Methanogenic Pathway Research Articles

Influence of inoculum-to-substrate ratio on biomethane production via anaerobic digestion of biomass.

The influence of the inoculum-to-substrate ratio (ISR) on anaerobic digestion (AD) of biomass in terms ofmethane yield and microbial community, was explored in this paper. The level of ISR can affect the AD performance in several ways. At extremely low ISR, volatile fatty acids (VFAs) accumuate, while inhibition occur at higher level of ISR. An ISR ranging from 1.0-2.0 was found optimal resulting in higher methane yield, organic matter removal and VFA degradation. Furthermore,a high ISR (2.0-4.0) is favourable to methanogenesis, while a lower ISR (<1.0) is prone to irreversible acidification. The range of ISR can shift the methanogenic pathway of AD to favour an acetoclastic or hydrogenotrophic response, indicated by the enriched group of microorganisms. The genus Methanosaeta (acetoclastic) and Methanobacterium (hydrogenotrophic) are the most enriched methanogens across all ISRs, while Firmicutes, Bacteroidetes, Proteobacteria and Spirochaetae are dominantin the bacterial community. Additionally, the interplay of substrate biodegradability and ISR potentially affects AD performance. Finally, novel equations are developed and proposed for characterizing the quantity of inoculum and substrate.

Impact of biochar prepared at different pyrolysis temperatures on the methane production and microbial community structure of food waste anaerobic digestion

Biochars were prepared through the pyrolysis of sawdust at 300 °C, 500 °C, and 700 °C, respectively, under oxygen-limited conditions. The basic physicochemical properties of biochars were explored by scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), surface area and porosity analyzer (BET), and Fourier-transform infrared spectrometer (FTIR). The effects of biochar addition on the methane yield and microbial community structure of anaerobic digestion of food waste were also investigated. SEM images showed that biochar had a honeycomb-like pore structure, EDS analysis showed that the C content in the biochar tended to increase, and the O contents tended to decrease with the increasing temperature. The specific surface area of biochars increased from 1.2014 m2/g (300 °C) to 326.8435 m2/g (700 °C). FTIR analysis showed that the number of different surface functional groups decreased with the increasing temperature. The addition of biochar could increase the cumulative methane volume by 11.63%–25.18%. High-throughput sequencing results showed that biochar addition could increase the relative abundance of Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Spirochaetota, which were associated with the degradation of refractory organic matters. Meanwhile, biochar addition could enrich the relative abundance of methanogens participating in direct electron transfer (Methanosaeta and Methanosarcina), and methanogens producing methane through multiple pathways (Methanobacterium and Methanosarcina). The addition of biochar derived at 700 °C significantly increased the relative abundance of Methanobacterium and Methanosarcina from 1.96% and 0.70% (control group) to 32.68% and 64.69%, respectively and improved methane production by transforming acetoclastic/hydrogenotrophic methanogenic pathways to more metabolically diverse methanogenic pathways.

All-in-One CO2 Capture and Transformation: Lessons from Formylmethanofuran Dehydrogenases.

ConspectusCarbon-one-unit (C1) feedstocks are generally used in the chemical synthesis of organic molecules, such as solvents, drugs, polymers, and fuels. Contrary to the dangerous and polluting carbon monoxide mostly coming from fossil fuels, formate and formamide are attractive alternative feedstocks for chemical synthesis. As these are currently mainly obtained from the oil industry, novel synthetic routes have been developed based on the transformation of the greenhouse gas CO2. Such developments are motivated by the urgent need for carbon chemical recycling, leading to a sustainable future. The inert nature of CO2 represents a challenge for chemists to activate and specifically convert the molecule through an affordable and efficient process. The chemical transformation could be inspired by biological CO2 activation, in which highly specialized enzymes perform atmospheric CO2 fixation through relatively abundant metal catalysts. In this Account, we describe and discuss the potential of one of the most efficient biological CO2-converting systems: the formylmethanofuran dehydrogenase (abbreviated as FMD).FMDs are multienzymatic complexes found in archaea that capture CO2 as a formyl group branched on the amine moiety of the methanofuran (MFR) cofactor. This overall reaction leading to formyl-MFR production does not require ATP hydrolysis as compared to the CO2-fixing microbes relying on the reductive Wood-Ljungdahl pathway, highlighting a different operative mode that saves cellular energy. FMD reaction represents the entry point in hydrogenotrophic methanogenesis (H2 and CO2 dependent or formate dependent) and operates in reverse in other methanogenic pathways and microbial metabolisms. Therefore, FMD is a key enzyme in the planetary carbon cycle. After decades of investigations, recent studies have provided a description of the FMD structure, reaction mechanism, and potential for the electroreduction of CO2, to which our laboratory has been actively contributing.FMD is an "all-in-one" enzyme catalyzing a redox-active transformation coupled to a redox-neutral transformation at two very different metal cofactors where new C-H and C-N bonds are made. First, the principle of the overall reaction consisting of an exergonic CO2 reduction coupled with an endergonic formate condensation on MFR is resumed. Then, this Account exposes the molecular details of the active sites and provides an overview of each catalytic mechanism. It also describes the natural versatility of electron-delivery modules fueling CO2 reduction and extends it to the possibilities of using artificial systems such as electrodes.A perspective concludes on how the mechanistic of FMD could be applied to produce CO2-based chemical intermediates to synthesize organic molecules. Indeed, through its biochemical properties, the enzyme opens opportunities for CO2 electroreduction to generate molecules such as formate and formamide derivatives, which are all intermediates for synthesizing organic compounds. Transferring the chemical knowledge acquired from these biological systems would provide coherent models that can lead to further development in the field of synthetic biology and bio-inspired synthetic chemistry to perform large-scale CO2 conversion into building blocks for chemical synthesis.

A metagenomic catalogue of the ruminant gut archaeome

While the ruminant gut archaeome regulates the gut microbiota and hydrogen balance, it is also a major producer of the greenhouse gas methane. However, ruminant gut archaeome diversity within the gastrointestinal tract (GIT) of ruminant animals worldwide remains largely underexplored. Here, we construct a catalogue of 998 unique archaeal genomes recovered from the GITs of ruminants, utilizing 2270 metagenomic samples across 10 different ruminant species. Most of the archaeal genomes (669/998 = 67.03%) belong to Methanobacteriaceae and Methanomethylophilaceae (198/998 = 19.84%). We recover 47/279 previously undescribed archaeal genomes at the strain level with completeness of >80% and contamination of <5%. We also investigate the archaeal gut biogeography across various ruminants and demonstrate that archaeal compositional similarities vary significantly by breed and gut location. The catalogue contains 42,691 protein clusters, and the clustering and methanogenic pathway analysis reveal strain- and host-specific dependencies among ruminant animals. We also find that archaea potentially carry antibiotic and metal resistance genes, mobile genetic elements, virulence factors, quorum sensors, and complex archaeal viromes. Overall, this catalogue is a substantial repository for ruminant archaeal recourses, providing potential for advancing our understanding of archaeal ecology and discovering strategies to regulate methane production in ruminants.

Coal-straw co-digestion-induced biogenic methane production: perspectives on microbial communities and associated metabolic pathways

This study assessed the impacts of wheat straw as a cosubstrate on coal biocoverion into methane and the associated mechanism within methane metabolic pathways. Co-digestion of coal with varying wheat straw concentrations resulted in a remarkable (1246.05%) increase in methane yield compared to that of the control (CK). Moreover, microbial analysis revealed a uniform distribution of Methanosarcinaceae (51.14%) and Methanobacteriaceae (39.90%) in the co-digestion of coal and wheat straw (CWS1) at a ratio of 3:1 (w/w) compared to other treatments such as coal and wheat straw (CWS2) at a ratio of 3:0.5. In addition, Hungatieclostridiaceae and Rhodobacteriaceae were abundant in both co-digesters, whereas the bacterial communities in the CK group were significantly different and more abundant than those in the Peptostreptococcaceae and Enterobacteriaceae groups. The key enzymes related to methanogenic metabolic pathways, including EC: 1.2.99.5 and EC: 2.1.1.86 (facilitating the conversion of CO2 into methane), and EC:1.12.98.1 exhibited significant abundance within CWS1. Aromatic compounds such as 4-(2-chloroanilino)-4-oxobutanoic acid and phthalic acid were substantially more abundant in CWS1 and CWS2 than in CK, indicating the increased bioavailability of coal to microbial activities. This novel approach demonstrates that wheat straw co-digestion with coal during anaerobic digestion modulates microbial communities and their metabolic pathways to enhance methane production from complex substrates such as coal.

Sulfate availability affect sulfate reduction pathways and methane consumption in freshwater wetland sediments

Methane (CH4) emissions from wetlands significantly contribute to global greenhouse gas fluxes, yet the mechanisms regulating CH4 production and oxidation in freshwater wetlands remain underexplored, particularly in the role of sulfate in the anaerobic oxidation of CH4. This study investigated the production, consumption, and release of CH4 in the sediments of the Qilihai wetland, focusing on the roles of hydrogenotrophic methanogenesis and sulfate dynamics across different seasons. CH4 concentrations ranged from 2.42 to 2290.52 μmol L⁻1, and δ13C–CH4 values became progressively more negative with depth, ranging from −87.37‰ to −57.18‰. Results indicate that hydrogenotrophic methanogenesis is the dominant pathway for CH4 production, particularly in sulfate-depleted environments, with CH4 concentrations in the sulfate-methane transition zone (SMTZ) strongly correlated with sulfate availability. Sulfate consumption through Sulfate Anaerobic Oxidation of Methane (SAOM) and Organoclastic Sulfate Reduction (OSR) demonstrated significant seasonal variation, with OSR accounting for up to 73% of sulfate consumption in the SMTZ. The SMTZ exhibited variations ranging from 2 to 40 cm in October, narrowing to 2–4 cm in July. These findings emphasize the complex interactions between sulfate availability, methanogenic pathways, and CH4 emissions in freshwater wetlands, highlighting the need for further research on sulfate dynamics and their implications for greenhouse gas emissions in the context of global climate change.

Methanogenesis—General Principles and Application in Wastewater Remediation

The first discovery of methanogens led to the formation of a new domain of life known as Archaea. The Archaea domain exhibits properties vastly different from previously known Bacteria and Eucarya domains. However, for a certain multi-step process, a syntrophic relationship between organisms from all domains is needed. This process is called methanogenesis and is defined as the biological production of methane. Different methanogenic pathways prevail depending on substrate availability and the employed order of methanogenic Archaea. Most methanogens reduce carbon dioxide to methane with hydrogen through a hydrogenotrophic pathway. For hydrogen activation, a group of enzymes called hydrogenases is required. Regardless of the methanogenic pathway, electrons are carried between microorganisms by hydrogen. Naturally occurring processes, such as methanogenesis, can be engineered for industrial use. With the growth and emergence of new industries, the amount of produced industrial waste is an ever-growing environmental problem. For successful wastewater remediation, a syntrophic correlation between various microorganisms is needed. The composition of microorganisms depends on wastewater type, organic loading rates, anaerobic reactor design, pH, and temperature. The last step of anaerobic wastewater treatment is production of biomethane by methanogenesis, which is thought to be a cost-effective means of energy production for this renewable biogas.

Enhanced anaerobic digestion of freezing and thawing pretreated cow manure with increasing solid content: kinetics and microbial community dynamics

High solid anaerobic digestion has proved the mainstream technology for the treatment of organic wastes. However, the high molecular weight and complex lignocellulosic structure of cow manure (CM) make it indigestible and inefficient, leading to limit the hydrolysis step of anaerobic digestion at high solid content. To mitigate this bottleneck, an improved cost-effective freezing and thawing pretreatment technique was proposed in this study. The freezing and thawing pretreatment of raw CM without any dilution was done for 20 days. The maximum cumulative methane yield (487 mL CH4 g− 1VS) was achieved at a total solid (TS) of 5% followed by TS of 10% and 15%, which was 13%, 20% and 21% higher than obtained from untreated CM, respectively. The kinetic results revealed that the biodegradable materials could be utilized at increasing TS with decreasing hydrolysis rate. The pretreatment significantly enhanced the methylotrophic methanogenic pathway during high solid anaerobic digestion, which was contrary to the general concept that the process is usually dominated by acetoclastic and hydrogenotrophic methanogens. This study is very important to understand the effect of solid content but also important to understand the effect of freezing and thawing pretreatment on process parameters and microbial community dynamics in high solid anaerobic digestion.

The geothermal gradient shapes microbial diversity and processes in natural-gas-bearing sedimentary aquifers

Abstract. Deep subsurface microorganisms constitute over 80 % of Earth's prokaryotic biomass and play an important role in global biogeochemical cycles. Geochemical processes driven by geothermal heating are key factors influencing their biomass and activities, yet their full breadth remains uncaptured. Here, we investigated the microbial community composition and metabolism in microbial-natural-gas-bearing aquifers at temperatures ranging from 38 to 81 °C, situated above nonmicrobial-gas- and oil-bearing sediments at temperatures exceeding 90 °C. Cultivation-based and molecular gene analyses, including radiotracer measurements, of formation water indicated variations in predominant methanogenic pathways across different temperature regimes of upper aquifers: high potential for hydrogenotrophic–methylotrophic, hydrogenotrophic, and acetoclastic methanogenesis at temperatures of 38, 51–65, and 73–81 °C, respectively. The potential for acetoclastic methanogenesis correlated with elevated acetate concentrations with increasing depth, possibly due to the decomposition of sedimentary organic matter. In addition to acetoclastic methanogenesis, in aquifers at temperatures as high as or higher than 65 °C, acetate is potentially utilized by microorganisms responsible for the dissimilatory reduction of sulfur compounds other than sulfate because of their high relative abundance at greater depths. The stable sulfur isotopic analysis of sulfur compounds in water and oil samples suggested that hydrogen sulfide, generated through the thermal decomposition of sulfur compounds in oil, migrates upward and is subsequently oxidized with iron oxides present in sediments, yielding elemental sulfur and thiosulfate. These compounds are consumed by sulfur-reducing microorganisms, possibly reflecting elevated microbial populations in aquifers at temperatures as high as or higher than 73 °C. These findings reveal previously overlooked geothermal-heat-driven geochemical and microbiological processes involved in carbon and sulfur cycling in the deep sedimentary biosphere.

Enhancing effect of conductive materials and rumen microorganisms on the anaerobic digestion performance of a real traditional Chinese medicine wastewater

This work evaluated the effect of different concentrations of conductive materials and rumen microorganisms on the anaerobic digestion (AD) performance of real traditional Chinese medicine (TCM) wastewater. The experiments first determined the optimal organic concentration of 0.25 L/L for the AD system and removed suspended solids from real TCM wastewater to reduce the inhibition effect on methanogenesis. Analysis of the modified Gompertz model showed that the addition of activated carbon, biochar, cow manure, and rumen fluid at doses of 12 g/L, 12 g/L, 12 mL/L, and 125 mL/L, respectively, had the greatest effect on CH4 production, increasing the cumulative CH4 yield by 13.5 %, 10.4 %, 26.8 %, and 32.9 %. Through microbial community and metabolism pathway analyses, the conductive materials promoted direct interspecies electron transfer (DIET) between Clostridium and Methanothrix, and the acetoclastic methanogenic pathway dominated by acetyl-CoA synthase. Rumen microorganisms enhanced AD performance by promoting the growth of hydrogenotrophic methanogens and the abundance of genes dominated by formylmethanofuran dehydrogenase in the hydrogenotrophic methanogenic pathway, illustrating the relationship between microbial community and metabolism pathway. Rumen microorganisms increased CH4 production more than conductive materials in real TCM wastewater. This study helps to better understand the internal mechanisms by which different materials enhance AD performance.

Comparing the effects and mechanisms of granular activated carbon and magnetite nanoparticles in dry anaerobic digestion: Focusing on microbial electron transfer and metagenomic evidence

Enhancing direct interspecies electron transfer (DIET) via granular activated carbon (GAC) and magnetite nanoparticles (MNPs) to promote methanation performance in dry anaerobic digestion (AD) systems has been extensively studied in recent years. However, the underlying mechanisms, particularly how GAC and MNPs influence DIET and extracellular polymeric substances (EPSs), remain poorly understood. In this study, the different effects and mechanisms of GAC and MNPs on dry AD fed with pig manure (PM) and corn straw were compared, with additions of 10, 20, and 50% of the volatile solid (VS) of PM. The results indicated that GAC improved the cumulative specific methane yields (CSMYs) by 18.8–20.9%, while a high dose of MNPs (50% of the VSPM) decreased the CSMY and hydrolysis rate constant by 27.0 and 75.4%, respectively. GAC provided microorganism attachment sites and functional groups for electron transfer, improving hydrolysis by enriching fermenters such as Fastidiosipila and Rikenellaceae. Nevertheless, MNPs stimulated intense dissimilatory iron reduction by Clostridium sp. in the early stage, competing with methanogens for substrates. Low and medium GAC doses (10 and 20% of the VSPM) increased the utilization of redox-active substances (e.g., humic substances and proteins) in EPS, while the high MNP dose stimulated their secretion due to microbial self-protection, impairing electron transfer. Metagenomic evidence showed that the medium GAC dose improved four methanogenic pathways and DIET mediated by microbial nanowires, while a high MNP dose impeded the final step of methanogenesis. These results provide deeper insights into the mechanisms of different conductive materials on dry AD.

Methanogenic degradation of long-chain fatty acids associated with syntrophic microbial dynamics during thermophilic AnMBR treatment of lipid-rich dairy wastes

Anaerobic membrane reactors (AnMBRs) have been applied to effectively treat dairy wastes because they can address the drawbacks of sludge flotation and biomass washout. However, when dairy wastes contain considerable proportion of lipids, any anaerobic systems will likely encounter challenges with long-chain fatty acid (LCFA) inhibition. In this study, a thermophilic AnMBR was operated to treat dairy processing wastewater (DPW) plus ice cream waste (ICW). To evaluate the impact of LCFAs, the lipid-rich ICW was gradually introduced at different levels (0, 5, 10, and 20 %) into the AnMBR, along with a stepwise increase in lipid loading rate (LLR) from 0.22 to 11.24 g lipid/L/d. The results of biomethanation revealed that the AnMBR could be successfully operated when the LLR was maintained at 5.73 g lipid/L/d with a lipid/VS ratio of 50.22 % in the feedstock. However, at ultra-high LLR of 11.24 g lipid/L/d (20 % ICW added), a significant inhibitory effect on methane yields was observed, resulting in a reduction of 56.90 %. Dynamic analysis of the LCFAs profiled that a lower degradation rate was an important factor contributing to LCFA accumulation, and the inhibition concentration of various LCFAs was identified. Based on microbial evolution and co-occurrence network results, potential syntrophic partnerships between LCFA-degrading bacteria and methanogens were proposed. The responsible enzyme genes in the LCFA-degrading, acetoclastic, and hydrogenotrophic methanogenic pathways played crucial roles in LCFA inhibition and degradation. These findings offer practical insights for the efficient methanogenic treatment of lipid-rich wastes.

An in-depth analysis of microbial response to exposure to high concentrations of microplastics in anaerobic wastewater fermentation

The impact of microplastics (MPs) in anaerobic wastewater treatment on microbial metabolism is significant. Anaerobic granular sludge (AS) and biofilm (BF) are two common ways, and their responses to microplastics will have a direct impact on their application potential. This study investigated the microbial reactions of AS and BF to three types of MPs: polyethylene (PE), polyvinyl chloride (PVC), and a mixture of both (MIX). Results exhibited that MPs reduced methane output by 44.65 %, 55.89 %, and 53.18 %, elevated short-chain fatty acid (SCFA) levels by 95.93 %, 124.49 %, and 110.78 %, and lowered chemical oxygen demand (COD) removal by 28.77 %, 36.78 %, and 33.99 % for PE-MP, PVC-MP, and MIX-MP, respectively, with PVC-MP showing the greatest inhibition. Meanwhile, microplastics also facilitated the relative production of reactive oxygen species (ROS, 40.29 %–96.99 %), lactate dehydrogenase (LDH, 20.01 %–75.02 %), and adenosine triphosphate (ATP, 26.64 %–43.80 %), while reducing cytochrome c (cyt c, 23.60 %–49.02 %) and extracellular polymeric substances (EPS, 17.44 %–26.58 %). AS and BF displayed distinct enzymatic activities under MPs exposure. Correspondingly, 16S-rRNA sequencing indicated that AS was mainly involved in acetate generation by Firmicutes, while BF performed polysaccharide degradation by Bacteroidota. Metatranscriptomic analysis showed AS to be rich in acetogens (Bacillus, Syntrophobacter) and methanogens (Methanothrix, Methanobacterium), while BF contained more fermentation bacteria (Mesotoga, Lentimicrobium) and electroactive microorganisms (Clostridium, Desulfuromonas) under MIX-MP. Moreover, BF exhibited higher glycolysis gene expression, whereas AS was more active in methane metabolism, primarily through the acetoclastic methanogenic pathway's direct acetate conversion. This study provides new insights into understanding the microbial response produced by microplastics during anaerobic wastewater digestion.

Biodegradable microplastics aggravate greenhouse gas emissions from urban lake sediments more severely than conventional microplastics

Freshwater ecosystems, such as urban lake sediments, have been identified as important sources of greenhouse gases (GHGs) to the atmosphere, as well as persistent sinks for ubiquitous microplastics due to the high population density and frequent anthropogenic activity. The potential impacts of microplastics on GHG production, however, remain underexplored. In this study, four types of common biodegradable microplastics (BMPs) versus four conventional non-biodegradable microplastics (NBMPs) were artificially exposed to urban lake sediments to investigate the responses of nitrous oxide (N2O) and methane (CH4) production, and make a comparison regarding how the biodegradability of microplastics affected GHG emissions. Importantly, results suggested that BMPs aggravated N2O and CH4 production in urban lake sediments more severely than conventional NBMPs. The production rates of N2O and CH4 increased by 48.78–71.88 % and 30.87–69.12 %, respectively, in BMPs groups, while those increased by only 0–25.69 % and 6.46–10.46 % with NBMPs exposure. Moreover, BMPs insignificantly affected nitrification but facilitated denitrification, while NBMPs inhibited both processes. BMPs not only created more oxygen-limited microenvironment, greatly promoting N2O production via nitrifier denitrification pathway, but also provided dissolved organic carbon favoring heterotrophic denitrification, which was primarily supported by the enriched denitrifiers and functional genes. In contrast, NBMPs slightly upregulated nitrifier denitrification pathway to generate N2O, and showed a toxic inhibition on both nitrifiers and denitrifiers. In addition, both BMPs and NBMPs promoted hydrogen-dependent methanogenic pathway but suppressed acetate-dependent pathway. The greater enhancement of CH4 production with BMPs exposure was attributed to the additional organic carbon substrates derived from BMPs and the stimulated microbial methane metabolism activities.

Intensification of ex-situ biomethanation in a bubble column bioreactor by addition of colonized biochips

Biological methanation is a promising sustainable energy technology. To intensify ex-situ biomethanation, a 3-L bubble column reactor was operated continuously under thermophilic conditions, with and without colonized biochips. Studies in batch reactors showed biofilm formation on biochips, with an archaea:bacteria ratio of 5.7 compared to 3.2 in planktonic phase with Methanothermobacter being the dominant archaea. Using colonized biochips in the bubble column increased methane production rate (MPR) nearly threefold, achieving a steady MPR of 15.7 ± 0.5 NLCH4/Lr.d at 84.4 ± 0.9 % methane content. Gas retention time (GRT) was 0.3 h, with 97.4 % and 96.5 % conversion of H2 and CO2, respectively. Volatile fatty acid (VFA) production was under 40 mg/L per day, indicating dominant hydrogenotrophic methanogenic (HM) pathway. The results suggest biofilm formation significantly enhances MPR in ex-situ methanation reactors, advancing towards industrial application.

Inhibiting methanogenesis by targeting thermodynamics and enzymatic reactions in mixed cultures of rumen microbes in vitro.

Mitigation of enteric methane (CH4) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH3) while the latter targets specific enzymes using structural analogs of CH4 and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH4 production by rumen microbes in batch culture. Sodium nitrate (NaNO3), sodium sulfate (Na2SO4), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO3 + Na2SO4 (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH4 concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH4 was highest in MF diet followed by HF and LF diets. Culture pH and NH3 in the control decreased in the order of HF, MF, to LF as expected. NaNO3 decreased (p < 0.001) CH4 and butyrate and increased acetate and propionate (p < 0.03 and 0.003, respectively). Cultures receiving NaNO3 had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH4 at 6h with no further decrease at 24 h (p < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors (p < 0.001) while enriching succinate producers and assimilators as well as propionate producers (p adj < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations (p < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H2 concentrations (p < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio (p < 0.001). Only the cultures treated with BCM in combination with other additives significantly (padj < 0.05) decreased the abundance of Methanobrevibacter expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation in-vitro, inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH3, and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition.

Mechanisms of biochar-mediated reduction of antibiotic-resistant bacteria and biogas production enhancement in anaerobic digesters

This study examined the impact of different biochar (BC) as an anaerobic digestion (AD) additive on antibiotic-resistant bacteria (ARB) survival and AD performance using dairy cow manure. Bamboo BC and Olive BC with different particle sizes were added into the mesophilic AD at 15 g/L and 30 g/L dosages (Bamboo-15, Bamboo-30, Olive-15, and Olive-30). The study provides a detailed analysis of biogas production, organic metabolism, and ARB and microbial dynamics, elucidating the mechanisms by which BC influences AD. Findings reveal significant reductions in CEZ-resistant bacteria (CEZ-r) across all reactors, ranging from 12.88 % to 76.47 %. Both Bamboo and Olive BC increased CEZ-r removal by 3.08–5.94 times compared to the control. Additionally, BC supplementation prevented the rise in CEZ-r percentage within the total bacteria count observed in the control reactor. Bamboo BC outperformed Olive BC in enhancing biogas yield, with Bamboo-15 and Bamboo-30 showing significant increases of 43.2 % and 48.0 %, respectively, compared to the control. Adding BC in AD regulates ARB by decreasing potential ARG hosts and impeding the transmission of resistance. It also enhances biogas production by improving the efficiency of methanogenic bacteria and optimizing the methanogenic pathway. This research provides insights into how BC can be used to enhance AD performance and mitigate ARB proliferation, offering a sustainable approach to waste management and energy production.

Microbial communities and functions are structured by vertical geochemical zones in a northern peatland

Northern peatlands are important carbon pools; however, differences in the structure and function of microbiomes inhabiting contrasting geochemical zones within these peatlands have rarely been emphasized. Using 16S rRNA gene sequencing, metagenomic profiling, and detailed geochemical analyses, we investigated the taxonomic composition and genetic potential across various geochemical zones of a typical northern peatland profile in the Changbai Mountains region (Northeastern China). Specifically, we focused on elucidating the turnover of organic carbon, sulfur (S), nitrogen (N), and methane (CH4). Three geochemical zones were identified and characterized according to porewater and solid-phase analyses: the redox interface (<10 cm), shallow peat (10–100 cm), and deep peat (>100 cm). The redox interface and upper shallow peat demonstrated a high availability of labile carbon, which decreased toward deeper peat. In deep peat, anaerobic respiration and methanogenesis were likely constrained by thermodynamics, rather than solely driven by available carbon, as the acetate concentrations reached 90 μmol·L−1. Both the microbial community composition and metabolic potentials were significantly different (p < 0.05) among the redox interface, shallow peat, and deep peat. The redox interface demonstrated a close interaction between N, S, and CH4 cycling, mainly driven by Thermodesulfovibrionia, Bradyrhizobium, and Syntrophorhabdia metagenome-assembled genomes (MAGs). The archaeal Bathyarchaeia were indicated to play a significant role in the organic carbon, N, and S cycling in shallow peat. Although constrained by anaerobic respiration and methanogenesis, deep peat exhibited a higher metabolic potential for organic carbon degradation, primarily mediated by Acidobacteriota. In terms of CH4 turnover, subsurface peat (10–20 cm) was a CH4 production hotspot, with a net turnover rate of ∼2.9 nmol·cm−3·d−1, while the acetoclastic, hydrogenotrophic, and methylotrophic methanogenic pathways all potentially contributed to CH4 production. The results of this study improve our understanding of biogeochemical cycles and CH4 turnover along peatland profiles.

Comparison of anaerobic co-digestion of vacuum toilet blackwater and kitchen waste under mesophilic and thermophilic conditions: Reactor performance, microbial response and metabolic pathway

Co-digestion of kitchen waste (KW) and black water (BW) can be considered as an attractive method to efficiently achieve the clean energy from waste. To find the optimal operation parameters for the co-digestion, the effects of different temperatures (35 and 55 °C) and BW:KW ratios on the reactor performances, microbial communities and metabolic pathways were studied. The results showed that the optimum BW:KW ratio was 1:3.6 and 1:4.5 for mesophilic and thermophilic optimal reactors, with methane production of 449.04 mL/g VS and 411.90 mL/g VS, respectively. Microbial communities showed significant differences between the reactors under different temperatures. For bacteria, increasing BW:KW ratio significantly promoted Defluviitoga enrichment (1.1%–9.5%) under thermophilic condition. For Archaea, the increase in BW:KW ratio promoted the enrichment of Methanosaeta (8.6%–56.4%) in the mesophilic reactor and Methanothermobacter (62.0%–89.2%) in the thermophilic reactor. The analysis of the key enzymes showed that, acetoclastic methanogenic pathway performed as the dominant under mesophilic condition, with high abundance of Acetate-CoA ligase (EC:6.2.1.1) and Pyruvate synthase (EC:1.2.7.1). Hydrogenotrophic methanogenic pathway was the main pathway in the thermophilic reactors, with high abundance of Formylmethanofuran dehydrogenase (EC:1.2.99.5).

Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion.

Over 20 years ago, the pyrrolysine encoding translation system was discovered in specific archaea. Our Review provides an overview of how the once obscure pyrrolysyl-tRNA synthetase (PylRS) tRNA pair, originally responsible for accurately translating enzymes crucial in methanogenic metabolic pathways, laid the foundation for the burgeoning field of genetic code expansion. Our primary focus is the discussion of how to successfully engineer the PylRS to recognize new substrates and exhibit higher in vivo activity. We have compiled a comprehensive list of ncAAs incorporable with the PylRS system. Additionally, we also summarize recent successful applications of the PylRS system in creating innovative therapeutic solutions, such as new antibody-drug conjugates, advancements in vaccine modalities, and the potential production of new antimicrobials.