How the acetate and propionate accumulation impact anaerobic syntrophy during methane formation is not well understood. To investigate such effect, continuous acetate (35 g/L), propionate (11.25 g/L) and bicarbonate (30 g/L) supplementation were used during mesophilic anaerobic digestion. The high throughput sequencing (16S rRNA and mcrA), Real-Time quantitative PCR, and stable carbon isotope fingerprinting were applied to investigate the structure and activity of microbial community members. The results demonstrated that the abundance of syntrophic acetate oxidizing bacteria exhibited a gradual decrease coupled with heavier stable carbon isotopic signature of methane (δ 13CH4) in the three reagents impacted reactors. The increased acetate and propionate concentrations exerted negative influence on biogas production but the relatively stable hydrogenotrophic methanogens together with syntrophic acetate/propionate oxidizing bacteria kept the stable methane formation facing acetate and propionate accumulation. The functional genes copy number of the hydrogenotrophic Methanocellaceae and Methanomicrobiaceae correlated significantly with δ 13CH4 (R2 > 0.74), but only the abundance of Methanocellaceae fitted well with δ 13CH4 (p < 0.05). The δ 13CH4 signatures can predict methanogenesis, as it directly reflects the main methanogenic pathway; yet, further investigation of isotope fractionation in acetate/propionate coupled with δ 13CH4 is needed. Collectively, these results provide deep insight into anaerobic syntrophy and reveal changes of synergistic relationships, both of which may contribute to the stability of biogas reactors.

Bioenergy Research Institute College of Biotechnology and Pharmaceutical Engineering Nanjing Tech University Nanjing 211816 China

Department of Applied Ecology Faculty of Environmental Sciences Czech University of Life Sciences Prague Kamýcká 129 16500 Prague Czech Republic

Institute of Agricultural Resources and Regional Planning Chinese Academy of Agricultural Sciences Beijing 100081 China

School of Life Jiangsu Normal University Shanghai Road 101 221116 Xuzhou China

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