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

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。

关键词:石油污染;固氮菌;寡氮营养菌;石油降解基因

Abstract:

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 petroleum- contaminated 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.

Key words:petroleum pollution;nitrogen-fixing bacteria;oligotrophic-nitrogen bacteria;petroleum degrading genes