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Isolation, Identification, and Fermentation Medium Optimization of a Caproic Acid‑Producing Enterococcus casseliflavus Strain from Pit Mud of Chinese Strong Flavor Baijiu Ecosystem

Hao Luo
Hao Luo
, 
Tao Li
Tao Li
, 
Jia Zheng
Jia Zheng
, 
Kaizheng Zhang
Kaizheng Zhang
, 
Zongwei Qiao
Zongwei Qiao
, 
Huibo Luo
Huibo Luo
 und 
Wei Zou
Wei Zou
   | 21. Dez. 2022
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Article Category: Original Paper
Online veröffentlicht: 21. Dez. 2022
Seitenbereich: 563 - 575
Eingereicht: 19. Sept. 2022
Akzeptiert: 11. Nov. 2022
DOI: https://doi.org/10.33073/pjm-2022-052
Schlüsselwörter
© 2022 Hao Luo et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

The yield of caproic acid of different isolated strains.
The yield of caproic acid of different isolated strains.

Fig. 2

Morphological and phylogenetic characteristics of the F6 strain. a) Colony morphology of strain BF-1, b) Gram staining and SEM image showing the coccoid shape of the BF-1 strain, c) phylogenetic analysis of strain BF-1 based on BLAST results.
Morphological and phylogenetic characteristics of the F6 strain. a) Colony morphology of strain BF-1, b) Gram staining and SEM image showing the coccoid shape of the BF-1 strain, c) phylogenetic analysis of strain BF-1 based on BLAST results.

Fig. 3

Genome annotation of strain BF-1. A) COG functional gene classification, B) KEGG functional gene classification.
Genome annotation of strain BF-1. A) COG functional gene classification, B) KEGG functional gene classification.

Fig. 4

Caproic acid production pathway of strain BF-1. Substrates are shown in purple, products are shown in red, annotated genes are shown in green, and non-annotated genes are shown in blue.
Caproic acid production pathway of strain BF-1. Substrates are shown in purple, products are shown in red, annotated genes are shown in green, and non-annotated genes are shown in blue.

Fig. 5

Single-omission growth experiments based on CDM.
Single-omission growth experiments based on CDM.

Fig. 6

Effects of adding carbon and nitrogen compounds on caproic acid yield of strain BF-1. a) Effects of different carbon sources, b) effects of different nitrogen sources, c) effect of glucose addition, d) effect of yeast extract addition.
Effects of adding carbon and nitrogen compounds on caproic acid yield of strain BF-1. a) Effects of different carbon sources, b) effects of different nitrogen sources, c) effect of glucose addition, d) effect of yeast extract addition.

Fig. 7

Effect of supplying essential nutrients to the fermentation medium on the cell growth and caproic acid production by E. casseliflavus BF-1 strain.
Effect of supplying essential nutrients to the fermentation medium on the cell growth and caproic acid production by E. casseliflavus BF-1 strain.

Fig. 8

Effect of adding of inorganic salts and growth factors on caproic acid production by strain BF-1. a) Sodium acetate, b) MgSO4 · 7H2O, c) KH2PO4, d) K2HPO4, e) (NH4)2SO4, f) L‑cysteine, g) biotin, h) ethanol.
Effect of adding of inorganic salts and growth factors on caproic acid production by strain BF-1. a) Sodium acetate, b) MgSO4 · 7H2O, c) KH2PO4, d) K2HPO4, e) (NH4)2SO4, f) L‑cysteine, g) biotin, h) ethanol.

Results of L16 (44) orthogonal experiment.

Glucose Yeast extract Sodium acetate Ethanol Caproic acid production (g/l)
1 1 1 1 1.76
1 2 2 2 1.99
1 3 3 3 1.81
1 4 4 4 1.63
2 1 2 3 2.33
2 2 1 4 2.22
2 3 4 1 2.79
2 4 3 2 1.96
3 1 3 4 1.87
3 2 4 3 2.43
3 3 1 2 2.12
3 4 2 1 1.96
4 1 4 2 2.44
4 2 3 1 2.56
4 3 2 4 2.08
4 4 1 3 1.56
K1 1.798 2.1 1.915 2.268
K2 2.325 2.3 2.09 2.127
K3 2.095 2.22 2.05 2.033
K4 2.16 1.777 2.322 1.95
R 0.527 0.523 0.407 0.318

Basic characteristics of the genome of strain BF-1.

Features BF-1
Read-Num 8,697,842 bp
HQ reads 8,663,546 bp
Genome size 2,968,377 bp
G + C content 43.52%
ORF number 3270
ORF density 0.967 genes per kb
ORF average length 907,76 bp
Intergenetic region length 414,643 bp
Coding percentage 87.74%
rRNA 3
tRNA 53
ncRNA 113
Q value 40
Q20-rate 98.84
Q30-rate 95.83
Contig N50 372,495 bp
Scaffold N50 570,209 bp
Contig total sequence length 3,383,020 bp
Scaffold total sequence length 3,383,020 bp

Orthogonal experiment of four factors and four levels.

Level Factor A (glucose g/l) Factor B (yeast extract g/l) Factor C (sodium acetate g/l) Factor D (ethanol %)
1 10 10 2 1
2 20 20 4 2
3 30 30 6 3
4 40 40 8 4

Variance analysis of the strain BF-1 orthogonal experiment.

Variance source Sum of squares Freedom Mean square F-value p-value Significance
Correction model   1.766a 12 0.147 14.537 0.025
Intercept 70.183 1 70.183 6,933.032 0.000
Factor A   0.583 3 0.194 19.182 0.018 *
Factor B   0.616 3 0.205 20.268 0.017 *
Factor C   0.345 3 0.115 11.354 0.038 *
Factor D   0.223 3 0.074 7.342 0.068 –
Error   0.030 3 0.010
Total 71.979 16
Corrected total   1.796 15

The occurrence of virulence factor encoding genes in the strain BF-1 genome.

Related genes VFclass
ebpA, ebpC, srtC, efaA, slrA Adherence
cpsA, cpsB, cpsJ Antiphagocytosis
bopD Biofilm formation
– Toxin
– Exoenzyme
ctpV Copper up take
capD, cps4I, cpsY Immune evasion
htrA/degP Protease
cheY Regulation

Results of physiological and biochemical experiments.

BF-1strain
Voges-Proskauer test – Mannitol +
MRtest + Fructose +
Gelatin hydrolysis – Stachyose +
Starch hydrolysis + Cellobiose +
Nitrate reduction – Xylose +
Lactose + Galactose +
Sucrose + Glycerin –
Raffinose + Mannose +
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