Synergistic Mechanism Of Methane Production In Anaerobic Microbial Consortium AMC-9

Anaerobic biogas fermentation technology is widely used in organic waste treatment,sewage treatment and biomass energy production.The anaerobic fermentation system is essentially driven by a combination of microorganisms with different functional types.Studying the biological process of how microorganisms synergistically degrade and convert substrates into biogas is of great significance for people to understand the scientific issues behind this natural phenomenon,and then effectively artificially regulate the fermentation process.However,the substrates in anaerobic fermentation are usually composed of complex substrates and complex microorganisms,which makes it difficult to clearly elucidate the mechanism of microbial synergy.If a simple single substrate is used as a carbon source to study,the biological process will be simplified,and it will be beneficial to analyze the mechanism of microbial synergy during anaerobic fermentation.Therefore,a biogas-producing complex bacterial system obtained in the early stage of this study using cow dung as a substrate was domesticated by using a simple and single compound as a substrate,trying to obtain a biogas fermentation complex bacterial system using a single compound as a carbon source.The synergistic mechanism of microbial methane production was studied by using this complex strain as a model flora.The gas production of the initial biogas fermentation compound bacteria reached the peak gas production on the 4th day.Methanol,sodium formate,sodium acetate,sodium formate+methanol were used as substrates instead of cow dung to domesticate the compound bacterial strains for continuous passage.The total methane production and OD₆₀₀nm were measured,and it was found that the bacteria with methanol as the substrate grew the fastest,had the best gas production effect,and could be stably passaged,while other treatments did not obtain a system that could be stably passaged,so methanol was used as the substrate.The domesticated biogas fermentation complex strain was used as the research object for follow-up research,and the strain was named AMC-9.Sampling was selected at the initial fermentation（FP）,peak fermentation（FF）,and final fermentation（FT）stages,and samples were sent to analyze the changes of microorganisms and infer the methanogenesis metabolic pathway to analyze the methanogenesis mechanism.The resul of metagenomic annotation showed that Desulfovibrio,Sporanaerobacter-acetigenes,Methanobacterium and Lentimicrobium were significantly higher in the peak gas production period than in the early and late periods.KEGG analysis found that map00680 was a methane metabolism pathway,and annotated the pathway of methanol metabolism to produce methane.The related enzymes and gene expressions were compared and analyzed,and it was found that the mcr A and hdr A2 gene contents were higher in the peak and late methane production stages,and the Other mtr A,hdr A1,mat A,mta B were annotated,these genes and enzymes are involved in various pathways of methanol metabolism and are key to methanol methanogenesis.The synergistic mechanism of methanol conversion to methane production was drawn by analyzing the annotation results,in which methanol（CH₃OH）was catalyzed by mta A,mta B and HS-Co M to generate Methy1-Co M,and then Methy1-Co M and Coenzyme B were catalyzed by mcr A to generate Co M-S-S-Co B and CH4.Among them,Co M-S-S-Co B can generate Coenzyme M and Coenzyme B under the action of hdr A1 and hdr A2,and Coenzyme M and Methy1-Co M can be converted into each other under the action of mtr A.There are 20 methanogenic archaea involved in the whole process.The methanol-substrate methanogenic complex strain AMC-9 was obtained through the domestication of the substrate.The abundance of methanogens and functional enzymes in the peak period of methanogenesis were significantly higher than those of the initial and final methanol conversion rates.The metabolic processes of different stages of methane production by the composite bacterial line AMC-9 were annotated,revealing the synergistic mechanism of the anaerobic composite bacterial line AMC-9 using methanol to produce methane.