Melle-boinot fermentation process can be used to increase the ethanol productivity in second-generation ethanol process (2G). However, bacterial contamination can result in decreased ethanol production and sugars consumption. The available literature on microbial contamination in the 2G at the secretome level, microbial interactions and their impacts on ethanol production are scarce. In this context, the cultivation of Spathaspora passalidarum was studied pure and in co-culture with Lactobacillus fermentum under conditions that mimic the Melle-boinot process. Glucose consumption and ethanol production by S. passalidarum were not affected by bacterial contamination. Xylose consumption was higher in pure culture (11.54 ± 2.62, 16.23 ± 1.76, 6.50 ± 1.68 g) than in co-culture fermentation (11.89 ± 0.38, 7.29 ± 0.49, 5.54 ± 2.63 g) in cycle 2. The protein profile of the fermented broth was similar in pure and co-culture fermentation. The low effect of L. fermentum on fermentation and protein profile may be associated with the inhibition of the bacteria by the low nutrient fermentation broth, with centrifugation and/or with sulfuric acid washing. Thereby, considering that research on microbial contamination in the 2G fermentation process is very limited, particularly at the omics level, these findings may contribute to the lignocellulosic biomass fermentation industry.
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