Keystone microbial taxa in the formation of stale aroma during pile fermentation of ripened Pu-erh tea

Ripened Pu-erh tea is prized for its distinctive stale aroma. Methoxy-phenolic compounds, key contributors to this aroma, are produced during pile fermentation; however, the specific microorganisms responsible for their synthesis remain unclear. In this study, we identified the dominant taxa (Aspergillus luchuensis, A. fumigatus, Staphylococcus gallinarum, and S. kloosii) during pile fermentation through morphological analysis and metagenomic profiling. Gas Chromatography-Mass Spectrometry (GC-MS) analysis demonstrated the pivotal role of methoxy-phenolic compounds in the stale aroma. Moreover, using a metagenomic-based Weighted Gene Co-expression Network Analysis (WGCNA) combined with bivariate correlation network analysis, we identified key microbial taxa (Trichomonascus ciferrii, Heyndrickxia coagulans and Enterococcus sp.) involved in the generation of these compounds. Finally, we found that solid-state fermentation involving both dominant and keystone microbial taxa produced the highest levels of methoxy-phenolic compounds. Our findings reveal an inconsistency between dominant high-abundance taxa and keystone microbial taxa responsible for methoxy-phenolic compound synthesis during pile fermentation.