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Investigating the effects of different kinds of inhibitors on the activity and structure of acetate-degrading microbial community involved in methane fermentation is critically important for developing countermeasures to make the fermentation process stable under different inhibitory conditions. In the present study, a mesophilic chemostat fed with acetate as the sole carbon source was constructed. Microbial community analysis based on high-throughput sequencing of 16S rRNA revealed that Methanothrix was the dominant methanogen and a variety of bacteria including acetate-oxidizing bacteria such as Tepidanaerobacter , Mesotoga , Geovibrio , and Geobacter were found. The activity and dynamic changes of the acetate-degrading microbial community under different inhibitory conditions were investigated. Addition of 600 mg L−1 ammonium and 150 mg L−1 sulfide reduced nearly half of the biogas production. The response of microbial community to sulfide inhibition was quicker than ammonium but the structure could recover in a short time. Addition of 8 mg L−1 chlortetracycline (CTC) and 160 mg L−1 enrofloxacin (EFX) exhibited a similar inhibitory effect on biogas production, with approximately 35% reduction. Compared to ammonium and sulfide, antibiotics showed stronger selective inhibition on some bacterial species. The genera related to acetate-oxidizing and sulfate-reducing bacteria showed stronger tolerance to CTC, which may be due to their low growth rates. Network analysis suggested that some genera which had close phylogenic relationship and similar functions showed constant positive correlation under different inhibitory conditions. [ABSTRACT FROM AUTHOR]