Improved fungal collection supports the identification and function evaluation of culturable but not sequenced (CBNS) fungi in high-temperature Daqu

Abstract

Next-generation sequencing technologies have significantly advanced our comprehension of microbial diversity and ecological roles within fermented foods. However, culture-based approaches remain essential for a comprehensive understanding of these complex ecosystems. This study integrated culture-dependent and culture-independent techniques to elucidate the microbial diversity and flavor-forming potential of fungi in high-temperature Daqu, a critical solid-state fermentation process in Maotai-flavor Baijiu production. Through iterative cultivation strategies, we successfully isolated and identified 660 pure eukaryotic colonies, representing 33 genera and 58 species, from Daqu samples. This approach significantly improved cultivation efficiency from 20.7 to 63.2%. In addition, despite the optimization of ITS rRNA primer sets to enhance the detection of eukaryotic microorganisms, we still identified 21 genera (comprising 27 species) that were culturable but not detected by high-throughput sequencing analysis. Fermentation experiments demonstrated the robust growth and substantial ethyl acetate production potential of these fungal species, particularly Kluyveromyces marxianus, which exhibited exceptional performance at elevated temperatures (45 °C). This study advanced culture-dependent techniques to improve the isolation of eukaryotic microorganisms from Daqu. These findings highlight the importance of culture-based approaches in characterizing microbial diversity and function.

Keywords

Eukaryotic microorganism / Microbial cultivability / Culturable but not sequenced / High-temperature Daqu / High-throughput sequencing / Biological Sciences / Microbiology

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Funding

Natural Science Research of Jiangsu Higher Education Institutions of China(BK20242077)