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.