BACTERIAL COMMUNITY STRUCTURE STUDY OF OIL WELL THROUGH SEQUENTIAL CULTIVATION AND ITS POTENTIAL IN IN-SITU MEOR WITH METAGENOMICS APPROACH

• Main Author: Fauzan Muttaqin S, Muhammad
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/62718
• Institution: Institut Teknologi Bandung

MEOR is a tertiary recovery technique that utilizes microbial activity that can help mobilize petroleum in an oil well. In-situ MEOR can be done by adding nutrients to the oil wells to stimulate the growth of hydrocarbonoclastic indigenous bacteria, especially those that degrade heavy fractions of crude oil. These nutrients can be formulated properly if the bacterial community in the well can be well-identified. Each well has a different diversity of bacteria, but in general, the light fraction degrading bacteria will have high activity at the beginning of the nutrient injection, followed by the activity of the heavy hydrocarbon-degrading bacteria. In this study, a culture-independent study of the bacterial community structure in oil wells was conducted and their potential, particularly in degrading heavy oil fractions, in a culture-independent manner. The initial bacterial community sample came from brine which was concentrated by centrifugation. Then DNA extraction from the bacterial isolates was carried out for further targeted sequencing with Illumina miseq targeting the V3-V4 region of 16S rRNA gene. Subsequently, cultivation was carried out 2 times in stages, in batches with the composition of the medium in the form of sterile brine and 0.1% yeast extract. Into the medium, 2% oil mixed with non-sterile brine was added as a source of inoculum and the main substrate. Cultivation was carried out in two stages, with each incubation for 14 days at 150 rpm at a temperature of 50°C. The bacteria that grew and the degraded oil in the first stage of cultivation were used as the source of inoculum and the main substrate in the second stage. At the end of stages I and II, 40 ml of medium was taken for DNA extraction and sequencing using the same method. Tests of the constituent fractions of the oil were also carried out using the silica column chromatography method. Metagenomic results showed that the bacterial community was dominated by the genus Tepidiphilus (49.76%), Lysinibacillus (5.78%), Paracoccus (3.57%), and Empedobacter (3.38%). Along with the gradual cultivation, there was a change in the structure of the bacterial community, which at the end of the first stage was dominated by the genus Bhargavaea (76.78%), Tepidiphilus (19.56%), and Bacillus (2.46%). It was also observed that there was a significant decrease in the percentage of the saturated fraction (35%), as well as the resin fraction (0.56%), and asphalt (0.58%). This is supported by the identification of gene markers that indicate the dominance of bacteria that have alkB and ladA genes which are important genes in the degradation of alkanes and long-chain alkanes at the beginning of stage I cultivation. At the end of stage II of cultivation, dominance was still found by the same genus, namely Bhargavaea (92.97%), Tepidiphilus (6.06%), and Bacillus (0.68%). The highest decrease occurred in the more complex fraction, namely the aromatic fraction (7.03%), followed by the asphalt fraction (3.72%). This is thought to be caused by the dominance of bacteria that have the dmpL and hpaD genes which are markers of the aromatic fraction degradation genes at the beginning of stage II cultivation. These results indicate that the indigenous bacteria in the oil well X Jatibarang proved to have the ability to degrade petroleum fractions, especially the heavy fraction needed in the application of in-situ MEOR. For this reason, in the future, further studies can be carried out regarding the optimization of the right nutritional formula to support the identified hydrocarbonoclastic indigenous bacteria, to be able to support the growth of these bacteria during in-situ MEOR, in order to obtain a higher heavyweight fraction degradation activity in its application in the field.