Methanogenesis Pathway Research Articles

Isolation and characterization of Methanosphaera sp. ISO3-F5, a member of a novel and widespread species of rumen methanogens growing with methanol plus hydrogen

Rumen methanogens predominantly fall into two physiological groups: hydrogenotrophs which use hydrogen (H2) to reduce carbon dioxide (CO2) to methane (CH4), and methylotrophs which use H2 to reduce methanol and methylamines as substrates for methanogenesis. We used a dilution to extinction approach to isolate two hydrogenotrophic Methanocatella spp. and four cultures of methylotrophic methanogens from sheep rumen contents. Three of the methylotrophs were stable mixed cultures containing methanogens belonging to different lineages of the order Methanomassiliicoccales and one was a pure Methanosphaera culture. Methanosphaera sp. ISO3-F5 has a comparatively large genome (2.68 Mb) comprised of two replicons, a chromosome and a megaplasmid. The genome has an average G + C content of 30.5 % and encodes 2360 putative protein-coding genes. Cells of ISO3-F5 have a spherical shape, 0.6–1.2 µm in diameter, usually occurring in pairs or loose clumps, and have no flagellum. Cells stain Gram positive, have a single thick cell wall and divide by the formation of a cross wall. The optimum temperature for growth was 39°C to 42°C and the optimum pH was 6.7–6.8. Acetate was required for growth, but CH4 was not produced from acetate, formate, ethanol, methylamine, or isopropanol with or without H2/CO2. Volatile fatty acids and rumen fluid were also found to enhance the growth of ISO3-F5, while coenzyme M did not. ISO3-F5 produced CH4 from methanol in the presence of H2 and the genes encoding the necessary methanogenesis pathway have been identified. Based on morphological, physiological, and genomic characteristics, ISO3-F5 is a new species of the genus Methanosphaera. Our study shows that simple isolation methods allowed us to culture diverse and significant members of the rumen methanogen community.

Strengthening the syntrophic pathway for acetate oxidation-hydrogenotrophic methanogenesis by biogas stirring for effectively mitigating acidification in anaerobic digestion

Strengthening the syntrophic pathway for acetate oxidation-hydrogenotrophic methanogenesis by biogas stirring for effectively mitigating acidification in anaerobic digestion

Cobalt-modified digestate-derived biochar enhances kitchen waste anaerobic dry digestion: Performance, microbial mechanisms, and metabolic pathways

To achieve simultaneous in-situ high-value utilization of digestate and efficient resource recovery of kitchen waste (KW). In this work, Co-modified digestate-derived biochar (DDB-Co) was prepared for the first time, the effects of DDB-Co on KW mesophilic dry anaerobic digestion were investigated, and the underlying mechanisms were elucidated. The results showed that the specific surface area of DDB-Co increased to 98.7 m2/g, which was 37.4 % higher than that of the unmodified group (DDB). DDB-Co exhibited a dose-dependent effect on biogas production from the KW dry digestion, with the optimal dose of 4.0 g/L (calculated on a solid content) DDB-Co yielding 205.3 mL/(g·VS) of biogas, representing an 80.9 % increase compared to the CK group. However, higher doses, such as 8.0 g/L DDB-Co, reduced the cumulative biogas production. Mechanistic analysis indicated that 4.0 g/L DDB-Co facilitated the dissolution of particulate organic matter and accelerates the conversion process of volatile fatty acids (VFA) in KW, thereby preventing excessive acidification. Microbial community structure analysis revealed that DDB-Co reduced microbial richness and diversity but selectively enriched microorganisms such as Lactobacillus and Methanothrix, accelerating the direct interspecific electron transfer (DIET) reaction rate and increasing biogas production. Metagenomic analysis demonstrated that DDB-Co primarily upregulated the relative abundances of key enzymes in the acetate decarboxylation methanogenesis pathway, such as acetate kinase (ackA), phosphate acetyltransferase (pta), and acetyl-CoA synthetase (acs), enhancing biogas yield and maintaining system stability. The findings of this work expand the high-value utilization pathways for digestate and provide an alternative solution for the efficient resource recovery of KW.

Gut microbiota predict retinopathy in patients with diabetes: A longitudinal cohort study

The gut microbiota has emerged as an independent risk factor for diabetes and its complications. This research aimed to delve into the intricate relationship between the gut microbiome and diabetic retinopathy (DR) through a dual approach of cross-sectional and prospective cohort studies. In our cross-sectional study cross-sectional investigation involving ninety-nine individuals with diabetes, distinct microbial signatures associated with DR were identified. Specifically, gut microbiome profiling revealed decreased levels of Butyricicoccus and Ruminococcus torques group, alongside upregulated methanogenesis pathways among DR patients. These individuals concurrently exhibited lower concentrations of short-chain fatty acids in their plasma. Leveraging machine learning models, including random forest classifiers, we constructed a panel of microbial genera and genes that robustly differentiated DR cases. Importantly, these genera also demonstrated significant correlations with dietary patterns and the molecular profiles of peripheral blood mononuclear cells. Building upon these findings, our prospective cohort study followed 62 diabetes patients over a 2-year period to assess the predictive value of these microbial markers. The results underlined the panel’s efficacy in predicting DR incidence. By stratifying patients based on the predictive genera and metabolites identified in the cross-sectional phase, we established significant associations between reduced levels of Butyricicoccus, plasma acetate, and increased susceptibility to DR. This investigation not only deepens our understanding of how gut microbiota influences DR but also underscores the potential of microbial markers as early indicators of disease risk. These insights hold promise for developing targeted interventions aimed at mitigating the impact of diabetic complications.Key points• Microbial signatures are differed in diabetic patients with and without retinopathy• DR-related taxa are linked to dietary habits and transcriptomic profiles• Lower abundances of Butyricicoccus and acetate were prospectively associated with DR

Enhanced Rock Weathering as a Source of Metals to Promote Methanogenesis and Counteract CO2 Sequestration.

Enhanced weathering of (ultra)mafic rocks has been proposed as a promising approach to sequester atmospheric CO2 and mitigate climate change. However, these silicate rocks contain varying amounts of trace metals, which are essential cofactors of metallaenzymes in methanogens. We found that weathering of crushed peridotite and basalt significantly promoted the growth and methanogenesis of a model methanogen─Methanosarcina acetivorans C2A under the condition of excess substrate. The released trace metals from peridotite and basalt, especially Fe, Ni, and Co, accounted for the promotion effect. Observation at different spatial scales showed a close association between the rocks and cells. Proteomic analysis revealed that rock amendment significantly enhanced the expression of core metalloenzymes in the methylotrophic methanogenesis pathway. Our study uncovers a previously unrecognized but important negative effect of enhanced rock weathering on methane production, which may counteract the carbon sequestration effort.

Coupling Fe3O4 and micro-hydrogen for enhancing the anaerobic bio-conversion of phenol: Methanogenesis pathway and interspecies electron transfer

Low anaerobic biological conversion rate is a challenge in the anaerobic biological treatment of phenol wastewater. The dominant syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway is expected to solve the problem. However, there are raw studies on artificially simulating a dominant SAO-HM pathway for enhancing the anaerobic bio-conversion of phenol. In this study, Fe3O4 and micro-hydrogen (Fe3O4/H2) was used to strengthen the anaerobic bio-conversion of phenol by simulating the dominant SAO-HM pathway. The results suggested Fe3O4 and H2 had a coupling promotion on the anaerobic bio-conversion of phenol and Fe3O4/H2 increased the relative abundance of Syntrophus (29.35%), Syntrophorhabdu (3.27%), Clostridium (3.01%) and Methanobacterium (65.74%), indicating Fe3O4/H2 formed a dominant SAO-HM pathway. However, Fe3O4/H2 obviously reduced the conductivity of sludge, the extracellular protein and the functional gens pilA, pilB and OmcS, implying direct interspecies electron transfer (DIET) generated by Fe3O4 was interfered by micro hydrogen in extracellular electron transport process which reduced the efficiency of extracellular electron transport. This study remind that extracellular electron transport cannot be ignored in the dominate SAO-HM pathway for the anaerobic bio-conversion of phenol.

Synchronously improving intracellular electron transfer in electron-donating bacteria and electron-accepting methanogens for facilitating direct interspecies electron transfer during anaerobic digestion of kitchen wastes

Two major issues generally limit the effectiveness of direct interspecies electron transfer (DIET) during anaerobic digestion: 1) lack of essential electrical connection component, electrically conductive pili (e-pili) or multi-heme c-type cytochrome (MHC); 2) the thermodynamic limitations of combining electrons with protons for reducing carbon dioxide to methane. To address both issues, a strategy for facilitating DIET via combining ethanol-type fermentation pretreatment (EFP) with NaCl addition during anaerobic digestion of kitchen wastes was proposed. Combining EFP with NaCl addition dramatically increased methane yield rates (531.3 ± 11.4 vs 410.5 ± 8.7 mL/gVSadded/d) and efficiencies of conversion of organic substrates to methane (571.2 ± 13.8 vs 488.7 ± 11.7 mL/gVSremoval). The increased performances by combining EFP with NaCl addition were higher than that by independent EFP or NaCl addition. Paludibacter sp., Petrimonas sp. and Syntrophomonas wolfei were the predominant and metabolically active electron-donating bacteria, and their expression of genes for e-pili/MHCs by combining EFP with NaCl addition was higher than that by independent EFP or NaCl addition. Methanothrix soehngenii and Methanoculleus bourgensis were the predominant and metabolically active electron-accepting methanogens, and their expression of genes for carbon dioxide reduction pathway for methanogenesis by combining EFP with NaCl addition was higher than that by independent EFP or NaCl addition. In addition, EFP specifically increased the transcript abundance of genes for NAD+/NADH transformation closely associated with the formation of e-pili/MHCs, while NaCl addition specifically increased the transcript abundance of genes for coenzyme F420/F420H2 transformation known to participate in reduction of carbon dioxide to methane.

Quorum-sensing molecules regulate biochar-assisted anaerobic digestion system for methane production: Single-stage vs. two-stage digestion

Promoting methane production performance from waste activated sludge is of great significance for energy self-sufficiency operation of wastewater treatment plants. This study evaluated the methane production enhancement strategies of biochar and quorum-sensing molecules like N-acylated homoserine lactone (AHL) in single-stage and two-stage anaerobic digestion systems. The methane yield increased to 134.9 mL/g volatile suspended solids in single-stage digestion system, which was 41.7 % higher than that of control group, while it in two-stage digestion system was not only lower than that obtained in single-stage digestion system but also less than that observed in groups adding AHL either at the beginning or on the third day of experiment period. The mechanism studies indicated that the single-stage digestion strategy promoted solubilization, hydrolysis, acetogenesis, and methanogenesis stages and improved the conversion and utilization of organic matters with the enhanced syntrophic metabolisms between microorganisms. In addition, the electron transfer activity between microorganisms was promoted, with an increase ratio of 54.7 %, meanwhile, the syntrophic relationships between methanogens such as Methanosarcina and electroactive bacteria such as norank_f__Bacteroidetes_vadinHA17 were promoted. As a result, both acetotrophic and hydrogenotrophic methanogenesis pathways were improved. This study provided some novel approaches for promoting anaerobic digestion performance while using biochar as the additive.

Methylotrophic substrates stimulated higher methane production than competitive substrates in mangrove sediments

Although mangrove forests can uptake atmospheric CO2 and store carbon as organic matter called “blue carbon”, it is also an important natural source of greenhouse gas methane. Methanogens are major contributors to methane and play important roles in the global carbon cycle. However, our understanding of the key microbes and metabolic pathways responsible for methanogenesis under specific substrates in mangrove sediments is still very limited. Here, we set an anaerobic incubation to evaluate the responses of methanogens in mangrove sediments from South China to the addition of diverse methanogenic substrates (H2/CO2, acetate, trimethylamine (TMA), and methanethiol (MT)) and further investigated the dynamics of the whole microbial community. Our results showed that diverse substrates stimulated methanogenic activities at different times. The stimulation of methanogenesis was more pronounced at early and late periods by the addition of methylotrophic substrates TMA and MT, respectively. The amplicon sequencing analysis showed that genus Methanococcoides was mainly responsible for TMA-utilized methanogenesis in mangrove sediment, while the multitrophic Methanococcus was most abundant in H2/CO2 and MT treatments. Apart from that, the bacteria enrichments of Syntrophotalea, Clostridium_sensu_stricto_12, Fusibacter in MT treatments might also be associated with the stimulation of methane production. In addition, the metagenomic analysis suggested that Methanosarcinaceae was also one of the key methanogens in MT treatments with different genomic information compared to that in TMA treatments. Finally, the total relative abundances of methanogenesis-related genes were also highest in TMA and MT treatments. These results will help advance our understanding of the contributions of different methanogenesis pathways and methanogens to methane emissions in mangrove sediments.

Experimental research on biogenic methane generation pathway and microflora constraints based on in-situ gas-water-microbe

Biogenic coalbed methane (CBM), generated through the biodegradation process of coal organic matter, is a relatively clean and environment-friendly unconventional natural gas resource. To investigate the differences in microbial communities (bacteria and archaea) as well as their environmental influencing factors between the Permian Shanxi Formation (P1s) and Taiyuan Formation (P1t), we systematically collected in-situ CBM samples along with coalbed water and microbial samples from a representative biogenic CBM field – the Baode block locked in the Ordos basin in China. The relative content of biogenic CBM was quantitatively calculated while analyzing gas composition, stable isotope, ion content, and high-throughput sequencing analysis to discuss the generation pathway for biogenic CBM and environmental control factors affecting microbial community structure. The results indicate that P1s and P1t are mixed with early thermogenic gas and secondary biogenic gas genetic types within their respective CBMs. The average proportion for biogenic gas within current coal seams is approximately 62.24 % for P1s, while it reaches around 64.37 % for P1t. Water quality type primarily consists of Na-HCO3 type with total dissolved solids (TDS) measuring at approximately 1015.86 mg/L for P1s, whereas P1t exhibits Na-Ca-HCO3-Cl water type with TDS content reaching about 2314.85 mg/L. Combining dominant Methanobacterium species, along with Δ13C(CO2-CH4) ratios and δD(CH4) versus δD(H2O) indexes, represents the biogenic CBM primarily generated through the hydrogenotrophic methanogenesis pathway. The in-situ bacterial communities of P1s are mainly influenced by Ni, pH, and TDS. The archaeal communities exhibit higher sensitivity to environmental factors (such as Co, pH, Ni, and dissolved organic carbon) than bacteria. These findings hold significant implications for understanding the generation process of biogenic CBM in middle and low-rank coal areas.

Optimization of the anaerobic digestion process and the metabolic pathway of methanogenesis of Jerusalem artichoke straw with the mixed addition of iron filings and biochar

This study explored the feasibility of the mixed addition of iron filings and biochar in the anaerobic digestion of Jerusalem artichoke straw (JAS), using iron filings and biochar as mixed additives for mid-temperature batch anaerobic digestion experiments, and applied response surface methodology to investigate the interactive effects of different ratios of iron filings and biochar (FE/BC), solid content, and initial pH on the cumulative methane production from JAS. The results showed that the optimal conditions for methane production were an FE/BC of 2.45:7.55, an initial pH of 7.59, and a solid content of 8.80 %. Under these conditions, the cumulative methane production was 278.11 mL g−1 volatile solid (VS) contents, which was very close to the predicted value (286.42 mL g−1 VS), with a relative error of less than 5 %, indicating the effectiveness of the model. The optimized conditions with mixed addition (HB) exhibited good synergistic promotion, increasing cumulative methane production by 48.69 % compared to the control without additives and by 22.29 % and 29.57 % compared to the single addition of iron filings and biochar, respectively. Moreover, the HB treatment under optimized conditions achieved good economic benefits. Metagenomic analysis showed that the relative abundances of bacterial taxa including Fermentimonas, DMER64, Bacteroidetes_VadinHA17, unclassified Synergistaceae, Clostridium, Ruminiclostridium, and unclassified Anaerolineaceae were higher in HB samples, and Methanothrix was also enriched. Methane metabolism pathway analysis indicated that the anaerobic digestion of JAS with the mixed addition of iron filings and biochar enhanced the acetoclastic methane production pathway, thereby increasing methane production.

Sludge bound-EPS solubilization enhance CH4 bioconversion and membrane fouling mitigation in electrochemical anaerobic membrane bioreactor: Insights from continuous operation and interpretable machine learning algorithms

Bound extracellular polymeric substances (EPS) are complex, high-molecular-weight polymer mixtures that play a critical role in pore clogging, foulants adhesion, and fouling layer formation during membrane filtration, owing to their adhesive properties and gelation tendencies. In this study, a novel electrochemical anaerobic membrane bioreactor (EC-AnMBR) was constructed to investigate the effect of sludge bound-EPS solubilization on methane bioconversion and membrane fouling mitigation. During the 150-days’ operation, the EC-AnMBR demonstrated remarkable performance, characterized by an exceptionally low fouling rate (transmembrane pressure (TMP) < 4.0 kPa) and high-quality effluent (COD removal > 98.2 %, protein removal > 97.7 %, and polysaccharide removal > 98.5 %). The highest methane productivity was up to 38.0 ± 3.1 mL/Lreactor/d at the applied voltage of 0.8 V with bound-EPS solubilization, 107.6 % higher than that of the control stage (18.3 ± 2.4 mL/Lreactor/d). Morphological and multiplex fluorescence labeling analyses revealed higher fluorescence intensities of proteins, polysaccharides, total cells and lipids on the surface of the fouling layer. In contrast, the interior exhibited increased compression density and reduced activity, likely attributable to compression effect. Under the synergistic influence of the electric field and bound-EPS solubilization, biomass characteristics exhibited a reduced propensity for membrane fouling. Furthermore, the bio-electrochemical regulation enhanced the electroactivity of microbial aggregates and enriched functional microorganisms, thereby promoting biofilm growth and direct interspecies electron transfer. Additionally, the potential hydrogenotrophic and methylotrophic methanogenesis pathways were enhanced at the cathode and anode surfaces, thereby increasing CH₄ productivity. The random forest-based machine learning model analyzed the nonlinear contributions of EPS characteristics on methane productivity and TMP values, achieving R² values of 0.879 and 0.848, respectively. Shapley additive explanations (SHAP) analysis indicated that S-EPSPS and S-EPSPN were the most critical factors affecting CH₄ productivity and membrane fouling, respectively. Partial dependence plot analysis further verified the marginal and interaction effects of different EPS layers on these outcomes. By combining continuous operation with interpretable machine learning algorithms, this study unveils the intricate impacts of EPS characteristics on methane productivity and membrane fouling behaviors, and provides new insights into sludge bound-EPS solubilization in EC-AnMBR.

Versatile enhancement for anaerobic moving bed biofilm (AnMBBR) treating pretreated landfill leachate by hydrochar: Energy recovery, greenhouse gas emission reduction and underlying microbial mechanisms

Hydrochars were prepared from fruit peels (HC-1) and vegetable waste (HC-2), and combined with fiber spheres, respectively, to form homogeneous biocompatible carriers, which were used for anaerobic moving bed biofilm reactor (AnMBBR) to enhance anaerobic digestion (AD) performance and energy recovery of landfill leachate treatment. Compared with the control AnMBBR with conventional fiber spheres as carriers, the chemical oxygen demand (COD) removal efficiency of the AnMBBR with HC-2 increased from 75 % to 88 %, methane yield increased from 77.7 mL/g-COD to 155.3 mL/g-COD, and achieved greenhouse gases (GHG) emission reductions of 1.74 t CO2 eq/a during long-term operation. HC-2-fiber sphere biocarriers provided more sites for attached-growth biomass (AGBS) and significantly enhanced the abundance of functional microbial community, with the relative abundance of methanogenic bacteria Methanothrix increased from 0.03 % to over 24.4 %. Moreover, the gene abundance of most the key enzymes encoding the hydrolysis, acidogenesis and methanogenesis pathways were up-regulated with the assistance of HC-2. Consequently, hydrochar-assisted AnMBBR were effective to enhance methanogenesis performance, energy recovery and carbon reduction for high-strength landfill leachate treatment.

Methyl-reducing methanogenesis by a thermophilic culture of Korarchaeia.

Methanogenesis mediated by archaea is the main source of methane, a strong greenhouse gas, and thus is critical for understanding Earth's climate dynamics. Recently, genes encoding diverse methanogenesis pathways have been discovered in metagenome-assembled genomes affiliated with several archaeal phyla1-7. However, all experimental studies on methanogens are at present restricted to cultured representatives of the Euryarchaeota. Here we show methanogenic growth by a member of the lineage Korarchaeia within the phylum Thermoproteota (TACK superphylum)5-7. Following enrichment cultivation of 'Candidatus Methanodesulfokora washburnenis' strain LCB3, we used measurements of metabolic activity and isotope tracer conversion to demonstrate methanol reduction to methane using hydrogen as an electron donor. Analysis of the archaeon's circular genome and transcriptome revealed unique modifications in the energy conservation pathways linked to methanogenesis, including enzyme complexes involved in hydrogen and sulfur metabolism. The cultivation and characterization of this new group of archaea is critical for a deeper evaluation of the diversity, physiology and biochemistry of methanogens.

Diverse and unconventional methanogens, methanotrophs, and methylotrophs in metagenome-assembled genomes from subsurface sediments of the Slate River floodplain, Crested Butte, CO, USA.

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling-particularly methane-cycling-microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and <10% contamination) from 50- to 150-cm depth below the sediment surface capturing the transition between oxic, unsaturated sediments and anoxic, saturated sediments in the Slate River (SR) floodplain (Crested Butte, CO, USA). We recovered genomes of putative methanogens, methanotrophs, and methylotrophs (n = 57). Methanogens, found only in deep, anoxic depths at SR, originate from three different clades (Methanoregulaceae, Methanotrichaceae, and Methanomassiliicoccales), each with a different methanogenesis pathway; putative methanotrophic MAGs originate from within the Archaea (Candidatus Methanoperedens) in anoxic depths and uncultured bacteria (Ca. Binatia) in oxic depths. Genomes for canonical aerobic methanotrophs were not recovered. Ca. Methanoperedens were exceptionally abundant (~1,400× coverage, >50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca. Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca. Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca. Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy.IMPORTANCEThe cycling of carbon by microorganisms in subsurface environments is of particular relevance in the face of global climate change. Riparian floodplain sediments contain high organic carbon that can be degraded into C1 compounds such as methane, methanol, and methylamines, the fate of which depends on the microbial metabolisms present as well as the hydrological conditions and availability of oxygen. In the present study, we generated over 1,000 MAGs from subsurface sediments from a montane river floodplain and recovered genomes for microorganisms that are capable of producing and consuming methane and other C1 compounds, highlighting a robust potential for C1 cycling in subsurface sediments both with and without oxygen. Archaea from the Ca. Methanoperedens genus were exceptionally abundant in one sample, indicating a potential C1/methane-cycling hotspot in the Slate River floodplain system.

Alkaline Pre-Fermentation Promotes Anaerobic Digestion of Enhanced Membrane Coagulation (EMC) Sludge: Performance and Microbial Community Response

Concentrating organic matter in sludge and converting it into methane through anaerobic bioconversion can improve resource recovery from domestic wastewater. Enhanced membrane coagulation (EMC) is highly efficient at concentrating organic matter, but residual coagulants (aluminum salts) can obstruct bioconversion by blocking microbial access. Limited research exists on evaluating EMC sludge bioconversion performance and addressing coagulant inhibition. This study proposes alkaline pre-fermentation to break down HO-Al-P backbones in coagulated sludge flocs, thereby improving hydrolysis and organic acid production for anaerobic digestion. Among the tested alkaline conditions (pH 9, pH 10, pH 11), pre-fermentation at pH 11 released the most organic matter (4710.0 mg/L SCOD), 20.4 times higher than without alkaline treatment. At pH 11, phosphate (61 mg/L PO43−–P) and organic acid production (2728.1 mg COD/L, with nearly 50% acetic acid) peaked, resulting in superior volatile solids removal (65.2%) and methane production (185.8 mL/g VS) during anaerobic digestion. Alkaline pre-fermentation favored alkali-tolerant bacteria such as Firmicutes and Actinobacteria, especially at pH 11, while neutrophilic Proteobacteria were suppressed. Trichococcus and Bifidobacterium, known acid producers, dominated under all conditions, with their abundance increasing at higher pH levels. Anaerobic digestion enriched fermentative bacteria like Chloroflexi and Synergistota (e.g., Thermovirga), especially in high pH reactors. Methanothrix, an acetoclastic methanogen, became the dominant methanogenic archaeon, indicating that methane production from EMC sludge primarily followed the acetoclastic methanogenesis pathway. Our findings demonstrate that alkaline pre-fermentation at pH 11 significantly enhances the hydrolysis efficiency of EMC sludge for methane recovery.

Differential effects of GAC and graphite on interspecies electron transfer during anaerobic digestion of waste activated sludge for methane production

This study investigated the effect of the particle size of granular activated carbon (GAC) and graphite on methane production efficiency. Additionally, differences in the mechanisms by which GAC and graphite-enhanced anaerobic digestion (AD) of waste activated sludge (WAS) were evaluated. The results showed that 150 μm GAC and 38 μm graphite had the best promoting effect, increasing cumulative biogas production by 14.6 % and 12.3 %, respectively, compared with blank control. GAC advanced the peak daily biogas production to day 8. Scanning electron microscopy revealed the formation of a dense bacterial biofilm on the GAC surface. Microorganisms on the graphite surface had more conductive pili than those on the GAC and blank surfaces. GAC enriched Methanobacterium (hydrogenotrophic methanotrophic archaea) while maintaining the relative abundance of existing acetate methanogenic bacteria. Conversely, graphite reduced the abundance of hydrogenotrophic methanogenic bacteria and enriched Methanosaeta, which participate in direct interspecies electron transfer. GAC increased the abundance of key enzymes in the hydrogenotrophic methanogenesis pathway and alleviated the inhibition of nitrate nitrogen and H2 in the methanogenesis reaction system. In contrast, graphite increased the abundance of pili genes and promoted electron exchange between symbiotic bacteria and methanogens, thereby accelerating substrate consumption and methane production.

Magnetite encapsulated in carbon shell particles (Fe3O4@C) to boost anaerobic methanogenesis of chloramphenicol wastewater

Magnetite (Fe3O4) is extensively applied to enhance efficacy of anaerobic biological treatment systems designed for refractory wastewater. However, the interaction between magnetite, organic pollutants and microorganisms in digestion solution is constrained by magnetic attraction. To overcome this limitation and prevent magnetite aggregation, the core-shell composite materials with carbon outer layer enveloping magnetite core particles (Fe3O4@C) were developed. The impact of Fe3O4@C with varying Fe3O4 mass ratios on the anaerobic methanogenesis capability in the treatment of chloramphenicol (CAP) wastewater was investigated. Experimental results demonstrated that Fe3O4@C not only enhanced chemical oxygen demand (COD) removal efficiency and biogas production by 2.42-13.18% and by 7.53%-23.25%, respectively, but also reduced the inhibition of microbial activity caused by toxic substances and the secretion of extracellular polymeric substances (EPS) by microorganisms responding to adverse environments. The reinforcing capability of Fe3O4@C increased with the rise in Fe3O4 content. Furthermore, High-throughput pyrosequencing illustrated that Fe3O4@C enhanced the relative abundance of Methanobacterium, a hydrogen-utilizing methanogen capable of participating in direct interspecies electron transfer (DIET), by 5%. Metagenomic analysis indicated that Fe3O4@C improved the decomposition of complex organics into simpler compounds by elevating functional genes encoding key enzymes associated with organic matter metabolism, acetogenesis, and hydrogenophilic methanogenesis pathways. These findings suggest that Fe3O4@C have the potential to strengthen both the hydrogenophilic methanogenesis and DIET processes. This insight offers a novel perspective on the anaerobic bioaugmentation of high-concentration refractory organic wastewater.

Synergic effect of thermo-chemical pretreatment of waste-activated sludge on bio-methane enhancement

Sustainable and environmentally friendly energy production is feasible via anaerobic digestion (AD) of organic wastes, such as waste-activated sludge (WAS). However, due to its limited degradation, a pretreatment strategy is applied to WAS to enhance its bio-degradation and, thus, biogas yield. Alkaline (0.5%–9% g NaOH/gTS, 30 min), microwave (MW) (90°C–175°C), and hybrid (0.5% g NaOH/gTS +125°C) pretreatments were applied to WAS. The characterization of untreated and pretreated WAS revealed that with higher alkaline and MW pretreatment, the soluble chemical oxygen demand (sCOD), carbohydrate, and protein increased; however, the readily biodegradable COD (rbCOD) rate was unlike the sCOD. The sCOD was 7%–18%, 8%–23%, and 37% for alkaline, MW, and hybrid pretreatments, respectively. Stronger alkaline and MW pretreatment induced higher turbidity, capillary suction time, and lower average particle size. AD of alkaline-, MW-, and hybrid-pretreated WAS produced 94% (0.5% NaOH), 125% (MW at 125°C), and 199% (0.5% NaOH and MW at 125°C) increased biogas, respectively, compared to the AD of untreated sludge. The AD data on the alkaline-, MW-, and hybrid-pretreated BMP assays fitted well with the modified Gompertz model with a coefficient of determination above 0.95. The PCA analysis showed that biogas production is closely correlated with pretreatment temperature, VFA production, rbCOD, sCOD, and soluble carbohydrates and protein. Microbial genome sequencing analysis showed an improvement in microbial abundance and diversity. Acetoclastic methanogen (Methanothrix) growth was improved by 37% (MW pretreatment). Abundances of Methanosarcina, using all three metabolic pathways for methanogenesis, were 17, 21, 11, and 48% in the control, alkaline-, MW-, and hybrid-pretreated digestate, respectively, corresponding to 186% improvement in hybrid pretreatment when compared to the control.

Biochar alleviates inhibition effects of humic acid on anaerobic digestion: Insights to performances and mechanisms

To recover methane from waste activated sludge through anaerobic digestion (AD) is one promising alternative to achieve carbon neutrality for wastewater treatment plants. However, humic acids (HAs) are one of the major compositions in waste activated sludge, and their accumulation performs inhibition effects on AD. This study investigated the potentials of biochar (BC) in alleviating inhibition effects of HAs on AD. Results showed that although the accumulated HAs reduced methane yield by 9.37% compared to control, the highest methane yield, 132.6 mL CH4/g VSS, was obtained after adding BC, which was 45.9% higher than that in HA group. Mechanism analysis showed that BC promoted the activities of hydrolase such as protease and α-glucosidase, which were 69.7% and 29.7% higher than those in HA group, respectively. The conversion of short-chain fatty acids was accelerated. In addition, the evolutions of electroactive microorganisms like Clostridium_sensu_stricto_13 and Methanosaeta were consistent with the activitiies of electron transfer and the contents of cytochrome c. Furthermore, parts of HAs rather than all of them were adsorbed by BC, and the remaining free HAs and BC formed synergistic effects on methanogenesis, then both CO2 reduction and acetoclastic methanogenesis pathways were improved. The findings may provide some solutions to alleviate inhibition effects of HAs on AD.