Period Covered by Report: June 1, 2002 through May 31, 2003
Date of Report: July 31, 2003
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, 2003
Project Amount: $150, 000
Research Category: Well-bore Cleanout
Keywords: Petroleum, bioremediation, microbiology
Objective: We aim 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 paraffintreatments represents a wise expenditure of investment dollars.
Progress Report/Accomplishments: We have completed the preliminary screening experiments for two crude oils (Alaska Oils A & B) selected by ConocoPhillips for this research project. These tests entailed incubating an oil with artificial brine medium in the absence (controls) or presence (tests) of a proprietary microbial product chosen specifically to treat the given oil. Tests were conducted at room temperature (~24°C) and/or at 60°C either in the presence or absence of oxygen. All incubations were in triplicate. Alaska Oil A was incubated for 3 days, whereas Alaska Oil B was incubated for 7 days (see below). After incubation with slow, end-over-end mixing, oil layers were removed to conduct the wax appearance temperature (WAT) test. Using cross-polarized microscopy, this test measures the temperature at which paraffin crystals begin to form when a given oil is cooled under controlled conditions. For the purposes of this research, microbial paraffin treatments which lowered the WAT by a minimum of 5% over that of the parallel microbial-free controls were considered successful for the preliminary screening assays. A lowering of the WAT values suggests that troublesome long-chain hydrocarbons have been "treated" by the microbial formulations.
For the preliminary screening of Alaska Oil A, all bottles were incubated for 3 days. Of the WAT tests conducted on Alaska Oil A, no bottles incubated under anaerobic conditions showed a reduction in the WAT. In contrast, one of the three replicates incubated aerobically at 60°C showed an 8% reduction in the WAT relative to a microbial-free control. However, conducting ANOVA analysis of all of the WAT data from Alaska Oil A preliminary experiments showed that there was no significant difference in the WAT values for any microbe-amended bottle incubated under any condition relative to the microbe-free (sterile) controls. These results suggested that the proprietary microbial product selected to treat this oil was ineffective, at least over the 3-day incubation period. This outcome was somewhat surprising since previous data had shown that this oil was amenable to microbial treatment (ConocoPhillips, unpublished data). Further, emulsions were observed in many of the microbe-amended bottles but not in the sterile controls, suggesting microbial action on the oil. Three factors were postulated to have had an effect on the unsuccessful results: (1) cultures were incubated for too short of a time, (2) aerobic incubations became oxygen-limited, and (3) WAT measurements for control incubations varied by as much as 10% which made it difficult to determine a 5% difference from microbe-amended incubations.
Thus, to address the first two points for the preliminary experiments with Alaska oil B, controls and cultures were incubated for 7 days instead of 3 days, and aerobic incubations were amended daily with sterile air to prevent oxygen deficiency. Further, WATs were conducted on controls at the onset of the analyses to evaluate the variability of oil samples removed from aqueous incubations. It was postulated that the wide differences in the WAT values observed in the control samples from Alaska Oil A may have been due to interferences from tiny emulsions with water droplets. For the controls in Alaska Oil B, at least duplicate measurements were made on the same drop of oil. The WAT values obtained from the controls incubated at 25°C (vary by ~28%) was much wider than that of the controls incubated at 60°C (vary by ~5%). Wax experts at ConocoPhillips suggested that the variability at 25°C is due to a phenomenon known as cold seeding in which paraffins drop out of the oil and move into an emulsion layer when the oil is below its cloud point resulting in irreproducible oil samples. Thus, we concluded that the WAT test can not accurately be performed on oils from room temperature incubations. Accordingly, incubations containing the microbial formulations were only established at 60°C. ANOVA tests performed on the WAT values measured at the end of the 7-day incubation period showed that there was a significant difference in the WAT value at 60°C under aerobic conditions relative to controls. We calculated a 7.2% reduction in the WAT value relative to controls. In contrast, under anaerobic conditions at 60°C, there were no statistically significant changes in the WAT values of the microbe-amended incubations relative to those of the controls (3.3% reduction in the WAT). The positive results observed at 60°C with Alaska Oil B has led us to establish Stage II experiments which will help pinpoint the mechanism of action of the microbial formulation which caused the significant reduction in the WAT values. These experiments are currently incubating. In this phase of the project, analytical methods for examining oils have been developed and will be used to determine whether changes in the long- chain hydrocarbons are evident as a result of microbial treatment at the end of Stage II experiments. High-temperature gas chromatography methods (e.g.simulated distillation) have been developed to analyze standard mixtures of long-chain hydrocarbons and paraffin-rich oils. Our analytical studies have shown that the proportion of the higher molecular waxes are small relative to the other hydrocarbons in the oil, and it will be difficult to quantitate their loss. Thus, we will have to perform a wax extraction procedure in order to concentrate these waxes for quantitative analysis. This procedure is currently being worked up and will follow the method of Thanh et al. (1999, Org. Geochem. 30: 119-132.). Stage II experiments will also involve surface tension measurements to determine whether biosurfactant production is a mechanism used by the microbes in the formulation to help treat or reduce paraffin accumulation. This measurement uses a DuNuoy ring detachment apparatus which has been calibrated using water and isopropanol as high- and low-end standards, respectively. Further, an emulsification assay (Trebbau and McInerney, 1996, J. Indust. Microbiol. 16: 1-7) will be carried out to determine whether the microbes in the formulation are acting to treat paraffin by secreting emulsifying agents.
Publications/Presentations: Not applicable at this time.
Future activites: At the end of a long incubation period (40 to 60 days), Stage II experiments with Alaska Oil B will be assayed for biosurfactant- or emulsifier-producing activities, and for changes in the hydrocarbon profiles relative to sterile controls. Further, supernatants of the incubations will also be analyzed for the presence of known hydrocarbon biodegradation products to in order to determine whether this is a mechanism used by microbes to treat paraffin-rich oil. We are also hoping to identify a third oil on which to carry out microbial paraffin-treatment studies.
Supplemental Keywords: paraffin, microbial treatment
Relevant Web Sites: None established for this project.