The goal is to investigate the production of linear alcohols and other intermediates using novel bacteria that transform long-chain alkanes without degrading aliphatic hydrocarbons of C8 or less.

Lawrence Berkeley National Laboratory
Berkeley, CA

The U.S. petroleum industry is increasingly dependent on heavy crude oil to meet domestic demand for gasoline and distillate fuels. Biocatalytic viscosity reduction (bioprocessing) uses bacteria to partially transform less-valuable crude oil components to surface-active compounds (alcohols and carboxylic acids) that reduce crude oil viscosity. Bioprocessing could be used to lowering heavy oil viscosity before introduction into a pipeline, thereby reduce pumping costs.

The goal of this project is to reduce viscosity in heavy crude oil. This will reduce the difficulty, and therefore the cost, of both transporting and processing heavy crude oil. This is important because much of U.S. oil production is heavy crude oil. Since heavy crude is difficult to transport and process, it sells at a discount to light, sweet crudes. Reducing viscosity could help increase profitability and production of this National resource.

Project Summary
Bacteria were isolated from contaminated environments and evaluated for their biocatalytic potential. A series of phylogenic, physiologic, and genetic assays were developed and applied to the strains and reference cultures from other laboratories. Biocatalysts showing novel or unique activity or genetic profiles were further evaluated. Using this approach, a "menu" of biocatalytic agents was developed for chemical processing and environmental applications.

A biocatalyst was identified that has a higher specificity for low-value alkanes than the higher-value light alkanes. Kinetic modeling of reaction processes using this biocatalyst indicates that the proper selection of catalyst will overcome a major challenge to crude oil bioprocessing's unintentional degradation of gasoline-range alkanes.

The project team selected 32 biocatalytic agents for evaluation. Initial evaluation identified specific strains as promising agents for the processing of crude oils and alkane mixtures. The majority of the selected strains have a broad substrate specificity encompassing C6-C16 alkanes. Some strains oxidize only alkanes of C8 and above.

The research has focused on completing evaluations of 11 strains selected as priorities from the complete culture collection. Each of these strains has been screened for fatty acid methyl ester (FAME) profile. Genomic DNA has been isolated for 16-DNA analysis. These strains have been tested for homology with known alkB gene sequences, and the results of the genetic analysis have been correlated with physiological tests measuring alkane oxidation profiles. Kinetic evaluation of these strains is in progress.

The results of the researchers' evaluation have demonstrated that the majority of the bacteria in their collection have homology with known alkB gene sequences. This sequence is correlated with the ability to transform low-molecular-weight (C8 and less) alkanes. Two strains that oxidize only select longer-chain alkanes do not show homology with alkB genes by project protocols. The lack of homology with alkB suggests that these strains may contain novel enzymes. One strain has a high specificity in target substrates and is considered of high potential value as a biocatalytic agent. Invention disclosure forms for this strain are being prepared.

Project Results
This research targeted the terminal oxidation of alkanes to alcohols as a specific mechanism for development. Biological conversion of alkanes to alcohols was carried out by alkane monooxygenase (AlkMO), an enzyme found in bacteria able to grow on alkanes. The most well-understood AlkMO is a diiron enzyme, AlkB, coded by the alkB gene.

For biocatalytic viscosity reduction to be economic, the process will need to incorporate an "open," non-sterile bioreactor design and use whole-cell biocatalysts. This precludes the use of genetically engineered organisms and requires that the process be controlled to maintain desirable biocatalytic agents and favorable kinetics in the presence of competing bacteria. Additionally, biocatalytic agents must target longer-chain alkanes (C8 and above) without oxidizing gasoline range alkanes of C7 or less.

Alkane-oxidizing bacteria are promising biocatalysts for crude oil bioprocessing. Five bacteria were developed with NGOTP funding that might be useful as biocatalysts, and two of them may harbor novel enzyme systems for alkane transformation.

This project is completed.

$732,000

$300,000 (29% of total)

NETL -Kathleen Stirling (kathy.stirling@netl.doe.gov or 918-699-2008)
LBNL - William Stringfellow (wstringfellow@lbl.gov or 510/486-7903)