Optimization of Bacillus velezensis FJ17-4 Fermentation
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摘要:目的 贝莱斯芽孢杆菌FJ17-4对许多病原菌具有较强的抑制作用，为提高其生防作用，开展FJ17-4发酵技术研究。方法 以发酵液的OD600值为评估指标，采用单因素和正交试验方法对发酵培养基和发酵条件进行筛选和优化，获得最佳发酵培养基和发酵条件后，进一步对优化后发酵液的菌体数、病原菌抑制率和室内盆栽防治效果进行测定和分析。结果 菌株FJ17-4的最佳培养基配方为黄豆粉12.5 g·L−1、玉米粉5.0 g·L−1、K2HPO4 12.5 g·L−1，最佳发酵条件为：初始pH 7.0，培养温度30 ℃，装液量20% （50 mL/250 mL），接种量12.5%，转速180 r·min−1，发酵培养时间40 h。优化后发酵液的OD600值和菌体数量分别为1.52×1010、1.03×1010 cfu·mL−1，比优化前分别提高了25.62%和21.95%。50倍优化发酵液对病原菌菌丝生长抑制率和室内盆栽防治效果分别为42.35%和 72.14%，比优化前分别提高了56.38%和13.46%。结论 优化后发酵培养基和发酵条件有效提高菌株FJ17-4的发酵效果，降低了发酵成本，研究结果为贝莱斯芽孢杆菌FJ17-4的开发和工业化生产及应用提供了理论依据。Abstract:Objective Fermentation of Bacillus velezensis FJ17-4 known with a high inhibitory activity against several pathogens was optimized for potential application as a biocontrol agent.Methods Spectrometric measurement at OD600 of the fermentation broth was used as the index for evaluation. Culture medium and conditions were optimized by single factor and orthogonal experiments. Bacterial quantity, pathogenic inhibition, and indoor potted control effect of the optimized fermentation product were determined.Results The optimized FJ17-4 fermentation used a medium that consisted of 12.5 g·L−1 soybean meal, 5.0 g·L−1 corn flour, and 12.5 g·L−1 potassium dihydrogen phosphate at an initial pH of 7.0 to ferment a liquid loading (medium volume) of 50 mL with an inoculum size of 12.5% for 40 h at 30 ℃ in a 250 mL flask that rotated at a constant speed of 180 r·min−1. The OD600 of the optimized fermentation broth was 1.52 representing a cell load of 1.03×1010 cfu·mL−1, which were 25.62% and 21.95%, respectively, higher than those prior to the optimization. The inhibition rate on the mycelial growth of Fusarium oxysporum f. sp. cucumerinum and the indoor potted control on cucumber fusarium wilt of the 50× optimized fermentation broth were 42.35% and 72.14%, representing 56.38% and 13.46% increases, respectively, over the original.Conclusion The FJ17-4 fermentation was significantly improved by the optimization. An operational cost reduction was also achieved.
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Keywords:
- Bacillus velezensis /
- culture medium /
- fermentation conditions /
- optimization
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图 2 不同C、N源和无机盐对菌株FJ17-4发酵的影响
A：碳源，B：氮源，C：无机盐。
Figure 2. Effects of C and N sources and inorganic salts on fermentation of FJ17-4
A: carbon source; B: nitrogen source; C: inorganic salt.
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图 3 不同初始pH值、温度、接种量、装液量、转速和培养时间对菌株FJ17-4发酵的影响
A：初始pH，B：温度，C：接种量，D：装液量，E：转速，F：培养时间。
Figure 3. Effects of initial pH, temperature, inoculum concentration, liquid loading, rotating speed, and culture time on FJ17-4 fermentation
A: initial pH; B: temperature; C: inoculum size; D: liquid loading; E: rotating speed; F: culture time.
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图 4 无菌滤液对黄瓜枯萎病菌FOC-1菌丝生长的抑制作用
a: 发酵体系优化后无菌滤液；b: 基础（原始）培养基无菌滤液；c: 无菌水。
Figure 4. Inhibitory effect of cell-free fermentation filtrate on mycelium growth of FOC-1
a: cell-free filtrate of optimized fermentation broth; b: cell-free filtrate of original fermentation broth; c: sterile water.
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图 5 发酵液对黄瓜枯萎病的防治效果
A: 50倍优化发酵液；B：10倍优化发酵液；C：2000倍98%噁霉灵可溶粉剂；D：无菌水；E: 50倍基础培养基发酵液。
Figure 5. Control effects of fermentation broth on cucumber fusarium wilt
A: 50× optimized fermentation broth; B: 10× optimized fermentation broth; C: 2000×98% hymexazol soluble powder; D: sterile water; F: 50× original fermentation broth.
表 1 培养基组分的正交试验因素与水平
Table 1 Factors and levels of orthogonal test for culture medium formulation
水平
Level各因素含量
Content of each factor /(g·L−1)A：玉米粉
CornmealB：黄豆粉
Soybean flourC：K2HPO4 1 5.0 10.0 10.0 2 7.5 12.5 12.5 3 10.0 15.0 15.0 
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表 2 培养基各组分比优化的正交试验
Table 2 Orthogonal test on all factors of medium
处理
TreatmentsA：玉米粉
A: Cornmeal
/(g·L−1)B：黄豆粉
B: Soybean
flour /(g·L−1)C：K2HPO4
C: K2HPO4
/（g·L−1）OD600值
OD600
Value1 5.0 10.0 10.0 0.98±0.01 2 5.0 12.5 12.5 1.02±0.02 3 5.0 15.0 15.0 1.12±0.00 4 7.5 10.0 12.5 1.28±0.01 5 7.5 12.5 15.0 0.86±0.01 6 7.5 15.0 10.0 0.96±0.02 7 10.0 10.0 15.0 1.03±0.01 8 10.0 12.5 10.0 0.95±0.01 9 10.0 15.0 12.5 1.06±0.02 K1 3.12 3.36 2.98 K2 3.16 3.12 3.12 K3 2.86 2.99 3.05 R 0.30 0.37 0.14 重要性顺序
Order of importanceB＞A＞C 优水平
Optimal levelsA2 B1 C2 优组合
Optimal combinationA2 B1 C2
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表 3 FJ17-4无菌滤液对黄瓜枯萎病菌菌丝生长的抑制效果
Table 3 Inhibitory effect of FJ17-4 cell-free fermentation filtrate on mycelial growth of Fusarium oxysporum f. sp. cucumerinum
处理
Treatments菌落直径
Colony
diameter/cm抑制效果
Inhibition
effect/%差异显著性
Difference significance
(P＜0.05)10倍优化后无菌发酵滤液 10 times optimized sterile fermentation filtrate 4.90±0.38 42.35±2.13 a 10倍基础培养基（LB）无菌滤液 10 times sterile fermentation filtrate of basic medium (LB) 6.2±0.29 27.08±1.45 b 无菌水对照 Sterile water 8.50±0.46 / /
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表 4 FJ17-4无菌滤液对黄瓜枯萎病菌分生孢子萌发的抑制效果
Table 4 Inhibitory effect of FJ17-4 cell-free fermentation filtrate on conidial germination of F. oxysporum f. sp. cucumerinum
处理
Treatments孢子萌发率
Spore germination
rate /%抑制效果
Inhibition
effect /%)差异显著性
Difference significance
(P＜0.05)50倍优化后无菌发酵滤液 50 times optimized sterile fermentation filtrate 13.13±0.76 85.32±0.35 a 50倍基础培养基（LB）无菌滤液 50 times sterile fermentation filtrate of basic medium (LB) 14.82±1.03 83.43±1.44 a 100倍优化后无菌发酵滤液 100 times optimized sterile fermentation filtrate 22.14±1.41 75.26±0.24 b 100倍基础培养基（LB）无菌滤液 100 times sterile fermentation filtrate of basic medium (LB) 24.93±1.22 72.12±0.36 c 无菌水对照 Sterile water 89.42±2.43 / /
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表 5 FJ17-4发酵液对黄瓜枯萎病的室内盆栽防治效果
Table 5 Control effect of FJ17-4 fermentation broth on cucumber fusarium wilt in potted experiment
处理
Treatments发病率
Incidence rate/%防治效果
Control effect /%差异显著性
Difference significance (P＜0.05)10倍优化发酵液 10 times optimized fermentation broth 24.02±0.86 73.68±0.52 a 50倍优化发酵液 50 times optimized fermentation broth 24.51±1.12 72.14±0.36 b 50倍基础培养基发酵液 50 times basic medium fermentation broth 33.24±2.13 63.58±1.23 d 2000倍98%噁霉灵可溶粉剂 2000 times 98% hymexazol soluble powder 27.62±1.79 69.73±0.54 c 无菌水对照 Sterile water 91.26±3.26 / /
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