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[Display omitted] • Acetoclastic pathway was most important in low-temperature MES. • Methahnosarcina and Methanothrix were enriched in DC and SC reactor, respectively. • Electroactive bacteria Syntrophomonas , Pseudomonas and Bacteroides were dominated. • The single-chamber MES achieve acetate to acetyl-CoA by acetate-CoA ligase. • The dual-chamber MES was by acetate kinase and phosphate acetyltransferase. In this study, single-chamber and dual-chamber Microbial electrosynthesis (MES) with carbon fiber brushes as electrodes were operated at 15°C to compare and analyze the difference in methanogenic performance. Metatranscriptomic analysis showed that the relative abundance of electroactive microorganisms Syntrophomonas , Pseudomonas and Bacteroides in each group exceeded 90%, while the abundance of Geobacter was less than 4%. Acetoclastic methanogens Methahnosarcina was more enriched in dual-chamber MES (61.74%~70.42%), and Methanothrix showed higher abundance in single-chamber MES (33.44%~51.71%). Methahnosarcina and Methanothrix could interact with electroactive microorganisms to improve the electron transfer efficiency through direct interspecies electron transfer (DIET). Analysis of the methane metabolic pathways of low-temperature MES found acetoclastic pathway was domination, and single-chamber MES achieved acetate to acetyl-CoA through acetate-CoA ligase (EC: 6.2.1.1), whereas dual-chamber MES was by acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8). These results are beneficial to further research on the treatment of low-temperature wastewater. [ABSTRACT FROM AUTHOR]