Heavy Crude Oil Biodegradation: Catechol Dioxygenase Gene Copy Number Variation Determination by Droplet Digital Polymerase Chain Reaction

The crude oil reserves in Oman mainly consist of heavy oil. Microbial enhanced heavy oil recovery (MEOR) has been proved to be an efficient technique in the tertiary heavy oil recovery. Five <i>Bacillus</i> species potential for enhanced heavy oil recovery (EHOR) were isolated and the biodegradation ability of these isolates was studied. As heavy crude oil comprises of aromatic hydrocarbons rather than aliphatic ones, the aromatic catabolism gene, catechol 2,3-dioxygenase (<i>C23O</i>) and catechol 1,2-dioxygenase (<i>C12O</i>) were the genes of interest in this study along with the reference gene, <i>16S</i> rDNA. The copy number variation of these genes was determined using droplet digital PCR (ddPCR). The primers and probes for ddPCR assay were designed targeting these genes. It was observed that the heavy crude oil biodegradation potential of the isolates correlated with the copy number of <i>C23O</i> gene in the microbial genomes. The isolate, <i>Paenibacillus ehimensis</i> BS1 had the highest <i>C23O</i> gene copy number (1.057) followed by <i>Bacillus firmus</i> BG4 (0.895) and <i>Bacillus halodurans</i> BG5 (0.031) as demonstrated by their biodegradation potential. This is one of the few studies deploying ddPCR in the field of heavy crude oil biodegradation by spore forming bacteria.

阿曼的原油储量以重质油为主。微生物强化重质油采收（Microbial enhanced heavy oil recovery, MEOR）已被证实是第三代重质油开采的高效技术。本研究分离得到5株具备强化重质油采收（enhanced heavy oil recovery, EHOR）潜力的芽孢杆菌属（Bacillus）菌株，并对这些菌株的生物降解能力展开研究。由于重质原油主要由芳香烃而非脂肪烃构成，因此本研究选取芳香烃分解代谢相关基因——儿茶酚2,3-双加氧酶（catechol 2,3-dioxygenase, C23O）与儿茶酚1,2-双加氧酶（catechol 1,2-dioxygenase, C12O），以及参考基因16S核糖体DNA（16S rDNA）作为目标基因。本研究采用液滴数字PCR（droplet digital PCR, ddPCR）技术测定上述基因的拷贝数变异，并针对这些基因设计了ddPCR检测所用的引物与探针。研究发现，受试菌株的重质原油生物降解能力与其基因组中C23O基因的拷贝数呈显著相关。其中，伊蒙类芽孢杆菌（Paenibacillus ehimensis）BS1的C23O基因拷贝数最高（1.057），其次是坚强芽孢杆菌（Bacillus firmus）BG4（0.895）与嗜盐芽孢杆菌（Bacillus halodurans）BG5（0.031），这一结果与各菌株的生物降解能力表现一致。本研究是为数不多将ddPCR技术应用于产芽孢细菌降解重质原油领域的研究之一。