WO2024215567A1

WO2024215567A1 - Compositions and methods for slurry preservation

Info

Publication number: WO2024215567A1
Application number: PCT/US2024/023205
Authority: WO
Inventors: Gabriela KNESEL; Ronney SILVA; Cathrine MONYAKE; Juan Cervantes
Original assignee: Locus Solutions Ipco, Llc
Priority date: 2023-04-10
Filing date: 2024-04-05
Publication date: 2024-10-17

Abstract

The subject invention provides safe, environmentally-friendly compositions and efficient methods for preserving slurries by reducing or eliminating the growth of organisms, particularly microorganisms. The methods further pertain to reducing or eliminating the growth of organisms in oil and gas production and other industrial activities. More specifically, the subject invention provides compositions derived from microorganisms for reducing or eliminating the growth of organisms.

Description

COMPOSITIONS AND METHODS FOR SLURRY PRESERVATION

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 63/495,123, filed April 10, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Mineral slurries are often difficult to preserve because of favorable conditions for microbial growth during production, storage and transportation. For example, high temperatures during the grinding process, poor hygienic conditions during transport and storage, or high pH values can lead to growth of organisms (e.g., microorganisms), greying, or product deterioration.

Slurry preservatives are used in mineral slurries to inhibit the growth of organisms, including yeasts, fungi, and bacteria. Slurry preservatives are added both during the mining and refining processes and in the final product. The selection of the preservative and dosage is based on laboratory analysis to measure efficacy and interactions with other slurry components. Additionally, regulatory, safety, and user requirements also impact the choice of slurry preservative.

There are a limited number of slurry preservatives for use in mineral slurries to inhibit bacterial growth, including, for example, glutaraldehyde or tetrahydro-3, 5-dimethyL 1,3, 5- thiadiazine-2-thione (Dazomet). Conventional slurry preservatives have numerous limitations, including sensitivity to pH and temperature, the presence of volatile organic compounds, a limited effectiveness at inhibiting the growth of organisms, and expense. Additionally, glutaraldehyde is hazardous to handle and has environmental concerns. Dazomet has limited application because it creates an odor and has other health and safety issues.

There is a need for effective slurry preservatives to address the growth of organisms in various slurry formulations, regulatory constraints, and to reduce or eliminate the use of toxic compounds. Therefore, novel, improved slurry preservatives and methods are needed.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides slurry preservatives and compositions thereof. The subject invention further provides methods of using these slurry preservatives for treating, disrupting and/or preventing the growth of organisms in slurries or on surfaces that contact slurries. More specifically, the subject invention provides environmentally-friendly slurry preservatives and methods for preserving slurries by reducing or eliminating the growth of organisms, such as, for example, in methods of mining, oil and gas production, and industrial production. In certain embodiments, existing slurry preservatives and methods of slurry preservation can be improved by incorporating the subject compositions and methods.

Advantageously, the compositions and methods of the subject invention increase the longevity and quality of slurries containing valuable substances, such as, for examples, slurries of ore, and can decrease the chemical usage, including synthetic or toxic slurry preservatives. Accordingly, the subject invention can be useful for eliminating organisms or reducing the rate of growth of organisms in a slurry.

In certain embodiments, the subject invention provides compositions comprising components that are derived from microorganisms. In certain embodiments, the composition comprises a microbial biosurfactant. In certain embodiments, the composition comprises one or more biosurfactants, and, optionally, other compounds, such as, for example, polymers, chemical surfactants, biocides, and conventional slurry preservatives, such as, for example, glutaraldehyde, dibromo-nitrilopropionamide (DBNPA), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), ortho-phenylphenol (OPP), 1,2- Benzisothiazol-3(2H)-one (BIT), tetrahydro-3, 5-dimethyl-l, 3, 5-thiadiazine-2-thione (Dazomet), bronopol, sodium ortho-phenylphenate, or alkyl dimethyl benzyl ammonium chloride (ADBAC).

In certain embodiments, the biosurfactant of the composition is utilized in crude form. The crude form can comprise, in addition to the biosurfactant, fermentation broth in which a biosurfactantproducing microorganism was cultivated, residual microbial cell matter or live or inactive microbial cells, residual nutrients, and/or other microbial growth by-products.

In some embodiments, the biosurfactant is utilized after being extracted from a fermentation broth and, optionally, purified.

The biosurfactant according to the subject invention can be a, for example, glycolipid (e.g., sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids and trehalose lipids), lipopeptide (e.g., surfactin, iturin, fengycin, arthrofactin, and lichenysin), flavolipid, phospholipid (e.g., cardiolipins), fatty acid ester compound, fatty acid ether compound, and/or high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide- protein-fatty acid complexes.

In certain specific embodiments, the biosurfactant is a sophorolipid (SLP), including linear SLP, lactonic SLP, acetylated SLP, de-acetylated SLP, salt-form SLP, esterified SLP derivatives, amino acid-SLP conjugates, and other SLP derivatives or isomers that exist in nature and/or are produced synthetically. In preferred embodiments, the SLP is a linear SLP or a derivatized linear SLP. In certain embodiments, the subject invention provides a method for eliminating organisms or reducing the rate of growth of organisms in a slurry, wherein the method comprises the following step: contacting a slurry preservative, according to the subject invention, to a mineral slurry.

Additionally, in preferred embodiments, the slurry preservative can be used to enhance the longevity or quality of the slurry. For example, in some embodiments, application of a sluny preservative, according to the subject invention, can inhibit the growth of organisms in the sluny, which can promote efficient processing, storage, and transportation of the slurry.

In some embodiments, the method comprises contacting a slurry preservative comprising a biosurfactant and, optionally, other components, such as, for example, water, chemical surfactants, polymers, conventional slurry preservatives, biocides, or any combination thereof to a slurry containing minerals. In certain embodiments, the slurry preservative can be applied to the slurry for a period of time and/or until a distinct volume or weight of the slurry preservative composition has been applied. The step can be repeated as many times as necessaiy to achieve a desired reduction in the amount of an organism or reduction of the growth rate of an organism in the slurry.

In certain embodiments, the methods of the subject invention result in at least a 25% decrease in the rate of growth of an organism, preferably at least a 50% decrease, after one treatment. In certain embodiments, the methods of the subject invention result in at least a 25% decrease in the amount of a living organism in a slurry, preferably at least a 50% decrease, after one treatment.

Advantageously, in certain embodiments, the slurry preservative, according to the subject invention, can be effective at increasing the efficiency of industrial processes, particularly processing of minerals from slurries. Furthermore, the methods of the subject invention do not require complicated equipment or high energy consumption, and production of the composition can be performed on site, for example, at a mine or at a refinery.

DETAILED DESCRIPTION

The subject invention relates generally to a method for reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry. More specifically, the subject invention provides environmentally-friendly compositions and methods for reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry, for example, in slurries that are derived from mining sites and/or used in industrial activities. Accordingly, the subject invention is useful for improving the efficacy of established compositions and methods used for reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry. Advantageously, the compositions and methods of the subject invention can increase the longevity of a slurry using safe, environmentally-friendly compositions. Selected Definitions

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, microbial growth and/or the action of a biosurfactant or other microbial growth by-product.

As used herein, a “biofilm” is a complex aggregate of microorganisms, such as bacteria, yeast, or fungi, wherein the cells adhere to each other and/or to a surface using an extracellular matrix. The cells in biofilms are physiologically distinct from planktonic cells of the same organism, which are single cells that can float or swim in liquid medium.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or organic compound such as a small molecule (e.g., those described below), is substantially free of other compounds, such as cellular material, with which it is associated in nature. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. An isolated microbial strain means that the strain is removed from the environment in which it exists in nature. Thus, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with a carrier.

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 98%, by weight the compound of interest. For example, a purified compound is one that is at least 80%, 85%, 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.

A “metabolite” refers to any substance produced by metabolism or a substance necessary for taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material, an intermediate in, or an end product of metabolism. Examples of metabolites include, but are not limited to, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, microelements, amino acids, biopolymers and biosurfactants.

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 of 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 a “reduction” means a negative alteration, and an “increase” means a positive alteration, wherein the negative or positive alteration is at least 0.001%, 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

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 using naturally-derived substrates.

Biosurfactants are a structurally diverse group of surface-active substances consisting of two parts: a polar (hydrophilic) moiety and non-polar (hydrophobic) group. Due to their amphiphilic structure, biosurfactants can, for example, increase the surface area of hydrophobic water-insoluble substances, increase the water bioavailability of such substances, and change the properties of bacterial cell surfaces. Biosurfactants can also reduce the interfacial tension between water and oil and, therefore, lower the hydrostatic pressure required to move entrapped liquid to overcome the capillary effect. Biosurfactants accumulate at interfaces, thus reducing interfacial tension and leading to the formation of aggregated micellar structures in solution. The formation of micelles provides a physical mechanism to mobilize, for example, oil in a moving aqueous phase.

The ability of biosurfactants to reduce the surface tension also permits their use as antibacterial, antifungal, and hemolytic agents to, for example, control pests and/or microbial growth.

Typically, the hydrophilic group of a biosurfactant is a sugar (e.g., a mono-, di-, or polysaccharide) or a peptide, while the hydrophobic group is typically a fatty acid. Thus, there are countless potential variations of biosurfactant molecules based on, for example, type of sugar, number of sugars, size of peptides, which amino acids are present in the peptides, fatty acid length, saturation of fatty acids, additional acetylation, additional functional groups, esterification, polarity and charge of the molecule.

These variations lead to a group of molecules comprising a wide variety of classes, including, for example, glycolipids (e.g., sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids and trehalose lipids), lipopeptides (e.g., surfactin, iturin, fengycin, arthrofactin and lichenysin), flavolipids, phospholipids (e.g., cardiolipins), fatty acid ester compounds, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein- fatty acid complexes. Each type of biosurfactant within each class can further comprise subtypes having further modified structures. Like chemical surfactants, each biosurfactant molecule has its own HLB value depending on its structure; however, unlike production of chemical surfactants, which results in a single molecule with a single HLB value or range, one cycle of biosurfactant production typically results in a mixture of biosurfactant molecules (e.g., subtypes and isomers thereof).

The phrases “biosurfactant” and “biosurfactant molecule” include all forms, analogs, orthologs, isomers, and natural and/or anthropogenic modifications of any biosurfactant class (e.g., glycolipid) and/or subtype thereof (e.g., sophorolipid).

As used herein, the term “sophorolipid,” “sophorolipid molecule,” “SLP” or “SLP molecule” includes all forms, and isomers thereof, of SLP molecules, including, for example, acidic (linear) SLP (ASL) and lactonic SLP (LSL). Further included are mono-acetylated SLP, di-acetylated SLP, esterified SLP, SLP with varying hydrophobic chain lengths, cationic and/or anionic SLP with fatty acid-amino acid complexes attached, esterified SLP, SLP-metal complexes, SLP-salt derivatives (e.g., a sodium salt of a linear SLP), and other, including those that are and/or are not described within in this disclosure.

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

(1)

(2)

where R¹ and R¹ independently represent saturated hydrocarbon chains or single or multiple, in particular single, unsaturated hydrocarbon chains having 8 to 20 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups, R² and R² 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 R³, R³ , R⁴ and R⁴ independently represent a hydrogen atom or COCi L. R⁵ is typically, but not limited to, —OH.

SLP are typically produced by yeasts, such as Starmerella spp. yeasts and/or Candida spp. yeasts, e.g., Starmerella (Candida) bombicola, Candida apicola, Candida batistae, Candida floricola, Candida riodocensis, Candida stellate and/or Candida kuoi. SLP have environmental compatibility, high biodegradability, low toxicity, high selectivity and specific activity in a broad range of temperature, pH and salinity conditions. Additionally, in some embodiments, SLP can be advantageous due to their small micelle size, which can help facilitate the movement of the micelle, and compounds enclosed therein, through nanoscale pores and spaces. In certain embodiments, the micelle size of a SLP is less than 100 nm, less than 50 nm, less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm.

As used herein, “beneficiation” refers to the process by which gangue materials are removed from the product of interest (e.g., element, compound, mineral).

As used herein, “ore” refers to a naturally occurring solid material from which a valuable substance, mineral and/or metal can be profitably extracted. Ores are often mined from ore deposits, which comprise ore minerals containing the valuable substance. “Gangue” minerals are minerals that occur in the deposit but do not contain the valuable substance. Examples of ore deposits include hydrothermal deposits, magmatic deposits, laterite deposits, volcanogenic deposits, metamorphically reworked deposits, carbonatite-alkaline igneous related deposits, placer ore deposits, residual ore deposits, sedimentary deposits, sedimentary hydrothermal deposits and astrobleme-related deposits. Ores, as defined herein, however, can also include ore concentrates or tailings.

As used herein, “leaching” refers to the process by which metal is extracted from ore by aqueous solutions including by, for example, ammonia leaching, alkali leaching, acid leaching, cyanidation (i.e., cyanide leaching), or thiosulfate leaching. As used herein “cyanidation” refers to the process of converting gold in ore to a water-soluble coordination complex using aqueous cyanide, including, for example, sodium cyanide, potassium cyanide, or calcium cyanide.

As used herein, the term “slurry” refers to a mixture of solids suspended in a liquid. The term slurry can refer to a mineral slurry in which a mineral of interest is suspended in water or any other liquid. Additionally, a slurry can also refer to a cement slurry, paper slurry, textile slurry, wood slurry, or slurry oil. 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.

Slurry Preservative Compositions

In certain embodiments, the subject invention provides slurry preservative compositions comprising components that are derived from microorganisms. In certain embodiments, the composition comprises a microbial biosurfactant. In certain embodiments, the composition comprises one or more biosurfactants, and, optionally, other compounds, such as, for example, water, chemical surfactants, polymers, conventional slurry preservatives, biocides, or any combination thereof.

In certain embodiments, the chemical surfactant of the slurry preservative composition is a detergent, wetting agent, emulsifier, foaming agent, and/or dispersant.

In certain embodiments, the polymer can include natural or synthetic polymers, water soluble polymers, cationic polymers, anionic polymers, or non-ionic polymers. The polymers can be, for example, polyacrylic acid polymers, co-polymers and multi-polymers containing various functional groups such as, for example, acrylic acid, hydroxypropyl acrylate (HPA), 2-acrylamido-2- methylpropane sulfonic acid (AMPS), and ter-butyl acrylate (TBA). In certain embodiments, the slurry preservative is a conventional slurry preservative used in the processing of mineral slurries, such as, for example, glutaraldehyde, dibromo-nitrilopropionamide (DBNPA), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), ortho-phenylphenol (OPP), 1,2- Benzisothiazol-3(2H)-one (BIT), tetrahydro-3, 5-dimethyl-l, 3, 5-thiadiazine-2-thione (Dazomet), bronopol, sodium ortho-phenylphenate (SOPP), or alkyl dimethyl benzyl ammonium chloride (ADBAC).

In certain embodiments, the biocide is, for example, antibiotics, including, for example, penicillins (such as penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, methicillin, piperacillin, and the like), tetracyclines (such as chlortetracycline, oxytetracycline, methacycline, doxycycline, minocycline and the like), cephalosporins (such as cefadroxil, cephalexin, cephradine, cephalothin, cephapirin, cefazolin, cefaclor, cefamandole, cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil, cefmetazole, cefprozil, loracarbef, ceforanide, cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, and the like), fluoroquinolones (e.g., levofloxacin), quinolones (such as nalidixic acid, cinoxacin, ciprofloxacin and norfloxacin and the like), lincomycins (e.g., clindamycin), macrolides (e.g., erythromycin, azithromycin), sulfones (e.g., dapsone), sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide, bactrim), lipopeptides (e.g., daptomycin), polypeptides (e.g., bacitracin), glycopeptides (e.g., vancomycin), aminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and the like), nitoimidazoles (e.g., metronidazole) and/or carbapenems (e.g., thienamycin).

In some embodiments, the biocide can include essential oils, botanicals, or other plant extracts with bactericidal and/or anti-bacterial effects. These can include oils/extracts at a concentration between 1-10% volume/volume (extract/invention), horseheal (Inula helenium, L. Asteraceae, elecampane), rose (Rosa damascena L., Rosaceae), lavender (Lavandula angustifolia L., Labiatae), chamomile (Matricaria recutica L., Asteraceae), orange (Rutaceae), grapefruit (Citrus paradisi), eucalyptus (Eucalyptus globulus L. ,Myrtaceae), geranium (Geranium robertianum L., Geraniaceae), juniper (Juniperus communis L., Cupressaceae), citrus (Citrus sinensis L., Rutaceae), tea tree (Melaceuca alternifolia), manuka bush (Leptospermum scoparium), neem tree (Azadirachta indica, A. Juss), tea plant (Camellia sinensis), rosemary (Rosmarinus officinalis L., Lamiaceae), lemon, oregano, cinnamon, eucalyptus, citronella, and thyme oils.

Other known biocides can also be utilized, such as, for example, alcohols, aldehydes, chlorine, and chlorine- releasing agents (e.g., sodium hypochlorite, chlorhexidine, chlorhexidine gluconate), iodine, peroxygen compounds (e.g., hydrogen peroxide, peracetic acid), phenolic type compounds, quaternary ammonium compounds (e.g., benzalkonium chloride), bases (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate), and acids (e.g., mineral and organic acids).

In certain embodiments, the slurry preservative comprises a microbe-based product comprising a biosurfactant utilized in crude form. The crude form can comprise, in addition to the biosurfactant, fermentation broth in which a biosurfactant-producing microorganism was cultivated, residual microbial cell matter or live or inactive microbial cells, residual nutrients, and/or other microbial growth by-products. The product may be, for example, at least, by weight, 1%, 5%, 10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product, by weight, may be, for example, anywhere from 0% to 100% inclusive of all percentages therebetween.

The biosurfactant according to the subject invention can be a glycolipid (e.g., sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids and trehalose lipids), lipopeptide (e.g., surfactin, iturin, fengycin, arthrofactin and lichenysin), flavolipid, phospholipid (e.g., cardiolipins), fatty acid ester compound, fatty acid ether compound, and/or high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

In certain specific embodiments, the biosurfactant is a sophorolipid (SLP), including linear SLP, lactonic SLP, acetylated SLP, de-acetylated SLP, salt-form SLP derivatives, esterified SLP derivatives, amino acid-SLP conjugates, and other SLP derivatives or isomers that exist in nature and/or are produced synthetically. In preferred embodiments, the SLP is a linear SLP or a derivatized linear SLP.

In some embodiments, the biosurfactant can be included in the composition at 0.01 to 99.9%, 0.1 to 90%, 0.5 to 80%, 0.75 to 70%, 1.0 to 50%, 1.5 to 25%, or 2.0 to 15%> by weight, with respect to the total slurry preservative.

In another embodiment, a purified biosurfactant may be added in combination with an acceptable carrier, in that the biosurfactant may be presented at concentrations of 0.001 to 50% (v/v), preferably, 0.01 to 20%> (v/v), more preferably, 0.02 to 5% (v/v).

In some embodiments, the biosurfactant can be included in the composition at, for example, 0.01 to 100,000 ppm, 0.05 to 10,000 ppm, 0.1 to 1,000 ppm, 0.5 to 750 ppm, 1.0 to 500 ppm, 2.0 to 250 ppm, or 3.0 to 100 ppm, with respect to the amount of liquid being treated.

In certain embodiments, the chemical surfactant of the slurry preservative composition is a detergent, wetting agent, emulsifier, foaming agent, and/or dispersant. In some embodiments, the chemical surfactant can be included in the composition at 0.01 to 99.9%, 0.1 to 90%, 0.5 to 80%, 0.75 to 70%, 1.0 to 50%, 1.5 to 25%, or 2.0 to 15% by weight, with respect to the total slurry preservative composition.

In some embodiments, the conventional slurry preservative or polymer can be included in the composition at 0.01 to 99.9%, 0.1 to 90%, 0.4 to 0.85%, 0.5 to 80%, 0.75 to 70%, 1.0 to 50%, 1.5 to 25%, 2.0 to 15%, or 0.4% to 4.5% by weight, with respect to the total amount of the slurry preservative composition.

The slurry preservative can further comprise other additives such as, for example, carriers, other microbe-based compositions, additional biosurfactants, enzymes, catalysts, solvents, salts, buffers, pH modifying agents, acids, emulsifying agents, lubricants, solubility controlling agents, preservatives, stabilizers, ultra-violet light resistant agents, viscosity modifiers, preservatives, tracking agents, chelating agents, and other microbes and other ingredients specific for an intended use.

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'-acetylcystcinc, n- hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordination group (e.g., rubeanic acid), STPP (sodiumtripolyphosphate, Na5P3OI0), 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 -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-hydroxy ethyliminodiacetic acid (HEIDA), pyridine-2,6-dicarboxylic acid (PDA), trimethyl glycine (TMG), Tiron, or any combination thereof.

In certain embodiments, the sluny preservative can be a liquid, a solid or a semi-solid, such as, for example, a powder or a paste.

Methods of Preserving Slurries

In certain embodiments, the subject invention provides a method for reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry, particularly at, for example, mining sites, quarrying sites, mineral processing sites, oil and gas extracting sites, oil and gas refining sites, and industrial sites. In certain embodiments, the microbe-containing and/or biosurfactant-containing composition can be applied to a slurry that requires reducing the presence or growth rate of organisms, particularly microorganisms, in the slurry.

The slurry preservatives of the subject invention can be applied to a variety of inorganic or organic objects such as, for example, steel, aluminum, copper, polyvinylchloride, polypropylene, polyvinylidene fluoride (PVDF), iron, wood, plastic, gypsum, paper, silk, glass, cotton, concrete, plaster, clay, stucco, or rubber. The compositions can be applied to objects that reside a range of temperatures, atmospheric pressures, or aquatic environments.

The composition can be applied to the surface by spraying using, for example, a spray bottle or a pressurized spraying device. The composition can also be applied using a cloth or a brush, wherein the composition is rubbed, spread or brushed onto the surface. Furthermore, the composition can be applied to the surface by dipping, dunking or submerging the surface into a container having the composition therein.

In preferred embodiments, the mining site can be a coal mine, iron ore mine, copper mine, copper-nickel mine, tin mine, nickel mine, gold mine, silver mine, molybdenum mine, aluminum mine (e.g., bauxite mine, kyanite mine), lead-zinc mine, tungsten mine, or zinc mine. The mine can be an underground mine, surface mine, placer mine or in situ mine.

In certain embodiments, the slurry can be derived from a mining or quarrying site that produces, for example, cobalt (e.g., erythrite, skytterudite, cobaltite, carrollite, linnaeite, and asbolite (asbolane)); copper (e.g., chalcopyrite, chalcocite, bornite, djurleite, malachite, azurite, chrysocolla, cuprite, tenorite, native copper and brochantite); gold (e.g., native gold, electrum, tellurides, calaverite, sylvanite and petzite); silver (e.g., sulfide argentite, sulfide acanthite, native silver, sulfosalts, pyrargyrite, proustite, cerargyrite, tetrahedrites); aluminum (e.g, bauxite, gibbsite, bohmeite, diaspore); antimony (e.g., stibnite); barium (e.g., barite, witherite); cesium (e.g., pollucite); chromium (e.g., chromite); iron (e.g., hematite, magnetite, pyrite, pyrrhotite, goethite, siderite); lead (e.g., galena, cerussite, anglesite); lithium (e.g., pegmatite, spodumene, lepidolite, petalite, amblygonite, lithium carbonate); magnesium (e.g., dolomite, magnesite, brucite, carnallite, olivine); manganese (e.g., hausmannite, pyrolusite, barunite, manganite, rhodochrosite); mercury (e.g., cinnabar); molybdenum (e.g., molybdenite); nickel (e.g., pentlandite, pyrrhotite, garnierite); phosphorus (e.g., hydroxylapatite, fluorapatite, chlorapatite); platinum group (platinum, osmium, rhodium, ruthenium, palladium) (e.g., native elements or alloys of platinum group members, sperrylite); potassium (e.g., sylvite, langbeinite); rare earth elements (cerium, dysprosium, erbium, europium, gadolinium, holmium, lanthanium, lutetium, neodymium, praseodymium, samarium, scandium, terbium, thulium, ytterbium, yttrium) (e.g., bastnasite, monazite, loparite); sodium (e.g., halite, soda ash); strontium (e.g., celestite, strontianite); sulfur (e.g., native sulfur, pyrite); tin (e.g., cassiterite); titanium (e.g., scheelite, huebnerite-ferberite); uranium (e.g., uraninite, pitchblende, coffinite, carnotite, autunite); vanadium; zinc (e.g., sphalerite, zinc sulfide, smithsonite, hemimorphite); and zirconium (e.g., zircon).

Additional elements and/or minerals that the slurry may include are, for example, arsenic, bertrandite, bismuthinite, borax, colemanite, kernite, ulexite, sphalerite, halite, gallium, germanium, hafnium, indium, iodine, columbite, tantalite-columbite, rubidium, quartz, diamonds, garnets (almandine, pyrope and andradite), corundum, barite, calcite, clays, feldspars (e.g., orthoclase, microcline, albite); gemstones (e.g., diamonds, rubies, sapphires, emeralds, aquamarine, kunzite); gypsum; perlite; sodium carbonate; zeolites; chabazite; clinoptilolite; mordenite; wollastonite; vermiculite; talc; pyrophyllite; graphite; kyanite; andalusite; muscovite; phlogopite; menatite; magnetite; dolomite; ilmenite; wolframite; beryllium; tellurium; bismuth; technetium; potash; rock salt; sodium chloride; sodium sulfate; nahcolite; niobium; tantalum and any combination of such substances or compounds containing such substances.

In certain embodiments, methods of reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry by contacting the slurry preservatives to various liquid streams, piping, pumps, drains, vessels, water storage areas, or other aquatic environments are provided. The compositions can be applied to liquids that reside at a range of temperatures and aquatic environments, such as, for example, a stream, waterway, pond, runoff area, containment pond, or water treatment/holding tank.

In certain embodiments, methods of reducing the presence or growth rate of organisms, particularly microorganisms, in a slurry are provided according to the subject methods by contacting the slurry preservatives to various solid surfaces, including, for example, conveyors, belts, piping, feeders, crushers, or grinders. The compositions can be applied to solid surfaces that reside at a range of temperatures and environments, such as, for example, a barge, railcar, hopper, tailings pile, or holding vessel.

The slurry preservative can be applied to a liquid slurry and, optionally, mixed by adding, pouring, agitating, or combining.

In certain embodiments, the time period in which the slurry preservative can be contacted to a slurry is for about 1 second to about 1 year, about 1 minute to about 1 year, about 1 minute to about 6 months, about 1 minute to about 1 month, about 1 minute to about 1 week, about 1 minute to about 48 hours, about 30 minutes to 40 hours, or preferably about 12 hours to 24 hours. In certain embodiments, the methods comprise applying a liquid or solid form of the slurry preservative to the liquid slurry for the period of time in which a slurry containing minerals is being produced, stored, and/or transported to, for example, inhibit the growth of organisms or eliminate the presence of growing organisms. In certain embodiments, the methods comprise applying a liquid or solid form of the slurry preservative to the liquid slurry until the amount of the organisms has been reduced to an amount that is determined to be satisfactory, which can be readily determined by one skilled in the art. The amount of living organisms that may be considered acceptable depends on the context. For example, removing the presence of fungi, yeast or bacteria that can be observed without the use of any equipment (e.g., a microscope) may be required. Alternatively, the rate of growth or the amount of a living organism may be reduced to a level at which the organism cannot be observed with a microscope or otherwise detected by, for example, using an assay to detect the presence of a nucleotide or protein of the organism.

In certain embodiments, the amount of the slurry preservative applied is about 0.00001 to 15%, about 0.00001 to 10%, about 0.0001 to 5%, about 0.001 to 3%, about 0.01%, or about 1 vol % based on an amount of slurry that is treated.

To slow or otherwise inhibit the growth of organisms in a slurry, the slurry preservatives can interact and modify the surfaces of cells. Due to their amphiphilic structure, biosurfactants increase the surface area of hydrophobic water- insoluble substances, increase the water bioavailabilify of such substances, and change the properties of cell surfaces. Biosurfactants accumulate at interfaces, thus reducing interfacial tension and leading to the formation of aggregated micellar structures in solution. In some embodiments, a biosurfactant (e.g., a sophorolipid) can form a micelle, wherein the micelle is less than 1 mm, 100 pm, 50 pm, 20 pm, 10 pm, 1 pm, 100 nm, less than 50 nm, less than 25 nm, less than 15 nm or less than 10 nm in size. The amphiphilic structure of biosurfactants allows for selfassociation and interaction with biological membranes. The ability of biosurfactants to form pores and destabilize biological membranes permits their use as antibacterial, antifungal, and hemolytic agents. Combined with the characteristics of low toxicity and biodegradability, biosurfactants are advantageous for use in a variety of application, including in the preservation of slurries.

In certain embodiments, the slurry preservatives can be used in methods of processing ores, ore slurries, or other products obtained via mining. In certain embodiments, the slurry preservatives can be used to inhibit the growth of an organism, particularly a microorganism, that can be found in the slurry. In certain embodiments, mined minerals require a preserved slurry for transportation in holding vessels or pipelines, as they are shipped over tens, hundreds, or thousands of miles or stored for days, weeks, or years. In certain embodiments, the slurry preservatives can be used in beneficiation processes. In order to extract the element or compound of interest, it can be necessary to crush and grind the ore and preconcentrate or separate the element or product of interest from the ore by flotation, settling, filtration, or gravity separation. In certain embodiments, during concentrating/purifying steps, and the slurry preservatives can be added during, before, or after the beneficiation process to limit or eliminate the presence of growing organisms. In certain embodiments, the slurry preservatives can be added after the beneficiation process to enhance the longevity of the storage of the liquids resulting from beneficiation.

In certain embodiments, the slurry preservatives can be used in methods of leaching, such as, for example, gold cyanidation. The process of extraction by leaching includes leaching (e.g., cyanide leaching), washing and filtering of leaching pulp, extraction of the metal from the leaching solution or pulp, and smelting of finished products. In certain embodiments, the slurry preservatives can be used in methods of leaching and washing and filtering leaching pulp, in which the slurry preservatives reduce the growth rate of organisms or eliminate organisms in the various solutions used in the leaching process.

In certain embodiments, the slurry preservative can be used in various industrial methods, including in the manufacturing or processing of food, beverages, oil and gas, and paper. In certain embodiments, the slurry preservatives can used to reduce the growth rate of organisms or eliminate organisms in a slurry, such as, for example, a paper slurry.

In certain embodiments, the subject slurry preservatives can be used in drilling fluids. In certain embodiments, high-angle and horizontal wells are used for hydraulic fracturing and the slurry preservatives can be to enhance the efficiency of the movement and/or storage of drilling fluids. In certain embodiments, the subject composition can be used in treating water or other liquids used in oil field applications. In certain embodiments, the oil field applications in which the subject compositions can be used include, for example, oil well drilling with oil field drilling muds, well stimulation, fracturing fluids, oil field processing applications, oil field water treatment systems, oil and gas production and transmission pipelines, gas storage fields and equipment. In certain embodiments, the equipment can include, for example, steam-injection water holding tanks, flood water, injection water, holding pond water, disposal-well water, water holding tanks, fuel storage tanks.

In certain embodiments, the presence and/or location of organisms in the slurry can be determined by imaging techniques such as, for example, visual observation by a person or X-ray and CT scans. In one embodiment, the presence of a growing organism can be detected by obtaining a sample from a slurry and measuring the presence of one or more biomarkers (e.g., exopolysaccharide, proteins, mRNA, DNA) that are associated with and/or expressed by the organism. In another embodiment, the organism can be detected by the presence of an extracellular polysaccharide (EPS) matrix, or chemicals contained in the EPS. Further, species of organisms that grow in the slurry can be determined by, for example, using antibodies that recognize antigens or peptides associated with the presence of organisms, or using probes that recognize nucleic acid molecules of the organisms. The presence and/or level of biomarkers useful according to the subject invention can be determined by techniques known in the art, such as for example, enzyme-linked immunosorbent assays (ELISA), western blot, northern blot, immunological assays, immunofluorescence, and nucleic acid hybridization techniques.

In certain embodiments, the organism targeted by the subject composition can be a bacterium or a eukaryote, such as, for example, a yeast or a fungi. In certain embodiments, the bacteria can be planktonic cells or a biofilm.

Advantageously, in certain embodiments, the slurry preservative, according to the subject invention, provides enhanced or increased storage times with limited negative environmental impacts. Additionally, the methods of the subject invention do not require complicated equipment or high energy consumption, and the production of the slurry preservative can be performed on site, including, for example, at a mine or at an industrial site. In certain embodiments, the subject slurry preservative can result in a decreased use of synthetic or toxic slurry preservatives or other potentially harmful chemicals used for reducing the growth rate of organisms or eliminating organisms in a slurry.

Production of Microbe-Based Products

In certain embodiments, the subject invention provides methods for cultivation of microorganisms and production of microbial metabolites and/or other by-products of microbial growth. The subject invention further utilizes cultivation processes that are suitable for cultivation of microorganisms and production of microbial metabolites on a desired scale. These cultivation processes include, but are not limited to, submerged cultivation/fermentation, solid state fermentation (SSF), and modifications, hybrids and/or combinations thereof.

The microorganisms can be, for example, bacteria, yeast and/or fungi. These microorganisms 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. chroococcuniy, 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 Blakeslea,' Candida spp. (e.g., C. albicans, C. rugosa, C. tropicalis,

C. lipolytica, C. torulopsis),' Clostridium (e.g., C. butyricum, C. tyrobutyricum, C. acetobutyricum, and C. beijerinckii Campylobacter spp.; Cornybacterium spp.; Cryptococcus spp.; Debaryomyces spp. (e.g., D. hansenii); 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. eulrophdy, Rhodococcus spp. (e.g., R. erythropolis),- Rhodospirillum spp. (e.g., R. rubruniy, Rhizobium spp.; Rhizopus spp.; Saccharomyces spp. (e.g., .S'. cerevisiae, S. boulardii sequela, S. torulay Sphingomonas spp. (e.g., S. paucimobilis); Starmerella spp. (e.g., S. bombicola),' Thraustochytrium spp.; Torulopsis spp.; Ustilago spp. (e.g., U. maydis); Wicker hamomyces spp. (e.g., W. anomalus); Williopsis spp.; and/or Zygosaccharomyces spp. (e.g., Z. bailii).

In preferred embodiments, 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.

As used herein “fermentation” refers to cultivation or growth of cells under controlled conditions. The growth could be aerobic or anaerobic. In preferred embodiments, the microorganisms are grown using SSF and/or modified versions thereof.

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

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, humidity, 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, a daily sample may be taken from the vessel and subjected to enumeration by techniques known in the art, such as dilution plating technique. Dilution plating is a simple technique used to estimate the number of organisms in a sample. The technique can also provide an index by which different environments or treatments can be compared.

In one embodiment, the method includes supplementing the cultivation with a nitrogen source. 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 a combination of two or more.

The method can provide oxygenation to the growing culture. One embodiment utilizes slow motion of air to remove low-oxygen containing air and introduce oxygenated air. In the case of submerged fermentation, the oxygenated air may be ambient air supplemented daily through mechanisms including impellers for mechanical agitation of liquid, and air spargers for supplying bubbles of gas to liquid for dissolution of oxygen into the liquid.

The method can further comprise supplementing the cultivation 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 glycerol; fats and oils such as soybean oil, canola oil, rice bran oil, olive oil, com oil, sesame oil, and/or linseed oil; etc. These carbon sources 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, and microelements can be included, for example, in the form of flours or meals, such as com flour, or in the form of extracts, such as 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.

In one embodiment, inorganic salts may also be included. Usable inorganic salts can 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, sodium chloride, calcium carbonate, and/or sodium carbonate. These inorganic salts may be used independently or in a combination of two or more.

In some embodiments, the method for cultivation may further comprise adding additional acids and/or antimicrobials in the medium before, and/or during the cultivation process. Antimicrobial agents or antibiotics are used for protecting the culture against contamination.

Additionally, antifoaming agents may also be added to prevent the formation and/or accumulation of foam during submerged cultivation.

The pH of the mixture should be suitable for the microorganism of interest. Buffers, and pH regulators, such as carbonates and phosphates, may be used to stabilize pH near a preferred value. When metal ions are present in high concentrations, use of a chelating agent in the medium may be necessary.

The microbes can be grown in planktonic form or as biofilm. In the case of biofilm, the vessel may have within it a substrate upon which the microbes can be grown in a biofilm state. The system may also have, for example, the capacity to apply stimuli (such as shear stress) that encourages and/or improves the biofilm growth characteristics.

In one embodiment, the method for cultivation of microorganisms is carried out at about 5° to about 100° C, preferably, 15 to 60° C, more preferably, 25 to 50° C. In a further embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivation process 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. 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 low water activity and low pH may be exploited to control undesirable bacterial growth.

In one embodiment, the subject invention further provides a method for producing microbial metabolites such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, beta-glucan, peptides, metabolic intermediates, polyunsaturated fatty acid, and lipids, by cultivating a microbe strain of the subject invention under conditions appropriate for growth and metabolite production; and, optionally, purifying the metabolite. The metabolite content produced by the method can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70 %, 80 %, or 90%.

The microbial growth by-product produced by microorganisms of interest may be retained in the microorganisms or secreted into the growth medium. The medium may contain compounds that stabilize the activity of microbial growth by-product. The biomass content of the fermentation medium may be, for example, from 5 g/1 to 180 g/1 or more, or from 10 g/1 to 150 g/1.

The cell concentration may be, for example, at least 1 x 10⁶ to 1 x 10¹², 1 x 10⁷ to 1 x 10”, 1 x 10⁸to 1 x 10¹⁰, or 1 x 10⁹ CFU/ml.

The method and equipment for cultivation of microorganisms and production of the microbial by-products can be performed in a batch, a quasi-continuous process, or a continuous process.

In one embodiment, all of the microbial cultivation composition is removed upon the completion of the cultivation (e.g., upon, for example, achieving a desired cell density, or density of a specified metabolite). In this batch procedure, an entirely new batch is initiated upon harvesting of the first batch.

In another embodiment, only a portion of the fermentation product is removed at any one time. In this embodiment, biomass with viable cells, spores, conidia, hyphae and/or mycelia remains in the vessel as an inoculant for a new cultivation batch. The composition that is removed can be a cell-free medium or contain cells, spores, or other reproductive propagules, and/or a combination of thereof. In this manner, a quasi-continuous system is created.

Advantageously, the method does not require complicated equipment or high energy consumption. The microorganisms of interest can be cultivated at small or large scale on site and utilized, even being still-mixed with their media.

In certain embodiments, the subject invention provides a “microbe-based composition,” meaning 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 by-products of microbial growth. 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. The microbes may be present in or removed from the composition. The microbes can be present, with broth in which they were grown, in the microbe-based composition. The cells may be present at, for example, a concentration of at least 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x ID¹¹, 1 x 10¹², 1 x IO¹³ or more CFU per milliliter of the composition.

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 a 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, acids, buffers, carriers, such as water, salt solutions, or any other appropriate carrier, added nutrients to support further microbial growth, non-nutrient 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, drying, purification and the like.

One microbe-based product of the subject invention is simply the fermentation medium containing the microorganisms and/or the microbial metabolites produced by the microorganisms and/or any residual nutrients. The product of fermentation may be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard extraction and/or purification methods or techniques described in the literature.

The microorganisms in the microbe-based products may be in an active or inactive form, or in the form of vegetative cells, reproductive spores, conidia, mycelia, hyphae, or any other form of microbial propagule. The microbe-based products may also contain a combination of any of these forms of a microorganism.

In one embodiment, different strains of microbe are grown separately and then mixed together to produce the microbe-based product. The microbes can, optionally, be blended with the medium in which they are grown and dried prior to mixing.

The microbe-based products may be used without further stabilization, preservation, and storage. Advantageously, direct usage of these microbe-based products preserves a high viability of the microorganisms, reduces the possibility of contamination from foreign agents and undesirable microorganisms, and maintains the activity of the by-products of microbial growth.

Upon harvesting the microbe-based composition from the growth vessels, further components can be added as the harvested product is placed into containers or otherwise transported for use. The additives can be, for example, buffers, carriers, other microbe-based compositions produced at the same or different facility, viscosity modifiers, preservatives, nutrients for microbe growth, surfactants, emulsifying agents, lubricants, solubility controlling agents, tracking agents, solvents, antibiotics, pH adjusting agents, chelators, stabilizers, ultra-violet light resistant agents, other microbes and other suitable additives that are customarily used for such preparations.

Optionally, the product can be stored prior to 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° C, 15° C, 10° C, or 5° C. On the other hand, a biosurfactant composition can typically be stored at ambient temperatures.

Claims

CLAIMS We claim:

1. A method of inhibiting the growth of an organism in a slurry, the method comprising contacting a slurry preservative composition comprising a biosurfactant with the slurry.

2. The method of claim 1, further comprising assaying the slurry for the presence of the organism.

3. The method of claim 2, wherein the assay comprises imaging the slurry, measuring a biomarker associated with and/or expressed by the organism, or a combination thereof.

4. The method of claim 3, wherein the biomarker is an exopolysaccharide, protein, mRNA, or DNA.

5. The method of claim 2, wherein the assaying is performed before the slurry preservative composition is contacted to the slurry.

6. The method of claim 1, wherein the slurry is a slurry of ore, mineral slurry, cement slurry, paper slurry, wood slurry, oil slurry, or textile slurry.

7. The method of claim 6, wherein the slurry of ore is from a coal mine, iron ore mine, copper mine, copper-nickel mine, tin mine, nickel mine, gold mine, silver mine, molybdenum mine, aluminum mine, phosphate mine, lead-zinc mine, tungsten mine, or zinc mine.

8. The method of claim 1, wherein the slurry preservative further comprises a chemical surfactant, polymer, conventional slurry preservative, biocide, or any combination thereof.

9. The method of claim 8, wherein the conventional slurry preservative is glutaraldehyde, dibromo-nitrilopropionamide (DBNPA), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), ortho-phenylphenol (OPP), l,2-Benzisothiazol-3(2H)-one (BIT), tetrahydro-3, 5-dimethyl- 1,3,5- thiadiazine-2-thione (Dazomet), bronopol, sodium ortho-phenylphenate (SOPP), alkyl dimethyl benzyl ammonium chloride (ADBAC), or any combination thereof.

10. The method of claim 1 , wherein the slurry preservative composition is in liquid or solid form, and wherein the contacting step comprises mixing the slurry preservative composition with the slurry for a time period of about 1 second to about 1 year.

1 1. The method of claim 10, wherein the contacting comprises mixing or agitating the slurry preservative composition and the slurry.

12. The method of claim 1, wherein the biosurfactant is a glycolipid and/or a yeast culture comprising a glycolipid.

13. The method of claim 12, wherein the yeast culture is a Starmerella sp. and/or a Candida sp. yeast.

14. The method of claim 12, wherein the glycolipid is a purified a glycolipid.

15. The method of claim 12, wherein the glycolipid is a sophorolipid, mannosy lerythritol lipid, trehalose lipid, rhamnolipid, or any combination thereof.

16. The method of claim 15, wherein the sophorolipid is a linear sophorolipid or a lactonic sophorolipid.

17. The method of claim 1, wherein the biosurfactant interacts with biological membranes of the organism.

18. The method of claim 17, wherein the interaction of the biosurfactant with biological membranes of the organism forms pores and destabilizes biological membranes of the organism.

19. A slurry preservative comprising a glycolipid and/or a yeast culture comprising a glycolipid and at least one conventional slurry preservative.

20. The composition of claim 19, wherein the yeast culture is a Starmerella sp. and/or a Candida sp. yeast.

21 . The method of claim 19, wherein the glycolipid is a sophorolipid, mannosylerythritol lipid, trehalose lipid, rhamnolipid, or any combination thereof.

22. The method of claim 21, wherein the sophorolipid is a linear sophorolipid or a lactonic sophorolipid.

23. The composition of claim 19, wherein the conventional slurry preservative is glutaraldehyde, DBNPA, THPS, OPP, BIT, Dazomet, bronopol, SOPP, ADBAC, or any combination thereof.