石油污染环境中固氮和寡氮营养细菌的分离鉴定及其特性  

Identification and characteration of nitrogen-fixing bacteria and oligotrophic-nitrogen bacteria from the polluted petroleum

作　　者：李方玲 张雅坤 梁立宝 王小通 杜显元 王磊 LI Fangling;ZHANG Yakun;LIANG Libao;WANG Xiaotong;DU Xianyuan;WANG Lei(State Key Laboratory of Agrobiotechnology,Department of Microbiology and Immunology,College of Biological Sciences,China Agricultural University,Beijing 100193,China;HKY Technology Company Limited,Beijing 100076,China;Xinjiang Petroleum Company Baikouquan Oil Production Plant Injection and Transportation Joint Station,Karamay 834000,Xinjiang,China;Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;State Key Laboratory of Petroleum and Petrochemical Pollutants Control and Treatment,Beijing 102206,China)

机构地区：[1]中国农业大学生物学院微生物学与免疫学系,农业生物技术国家重点实验室,北京100193 [2]北京华科仪科技股份有限公司,北京100076 [3]新疆油田公司百口泉采油厂注输联合站,新疆克拉玛依834000 [4]中国科学院微生物研究所,北京100101 [5]石油石化污染物控制与处理国家重点实验室,北京102206

出　　处：《微生物学报》2022年第2期661-671,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金(41977198)。

摘　　要：【背景】石油污染治理中的生物修复因无二次污染、处理成本低等优点受到人们的广泛关注,但由于石油烃向环境中大量输入,导致环境中氮源的相对不足成为制约生物修复效率的关键因素之一,因此筛选能够适应寡氮环境的微生物具有重要的生态意义。【目的】从辽河油田油藏水中筛选在不添加氮源培养基中生长的微生物,为石油污染环境生物修复提供候选菌株。【方法】利用改良无氮培养基分离菌株,根据16S rRNA基因序列同源性鉴定菌株,并对其进行固氮酶活性分析、固氮基因和石油降解基因扩增。【结果】筛选得到21株菌,分属于16个不同的属,其中假单胞菌属所占比例最高(23.8%)。固氮酶活性分析结果表明,8株细菌可检测到乙炔还原活性,从3株菌株中扩增到固氮基因nifH,其余13株细菌被鉴定为寡氮营养菌(oligotrophic-nitrogen bacteria)。对21株菌株的石油降解相关基因进行扩增,发现5株菌株基因组中具有烷烃单加氧酶基因alkB或细胞色素P450基因。【结论】本研究从辽河油田油藏水中分离到的细菌适应寡氮环境,具备降解石油潜能,丰富了石油污染地区的微生物多样性,为生物修复提供菌种基础。Bioremediation in petroleum pollution has attracted wide attention due to its advantages including no secondary pollution and low cost.However,due to the large input of petroleum hydrocarbons into the environment,the relative shortage of nitrogen sources in the environment has become one of the key factors that restricted the efficiency of bioremediation.Therefore,screening microorganisms that can grow in the oligotrophic-nitrogen environments has important ecological significance.[Objective]To screen microorganisms that can grow on nitrogen-free conditions from the Liaohe Oilfield reservoir,and to provide candidate strains for the bioremediation of petroleumcontaminated environments.[Methods]The strains were isolated using modified nitrogen-free medium,followed by the analysis of 16S rRNA gene sequence and nitrogenase activity,and amplication of nitrogen fixation genes and petroleum degradation genes.[Results]21 microorganisms were screened,belonging to 16 different genera,among which Pseudomonas accounted for the highest proportion.The analysis of nitrogenase activity showed that 8 microorganisms could detect acetylene reduction activity,3 microorganisms successfully amplified the nitrogen-fixing gene nifH,and the remaining 13 microorganisms defined them as oligotrophic-nitrogen bacteria.Amplification of petroleum degradation-related genes in these 21 strains revealed that 5 strains have alkB monooxygenase genes or cytochrome P450 genes in their genomes.[Conclusion]The bacteria isolated from the reservoir water of the Liaohe Oilfield in this study can adapt to the oligo-nitrogen environment,coupled with the potential to degrade oil.This study enrichs the microbial diversity in oil-contaminated areas and provide bacterial foundation for microbial remediation.

关 键 词：石油污染 固氮菌 寡氮营养菌 石油降解基因 

分 类 号：X172[环境科学与工程—环境科学]