HYDROCARBON

TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to the use of petroleum hydrocarbons to generate value added products by enzymatic biotransformation.

BACKGROUND OF THE INVENTION

[002] Lipases, cellulases and similar hydrolytic enzymes have been identified as useful indicators of hydrocarbon biodegradation in contaminated soils. It has been suggested in the literature that these enzymes degrade intermediate products produced during biodegradation as opposed to breaking down hydrocarbons directly.

[003] Recent research has demonstrated the ability of enzymatic solutions from known hydrocarbon-degrading microbial genus to biodegrade petroleum into fatty acids, saccharides, steroids and a mixture of natural organic compounds. Results in the literature reveal this conversion by enzymes applies over a wide range of petroleum compositions including motor oil and bitumen.

[004] This technique has the potential for industrial scale application either through the in-situ fermentation of petroleum reservoirs with enzyme flooding or through the industrial scale surface processing of produced hydrocarbons. As the societal response to global warming restricts further development of fossil fuel resources a significant fraction of reservoirs may become uneconomic or unfeasible to produce conventionally.

[005] The key issue constraining the over $5 trillion global chemical industry from moving away from a petrochemical foundation and towards a biochemical one has been the lack of sufficiently plentiful, cheap biological feedstocks which do not compete with the food supply. Over the last two centuries of organic chemistry, a considerable number of biotransformative pathways to value added chemical products have been discovered which build on these fundamental biomolecules. Most notably, the composition of certain fatty acids produced by hydrocarbon degradation closely resemble those produced by algae and consequently a wide body of literature exists with respect to transforming these products to biodiesel, biodegradable plastics, pharmaceuticals, nutritional products, industrial chemical feedstocks, printing inks, cosmetics, lubricants, cleaning products and potentially more possibilities through genetically modified fermentation and/or additional chemical processing. As the societal reaction to global warming progresses it will become increasingly important to find means of producing these products using more ecologically sustainable methods.

[006] Biodiesel from vegetable and animal oils is currently produced from the transesterification of C16 and Cl 8 fatty acids to produce fatty acid methyl esters (FAMEs), either through methanol alcholysis, lipase-mediated transesterification or a similar method. Glycerol is a common side- product which has been primarily produced petrochemically from propene via epichlorohydrin, but now is produced in surplus and increased research efforts have been exerted recently to find cost-effective transformative pathways for its commercial utilization, such as in acrolein synthesis for polyacrylic acid polymerization.

[007] Chemical and biochemical synthetic pathways exist by which natural fatty acids, saccharides and proteins can be polymerized to produce a wide range of polymers including biodegradable polyesters, polyurethanes, polyamides or new polymers through emerging pathways such as acyclic triene metathesis (ATMET) which produces highly branched polyesters with similar properties to HDPE from fatty acids.

[008] Microbial fermentation and bioreactor processing presents additional biotransformative pathways for natural biomolecules into fermentation products, organic acids, biosurfactants, pharmaceuticals, and other value-added products. For example, hydrocarbon-derived steroids could be metabolically transformed into pharmaceuticals such as immunosuppressants (such as prednisone), anti-inflammatory agents, diuretics, sedatives or vitamins such as Vitamin A.

[009] Enzyme Enhanced Oil Recovery (EEOR) and Microbial EOR have observed limited success in field trials wherein nutrients, warm water and readily metabolized carbon sources like molasses were injected into American and Soviet reservoirs in field trials from the 50s to the 80s. Personnel conducting these field trials observed production of biogases, organic acids, fermentation products and some reports of reduced oil viscosity with concomitant enhanced production. These techniques have traditionally focused on the mobilization and production of hydrocarbons as opposed to their biotransformation and relied on anaerobic enzymes or microbial communities indigenous to petroleum reservoirs to produce biosurfactants, fermentation gases, organic acids, and water thickening polysaccharides to improve mobility ratios, reduce interfacial tension, increase permeability or otherwise facilitate essentially a more biocompatible EOR water flood with additives.

SUMMARY OF THE INVENTION

[010] According to a broad aspect of the present invention, there is provided a method for producing value-added products from the processing of enzymatic biotransformation products of petroleum hydrocarbons.

[Oil] According to a first broad aspect of the present invention, there is a provided a method for generating bioconversion products from a petroleum hydrocarbon, the petroleum hydrocarbon located in a subsurface reservoir accessible by a well from surface, the method comprising the steps of: a. determining at least one hydrolytic enzyme suitable for biodegradation of the petroleum hydrocarbon based on characteristics of the subsurface and the petroleum hydrocarbon; b. injecting the at least one hydrolytic enzyme down the well and into the subsurface reservoir; c. allowing the at least one hydrolytic enzyme to contact the petroleum hydrocarbon and biodegrade at least a portion of the petroleum hydrocarbon to form at least one bioconversion product; and d. producing the at least one bioconversion product to the surface through the well.

[012] In some exemplary embodiments of the present invention, at least one solvent is co- injected with, or injected subsequent to the injection of, the at least one hydrolytic enzyme. The solvent injection may be used to mobilize the at least one bioconversion product for production to the surface.

[013] The at least one hydrolytic enzyme may be selected from the group consisting of lipases, cellulases, proteases, oxygenases, ligninases, saccharases, and combinations thereof. Additional injectants may be selected from the group consisting of bile acids, surfactants, oxygen, terminal electron acceptors, and combinations thereof.

[014] In some exemplary embodiments, the subsurface reservoir may be heated by injection of warm water through the well.

[015] The step of allowing the at least one hydrolytic enzyme to contact the petroleum

hydrocarbon and biodegrade the at least a portion of the petroleum hydrocarbon to form the at least one bioconversion product preferably comprises a period of time suitable to enable the biodegradation.

[016] In some exemplary embodiments of the preset invention, steps b. through d. are repeated at least once.

[017] In some exemplary embodiments of the present invention, at least one reactive component is injected after step c. and before step d. to enable further in-situ conversion of the at least one bioconversion product.

[018] According to some exemplary embodiments of the present invention, hydrolytic enzymes and suitable solvents containing bile acids, oxygen, and potentially other additives (such as hot water or pH modifiers) can be employed to biodegrade petroleum hydrocarbons (including diesels, crude oil, heavy oils, bitumen, shale oils, kerogen, etc.) into fermentation products such as fatty acids, sugars, proteins and other fermentation products. Solvents would be suitable in the sense that they would mainly dissolve or potentially react with the bioconversion products, e.g., rejection of asphaltenes from the bioconversion products, such as n-alkanes, alcohols and ketones.

[019] In some exemplary embodiments of the present invention, methods are provided to inject enzymes and other additives into petroleum reservoirs to convert the hydrocarbons into biomolecules which can then be transformed into secondary products in-situ by the subsequent injection of reactive agents, solvents, etc., followed by production. Furthermore, as will be clear to the skilled person, several other potential embodiments can exist depending on the desired product, the conditions necessary for adequate substrate conversion and how it may be necessary to mobilize the resulting aqueous, oleic or gaseous product to a production well. [020] Exemplary embodiments of the present invention may involve the application of mixtures of hydrolytic enzymes (for example, Lipases, Cellulases, Proteases, Oxygenases, Ligninases, Saccharases, etc.), bile acids or similar synergistic surfactants, oxygen and/or other terminal electron acceptors (TEAs) and other additives in solution with or without acid and/or base to produce a final composition which is both formation compatible while optimizing and maintaining enzymatic activity. The reservoir may be either lightly flooded or left to soak, either at the original reservoir temperature or slightly elevated 30 to 40 °C by warm water injection, for a period of months to years for the petroleum to biodegrade in-situ.

[021] After the soak period, which is typically once an adequate product conversion has been achieved, the biomolecules produced by enzymatic hydrocarbon biodegradation are pumped to surface. The produced stream will contain fermentation gases, water, residual hydrocarbons, solids and other components often found in oilfield produced water in addition to the bioproducts which must be removed in pre-processing. Ideally then the bioproducts are extracted from the produced water stream and concentrated by solvent extraction, as membrane permeate or by another known method to significantly reduce the process stream volume and minimize the size of subsequent process operations. As appropriate, water soluble hydrocarbon-derived products will have to be recovered from the water stream before it is sent to treatment for reinjection or disposal.

[022] Bioprocessing consists of reacting the hydrocarbon-derived biomolecular products in a reactor to produce a desired product with certain properties. As in traditional reactor engineering the industrial application depends on maintaining certain pH, temperature, pressure, phase volume fractions, phase compositions and other reactor conditions for effective conversion. The mixed product stream leaving the reactor must be fractionated to purify its products and unreacted substrates for sale. The biorefming process can be achieved using an appropriate combination of separation unit operations including solvent extraction, ultrafiltration and/or diafiltration, chromatography, precipitation, ion exchange, supercritical extraction and other techniques.

[023] Fatty acids have been identified as primary products of the enzymatic biodegradation of petroleum hydrocarbons observed in some embodiments of the present invention, including glycerin, C16 and Cl 8 saturated and unsaturated fatty acids similar to natural vegetable and animal oils in addition to a broader mixture of fatty acids, terpenes and steroids. Industrially, fatty acids can be transformed at their carboxyl groups to produce soaps, esters, amides, amines and other chemical products. Significant product peaks were observed in GC-MS data correlating to unsaturated fatty acids such as oleic acid to which traditional double-bond reactions apply such as hydrogenation, oxidative cleavage or epoxidation.

[024] Recent research has also extended to the application of fungal enzyme preparations from Aspergillus spp. to EOR as these mixtures have demonstrated the ability to partially biodegrade crude oils anaerobically, reducing their viscosity and thus potentially improving reservoir production. The present application identifies key enzymes from similar mixtures and from other hydrocarbon-degrading species which have demonstrated the ability to bioconvert a wide range of petroleum hydrocarbons.

[025] Embodiments according to the present invention may comprise at least some of the following features:

1. Procurement and mixing of enzyme powder or solution or suspension containing bile acids, oxygen, solvents and potentially other additives to create an enzyme solution. These enzymes can either be purchased, fermented or bio-refined on site for endogenous use or produced in-situ by injection and stimulation of appropriate enzyme-producing cultures or cell-free enzyme systems, natural or genetically-altered. Single enzymes or mixtures may be appropriate depending on substrate, products and environmental conditions.

2. Enzymatic hydrocarbon biotransformation can either be conducted at scale on surface or in the reservoir. Oil can be fermented with enzyme solution in tanks, vats, bioreactors and similar large vessels on surface to provide the residence time necessary for biodegradation. The enzyme solution can be added to pipelines for bioconversion while the oil is in transport. For reservoir- based application, drilling of horizontal, vertical, directional or other wells into the reservoir may be conducted to disperse the enzyme solution with oxygen, bile acids, solvents and other additives as appropriate, again followed by a residence time after which fermentation products may be pumped to surface. 3. Following initial enzymatic fermentation, for reservoir-based application, secondary floods may be conducted with reactive components to transform some or all of the primary bioproducts into secondary product molecules. This can include the injection of salts, acids/bases, microbes, cell-free enzyme systems, surfactants or other components necessary to create effective conditions for reaction in-situ. A final injection of organic solvent such as, but not limited to, hexane may be necessary to solubilize components such as fatty acids for easier mobilization to the surface. Alternatively, reactive components can be employed, at surface, to transform some or all of the primary bioproducts into secondary product molecules. Similarly, secondary solutions can be added to surface-based applications or in-pipeline applications.

4. Produced fluids will first have to be pre-processed to remove oil field contaminants including residual oil, salts, produced gases and other produced water compounds which may affect subsequent bioprocessing or contaminate the final product. Produced gases and residual oil can be primarily separated using traditional oilfield production facility techniques including settling vessels or centrifugation with the potential necessity for subsequent steps to meet purity specifications and subsequently processed traditionally. Lipid products can be separated from the produced water stream which should then be stripped of water-soluble hydrocarbon-derived bioproducts before being sent to water treatment.

5. Following initial separation of enzymatically produced bioproducts from oilfield contaminants these molecules can be subjected to further bioprocessing and synthetic

transformation into secondary products in bioreactors or similar.

6. The bioprocessing product stream is then sent to a product separation means to fractionate reaction products from reactants and prepare the final products for sale.

[026] In some exemplary embodiments of the present invention, enzymes are dispersed immobilized on a magnetic support to facilitate its recollection and reuse during bioprocessing. Immobilized enzymes have demonstrated increased activity relative to solutions due to increased local density of active enzymatic sites and can have been industrially implemented for solid-state fermentation in fluidized or fixed bed bioreactors. This enzyme could be the hydrolytic enzymes used to biodegrade hydrocarbons into biomolecules or other enzymes used to transform these primary biomolecules into secondary products.

[027] In some exemplary embodiments of the present invention, specifically formulated natural or genetically modified microbial communities, cell-free enzyme systems, fungi or similar could be co-introduced to the reservoir with the enzyme solution, during the soak period or after the enzyme degradation process. This can have the benefits of reduced enzymatic product inhibition when products are consumed by syntrophic microbes, additional metabolic biotransformative potential to target specific pollutants or further transform the enzyme-produced fermentation products into secondary biochemical compounds. As an example, the introduction of reservoir compatible microbial species which preferentially consume glycerin as a carbon source would enhance the overall conversion of hydrocarbon-derived fatty acid into biodiesel.

[028] In some exemplary embodiments of the present invention, specifically formulated natural or genetically modified microbial communities, cell-free enzyme systems, fungi or similar could be used in the bioprocessing operations to transform the primary hydrocarbon-derived biomolecules into secondary metabolite products during bioprocessing. This process requires unique operations for product separation including cell disruption via bead milling or similar, fractionation of waste biomass, etc.

[029] In some exemplary embodiments of the present invention, enzymes are dispersed immobilized on a magnetic support or nanoparticle to facilitate its dispersion and recollection. Immobilized enzymes have demonstrated increased activity relative to solutions due to increased local density of active enzymatic sites and can be injected into reservoirs as such in nanoparticle suspensions. Magnetic supports present the potential for using magnetic fields as an additional driving force to direct enzyme through the reservoir porous media. More importantly, magnetic supports facilitate the separation of immobilized enzymes from produced fluids and their recycling.

[030] As global markets begin to transition from fossil fuels in response to global warming there exists the potential for billions of dollars in reservoir leases to face the potential of becoming stranded assets if alternative uses for petroleum are not pursued. This presents an opportunity to find new transformative pathways into value-added products for the most plentiful feedstock in human history. The present invention described herein has the potential to create value from abandoned, depleted, thin, shallow, carbonate, clastic and other reservoirs with particular efficacy anticipated for reservoirs with high water cut and saturation. Performing the initial stages of bioprocessing in-situ has the potential to reduce the expense and number of unit operations on surface necessary to achieve the same outcome. The present invention has the added benefits of utilizing enzymes which are currently produced in industrial quantity and an injected solution which comprises relatively biocompatible agents which produce products that are generally less toxic than the original hydrocarbons.

[031] While algal oil and similar biomass dependent based processes have found success in producing valuable products, they have always been limited by the ability to produce cheap microbial biomass in sufficient abundance for scale. The present invention intends to extend the existing practice of bioprocessing and biorefming to the use of biological feedstocks produced from enzymatically biotransformed petroleum hydrocarbons, which might achieve the necessary scale to shift the over $5 trillion global chemical industry from a petrochemical basis to a less energy-intensive, more ecologically sound biochemical foundation.

[032] The enzymatic products of petroleum biotransformation which have been analytically determined has suggested they principally comprise fatty acids and saccharides, similar in composition to algal oils and more traditional biological feedstocks. Consequently, there exists a wide body of literature with respect to transforming these biomolecules into biodiesel, biodegradable plastics, pharmaceuticals, nutritional products, industrial chemical feedstocks, printing inks, cosmetics, lubricants, cleaning products and potentially more possibilities through genetically modified fermentation and/or additional chemical processing. The present invention intends to relate to the extent to which these processes can be conducted in-situ within reservoirs through the design of multi-stage reactive floods.

[033] A detailed description of exemplary embodiments of the present invention is given in the following. It is to be understood, however, that the invention is not to be construed as being limited to these embodiments. The exemplary embodiments are directed to particular applications of the present invention, while it will be clear to those skilled in the art that the present invention has applicability beyond the exemplary embodiments set forth herein BRIEF DESCRIPTION OF THE DRAWINGS

[034] In the accompanying drawings, which illustrate exemplary embodiments of the present invention:

[035] Figure 1 illustrates potential applications of the methods herein described to treat environmental petroleum hydrocarbon contamination sites.

[036] Fig. 2 illustrates primary biodegradation to generate bioconversion products.

[037] Fig. 3 shows an example of an additional step which may be necessary to mobilize biodegraded hydrocarbons for production.

[038] Fig. 4 shows an example of a particular embodiment to convert petroleum hydrocarbons into biodiesel in-situ.

[039] Fig. 5 illustrates an exemplary embodiment for the in-situ conversion of enzymatically biodegraded hydrocarbons into polymer products.

[040] Fig. 6 illustrates an exemplary production step along with an example of potential surface facilities for processing.

[041] Fig. 7(a) to 7(b) illustrate some exemplary unit operations to implement the enzymatic biotransformation of hydrocarbons in process facilities.

[042] Fig. 8 illustrates an exemplary FAME biodiesel production process from enzymatically generated bioproducts.

[043] Fig. 9 illustrates an example of a chemoenzymatic epoxidation process for converting hydrocarbon-derived fatty acid bioproducts into polyester.

[044] Fig. 10 illustrates an exemplary microbial fermentation process for converting

enzymatically produced biomolecules into pharmaceuticals.

[045] Exemplary embodiments will now be described with reference to the accompanying drawings. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[046] Throughout the following description, specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. The following description of examples of the invention is not intended to be exhaustive or to limit the invention of the precise forms of any exemplary embodiment. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[047] The present invention is directed to the generation and processing of enzymatically biodegraded petroleum hydrocarbons into value-added products.

[048] These are processes which can be used to generate value from petroleum reservoirs which might be otherwise uneconomic to produce or have been depleted with respect to traditional reservoir production and EOR. Specifically, at least two classes of hydrolytic enzyme have been shown to biodegrade petroleum hydrocarbons (including crude oil, bitumen) into biomolecules including fatty acids, saccharides, steroids, and proteins. These primary bioproducts can be further transformed into secondary value-added products in-situ by the injection of reactive solutions followed by production and surface processing.

[049] Enzyme solutions will have to be prepared to be formation compatible in that it cannot plug the porous media upon reacting with reservoir water chemistry and mineralogy while also providing the appropriate pH, co-reactants, solvents, surfactants and similar necessities for improved enzymatic activity and conversion.

[050] The appropriate composition of enzyme mixture to apply will depend on the source rock composition and diagenic history of each petroleum deposit. Different source kerogens derive from particular types of deposited biomass, for example Type II kerogen is derived largely from lipid-rich planktonic biomass deposited in marine conditions and thus may be susceptible to lipase treatment. Petroleum deposits derived from lacustrine and fluvial deltas often contain more terrestrial organic matter including cellulose and lignin, defined as Type III kerogen and thus cellulase/ligninase may be applicable for enzyme flooding. In general, any hydrolase or other digestive enzyme might be applicable towards the viscosity reduction of heavy and shale oils depending on their relative biodegradation state. [051] The enzymatic biotransformation process described herein produces fatty acids which closely resemble palm oil and similar plant/animal vegetable oils. Consequently, this technology has potential to disrupt the greater than$l9 billion global oleochemical industry and encourage palm plantations to return to natural ecosystems.

[052] Residual oil will likely be produced along with the desired products and will have to be separated to produce pure, safe products. This can be done through traditional methods such as settling tanks, froth flotation tanks, filter beds, membranes, etc. Produced gases can include C0 ₂ , H ₂ and CH ₄ which can be separated at the process inlet and should be accounted for in production well casing and pad designs. Ideally this process would be conducted in sweet reservoirs with relatively fresh, low TDS water compositions which minimize enzyme deactivation but can be potentially mitigated with enzyme co-injectants.

[053] Ideally the bioproducts are extracted from the produced water stream and concentrated to significantly reduce the product stream volume and minimize the size of separation operations. Secondary processing may be conducted on surface to remove contaminants like intermediate products, or to further modify the product stream to achieve a final product with desired properties. After processing the product stream should be separated by methods such as solvent extraction, diafiltration, ion exchange, chromatography, crystallization, etc. and then spray dried or otherwise prepared for shipment.

[054] This present invention would be particularly well suited to the retrofitting of abandoned or depleted reservoir facilities as in some embodiments it can use the same water and oil processing units with additional bioprocessing and refining units added after in series. Similarly, facilities for mixing, storing, fermenting, or potentially generating enzyme and similar injectants on site involve unit operations which are incremental evolutions of existing petrochemical and wastewater processes but can produce potentially valuable chemical products at large scale with less energy consumption and a few biocompatible inputs.

[055] Fig 1 illustrates some exemplary value-added products which can be produced from fatty acids, sugars, proteins, steroids and similar biomolecules.

[056] Fig. 2 shows the initial stages of in-situ enzymatic conversion. An enzyme solution is prepared on surface potentially containing bile acids, organic acids, and oxygen when utilizing aerobic enzymes. This solution can then be injected into a hydrocarbon reservoir via a horizontal, vertical or other well and left to ferment with ongoing passive or active oxygen introduction. The enzyme solution biodegrades petroleum hydrocarbons into a range of biomolecular products including fatty acids, steroids and saccharides depending on the choice of enzymes and substrate composition. As the enzyme solution is generally heavier than water it may be preferential to place wells near the top of formations and recover enzymatically produced bioproducts using organic solvents which are lighter than water. Enzyme solutions will have to be formulated with reservoir water chemistry and reactive mineralogy in mind to avoid acidification damage from mineral dissolution while also attempting to create optimal conditions for enzymatic activity in- situ which often range in a pH of 5 to 9. Once the petroleum has been sufficiently biodegraded it is ready for additional reactive floods to convert the biomolecular products into secondary value- added products.

[057] Fig. 3 demonstrates an additional stage which may be necessary in the case that primary enzymatically-produced bioproducts or secondary products are mostly insoluble in water and present a formation plugging issue. Organic, aqueous or other solvents as appropriate can be injected after the initial fermentation period and potentially subsequent reactive flood steps depending on the desired final product. Additional driving forces can be applied to enhance recovery such as flooding with injection wells, fracking, gas injection, etc.

[058] Fig. 4 illustrates an exemplary embodiment for the in-situ transesterification of

enzymatically produced fatty acids into fatty acid methyl ester (FAME) biodiesel. The illustrated process employs alcoholysis by the injection of an appropriate solution containing methanol, ethanol, ethyl acetate or similar which can react with the fatty acids. It will be known to the skilled person that additional additives may be necessary to dilute this reactive solution, make the reactive solution formation compatible, mitigate enzyme deactivation or otherwise improve conversion and recovery. Injection of microbial communities or similar biological systems which can survive in the reservoir environment and metabolize glycerin might be beneficial to reduce product inhibition and generate tertiary products.

[059] Fig. 5 illustrates an exemplary embodiment for the conversion of enzyme-produced fatty acid products into polyesters by the Prilezhaev reaction. This pathway takes advantage of the presence of necessary enzymes in the reservoir to catalyze fatty acid epoxidation with the addition of hydrogen peroxide. Organic solvents or other carriers should preferably be co- injected to decelerate the introduction of hydrogen peroxide into the aqueous phase to minimize enzyme deactivation. Additional co-substrates may be necessary to facilitate peroxy acid formation for epoxidation such as ethyl acetate, dimethyl carbonate or lactone. If not already introduced, additional solvent may need to be injected to mobilize this product.

[060] Fig. 6 illustrates an exemplary final production step of some methods according to the present invention, whereby produced wells pump aqueous and oleic product from the subsurface biodegradation and any subsequent in-situ conversions to surface for subsequent processing. Typical oilfield contaminants such as produced gases, residual oil, salts, and excess produced water will have to be separated from the fermentation products via separation tanks, diafiltration, froth flotation, solvent extraction or otherwise. The bioprocessing stage shown in Fig. 6 refers to any additional reactions or fermentations which may be conducted on surface to bring the produced molecules closer to desired product specifications, conduct tertiary biotransformations, achieve certain properties or otherwise treat the production stream. This may be conducted in conjunction with separation processes such as ion exchange, ultrafiltration, chromatography, precipitation, solvent extraction and similar techniques to purify or fractionate streams.

[061] Fig 7(a) illustrates the enzymatic biotransformation of hydrocarbons in solution by a fixed bed reactor with a support covered with immobilized enzyme. Such a system may be ideal for relatively dilute hydrocarbon solutions. Fig 7(b) illustrates the surface enzymatic fermentation of petroleum hydrocarbons in vessels such as aerated storage tanks. This technique may be more economically feasible for smaller batch operations as the necessary reaction residence time for reasonable hydrocarbon quantities is on the order of weeks to months depending on pH, temperature, enzyme activity, substrate composition, etc. Initial lab results have demonstrated sufficient density difference between fatty acid, proteinaceous and other enzymatic bioproducts which primarily sink in water and can be thus separated from biodegraded hydrocarbons which float. Subsequent purification of these settled bioproducts may be necessary to remove contaminants as per specification. [062] Fig 8 illustrates an exemplary process for the separation of enzymatically produced biomolecules from produced fluids and the transesterification of the fatty acid components to produce fatty acid methyl ester (FAME) biodiesel. Lipid and other non-water soluble products are separated from the produced water stream after pre-treatment and is reacted with methanol to produce FAMEs via transesterification. This reaction is product limited particularly with respect to glycerin and an advantage of conducting the process in a surface bioreactor instead of in-situ is the ability to employ membrane reactors, reactive extraction or similar methods to

continuously remove glycerin from the reaction zone and improve conversion. The glycerin side product can then be sold as-is or further processed and/or fermented into secondary products.

[063] Fig 9 illustrates an exemplary process for the chemoenzymatic epoxidation production of polyester from fatty acids derived from biotransformed hydrocarbons. This process takes advantage of the presence of enzyme in the feedstock solution, which may be introduced with the hydrogen peroxide and solvent for enzyme-catalyzed epoxidation. Depending on reaction conditions such as the use of organic solvents, urea hydrogen peroxide or other techniques to decelerate hydrogen peroxide into the aqueous phase it should be possible to minimize enzymatic deactivation and recycle enzyme for reutilization. Finally, the polymer product will be separated from unreacted fatty acids and other reactants with solvents, precipitation or other techniques before being further extruded, stripped, dried and shipped.

[064] Fig 10 illustrates an exemplary process to produce pharmaceutical or similarly therapeutic molecules from enzymatically biotransformed petroleum using microbial fermentation. In this embodiment the biological products of enzymatic hydrocarbon biodegradation are transformed into secondary metabolic products by a single or multiple fermentation steps in series after being pre-treated to remove produced oilfield contaminants. Depending on the toxic tolerance of the microbial culture and produced biomolecule composition it may be necessary to perform additional separation and biorefming before fermentation to purify the feedstock. Fermentation products can then be sent for product separation by conventional techniques. The desired pharmaceutical product could end up in different final process streams depending on its nature. This process can be extended to similar value-added bioproduct processes such as the production of cosmetics, therapeutics, nutritional products, agrifeedstocks, lubricants, biosurfactants, biofuels, chemical feedstocks, inks, enzymes, proteins and other health products. [065] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention.

[066] The foregoing is considered as illustrative only of the principles of the present invention. The scope of the claims should not be limited by the exemplary embodiments set forth in the foregoing, but should be given the broadest interpretation consistent with the specification as a whole.