EA041808B1 - MICROORGANISM PRODUCTS AND THEIR APPLICATIONS TO IMPROVE OIL PRODUCTION - Google Patents

Description

Cross-reference to related application

This application claims priority to US Provisional Application No. 62/483425, filed April 9, 2017, which is incorporated herein by reference in its entirety.

State of the art invention

High demand for fossil fuels requires efficient oil extraction. As oil wells mature, it becomes more difficult and costly to continue pumping oil at an economically viable rate. Thus, there is a need to develop improved oil recovery methods. One such mechanism uses microorganisms and their by-products.

Oil exists in small pores and narrow fissures in reservoir rock below the earth's surface. The natural pressure in the reservoir forces the oil to flow to the surface, thereby providing primary production; however, as oil is produced, reservoir pressure decreases to the point where artificial lift or pumping is required to maintain the optimum oil production rate.

When it is necessary to bring external energy to the reservoir to achieve additional oil recovery (secondary recovery), additional energy can be introduced by supplying gas (gas injection) and/or water (flooding). After several years of operation in the field, the injected fluids flow predominantly along high permeability layers, which cause these fluids to bypass oil-saturated areas in the reservoir. Thus, an increasing amount of water (or gas) rises with the oil, and due to the decrease in the oil-to-water ratio, it eventually becomes uneconomic to continue the process; during this time the deposit should be abandoned.

Primary recovery typically results in an average recovery of only the fraction of oil originally present in the reservoir. Secondary production, such as waterflooding, typically provides another 10% of production by the time it becomes uneconomic to continue. It is not uncommon to leave 60 to 70% of the oil originally present in the reservoir, even after secondary recovery reaches the economic limit. In this situation, the third stage of oil production, the so-called tertiary production, can be considered. This tertiary stage uses technically advanced techniques to modify the properties of either the formation fluids or the characteristics of the formation rocks. In general, methods can be divided into four main categories: thermal methods, chemical methods, solvent mixing or injection, and microbiological methods.

Microbiological reservoir enhancement (MEOR) is an interdisciplinary field including, but not limited to, geology, chemistry, microbiology, fluid mechanics, petroleum recovery technology, field simulation technology, and chemical engineering. MEOR uses microorganisms and/or their metabolites to enhance oil recovery. Microbiological methods used in MEOR can be directed to cleaning the wellbore to remove dirt and other debris that blocks the channels through which oil flows; well stimulation that improves the flow of oil from the drainage area into the wellbore; and increased water injection, which increases microbiological activity by introducing selected microorganisms and sometimes nutrients.

In MEOR, nutrients and suitable microorganisms that preferentially grow under the conditions of an anaerobic reservoir are introduced into the reservoir. By-products of microbial activity, which may include biological surfactants (bio-surfactants), biopolymers, acids, solvents, gases and enzymes, for example, may alter the properties of oil and the exchange between oil, water and porous media, alter the permeability of subterranean formations and ultimately increase mobility and oil production.

Interest in microbial surfactants has steadily increased in recent years due to their diversity, environmentally friendly nature, large-scale production capability, selectivity, performance under extreme conditions, and potential environmental applications. Microbiologically obtained surfactants, i.e. Bio-surfactants reduce the interfacial tension between water and oil, and therefore lower hydrostatic pressure is required to move the fluid trapped in the pores to overcome the capillary effect. Second, bio-surfactants promote the formation of micelles, which provide the physical mechanism for mobilizing oil in the moving water phase.

There is a continuing need for improved oil recovery methods, specific methods that can be sustainable over long periods of time. This includes the need for improved methods for increasing oil recovery, such as using, for example, microorganisms and/or their by-products. Bio-surfactants increase the emulsification of hydrocarbons, have the ability to solubilize hydrocarbon contaminants and increase their availability for degradation by microorganisms. These compounds can also be used to improve oil production.

Brief summary of the invention

In some embodiments, this invention provides microorganisms, as well as substances such as bio-surfactants, solvents and/or enzymes derived from these microorganisms,

- 1 041808 and the fermentation broth in which they are obtained. The claimed invention also provides methods for using these microorganisms and their by-products in improved oil recovery.

In particular, the present invention provides cost-effective, environmentally friendly approaches to improve oil production. Advantageously, these methods can be carried out over a wide range of temperatures, including from 20 to 70°C and above.

In some embodiments, the present invention provides materials and methods for improving oil recovery by treating an oil producing site, such as an oil reservoir or oil well, with microorganisms and/or their by-products. In one embodiment, the present invention may be useful for increasing oil production from an oil well by, for example, stimulating the flow of oil from the well while sediments are being dissolved within the formation.

In some embodiments, the present invention utilizes yeast by-products such as, for example, bio-surfactants. Bio-surfactants are useful in the oil and gas industry due to their ability to increase oil recovery. Bio-surfactants can alter the properties of the oil and the exchange between oil, water and the porous media in which the oil and gas are originally located, thereby increasing the mobility and hence the recovery of the oil. Thus, the compositions and methods of the present invention can increase the recovery of crude oil and natural gas from oil and gas containing formations by dramatically reducing both surface and interfacial tension between substances within the formations and altering the wettability of the formations.

In one embodiment, the present invention provides yeast fermentation products for enhancing oil recovery from an oil reservoir. In one embodiment, a yeast fermentation product is obtained by culturing a bio-surfactant-producing yeast using processes ranging from small to large scale. The culture process may be, for example, submerged culture, solid phase fermentation (SSF), and/or a combination thereof. In one embodiment, yeast products are grown using a simplified yeast fermentation technique that reduces culture time by 50% and reduces the addition of a carbon source.

A yeast fermentation product can be obtained by cultivating a biochemical producing yeast such as, for example, Pichia anomala (Wickerhamomyces anomalus). The fermentation broth after 7 days of cultivation at 25-30°C may contain a suspension of yeast cells and, for example, 4 g/l or more glycolipid bio-surfactants.

The yeast fermentation product can also be obtained by cultivating the bio-surfactant producing yeast, Starmerella bombicola. The fermentation broth after 5 days of cultivation at 25°C may contain a suspension of yeast cells and, for example, 150 g/l or more glycolipid bio-surfactants.

The yeast fermentation product may contain a fermentation broth separated from the yeast cells. In one embodiment, the bio-surfactant or other by-products in the broth are further separated from the broth and purified.

In some embodiments, the present invention uses strains of bacteria and their by-products. These by-products may include, for example, metabolites, polymers, biosurfactants, enzymes, organic acids, and solvents. In some embodiments, the bacteria are strains of Bacillus that thrive in high salt conditions, such as those often found in an oilfield. In some embodiments, the bacteria are surfactant-producing Bacillus strains, meaning that such strains are characterized by increased production of bio-surfactants compared to wild-type Bacillus strains. In certain embodiments, the Bacillus strains are characterized by increased production of the enzyme.

In one embodiment, the microorganisms are strains of Bacillus subtilis, Bacillus licheniformis and/or Bacillus amyloliquefaciens. In preferred embodiments, the bacteria are in the form of spores.

In some embodiments, strains of Bacillus subtilis are able to actively grow under low oxygen conditions, which promotes growth under microaerophilic and anaerobic conditions. Under anaerobic conditions, nitrate salts can be added to replace oxygen as an electron acceptor to support microaerophilic and/or anaerobic respiration.

In one embodiment, the strains of Bacillus subtilis are, for example, strains of B. subtilis var. locuses B1 and B2, which are effective producers of amphiphilic lipopeptide surfactin.

In one embodiment, the present invention provides a method for improving oil recovery by applying one or more microorganisms capable of producing beneficial biochemical by-products to an oil producing area, such as an oil reservoir.

- 2 041808 and/or oil well. The method optionally includes adding nutrients and/or other agents to the site. In some embodiments, the microorganisms are selected from strains

Bacillus, including but not limited to strains of Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens. In preferred embodiments, the bacteria are in the form of spores.

The method may also include the addition of a yeast fermentation product, such as a fermentation broth obtained from a culture of, for example, Starmerella bombicola or Wickerhamomyces anomalus. In one embodiment, the yeast cells can be removed from the yeast fermentation product and only the broth containing the bio-surfactant and other cell exudates is used. In one embodiment, the yeast fermentation product contains bio-surfactants that have been separated from the fermentation broth and purified.

The method may also include the use of microorganisms and/or microbial by-products with one or more alkaline compounds. The alkaline compound may be, for example, ammonium hydroxide.

In some embodiments, the implementation of the method may also include the use of microorganisms and/or by-products of vital activity of microorganisms with one or more polymeric compounds. The polymeric compounds may be selected from biopolymers such as, for example, hydrogels, polysaccharides, xanthan gum, guar gum, and cellulose polymers.

In some embodiments, the implementation of the method may also include the use of microorganisms and/or by-products of vital activity of microorganisms with one or more non-biological surfactants. Surfactants can be, for example, anionic, cationic, nonionic or zwitterionic.

In one embodiment, microorganisms and/or microbial by-products may be used with one or more chelating agents to reduce, for example, dissolve, sediments that have accumulated within the reservoir. These chelating agents may be, for example, citric acid, EDTA, sodium citrate, and/or combinations thereof.

In one embodiment, the microorganisms can grow and develop in situ within an oil reservoir or oil well and produce bio-surfactants therein. Therefore, a high concentration of bio-surfactants and bio-surfactant-producing microorganisms at a treatment site (eg, an oil well) can be achieved easily and continuously.

In some embodiments, the microbial products and methods of the present invention can additionally be used to remove wax, liquefy hard asphaltenes, and bioremediate hydrocarbon contaminated waters, soils, and other sites. For such applications, the methods may further include the addition of solvents, such as isopropyl alcohol or ethanol, with microorganisms and/or by-products of microorganisms.

In one embodiment, the present invention provides methods for producing a bio-surfactant by cultivating a microorganism strain of the present invention under conditions suitable for growth and production of the bio-surfactant and purifying the bio-surfactant. The present invention also relates to methods for obtaining solvents, enzymes or other proteins by culturing the microorganism strain of the present invention under conditions suitable for growth, obtaining solvents, enzymes or protein expression; and purifying the solvent, enzyme, or other protein.

The microbial products of the present invention can be used in a variety of unique settings due to, for example, the ability to efficiently deliver: fresh fermentation broth with active metabolites; cell mixture and fermentation broth; compositions with high cell density; products based on microorganisms that do not require time to obtain them; and microorganism-based products to remote locations.

Advantageously, the present invention can be used without releasing large amounts of inorganic compounds into the environment. In addition, the claimed compositions and methods use components that are biodegradable and toxicologically safe. Thus, the present invention can be used in oil and gas (and other industries) as an environmentally friendly treatment.

Detailed description of the invention

In some embodiments, this invention provides a microorganism, as well as substances such as bio-surfactants, solvents and/or enzymes obtained from these microorganisms, and the fermentation broth in which they are obtained. The claimed invention also provides methods for using these microorganisms and their by-products in improved oil recovery.

In particular, the present invention provides cost-effective, environmentally friendly approaches to improve oil recovery. Advantageously, these methods can be carried out over a wide range of temperatures, including from 20 to 70°C and above.

- 3 041808

In some embodiments, the present invention provides materials and methods for improving oil recovery by treating an oil producing site with microorganisms and/or their by-products. In one embodiment, the present invention may be useful for enhancing oil production from an oil reservoir or oil well by, for example, stimulating the flow of oil from the reservoir or well while sediments are being dissolved therein.

In some embodiments, the present invention utilizes yeast by-products such as, for example, bio-surfactants. Bio-surfactants are useful in the oil and gas industry due to their ability to increase oil recovery. Bio-surfactants can alter the properties of the oil and the exchange between oil, water and the porous media in which the oil and gas are originally located, thereby increasing the mobility and hence the recovery of the oil. Thus, the compositions and methods of the present invention can increase the recovery of crude oil and natural gas from oil and gas containing formations by dramatically reducing both surface and interfacial tension between substances within the formations and altering the wettability of the formations.

In specific embodiments, the methods and compositions described herein use microorganisms to improve oil recovery. Microorganisms can improve the quantity and quality of oil produced from oil reservoirs, including those considered mature. In addition, microorganisms can remove toxic substances from oil production sites.

In some embodiments, the present invention provides materials and methods for improving oil recovery by treating an oil producing site with microorganisms and/or their by-products. In one embodiment, the present invention may be useful for enhancing oil production from an oil reservoir or oil well by, for example, stimulating the flow of oil from the well or reservoir.

In one embodiment, the present invention provides a method for improving oil recovery by applying one or more microorganisms capable of producing useful biochemical by-products to an oil producing area, such as an oil reservoir and/or an oil well. The method optionally includes adding nutrients and/or other agents to the site. In some embodiments, the microorganisms are selected from Bacillus strains, including, but not limited to, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens strains. In preferred embodiments, the bacteria are in the form of spores.

In some embodiments, the bacteria are strains of Bacillus that thrive in high salt conditions, such as those often found in an oilfield. In some embodiments, the bacteria are surfactant-producing Bacillus strains, meaning that such strains are characterized by increased production of bio-surfactants compared to wild-type Bacillus strains. In certain embodiments, the Bacillus strains are characterized by increased production of the enzyme.

The method may also include the addition of yeast fermentation products, such as a fermentation broth obtained from the cultivation of yeast producing biochemicals, such as Starmerella bombicola or Wickerhamomyces anomalus. In one embodiment, the yeast fermentation product contains purified bio-surfactants produced by the yeast.

The method may also include the use of microorganisms with one or more alkaline compounds, polymers, surfactants and/or chelating agents.

Selected definitions.

As used herein, the term microorganism-based composition means a composition that contains components that have been derived from the activity of microorganisms or other cell cultures. Thus, the composition based on microorganisms may contain the microorganisms themselves and/or by-products of vital activity of microorganisms. The microorganisms may be in a vegetative state, in the form of spores, in the form of mycelium, in any other form of propagules, or mixtures thereof. The microorganisms may be in the form of plankton or in the form of a biofilm or a mixture of both. By-products can be, for example, metabolites (eg, bio-surfactants), cell membrane components, expressed proteins, and/or other cellular components. Microorganisms may be whole or lysed. Cells may be absent or cells may be present, for example, at a concentration of 1x10 ⁴ , 1x105, 1x10, 1x10 ⁷ , 1x10 ⁸ , 1x10 ⁹ , 1x10 ¹⁰ or 1x1011 or more cells or propagules per ml of composition. As used herein, the term propagule means any part of a microorganism from which a new and/or mature organism can develop, including but not limited to cells, conidia, cysts, spores (e.g., reproductive spores, endospores, and exospores), mycelium, buds, and seeds. them.

The claimed invention further provides products based on microorganisms, which are products that must be applied in practice to achieve the desired result. The microbial product may simply be a composition

- 4 041808 on the basis of microorganisms, collected in the process of cultivation of microorganisms. Alternatively, the microorganism-based product may contain additional ingredients that have been added. Said additional ingredients may include, for example, stabilizers, buffers, suitable carriers such as water, saline solutions or any other suitable carrier, added nutrients to support the continued viability of microorganisms, non-nutrient growth enhancers such as plant hormones, and /or agents that facilitate the tracking of microorganisms and/or compositions in the environment in which they are used. The microbial product may also contain mixtures of microbial compositions. The microorganism-based product may also contain one or more microorganism-based composition components that have been processed in some way, such as, but not limited to, filtration, centrifugation, lysis, drying, purification, and the like.

As used herein, the term harvested refers to the removal of part or all of the microbial composition from the growth vessel.

In some embodiments, the implementation of the microorganisms used in accordance with the claimed invention, are superproducers of surfactants. For example, the strain may produce at least 0.1-10 g/l, such as 0.5-1 g/l of surfactant. For example, bacteria produce at least 10%, 25%, 50%, 100%, 2 times, 5 times, 7.5 times, 10 times, 12 times, 15 times or more compared to other strains of microorganisms for oil production . Specifically, Bacillus subtilis ATCC 39307 is used herein as a reference strain.

As used herein, the term isolated or purified nucleic acid molecule, polynucleotide, polypeptide, protein, or organic compound such as a small molecule (such as those described below) is substantially free of other compounds, such as cellular material, with which it is bound in a natural state. The term isolated in relation to a strain of a microorganism means that the strain is isolated from the environment in which it exists in its natural state. Thus, the isolated strain may exist as, for example, a biologically pure culture or as spores (or other forms of the strain) in combination with a carrier. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain genes or sequences that flank it in its natural state. A purified or isolated polypeptide does not contain amino acids or sequences that flank it in its natural state.

In certain embodiments, the purified compounds comprise at least 60 wt.% (dry weight) of the compound of interest. Preferably, the drug comprises at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is a compound that is at least 90 wt.%, 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 98 wt.%, 99 wt. .% or 100 wt.% of the desired compound by weight. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC).

The term metabolite refers to any substance produced in metabolism, or a substance required to participate in a particular metabolic process. The metabolite can be an organic compound that is a starting material (eg glucose), an intermediate (eg acetyl-CoA) or an end product (eg n-butanol) of metabolism. Examples of metabolites may include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, trace elements, amino acids, polymers, and surfactants.

Modulation refers to change (increase or decrease). Such changes are detected by standard methods known in the art, such as those described herein.

The ranges presented in this document are considered to be a shorthand for all values in the specified range. For example, a range of 1 to 50 is any value, combination of values, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, as well as all intermediate decimal values between the above 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 subranges, covered subranges are specifically provided that are within any endpoint of the range. For example, a covered subrange of the exemplary range 1 to 50 may include 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.

The term reduces means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75% or 100%.

The term reference refers to a standard or control condition.

The term salt-tolerant means that the microorganism strain is capable of growing at a concentration

- 5 041808 sodium chloride 15% or more. In a specific embodiment, salt tolerant refers to the ability to grow at 150 g/l NaCl or more.

As used herein, the term biofilm is a complex aggregate of microorganisms, such as bacteria, in which cells are adhered to each other. Cells in biofilms are physiologically different from planktonic cells of the same organism, which are single cells that can float on the surface or float in the depth in a liquid culture medium.

As used herein, the term surfactant refers to a compound that reduces surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants act as detergents, wetting agents, emulsifiers, foaming agents and/or dispersants. A surfactant produced by microorganisms is called a bio-surfactant.

As used herein, the term oil production refers to any and all operations associated with the extraction of hydrocarbons, such as crude oil or natural gas, from a reservoir, its subsequent processing and use by consumers. Oil production may include, but is not limited to drilling, pumping, production, pumping, processing, processing, transporting and storing hydrocarbons.

The term oil producing site refers to any environment or structure, whether natural or man-made, in which one or more hydrocarbon, oil and/or natural gas production processes take place, including, but not limited to, subterranean oil reservoirs, oil and gas reservoirs, wells and wellbores. them.

As used herein, the term precipitation refers to accumulations formed, for example, by deposits of precipitated mineral salts, which may result from, for example, changes in the pressure, composition and/or temperature of crude oil. Precipitation may result from precipitation, for example, barium sulfate, calcium carbonate, strontium sulfate, calcium sulfate, sodium chloride, silica, iron sulfide, iron oxides, iron carbonate, silicates, phosphates and oxides, or any of a number of compounds that are insoluble or slightly soluble in water.

As used herein, the term improving oil production includes increasing the production of oil and hydrocarbons, and also means increasing the amount of hydrocarbons produced and/or increasing the rate at which they are produced, for example, by stimulating the flow of oil from the well.

The transitional term containing, which is synonymous with comprising or containing, is inclusive or open, and does not exclude additional omitted elements or method steps. In contrast, the transition phrase consisting of excludes any element, step, or ingredient not listed in the claims. The transitive phrase consisting essentially of limits the scope of the claims to the materials or steps indicated and those that do not materially affect the essential and novel feature(s) of the claimed invention.

Unless specifically stated or obvious from the context, the term or used in this document is understood to be inclusive. Unless specifically stated or obvious from the context, the terms used in this document in the singular also include the plural.

Unless specifically indicated or obvious from the context, the term used herein is roughly understood to be within the range of normal tolerances in the art, for example, within 2 standard deviations of the mean. The term can roughly be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0 .05% or 0.01% of the specified value.

The statement of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the groups specified in the list. The statement of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or parts thereof.

Any compositions or methods provided herein may be combined with one or more of any other compositions and methods provided herein.

Other features and advantages of the present invention will be apparent from the following description of its preferred embodiments and from the claims. All references cited in this document are incorporated herein by reference.

By-products of vital activity of microorganisms in accordance with the claimed invention.

In preferred embodiments, the composition is intended to improve and/or increase oil production from an oil production site, wherein the composition contains one or more microorganisms and/or their by-products. In specific embodiments, the implementation of microorganisms are capable of producing one or more bio-surfactants and are used for this.

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Bio-surfactants are a structurally diverse group of surfactants produced by microorganisms. Bio-surfactants are biodegradable and can be easily and cheaply produced using selected organisms on renewable substrates. Most bio-surfactant-producing organisms produce bio-surfactants in response to the presence of a hydrocarbon source (eg, oils, sugar, glycerol, etc.) in the growth medium. Other environmental components, such as iron concentration, can also significantly affect bio-surfactant production.

All bio-surfactants are amphiphilic. They consist of two parts: a polar (hydrophilic) fragment and a non-polar (hydrophobic) group. Due to their amphiphilic structure, bio-surfactants increase the surface area of hydrophobic water-insoluble substances, increase the bioavailability of such substances in water, and change the surface properties of bacterial cells.

Bio-surfactants accumulate at interfaces, thereby reducing interfacial tension and leading to the formation of aggregated micellar structures in solution. The ability of bio-surfactants to form pores and destabilize biological membranes allows them to be used as antibacterial, antifungal and hemolytic agents. Combined with characteristics such as low toxicity and biodegradability, bio-surfactants are advantageous for use in the oil and gas industry for a variety of applications. These applications include improving crude oil production, reducing oil viscosity, removing wax from rods, pipelines, liners and pumps, preventing corrosion of petroleum equipment, fracturing fluids, reducing the concentration of H2S in produced crude oil, and cleaning tanks, flow lines and pipelines, but not limited to them.

Safe, effective bio-surfactants of microorganisms reduce surface and interfacial tension between the molecules of liquids, solids and gases. As discussed herein, this activity can be very beneficial in the context of oil production.

Bio-surfactants obtained in accordance with the claimed invention can be used for other purposes not related to oil production, including, for example, the cleaning of pipelines, reactors and other mechanisms or surfaces.

Bio-surfactants include low molecular weight glycolipids (GL), lipopeptides (LP), flavolipids (FL), phospholipids, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. The fatty acid hydrocarbon chain acts as a normal lipophilic fragment of the bio-surfactant molecule, while the hydrophilic portion is formed by the ester or alcohol groups of neutral lipids, the carboxylate group of fatty acids or amino acids (or peptides), an organic acid in the case of flavolipids, or a carbohydrate in the case of glycolipids.

In one embodiment, the microbial bio-surfactants of the present invention include glycolipids such as rhamnolipids (RLP), sophorolipids (SLP), trehalose lipids, or mannosylerythritol (MEL) lipids.

In one embodiment, the microbial bio-surfactant is a lipopeptide such as, for example, surfactin or iturin A.

Bio-surfactant microorganisms are produced by various microorganisms such as bacteria, fungi and yeasts. Exemplary bio-surfactant producing microorganisms include Pseudomonas species (P. aeruginosa, P. putida, P. florescens, P. fragi, P. syringae); Pseudozyma (P. aphidis); Flavobacterium spp.; Pichia spp. (P. anomala, P. lynferdii, P. guilliermondii, P. sydowiorum), Bacillus spp. (B. subtilis, B. amyloliquefaciens, B. pumillus, B. cereus, B. licheniformis); Wickerhamomyces spp. (W. anomalus), Starmerella spp. (S. bombicold), Candida spp. (C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. torulopsis); Rhodococcus spp.; Arthrobacter spp.; Campylobacter spp.; Cornybacterium spp. and so on. Bio-surfactants can be produced by fermentation methods known in the art.

Cultivation of microorganisms in accordance with the claimed invention.

The claimed invention uses methods for cultivating microorganisms and producing microbial metabolites and/or other by-products of vital activity of microorganisms. The present invention further utilizes culture processes that are suitable for culturing microorganisms and obtaining microbial metabolites at the desired scale. Microorganism culture systems typically use submerged cultures; however, surface cultures and hybrid systems can also be used. As used herein, the term culture refers to growing cells under controlled conditions. Cultivation may be aerobic or anaerobic.

In one embodiment, the present invention provides materials and methods for producing biomass (e.g., viable cellular material), extracellular metabolites (e.g., small molecules and isolated proteins), residual nutrients, and/or intracellular components (e.g., enzymes and other proteins) .

The microorganism growth vessel used in accordance with the present invention may be any fermenter or culture reactor for industrial use. In one embodiment, the container may have functional elements

- 7 041808 controls/sensors or can be linked to functional controls/sensors to measure important factors in the cultivation process such as pH, oxygen, pressure, temperature, agitator power, humidity, viscosity and/or density of microorganisms and/or concentration of metabolites.

In a further embodiment, the container may also be capable of monitoring the growth of microorganisms within the container (eg, measuring the number of cells and growth phases). Alternatively, a daily sample may be taken from the vessel and counted by methods known in the art, such as dilution inoculation. Dilution inoculation is a simple method used to count the number of bacteria in a sample. With this method, it is also possible to calculate an index by which different media or processing methods can be compared.

In one embodiment, the method includes supplementing the culture with a source of nitrogen. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea and/or ammonium chloride. These nitrogen sources may be used independently or in combination of two or more.

The culture method can provide oxygenation to the growing culture. In one embodiment, slow air movement is used to remove low oxygen air and introduce oxygenated air. The oxygenated air may be ambient air replenished daily through mechanisms including turbine agitators to mechanically agitate the liquid and air distributors to introduce gas bubbles into the liquid to dissolve oxygen into the liquid.

The method may further include supplementing the culture with a carbon source. The carbon source is typically a carbohydrate such as glucose, 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 glycerin; fats and oils such as soybean oil, rice bran oil, olive oil, canola oil, coconut oil, corn oil, sesame oil and/or linseed oil. These carbon sources may be used independently or in combination of two or more.

In one embodiment, growth factors and micronutrients for microorganisms are included in the growth medium. This is particularly advantageous when cultivating microorganisms that are not capable of producing all the vitamins they need. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt, may also be included in the nutrient medium. In addition, sources of vitamins, essential amino acids and trace elements may be included, for example, in the form of flour or meal, such as cornmeal, or in the form of extracts, such as yeast extract, potato extract, beef extract, soybean extract, banana peel and the like or in purified forms. Amino acids, such as, for example, those useful for protein biosynthesis, may also be included, such as L-alanine.

In one embodiment, inorganic salts may also be included. Suitable inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate and/or carbonate sodium. These inorganic salts may be used independently or in combination of two or more.

In some embodiments, the culture method may further include adding additional acids and/or antimicrobials to the liquid nutrient medium before and/or during the culture process. Antimicrobial agents or antibiotics may be used to protect the culture from contamination. In addition, defoamers can be added to prevent the formation and/or accumulation of foam when gas is generated during cultivation.

The pH value of the mixture should be appropriate for the microorganism of interest. Buffers and pH adjusters such as carbonates and phosphates can be used to stabilize the pH at about the preferred value. When metal ions are present in high concentrations, it may be necessary to use a chelating agent in the liquid nutrient medium.

The method and equipment for cultivating microorganisms and producing microbial by-products can be performed in batch, semi-continuous or continuous culture processes.

Microorganisms can be grown in planktonic form or as a biofilm. In the case of a biofilm, the vessel may contain a substrate on which the microorganisms can be grown in the biofilm state. The system may also have, for example, the ability to apply stimuli (such as mechanical stimulation) that stimulate and/or improve biofilm growth characteristics.

In one embodiment, the method for cultivating microorganisms is carried out at a temperature of from about 5 to about 100°C, preferably from 15 to 60°C, more preferably

- 8 041808 from 25 to 50°C. In an additional embodiment, the cultivation can be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subjected to temperature changes.

In one embodiment, the equipment used in the culture method and process is sterile. Culture equipment such as a reactor/vessel may be separate from, but connected to, a sterilization device such as an autoclave. The culture equipment may also have a sterilization unit that sterilizes in situ prior to inoculation. Air can be sterilized by methods known in the art. For example, ambient air may pass through at least one filter before entering the container. In other embodiments, the culture medium may be pasteurized or optionally not heated at all, and low water activity and low pH may be used to control bacterial growth.

The content of biomass in the fermentation broth may be, for example, from 5 to 180 g/l or more. In one embodiment, the solids content of the broth is between 10 and 150 g/l.

In one embodiment, the present invention further relates to a method for producing microbial metabolites such as ethanol, lactic acid, β-glucan, proteins, peptides, intermediate metabolites, polyunsaturated fatty acids, and lipids. The content of the metabolite obtained by this method may be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

A microbial by-product produced by the microorganism of interest may be retained in the microorganism or secreted into the liquid nutrient medium. In another embodiment, the method for producing the microbial by-product may further comprise the steps of concentrating and purifying the microbial by-product of interest.

In a further embodiment, the liquid growth medium may contain compounds that stabilize the activity of the microbial by-product.

In one embodiment, the entire microorganism culture composition is removed at the completion of the culture (eg, after reaching the desired cell density or broth density of the indicated metabolite). In said batch culture mode, a completely new batch is started after receiving the first batch.

In another embodiment, only a portion of the fermentation product is removed at a time. In this embodiment, the viable cell biomass remains in the vessel as an inoculant for a new culture batch. The composition that is removed may be a cell-free broth or contain cells. Thus, a semi-continuous system is created.

Advantageously, the method does not require complex equipment or high energy consumption. The microorganisms of interest can be cultured on a small or large scale in situ and used even while still mixed with the growth medium. Likewise, microbial metabolites can also be produced in large quantities in the right place.

Advantageously, microorganism-based products can be produced in remote locations. The means for growing microorganisms can be operated off-grid using, for example, solar, wind and/or hydropower.

Microorganisms used in accordance with the systems and methods of the present invention may be, for example, bacteria, yeast and/or fungi. These microorganisms may be natural or genetically modified microorganisms. For example, microorganisms can be transformed with specific genes to display specific characteristics. The microorganisms can also be mutants of the desired strain. As used herein, the term mutant means a strain, genetic variant, or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation, or repeat expansion) compared to a reference microorganism. Procedures for obtaining mutants are well known in the field of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

In one embodiment, the microorganisms are bacteria, including Gram-positive and Gram-negative bacteria. The bacteria may be endospore-forming or exospore-forming bacteria. The bacteria can be, for example, Agrobacterium radiobacter, Alcanivora borkumensis, Azobacter (A. vinelandii, A. chroococcum), Azospirillum brasiliensis, Bacillus (for example, B. subtilis, B. licheniformis, B. firmus, B. laterosporus, B. megaterium, B. amyloliquifaciens), Clostridium (C. butyricum, C. tyrobutyricum, C. acetobutyricum, Clostridium NIPER 7 and C beijerinckii), Lactobacillus fermentum, Norcardia sp., Pseudomonas (P. chlororaphis subsp. aureofaciens (Kluyver), P. aeruginosa) , Rhizobium, Rhodospirillum rubrum. Sphingomonas paucimobilis, Ralstonia eulropha, Serratia marcescens and/or Tsukamurella sp.

- 9 041808

In preferred embodiments, the microorganism is a Bacillus strain selected from B. subtilis, B. amyloliquefaciens, and B. licheniformis. Even more preferably, the Bacillus strain is in the form of spores.

In certain embodiments, the present invention uses strains of Bacillus subtilis with increased production of bio-surfactants compared to wild-type Bacillus subtilis, as well as compared to other microorganisms used in oil production. In certain embodiments, the Bacillus subtilis strains increase the production of the biopolymer solvent and/or enzyme. Such Bacillus subtilis have been referred to as members of the B series, including but not limited to B1, B2 and B3.

In one embodiment, the microorganism is B. subtilis var. locuses B1 or B2, which are efficient producers of, for example, surfactin and other bio-surfactants, as well as biopolymers. This description incorporates international publication WO 2017/044953 A1 by reference to the extent that it corresponds to the disclosure of this application.

A culture of the microorganism B. subtilis B1 has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA. The deposit was assigned the depositary number АТСС РТА-123459 by the depository and the deposit was deposited on August 30, 2016.

The vegetative cells of the Bacillus subtilis B1 strain are rods that are 0.7 to 0.9 µm wide, 1.6 to 3.3 µm long and are isolated. It is motile, Gram positive, and produces biopolymers on nutrient agar and potato dextrose agar. It also produces ellipsoidal spores centrally or paracentrally in non-swollen sporangia. Mature spores are 0.8 to 1.0 µm wide and 1.6 to 1.9 µm long. After 16 hours at 40° C. on a nutrient agar plate, colonies on agar are cream/beige, embossed, mucoid, round, solid, smooth, shiny, and 3.0 to 7.0 mm in diameter. It is a facultative aerobe with a growth temperature range of 25-55°C, with an optimum growth temperature at 35°C. It hydrolyses starch, is positive in the Voges-Proskauer test, can use citrate, and can grow at 15% NaCl.

In certain embodiments, the Bacillus subtilis strains are salt tolerant. Salt tolerance can be with respect to any one or more salts. For example, the salt may be a monovalent salt such as a sodium or potassium salt, eg NaCl or KCl, or a divalent salt such as a magnesium or calcium salt, eg MgCl ₂ or CaCl ₂ , or a trivalent salt. Given the geographic location of the areas to be treated, salts of zinc, iron, bromine or lithium are present in the composition or area. In preferred embodiments, the bacteria described herein are tolerant of NaCl as well as the other salts mentioned above and are therefore widely used in oil recovery.

In some embodiments, strains of Bacillus subtilis are capable of actively growing under low oxygen conditions. In some embodiments, the Bacillus subtilis strain is grown under microaerophilic and anaerobic conditions. Under microaerophilic and/or anaerobic conditions, nitrate salts can be added as an electron acceptor to support anaerobic respiration.

Bacillus subtilis series B strain produced more bio-surfactants compared to Bacillus subtilis reference strains. In addition, strains of Bacillus subtilis survive under high salt and anaerobic conditions better than other known strains. The strains are also able to grow under anaerobic conditions. Series B strains of Bacillus subtilis can also be used to produce enzymes that break down or metabolize oil or other petroleum products.

In one embodiment, the claimed methods may use yeast or fungal fermentation products. Suitable yeast and fungal species for use in the present invention include, for example, Candida,

Saccharomyces (S. cerevisiae, S. boulardii sequela, S. torula), Issatchenkia, Kluyveromyces, Pichia, Wickerhamomyces (e.g. W. anomalus), Starmerella (e.g. S. bombicola), Rhodotorula (e.g. R. glutinous and R. graminus), Mycorrhiza, Mortierella, Phycomyces, Blakeslea, Thraustochytrium, Phythium, Entomophthora, Aureobasidium pullulans, Pseudozyma aphidis, Aspergillus and/or Rhizopus spp.

In one embodiment, the yeast is a killer yeast. As used herein, the term killer yeast refers to a strain of yeast characterized by the secretion of toxic proteins or glycoproteins against which the strain itself is immune. The exotoxins produced by killer yeast are capable of killing other strains of yeast, fungi, or bacteria. For example, microorganisms that can be controlled by killer yeast include Fusarium and other filamentous fungi. Such yeasts may include, Wickerhamomyces (e.g. W. anomalus), Pichia (e.g. P. anomala, P. guielliermondii, P. occidentalis, P. kudriavzevii), Hansenula, Saccharomyces, Hanseniaspora, (e.g. H. uvarum), Ustilago (eg, U. maydis), Debaryomyces hansenii, Candida, Cryptococcus, Kluyveromyces, Torulopsis, Williopsis, Zygosaccharomyces (eg, Z. bailii), and others, but are not limited to.

In one embodiment, a yeast fermentation product can be obtained by culturing a biochemical producing yeast such as, for example, Pichia anomala (Wickerhamomyces anomalus). Wickerhamomyces anomalus is often associated with food and cereal production and is an efficient producer of various solvents, enzymes, toxins as well as glycolipid bio-surfactants such as SLP. The fermentation broth after 7 days of cultivation at 25-30°C may contain a suspension of yeast cells and, for example, 4 g/l or more glycolipid bio-surfactants.

In one embodiment, a yeast fermentation product can also be obtained by culturing a bio-surfactant producing yeast, Starmerella bombicola. This species is an efficient producer of bio-surfactant glycolipids such as SLP. The fermentation broth after 5 days of cultivation at 25°C may contain a suspension of yeast cells and, for example, 150 g/l or more glycolipid bio-surfactants.

In one embodiment, the yeast fermentation product may contain a fermentation broth separated from the yeast cells. In one embodiment, the bio-surfactant or other by-products in the broth are further separated from the broth and purified.

Other microorganism strains, including, for example, other fungal strains capable of accumulating significant amounts of, for example, glycolipid or lipopeptide bio-surfactants or other metabolites, can be used in accordance with the present invention. Other metabolites useful in accordance with the present invention include mannoprotein, β-glucan and other substances that have bioemulsifying properties and help reduce surface/interfacial tension.

Preparation of products based on microorganisms.

One of the microbial products of the present invention is simply a fermentation broth containing the microorganism and/or metabolites of the microorganism produced by the microorganism and/or any residual nutrients. The fermentation product can be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard or extraction techniques known to those skilled in the art.

The microorganisms in a microorganism-based product may be in active or inactive form. Products based on microorganisms can be used without further stabilization, conservation and storage. Advantageously, the direct use of these microbial-based products maintains high microbial viability, reduces the potential for contamination by foreign agents and unwanted micro-organisms, and maintains the activity of microbial by-products.

The microorganisms and/or the broth resulting from the growth of the microorganisms can be removed from the growth vessel and transferred, for example, through a pipeline for immediate use.

Advantageously, according to the invention, the microbial product may contain a broth in which the microorganism has been grown. The product may be, for example, at least 1 wt.%, 5 wt.%, 10 wt.%, 25 wt.%, 50 wt.%, 75 wt.% or 100 wt.% broth. The amount of biomass in the product may be, for example, from 0 to 100 wt.%, including all percentages in the range between them.

In other embodiments, the composition (microorganisms, broth, or microorganisms and broth) can be placed in containers of appropriate size, taking into account, for example, the intended use, the intended method of use, the size of the fermentation vessel, and any method of transportation from the installation for growing the microorganism to the place of use. . Thus, the containers in which the microbial composition is placed can have a volume of, for example, 1 to 1000 gallons or more. In certain embodiments, the containers have a volume of 2 gallons, 5 gallons, 25 gallons or more.

Once the microbial composition has been obtained from the growth vessel, additional components may be added when the resulting product is containerized and/or piped (or otherwise transported for use). Additives may be, for example, buffers, carriers, other microbial compositions produced in the same or different plant, viscosity modifiers, preservatives, microbial growth nutrients, tracers, pesticides, and other ingredients specific to the intended use.

For example, up to 50 wt.% or more additives can be added as needed for specific applications, such as, for example, changing the levels of volatile organic compounds, increasing the penetration of the mixture, reducing the viscosity of the mixture, as connectors for insoluble solvents in the mixture, and to provide solvents. All additives must have a flash point greater than 100°F, preferably greater than 150°F, and more preferably 195°F of thermal catalytic combustion to achieve a final product ignition temperature of greater than 200°F.

- 11 041808

If desired, the product can be stored until use. The storage time is preferably short. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at a cool temperature such as, for example, less than 20, 15, 10 or 5°C. On the other hand, the bio-PVA composition can usually be stored at ambient temperature.

Local production of products based on microorganisms.

In preferred embodiments of the present invention, the microorganism growth facility produces novel, high-density micro-organisms and/or microbial by-products of interest at the desired scale. The microbial growth facility may be located at or near the application site. The facility produces high-density microbial compositions in batch, semi-continuous or continuous cultivation.

The claimed invention utilizes culture processes that range from small scale (eg laboratory plant) to large scale (eg industrial plant). These culture processes include, but are not limited to, submerged culture/fermentation, solid phase fermentation (SSF), and combinations thereof.

The microbial growth apparatus of the present invention produces novel microorganism-based compositions containing the microorganism itself, metabolites of the microorganism and/or other components of the broth in which the microorganism is grown. If desired, the compositions may have a high density of vegetative cells or a mixture of vegetative cells, spores, mycelium, conidia, or other microbial propagules.

Advantageously, the claimed microorganism-based products can be adapted for use in a given location. In one embodiment, the microbial growth facility is located at or near the site where the microbial products will be used. For example, the microorganism growth facility may be located less than 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from the point of use.

Since the microorganism-based product is produced locally, at or near the site of use, without resorting to the processes of microorganism stabilization, preservation, storage and transportation of conventional microbial production, a much higher density of live microbes can be obtained. Thus, a smaller volume of microbial product is required for on-site application. In addition, it allows the use of microorganisms with a higher density, where necessary to achieve the desired efficiency.

Advantageously, this allows the use of a smaller bioreactor (eg, a smaller fermentation tank and smaller stocks of feedstock, nutrients, pH adjusters and defoamers, etc.), making the system efficient and facilitating product portability. Local preparation of the microbial product also facilitates incorporation of the growth broth into the product, thus eliminating the need for cell stabilization or separation from the culture broth. The broth may contain agents formed during fermentation which are particularly well suited for topical application.

Locally produced, high-density and resistant cultures of microorganisms are more effective in the field than those that have undergone cell stabilization or have been in the supply chain for some time. The microorganism-based products of the present invention are particularly advantageous over conventional products in which cells, spores, mycelium, conidia or other microbial propagules have been separated from the metabolites and nutrients present in the fermentation broth. Reduced transport times allow new batches of microorganisms and/or their metabolites to be produced and delivered at the right time and in the amount required by local demand.

Predominantly, these microbial growth facilities provide a solution to the current problem of relying on large industrial manufacturers whose product quality suffers due to processing delays, supply chain bottlenecks, improper storage, and other contingencies that prevent on-time delivery. and using, for example, a viable cell-rich product and its associated broth and metabolites in which the cells were originally grown.

Microbial growth facilities provide manufacturing versatility through the ability to tailor microbial products to improve synergy with geographic destinations. Advantageously, in preferred embodiments, the systems of the present invention utilize the capabilities of naturally occurring microorganisms and their by-products of their metabolism to improve oil recovery. Indigenous microorganisms can be identified based on, for example, salt tolerance or ability to grow at high temperatures.

The culture time for individual containers can be, for example, 1 to 7 days or longer. The culture product can be obtained by any of a variety of methods.

- 12 041808

Local production and delivery within, for example, 24 hours after fermentation leads to pure compositions with high cell density and significantly reduces the cost of delivery. With the promise of rapidly advancing the development of more efficient and potent micro-organisms for inoculation, consumers will greatly benefit from this ability to rapidly deliver microbial-based products.

Methods for increasing oil production.

In some embodiments, the present invention provides materials and methods for improving oil recovery by treating an oil producing site, such as an oil reservoir or oil well, with microorganisms and/or their by-products. In one embodiment, the present invention may be useful for increasing oil production from an oil well by, for example, stimulating the flow of oil from the well while sediments are being dissolved within the formation.

As used herein, the term application of a composition or product refers to contact with a target or site such that the composition or product can affect that target or site. The effect may be due, for example, to the growth of microorganisms and/or the action of a bio-surfactant or other by-product of vital activity. For example, microorganism-based compositions or products may be introduced into oil wells and/or pipelines, casing, annulus between pipeline and casing, pumps, reservoirs, etc. associated with oil producing areas and oil reservoirs.

In one embodiment, the present invention provides a method for improving oil recovery by applying one or more microorganisms capable of producing beneficial biochemical by-products to an oil producing area, such as an oil reservoir and/or an oil well. The method optionally includes adding nutrients and/or other agents to the site. In preferred embodiments, the microorganism is a bio-surfactant-producing bacterial species.

In some embodiments, the microorganisms are selected from Bacillus strains, including, but not limited to, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens strains. In preferred embodiments, the bacteria are in the form of spores.

In one embodiment, the method further comprises adding nutrients and/or growth enhancers to promote the growth and development of microorganisms. For example, nutrients such as sources of carbon, nitrogen, magnesium, phosphorus and protein can be added. Growth enhancers such as L-alanine and manganese may also be added.

The method may also include the addition of a yeast fermentation product, such as a fermentation broth obtained from a culture of, for example, Starmerella bombicola or Wickerhamomyces anomalus. In one embodiment, the yeast is a bio-surfactant producing yeast. In one embodiment, the fermentation broth contains yeast by-products such as, for example, glycolipid bio-surfactants and other metabolites.

In one embodiment, the yeast cells can be removed from the yeast fermentation product and only the broth containing the bio-surfactant and other metabolites is used. In one embodiment, the yeast fermentation product contains bio-surfactants that have been separated from the fermentation broth and purified.

In some embodiments, the yeast fermentation products of the present invention have advantages over, for example, only bio-surfactants, including one or more of the following: high concentrations of mannoprotein as part of the outer surface of the yeast cell wall; the presence of β-glucan in yeast cell walls; and the presence of bio-surfactants and other metabolites (eg lactic acid, ethanol, ethyl acetate, etc.) in culture.

The method may also include the use of microorganisms and/or microbial by-products with one or more alkaline compounds. The alkaline compound may be, for example, ammonium hydroxide.

In some embodiments, the implementation of the method may also include the use of microorganisms and/or by-products of vital activity of microorganisms with one or more polymeric compounds. The polymeric compounds may be selected from biopolymers such as, for example, hydrogels, polysaccharides, xanthan gum, guar gum, and cellulose polymers.

In some embodiments, the implementation of the method may also include the use of microorganisms and/or by-products of vital activity of microorganisms with one or more non-biological surfactants. Surfactants can be, for example, anionic, cationic, nonionic or zwitterionic.

In one embodiment, microorganisms and/or microbial by-products may be used with one or more chelating agents to reduce, for example, dissolve, deposits that have accumulated within the reservoir. These chelating agents may be, for example, citric acid, EDTA and/or sodium citrate.

- 13 041808

In some embodiments, the microbial products and methods of the present invention can additionally be used to remove wax, liquefy hard asphaltenes, and bioremediate hydrocarbon contaminated waters, soils, and other sites.

In one embodiment, microbial based products are applied to a production well, including the surrounding formation. In this embodiment, the product may be poured or introduced into the wellbore casing (backlines) and allowed to mix with the fluid already present in the wellbore. When sufficient liquid is present, the composition can optionally be circulated, for example by means of a pump, for 24-72 hours, preferably 48-72 hours. The composition can be allowed to sink before circulation, for example, for 8-24 hours circulation and dosage depend on the depth and size of the well. The base starting dose may be 20 gallons of composition and at least about 5 gallons of composition per well on an intermittent basis, eg, biweekly, monthly, bimonthly, but not limited to.

In one embodiment, the microorganisms can grow and develop in situ and produce bio-surfactants in the oilfield. Therefore, a high concentration of bio-surfactants and bio-surfactant-producing microorganisms at a treatment site (eg, an oil well) can be achieved easily and continuously.

In one embodiment, it is desirable to introduce the composition through openings in the casing into the surrounding reservoir. The composition may be pressurized into the surrounding formation or, if the composition is allowed to sink to the bottom of the casing, the composition may infiltrate into the formation without additional pressure. The composition penetrates the formation, dissolving blockages in the formation to allow for more efficient oil and gas recovery.

In additional embodiments, the implementation of the composition of the present invention can be applied directly to the equipment. For example, prior to placing the rods and casing strings in gas and/or oil wells, these parts may be sprayed or impregnated with the composition. Parts may also be immersed in tanks filled with the composition.

The composition can be injected using injection pumps into offshore gas or oil wells to increase oil production. For pipeline treatment from 1-500 gallons to 1000 bbl, for example, 10,000 bbl or more of the composition can be applied to the composition at an injection rate of, for example, 1 to 20 gpm or 1 to 20 bpm.

The claimed treatment may be effective in a number of different geological formations. For example, the claimed invention may be useful in formations as deep as about 7000 feet or deeper and as shallow as about 1500 feet or less. In addition, the invention may be useful in formations having a range of porosity and/or permeability, for example, from about 0.1% to about 20% or more. The invention may also be useful in formations having a wide range of temperatures, pH and salinity.

In one embodiment, enhanced oil recovery is achieved by selective plugging, in which fluid flow through the formation is shifted from zones of high permeability of the formation to zones of moderate or low permeability. Drive efficiency can be increased, for example, by injecting water with force to pass through previously bypassed oil zones of the formation. Changes in the flow pattern can be achieved by increasing the microbial cell mass within the formation, for example by injecting the microorganism along with nutrients. Introduced nutrients and microorganisms preferentially flow into high permeability zones of the formation and as a result of cell growth, the biomass selectively plugs these zones to a greater extent than moderate or low permeability zones. In one embodiment, the microorganisms are introduced in spore form and they develop while inside the formation.

A method for increasing oil production using a flooding method using alkalis, surfactants and polymers (ASP).

In one embodiment, methods are provided for enhancing oil recovery, wherein the microorganism product of the claimed invention is applied to an oil production site in combination with one or more alkaline compounds, polymers, surfactants, or combinations thereof.

In surfactant flooding, by reducing the interfacial tension at the interface between oil and displacing water, as well as the interfacial tension between oil and rock at the interface, residual oil can be displaced and recovered.

In alkaline flooding, the reaction of alkaline compounds with organic acids in oil forms in situ natural surfactants that lower the water-oil interfacial tension.

In addition to surfactant and caustic flooding, polymers are used to increase the viscosity of the drive water to improve the efficiency of the oil drive.

ASP flooding is a process in which a combination of alkali, surfactant and polymer is introduced. ASP involves the introduction of a solution containing polymer, alkali and surfactant into a depleted or mature oil field in order to achieve an optimal chemical composition at high

- 14 041808 volumes of introduction for the minimum cost. The alkali-surfactant mixture forms an emulsion with the oil, which is then driven out of the formation using a polymer stimulus. ASP flooding improves microscopic displacement efficiency by reducing the interfacial tension (IFT) between water and oil by adding a surfactant to the water while adjusting the mobility of the oil and water by adding a polymer. Alkali is also added to the water to reduce surfactant adsorption in wall pores and to control local salinity to ensure minimum IFT and change rock wettability.

The use of microorganisms with surfactants in oil production.

In some embodiments, the oil recovery methods described herein use one or more micro-organisms and/or microbial by-products (eg, bio-surfactants) in combination with other compositions. In one embodiment, the other compositions are non-biological surfactants.

A surfactant (surfactant) molecule has two functional groups, namely a hydrophilic (water-soluble) or polar group and a hydrophobic (oil-soluble) or non-polar group. The hydrophobic group is typically a long hydrocarbon chain (C8C18) which may or may not be branched, while the hydrophilic group is formed by residues such as carboxylates, sulfates, sulfonates (anionic), alcohols, polyoxyethylated chains (nonionic) and Quaternary ammonium salts (cationic).

Surfactants work in ASP flooding to reduce interfacial tension (IFT) between entrained oil and brine to aid in mobilization and ensure the formation of oil rolls. Reducing the IFT reduces capillary forces and allows the oil roll to flow more freely without re-entrainment. The selection of an appropriate surfactant for oil recovery purposes is based on the ability to reduce IFT between crude oil and brine, thermal stability, resistance to salinity and brine hardness, solubility in brine, phase behavior parameters, adsorption testing under static and dynamic conditions, and displacement studies. in reservoir conditions.

Surfactants according to the claimed methods include anionic surfactants, ammonium lauryl sulfate, sodium lauryl sulfate (also referred to as SDS, sodium dodecyl sulfate), sodium laureth sulfate alkyl ether sulfates (also known as sodium laureth sulfate ether (SLES)), sodium myreth sulfate; docusates, sodium dioctylsulfosuccinate, perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, linear alkylbenzenesulfonates (LAB), alkylaryl ether phosphates, alkylether phosphate; carboxylates, alkyl carboxylates (soaps), sodium stearate, sodium lauryl sarcosinate, carboxylate-based fluorine-containing surfactants, perfluoronanoate, perfluorooctanoate; cationic surfactants, pH dependent primary, secondary or tertiary amines, octenidine dihydrochloride, permanently charged quaternary ammonium cations, alkyltrimethylammonium salts, cetyltrimethylammonium bromide (CTAB) (also known as hexadecyltrimethylammonium bromide), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC) , benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide (DODAB); zwitterionic (amphoteric) surfactants, sultaines CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate), cocamidopropyl hydroxysultaine, betaines, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins, ethoxylate, long chain alcohols, fatty alcohols, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, oleyl alcohol, polyoxyethylene glycol alkyl ethers (Brij) CH3-(CH2)10-16-(O-C2H4)1-25-OH (octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether), polyoxypropylene glycol alkyl ethers: CH3-(CH2 )10-16-(O-C3H6)1-25-OH, glucoside alkyl esters: CH3-(CH2)10-16-(O-glucoside)1-3-OH (decyl glucoside, lauryl glucoside, octyl glucoside), esters polyoxyethylene glycol octylphenol: C8H17 (C6H4) - (O-C2H4) 1-25-OH (triton X-100), polyoxyethylene glycol alkylphenol esters: C9H19- (C6H4) (O-C2H4) 1-25-OH (nonoxynol-9), glycerol alkyl esters (glyceryl laurate), polyoxyethylene glycol sorbitan alkyl ethers (polysorbate), sorbitan alkyl esters (spans), cocamidmon but not limited to oethanolamide, cocamide diethanolamide, dodecyldimethylamine oxide, polyethylene glycol-polypropylene glycol copolymers (poloxamers), and polyethoxylated tallow amine (POEA).

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

Surfactants with cationic groups at the head of the molecule include pH-dependent primary, secondary, or tertiary amines; octenidine dihydrochloride, permanently charged quaternary ammonium cations such as alkyltrimethylammonium salts of cetyltrimethylammonium bromide (CTAB) also known as hexadecyltrimethylammonium bromide, cetyltrimethylammonium chloride (STAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5 -bromo-5-nitro-1,3dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide and dioctadecyldimethylammonium bro

- 15 041808 mid (DODAB).

Zwitterionic (amphoteric) surfactants have both cationic and anionic centers attached to the same molecule. The cationic portion is based on primary, secondary or tertiary amines or quaternary ammonium cations. The anionic part may be more varied and include sulfonates. The most common biological zwitterionic surfactants contain an amine or ammonium phosphate anion such as phosphatidylserine phospholipids, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

A surfactant with an uncharged hydrophilic moiety, such as ethoxylate, is not ionic. Many long chain alcohols have some surfactant properties.

The use of microorganisms with polymers in oil production.

The present invention provides methods for enhancing oil recovery using one or more micro-organisms and/or microbial by-products in combination with one or more polymeric compounds. The polymeric compounds used for oil recovery in combination with the microorganisms of the present invention include, but are not limited to, hydrogels, acrylic acid, acrylamide, polyacrylamide (PAM), hydrolyzed polyacrylamide (HPAM), polysaccharide, xanthan gum, guar gum, and cellulose polymer. In preferred embodiments, the polymer is a biopolymer selected from, for example, hydrogels, xanthan gum, guar gum, cellulose polymers, polysaccharides, and others.

An associative water soluble polymer is a relatively new class of polymers that has recently been proposed for oilfield applications. These polymers are composed of a hydrophilic long chain backbone, with few hydrophobic groups located either randomly along the chain or at the ends of the chain. When these polymers are dissolved in water, the hydrophobic groups aggregate to minimize their exposure to water. The introduced groups associate due to intramolecular hydrophobic interactions and intermolecular hydrophobic interactions. The functional groups of this polymer are less sensitive to the salinity of the brine compared to polyacrylamide, the viscosity of which drops sharply with increasing salinity.

Polymer flooding may involve adding a polymer to the flood water to reduce its mobility. The polymers increase the viscosity of the aqueous phase and also reduce the permeability of the water due to mechanical retention, thereby leading to a more favorable mobility ratio. With a more viscous phase, the collected oil roll can be moved more easily through the formation and eventually into the production well.

The polymers according to these embodiments can also be removed and/or destroyed using the microbial composition of the present invention once their function in the well is no longer needed.

In one embodiment, the present invention provides a method of improving hydrocarbon production from a hydraulically fractured well by applying a microorganism-based composition containing one or more microorganism strains to a drilling site. In some embodiments, polymers accumulate downhole after they have performed their required function downhole.

Microorganisms in the microbial composition and/or their by-products can rapidly degrade polymers such as polylactic acid (PLA); thus, the method improves the ability to recover hydrocarbon resources by reducing the accumulation of PLA and other resins within fractures and fracturing wellbores once their usefulness has been exhausted. The method optionally includes adding nutrients and/or other agents to the site in order to stimulate the growth of microorganisms. The method may further include adding polymer degrading enzymes to the site in order to enhance polymer degradation.

In one embodiment, the claimed invention provides methods for recovering polymeric substances that remain in wells, including hydraulically fractured wells after their usefulness has been exhausted. For example, bio-surfactants produced by the methods and microorganisms of the present invention can reduce the interfacial tension of fluids used to lift polymeric materials such as PAM gel friction reducers. In another embodiment, bio-surfactants can be used to cleave the PAM gel prior to lifting.

The use of microorganisms with alkaline compounds in oil production.

The present invention provides methods for enhancing oil recovery using one or more micro-organisms and/or microbial by-products in combination with one or more alkaline compounds. Alkaline compounds used in oil recovery in combination with the microorganisms of the present invention include, but are not limited to, ammonium hydroxide.

Alkali is the main ionic salt of an alkali metal or alkaline earth element.

- 16 041808 metal element. The use of caustic in chemical flooding offers several advantages including promoting crude oil emulsification, increasing the ionic strength of the aqueous phase leading to control of the phase behavior of the injected surfactant, and lowering the IFT to ultra-low values in the presence of surfactant.

Alkali can also reduce costs by limiting the amount of surfactant needed in the two ways. In the first, alkali reduces surfactant adsorption by increasing the negative charge density of the rock surface, making it predominantly wetted by water. In the second, the alkali reacts with the acids in the crude oil to produce soap in situ, which in turn expands the optimum salinity range. The soap formed creates a microemulsion phase that can coexist with oil and water, thus expanding the three-phase region (or ultra-low IFT region).

The choice of alkali depends on the type of formation, type of clay and divalent cations. Common alkaline agents include sodium hydroxide (NaOH or caustic soda), sodium carbonate (Na2CO ₃ or soda ash), sodium bicarbonate (NaHCO ₃ ), and sodium metaborate (NaBO2). Sodium hydroxide solutions have been reported to react strongly with sandstone at elevated temperatures (185°F), resulting in sandstone weight loss and increased porosity. Alkali consumption resulting from the dissolution of NaOH from silicate minerals can be a significant and costly factor during field application. Anionic surfactants showed significantly lower adsorption in the presence of Na2CO ₃ compared to NaOH. Hydroxide is not an ion that determines the potential for carbonate surfaces. Calcium and other divalent cations can cause precipitation of alkalis such as Na2CO ₃ unless a weak brine is used. This is a limitation for Na2CO ₃ . NaBO2 has been reported as a replacement for Na2CO ₃ . This alkali gives pH values of about 11 at an alkali concentration of 1 wt.% and forms a soap with crude oil with a high content of hydrogen sulfide. Another important advantage of NaBO2 (sodium metaborate) species is their resistance to divalent cations. In carbonate formations, sodium metaborate is used instead of other alkalis. If the formation contains clays, NaHCO3 is preferred. Na2CO3 is the most commonly used alkali because it is inexpensive and transports better in porous media.

The preferred oil reservoirs for alkali flooding are sand reservoirs rather than carbonate reservoirs that contain anhydrite (calcium sulfate) (CaSO ₄ ) or gypsum (calcium sulfate dihydrate) (CaSO ₄ -2H2O), which can consume large amounts of alkaline chemicals . In addition, in carbonate formations, precipitation of calcium carbonate (CaCO ₃ ) or calcium hydroxide (Ca(OH) ₂ ) occurs when Na ₂ CO ₃ or NaOH is added. Carbonate formations also contain brine with a higher concentration of divalent compounds and can cause sedimentation. To overcome this problem, the proposed NaHCO ₃ and sodium sulfate (Na2SO4) are used. NaHCO3 has a significantly lower concentration of carbonate ions, and additional sulfate ions can reduce the concentration of calcium ions in solution. These chemicals are also consumed by clays, minerals or silica, and the higher the formation temperature, the higher the alkali consumption. Another common problem during alkali flooding is the formation of scale in live wells. During alkaline flooding, the injection sequence typically includes: (1) flushing to improve reservoir conditions prior to initial water plug injection, (2) initial water plug (alkaline chemicals), (3) polymer as buffer water polymer solution for displacement of the initial water plug. Alkali flooding can be modified as AP (alkali and polymer), AS (alkali and surfactant), and alkali, surfactant and polymer (ASP) methods. Soap, produced by the reaction between the acidic components of crude oil and injected alkali, is the main mechanism for oil recovery in alkali flooding.

Application of microorganisms with chelating agents.

In some embodiments, the implementation of microorganisms and/or by-products of their vital activity can be used with a chelator or chelating agent. Preferably, the use of chelating agents aids in increasing oil recovery by dissolving deposits in the formation. Sediments can block pores and other flow paths of the reservoir, thereby slowing and/or blocking the flow of oil from the reservoir.

As used herein, the term chelator or chelating agent means an active agent capable of removing a metal ion from a system by complex formation such that the metal ion, for example, cannot readily participate in or catalyze the formation of an oxygen radical.

Examples of chelating agents suitable for the present invention include dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropanesulfonic acid (DMPS), α-lipoic acid (ALA), thiaminetetrahydrofurfuryl disulfide (TTFD), penicillamine, ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium citrate and citric acid, but are not limited to.

In preferred embodiments, the chelating agent is citrate per-

Claims (15)

sodium, citric acid, EDTA, or a combination thereof. Examples A deeper understanding of the present invention and its many advantages can be obtained from the following examples given by way of illustration. The following examples illustrate some of the methods, uses, embodiments, and variations of the present invention. They are not to be construed as limiting the invention. Numerous changes and modifications can be made to the invention. Example 1 Preparation of Bacillus subtilis. Fermentation of Bacillus subtilis var. locuses can be carried out in a 500 L reactor with 350 L of culture medium containing (g/L): Glucose 18 Powdered molasses 2 sucrose 1 KN ₂ RO ₄ 0.5 Na ₂ HPO ₄ -7H ₂ O 2.1 KS1 0.1 _MgSO4 0.5 CaCl ₂ 0.05 Urea 2.5 _NH4C1 1.24 Yeast extract 2 Corn peptone 0.5 Micronutrients TekNova (ml) 1 The cultivation temperature is 40°C, the pH stabilization is 6.8-7.0, the DO stabilization is 20-30% (oxygen concentration in the air is taken as 100%). The duration of cultivation is 24-36 hours or until at least 95% of the bacteria reach the stage of sporulation. The final concentration of the bacterial culture is not less than 1x10 ⁹ CFU/ml. The amount of culture produced in one round of fermentation allows to obtain more than 2000 barrels of the final treatment composition containing 10 ⁶ CFU of the indicated strain of Bacillus. Example 2 Fermentation of Starmerella bombicola to produce a bio-surfactant. The fermenter is an autoclavable stainless steel vessel designed specifically for yeast cultivation and bio-surfactant production. The fermenter is equipped with a micro atomizer and a turbine agitator, as well as sensors for dissolved oxygen, pH, temperature and foam. It has an integrated control station with a color touch interface, built-in pumps for increased broth agitation, gas flow controllers, and pH, dissolved oxygen, and foam level controllers. The working volume of the 550 gallon reactor is 500 gallons. The nutrient medium contains sources of carbon, protein, nitrogen and unsaturated butyric or fatty acids. A one-day culture of Starmerella bombicola (60-70 L) is used to inoculate the reactor. The initial pH value of the culture is 5.0-6.0 before the growth of microorganisms, and the pH begins to decrease. The duration of cultivation and selection of the finished product continues for 5 days at 25°C, and the pH is stabilized at 3.5. The final content of sophorolipids can reach at least 40% of the working volume per cycle or 150 g/l or higher. CLAIM 1. A method of increasing the amount of oil recovered from an oil reservoir by dissolving sediment deposits present in the reservoir, characterized in that the method includes applying to the reservoir a yeast fermentation product that contains a broth obtained from the fermentation of yeasts selected from Wickerhamomyces anomalus and Starmerella bombicola ; ammonium hydroxide; a solvent selected from isopropyl alcohol and ethanol; a chelating agent containing citric acid, sodium citrate, ethylenediaminetetraacetic acid (EDTA), or a combination of one or more of them; and water; while the yeast fermentation product does not contain active yeast cells. 2. The method of claim 1, further comprising introducing one or more polymeric compounds into the formation. 3. The method of claim 2, wherein the one or more polymeric compounds are selected from xanthan gum, guar gum, hydrogels, polysaccharide, and cellulose polymers. - 18 041808 4. The method of claim 1, further comprising introducing one or more additional non-biological surfactants (surfactants) into the formation. 5. The method of claim 1, wherein the yeast fermentation product contains a glycolipid biological surfactant (bio-surfactant). 6. The method of claim 5 wherein the bio-surfactant is sophorolipid. 7. The method of claim 1 wherein the yeast fermentation product contains a phospholipid bio-surfactant. 8. The method of claim 1, wherein the yeast fermentation product is produced 482.8 km (300 miles) or less from the formation. 9. The method of claim 1, further comprising applying one or more purified bio-surfactants to the formation. 10. The method of claim 9 wherein the one or more purified bio-surfactants are glycolipids. 11. A composition for increasing the amount of oil recovered from an oil-bearing formation by dissolving sediment deposits, containing a yeast fermentation product containing a broth obtained as a result of the fermentation of the yeast Wickerhamomyces anomalus and Starmerella bombicola, ammonium hydroxide and one or more chelating agents selected from citric acid, sodium citrate and EDTA. 12. Composition according to claim 11, wherein the yeast fermentation product does not contain active yeast cells. 13. The composition according to claim 11, additionally containing a bio-surfactant selected from glycolipids, lipopeptides and phospholipids. 14. The composition according to claim 11, additionally containing a solvent. 15. The composition of claim 14 wherein the solvent is isopropyl alcohol or ethanol. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2