Abstract: In this study, metagenomics combined with headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was employed to investigate the dynamic changes in four ethyl esters (ethyl lactate, ethyl acetate, ethyl butyrate, and ethyl caproate), microbial communities, and ester synthase-encoding genes during the fermentation of qingxiangxing baijiu. The results showed that ethyl acetate, ethyl butyrate, and ethyl caproate accumulated rapidly in the early stage of fermentation (0–3 days), while ethyl lactate started to accumulate rapidly on the 5th day. Based on the characteristics of microbial communities, the fermentation process could be divided into two stages (day 0 and days 3–28). Saccharomyces, Lactobacillus, and Rhizopus were not only the dominant genera in the early fermentation stage but also the differential genera between the two fermentation stages. Correlation analysis revealed that Saccharomyces and Lactobacillus were significantly correlated with ethyl lactate (P < 0.05), while Rhizopus was significantly correlated with ethyl acetate (P < 0.05). According to the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, the genes encoding hemiacetal dehydrogenation, alcohol acetyltransferases, and esterase dominated throughout the fermentation process. In the early fermentation stage, hemiacetal dehydrogenases and alcohol acetyltransferases were the predominant enzymes, which were also primarily responsible for the differences between the two stages. Further analysis of species contribution indicated that hemiacetal dehydrogenases were mainly derived from Leuconostoc and Weissella in the early fermentation stage, whereas they were primarily sourced from Lactobacillus in the late fermentation stage. Alcohol acetyltransferases were mainly produced by Saccharomyces and Wickerhamomyces in the early fermentation stage, whereas they were primarily sourced from Lactobacillus and Kazachstania in the late fermentation stage. Esterases were mainly derived from the genera Pachysolen and Weissella during the early stage of fermentation, while they were predominantly produced by Lactobacillus, Kazachstania, and Pachysolen in the late fermentation stage. Based on the above results, it can be inferred that the genera Lactobacillus, Kazachstania, Rhizopus, Saccharomyces, Wickerhamomyces, and Weissella are the key microorganisms involved in ester synthesis. The results of correlation analysis also demonstrated a significant correlation between ethyl acetate and alcohol acetyltransferases (P < 0.05), and the genera Saccharomyces, Wickerhamomyces, Lactobacillus and Kazachstania were proposed to be the primary taxa responsible for ethyl acetate synthesis via the alcohol acetyltransferases pathway.

Key words: metagenomics; ester compounds; microbial community; enzyme-encoding genes; fermentation process; variation patterns