Abstract:A fermentation system with binary mixed bacteria was constructed with the acid-producing bacteria and the esterase-producing strains screened in the preliminary study. The competitive inhibition of the system was evaluated based on OD600 nm. The fatty acid ethyl esters and other flavor substances in the system without competitive inhibition were quantified by gas chromatography-mass spectrometry. The results showed that the yield of fatty acid ethyl esters was the highest in the fermentation system consisting of Sphingomonas paucimobilis ARUV3-26 and Clostridium butyricum GD1-1，including ethyl acetate at 1.44 g/L，ethyl butyrate at 1.32 g/L，ethyl lactate at 1.17 g/L，and ethyl hexanoate at 1.04 g/L. The fermentation conditions of the system were optimized by single factor and response surface methods. The optimized conditions were as follows：inoculation of the esterase-producing strain at the time point of 3.4 d and an amount of 5.3% and the fermentation time of 5.3 d. Under these conditions，the yields of ethyl acetate，ethyl butyrate，ethyl lactate，and ethyl hexanoate produced by the system were （0.87±0.02），（0.91±0.03），（1.16±0.05），and（1.48±0.11） g/L，respectively. The fermentation system with binary mixed bacteria was characterized by easy construction，fermentation broth rich in flavor substances，and esterification without the need for cofactors，thus ensuring the flavor and quality of the product.