As3+胁迫下Bacillus sp. ZJS3菌株的生理生化响应特性

doi:10.13560/j.cnki.biotech.bull.1985.2021-1234

摘要/Abstract

摘要：

研究菌株Bacillus sp. ZJS3在适宜生长条件下对As³⁺胁迫的响应机制。本研究通过菌株Bacillus sp. ZJS3在固体培养基的长势确定耐受范围。在不同砷浓度影响条件下测定该菌株抗氧化酶活、丙二醛（malondialdehyde,MDA）含量及胞外聚合物（extracellular polymeric substances,EPS）中蛋白质和多糖含量。通过拍摄生物扫描电镜（scanning electron microscope,SEM）,测定菌株的吸附率和氧化还原性以及检测耐受基因来研究菌株耐受机制。本研究确定了菌株Bacillus sp. ZJS3砷的耐受范围为100-300 mg/L。根据单因素实验结果,比较适宜培养条件pH为7.0、转速为200 r/min、温度为30℃。通过硝酸银染色实验和砷钼蓝法,检测到菌株能将As³⁺氧化成As⁵⁺。不同砷浓度胁迫下,过氧化物酶（peroxidase,POD）、过氧化氢酶（catalase,CAT）、超氧化物歧化酶（superoxide dismutase,SOD）和MDA含量明显升高且EPS内多糖含量持续升高,而蛋白质是先升高后降低。通过SEM观察到砷暴露后菌体形态逐渐变长且表面出现颗粒状的物质。在24 h内,该菌株对砷的最高吸附率为25.00%,吸附量为3.23 mg/g。Bacillus sp. ZJS3可将高毒性的As³⁺氧化为低毒性的As⁵⁺,且对砷具有吸附作用,其抗氧化酶和胞外聚合物在砷胁迫过程发挥着重要作用。

关键词: As³⁺, 氧化还原性, 胞外多糖, 抗氧化酶活, 吸附率

Abstract:

We aim to investigate the response mechanism of strain Bacillus sp. ZJS3 to As³⁺ stress under optimal growth conditions. The tolerance range was determined by the growth of the strain Bacillus sp. ZJS3 in solid medium. The activities of antioxidant enzymes,the content of malondialdehyde（MDA）and the contents of protein and polysaccharide in extracellular polymeric substances（EPS）were determined under the influence of different arsenic concentrations. The mechanism of tolerance of Bacillus sp. ZJS3 was studied by photographing biological scanning electron microscope（SEM）,measuring the adsorption rate and redox activity,detecting tolerance genes. The tolerance range of Bacillus sp. ZJS3 to As³⁺was 100-300 mg/L. According to the single factor experiments,the suitable culture conditions were pH 7.0,rotation speed 200 r/min,and temperature 30℃. Through silver nitrate staining and arsenic molybdenum blue method,it was detected that Bacillus sp. ZJS3 oxidized As³⁺ to As⁵⁺. Under different arsenic concentration stress,the contents of peroxidase（POD）,catalase（CAT）,superoxide dismutase（SOD）and MDA increased significantly,and the polysaccharide content in EPS increased continuously,while the protein increased first and then decreased. It was observed by SEM that the cell morphology gradually lengthened and granular substances appeared on the surface after arsenic pollution. Within 24 h,the maximum adsorption rate of arsenic by the strain was 25.00% and the adsorption capacity was 3.23 mg/g. In conclusion,Bacillus sp. ZJS3 can oxidize high toxic As³⁺ to low toxic As⁵⁺,and has adsorption effect on arsenic. Its antioxidant enzymes and extracellular polymers play an important role in the process of arsenic stress.

Key words: As³⁺, redox activity, exopolysaccharides, anti-oxidative enzyme activity, adsorption rate

袁存霞, 李艳楠, 张肖冲, 杨瑞, 刘建利, 李靖宇. As³⁺胁迫下Bacillus sp. ZJS3菌株的生理生化响应特性[J]. 生物技术通报, 2022, 38(7): 236-246.

YUAN Cun-xia, LI Yan-nan, ZHANG Xiao-chong, YANG Rui, LIU Jian-li, LI Jing-yu. Physiological and Biochemical Response Characteristics of Bacillus sp. ZJS3 Under As³⁺ Stress[J]. Biotechnology Bulletin, 2022, 38(7): 236-246.

图/表 10

图1 菌株Bacillus sp. ZJS3在不同As3+浓度胁迫下的生长曲线

Fig.1 Growth curve of strain Bacillus sp. ZJS3 under stress of different As3+ concentrations

图2 不同条件对As3+胁迫Bacillus sp. ZJS3生长的影响 A：pH;B：转速;C：温度;不同字母表示具有显著性差异（P < 0.05）

Fig.2 Effects of different conditions on the growth of Bacillus sp. ZJS3 under As3+ stress A：pH. B：Rotation speed. C：Temperature. Different letters indicate significant differences（P < 0.05）

图3 As3+和Na+共同胁迫下Bacillus sp. ZJS3的生长 A-H表示As1-As8和Na+（1%-8%）共同胁迫下的OD值;*P < 0.05;** P < 0.01;*** P < 0.001;ns表示无显著性差异,下同

Fig.3 Growth of Bacillus sp. ZJS3 under the combined stress of As3+ and Na+ A-H refers to the OD value of As1-As8 under the common stress of Na+（1%-8%）;* indicates P < 0.05;** P < 0.01;*** P < 0.001; ns means there is no significant difference. The same below

图4 定性硝酸银（AgNO3）染色

Fig.4 Qualitative silver nitrate（AgNO3）staining

图5 砷钼蓝法测定砷含量 A、B和C、D分别是100 mg/L和300 mg/L As3+胁迫24 h、48 h培养基中砷的含量

Fig.5 Determination of arsenic content by arseno-molybdenum blue method A,B and C,D are the content of arsenic in the medium under 100 mg/L and 300 mg/L As3+ stress for 24 h and 48 h,respectively.

图6 As3+胁迫下Bacillus sp. ZJS3胞内的ROS含量

Fig.6 Content of ROS in Bacillus sp. ZJS3 cell under As3+ stress

图7 As3+胁迫对Bacillus sp. ZJS3的抗氧化酶活及MDA含量的影响

Fig.7 Effects of As3+ stress on the antioxidant enzyme activity and MDA content of Bacillus sp. ZJS3

图8 不同As3+浓度胁迫下Bacillus sp. ZJS3的扫描电镜图 A：对照;B：140 mg/L;C：260 mg/L

Fig.8 SEM of Bacillus sp. ZJS3 under different As3+ stress A：Control. B：140 mg/L. C：260 mg/L

图9 As3+胁迫下菌株Bacillus sp. ZJS3 EPS中多糖和蛋白质的含量

Fig.9 Contents of polysaccharides and proteins in EPS of Bacillus sp. ZJS3 under As3+ stress

图10 不同条件下ZJS3对As3+的吸附率

Fig.10 Adsorption rates of As3+ by ZJS3 under different conditions

参考文献 30

[1]	Sharma B, Shukla P. Lead bioaccumulation mediated by Bacillus cereus BPS-9 from an industrial waste contaminated site encoding heavy metal resistant genes and their transporters[J]. J Hazard Mater, 2021, 401:123285. doi: 10.1016/j.jhazmat.2020.123285 URL
[2]	党政, 李成, 程丽媛, 等. LC-ICP-MS联用测定As元素形态的分析[J]. 广州化工, 2017, 45(17):102-103, 116.
	Dang Z, Li C, Cheng LY, et al. Analysis of arsenic speciation with LC-ICP-MS[J]. Guangzhou Chem Ind, 2017, 45(17):102-103, 116.
[3]	Sher S, Rehman A. Use of heavy metals resistant bacteria-a strategy for arsenic bioremediation[J]. Appl Microbiol Biotechnol, 2019, 103(15):6007-6021. doi: 10.1007/s00253-019-09933-6 URL
[4]	Wu D, Zhang Z, Gao Q, et al. Isolation and characterization of aerobic, culturable, arsenic-tolerant bacteria from lead-zinc mine tailing in Southern China[J]. World J Microbiol Biotechnol, 2018, 34(12):177. doi: 10.1007/s11274-018-2557-x URL
[5]	Yuan X, Xue N, Han Z. A meta-analysis of heavy metals pollution in farmland and urban soils in China over the past 20 years[J]. J Environ Sci:China, 2021, 101:217-226. doi: 10.1016/j.jes.2020.08.013 URL
[6]	李韵诗, 冯冲凌, 吴晓芙, 等. 重金属污染土壤植物修复中的微生物功能研究进展[J]. 生态学报, 2015, 35(20):6881-6890.
	Li YS, Feng CL, Wu XF, et al. A review on the functions of microorganisms in the phytoremediation of heavy metal-contaminated soils[J]. Acta Ecol Sin, 2015, 35(20):6881-6890.
[7]	苏世鸣, 曾希柏, 蒋细良, 等. 高耐砷真菌的分离及其耐砷能力[J]. 应用生态学报, 2010, 21(12):3225-3230.
	Su SM, Zeng XB, Jiang XL, et al. High arsenic-tolerant fungi:Their isolation and tolerant ability[J]. Chin J Appl Ecol, 2010, 21(12):3225-3230.
[8]	Gu YF, Wang YY, Sun YH, et al. Genetic diversity and characterization of arsenic-resistant endophytic bacteria isolated from Pteris vittata, an arsenic hyperaccumulator[J]. BMC Microbiol, 2018, 18(1):42. doi: 10.1186/s12866-018-1184-x URL
[9]	Dunivin TK, Yeh SY, Shade A. A global survey of arsenic-related genes in soil microbiomes[J]. BMC Biol, 2019, 17(1):45. doi: 10.1186/s12915-019-0661-5 pmid: 31146755
[10]	Tong H, Liu CS, Hao LK, et al. Biological Fe(II)and As(III)oxidation immobilizes arsenic in micro-oxic environments[J]. Geochimica et Cosmochimica Acta, 2019, 265:96-108. doi: 10.1016/j.gca.2019.09.002
[11]	Jia MR, Tang N, Cao Y, et al. Efficient arsenate reduction by As-resistant bacterium Bacillus sp. strain PVR-YHB₁-1:Characterization and genome analysis[J]. Chemosphere, 2019, 218:1061-1070. doi: 10.1016/j.chemosphere.2018.11.145 URL
[12]	韩永和, 王珊珊. 微生物耐砷机理及其在砷地球化学循环中的作用[J]. 微生物学报, 2016, 56(6):901-910.
	Han YH, Wang SS. Arsenic resistance mechanisms in microbes and their roles in arsenic geochemical cycling-A review[J]. Acta Microbiol Sin, 2016, 56(6):901-910.
[13]	Fakhar A, Gul B, Gurmani AR, et al. Heavy metal remediation and resistance mechanism of Aeromonas, Bacillus, and Pseudomonas:a review[J]. Crit Rev Environ Sci Technol, 2020:1-48.
[14]	Ding P, Song W, Yang Z, et al. Influence of Zn(II)stress-induction on component variation and sorption performance of extracellular polymeric substances(EPS)from Bacillus vallismortis[J]. Bioprocess Biosyst Eng, 2018, 41(6):781-791. doi: 10.1007/s00449-018-1911-6 URL
[15]	Titah HS, Abdullah SRS, Idris M, et al. Arsenic resistance and biosorption by isolated rhizobacteria from the roots of Ludwigia octovalvis[J]. Int J Microbiol, 2018, 2018:1-10.
[16]	文雅, 冷艳, 李师翁. 微生物重金属耐受性及其机制的研究进展[J]. 环境科学与技术, 2020, 43(9):79-86.
	Wen Y, Leng Y, Li SW. Research progress on microbial tolerance to heavy metals and its mechanisms[J]. Environ Sci Technol, 2020, 43(9):79-86.
[17]	汤思敏, 朱健, 林晓敏, 等. 一株耐As细杆菌对As³⁺的吸附特征与机理[J]. 中南林业科技大学学报, 2020, 40(2):148-155.
	Tang SM, Zhu J, Lin XM, et al. Biosorption characteristic and mechanism of As³⁺ from aqueous solution by a high arsenic-resistant strain of Microbacterium[J]. J Central South Univ For Technol, 2020, 40(2):148-155.
[18]	杨俐, 邓阳川, 苏燕燕, 等. 冬虫夏草产地土壤中耐砷细菌的分离、鉴定及耐砷能力测定[J]. 世界科学技术-中医药现代化, 2020, 22(7):2563-2571.
	Yang L, Deng YC, Su YY, et al. Isolation, identification and arsenic tolerance of arsenic-resistant soil bacteria of Cordyceps sinensis[J]. Mod Tradit Chin Med Mater Med World Sci Technol, 2020, 22(7):2563-2571.
[19]	陈殿耿, 肖一然, 李文莉, 等. 砷钼蓝法测定某含金多金属矿碱浸液中砷的含量及其价态[J]. 中国无机分析化学, 2019, 9(1):14-16.
	Chen DG, Xiao YR, Li WL, et al. Determination of arsenic and its valence in alkali leaching liquid of a gold containing polymetallic ore by arseno-molybdenum blue method[J]. Chin J Inorg Anal Chem, 2019, 9(1):14-16.
[20]	李金璞, 张雯雯, 杨新萍. 活性污泥污水处理系统中胞外多聚物的作用及提取方法[J]. 生态学杂志, 2018, 37(9):2825-2833.
	Li JP, Zhang WW, Yang XP. The roles and extraction methods of extracellular polymeric substances in activated sludge wastewater treatment system[J]. Chin J Ecol, 2018, 37(9):2825-2833.
[21]	李宁杰, 兰琪, 陈中维, 等. 黄孢原毛平革菌BKMF-1767产絮凝剂PCF-1767的絮凝特性及其机理解析[J]. 微生物学通报, 2020, 47(2):431-439.
	Li NJ, Lan Q, Chen ZW, et al. Flocculation characteristics and mechanism of flocculant PCF-1767 produced by Phanerochaete chrysosporium BKMF-1767[J]. Microbiol China, 2020, 47(2):431-439.
[22]	Podder MS, Majumder CB. Bioaccumulation of As(III)/As(V)ions by living cells of Corynebacterium glutamicum MTCC 2745[J]. Sep Sci Technol, 2016, 51(18):2970-2990. doi: 10.1080/01496395.2016.1238485 URL
[23]	胡玉婕, 朱秀玲, 丁延芹, 等. 芽孢杆菌的耐盐促生机制研究进展[J]. 生物技术通报, 2020, 36(9):64-74. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0746
	Hu YJ, Zhu XL, Ding YQ, et al. Research progress on salt tolerance and growth-promoting mechanism of Bacillus[J]. Biotechnol Bull, 2020, 36(9):64-74.
[24]	Patel M, Parida AK. Salinity alleviates the arsenic toxicity in the facultative halophyte Salvadora persica L. by the modulations of physiological, biochemical, and ROS scavenging attributes[J]. J Hazard Mater, 2021, 401:123368. doi: 10.1016/j.jhazmat.2020.123368 URL
[25]	苏世鸣, 曾希柏, 白玲玉, 等. 微生物对砷的作用机理及利用真菌修复砷污染土壤的可行性[J]. 应用生态学报, 2010, 21(12):3266-3272.
	Su SM, Zeng XB, Bai LY, et al. Action mechanisms of microorganisms on arsenic and the feasibility of utilizing fungi in remediation of arsenic-contaminated soil[J]. Chin J Appl Ecol, 2010, 21(12):3266-3272.
[26]	张海欧, 周维芝, 马玉洪, 等. 微生物胞外聚合物对重金属镉的解毒作用及红外光谱分析[J]. 光谱学与光谱分析, 2013, 33(11):3041-3043.
	Zhang HO, Zhou WZ, Ma YH, et al. FTIR spectrum and detoxication of extracellular polymeric substances secreted by microorganism[J]. Spectrosc Spectr Anal, 2013, 33(11):3041-3043.
[27]	邹春艳, 于杨格, 连宾. 胶质芽孢杆菌对重金属离子Pb²⁺、Zn²⁺、Cd²⁺、Cu²⁺和Cr³⁺的吸附与解吸特征[J]. 南京师大学报:自然科学版, 2018, 41(1):68-75.
	Zou CY, Yu YG, Lian B. Adsorption and desorption of Pb²⁺, Zn²⁺, Cd²⁺, Cu²⁺ and Cr³⁺ by Bacillus mucilaginosus[J]. J Nanjing Norm Univ:Nat Sci Ed, 2018, 41(1):68-75.
[28]	沈秋悦, 曹志强, 朱月芳, 等. 一株耐镉细菌的分离鉴定及其吸附条件的优化[J]. 土壤, 2016, 48(3):615-620.
	Shen QY, Cao ZQ, Zhu YF, et al. Isolation of a Cd-resistant bacterium and optimization of its bio-accumulation condition[J]. Soils, 2016, 48(3):615-620.
[29]	潘攀, 杨俊诚, 邓仕槐, 等. 土壤-植物体系中农药和重金属污染研究现状及展望[J]. 农业环境科学学报, 2011, 30(12):2389-2398.
	Pan P, Yang JC, Deng SH, et al. Proceedings and prospects of pesticides and heavy metals contamination in soil-plant system[J]. J Agro Environ Sci, 2011, 30(12):2389-2398.
[30]	Niu AP, Bian WP, Feng SL, et al. Role of manganese superoxide dismutase(Mn-SOD)against Cr(III)-induced toxicity in bacteria[J]. J Hazard Mater, 2021, 403:123604. doi: 10.1016/j.jhazmat.2020.123604 URL

As³⁺胁迫下Bacillus sp. ZJS3菌株的生理生化响应特性

Physiological and Biochemical Response Characteristics of Bacillus sp. ZJS3 Under As³⁺ Stress

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王珊珊, 孙敏, 王永霞, 李惟栋, 韩春超. 鸡腿蘑胞外多糖的形貌结构及分子量动态变化与抗氧化的相关性研究[J]. 生物技术通报, 2021, 37(2): 129-137.
[2]	范敏, 王丽宁. 等离子体诱变选育高产胞外多糖花脸香蘑菌株[J]. 生物技术通报, 2021, 37(11): 119-124.
[3]	邓超, 杜秀娟, 黄涛, 郭英, 李炳学, 卜宁. 碳氮比对固氮菌株WN-F合成胞外多糖的影响[J]. 生物技术通报, 2018, 34(3): 194-199.
[4]	李彬, 陈向楠, 张建法, 王世明. 产胞外多糖菌株的筛选及胞外多糖结构分析[J]. 生物技术通报, 2016, 32(5): 165-171.
[5]	龙寒,陈盛峰,陈佳,杨迪,李晓燕,黄玉油,何秀苗，禤金彩. 一株产胞外多糖海洋弧菌的分离鉴定及其多糖抗肿瘤活性初步研究[J]. 生物技术通报, 2016, 32(12): 166-171.
[6]	辛跃强, 梁荣荣, 王瑞明. 低聚半乳糖对肠道益生菌产胞外多糖作用的研究[J]. 生物技术通报, 2015, 31(6): 144-150.
[7]	苗自利,袁佐清. PFOS对再生东亚三角涡虫抗氧化酶活性的影响[J]. 生物技术通报, 2015, 31(10): 211-215.
[8]	李明源, 王继莲, 魏云林, 季秀玲. 细菌胞外多糖的特性及应用研究[J]. 生物技术通报, 2014, 0(6): 51-56.
[9]	黄怡诚, 刘旸, 庞昕, 马中瑞, 韩东雷, 陈敏. 产胞外多糖植物内生菌的分离和鉴定[J]. 生物技术通报, 2014, 0(4): 147-151.
[10]	曹春蕾;崔宝凯;戴玉成;. 桑木层孔菌液体培养条件的研究[J]. , 2012, 0(02): 176-181.
[11]	王正荣;生吉萍;申琳;. 细菌胞外多糖的生物合成与基因控制[J]. , 2010, 0(11): 48-55.
[12]	郑世英;商学芳;王景平;. 可见分光光度法测定盐胁迫下玉米幼苗抗氧化酶活性及丙二醛含量[J]. , 2010, 0(07): 106-109.
[13]	王斌;2连宾;潘牧;. 超滤法提取裂褶菌胞外多糖几个主要参数的研究[J]. , 2006, 0(S1): 490-493.
[14]	崔艳红;黄现青;. 微生物胞外多糖研究进展[J]. , 2006, 0(02): 25-28.
[15]	. 国外动态[J]. , 2002, 0(03): 10-14.