The increasing demand for renewable energy sources has prompted a search for innovative and efficient approaches for biogas production. This study investigates the potential of achieving sustainable biogas production through a synergistic combination of biological pre-treatment and co-digestion of rice straw (RS) and sewage sludge (SS). The goal is to enhance the overall methane yield, mitigate substrate limitations, and optimize the biogas production process. In this study, rice straw and sewage sludge were anaerobically co-digested in ratios of 100:0, 70:30, 50:50, 30:70, and 30:70. It was observed that the co-digestion ratio of 70:30 is optimal to achieve maximum methane yield of 0.3 L CH4/(g VS added). Biological pre-treatment with five different organisms Sphingobium sp., Paenibacillus sp., Microbacterium sp., Pseudomonas sp., and Stenotrophomonas sp. was also examined for RS and co-digested with SS at the optimized ratio to improve the degradability of RS. This pre-treatment strategy is anticipated to enhance the accessibility of microorganisms to substrates and accelerate the rates of hydrolysis. The biological pre-treatment resulted in a 20–23% improvement in methane yield compared to untreated substrates. Metagenomic studies revealed the dominance of Bacteroidetes, Firmicutes, and Proteobacteria, which are capable of utilizing lignocellulosic biomass and ultimately converting it to methane. The acetoclastic methanogensis is the major methane generating pathway which is supported by the complete dominance of genus Methanosaeta.
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