Development of evaluation method for spilled-oil bioremediation using functional genes involved in oil degradation pathway

生物修复已被部分用作清除某些沿海和土壤环境中溢油的技术选择。在几乎所有情况下，生物刺激，其中土著污染物分解微生物富集和/或激活的外源性供应的营养素已被应用，而不是生物强化。溢油生物修复的实质是外源性地向内源性烃类降解微生物提供大量营养物质。然而，大量营养素外源供给的有效性仍存在争议。近年来，DGGE和16 SrDNA克隆文库分析等方法被广泛应用于石油生物修复过程中关键降解菌的行为和出现情况的研究。不幸的是，这些工具给我们的是定性信息，而不是定量信息。因此，本研究构建了石油降解菌特异性检测的PCR引物，并利用实时荧光PCR技术对石油降解菌在生物刺激过程中的转变进行了追踪，成功构建了石油降解途径中烷烃羟化酶、邻苯二酚1，2双加氧酶和邻苯二酚2的代表基因alkB、C120和C230的PCR引物，3双加氧酶。现场试验结果表明，具有alkB和C120的细菌在石油降解过程中占优势。这3对引物对土壤样品也是有效的。这些结果表明，我们的引物组合是可行的，用于评价生物修复的有效性和安全性。

Bioremediation has been partially employed as a technical option to remove spilled oil at some coastal and soil environments. In almost of all cases, biostimulation in which indigenous pollutants-decomposing microorganisms are enriched and/or activated by the exogenous supply of nutrients has been applied instead of bioaugmentation. The essence of spilled oil bioremediation is exogenous supply of macronutrients to indigenous hydrocarbon-degrading microorganisms. However, the efficacy to exogenously supply of macronutrients remains to be controversial. Recently some methods like DGGE and clone library analysis of 16S rDNA are widely used to chase the behaviors and occurrences of the key hydrocarbon-degrading bacteria in evaluating oil bioremediation processes. Unfortunately, these tools give us qualitative information, not quantitative one. Therefore, in this research, we constructed PCR primer sets to detect oil-degrading bacteria specifically, and traced the transition of oil-degrading bacteria in biostimulation process by using real-time PCR.Consequently, we succeeded to construct the PCR primer sets to detect representative genes in oil-degrading pathway, alkB, C120, and C230, encoding alkane hydroxylase, catechol 1,2 dioxygenase, and catechol 2,3 dioxygenase, respectively. In the field experiments, it was shown that bacteria possessing alkB and C120 were dominant in oil degradation process. These 3 primer sets were also effective for soil samples. These results suggest that our primer sets are feasible for the use in evaluating effectiveness and safety of bioremediation.