Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16

doi:10.3969/j.issn.1004-1524.20240710

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (12): 2563-2573.DOI: 10.3969/j.issn.1004-1524.20240710

• Plant Protection • Previous Articles Next Articles

Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16

YANG Xueyu¹^,²(), NIU Li¹, TAN Lin³, DENG Yulian¹, BAO Qiang⁴^,^*(), HU Qiulong¹^,^*()

1. College of Horticulture, Hunan Agricultural University, Changsha 410128, China
2. Chenzhou Branch of Hunan Tobacco Company, Chenzhou 423000, Hunan, China
3. College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
4. Tea Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China

Received:2024-08-06 Online:2025-12-25 Published:2026-01-09

Abstract

Abstract:

This study aimed to optimize the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16 to enhance its biomass and antimicrobial activity. The optimal carbon source, nitrogen source, types of inorganic salts, and their corresponding mass fraction ranges were preliminarily screened by single-factor experiments. Subsequently, the Plackett-Burman experiment was performed based on the D₆₀₀ (absorbance at 600 nm) as an important factor from the single-factor screening results, a non-linear equation between significant factors and biomass was fitted and solved with Box-Behnken Design to predict the optimal culture conditions for strain kc-16, and the predicted results were validated. Results showed that the optimized culture medium components (mass fraction) were determined to be 0.5% lactose, 4.0% peptone, 0.1% magnesium sulfate, 0.6% sodium chloride, and 0.2% calcium carbonate. The optimal culture conditions were identified as 200 r·min^-1 agitation speed, initial pH value of 7.7, temperature of 30 ℃, and inoculation amount of 0.6%. Under these conditions, the model predicted an D₆₀₀ value of 0.112, which closely matched with the validated value of 0.307. Plate count results indicated that the viable cell count after optimization reached 6.7×10⁹ CFU·mL^-1, which was 16.75 times that the initial count (4.0×10⁸ CFU·mL^-1). This study achieved optimized culture conditions for strain kc-16 through a combination of single-factor experiments and response surface methodology, providing support for its large-scale production and application.

Key words: Bacillus subtilis subsp. inaquosorum, culture medium, response surface methodology, fermentation optimization

CLC Number:

S435.711

YANG Xueyu, NIU Li, TAN Lin, DENG Yulian, BAO Qiang, HU Qiulong. Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16[J]. Acta Agriculturae Zhejiangensis, 2025, 37(12): 2563-2573.

Figures/Tables 10

References 29

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水平Level	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%
高水平	1.0	5	0.2	0.8	0.4	8	220	32	1.0
低水平	0.5	3	0.1	0.4	0.1	7	180	28	0.5

水平Level	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%
高水平	1.0	5	0.2	0.8	0.4	8	220	32	1.0
低水平	0.5	3	0.1	0.4	0.1	7	180	28	0.5

组别Group	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%	D₆₀₀
1	1.0	5	0.1	0.8	0.4	8	180	28	0.5	1.68
2	0.5	5	0.2	0.4	0.4	8	220	28	0.5	0.32
3	1.0	3	0.2	0.8	0.1	8	220	32	0.5	0.10
4	0.5	5	0.1	0.8	0.4	7	220	32	1.0	0.16
5	0.5	3	0.2	0.4	0.4	8	180	32	1.0	0.80
6	0.5	3	0.1	0.8	0.1	8	220	28	1.0	0.18
7	1.0	3	0.1	0.4	0.4	7	220	32	0.5	1.90
8	1.0	5	0.1	0.4	0.1	8	180	32	1.0	0.33
9	1.0	5	0.2	0.4	0.1	7	220	28	1.0	1.98
10	0.5	5	0.2	0.8	0.1	7	180	32	0.5	1.97
11	1.0	3	0.2	0.8	0.4	7	180	28	1.0	1.89
12	0.5	3	0.1	0.4	0.1	7	180	28	0.5	1.77

组别Group	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%	D₆₀₀
1	1.0	5	0.1	0.8	0.4	8	180	28	0.5	1.68
2	0.5	5	0.2	0.4	0.4	8	220	28	0.5	0.32
3	1.0	3	0.2	0.8	0.1	8	220	32	0.5	0.10
4	0.5	5	0.1	0.8	0.4	7	220	32	1.0	0.16
5	0.5	3	0.2	0.4	0.4	8	180	32	1.0	0.80
6	0.5	3	0.1	0.8	0.1	8	220	28	1.0	0.18
7	1.0	3	0.1	0.4	0.4	7	220	32	0.5	1.90
8	1.0	5	0.1	0.4	0.1	8	180	32	1.0	0.33
9	1.0	5	0.2	0.4	0.1	7	220	28	1.0	1.98
10	0.5	5	0.2	0.8	0.1	7	180	32	0.5	1.97
11	1.0	3	0.2	0.8	0.4	7	180	28	1.0	1.89
12	0.5	3	0.1	0.4	0.1	7	180	28	0.5	1.77

方差来源 Soruce	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	p	标准化效应 Standardization effect	系数 Coefficient	贡献率 Contribution rate/%	重要性排序 Importance rank
A	0.145 200	1	0.145 200	256.235 300	0.003 880	-0.220 000	-0.110 000	14.508 390	4
B	0.003 333	1	0.003 333	5.882 353	0.136 132	-0.033 330	-0.016 670	0.333 067	8
C	0.090 133	1	0.090 133	159.058 800	0.006 228	0.173 333	0.086 667	9.006 128	6
D	0.104 533	1	0.104 533	184.470 600	0.005 377	-0.186 670	-0.093 330	10.444 980	5
E	0.014 700	1	0.014 700	25.941 180	0.036 454	0.070 000	0.035 000	1.468 825	7
F	0.252 300	1	0.252 300	445.235 300	0.002 238	0.290 000	0.145 000	25.209 830	1
G	0.003 333	1	0.003 333	5.882 353	0.136 132	0.033 333	0.016 667	0.333 067	9
H	0.172 800	1	0.172 800	304.941 200	0.003 263	0.240 000	0.120 000	17.266 190	3
I	0.213 333	1	0.213 333	376.470 600	0.002 646	0.266 667	0.133 333	21.316 280	2
残差Residual	0.001 133	2	0.000 567	—	—	—	—	—	—
总离差平方和	1.000 798	11	—	—	—	—	—	—	—
Cor Total
模型Model	0.999 665	9	0.111 074	196.013 100	0.005 086	Significant	—	—	—

Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16

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组别Group	F	I/%	H/℃	D₆₀₀
1	7.0	0.10	30	1.441
2	8.0	0.10	30	1.868
3	7.0	1.00	30	1.913
4	8.0	1.00	30	1.916
5	7.0	0.55	28	1.929
6	8.0	0.55	28	1.994
7	7.0	0.55	32	1.929
8	8.0	0.55	32	1.994
9	7.5	0.10	28	1.741
10	7.5	1.00	28	1.864
11	7.5	0.10	32	1.761
12	7.5	1.00	32	1.864
13	7.5	0.55	30	0.200
14	7.5	0.55	30	0.005
15	7.5	0.55	30	0.095
16	7.5	0.55	30	0.136
17	7.5	0.55	30	0.027

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	p
模型Model	0.430	9	0.048	7.100	0.008 5
F	0.039	1	0.039	5.780	0.047 2
I	0.070	1	0.070	10.250	0.015 0
H	5.000×10^-5	1	5.000×10^-5	7.368×10^-3	0.934 0
FI	0.045	1	0.045	6.620	0.036 8
FH	5.551×10^-17	1	5.551×10^-17	8.180×10^-15	1.000 0
IH	1.000×10^-4	1	1.000×10^-4	0.015	0.906 8
F²	0.025	1	0.025	0.680	0.096 4
I²	0.220	1	0.220	0.120	0.000 7
H²	0.012	1	0.012	1.810	0.220 2
残差Residual	0.048	7	6.787×10^-3	—	—
失拟项Lack of fit	0.022	3	7.368×10^-3	1.160	0.428 0
纯误差Pure error	0.025	4	6.350×10^-3	—	—
总和Cor total	0.936	32	—	—	—

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