digestion with more balanced nutrients and higher buffering capacity enriched Actinobacteriota, Firmicutes, and Synergistota, thereby facilitating the substrate degradation. Furthermore, the predominant acetoclastic methanogens, increased hydrogenotrophic methanogens, and decreased methylotrophic methanogens in the digester combined to prompt the synergy.

Methanolobus use unspecific methyltransferases to produce methane from dimethylsulphide in Baltic Sea sediments

BackgroundIn anoxic coastal and marine sediments, degradation of methylated compounds is the major route to the production of methane, a powerful greenhouse gas. Dimethylsulphide (DMS) is the most abundant biogenic organic sulphur compound in the environment and an abundant methylated compound leading to methane production in anoxic sediments. However, understanding of the microbial diversity driving DMS-dependent methanogenesis is limited, and the metabolic pathways underlying this process in the environment remain unexplored. To address this, we used anoxic incubations, amplicon sequencing, genome-centric metagenomics and metatranscriptomics of brackish sediments collected along the depth profile of the Baltic Sea with varying sulphate concentrations.ResultsWe identified Methanolobus as the dominant methylotrophic methanogens in all our DMS-amended sediment incubations (61–99%) regardless of their sulphate concentrations. We also showed that the mtt and mta genes (trimethylamine- and methanol-methyltransferases) from Methanolobus were highly expressed when the sediment samples were incubated with DMS. Furthermore, we did not find mtsA and mtsB (methylsulphide-methyltransferases) in metatranscriptomes, metagenomes or in the Methanolobus MAGs, whilst mtsD and mtsF were found 2–3 orders of magnitude lower in selected samples.ConclusionsOur study demonstrated that the Methanolobus genus is likely the key player in anaerobic DMS degradation in brackish Baltic Sea sediments. This is also the first study analysing the metabolic pathways of anaerobic DMS degradation in the environment and showing that methylotrophic methane production from DMS may not require a substrate-specific methyltransferase as was previously accepted. This highlights the versatility of the key enzymes in methane production in anoxic sediments, which would have significant implications for the global greenhouse gas budget and the methane cycle.47Ug-8P75YQNwjurZUgoHpVideo

Microbial communities in the fluid migration zone in the sediments of the Krasny Yar methane seep (South Baikal)

The diversity and structure of microbial communities were investigated using 16S rRNA gene barcoding in the sediments of the Krasny Yar methane seep, in the zone of near-surface occurrence of gas hydrates and the presence of oxidized and restored channels. The diversity of both bacteria and archaea along the core depth was similar to the diversity found in sediments from other areas: methylotrophic methanogens and microorganisms involved in different stages of the organic matter fermentation were detected in the sediment strata of all depths investigated. Migration flows of oxygen-rich and aerobic bacteria-rich near-bottom water influenced greatly the diversity of microbial communities in oxidized channels. Fluids migrating from the deep zone to the bottom surface provided transport from anoxygenic sediments of anaerobic archaea involved in the AOM process. The data obtained are consistent with geochemical and geothermal indicators defining the zone of active migration of near-bottom waters.

Highly diverse-Low abundance methanogenic communities in hypersaline microbial mats of Guerrero Negro B.C.S., assessed through microcosm experiments.

Methanogenic communities of hypersaline microbial mats of Guerrero Negro, Baja California Sur, Mexico, have been recognized to be dominated by methylotrophic methanogens. However, recent studies of environmental samples have evidenced the presence of hydrogenotrophic and methyl-reducing methanogenic members, although at low relative abundances. Physical and geochemical conditions that stimulate the development of these groups in hypersaline environments, remains elusive. Thus, in this study the taxonomic diversity of methanogenic archaea of two sites of Exportadora de Sal S.A was assessed by mcrA gene high throughput sequencing from microcosm experiments with different substrates (both competitive and non-competitive). Results confirmed the dominance of the order Methanosarcinales in all treatments, but an increase in the abundance of Methanomassiliiccocales was also observed, mainly in the treatment without substrate addition. Moreover, incubations supplemented with hydrogen and carbon dioxide, as well as the mixture of hydrogen, carbon dioxide and trimethylamine, managed to stimulate the richness and abundance of other than Methanosarcinales methanogenic archaea. Several OTUs that were not assigned to known methanogens resulted phylogenetically distributed into at least nine orders. Environmental samples revealed a wide diversity of methanogenic archaea of low relative abundance that had not been previously reported for this environment, suggesting that the importance and diversity of methanogens in hypersaline ecosystems may have been overlooked. This work also provided insights into how different taxonomic groups responded to the evaluated incubation conditions.

Potential of acetic acid to restore methane production in anaerobic reactors critically intoxicated by ammonia as evidenced by metabolic and microbial monitoring

BackgroundBiogas and biomethane production from the on-farm anaerobic digestion (AD) of animal manure and agri-food wastes could play a key role in transforming Europe’s energy system by mitigating its dependence on fossil fuels and tackling the climate crisis. Although ammonia is essential for microbial growth, it inhibits the AD process if present in high concentrations, especially under its free form, thus leading to economic losses. In this study, which includes both metabolic and microbial monitoring, we tested a strategy to restore substrate conversion to methane in AD reactors facing critical free ammonia intoxication.ResultsThe AD process of three mesophilic semi-continuous 100L reactors critically intoxicated by free ammonia (> 3.5 g_N L−1; inhibited hydrolysis and heterotrophic acetogenesis; interrupted methanogenesis) was restored by applying a strategy that included reducing pH using acetic acid, washing out total ammonia with water, re-inoculation with active microbial flora and progressively re-introducing sugar beet pulp as a feed substrate. After 5 weeks, two reactors restarted to hydrolyse the pulp and produced CH4 from the methylotrophic methanogenesis pathway. The acetoclastic pathway remained inhibited due to the transient dominance of a strictly methylotrophic methanogen (Candidatus Methanoplasma genus) to the detriment of Methanosarcina. Concomitantly, the third reactor, in which Methanosarcina remained dominant, produced CH4 from the acetoclastic pathway but faced hydrolysis inhibition. After 11 weeks, the hydrolysis, the acetoclastic pathway and possibly the hydrogenotrophic pathway were functional in all reactors. The methylotrophic pathway was no longer favoured. Although syntrophic propionate oxidation remained suboptimal, the final pulp to CH4 conversion ratio (0.41 ± 0.10 LN_CH4 g_VS−1) was analogous to the pulp biochemical methane potential (0.38 ± 0.03 LN_CH4 g_VS−1).ConclusionsDespite an extreme free ammonia intoxication, the proposed process recovery strategy allowed CH4 production to be restored in three intoxicated reactors within 8 weeks, a period during which re-inoculation appeared to be crucial to sustain the process. Introducing acetic acid allowed substantial CH4 production during the recovery period. Furthermore, the initial pH reduction promoted ammonium capture in the slurry, which could allow the field application of the effluents produced by full-scale digesters recovering from ammonia intoxication.

Methanolobus mangrovi sp. nov. and Methanolobus sediminis sp. nov, two novel methylotrophic methanogens isolated from mangrove sediments in China.

Two methylotrophic methanogens, designated strains FTZ2T and FTZ6T, were isolated from mangrove sediment sampled in Futian Mangrove Nature Reserve in Shenzhen, PR China. Cells of strains FTZ2T and FTZ6T were cocci, with diameters of 0.6-1.0 µm and 0.6-0.9 µm, respectively. Both strains grew on methanol, methylamine, dimethylamine and trimethylamine, but not on acetate, formate, H2/CO2, choline, betaine or dimethyl sulphide. Strain FTZ2T grew at 10-37 °C (optimally at 33 °C), pH 5.5-8.0 (optimally at pH 7.0) and 0-1.03 M NaCl (optimally at 0.17 M NaCl). In contrast, strain FTZ6T grew at 15-42 °C (optimally at 37 °C), pH 5.0-7.5 (optimally pH 6.5) and 0-1.03 M NaCl (optimally at 0.17 M NaCl). Both strains required magnesium for growth and were susceptible to sodium dodecyl sulphate. Biotin was required for the growth of strain FTZ2T but not of strain FTZ6T. The genomic G+C contents of strains FTZ2T and FTZ6T were 41.6 and 40.9 mol%, respectively. Phylogenetic analyses revealed that strain FTZ2T was mostly related to Methanolobus psychrotolerans YSF-03T, with 16S rRNA gene similarity of 98.6 %, an average nucleotide identity (ANI) of 82.5 %, and a digital DNA-DNA hybridization (dDDH) of 24.6 %. While strain FTZ6T was mostly related to Methanolobus vulcani PL-12/MT, with 16S rRNA gene similarity of 99.4 %, an ANI of 88.6% and a dDDH of 34.6 %. Based on phenotypic, phylogenetic and genotypic evidence, two novel species of the genus Methanolobus, Methanolobus mangrovi sp. nov. and Methanolobus sediminis sp. nov., are proposed. The type strain of M. mangrovi sp. nov. is FTZ2T (=CCAM 1276T=JCM 39396T) and the type strain of M. sediminis sp. nov. is FTZ6T (=CCAM 1277T=JCM 39397T).

Microbial drivers of DMSO reduction and DMS-dependent methanogenesis in saltmarsh sediments

Saltmarshes are highly productive environments, exhibiting high abundances of organosulfur compounds. Dimethylsulfoniopropionate (DMSP) is produced in large quantities by algae, plants, and bacteria and is a potential precursor for dimethylsulfoxide (DMSO) and dimethylsulfide (DMS). DMSO serves as electron acceptor for anaerobic respiration leading to DMS formation, which is either emitted or can be degraded by methylotrophic prokaryotes. Major products of these reactions are trace gases with positive (CO2, CH4) or negative (DMS) radiative forcing with contrasting effects on the global climate. Here, we investigated organic sulfur cycling in saltmarsh sediments and followed DMSO reduction in anoxic batch experiments. Compared to previous measurements from marine waters, DMSO concentrations in the saltmarsh sediments were up to ~300 fold higher. In batch experiments, DMSO was reduced to DMS and subsequently consumed with concomitant CH4 production. Changes in prokaryotic communities and DMSO reductase gene counts indicated a dominance of organisms containing the Dms-type DMSO reductases (e.g., Desulfobulbales, Enterobacterales). In contrast, when sulfate reduction was inhibited by molybdate, Tor-type DMSO reductases (e.g., Rhodobacterales) increased. Vibrionales increased in relative abundance in both treatments, and metagenome assembled genomes (MAGs) affiliated to Vibrio had all genes encoding the subunits of DMSO reductases. Molar conversion ratios of <1.3 CH4 per added DMSO were accompanied by a predominance of the methylotrophic methanogens Methanosarcinales. Enrichment of mtsDH genes, encoding for DMS methyl transferases in metagenomes of batch incubations indicate their role in DMS-dependent methanogenesis. MAGs affiliated to Methanolobus carried the complete set of genes encoding for the enzymes in methylotrophic methanogenesis.

Changes in stoichiometric ratio of carbon and sulfate affect methanogenesis pathways in sulfate-rich sewers

Methane emissions from sewer systems have been a major concern in terms of posing safety risks and aggravating the greenhouse effect. Discharging a large amount of sulfate-rich wastewater produced by industrial manufacture into municipal sewers breaks the balance between organic matter and sulfate, and has a significant influence on methanogenesis. However, the direct impact of sulfate-rich wastewater on the interactions between methane and sulfate metabolism in sewers remains unclear. In this study, methane production, microbial community and functional genes of the sewers with different chemical oxygen demand/sulfate ratios (C/S) were studied by 33 sampling sites collected in full-scale field investigation. The concentrations of chemical oxygen demand in the sewers were 10.0–378.0 mg/L, while the concentrations of sulfate fluctuated in a wider range of 4.1–758.4 mg/L, which distinguished the sewers into different C/S of 0.3, 2.5 and 5.8. With the average sulfate concentrations of 37.9 mg/L, the methane-production capabilities of sewers were reduced as the decreased C/S from 5.8 to 2.5. In contrast, the sewers with low C/S of 0.3 and high sulfate concentration of 368.2 mg/L exhibited twice as much methane concentrations (1.2 mL/L) than the sewers (0.6 mL/L) with C/S of 2.5 and 5.8. Genera showing a positive correlation (Enterobacter, Prevotella_9 and Acinetobacter) and a negative correlation (Bacteroidetes vadinHA17_norank, Smithella and Thiobacillus) with methanogenesis contributed significant variations of methane in the sewers with different C/S. The results of functional gene prediction suggested that the main pathways of methanogenesis in sewers with C/S of 5.8 and 2.5 were acetoclastic (ACSS and ackA) and hydrogenotrophic methanogenesis (mtrA, mtrH and K00400), whereas methylotrophic methanogenesis (mttB) was dominant in sewers with C/S of 0.3. The genes expressed by methylotrophic methanogens in sewer with low C/S and higher sulfate concentration refrained from the competitive inhibition by SRB, which contributed to the superior methane-production of the sewers. These results bridge the knowledge gap of methanogenesis with different sulfate levels and improve the understanding of methane cycle driven by microorganisms, facilitating more accurate prediction and assessment of methane in sewers.

Crystal Structures of Bacterial Pectin Methylesterases Pme8A and PmeC2 from Rumen Butyrivibrio.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed β-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

Methylotrophic methanogens and bacteria synergistically demethylate dimethylarsenate in paddy soil and alleviate rice straighthead disease.

Microorganisms play a key role in arsenic (As) biogeochemistry, transforming As species between inorganic and organic forms and different oxidation states. Microbial As methylation is enhanced in anoxic paddy soil, producing primarily dimethylarsenic (DMAs), which can cause rice straighthead disease and large yield losses. DMAs can also be demethylated in paddy soil, but the microorganisms driving this process remain unclear. In this study, we showed that the enrichment culture of methylotrophic methanogens from paddy soil demethylated pentavalent DMAs(V) efficiently. DMAs(V) was reduced to DMAs(III) before demethylation. 16S rRNA gene diversity and metagenomic analysis showed that Methanomassiliicoccus dominated in the enrichment culture, with Methanosarcina and Methanoculleus also being present. We isolated Methanomassiliicoccus luminyensis CZDD1 and Methanosarcina mazei CZ1 from the enrichment culture; the former could partially demethylate trivalent DMAs(III) but not DMAs(V) and the latter could demethylate neither. Addition of strain CZDD1 to the enrichment culture greatly accelerated DMAs(V) demethylation. Demethylation of DMAs(V) in the enrichment culture was suppressed by ampicillin, suggesting the involvement of bacteria. We isolated three anaerobic bacterial strains including Clostridium from the enrichment culture, which could produce hydrogen and reduce DMAs(V) to DMAs(III). Furthermore, augmentation of the Methanomassiliicoccus-Clostridium coculture to a paddy soil decreased DMAs accumulation by rice and alleviated straighthead disease. The results reveal a synergistic relationship whereby anaerobic bacteria reduce DMAs(V) to DMAs(III) for demethylation by Methanomassiliicoccus and also produce hydrogen to promote the growth of Methanomassiliicoccus; enhancing their populations in paddy soil can help alleviate rice straighthead disease.

Impact of Varying Mass Concentrations of Ammonia Nitrogen on Biogas Production and System Stability of Anaerobic Fermentation

High ammonium release from chicken manure poses a significant limitation to aerobic digestion, impeding microbial processes and inhibiting biogas production. In this study, we conducted anaerobic digestion of a mixture consisting of chicken manure and corn straw as the fermented raw material. The inoculum used was obtained from the residue of previously fermented chicken manure. To assess the inhibitory effect, we varied the ammonia levels within the range of 750–4250 mg/L by introducing ammonium chloride. The efficiency of aerobic digestion was monitored through the measurement of volatile fatty acids (VFA), chemical oxygen demand (COD), total inorganic carbon (TOC), and methane yield. Our results indicated that elevated levels of ammonia nitrogen had a suppressive impact on methane release, and this decrease followed a linear relationship with the increasing ammonia nitrogen load. Moreover, the addition of ammonia led to a slower release, with the maximum daily ammonia concentration observed at 15 days compared to the 6th day at lower ammonia levels. Furthermore, on the 40th day of aerobic digestion, the cumulative methane production at 4250 mg/L was inhibited by 41% compared to the 750 mg/L condition. The patterns of VFA, inorganic carbon, and COD reduction were consistent across all ammonia levels, with VFA and TOC levels being highest at the highest ammonia concentration and lowest at the lowest ammonia concentration. The accumulation of VFA resulted in a decrease in pH and a decline in methanogenic activity. Additionally, high ammonia levels altered the relative abundance of methanogens. Acetoclastic methanogens (Methanosaeta) exhibited a decrease in abundance, while hydrogenotrophic methanogens (Methanosaeta, Methanoculleus) and methylotrophic methanogens (Candidatus Methanoplasma) demonstrated an increase in abundance. Overall, our findings highlight the inhibitory effects of high ammonia concentrations on biogas production, providing insights into the changes in microbial composition and activity during anaerobic fermentation.

Well-hidden methanogenesis in deep, organic-rich sediments of Guaymas Basin

Deep marine sediments (>1mbsf) harbor ~26% of microbial biomass and are the largest reservoir of methane on Earth. Yet, the deep subsurface biosphere and controls on its contribution to methane production remain underexplored. Here, we use a multidisciplinary approach to examine methanogenesis in sediments (down to 295 mbsf) from sites with varying degrees of thermal alteration (none, past, current) at Guaymas Basin (Gulf of California) for the first time. Traditional (13C/12C and D/H) and multiply substituted (13CH3D and 12CH2D2) methane isotope measurements reveal significant proportions of microbial methane at all sites, with the largest signal at the site with past alteration. With depth, relative microbial methane decreases at differing rates between sites. Gibbs energy calculations confirm methanogenesis is exergonic in Guaymas sediments, with methylotrophic pathways consistently yielding more energy than the canonical hydrogenotrophic and acetoclastic pathways. Yet, metagenomic sequencing and cultivation attempts indicate that methanogens are present in low abundance. We find only one methyl-coenzyme M (mcrA) sequence within the entire sequencing dataset. Also, we identify a wide diversity of methyltransferases (mtaB, mttB), but only a few sequences phylogenetically cluster with methylotrophic methanogens. Our results suggest that the microbial methane in the Guaymas subsurface was produced over geologic time by relatively small methanogen populations, which have been variably influenced by thermal sediment alteration. Higher resolution metagenomic sampling may clarify the modern methanogen community. This study highlights the importance of using a multidisciplinary approach to capture microbial influences in dynamic, deep subsurface settings like Guaymas Basin.

Investigate the anaerobic degradation of high-acetone latex wastewater with magnetite supplement

This study evaluated the effects of acetone on the anaerobic degradation of synthetic latex wastewater, which was simulated from the wastewater of the deproteinized natural rubber production process, including latex, acetate, propionate, and acetone as the main carbon sources, at a batch scale in 5 cycles of a total of 60 days. Fe3O4 was applied to accelerate the treatment performance from cycle 3. Acetone was added in concentration ranges of 0%, 0.05%, 0.1%, 0.15%-included latex, and 0.15%-free latex (w/v). In the Fe3O4-free cycles, for latex-added vials, soluble chemical oxygen demand (sCOD) was removed at 43.20%, 43.20%, and 12.65%, corresponding to the input acetone concentrations varying from 0.05% to 0.15%, indicating the interference of acetone for COD reduction. After adding Fe3O4, all flasks reported a significant increase in COD removal efficiency, especially for acetone-only and latex-only vials, from 36.9% to 14.30%–42.95% and 83.20%, respectively. Other highlighted results of COD balance showed that Fe3O4 involvement improved the degradation process of acetate, propionate, acetone, and the other COD parts, including the intermediate products of latex reduction. Besides, during the whole batch process, the order of reduction priority of the carbon sources in the synthetic wastewater was acetate, propionate and acetone. We also found that the acetate concentration appeared to be strongly related to reducing other carbon sources in natural rubber wastewater. Microbial community analysis revealed that protein-degrading bacteria Bacteroidetes vadinHA17 and Proteinniphilum and methylotrophic methanogens might play key roles in treating simulated deproteinized-natural-rubber wastewater.

Mcr-dependent methanogenesis in Archaeoglobaceae enriched from a terrestrial hot spring.

The preeminent source of biological methane on Earth is methyl coenzyme M reductase (Mcr)-dependent archaeal methanogenesis. A growing body of evidence suggests a diversity of archaea possess Mcr, although experimental validation of hypothesized methane metabolisms has been missing. Here, we provide evidence of a functional Mcr-based methanogenesis pathway in a novel member of the family Archaeoglobaceae, designated Methanoglobus nevadensis, which we enriched from a terrestrial hot spring on the polysaccharide xyloglucan. Our incubation assays demonstrate methane production that is highly sensitive to the Mcr inhibitor bromoethanesulfonate, stimulated by xyloglucan and xyloglucan-derived sugars, concomitant with the consumption of molecular hydrogen, and causing a deuterium fractionation in methane characteristic of hydrogenotrophic and methylotrophic methanogens. Combined with the recovery and analysis of a high-quality M. nevadensis metagenome-assembled genome encoding a divergent Mcr and diverse potential electron and carbon transfer pathways, our observations suggest methanogenesis in M. nevadensis occurs via Mcr and is fueled by the consumption of cross-fed byproducts of xyloglucan fermentation mediated by other community members. Phylogenetic analysis shows close affiliation of the M. nevadensis Mcr with those from Korarchaeota, Nezhaarchaeota, Verstraetearchaeota, and other Archaeoglobales that are divergent from well-characterized Mcr. We propose these archaea likely also use functional Mcr complexes to generate methane on the basis of our experimental validation in M. nevadensis. Thus, divergent Mcr-encoding archaea may be underestimated sources of biological methane in terrestrial and marine hydrothermal environments.