一株耐铀菌株的鉴定及其促生特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1031

摘要/Abstract

摘要：

从退役铀矿区土壤中筛选获得耐铀促生菌株，为铀污染土壤的微生物-植物联合修复技术提供优良菌种资源，以解决退役铀矿区污染治理问题。梯度稀释某退役铀矿区污染土壤，涂布含铀培养基，分离筛选出一株具有耐铀性能菌株B2。通过形态学观察、生理生化实验及16S rDNA序列比较分析，对其进行初步鉴定。采用分光光度法测定菌株在铀胁迫下的生长曲线和培养基铀含量，分析其耐铀能力和铀吸附或吸收能力。通过平板法测定其固氮、解磷、产纤维素酶、合成铁载体能力。用Salkowski比色法测定其产吲哚乙酸（3-indole acetic acid, IAA）能力及产量。通过种子萌发和盆栽实验，验证该菌株的促生能力。综合形态观察结果、生理生化特征和基于16S rDNA序列的进化分析，确定菌株B2为微枝形杆菌属细菌（Microvirga makkahensis sp.），在铀浓度为0-400 mg/L时，其生长曲线符合S型生长曲线模型，当铀浓度达到600 mg/L后生长受抑制，其对培养基中的铀无吸附或吸收作用。菌株B2具有固氮、解磷、产纤维素酶、合成铁载体和产IAA的促生特性，培养48 h后IAA产量可达到24.39 μg/mL。菌株B2可显著提高上海青（Brassica chinensis L.）种子的发芽率，促进幼苗的生长发育，显著增加上海青生物量。菌株B2为一株具有产IAA等促生能力、耐铀的微枝形杆菌属细菌，能有效促进上海青种子萌发和幼苗生长，可作为微生物-植物联合修复铀污染土壤的候选菌株。

关键词: 耐铀, 植物促生, 微枝形杆菌, 吲哚乙酸（IAA）, 种子萌发, 幼苗生长, 生物量, 上海青

Abstract:

To provide excellent strain resources for the microbial-phytoremediation technologies of uranium-contaminated soil, and to solve the problem of uranium pollution treatment in the decommissioned mining area, the uranium-resistant and plant growth-promoting bacteria were isolated from the soil collected from the decommissioned uranium mine. Gradient dilution of contaminated soil in a decommissioned uranium mine was conducted, and a uranium-resistant strain B2 was isolated on uranium-containing nutrient agar. Then the preliminary identification of strain B2 was carried out by morphological observation, physiological, and biochemical experiments, and 16S rDNA sequence analysis. To analyze the uranium tolerance and uranium adsorption capacity of strain B2, the growth curves and uranium content of the mediums under uranium stress were measured by spectrophotometric method. The abilities of nitrogen fixation, phosphate solubilization, cellulase production, and siderophore were determined by plate methods. The production of 3-indole acetic acid（IAA）was determined by Salkowski colorimetric method, and the growth-promoting ability was verified by seed germination and potting experiment. The morphological observations, physiological and biochemical characteristics, and evolutionary analysis based on 16S rDNA sequences showed that strain B2 was a Microvirga makkahensis sp., and its growth curves conformed to the S-shaped growth curve model at a uranium concentration of 0-400 mg/L. Its growth was inhibited when the uranium concentration reached 600 mg/L. It showed no adsorption effect on uranium. Strain B2 had the probiotic properties of nitrogen fixation, phosphate solubilization, cellulase production, siderophore, and IAA production. IAA production reached 24.39 μg/mL after 48 h incubation. Strain B2 significantly improved the seed germination rate of Brassica chinensis L. and promoted the growth and development of seedlings, significantly increased the biomass of B. chinensis L. Strain B2 is a uranium-resistant M. makkahensis sp. with IAA production and other growth-promoting abilities, which can effectively promote the germination of B. chinensis L. seeds and seedling growth, and can be a candidate strain for the in plant-microbe remediation of uranium-contaminated soil.

Key words: uranium-resistant, plant growth-promoting, Microvirga makkahensis, 3-indole acetic acid（IAA）, seed germination, seedling growth, biomass, Brassica chinensis L.

罗义, 张丽娟, 黄伟, 王宁, 吾尔丽卡·买提哈斯木, 施宠, 王玮. 一株耐铀菌株的鉴定及其促生特性研究[J]. 生物技术通报, 2023, 39(5): 286-296.

LUO Yi, ZHANG Li-juan, HUANG Wei, WANG Ning, Wuerlika MAITIHASEM, SHI Chong, WANG Wei. Identification of a Uranium-resistant Strain and Its Growth-promoting Properties[J]. Biotechnology Bulletin, 2023, 39(5): 286-296.

图/表 8

图1 菌株B2菌落形态（A、B）和革兰氏染色结果（C）

Fig. 1 Colony morphology (A、B) and gram stain result (C) of strain B2

表1 菌株B2的生理生化特征

Table1 Physiological characteristics of strain B2

生化特征Biochemical characteristics	B2	Microvirga makkahensis SV1470^T[26]	M. lupini Lut6^T[27]	M. aerophila DSM 21344^T[28]	M. flavescens c27j1^T[29]
菌株来源Source of strain	土壤	土壤	根瘤	空气	土壤
培养基Medium	在NA、R2A、LB生长良好	在改良的Bennett's、察氏、NA、ISP2、YMA、Rauff、R2A生长良好	在1/2LA、YMA、TY、NA生长良好	在R2A、NA生长良好，在TSA、2216E不生长	在R2A、PYE生长良好，在TSA、NA不生长
菌落颜色Color of colony	浅粉色	浅粉色	淡橙色	浅粉色	淡黄色
革兰氏染色Gram stain	G-	G-	G-	G-	G-
细胞形状Cell shape	棒状	棒状	棒状	棒状	棒状
好氧性试验 Aerobic test	严格好氧	严格好氧	严格好氧	严格好氧	严格好氧
产孢子Producing spores	-	-	-	-	-
运动性Motility	-	+	-	-	+
细胞长×细胞宽Cell length（μm）× Cell width（μm）	(1.1-1.9)×(0.5-0.7)	(1.0-1.6)×(0.8-1.3)	(1.0-2.2)×(0.4-0.5)	(1.6-4.2)×(0.8-1.1)	(1.8-4.2)×(0.4-0.7)
细胞色素氧化酶 Cytochrome oxidase	+	/	-	+	+
过氧化氢酶Oxidase	+	+	-	+	+
产吲哚Producing indoles	+	/	w	-	-
产H₂S Producing hydrogen sulphide	-	/	-	/	/
明胶液化Hydrolysis of gelatin	+	+	-	-	-
硝酸盐还原Nitrate reduction	+	-	-	-	+
柠檬酸盐利用Citrate utilization	+	/	-	/	/
脲酶Urease	-	-	+	-	-
淀粉酶Amylase	/	-	-	+	-
β-半乳糖苷酶 β-galactosidase	-	/	-	-	/
精氨酸双水介酶Arginine hydrophilic enzyme	-	+	-	-	-

图2 基于菌株B2 16S rDNA 序列采用邻接法构建的系统发育树分支上的数值表示采用Neighbor-joining方法构建系统发育树时，1 000次计算时形成该节点的百分比；括号中序号为GenBank登录号；分支点上的数字代表Bootstrap值；标尺0.010 0代表1%的核苷酸差异

Fig. 2 Phylogenetic tree reconstructed by the neighbor-joining method, based on the 16S rDNA sequence of strain B2 The value on the branch indicates the percentage of the node formed in 1 000 calculations when the neighbor-joining method is used to construct the phylogenetic tree. Numbers in parentheses are the GenBank accession numbers. Numbers at the nodes indicate the Bootstrap value. The scale bar indicates 1% nucleotide substitution

图3 菌株B2在不同铀浓度下的生长曲线

Fig. 3 Growth curves of strain B2 under different uranium concentrations

图4 不同培养基中菌株B2的IAA产量不同小写字母表示差异显著（P < 0.05）

Fig. 4 IAA production by strain B2 in different media Different lower letters indicate significant differnce（P < 0.05）

图5 菌株B2处理后对上海青种子生长影响 A：菌株B2对上海青发芽势和发芽率的影响；B：菌株B2对上海青发芽指数和活力指数的影响；C：菌株B2处理后对上海青根长和芽长的影响。*P < 0.05，**P < 0.01，下同

Fig. 5 Effects of strain B2 on the seed growth of B. chine-nsis L. A: Effects of strain B2 on germination potential and percentage of B. chinensis L. B: Effects of strain B2 on germination index and vitality index of B. chinensis L.. C: Effect of strain B2 on root and bud length of B. chinensis L. * P < 0.05, and ** P < 0.01. The same below

图6 菌株B2处理对上海青的促生效应 A：对照组；B：菌株B2处理组

Fig. 6 Promoting effects of strain B2 on B. chinensis L. A: Control group. B: Strain B2 treatment group

表2 菌株B2处理对上海青生长性状的影响

Table 2 Effects of strain B2 treatment on growth traits of B. chinensis L.

处理Treatment	株高Plant height/cm	根长Root length/cm	茎粗Stem thickness/mm	叶片数Number of leaves	叶宽Leaf width/cm	叶长Leaf length/cm	鲜重Fresh weight		干重 Dry weight
处理Treatment	株高Plant height/cm	根长Root length/cm	茎粗Stem thickness/mm	叶片数Number of leaves	叶宽Leaf width/cm	叶长Leaf length/cm	地上Above ground/g	地下Underground/g	地上Above ground/g	地下Underground/g
CK	13.68±1.38	12.12±2.45	3.50±0.50	4.40±0.89	3.38±0.69	5.51±1.16	2.53±0.94	0.44±0.25	0.38±0.16	0.09±0.02
B2	17.35±0.97**	18.70±4.51*	5.25±0.50**	6.75±0.50**	5.23±0.59**	7.58±1.19**	9.23±2.54**	2.40±0.75**	0.86±0.12**	0.28±0.07**

参考文献 43

[1]	Wei GL, Han WH, Shu XY, et al. Heavy-ion irradiation effects on uranium-contaminated soil for nuclear waste[J]. J Hazard Mater, 2021, 405: 124273. doi: 10.1016/j.jhazmat.2020.124273 URL
[2]	Cheng CH, Chen LY, Guo KX, et al. Progress of uranium-contaminated soil bioremediation technology[J]. J Environ Radioact, 2022, 241: 106773. doi: 10.1016/j.jenvrad.2021.106773 URL
[3]	钟娟, 刘兴宇, 张明江, 等. 铀污染的微生物修复技术研究进展[J]. 稀有金属, 2021, 45(1): 93-105.
	Zhong J, Liu XY, Zhang MJ, et al. Research progress of bioremediation technology for uranium contamination[J]. Chin J Rare Met, 2021, 45(1): 93-105.
[4]	Li XL, Ding CC, Liao JL, et al. Microbial reduction of uranium(VI)by Bacillus sp. dwc-2: a macroscopic and spectroscopic study[J]. J Environ Sci(China), 2017, 53: 9-15.
[5]	Liang XJ, Csetenyi L, Gadd GM. Uranium bioprecipitation mediated by yeasts utilizing organic phosphorus substrates[J]. Appl Microbiol Biotechnol, 2016, 100(11): 5141-5151. doi: 10.1007/s00253-016-7327-9 pmid: 26846744
[6]	梁朱明. 超富集植物体微生物减容减重应用基础研究[D]. 绵阳: 西南科技大学, 2020.
	Liang ZM. Basic research on the applation of microorganism in volume and weight reduction of hyperconcentration plants[D]. Mianyang: Southwest University of Science and Technology, 2020.
[7]	Yuan Y, Liu N, Dai Y, et al. Effective biosorption of uranium from aqueous solution by cyanobacterium Anabaena flos-aquae[J]. Environ Sci Pollut Res Int, 2020, 27(35): 44306-44313. doi: 10.1007/s11356-020-10364-4
[8]	Chen L, Liu JR, Zhang WX, et al. Uranium(U)source, speciation, uptake, toxicity and bioremediation strategies in soil-plant system: a review[J]. J Hazard Mater, 2021, 413: 125319. doi: 10.1016/j.jhazmat.2021.125319 URL
[9]	Zheng XY, Shen YH, Wang XY, et al. Effect of pH on uranium(VI)biosorption and biomineralization by Saccharomyces cerevisiae[J]. Chemosphere, 2018, 203: 109-116. doi: S0045-6535(18)30588-5 pmid: 29614403
[10]	杨晓玫, 冯起, 吕丹彤, 等. 植物根际促生菌Bacillus mycoides Gnyt1菌株生物学特性比较研究[J]. 草地学报, 2022, 30(3): 553-559. doi: 10.11733/j.issn.1007-0435.2022.03.006
	Yang XM, Feng Q, Lyu DT, et al. Comparative study on the biological characteristics of plant rhizosphere growth-promoting bacteria Bacillus mycoides Gnyt1[J]. Acta Agrestia Sin, 2022, 30(3): 553-559.
[11]	Ke T, Guo GY, Liu JR, et al. Improvement of the Cu and Cd phytostabilization efficiency of perennial ryegrass through the inoculation of three metal-resistant PGPR strains[J]. Environ Pollut, 2021, 271: 116314. doi: 10.1016/j.envpol.2020.116314 URL
[12]	亓琳, 杨莹博, 张博, 等. 丛枝菌根真菌强化高粱幼苗修复锶污染土壤的研究[J]. 草业学报, 2018, 27(12): 103-112. doi: 10.11686/cyxb2018333
	Qi L, Yang YB, Zhang B, et al. Arbuscular mycorrhizal fungi(AMF)enhance phytoremediation of strontium-contaminated soil by Sorghum bicolor seedlings[J]. Acta Prataculturae Sin, 2018, 27(12): 103-112.
[13]	王焯, 罗学刚, 丁翰林, 等. 一种耐铀植物促生菌的筛选及促生特性研究[J]. 生物技术通报, 2019, 35(1): 42-50. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0577
	Wang Z, Luo XG, Ding HL, et al. Isolation and identification of a uranium-resistant strain and effect of its characteristics on growth promoting[J]. Biotechnol Bull, 2019, 35(1): 42-50. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0577
[14]	Bhakat K, Chakraborty A, Islam E. Characterization of arsenic oxidation and uranium bioremediation potential of arsenic resistant bacteria isolated from uranium ore[J]. Environ Sci Pollut Res Int, 2019, 26(13): 12907-12919. doi: 10.1007/s11356-019-04827-6
[15]	蒋小梅. 土著微生物菌群的选育及除铀效能试验研究[D]. 衡阳: 南华大学, 2018.
	Jiang XM. The selection and effectiveness studies to uranium of native microbial consortium[D]. Hengyang: University of South China, 2018.
[16]	胡南, 陈思羽, 胡劲松, 等. 一株耐铀镉真菌菌株的筛选及其耐铀镉特性的研究[J]. 南华大学学报: 自然科学版, 2019, 33(2): 16-21.
	Hunan, Chen SY, Hu JS, et al. Screening of a uranium-cadmium tolerant fungal strain and its uranium-cadmium tolerance[J]. J Univ South China Sci Technol, 2019, 33(2): 16-21.
[17]	张健, 宋晗, 邓洪, 等. 铀与微生物相互作用研究进展[J]. 矿物岩石地球化学通报, 2018, 37(1): 55-62, 158.
	Zhang J, Song H, Deng H, et al. Research progress on interaction between uranium and microorganism[J]. Bull Mineral Petrol Geochem, 2018, 37(1): 55-62, 158.
[18]	张远科. 纤维素酶产生菌的筛选、产酶特性及酶基因克隆表达[D]. 株洲: 湖南工业大学, 2022.
	Zhang YK. Screening, enzyme production characteristics, and gene cloning and expression of cellulase-producing strain[D]. Zhuzhou: Hunan University of Technology, 2022.
[19]	周贝贝. 植物根际促生菌的筛选及其在草莓上的应用研究[D]. 泰安: 山东农业大学, 2018.
	Zhou BB. Screening of plant growth-promoting rhizobacteria and its application in strawberry[D]. Tai'an: Shandong Agricultural University, 2018.
[20]	王君. 蓝莓根际促生细菌的筛选、鉴定及其促生效果[D]. 泰安: 山东农业大学, 2016.
	Wang J. Screening, identification and growth-promoting effects of PGPR from blueberry rhizosphere[D]. Tai'an: Shandong Agricultural University, 2016.
[21]	Yong P, Eccles H, Macaskie LE. Determination of uranium, thorium and lanthanum in mixed solutions using simultaneous spectrophotometry[J]. Anal Chimica Acta, 1996, 329(1/2): 173-179. doi: 10.1016/0003-2670(96)00101-8 URL
[22]	杜浪, 李玉香, 马雪, 等. 偶氮胂Ⅲ分光光度法测定微量铀[J]. 冶金分析, 2015, 35(1): 68-71.
	Du L, Li YX, Ma X, et al. Determination of micro uranium by arsenazo Ⅲ spectrophotometry[J]. Metall Anal, 2015, 35(1): 68-71.
[23]	张垚, 张芝, 王志刚, 等. 辣椒根际促生菌筛选鉴定及其促生效应初探[J]. 浙江农业科学, 2022, 63(5): 958-963.
	Zhang Y, Zhang Z, Wang ZG, et al. Screening, identification of growth-promoting rhizobacteria in pepper and preliminary study on its promoting effect[J]. J Zhejiang Agric Sci, 2022, 63(5): 958-963.
[24]	张凯晔. 田菁种子内生菌的分离及其促生功能研究[D]. 太谷: 山西农业大学, 2020.
	Zhang KY. Isolation of endophytes from Sesbania cannabina and plant growth-promoting characteristics of endophyte[D]. Taigu: Shanxi Agricultural University, 2020.
[25]	李柯, 施宠, 王文全, 等. 重金属Pb胁迫下内生真菌侵染对德兰臭草种子萌发及生长的影响[J]. 农业资源与环境学报, 2020, 37(2): 280-286.
	Li K, Shi C, Wang WQ, et al. Seed germination and growth effects of endophyte infection on Melica transsilvanica under Pb stress[J]. J Agric Resour Environ, 2020, 37(2): 280-286.
[26]	Veyisoglu A, Tatar D, Saygin H, et al. Microvirga makkahensis sp. nov., and Microvirga arabica sp. nov., isolated from sandy arid soil[J]. Antonie Van Leeuwenhoek, 2016, 109(2): 287-296. doi: 10.1007/s10482-015-0631-z pmid: 26671415
[27]	Ardley JK, Parker MA, de Meyer SE, et al. Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts[J]. Int J Syst Evol Microbiol, 2012, 62(Pt 11): 2579-2588. doi: 10.1099/ijs.0.035097-0 URL
[28]	Weon HY, Kwon SW, Son JA, et al. Description of Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov., isolated from air, reclassification of Balneimonas flocculans Takeda et al. 2004 as Microvirga flocculans comb. nov. and emended description of the genus Microvirga[J]. Int J Syst Evol Microbiol, 2010, 60(Pt 11): 2596-2600. doi: 10.1099/ijs.0.018770-0 URL
[29]	Zhang XJ, Zhang J, Yao Q, et al. Microvirga flavescens sp. nov., a novel bacterium isolated from forest soil and emended description of the genus Microvirga[J]. Int J Syst Evol Microbiol, 2019, 69(3): 667-671. doi: 10.1099/ijsem.0.003189 URL
[30]	Pramanik K, Mitra S, Sarkar A, et al. Alleviation of phytotoxic effects of cadmium on rice seedlings by cadmium resistant PGPR strain Enterobacter aerogenes MCC 3092[J]. J Hazard Mater, 2018, 351: 317-329. doi: 10.1016/j.jhazmat.2018.03.009 URL
[31]	Tapase SR, Kodam KM. Assessment of arsenic oxidation potential of Microvirga indica S-MI1b sp. nov. in heavy metal polluted environment[J]. Chemosphere, 2018, 195: 1-10. doi: 10.1016/j.chemosphere.2017.12.022 URL
[32]	刘清, 徐伟昌, 招国栋, 等. 铀和铅对枯草杆菌毒性的研究[J]. 环境污染与防治, 2007, 29(4): 247-249, 253.
	Liu Q, Xu WC, Zhao GD, et al. Toxic effects of uranium and lead to B. subtilis[J]. Environ Pollut ＆ Control, 2007, 29(4): 247-249, 253.
[33]	郭俊, 潘虎, 张晓明, 等. 微生物耐受重金属的作用机制[J]. 东北农业科学, 2023, 48(1):136-139.
	Guo J, Pan H, Zhang XM, et al. Advance on mechanisms for heavy metals tolerance of microorganisms[J]. J Northeast Agric Sci, 2023, 48(1):136-139.
[34]	Liu ZT, Xian WD, Li MM, et al. Microvirga arsenatis sp. nov., an arsenate reduction bacterium isolated from Tibet hot spring sediments[J]. Antonie Van Leeuwenhoek, 2020, 113(8): 1147-1153. doi: 10.1007/s10482-020-01421-6
[35]	Mouad L, Hanane L, Omar B, et al. Nodulation of Retama species by members of the genus Microvirga in Morocco[J]. Symbiosis, 2020, 82(3): 249-258. doi: 10.1007/s13199-020-00725-5
[36]	Jiménez-Gómez A, Saati-Santamaría Z, Igual JM, et al. Genome insights into the novel species Microvirga brassicacearum, a rapeseed endophyte with biotechnological potential[J]. Microorganisms, 2019, 7(9): 354. doi: 10.3390/microorganisms7090354 URL
[37]	李福艳, 刘晓玉, 颜静婷, 等. 三株产吲哚乙酸根际促生芽孢杆菌的筛选鉴定及其促生作用[J]. 浙江农业学报, 2021, 33(5): 873-884. doi: 10.3969/j.issn.1004-1524.2021.05.13
	Li FY, Liu XY, Yan JT, et al. Isolation and identification of three indole-3-acetic acid producing plant-growth-promoting rhizosphere Bacillus sp. and their growth-promoting effects[J]. Acta Agric Zhejiangensis, 2021, 33(5): 873-884.
[38]	Bruno LB, Anbuganesan V, Karthik C, et al. Enhanced phytoextraction of multi-metal contaminated soils under increased atmospheric temperature by bioaugmentation with plant growth promoting Bacillus cereus[J]. J Environ Manage, 2021, 289: 112553. doi: 10.1016/j.jenvman.2021.112553 URL
[39]	Pal AK, Sengupta C. Isolation of cadmium and lead tolerant plant growth promoting rhizobacteria: Lysinibacillus varians and Pseudomonas putida from Indian agricultural soil[J]. Soil Sediment Contam Int J, 2019, 28(7): 601-629. doi: 10.1080/15320383.2019.1637398 URL
[40]	Gheidary S, Akhzari D, Pessarakli M. Effects of salinity, drought, and priming treatments on seed germination and growth parameters of Lathyrus sativus L[J]. J Plant Nutr, 2017, 40(10): 1507-1514. doi: 10.1080/01904167.2016.1269349 URL
[41]	赵继武. 土壤—植物系统中铀生物有效性和迁移模型的研究[D]. 绵阳: 西南科技大学, 2020.
	Zhao JW. Study on uranium bioavailability and migration model in soil-plant system[D]. Mianyang: Southwest University of Science and Technology, 2020.
[42]	Santoyo G, Urtis-Flores CA, Loeza-Lara PD, et al. Rhizosphere colonization determinants by plant growth-promoting rhizobacteria(PGPR)[J]. Biology, 2021, 10(6): 475. doi: 10.3390/biology10060475 URL
[43]	黄一绥, 邱健斌, 佘晨兴, 等. 土壤重金属污染对上海青根伸长的抑制效应研究[J]. 热带作物学报, 2011, 32(11): 2133-2137.
	Huang YS, Qiu JB, She CX, et al. Influence of soil heavy metal pollution on the root elongation of Brassica rapa L[J]. Chin J Trop Crops, 2011, 32(11): 2133-2137.