Methylotrophic Methanogenesis Research Articles

Picoplanktonic methane production in eutrophic surface waters

Abstract. Over the past decade, extensive research has delved into the methane (CH4) paradox, which involves aerobic CH4 production. We present noteworthy observations of CH4 oversaturation within the surface layer of the central Chile upwelling zone (36° S, 73° W) over two consecutive seasonal cycles (2018–2021). Complementing these observations, CH4 cycling experiments were conducted, utilizing distinct plankton fractions (encompassing the natural planktonic community, fractions < 150, < 3 and < 0.2 µm), in different productivity periods of phytoplanktonic production and composition throughout the year. Our findings underscore the pivotal role of picoplankton (< 3 µm) in CH4 production on the ocean surface, contrasting with the limited contribution of larger microorganisms (< 150 µm). Notably, incubations with methylated substrates, such as methylphosphonic acid (MPn) and trimethylamine (TMA), induce heightened CH4 production within the picoplanktonic fraction. This phenomenon is consistently observed during both upwelling (austral spring–summer) and non-upwelling (winter) seasons, with significance in the latter period, when Synechococcus sp. exhibits notably high relative abundance. Long-term microcosm experiments highlight the crucial roles played by heterotrophic bacteria and cyanobacteria in methylotrophic methanogenesis. This process enhances CH4 production, facilitated by the recycling of dissolved organic carbon (DOC). Picoplankton emerges as a pivotal factor influencing the recycling of methylated substrates, and it is responsible for maintaining CH4 supersaturation. These findings provide valuable insights into the biogeochemical processes driving CH4 dynamics, particularly in highly productive upwelling areas.

Long-term temporal dynamics of total and potentially active microbiota affect the biogas quality from the anaerobic digestion of vinasse in a pilot-scale hybrid anaerobic reactor

The microbiota was evaluated through a time series of the 16S rrna gene in a pilot-scale Hybrid Anaerobic Reactor treating vinasse. The microbial composition, evenness and diversity differed between the DNA and cDNA, the latter showing a better response for %CH4 along monitoring of the microbiota. Moreover, the microbial composition and richness were distinct between the reactor's compartments, possibly contributing to the COD removal in the sludge blanket. The abundant Methanospirillum, Methanomassiliicoccaceae, Bacteroidetes-vadinHA17, Paludibacteraceae-H1, Prolixibacteraceae, Ruminococcaceae, Syntrophobacter and Desulfovibrio contributed to the %CH4, as well as the redundancy of fermentation and hydrogenotrophic and methylotrophic methanogenesis, and the evenness and diversity of potentially active microbiota and metabolisms. The microbial changes after the middle of reactor's operation required new adaptations and impacted the biogas quality, but did not affect COD removal. This study brings important knowledge about the microbial temporal dynamics, plasticity and contribution to the anaerobic digestion in pilot-scale reactors.

Time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies predominates the methanogen dynamics in Philippine rice field soil

BackgroundThe final step in the anaerobic decomposition of biopolymers is methanogenesis. Rice field soils are a major anthropogenic source of methane, with straw commonly used as a fertilizer in rice farming. Here, we aimed to decipher the structural and functional responses of the methanogenic community to rice straw addition during an extended anoxic incubation (120 days) of Philippine paddy soil. The research combined process measurements, quantitative real-time PCR and RT-PCR of particular biomarkers (16S rRNA, mcrA), and meta-omics (environmental genomics and transcriptomics).ResultsThe analysis methods collectively revealed two major bacterial and methanogenic activity phases: early (days 7 to 21) and late (days 28 to 60) community responses, separated by a significant transient decline in microbial gene and transcript abundances and CH4 production rate. The two methanogenic activity phases corresponded to the greatest rRNA and mRNA abundances of the Methanosarcinaceae but differed in the methanogenic pathways expressed. While three genetically distinct Methanosarcina populations contributed to acetoclastic methanogenesis during the early activity phase, the late activity phase was defined by methylotrophic methanogenesis performed by a single Methanosarcina genomospecies. Closely related to Methanosarcina sp. MSH10X1, mapping of environmental transcripts onto metagenome-assembled genomes (MAGs) and population-specific reference genomes revealed this genomospecies as the key player in acetoclastic and methylotrophic methanogenesis. The anaerobic food web was driven by a complex bacterial community, with Geobacteraceae and Peptococcaceae being putative candidates for a functional interplay with Methanosarcina. Members of the Methanocellaceae were the key players in hydrogenotrophic methanogenesis, while the acetoclastic activity of Methanotrichaceae members was detectable only during the very late community response.ConclusionsThe predominant but time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies represents a novel finding that expands our hitherto knowledge of the methanogenic pathways being highly expressed in paddy soils.7k5euw-hvBknz_jds4sXXfVideo

Insight into using hydrochar to alleviate ammonia nitrogen inhibition during anaerobic digestion of waste activated sludge: Performance, metagenomic and metabolomic signatures

In this study, hydrochar (HCR) was used to alleviate high ammonia inhibition to the anaerobic digestion (AD) of waste activated sludge (WAS) and to elucidate the inner microorganism mechanism. After HCR addition, the cumulative methane yield increased by 73.6 % and 35.6 % under ammonia inhibition levels of 3000 and 6000 mg/L, respectively. Metagenomic analysis showed that HCR enriched the diversity of hydrogenotrophic methanotrophs, and the relative abundances of functional microorganisms with electron transfer capabilities (Geobacteraceae bacterium etc.) were 1.5–7.8 times higher than those without HCR addition. Metabolomics analysis implied that metabolites related to fatty acid degradation, such as glutaric acid and hexadecanal, were downregulated (2.9–15.7 %) under ammonia inhibition conditions and that HCR regulates metabolites in the methane metabolic pathway. Moreover, HCR changed the methanogenic pathway from hydrogenotrophic methanogenesis to multiple pathways under ammonia inhibition conditions, especially methanolic and methylotrophic methanogenesis, which facilitated the methane yield. This study provides valuable information for understanding the inner microbial mechanism of HCR addition on alleviating high ammonia inhibition to AD of WAS, and gives basic knowledge for the application of AD of WAS under ammonia inhibition conditions.

Methylotrophic methanogenesis in the Archaeoglobi revealed by cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring.

Over the past decade, environmental metagenomics and polymerase chain reaction-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of thermophilic Euryarchaeota that have long been considered to live non-methanogenic lifestyles. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64-70°C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park, as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems.

Nitrous oxide inhibition of methanogenesis represents an underappreciated greenhouse gas emission feedback.

Methane (CH4) and nitrous oxide (N2O) are major greenhouse gases that are predominantly generated by microbial activities in anoxic environments. N2O inhibition of methanogenesis has been reported, but comprehensive efforts to obtain kinetic information are lacking. Using the model methanogen Methanosarcina barkeri strain Fusaro and digester sludge-derived methanogenic enrichment cultures, we conducted growth yield and kinetic measurements and showed that micromolar concentrations of N2O suppress the growth of methanogens and CH4 production from major methanogenic substrate classes. Acetoclastic methanogenesis, estimated to account for two-thirds of the annual 1 billion metric tons of biogenic CH4, was most sensitive to N2O, with inhibitory constants (KI) in the range of 18-25μM, followed by hydrogenotrophic (KI, 60-90μM) and methylotrophic (KI, 110-130μM) methanogenesis. Dissolved N2O concentrations exceeding these KI values are not uncommon in managed (i.e. fertilized soils and wastewater treatment plants) and unmanaged ecosystems. Future greenhouse gas emissions remain uncertain, particularly from critical zone environments (e.g. thawing permafrost) with large amounts of stored nitrogenous and carbonaceous materials that are experiencing unprecedented warming. Incorporating relevant feedback effects, such as the significant N2O inhibition on methanogenesis, can refine climate models and improve predictive capabilities.

Microbial adaptation to high ammonia environment in submerged anaerobic membrane bioreactor under volatile fatty acids and ammonia stresses

Anaerobic membrane bioreactor (AnMBR) is a high-rate bioreactor which is preferred over other configurations due to its potential to completely decouple solid retention time from hydraulic retention time. However, limited information is available on the bioreactor behavior under stressed environment, particularly during the startup period. The objective of this study was to investigate the effects of ammonium (NH4+) and organic loading rate (OLR) on the digestion performance and microbial population of submerged AnMBR. The findings revealed that high NH4+ was positively correlated with high methane concentration while high OLR increased methane production. The microbial analysis showed that both bacteria and archaea were influenced by the change of NH4+ and OLR. Particularly, at high OLR, Bacteroidetes were shown to be more sensitive to high NH4+ than Firmicutes, and Chloroflexi tolerated NH4+ even up to 2.5 g/L. Moreover, NH4+ was possibly the first driving factor in the change of methane production pathways from acetoclastic methanogenesis to hydrogenotrophic and methylotrophic methanogenesis. Membrane biofouling was also caused mainly by bacterial community (Firmicutes and Chloroflexi), while the contribution of archaeal community was minimal (Methanosaetaceae).

Methylotrophy in the Mire: direct and indirect routes for methane production in thawing permafrost.

While wetlands are major sources of biogenic methane (CH4), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site's methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH4) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH4 formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH4 budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevant in situ and have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle.

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.

High methane concentrations in tidal salt marsh soils: Where does the methane go?

Tidal salt marshes produce and emit CH4 . Therefore, it is critical to understand the biogeochemical controls that regulate CH4 spatial and temporal dynamics in wetlands. The prevailing paradigm assumes that acetoclastic methanogenesis is the dominant pathway for CH4 production, and higher salinity concentrations inhibit CH4 production in salt marshes. Recent evidence shows that CH4 is produced within salt marshes via methylotrophic methanogenesis, a process not inhibited by sulfate reduction. To further explore this conundrum, we performed measurements of soil-atmosphere CH4 and CO2 fluxes coupled with depth profiles of soil CH4 and CO2 pore water gas concentrations, stable and radioisotopes, pore water chemistry, and microbial community composition to assess CH4 production and fate within a temperate tidal salt marsh. We found unexpectedly high CH4 concentrations up to 145,000 μmol mol-1 positively correlated with S2- (salinity range: 6.6-14.5 ppt). Despite large CH4 production within the soil, soil-atmosphere CH4 fluxes were low but with higher emissions and extreme variability during plant senescence (84.3 ± 684.4 nmol m-2  s-1 ). CH4 and CO2 within the soil pore water were produced from young carbon, with most Δ14 C-CH4 and Δ14 C-CO2 values at or above modern. We found evidence that CH4 within soils was produced by methylotrophic and hydrogenotrophic methanogenesis. Several pathways exist after CH4 is produced, including diffusion into the atmosphere, CH4 oxidation, and lateral export to adjacent tidal creeks; the latter being the most likely dominant flux. Our findings demonstrate that CH4 production and fluxes are biogeochemically heterogeneous, with multiple processes and pathways that can co-occur and vary in importance over the year. This study highlights the potential for high CH4 production, the need to understand the underlying biogeochemical controls, and the challenges of evaluating CH4 budgets and blue carbon in salt marshes.

Optimization of methane production from co-digestion of citrus agro-industrial waste in a two-stage system: Investigation of the effects of organic matter concentration and alkalinization

The essential oils in citrus waste poses challenges for the anaerobic digestion application to energy recovery from these wastes due to the potential to inhibit the bioprocess. The two-stage system is a practical alternative to overcome these limitations of anaerobic co-digestion of citrus peel waste and wastewater. This study proposes to overcome drawbacks associated with the use of these substrates in anaerobic digestion by separating the phases, performing parameters optimization of the methanogenic stage. To obtain optimized conditions for the methanogenic stage, the Rotational Central Composite Design (RCCD) and Response Surface Methodology (RSM) were applied, evaluating the effects of diluting the Fermentation Reactor Content (FRC) of the first stage (35.7%–100%) and alkalinization (up to 0.60 g sodium bicarbonate.gCOD−1), in methane production and organic matter removal. Values from 2100.4 to 6320.6 mLCH4.L−1 were obtained in the experimental design assays, with no inhibition identified by the high substrate concentration, nor by the limonene content (1.5–5.0 mg.L−1). Lower dilution of the fermentation reactor content (90%–100%) resulted in higher accumulated methane production, while the variation in the alkalinization did not imply significant effects on this response. The optimized condition for maximum production potential was obtained with 99.3% of fermentation reactor content and 0.58 gNaCHO3.gCOD−1, reaching a production of 6549.7 mLCH4.L−1, with organic matter removal (rCOD) of 87.1%, and 58.6% of reduction in solid waste content. For this condition, acetogenic metabolism predominance was observed, in the conversion of butyric acid and propionic acid into acetic acid. In the analysis of the microbial community of the optimized condition assay, a high relative abundance of hydrolytic and acidogenic bacteria (AUTHM297, Lachnospiraceae, and Hungateiclostridiaceae) was observed, as well as methanogenic archaea related to acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis (Methanoregula, Methanolinea, Methanomassiliicoccus, Candidatus Methanomethylicus, and Methanofolis).

Evidence of cryptic methane cycling and non-methanogenic methylamine consumption in the sulfate-reducing zone of sediment in the Santa Barbara Basin, California

Abstract. The recently discovered cryptic methane cycle in the sulfate-reducing zone of marine and wetland sediment couples methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). Here we present evidence of cryptic methane cycling activity within the upper regions of the sulfate-reducing zone, along a depth transect within the Santa Barbara Basin, off the coast of California, USA. The top 0–20 cm of sediment from each station was subjected to geochemical analyses and radiotracer incubations using 35S–SO42-, 14C–mono-methylamine, and 14C–CH4 to find evidence of cryptic methane cycling. Methane concentrations were consistently low (3 to 16 µM) across the depth transect, despite AOM rates increasing with decreasing water depth (from max 0.05 nmol cm−3 d−1 at the deepest station to max 1.8 nmol cm−3 d−1 at the shallowest station). Porewater sulfate concentrations remained high (23 to 29 mM), despite the detection of sulfate reduction activity from 35S–SO42- incubations with rates up to 134 nmol cm−3 d−1. Metabolomic analysis showed that substrates for methanogenesis (i.e., acetate, methanol and methylamines) were mostly below the detection limit in the porewater, but some samples from the 1–2 cm depth section showed non-quantifiable evidence of these substrates, indicating their rapid turnover. Estimated methanogenesis from mono-methylamine ranged from 0.2 to 0.5 nmol cm−3 d−1. Discrepancies between the rate constants (k) of methanogenesis (from 14C–mono-methylamine) and AOM (from either 14C–mono-methylamine-derived 14C–CH4 or from directly injected 14C–CH4) suggest the activity of a separate, concurrent metabolic process directly metabolizing mono-methylamine to inorganic carbon. We conclude that the results presented in this work show strong evidence of cryptic methane cycling occurring within the top 20 cm of sediment in the Santa Barbara Basin. The rapid cycling of carbon between methanogenesis and methanotropy likely prevents major build-up of methane in the sulfate-reducing zone. Furthermore, our data suggest that methylamine is utilized by both methanogenic archaea capable of methylotrophic methanogenesis and non-methanogenic microbial groups. We hypothesize that sulfate reduction is responsible for the additional methylamine turnover, but further investigation is needed to elucidate this metabolic activity.

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.

Studying the isotopic composition of microbial methane with a genetically-tractable methanogen

Nearly all biogenic methane is produced by a group of microorganisms called methanogenic archaea (or methanogens). Methanogens can use a variety of substrates, such as H2 + CO2, acetate, and methylated compounds, for methanogenesis. Previous studies have shown that the stable carbon and hydrogen isotopic compositions of methane produced by methanogens can vary drastically depending on the substrate composition and concentration in the environment. For instance, the concentration of H2 in the environment has a substantial impact on the isotopic composition of methane derived from hydrogenotrophic methanogenesis (reduction of CO2 to methane using H2 as the electron donor) (Valentine et al. 2004, Penning et al. 2005). While there is substantial empirical data on isotopic signatures of methane from different substrates and under different conditions, the physiological and molecular features that control these values are not as well understood. To address this, we are using the metabolically diverse and genetically tractable methanogen, Methanosarcina acetivorans as a model system to uncover key cellular processes that control the stable bulk isotopic composition of methane (i.e., 13C/12C and D/H ratios), and the distributions of the “clumped” 13CH3D and 12CH2D2 isotopologues. The methanogen M. acetivorans grows on a wide variety of compounds such as acetate, methanol, methylamines, and methylsulfides. We found that the methylotrophic pathways (for methanol and trimethylamine) and the aceticlastic pathway have large and similar primary hydrogen isotopic effects (α of ~0.45). These data are in contrast to previous findings and imply a minor isotopic exchange between CH4 and H2O (Valentine et al. 2004, Gruen et al. 2018). Focusing first on the methylotrophic pathway, we generated mutants of two key enzymes in the methylotrophic pathway: a) methyl coenzyme M reductase (Mcr) that catalyzes the last step in methanogenesis and b) methyltransferases that catalyze the first step in methylotrophic methanogenesis from methanol (Mta). A mutant with reduced Mcr expression had no observable change in the hydrogen isotopic effect relative to the wild-type, validating the initial observation of minimal H2O-CH4 hydrogen isotopic exchange. One of the Mta mutants, which only expressed a specific methyltransferase isoform, had a smaller carbon isotopic effect relative to the other isoforms (α of ~1.074 vs. ~1.080). Since the isoforms are thought to be identical in structure, the different isotopic effects could result from differential expression of each isoform, or from different kinetic properties. By combining our genetic approaches with traditional and high-resolution isotopic analytical methods, we aim to develop a quantitative understanding of the mechanisms that control the isotopic compositions of biological methane. Our preliminary results show that M. acetivorans would be an ideal candidate for such research, which could help in understanding methanogens’ physiology in natural environments in past, present, and future Earth.

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.

Aerobic methanotrophy increases the net iron reduction in methanogenic lake sediments.

In methane (CH4) generating sediments, methane oxidation coupled with iron reduction was suggested to be catalyzed by archaea and bacterial methanotrophs of the order Methylococcales. However, the co-existence of these aerobic and anaerobic microbes, the link between the processes, and the oxygen requirement for the bacterial methanotrophs have remained unclear. Here, we show how stimulation of aerobic methane oxidation at an energetically low experimental environment influences net iron reduction, accompanied by distinct microbial community changes and lipid biomarker patterns. We performed incubation experiments (between 30 and 120 days long) with methane generating lake sediments amended with 13C-labeled methane, following the additions of hematite and different oxygen levels in nitrogen headspace, and monitored methane turnover by 13C-DIC measurements. Increasing oxygen exposure (up to 1%) promoted aerobic methanotrophy, considerable net iron reduction, and the increase of microbes, such as Methylomonas, Geobacter, and Desulfuromonas, with the latter two being likely candidates for iron recycling. Amendments of 13C-labeled methanol as a potential substrate for the methanotrophs under hypoxia instead of methane indicate that this substrate primarily fuels methylotrophic methanogenesis, identified by high methane concentrations, strongly positive δ13CDIC values, and archaeal lipid stable isotope data. In contrast, the inhibition of methanogenesis by 2-bromoethanesulfonate (BES) led to increased methanol turnover, as suggested by similar 13C enrichment in DIC and high amounts of newly produced bacterial fatty acids, probably derived from heterotrophic bacteria. Our experiments show a complex link between aerobic methanotrophy and iron reduction, which indicates iron recycling as a survival mechanism for microbes under hypoxia.

Fe/Mn modified biochar as electrode particles in electrochemical system for efficient anaerobic sludge digestion

Electrochemical pretreatment (EPT) of sewage sludge demonstrates an aggressive effect on extracellular polymeric substance destruction and sludge solubilization. While the low efficiency of methane production does not match the initial dissolution of the sludge after EPT. Here we proposed a novel method, using Fe-Mn-modified biochar (FMBC) strengthening electrochemical pretreatment, aiming at improving the methanogenic efficiency. The strengthening mechanism of FMBC was studied according to the physicochemical parameters of sludge at different concentrations and the change of microbial community. Experimentally, with the addition of 2 g/L FMBC, the methane production increased by 7.96% and 10.06% compared with electrochemical pretreatment alone and the control group. The sludge organic matter solubilization varied from 344.6 mg/L in E0 to 383.4 mg/L in E2 after pretreatment. Simultaneously, analysis of microbial communities showed that the addition of 2 g/L FMBC obviously increased the relative abundance of Methanofastidiosum and Methanosaeta from 5.5% and 20.9% in CK to 16% and 28.7% in E2, respectively. The addition of FMBC also intensified the acetoclastic methanogenesis and methylotrophic methanogenesis. This study provides new ideas for the enhancement of electrochemical pretreatment of sludge.

Conversion of natural coastal wetlands to mariculture ponds dramatically decreased methane production by reducing substrate availability

Coastal wetlands are increasingly being converted into aquaculture ponds to meet growing global demand for fish protein. Coastal wetlands conversion has been predicted to result in significant carbon (C) emissions; however, a mechanistic understanding of the effects of coastal wetland conversion on soil’s capacity to produce greenhouse gases remains lacking. Here, integrated biogeochemical investigations on methanogenic substrates, community structures, and activities were conducted in a coastal Spartina alterniflora saltmarsh and three saltmarsh-converted mariculture ponds aged 6, 13, and 20 years. Saltmarsh conversion into mariculture ponds decreased CH4 production potential from 353 to 16.3–78.3 µg CH4 kg−1 d−1. The concentrations of dissolved organic C and non-competitive methanogenic substrate, trimethylamine, were reduced by 84.8% and 79.7%, respectively, whereas methanogens abundance decreased by 43.7%, probably due to the decreased C input following conversion. Among the ponds, CH4 production potential, methanogenic substrates (acetate and trimethylamine), and methanogen abundance decreased with conversion chronosequence. However, the dominant methanogen group did not shift after land conversion, and the potential methylamine-utilizing Methanosarcinaceae accounted for 86.5% and 85.3–89.7% of the total populations in the S. alterniflora saltmarsh and mariculture ponds, respectively, implying that methylotrophic methanogenesis was the predominant pathway of CH4 production in both ecosystems. The results indicated that the conversion of natural coastal saltmarsh to mariculture ponds decreased CH4 production by reducing substrate availability and methanogen abundance, and CH4 production potential in mariculture ponds was more efficiently suppressed with conversion chronosequence.

Genetic and Physiological Probing of Cytoplasmic Bypasses for the Energy-Converting Methyltransferase Mtr in Methanosarcina acetivorans.

Methanogenesis is a unique energy metabolism carried out by members of the domain Archaea. Unlike most other methanogens, which reduce CO2 to methane with hydrogen as the electron donor, Methanosarcina acetivorans is able to grow on methylated compounds, on acetate, and on carbon monoxide (CO). These substrates are metabolized via distinct yet overlapping pathways. For the use of any single methanogenic substrate, the membrane-integral, energy-converting N5-methyl-tetrahydrosarcinapterin (H4SPT):coenzyme M (HS-CoM) methyltransferase (Mtr) is required. It was proposed that M. acetivorans can bypass the methyl transfer catalyzed by Mtr via cytoplasmic activities. To address this issue, conversion of different energy substrates by an mtr deletion mutant was analyzed. No significant methyl transfer from H4SPT to HS-CoM could be detected with CO as the electron donor. In contrast, formation of methane and CO2 in the presence of methanol or trimethylamine was indicative of an Mtr bypass in the oxidative direction. As methane thiol and dimethyl sulfide were transiently produced during methylotrophic methanogenesis in the mtr mutant, involvement in this process of methyl sulfide-dependent methyltransferases (Mts) was analyzed in a strain lacking both the Mts system and Mtr. It could be unequivocally demonstrated that the Mts system is not involved in bypassing Mtr, thereby ruling out previous proposals. Conversion of [13C]methanol indicated that in the absence of Mtr M. acetivorans provides the reducing equivalents for methyl-S-CoM reduction to methane by oxidizing (an) intracellular compound(s) to CO2 rather than disproportioning the source of methyl groups. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans. IMPORTANCE Methanogenic archaea possess only a limited number of chemiosmotic coupling sites in their respiratory chains. Among them, N5-methyl-H4SPT:HS-CoM methyltransferase (Mtr) is the most widely distributed. Previous observations led to the conclusion that Methanosarcina acetivorans is able to bypass this reaction via methyl sulfide-dependent methyltransferases (Mts). However, strains lacking Mtr are not able to produce methane from CO. Also, these strains are unable to oxidize methylated substrates to CO2, in contrast to observations in the close relative Methanosarcina barkeri. The results also highlight the sole function of the Mts system in methyl sulfide metabolism. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans.

Long-Term Cultivation and Meta-Omics Reveal Methylotrophic Methanogenesis in Hydrocarbon-Impacted Habitats

The microbial conversion of alkanes to methane in hydrocarbon-contaminated environments is an intrinsic bioremediation strategy under anoxic conditions. However, the mechanism of microbial methanogenic alkane degradation is currently unclear. Under ten-years of continuous efforts, we obtained a methanogenic n-alkane-degrading (C15–C20) enrichment culture that exhibited sustained improvements in the kinetic properties of methane production. The integrated metagenomic and metatranscriptomic analyses revealed that n-alkanes were mainly attacked by members of Desulfosarcinaceae, Firmicutes, and Synergistetes using the fumarate addition strategy, and were then further degraded in a common effort by Tepidiphilus members. Meanwhile, the abundant members of Anaerolineaceae were mainly responsible for cell debris recycling. However, according to the metatranscriptomic analyses, methane was predicted to be produced mainly via H2-dependent methylotrophic methanogenesis, primarily from necromass-derived trimethylamine mediated by Methanomethyliaceae within the candidate phylum Verstraetearchaeota. These findings reveal that H2-dependent methylotrophic methanogens, as well as methylotrophic methanogens, may play important ecological roles in the carbon cycle of hydrocarbon enriched subsurface ecosystems.