CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 63/347,230, filed May 31, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Dust can create serious health and environmental issues. The source of the dust can be a variety of particulate sources, including mines, roadways, and farms. In the minerals industry, there are two major types of dust problems — road dust and fugitive workplace dust. Excessive dust can cause equipment fouling, respiratory ailments, obstructed road visibility, and general air pollution. Without a method to inhibit dust, the dust can create pollution, often comprising respirable airborne particulates that are less than 10 pm in diameter. The airborne particulates can lead to long-term health consequences, such as, cancer and lung diseases, including, for example, coal worker’s pneumonoconiosis (CWP).

Existing dust suppression methods and compositions can contain toxic components, including asphalt emulsions or material that is ineffective for long-term dust suppression. The city of Times Beach, Missouri was permanently evacuated in 1985 and later disincorporated due, in part, to the use of sprayed waste oil containing dioxin on city streets and in barns as a means of dust suppression. Furthermore, wastewater from hydraulic fracturing is permitted and used as a dust suppressant and deicer in numerous areas of the United States, which can spread radium and organic micropollutants throughout the environment (see Tasker TL, Burgos WD, Piotrowski P, Castillo-Meza L, Blewett TA, Ganow KB, Stallworth A, Delompre PLM, Goss GG, Fowler LB, Vanden Heuvel JP, Dorman F, Warner NR. Environmental and Human Health Impacts of Spreading Oil and Gas Wastewater on Roads. Environ Sci Technol. 2018 Jun 19; 52( 12):7081 - 7091. doi: 10.1021/acs.est.8b00716. Epub 2018 May 30. PMID: 29845864.), establishing the need for improving compositions and methods for long-term and effective dust suppression.

There are four main types of water-based dust suppression applications — water/surfactant spray, foam, water fog, and plain water spray. Water/surfactant spray relies on reducing the surface tension of water droplets and fugitive dust particles, altering the inherent hydrophobic and friction-generated surface charges, enabling fine particle agglomeration, and increasing particle weight/surface area ratio to the point at which fugitive dust particles fail to remain airborne. In addition, hygroscopic inorganic salts, such as, for example, calcium chloride and magnesium chloride, are being used in mines and elsewhere to reduce the formation of dust and to stabilize surfaces. Therefore, novel, improved compositions and methods are needed for the suppression of dust.

BRIEF SUMMARY OF THE INVENTION

The subject invention relates generally to the suppression of dust. More specifically, the subject invention provides environmentally-friendly compositions and methods for suppressing dust, such as, for example, dust that is produced at mining sites, construction sites, roadways, agricultural sites, and during transport of goods. In certain embodiments, the production of the dust can be inhibited and/or existing dust can be settled with the subject compositions and methods.

Advantageously, the compositions and methods of the subject invention increase the efficiency of suppressing dust and can decrease the chemical usage, including chemical surfactant usage, required for suppressing dust. Accordingly, the subject invention can be useful for reducing the pollution produced by the production of dust, including during mining operations, creation and use of roadways, transport of goods, construction, farming, and ranching.

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, salts (e.g., magnesium chloride, ammonium chloride, calcium chloride, sodium chloride), clays, plant oils (e.g., pine oil, vegetable oil), petroleum oils, lignin, asphalt, binders, tackifiers, humectants, or any combination thereof. In certain embodiments, the biosurfactant can produce a foam when applied to a surface or to airborne dust.

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 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 suppressing dust and/or for inhibiting the creation of dust, wherein the method comprises the following step: contacting a dust suppressing composition according to the subject invention to an object and/or surface or to airborne dust.

In some embodiments, the method reduces airborne dust particles or inhibits dust production of particles that can be less than about 1 mm, about 500 pm, about 100 pm, about 10 pm, about 1 pm, about 100 nm, about 10 nm, or about 1 nm in diameter.

In some embodiments, contacting a dust suppressing composition comprising a biosurfactant and, optionally, other components, such as, for example, water, chemical surfactants, polymers, salts (e.g., magnesium chloride, ammonium chloride, calcium chloride, sodium chloride), clays, plant oils (e.g., pine oil, vegetable oil), petroleum oils, lignin, cement, asphalt to the object or surface. In certain embodiments, the dust suppressing composition can be applied to the object and/or surface or the airborne dust for a period of time and/or until a distinct volume of the composition has been applied. The step can be repeated as many times as necessary to achieve a desired reduction of airborne dust or inhibition or dust production.

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

In certain embodiments, the salt of the dust suppression composition is magnesium chloride, ammonium chloride, calcium chloride, sodium chloride, or any combination thereof.

In certain embodiments, the dust suppressing composition according to the subject invention is effective due to enhancing the wettability and/or increasing the aggregation of the particles from the source of the dust. In some embodiments, the sophorolipid or other biosurfactant serves as a vehicle for preventing the scattering of dust particles into the air. For example, in some embodiments, a sophorolipid will form a micelle containing the dust, wherein the micelle is less than 500 pm, less than 100 pm, less than 10 pm, less than 1 pm, less than 100 nm, less than 50 nm, less than 25 nm, less than 15 nm or less than 10 nm in size. The small size and amphiphilic properties of the micelle allow for enhanced penetration into the source of the dust so that greater contact can be made with particles therein, allowing for enhanced wettability or adhesion of particles.

In certain embodiments, the effectiveness of foam dust suppression results from the foaming surfactant’s bubbles contacting and collapsing around dust particles, treating the dust particle surface tension rather than adding moisture to the process. This surface tension reduction allows dust particles to agglomerate or adhere into a material mass. In preferred embodiments, the subject compositions can be applied within material freefall areas, such as, for example, transfer chutes and crushers. The compositions can produce a substantial level of dust control through several minutes of conveyor time and downstream transfer points through the material handling system.

In certain embodiments, the methods of the subject invention result in at least a 25% reduction in dust production or presence of airborne dust, preferably at least a 50% reduction, after one treatment. In certain embodiments, the airborne dust or source of the dust can be treated multiple times to further reduce the dust.

Advantageously, in certain embodiments, the dust suppressing composition according to the subject invention can be effective at inhibiting the production of dust before particulate matter becomes respirable. 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 roadway.

DETAILED DESCRIPTION

The subject invention relates generally to the suppression of dust. More specifically, the subject invention provides environmentally-friendly compositions and methods for suppressing dust, such as, for example, dust that is produced at mining sites, construction sites, roadways, agricultural sites, and during transport of goods. In certain embodiments, the production of the dust can be inhibited and/or existing dust can be settled with the subject compositions and methods.

Accordingly, the subject invention is useful for improving the efficiency and efficacy of methods of suppressing dust. Advantageously, the compositions and methods of the subject invention increase the suppression of dust 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 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 “biosurfactanf ’ 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 preferred embodiments, the SLP molecules according to the subject invention are represented by General Formula (1) and/or General Formula (2), and include 30 or more compounds having different fatty acid chain lengths (R ³ ), and, in some instances, having an acetylation or protonation at R ¹ and/or R ² .

In General Formula (1) or (2), R° can be either a hydrogen atom or a methyl group. R ¹ and R ² are each independently a hydrogen atom or an acetyl group. R ³ is a saturated aliphatic hydrocarbon chain, or an unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more Substituents.

Examples of the Substituents include halogen atoms, hydroxyl, lower (Cl -6) alkyl groups, halo lower (Cl -6) alkyl groups, hydroxy lower (Cl -6) alkyl groups, halo lower (Cl -6) alkoxy groups, and others. R ³ typically has 11 to 20 carbon atoms. In certain embodiments of the subject invention, R ³ has 18 carbon atoms.

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, “dust suppression” refers to the process by which dust is prevented or inhibited from being scattered or suspended into the air (i.e., airborne dust). Dust suppression can further comprise a process of settling dust that is suspended in the air (i.e., airborne).

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.

Dust Suppressing Compositions

In certain embodiments, the subject invention provides dust suppression compositions comprising components that are derived from microorganisms. In certain embodiments, the dust suppressing composition comprises a microbial biosurfactant. In certain embodiments, the composition comprises a biosurfactant, and, optionally, water, chemical surfactants, polymers, salts (e.g., magnesium chloride, ammonium chloride, calcium chloride, sodium chloride), clays, plant oils (e.g., pine oil, vegetable oil), petroleum oils, lignin, asphalt, binders, tackifiers, humectants, or any combination thereof.

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

In 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 dust suppressing composition.

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 dust and/or surface being treated.

In certain embodiments, the chemical surfactant of the dust suppressing 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 dust suppressing composition.

The dust suppressing 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, biocides, 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-acetylcysteine, n- hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordination group (e.g., rubeanic acid), STPP (sodiumtripolyphosphate, Na5P30lO), trisodium phosphate (TSP), water, carbohydrates, organic acids with more than one coordination group (e.g., citric acid), lipids, steroids, amino acids or related compounds (e.g., glutathione), peptides, phosphates, nucleotides, tetrapyrrols, ferrioxamines, ionophores, orphenolics, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid diacetic Acid (GLDA), GLDA-Na4, methyl glycindiacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic P-diketone, and (14,16)-hentriacontanedione, ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-dimalonic acid (EDDM), 3-hydroxy-2,2-iminodisuccinic acid (HIDS), 2-hydroxyethyliminodiacetic acid (HEIDA), pyridine-2,6-dicarboxylic acid (PDA), trimethyl glycine (TMG), Tiron, or any combination thereof.

Methods of Suppressing Dust

In certain embodiments, the subject invention provides a method for suppressing dust from various sources, including mining sites, tunneling sites, quarrying sites, construction sites, roadways, agricultural sites, and during transport of goods. In certain specific embodiments, the dust source is a mine, including coal dust, asbestos dust, metalliferous dust, crystalline silica dust; roadway dust from, for example, soil, exhaust gas, tires, brake pads, oil; agricultural dust from, for example, burning of vegetation, movement of animals, or tilling of soil; during the transport of goods, including movement of particulate matter by trucks or trains. The described elements of the subject invention are not an exhaustive examination of all applications.

In certain embodiments, the mining site can be a coal mine, asbestos mine, iron ore mine, copper mine, tin mine, nickel mine, gold mine, silver mine, or zinc mine. The mine can be an underground mine, surface mine, placer mine or in situ mine. In certain embodiments, the agricultural site can be a ranch, farmland, pastureland, rangeland, barn, pen, or animal racing track. In certain embodiments, the roadway can be asphalt, tar and chip, earthen roads (e.g., dirt and gravel), or concrete. The road surfaces can be intended for use for semi-trucks or other industrial machinery, including for example, excavator or dump trucks, or for use as an automobile, bicycle, or motorcycle racetrack. In certain embodiments, the construction site can be the site of producing a commercial building, including, for example, an office or warehouse; residential building, including, for example, an single family or multifamily home; an industrial facility, including for example, a factory; or an infrastructure project, such as, for example, a roadway, bridge, waterway, or sewer. In certain embodiments, the microbe-containing and/or biosurfactant-containing composition can improve agglomeration between the dust particles or dust particles and a surface, such as, for example, agglomerating coal dust particles to each other.

In certain embodiments, the microbe-containing and/or biosurfactant-containing composition can form a layer of aggregated particulate matter over a source of dust. The source of dust can be sealed from penetration by gases, liquids, or solid substances, thereby inhibiting dust production. In certain embodiments, the microbe-containing and/or biosurfactant-containing composition can be added to the water to extend its life as a dust control agent. Surfactants work by reducing the surface tension of water. This allows the particles from the surface to better penetrate the water droplets, thereby increasing the potential surface area coverage of the droplet. The reduced surface tension also permits easier penetration of the treated water into a few inches of the subsurface. This action allows the wetted subgrade to act as a reservoir of water such that once the surface moisture evaporates, the subgrade can provide moisture for dust control through capillary action, thus extending the life of the water.

In certain embodiments, the composition can reduce the effects of wind, rain, or physical movement of the source of the dust on the production of dust. Examples of physical movement of the source of the dust include tilling, digging, tunneling, excavating, transporting, dumping, crushing, blasting, separating, pelleting, cutting, casting, quarrying, dredging, fracturing, or any combination thereof. Other examples of physical movements of the source of the dust include frictional forces, such as, for example, driving or walking over the source of the dust or transporting the source of the dust by, for example, conveyor belt, truck, train, chute, or excavator bucket.

In certain embodiments, the microbe-containing and/or biosurfactant-containing composition has increased longevity versus traditional dust suppressing composition due to its reduced need for wetting the dust or source of the dust. As sources of dust become dry, there is an increased risk for the production of dust. By prolonging the wetting of the source of the dust, the production of dust can be reduced.

The dust suppressing compositions of the subject invention can be applied to a variety of inorganic or organic objects such as, for example, roadway, trail, rock, ore (e.g., coal ore, asbestos ore, iron ore, copper ore, tin ore, nickel ore, gold ore, silver ore, or zinc ore), wood, steel, iron, paint, plastic, paper, silk, glass, cotton, concrete, plaster, clay, stucco, plastic, rubber, or soil. The compositions can be applied to objects that reside at a range of temperatures, aquatic environments, or other dustproducing conditions.

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 certain embodiments, the time period in which the dust suppressing composition can be applied to dust or a source of dust is from 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 foam form of the dust suppressing composition to the airborne dust or source of the dust for the period of time in which dust in being produced or until the amount of airborne dust has been reduced to an amount that is determined to be satisfactory or safe, which can be readily determined by one skilled in the art. The amount of airborne dust may be considered safe depending on the site of the measurement. For example, the amount of dust may be acceptable in higher amounts at mining sites that require personal protective equipment than at primarily residential sites.

In certain embodiments, the amount of the dust suppressing 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 dust or the source of dust.

In certain embodiments, the methods of the subject invention result in at least a 25% reduction in dust production or presence of airborne dust, preferably at least a 50% reduction, after one treatment. In certain embodiments, the methods of the subject invention result in at least a 25% increase in the length of time in which the production of dust or presence of airborne dust is suppressed, preferably at least a 50% reduction, after one treatment. In certain embodiments, the airborne dust or source of the dust can be treated multiple times to further reduce the dust content.

In certain embodiments, the dust suppressing composition according to the subject invention is effective due to amphiphiles-mediated adhesion of the dust. 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. For example, in some embodiments, a sophorolipid will form a micelle containing the dust, wherein the micelle is less than 1 mm, 100 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 small size and amphiphilic properties of the micelle allow for enhanced adhesion of the dust particle so that greater suppression can be occur.

Advantageously, in certain embodiments, the dust suppressing composition according to the subject invention provides enhanced or increased efficiency at suppressing dust 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 dust suppressing composition can be performed on site, including, for example, at a mine or at a roadway. In certain embodiments, the subject dust suppressing composition can result in a decreased use of chemical surfactants or other potentially harmful chemicals used to suppress dust.

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. pullulansy 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. lichenifarmis, B. firmus, B. laterosporus, B. megateriumy, Blakeslea,' Candida spp. (e.g., C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. lorulopsisy, Clostridium (e.g., C. butyricum, C. tyrobutyricum, C. acetobutyricum, and C. beijerinckii); Campylobacter spp.; Cornybacierium 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. 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. erythropolisy, Rhodospirillum spp. (e.g., R. rubrurriy, Rhizobium spp.; Rhizopus spp.; Saccharomyces spp. (e.g., S'. cerevisiae, S. boulardii sequela, S. torula),' Sphingomonas spp. (e.g., S. paucimobilis); Starmerella spp. (e.g., S. bombicola),- Thraustochytrium spp.; Torulopsis spp.; Uslilago spp. (e.g., U. maydis),- Wickerhamomyces 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 10 ⁶ to 1 x 10 ¹² , 1 x 10 ⁷ to 1 x 10”, 1 x 10 ⁸ to 1 x 10 ¹⁰ , or 1 x 10 ⁹ 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 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 microbebased 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, 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.