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The dynamics of methane (CH4) cycling in high-latitude peatlands through different pathways of methanogenesis and methanotrophy are still poorly understood due to the spatiotemporal complexity of microbial activities and biogeochemical processes. Additionally, long-term in situ measurements within soil columns are limited and associated with large uncertainties in microbial substrates (e.g. dissolved organic carbon, acetate, hydrogen). To better understand CH4 cycling dynamics, we first applied an advanced biogeochemical model, ecosys, to explicitly simulate methanogenesis, methanotrophy, and CH4 transport in a high-latitude fen (within the Stordalen Mire, northern Sweden). Next, to explore the vertical heterogeneity in CH4 cycling, we applied the PCMCI/PCMCI+ causal detection framework with a bootstrap aggregation method to the modeling results, characterizing causal relationships among regulating factors (e.g. temperature, microbial biomass, soil substrate concentrations) through acetoclastic methanogenesis, hydrogenotrophic methanogenesis, and methanotrophy, across three depth intervals (0–10 cm, 10–20 cm, 20–30 cm). Our results indicate that temperature, microbial biomass, and methanogenesis and methanotrophy substrates exhibit significant vertical variations within the soil column. Soil temperature demonstrates strong causal relationships with both biomass and substrate concentrations at the shallower depth (0–10 cm), while these causal relationships decrease significantly at the deeper depth within the two methanogenesis pathways. In contrast, soil substrate concentrations show significantly greater causal relationships with depth, suggesting the substantial influence of substrates on CH4 cycling. CH4 production is found to peak in August, while CH4 oxidation peaks predominantly in October, showing a lag response between production and oxidation. Overall, this research provides important insights into the causal mechanisms modulating CH4 cycling across different depths, which will improve carbon cycling predictions, and guide the future field measurement strategies.

The dynamics of methane (CH4) cycling in high-latitude peatlands through different pathways of methanogenesis and methanotrophy are still poorly understood due to the spatiotemporal complexity of microbial activities and biogeochemical processes. Additionally, long-term in situ measurements within soil columns are limited and associated with large uncertainties in microbial substrates (e.g. dissolved organic carbon, acetate, hydrogen). To better understand CH4 cycling dynamics, we first applied an advanced biogeochemical model, ecosys, to explicitly simulate methanogenesis, methanotrophy, and CH4 transport in a high-latitude fen (within the Stordalen Mire, northern Sweden). Next, to explore the vertical heterogeneity in CH4 cycling, we applied the PCMCI/PCMCI+ causal detection framework with a bootstrap aggregation method to the modeling results, characterizing causal relationships among regulating factors (e.g. temperature, microbial biomass, soil substrate concentrations) through acetoclastic methanogenesis, hydrogenotrophic methanogenesis, and methanotrophy, across three depth intervals (0–10 cm, 10–20 cm, 20–30 cm). Our results indicate that temperature, microbial biomass, and methanogenesis and methanotrophy substrates exhibit significant vertical variations within the soil column. Soil temperature demonstrates strong causal relationships with both biomass and substrate concentrations at the shallower depth (0–10 cm), while these causal relationships decrease significantly at the deeper depth within the two methanogenesis pathways. In contrast, soil substrate concentrations show significantly greater causal relationships with depth, suggesting the substantial influence of substrates on CH4 cycling. CH4 production is found to peak in August, while CH4 oxidation peaks predominantly in October, showing a lag response between production and oxidation. Overall, this research provides important insights into the causal mechanisms modulating CH4 cycling across different depths, which will improve carbon cycling predictions, and guide the future field measurement strategies.

Carbon capture and storage (CCS) is a key technology to mitigate the environmental impact of carbon dioxide (CO.sub.2) emissions. An understanding of the potential trapping and storage mechanisms is required to provide confidence in safe and secure CO.sub.2 geological sequestration.sup.1,2. Depleted hydrocarbon reservoirs have substantial CO.sub.2 storage potential.sup.1,.sup.3, and numerous hydrocarbon reservoirs have undergone CO.sub.2 injection as a means of enhanced oil recovery (CO.sub.2-EOR), providing an opportunity to evaluate the (bio)geochemical behaviour of injected carbon. Here we present noble gas, stable isotope, clumped isotope and gene-sequencing analyses from a CO.sub.2-EOR project in the Olla Field (Louisiana, USA). We show that microbial methanogenesis converted as much as 13-19% of the injected CO.sub.2 to methane (CH.sub.4) and up to an additional 74% of CO.sub.2 was dissolved in the groundwater. We calculate an in situ microbial methanogenesis rate from within a natural system of 73-109 millimoles of CH.sub.4 per cubic metre (standard temperature and pressure) per year for the Olla Field. Similar geochemical trends in both injected and natural CO.sub.2 fields suggest that microbial methanogenesis may be an important subsurface sink of CO.sub.2 globally. For CO.sub.2 sequestration sites within the environmental window for microbial methanogenesis, conversion to CH.sub.4 should be considered in site selection.

Extremophilic biological process (EBP) pretreatment increases substrate availability in anaerobic digestion, but the effect on downstream microbial community composition in industrial systems is not characterized. Changes in microbial communities were determined at an industrial facility processing dairy manure in a modified split-stream system with three reactor types: (1) EBP tanks at 70–72 °C; (2) mesophilic Continuously Stirred Tank Reactors (CSTRs); (3) mesophilic Induced Bed Reactors (IBRs) receiving combined CSTR and EBP effluent. All reactors had a two-day hydraulic retention time. Samples were collected weekly for 60 days. pH, volatile fatty acid and bicarbonate concentrations, COD, and methane yield were measured to assess tank environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing. EBP pretreatment increased acetate availability but led to a decline in the relative abundance of acetoclastic Methanosarcina species in downstream IBRs. Rather, syntrophic methanogens, e.g., members of Methanobacteriaceae, increased in relative abundance and became central to microbial co-occurrence networks, particularly in association with hydrogen-producing bacteria. Network analysis also demonstrated that these syntrophic relationships were tightly coordinated in pretreated digestate but absent in the untreated CSTRs. By promoting syntrophic methanogenesis while increasing acetate concentrations, EBP pretreatment requires system configurations that enable acetoclast retention to prevent acetate underutilization and maximize methane yields.

Anaerobic digestion (AD) is an effective biological treatment for stabilizing organic compounds in waste/wastewater and in simultaneously producing biogas. However, it is often limited by the slow reaction rates of different microorganisms’ syntrophic biological metabolisms. Stable and fast interspecies electron transfer (IET) between volatile fatty acid-oxidizing bacteria and hydrogenotrophic methanogens is crucial for efficient methanogenesis. In this syntrophic interaction, electrons are exchanged via redox mediators such as hydrogen and formate. Recently, direct IET (DIET) has been revealed as an important IET route for AD. Microorganisms undergoing DIET form interspecies electrical connections via membrane-associated cytochromes and conductive pili; thus, redox mediators are not required for electron exchange. This indicates that DIET is more thermodynamically favorable than indirect IET. Recent studies have shown that conductive materials (e.g., iron oxides, activated carbon, biochar, and carbon fibers) can mediate direct electrical connections for DIET. Microorganisms attach to conductive materials’ surfaces or vice versa according to particle size, and form conductive biofilms or aggregates. Different conductive materials promote DIET and improve AD performance in digesters treating different feedstocks, potentially suggesting a new approach to enhancing AD performance. This review discusses the role and potential of DIET in methanogenic systems, especially with conductive materials for promoting DIET.

Background Commercial biogas upgrading facilities are expensive and consume energy. Biological biogas upgrading may serve as a low-cost approach because it can be easily integrated with existing facilities at biogas plants. The microbial communities found in anaerobic digesters typically contain hydrogenotrophic methanogens, which can use hydrogen (H2) as a reducing agent for conversion of carbon dioxide (CO2) into methane (CH4). Thus, biological biogas upgrading through the exogenous addition of H2 into biogas digesters for the conversion of CO2 into CH4 can increase CH4 yield and lower CO2 emission. Results The addition of 4 mol of H2 per mol of CO2 was optimal for batch biogas reactors and increased the CH4 content of the biogas from 67 to 94%. The CO2 content of the biogas was reduced from 33 to 3% and the average residual H2 content was 3%. At molar H2:CO2 ratios > 4:1, all CO2 was converted into CH4, but the pH increased above 8 due to depletion of CO2, which negatively influenced the process stability. Additionally, high residual H2 content in these reactors was unfavourable, causing volatile fatty acid accumulation and reduced CH4 yields. The reactor microbial communities shifted in composition over time, which corresponded to changes in the reactor variables. Numerous taxa responded to the H2 inputs, and in particular the hydrogenotrophic methanogen Methanobacterium increased in abundance with addition of H2. In addition, the apparent rapid response of hydrogenotrophic methanogens to intermittent H2 feeding indicates the suitability of biological methanation for variable H2 inputs, aligning well with fluctuations in renewable electricity production that may be used to produce H2. Conclusions Our research demonstrates that the H2:CO2 ratio has a significant effect on reactor performance during in situ biological methanation. Consequently, the H2:CO2 molar ratio should be kept at 4:1 to avoid process instability. A shift toward hydrogenotrophic methanogenesis was indicated by an increase in the abundance of the obligate hydrogenotrophic methanogen Methanobacterium.

1. Plant invasion can strongly influence carbon (C) cycling processes, thus it may affect climate change by altering C sequestration and greenhouse gas emissions in the invaded ecosystem. Since 1979, the exotic Spartina alterniflora has rapidly expanded in China's coastal areas, where significant increase in methane (CH₄) emissions has been documented from post-invaded sites. However, a mechanistic understanding of the structural and functional changes of associated methanogens accompanying this invasion remains elusive. 2. Here we conducted integrated biogeochemical investigations on methanogenic substrates, activity, and diversity to identify implications of S. alterniflora invasion for methanogenesis in coastal wetlands. To do this, we collected and analysed 0-50 cm soil profiles from an uncolonised tidal flat (TF) and salt marshes that S. alterniflora has invaded for 1 year (SA-1) and 12 years (SA-12) in Jiangsu, China. Methanogenic community composition was characterised by massive parallel sequencing. The rates and pathways of methanogenesis were determined by adding trace concentrations of ¹³C-labelled substrates to anaerobic incubated samples. 3. Our results revealed that 12-year invasion of S. alterniflora drastically increased CH₄ production potential by one order of magnitude over that of TF. This substantial increase was primarily attributed to methanogenesis from trimethylamine; its rates increased by two orders of magnitude over TF whereas those from acetate and H₂/CO₂ increased far less. Hydrogenotrophic methanogenesis was the dominant pathway operating in the TF, but methanotrophic pathway contributed most to CH₄ production in the surface layer of SA-1 and uppermost 40-cm layers of SA-12. Consistent with these observations, the dominant methanogens shifted from obligate hydrogenotrophic Methanococcales in TF to potential methylamineutilising Methanosarcinaceae in SA-12. Our Mantel analysis indicated that 'non-competitive' trimethylamine, derived from cytoplasmic osmolytes of S. alterniflora, was the major driver of this change in methanogenic community composition. 4. Synthesis. Our results suggest that invasive Spartino alterniflora plants gradually facilitated the local dominance of methylotrophic Methanosarcinaceae by changing the key type of methanogenic substrate in coastal marshes. Shifts in methanogen communities and enhanced availability of trimethylamine elevated the rates and importance of methylotrophic methanogenesis, thereby markedly increasing CH₄ production potential and emission rates in this type of ecosystem.

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day −1 ) and ATH (0.05 day −1 ) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day −1 ) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter , a known SAOB, species of Thauera , Thermodesulfovibrio , Anaerobaculum , Ruminiclostridium , Comamonas , and uncultured bacteria belonging to Lentimicrobiaceae , o_MBA03, Thermoanaerobacteraceae , Anaerolineaceae , Clostridiales , and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F 420 , majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.

Background Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones. Results Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl 3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl 3 . When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using 13 C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing. Conclusions We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.

This study applied Illumina high-throughput sequencing to explore the microbial communities and functions in anaerobic digestion sludge (ADS) from two wastewater treatment plants based on a metagenomic view. Taxonomic analysis using SILVA SSU database indicated that Proteobacteria (9.52–13.50 %), Bacteroidetes (7.18 %–10.65 %) and Firmicutes (7.53 %–9.46 %) were the most abundant phyla in the ADS. Differences of microbial communities between the two types of ADS were identified. Genera of Methanosaeta and Methanosarcina were the major methanogens. Functional analysis by SEED subsystems showed that the basic metabolic functions of metagenomes in the four ADS samples had no significant difference among them, but they were different from other microbial communities from activated sludge, human faeces, ocean and soil. Abundances of genes in methanogenesis pathway were also quantified using a methanogenesis genes database extracted from KEGG. Results showed that acetotrophic was the major methanogenic pathway in the anaerobic sludge digestion.