微生物学报

Number	Incubation time (h)	Incubation temperature (℃)	Incubator speed (r/min)
1	24	20	120
2	24	28	240
3	24	37	180
4	48	20	240
5	48	28	180
6	48	37	120
7	72	20	180
8	72	28	120
9	72	37	240

Number	Incubation time (h)	Incubation temperature (℃)	Incubator speed (r/min)
1	24	20	120
2	24	28	240
3	24	37	180
4	48	20	240
5	48	28	180
6	48	37	120
7	72	20	180
8	72	28	120
9	72	37	240

Strain	LB	PDA	NA	Glucose-yeast powder
Different small letters in the same vertical column indicate that there are significant differences in the EPS content among different strains in the same medium (P < 0.05). Different capital letters in the same row indicate that there are significant differences in the EPS content of the same strain in different medium (P < 0.05).
G20	50.22±4.25Ccd	562.72±2.57Ab	22.64±3.97Dde	121.49±11.93Bde
F1	113.82±4.38Ca	867.54±12.14Aa	57.45±5.53Da	305.85±16.52Ba
G51	33.99±2.55Cd	322.37±5.88Ae	17.54±3.91Ce	132.02±6.24Bcde
F36	58.77±7.04Cbc	512.28±16.44Ac	32.46±3.01Cbc	143.04±17.94Bcd
G36	73.99±6.12Cb	481.14±14.84Ad	24.95±4.35Dcde	218.14±13.34Bb
G55	33.99±0.51Cd	328.07±7.12Ae	32.02±4.57Cbc	98.25±5.17Be
F2	63.16±3.02Cbc	500.00±12.96Acd	35.53±3.30Db	163.53±29.98Bc
G46	40.35±1.02Bcd	135.53±10.13Af	31.14±2.36Bbcd	111.40±23.06Ade

Strain	LB	PDA	NA	Glucose-yeast powder
Different small letters in the same vertical column indicate that there are significant differences in the EPS content among different strains in the same medium (P < 0.05). Different capital letters in the same row indicate that there are significant differences in the EPS content of the same strain in different medium (P < 0.05).
G20	50.22±4.25Ccd	562.72±2.57Ab	22.64±3.97Dde	121.49±11.93Bde
F1	113.82±4.38Ca	867.54±12.14Aa	57.45±5.53Da	305.85±16.52Ba
G51	33.99±2.55Cd	322.37±5.88Ae	17.54±3.91Ce	132.02±6.24Bcde
F36	58.77±7.04Cbc	512.28±16.44Ac	32.46±3.01Cbc	143.04±17.94Bcd
G36	73.99±6.12Cb	481.14±14.84Ad	24.95±4.35Dcde	218.14±13.34Bb
G55	33.99±0.51Cd	328.07±7.12Ae	32.02±4.57Cbc	98.25±5.17Be
F2	63.16±3.02Cbc	500.00±12.96Acd	35.53±3.30Db	163.53±29.98Bc
G46	40.35±1.02Bcd	135.53±10.13Af	31.14±2.36Bbcd	111.40±23.06Ade

Test items	Results	Test items	Results
+: Positive; ‒: Negative.
β-xylosidase	‒	Cyclodextrin	‒
l-lysine arylaminase	‒	d-galactose	+
l-aspartate arylaminase	+	Glycogen	+
Leucine arylaminase	+	Inositol	‒
Phenylalanine arylaminase	+	Methyl-α-d-glucopyranosidation	‒
l-proline arylaminase	‒	Ellman	‒
β-galactosidase	+	Methyl-d-xyloside	‒
l-pyrrolidone arylaminase	+	Maltotriose	+
α-galactosidase	+	d-mannitol	‒
Alanine arylaminase	+	d-mannose	+
Tyrosine arylaminase	+	d-pinotriose	‒
β-N-acetylglucosaminidase	+	N-acetyl-d-glucosamine	‒
Alanine-phenylalanine-proline arylaminase	‒	Ancient sugar	+
α-mannosidase	‒	l-rhamnose	‒
β-glucosidase	+	Phosphorylcholine	‒
β-mannosidase	‒	Pyruvate	+
Triphenyl tetrazolium chloride	‒	d-tagatose	‒
α-glucosidase	+	d-trehalose	+
Resistant to kanamycin	‒	Inulin	‒
Resistant to bambusamycin	‒	d-glucose	+
Resistant to polymyxin B	‒	d-ribose	+
Esculin hydrolysis	+	Putrescine assimilation	‒
6.5% sodium chloride growth	+

Test items	Results	Test items	Results
+: Positive; ‒: Negative.
β-xylosidase	‒	Cyclodextrin	‒
l-lysine arylaminase	‒	d-galactose	+
l-aspartate arylaminase	+	Glycogen	+
Leucine arylaminase	+	Inositol	‒
Phenylalanine arylaminase	+	Methyl-α-d-glucopyranosidation	‒
l-proline arylaminase	‒	Ellman	‒
β-galactosidase	+	Methyl-d-xyloside	‒
l-pyrrolidone arylaminase	+	Maltotriose	+
α-galactosidase	+	d-mannitol	‒
Alanine arylaminase	+	d-mannose	+
Tyrosine arylaminase	+	d-pinotriose	‒
β-N-acetylglucosaminidase	+	N-acetyl-d-glucosamine	‒
Alanine-phenylalanine-proline arylaminase	‒	Ancient sugar	+
α-mannosidase	‒	l-rhamnose	‒
β-glucosidase	+	Phosphorylcholine	‒
β-mannosidase	‒	Pyruvate	+
Triphenyl tetrazolium chloride	‒	d-tagatose	‒
α-glucosidase	+	d-trehalose	+
Resistant to kanamycin	‒	Inulin	‒
Resistant to bambusamycin	‒	d-glucose	+
Resistant to polymyxin B	‒	d-ribose	+
Esculin hydrolysis	+	Putrescine assimilation	‒
6.5% sodium chloride growth	+

	F1	Bacillus zanthoxyli	Bacillus aryabhattai	Bacillus megaterium
F1	100.00
Bacillus zanthoxyli	95.69	100.00
Bacillus aryabhattai	95.69	98.64	100.00
Bacillus megaterium	99.54	95.67	95.68	100.00

	F1	Bacillus zanthoxyli	Bacillus aryabhattai	Bacillus megaterium
F1	100.00
Bacillus zanthoxyli	95.69	100.00
Bacillus aryabhattai	95.69	98.64	100.00
Bacillus megaterium	99.54	95.67	95.68	100.00

Number	Incubation time (h)	Incubation temperature (℃)	Rotate speed (r/min)	EPS content (μg/mL)
1	24	20	120	657.83±31.26
2	24	28	240	1 097.67±36.51
3	24	37	180	1 088.60±34.15
4	48	20	240	917.08±42.14
5	48	28	180	889.91±63.16
6	48	37	120	647.98±9.73
7	72	20	180	473.80±9.26
8	72	28	120	364.52±11.74
9	72	37	240	387.34±23.70
𝐾₁	948.033	682.903	556.777
𝐾₂	818.323	784.033	800.697
𝐾₃	408.553	707.973	817.437
Range (R)	539.48	101.13	260.66
Factor optimum level	24	28	180

Number	Incubation time (h)	Incubation temperature (℃)	Rotate speed (r/min)	EPS content (μg/mL)
1	24	20	120	657.83±31.26
2	24	28	240	1 097.67±36.51
3	24	37	180	1 088.60±34.15
4	48	20	240	917.08±42.14
5	48	28	180	889.91±63.16
6	48	37	120	647.98±9.73
7	72	20	180	473.80±9.26
8	72	28	120	364.52±11.74
9	72	37	240	387.34±23.70
𝐾₁	948.033	682.903	556.777
𝐾₂	818.323	784.033	800.697
𝐾₃	408.553	707.973	817.437
Range (R)	539.48	101.13	260.66
Factor optimum level	24	28	180

Treatment	Content of > 0.25 mm water stable aggregate (%)	Content of soil polysaccharide (μg/g)
Different small letters in the same column indicate that there are significant differences for the same index among different treatments; ^**: P < 0.01.
CK	1.79±0.43d	1.62±0.45d
PDA	3.24±0.34c	5.93±0.33c
EPS	5.20±0.24b	10.99±0.71b
F1	9.74±0.62a	18.54±5.04a
Pearson correlation coefficient	0.825^**

Treatment	Content of > 0.25 mm water stable aggregate (%)	Content of soil polysaccharide (μg/g)
Different small letters in the same column indicate that there are significant differences for the same index among different treatments; ^**: P < 0.01.
CK	1.79±0.43d	1.62±0.45d
PDA	3.24±0.34c	5.93±0.33c
EPS	5.20±0.24b	10.99±0.71b
F1	9.74±0.62a	18.54±5.04a
Pearson correlation coefficient	0.825^**

一株高产胞外多糖巨大芽孢杆菌的筛选鉴定、发酵条件优化及其对土壤团聚体的影响

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刘丙花 ¹ , 丁延芹 ² , 刘方春 ¹ , 刘幸红 ¹ , 马胜国 ³ , 彭琳 ¹ , 孙铭婕 ¹ , 于连家 ¹ , 马海林 ¹^,^*

微生物学报 | 研究报告 2024,64(10): 3968-3979

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微生物学报 | 研究报告 2024, 64(10): 3968-3979

一株高产胞外多糖巨大芽孢杆菌的筛选鉴定、发酵条件优化及其对土壤团聚体的影响

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刘丙花¹, 丁延芹², 刘方春¹, 刘幸红¹, 马胜国³, 彭琳¹, 孙铭婕¹, 于连家¹, 马海林¹^,^*

作者信息

1 山东省林业科学研究院, 山东济南 250014

2 山东农业大学生命科学学院, 山东泰安 271018

3 山东省林业保护和发展服务中心, 山东济南 250014

Screening, identification, fermentation condition optimization of an exopolysaccharide-producing strain and the influence on soil macro-aggregate formation

Binghua LIU¹, Yanqin DING², Fangchun LIU¹, Xinghong LIU¹, Shengguo MA³, Lin PENG¹, Mingjie SUN¹, Lianjia YU¹, Hailin MA¹^,^*

Affiliations

1 Shandong Academy of Forestry, Jinan 250014, Shandong, China

2 College of Life Sciences, Shandong Agricultural University, Tai'an 271018, Shandong, China

3 Shandong Forestry Protection and Development Service center, Jinan 250014, Shandong, China

出版时间: 2024-07-24 doi: 10.13343/j.cnki.wsxb.20240262

文章导航

摘要

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【目的】筛选具有高产胞外多糖(exopolysaccharides, EPS)特性的微生物菌株，为功能性土壤改良菌剂的研制提供菌种资源。【方法】采用LB-苯胺蓝平板结合菌丝拉丝法定性筛选获得具有产EPS特性的植物根际促生细菌(plant growth promoting rhizobacteria, PGPR)功能菌株；采用低温醇沉分离、硫酸-蒽酮比色法测定PGPR功能菌株经4种培养基发酵后发酵液中EPS含量，获得具有高产EPS特性的PGPR功能菌株；以发酵液中EPS含量为衡量指标，采用正交试验对高产EPS菌株发酵条件进行优化；采用培养皿土壤培养试验，分析F1发酵液对砂壤土土壤大团聚体含量的影响。【结果】初选获得8株具有产EPS特性的PGPR功能菌株；经复选确定菌株F1具有高产EPS特性，PDA是F1产EPS的最优培养基，EPS含量为867.54 μg/mL。基于形态学、生理生化特性测定，以及16S rRNA基因序列系统发育分析、全基因组平均核苷酸相似度分析，确定F1为一株巨大芽孢杆菌(Bacillus megaterium)。F1产EPS的最优培养条件：28 ℃、180 r/min培养24 h，在该条件下EPS产量为1 123.39 μg/mL。F1应用于砂壤土进行土壤培养40 d后，粒径 > 0.25 mm土壤水稳性大团聚体含量比对照提高了4.44倍。【结论】F1菌株具有较强的产EPS能力，能够促进砂壤土水稳性大团聚体的形成；F1产EPS最优培养基为PDA，最优发酵条件为28 ℃、180 r/min培养24 h。

关键词

胞外多糖 / 巨大芽孢杆菌 / 产胞外多糖发酵条件 / 土壤团聚体

Abstract

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[Objective] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents. [Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production. After fermentation with each strain in four media, the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method, on the basis of which the PGPR strain with high EPS production was screened out. The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator. The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment. [Results] Eight EPS-producing PGPR strains were primarily screened out, among which strain F1 had the highest EPS production. PDA was the best medium for F1 to produce EPS, with the EPS content of 867.54 μg/mL. Based on morphological, physiological and biochemical characteristics, phylogenetic analysis based on 16S rRNA gene sequence, and also average nucleotide identity analysis, F1 was identified as a strain of Bacillus megaterium. The optimal culture conditions for F1 to produce EPS were 28 ℃ and 180 r/min for 24 h, under which the EPS yield reached 1 123.39 μg/mL. After F1 was incubated in sandy loam soil for 40 days, the content of water-stable macro-aggregates with the grain diameter > 0.25 mm in the soil increased by 4.44 times compared with that of the control. [Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil. The optimal conditions for F1 to produce of EPS was incubation in PDA at 28 ℃ and 180 r/min for 24 h.

Key words

exopolysaccharides / Bacillus megaterium / fermentation conditions for exopolysaccharide production / soil aggregate

引用本文

刘丙花, 丁延芹, 刘方春, 刘幸红, 马胜国, 彭琳, 孙铭婕, 于连家, 马海林. 一株高产胞外多糖巨大芽孢杆菌的筛选鉴定、发酵条件优化及其对土壤团聚体的影响. 微生物学报, 2024 , 64 (10) : 3968 -3979 . DOI: 10.13343/j.cnki.wsxb.20240262

Binghua LIU, Yanqin DING, Fangchun LIU, Xinghong LIU, Shengguo MA, Lin PENG, Mingjie SUN, Lianjia YU, Hailin MA. Screening, identification, fermentation condition optimization of an exopolysaccharide-producing strain and the influence on soil macro-aggregate formation[J]. Acta Microbiologica Sinica, 2024 , 64 (10) : 3968 -3979 . DOI: 10.13343/j.cnki.wsxb.20240262

正文

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微生物胞外多糖(exopolysaccharides, EPS)是一些特殊微生物生长代谢过程中分泌到细胞壁外或结合在细胞表面的水溶性生物大分子物质，能够促进土壤大团聚体的形成和维持其稳定性，具有改善土壤特性、提高植物抗逆性、吸附重金属等复合效应，而且环境友好，在土壤环境修复及农业生产中具有良好的应用前景^[1-5]。自然界中，能够合成多糖的微生物种类繁多，如细菌、真菌、放线菌均能合成多糖，其中细菌在土壤中的占比达70%−90%，常见的能够产多糖的细菌属有土壤杆菌属、固氮菌属、芽孢杆菌属、假单胞菌属、根瘤菌属等^[6-9]。植物根际促生细菌(plant growth promoting rhizobacteria, PGPR)是指能够在植物根际持续稳定定殖，对植物生长、生理代谢和环境适应等方面起到积极作用的有益细菌。从PGPR中筛选具有高产EPS特性的细菌，研制PGPR复合功能菌剂，对促进农业可持续发展有重要意义。

近年来，作者所在单位在功能PGPR菌株的筛选、鉴定及菌剂化应用方面取得了突出成绩，从滨海盐碱地分离获得一批具有促生、抗逆、土壤修复等复合效应的功能PGPR菌株。本研究对山东省东营市孤岛镇刺槐林场刺槐林根际土壤分离的PGPR菌种，采用LB-苯胺蓝平板结合菌丝拉丝法定性筛选具有产EPS性质的菌株；随后，采用低温醇沉分离、硫酸-蒽酮比色法测定初筛菌株EPS产量，筛选高产EPS菌株，并对其进行形态学、生理生化特性和分子学鉴定；以发酵液中EPS含量为衡量指标，对其产EPS发酵条件进行优化，以期为功能性土壤改良菌剂的研制提供菌种资源及其后期的推广应用奠定基础。

1 材料与方法

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1.1 材料

1.1.1 菌种

试验所用菌种均分离自山东省东营市孤岛镇刺槐林场刺槐林根际土壤，保存于山东农业大学生命与科学学院PGPR-植物相互作用研究室菌种资源库。

1.1.2 培养基

LB-苯胺蓝培养基(g/L)：蛋白胨10.0，酵母粉5.0，氯化钠10.0，苯胺蓝5.0，琼脂粉15.0，pH 7.0。121 ℃灭菌20 min。

PDA培养基(g/L)：土豆200.0，葡萄糖20.0，琼脂15.0，pH 7.0。115 ℃灭菌20 min。

LB培养基(g/L)：蛋白胨10.0，酵母粉5.0，氯化钠10.0，琼脂15.0，pH 7.0。121 ℃灭菌20 min。

NA培养基(g/L)：蛋白胨10.0，牛肉膏3.0，氯化钠5.0，琼脂15.0，pH 7.0。121 ℃灭菌20 min。

葡萄糖-酵母粉培养基(g/L)：葡萄糖50.0，酵母粉5.0，氯化钙3.0，琼脂15.0，pH 7.0。115 ℃灭菌20 min。

1.2 菌株筛选

1.2.1 初筛

采用LB-苯胺蓝平板结合菌丝拉丝法定性筛选具有产EPS性质的菌株。将菌种资源库中由甘油管保藏的菌株在LB培养基上活化；挑取单菌落接种于LB-苯胺蓝筛选培养基上，置于恒温恒湿培养箱(上海龙跃仪器设备有限公司) 37 ℃条件下培养24 h后，观察筛选培养基上菌体形态。将平板上菌体被水溶性苯胺蓝浸润，呈蓝色液滴状且采用灭菌牙签挑起时能拉丝的菌株认定具有产EPS性质。

1.2.2 复筛

采用低温醇沉分离、硫酸-蒽酮比色法测定初筛菌株EPS产量，筛选高产EPS菌株。将初筛获得的菌株从甘油管中接种到LB液体培养基中，置于恒温振荡器(常州市国旺仪器制造有限公司) 37 ℃、180 r/min培养24 h获得种子液。种子液经4 ℃、4 000 r/min离心5 min，收集菌体并重悬于生理盐水中。以空白生理盐水为对照，将菌悬液OD₆₀₀调整到1.0。将体积分数为1.0%的菌悬液分别接种到LB、PDA、NA和葡萄糖-酵母粉液体培养基中，置于恒温振荡器(常州市国旺仪器制造有限公司) 37 ℃、180 r/min培养48 h获得细菌发酵液。依据单位发酵液中EPS含量高低筛选具有高产EPS特性的菌株。

1.3 EPS提取及测定

采用低温醇沉分离法提取发酵液EPS^[10]。将复选菌株发酵液于4 ℃、10 000 r/min离心10 min，将上清液转移到新离心管中。上清液置于沸水中水浴10 min，使蛋白质变性，自然冷却后在4 ℃、10 000 r/min离心15 min去蛋白，保留上清液。取2.0 mL上清液，加入6 mL的预冷的无水乙醇，放置在4 ℃条件下低温萃取24 h，使EPS充分析出。将析出EPS的上清液在4 ℃、10 000 r/min离心15 min，去除上清液，保留EPS沉淀；将含有EPS沉淀离心管置于烘箱中50 ℃烘干，保存于−20 ℃冰箱备用。

采用蒽酮-硫酸法测定EPS含量^[11]。将提取的EPS样品溶解于1.0 mL蒸馏水中，充分振荡混匀；吸取0.2 mL于洁净试管中，用蒸馏水补充到2.5 mL，混匀后加入5.0 mL硫酸-蒽酮试剂，立刻塞上橡胶塞，防止水分蒸发。将试管放入沸水中水浴保温10 min，取出后自然冷却至室温并摇匀。以加2.5 mL蒸馏水的试管为空白，采用紫外分光光度计(北京普析通用仪器有限责任公司)测定620 nm处的吸光度值。

1.4 形态学鉴定

将高产EPS菌株在PDA培养基上进行三区划线，37 ℃条件下培养至出现单菌落。参考《伯杰细菌鉴定手册》^[12]观察单菌落的菌落形状、大小、透明度、颜色以及菌落边缘形态。

挑取平板上的单菌落，接种到LB液体培养基中，37 ℃培养至对数生长期，分别进行革兰氏染色和芽孢染色，用油镜观察菌体及芽孢的形态^[13]。

1.5 生理生化特性检验

采用VITEK全自动微生物鉴定分析系统(法国生物梅里埃股份有限公司)对高产EPS菌株的生理生化特性进行测定。经充填机将菌悬液注入试卡内，封口后放入带有读数器的恒温培养箱，根据试卡各生化反应孔中的变化情况，由读数器按光学扫描原理定时测定各生化介质中指示剂的显色(或浊度)反应，把读出信息与预定阈值进行分析比较，判定鉴定结果。

1.6 分子鉴定

采用细菌DNA提取试剂盒[生工生物工程(上海)股份有限公司]提取高产EPS菌株的总DNA，委托生工生物工程(上海)股份有限公司进行高产EPS菌株的16S rRNA基因测序和全基因组测序。将高产EPS菌株的16S rRNA基因测序结果导入美国国家生物技术信息中心(NCBI)，选用rRNA/ITS databases进行序列比对。在LPSN数据库下载比对结果中标准菌株的16S rRNA基因序列，并导入MEGA 11.0中进行比对建树，进行系统发育树分析。

使用程序JspeciesWS在线对高产EPS菌株全基因组进行平均核苷酸相似度(average nucleotide identity, ANI)值的计算。选择在16S rRNA基因序列对比中相似性高且具有全基因组序列数据的菌株进行ANI分析。

1.7 高产EPS菌株发酵条件优化

1.7.1 生长曲线测定

将高产EPS菌株接种于PDA固体培养基，37 ℃条件下培养18 h进行活化。用接菌环挑取活化的高产EPS菌株接种至装液量为50.0 mL (250 mL三角瓶)的PDA液体培养基中，28 ℃、180 r/min摇床振荡发酵培养，每隔2 h取1次样，测定发酵液在600 nm处的吸光度，绘制菌株的生长曲线。

1.7.2 发酵条件优化

以PDA液体培养基为发酵培养基，以发酵液中EPS含量为衡量指标，设计正交试验，对高产EPS菌株发酵条件(培养时间、培养温度和培养箱转速)进行优化(表1)。

1.8 高产EPS菌株对土壤团聚体的影响

供试土壤为砂壤土，过0.25 mm筛，采用压力蒸汽灭菌器[致微(厦门)仪器有限公司] 121 ℃灭菌20 min，共2次。分别将高产EPS菌株发酵液(F1)、EPS水溶液(EPS，由1.3提取的EPS加入无菌水充分振荡溶解制成)、PDA和对照(无菌水)按照10.0%的接种量接入装有80.0 g土样中培养皿中。将培养皿置于(28±2) ℃培养箱(上海博迅实业有限公司)中培养40 d。培养过程中，定期补充无菌水，保持土壤表面相对湿润。

于试验结束时采用湿筛法测定 > 0.25 mm土壤水稳性团聚体含量^[9]。采用改良热水法提取多糖法提取土壤多糖^[14]，采用蒽酮-硫酸法测定土壤多糖含量^[11]。

2 结果与分析

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2.1 菌株筛选

采用LB-苯胺蓝平板培养结合菌丝拉丝法定性筛选获得8株具有产EPS性质的菌株，分别为G20、F1、G51、F36、G36、G55、F2、G46，各菌株在LB-苯胺蓝培养基上单菌落形态如图1所示。

将初筛获得的8株产EPS菌株分别经LB、PDA、NA和葡萄糖-酵母粉4种液体培养基发酵后，测定发酵液中EPS含量，结果如表2所示。8株细菌在4种培养基中均产EPS，但不同菌株在相同培养基中的EPS产量不同，同一菌株在不同培养基中的EPS产量也不同。菌株F1在LB、PDA、NA和葡萄糖-酵母粉培养基中EPS含量分别为113.83、867.54、57.45和305.85 μg/mL，均显著高于其他菌株(P < 0.05)，并且F1在PDA培养基中EPS产量显著高于其他3种培养基，表明F1具有高产EPS特性，PDA是F1产EPS的最适合培养基。

2.2 菌株鉴定

2.2.1 形态学特征

由图2可知，F1菌落呈规则的圆形，表面凸起较湿润，乳白色，半透明。菌体较小，呈杆状，末端圆，单个或呈短链排列，革兰氏染色呈阳性。芽孢染色结果显示菌体呈红色，产芽孢，芽孢末端生，呈绿色椭圆形。

2.2.2 生理生化特性

参考芽孢杆菌VITEK鉴定方法鉴定菌株F1生理生化特性，结果见表3。菌株F1可利用d-半乳糖、糖原、麦芽三糖、d-甘露糖、古老糖、d-海藻糖、d-葡萄糖、d-核糖作为碳源产酸发酵，不能利用环糊精、肌醇、甲基-d-木糖甘、d-甘露醇、d-松三糖、N-乙酰-d-氨基葡萄糖、l-鼠李糖、d-塔格糖和菊粉。菌株F1初步鉴定为巨大芽孢杆菌(Bacillus megaterium)，鉴定率为94.00%。

2.2.3 分子生物学鉴定

将菌株F1的16S rRNA基因序列在NCBI数据库中进行比对，运用MEGA 11.0软件构建系统发育树。结果如图3所示，菌株F1为芽孢杆菌属，与花椒芽孢杆菌(Bacillus zanthoxyli^T，登录号KX865140)、阿氏芽孢杆菌(Bacillus aryabhattai，登录号B8W22^T)、巨大芽孢杆菌(Bacillus megaterium，登录号IAM 13418^T)亲缘关系较近，但是无法借助16S rRNA基因序列进行准确鉴定。因此，将F1全基因组序列与16S rRNA基因序列对比中相似性较高且具有全基因组序列数据的菌株进行ANI分析，结果表明F1与Bacillus megaterium的ANI值最高为99.54% (表4)。因此，结合菌落形态及生理生化特征，将菌株F1鉴定为巨大芽孢杆菌(Bacillus megaterium)。

2.3 F1产EPS发酵条件优化

2.3.1 生长曲线

F1在PDA液体培养基中的生长曲线如图4所示，0−4 h为延迟期，从第6 h开始菌体进入对数生长期，发酵液OD₆₀₀值快速升高，从第14 h开始进入生长平台期。因此，选择培养12−14 h的菌体作为种子液。

2.3.2 发酵条件优化

以培养时间、培养温度和培养箱转速为因素，以F1发酵液EPS含量为指标，设计三因素三水平正交试验，结果见表5。根据极差R的大小确定三因素对发酵液EPS含量影响的权重关系为培养时间 > 培养箱转速 > 培养温度。根据K值大小，获得F1产EPS最佳发酵条件为28 ℃、180 r/min发酵24 h，在此条件下，F1发酵液EPS含量为1 123.39 μg/mL。

2.4 F1对土壤团聚体的影响

如表6所示，经过40 d培养后，F1发酵液和EPS水溶液处理组土壤 > 0.25 mm水稳性大团聚体含量和多糖含量分别为9.74%和18.54 μg/g、5.20%和10.99 μg/g，均显著高于PDA处理组和无菌水对照处理组(P < 0.05)。粒径 > 0.25 mm土壤水稳性大团聚体含量与土壤多糖含量的皮尔逊相关性系数为0.825，呈极强的正相关关系。结果说明，土壤中添加F1发酵液或其EPS水溶液均可通过提高土壤多糖含量进而促进 > 0.25 mm土壤水稳性大团聚体的形成。

3 讨论与结论

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土壤团聚体是构成土壤结构的基本单元，是由土壤中的原生颗粒经过胶结物的胶结、凝结、黏结等作用形成，主要通过改变土壤的物理性质、土壤肥力、土壤微生物活性与丰度等，影响土壤质量^{[6-7, 15-18]}。微生物EPS由于其本身结构的特殊性(线性大分子结构)和分子内活性基团的多样性(富含羟基、羧基等官能团及大量的酸性基团)，能够胶结土壤颗粒，促进大粒径土壤团聚体的形成，并且能够在颗粒表面形成衣膜，起到稳定团聚体的作用^{[3, 5, 19-20]}。可见，EPS是促进团聚体形成与稳定的重要助力，高产EPS的菌株在土壤结构改良方面具有较好的应用前景。然而，目前应用于土壤结构改良的菌种资源十分有限，迫切需要筛选EPS产量高，兼具促生抗逆特性，而且环境友好的功能菌株，为复合功能菌剂的研发和产业化应用提供候选菌种资源。

菌种的筛选鉴定及发酵条件优化是功能微生物菌剂研发和生产应用的重要环节。细菌菌株类型、生长阶段、底物可利用性及发酵条件等均会影响细菌EPS的产生及其组成^{[5, 21-24]}。不同的细菌细胞对培养基碳源和氮源有不同的喜好，培养基碳氮来源及其浓度影响EPS的产生效率，速效碳源(例如葡萄糖)能有效促进细菌代谢产生EPS，而过量的无机氮阻碍EPS的产生^{[2, 23, 25]}，因此，合适的培养基碳氮比可确保EPS的最大产量。发酵条件(如温度、培养时间、接种量、pH等)的确定是菌液发酵的重要环节，直接影响发酵液的质量。研究表明，大多数细菌产生EPS的最佳生长温度为25−30 ℃^[23]，最佳pH条件为中性^{[2, 24]}，这主要取决于菌株本身的性质。本研究从滨海盐碱土筛选获得一株具有高产EPS特性的PGPR功能菌株F1，28 ℃、180 r/min条件下经PDA发酵培养24 h，EPS产量为1 123.39 μg/mL，显著高于其他条件，这可能是该培养条件下最有利于F1利用碳源，促进生长代谢诱导EPS的产生。为深入研究F1菌株的功能特性，促进其菌剂研发与应用，我们将继续深入研究F1不同生长阶段，培养基碳氮来源、碳氮比及发酵条件(如pH、接种量等)对EPS生成的影响。

近年来，随着连续耕作及化肥的过度使用，土壤结构受到极大的破坏，导致土壤持水保肥能力急剧下降，微生物群落稳定性降低，严重影响了农业生产效率。EPS在驱动土壤颗粒团聚和促进团聚体稳定方面的研究受到广泛关注，细菌EPS作为土壤颗粒的有效胶结剂，在改善土壤结构、提升土壤质量方面成效显著^{[5, 20]}。研究表明，向土壤中施加产EPS的细菌发酵液或其EPS水溶液均能提高土壤多糖含量，促进土壤水稳性团聚体的形成和稳定性提高，起到改善土壤结构的作用^{[21, 26-27]}。本研究将高产EPS菌株F1的发酵液和EPS水溶液应用于砂壤土40 d后，F1发酵液和EPS水溶液处理组 > 0.25 mm土壤水稳性大团聚体含量分别比对照提高了4.44倍和1.91倍，而且 > 0.25 mm土壤水稳性大团聚体含量与土壤多糖含量呈极强的正相关性。结果表明，F1可通过提高土壤多糖含量进而促进 > 0.25 mm土壤水稳性大团聚体的形成。EPS的单糖片段网状分子结构使其具有强大的黏附性，能黏合在土壤颗粒表面形成一个被大量土壤颗粒包围的中间区域^{[3, 19]}，土壤颗粒与EPS在该区域不断黏合逐渐形成 > 0.25 mm的大团聚体，最终使分散的砂壤土颗粒聚集在一起，提高砂质土壤的土壤团聚体比例及其稳定性，提高砂质土壤的水肥持有能力。本研究的不足之处是未设置原始土壤处理组，产EPS菌株是否通过影响土壤原有微生物群落结构而影响土壤多糖含量，最终对土壤的水稳性团聚体含量产生影响将是我们下一步工作重点之一。

综上所述，本研究从滨海盐碱土分离筛选获得一株具有高产EPS特性的巨大芽孢杆菌(Bacillus megaterium) F1，可通过提高土壤多糖含量促进砂壤土水稳性大团聚体的形成并维持其稳定性，具有进一步开发推广价值。

基金

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山东省重点研发计划(重大科技创新工程)项目(2021CXGC010804)
2024年中央财政林业科技推广示范项目
济南市科技型中小企业创新能力提升工程项目

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2024年第64卷第10期

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doi: 10.13343/j.cnki.wsxb.20240262

接收时间：2024-04-25
首发时间：2026-03-21
出版时间：2024-07-24

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文章信息

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出版历史

收稿日期：2024-04-25
录用日期：2024-07-22

基金

Key Research and Development Program of Shandong Province (Major Scientific and Technological Innovation Project)(2021CXGC010804)

山东省重点研发计划(重大科技创新工程)项目(2021CXGC010804)

2024 Central Finance Forestry Technology Promotion Demonstration Project

2024年中央财政林业科技推广示范项目

Innovation Ability Improvement Project of Small and Medium-sized Technology-based Enterprise of Jinan

济南市科技型中小企业创新能力提升工程项目

作者信息

1 山东省林业科学研究院, 山东济南 250014

2 山东农业大学生命科学学院, 山东泰安 271018

3 山东省林业保护和发展服务中心, 山东济南 250014

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Number	Incubation time (h)	Incubation temperature (℃)	Incubator speed (r/min)
1	24	20	120
2	24	28	240
3	24	37	180
4	48	20	240
5	48	28	180
6	48	37	120
7	72	20	180
8	72	28	120
9	72	37	240

Number

Incubation time (h)

Incubation temperature (℃)

Incubator speed (r/min)

120

240

180

240

180

120

180

120

240

Strain	LB	PDA	NA	Glucose-yeast powder
Different small letters in the same vertical column indicate that there are significant differences in the EPS content among different strains in the same medium (P < 0.05). Different capital letters in the same row indicate that there are significant differences in the EPS content of the same strain in different medium (P < 0.05).
G20	50.22±4.25Ccd	562.72±2.57Ab	22.64±3.97Dde	121.49±11.93Bde
F1	113.82±4.38Ca	867.54±12.14Aa	57.45±5.53Da	305.85±16.52Ba
G51	33.99±2.55Cd	322.37±5.88Ae	17.54±3.91Ce	132.02±6.24Bcde
F36	58.77±7.04Cbc	512.28±16.44Ac	32.46±3.01Cbc	143.04±17.94Bcd
G36	73.99±6.12Cb	481.14±14.84Ad	24.95±4.35Dcde	218.14±13.34Bb
G55	33.99±0.51Cd	328.07±7.12Ae	32.02±4.57Cbc	98.25±5.17Be
F2	63.16±3.02Cbc	500.00±12.96Acd	35.53±3.30Db	163.53±29.98Bc
G46	40.35±1.02Bcd	135.53±10.13Af	31.14±2.36Bbcd	111.40±23.06Ade

Strain

PDA

Glucose-yeast powder

Different small letters in the same vertical column indicate that there are significant differences in the EPS content among different strains in the same medium (P < 0.05). Different capital letters in the same row indicate that there are significant differences in the EPS content of the same strain in different medium (P < 0.05).

G20

50.22±4.25Ccd

562.72±2.57Ab

22.64±3.97Dde

121.49±11.93Bde

113.82±4.38Ca

867.54±12.14Aa

57.45±5.53Da

305.85±16.52Ba

G51

33.99±2.55Cd

322.37±5.88Ae

17.54±3.91Ce

132.02±6.24Bcde

F36

58.77±7.04Cbc

512.28±16.44Ac

32.46±3.01Cbc

143.04±17.94Bcd

G36

73.99±6.12Cb

481.14±14.84Ad

24.95±4.35Dcde

218.14±13.34Bb

G55

33.99±0.51Cd

328.07±7.12Ae

32.02±4.57Cbc

98.25±5.17Be

63.16±3.02Cbc

500.00±12.96Acd

35.53±3.30Db

163.53±29.98Bc

G46

40.35±1.02Bcd

135.53±10.13Af

31.14±2.36Bbcd

111.40±23.06Ade

Test items	Results	Test items	Results
+: Positive; ‒: Negative.
β-xylosidase	‒	Cyclodextrin	‒
l-lysine arylaminase	‒	d-galactose	+
l-aspartate arylaminase	+	Glycogen	+
Leucine arylaminase	+	Inositol	‒
Phenylalanine arylaminase	+	Methyl-α-d-glucopyranosidation	‒
l-proline arylaminase	‒	Ellman	‒
β-galactosidase	+	Methyl-d-xyloside	‒
l-pyrrolidone arylaminase	+	Maltotriose	+
α-galactosidase	+	d-mannitol	‒
Alanine arylaminase	+	d-mannose	+
Tyrosine arylaminase	+	d-pinotriose	‒
β-N-acetylglucosaminidase	+	N-acetyl-d-glucosamine	‒
Alanine-phenylalanine-proline arylaminase	‒	Ancient sugar	+
α-mannosidase	‒	l-rhamnose	‒
β-glucosidase	+	Phosphorylcholine	‒
β-mannosidase	‒	Pyruvate	+
Triphenyl tetrazolium chloride	‒	d-tagatose	‒
α-glucosidase	+	d-trehalose	+
Resistant to kanamycin	‒	Inulin	‒
Resistant to bambusamycin	‒	d-glucose	+
Resistant to polymyxin B	‒	d-ribose	+
Esculin hydrolysis	+	Putrescine assimilation	‒
6.5% sodium chloride growth	+

Test items

Results

Test items

Results

+: Positive; ‒: Negative.

β-xylosidase

‒

Cyclodextrin

‒

l-lysine arylaminase

‒

d-galactose

l-aspartate arylaminase

Glycogen

Leucine arylaminase

Inositol

‒

Phenylalanine arylaminase

Methyl-α-d-glucopyranosidation

‒

l-proline arylaminase

‒

Ellman

‒

β-galactosidase

Methyl-d-xyloside

‒

l-pyrrolidone arylaminase

Maltotriose

α-galactosidase

d-mannitol

‒

Alanine arylaminase

d-mannose

Tyrosine arylaminase

d-pinotriose

‒

β-N-acetylglucosaminidase

N-acetyl-d-glucosamine

‒

Alanine-phenylalanine-proline arylaminase

‒

Ancient sugar

α-mannosidase

‒

l-rhamnose

‒

β-glucosidase

Phosphorylcholine

‒

β-mannosidase

‒

Pyruvate

Triphenyl tetrazolium chloride

‒

d-tagatose

‒

α-glucosidase

d-trehalose

Resistant to kanamycin

‒

Inulin

‒

Resistant to bambusamycin

‒

d-glucose

Resistant to polymyxin B

‒

d-ribose

Esculin hydrolysis

Putrescine assimilation

‒

6.5% sodium chloride growth

	F1	Bacillus zanthoxyli	Bacillus aryabhattai	Bacillus megaterium
F1	100.00
Bacillus zanthoxyli	95.69	100.00
Bacillus aryabhattai	95.69	98.64	100.00
Bacillus megaterium	99.54	95.67	95.68	100.00

Bacillus zanthoxyli

Bacillus aryabhattai

Bacillus megaterium

100.00

Bacillus zanthoxyli

95.69

100.00

Bacillus aryabhattai

95.69

98.64

100.00

Bacillus megaterium

99.54

95.67

95.68

100.00

Number	Incubation time (h)	Incubation temperature (℃)	Rotate speed (r/min)	EPS content (μg/mL)
1	24	20	120	657.83±31.26
2	24	28	240	1 097.67±36.51
3	24	37	180	1 088.60±34.15
4	48	20	240	917.08±42.14
5	48	28	180	889.91±63.16
6	48	37	120	647.98±9.73
7	72	20	180	473.80±9.26
8	72	28	120	364.52±11.74
9	72	37	240	387.34±23.70
𝐾₁	948.033	682.903	556.777
𝐾₂	818.323	784.033	800.697
𝐾₃	408.553	707.973	817.437
Range (R)	539.48	101.13	260.66
Factor optimum level	24	28	180

Number

Incubation time (h)

Incubation temperature (℃)

Rotate speed (r/min)

EPS content (μg/mL)

120

657.83±31.26

240

1 097.67±36.51

180

1 088.60±34.15

240

917.08±42.14

180

889.91±63.16

120

647.98±9.73

180

473.80±9.26

120

364.52±11.74

240

387.34±23.70

𝐾₁

948.033

682.903

556.777

𝐾₂

818.323

784.033

800.697

𝐾₃

408.553

707.973

817.437

Range (R)

539.48

101.13

260.66

Factor optimum level

180

Treatment	Content of > 0.25 mm water stable aggregate (%)	Content of soil polysaccharide (μg/g)
Different small letters in the same column indicate that there are significant differences for the same index among different treatments; ^**: P < 0.01.
CK	1.79±0.43d	1.62±0.45d
PDA	3.24±0.34c	5.93±0.33c
EPS	5.20±0.24b	10.99±0.71b
F1	9.74±0.62a	18.54±5.04a
Pearson correlation coefficient	0.825^**

Treatment

Content of > 0.25 mm water stable aggregate (%)

Content of soil polysaccharide (μg/g)

Different small letters in the same column indicate that there are significant differences for the same index among different treatments; ^**: P < 0.01.

1.79±0.43d

1.62±0.45d

PDA

3.24±0.34c

5.93±0.33c

EPS

5.20±0.24b

10.99±0.71b

9.74±0.62a

18.54±5.04a

Pearson correlation coefficient

0.825^**