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Characterization of Microbial Diversity and Community Structure in Fermentation Pit Mud of Different Ages for Production of Strong-Aroma Baijiu

XU-JIA WANG
XU-JIA WANG
, 
HONG-MEI ZHU
HONG-MEI ZHU
, 
ZHI-QIANG REN
ZHI-QIANG REN
, 
ZHI-GUO HUANG
ZHI-GUO HUANG
, 
CHUN-HUI WEI
CHUN-HUI WEI
 y 
JIE DENG
JIE DENG
   | 12 may 2020
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Article Category: original-paper
Publicado en línea: 12 may 2020
Páginas: 151 - 164
Recibido: 06 ene 2020
Aceptado: 29 mar 2020
DOI: https://doi.org/10.33073/pjm-2020-018
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© 2020 Xu-Jia Wang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

The production (A) and the FP (B) of strong-aroma Baijiu. A: The concentration of rice husks is about 20% of sorghum (dry weight); the main chemical composition of the fresh grain (sorghum and rice husks) are starch (48.1–50.4%), protein (5.3–8.3%), fat (3.2–3.7%), cellulose (8.3–10.2%), lignin (4.8–6.4%), pentosan (3.2–3.4%), tannin (0.1–0.5%), etc; the concentration of fresh grain is about 20% of mixed grain (dry weight); the concentration of Daqu is about 20% of sorghum; during fermentation, the moisture content of the grain is about 55–60%. B: The solid circle (●) represent sampling sites of FPM, the FP is surrounded by FPM.
The production (A) and the FP (B) of strong-aroma Baijiu. A: The concentration of rice husks is about 20% of sorghum (dry weight); the main chemical composition of the fresh grain (sorghum and rice husks) are starch (48.1–50.4%), protein (5.3–8.3%), fat (3.2–3.7%), cellulose (8.3–10.2%), lignin (4.8–6.4%), pentosan (3.2–3.4%), tannin (0.1–0.5%), etc; the concentration of fresh grain is about 20% of mixed grain (dry weight); the concentration of Daqu is about 20% of sorghum; during fermentation, the moisture content of the grain is about 55–60%. B: The solid circle (●) represent sampling sites of FPM, the FP is surrounded by FPM.

Fig. 2.

OTUs rarefaction curves at a 97% sequence similarity cutoff. OTUs from the amplicon libraries of bacteria (A), and archaea (B).
OTUs rarefaction curves at a 97% sequence similarity cutoff. OTUs from the amplicon libraries of bacteria (A), and archaea (B).

Fig. 3.

Phylum level prokaryotes taxonomic classification in the FPM samples as determined with the RDP classifier using an 85% confidence threshold.
Phylum level prokaryotes taxonomic classification in the FPM samples as determined with the RDP classifier using an 85% confidence threshold.

Fig. 4.

The relative FPM prokaryotic genera abundance, based on Roche 454 pyrosequencing. It includes the genera of bacteria (A) and genera of archaea (B). Variations in log2 scale-based genera abundance are shown by the scale bar, with “*” indicating the genera shared among all FPM samples that had the relative abundance >1%.
The relative FPM prokaryotic genera abundance, based on Roche 454 pyrosequencing. It includes the genera of bacteria (A) and genera of archaea (B). Variations in log2 scale-based genera abundance are shown by the scale bar, with “*” indicating the genera shared among all FPM samples that had the relative abundance >1%.

Fig. 5.

Concentration of phospholipid fatty acids (PLFAs) in different FPM samples. The concentration of each sample was determined by an internal standard (19:0). Error bars indicate standard deviations (n = 3).
Concentration of phospholipid fatty acids (PLFAs) in different FPM samples. The concentration of each sample was determined by an internal standard (19:0). Error bars indicate standard deviations (n = 3).

Fig. 6.

Concentrations of phospholipid fatty acids (PLFA) in different samples at the microbial groups level. Error bars show standard deviations (n = 3).
Concentrations of phospholipid fatty acids (PLFA) in different samples at the microbial groups level. Error bars show standard deviations (n = 3).

Fig. 7.

Prokaryotic community redundancy analysis (RDA) based upon Roche 454 pyrosequencing and chemical properties. The dominant community structure-related genera and the direction/magnitude of chemical properties are represented by arrows. It includes the genera of bacteria (A) and genera of archaea (B).
Prokaryotic community redundancy analysis (RDA) based upon Roche 454 pyrosequencing and chemical properties. The dominant community structure-related genera and the direction/magnitude of chemical properties are represented by arrows. It includes the genera of bacteria (A) and genera of archaea (B).

The preference of sequencing results based on different variable regions V2-V3 and V6-V8 a of the 16S rRNA gene.

Variable region FPM age (yr) Total Bacteria Archaea
reads OTU reads OTU reads OTU
V2-V3     5 10977 ± 684 126 ± 12 1267 ± 104 51 ± 9 9710 ± 562 75 ± 7
  30 10562 ± 513 100 ± 11 1009 ± 84 47 ± 8 9553 ± 410 53 ± 5
100 11172 ± 751 75 ± 6 938 ± 53 27 ± 5 10233 ± 720 48 ± 4
V6-V8     5 18893 ± 886 594 ± 17 17826 ± 844 583 ± 13 1067 ± 89 11 ± 3
  30 17409 ± 395 428 ± 15 16543 ± 354 419 ± 11 866 ± 68   9 ± 2
100 16894 ± 806 319 ± 24 15537 ± 758 312 ± 20 1357 ± 133   7 ± 2

Prokaryotic diversity indices based on a cutoff of 97% similarity of the 16S rRNA gene sequences a .

FPM age (yr) Bacteria Archaea
Chao1 Observed OTU Shannon Chao1 Observed OTU Shannon
    5 634.0 ± 84.4 583 ± 13 2.49 ± 0.56 97.3 ± 12.2 75 ± 7 1.374 ± 0.049
  30 501.6 ± 64.6 419 ± 11 2.16 ± 0.31 78.5 ± 6.3 53 ± 5 1.159 ± 0.068
100 435.8 ± 62.1 312 ± 27 1.93 ± 0.34 74.2 ± 4.7 48 ± 4 1.068 ± 0.087

Basic physiocochemical properties of fermentation pit mud.

Variable Values for fermentation pit age (yr) a
5 30 100
Moisture(%) 35.15 ± 6.42 39.62 ± 1.11 44.97 ± 2.83
Humic matter (%) 10.54 ± 1.59 13.82 ± 0.09 10.90 ± 0.52
Total acidity(%)   1.54 ± 0.09   1.81 ± 0.05   1.13 ± 0.15
Acetic acid(mg/g)   3.98 ± 0.36   3.44 ± 0.46   4.21 ± 0.67
Lactic acid(mg/g) 94.22 ± 1.30 28.81 ± 7.05 21.33 ± 3.15
Butyric acid(mg/g)   2.87 ± 0.34   2.36 ± 0.26   2.79 ± 0.38
Caproic acid(mg/g) 15.15 ± 5.12 29.62 ± 4.19 45.97 ± 5.13
Total nitrogen(%)   0.94 ± 0.09   1.41 ± 0.08   2.16 ± 0.11
NH4 +(mg/g) 14.12 ± 1.77 21.02 ± 2.03 25.32 ± 1.66
Available phosphorus(mg/g)   4.91 ± 0.52   9.15 ± 0.78   9.15 ± 0.41
Available calcium(mg/g)   9.51 ± 1.02   8.18 ± 1.91   7.41 ± 0.52

PLFA markers used for taxonomic groups.

Taxonomic group PLFA group Specific PLFA markers Reference
Bacteria Multiple groups Sum of i15:0, a15:0, i16:0, i17:0, a17:0, cy17:0, cy19:0, 16:1ω7, 18:1ω7, and 17:1ω9 (Frostegård and Bååth 1996)
Gram-positive bacteria Branched PLFAs Sum of i15:0, a15:0, i16:0, i17:0, and a17:0 (O’Leary 1988)
Gram-negative bacteria Cyclopropyl and mono PLFAs Sum of cy17:0, 16:1ω7, 18:1ω7, and 17:1ω9 (Moore-Kucera and Dick 2008)
Actinomycetes 10Me-PLFAs Sum of 10Me16:0, 10Me17:0, and 10Me18:0 (Moore-Kucera and Dick 2008)
Anaerobes Cyclopropyl Sum of cy17:0, cy19:0ω7, cy19:0ω9 (Li et al. 2017)
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