The Effect of Spatial Location of Fermentation Pit on Prokaryotic Community Diversity in Pit Mud for Chinese Strong-Flavor Baijiu Production

Chinese strong-flavor baijiu (CSFB) accounts for more than 70% of all Chinese liquor 18 markets. Diverse microbes in pit mud found in the fermentation pit play a key role in 19 CSFB production. However, the effect of spatial location on the diversity and 20 structure of the microbial community in pit mud is still poorly understood Prokaryotic microbes in different pit mud （ 4- and 40-year ） were analyzed by using 23 Illumina MiSeq sequencing of 16S rRNA gene. The samples were collected from pit 24 mud that was 4 and 40 years old in the top, middle, and under parts of the cellar walls 25 and at the bottom of them. The results showed there was no significant difference 26 (p>0.05) in the physicochemical factors, the Observed OTU, and α -diversity between 27 the pit mud of two different ages. The index of the 4-year-old pit mud was lower 28 (except for total acid, which was higher than) than that of the 40-year-old pit mud. The pH, total acid, Observed OTU, Chao 1, ACE in the 4-year-old pit mud (in the top 30 and middle part of the cellar wall) and the 40-year-old pit mud(in the top part of the 31 cellar wall) had significant (p<0.05) difference. At the phylum level, Firmicutes 32 (56.70%) and Bacteroidetes (26.56%) accounted for more than 80% of the overall 33 level. For the 4-year-old pit mud sample, the dominant genuses at each location were 34 Proteiniphilum , Lactobacillus , and Caproiciproducens , while for the 40-year-old pit 35 mud, there were 10 common genuses accounted for more than 50% of the dominant 36 genuses in different pit mud belong to Clostridia. The RDA analysis results showed 37 that 85.71% of the dominant bacteria positively correlated with pH, and especially the most correlated with the total acid and available phosphorus, so its content was significantly related to the dominant bacteria (p<0.01). Therefore, it inferred that the total acid and available phosphorus were the main physicochemical factors that affected the spatial distribution of prokaryotic microbial communities in the pit mud 42 of cellars from the liquor distillery. Comparing the young pit mud and the old pit mud, the structure and physicochemical 45 factors of the prokaryotic microbial community in pit mud from Henan liquor-making 46 company changed, and the changes occurred in the spatial location of different pits. 47 Clostridia, which accounted for more than 50% of all types of pit mud, were 48 responsible for the effect of total acid and available phosphorus on the microbial 49 community. Based on the above, it provides a theoretical basis for Henan liquor- 50 making companies in maintaining pit mud. 51 52

Prokaryotic microbes in different pit mud(4-and 40-year)were analyzed by using (except for total acid, which was higher than) than that of the 40-year-old pit mud. 29 The pH, total acid, Observed OTU, Chao 1, ACE in the 4-year-old pit mud (in the top In Table 1, compared with the 40-year pit mud, the water content, pH value,   94 ammonium nitrogen, and available phosphorus content of the 4-year-old pit mud were 95 lower but its total acid content was significantly higher (p<0.05). There was a 96 significant difference (p>0.05) neither in the moisture content of different aged pit 97 mud nor in the physicochemical factors of 40-year-old pit mud in different locations.

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In comparison with the 40-year-old pit mud, the pH value of the 4-year-old pit mud 99 was lower (p<0.05) (on the top and middle cellar wall) but the same at the bottom 100 while its total acid content at all locations was higher (p<0.05). Furthermore, the contents of NH4+ and AP were the lowest in the top 4-year-old pit mud. In summary, 102 the physicochemical factors of the top 4-year-old pit mud were different from those of 103 the under 4-year-old pit mud and the 40-year-old pit mud.

Alpha diversity of microbial communities in different pit muds 107
As shown in Table 1，the   The cluster analysis of species UniFrac (with species and its abundance taken into 124 account) showed that the samples were classified into two categories (Fig. 1). The  At the genera level, 301 genera were found. In this study, the top 10 genera (Top 10, 158 38 in total) of each sample were selected to reveal the diversity of microbial 159 community composition in the pit mud from the genus classification level (Fig. 3). 160 The top 10 genera of each sample contained dominant genera(relative abundance> 1%,  were detected (Fig. 5a). All nodes belonged to 12 classes, mainly concentrated in 211 Clostridia (50% of the total number of nodes), Bacteroides (19.05%), 212 Methanobacteria (7.14%), and Gammaproteobacteria (4.76%), and Bacillus (4.76%). Bacillus (Figure 6a). They belonged to 6 classes: Clostridia (4), Bacteroidia (2),

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The total content of the 40-year pit mud hub (0.047%) was much lower than that of 220 the 4-year pit mud hub (11.7%).  40-year-old pit mud from different locations of the cellar could be clustered together 241 and its prokaryotic microbial community had obvious spatial structure characteristics, 242 which were greatly affected by the age of the pit mud.

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Conditional Term Effects was used to analyze comprehensively how each 244 physicochemical factors contributed, which was total acid (30.9%), AP (15.5%), pH 245 (6.1%), moisture content (2.9%), and NH4+ (2.4%), had influenced the flora structure 246 (Fig 6a), The total acid and AP had the greatest influence on the prokaryotic microbial 247 community and showed a significant correlation (P<0.01). There was no significant 248 correlation between pH, water content, and ammonium nitrogen (P>0.05). the bacteria content in the pit mud (p<0.01) [11]. In this study (Table 1)   Axis1, but only two genera, Clostridium_sensu_stricto_12 and Lactobacillus, were 332 distributed on the right side and were positively correlated with total acid (Fig. 6a).

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Lactobacillus was positively correlated with total acid content and negatively with pH.
Because Lactobacillus metabolites were mainly lactic acid, the accumulation of lactic 335 acid could lead to an increase in total acid content and a decrease in pH, which in turn 336 gave rise to a deterioration in the quality of pit mud. were principal. The proportion of endemic bacteria was higher in 4-year-old pit mud, 354 and the number of endemic bacteria in 4-year-old pit mud on the top cellar wall was the highest. In 40-year-old pit mud, the dominant bacterial genuses were common in walls. (Figure 1). 16 representative samples (

Determination of physicochemical factors 384
Referring to drying methods and potentiometric methods employed by Li Junhui and   The Illumina MiSeq sequence was controlled using Cutadapt (v1.9.1), Vsearch (1.9.6), 420 and Qiime (1.9.1) software. These quality controls included sequence splicing, 421 removal of sequences containing N, primers, and linkers, bases with a quality value 422 <20 and sequences with a length of <200 bp, and chimeras. VSEARCH (1.9.6) was Origin9.0 was applied to draw a histogram of the percentage of phylum in the 432 advantages of pit mud samples (relative abundance was more than 1%). HemI and 433 software Canoco 5 were used to draw Heat maps and RDA maps separately.