Period Covered by Report: January 1, 2004 through March 31, 2004
EPA Agreement Number: R 827015-01-0
Title: Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
Investigators: L. M. Gieg, M. J. McInerney, and J. M. Suflita
Institution: University of Oklahoma
EPA Project Officer: Bala Krishnan
Project Period: June 1, 2002 through May 31, 2004
Project Amount: $150, 000 + no cost and cost extension
Research Category: Well-Bore Cleanout
Keywords: Petroleum, bioremediation, microbiology
Objective: We aimed to determine the mechanism(s) of action of commercially available, microbial formulations used to treat paraffin deposition in the oil field. Because there are many conflicting reports by producers on the efficacy of microbial treatments to remedy paraffin deposits, it is not known why microbial treatments work under some conditions but not others. Knowledge of the mechanism(s) used by microorganisms to remediate paraffin deposits is a critical first step to understanding how the application of microbial treatments for paraffin removal can be optimized in the oil field. The results of this study will benefit the domestic oil industry because understanding the mechanisms of action of these products will allow the independent producer to determine the conditions under which they are likely to succeed and to determine if and when the purchase of microbial commercial paraffin treatments represents a wise expenditure of investment dollars.
Progress Report/Accomplishments: In the previous reporting period, we described detailed mechanism experiments that we carried out in the laboratory in order to help pinpoint mechanisms by which microbial paraffin treatment might be occurring in Alaska Oil B. Preliminary screening experiments with this oil had shown a significant reduction of the wax appearance temperature (WAT) in microbe-amended incubations relative to sterile controls under aerobic conditions at 60°C. Thus, a more detailed mechanism experiment was begun by setting up incubations with Alaska Oil B at 60°C under 6 different conditions. These included the following amendments: (1) whole formulation; (2) cells only; (3) supernatant only; (4) whole formulation plus chloramphenicol; (5) heat-killed whole formulation; and (6) no microbes added (sterile control). All incubations were done in triplicate under both aerobic and anaerobic conditions. Surface tension measurements and emulsification assays performed over time up to 62 days of incubation showed no significant differences between microbe-amended incubations and microbe-free controls. These results suggested that biosurfactant or bioemulfisifier production was not a predominant mechanism occurring during paraffin treatment of Alaska Oil B at 60°C under either aerobic or anaerobic conditions. Thus, in order to determine whether paraffin biodegradation was a mechanism of microbial treatment, we examined the oil layers in the incubations for evidence of paraffin decomposition. Oils were sub-sampled from the incubations and analyzed by high-temperature gas chromatography (HT-GC) to determine whether decreases in paraffin concentrations were evident or whether the paraffin profile shifted from one of higher molecular weight to lower molecular weight alkanes. Prior to HT-GC analysis, oil samples were amended with C24D50 as an internal standard for quantification. To this end, we compared the resulting total paraffin GC peak areas to internal standard GC peak area ratio in oils removed from sterile control versus whole formulation incubations under aerobic and anaerobic conditions. We found no significant differences in the loss of paraffin components in the microbe-amended treatments incubated under anaerobic conditions relative to sterile controls, but did find significant losses of paraffins in those microbe-amended bottles incubated under aerobic conditions. These seemingly positive results were shown in the previous report (Quarterly Report #6). However, when we repeated the analysis of the whole formulation and sterile control samples along with the analysis of the other incubations, we found no significant differences in any of the incubations. Table 1 shows the oil to internal standard peak area ratio of the paraffins quantified (C10 to C40) for aerobic incubations. Using this method of quantification, the oil-to-internal standard peak area ratio should decrease if a reduction in the paraffins occurred. As can be seen in Table 1, the oil-to-internal standard peak area ratios were remarkably similar in all of the detailed mechanism incubations as described above.
Based on these results, it appeared that the incubations of the microbial formulation did not have a biological effect (biosurfactant production, bioemulsification, or biodegradation) on paraffin reduction in controlled laboratory studies. However, in all previous experiments, a minimal salts medium was used for incubations of the microbial formulations and oil was supplied as the sole carbon and energy source. In field application of the microbial formulations, a nutrient solution is often added to help stimulate the in situ activity of the microbes (personal communication, proprietary formulation provider). Thus, during this reporting period, we set up another series of experiments in order to determine the effects of adding a commercial nutrient solution to the incubations. To this end, test bottles containing microbial formulations plus nutrients were incubated alongside those containing only the microbial formulations were established under aerobic and anaerobic conditions and incubated at 60°C. Appropriate sterile controls were also established. After a lengthy incubation (e.g., at least one month), supernatants will be tested for biosurfactant-producing or bioemulsification activity and the oil layers will be analyzed for paraffin losses. Further, we plan on conducting cold-finger tests in order to determine whether the microbial cells added to treat paraffinic reservoirs are functioning in a more physical capacity, such as minimizing deposition, rather than in a more expected biological capacity.