English abstract
Diminishing light oil resources and minimal change in energy consumption has led explorers to develop the low quality heavy crude oil (HCO) resources that are estimated at seven times that of conventional crude oils through numerous means of enhanced oil recovery (EOR) methods. Microbial EOR (MEOR) methods depend on bacteria and bacterial bioproducts to extract remaining oil volumes at petroleum reservoirs and they are environmentally friendly. In this context, the study aimed to isolate and characterize local microbes that can degrade heavy crude oil and evaluate the effectiveness of the biotransformation process. The study was conducted on bacteria isolated from oil-contaminated soil samples collected from oil sludge pits and heavy crude oil of an Omani oil field.
DNA from the soil samples was extracted from the V3-V4 region of 16S rRNA and sequenced using Illumina MiSeq sequencer in order to perform the biodiversity analyses. Firmicutes and Proteobacteria were the most abundant phyla at the samples. At genus level, Halanaerobium dominated across all the samples followed by Deferribacter and Desulfovermiculus. As expected at such case of HCO contaminated that naturally contains sulfur, sulfate-reducing bacteria (SRB) were abundant. The findings supported the use of contaminated soil with heavy crude oil as a source for bacteria that are able to survive harsh conditions and degrade crude oil for bioremediation and enhanced oil recovery purposes.
Collected soil samples were heated to narrow the bacterial pool and focus the research on spore forming bacteria only. Bacteria were grown in five different minimal salts media and were isolated from the soils samples and the HCO sample. The isolates were identified by MALDI biotyper and 16S rRNA sequencing. The nucleotide sequences were registered in GenBank (NCBI) database (accession numbers: KJ729814 to KJ729828). Microbial growth and HCO biodegradation were assessed by growth at flasks, well-assays on agar plates and GC-FID of extracted crude oil. Among the five selected minimal salts media, the M2 medium was the best medium for growth and biotransformation. Among the isolates, B. licheniformis AS5 and B. subtilis AS2 were the most efficient isolate in biotransformation. Biotransformation of HCO by AS1 to AS10 isolates was evaluated by GC-FID according to mixed n-alkane gas chromatography standard after 30 days of incubation. More than 50% of C24 was degraded and C16, C18 and C26 were degraded by most isolates. A significant increase of C12 and C14 by several isolates indicated HCO biotransformation from heavier to lighter compounds.
The key challenge of experimental in-situ anaerobic biotransformation of HCO is time as a single in-situ flooding experiment could take at least three weeks including incubation of bacteria at the cores. Thus, sand pack column flooding experiments were conducted in order to save time and cost. Regrettably, the sand pack columns did not work effectively due to brine bypassing oil.
Effects of different nitrogen sources, glucose and sodium thiosulfate on growth and biotransformation were investigated. Yeast extract was the most favorable nitrogen source for our bacteria as it resulted in the highest growth level in comparison to urea and ammonium nitrate nitrogen sources. Glucose addition to M2 medium led to increased bacterial growth at aerobic and anaerobic conditions while sodium thiosulfate resulted in reduced growth.
In core flooding experiments, AS5 showed promising results by increasing HCO recovery factor (RF) by 16% after 5 days of incubation. In subsequent flooding experiments, biotransformation RF dropped to 3% due to slowed bacterial growth. By increasing incubation time and inoculation percentages, RF increased to 5%. The addition of 2%m/v (2 g/100 ml) glucose into M2 medium increased RF to 16.4% after 2 weeks of incubation and to 18.3% after 6 weeks of incubation.
Whole genome sequencing analysis and annotation were conducted on AS2 and A$5 and were compared to type strains B. subtilis str. 168 and B. licheniformis DSM 13. On biodegradation, aromatic compounds degradation gene: pcaC and aminobenzoate degradation gene: atoD were found at AS2 but not at type strain B. subtilis str. 168. A$5 and type strain B. licheniformis DSM 13 had similar degradation genes except these of the styrene where AS5 had E3.5.1.4/amiE and catE genes while B. licheniformis DSM 13 had feaB and catE genes. AS2, A$5 and type strains contained many similar degradation genes of 13 other hydrocarbon compounds involving various genes. On biosurfactants, AS2 had surfactin and iturin biosurfactants family genes while B. subtilis str. 168 had surfactin and plipastatin biosurfactants family genes. AS5 and B. licheniformis DSM 13 had similar lichenysin genes. On biopolymers, AS2 and AS5 had the race, ywtB, ywtD and ggt genes; however, did not contain the polygamma-glutamate biosynthesis protein pgsC observed at the type strains.
Bacteria of this study did not produce any bioproducts. Thus, other media were tested which were used in literature to produce metabolites from bacteria similar to this study's isolated bacteria. At the experiments, biosurfactant was produced by AS2 while A$5 did not produce neither biosurfactant nor biopolymer. The lowest observed surface tension was 28.8+0.8 mN/m at MP8 by AS2, AS2 MP8 interfacial tension to hexadecane was 4.240.6 mN/m. The performance of the AS2 biosurfactant was comparable to biosurfactants similar to its kind. The IR spectrum showed similarity to the standard surfactin biosurfactant. The sand pack flooding results showed 4-5% additional recovery. This is a good indication that the biosurfactant could work at core flooding experiments.
