## 4 Discussion
### Influence Mechanism of Physicochemical Factors on Microorganisms  
The changes in the community were mainly attributed to the variations in relevant physicochemical parameters during the stacked fermentation process [25]. According to the results of the redundancy analysis (RDA), glucose, starch, ethanol, and temperature were identified as the key factors driving the succession of microbial communities. As the fermentation time extended, the dynamic change trends of the physicochemical parameters of the fermented grains in the two workshops were basically consistent. The overall content of glucose showed an upward trend, while that of starch showed a downward trend. Correlation analysis indicated that _Ascomyca_ was significantly positively correlated with starch and significantly negatively correlated with glucose. Moreover, during the stacked fermentation process, the succession pattern of _Ascomyca_ was the same as the change pattern of starch. The abundance of _Ascomyca_gen_Incerta_sed_ and the content of starch in workshop L were significantly higher than those in workshop H. This suggests that during the stacked fermentation process, the amylase and glucoamylase produced by _Ascomyca_gen_Incerta_sed_ play an important role in the conversion of starch into glucose and meanwhile provide sufficient energy for subsequent alcohol fermentation [42]. However, the driving effects of these physicochemical factors on microorganisms varied between the two workshops. In workshop H, _Saccharomyces_ and _Thermomyces_ were significantly correlated with starch, while in workshop L, _Monascus_ was significantly negatively correlated with starch. This may be related to the differences in the starch content of the fermented grains under the initial conditions of the two workshops, which drive the growth of different microorganisms. Meanwhile, we found that the change in glucose content was consistent with the succession pattern of _Saccharomyces_, indicating that there is a certain relationship between the rate of starch conversion into glucose and the growth and metabolism of _Saccharomyces_[37]. Among the major bacterial genera,_Kroppenstedtia_ in workshop L was significantly positively correlated with glucose. This may be because the fermented grains at the initial fermentation stage in workshop L contained more starch, which was more conducive to driving _Kroppenstedtia_ to produce some enzymes that act on the polysaccharides in the fermented grains and gradually decompose them into glucose [43]. Meanwhile, we also found that although there were significant differences in the contents of glucose and starch between the two workshops at the beginning of fermentation, the differences were minimal at the end of stacked fermentation. This might be the result of the joint metabolism of multiple microorganisms, indicating that after the cooperative fermentation by multiple microorganisms, the differences in glucose and starch between the two workshops have decreased, respectively. Also worth our attention was that both temperature and ethanol showed an upward trend during the stacked fermentation process. They were both significantly negatively correlated with _Ascomycota_ and _Aspergillus_ and positively correlated with _Saccharomyces_ and _Kroppenstedtia_. Moreover, the abundance of _Saccharomyces_ increased during the fermentation process. This indicates that _Saccharomyces_ can convert the glucose produced by the metabolism of _Ascomycota_gen_Incerta_sed_ and _Aspergillus_ into alcohol [37]. The biofermal energy generated during its metabolism gradually raises the temperature of the fermented grains [33], making the fermentation conditions more and more suitable for its growth. These microorganisms work synergistically in the fermented grains to convert and utilize the starch in the raw materials, jointly promoting the progress of fermentation. Notably, although the temperatures of the fermented grains in the two workshops were different at the beginning of fermentation, the difference was negligible at the end of fermentation.