Compositions and methods for clarification

Abstract

The subject invention provides safe, environmentally-friendly compositions and efficient methods of clarification. More specifically, the subject invention provides compositions derived from microorganisms for clarification, which can be used to increase the rate of clarification of liquids.

Description

[0001] DESCRIPTION

[0002] COMPOSITIONS AND METHODS FOR CLARIFICATION

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 583,635, filed September 19, 2023, which is incorporated herein by reference in its entirety.

[0005] BACKGROUND OF THE INVENTION

[0006] Efficiently processing deposits from the earth and reducing pollution associated with various mining and quarrying practices commonly employs the use of clarifiers. During mining, clarifiers are used for the removal of solids from a liquid. Clarifiers are large settling tanks that separate liquids from solids. The clarification process is often aided by the addition of flocculants and coagulants, such as, for example, polyelectrolytes and ferric sulfate. Inside the clarifier, solid contaminants settle to the bottom of the tank and are collected. The contaminants can then be concentrated and removed from the bottom of the tank. The particles that float to the surface of the liquid are called scum. The clarification process differs from that of a thickener because the clarifier is used to purify liquids, and thickeners concentrate solids. Thickeners and clarifiers can be used together to treat a suspension.

[0007] Clarifiers are useful tools in multiple industries including mining, water treatment, steel processing, automotive, plastic, and municipal water treatment plants.

[0008] There are multiple types of clarifiers: conventional, sludge blanket, and suction. Conventional clarifiers are rectangular or circular gravity sedimentation tanks. Sludge blanket clarifiers are up-flow products, in which the water flows upward from the bottom of the clarifier through a blanket of suspended solids that acts as a filter. Suction clarifiers use a vacuum system to collect sludge from the bottom of the tank. Suction clarifiers can be used for secondary and final clarification processes to further separate the suspension.

[0009] The clarification process can also be made more efficient through the use of chemical clarifying agents. Clarifying agents aggregate particles together in a liquid. After the particles are aggregated, they can be captured by a filter.

[0010] Clarification is a helpful tool in many industries, but it requires expensive equipment and yields unpredictable results. Therefore, novel, improved methods and compositions of clarification are needed. BRIEF SUMMARY OF THE INVENTION

[0011] The subject invention relates generally to clarification compositions and methods of using these compositions. More specifically, the subject invention provides environmentally-friendly clarification compositions and methods of clarification, such as, for example, methods of mining, wastewater treatment, steel processing, and household uses, including in swimming pools. In certain embodiments, existing methods can incorporate the subject compositions and methods. In certain embodiments, the subject composition and methods can replace the use of existing clarification methods.

[0012] Advantageously, the compositions and methods of the subject invention increase the efficiency of clarification and decrease the chemical usage, including chemical surfactant usage. Accordingly, the subject invention can be useful for reducing the time needed for clarification in mining and water treatment (e.g., mining wastewater, municipal wastewater, stormwater, or swimming pool water).

[0013] 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, water; chemical surfactants; polymers; pH adjusters; water softeners; ion exchangers; corrosion inhibitors; biocides, including, for example, algaecides; clarifying agents; coagulants; quaternary ammonium chlorides; polyacrylamides; natural clarifying agents, including, for example, chitin, egg albumen, gelatin, and diatomaceous earth; inorganic salts, including, aluminum, iron, magnesium, and calcium salts; or any combination thereof.

[0014] 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 biosurfactant-producing microorganism was cultivated, residual microbial cell matter or live or inactive microbial cells, residual nutrients, and / or other microbial growth by-products.

[0015] In some embodiments, the biosurfactant is utilized after being extracted from a fermentation broth and, optionally, purified.

[0016] 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, 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.

[0017] In certain embodiments, the subject invention provides a method for clarification, wherein the method comprises the following step: contacting the clarification composition according to the subject invention to a liquid.

[0018] In some embodiments, the subject method enhances the rate of clarification and / or the amount of clarified liquid.

[0019] In certain embodiments, the clarification composition according to the subject invention is effective due to enhancing and / or increasing the rate of agglomeration or total amount of the agglomerated particles from a liquid containing a colloidal suspension of said particles. For example, in some embodiments, a sophorolipid will form a micelle containing, linking, or neutralizing the particles, wherein the micelle is less than about 500 pm, less than about 100 pm, less than about 10 pm, less than about 1 pm, less than about 100 nm, less than about 50 nm, less than about 25 nm, less than about 15 nm or less than about 10 nm in size.

[0020] In certain embodiments, the methods of the subject invention result in at least a 25% increase in a clarified liquid, preferably at least a 50% increase, after one treatment when compared to a liquid that has not be contacted to the subject clarifying compositions. In certain embodiments, the liquid can be treated multiple times to further increase the amount of clarified liquid.

[0021] Advantageously, in certain embodiments, the clarifying composition according to the subject invention can be effective at clarifying liquids containing toxic particles. 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 wastewater treatment facility.

[0022] DETAILED DESCRIPTION

[0023] The subject invention relates generally to the clarification of a liquid. More specifically, the subject invention provides environmentally-friendly compositions and methods for clarification of liquids that are produced at, for example, mining sites, oil wells, or hydraulic fracturing sites, or water derived from industrial activities. Accordingly, the subject invention is useful for improving the efficiency and efficacy of methods of clarification. Advantageously, the compositions and methods of the subject invention increase the clarity of a liquid using safe, environmentally-friendly compositions. Selected Definitions

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub- ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0030] 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%.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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-faty acid complexes. Each type of biosurfactant within each class can further comprise subtypes having further modified structures.

[0036] 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).

[0037] 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).

[0038] 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). Lurther included are mono-acetylated SLP, diacetylated SLP, esterified SLP, SLP with varying hydrophobic chain lengths, cationic and / or anionic SLP with faty acid-amino acid complexes atached, 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.

[0039] In certain embodiments, the glycolipid biosurfactant is a sophorolipid (SLP). Sophorolipids are glycolipid biosurfactants produced by, for example, various yeasts of the Starmerella clade when cultivated in the presence of a hydrocarbon-based source of one or more faty acids. SLP typically consist of a disaccharide sophorose linked to long chain hydroxy faty acids. They can comprise a partially acetylated 2-O-P-D-glucopyranosyl-D-glucopyranose unit atached P-glycosidically to 17- L-hydroxyoctadecanoic or 17-L-hydroxy-A9-octadecenoic acid. The hydroxy faty acid is generally 16 or 18 carbon atoms and may contain one or more unsaturated bonds. Lurthermore, the sophorose residue can be acetylated on the 6- and / or 6’-position(s). The faty acid carboxyl group can be free (acidic or linear form (General Lormula 2)) or internally esterified at the 4"-position (lactonic form (General Lormula 1)). S. bombicola produces a specific enzyme, called S. bombicola lactone esterase, which catalyzes the esterification of linear SLP to produce lactonic SLP.

[0040] In preferred embodiments, the SLP according to the subject invention are represented by General Formula (1) and / or General Formula (2), and include 30 or more structural homologs: <O where R1and R1independently represent saturated hydrocarbon chains or single or multiple, in particular single, unsaturated hydrocarbon chains having 8 to 20, in particular 12 to 18 carbon atoms, more preferably 14 to 18 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups, R2and R2independently 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, R4and R4independently represent a hydrogen atom or -COCHs. R5is typically, but is not limited to, -H or -OH.

[0041] The composition utilized according to the subject methods can comprises more than one form of SLP, including linear SLP and lactonic SLP. The SLP can be non-acetylated, monoacetylated and / or di -acetylated SLP.

[0042] In certain specific embodiments, the composition comprises SLP according to General Formula (1) (linear SLP) wherein R1and / or R2are an acetyl group, and wherein R3is derived from a stearic, oleic and / or linoleic fatty acid.

[0043] 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 about 100 nm, less than about 50 nm, less than about 20 nm, less than about 15 nm, less than about 10 nm, or less than about 5 nm.

[0044] In certain embodiments, the glycolipid is a rhamnolipid. Rhamnolipids comprise a glycosyl head group (i.e., a rhamnose) moiety, and a 3-(hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail, such as, e.g., 3 -hydroxy decanoic acid. Two main subtypes of rhamnolipids exist, mono- and dirhamnolipids, which comprise one or two rhamnose moieties, respectively. The HAA moiety can vary in length and degree of branching, depending on, for example, the growth medium and the environmental conditions. The highest accumulation of rhamnolipids (RLP) has been shown by submerged cultivation of Pseudomonas spp., such as P. aeruginosa.

[0045] Rhamnolipids according to the subject invention can have the following structure, according to General Formula (3): wherein m is 2, 1 or 0, n is 1 or 0,

[0046] R1and R2are, independently of one another, the same or a different organic functional group having 2 to 24, preferably 5 to 13 carbon atoms, in particular a substituted or unsubstituted, branched, or unbranched alkyl functional group, which can also be unsaturated or saturated.

[0047] In certain embodiments, the alkyl functional group is a linear saturated alkyl functional group having 8 to 12 carbon atoms or is a nonyl or a decyl functional group or a mixture thereof.

[0048] Salts of these compounds are also included according to the invention. In the present invention, the term “di -rhamnolipid” is understood to mean compounds of the above formula or the salts thereof in which n is 1. Accordingly, “mono-rhamnolipid” is understood in the present invention to mean compounds of the general formula or the salts thereof in which n is 0. In certain specific embodiments, the composition comprises a mixture of mono- and di -rhamnolipids. As used herein, “beneficiation” refers to the process by which gangue materials are removed from the product of interest (e.g., element, compound, mineral).

[0049] 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.

[0050] As used herein, “colloid” or “colloidal particle” refers to a mixture in which one insoluble substance is dispersed or suspended throughout another substance. The insoluble substance is generally dispersed in a liquid.

[0051] 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).

[0052] 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.

[0053] 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.

[0054] 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.

[0055] All references cited herein are hereby incorporated by reference in their entirety. Clarification Compositions

[0056] 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, water; chemical surfactants; polymers; pH adjusters; water softeners; ion exchangers; corrosion inhibitors; biocides; clarifying agents; coagulants; quaternary ammonium chlorides; inorganic salts; or any combination thereof.

[0057] In certain embodiments, the chemical surfactant of the clarification composition is a detergent, wetting agent, emulsifier, foaming agent, and / or dispersant.

[0058] In certain embodiments, the polymers can include natural or synthetic polymers, water soluble polymers, cationic polymers, anionic polymers, or non-ionic polymers. The polymers can be, for example, polyacrylamide, anionic polyacrylamide, modified polyacrylamide, nonionic polyacrylamide, starch, guar gum, Moringa oleifera seed extract, Strychnos potatorum seed extract, gelatin (e.g., isinglass), alginate (e.g., sodium alginate), or chitosan. In certain embodiments, the polyacrylamide can be, for example, poly(N,N-dimethylacrylamide) (PDMA), poly(dimethyldiallyammonium chloride) (PDMDAAC), poly(acrylooxyethyltrimethyl ammonium chloride) (PDAC), and any combinations or copolymers thereof. Copolymers of acrylamides comprise any polymer with an acrylamide present as one of the monomers, which are polymers derived from acrylic acid. Copolymers derived from acrylic acid comprise ethylene acrylic acid copolymers, acylates copolymer, and butyl acrylate. In some embodiments, the polymer 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 clarification composition.

[0059] In certain embodiments, the pH adjuster can be any compound that can yield a pH in a liquid of about 4 to about 10, about 5 to about 9, about 6 to about 8, about 6 to about 7.5, about 6.5 to about 7.5, or about 7 upon the addition of said compound. In certain embodiments, the compound is carbon dioxide, sulfuric acid, or sodium hydroxide. In some embodiments, the pH adjuster 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 clarification composition.

[0060] In certain embodiments, the water softener is ammonia, borax, calcium hydroxide (slaked lime), trisodium phosphate, or sodium carbonate (soda ash). In certain embodiments, the water softener 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 clarification composition.

[0061] In certain embodiments, the corrosion inhibitor is, for example, a chromate, nitrate, molybdate, or tungstate. In certain embodiments, the corrosion inhibitor 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 clarification composition.

[0062] In certain embodiments, the biocide is an herbicide, insecticide, algaecide, molluscicide, miticide, piscicide, rodenticide, slimicide, germicide, antibiotic, antibacterial, antiviral, antifungal, antiprotozoal, or antiparasitic compound. In certain embodiments, the biocide 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 clarification composition.

[0063] In certain embodiments, inorganic salts comprise aluminum, iron, magnesium, and calcium salts, or any combination thereof. In some embodiments, the inorganic salt 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 clarification composition. In certain embodiments, the aluminum salt is, for example, aluminum sulfate, aluminum chloride, sodium aluminate, alum (a hydrated double sulfate salt of aluminum), aluminum chlorohydrate, polyaluminum chloride, polyaluminum sulfate chloride, polyaluminum silicate chloride, or polyaluminum chloride with organic polymers. In certain embodiments, the iron salts is, for example, ferric sulfate, ferrous sulfate, ferric chloride, ferric chloride sulfate, polyferric sulfate and ferric salts with polymers. In certain embodiments, the magnesium salt is, for example, magnesium carbonate or magnesium sulfate. In certain embodiments, the inorganic salts can be polymerized aluminum-iron blends.

[0064] In certain embodiments, the coagulants can be lime (e.g., calcium carbonate, calcium oxide) or hydrated lime (i.e., calcium hydroxide). In certain embodiments, the coagulant 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 clarification composition.

[0065] In certain embodiments, the quaternary ammonium chloride is, for example, benzalkonium chloride, benzethonium chloride, methylbenxethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium cetrimide, dofanium chloride, tetraethylammonium bromide, didecyldimethylammonium chloride, domiphen bromide, and any combination thereof. In certain embodiments, the quaternary ammonium chloride 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 clarification composition.

[0066] In certain embodiments, the clarifying is, for example, a natural clarifying agent, such as, for example, chitin, egg albumen, gelatin, and diatomaceous earth. In some embodiments, the clarifying agent 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 clarification composition. In certain embodiments, the clarification composition 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.

[0067] In some embodiments, the biosurfactant is utilized after being extracted from a fermentation broth and, optionally, purified.

[0068] 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.

[0069] 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 certain embodiments, the subject compositions can comprise lactonic and linear SLP, with at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the SLP comprising linear forms, and the remainder comprising lactonic forms.

[0070] 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 clarification composition.

[0071] 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).

[0072] 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.

[0073] In certain embodiments, the chemical surfactant of the clarification 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. O to 50%, 1.5 to 25%, or 2. O to 15% by weight, with respect to the total clarification composition.

[0074] The clarification composition can further comprise other additives such as, for example, carriers, other microbe-based compositions, additional biosurfactants, enzymes, catalysts, solvents, salts, buffers, chelating agents, acids, emulsifying agents, lubricants, solubility controlling agents, preservatives, stabilizers, ultra-violet light resistant agents, viscosity modifiers, preservatives, tracking agents, and other microbes and other ingredients specific for an intended use.

[0075] In certain embodiments, chelating agents can be, but are not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), nitrilotris(methylene)triphosphonic acid (NTTA), trimethylenedinitrilotetraacetic acid (TMDTA), L-5-glutamyl-L-cysteinylglycine (GCG), calcium trisodium diethylenetriaminepentaacetic acid, sodium nitrilotriacetic acid, a phosphonate, succimer (DMSA), diethylenetriaminepentaacetate (DTP A), A-acctylcystcinc. n-hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordination group (e.g., rubeanic acid), STPP (sodiumtripolyphosphate, NasPsOio), 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, tetrapyrroles, ferrioxamines, ionophores, orphenolics, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid, N,N diacetic Acid (GLDA), GLDA-Na4, methyl glycinediacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic - diketone, (14,16)-hentriacontane dione, ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-dimalonic acid (EDDM), 3 -hydroxy-2, 2-iminodisuccinic acid (HIDS), 2- hydroxyethyliminodiacetic acid (HEID A), pyridine-2,6-dicarboxylic acid (PDA), trimethyl glycine (TMG), Tiron, or any combination thereof.

[0076] Methods of Clarification

[0077] In certain embodiments, the subject invention provides a method for clarifying liquids from various sources, including, for example, mining sites, quarrying sites, wastewater sites, agricultural sites, and industrial sites.

[0078] In certain embodiments, the subject invention provides a method for clarifying liquids containing minerals, elements, or other target materials derived from mines. In certain embodiments, the subject method comprises contacting the subject compositions to the liquid and then removing agglomerated particles or the clarified liquid. In certain embodiments, the method of the subject invention can include the use of a clarifier machine before, during, or after the subject compositions contact the liquids containing materials derived from mines. A variety of minerals, elements, or other target materials can be derived from mining activities. In certain embodiments, methods of removing said materials from a liquid are provided according to the subject methods by contacting the clarifying compositions to various water streams, piping, pumps, water storage areas, or other aquatic environments. The compositions can be applied to liquids that reside at a range of temperatures and aquatic environments, such as, for example, a stream, river, waterway, ocean, sea, lake, pond, runoff area, containment pond, or wastewater treatment / holding tank.

[0079] In certain embodiments, the microbe-containing and / or biosurfactant-containing composition can form a layer of agglomerated particulate around and / or between particles suspended in a liquid. In certain embodiments, the microbe-containing and / or biosurfactant-containing composition can neutralize charged particles, particularly negatively charged molecules on the surface of the particles.

[0080] The clarifying composition can be applied to a liquid and, optionally, mixed by adding, pouring, or combining.

[0081] In certain embodiments, the time period in which the clarifying composition can be contacted to a liquid 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 form of the clarifying composition to the liquid for the period of time in which liquid is being produced or until the amount of clarified liquid has been increased to an amount or relative clarity that is determined to be satisfactory, which can be readily determined by one skilled in the art. The relative clarity of the clarified liquid may be considered acceptable depending on the context. For example, the relative clarity of a liquid may be acceptable in lower amounts (i.e., less clear) at mining sites that is stored in a pond at the mining site than in the treated water from municipal wastewater, in which the treated water is injected into aquifers or other waterways.

[0082] In certain embodiments, the subject method can comprise measuring the clarity of a liquid before, after, or during clarification, according to the subject invention. In certain embodiments, the subject method uses a clarity meter (i.e., transparency meter) to measure clarity. A clarity meter measures the optical distinctness with which an object can be seen through a liquid, such as, for example, the distance from the water’s surface to the depth where sunlight can no longer penetrate the water. The subject method can include other methods to measure clarity, such as, using a Secchi disk, using a device that measures the strength of a collimated beam of light, or measuring the amount of total suspended solids. A Secchi disk is used to take vertical measurements through a liquid. The measurement of the amount of total suspended solids in a water sample is performed by filtering a water sample and then drying and weighing the filter. The difference in filter weight before and after drying can provide an accurate quantity of the amount of material that was suspended in the water column when the sample was taken.

[0083] In certain embodiments, the amount of the clarifying composition 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 liquid that is treated.

[0084] In certain embodiments, the methods of the subject invention result in at least a 25% increase in the rate of clarification and / or amount of clarified liquid, preferably at least a 50% increase, after one treatment when compared to a liquid that is not treated with the subject clarifying compositions. In certain embodiments, the liquid can be treated multiple times to further increase the amount of clarification of the liquid.

[0085] In certain embodiments, the clarifying composition according to the subject invention is effective due to biosurfactant-mediated neutralization of the suspended particles. In some embodiments, the sophorolipid, or other biosurfactant, serves as a vehicle for facilitating agglomeration of particulate matter and / or adhesion of particulate matter to a surface and / or object by altering the charge of the molecules of the particles. For example, in some embodiments, a sophorolipid will form a micelle containing a particle, wherein the micelle is less than about 1 mm, less than about 100 pm, less than about 50 pm, less than about 20 pm, less than about 10 pm, less than about 1 pm, less than about 100 nm, less than about 50 nm, less than about 25 nm, less than about 15 nm or less than about 10 nm in size. The small size and amphiphilic and neutralizing properties of the micelle allow for enhanced adhesion of the particle so that greater agglomeration of particles can occur.

[0086] In some embodiments, the subject method comprises clarifying compositions that can be used in methods of processing ores, ore slurries, or other products obtained via mining. In certain embodiments, the clarifying compositions can be used for settling particles before treating the minerals or suspension.

[0087] In certain 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 clarified water can be reused in the mine, discharged into a waterway, or consumed for agricultural purposes.

[0088] In certain embodiments, in coal mines, water is used to extract and wash the coal. After the water is used to extract and wash coal, the subject methods can be used to clarify this water by separating the water from oil, grease, or other compounds that remain after the extracting and / or coal washing processes.

[0089] In certain embodiments, the subject methods can use clarification machines to treat suspended solids in mining wastewater. In certain embodiments, these methods comprise sedimentation, floc blanket clarification, and / or filtration. The subject methods use sedimentation to refine raw ore into, for example, refined borates. After dissolving the ore, the saturated borate solution is pumped into a large settling tank, then borates float to the top of the clarifier while rock and clay settle to the bottom.

[0090] The subject methods can use clarification machines (i.e., clarifiers). Clarifiers are settling tanks that continuously remove deposited solids. In certain embodiments, the clarifiers used in the subject method can be used to remove solid particles or suspended solids from liquids for clarification and / or thickening. The subject method can use different types of clarifiers, such as, for example, a sludge blanket clarifier, suction clarifier, suction header clarifier, lift suction clarifier, primary clarifier, secondary clarifier, rectangular clarifier, lamella clarifier, solids contact clarifier, or any combination thereof.

[0091] In certain embodiments, the subject methods use a sludge blanket clarifier to combine flocculation and an upward flow of solids in a liquid. In certain embodiments, the liquid containing the solids moves through a filter, clarifying the liquid. In certain embodiments, the subject methods use a suction clarifier, which has a suction duct at the bottom that applies negative pressure to the particles at the bottom of the suction clarifier. In certain embodiments, the subject methods can use a suction header clarifier, which has a sealed tube at the bottom of the clarifier, the sealed tube helps ensure the even removal of solids over the entire base of the clarifier. In certain embodiments, the subject methods can use a lift suction clarifier, which comprises vertical draft tubes and rotating sludge collection troughs. In certain embodiments, the subject methods can use primary clarifiers, which are vessels that contain skimmers or filters that remove floating material from a liquid. In certain embodiments, the subject methods can use secondary clarifiers, which are clarifiers used after a primary clarifier to remove suspended solids from a liquid. In certain embodiments, secondary clarifiers are constructed downstream of a biological treatment or activated sludge facility and work to separate wastewater from sludge. In certain embodiments, the subject methods can use a rectangular clarifier, which is a clarifier that is rectangular in shape. In certain embodiments, the subject methods can use a lamella clarifier (i.e., an inclined plate settler), which uses a cone-shaped base to separate solids from liquids. In certain embodiments, the subject methods can use a solids contact clarifier, which combines the process of mixing, flocculation, and sedimentation in a single tank. In certain embodiments, the subject methods of clarification can be used with a leaching process, such as, for example, gold cyanidation. In certain embodiments, the subject methods can be performed before or after leaching. In certain embodiments, the clarification eliminates or reduces the amount of suspended solids in the water used in the leaching process, as water that has suspended solids in it can reduce the effectiveness of the leaching process and require the use of additional leaching solution.

[0092] In certain embodiments, the clarifying compositions can be used to remove heavy metals and metalloids including, for example, As, Cd, Co, Cu, Hg, Mn, Ni, U, and Zn, from mining wastewater, which can present a significant potential ecological and human health risk associated with metal and metalloid exposure from contaminated waterways.

[0093] In certain embodiments, the clarifying composition can be used in methods of treating industrial sewage and sewage water containing water-soluble organic substances. The purification process uses activated sludge treatment for the removal of soluble organic substances. In certain embodiments, the activated sludge treatment uses the growth of microorganisms for processing, and a coagulation treatment of sludge is often used to reduce sludge volumes. In certain embodiments, the subject clarifying compositions can be used instead of conventional coagulants (e.g., inorganic salts) or in conjunction with conventional coagulants to clarify wastewater. In certain embodiments, the clarification methods can reduce the amount of unwanted particles in the wastewater by about at least about 50%, about 60%, about 70%, or about 80%.

[0094] In certain embodiments, the clarification composition can be used in various industrial methods, including in manufacturing of food, steel, oil, chemicals, petrochemicals, cars, and plastics. In certain embodiments, the subject methods can be used during the manufacturing of products in which clarifiers are used primarily in treating wastewater (i.e., to separate solids and liquids). In certain embodiments, a suspension comprising particles and a liquid are added to a clarifier, and the clarifying composition of the subject invention can be added. Then the clarifier, with the help of the clarifying composition, agglomerates the particles to form a solid that sinks to the bottom and / or floats to the top of the clarifying tank.

[0095] In certain embodiments, the subject methods can be used to treat industrial wastewater. Industrial wastewater is produced water from, for example, an oil or gas well, a hydraulic fracturing site, or an offshore oil drilling rig or water derived from chemical production plant or a petrochemical production plant. Geological features that contain oil and gas can also contain water and naturally occurring toxic substances, including arsenic and radium. In certain embodiments, the subject compositions and methods can be used to clarify the water so that, for example, the water can be recycled. In certain embodiments, the subject methods use clarifiers that are designed to clarify water by removing heavy suspended solids and dispersed, non-emulsified oil. In certain embodiments, the resulting clarified water can be used in the industrial site (e.g., oil or gas well, a hydraulic fracturing site, an offshore oil drilling rig, or a chemical production plant) or for domestic use, such as, for example, discharged into aquifers or other waterways.

[0096] In certain embodiments, the subject method can use other additives to further clarify a liquid. After the agglomeration of the particles in the liquid or in combination with the agglomeration of the particles in the liquid, a second composition comprising a coagulant, flocculant, clarifying agent, polymer, filtration aid, or any combination thereof is contacted to the liquid. In certain embodiments, the flocculant, clarifying agent, polymer, and / or filtration aid can further agglomerate the particles in the liquid by, for example, flocculating the particles in the liquid.

[0097] Advantageously, in certain embodiments, the clarification composition according to the subject invention provides enhanced or increased efficiency of clarifying a liquid 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 clarification composition can be performed on site, including, for example, at a mine or at an industrial site. In certain embodiments, the subject clarification composition can result in a decreased use of chemical surfactants or other potentially harmful chemicals used for clarifying.

[0098] Production of Microbe-Based Products

[0099] 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.

[0100] 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.

[0101] 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. radiobacie ). Arihrobacie r spp.; Aspergillus spp.; Aureobasidium spp. (e.g., A. pullulans),' Azotobacter (e.g., A. vinelandii, A. ch oococcum). 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. megaierium). Blakeslecr. Candida spp. (e.g., C. albicans, C. rugosa, C. tropicalis, C. lipolylica. C. torulopsisy, Clostridium (e.g., C. buiyricum. C. iyrobuiyricum. 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. uvaruiriy, 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. kudriavzeviiy, Phycomyces spp.; Phythium spp.; Pseudomonas spp. (e.g., P. aeruginosa, P. chlororaphis, P. putida, P. florescens, P. fragi, P. syringae),' Pseudozyma spp. (e.g., P. aphidis),' Ralslonia spp. (e.g., R. eulropha , Rhodococcus spp. (e.g., R. erythropolis),' Rhodospirillum spp. (e.g., R. rub rum). Rhizobium spp.; Rhizopus spp.; Saccharomyces spp. (e.g., .S', cerevisiae, S. boulardii sequela, S. iorid y. Sphingomonas spp. (e.g., .S', paucimobilis); Starmerella spp. (e.g., .S', bombicola , Thraustochytrium spp.; Torulopsis spp.; Ustilago spp. (e.g., U. maydisy. Wickerhamomyces spp. (e.g., W. anomalus),' Williopsis spp.; and / or Zygosaccharomyces spp. (e.g., Z. baiUi).

[0102] 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.

[0103] 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.

[0104] 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).

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Additionally, antifoaming agents may also be added to prevent the formation and / or accumulation of foam during submerged cultivation.

[0113] 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.

[0114] The microbes can be grown in planktonic form or as biofdm. In the case of biofilm, the vessel may have within it a substrate upon which the microbes can be grown in a biofdm state. The system may also have, for example, the capacity to apply stimuli (such as shear stress) that encourages and / or improves the biofdm growth characteristics.

[0115] 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.

[0116] 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 fdter 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.

[0117] 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.

[0118] 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.

[0119] The cell concentration may be, for example, at least 1 x 106to 1 x 1012, 1 x 107to 1 x 1011, 1 x 108to 1 x 1010, or 1 x 109CFU / ml.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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 1011, 1 x 1012, 1 x 1013or 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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, biocides, 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

CLAIMSWe claim:

1. A method of clarification, the method comprising contacting a clarification composition comprising a biosurfactant with a liquid containing suspended particles to yield an agglomerated particle and a clarified liquid.

2. The method of claim 1, further comprising removing said agglomerated particle from the clarified liquid.

3. The method of claim 2, wherein the agglomerated particle can be removed from the liquid by sedimentation, filtration, or thickening.

4. The method of claim 1, wherein the agglomerated particle comprises gangue.

5. The method of claim 1 , wherein the method further comprises measuring the clarity of the liquid before and / or after contacting the clarification composition to the liquid.

6. The method of claim 1, wherein the liquid is wastewater or a slurry of ore.

7. The method of claim 6, wherein the wastewater is municipal wastewater, stormwater, mining wastewater, quarrying wastewater, industrial wastewater, or produced water.

8. The method of claim 6, wherein the wastewater or 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, lead-zinc mine, tungsten mine, zinc mine, or any combination thereof.

9. The method of claim 1, wherein the clarification composition further comprises a chemical surfactant, polymer, filtration aid, coagulant, inorganic salt, quaternary ammonium chloride, polyacrylamide, a natural clarifying agent, or any combination thereof.

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

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

12. The method of claim 3, wherein the sedimentation, filtration, or thickening occurs in a clarifier.

13. The method of claim 12, wherein clarifier comprises a sludge blanket clarifier, suction clarifier, suction header clarifier, lift suction clarifier, primary clarifier, secondary clarifier, rectangular clarifier, lamella clarifier, solids contact clarifier, and any combination thereof.

14. The method of claim 1, further comprising flocculating the agglomerated particle, wherein the flocculating comprises mixing the agglomerated particle with a flocculant.

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

16. The method of claim 15, wherein the inactive yeast culture is a Starmerella sp. and / or a Candida sp. yeast.

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

18. The method of claim 17, wherein the sophorolipid is a linear sophorolipid or a lactonic sophorolipid.

19. A clarification composition comprising a glycolipid and / or an inactive yeast culture comprising a glycolipid and one or more traditional clarifying components.

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

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

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

23. The composition of claim 19, wherein the traditional clarifying components are selected from one or a combination of the following: a) chemical surfactant; b) polymer; c) fdtration aid; d) coagulant; e) inorganic salt; f) quaternary ammonium chloride; g) polyacrylamide; and h) clarifying agent.

24. The composition of claim 23, wherein the inorganic salt is aluminum sulfate, aluminum chloride, sodium aluminate, alum, aluminum chlorohydrate, polyaluminum chloride, polyaluminum sulfate chloride, polyaluminum silicate chloride, polyaluminum chloride with organic polymers, ferric sulfate, ferrous sulfate, ferric chloride, ferric chloride sulfate, polyferric sulfate, ferric salts with polymers, magnesium carbonate, magnesium sulfate, polymerized aluminum-iron blends, lime, or any combination thereof.

25. The composition of claim 23, wherein the quaternary ammonium chloride is benzalkonium chloride, benzethonium chloride, methylbenxethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium cetrimide, dofanium chloride, tetraethylammonium bromide, didecyldimethylammonium chloride, domiphen bromide, and any combination thereof.

26. The composition of claim 23, wherein the polyacylamide is poly(N,N-dimethylacrylamide) (PDMA), poly(dimethyldiallyammonium chloride) (PDMDAAC), poly(acrylooxyethyltrimethyl ammonium chloride) (PDAC), and any combinations or copolymers thereof.

27. The composition of claim 23, wherein the clarifying agent is chitin, egg albumen, gelatin, diatomaceous earth, or any combination thereof.

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