一株镍抗性和石油烃降解菌的分离鉴定及其生物学特性

doi:10.13560/j.cnki.biotech.bull.1985.2017-0687

摘要/Abstract

摘要： 从四川省长宁-威远地区页岩气开发井场重度污染区的土壤中，筛选出一株镍抗性和石油高效降解菌株，研究其生物学特性和部分生理生化指标。将种子液接种在原油重金属液体培养基中，30℃、130 r/min条件下分别培养7 d探究该菌株对金属镍离子的抗性和对总石油烃（Total petroleum hydrocarbon，TPH）的降解率，以及经生理生化实验、形态观察和16S rDNA对该菌株鉴定分析。结果显示，该菌株对Ni²⁺的耐受性可达300 mg/L，对Ni²⁺的去除率和吸附率分别达到了56.64%和52.16%，同时对总TPH的降解率达到35.65%。确定该菌种属柠檬酸杆菌属（Citrobacter sp.），命名为Citrobacter farmeri strain M1。该菌株的最适生长温度为30℃，最适生长pH范围为7-9，最适C∶N为10∶1和盐最高耐受度可达5%。该菌株有较好的石油降解能力并对金属镍有较强的抗性，适用于石油重金属混合污染土壤的修复。

关键词: 石油降解菌, 镍抗性, 分离鉴定, 生物学特性

Abstract: : A bacterial strain with high ability of nickel resistance and petroleum-degrading was isolated from the soil of heavily-polluted areas around the shale gas wells in Changning-Weiyuan，Sichuan Province，and its biological characteristics and physiological-biochemical indexes were studied. The seed liquid was inoculated into the liquid medium containing heavy metal and crude oil，cultured at 30℃ and 130 r/min for 7 days to explore the resistance of the strain to nickel ions（Ni²⁺）and the rate of degrading total petroleum hydrocarbon（TPH）. Further，the strain was identified based on the experimental results of physiology and biochemistry，morphological observation and 16S rDNA sequences. As results，the tolerance of the strain to Ni²⁺ reached 300 mg/L，and the removal rate and adsorption rate of Ni²⁺ reached 56.64% and 52.16%，respectively; meanwhile，the degradation rate of TPH reached 35.65%. The species of the strain belonged to Citrobacter（Citrobacter sp.）and named Citrobacter farmeri strain M1. The optimal growth temperature of this strain was 30℃，the optimal growth pH was from 7 to 9，the optimal C∶N was 10∶1，and the maximum salt tolerance was up to 5%. In conclusion，solid nickel resistance and petroleum degradability of the strain contributes to its applicability in the remediation of heavy metal and oil-contaminated soil.

Key words: petroleum-degrading bacterium, nickel resistance, isolation and identification, biological characteristic

马永松, 李琋, 李珍珍, 王佩洁. 一株镍抗性和石油烃降解菌的分离鉴定及其生物学特性[J]. 生物技术通报, 2017, 33(10): 169-177.

MA Yong-song, LI Xi, LI Zhen-zhen, WANG Pei-jie. Isolation and Identification of a Nickel-resistant and Petroleum Hydrocarbon Degrading Strain and Its Biological Characteristics[J]. Biotechnology Bulletin, 2017, 33(10): 169-177.

参考文献

[1] 吴孟余. 我国页岩气开发现状与展望[J]. 电力与能源, 2014, 35(4)：405-407.
[2] 胡颖, 彭博. 美国页岩气开发现状及其能源发展趋势[J]. 广东化工, 2014, 41(23)：82-83.
[3] 杜群, 万丽丽. 美国页岩气能源开发的环境法律管制及对中国的启示[J]. 中国政法大学学报, 2015(6)：146-158.
[4] 郭亦成. 四川省页岩气开发研究[D]. 成都：四川省社会科学院, 2016.
[5] 于慧龙. 论页岩气开发利用的革命性影响和作用[D]. 重庆：重庆师范大学, 2013.
[6] 王景艺. 非传统能源开发对气藏区周边水系统的影响分析[D]. 北京：华北电力大学, 2016.
[7] 孟永涛. 页岩气水平井油基泥浆体系的研究及应用[D]. 荆州：长江大学, 2013.
[8] 朱天菊, 解艺平, 吴波, 等. 页岩气开发井场污染土壤可生化性研究[J]. 广州化工, 2017(3)：60-62.
[9] 卢邦俊. 页岩气钻屑中的重金属成分究[J]. 能源环境保护, 2015, 29(5)：33-34.
[10] 骆永明. 污染土壤修复技术研究现状与趋势[J]. 化学进展, 2009, 21(203)：558-565.
[11] Lu M. The use of goosegrass(Eleusine indica)to remediate soil contaminated with petroleum[J]. Water, Air, & Soil Pollution, 2010, 209(1)：181-189.
[12] Xu Y, Lu M. Bioremediation of crude oil-contaminated soil：comparison of different biostimulation and bioaugmentation treatments[J]. Journal of Hazardous Materials, 2010, 183(1-3)：395-401.
[13] Alisi C, Musella R, Tasso F, et al. Bioremediation of diesel oil in a co-contaminated soil by biougmentation with a microbial formula tailored with native strains selected for heavy metals resistance[J]. Science of the Total Environment, 2009, 407(8)：3024-3022.
[14] Atagana HI. Bioremediation of co-contamination of crude oil and heavy metals in soil by phytore-mediation using chromolaena odorata, (L)King & H. E. Robinson[J]. Water, Air, & Soil Pollution, 2011, 215(1)：261-271.
[15] PéREZ RM. Combined strategy for the precipitation of heavy metals and biodegradation of petroleum in industrial waste-waters[J]. Journal of Hazardous Materials, 2010, 182(1-3)：896-902.
[16] Yang W, Zhang T, Li S, et al. Metal removal from and microbial property improvement of a multiple heavy-metals contaminated soil by phytoextraction with a cadmium hyperaccumulator Sedum alfredii, H[J]. Journal of Soils and Sediments, 2014, 14(8)：1385-1396.
[17] 李亭亭. 石油-重金属复合污染盐渍土生物修复中MCB对重金属的钝化研究[D]. 济南：山东师范大学, 2014.
[18] Reddy KR. Technical challenges to in-situ remediation of polluted Sites[J]. Geotechnical and Geological Engineering, 2010, 28(3)：211-221.
[19] 杜连祥, 路福平. 微生物学实验技术[M]. 北京：中国轻工业出版社, 2010.
[20] 马前, 曹同成. 新型耐金属微生物的筛选研究[C]// 2013中国环境科学学会学术年会论文集. 中国环境科学学术年会, (第五卷), 2013.
[21] 王慧萍. 耐锌细菌的筛选、抗锌特性及其对苯酚的降解研究[D]. 上海：东华大学, 2011.
[22] 付瑾. 镉抗性菌DX-T3-01的筛选鉴定与吸附镉机理及其降解苯酚特性研究[D]. 上海：东华大学, 2011.
[23] 金忠民, 郝宇, 刘丽杰, 等. 一株铅镉抗性菌株的分离鉴定及其生物学特性[J]. 环境工程学报, 2015(7)：3551-3557.
[24] 孙士顺. 重金属铜抗性细菌的筛选鉴定、吸附特性与机理研究[D]. 长春：东北师范大学, 2016.
[25] 曹德菊, 王方, 胡海荣. 抗铅微生物的筛选及对水中铅的去除效果研究[J]. 环境与健康杂志, 2011, 28(1)：66- 68.
[26] 张汉波, 郑月, 曾凡, 等. 几株细菌的重金属抗性水平和吸附量[J]. 微生物学通报, 2005, 32(3)：24-29.
[27] 悉旦立. 环境监测(修订版)[M]. 北京：高等教育出版社, 1995.
[28] Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain specific 16Sprimers[J]. Journal of Microbiological Methods, 2003, 55(3)：541-555.
[29] 林辰壹, 马娟, 杨婷婷, 等. 优化氮源种类及碳氮比对阿魏菇液体种生长的效应[J]. 新疆农业科学, 2012, 49(11)：2042-2047.
[30] 赵本良, 仇荣亮, 刘金芩, 等. 一株硫酸盐还原细菌的筛选及其功能研究[C]// 第十次全国环境微生物学术研讨会论文摘要集. 2007.
[31] Chen C, Lei W, Min L, et al. Characterization of Cu(II)and Cd(II)resistance mechanisms in Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHEOCH and their potential application in the bioremediation of heavy-Metal phenanthrene co-contaminated sites[J]. Environmental Science and Pollution Research, 2016, 23(7)：6861.
[32] Dave S, Damani M, Tipre D. Copper remediation by Eichhornia spp. and sulphate-reducing bacteria[J]. Journal of Hazardous Materials, 2010, 173(13)：231-235.
[33] Limcharoensuk T, Sooksawat N, Sumarnrote A, et al. Bioaccumulation and biosorption of Cd²⁺ and Zn²⁺ by bacteria isolated from a zinc mine in Thailand[J]. Ecotoxicol Environ Saf, 2015, 122：322-330.
[34] 赵晓秀. 重金属铜复合污染土壤中石油的微生物降解[D]. 大连：大连理工大学, 2008.
[35] 谢鲲鹏, 周集体, 曲媛媛, 等. 一株耐盐原油降解菌的分离鉴定及其降解特性研究[J]. 海洋环境科学, 2009, 28(6)：680-683.