English abstract
The increasing energy demands due to global population growth, the difficulty in discovering new oilfields and the maturity of existing oil fields demand for alternative technologies since fossil fuels are the main source of energy. Oman is continuously applying efforts to increase oil recovery. The residual oil that is left behind in the reservoir after primary and secondary recovery is the target for Enhanced Oil Recovery (EOR). The conventional tertiary oil recovery methods include chemical flooding, miscible CO2 injection and thermally enhanced oil recovery. Microbial Enhanced Oil Recovery (MEOR) is a low cost, environment friendly tertiary technique. The exploration, production and transport of crude oil lead to oil spills, the disposal of which is expensive. The conventional methods of oil spill clean-up include land filling, incineration, natural remediation and chemical method. Bioremediation is an environmental friendly acceptable method of elimination of crude oil pollution since most of the hydrocarbons present in the crude oil are biodegradable.
Inhabitant spore-forming bacteria that can utilize crude heavy oil were isolated and screened for their potential for heavy oil bio-fractionation. The crude oil biodegradation potential was initially assessed by their growth characteristics in Bushnell-Haas (BH) medium containing crude heavy oil (API° 4.57) as the sole carbon source. The five isolates, Paenibacillus ehimensis, Bacillus firmus, Bacillus halodurans, Bacillus subtilis and Bacillus licheniformis which showed maximum growth were selected for the study and their crude heavy oil tolerance (upto 7%) was determined. Gas-chromatography Mass Spectroscopy (GC-MS) analysis of the biofractionated heavy crude oil acted upon by the isolates in BH medium for a period of 9 days further proved the efficacy of the isolates. The GC-MS analysis of bio-transformed heavy crude oil by P. ehimensis showed 67.12% biotransformation of total crude heavy oil with 85.3% reduction in aromatic fractions and the aliphatic fractions to 45.9% reduction. The biotransformation studies using GC-MS showed 81.36% biotransformation of heavy crude oil for B. firmus and 81.93% for B. halodurans compared to the abiogenic control. B. subtilis and B. licheniformis biodegraded crude heavy oil, utilizing both aliphatic compounds and aromatic compounds in the crude heavy oil and have proved to be promising candidates for bioremediation.
An attempt to characterize the genes responsible for the biofractionation was done. Among the 20 sets of primers used, the sequencing of the bands from two sets of primers, C230 (product size: 700bp) and Acci20 (product size: 700bp) showed the results as heme ABC transporter gene, which is the flanking region of catechol dioxygenase gene and cation.proton antiporter gene, which was reported for alkaline pH homeostasis. The QX100 ddPCR analyses revelaed that the copy number variation of these genes in the 5 isolates were as follows: the catechol dioxygenase gene copy number is highest in P. ehimensis, followed by B. firmus and B. halodurans; and the copy of cation:proton antiporter gene is the least in P. ehimensis. These findings suggest that the isolates use different mechanisms or variant of the genes to maintain the pH of cells. For the microbial growth, activity and survival, metals and minerals play a major role. The utilization of minerals by the isolates in BH medium containing crude heavy oil during the study showed that the most utilized element being Mg, followed by Fe. The source of soil from where the isolates contained Fe, Al, Si as major elements whereas Mg was seen only among 3 or 4 soil samples. The most utilized element by the isolates was supplemented through BH medium. Some of the elements like Ca, Be, B, Al, Mn, Cu, Zn and Sr were found to be utilized by the isolates in the medium, which were not supplemented, suggesting their contribution by the crude oil in the medium.
Core-flooding experiment using sandstone cores simulating the oil field conditions resulted in 10-13% extra recovery by P. ehimensis; 9.5-10.5% and 7-8% for B. firmus and B. halodurans after the 10 days shut in period. The detailed microscopic study of the sections of the core plug used in the study showed microbial growth inside the cores. GC-MS analysis of the extra recovered oil resulted in higher percentage of lighter fractions suggesting the increase in the flow of the crude oil. No biosurfactant production was observed during the study period. The metagenomics analysis of the soil samples indicated the abundance of Bacillus sp., followed by Paenibacillus sp. The extreme temperature conditions along with low moisture content (~0.3 m2/m?) and a slightly alkaline pH might be the reason for the abundance of Bacillus sp. These findings suggest that P. ehimensis; B. firmus and B. halodurans can be used as potential candidates for MEOR and B. subtilis and B. licheniformis for bioremediation. The choice of isolates that are abundantly found in the environment will increase the applicability in diverse geographical areas.
