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Medium chain carboxylates (MCCs) are important precursors for biodiesel production. Using chain elongation to produce MCCs is an emerging bioenergy technology. In this study, batch tests were conducted to investigate fermentative MCC production through chain elongation from acetate, propionate, n-butyrate, and ethanol. The effect of the acid/ethanol ratio on MCC production by mixed culture was investigated. Better MCC production, especially n-caproate production, was achieved at optimal acid/ethanol ratios of 1:4, 1:3, and 1:2 with acetate, propionate, and n-butyrate as the electron acceptor, respectively. The n-caproate concentration was high, up to 41.54 mmol/L, and the highest n-caproate production efficiency was 57.96% with the n-butyrate/ethanol ratio of 1:2. The higher concentration of ethanol might stimulate the growth of chain elongation bacteria to promote chain elongation. The highest MCC production efficiency with different electron acceptors corresponded to less carbon loss and a higher chain elongation degree. In addition, with the optimal acid/ethanol ratio, the substrate was maximally utilized for chain elongation. The microbial community analysis confirmed the carbon balance analysis with the maximum relative abundance of 52.66–60.55% of the n-caproate producer Clostridium_sensu_stricto_12 enriched by the optimal acid/ethanol ratios with different volatile fatty acids (VFAs) as electron acceptors.

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