Optimizing Fermentation of Biocontrol Bacterium Bacillus cereus BCCY-22
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摘要:
目的 蜡样芽孢杆菌BCCY-22是有效防治多种线虫病害的生防细菌。本研究旨在探明生防菌蜡样芽孢杆菌BCCY-22的最优培养条件，为实现该菌株的高效发酵奠定基础。 方法 以蜡样芽孢杆菌BCCY-22的OD600为指标，采用单因素试验和响应面法对该菌株发酵条件进行优化。 结果 高温、低初始pH和供氧不足显著影响蜡样芽孢杆菌BCCY-22的生长，最适发酵条件：发酵温度21.5 ℃、初始pH 7.3、种子液接种量3%、装瓶量23%、发酵时间36 h。 结论 蜡样芽孢杆菌BCCY-22优化发酵条件后，缩短了发酵时间6 h，生物量是优化前的135.94%，研究为蜡样芽孢杆菌BCCY-22生防菌的发酵生产和开发利用提供了依据。 Abstract:Objective Conditions for optimal fermentation of Bacillus cereus BCCY-22, which is known to be capable of effectively controlling a variety of nematode diseases, were determined. Methods Using OD600 of the fermentation broth as the indicator, optimal culture conditions for the process were determined with a single factor and response surface experimentation. Results High temperature, low initial pH, and restricted oxygen supply significantly governed the bacterial growth. The finalized process was carried out at 21.5 ℃ with an initial pH at 7.3 and inoculation of the seeding broth at 3% to fill 23% in a flask for 36 h. Conclusion The optimization shortened the fermentation time by 6 h and increased the biomass by 135.94%. The improvements materially provided the basis for industrialized utilization of B. cereus BCCY-22 as a biocontrol agent. -
图 2 菌株BCCY-22在不同培养温度下的生长状况
同组柱形图上标注的不同小写字母，表示同一时间不同组别间差异显著（P＜0.05）；下图同。
Figure 2. Growth of BCCY-22 under varied temperatures
Different lowercase letters marked on the columns of the same group showed significant differences among different groups at the same time (P ＜ 0.05); Same for thefollowing.
图 3 菌株BCCY-22在不同初始pH培养基中生长状况
Figure 3. Growth of BCCY-22 in media with varied initial pH
图 4 菌株BCCY-22在不同接种量下的生长状况
Figure 4. Growth of BCCY-22 with varied inoculum concentration
图 6 pH、温度的交互作用对OD600的响应曲面
Figure 6. Response surface diagram on OD600 affected by interaction between initial pH and temperature
图 7 pH、温度的交互作用对OD600的等值线
Figure 7. Contour lines of OD600 affected by interaction between initial pH and temperature
图 8 温度、装瓶量的交互作用对OD600的响应曲面
Figure 8. Response surface diagram on OD600 affected by interaction between temperature and flask- filling
图 9 装瓶量、温度的交互作用对OD600的等值线
Figure 9. Contour lines of OD600 affected by interaction between temperature and flask-filling
图 10 pH、装瓶量的交互作用对OD600的响应曲面
Figure 10. Response surface diagram on OD600 affected by interaction between initial pH and flask-filling
图 11 OD600和pH、装瓶量的等值线
Figure 11. Contour lines of OD600 affected by interaction between initial pH and flask-filling
表 1 试验因素水平
Table 1. Factor level of optimization experimentation
编号
Code变量
Variable水平 Level α +1 0 −1 −α A 温度 Temperature/ ℃ 33.41 30 25 20 16.59 B pH 8.86 7.5 5.5 3.5 2.14 C 装瓶量 Volume/% 73.64 60 40 20 6.36 
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表 2 中心复合设计及试验结果
Table 2. Design and results of experiment
编码类型
PtType区组
GroupA B C 温度
Temperature/ ℃pH 装瓶量
Volume/%OD600 1 1 1 1 −1 30.00 7.50 20.00 0.632 1 1 −1 −1 −1 20.00 3.50 20.00 0.048 1 1 1 −1 −1 30.00 3.50 20.00 0.050 −1 1 1.681792831 0 0 33.41 5.50 40.00 0.612 −1 1 −1.681792831 0 0 16.59 5.50 40.00 0.820 −1 1 0 0 1.681792831 25.00 5.50 73.64 0.535 1 1 −1 −1 1 20.00 3.50 60.00 0.056 −1 1 0 0 −1.681792831 25.00 5.50 6.36 0.915 0 1 0 0 0 25.00 5.50 40.00 0.782 1 1 −1 1 −1 20.00 7.50 20.00 0.854 1 1 1 1 1 30.00 7.50 60.00 0.614 0 1 0 0 0 25.00 5.50 40.00 0.737 1 1 −1 1 1 20.00 7.50 60.00 0.959 1 1 1 −1 1 30.00 3.50 60.00 0.043 0 1 0 0 0 25.00 5.50 40.00 0.788 0 1 0 0 0 25.00 5.50 40.00 0.755 −1 1 0 −1.681792831 0 25.00 2.14 40.00 0.060 0 1 0 0 0 25.00 5.50 40.00 1.012 −1 1 0 1.681792831 0 25.00 8.86 40.00 0.804 0 1 0 0 0 25.00 5.50 40.00 0.805
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表 3 二次多项式回归方差分析
Table 3. Quadratic polynomial regression variance test results
来源
Source自由度
dfSeqSS AdjSS AdjMS F P 回归 Regression 9 1.91598 1.91598 0.21289 7.11 0.003 线性 Linear 3 1.32442 1.32442 0.44147 14.74 0.001 平方 Square 3 0.54962 0.54962 0.18321 6.12 0.012 交互作用 Interaction 3 0.04195 0.04195 0.01398 0.47 0.712 残差误差 Residual 10 0.29956 0.29956 0.02996 失拟 Misfit 5 0.24917 0.24917 0.04983 4.94 0.052 纯误差 Pure error 5 0.05039 0.05039 0.01008 合计 Total 19 2.21554
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表 4 二次多项式回归系数及其显著性检验
Table 4. Quadratic polynomial regression coefficient and significance test
项
Item系数
Coefficient标准误
Standard errorT P 常量 Constant 0.81986 0.07059 11.615 0.000 A −0.11426 0.07877 −1.451 0.178 B 0.50628 0.07872 6.432 0.000 C −0.06787 0.07877 −0.862 0.409 A×A −0.21984 0.12896 −1.705 0.119 B×B −0.50411 0.12890 −3.911 0.003 C×C −0.21084 0.12896 −1.635 0.133 A×B −0.19639 0.17291 −1.136 0.283 A×C −0.04880 0.17312 −0.282 0.784 B×C 0.03038 0.17291 0.176 0.864
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