Evaluation of Commercial, Microbial-Based Products to Treat Paraffin
Deposition in Tank Bottoms and Oil Production Equipment
Period Covered by Report: October 1, 2003 through December 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, 2004
Project Amount: $150, 000 + 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
began detailed mechanism experiments designed to pinpoint mechanisms by
which microbial paraffin treatment might be occurring. Preliminary
screening experiments with Alaska Oil B 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 both the oil and aqueous 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. Also, aqueous layers
of the incubations were acidified, extracted with organic solvent, and
analyzed following silylation by gas chromatography-mass spectrometry (GC-
MS) to determine whether any known microbial hydrocarbon metabolites were
present. Prior to HT-GC analysis, oil samples were amended with C24D50 as
an internal standard for quantification. Table 1 shows 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:
Table 1.
|Incubation |Oil TO Internal Standard RATIO |
| |+ oxygen |- oxygen |
|Sterile control 1 |32.6 |50.8 |
|Sterile control 2 |32.4 |48.1 |
|Sterile control 3 |35.9 |46.5 |
| | | |
|Whole formulation 1 |21.1 |47.9 |
|Whole formulation 2 |23.1 |36.7 |
|Whole formulation 3 |24.0 |45.0 |
Using this method of quantification, the oil to internal standard peak area
ratio should decrease if a reduction in the paraffins occurred. Indeed, we
observed an approximately one-third decrease in this ratio in those
incubations containing the whole microbial formulation versus the sterile
controls under aerobic conditions. In contrast, such a decrease was not
observed in the absence of oxygen (Table 1). Thus, total loss of
hydrocarbons was apparent when microbial formulations were incubated with
Alaska Oil B under aerobic conditions, which may be occurring by
biodegradation. Oil analyses are ongoing in the other incubations described
above to verify this observation. It is also possible that paraffin losses
are occurring by another mechanism, such as sorption to cells, but this is
yet unclear and will be tested in future experiments. Metabolite analyses
are also ongoing. To date, we have not been able to detect predicted
alkane metabolites, such as long-chain alkanoic acids or alcohols, but have
detected some alkanedioic acids. Future activities will include ongoing
oil and metabolite analysis. It should be noted that all experiments we
have conducted to date have used a minimal brine medium, but field success
in using microbial formulations to treat paraffins has often required the
addition of extra nutrients in the treatment train. Thus, future studies
will also examine the effect of adding nutrients to enhance the ability of
microbial formulations to eradicate paraffins. The remaining work for this
project will continue to focus on Alaska Oil B, since the most positive
results have been observed with this oil.