FROM STORAGE AND TRANSPORT VESSELS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 63/394,792, filed August 3, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Hydraulic fracturing, or “fracing,” of earth formations around a wellbore is a process used to increase a well’s productivity. Standard vertical wells undergo fracturing during original production or to stimulate production. Other applications involve the use of horizontal wells, wherein a vertical well is drilled to a desired depth, at which point the drill is turned to begin drilling horizontally. The horizontal portion of these wells can extend several thousands of feet in length.

Once drilling has occurred, thousands of gallons of “pad” fluid, an oil-based or water-based fluid, are injected into a formation at extreme pressures. This causes cracks or fractures to develop in the face of the rock at the wellbore. Continued fluid injection into the well then causes the fractures to increase in length and width. After a sufficient width is achieved, particles called “proppant” are added to the fluid, often coated with polymeric materials to aid in proper functioning in tight subterranean formations. Silica sand is commonly used as a proppant in fracing applications.

After fluid injection has ceased, fracturing fluid flows out of the fractures, allowing the walls of the fractures to close on the proppant. The proppant particles then “prop” the walls of the fractures apart. Because proppant particles are normally much larger than the pores of the formation, the fluid permeability of a propped fracture is much greater than that of the natural formation; hence, the flow capacity of the well is increased. At the end of a fracturing treatment, proppant-laden fluid is “flushed” from the wellbore into the formation by a proppant-free displacement fluid.

Despite the increases in oil and gas productivity associated with the use of fracing, certain drawbacks and complications can arise. For example, fracturing jobs utilize highly pressurized fluids that must be viscous enough to carry proppant into the fractures in the rock formation to keep them open yet fluid enough to flow. Water is mixed with various chemical additives, including crosslinked gelling agents and friction reducers (FR) to increase the ability to carry proppant and the displacement efficiency.

Standard FRs were historically designed to carry proppant into the reservoir by pumping fluids at a high flow rate. To maximize proppant loading into these unconventional wells, High Viscosity Friction Reducers (HVFRs) containing high molecular weight polymers, such as polyacrylamide (PAM), have been developed. HVFRs can reduce water consumption, minimize chemical usage, enhance proppant carrying capacity, and require less operating equipment on location; however, these heavy compounds, which are utilized in very high quantities across the oil and gas industry, can also cause operational obstacles once recovered from formations as flowback.

When shipped in tankers, the tankers must be cleaned of the polymers before re-use to avoid cross-contamination upon refilling. Water-soluble polymers can be difficult to degrade, however, often leaving a sticky, highly viscous emulsion on tank surfaces. To clean buildup of HVFRs from a tank often requires manual excavation, collection and removal of the substances. This is time consuming, labor intensive and expensive. Furthermore, the personnel working within the tank are exposed to potential health risks, as well as possible injury.

An additional drawback of post-production polymer-containing fluids is disposal. Large volumes of rinsate from the cleaning of tanks must be shipped to designated locations for disposal, for example, in underground injection wells. The result is significant costs associated with transportation, as well as the wasting of potentially usable product leftover in the wastewater.

Efficient production of oil depends upon the proper functioning of equipment and the ability of operators to utilize resources efficiently. Thus, with the growing problems associated with disposal of flowback fluids containing FRs, improved systems, methods and compositions are needed for cleaning highly viscous flowback emulsions in storage tanks and other storage and/or transport vessels.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides compositions and methods for cleaning highly viscous substances from storage and/or transport vessels. More specifically, the subject invention provides materials and methods for improving oil and gas production by treating storage and/or transport vessels with a composition capable of emulsifying highly viscous polymeric substances for enhancing the removal and/or recycling thereof. Advantageously, the compositions and methods of the subject invention are environmentally-friendly, operational-friendly and cost-effective.

In preferred embodiments, the subject invention provides a cleaning composition comprising a carrier or diluent and a detersive agent. The composition can further comprise a co-solvent and/or coupling agent for the detersive agent.

The carrier is preferably a base oil, such as one of the following non-limiting examples: hydrotreated petroleum; light distillate; hydro-treated distillate that can comprise dodecane, tridecane, tetradecane, and undecane; fatty acid methyl esters; soy methyl esters; and mineral oils. In certain embodiments, the detersive agent is a surface-active agent or a solvent. The detersive agent is preferably non-ionic to ensure compatibility with anionic, cationic and amphoteric polymers.

Even more preferably, in certain embodiments, the detersive agent is a biosurfactant. Biosurfactants can include, for example, glycolipids, lipopeptides, flavolipids, phospholipids, and high-molecular-weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. In some embodiments, blends of more than one biosurfactant molecule are utilized. In some embodiments, the biosurfactant molecules can be subjected to chemical derivatization.

The coupling agent and/or co-solvent can be a surface-active agent and/or a solvent that is not the same surface-active agent or solvent used as the detersive agent. The selection of the coupling agent and/or co-solvent is dependent upon the composition of the detersive agent, wherein the coupling agent and/or co-solvent serves to keep the detersive agent in solution. In some embodiments, a coupling agent and/or co-solvent is not needed.

The compositions and methods of the subject invention can remove highly viscous polymeric substances from storage containers, pumps, piping, drains, drills, tanks, and tubing. For example, the subject invention can be used to remove residual highly viscous polymeric substances from container walls without requiring heat. Advantageously, in certain embodiments, this can be done in only a few hours. Highly viscous polymeric substances can then be recovered from the container and disposed of or reused in hydraulic fracturing operations, thus increasing oil and gas production efficiency. The composition can be prepared and mixed prior to being applied to the container or surface, or the individual components of the composition can be added separately to the storage container or surface and mixed therein or thereon. In preferred embodiments, the storage container is a storage tank for highly viscous polymeric substances.

In preferred embodiments, the subject invention provides a method for maintaining or improving oil and gas production efficiency by applying the cleaning composition containing the residual emulsified highly viscous polymeric substance to an oil well or hydraulic fracturing site.

In one embodiment, the highly viscous polymeric substance cleaning composition is mixed within a storage tank containing the highly viscous polymeric substance for a period of time sufficient to remove a highly viscous polymeric substance and form an emulsion comprising the composition and the highly viscous polymeric substance. Mixing can be performed using any mixing device or technique, for example, spraying the composition onto a surface covered in the highly viscous polymeric substance. In one embodiment, the storage tank has a built-in mixing system, such as, for example, rolling of the storage tank or surface, agitating the storage tank or surface, and/or spraying the highly viscous polymeric substance and/or the cleaning composition. The amount of the highly viscous polymeric substance cleaning composition that can be added to the container is preferably at a ratio of from 10: 1, 9:1, 8:1, 7:1, 6: 1, 5: 1 , 4: 1, 3: 1, 2: 1, 1:1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1:9, or 1 : 10 units of composition to the highly viscous polymeric substance. In specific preferred embodiments, the composition to the highly viscous polymeric substance ratio is 1 : 1 or 2: 1.

Preferably, the mixing takes place for about 1 min, about 5 min, about 10 min, about 15 min, about 30 min, about 45 min, about 1 h, about 2 h, about 3 h, about 4 h, about 6 h, or about 12 h, or any time period within that range depending on the size of the container to be cleaned and the volume of residual highly viscous polymeric substance remaining in the vessel. Then, the emulsion can be allowed to sit for 1 min to 3 hours, up to 12 to 24 hours, or more, or until a single emulsion forms containing both the composition and the highly viscous polymeric substance. Preferably, the emulsion can be immediately removed from the container after mixing.

In one embodiment, the highly viscous polymeric substance storage container is a large storage tank, for example, having a diameter of about 5 feet to about 500 feet. A portion of highly viscous polymeric substance can be removed from the large storage tank via robotic or manual methods (e.g., draining) and placed into a smaller storage tank, for example, a tank 1/3 or 1/2 the size of the large storage tank. Then, the methods according to the subject invention can be carried out in the smaller storage tank, which can reduce the time required for highly viscous polymeric substance removal.

In one embodiment, the method further comprises, after the composition has been applied to the highly viscous polymeric substance, by, for example, pumping the composition into a vessel containing the highly viscous polymeric substance, applying a gas (e.g., air) to the composition and the highly viscous polymeric substance. Advantageously, in one embodiment, the container has less highly viscous polymeric substance remaining in it than it would have if it were not treated with the subject composition. Thus, the processing of the highly viscous polymeric substance can operate at a faster speed, and the overall process can take less time. Accordingly, the highly viscous polymeric substance can be cleaned in a matter of hours or minutes to be disposed of or reused in hydraulic fracturing operations. Furthermore, the residual highly viscous polymeric substance that is recovered from the container can be processed and/or used directly in hydraulic fracturing operations. At certain ratios of cleaning composition to highly viscous polymeric substance, the emulsion will phase separate upon sitting for at least about 30 min, about 1 h, about 2 h, about 3 h, about 6 h, about 9 h, about 12 h, about 16 h, about 1 day, about 2 days, about 3 days, or about 1 week, allowing a portion of the cleaning solution to be removed from the liquid containing the highly viscous polymeric substance. The cleaning composition can be recycled to clean a different vessel or surface, while the composition containing the highly viscous polymeric substance can be processed and/or used directly in hydraulic fracturing operations.

After the highly viscous polymeric substance emulsion has been removed from the storage tank, the storage tank and any remaining highly viscous polymeric substance therein can be treated at least 1, 2, 3, 4 5, 6, 7, 8, 9, or 10 more times with the cleaning composition. For example, the cleaning composition can be applied to the tank, mixed, and then allowed to sit and remove the remaining highly viscous polymeric substance.

In one embodiment, methods are provided for treating, for example, a borehole, oilfield, and/or oil and gas transportation, transmission and/or refinery equipment. In certain embodiments, the methods are used to improve oil production, as well as maintenance of, for example, pipes, pumps, drains, drills, tanks and other structures and equipment involved in oil and/or gas production, transportation, storage and/or refining. In certain embodiments, the cleaning compositions can be used in the flowback process to inhibit the buildup of a highly viscous polymeric substance in various tubing and tanks after the hydraulic fracturing process is complete.

The application of the biosurfactant based products of the present invention can be performed during transportation and/or storage of highly viscous polymeric substance. For example, the subject products can be applied to a storage tank at the site of recovery, e.g., at or near an oil field.

Advantageously, the present invention can be used without using large quantities of toxic compounds and, therefore, without the need to dispose of said toxic compounds. The biosurfactant based cleaning composition has no added water, preventing the inversion of the polymer and reducing the volume of waste to be disposed or reused. Additionally, the compositions and methods utilize components that are biodegradable and toxicologically safe. Thus, the present invention can be used in all possible operations of oil and gas production as an environmentally-friendly treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows samples of hydrolyzed polyacrylamide (HPAM) and the subject cleaning composition (e.g., AssurClean™) residual washing waste (Figure 1A); based on viscosity, the subject cleaning composition appears to not affect the overall HPAM emulsion at levels up to 50%. Figure IB shows a slight separation after 16 hours but can be reconstituted with slight agitation or separated for recycling.

Figure 2 shows either water, a surfactant, or the subject composition (e.g., AssurClean™) added to a high viscosity polymeric substance. With the addition of water, a thick polymeric film is adhered to the glass walls and the emulsion is inverted, creating a highly viscous gel. With the addition of a surfactant, a viscous liquid with suspended particulates (polymer) is adhered to the glass walls and the emulsion tended to invert. With the addition of the subject cleaning composition, a low viscosity liquid with suspended particles exists in the container and the emulsion did not invert.

Figure 3 shows the shake detergency test on glass bottles using the subject compositions (AssurClean 2120S), water, chemical cleaner (M-152, DFW-6590), MeOH, and carrier fluid to clean anionic friction reducer Al (FR-A1) HVPAM polymeric emulsion.

Figure 4 shows the shake detergency test on glass bottles using the subject composition (AssurClean 2120S) to clean anionic friction reducers FR-A1, FR-A2, FR-A3 and cationic friction reducers FR-C1 , and FR-C2 HVPAM polymeric emulsions.

DETAILED DESCRIPTION

The subject invention provides compositions and methods for cleaning highly viscous substances from storage and/or transport vessels. More specifically, the subject invention provides materials and methods for improving oil and gas production by treating storage and/or transport vessels with a composition capable of emulsifying highly viscous polymeric substances for enhancing the removal and/or recycling thereof.

Selected Definitions

As used herein, “impurity” or “contaminant” refers to any substance that causes another substance or object to become fouled, contaminated or impure. Impurities can be living or non-living and can be inorganic or organic substances or deposits. Furthermore, impurities can include, but are not limited to, hydrocarbons, such as petroleum or asphaltenes; fats, oils and greases (FOG), such as cooking grease and lard; lipids; waxes, such as paraffin; resins; biofilms; or any other substances referred to as, for example, dirt, dust, scale, sand, crud, slag, grime, scum, plaque, buildup, or residue. Sludge and its individual components can be included in the phrase “impurity.”

As used herein, “cleaning” as used in the context of highly viscous polymeric substance removal, means removal or reduction of the highly viscous polymeric substance and its components. Cleaning can include remediating, separating, purifying, defouling, decontaminating, dissolving, clearing, treating and/or unclogging. Cleaning can further include controlling, inhibiting or preventing further collection of the highly viscous polymeric substance.

As used herein, a “friction reducer” refers to a chemical used during the hydraulic fracturing process to reduce the frictional effects (i.e., pressure) that occurs as water is pumped down the wellbore. A friction reducer often comprises a polymer, such as, for example, polyacrylamide and a carrier fluid, such as, for example, a mineral oil or surfactant.

As used herein, the term “residual” refers to any amount of material (e.g., highly viscous polymeric substance) remaining in a container or on a surface that can be cleaned and/or removed. The material may be a small volume, coating the container walls, that is difficult to see with the eye or, alternatively, a large volume in the bottom of a container.

As used herein, the term “flowback” refers to the process of allowing fluids to flow from an oil or gas well following a hydraulic fracturing treatment, either in preparation for a subsequent phase of treatment or in preparation for cleanup and returning the well to production.

As used herein, “prevention” means avoiding, delaying, forestalling, inhibiting or minimizing the onset or progression of an occurrence or situation. Prevention can include, but does not require, absolute or complete prevention, meaning the occurrence or situation may still develop at a later time and/or with a lesser severity than it would without preventative measures. Prevention can include reducing the severity of the onset of an occurrence or situation, and/or inhibiting the progression thereof to one that is more severe. A highly viscous polymeric substance “inhibitor” is an agent that prevents the accumulation of a highly viscous polymeric substance onto surfaces.

As used herein, reference to a “microbe-based composition” means a composition that comprises components that were produced as the result of the growth of microorganisms or other cell cultures. Thus, the microbe-based composition may comprise the microbes themselves and/or byproducts of microbial growth. Preferably, the compositions according to the subject invention comprise inactivated microbes, or have been separated from the microbes altogether. The by-products of microbial growth may be, for example, metabolites (e.g., biosurfactants), cell membrane components, expressed proteins, and/or other cellular components.

The subject invention further provides “microbe-based products,” which are products that are to be applied in practice to achieve a desired result. The microbe-based product can be simply the microbe-based composition harvested from the microbe cultivation process. Alternatively, the microbe-based product may comprise further ingredients that have been added. These additional ingredients can include, for example, stabilizers, buffers, appropriate carriers, such as water, salt solutions, or any other appropriate carrier, added nutrients to support further microbial growth, nonnutrient growth enhancers, and/or agents that facilitate tracking of the microbes and/or the composition in the environment to which it is applied. The microbe-based product may also comprise mixtures of microbe-based compositions. The microbe-based product may also comprise one or more components of a microbe-based composition that have been processed in some way such as, but not limited to, filtering, centrifugation, lysing, diying, purification and the like.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or organic compound, such as a small molecule, is substantially free of other compounds, such as cellular material, with which it is associated in nature. In certain embodiments, purified compounds are at least 60% by weight the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination of numbers, or subrange from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 and 20, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

As used herein, “reduces” means a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

As used herein, “reference” means a standard or control condition.

A “metabolite” refers to any substance produced by metabolism (e.g., a growth by-product) or a substance necessary for taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material (e.g., glucose), an intermediate (e.g., acetyl-CoA) in, or an end product (e.g., n-butanol) of metabolism. Examples of metabolites include, but are not limited to, biosurfactants, enzymes, acids, solvents, gasses, alcohols, proteins, vitamins, minerals, microelements, amino acids, and polymers.

As used herein, “surfactant” means a compound that lowers the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants act as, e.g., detergents, wetting agents, emulsifiers, foaming agents, and/or dispersants. A “biosurfactant” is a surface-active substance produced by a living cell and/or derived from naturally-occurring substrates.

The transitional term “comprising,” which is synonymous with “including,” or “containing,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Use of the term “comprising” contemplates other embodiments that “consist” or “consist essentially of’ the recited component(s).

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “and” and “the” are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All references cited herein are hereby incorporated by reference in their entirety.

Cleaning Compositions

The subject invention provides environmentally-friendly, non-toxic compositions and methods for removing highly viscous polymeric substances from a surface. Specifically, the subject invention provides cleaning compositions comprising microbial surfactants, or biosurfactants, as well as methods of their use for removing highly viscous polymeric substances on the surfaces of, for example, reservoirs, valves, pipelines, tubes, pumps, containers, tanks, drains, and other equipment. In certain embodiments, the compositions and methods can also enhance or maintain oil recovery efficiency.

In certain embodiments, the compositions and methods can be useful for removing highly viscous polymeric substances on equipment, such as, for example, tubing, pipes, pipelines, drills, pumps, valves and tanks associated with all aspects of oil and/or gas production. This can be achieved via, for example, emulsification. The removing can comprise mechanical or manual techniques or draining.

In certain embodiments, the compositions and methods can be useful for emulsifying highly viscous polymeric substances and/or for maintaining an emulsification of highly viscous polymeric substances.

In certain embodiments, the compositions and methods function surprisingly better than chemical surfactants at emulsifying highly viscous polymeric substances. Thus, in one embodiment, the subject invention can replace, or reduce the amount used of, compositions that utilize synthetic or chemical surfactants. Furthermore, the subject methods can reduce or replace the need for physical cleaning of highly viscous polymeric substances from a surface.

The biosurfactants utilized according to the subject invention can be one or more glycolipids such as, for example, rhamnolipids, rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates, trehalose monomycolates, mannosylerythritol lipids, cellobiose lipids, ustilagic acids and/or sophorolipids. In one embodiment, the biosurfactants comprise one or more lipopeptides, such as, for example, surfactin, iturin, fengycin, arthrofactin, viscosin, amphisin, syringomycin, and/or lichenysin.

In certain preferred embodiments, the biosurfactants are glycolipids selected from sophorolipids and rhamnolipids.

In certain embodiments, the cleaning composition comprises components that were produced as the result of the growth of microorganisms or other cell cultures. Thus, the composition may comprise the microbes themselves and/or by-products of microbial growth. In some embodiments, the glycolipids are purified from the cell culture. In some embodiments, they are utilized in crude form, wherein the crude form comprises residual cells, fermentation broth, growth by-products, and/or nutrients from fermentation. The microbes may be in a vegetative state, in spore form, in mycelial form, in any other form of propagule, or a mixture of these. The microbes may be planktonic or in a biofilm form, or a mixture of both. The by-products of growth may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. The microbes may be intact or lysed, active or inactive. In some embodiments, the microbes are present, with medium in which they were grown, in the composition. The microbes may be present at, for example, a concentration of 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , 1 x 10 ⁹ , 1 x 10 ¹⁰ , 1 x 10 ¹¹ , 1 x 10 ¹² , or 1 x 10 ¹³ (or any concentration there-between) or more CFU per milliliter of the composition. If present, the microbe cells are preferably inactivated prior to use by, for example, heat inactivation.

In certain embodiments, use of fermentation products according to the subject invention can be superior to, for example, purified microbial metabolites alone, due to, for example, the advantageous properties of the microbial cell walls. These properties include high concentrations of mannoprotein as a part of yeast cell wall’s outer surface (mannoprotein is a highly effective bioemulsifier) and the presence of biopolymer beta-glucan (also an effective emulsifier) in yeast cell walls. Additionally, the fermentation product can further comprise biosurfactants capable of reducing both surface and interfacial tension, enzymes and other metabolites (e.g., lactic acid, ethyl acetate, ethanol, etc.) that are capable of removing highly viscous polymeric substances from a surface.

In certain embodiments, the glycolipid biosurfactant is a sophorolipid (SLP). Sophorolipids are glycolipid biosurfactants produced by, for example, various yeasts of the Starmerella clade when cultivated in the presence of a hydrocarbon-based source of one or more fatty acids. SLP typically consist of a disaccharide sophorose linked to long chain hydroxy fatty acids. They can comprise a partially acetylated 2-O-P-D-glucopyranosyl-D-glucopyranose unit attached p-glycosidically to 17-L- hydroxyoctadecanoic or 17-L-hydroxy-A9-octadecenoic acid. The hydroxy fatty acid is generally 16 or 18 carbon atoms, and may contain one or more unsaturated bonds. Furthermore, the sophorose residue can be acetylated on the 6- and/or 6’-position(s). The fatty acid carboxyl group can be free (acidic or linear form (General Formula 2)) or internally esterified at the 4"-position (lactonic form (General Formula 1)). 5. bombicola produces a specific enzyme, called S. bombicola lactone esterase, which catalyzes the esterification of linear SLP to produce lactonic SLP.

In preferred embodiments, the SLP according to the subject invention are represented by General Formula ( 1 ) and/or General Formula (2), and are obtained as a collection of 30 or more types of structural homologues:

(i) where R ¹ and R ¹ independently represent saturated hydrocarbon chains or single or multiple, in particular single, unsaturated hydrocarbon chains having 8 to 20, in particular 12 to 18 carbon atoms, more preferably 14 to 18 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups, R ² and R ² independently represent a hydrogen atom or a saturated alkyl functional group or a single or multiple, in particular single, unsaturated alkyl functional group having 1 to 9 carbon atoms, more preferably 1 to 4 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups, and R ³ , R ³ , R ⁴ and R ⁴ independently represent a hydrogen atom or -COCH3.

The composition utilized according to the subject methods can comprises more than one form of SLP, including linear SLP and lactonic SLP. The SLP can be non-acetylated, monoacetylated and/or di-acetylated SLP. In certain embodiments, the subject compositions can comprise lactonic and linear SLP, with at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the SLP comprising linear forms, and the remainder comprising lactonic forms.

In certain embodiments, the glycolipid is a rhamnolipid. Rhamnolipids comprise a glycosyl head group (i.e., a rhamnose) moiety, and a 3-(hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail, such as, e.g., 3-hydroxydecanoic acid. Two main subtypes of rhamnolipids exist, mono- and dirhamnolipids, which comprise one or two rhamnose moieties, respectively. The HAA moiety can vary in length and degree of branching, depending on, for example, the growth medium and the environmental conditions. The highest accumulation of rhamnolipids (RLP) has been shown by submerged cultivation of Pseudomonas spp., such as P. aeruginosa.

Rhamnolipids according to the subject invention can have the following structure, according to General Formula (3): wherein m is 2, 1 or 0, n is 1 or 0,

R ¹ and R ² are, independently of one another, the same or a different organic functional group having 2 to 24, preferably 5 to 13 carbon atoms, in particular a substituted or unsubstituted, branched or unbranched alkyl functional group, which can also be unsaturated, wherein the alkyl functional group is a linear saturated alkyl functional group having 8 to 12 carbon atoms, or is a nonyl or a decyl functional group or a mixture thereof.

Salts of these compounds are also included according to the invention. In the present invention, the term “di-rhamnolipid” is understood to mean compounds of the above formula or the salts thereof in which n is 1. Accordingly, “mono-rhamnolipid” is understood in the present invention to mean compounds of the general formula or the salts thereof in which n is 0. In certain specific embodiments, the composition comprises a mixture of mono- and di-rhamnolipids. In some embodiments, the subject compositions have enhanced highly viscous polymeric substances cleaning properties over chemical or synthetic surfactants due to the small size of the glycolipid biosurfactants and/or micelles formed by the biosurfactants. The small size of the micelles allows for penetration of the composition into nano-sized spaces and pores on surfaces that may otherwise be passed over by larger-sized synthetic materials. For example, in some embodiments, the size of the glycolipid biosurfactant micelle is less than about 500 nm, preferably less than about 100 nm, and more preferably, less than about 5 nm. In one example, the glycolipid has a micelle size of about 1 nm to about 5 nm.

In preferred embodiments, the biosurfactants are isolated from the microbial culture in which they were produced and purified to at least 90%, more preferably at least about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, or about 98% purity. In certain embodiments the concentration of the biosurfactant(s) is at or above minimum inhibitory concentration, which can range from 1 to 20 ppm, 5 ppm to about 15,000 ppm, about 10 ppm to about 10,000 ppm, or about 100 ppm to about 600 ppm SLP, relative to the total fluid being applied and/or treated.

In certain embodiments, the composition can further comprise other substances that are useful for emulsifying and/or cleaning and/or that are useful for enhancing oil recovery, such as, for example, solvents, non-biological surfactants, enzymes, polymers, carriers and/or diluents, and/or chelating agents. Preferably, however, the additional substances are non-toxic and/or biodegradable. These additional compounds can be added in amounts ranging from, for example, 0.001% to 20% or greater, by weight or volume.

In certain embodiments, chelating agents can be, but are not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), a phosphonate, succimer (DMSA), diethylenetriaminepentaacetate (DTPA), A-acetylcysteine, n- hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordination group (e.g., rubeanic acid), STPP (sodiumtripolyphosphate, Na5P3O10), trisodium phosphate (TSP), water, carbohydrates, organic acids with more than one coordination group (e.g., citric acid), lipids, steroids, amino acids or related compounds (e.g., glutathione), peptides, phosphates, nucleotides, tetrapyrrols, ferrioxamines, ionophores, orphenolics, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid diacetic Acid (GLDA), GLDA-Na4, methyl glycindiacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic P-diketone, and (14,16)-hentriacontanedione, ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-dimalonic acid (EDDM), 3-hydroxy-2,2-iminodisuccinic acid (HIDS), 2-hydroxyethyliminodiacetic acid (HEIDA), pyridine-2,6-dicarboxylic acid (PDA), trimethyl glycine (TMG), Tiron, or any combination thereof. In one embodiment, the subject compositions and methods can be used alongside and/or to enhance or supplement other compositions and methods substances for emulsifying and/or removal of highly viscous polymeric substances, e.g., other microbial, mechanical, thermal and/or chemical treatments.

In some embodiments, the methods can comprise applying the composition alongside a non- biological surfactant.

Surfactants are surface active agents having two functional groups, namely a hydrophilic (water-soluble) or polar group and a hydrophobic (oil-soluble) or non-polar group. The hydrophobic group is usually a long hydrocarbon chain (C8-C18), which may or may not be branched, while the hydrophilic group is formed by moieties such as carboxylates, sulfates, sulfonates (anionic), alcohols, polyoxyethylenated chains (nonionic) and quaternary ammonium salts (cationic).

Non-biological surfactants according to the subject compositions and methods include, but are not limited to: ethoxylated nonyl phenol phosphate esters, alkyl glucoside, alkyl phosphonium chloride, alkyl phosphonate surfactants, linear alcohols, nonylphenol compounds, quaternary amine, alkyoxylated fatty acids, alkylphenol alkoxylates, ethoxylated amides, methyl ester sulfonates, hydrolyzed keratin, sulfosuccinates, taurates, trimethyltallowammonium chloride, trimethylcocoammonium chloride, quaternary alkyl ammonium chloride, propargyl alcohol, acetylenic alcohol, phosphate esters, imidazolines, amine salts, amide salts, amine oxides, alkoxylated alcohols, lauryl alcohol ethoxylate, ethoxylated nonyl phenol, ethoxylated fatty amines, ethoxylated alkyl amines, cocoalkylamine ethoxylate, modified betaines, alkylamidobetaines, cocoamidopropyl betaine, sulfonated olefins, anionic surfactants, ammonium lauryl sulfate, sodium lauryl sulfate (also called SDS, sodium dodecyl sulfate), alkyl-ether sulfates sodium laureth sulfate (also known as sodium lauryl ether sulfate (SLES)), sodium myreth sulfate; docusates, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, linear alkylbenzene sulfonates (LABs), alkyl-aryl ether phosphates, alkyl ether phosphate; carboxylates, alkyl carboxylates (soaps), sodium stearate, sodium lauroyl sarcosinate, carboxylate-based fluorosurfactants, perfluorononanoate, perfluorooctanoate; cationic surfactants, pH-dependent primary, secondary, or tertiary amines, octenidine dihydrochloride, permanently charged quaternary ammonium cations, alkyltrimethylammonium salts, cetyl trimethylammonium bromide (CTAB) (a.k.a. hexadecyl trimethyl ammonium bromide), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-Bromo-5-nitro-l,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldi-methylammonium bromide (DODAB); zwitterionic (amphoteric) surfactants, sultaines CHAPS (3-[(3- Cholamidopropyl)dimethylammonio]- 1 -propanesulfonate), cocamidopropyl hydroxysultaine, betaines, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins; nonionic surfactants, ethoxylate, long chain alcohols, fatty alcohols, cetyl alcohol, stearyl alcohol, cetosteaiyl alcohol, oleyl alcohol, polyoxyethylene glycol alkyl ethers (Brij): CH3 (CH2)10- 16-(O-C2H4)l-25-OH (octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether), polyoxypropylene glycol alkyl ethers: CH3-(CH2)10 16 (O-C3H6)l-25-OH, glucoside alkyl ethers: CH3-(CH2)10-16-(O-Glucoside)l-3-OH (decyl glucoside, lauryl glucoside, octyl glucoside), polyoxyethylene glycol octylphenol ethers: C8H17- (C6H4)-(O-C2H4)l-25-OH (Triton X-100), polyoxyethylene glycol alkylphenol ethers: C9H19- (C6H4)-(O-C2H4)1 25 OH (nonoxynol-9), glycerol alkyl esters (glyceryl laurate), polyoxyethylene glycol sorbitan alkyl esters (polysorbate), sorbitan alkyl esters (spans), cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, copolymers of polyethylene glycol and polypropylene glycol (poloxamers), and polyethoxylated tallow amine (POEA).

Anionic surfactants contain anionic functional groups at their head, such as sulfate, sulfonate, phosphate, and carboxylates. Prominent alkyl sulfates include ammonium lauryl sulfate, sodium lauryl sulfate (also called SDS, sodium dodecyl sulfate) and the related alkyl-ether sulfates sodium laureth sulfate, also known as sodium lauiyl ether sulfate (SLES), and sodium myreth sulfate. Carboxylates are the most common surfactants and comprise the alkyl carboxylates (soaps), such as sodium stearate.

Surfactants with cationic head groups include: pH-dependent primary, secondary, or tertiary amines; octenidine dihydrochloride; permanently charged quaternary ammonium cations such as alkyltrimethylammonium salts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC); benzalkonium chloride (BAC); benzethonium chloride (BZT); 5-Bromo-5-nitro-l,3-dioxane; dimethyldioctadecylammonium chloride; cetrimonium bromide; and dioctadecyldi-methylammonium bromide (DODAB).

Zwitterionic (amphoteric) surfactants have both cationic and anionic centers attached to the same molecule. The cationic part is based on primaty, secondary, or tertiary amines or quaternary ammonium cations. The anionic part can be more variable and include sulfonates. Zwitterionic surfactants commonly have a phosphate anion with an amine or ammonium, such as is found in the phospholipids phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

A surfactant with a non-charged hydrophilic part, e.g., ethoxylate, is non-ionic. Many long chain alcohols exhibit some surfactant properties.

Other suitable additives include, for example, emulsifying agents, lubricants, buffering agents, solubility controlling agents, pH adjusting agents, preservatives and, stabilizers. In some embodiments, diluents or carrier fluids can be added to the composition to aid in high viscosity polymeric substance removal and, for example, reducing the viscosity of the high viscosity polymeric substance produced from the subject methods, by, for example, diluting the high viscosity polymeric substance removal. Diluents can include, for example, naphthas, gas condensates, light crude oil, synthetic crude oil, gasoline, kerosene, methyl tert-butyl ether (MTBE), tert-Amyl methyl ether (TAME), Dimethoxyethane (DME), alcohols (e.g., ethyl alcohol), ethane, propane, heptane, toluene, butanone, hydrotreated petroleum, light distillate, hydro-treated distillate that can comprise dodecane, tridecane, tetradecane, and undecane, fatty acid methyl ester, soy methyl esters, n-Butyl acetate, and minerals oils.

Use of Cleaning Compositions

In preferred embodiments, the subject invention provides a method for cleaning high viscosity polymeric substances from a surface by applying to a surface, a composition comprising one or more biosurfactant-producing microorganisms and/or biosurfactant(s). The composition can be prepared and mixed prior to being applied to the surface, or the individual components of the composition can be added separately to the container and mixed therein.

In certain embodiments, the high viscosity polymeric substance that can be cleaned include, for example, hydrolyzed polyacrylamide (HPAM) and polyacrylamide (PAM) including, for example, polyacrylamide emulsions, slurries, and dry polymers - cationic and anionic; emulsion type PAMs including, for example, KemFlow A-4251 , KemFlow A4355, KemFlow A-4356, KemFlow A-4358, KemFlow A-4361. KemFlow A-4366 (Kemira, Atlanta, Ga., USA); cationic substances, including, for example, FLOJET DR-7000, FLOJET DR-3046 (SNF, Riceboro, Ga., USA); anionic substances, including, for example, Sedifloc 320A, and Sedifloc, 331 A (3F Chimica, Charlotte, N.C., USA); cationic PAM emulsions including, for example, Alcomer-788 and Alcomer-889 (BASF, Florham Park, N.J., USA); solid (powder) PAMs include, for example, KemFlow A-5156, KemFlow A-5157, KemFlow A-5251, KemFlow A-5252. KemFlow A-5253, KemFlow A-5254, KemFlow A-5351 , KemFlow A-5352, KemFlow A-5353, KemFlow A-5354, KemFlow A-5356 (Kemira, Atlanta, Ga., USA); Sedifloc 7030HM; Sedifloc 7030HHM (3F Chimica, Charlotte, N.C., USA); drag reducers (alpha olefin polymers); alpha olefin homopolymers, including, for example, polyhexene- 1, polyoctene- 1, polyde-cene- 1 , polyhexadecene- 1, and polyeicosene-1; a-olefin copolymers, including, for example, hexene- 1 -dodecene- 1 copolymer, octene- 1 -tetradecene- 1 copolymer, polyhexene- 1 copolymer, polyoctene-1 copolymer, poly decene- 1 copolymer, poly dodecene- 1 copolymer, polytetradecene- 1 copolymer, hexene- 1 -dodecene- 1 copolymer, polyhexene- 1 , and polyoctene-1.

As used herein, “applying” a composition or product refers to contacting it with a target or site such that the composition or product can have an effect on that target or site. The effect can be due to, for example, the action of a biosurfactant. Compositions according to the subject invention can be used for cleaning pipes, tanks, tubes, rods, pumps, equipment and/or surfaces. For example, the cleaning composition can also be poured, sprayed, or injected into storage tanks and/or the piping, tubing, pumps, etc. associated with high viscosity polymeric substances.

In preferred embodiments, the cleaning compositions are applied to storage tanks. The composition of the subject invention may be applied directly to equipment. For example, the composition can be poured into the tank, or the inner surfaces of the tank can be sprayed or coated with the composition, to aid in the removal of high viscosity polymeric substances.

The equipment that can be remediated according to the subject invention includes all types and varieties of equipment associated with gas and oil recovery, transmission, transportation and processing where water-in-oil emulsions might occur, and particularly, where high viscosity polymeric substances accumulate. This includes, for example, well casings, pumps, rods, pipes, lines, tanks, separators, and the like. It is contemplated that the present composition may be used with all such equipment.

In one embodiment, the cleaning composition is mixed within the storage tank or on the surface for a period of time sufficient to emulsify the high viscosity polymeric substances and form an emulsion comprising the composition and the high viscosity polymeric substance components. Mixing can be performed using any mixing device, for example, an air pump to continuously pump air/gas throughout the tank, for example, in the form of bubbles. In one embodiment, the storage tank has a or uses a mixing system, such as, for example, rolling of the storage tank or surface, agitating the storage tank or surface, spraying the highly viscous polymeric substance and/or the cleaning composition, or any other method of mixing.

The amount of the highly viscous polymeric substance cleaning composition that can be added is preferably at a ratio of from 10:1, 9: 1, 8: 1, 7:1, 6:1, 5: 1, 4: 1, 3:1, 2:1, 1 :1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1:7, 1 :8, 1 :9, or 1: 10 units of composition to the highly viscous polymeric substance. In specific preferred embodiments, the composition to highly viscous polymeric substances ratio is 1 : 1 or 2: 1 .

Preferably, the mixing takes place for about 1 min, about 5 min, about 10 min, about 15 min, about 30 min, about 45 min, about 1 h, about 2 h, about 3 h, about 4 h, about 5 h, or about 6 h, or any time period within that range. Then, the emulsion can be allowed to sit for 1 min to 3 hours, up to 12 to 24 hours, or more, or until a single emulsion forms containing both the composition and the highly viscous polymeric substance. Preferably, the emulsion can be immediately removed from the container after mixing.

In one embodiment, a method of removing highly viscous polymeric substance from a surface or container, includes the steps of pouring, spraying, or injecting the composition into a storage tank or on a surface and allowing it to mix with the fluid that is already in the tank or on the surface. The composition can then optionally be circulated by, for example, a pump for about 1 min to about 1 week, about 5 min to about 72 h, preferably about 5 min to about 6 h. Prior to circulating, the composition may be allowed to sit for about 1 min to about 1 week, about 5 min to about 1 day, about 8 h to 1 day, for example. The setting time, circulating time and applied amount depend on the amount of the highly viscous polymeric substance present, as well as the size of the tank or surface.

In one embodiment, the highly viscous polymeric substance storage container is a large storage tank, for example, having a diameter of about 5 feet to about 500 feet. A portion of highly viscous polymeric substance can be removed from the large storage tank via robotic or manual methods (e.g., draining) and placed into a smaller storage tank, for example, a tank 1/3 or 1/2 the size of the large storage tank. Then, the methods according to the subject invention can be carried out in the smaller storage tank, which can reduce the time required for highly viscous polymeric substance removal.

In one embodiment, the method further comprises applying air to the composition and highly viscous polymeric substance after the composition has been applied to the highly viscous polymeric substance, by, for example, pumping the composition into a vessel containing the highly viscous polymeric substance.

Advantageously, in one embodiment, the container has less highly viscous polymeric substance remaining in it than it would have if it were not treated with the subject composition. Thus, the processing of the highly viscous polymeric substance can operate at a faster speed, and the overall process can take less time. Accordingly, the highly viscous polymeric substance can be cleaned in a matter of hours or minutes to be disposed of or reused in hydraulic fracturing operations. Furthermore, the residual highly viscous polymeric substance that is recovered from the container can be processed (e.g., additional highly viscous polymeric substances can be added to the recovered highly viscous polymeric substance) and/or used directly in hydraulic fracturing operations. At certain ratios of cleaning composition to highly viscous polymeric substance, the emulsion will phase separate upon sitting (i.e., settle) for at least about 30 min, about 1 h, about 2 h, about 3 h, about 6 h, about 9 h, about 12 h, about 16 h, about 1 day, about 2 days, about 3 days, or about 1 week, allowing a portion of the cleaning solution to be removed from the liquid containing the highly viscous polymeric substance. The cleaning composition can be recycled to clean a different vessel or surface, while the composition containing the highly viscous polymeric substance can be processed and/or used directly in hydraulic fracturing operations.

After the highly viscous polymeric substance emulsion has been removed from the storage tank or surface, the storage tank or surface and any remaining highly viscous polymeric substance therein can be treated at least 1, 2, 3, 4 5, 6, 7, 8, 9, or 10 more times with the cleaning composition. For example, the cleaning composition can be applied to the tank or surface, mixed, and then, optionally, allowed to sit and remove the remaining highly viscous polymeric substance.

In one embodiment, methods are provided for applying the cleaning composition to, for example, a borehole, oilfield, and/or oil and gas transportation, transmission and/or refineiy equipment. In certain embodiments, the methods are used to improve oil production, as well as maintenance of, for example, pipes, pumps, drains, drills, tanks and other structures and equipment involved in oil and/or gas production, transportation, storage and/or refining.

The application of the microbe-based products of the present invention can be performed during production, transportation, storage, and/or refining of crude oil. For example, the subject products can be applied to a wellbore at the site of hydraulic fracturing, e.g., at an oil field, where fracking fluid is injected into a geological formation. In certain embodiments, the cleaning compositions can be used in the flowback process to inhibit the buildup of a highly viscous polymeric substance in various tubing and tanks after the hydraulic fracturing process is complete by applying the cleaning composition to a surface or container.

In one embodiment, the method further comprises, after the removing the highly viscous polymeric substance from the surface or container, applying the emulsified highly viscous polymeric substance to an oil or gas well. Advantageously, in one embodiment, the highly viscous polymeric substance emulsion can be applied directly to the oil or gas well without any processing for use as a friction reducer or a single processing step comprising adding additional highly viscous polymeric substances to the recovered highly viscous polymeric substance can be performed. Thus, the highly viscous polymeric substance can be used for hydraulic fracturing instead of disposing of the highly viscous polymeric substance. Additionally, the lack of processing of the highly viscous polymeric substance can create a hydraulic fracturing process operating at a greater efficiency than if the highly viscous polymeric substance was disposed of or required further processing after removal from a surface or storage tank.

Advantageously, the volume of cleaning composition used to remove the highly viscous polymeric substance is less than with conventional cleaning compositions. The amount of hours to clean the surface or tank can also be reduced by reducing or eliminating the need for a person to go into a storage tank, reduce or eliminate the need for soaking the highly viscous polymeric substance in the cleaning composition, and reduced or eliminate any waste and transferring said waste.

Production of Biosurfactants

In one embodiment, the subject invention provides methods of producing a microbial metabolite by cultivating a microbe under conditions appropriate for growth and production of the metabolite; and, optionally, purifying the metabolite. In a specific embodiment, the metabolite is a biosurfactant. The metabolite may also be, for example, ethanol, lactic acid, beta-glucan, proteins, amino acids, peptides, metabolic intermediates, polyunsaturated fatty acids, and lipids. The metabolite content produced by the method can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70 %, 80 %, or 90%.

The microorganisms utilized according to the subject invention may be natural, or genetically modified microorganisms. For example, the microorganisms may be transformed with specific genes to exhibit specific characteristics. The microorganisms may also be mutants of a desired strain. As used herein, “mutant” means a strain, genetic variant or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion) as compared to the reference microorganism. Procedures for making mutants are well known in the microbiological art. For example, UV mutagenesis and nitrosoguanidine are used extensively toward this end.

In certain embodiments, the microbes are capable of producing amphiphilic molecules, enzymes, proteins and/or biopolymers. Microbial biosurfactants, in particular, are produced by a variety of microorganisms such as bacteria, fungi, and yeasts, including, for example, Agrobacterium spp. (e.g., A. radiobacter),' Arthrobacter spp.; Aspergillus spp.; Aureobasidium spp. (e.g., A. pullulans),' Azotobacter (e.g., A. vinelandii, A. chroococcum); Azospirillum spp. (e.g., A. brasiliensis),' Bacillus spp. (e.g., B. subtilis, B. amyloliquefaciens, B. pumillus, B. cereus, B. licheniformis, B. firmus, B. laterosporus, B. megaterium, NRRL B-67928, NRRL B-68031, ATCC PTA- 123459); Blakeslea'. Candida spp. (e.g., C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. torulopsis),' Clostridium (e.g., C. butyricum, C. tyrobutyricum, C. acetobutyr icum, and C. beijerinckii),' Campylobacter spp.; Comybacterium spp.; Cryptococcus spp.; Debaryomyces spp. (e.g., D. hanseniiy, Entomophthora spp.; Flavobacterium spp.; Gordonia spp.; Hansenula spp.; Hanseniaspora spp. (e.g., H. uvarum),' Issatchenkia spp; Kluyveromyces spp.; Meyerozyma spp. (e.g., M. guilliermondiiy, Mortierella spp.; Mycorrhiza spp.; Mycobacterium spp.; Nocardia spp.; Pichia spp. (e.g., P. anomala, P. guilliermondii, P. occidentalis, P. kudriavzevii); Phycomyces spp.; Phythium spp.; Pseudomonas spp. (e.g., P. aeruginosa, P. chlororaphis, P. putida, P. florescens, P. fragi, P. syringae); Pseudozyma spp. (e.g., P. aphidis),' Ralslonia spp. (e.g., R. eulropha),' Rhodococcus spp. (e.g., R. erythropolis)', Rhodospirillum spp. (e.g., R. rubrum)', Rhizobium spp.; Rhizopus spp.; Saccharomyces spp. (e.g., S. cerevisiae, S. boulardii sequela, S. torula)'. Sphingomonas spp. (e.g., .S' paucimobilis); Starmerella spp. (e.g., A bomhicola)'. Thraustochytrium spp.; Torulopsis spp.; Ustilago spp. (e.g., U. maydis).' Wickerhamomyces spp. (e.g., W. anomalus, NRRL Y-68030); Williopsis spp.; and/or Zygosaccharomyces spp. (e.g., Z. bailii).

In certain embodiments, the microorganism is a Starmerella spp. yeast and/or Candida spp. yeast, e.g., Starmerella (Candida) bombicola, Candida apicola, Candida batistae, Candida floricola, Candida riodocensis, Candida stellate and/or Candida kuoi. In a specific embodiment, the microorganism is Starmerella bombicola, e.g., strain ATCC 22214. These yeasts are known sophorolipid producers.

The microbe growth vessel used according to the subject invention can be any fermenter or cultivation reactor for industrial use. In one embodiment, the vessel may have functional controls/sensors or may be connected to functional controls/sensors to measure important factors in the cultivation process, such as pH, oxygen, pressure, temperature, agitator shaft power, humidity, viscosity and/or microbial density and/or metabolite concentration.

In a further embodiment, the vessel may also be able to monitor the growth of microorganisms inside the vessel (e.g., measurement of cell number and growth phases). Alternatively, samples may be taken from the vessel for enumeration, purity measurements, biosurfactant concentration, and/or visible oil level monitoring. For example, in one embodiment, sampling can occur every 24 hours.

The microbial inoculant according to the subject methods preferably comprises cells and/or propagules of the desired microorganism, which can be prepared using any known fermentation method. The inoculant can be pre-mixed with water and/or a liquid growth medium, if desired.

In certain embodiments, the cultivation method utilizes submerged fermentation in a liquid growth medium. In one embodiment, the liquid growth medium comprises a carbon source. The carbon source can be a carbohydrate, such as glucose, dextrose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; fats and oils such as canola oil, soybean oil, rice bran oil, olive oil, com oil, sunflower oil, sesame oil, and/or linseed oil; powdered molasses, etc. These carbon sources may be used independently or in a combination of two or more. In preferred embodiments, a hydrophilic carbon source, e.g., glucose, and a hydrophobic carbon source, e.g., oil or fatty acids, are used.

In one embodiment, the liquid growth medium comprises a nitrogen source. The nitrogen source can be, for example, yeast extract, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride. These nitrogen sources may be used independently or in a combination of two or more.

In one embodiment, one or more inorganic salts may also be included in the liquid growth medium. Inorganic salts can include, for example, potassium dihydrogen phosphate, monopotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate, calcium nitrate, magnesium sulfate, sodium phosphate, sodium chloride, and/or sodium carbonate. These inorganic salts may be used independently or in a combination of two or more.

In one embodiment, growth factors and trace nutrients for microorganisms are included in the medium. This is particularly preferred when growing microbes that are incapable of producing all of the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. Furthermore, sources of vitamins, essential amino acids, proteins and microelements can be included, for example, com flour, peptone, yeast extract, potato extract, beef extract, soybean extract, banana peel extract, and the like, or in purified forms. Amino acids such as, for example, those useful for biosynthesis of proteins, can also be included.

The method of cultivation can further provide oxygenation to the growing culture. One embodiment utilizes slow motion of air to remove low-oxygen containing air and introduce oxygenated air. The oxygenated air may be ambient air supplemented daily through mechanisms including impellers for mechanical agitation of the liquid, and air spargers for supplying bubbles of gas to the liquid for dissolution of oxygen into the liquid. In certain embodiments, dissolved oxygen (DO) levels are maintained at about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, or about 50% of air saturation.

In some embodiments, the method for cultivation may further comprise adding additional acids and/or antimicrobials in the liquid medium before and/or during the cultivation process. Antimicrobial agents or antibiotics (e.g., streptomycin, oxytetracycline) are used for protecting the culture against contamination. In some embodiments, however, the metabolites produced by the yeast culture provide sufficient antimicrobial effects to prevent contamination of the culture.

In one embodiment, prior to inoculation, the components of the liquid culture medium can optionally be sterilized. In one embodiment, sterilization of the liquid growth medium can be achieved by placing the components of the liquid culture medium in water at a temperature of about 85-100°C. In one embodiment, sterilization can be achieved by dissolving the components in 1 to 3% hydrogen peroxide in a ratio of 1:3 (w/v).

In one embodiment, the equipment used for cultivation is sterile. The cultivation equipment such as the reactor/vessel may be separated from, but connected to, a sterilizing unit, e.g., an autoclave. The cultivation equipment may also have a sterilizing unit that sterilizes in situ before starting the inoculation. Gaskets, openings, tubing and other equipment parts can be sprayed with, for example, isopropyl alcohol. Air can be sterilized by methods know in the art. For example, the ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the medium may be pasteurized or, optionally, no heat at all added, where the use of pH and/or low water activity may be exploited to control unwanted microbial growth. The pH of the culture should be suitable for the microorganism of interest. In some embodiments, the pH is about 2.0 to about 7.0, about 3.0 to about 5.5, about 3.25 to about 4.0, or about 3.5. Buffers, and pH regulators, such as carbonates and phosphates, may be used to stabilize pH near a preferred value. In certain embodiments, a base solution is used to adjust the pH of the culture to a favorable level, for example, a 15% to 30%, or a 20% to 25% NaOH solution. The base solution can be included in the growth medium and/or it can be fed into the fermentation reactor during cultivation to adjust the pH as needed.

In one embodiment, the method of cultivation is carried out at about 5° to about 100° C, about 15° to about 60° C, about 20° to about 45° C, about 22° to about 35 °C, or about 24° to about 28°C. In one embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to changing temperatures.

According to the subject methods, the microorganisms can be incubated in the fermentation system for a time period sufficient to achieve a desired effect, e.g., production of a desired amount of cell biomass or a desired amount of one or more microbial growth by-products. The microbial growth by-product(s) produced by microorganisms may be retained in the microorganisms and/or secreted into the growth medium. The biomass content may be, for example from 5 g/1 to 180 g/1 or more, or from 10 g/1 to 150 g/1.

In certain embodiments, fermentation of the microbial culture occurs for about 100 to 150 hours, or about 115 to about 125 hours, or about 120 hours. The biosurfactants resulting from cultivation can be extracted and, optionally purified. Alternatively, the broth containing the biosurfactant and residual products of fermentation, including live, inactive and/or lysed microorganisms can be present.

EXAMPLES

A greater understanding of the present invention and of its many advantages may be had from the following examples, given by way of illustration. The following examples are illustrative of some of the methods, applications, embodiments and variants of the present invention. They are not to be considered as limiting the invention. Numerous changes and modifications can be made with respect to the invention.

EXAMPLE 1 - SHAKED DETERGENCY LAB TEST

2 g of FR-A1 (friction reducer Al ; referred to as Al) HVPAM polymeric emulsion were dosed into a 20 mL glass vial. 2 g or 4 g of the subject cleaning composition was added on the top of the emulsion. The glass vial was capped and moderately shaken for 1 min. Each vial was allowed to soak in an upright position for 30 mins at room temperature. After 30 min, the vial was turned upside down to compare mobility of the residue and the cleanliness of the inside surface of the vial. The liquid was drained, and vials were capped then placed on their side to take a photo of the bottom of the vial. The subject cleaning composition (e.g., AssurClean 2120S) had the best cleaning attributes, maintaining the emulsion but emulsifying the polymer to generate a low viscosity, free flowing fluid and cleaning the walls (Figure 3). The effective cleaning properties of the subject composition is maintained for FR-A1, FR-A2, FR-A3 FR-C1, and FR-C2 HVPAM polymeric emulsions (Figure 4).