Revealing the influence of exogenously inoculated Bacillus spp. on the microbiota and metabolic potential of medium-temperature Daqu: A meta-omics analysis.

To determine the unique contribution of the bioturbation to the properties of the medium-temperature Daqu, we investigated the differences in microbiota and metabolic composition using the meta-omics approach. Bioturbation increased the amounts of microbial specie and influenced the contribution of the core microbiota to the metabolome. Specifically, inoculated synthetic microbiota (MQB) enhanced the abundance of Bacillus amyloliquefaciens, while Bacillus licheniformis (MQH) increased the abundance of the two Aspergillus species and four species level of lactic acid bacteria. These changes of the microbial profiles significantly increased the potentials of carbohydrate metabolism, amino acid metabolism, and biosynthesis of ester compounds. Consequently, both patterns significantly increased the content of volatile compounds and free amino acids, which were 27.61% and 21.57% (MQB), as well as 15.14% and 17.83% (MQH), respectively. In addition, the contents of lactic acid in MQB and MQH decreased by 65.42% and 42.99%, respectively, closely related to the up- or down-regulation of the expression of their corresponding functional enzyme genes. These results suggested that bioturbation drove the assembly of the core microbiota, rather than becoming critical functional species. Overall, our study provides new insights into the functional role of exogenous isolates in the Daqu microecosystem.

Engineering the synthesis of unsaturated fatty acids by introducing desaturase improved the stress tolerance of yeast.

BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild-type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry.

Improvement of the viability of Tetragenococcus halophilus under acidic stress by forming the biofilm cell structure based on RNA-Seq and iTRAQ analyses.

BACKGROUND: Tetragenococcus halophilus is a halophilic lactic acid bacterium (LAB) isolated from soya sauce moromi. During the production of these fermented foods, acid stress is an inevitable environmental stress. In our previous study, T. halophilus could form biofilms and the cells in the biofilms exhibited higher cell viability under multiple environmental stresses, including acid stress. RESULTS: In this study, the effect of preformed T. halophilus biofilms on cell survival, cellular structure, intracellular environment, and the expression of genes and proteins under acid stress was investigated. The result showed that acid stress with pH 4.30 for 1.5 h reduced the live T. halophilus cell count and caused cellular structure damage. However, T. halophilus biofilm cells exhibited greater cell survival under acid stress than the planktonic cells, and biofilm formation reduced the damage of acid stress to the cell membrane and cell wall. The biofilm cells maintained a higher level of H+ -ATPase activity and intracellular ammonia concentration after acid stress. The RNA-Seq and iTRAQ technologies revealed that the genes and proteins associated with ATP production, the uptake of trehalose and N-acetylmuramic acid, the assembly of H+ -ATPase, amino acid biosynthesis and metabolism, ammonia production, fatty acid biosynthesis, CoA biosynthesis, thiamine production, and acetoin biosynthesis might be responsible for the stronger acid tolerance of T. halophilus biofilm cells together. CONCLUSION: These findings further explained the mechanisms that allowed LAB biofilm cells to resist environmental stress. © 2023 Society of Chemical Industry.

Engineering acetyl-CoA metabolism to enhance stress tolerance of yeast by regulating membrane functionality.

Zygosaccharomyces rouxii has excellent fermentation performance and good tolerance to osmotic stress. Acetyl-CoA is a crucial intermediate precursor in the central carbon metabolic pathway of yeast. This study investigated the effect of engineering acetyl-CoA metabolism on the membrane functionality and stress tolerance of yeast. Firstly, exogenous supplementation of acetyl-CoA improved the biomass and the ability of unsaturated fatty acid synthesis of Z. rouxii under salt stress. Q-PCR results suggested that the gene ACSS (coding acetyl-CoA synthetase) was significantly up-expressed. Subsequently, the gene ACSS from Z. rouxii was transformed and heterologously expressed in S. cerevisiae. The recombinant cells exhibited better multiple stress (salt, acid, heat, and cold) tolerance, higher fatty acid contents, membrane integrity, and fluidity. Our findings may provide a suitable means to enhance the stress tolerance and fermentation efficiency of yeast under harsh fermentation environments.

Succession of microbial community of the pit mud under the impact of Daqu of Nongxiang Baijiu.

Complex microbiomes of pit mud play significant roles in imbuing flavors and qualities of Nongxiang Baijiu during fermentation. However, pit mud microbial enrichment and succession is a long process that is also accompanied by aging. Development of high-quality artificial pit mud becomes an urgent problem. In this study, a new medium based on space (TK) Daqu was used to effectively enrich the dominant microorganisms in pit mud. The results showed that Caproiciproducens was the most preponderance in the cultures unadded Daqu, whereas Clostridium sensu stricto 12 was the most preponderance, followed by Caproiciproducens in the enrichment cultures added TK Daqu. It is worth noting that TK Daqu balanced the relative abundance of Caproiciproducens and Clostridium sensu stricto 12 in 100-year pit mud culture (S100), which was more conducive to the increase of methanogens. PICRUSt2 prediction results showed that hydrogenotrophic methanogens could promote the synthesis of caproic acid by using the product H2 as the metabolic substrate and increased significantly in the pit mud enrichment cultures with TK Daqu. The increase of lactate dehydrogenase (EC 1.1.1.27) content in S100 contributed to the degradation of lactic acid and the increase of caproic acid. Adding TK Daqu enrichment cultures is more conducive to the enrichment and metabolic balance of pit mud microorganisms.

Exploring the Successions in Microbial Community and Flavor of Daqu during Fermentation Produced by Different Pressing Patterns.

Daqu can be divided into artificially pressed daqu (A-Daqu) and mechanically pressed daqu (M-Daqu) based on pressing patterns. Here, we compared the discrepancies in physicochemical properties, volatile metabolites, and microbiota features between A-Daqu and M-Daqu during fermentation and further investigated the factors causing those differences. A-Daqu microbiota was characterized by six genera (e.g., Bacillus and Thermoactinomyces), while five genera (e.g., Bacillus and Thermomyces) dominated in M-Daqu. The flavor compounds analysis revealed that no obvious difference was observed in the type of esters between the two types of daqu, and M-Daqu was enriched with more alcohols. The factors related to differences between the two types of daqu were five genera (e.g., Hyphopichia). The functional prediction of microbial communities revealed that the functional discrepancies between the two types of daqu were mainly related to ethanol metabolism and 2,3-butanediol metabolism. This study provided a theoretical basis for understanding the heterogeneity of daqu due to the different pressing patterns.

Metaproteomic investigation of enzyme profile in daqu used for the production of Nongxiangxing baijiu.

Enzymes and microbiota in daqu are essential for the brewing of Nongxiangxing baijiu. Uncover the key enzymes and functional strains in daqu is beneficial to improve the flavor and quality of Nongxiangxing baijiu. In this study, metaproteome technology was employed to determine the enzyme profiles in Nongxiangxing daqu, and strains with high saccharification activity were screened and identified. 933 proteins were identified in daqu, of which 463 belonged to enzymes, including 140 oxidoreductases, 98 transferases, 91 hydrolases, 49 ligases, 41 lyases and 27 isomerases, and hydrolase is the enzyme with the highest abundance in baijiu brewing. Among hydrolases, a total of 36 carbohydrate metabolism-related enzymes (CMEs) were identified, and 12 of them were key enzymes related to glycoside hydrolysis. Four major glycoside hydrolysis enzymes glucoamylase (EC 3.2.1.3), glucan 1,4-alpha-glucosidase (EC 3.2.1.3), glucanase (EC 3.2.1.-) and ß-glucosidase (EC 3.2.1.21) were revealed, and their sources were Byssochlamys spectabilis, Lichtheimia ramosa and Thermoascus aurantiacus, respectively. Then, strains Aspergillus A2, A3, A7, Lichtheimia L1, L4, L5, and Saccharomycopsis S2, S4, S6 with high saccharifying enzyme-producing capacity were screened through culture-dependent approach. Resents presented in this study can further reveal the enzyme profiles and identify the main functional strains in daqu, which can provide theoretical support for the brewing of Nongxiangxing baijiu.

Characterization of the differences in aroma-active compounds in strong-flavor Baijiu induced by bioaugmented Daqu using metabolomics and sensomics approaches.

Bioaugmenting Daqu is an effective strategy to improve the quality of Baijiu, but its effect on overall flavor profiles and aroma-active compounds is unknown. Here, 168 volatiles were determined from fresh strong-flavor Baijiu (SFB) and bioaugmented Daqu increased their diversity and altered the flavor characteristics. Among 49 odorants identified by aroma extraction dilution analysis, 29 aroma-active compounds had odor activity values ≥1, of which 18, 8, and 3 components exhibited the highest content in the SFB fermented by fortified-, space- and conventional-Daqu, respectively. The contribution of increasing ethyl hexanoate and decreasing ethyl lactate of fresh SFB by bioaugmented Daqu was confirmed, and their content changed from 4650 and 1890 mg/L (conventional-SFB) to 6680 and 1760 mg/L (fortified-SFB) and 6130 and 1710 mg/L (space-SFB). Meanwhile, the discriminators among different samples were determined by multivariate statistical analysis. These findings are beneficial for the optimization and improvement of Baijiu aroma.

Arginine deiminase pathway of Tetragenococcus halophilus contributes to improve the acid tolerance of lactic acid bacteria.

Arginine deiminase pathway, controlled by arginine deiminase, ornithine carbamoyltransferase and carbamate kinase, could affect and modulate the intracellular pH homeostasis of lactic acid bacteria under acid stress. Herein, strategy based on exogenous addition of arginine had been proposed to improve the robustness of Tetragenococcus halophilus during acid stressed condition. Results indicated cells cultured in the presence of arginine acquired high tolerance to acid stress mainly through maintaining the homeostasis of intracellular microenvironment. Additionally, metabolomic analysis and q-PCR showed the content of intracellular metabolites and expression levels of genes involved in ADI pathway significantly increased when cells encountered acid stress with the presence of exogenous arginine. Furthermore, Lactococcus lactis NZ9000 with heterologous overexpression of arcA and arcC from T. halophilus exhibited high stress tolerance to acidic condition. This study may provide an insight into the systematical understanding about the mechanism underlying acid tolerance and improve the fermentation performance of LAB during harsh condition.

Synergistic effect of biotic and abiotic factors drives microbiota succession and assembly in medium-temperature Daqu.

BACKGROUND: The feasibility of fortification techniques to improve the quality of medium-temperature Daqu (MTD) by inoculation functional isolates has been demonstrated. However, it is unclear what is the effect of inoculation on the controllability during the MTD fermentation process. Here, inoculated a single strain of Bacillus licheniformis, and the microbiota composed of Bacillus velezensis and Bacillus subtilis, were used to investigate the synergistic effect of biotic and abiotic factors on the succession and assembly of the MTD microbiota during the process. RESULTS: The biotic factors promoted the proliferation of microorganisms that arrived early at the MTD. Subsequently, this alteration might inhibit microorganisms that colonized later in the MTD microecosystem, thereby assembling a different but more stable microbial community. Moreover, the biotic factors making bacterial community assembly were dominated by variable selection earlier, whereas the fungal community assembly was dominated mainly by extreme abiotic factors rather than biotic factors. Interestingly, fermentation temperature and moisture were significantly associated with the succession and assembly of the fortified MTD community. Meanwhile, the effect of the environmental variables on endogenous variables was also significant. Thus, changes in endogenous variables could be mitigated by adjusting environmental variables to regulate the process of MTD fermentation. CONCLUSION: Biotic factors cause rapid changes of the microbiota during the MTD fermentation process, which could be controlled indirectly by regulating environmental variables. Meanwhile, a more stable MTD ecological network might be beneficial for enhancing the stability of MTD quality. © 2023 Society of Chemical Industry.

Effects of Daqu properties on the microbial community and their metabolites in fermented grains in Baijiu fermentation system.

Daqu is the natural starter for Nong-flavor Baijiu brewing. The effects of Daqu properties on the microbial community succession and their metabolites in fermented grains (FG) during Baijiu brewing were determined. These results showed that the effect of Daqu on the bacterial communities was stronger than that of the fungal communities. Compared with the conventional Daqu (DZ), Taikong (TK), and Qianghua (QH), Daqu significantly enhanced the content of volatile metabolites (especially esters) and ethanol when they were used, respectively, for FG fermentation. In the second round of fermentation, the relative abundance of Lactobacillus decreased, the content of lactic acid decreased, and that of caproic acid increased. In particular, the abundance of Lactobacillus was also reduced by 20% in FGs of the second round when TK Daqu was used than that in the respective first round. Partial least squares structural equation model analysis also showed that physicochemical parameters and Daqu properties significantly affected FG community structure and metabolism. This study provides a theoretical basis for further study on the effect of high-quality Daqu on the quality of fresh Baijiu and lays an important theoretical foundation for the stabilization of the Baijiu fermentation system based on Daqu.

Mechanism of Enhancing Pyrazines in Daqu via Inoculating Bacillus licheniformis with Strains Specificity.

Despite the importance of pyrazines in Baijiu flavor, inoculating functional strains to increase the contents of pyrazine in Daqu and how those interact with endogenic communities is not well characterized. The effects of inoculating Bacillus licheniformis with similar metabolic capacity on pyrazine and community structure were assessed in the Daqu complex system and compared with traditional Daqu. The fortification strategy increased the volatile metabolite content of Daqu by 52.40% and the pyrazine content by 655.99%. Meanwhile, results revealed that the pyrazine content in Daqu inoculated isolate J-49 was 2.35-7.41 times higher than isolate J-41. Both isolates have the almost same capability of 2,3-butanediol, a key precursor of pyrazine, in pure cultured systems. Since the membrane fatty acids of isolate J-49 contain unsaturated fatty acids, it enhances the response-ability to withstand complex environmental pressure, resulting in higher pyrazine content. PICRUSt2 suggested that the increase in pyrazine was related to the enzyme expression of nitrogen metabolism significantly increasing, which led to the enrichment of NH4+ and 2,3-butanediol (which increased by 615.89%). These results based on multi-dimensional approaches revealed the effect of functional bacteria enhancement on the attribution of Daqu, laid a methodological foundation regulating the microbial community structure and enhanced the target products by functional strains.

Profiling the composition and metabolic functions of microbial community in pellicle-forming radish paocai.

Pellicle formation is an obvious indicator of spoilage and is followed by a loss of flavor in a variety of fermented vegetables. In this study, the pellicle-forming microorganisms were isolated using culture-dependent approaches, then a comparative analysis between the pellicle-forming (PF) radish paocai and normal fermented paocai in the diversity and function of microbial community was conducted by metagenome sequencing. Based on a pairwise t-test and OPLS-DA analysis, diallyl sulfide, (z)-1-allyl-2-(prop-1-en-1-yl) disulfane, and terpineol were considered to be the main components responsible for the unpleasant flavor of PF paocai. Yarrowia spp., Enterobacter spp., and Pichia spp. were the main pellicle-forming microorganisms. All 17 isolated Enterobacter strains showed pectinase-producing and cellulase-producing abilities, and 3 isolated Pichia strains showed gas-producing capacity. According to LEfSe analysis based on metagenomes, unclassified_g__Citrobacter and Yarrowia lipolytica were the uppermost biomarkers that distinguished the PF paocai from normal paocai. Unclassified_g__Lactobacillus and Lactobacillus plantarum were found to be actively engaged in starch and sucrose metabolism, cysteine and methionine metabolism, galactose metabolism, fructose and mannose metabolism, lysine biosynthesis, fatty acid biosynthesis, and arginine biosynthesis, all of which contributed to the flavor formation of paocai. Combining the results of metagenome sequencing with the data obtained based on the culture-dependent method, we could deduce that the growth of Yarrowia lipolytica first promoted the increase of pH and the formation of pellicle, which provided a suitable niche for the growth of some harmful bacteria such as Enterobacter, Citrobacter, and Serratia. These hazardous bacteria then worked in concert to induce the odorous stench and texture softening of paocai, as well as more pellicle formation.

Comprehensive analysis for the bioturbation effect of space mutation and biofortification on strong-flavor Daqu by high-throughput sequencing, volatile analysis and metabolomics.

Daqu, an indispensable starter, significantly affects the quality and yield of Chinese baijiu. To explore the bioturbation effect of space mutation and biofortification on strong-flavor Daqu, the microbial community, physicochemical properties and overall metabolic profile were investigated. Lactobacillus and Aspergillus were significantly reduced after bioturbation, while seven genera (e.g., Bacillus and Pichia) and five genera (e.g., Enterococcus and Thermomyces) were enriched by space mutation and biofortification, respectively, resulting in improved enzymatic activities. Among the 14 aroma-active volatiles and 51 differential metabolites identified, most had a higher content after bioturbation, including pyrazines, alcohols, small peptides and carbohydrates. Moreover, correlation analysis and integrated metabolic pathway showed that differences in flavor quality among these Daqu were mainly associated with bacterial communities, especially Bacillus, Kroppenstedtia, Thermoactinomyces and Scopulibacillus. These results are useful for elucidating the mechanism of flavor formation in Daqu and promoting the further application of bioturbation technology in the traditional fermentation industry.

Response of microbiota to exogenous inoculation improved the enzymatic activities of medium-temperature Daqu.

To explore the potential mechanism of improving enzymatic activities in medium-temperature Daqu (MTD) by inoculation functional isolates, we inoculated a single strain of Bacillus licheniformis, and the microbiota composed of Bacillus velezensis and Bacillus subtilis in MTD to investigate the association between the response of the functional microbiota and the enzymatic activity. The results showed that the bacterial community of MTD might be more sensitive to bioturbation than the fungal community, and the indigenous microbiota responded to the single strain more than to the microbiota. Moreover, the differential microorganisms mainly included Lactobacillales, Bacillales, and Saccharomycetales between the conventional and fortified samples. Notably, the composition of functional microbiota related to liquefying activity (LA) and saccharifying activity (SA) were significantly different, changing from Lactobacillus and Rhizomucor to Bacillus, Weissella, and Hyphopichia. That might be closely related to the effect of the bioturbation on LA (31.33%) and SA (43.54%) associated microorganisms was more tellingly. Furthermore, the relative abundance changes of bioturbation-sensitive modules in the co-occurrence network might also lead to the difference in enzymatic activities. Therefore, the LA and SA of MTD were improved by bioturbation significantly. These results provide diverse insights into the exogenous functional isolates to regulate the MTD microbiota and improve enzymatic activities.

Characterizing the correlation between species/strain-specific starter with community assembly and metabolic regulation in Xiaoqu Pei.

Studying the correlation between microbiome metabolism and flavor of fermented foods has garnered significant attention recently. Understanding the contribution of metabolic regulation and environmental stress to microecosystems is essential for exploring the mechanisms of action of traditional fermented foods. Here, the interaction between microbial communities was investigated using a Xiaoqu fermentation system, processed as "simulative microecosystems," in which starters were composed of Rhizopus-specific species/strains, Meyerozyma guilliermondii, and Bacillus licheniformis. The differences between community succession and metabolites were also explored. The results indicated that Rhizopus species/strain specificity affected starch hydrolyzation, resulting in a remarkable difference in the type and content of organic acids. This further suggested that the differences in nutrient abundance and organic acids influenced the colonization of microorganisms in the fermentation system, thereby influencing the succession of their communities. The fungi in the community predominantly originated from starters, whereas the bacteria were derived from both the environment and starter. Environmentally colonized microbes were the major contributors to the co-occurrence network and were strongly correlated with network. Regional characteristics of fermented foods were closely related to environmental microbes. These results contribute to the understanding of microbial assembly and flavor metabolism in fermented foods and provide strategies for quality regulation.

Phyllosphere microbial community of cigar tobacco and its corresponding metabolites.

Cigar is made of a typical fermented tobacco where the microbiota inhabits within an alkaline environment. Our current understanding on cigar fermentation is far from thorough. This work employed both high-throughput sequencing and chromatography-mass spectrometric technologies to provide new scientific reference for this specific fermented system. Typical cigar samples from different regions (the Caribbeans, South America, East Asia, and Southeast Asia) were investigated. The results show that Firmicutes, Actinobacteria, Proteobacteria, Ascomycota, and Basidiomycota were the predominant phyla in the cigar samples. Rather than the fungal community, it was the bacterial community structures that played vital roles to differentiate the cigar from different regions: Staphylococcus was the dominant genus in the Americas; Bacillus was the dominant genus in Southeast Asia; while in East Asia, there was no dominant genus. Such differences in community structure then affected the microflora metabolism. The correlation between microbiota and metabolites revealed that Aspergillaceae, Cercospora, and Staphylococcus were significantly correlated with sclareolide; Bacillus were positively associated with isophorone. Alcaligenaceae was significantly and positively correlated with L-nicotine and hexadecanoic acid, methyl ester. GRAPHICAL ABSTRACT.

Formation of biofilm changed the responses of Tetragenococcus halophilus to ethanol stress revealed by transcriptomic and proteomic analyses.

Biofilms were found to promote the survival of Tetragenococcus halophilus, a functional halophilic lactic acid bacterium in the production of high-salt fermented foods under various environmental stresses including ethanol stress. Here, a comprehensive exploration of the response of T.halophilus biofilms and planktonic cells to ethanol stress was performed. Biofilms showed an ability to reduce death and damage of cell membrane and wall under 12% ethanol stress The formation of biofilm changed the characteristic of Fourier transformed infrared spectroscopy (FT-IR). RNA-seq technology and iTRAQ technology revealed the differential expression of genes and proteins in biofilm and planktonic cells with or without ethanol treatment. The differentially expressed genes and proteins played positive roles in the biosynthesis of polysaccharides, proteins, and DNA, benefitting biofilm matrix production. The shelter provided by biofilms and the differential expression of genes and proteins involved in citrate formation, malate utilization, and the biosynthesis of tryptophan, fatty acid, lipoteichoic acid, and peptidoglycan might contribute to the stress tolerance of biofilm cells together. Results presented in this study may contribute to our understanding of biofilm formation by T. halophilus and the roles of bacterial biofilm in stress tolerance.

Effects of Daqu Attributes on Distribution and Assembly Patterns of Microbial Communities and Their Metabolic Function of Artificial Pit Mud.

Daqu provides functional microbiota and various nutrients for artificial pit mud (APM) cultivation. However, little is known about whether its attributes affect the microbiome and metabolome of APM. Here, two types of APM were manufactured by adding fortified Daqu (FD) and conventional Daqu (CD); they were comprehensively compared by polyphasic detection methods after being used for two years. The results showed that FD altered the prokaryotic communities rather than the fungal ones, resulting in increased archaea and Clostridium_sensu_stricto_12 and decreased eubacteria and Lactobacillus. Correlation analysis suggested that these variations in community structure promoted the formation of hexanoic acid, butyric acid, and the corresponding ethyl esters, whereas they inhibited that of lactic acid and ethyl lactate and thus improved the flavor quality of the APM. Notably, pH was the main driving factor for the bacterial community variation, and the total acid mediated the balance between the stochastic and the deterministic processes. Furthermore, the results of the network analysis and PICRUSt2 indicated that FD also enhanced the modularity and robustness of the co-occurrence network and the abundance of enzymes related to hexanoic acid and butyric acid production. Our study highlights the importance of Daqu attributes in APM cultivation, which are of great significance for the production of high-quality strong-flavor Baijiu.

Dynamic succession of microbial community in Nongxiangxing daqu and microbial roles involved in flavor formation.

Daqu, the fermentation starter of Chinese baijiu, is produced by barley and/or peas during open fermentation using natural inoculation. However, the succession of microbial communities and their roles during daqu manufacture remain unclear, which are closely related to the flavor and quality of baijiu. In this study, the dynamics of physicochemical properties, microbial communities, and volatile compounds were investigated at both fermentation (FM) and storage (ST) stages during the manufacture of Nongxiangxing daqu. High-throughput sequencing analysis showed that Weissella and Lactobacillus were the predominant bacterial genera, at the end of storage, the proportion was 31.45% and 13.14% respectively. Thermoascus, Rhizopus, Pichia and Rhizomucor were dominant fungi in daqu, and Thermoascus, Pichia and Rhizopus still occupied a large proportion at the end of daqu storage, accounting for 67.65%, 23.94% and 7.05% respectively. Redundancy analysis was employed to investigate the relationship between microorganisms and physicochemical indexes, and the results showed that acidity and Lactobacillus were positively correlated, saccharification power was positively correlated with Aspergillus, Rhizomucor, Rasamsonia and Thermoascu. The correlation networks between microorganisms and volatile substances were analyzed, a total of 60 compounds including 42 esters, 4 alcohols, 5 ketones and 5 aldehydes, 2 acids, 1 amine, 1 pyrazine were detected, the results suggested that esters are the main aroma components in daqu, and most of esters are positively correlated with fungi. In addition, key enzymes involved in carbohydrate hydrolysis, ethanol fermentation, and flavor formation were revealed. This study may further improve our understanding concerning the microbial system and microbial roles of Nongxiangxing daqu and may contribute to optimize the process during daqu manufacture.