产胞外多糖多功能促生菌的筛选鉴定及促生评价

doi:10.13560/j.cnki.biotech.bull.1985.2023-0942

摘要/Abstract

摘要：

【目的】 探究产胞外多糖（exopolysaccharides, EPS）菌株在促进盐碱土中植物生长，改善土壤性能等方面的影响，为研制适用于盐碱土壤修复的微生物菌剂奠定基础。【方法】 从新疆喀什地区盐碱草地根际土壤中筛选产胞外多糖菌株，基于16S rRNA基因序列比对及系统发育分析对产胞外多糖菌株进行种属鉴定，采用单因素和响应面法优化最高产菌株的培养基组分，通过接种试验评估其对盐碱土栽培植物的促生效果及土壤团聚体的影响。【结果】 共筛选出19株具有产胞外多糖能力的菌株，隶属于7个菌属，以假单胞菌属（Pseudomonas）占绝对优势。菌株产糖量介于236-1 544 mg/L，尤以泛菌属Pantoea MQ A0产糖量最高，且兼具固氮、产吲哚乙酸（IAA）和铁载体、解磷等功能。MQ A0的最适产糖发酵培养基为甘油12.5 mL/L，蛋白胨9.0 g/L，酵母粉5.5 g/L，CaCO₃5.1 g/L，在此条件下菌株产糖量达2 436 mg/L，较优化前增加57.8%。接种MQ A0发酵液使盐碱土栽培的玉米株高、鲜重、茎粗、须根数和总根长分别提高41.2%、203.0%、42.7%、30.4%和99.7%；粗多糖提取液对土壤团聚体的形成也有显著促进作用（P<0.05）。【结论】 高产胞外多糖菌株MQ A0兼具多种促生特性，在促进盐碱土中植物生长和土壤改良方面效果显著。

关键词: 产胞外多糖菌株, 盐碱草地, 响应面法, 盐碱土, 土壤团聚体

Abstract:

【Objective】 To explore the effect of exopolysaccharides-producing strains in promoting plant growth and improving soil properties in saline soils, which may lay a foundation for the development of microbial bacterial agents applicable to saline soil remediation.【Method】 Strains with high-yield exopolysaccharide were isolated from the rhizosphere soil of saline-alkali grassland in Kashi, Xinjiang, and species identification was performed based on 16S rRNA gene sequence comparison and phylogenetic analysis. Moreover, the single factor and response surface method were used to optimize the medium components of the excellent strains to increase the yield of exopolysaccharides, and its effects on plant seedlings cultivated in saline-alkali soil and soil aggregate formation were verified by inoculation test.【Result】 A total of 19 exopolysaccharides-producing strains were screened, which were divided into 7 genera, of which Pseudomonas were the dominant. The exopolysaccharides yield was 236-1 544 mg/L, especially Pantoea sp. MQ A0. In addition, MQ A0 was capable of nitrogen fixation, indole-3-acetic acid（IAA）secretion, siderophores production and phosphorus solubilization. The optimal fermentation medium of MQ A0 was 12.5 mL/L glycerol, 9.0 g/L peptone, 5.5 g/L yeast exact and 5.1 g/L CaCO₃. Compared with the initial fermentation process, the exopolysaccharides yield reached 2 436 mg/L, which increased by 57.8%. Pot experiments showed that the inoculation of Zea mays with the fermentation broth of MQ A0 had a significant promoting effect on seedling in saline-alkali soil, including the development of plant height, fresh weight, stem diameter, root number and total root length, which increased by 41.2%, 203.0%, 42.7%, 30.4%, 99.7%, respectively. And the formation of soil aggregate was significantly promoted by the EPS（P<0.05）.【Conclusion】 The high-yield exopolysaccharide strain MQ A0 had various growth-promoting properties, and showed positive effects on plant growth and soil improvement in saline-alkali soil.

Key words: exopolysaccharides-producing strains, saline-alkali grassland, response surface method, saline-alkali soil, soil aggregate

常海霞, 李明源, 麦日艳古·亚生, 周茜, 王继莲. 产胞外多糖多功能促生菌的筛选鉴定及促生评价[J]. 生物技术通报, 2024, 40(3): 273-285.

CHANG Hai-xia, LI Ming-yuan, MAIRIYANGU·Yasheng , ZHOU Qian, WANG Ji-lian. Screening and Growth-promoting Characteristics of Multifunctional Exopolysaccharides-producing Bacteria[J]. Biotechnology Bulletin, 2024, 40(3): 273-285.

图/表 10

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编号 Strain No.	多糖产量Exopolysaccharides yield/（mg·L^-1）	固氮Nitrogen fixation	IAA分泌量 IAA concentration/ （µg·mL^-1）	产铁载体Siderophore	无机磷含量Inorganic phosphate solubility /（mg·L^-1）	有机磷含量 Organic phosphate solubility/ （mg·L^-1）
A1	302±2.83	-	7.77±2.39	+	2.61±1.63	1.26±0.55
B2	241±1.92	+	7.62±1.67	+	1.54±0.56	0.84±0.01
D1	256±0.87	+	8.38±2.26	+	2.24±1.01	0.60±0.03
MQ A0	1544±3.21	+	7.15±2.83	+	9.39±2.34	12.33±3.24
MQ 2-1-1	443±1.44	-	7.00±3.64	-	-	-
MQ 2-1-2	236±2.59	+	5.77±2.11	+	8.92±2.46	11.82±0.94
MQ 2-2	386±4.13	+	6.54±3.39	+	8.59±1.16	6.63±1.64
MQ 2-3	464±0.91	+	3.62±2.71	-	1.63±0.29	0.84±0.22
MQ 2-4	442±1.67	+	10.08±0.84	+	8.59±0.06	14.11±3.51
MQ 5-1	513±4.11	+	7.92±1.94	+	7.33±2.21	7.14±1.61
MQ 5-2	462±3.82	+	11.15±5.36	+	1.91±0.66	6.49±0.34
MQ 5-3	507±4.92	+	7.00±4.15	+	1.49±0.59	7.75±0.93
MQ 5-4	513±5.14	+	5.62±2.89	+	0.65±0.11	7.33±0.36
MQ 5-5	331±3.33	+	5.31±2.14	+	0.44±0.21	8.27±1.24
MQ 5-6	572±1.49	+	5.92±1.27	+	4.06±1.11	8.59±0.94
MQ 5-7	459±3.08	+	5.92±2.98	+	1.82±0.04	10.88±1.07
MQ 7-1	623±3.42	-	4.85±0.13	+	0.33±0.02	2.43±0.04
MQ 7-5	369±1.53	-	10.23±3.64	+	-	2.66±0.26
MQ 7-6	1342±2.67	-	7.31±1.69	+	-	-

编号 Strain No.	多糖产量Exopolysaccharides yield/（mg·L^-1）	固氮Nitrogen fixation	IAA分泌量 IAA concentration/ （µg·mL^-1）	产铁载体Siderophore	无机磷含量Inorganic phosphate solubility /（mg·L^-1）	有机磷含量 Organic phosphate solubility/ （mg·L^-1）
A1	302±2.83	-	7.77±2.39	+	2.61±1.63	1.26±0.55
B2	241±1.92	+	7.62±1.67	+	1.54±0.56	0.84±0.01
D1	256±0.87	+	8.38±2.26	+	2.24±1.01	0.60±0.03
MQ A0	1544±3.21	+	7.15±2.83	+	9.39±2.34	12.33±3.24
MQ 2-1-1	443±1.44	-	7.00±3.64	-	-	-
MQ 2-1-2	236±2.59	+	5.77±2.11	+	8.92±2.46	11.82±0.94
MQ 2-2	386±4.13	+	6.54±3.39	+	8.59±1.16	6.63±1.64
MQ 2-3	464±0.91	+	3.62±2.71	-	1.63±0.29	0.84±0.22
MQ 2-4	442±1.67	+	10.08±0.84	+	8.59±0.06	14.11±3.51
MQ 5-1	513±4.11	+	7.92±1.94	+	7.33±2.21	7.14±1.61
MQ 5-2	462±3.82	+	11.15±5.36	+	1.91±0.66	6.49±0.34
MQ 5-3	507±4.92	+	7.00±4.15	+	1.49±0.59	7.75±0.93
MQ 5-4	513±5.14	+	5.62±2.89	+	0.65±0.11	7.33±0.36
MQ 5-5	331±3.33	+	5.31±2.14	+	0.44±0.21	8.27±1.24
MQ 5-6	572±1.49	+	5.92±1.27	+	4.06±1.11	8.59±0.94
MQ 5-7	459±3.08	+	5.92±2.98	+	1.82±0.04	10.88±1.07
MQ 7-1	623±3.42	-	4.85±0.13	+	0.33±0.02	2.43±0.04
MQ 7-5	369±1.53	-	10.23±3.64	+	-	2.66±0.26
MQ 7-6	1342±2.67	-	7.31±1.69	+	-	-

方差来源Source	平方和Sum of squares	自由度Degree of freedom	均方Mean square	F	P	显著性Significant
Model	4230000	14	302000	41.5	< 0.0001	**
A	1000000	1	1000000	137.23	< 0.0001	**
B	0.75	1	0.75	0.000103	0.992
C	125000	1	125000	17.11	0.001	**
D	4961.33	1	4961.33	0.68	0.4231
AB	421000	1	421000	57.8	< 0.0001	**
AC	15129	1	15129	2.08	0.1716
AD	36	1	36	0.00494	0.945
BC	729	1	729	0.1	0.7564
BD	71022.25	1	71022.25	9.75	0.0075	**
CD	45582.25	1	45582.25	6.26	0.0254	*
A²	790000	1	790000	108.36	< 0.0001	**
B²	1150000	1	1150000	157.42	< 0.0001	**
C²	1200000	1	1200000	164.04	< 0.0001	**
D²	836000	1	836000	114.66	< 0.0001	**

方差来源Source	平方和Sum of squares	自由度Degree of freedom	均方Mean square	F	P	显著性Significant
Model	4230000	14	302000	41.5	< 0.0001	**
A	1000000	1	1000000	137.23	< 0.0001	**
B	0.75	1	0.75	0.000103	0.992
C	125000	1	125000	17.11	0.001	**
D	4961.33	1	4961.33	0.68	0.4231
AB	421000	1	421000	57.8	< 0.0001	**
AC	15129	1	15129	2.08	0.1716
AD	36	1	36	0.00494	0.945
BC	729	1	729	0.1	0.7564
BD	71022.25	1	71022.25	9.75	0.0075	**
CD	45582.25	1	45582.25	6.26	0.0254	*
A²	790000	1	790000	108.36	< 0.0001	**
B²	1150000	1	1150000	157.42	< 0.0001	**
C²	1200000	1	1200000	164.04	< 0.0001	**
D²	836000	1	836000	114.66	< 0.0001	**

处理 Treatment group	株高 Plant height/cm	根长 Root length/cm	须根数 Lateral root number	茎粗 Stem diameter/mm	鲜重 Fresh weight/g	叶绿素含量 Chlorophyll content/（mg·g^-1）
发酵液Fermentation broth	56.60±6.076a	52.78±5.926a	7.50±1.732a	6.25±0.311a	5.03±0.471a	20.80±0.901a
粗多糖 EPS	54.88±5.393a	45.88±2.732b	7.25±1.500a	6.28±0.359a	4.16±0.334a	22.10±1.105a
对照 Control check	40.10±3.340b	26.43±4.378c	5.75±0.957b	4.38±0.427b	1.66±0.236b	13.20±2.150b