Constructing metabolic pathways for biogas production at low temperatures through metagenomic and metatranscriptomic analyses

This study aimed to determine the metabolic pathways based on the abundance of microorganisms and the activity of genetic clusters of orthologous groups of proteins (COGs) involved in biogas production. Cow manure and tofu waste were used as substrates in a 1:1 ratio. The fermentation process was carried out in a glass reactor at 10-12 °C using long-term inoculum derived from active sludge. Metagenomic analysis based on 16S rDNA sequences and metatranscriptomic analysis (16S rRNA) were performed. The results showed that the average biogas production at low temperatures was only 0.73 L/day, with CH₄ levels exceeding 0.517 L/g VS. Clostridium cellulovorans dominated the microbial community with a relative abundance of 24%. COGs activities such as glucosidase and periplasmic β-galactosidase were predominant during the hydrolysis stage, while 3-hydroxyacyl-CoA dehydrogenase dominated acidogenesis, and acetate kinase was exclusively found in the acetogenesis stage. Biogas (CH₄) formation was primarily driven by the H₂/CO₂ pathway (49.2%), involving formylmethanofuran dehydrogenase and H₄MPT S-methyltransferase from archaeal genera Methanocorpusculum, Methanobrevibacter, and Methanobacterium. These findings provide insights into the microbial and enzymatic mechanisms underlying biogas production at low temperatures. Enhancing biogas yield under such conditions can be achieved by prioritizing the hydrogenotrophic pathway during methanogenesis and managing VFA accumulation during acidogenesis. Genetic engineering to enhance the expression of key enzymes and control specific microbes such as Syntrophomonas zehnderi may overcome enzymatic limitations at low temperatures, accelerate the conversion of CO₂ and H₂ into methane, and minimize inhibition of the methanogenesis process.