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Procedures. A cell concentration of 12 to 50 g wet cell paste (WCP) per liter of total liquid
volume contained in the reactor was utilized. Frozen cells were first added to sodium phosphate
buffer (pH 7.5 at 0.156 mM) with 3% glucose. The cell slurry was placed in the reactor, agitated
at 1000 rpm and sparged with 0.2 vvm air. The oil was added in the ratio of 1 part oil to 3 parts
buffer. Shake flasks were taken down at specified times and BSRs were sampled at regular
intervals lo monitor sulfur concentrations. Samples were centrifuged at 39,000 x g for IO minutes
in order to separate the mixture of oil, water and cells. To ensure that any observed change was
strictly cell-dependent, parallel experiments were performed without cells.
18
Analytical Methods. An HP 6890 gas chromatograph with electronic pressure control and
detection by flame ionization detector (FID)and a Sieves model 350 flameless SCD was employed
for crude oil analysis. The column was a Restek RTX-5, 15 meter. 0.25 mm ID with a 0.25 pm
film thickness. The injection port was held at 340C. The oven temperature program bcgan at
50C and was held for 2 minutes. The temperature was then increased by 15C/minute lo 320C
and was held for IO minutes. A typical SCD chromatogram consists of a group of resolved peaks
above a broad envelope of sulfur compounds (Figure I).
A GCMS SIM method for the quantitation of Cx-DBTs and Cx-benzonaphthothiophenes (CxBNTs) in crude oil was performed using a HP 5890 Series I1 plus gas chromatograph with
electronic pressure control and mass spectrometric detection performed with an HP 5972 MSD.
The column was a Restek RTX-1, 30 meter, 0.25 mm ID with a 0.5 pm film thickness. The
injection pori was held at 290C. The oven temperature program began at I00C. increased at a
rate of 4Clmin to 3 15OC and held for 20 minutes.
Total sulfur quantitation of crude oils was performed with either a Horiba SLFA-1800H x-ray
fluorescence analyzer or a Leco SC-444 Sulfur and Carbon Combustion Analyzer with infrared
I
detection.
Sulfur XANES analyses were obtained at beam-line X-19A of the National Synchrotron Light
source (NSLS). Brookhaven National Laboratory. Work was performed under contract with the
University of Kentucky.
19
Removal of nitrogen is also being investigated as an additional approach to fuel upgrading. This
work is being performed at the University of Houston [6] and has been subsidized by EBC. Work
has focused on the carbazole-degrading Pseudomonad LD2. Approaches in progress include
isolation and characterization of the carbazole degradation enzymes, as well as the characterization
and cloning of the genes encoding these enzymes.
The goal is to put all these catalytic activities together either in a single biocatalyst or a consortia to
upgrade the petroleum by removing the sulfur, nitrogen. metals and reducing the viscosity in a
single process step.
Process Development
Shake flask and BSR experiments were performed to address process concerns, such as reaction
characteristics, separation characteristics, and catalyst stability and effectiveness. The effect of
these parameters were determined in a series of assays designed to compare initial rates of
desulfurization under a variety of process conditions. The assay results determined the optimum
process conditions for BDS. The key parameters evaluated were water to oil ratio (WOR), catalyst
to oil ratio (COR), mixing effects, oxygen demand, and temperature and pH optimum.
A process concept for the biodesulfurization of crude oil was developed based on the knowledge
generated. a bench scale unit was constructed and proof-ofconcept experiments were performed to
develop a design basis specifying performance criteria, unit operations and process parameters for
the biodesulfurization of crude oil. As part of the process flow diagram (PFD) development, a
general description of the expected site conditions and product stream attributes was compiled. In
addition, the process assumptions and equipment issues were delineated that were crucial to the
design.
At this timc, we envision a simple system capable of running with minimum ooeratnr ;?!cn.tc:icr,
in the oilfield. The base case scenario for a field ycre:: i; u Laich reaction utilizing a pump and
inductor for mixicg xi icration. separations will be performed with standard oilfield equipment,
with desulfurized oil returning to storage and process water reinjected into a disposal well in the
field. The spent catalyst will be inactivated and landfilled. The stored product oil would be tested
for sulfur and other oil quality specifications prior to transport. Based on this process concept, the
identified process parameters and assumptions, the attached PFD (Figure 5) was developed.
CONCLUSIONS
Significant progress has been made toward the commercialization of crude oil biodesulfurization.
This progress includes the characterization of crude oil candidates for the BDS process; improved
biocatalyst performance that directly relates lo crude oil biodesulfurization; development of
analytical methodology, which led to breakthroughs in the characterization of DRM; development
of a process concept for crude oil BDS; and construction and testing of a prototype bench unit.
Technical hurdles still need to be overcome to achieve commercialization. The major obstacles to
the economical biodesulfurization of crude oil include catalyst specificity and rate. Work continues
to modify the catalyst to increase its effectiveness and to screen other organisms for additional
desulfurization capabilities. In addition, mass transfer and separations hurdles must be overcome
in crude oils with increased oil viscosity and density.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the National Institute of Standards and Technology (NIST)
for support received through an Advanced Technology Program (ATP) grant for crude oil BDS.
Phil Gibbs and Richard Willson of the University of Houston for their work on denitrogenation.
Texaco Exploration and Production Technology for providing the crude oils and the many
dedicated people at Energy BioSystems Corporation.
REFERENCES
[I] Kilbane, J. J. and K. Jackowski. 1992. Biodesulfurization of water-soluble coal derivcd
material by Rhodococcus eryflrropolis IGTSB. Biotechnol. Bioeng. 4 0 1107-1 1 14.
[3] Gray, Kevin A,, 0. Pogrebinsky, G. Mrachko, L. Xi, D. J. Monticello and C. Squires. 1996.
Molecular mechnanisms of biocatalytic desulfurization of fossil fuels. Nature Biotechnology.
14:1705- 1709.
[4] Monticello, D. J. and W. M. Haney. 1996. Biocatalytic process for reduction of petroleum
viscosity. U. S. Patent #5.529,930.
20
[SI XU. G., K. Mitchell and D. J. Monticello. 1997. Process for demetalizing a fossil fuel. U.S.
Patent #5.624,844.
[61 Gibbs. P. R., R. R. Riddle, M. J. Benedik and R. C. Willson. Biochemistry of carbazole
degradation. ACS Biotechnology Secretariat Symposium on Environmentally Benign Synthesis
and Biocatalysis in Remediation. Boston, MA, USA, August 23-27, 1998.
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