Objective  Changes of the microbial community in fermentation of substrates for cultivating Agaricus bisporus were investigated.

  Method  The microbial community characteristics of a compound A. bisporus culture substrates containing spent Flammulina velutiper and Pleurotus eryngii materials were monitored during the 7 stages, Ag1 to Ag7 from pile building to 1^st and 2^nd fermentation, using the high throughput 16S rDNA full-length sequencing of Pacbio platform.

  Result  A total of 715 OTUs were obtained in the fermentation process (i.e., 328, 340, 294, 377, 364, 166, and 174 for each stage) with 161 OTUs commonly found in all stage. The microbes included 21 phyla, 299 genera, and 399 species. At phylum level, Fimicutes, Proteobacteria, Bacteroidetes, and Gemmatimonadetes had higher abundance in all 7 stages. The dominant genus in the substrate during the pile building and 1^st fermentation stages was Ureibacillus, while Limnochordaceae, S0134_terrestrial_group, Thermobacillu, and Ruminiclostridium stood out in the 2^nd fermentation stage. At species level, Ureibacillus thermophilus and Ureibacillus terrenus were dominant in the pile building and 1^st fermentation stages. In the 2^nd fermentation stage, the species in the genus of Limnochordaceae were the greatest in relative abundance. It appeared that the microbial abundance and diversity increased gradually before the onset of 2^nd fermentation. The community structure of the substrate varied significantly between the 1^st and the 2^nd fermentation, but the deviation diminished significantly after the 2^nd fermentation leaving mostly the dominant species that basically functioned to degrade the waste mushroom material enriching the fertilization effect.

  Conclusion  The full-length sequencing technology clearly identified the dominant species unveiling many previously unclassified microorganisms. The results helped to better optimize the substrate fermentation process for an improved cultivation of A. bisporus.