## 3 Results
### Microbial Diversity and Community Composition During Stacked Fermentation  
Table 1 summarizes the alpha diversity indices of the bacterial and fungal communities across different stages of the fourth round of stacked fermentation in workshop L (L.4R.1 to L.4R.5) and workshop H (H.4R.1 to H.4R.5).  
For fungi, the Shannon and Simpson indices exhibited parallel trends, initially increasing and subsequently decreasing in both workshops, indicating greater fungal diversity during the middle and later stages of stacked fermentation. The Chao1 index exhibited continuous fluctuations throughout stacked fermentation, with a general increasing trend observed in workshop H and a decreasing trend in workshop L, reflecting divergent changes in fungal community structure between workshops. A total of 5,187,042 original sequences were obtained, including 1,273,411 bacterial sequences and 3,913,631 fungal sequences. After quality filtering, 4,556,138 valid sequences were retained, comprising 1,019,142 bacterial sequences and 3,536,996 fungal sequences. These sequences were clustered into 951 bacterial OTUs and 1120 fungal OTUs at a 97% similarity threshold.  
As shown in Table 1, the sequencing coverage for both bacterial and fungal communities indicated sufficient sequencing depth and an accurate representation of microbial diversity in the fermented grains. For fungal communities, the Shannon and Simpson indices exhibited an increasing trend during the middle stages of fermentation, followed by a decline, indicating greater fungal diversity during these stages. The Chao1 index fluctuated but showed an overall increase in workshop H, reflecting more dynamic fungal community changes compared to the declining trend observed in workshop L.  
For bacterial communities, the Shannon and Simpson indices showed a similar decreasing-then-increasing pattern in both workshops. In workshop H, bacterial diversity began to recover on the fourth day, while in workshop L, this recovery was observed on  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline
**Samples** & \multicolumn{2}{c}{**Shannon**} & \multicolumn{2}{c}{**[PERSON]**} & \multicolumn{2}{c}{**[PERSON]**} & \multicolumn{2}{c}{**[PERSON]**} & \multicolumn{2}{c}{**Goods\_Coverage**} \\ \hline  & **Bacteria** & **Fungi** & **Bacteria** & **Fungi** & **Bacteria** & **Fungi** & **Bacteria** & **Fungi** & **Bacteria** & **Fungi** \\ \hline L.4R.1 & 3.642 & 2 & 0.85 & 0.629 & 134.071 & 177.75 & 135.01 & 179.598 & 0.9999 & 0.9999 \\ L.4R.2 & 3.614 & 2.424 & 0.816 & 0.696 & 131.765 & 170.85 & 132.527 & 174.381 & 0.9999 & 0.9999 \\ L.4R.3 & 3.526 & 2.838 & 0.838 & 0.786 & 106.248 & 171.13 & 105.793 & 173.517 & 0.9998 & 0.9997 \\ L.4R.4 & 2.996 & 2.621 & 0.796 & 0.72 & 124.012 & 176.23 & 119.945 & 183.195 & 0.9999 & 0.9999 \\ L.4R.5 & 3.521 & 2.925 & 0.83 & 0.79 & 140.376 & 187.04 & 137.677 & 187.074 & 0.9999 & 0.9998 \\ H.4R.1 & 3.647 & 1.807 & 0.844 & 0.529 & 142.999 & 217.863 & 143.95 & 220.22 & 0.9999 & 0.9999 \\ H.4R.2 & 3.485 & 2.396 & 0.844 & 0.645 & 144.424 & 254.32 & 146.77 & 235.034 & 0.9997 & 0.9998 \\ H.4R.3 & 3.401 & 2.908 & 0.791 & 0.777 & 124.437 & 222.557 & 123.038 & 230.22 & 0.9998 & 0.9999 \\ H.4R.4 & 4.027 & 2.517 & 0.896 & 0.718 & 107.333 & 170.113 & 106.704 & 177.512 & 0.9998 & 0.9999 \\ H.4R.5 & 3.548 & 2.855 & 0.846 & 0.76 & 83.032 & 234.322 & 83.607 & 234.205 & 0.9999 & 0.9999 \\ \hline \end{tabular} Note: Samples for workshop L were collected from L.4R.1, L.4R.2, L.4R.3, L.4R.4, and L.4R.5, and samples for workshop H were collected from H.4R.1, H.4R.2, H.4R.3, H.4R.4, and H.4R.5.  
\end{table}
Table 1: \(\alpha\)-Diversity indexes of bacterial and fungal communities during different times of fourth round of stacked fermentation in workshop L and workshop H.  
the fifth day. The lowest bacterial diversity was recorded on the third day in workshop H and on the fourth day in workshop L, reflecting different successional dynamics between the workshops.