Team:Lethbridge/projectbackground

Project

Background and Rationale

Alberta’s bitumen oil deposits represent one of the largest oil reservoirs in the world1. Bitumen-bearing carbonate rock, or carbonate rich oil shale, represents 26% percent of Alberta’s total bitumen resources, or approximately 447 billion barrels of unprocessed heavy oil1,2. The majority of this carbonate-based oil shale is found within what is called the Carbonate Triangle where the oil is trapped within carbonate rock and therefore poses additional difficulties for extraction when compared to conventional oil reserves. Although carbonate oil reservoirs have been successfully exploited in countries such as Venezuela and Mexico3, unstable oil prices in Western Canada have prevented large-scale industrial exploitation of Alberta’s Carbonate Triangle oil reservoirs. Since the utilization of unconventional oil can emit 5-15% more CO2 than the production of conventional oil4 it is necessary to develop alternative technologies to facilitate extraction of unconventional heavy oil and mitigate the subsequent negative environmental impact. Our goal is to use a synthetic biology-based approach to develop a proof-of-principle method for extracting carbonate oil and to evaluate the associated economic and environmental issues.

Many carbonate oil deposits around the globe are being extracted through the use of in situ application of heat and steam1. While steam-based methods have been successful in places such as Egypt, Oman, and Italy1, these methods are taxing on the environment both through the use of large quantities of water and the energy needed to generate the steam. Microbial enhanced oil recovery (MEOR) uses natural bacterial products to enhance oil recovery in tertiary extraction processes and has been successfully tested in Germany and Russia to enhance the productivity of carbonate oil deposits5. Typically, microbes are directly injected into the well site where they proliferate and disperse into the matrix to deliver acids, biosurfactants, or other useful products6. A major problem associated with current MEOR technologies is the difficulty in isolating or engineering bacteria that can survive in the extreme environments of oil reservoirs6. The project outlined here will circumvent this issue by having MEOR products secreted from bacterial cells for collection thereby removing the need for direct application of bacteria to the carbonate deposits.

Carbon fixation to fuel cell growth: To address environmental concerns regarding unconventional oil extraction, carbon capture can be used to help limit greenhouse gas emission. Cyanobacteria are photosynthetic microorganisms with the capability of fixing both nitrogen and carbon. The cyanobacteria Synechococcus elongatus PCC7942 has been engineered to convert CO2 into glucose and fructose for export out of the cell to exclusively sustain the growth of Escherichia coli7. This system can be used to efficiently fix carbon and support the growth of E. coli engineered to produce useful MEOR products. An alternative method for carbon fixation is the use of a carboxysome bacterial microcompartment that enhances CO2 fixation in microbes by co-localizing carbon fixation enzymes within a proteinaceous shell8. The sulphur-oxidizing bacterium Halothiobacillus neapolitanus uses reduced sulphur compounds such as hydrogen sulphide, a toxic compound commonly produced by sulphur-reducing bacteria in petrochemical operations, as an energy source for fixing CO2. H. neapolitanus, like S. elongatus, fixes carbon within an α-carboxysome using the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) to convert CO2 into 3-phosphoglycerate (3PG)8. 3PG diffuses out of the carboxysome and enters the cell’s central metabolism9. Although RuBisCO is a relatively inefficient enzyme, localization within the carboxysome increases the local CO2 concentration to help RuBisCO perform near its maximum rate with increased substrate specificity9,10. To further enhance carbon fixation, models of synthetic carbon fixation pathways suggest that alternative carbon fixation pathways could have quantitative advantages over natural pathways11. Ultimately, natural or synthetic carbon fixation pathways can be optimized as a module to fuel bacterial cultures and the production of useful bioproducts.

Acetic acid for breakdown of carbonate rock: Stimulation of oil wells using organic acids, as opposed to stronger, inorganic acids, is frequently used to increase pore size while retaining the acid at the injection site and preventing oil/acid emulsions12. Acetic acid has been used successfully with other organic acids to treat carbonate oil deposits to increase porosity of the rock and enhance flow rates5. The natural E. coli enzymes phosphotransacetylase (PTA) and acetate kinase (ACK) produce acetic acid as a part of normal metabolic processes13,14. The acetic acid efflux protein Aata from Acetobacter aceti pumps acetic acid out of the cell and helps confer acetic acid resistance15. Optimizing production and secretion of acetic acid by E. coli can provide an inexpensive method for generating large amounts of acetic acid for MEOR.

Biosurfactants for MEOR: Chemically synthesized surfactants have previously been used successfully for EOR. Biosurfactants are currently costly to produce but have higher biodegradability and lower toxicity16,17. Rhamnolipid produced by Pseudomonas aeruginosa has been extensively studied as a biosurfactant and causes significant reduction in interfacial surface tension between oil and water to enhance oil recovery16. One of the major costs associated with biosurfactant production is sustaining culture growth, which can be overcome by using sugars produced through carbon fixation or through the use of inexpensive sugars in the culture media, such as molasses17. Increasing the commercial viability of rhamnolipid production with engineered E. coli can be a beneficial addition to using acetic acid for MEOR.

Preventing environmental contamination: Safety measures will be taken to eliminate any risk of environmental contamination by genetically modified organisms used in MEOR. Enzymes called proteases act to degrade proteins in the cell, preventing cell proliferation. Incorporating proteases into the bacterial genome will allow us to control growth with specific culture conditions. Using a light-repressible system, the expression of the proteases will be repressed under a specific wavelength of light that can be supplied in controlled growth conditions. Any bacteria released into the environment and no longer exposed to the required wavelength of light will begin to express the proteases and will be rendered inert.