Traditional Chinese Baijiu has a variety of flavor styles such as Jiang flavor, light flavor, and Nong flavor, and a long history (Jia et al., 2020). Jiang-flavor Baijiu is one of the most popular and typical flavor styles in traditional Chinese Baijiu, which is represented by the famous Maotai from Huairen city, Guizhou Province, and Danquan Baijiu from Hechi city, Guangxi Autonomous Region.

In the past decades, there were some important discoveries about the origin of Chinese Baijiu based on the study of historical documents and the important physical evidence for Baijiu brewing (i.e., distillers). Different dynasties were suggested about the origin of Chinese Baijiu such as the Eastern Han Dynasty, Tang Dynasty, Song Dynasty, and Yuan Dynasty (Fu, 2005; Feng, 2015). However, regardless of scholars' perspectives, there is a completely consistent consensus. Chinese distilled liquor entered the consumer market and rapidly developed in the Yuan Dynasty (Mao, 2014). Chinese Baijiu, brandy, whisky, vodka, rum, and gin are known as the six major distilled spirits in the world, which highlighted the importance of Chinese Baijiu (Zheng et al., 2017). In 2020 and 2021, the export volumes of Chinese Baijiu were 14,246 and 16,023 kiloliters (with $459.912 and $564.827 million of sales), respectively, which accounted for only 0.19 and 0.22% of the domestic Chinese Baijiu yield. In 2020 and 2021, the total sales of Chinese Baijiu were 583.6 and 603.3 billion. The huge sales show the importance of Baijiu in the economy and society. However, the very low export proportion of Chinese Baijiu indicates that there is much room for expansion in the foreign market.

The production of Danquan Baijiu utilizes traditional and spontaneous multi-strain (including bacteria and fungi) solid-state fermentation (Liu et al., 2019; Jiang et al., 2020). The quality and yield of Baijiu are closely associated with microbes during the fermentation process (Yang et al., 2018; Jiang et al., 2020). The correlation between the microbes producing Taorong-type Baijiu and the major volatile substances in the pit mud was analyzed, which revealed the importance of the related microbes (Liu et al., 2022). Li et al. explored the effect of brewing ingredients in pit muds on the yield of Luzhou-flavor Baijiu and the specific effect of pit mud microbes in different locations on Baijiu production (Li et al., 2022). Luzhou-flavor baijiu is yielded by the combined activities of various microbes, and its flavor is affected by specific microbial community structures and the expression of specific genes (Zhou et al., 2022). The aforementioned studies fully clarified the importance of microorganisms in the Baijiu fermentation process. Chinese Baijiu is one of the world's oldest distilled liquors, which typically originated from the use of Daqu fermentation starters. Daqu is a saccharifying and fermenting agent, which has a significant effect on the flavor of Chinese Baijiu (Zheng et al., 2011). The microbes in Danquan Baijiu brewing primarily originates from high-temperature Daqu (HTD) which is responsible for saccharification and fermentation processes (Yang et al., 2018; Li et al., 2019). For Danquan Baijiu, HTD is made from wheat and old Daqu, then subjected to shaping, fermenting, and ripening, which involves numerous microbes and enzymes (Wang et al., 2022). HTD significantly affects the yield and quality of Baijiu (Liu et al., 2019). With the higher HTD-making temperature, most yeasts and molds are destroyed, and the microbial community of HTD primarily propagates bacteria, leading to the microbial composition dominated by thermophilic bacteria (Gan et al., 2019; Xie et al., 2020). Moreover, as an important functional community in HTD, bacteria can produce various enzymes and plentiful Baijiu aroma substances or their precursors, which give Baijiu a unique flavor (Zhao et al., 2020). Therefore, systematic research on the diversity of bacterial community in HTD is necessary for fully understanding the bacterial resources and fermentation mechanism of Baijiu such as Danquan Baijiu and improving the quality and yield of Baijiu.

At present, high-throughput sequencing technology has been widely utilized for the analysis of microbial community of diverse environmental samples (Dreier et al., 2022). Compared with traditional fingerprint technology such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD), high-throughput sequencing by Illumina MiSeq sequencing platform has many advantages such as high-throughput, fast rate, and accurate result (Hu et al., 2022). High-throughput sequencing has also been widely used to analyze the microbial community of various Daqu samples (Deng et al., 2020; Chen et al., 2021). Deng et al. investigated the differences of microbial communities and potential functions among four Jiang-flavor Daqu with different colors (named QW, QB, QY, and QR) by amplicon sequencing (Deng et al., 2020). Chen et al. used Illumina MiSeq sequencing to analyze the microbial community of special-flavor Baijiu Daqu in Jiangxi Province (Chen et al., 2021).

In this study, three samples (two HTDs and one fermented grains) were collected from the Danquan distillery. Through Illumina MiSeq high-throughput sequencing technology, the bacterial community in three samples was observed and compared. Then, the bacterial function was predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSTs) (Douglas et al., 2018). Therefore, this study is aimed to (a) fully reveal the bacterial community of three samples, (b) identify the most abundant species of three samples, and (c) understand the bacterial function in three samples.

High-temperature Daqu is manufactured by traditional spontaneous solid-state fermentation in the open environment (Huang et al., 2017). The polymicrobial co-fermentation process under the high-temperature condition is a distinct character of HTD, which is obviously different from other types of Daqu such as low-temperature Daqu for light-flavor Baijiu (Cai et al., 2021). Bacteria are important microbes, which produce a variety of aroma substances and enzymes in HTD.

In this study, Illumina MiSeq high-throughput sequencing was used to examine bacterial diversity of two HTDs (dqjq_ck and dqjqcp) and one fermented grain (dqjp3), respectively. Figure 2B shows that the dominant genera in the three samples were Bacillus, Comamonas, Myroides, Paenibacillus, Acetobacter, and unclassified_Lactobacillales. Different from our results, Jin et al. (2019) revealed dominant bacterial genera in HTD from Moatai and Xijiu towns, Guizhou province, China, were Bacillales, Enterobacteriales, and Lactobacillales, and Wang Y. et al. (2020) found that dominant bacterial genera in HTD from a traditional Maotai-flavor liquor production factory in Xiangyang, Hubei province, China, were Thermoactinomyces, Staphylococcus, Lentibacillus, Kroppenstedtia, and Saccharopolyspora (Jin et al., 2019; Wang Y. et al., 2020). This phenomenon suggested that HTD from different regions had a distinct bacterial community due to different environmental conditions.

The result of Figure 2B indicated that the most abundant species in dqjq_ck was Comamonas, and its relative abundance was highest in sample dqjq_ck. As the denitrifying bacteria, Comamonas can use oxygen present in nitrate or nitrite to oxidize the carbon, which leads to the formation of nitrogen gas from harmful nitrate or nitrite for human health when there is no oxygen available (Chu et al., 2022). Comamonas could be used as the indole-degrading genus (Yang et al., 2022). The dominant microbe Comamonas sp. was linked to the yield of a unique PHA member poly(3-hydroxyisobutyrate) with an isobutyrate-fed enrichment culture (Vermeer et al., 2022). Comamonas played a dominant role in tetracycline hydrochloride degradation and power generation in a microbial fuel cell (Zhang et al., 2021). Comamonas testosteroni YAZ2 could be used for the cleanup of polychlorinated biphenyl water pollution (Hara et al., 2021). The laccase enzymatic traits and delignification procedures of rice straw by Comamonas testosteroni FJ17 were investigated (Wang et al., 2021). Comamonas thiooxydans CG-1 had the ability for biodegradation of polyethylene terephthalate (a general plastic with non-degradable property) (Huang et al., 2022). Comamonas genus can degrade the raw materials in fermented grains, provide carbon source for the growth of other microorganisms, and participate in the formation of organic acids (Li and Qiu, 2021). Comamonas acidovorans has the ability of inhibiting spoilage mold. The existence of these bacteria can reduce the side effects caused by the invasion of spoilage mold in the brewing system (Yu et al., 2016). In brief, genus Comamonas could degrade a variety of pollutants in liquor production process.

The result of Figure 2B indicated that the most abundant species in dqjqcp was Bacillus, and its relative abundance was highest in sample dqjqcp. Genus Bacillus had been previously thought as a most represented bacterium with importance in a variety of Daqu (Zhang et al., 2014; Zheng et al., 2014; Hu et al., 2017). Genus Bacillus had been demonstrated to be the major Jiang flavor-producing bacterium for the yield of Jiang-flavor Baijiu, which could produce a variety of degrading enzymes such as protease, cellulase, amylase, and glucoamylase. These enzymes could hydrolyze macromolecules (protein, starch, etc.) to produce many flavor compounds during the production of Jiang-flavor Baijiu (Cai et al., 2021; Wu et al., 2021). Specific species of genus Bacillus and its quantity obviously affect the quality of Daqu and liquor style (Fan et al., 2018). For example, previous studies indicated that Bacillus subtilis and Bacillus sarcina could produce a red pigment, which might play an important role in Baijiu production (Zhao et al., 2009; Wei et al., 2014; Deng et al., 2020). Flavor metabolism of Saccharomyces cerevisiae by mixed culture with Bacillus licheniformis was improved during Chinese Maotai-flavor Baijiu fermentation, and the content of 12 flavor compounds, containing fatty acids and their esters, terpene, and aromatic compounds, was improved (Meng et al., 2015). Bacillus licheniformis stains MT-6 and MT-15 could produce the volatile compounds including C4 compounds, volatile acids, pyrazines, and aromatic and phenolic compounds, which could improve aroma concentration during the fermentation of Moutai-flavor Baijiu (Zhang et al., 2013). Bacillus cereus could make flavor compound ethyl hexanoate during the production process of Baijiu (Zhao et al., 2017). B. cereus and B. licheniformis were the most abundant bacteria to produce α-amylase in Jiuqu (Wang Z. M. et al., 2020). As the flavor-producing strains, Bacillus amyloliquefaciens and Bacillus subtilis could secrete α-amylase to enhance the saccharification process of raw materials and improve the contents of alcohols, esters, and diverse organic acids (Li et al., 2014; Yao et al., 2015). The relative abundance of genus Bacillus was higher at sites rich in oxygen in Daqu (He et al., 2019). Therefore, the most abundant species in sample dqjqcp, which was distributed in a well-ventilated area with more oxygen, is genus Bacillus. However, sample dqjq_ck was covered with thick rice straw with the least oxygen concentration, so relative abundance of genus Bacillus was the lowest. In addition, Bacillus could adapt to various unfavorable conditions such as heat and acid owing to the formation of spores (Wang W. et al., 2018; Mirza et al., 2022). In addition, genus Bacillus was the dominant genus in wheat (Zheng et al., 2015). Therefore, wheat as one of raw materials of two HTD samples (dqjqcp and dqjq_ck) was an important source of genus Bacillus (Du et al., 2019).

The result of Figure 2B indicated that the most abundant species in dqjp3 was unclassified Lactobacillales, and its relative abundance was highest in sample dqjp3. Lactobacillales is in the order of lactic acid bacteria. Lactobacillales was the dominant bacterium in Daqu owing to its extraordinary ability to adapt to various temperature and moisture, which helped it survive in high-temperature environments (Zhang et al., 2014; Wang and Xu, 2015). Lactobacillales can produce organic acids which may affect the growth of other microbes. For example, acetic acid can inhibit the growth of microbes and affect the structure of microbial community during the process of high-temperature fermentation (Nie et al., 2013). Furthermore, most genera of lactic acid-producing bacteria such as Lactobacillales could produce antibiotics such as bacteriocin, tetracycline, erythromycin, aminoglycoside, and vancomycin (Wu et al., 2017; Anisimova and Yarullina, 2019; Campedelli et al., 2019; Todorov et al., 2019; Arellano et al., 2020; Colautti et al., 2022). Lactobacillales and Acetobacter were found to affect the biomarkers through producing organic acids and bacteriocins and advance the occurrence of microbial community succession (Ma et al., 2022). In summary, Lactobacillales played an important role in flavor formation, microorganism antagonism, and microbial community succession.

Danquan Baijiu production involved three stages: the first stage (45 d of the first Daqu fermentation), the second stage (90 d of the second Daqu fermentation for the mature Daqu), and the third stage (mixing the crushed mature Daqu with the steamed sorghum for Baijiu brewing). After the end of each stage, the samples were collected, which in turn were dqjq_ck, dqjqcp, and dqjp3. Numerous studies had shown that Bacillus genus had the potential to produce alcohol. Bacillus subtilis can survive in different substrates because of its metabolic diversity and robust systems for the yield and secretion of a diversity of enzymes (Stülke and Hillen, 2000; Zhang and Zhang, 2010; Gu et al., 2018; Banerjee et al., 2019). Bacillus subtilis with innate ability to ferment a diversity of carbohydrates from mono-, di-, oligo-, and polysaccharides (such as starch, xylan, galactan, pullulan, arabinan, rhamnogalacturonan, and pectin) seems promising for inexpensive consolidated bioprocessing of bioethanol production using renewable plant biomass and wastes (Maleki et al., 2021). In addition, the ethanologenic Bacillus subtilis AP was estimated without and with Xyn-2 for bioethanol production from wheat bran. The strain Bacillus subtilis AP could produce 5.5 g/L ethanol, with a yield of 22.6% in consolidated bioprocessing (Rajabi et al., 2022). The proportions of Bacillus genus in dqjq_ck, dqjqcp, and dqjp3 were 1.43, 57.88, and 9.04%, respectively. With the higher HTD-making temperature, most yeasts and molds are destroyed, and the microbial community of HTD primarily propagates thermophilic bacteria (Gan et al., 2019; Xie et al., 2020). Therefore, we inferred that as a kind of thermophilic bacteria, Bacillus genus might play an important role in the bioethanol production for Danquan Baijiu brewing. Through our study, the succession of microbiota in the three samples representing the three important stages of Danquan Baijiu brewing were revealed. Our results will provide an important theoretical basis for enhancing the brewing process and the quality of Jiang-flavor Danquan Baijiu.

The result of functional potential and microbiome phenotype prediction is shown in Figures 5, 6. The predicted COG functional profile by PICRUSt showed that four functional categories with higher relative abundance in the three samples included K (transcription), E (amino acid transport and metabolism), G (carbohydrate transport and metabolism), and C (energy production and conversion) among all functional categories. In the meantime, amino acid transport and metabolism also require energy. In HTD, amino acids took part in Maillard reaction at high temperature, which gave HTD unique Jiang flavor (Gan et al., 2019; Feng et al., 2020). The noticeable expression of the functional category G (carbohydrate transport and metabolism) could provide a lot of energy for material transport, the reproduction and growth, and respiration in microbes. Carbohydrate metabolism and amino acid metabolism are very vital for microbes, which play an important role in the survival of microbes and the implementation of related functions. The KEGG-based analysis indicated that Baijiu Daqu and huangjiu wheat Qu microbiota were enriched with genes related to carbohydrate metabolism, amino acid metabolism, and energy metabolism. This result suggested that Daqu and wheat Qu microbiota had huge potential for the degradation of raw materials (such as wheat, rice, and barley) and flavor compound metabolism, which was consistent with our study (Zhang et al., 2022). Cai et al. suggested that the dominant microbial functions (such as E, G, and C) were the most vital functions in low-temperature Daqu (LTD), which indicated that the fermentation of LTD depended on the transport and metabolism of carbohydrates and amino acids; meanwhile, the life activity of microbes relied on energy metabolism (Cai et al., 2021). Given all that, categories E, G, and C were vital for different types of Qu.

Simultaneously, all samples also had a higher relative abundance in categories K (transcription), J (translation, ribosomal structure, and biogenesis), L (replication, recombination, and repair), and M (cell wall/membrane/envelope biogenesis), which indicated that bacteria in all samples maintained the frequent metabolism and had a high ability of growth and reproduction. Furthermore, all samples also had a high relative abundance in categories P (inorganic ion transport and metabolism) and H (coenzyme transport and metabolism). The transport of different inorganic ions is essential for bacterial metabolism, which can be utilized as cofactors of a variety of enzymes to advance different biochemical reactions. About coenzyme transport and metabolism, enzymes in HTD could catalyze the metabolism of amino acids to enhance the fermentation rate and provide substrates for Maillard reaction under high-temperature conditions, which caused Jiang flavor of HTD [7, 8] (Gan et al., 2019; Xie et al., 2020).

Traditional fermented foods such as Baijiu are frequently produced by spontaneous fermentation with multiple microbes. Environmental factors play important roles in microbial succession. Models were constructed to predict the population of core microbiota by processing parameters (room humidity and room temperature) (Ban et al., 2022). This research will be helpful for regulating microbes via controlling processing parameters in spontaneous fermentation such as Daqu fermentation (Ban et al., 2022). The microbial community succession (MCS) was driven by environmental factors such as temperature, humidity, CO₂, O₂, acidity, and moisture during fermentation of Nongxiangxing Daqu, and each environmental factor drove the occurrence of MCS by different mechanisms (Ma et al., 2022). The primary driving factors of MCS were acidity, moisture, and temperature for fermentation of Nongxiangxing Daqu (Ma et al., 2022). In our study, the fermentation temperature and humidity of dqjq_ck and dqjqcp were 65–70°C/80–90% and about 40°C/natural humidity, respectively. We inferred that the significant differences in environmental factors such as temperature and humidity affected MCS in samples dqjq_ck and dqjqcp. Traditional spontaneous fermentation with MCS and the changes of environmental factors result in an unstable quality of fermented foods in the microecosystem (He et al., 2020). He et al. found that a higher abundance affected the yield of flavor metabolites and changed the interspecies interactions of microbial community (He et al., 2020). Then He et al. investigated the bioturbation effect of fortified Daqu inoculating Bacillus strains on microbial composition and flavor metabolite and found that fortified Daqu could reduce the yield of lactic acid (He et al., 2020). More studies had explained that the acidification of fermentation cellar was primarily derived from the accumulation of lactic acid bacteria. In our study, as lactic acid bacterium, unclassified Lactobacillales had the highest abundance in fermented grains (i.e., sample dqjp3), which perhaps affected the flavor and quality of Baijiu. The fortified Daqu could help improve the flavor and quality of Baijiu. The indigenous microorganisms can better adapt to the local environment. Therefore, we isolated the suitable microorganisms from fermented grains such as Bacillus strains to improve the flavor and quality of Danquan Baijiu.

In summary, the bacterial communities of the three samples from the Danquan distillery were analyzed. This article discussed the dominant genera with the potential for the improvement of the quality of Danquan Baijiu in the three samples. The most abundant species in dqjq_ck, dqjqcp, and dqjp3 were Comamonas, Bacillus, and unclassified Lactobacillales, respectively. Genus Comamonas could degrade a variety of pollutants in the fermentation process. Genus Bacillus could adapt to various unfavorable conditions, which was the major Jiang flavor-producing bacterium for the yield of Jiang-flavor Baijiu. The unclassified Lactobacillales played an important role in flavor formation, microorganism antagonism, and microbial community succession. With the changes in environmental conditions (fermentation temperature and humidity) and sample types, the microflora also underwent the corresponding adaptive changes. The results would lay the foundation for the isolation of some excellent indigenous bacteria and the construction of their genetic engineering modification to prepare fortified Daqu and improve the quality of Danquan Baijiu in future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioSample - SAMN26292234, SAMN26292235, and SAMN26292236.

CS: conceptualization, writing-review and editing, supervision, project administration, funding acquisition, and methodology. YL: software. YL and JZ: validation. CS, YL, and JZ: formal analysis. YL, JZ, and SG: investigation and data curation and writing-original draft preparation. JZ and SG: visualization. All authors have read and agreed to the published version of the manuscript.

This research was financially supported by Guangxi Key Research and Development Program (AB21220057 and 2021AB27009), Research Funds of the Guangxi Key Laboratory of Landscape Resources Conservation and Sustainable Utilization in Lijiang River Basin, Guangxi Normal University (LRCSU21Z0207), Research Funds of Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, China (ERESEP2022Z11), National Training Program of Innovation and Entrepreneurship for Undergraduates (202210602064), and Innovation Project of Guangxi Graduate Education (YCSW2022178 and XJCY2022011).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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