陕北地区高效石油降解菌的筛选及其对油污土壤的修复研究
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摘要
通过筛选陕北地区的高效石油降解菌,为后续的土壤修复提供优良的菌种资源。本研究的石油降解菌分离来自延安市延长县某油井的土壤样品,通过以石油为唯一碳源,进行筛选、富集培养和平板划线分离,得到可降解石油的菌株,并采用紫外可见分光光度法测量其对富集培养基中的石油降解率。利用PCR扩增技术对筛选的石油降解菌的16S r DNA序列进行扩增,通过对16S r DNA序列的测定和NCBI数据库集进行基因序列比对确定其种属;在被石油污染过土壤中加入筛选出的石油降解菌进行修复试验,经50 d的修复反应,测定石油降解菌对油污土壤中石油的降解效率;最后通过种植小麦检验石油降解菌的降解效果。共筛选出3株高效的可降解石油的菌株:W1、W3、N4,三株菌均可以在以石油为唯一碳源的环境中生长,它们在富集培养基中的石油降解率分别为42. 55%,37. 18%和33. 57%,利用分子生物学技术对三株菌进行鉴定,结果是W1菌株为假单胞菌属,W3菌株为芽孢杆菌属,N4菌株为红球菌属。在菌株对油污土壤修复的研究中,菌W1和菌W3分别对油污土壤进行50 d的降解,土壤中石油量得到很大程度的降解,W1菌株的降油率为52. 20%,W3菌株的降油率为47. 84%,修复后土壤的质量对于小麦的生长没有影响。通过本研究课题,为陕北地区石油污染土壤修复提供了优良的菌种资源,同时为陕北地区的石油污染土壤的微生物修复提供了一定的科学依据。
To select the efficient oil degradation bacteria in northern Shaanxi,and to provide excellent bacterial resources for subsequent soil restoration. The research subject of oil degradation bacteria isolated from Yanan Yanchang oil Wells of soil samples,by screening with oil as the sole carbon source separation,enrichment culture and plate line,select biodegradable oil bacteria,and measured with UV-VIS spectrophotometry for the enrichment of the culture medium of oil degradation rate. The 16 S r DNA sequences of the selected oil-degrading bacteria were amplified by PCR amplification technology,and their species were determined by comparing the sequence of the16 S r DNA sequence with the NCBI database set; the oil degradation bacteria was added to the oil contaminated soil for repair trials,and 50 d will be used to determine the efficiency of the oil in the soil. Finally,the degradation effect of the oil degradation bacteria was tested by planting wheat. A total of 3 strains screened out efficient strains of biodegradable oil: W1,W3,N4 interchange,all can be in three bacteria grow with oil as sole carbon source environment,they are in the enrichment medium oil degradation rate were 42. 55%,37. 18% and 33. 57%. The three strains were identified by using molecular biology technique,and the result was the bacterium W1,which is a pseudomonas,and the bacterium W3 was a bacillus,and the N4 was a red coccus. In strains of oil pollution in the study of soil restoration,bacteria W1 and bacteria W3,respectively,the oil pollution of soil degradation in the 50 d,bacterium of W1 drop oil rate was 52. 20%,the bacteria of W3 oil at a rate of 47. 84%,reducing the degree of degradation of oil amount in the soil,to achieve the soil that wheat growth. Through this research topic. For petroleum contaminated soil restoration in northern Shaanxi,this provides excellent species resources,at the same time,in northern Shaanxi area of petroleum contaminated soil microbial remediation provides certain scientific basis.
To select the efficient oil degradation bacteria in northern Shaanxi,and to provide excellent bacterial resources for subsequent soil restoration. The research subject of oil degradation bacteria isolated from Yanan Yanchang oil Wells of soil samples,by screening with oil as the sole carbon source separation,enrichment culture and plate line,select biodegradable oil bacteria,and measured with UV-VIS spectrophotometry for the enrichment of the culture medium of oil degradation rate. The 16 S r DNA sequences of the selected oil-degrading bacteria were amplified by PCR amplification technology,and their species were determined by comparing the sequence of the16 S r DNA sequence with the NCBI database set; the oil degradation bacteria was added to the oil contaminated soil for repair trials,and 50 d will be used to determine the efficiency of the oil in the soil. Finally,the degradation effect of the oil degradation bacteria was tested by planting wheat. A total of 3 strains screened out efficient strains of biodegradable oil: W1,W3,N4 interchange,all can be in three bacteria grow with oil as sole carbon source environment,they are in the enrichment medium oil degradation rate were 42. 55%,37. 18% and 33. 57%. The three strains were identified by using molecular biology technique,and the result was the bacterium W1,which is a pseudomonas,and the bacterium W3 was a bacillus,and the N4 was a red coccus. In strains of oil pollution in the study of soil restoration,bacteria W1 and bacteria W3,respectively,the oil pollution of soil degradation in the 50 d,bacterium of W1 drop oil rate was 52. 20%,the bacteria of W3 oil at a rate of 47. 84%,reducing the degree of degradation of oil amount in the soil,to achieve the soil that wheat growth. Through this research topic. For petroleum contaminated soil restoration in northern Shaanxi,this provides excellent species resources,at the same time,in northern Shaanxi area of petroleum contaminated soil microbial remediation provides certain scientific basis.
引文
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[11]姜淑梅,张龙,戴世鲲,等.一种简单、有效的适于PCR操作的放线菌DNA提取方法[J].生物技术,2007(1):39-41.
[12]汪杰,郑维爽,礼晓,等.高效石油降解菌的筛选鉴定及修复能力研究[J].环境科学学报,2010,30(6):1228-1234.
[13]丁艺.石油烃降解菌的筛选及其对油污土壤的修复研究[D].西安建筑科技大学,2013.
[14]卢丽丽.石油污染土壤的植物修复研究[D].西安建筑科技大学,2008.
[15]王彦杰,李志国,洪秀杰,等.三株假单胞菌对石油降解特性的研究[J].黑龙江八一农垦大学学报,2007(1):1-5.
[16]梁生康,王修林,汪卫东,等.高效石油降解菌的筛选及其在油田废水深度处理中的应用[J].化工环保,2004(1):41-46.
[17]周际海,袁颖红,朱志保,等.土壤有机污染物生物修复技术研究进展[J].生态环境学报,2015,24(2):343-351.
[2]孙远军.降解石油烃优势菌的筛选、分离及性能研究[D].西安建筑科技大学,2006.
[3]马强,林爱军,马薇,等.土壤中总石油烃污染(TPH)的微生物降解与修复研究进展[J].生态毒理学报,2008(1):1-8.
[4]田苗.石油降解菌的筛选及对石油污染土壤的修复实验[D].西北大学,2010.
[5]何翊,吴海,魏薇.石油污染土壤菌剂修复技术研究[J].土壤,2005(3):338-340.
[6]姚瑶,刘兆普,郑青松,等.胜利油田土壤中石油降解菌分离和鉴定及代谢产物的研究[J].南京农业大学学报,2013,36(1):65-71.
[7]押辉远,易力,周胜利,等.南阳油污土壤微环境分析及石油降解菌筛选[J].湖北农业科学,2011,50(6):1132-1136.
[8]王伟平,李伟,邱雁临,等.高效石油降解菌的筛选及初步鉴定[J].化学与生物工程,2009,26(7):64-66.
[9]吴玉新.紫外分光光度法测定污水中油含量的研究[J].石化技术,1998(2):50-52.
[10]徐平,李文均,徐丽华,等.微波法快速提取放线菌基因组DNA[J].微生物学通报,2003(4):82-84.
[11]姜淑梅,张龙,戴世鲲,等.一种简单、有效的适于PCR操作的放线菌DNA提取方法[J].生物技术,2007(1):39-41.
[12]汪杰,郑维爽,礼晓,等.高效石油降解菌的筛选鉴定及修复能力研究[J].环境科学学报,2010,30(6):1228-1234.
[13]丁艺.石油烃降解菌的筛选及其对油污土壤的修复研究[D].西安建筑科技大学,2013.
[14]卢丽丽.石油污染土壤的植物修复研究[D].西安建筑科技大学,2008.
[15]王彦杰,李志国,洪秀杰,等.三株假单胞菌对石油降解特性的研究[J].黑龙江八一农垦大学学报,2007(1):1-5.
[16]梁生康,王修林,汪卫东,等.高效石油降解菌的筛选及其在油田废水深度处理中的应用[J].化工环保,2004(1):41-46.
[17]周际海,袁颖红,朱志保,等.土壤有机污染物生物修复技术研究进展[J].生态环境学报,2015,24(2):343-351.
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