## 3 Results and Discussion
### Discussion  
Currently, the factory cultivation of _A. bisporus_ is developing rapidly, and the process mainly includes the secondary fermentation method and the tertiary fermentation method. The production of _A. bisporus_ is a process of transforming raw materials into substrates that will promote the growth of _A. bisporus_ (divided into two stages: PI and PII) [25]. It was found that the phase I (PI) stage lasted for 17 days in the fermentation process with millet straw as the culture material, which was approximately 2-3 times that of wheat straw and wheat-rice mixed straw, which is consistent with the observations of [PERSON] et al. [8], [PERSON] et al. [10], [PERSON] et al. [11] and [PERSON] et al. [12]. In the PI stage, we observed that thermophilic microorganisms proliferated and degraded carbohydrates and proteins when the temperature was up to 80 \({}^{\circ}\)C, which resulted in the release of heat and ammonia, consistent with the observations of [PERSON] et al. [13]. These reactions led to the softening of raw materials such as wheat straw and rice straw, which is consistent with the observations of [PERSON] et al. [7; 26]. In phase II, pasteurization was mainly used to eliminate insects, nematodes, competitive species and pathogens, while conditioning was mainly used to remove ammonia generated in the first stage, decompose carbohydrate sources (including hemicellulose and cellulose) and establish beneficial microflora that can feed and/or stimulate mushroom growth [13; 27]. The activity and transcript levels decrease during mushroom formation, and therefore, lactose can only be used as a biomarker during phase III. In the secondary fermentation method, the temperature and time during the fermentation process were not easy to control, and it tended to cause problems such as compost retting and a high free ammonia content, resulting in fermentation failure according to [PERSON] et al. [18]. These problems of the secondary fermentation method can be solved by continuous steam heating and heat preservation after secondary fermentation. According to the yield measurement, the average yield of _A. bisporus_ in the Shanghai Lianzhong secondary fermentation method was 22.5 kg/m\({}^{2}\) and that in the Jiangsu Yuguan tertiary fermentation method was 33.5 kg/m\({}^{2}\) (Unreported), which may demonstrate that the tertiary fermentation method could significantly increase the yield of _A. bisporus_. The reason for the difference may be related to the change in metabolites in the fermentation process of the compost. Reports on changes in metabolites in the fermentation stage of secondary fermentation and tertiary fermentation method have not yet been published. We believe these metabolites may be important substances that reflect the quality of fermentation. Figure 8: _Cont._  
Figure 8: Conf. Figure 8: Significant enrichment KEGG pathway (**a**) synthesis and degradation of ketone bodies; (**b**) glycine, serine and threonine metabolism; (**c**) lysine biosynthesis; (**d**) vitamin B6 metabolism; (**e**) arginine biosynthesis). Based on the VIP \(>\) 1 and \(p\) value \(<\) 0.05 of PLS-DA analysis, 92 differential metabolites between the N1F and TS stages in the secondary fermentation method of Shanghai Lanzhong were identified, while 69 differential metabolites between phase I and phase II were determined in the tertiary fermentation method of Jiangsu Yutan, and 123 differential metabolites were found between phase II and phase III, indicating that more differential metabolites were produced by the tertiary fermentation method than the second fermentation process (Table S2). (R)-3-Hydroxyudyric acid was found at different stages. This compound mainly exists as a carbon source and energy storage material in organisms and is a commonly used material in solid-state fermentation. The difference in metabolites between the secondary fermentation method and Jiangsu Yuguan tertiary fermentation method was mainly involved in carbohydrate metabolism and amino acid metabolism.