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一些沿海和土壤环境中溢出的石油的技术选择。在几乎所有的情况下，使用的是生物刺激，即分解本地污染物的微生物通过外源营养物质的供应而得到丰富和/或激活，而不是生物强化。溢油生物修复的实质是向本土碳氢化合物降解菌提供外源常量营养素。然而，外源性供应大量营养素的效果仍然存在争议。近年来，DGGE和16S rDNA克隆文库分析等方法被广泛用于追踪石油生物修复过程中关键降解菌的行为和出现情况。不幸的是，这些工具给了我们定性的信息，而不是定量的信息。因此，在本研究中，我们构建了特异检测石油降解菌的聚合酶链式反应引物组，并利用实时荧光定量聚合酶链式反应跟踪石油降解菌在生物刺激过程中的变迁，从而成功地构建了检测石油降解途径中具有代表性的基因：烷烃羟基酶、儿茶酚1，2双加氧酶和儿茶酚2，3双加氧酶基因的聚合酶链式反应引物组。现场实验表明，在原油降解过程中，产AlB和C120的细菌占主导地位。这3套引物对土壤样品同样有效。这些结果表明，我们的引物组用于评估生物修复的有效性和安全性是可行的。

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.