Bio-Heat Is a Key Environmental Driver Shaping the Microbial Community of Medium-Temperature Daqu

Abstract

"Daqu" is a saccharifying and fermenting agent commonly used in the traditional solid-state fermentation industry (e.g., baijiu and vinegar). The patterns of microbial community succession and flavor formation are highly similar among batches, yet the mechanisms promoting temporal succession in the Daqu microbial ecology remain unclear. Here, we first correlated temporal profiles of microbial community succession with environmental variables (temperature, moisture, and titratable acidity) in medium temperature Daqu (MT-Daqu) throughout fermentation. Temperature dynamics significantly correlated (P < 0.05) with the quick succession of MT-Daqu microbiota in the first 12 d of fermentation, while the community structure was relatively stable after 12 d. Then, we explored the effect of temperature on the MT-Daqu community assembly. In the first 4 d of fermentation, the rapid propagation of most bacterial taxa and several fungal taxa, including Candida, Wickerhamomyces, and unclassified Dipodascaceae and Saccharomycetales species, significantly increased MT-Daqu temperature to 55°C. Subsequently, sustained bio-heat generated by microbial metabolism (53 to 56°C) within MT-Daqu inhibited the growth of most microbes from day 4 to day 12, while thermotolerant taxa, including Bacillus, unclassified Streptophyta, Weissella, Thermoactinomyces, Thermoascus, and Thermomyces survived or kept on growing. Furthermore, temperature as a major driving force on the shaping of MT-Daqu microbiota was validated. Lowering the fermentation temperature by placing the MT-Daqu in a 37°C incubator resulted in decreased relative abundances of thermotolerant taxa, including Bacillus, Thermoactinomyces, and Thermoascus, in the MT-Daqu microbiota. This study revealed that bio-heat functioned as a primary endogenous driver promoting the formation of functional MT-Daqu microbiota.IMPORTANCE Humans have mastered the Daqu preparation technique of cultivating functional microbiota on starchy grains over thousands of years, and it is well known that the metabolic activity of these microbes is key to the flavor production of Chinese baijiu. The pattern of microbial community succession and flavor formation remains highly similar between batches, yet mechanistic insight into these patterns and into microbial population fidelity to specific environmental conditions remains unclear. Our study revealed that bio-heat was generated within Daqu bricks in the first 4 d of fermentation, concomitant with rapid microbial propagation and metabolism. The sustained bio-heat may then function as a major endogenous driving force promoting the formation of the MT-Daqu microbiota from day 4 to day 12. The bio-heat-driven growth of thermotolerant microorganisms might contribute to the formation of flavor metabolites. This study provides useful information for the temperature-based modulation of microbiota function during the fermentation of Daqu.

Keywords: Daqu; bio-heat; metagenomics; microbial communities.

FIG 1

Dynamics of environmental variables, microbial cell number, and amylase and glucoamylase activities in MT-Daqu throughout fermentation. Abundance of bacterial and fungal cell numbers in the MT-Daqu are expressed as the log (10) of 16S rRNA gene and ITS rRNA gene copy numbers, respectively.

FIG 2

Temporal patterns of microbial community structure during the fermentation process of MT-Daqu. (A) Temporal profile for the relative abundance of bacterial taxa represented at the genus level; (B) distance tree based on V1-V3 16S rRNA gene amplicon sequences of bacteria in MT-Daqu constructed by the unweighted pair group method with arithmetic mean (UPGMA); (C) temporal profile for the relative abundance of fungal taxa represented at the genus level; and (D) distance tree based on ITS1 ITS rRNA gene amplicon sequences of fungi in MT-Daqu constructed by UPGMA.

FIG 3

Variation of temperature in the center of MT-Daqu in 10 batches of fermentation. Temperature data in two different phases (day 0 to 4 and day 4 to 12) were fitted separately by the weighted least square method.

FIG 4

Heatmaps showing the succession of microbial phylotypes (OTUs clustered at 97%) and its correlation with temperature dynamics in the microbial community of MT-Daqu in the first 12 d of fermentation. (A) Bacterial community succession from day 0 to day 4; (B) bacterial community succession from day 4 to day 12; (C) fungal community succession from day 0 to day 4; and (D) fungal community succession from day 4 to day 12. Only OTUs above 0.05% at total relative abundance are shown.

FIG 5

Effect of environmental temperature on the structures of bacterial community (A), fungal community (B), titratable acidity and moisture content (C), and glucoamylase and amylase activities (D) within MT-Daqu after 12 d of fermentation. MT-Daqu bricks in the fermentation room after 4 d of fermentation (Ctrl group, 52.2 ± 0.8°C) were moved to two different fermentation rooms with controlled temperatures at 50°C (T50 group) and 37°C (T37 group), respectively. Values are compared by Duncan's multiple-range test. Columns marked with different letters possess significantly different values (P < 0.05).