Chinese strong-flavour liquor is prepared through the fermentation of a mixture of sorghum, rice, and wheat known as Zaopei in a rectangular cellar composed of pit mud. This pit mud is an ideal habitat for microbes that are integral to the distillation process, serving as key determinants of the flavour of the resultant liquor. The quality of pit mud is thus an important regulator of the quality and taste of the liquor produced.
Pit mud tends to age with increasing cellar usage, and the microbial communities present within this mud vary based upon their spatial location within the walls or bottom of the cellar. A range of sensory descriptions and physicochemical indices have been used to describe pit mud from different locations within these fermentation cellars. For example, pit mud from the bottom of these cellars is often described as smooth, fine, soft, moist, and sooty with an aroma of esters, ammonia, and hydrogen sulfide. In contrast, pit mud from the top of these cellars is rough, hard, dry, and light grey with white lumps or aciform crystals and no aroma. While pit mud from the bottom layer can support the production of good-quality liquor, that from the upper layer cannot. As such, studying the microbial communities present within pit mud is essential in order to understand the molecular mechanisms governing the flavor and aroma of Chinese strong-flavour liquor in an effort to improve the quality of this popular and culturally important beverage.
In prior studies, researchers have utilized both culture-dependent and –independent strategies to determine that bacteria, fungal, archaea, and actinomycetes species are present within pit mud samples, with bacteria and archaea being dominant in this environment [5]. At the family level, common pit mud-resident bacteria include haloplasmatacea, Bacillaceae, planococcaceae, synergistaceae, staphylococcaceae, Thermoanaerobacter, and clostridiaceae species. Archaea present within pit mud are largely consistent across regions, and primarily include methanobacteria (Methanobacteriaceae), Methanococcus (Methanococcus), and thermoplasmataceae (thermoplasmata) species [5]. Microbes in the Clostridia class are thought to be primary producers of short- and medium-chain fatty acids including butanoic acid and hexanoic acid, which are directly relevant to the liquor production process [4]. Liu et al. (2018) isolated the Lysi-nibacillussphaerieus, Brevibacillusbrevis, and Paenibacilluslarvae subsup.pulvifaciens strains from pit mud and found that these microbes were important mediators of fermentation and producers of aromatic compounds in the context of Chinese strong-flavour liquor distillation [8]. Wang et al. (2019) explored bacterial community structures in samples of pit mud from a 16-year-old Chinese strong-flavour liquor cellar, and found that Clostridium, Aminobacterium, Petrimonas, Syntrophmonas, and Sedimentibacter species were the dominant microbes therein [9]. Ding et al. (2014) employed a PCR-DGGE approach to characterize the eubacterial pit mud communities associated with Luzhou-flavour liquor and consistently detected higher levels of eubacterial diversity in samples from the bottom of the cellar relative to samples from the cellar walls [6]. Most of these past studies have specifically focused on prokaryotic species, and there have been few comparable analyses of the fungal communities found within pit mud.
Herein, we explored the structures of fungal communities in multidimensional pit mud environments via a DGGE approach, revealing clear discrimination between the communities present in different locations within the fermentation cellar. Penicillium fuscoglaucum, Penicillium glandicola, Aotearoamyces nothofagi, Malassezia restricta, Penicillium lanosocoeruleum, Penicillium crustosum, and Aspergillus tonophilus were only present in the upper cellar wall pit mud layer, whereas Alternaria alstroemeriae, Trichosporon insectorum, Fusarium equiseti, Calonectria pseudoreteaudii, Penicillium clavigerum, Penicillium compactum, Ascochyta phacae, Metarhizium frigidum, Alternaria burnsii, Fusarium nurragi, and Didymella keratinophila were presented only detected in the middle wall layer. Similarly, Alternaria zantedeschiae and Ilyonectria cyclaminicola were only identified in the lower cellar wall pit mud layer, while Leptobacillium leptobactrum, Calonectria queenslandica, Aspergillus appendiculatus, and Candida pseudolambica were only detected in the pit mud found on the bottom of the fermentation cellar. These differences may explain why the quality of strong-flavour liquor varies with cellar position. We found that fungal abundance in the upper and middle layers was significantly higher than that in the lower wall and bottom layers, potentially due to the lower oxygen levels in these latter two environments, as such oxygen deficiency may have compromised fungal survival [10]. This, in turn, may explain the higher saccharification efficiency that is typically detected in the upper and middle Zaopei layers in the context of liquor fermentation.
With respect to pit mud physicochemical properties, we found that moisture, pH, PO43-, acetic acid, Humus, K+, Mg2+, Ca2+, acetic acid, butyric acid, and caproic acid levels rose with sample position from the upper wall to the bottom of the fermentation cellar, suggesting that organic compounds were gradually degraded with position. The maximal moisture levels in the bottom pit mud layer may be associated with the high levels of Huangshui present in this setting. The higher pH levels lower in the cellar may be linked to the degradation of various acids such as lactic acid [9], and to the synthesis of ammonium nitrogen, consistent with the observed trends in NH4+-N levels. The lower acetic acid levels with the upper wall pit mud layer are consistent with less robust prokaryotic metabolism in this location, given that acetic acid is a metabolic end product produced by many bacterial species [4]. The rising lactic acid levels detected from the bottom of the pit to the upper pit may correspond to the different Lactobacillus activity levels in these positions.
We then conducted an RDA analysis to explore relationships between the 51 detected fungal genera and the 12 measured environmental variables (Fig. 4). This revealed that moisture, pH, and NH4+-N levels were the most significant environmental factors, accounting for 67.1% of microbial community variability, indicating that these physicochemical factors are closely linked to pit mud microbe growth. Fungal community structure was moderately correlated with these physicochemical variables. For example, Alternaria zantedeschiae, Ilyonectria cyclaminicola, Calonectria pseudoreteaudii, Leptobacillium leptobactrum, Calonectria queenslandica, Aspergillus appendiculatus, Aspergillus heterocaryoticus, Penicillium argentinense, Antarctomyces psychrotrophicus, and Ramgea ozimecii levels were strongly positively correlated with moisture, pH, NH4+-N, PO43-, Humus, K+, Mg2+, Ca2+, acetic acid, butyric acid, and caproic acid levels, whereas for Metarhizium robertsii, Alternaria destruens, Bipolaris axonopicola, Thermomyces lanuginosus, and Cladosporium chasmanthicola levels these correlations were more moderate.
Many prior studies have sought to understand the relationship between pit mud physicochemical properties and the microbial communities therein. Meng et al. (2020), for example, found that these properties were significantly influenced by depth within the fermentation cellar [7]. Zhang et al. (2020) found that acid and amino nitrogen concentrations were higher in the bottom pit mud layers relative to other positions, suggesting that these compounds may influence the overall diversity of the microbial communities found within this bottom layer [4]. We similarly detected a clear relationship between fungal community structure and physicochemical variables in pit mud samples. However, further research will be essential to develop the efficient cultivation strategies necessary to delineate the independent contributions of different fungi to the production of Chinese strong-flavour liquor production.