一株石油降解菌Lysinibacillus fusiformis 23-1的筛选鉴定及原油降解特性
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摘要
石油泄露造成了严重的环境问题和重大经济损失,微生物修复是解决石油污染的最为有效的途径。从青藏高原石油污染土样筛选产生物表面活性剂的低温石油降解菌,并研究了该菌株对石油的乳化性能、降解特性、降解条件以及对不同碳链烃类的利用。本实验用血平板法分离到一株产表面活性剂石油降解菌23-1,形态学和16S rRNA基因序列分析鉴定其为纺锤形赖氨酸芽孢杆菌(Lysinibacillus fusiformis 23-1),比色法测定该菌株的乳化性能为19.6%,超声波法测定乳化稳定性为37.5%,表明其能够降低油水界面张力,具有增溶作用。质量法测定L.fusiformis 23-1对石油的降解率为57%,适宜降解条件为:初始pH为7.5,温度为25℃,培养8 d后获得最佳降解效果。GC-MS方法测定该菌株的石油降解特性,结果表明,该菌对石油中不同碳链的烃类降解能力不同。L.fusiformis 23-1能产表面活性剂,具有较强的降解石油能力,可用于石油污染修复。
Petroleum spill has caused serious environmental problems and enormous economic losses. The microbial bioremediation have been shown to be the most effective way for the removal of petroleum pollution in environments.We isolated a biosurfactant producing and petroleum degrading bacteria from petroleum contaminated soil in the region of Qilian Mountain Baoping River in the Qinghai-Tibet Plateau. We further investigated the petroleum emulsifiability,degradation properties,and degradation conditions of the isolated strain,as well as the uses of hydrocarbons with different carbon chain by the isolated strain. A biosurfactant producing and petroleum degrading strain,assigned as 23-1,was successfully isolated on the medium with petroleum as sole carbon source using the blood plate screening method. Morphological and 16 S rRNA gene sequence analysis identified this isolated strain as Lysinibacillus fusi-formis strain 23-1. The colorimetric and ultrasonic analysis indicated that the emulsifiability and emulsion stability of strain 23-1 were 19. 6% and 37. 5%,respectively,suggesting that L. fusiformis strain 23-1 can reduce the oil-water interfacial tension and enhance the oil solubilization. The gravimetric method analysis showed that 57% petroleum added in the medium could be degraded by L. fusiformis 23-1 after 8-d incubation,with pH 7. 5 and 25 ℃. Further GC-MS measurement indicated that L. fusiformis 23-1 strongly degraded direct chain saturated alkanes,partly degraded the short chain alkanes and long chain alkanes,but weakly degraded the aromatic hydrocarbons. L. fusiformis23-1 can produce certain biosurfactant and degrade petroleum during growth in the medium added with petroleum as sole carbon source. This strain could be used for bioremediation of petroleum contaminated environments.
Petroleum spill has caused serious environmental problems and enormous economic losses. The microbial bioremediation have been shown to be the most effective way for the removal of petroleum pollution in environments.We isolated a biosurfactant producing and petroleum degrading bacteria from petroleum contaminated soil in the region of Qilian Mountain Baoping River in the Qinghai-Tibet Plateau. We further investigated the petroleum emulsifiability,degradation properties,and degradation conditions of the isolated strain,as well as the uses of hydrocarbons with different carbon chain by the isolated strain. A biosurfactant producing and petroleum degrading strain,assigned as 23-1,was successfully isolated on the medium with petroleum as sole carbon source using the blood plate screening method. Morphological and 16 S rRNA gene sequence analysis identified this isolated strain as Lysinibacillus fusi-formis strain 23-1. The colorimetric and ultrasonic analysis indicated that the emulsifiability and emulsion stability of strain 23-1 were 19. 6% and 37. 5%,respectively,suggesting that L. fusiformis strain 23-1 can reduce the oil-water interfacial tension and enhance the oil solubilization. The gravimetric method analysis showed that 57% petroleum added in the medium could be degraded by L. fusiformis 23-1 after 8-d incubation,with pH 7. 5 and 25 ℃. Further GC-MS measurement indicated that L. fusiformis 23-1 strongly degraded direct chain saturated alkanes,partly degraded the short chain alkanes and long chain alkanes,but weakly degraded the aromatic hydrocarbons. L. fusiformis23-1 can produce certain biosurfactant and degrade petroleum during growth in the medium added with petroleum as sole carbon source. This strain could be used for bioremediation of petroleum contaminated environments.
引文
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[24]WANG Z,FINGAS M F.Development of oil hydrocarbon fingerprinting and identification techniques[J].Marine Pollution Bulletin,2003,47(9):423-452.
[25]HE L,DANG Z,TANG X,et al.Biodegradation characteristics of crude oil by mixed bacterial strains[J].Acta Scientiae Circumstantiae,2010,30(6):1220-1227.
[26]齐永强,王红旗,刘敬奇,等.土壤中石油污染物微生物降解过程中各石油烃组分的演变规律[J].环境科学学报,2003,23(6):834-836.QI Y Q,WANG H Q,LIU J Q,et al.Component changes of hydrocarbon during the precess of bioremediation of hydrocarbon-contaminatel soil[J].Acta Scientiae Circumstantiae,2003,23(6):834-836.(in Chinese with English abstract)
[27]CHEN L,XIAO C,LUO X,et al.Study on biological degradation and transform characteristics of different components in petroleum hydrocarbon used by bacterial consortium[J].Environmental Earth Sciences,2016,75(9):1-12.
[28]杨丽芹,蒋继辉.微生物对石油烃类的降解机理[J].油气田环境保护,2011,21(2):24-26.YANG L Q,JIANG J H.The microorganism degradation mechanism of petroleum hydrocarbons[J].Environmental Protection of Oil&Gas Fields,2011,21(2):24-26.(in Chinese with English abstract)
[2]KING T L,ROBINSON B,CUI F,et al.An oil spill decision matrix in response to surface spills of various bitumen blends.[J].Environmental Science Processes&Impacts,2017,19(7):928-938.
[3]樊林.用生物法处理石油烃类污染物的初步研究[D].重庆:重庆大学,2005.FAN L.The initial research of breaking down petroleum hydrocarbon pollutants with biological method[D].Chongqing:Chongqing University,2005.(in Chinese with English abstract)
[4]BANU J R,USHANI U,MERRYLIN J,et al.Evaluation of operational parameters for biodegration of bacterially disintegrated sludge[J].Desalination&Water Treatment,2016,57(11):25018-25027.
[5]张文.应用表面活性剂强化石油污染土壤及地下水的生物修复[D].北京:华北电力大学,2012.ZHANG W.Application of surfactant in enhancing bioremediation of petroleum-contaminated soil and groundwater[D].Beijing:North China Electric Power University,2012.(in Chinese with English abstract)
[6]KOSARIC N.Biosurfactants and their application for soil bioremediation[J].Food Technology&Biotechnology,2001,39(4):295-304.
[7]ATLAS R M.Microbial degradation of petroleum hydrocarbons:an environment perspective[J].Microbiological Reviews,1981,45(1):180-209.
[8]FULEKAR M H.Microbial degradation of petrochemical waste-polycyclic aromatic hydrocarbons[J].Bioresources&Bioprocessing,2017,4(1):28-30.
[9]BALSEIRO-ROMERO M,GKOREZIS P,KIDD P S,et al.Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation[J].International Journal of Phytoremediation,2017,29(1):1214-1217.
[10]ARIMA K,SAKURA Y,IZAKI K.Studies on comenic acid and 5-oxymaltol accumulations by microorganism isolated from soil[J].Journal of the Agricultural Chemical Society of Japan,1968,42(3):103-108.
[11]杨蕊琪,薛林贵,常思静,等.一株耐低温原油降解菌的分离鉴定及降解特性[J].浙江农业学报,2016,28(10):1781-1789.YANG R Q,XUE L G,CHANG S J,et al.Studies on isolation,identification of cold-resistant petroleum-degrading strain and its degradation capability[J].Acta Agriculturae Zhejiangensis,2016,28(10):1781-1789.(in Chinese with English abstract)
[12]HUANG X F,LIU J,LU L J,et al.Evaluation of screening methods for demulsifying bacteria and characterization of lipopeptide biodemulsifier produced by Alcaligenes sp.[J].Bioresource Technology,2009,100(3):1358-1365.
[13]LIANG J Z,MEI J Q,WANG Y X.Screening and identification of the degradation bacteria of residues chlorpyrifos pesticide on fruits and vegetables[J].Science&Technology of Food Industry,2017,10(2):451-456.
[14]ALENEZI F N,REKIK I,CHENARI BOUKET A,et al.Increased biological activity of Aneurinibacillus migulanus strains correlates with the production of new gramicidin secondary metabolites[J].Frontiers in Microbiology,2017,8(4):517-522.
[15]DUNBAR J,TAKALA S,BARNSS M,et al.Levels of bacterial community diversity in four arid soils compared by cultivation and 16S r RNA gene cloning[J].Applied and Environmental Microbiology,1999,65(4):1662-1669.
[16]DULBECCO R,VOGT M.One-step growth curve of western equine encephalomyelitis virus on chicken embryo cells grown in vitro and analysis of virus yields from single cells[J].Journal of Experimental Medicine,1954,99(2):183.
[17]章慧,郭楚玲,卢桂宁,等.具有产表面活性剂功能石油降解菌的筛选及其发酵条件优化[J].农业环境科学学报,2013,32(11):2185-2191.ZHANG H,GUO C L,LU G N,et al.Screening and fermentation optimization of a biosurfactant-producing oil-degeading bacterium[J].Journal of Agro-Environment Science,2013,32(11):2185-2191.(in Chinese with English abstract)
[18]表面活性剂乳化力的测定比色法:GB/T 6369—2008[S].北京:中国标准出版社,2008.
[19]XU F.Study on the screening and degradation characteristics of highly efficient petroleum degrading bacteria[J].Biotechnology Bulletin,2010,20(7):221-226.
[20]崔中利,刘卫东,齐耀程,等.生物表面活性剂产生菌的分离培养及其产物特性研究[J].土壤,2005,37(6):607-612.CUI Z L,LIU W D,QI Y C,et al.Studies on isolation biosurfactant and its degradation capability[J].Soil,2005,37(6):607-612.(in Chinese with English abstract)
[21]WANG X B,CHI C Q,NIE Y,et al.Degradation of petroleum hydrocarbons(C6-C40)and crude oil by a novel Dietzia strain[J].Bioresource Technology,2011,102(17):7755-7759.
[22]JIAN L U.Isolation,identification and characterization of a high-efficiency alkane-degrading bacterium xcz[J].Soils,2008,40(3):460-464.
[23]ZAMPOLLI J,COLLINA E,LASAGNI M,et al.Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism[J].Parallel Processing Letters,2014,4(1):1-9.
[24]WANG Z,FINGAS M F.Development of oil hydrocarbon fingerprinting and identification techniques[J].Marine Pollution Bulletin,2003,47(9):423-452.
[25]HE L,DANG Z,TANG X,et al.Biodegradation characteristics of crude oil by mixed bacterial strains[J].Acta Scientiae Circumstantiae,2010,30(6):1220-1227.
[26]齐永强,王红旗,刘敬奇,等.土壤中石油污染物微生物降解过程中各石油烃组分的演变规律[J].环境科学学报,2003,23(6):834-836.QI Y Q,WANG H Q,LIU J Q,et al.Component changes of hydrocarbon during the precess of bioremediation of hydrocarbon-contaminatel soil[J].Acta Scientiae Circumstantiae,2003,23(6):834-836.(in Chinese with English abstract)
[27]CHEN L,XIAO C,LUO X,et al.Study on biological degradation and transform characteristics of different components in petroleum hydrocarbon used by bacterial consortium[J].Environmental Earth Sciences,2016,75(9):1-12.
[28]杨丽芹,蒋继辉.微生物对石油烃类的降解机理[J].油气田环境保护,2011,21(2):24-26.YANG L Q,JIANG J H.The microorganism degradation mechanism of petroleum hydrocarbons[J].Environmental Protection of Oil&Gas Fields,2011,21(2):24-26.(in Chinese with English abstract)