Analysis of Microbial Community Structure and Volatile Compounds in the Pit Mud Used for Manufacturing Taorong-type Baijiu Based on High-Throughput Sequencing

In this study, the pit mud used in manufacturing Taorong-type Baijiu was collected from the upper, middle, lower and bottom layers of pits in Henan Yangshao Liquor Co., LTD. Besides, high-throughput sequencing (HTS) technology was adopted to analyze the microbial community structure of the pit mud. In addition, the volatile compounds in the pit mud were subjected to preliminarily qualitative analysis through headspace-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The results of HTS demonstrated that there were 5, 3, 5 and 5 dominant bacterial phyla (including 11, 11, 9 and 8 dominant bacterial genera) and 3, 3, 3 and 3 dominant fungal phyla (including 4, 7, 7 and 5 dominant fungal genera) in the pit mud from F-S (upper), G-Z (middle), H-X (lower) and I-D (bottom), respectively. The qualitative analysis results of volatile compounds demonstrated that a total of 78 kinds of volatile compounds were detected in the pit mud, including 46, 45, 39 and 49 kinds in the pit mud from F-S, G-Z, H-X and I-D, respectively. Ester and acid were the two main components in the pit mud. Meanwhile, the correlation between microorganisms and main volatile compounds in the pit mud was analyzed. Moreover, Lentimicrobium, Syner-01 and Blvii28_wastewater-sludge group were found for the rst time in the pit mud used for manufacturing Taorong-type Baijiu. The ndings of this study could provide a theoretical foundation for improving the quality of pit mud and the avor of Taorong-type Baijiu.


Introduction
Baijiu has a long history in China, and it is one of China's national cultural characteristics [1] . As an innovative avor type in this industry in China, Taorong-type Baijiu is uniquely characterized by "yellowish and transparent color, delicate, mellow and harmonious avor and lasting fragrance" [2] . There is an old saying, namely, "pits throughout years and distillers' grains throughout centuries". Taorong-type Baijiu is fermented in a solid anaerobic environment [3] . Pit mud is the basis for the fermentation of traditional solid-state Baijiu [4] . As one of the crucial in uencing factors, pit mud determines the quality and avor of Baijiu produced to a great extent [5] , and plays a vital role in the brewing process of Taorong-type Baijiu [6] .
Besides, pit mud provides a comfortable habitat for fermentation microorganisms [5] , who need to grow and reproduce in pit mud [7] . In addition, different microbial communities constitute a complex ecosystem [8] . The avor substances in Taorong-type Baijiu are mainly generated in pit mud. There are different living environments for the microorganisms in different spatial positions of the pits [9] . Various microbial communities contribute to generating speci c avor compounds, which determine the avor and quality of Baijiu [10] .
The microbial diversity of pit mud has a signi cant in uence on the avor of Baijiu [11] . Bacteria and fungi are important functional ora, which can produce abundant enzymes and avor substances contained in Baijiu, thus bestowing it with unique avor [12] . Bacterial metabolism in pit mud would generate important organic acids (such as butyric acid and caproic acid) in Taorong-type Baijiu, which will increase its avor and reduce its stimulation. Fungi, such as Saccharomyces cerevisiae (S. cerevisiae) can be involved in alcoholic fermentation, and Aspergillus can be involved in the generation of glucoamylase. The composition and quantity of microorganisms in pit mud are two of the factors affecting the avor of Taorong-type Baijiu [13] . Pit mud in different spatial positions could affect the reproduction and metabolism of microorganisms and the avor composition of Taorong-type Baijiu [14] . Therefore, it is necessary to explore the microbial community in pit mud.
Traditionally, isolation and culture methods are commonly adopted in the study of microorganisms [15] .
However, the species and quantity of isolated microorganisms are limited, many microbial species are usually missed, and even key functional microorganisms may be omitted [16] . Due to the fact that the closed environment of pit mud is strictly anaerobic in essence, many microorganisms cannot be cultivated or it is di cult to cultivate them. The non-culture method can be employed to detect more microorganisms compared with the culture method, which objectively re ects the microbial composition of samples [17] . In contrast, high-throughput sequencing (HTS) technology is the preferred method for the analysis of pit mud [18,19] . HTS methods have been extensively used, in an attempt to obtain a more comprehensive analysis of microbial diversity [20] . HTS, also known as the next-generation sequencing (NGS) technology, features high throughput, high sensitivity, high resolution, low cost and simple operation, which can be employed to generate plenty of data in a short time [21] , and can comprehensively present the microbial community composition and diversity information of samples. Headspace solidphase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) is an advanced technology [22] , which can be considerably effective in the analysis of volatile substances [23] .
Solid-phase microextraction (SPME) is a rapid analysis technology, which integrates the pretreatment, adsorption and extraction of samples. Owing to the emergence of different types of adsorbents with multiple polarities, it is possible to separate trace compounds with different substrates, which has been used in a series of elds. Besides, it is suitable for the detection and analysis of trace components, process monitoring and so forth in Baijiu and related production process [24] . Gas chromatography-mass spectrometry (GC-MS) has been regarded as the gold standard for the analysis of many compounds [25] .
Further, it is a common and mature technique in the analysis of volatile and semi-volatile aroma components in the world. This technology has such advantages as high sensitivity, high separation e ciency, high selectivity and rapid analysis. It has a high and excellent performance in the separation, detection and data processing, and hence it can be applied in the accurate qualitative analysis [2] .
Currently, there is no report on the microbial community structure, volatile compounds and their correlation in the pit mud used for manufacturing Taorong-type Baijiu. Therefore, HTS technology was adopted in this study to analyze the microbial community structure of the upper, middle, lower and bottom layers of these pits used in manufacturing Taorong-type Baijiu. Moreover, the volatile compounds in the pit mud from different layers were investigated by a combined method with HS-SPME and GC-MS.
Meanwhile, the correlation between the microbial community structure and volatile compounds was explored, which provided resources for establishing the microbial information database of Taorong-type Baijiu. Additionally, it could provide theoretical support for improving the quality of pit mud, cultivating arti cial pit mud, and improving the avor and quality of Baijiu, which conduces to thoroughly understanding the contribution of microbes in pit mud to the avor during the brewing of Baijiu.

Materials
The pit mud samples adopted in this experiment were from the 30-year-old pit mud in Henan Yangshao Liquor Co., LTD. Three pits were randomly selected, and 10g pit mud was collected from the center of the four pit walls of the upper layer (F-S, 50cm away from the pit mouth), the middle layer (G-Z, the pit center) and the lower layer (H-X, 50cm away from the pit bottom) of each pit, as well as the center of the bottom layer (I-D). Subsequently, they were mixed evenly. These collected samples were marked as F-S-1, F-S-2, F-S-3, G-Z-1, G-Z-2, G-Z-3, H-X-1, H-X-2, H-X-3, I-D-1, I-D-2 and I-D-3, respectively. They were stored in a refrigerator at -20℃ [26] .

Reagents and Instruments
Reagent: D3141 HiPure Soil DNA Kits (Soil DNA Extraction Kit) were purchased from Guangzhou Magen Biotechnology Co., Ltd.; PCR related reagents were purchased from TOYOBO (SHANGHAI) BIOTECH CO., LTD.; AMPure XP magnetic beads were purchased from Beckman Coulter, USA; Anhydrous ethanol was purchased from Guangzhou Chemical Reagent Factory (GCRF); Agarose (BiowestAgarose) was purchased from Beijing Mengyimei Business Center; The goldview (Goldview I) was purchased from Beijing Mengyimei Business Center; NaCl was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.

Sample DNA Extraction
According to the instruction manual for HiPure Soil DNA Kits (Soil DNA Extraction Kit) of Guangzhou Magen Biotechnology Co., Ltd., the genomic DNA of bacteria was extracted, and the DNA integrity was detected by 1% agarose gel electrophoresis.

Library Quanti cation and Sequencing
AMPure XP Beads were adopted to purify the products of the second round of ampli cation, and ABI StepOnePlus Real-Time PCR System (Life Technologies) was adopted to conduct the quanti cation.
Relying on Guangzhou Gene Denovo Biotechnology Co., Ltd., the products were sequenced according to PE250 mode pooling of Novaseq 6000.

Pretreatment of Pit Mud Samples
1g pit mud was put into a headspace bottle, and 2g NaCl and 5mL distilled water were added. Subsequently, the stopper was tightened and the mixture was shaken properly.

Conditions of Headspace Solid-phase Microextraction (HS-SPME)
1g pit mud sample was weighed and placed in a headspace bottle, which was preheated in a water bath at 50℃ for 10min. Solid-phase CAR/PDMS (75µm CAR/PDMS, carbon molecular sieve/polydimethylsilane) extraction ber head was inserted into the silica gel stopper of the headspace bottle, and inserted into the sample for headspace adsorption for 30min.

Results
Basic Sequencing Data and Alpha Diversity Analysis As can be seen from Table 1, effective sequences are obtained by the quality control of original sequences and the removal of chimeras. The average remaining effective sequences of pit mud samples in F-S, G-Z, H-X and I-D are 116693, 122327, 115977 and 121991, respectively. The coverage rate is more than 0.99, thus indicating that the sequencing depth is su cient, the sequences in the samples are basically completely detected, and the results are true and reliable, which can be used for the subsequent analysis.
According to the Alpha diversity analysis of pit mud samples, Chao 1 index is mainly related to the abundance of samples, and the larger the index, the higher the abundance; Shannon index is mainly related to the diversity information of samples, which not only re ects the abundance of species, but also re ects the evenness of species. The larger the index, the higher the diversity. As can be seen from Table  1

Analysis of Fungal Community Structure at the Phylum Level
As can be seen from Fig. 3

Analysis of Bacterial Community Structure at the Genus Level
As can be seen from Fig. 4

Composition and Content of Volatile Compounds in Pit Mud
The composition and content of volatile compounds in pit mud are listed in Table 2.  being 0.67%, 3.94%, 0.06% and 9.23% respectively. It is mainly generated under the synergistic action of various bacteria and enzymes [11] . Ethanol and acetic acid combine to form butyric acid, followed by the synthesis of caproic acid through esterase. Subsequently, caproic acid is synthesized through ethanol [27] .
Ethyl caproate is considered as the key component contributing to the avor and quality of Baijiu [28] . produced by ethanol oxidation, and then ethanol reacts with butyric acid to produce caproic acid [29] .
Caproic acid and ethyl caproate produced by pit mud fermentation are the main aroma components of Taorong-type Baijiu [11] . A large amount of caproic acid is produced during fermentation, and then reacts with ethanol to produce ethyl caproate, the main aroma component of Baijiu [30] . Acetic acid, butyric acid, heptanoic acid and caprylic acid have a second higher content, and they are the main organic acid components of Taorong-type Baijiu. The correlation analysis between the main volatile compounds and bacteria and fungi in pit mud was conducted respectively, and the correlation heat map was obtained. As can be seen in Fig. 6, the bacterial genera that are closely correlated with the main volatile compounds in pit mud include Hydrogenispora, Aminobacterium, Lentimicrobium, Sedimentibacter, Rumino libacter, Christensenellaceae_R-7_group and Syner-01. As can be seen from Fig. 7, the fungal genera that are closely correlated with the main volatile compounds in pit mud include Rhizopus, Thermomyces, Monascus and Penicillium. Ethyl caproate is the main aroma substance in Baijiu, which has the highest correlation with Sedimentibacter and Monascus, followed by Hydrogenispora and Rhizopus. Sedimentibacter can synthesize caproic acid, butyric acid, acetic acid, hexanol, ethanol and butanol with carbon source and protein as substrates, and can generate ethyl caproate. Acetic acid is positively correlated with Syner-01, Rumino libacter, Lentimicrobium, Caproiciproducens and Thermomyces, and negatively correlated with Aminobacterium, Monascus and Penicillium. Hexanol, octanol, 6-hendecanol and benzyl alcohol are positively correlated with Syner-01, Christensenellaceae_R-7_group, Rumino libacter and Thermomyces. Therefore, the microbial community structure in pit mud has a certain in uence on the avor and quality of Baijiu.

Conclusion And Discuss
In this study, HTS technology was employed to analyze the bacterial community structure of the pit mud used for manufacturing Taorong caproic acid and reducing lactic acid [31] ; The content of Synergistetes shows a decrease-increase trend, with the highest content of 10.3% in I-D. These bacteria could degrade amino acids, and some of them are speci c anaerobic. Deng et al. [32] adopted HTS technology to explore the microbial community structure in the 5-year-old and 30-year-old pit mud from a wine company in Sichuan, and they found that Firmicutes, Bacteroidetes, Synergistetes, Spirochaetes and Chloro exi were the dominant bacterial genera. There were 3 dominant fungal phyla in the pit mud from F-S, G-Z, H-X and I-D, including Ascomycota, Mucoromycota and Basidiomycota. It can be seen that, at the phylum level, there is the same structure composition of fungal phyla in different structure layers in pit mud. The content of Ascomycota and Mucoromycota is higher, and they are absolutely dominant fungal phyla in each layer of pit mud. On the whole, with an increase in the depth of pit mud, the relative abundance of Ascomycota shows a downward trend, while that of Mucoromycota shows an upward trend.
There are 11, 11, 9 and  At the genus level, there is spatial heterogeneity in the microbial community structure of pit mud at different spatial levels. Hydrogenispora, Caproiciproducens, Sedimentibacter and Syntrophomonas belong to Clostridia,Clostridia ora can synthesize caproic acid, butyric acid, acetic acid, hexanol, ethanol and butanol with carbon source and protein as substrates, and it can also generate ethyl caproate [33] . In the pits, Clostridia and Bacteroidia have a considerably higher proportion (51.1% and 29.5%, respectively), and the content of Clostridia is the highest in the pit mud from I-D, reaching 63.86%, indicating that the tested pit mud samples are in the mature state. Clostridia can use ethanol and acetic acid to generate acetoacetic acid [34] , and it is one of the key microbial groups to promote the synthesis of short-chain and medium-chain fatty acids, such as butyric acid and caproic acid [35] . These microbes can be easily screened from pit mud. Hydrogenispora, Sedimentibacter, Petrimonas, Syntrophomonas and Aminobacterium have a high content in the pit mud from I-D. Hydrogenispora can produce acetate, ethanol and H 2 [36] . Sedimentibacter and Aminobacterium can ferment amino acids [33] to generate ammonium nitrogen through metabolism, which could provide nitrogen sources for the growth of other microbes [37] . In addition, they have the function of degrading lactic acid; Most members of Petrimonas can use glucose to produce H 2 and CO 2 , or acetic acid and propionic acid. Aminobacterium, Syntrophomonas and Petrimonas play a positive role in the maturing of pit mud [38] . Caproiciproducens is a signi cant ora in pit mud. Due to the fact that the caproic acid produced by Caproiciproducens can inhibit lactic acid bacteria, and the quality of pit mud is closely correlated with pH, the degradation of lactic acid bacteria can improve the quality of pit mud. Lactobacillus is the uniquely dominant bacterial genera in the pit mud from G-Z, and its metabolism can produce aroma substances of Nongxiang-type (strong aroma) Baijiu. However, the content of Lactobacillus in the pit mud from G-Z is too high, reaching 10.87%. The accumulation of lactic acid will increase the content of ethyl lactate in Baijiu and reduce its quality [30] . Besides, the content of Lactobacillus in mature pit mud will decrease signi cantly [39] . Hu et al. [40] also found that the content of Lactobacillus in high-quality pit mud decreased signi cantly, and the content of such core bacteria as Aminobacterium increased signi cantly. Therefore, there should be a moderate amount of lactic acid bacteria in pit mud [37] .
There are 4, 7, 7 and 5 dominant fungal genera in the pit mud from F-S, G-Z, H-X and I-D, respectively. Thermomyces is the absolutely dominant fungal genera in the pit mud from F-S, and its content decreases with the depth increase of pit mud. Rhizopus is the absolutely dominant fungal genera in the pit mud from G-Z, H-X and I-D, and its content shows an upward trend with the depth increase of pit mud. Thermomyces is the dominant mold in the pit mud used for manufacturing Daqu jiu and the brewing environment. It has favorable comprehensive enzyme activity characteristics, can be used as an important microbial index for brewing environment evaluation, monitoring, storage period and quality evaluation of Daqu products [41] . In addition, it has strong thermal stability and can maintain stable catalytic e ciency under the condition of high temperature in the fermentation process of Baijiu [42] ; Rhizopus is the main contributor to protein analysis in metabonomics. It plays a sacchari cation role by secreting three glycosidases and two glycosyltransferases [43] . It is the crucial ora for the sacchari cation of distiller's yeast and contributes to improving the sacchari cation agent for food fermentation. Aspergillus has been extensively applied in the brewing industry. It has certain acid resistance and a strong ability to produce amylase, protease and metabolize organic acids, and could secrete glucoamylase [44] . Therefore, it plays an important role in the production and aroma generation of Baijiu.
A total of 78 volatile compounds are detected from these pit mud samples, including 46, 45, 39 and 49 kinds in the pit mud from F-S, G-Z, H-X and I-D, respectively. These volatile compounds mainly include esters, acids and alcohols. Esters and acids are two main components in these pit mud samples. There are signi cant differences in esters and acids, as well as their content in different layers of pit mud; while, the differences in alcohols are not signi cant. Ethyl caproate, 1-hexanol and caproic acid are esters, alcohols and acids with the highest content in these pit mud samples, respectively. Meanwhile, the correlation analysis between microbes and volatile compounds in these pit mud samples is also conducted, and the correlation heat map could clearly present the correlation between microbes and compounds. Moreover, Lentimicrobium, Syner-01 and Blvii28_wastewater-sludge group are found in the pit mud used for manufacturing Taorong-type Baijiu for the rst time, which provides resources for establishing the microbial information database of Taorong-type Baijiu. Furthermore, the ndings of this study also provide theoretical support for improving the quality of pit mud and enhancing the avor and quality of Taorong-type Baijiu.