Microbial degradation of recalcitrant alkanes under anaerobic conditions results in the accumulation of heavy oil fraction in oil reservoirs. Hydroxylation of alkanes is an important activation mechanism under anaerobic conditions, but the diversity and distribution of the responsible microorganisms in the subsurface environment are still unclear. The lack of functional gene polymerase chain reaction (PCR) primers and commercially available intermediate degradation chemical compounds are the major obstacles for this research. In this investigation, PCR primers for the ahyA gene (encoding alkane hydroxylase) were designed, evaluated, and improved based on the nucleotide sequences available. Using microbial genomic DNA extracted from oil-contaminated soil and production water samples of oil reservoirs, ahyA gene nucleotide sequences were amplified and retrieved successfully from production water sample Z3-25 of Shengli oilfield. Additionally, the signature biomarker of 2-acetylalkanoic acid was detected in both Shengli and Jiangsu oilfields. These results demonstrate that anaerobic hydroxylation is an active mechanism used by microorganisms to degrade alkanes in oxygen-depleted oil reservoirs. This finding expands the current knowledge of biochemical reactions about alkane degradation in subsurface ecosystems. In addition, the PCR primers designed and tested in this study serve as an effective molecular tool for detecting the microorganisms responsible for anaerobic hydroxylation of alkanes in this and other ecosystems.

在厌氧条件下难降解烷烃的微生物降解导致油层中重油馏分的积累。烷烃的羟基化是厌氧条件下的重要激活机制，但地下环境中负责任微生物的多样性和分布仍不清楚。缺乏功能基因聚合酶链反应（PCR）引物和可商购的中间降解化合物是该研究的主要障碍。在这项研究中，基于可用的核苷酸序列，设计，评估和改进了ahyA基因（编码烷烃羟化酶）的PCR引物。利用从受油污染的土壤和储油层采出水样品中提取的微生物基因组DNA，从胜利油田采出水样品Z3-25中成功扩增并检索了ahyA基因核苷酸序列。此外，在胜利油田和江苏油田均检测到2-乙酰基链烷酸的标志性生物标志物。这些结果表明厌氧羟基化是微生物用来降解贫氧油藏中烷烃的一种活性机制。这一发现扩展了有关地下生态系统中烷烃降解的生化反应的当前知识。此外，在本研究中设计和测试的PCR引物可作为一种有效的分子工具，用于检测导致该生态系统和其他生态系统中烷烃厌氧羟基化的微生物。