一种耐盐复合菌剂的制备和促生作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0123

摘要/Abstract

摘要：

土壤盐渍化是影响作物生长发育和产量的主要环境因素，利用土壤有益微生物治理盐渍化土壤是盐碱地改良的有效途径。本实验室前期从盐渍化土壤中筛选到两株耐盐菌株氧化微杆菌（Microbacterium oxydans）C8和嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）B4。菌株C8具有解钾、溶有机磷和无机磷及产生长素的能力，菌株B4具有溶有机磷和产生长素的功能。本文研究了C8和B4混合发酵对其功能的影响，结果表明，C8和B4混合培养后，其解钾、溶磷及产生长素的能力显著高于单一菌株。利用正交试验和响应面优化试验对其混合发酵培养基和培养条件进行优化，C8和B4混合发酵最适培养基配方为：葡萄糖10 g/L，酵母膏10 g/L，NaCl 4.5 g/L；最适培养条件为：pH 7.4，温度28.8℃，转速129 r/min，接种量2%，装瓶量20%，培养时间23 h。以烟草为材料，检测复合菌剂对植株生长的作用，结果显示，盐胁迫下C8和B4复合菌剂处理显著促进植株生长。

关键词: 耐盐促生混合菌剂, 发酵优化, 烟草, 促生作用

Abstract:

Soil salinization is a major factor seriously limiting crop growth and yield. Using soil beneficial microorganisms for saline field amendment is the reliable and sustainable approach. Two halotolerant bacteria strains, Microbacterium oxydans C8 and Stenotrophomonas maltophilia B4, were isolated from saline field in our previous study. Strain C8 has the ability to dissolve potassium, organic and inorganic phosphorus as well as produce auxin. Strain B4 is capable of solubilizing organic phosphorus and producing auxin. In this paper, the effect of mixed culture of C8 and B4 on their functions was studied. The results showed that the mixed culture of C8 and B4 demonstrated significantly stronger ability of dissolving potassium, phosphorus and produce auxin than a single strain. Orthogonal experiments and response surface test were used to optimize the fermentation medium and condition of C8 and B4 mixture, the results showed that the optimized fermentation medium was as follows: glucose 10 g/L, yeast extract 10 g/L, sodium chloride 4.5 g/L. The optimized fermentation conditions were: pH 7.4, temperature 28.8℃, rotating speed 129 r/min, inoculation amount 2%, the liquid content 20% and culture time 23 h. Using tobacco as material, the growth-promoting effects of compound bioinoculant of C8 and B4 were studied. The results showed that, the compound bioinoculant significantly prompted plant growth under salt stress.

Key words: salt-tolerant and growth-promoting bacteria inoculant, optimization of fermentation, tobacco, growth-promoting effect

车永梅, 刘广超, 郭艳苹, 叶青, 赵方贵, 刘新. 一种耐盐复合菌剂的制备和促生作用研究[J]. 生物技术通报, 2023, 39(11): 217-225.

CHE Yong-mei, LIU Guang-chao, GUO Yan-ping, YE Qing, ZHAO Fang-gui, LIU Xin. Preparation of Compound Halotolerant Bioinoculant and Study on Its Growth-promoting Effect[J]. Biotechnology Bulletin, 2023, 39(11): 217-225.

图/表 11

表1 正交设计因素及水平

Table 1 Factors and levels of orthogonal test

水平 Level	葡萄糖 Glucose/（g·L^-1）	酵母膏 Yeast extract/（g·L^-1）	NaCl/（g·L^-1）
1	8	5	2.5
2	10	10	4.5
3	12	15	6.5

表2 Plackett-Burman设计因素及水平

Table 2 Factors and levels of Plackett-Burman design

因素Factor	水平Level
因素Factor	-1	1
pH	5	9
温度 Temperature/^oC	20	30
装瓶量 Bottling amount/%	10	30
转速 Rotation speed/（r·min^-1）	130	180
接种量 Inoculation amount/%	1	3

表3 CCD试验因素与水平

Table 3 Factors and levels of CCD

水平 Level	温度 Temperature/℃	pH	转速 Rotation speed/（r·min^-1）
α	33.4	10.4	200.5
1	30	9	180
0	25	7	150
-1	20	5	120
-α	16.6	3.6	99.5

图1 混合发酵对菌株C8和B4 解钾、解有机磷、无机磷和产生长素能力的影响结果为平均值±标准差，图中柱子上不同小写字母表示不同处理间差异显著（P <0.05），下同

Fig. 1 Effects of mixed culture on the abilities of C8 and B4 dissolving potassium, organic and inorganic phosphorus as well as producing auxin The result is the mean ± standard deviation. Different lowercase letters on the column in the figure indicate significant difference between different treatments（P<0.05）, the same below

表4 正交试验设计和结果

Table 4 Design and results of orthogonal experiment

序号 No.	葡萄糖Glucose（A）/（g·L^-1）	酵母膏Yeast extract（B）/（g·L^-1）	NaCl（C）/（g·L^-1）	OD₆₀₀
1	1	1	1	1.17
2	1	2	2	1.28
3	1	3	3	1.12
4	2	1	2	1.30
5	2	2	3	1.35
6	2	3	1	1.32
7	3	1	3	1.34
8	3	2	1	1.27
9	3	3	2	1.32
k₁	1.27	1.28	1.28
k₂	1.30	1.30	1.31
k₃	1.25	1.27	1.23
R	0.05	0.03	0.09

表5 Plackett-Burman实验设计与结果

Table 5 Plackett-Burman design and results

序号 No.	因素Factor					OD₆₀₀
序号 No.	pH	温度Temperature/℃	装瓶量Bottling volunm/%	转速Rotating speed/（r·min^-1）	接种量Inoculation amount/%	OD₆₀₀
1	-1	1	1	1	-1	0.79
2	1	-1	1	1	-1	0.81
3	-1	1	-1	1	1	1.12
4	-1	1	1	-1	1	1.29
5	1	-1	-1	-1	1	1.37
6	-1	-1	-1	-1	-1	1.14
7	-1	-1	-1	1	-1	0.80
8	-1	-1	1	-1	1	1.11
9	1	1	-1	1	1	1.30
10	1	-1	1	1	1	1.48
11	1	1	1	-1	-1	1.20
12	1	1	-1	-1	-1	1.10

表6 CCD设计与结果

Table 6 Results of CCD design

运行序 Sequence	温度 Temperature/℃	pH	转速Rotational speed/（r·min^-1）	OD₆₀₀
1	1	-1	1	1.035
2	-1	-1	-1	1.028
3	1	1	1	1.173
4	0	0	1	1.320
5	33.409	0	1	1.241
6	0	0	99.546	1.273
7	0	0	1	1.350
8	0	0	200.454	0.984
9	-1	1	-1	1.126
10	-1	1	1	1.011
11	0	0	1	1.363
12	0	0	1	1.285
13	0	10.364	1	1.118
14	1	1	-1	1.329
15	1	-1	-1	1.285
16	16.591	0	1	0.925
17	0	3.636	1	1.044
18	0	0	1	1.387
19	-1	-1	1	0.998
20	0	0	1	1.387

表7 CCD设计方差分析

Table 7 Variance analysis of CCD design

来源Source	自由度Degree of freedom	Seq SS	Adj SS	Adj MS	F	P
回归	9	0.439 222	0.439 222	0.048 802	25.01	0.000
线性	3	0.195 425	0.123 644	0.041 215	21.12	0.000
平方	3	0.234 599	0.234 599	0.078 200	40.07	0.000
交互作用	3	0.009 197	0.009 197	0.003 066	1.57	0.257
残差误差	10	0.019 517	0.019 517	0.001 952
失拟	5	0.011 442	0.011 442	0.002 288	1.42	0.356
纯误差	5	0.008 075	0.008 075	0.001 615
合计	19	0.458 739

图2 各因素交互作用对菌株生物量的响应面 A：转速和pH对生物量的影响；B：转速和温度对生物量的影响；C：温度和pH对生物量的影响

Fig. 2 Response surface of the interactive effect of different factors on the biomass of bacteria strain A: Effect of rotating speed and pH on the biomass of bacteria strain. B: Effect of rotating speed and temperature on the biomass of bacteria strain. C: Effect of temperature and pH on the biomass of bacteria strain

图3 发酵优化对C8和B4混合菌株生物量、解钾、解无机磷、有机磷和产生长素能力的影响

Fig. 3 Effect of fermentation optimization on biomass, function of dissolving potassium, inorganic phosphorus and organic phosphorus as well as producing auxinof mixed strains of C8 and B4

图4 C8和B4复合菌剂对盐胁迫下烟草地上部分和地下部分生长的影响

Fig. 4 Effect of compound bioinoculant of C8 and B4 on the growth of aboveground and underground parts of tobacco under salt stress

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