Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (4): 116-126.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0989
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ZHOU Jing(), HUANG Wen-mao, QIN Li-jun, HAN Li-zhen()
Received:
2020-08-07
Online:
2021-04-26
Published:
2021-05-13
Contact:
HAN Li-zhen
E-mail:1846794394@qq.com;lzhan1@gzu.edu.cn
ZHOU Jing, HUANG Wen-mao, QIN Li-jun, HAN Li-zhen. Construction of Mixed Fermentation System of Four PGPR Strains and Evaluation of Its Promoting Effect[J]. Biotechnology Bulletin, 2021, 37(4): 116-126.
碳源 Carbon source | 碳源添加量Dosage/(g·L-1) | 氮源 Nitrogen source | 氮源添加量Dosage/(g·L-1) |
---|---|---|---|
果糖 Fructose | 20.0 | 牛肉膏 Beef extract | 12.08 |
蔗糖 Sucrose | 19.0 | 酵母浸出物 Yeast extract | 12.53 |
乳糖 Lactose | 19.0 | 硫酸铵 Ammonium sulfate | 6.84 |
麦芽糖 Maltose | 19.0 | 硝酸钠 Sodium nitrate | 8.79 |
Table 1 Type and dosage of carbon sources and nitrogen sources
碳源 Carbon source | 碳源添加量Dosage/(g·L-1) | 氮源 Nitrogen source | 氮源添加量Dosage/(g·L-1) |
---|---|---|---|
果糖 Fructose | 20.0 | 牛肉膏 Beef extract | 12.08 |
蔗糖 Sucrose | 19.0 | 酵母浸出物 Yeast extract | 12.53 |
乳糖 Lactose | 19.0 | 硫酸铵 Ammonium sulfate | 6.84 |
麦芽糖 Maltose | 19.0 | 硝酸钠 Sodium nitrate | 8.79 |
水平 Level | 因素Factors | |||
---|---|---|---|---|
A碳源 Carbon source | B氮源Nitrogen source | C碳氮比 Carbon/Nitrogen | ||
1 | 葡萄糖Glucose | 蛋白胨Peptone | 1∶1 | |
2 | 蔗糖Sucrose | 牛肉膏 Beef extract | 5∶1 | |
3 | 乳糖Lactose | 酵母浸出物Yeast extract | 10∶1 | |
4 | 麦芽糖Maltose | 硫酸铵Ammonium sulfate | 15∶1 |
Table 2 Orthogonal experiment for carbon sources and nitrogen sources
水平 Level | 因素Factors | |||
---|---|---|---|---|
A碳源 Carbon source | B氮源Nitrogen source | C碳氮比 Carbon/Nitrogen | ||
1 | 葡萄糖Glucose | 蛋白胨Peptone | 1∶1 | |
2 | 蔗糖Sucrose | 牛肉膏 Beef extract | 5∶1 | |
3 | 乳糖Lactose | 酵母浸出物Yeast extract | 10∶1 | |
4 | 麦芽糖Maltose | 硫酸铵Ammonium sulfate | 15∶1 |
因素Factor | 水平Level | |||
---|---|---|---|---|
-1 | 0 | +1 | ||
A. 培养温度 Temperature/℃ | 20 | 30 | 40 | |
B. 起始pH Initial pH | 6.0 | 7.0 | 8.0 | |
C. 接种比 Inoculation proportion /% | 1 | 3 | 5 | |
D. 装液量 Volume/mL | 50 | 100 | 150 |
Table 3 Factors and levels designed by response-surface methodology
因素Factor | 水平Level | |||
---|---|---|---|---|
-1 | 0 | +1 | ||
A. 培养温度 Temperature/℃ | 20 | 30 | 40 | |
B. 起始pH Initial pH | 6.0 | 7.0 | 8.0 | |
C. 接种比 Inoculation proportion /% | 1 | 3 | 5 | |
D. 装液量 Volume/mL | 50 | 100 | 150 |
Fig.2 Single level studying of effect on the growth of compound agent under the conditions of different carbon, nitrogen sources and ratio Different lowercase letters refer to the same condition, and the difference between different treatments is significant (P < 0.05), the same below
试验号 Experiment No | A碳源 Carbon source | B氮源 Nitrogen source | C碳氮比 Carbon-Nitrogen ratio | D空白列 Blank | OD600值 OD600 value |
---|---|---|---|---|---|
1 | 1(葡萄糖Glucose) | 1(蛋白胨Peptone) | 1(1∶1) | 1 | 0.741 |
2 | 1 | 2(牛肉膏Beef extract) | 3(10∶1) | 2 | 1.022 |
3 | 1 | 3(酵母浸出物Yeast extract) | 4(15∶1) | 3 | 1.747 |
4 | 1 | 4(硫酸铵Ammonium sulfate) | 2(5∶1) | 4 | 0.428 |
5 | 2(蔗糖Surcose) | 1 | 2 | 4 | 1.428 |
6 | 2 | 2 | 4 | 3 | 1.619 |
7 | 2 | 3 | 3 | 2 | 1.805 |
8 | 2 | 4 | 1 | 1 | 0.228 |
9 | 3(乳糖Lactose) | 1 | 3 | 2 | 1.463 |
10 | 3 | 2 | 1 | 1 | 1.509 |
11 | 3 | 3 | 2 | 4 | 1.628 |
12 | 3 | 4 | 4 | 3 | 0.154 |
13 | 4(麦芽糖Maltose) | 1 | 4 | 3 | 1.313 |
14 | 4 | 2 | 2 | 4 | 1.590 |
15 | 4 | 3 | 1 | 1 | 1.607 |
16 | 4 | 4 | 3 | 2 | 0.250 |
K1 | 3.938 | 4.946 | 4.085 | 4.085 | |
K2 | 5.080 | 5.740 | 5.075 | 4.540 | |
K3 | 4.755 | 6.787 | 4.540 | 4.833 | |
K4 | 4.760 | 1.060 | 4.833 | 5.075 | |
k1 | 0.985 | 1.237 | 1.021 | 1.021 | |
k2 | 1.270 | 1.435 | 1.269 | 1.135 | |
k3 | 1.189 | 1.697 | 1.135 | 1.208 | |
k4 | 1.190 | 0.265 | 1.208 | 1.269 | |
R | 1.142 | 5.727 | 0.990 | 0.990 | |
因素主次Order of factors | B﹥A﹥C | ||||
最优组合Optimal combination | A2B3C2 |
Table 4 Optimized results of orthogonal experiment
试验号 Experiment No | A碳源 Carbon source | B氮源 Nitrogen source | C碳氮比 Carbon-Nitrogen ratio | D空白列 Blank | OD600值 OD600 value |
---|---|---|---|---|---|
1 | 1(葡萄糖Glucose) | 1(蛋白胨Peptone) | 1(1∶1) | 1 | 0.741 |
2 | 1 | 2(牛肉膏Beef extract) | 3(10∶1) | 2 | 1.022 |
3 | 1 | 3(酵母浸出物Yeast extract) | 4(15∶1) | 3 | 1.747 |
4 | 1 | 4(硫酸铵Ammonium sulfate) | 2(5∶1) | 4 | 0.428 |
5 | 2(蔗糖Surcose) | 1 | 2 | 4 | 1.428 |
6 | 2 | 2 | 4 | 3 | 1.619 |
7 | 2 | 3 | 3 | 2 | 1.805 |
8 | 2 | 4 | 1 | 1 | 0.228 |
9 | 3(乳糖Lactose) | 1 | 3 | 2 | 1.463 |
10 | 3 | 2 | 1 | 1 | 1.509 |
11 | 3 | 3 | 2 | 4 | 1.628 |
12 | 3 | 4 | 4 | 3 | 0.154 |
13 | 4(麦芽糖Maltose) | 1 | 4 | 3 | 1.313 |
14 | 4 | 2 | 2 | 4 | 1.590 |
15 | 4 | 3 | 1 | 1 | 1.607 |
16 | 4 | 4 | 3 | 2 | 0.250 |
K1 | 3.938 | 4.946 | 4.085 | 4.085 | |
K2 | 5.080 | 5.740 | 5.075 | 4.540 | |
K3 | 4.755 | 6.787 | 4.540 | 4.833 | |
K4 | 4.760 | 1.060 | 4.833 | 5.075 | |
k1 | 0.985 | 1.237 | 1.021 | 1.021 | |
k2 | 1.270 | 1.435 | 1.269 | 1.135 | |
k3 | 1.189 | 1.697 | 1.135 | 1.208 | |
k4 | 1.190 | 0.265 | 1.208 | 1.269 | |
R | 1.142 | 5.727 | 0.990 | 0.990 | |
因素主次Order of factors | B﹥A﹥C | ||||
最优组合Optimal combination | A2B3C2 |
变异来源 Source | 平方和 Sum of square | df | 均方 Mean square | F值 F value | P值 P value |
---|---|---|---|---|---|
碳源 Carbon source | 0.178 | 3 | 0.059 | 1.093 | 0.422 |
氮源 Nitrogen source | 4.682 | 3 | 1.561 | 28.679 | 0.001 |
碳氮比 Carbon-Nitrogen ratio | 0.136 | 3 | 0.045 | 0.832 | 0.523 |
误差Error | 0.327 | 6 | 0.054 | ||
总计Total | 26.788 |
Table 5 ANOVA analysis results of variance
变异来源 Source | 平方和 Sum of square | df | 均方 Mean square | F值 F value | P值 P value |
---|---|---|---|---|---|
碳源 Carbon source | 0.178 | 3 | 0.059 | 1.093 | 0.422 |
氮源 Nitrogen source | 4.682 | 3 | 1.561 | 28.679 | 0.001 |
碳氮比 Carbon-Nitrogen ratio | 0.136 | 3 | 0.045 | 0.832 | 0.523 |
误差Error | 0.327 | 6 | 0.054 | ||
总计Total | 26.788 |
序号 No | A 发酵温度 Temperature | B 起始pH值 Initial pH value | C 接种比 Inoculation proportion | D 装液量 Volume | OD600值 OD600 value | ||
---|---|---|---|---|---|---|---|
实测值 Measured value | 预测值 Predicted value | ||||||
1 | -1 | -1 | 0 | 0 | 1.65 | 1.64 | |
2 | 1 | -1 | 0 | 0 | 1.66 | 1.66 | |
3 | -1 | 1 | 0 | 0 | 1.67 | 1.65 | |
4 | 1 | 1 | 0 | 0 | 1.77 | 1.75 | |
5 | 0 | 0 | -1 | -1 | 1.88 | 1.88 | |
6 | 0 | 0 | 1 | -1 | 1.89 | 1.88 | |
7 | 0 | 0 | -1 | 1 | 1.50 | 1.49 | |
8 | 0 | 0 | 1 | 1 | 1.54 | 1.53 | |
9 | -1 | 0 | 0 | -1 | 1.86 | 1.87 | |
10 | 1 | 0 | 0 | -1 | 1.85 | 1.86 | |
11 | -1 | 0 | 0 | 1 | 1.41 | 1.43 | |
12 | 1 | 0 | 0 | 1 | 1.54 | 1.55 | |
13 | 0 | -1 | -1 | 0 | 1.65 | 1.66 | |
14 | 0 | 1 | -1 | 0 | 1.70 | 1.71 | |
15 | 0 | -1 | 1 | 0 | 1.66 | 1.68 | |
16 | 0 | 1 | 1 | 0 | 1.73 | 1.74 | |
17 | -1 | 0 | -1 | 0 | 1.65 | 1.65 | |
18 | 1 | 0 | -1 | 0 | 1.72 | 1.72 | |
19 | -1 | 0 | 1 | 0 | 1.69 | 1.68 | |
20 | 1 | 0 | 1 | 0 | 1.74 | 1.73 | |
21 | 0 | -1 | 0 | -1 | 1.88 | 1.86 | |
22 | 0 | 1 | 0 | -1 | 1.86 | 1.87 | |
23 | 0 | -1 | 0 | 1 | 1.46 | 1.44 | |
24 | 0 | 1 | 0 | 1 | 1.53 | 1.54 | |
25 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
26 | 0 | 0 | 0 | 0 | 1.76 | 1.76 | |
27 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
28 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
29 | 0 | 0 | 0 | 0 | 1.77 | 1.76 |
Table 6 Experimental design and results of Box-Benhnken central combination
序号 No | A 发酵温度 Temperature | B 起始pH值 Initial pH value | C 接种比 Inoculation proportion | D 装液量 Volume | OD600值 OD600 value | ||
---|---|---|---|---|---|---|---|
实测值 Measured value | 预测值 Predicted value | ||||||
1 | -1 | -1 | 0 | 0 | 1.65 | 1.64 | |
2 | 1 | -1 | 0 | 0 | 1.66 | 1.66 | |
3 | -1 | 1 | 0 | 0 | 1.67 | 1.65 | |
4 | 1 | 1 | 0 | 0 | 1.77 | 1.75 | |
5 | 0 | 0 | -1 | -1 | 1.88 | 1.88 | |
6 | 0 | 0 | 1 | -1 | 1.89 | 1.88 | |
7 | 0 | 0 | -1 | 1 | 1.50 | 1.49 | |
8 | 0 | 0 | 1 | 1 | 1.54 | 1.53 | |
9 | -1 | 0 | 0 | -1 | 1.86 | 1.87 | |
10 | 1 | 0 | 0 | -1 | 1.85 | 1.86 | |
11 | -1 | 0 | 0 | 1 | 1.41 | 1.43 | |
12 | 1 | 0 | 0 | 1 | 1.54 | 1.55 | |
13 | 0 | -1 | -1 | 0 | 1.65 | 1.66 | |
14 | 0 | 1 | -1 | 0 | 1.70 | 1.71 | |
15 | 0 | -1 | 1 | 0 | 1.66 | 1.68 | |
16 | 0 | 1 | 1 | 0 | 1.73 | 1.74 | |
17 | -1 | 0 | -1 | 0 | 1.65 | 1.65 | |
18 | 1 | 0 | -1 | 0 | 1.72 | 1.72 | |
19 | -1 | 0 | 1 | 0 | 1.69 | 1.68 | |
20 | 1 | 0 | 1 | 0 | 1.74 | 1.73 | |
21 | 0 | -1 | 0 | -1 | 1.88 | 1.86 | |
22 | 0 | 1 | 0 | -1 | 1.86 | 1.87 | |
23 | 0 | -1 | 0 | 1 | 1.46 | 1.44 | |
24 | 0 | 1 | 0 | 1 | 1.53 | 1.54 | |
25 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
26 | 0 | 0 | 0 | 0 | 1.76 | 1.76 | |
27 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
28 | 0 | 0 | 0 | 0 | 1.75 | 1.76 | |
29 | 0 | 0 | 0 | 0 | 1.77 | 1.76 |
变异源 Source | 平方和 Sum of square | df | 均方 Mean square | F值 F value | P值 P value |
---|---|---|---|---|---|
Model | 0.47 | 14 | 0.033 | 180.48 | <0.0001 |
A | 0.011 | 1 | 0.011 | 59.66 | <0.0001 |
B | 7.550E-003 | 1 | 7.550E-003 | 40.80 | <0.0001 |
C | 1.657E-003 | 1 | 1.657E-003 | 8.95 | 0.0097 |
D | 0.42 | 1 | 0.42 | 2247.41 | <0.0001 |
AB | 1.764E-003 | 1 | 1.764E-003 | 9.53 | 0.0080 |
AC | 1.960E-004 | 1 | 1.960E-004 | 1.06 | 0.3209 |
AD | 4.356E-003 | 1 | 4.356E-003 | 23.54 | 0.0003 |
BC | 7.225E-005 | 1 | 7.225E-005 | 0.39 | 0.5421 |
BD | 1.849E-003 | 1 | 1.849E-003 | 9.99 | 0.0069 |
CD | 2.560E-003 | 1 | 2.560E-003 | 1.38 | 0.2591 |
A2 | 0.010 | 1 | 0.010 | 56.53 | <0.0001 |
B2 | 0.010 | 1 | 0.010 | 54.09 | <0.0001 |
C2 | 3.149E-003 | 1 | 3.149E-003 | 17.02 | 0.0010 |
D2 | 0.011 | 1 | 0.011 | 58.66 | <0.0001 |
Residual | 2.591E-003 | 14 | 1.851E-004 | ||
Lack of fit | 2.258E-003 | 10 | 2.258E-004 | 2.71 | 0.1745 |
Pure error | 3.332E-003 | 4 | 8.330E-003 | ||
Cor total | 0.47 | 28 | |||
R2 | 99.45% |
Table 7 ANOVA analysis of quadratic regression model of response surface
变异源 Source | 平方和 Sum of square | df | 均方 Mean square | F值 F value | P值 P value |
---|---|---|---|---|---|
Model | 0.47 | 14 | 0.033 | 180.48 | <0.0001 |
A | 0.011 | 1 | 0.011 | 59.66 | <0.0001 |
B | 7.550E-003 | 1 | 7.550E-003 | 40.80 | <0.0001 |
C | 1.657E-003 | 1 | 1.657E-003 | 8.95 | 0.0097 |
D | 0.42 | 1 | 0.42 | 2247.41 | <0.0001 |
AB | 1.764E-003 | 1 | 1.764E-003 | 9.53 | 0.0080 |
AC | 1.960E-004 | 1 | 1.960E-004 | 1.06 | 0.3209 |
AD | 4.356E-003 | 1 | 4.356E-003 | 23.54 | 0.0003 |
BC | 7.225E-005 | 1 | 7.225E-005 | 0.39 | 0.5421 |
BD | 1.849E-003 | 1 | 1.849E-003 | 9.99 | 0.0069 |
CD | 2.560E-003 | 1 | 2.560E-003 | 1.38 | 0.2591 |
A2 | 0.010 | 1 | 0.010 | 56.53 | <0.0001 |
B2 | 0.010 | 1 | 0.010 | 54.09 | <0.0001 |
C2 | 3.149E-003 | 1 | 3.149E-003 | 17.02 | 0.0010 |
D2 | 0.011 | 1 | 0.011 | 58.66 | <0.0001 |
Residual | 2.591E-003 | 14 | 1.851E-004 | ||
Lack of fit | 2.258E-003 | 10 | 2.258E-004 | 2.71 | 0.1745 |
Pure error | 3.332E-003 | 4 | 8.330E-003 | ||
Cor total | 0.47 | 28 | |||
R2 | 99.45% |
Fig.5 Effects of mixed fermentation culture and single fermentation culture on the growth of pepper seedlings A is control. B is the mixture of single strain fermentation culture. C is the culture of mixed fermentation systme
不同处理Treatment | 株高Plant height/cm | 根重Root weight/g | 鲜重Fresh weight/g | 叶绿素含量Chlorophyll content/(mg·g-1) |
---|---|---|---|---|
CK | 25.60±2.31c | 1.22±0.13c | 4.96±0.27c | 1.97±0.004c |
单菌混合菌液 Mixture of single strain fermentation | 32.10±1.27b | 2.27±0.28b | 7.81±0.42b | 2.16±0.034b |
混菌发酵菌液 Culture of mixed fermentation system | 34.70±1.19a | 3.41±0.40a | 11.22±1.07a | 2.31±0.114a |
Table 8 Effects of two different compound bacterial agents on the growth of pepper seedlings
不同处理Treatment | 株高Plant height/cm | 根重Root weight/g | 鲜重Fresh weight/g | 叶绿素含量Chlorophyll content/(mg·g-1) |
---|---|---|---|---|
CK | 25.60±2.31c | 1.22±0.13c | 4.96±0.27c | 1.97±0.004c |
单菌混合菌液 Mixture of single strain fermentation | 32.10±1.27b | 2.27±0.28b | 7.81±0.42b | 2.16±0.034b |
混菌发酵菌液 Culture of mixed fermentation system | 34.70±1.19a | 3.41±0.40a | 11.22±1.07a | 2.31±0.114a |
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