Microbial-enhancing composition that overcomes incompatibility with antagonistic surfactants

Abstract

A composition is disclosed comprising one or more surfactants that adversely affect the viability, growth, and / or biological activity of beneficial microorganisms, and a specific additive that promotes the viability, growth, and biological activity of beneficial microorganisms by overcoming the adverse effects of the surfactants. The additive capable of overcoming the adverse effects of the surfactant comprises, in specific amounts and ratios, at least one L-amino acid combined with at least one of (a) an inorganic divalent metal salt or (b) a monovalent salt, and optionally at least one carbohydrate. A method for overcoming the adverse effects that a particular surfactant has on beneficial microorganisms by combining the surfactant with a specific additive is also disclosed. The composition and method can be used in a variety of end uses involving beneficial microorganisms, including surface cleaning compositions, environmental remediation, and agricultural applications.

Description

[Technical Field]

[0001] This technology relates to a composition comprising one or more surfactants that adversely affect the viability, growth, and / or biological activity of beneficial microorganisms, and specific additives that promote the viability, growth, and biological activity of beneficial microorganisms by overcoming the adverse effects of the surfactants. This technology also relates to a method for overcoming the adverse effects that a particular surfactant has on beneficial microorganisms by combining the surfactant with a particular additive. [Background technology]

[0002] To enable improved results, there has been a recent trend to formulate products using whole cells of beneficial microorganisms, such as various Bacillus species. For example, bacterial spores may be added to cleaning formulations to obtain long-lasting cleaning effects. Bacterial spores can also be used in agricultural applications to manage pests and diseases and enhance plant health and vitality, as well as in bioremediation compositions to further accelerate the biodegradation of organic pollutants. In spore form, beneficial microorganisms do not provide any specific benefits to a composition. To obtain performance benefits, spores must germinate, multiply, and exhibit biological activity when needed. If environmental conditions such as pH, ionic strength, and nutrients (such as proteins and oils) are favorable, beneficial microbial spores can germinate and transform into vegetative cells, at which point the microorganisms produce enzymes and metabolites, digest organic matter (e.g., buried soil), solubilize inorganic plant nutrients, promote a healthy microbiome, and displace and eliminate undesirable and pathogenic microorganisms.

[0003] A crucial issue for germination, growth, and biological activity is the compatibility between beneficial microorganisms and other components in the formulation. Currently, many surfactants used in, for example, cleaning compositions, agricultural compositions, and bioremediation compositions have been found to create unfavorable environments for microorganisms. Surfactants that can adversely affect the viability of beneficial microorganisms include many common anionic surfactants such as alkyl sulfates and alkyl ether sulfates, as well as most amphoteric surfactants. Another surfactant that adversely affects beneficial microorganisms is rhamnolipid, a surfactant glycolipid produced by various bacterial species. Adding beneficial microorganisms to compositions containing these surfactants does not yield the desired benefits because the beneficial microorganisms are unable to germinate, or, after germination, cannot grow, reproduce, or survive.

[0004] Another important consideration for compositions containing beneficial microorganisms is their ability to maintain a stable suspension of microorganisms during storage and use. Microorganisms that settle at the bottom of the container may not be properly dispersed by shaking or stirring, resulting in uneven or inconsistent application of the microorganisms.

[0005] Therefore, there is a need for a composition containing beneficial microorganisms that can maintain their viability during storage and allow beneficial microorganisms to germinate and proliferate when used. Furthermore, there is a need for a composition that is stable during storage and can maintain the suspension state of microorganisms.

[0006] The applicants have demonstrated that specific additives in specific amounts and ratios can be combined with surfactants that adversely affect beneficial microorganisms, and that these additives can overcome the adverse effects of the surfactants on beneficial microorganisms, thereby promoting the viability and growth of the microorganisms. The use of these additives in microbial-enhancing compositions maintains the viability of beneficial microorganisms while simultaneously advancing the United Nations Sustainable Development Goals ("SDGs"). Many biosurfactants, such as rhamnolipids and other bio-based surfactants, are antagonistic to beneficial microorganisms, and the additives described herein enable the broader use of such bio-based surfactants in a variety of different applications. Both rhamnolipids and beneficial microorganisms, such as Bacillus species, are produced by fermentation, a less energy-intensive bio-based manufacturing process that uses renewable resources and produces biodegradable waste products with reduced environmental impact. These benefits also align with SDG Goal 12 (Responsible Consumption and Production). [Overview of the Initiative]

[0007] In one embodiment, the present technology provides a composition for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms, the composition comprising: (a) at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; (b) an additive comprising (i) at least one L-amino acid combined with at least one inorganic divalent metal salt or monovalent salt in a weight ratio of 0.2:1 to 1:1 of the additive component (i) to the surfactant; and (ii) optionally at least one carbohydrate; and (c) water in an amount totaling 100% by weight of the composition, wherein when the composition is combined with beneficial living microorganisms, the additive in the composition overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial living microorganisms and promotes the viability, growth, and biological activity of the beneficial microorganisms.

[0008] In a further embodiment, the present technology provides a method for overcoming surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms, the method comprising: (a) providing an aqueous composition comprising at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; (b) combining an additive with the aqueous composition, wherein the additive comprises (i) at least one L-amino acid combined with at least one inorganic divalent metal salt or monovalent salt in an amount that results in a weight ratio of additive component (i) to surfactant of 0.2:1 to 1:1; and (ii) optionally at least one carbohydrate; and (c) combining beneficial living microorganisms with the aqueous composition before or after step (b), wherein the additive overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial living microorganisms and promotes the viability, growth, and biological activity of beneficial microorganisms.

[0009] In another embodiment, the present technology provides a method for environmental remediation of soil suitable for bioremediation applications. The method comprises (a) applying an aqueous composition containing 0.1% to 25% by weight of rhamnolipid as an active ingredient to soil to be remediated, and (b) applying the composition to soil to be remediated, wherein the composition comprises (i) an additive comprising a blend of L-amino acids and divalent metal salts in an amount of 0.1% to 25% by weight relative to the composition, and optionally, at least one carbohydrate in an amount not exceeding the weight of the blend of L-amino acids and divalent metal salts in the composition, and (ii) 1 × 10 6 ~1 × 10 10 The process comprises (iii) a beneficial amount of live microbial spores in CFU / g, and (iii) a carrier in total amounting to 100% by weight of the composition.

[0010] In another embodiment, the technology comprises (a) at least one surfactant in an amount of about 0.05% to 1.0% by weight of active ingredient that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; (b) an additive in an amount of about 0.05% to 1.0% by weight that overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial living microorganisms and promotes the viability, growth, and biological activity of beneficial microorganisms, wherein the additive comprises (i) at least one L-amino acid; (ii) at least one inorganic divalent metal salt or at least one monovalent salt or a combination thereof; and (iii) optionally at least one carbohydrate, wherein additive components (i) and (ii) are present in the composition in an amount such that the weight ratio of additive component (i) to (ii) to surfactant is 0.2:1 to 1:1; and (c) 1 × 10 4 ~Approx. 1×10 8 The present invention provides a microbially enhanced cleaning composition comprising (d) microbial spores in an amount of CFU / g and (f) water in an amount totaling 100% by weight of the composition.

[0011] In a further aspect, the technology provides a composition for overcoming antagonistic surfactant incompatibility between an alkyl ether sulfate surfactant and beneficial live microorganisms, wherein the surfactant incompatibility has an adverse effect on the viability, growth, or biological activity of the beneficial live microorganisms. The composition comprises: (a) an alkyl ether sulfate surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial live microorganisms and has an adverse effect on the viability, growth, or biological activity of the beneficial live microorganisms; (b) an additive comprising (i) at least one monovalent salt and (ii) at least one inorganic divalent metal salt, wherein the additive and the alkyl ether sulfate surfactant are present in a weight ratio of alkyl ether sulfate surfactant to additive of 0.2:1 to 1:1; (c) an alkoxylated castor oil; and (d) water in an amount such that the total is 100% by weight of the composition. When the composition is combined with beneficial live microorganisms, the additive and the alkoxylated oil in the composition overcome the antagonistic surfactant incompatibility between the alkyl ether sulfate surfactant and the beneficial live microorganisms and promote the viability, growth, and biological activity of the beneficial microorganisms.

[0012] In some embodiments, the composition can be in the form of a liquid concentrate that is diluted, especially at dilution ratios of 1:1000, 1:400, 1:100, 1:64, 1:32, 1:16, or 1:10, before use. In other embodiments, the composition can be a ready-to-use (RTU) composition in which the active ingredients are in an amount suitable for use.

Brief Description of the Drawings

[0013] [Figure 1] FIG. 1 is a contour plot created from the test composition of Example 1 containing rhamnolipid and various amounts of additive without glucose. [Figure 2] FIG. 2 is a contour plot created from the test composition of Example 1 containing rhamnolipid and various amounts of additive with glucose. [Figure 3]Figure 3 shows contour plots prepared from the test compositions of Example 2, which contain alkyl sarcosinate surfactants and various amounts of additives. [Figure 4] Figure 4 shows contour plots prepared from the test compositions of Example 3, which contain alpha-sulfonated alkyl esters and various amounts of additives that do not contain glucose. [Figure 5] Figure 5 shows contour plots prepared from the test compositions of Example 3, which contain alpha-sulfonated alkyl esters and various amounts of additives including glucose. [Figure 6] Figure 6 is a photograph comparing the biological activity of test samples with and without additives containing a moderately hydrophilic-lipophilic ("HLB") surfactant. [Figure 7] Figure 7 is a photograph comparing the biological activity of test samples with and without additives containing low-HLB surfactants. [Figure 8] Figure 8 is a photograph comparing the biological activity of test samples with and without additives, including a dispersant. [Figure 9] Figure 9 is a photograph comparing the biological activity of test compositions with and without additives containing a hydroxysultaine surfactant. [Figure 10] Figure 10 is a contour plot showing the results from stability and oil digestion tests for compositions containing alpha-sulfonated alkyl esters and various amounts of additives. [Figure 11] Figure 11 is a photograph comparing the biological activity of test compositions containing various amounts of additives. [Figure 12] Figure 12 is a photograph comparing the biological activity of test compositions containing different potassium salts. [Figure 13] Figure 13 is a photograph comparing the biological activity of test compositions containing different magnesium salts. [Figure 14] Figure 14 is a photograph comparing the biological activity of test samples with and without additives, containing different propylene oxide-containing alcohol alkoxylate surfactants. [Modes for carrying out the invention]

[0014] definition "Beneficial microorganisms" refer to microorganisms that have a positive effect on the health and well-being of living organisms and ecosystems.

[0015] "Antagonistic surfactant incompatibility" refers to surfactants that adversely affect the survival ability, growth, and biological activity of beneficial microorganisms. "Adverse effect" or "adverse effect" means interference with the biological capabilities (such as germination), growth capacity, or biological activity of beneficial microorganisms. Such interference includes interference with any of the processes involved in the microbial life cycle, such as spore formation or germination of spores and conidia, production of metabolites and enzymes for digesting food sources, metabolism of these food sources, and maintenance and / or growth of beneficial biological populations.

[0016] "Bioactivity" refers to the various processes and functions that microorganisms perform in their environment, including their ability to grow, replicate, metabolize nutrients, release enzymes and metabolites, move or bind molecules internally, generate and respond to signals, facilitate the cycling and availability of nutrients, and interact with other organisms.

[0017] "Microbial-enhanced composition" means a composition comprising at least one surfactant that provides the main benefits of reducing aqueous surface tension and wetting substrates such as soil, leaves, or hard and soft surfaces, and at least one beneficial live microorganism.

[0018] "Inhibitory surfactants" refer to surfactants that, when used in a manner that reduces aqueous surface tension and results in wetting of substrates such as soil, leaves, and hard and soft surfaces, adversely affect the germination, growth, and / or biological activity of added beneficial microorganisms when used or combined in a common composition.

[0019] "Overcoming surfactant incompatibility" means the ability of an additive to promote the biological activity of beneficial microorganisms in the presence of an inhibitory surfactant, as indicated by a decrease in droplet size or the appearance of turbidity after 2 to 6 days, as determined according to the test methods described in the Examples.

[0020] As used herein, "carbohydrates" include, but are not limited to, simple sugars and complex molecules, monosaccharides and disaccharides including glucose, fructose, galactose, sucrose, lactose, maltose, and xylose; polysaccharides such as starch, fiber, maltodextrin, amylose, amylopectin, and glycogen; and complex sugar sources such as molasses.

[0021] The Bio-Renewable Carbon Index (BCI) refers to the calculation of the percentage of carbon derived from bio-renewable resources, and is calculated based on dividing the number of bio-renewable carbons by the total number of carbons in the entire molecule.

[0022] "Bio-renewable" is defined herein as derived from animal, plant, or marine biological materials. The terms "active ingredient," "active ingredient %," and "active ingredient by weight %" refer to the amount of the active ingredient, without considering the amount of water or other solvents that may be present with the ingredient.

[0023] The terms "ready to use" or "RTU" products, compositions, or formulations of this technology mean products, compositions, or formulations that can be applied or used immediately as is. The “Dilution,” “Concentrate,” or “Dilution Concentrate” products, compositions, or formulations of this Technology mean products, compositions, or formulations that need to be diluted with a diluent (e.g., water) in a ratio of, for example, 1:1000, 1:400, 1:100, 1:64, 1:32, 1:16, or 1:10 before they can be applied or used for their intended purpose.

[0024] As defined herein, “rhamnolipid” is a glycolipid having one or more typically linear, saturated or unsaturated β-hydroxycarboxylic acid moieties in a lipid moiety and one or more units of rhamnose in a sugar moiety. The sugar moiety and the lipid moiety are linked via β-glycosidic bonds between the 1-OH group of the rhamnose moiety of the sugar moiety and the 3-OH group of the β-hydroxycarboxylic acid in the lipid moiety. Thus, the carboxylic acid of one carboxylic acid moiety defines the terminus of the rhamnolipid. If two or more rhamnose moieties are present in the rhamnolipid, each of the rhamnose moieties not linked to the lipid moiety is linked to another rhamnose moiety via a 1,4β-glycosidic bond. In embodiments where two or more β-hydroxycarboxylic acids are present in the rhamnolipid, the β-hydroxycarboxylic acid moieties are selected independently of each other. In some embodiments, the β-hydroxycarboxylic acid moieties may be identical. In some embodiments, they may be different from each other.

[0025] This technology is based on the discovery that many useful and important classes of surfactants are antagonistic to beneficial microorganisms at surfactant usage levels considerably lower than those required for typical product performance. However, it has been found that by combining specific additives with surfactants, surfactant incompatibility can be overcome, making surfactants usable in compositions containing beneficial microorganisms. As a result, beneficial microorganisms in the composition can germinate, grow, and exhibit biological activity when the additives are used in combination with one or more of these incompatible surfactants. While not bound by any particular theory, it is believed that the desired effect of overcoming antagonistic surfactant incompatibility can be achieved by combining specific additives in selected amounts and ratios.

[0026] The composition of this technology contains at least one surfactant that is incompatible with beneficial microorganisms. Incompatibility between surfactants and beneficial microorganisms has been investigated by various methods, including the standard zone of inhibition test, the standard agar plate test, or the standard resazurin (7-hydroxy-10-oxidephenoxazine-10-ium-3-one, sodium) (blue fluorescent dye) compatibility test. In the zone of inhibition test, microorganisms are grown, cultured, diluted to the target level, and then applied to a petri dish containing agar nutrient medium. The test sample containing the target surfactant is diluted and packed into agar wells prepared in the agar nutrient medium. The plate is incubated to allow microbial growth to proceed. The test sample diffuses through the agar, and if the surfactant in the test sample has an inhibitory effect on the microorganism, a ring-shaped area of ​​non-growth is observed around the well filled with the sample. This ring-shaped area is called the zone of inhibition. The larger the zone of inhibition, the greater the inhibitory effect. The main limitations of this test method for non-antibiotic test samples are the inherent dilution of the active ingredient, the challenges of agar diffusion, and the incompatibility of the nutrient medium (neutralization, precipitation, etc.). While this method has been very well used in the medical field for antibiotics for decades, its suitability for testing non-antibiotic samples is low. In the standard agar plate test, a test sample containing the target surfactant and microorganism is exposed, diluted by several orders of magnitude, and incubated on a nutrient agar plate. After an incubation period appropriate for the bacterial species, the microbial growth pattern is evaluated against a suitable control. This method is suitable for evaluating viable cell counts over time, but is not very suitable for inhibition testing due to the significant serial dilution required to remove all coformulants from the test microbial species on the evaluation agar plate. In the resazurin-based test, a test sample containing the target surfactant is mixed with reduced microbial nutrients, beneficial microorganisms, and resazurin in a suspension. The test sample is incubated, and then the colorimetric and fluorescence changes are examined. A test sample that is blue and has low fluorescence indicates a lack of biological activity and incompatibility between the surfactant and microorganisms.Test samples that are pink and highly fluorescent are bioactive and indicate compatibility between the surfactant and microorganisms. This test and other similar suspension tests address the limitations of the conventional tests mentioned above and provide a suitable, high-throughput, microorganism-independent platform for evaluating the inhibitory effects of surfactants on bioagents.

[0027] Surfactants found to be incompatible with beneficial microorganisms at surfactant concentrations of 1 g / L or less include some anionic surfactants, some nonionic surfactants, most, if not all, amphoteric surfactants, and most, if not all, cationic surfactants. Specific examples of incompatible surfactants include alkyl sulfates, alkyl ether sulfates, alkyl sarcosinates, alkyl glutamates, alpha-sulfonated alkyl esters, alkyl sulfonates, alkyl sulfoacetates, some alkyl phosphate esters, sulfosuccinates, rhamnolipids, hydroxysultaines, alkyl betaines, alkylamidopropyl betaines, alkylamine oxides, alkylamine alkoxylates, quaternized alkylamine alkoxylates, some alcohol alkoxylates, monoglycerides and / or diglycerides, and some EO / PO block copolymers. Any of these incompatible surfactants may be used in the compositions of this technology, and it is intended that they can be made compatible with beneficial microorganisms through the combination of additives and surfactants described herein.

[0028] The amount of surfactant in a composition varies depending on whether the composition is a concentrate intended to be diluted with water or other diluents before use, or whether the composition is a ready-to-use composition where the active ingredient is already at its final use concentration. The amount of surfactant may also depend on the specific inhibitory surfactant in the composition and the end use. In concentrated compositions, the amount of surfactant may range from about 5% to about 20% by weight of the active ingredient, based on the total weight of the composition. In ready-to-use compositions, the amount of surfactant may range from 0.05% to about 1% by weight of the active ingredient, based on the total weight of the composition.

[0029] The compositions of this technology also include additives that can be combined with surfactants to overcome antagonistic surfactant incompatibility between surfactants and beneficial microorganisms and to promote the viability, growth, and biological activity of beneficial microorganisms. In some embodiments, the additive comprises at least one L-amino acid in combination with at least one inorganic divalent metal salt or monovalent salt. The additive may, at least in part, be a combination of L-amino acid and a divalent metal salt, a combination of L-amino acid and a monovalent salt, or a combination of L-amino acid and both a divalent metal salt and a monovalent salt, depending on the specific surfactant used in the composition. The additive may also contain carbohydrates as an optional component. In other embodiments, depending on the specific surfactant used in the composition, the additive may comprise a combination of a divalent metal salt and a monovalent salt that does not contain an L-amino acid.

[0030] The L-amino acid may be one or more L-amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-serine, L-arginine, L-cysteine, L-glutamine, L-glycine, L-proline, and L-tyrosine. In some embodiments, the L-amino acid is L-alanine.

[0031] The inorganic divalent metal salt may be one or more of the following: magnesium chloride, calcium chloride, manganese chloride, iron chloride, copper chloride, zinc chloride, cobalt chloride, magnesium nitrate, magnesium sulfate, calcium sulfate, manganese sulfate, iron sulfate, copper sulfate, zinc sulfate, cobalt sulfate, magnesium citrate, calcium citrate, any of the above hydrates, or any combination thereof. In some embodiments, the divalent metal salt is magnesium chloride or its hydrate.

[0032] The monovalent salt may be one or more of the following: potassium nitrate, sodium nitrate, potassium chloride, potassium iodide, potassium manganese oxide, potassium sulfate, sodium bicarbonate, sodium sulfate, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium citrate, potassium citrate, ammonium citrate, or a combination thereof. In some embodiments, the monovalent metal salt is potassium nitrate.

[0033] The additive may optionally contain carbohydrates, which can provide a food source for beneficial microorganisms and enhance their growth. The carbohydrates may be one or more of glucose, maltose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan. In some embodiments, the carbohydrate is glucose. If carbohydrates are included in the additive, their amount is preferably less than or equal to the total weight of the other additive components in the additive (L-amino acids, divalent metal salts, and / or monovalent salts).

[0034] The total amount of additives in a composition of this technology varies depending on whether the composition is a concentrate or a ready-to-use composition. In a concentrated composition, the total amount of additives may be in the range of about 5% to about 20% by weight of active ingredients, or alternatively, about 5% to about 10% by weight, based on the total weight of the composition. In the case of a ready-to-use composition, the total amount of additives may be in the range of 0.05% to about 1% by weight of active ingredients, based on the total weight of the composition. The amount of additives also depends on the amount of surfactant in the composition. The weight ratio of additives (L-amino acids, divalent metal salts, and / or monovalent salts) to surfactant in the composition may be in the range of 0.20:1 to about 1:1. In some embodiments, a composition of this technology contains about 1% by weight of additives (L-amino acids, divalent metal salts, and / or monovalent salts) per 1% by weight of surfactant active ingredients in the composition, and the weight ratio of surfactant to additives (excluding the weight of carbohydrates, if present) may be about 1:1. In some embodiments, a composition of this technology may have a surfactant to additive weight ratio of about 5:1. The specific amounts and relative ratios of L-amino acids, divalent metal salts, and monovalent salts in additives can vary, at least in part, depending on whether the surfactant and additives in the composition contain carbohydrates. In the case of inhibitory surfactants containing carboxylate moieties, such as rhamnolipids and sophorolipids, a divalent metal salt should be included as one of the additives to overcome the inhibitory effect on beneficial microorganisms. In the case of inhibitory surfactants that do not contain carboxylate moieties, the inhibitory effect can be overcome if the additive does not contain a divalent metal salt, or contains only a small amount.

[0035] In some embodiments, the surfactant in the composition is a rhamnolipid surfactant, and the additives include L-amino acids and divalent metal salts. Since rhamnolipid is naturally derived, its BCI is 100. The L-amino acids and divalent metal salts may be combined in any weight ratio, but a preferred weight ratio of L-amino acids to divalent metal salts is 1:1 or greater. If carbohydrates such as glucose are included along with the L-amino acids and divalent metal salt additives, a preferred weight ratio of divalent metal salts to L-amino acids is 1:1 or greater. In other embodiments where rhamnolipid is the surfactant, the additives may include a combination of L-amino acids, divalent metal salts, monovalent salts, and optionally carbohydrates. If the additives do not include carbohydrates, a preferred weight ratio of the additive components is 4:1 or greater, in the weight ratio of (divalent metal salt + L-amino acid) to monovalent salt. A particularly preferred additive blend for use with rhamnolipid surfactants comprises 0.7% by weight of a divalent metal salt, e.g., magnesium chloride hydrate, 0.15% by weight of a monovalent salt, e.g., potassium nitrate, and 0.15% by weight of an L-amino acid, e.g., L-alanine, per 1% by weight of the active ingredient amount of rhamnolipid surfactant. If carbohydrates are present in the additive, the preferred weight ratio of the additive components is 1:1 or greater for divalent metal salts to (L-amino acid + monovalent salt).

[0036] In some embodiments, the surfactant in the composition comprises an alkyl sarcosinate surfactant, and the additive comprises an L-amino acid, a monovalent salt, and a carbohydrate. Preferably, the alkyl sarcosinate surfactant is of natural origin and has a BCI of at least 80, alternatively at least 90, alternatively at least 95, and preferably 100. Since divalent metal salts can co-precipitate with alkyl sarcosinates, it may be desirable to use additives that do not contain divalent metal salts when the surfactant contains an alkyl sarcosinate. However, divalent metal salts may be included in amounts up to about 10% by weight based on the total weight of the additive, and this can still overcome the inhibitory effect of the alkyl sarcosinate surfactant. In some embodiments, the additive may contain an L-amino acid, a monovalent salt, and a carbohydrate such as glucose in a weight ratio of (monovalent salt + L-amino acid) to carbohydrate of 1:1 or more. Alternatively, the additive may contain an L-amino acid, a monovalent salt, and a carbohydrate in a weight ratio of monovalent salt to (L-amino acid + carbohydrate) of 2:1 or more. In one embodiment, the additive blend for use with an alkyl sarcosinate surfactant comprises 0.45% by weight of a monovalent salt, such as potassium nitrate, 0.45% by weight of an L-amino acid, such as L-alanine, and 0.1% by weight of a divalent metal salt, such as magnesium chloride hydrate, per 1% by weight of the alkyl sarcosinate surfactant as an active ingredient.

[0037] In some embodiments, the surfactant in the composition comprises an alpha-sulfonated alkyl ester, and the additive comprises an L-amino acid, a divalent metal salt, a monovalent salt, and optionally a carbohydrate. Preferably, the alpha-sulfonated alkyl ester surfactant is of natural origin, and its BCI is at least 80, alternatively at least 90, alternatively at least 95, and preferably 100. If the additive does not contain a carbohydrate, the preferred weight ratio of the additive components is 3:1 or greater, in the weight ratio of (L-amino acid + monovalent salt) to divalent metal salt. If a carbohydrate is present in the additive, the preferred weight ratio of the additive components is 1:1 or greater, in the ratio of monovalent salt to (L-amino acid + divalent metal salt). The amount of carbohydrate, if present, may not exceed the total amount of other additives in the composition. In one embodiment, the additive blend for use with the alpha-sulfonated alkyl ester surfactant comprises 0.45% by weight of a monovalent salt, such as potassium nitrate, 0.45% by weight of an L-amino acid, such as L-alanine, and 0.1% by weight of a divalent metal salt, such as magnesium chloride hydrate, per 1% by weight of the active ingredient amount of the alpha-sulfonated alkyl ester surfactant.

[0038] The compositions of this technology, when combined with beneficial microorganisms, promote the viability, growth, and biological activity of those beneficial microorganisms. The beneficial microorganisms that can be used in combination with the compositions may be bacteria, fungi, yeasts, or molds, and may be pores (also called spores), vegetative cells, conidial cells, or mycelial cells. In some embodiments, the bacterial endospores are of the genus Bacillus.Bacillus spores include those of Bacillus amyloliquefaciens, Bacillus brevis, non-pathogenic mutants of Bacillus cereus (such as toyoi), Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, and Bacillus megatherium. megaterium), Bacillus methylotrophicus, Bacillus mycoides, Bacillus pasteurii, Bacillus polyfermenticus, Bacillus polymyxa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thiaminolyticus, Bacillus thuringiensis (Bacillus It may be one or more of the following: Bacillus thuringiensis, and combinations thereof. In some embodiments, the beneficial microorganism is a blend of bacterial spores of two or more species, in particular a blend of two or more Bacillus species. Blends of Bacillus spores of different Bacillus species are commercially available from various sources.The blend of Bacillus spores can include spores derived from one or more of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus thuringiensis, and Bacillus pumilus.

[0039] The amount of beneficial microorganisms that can be combined with the compositions of the present technology varies depending on the end use of the composition, but generally, it is at least 1×10 4 CFU / g, more preferably at least 1×10 5 CFU / g. When the composition is a concentrate that is diluted before use, the amount of beneficial microorganisms can be in the range of 1×10 7 ~1×10 10 CFU / g, preferably 1×10 9 ~5×10 9 CFU / g. When the composition is a ready-to-use composition, the amount of beneficial microorganisms can be in the range of 1×10 4 ~1×10 9 CFU / g, or alternatively 1×10 4 ~1×10 8 CFU / g, or alternatively 1×10 5 ~1×10 9 CFU / g, or alternatively 1×10 5 ~1×10 8 CFU / g.

[0040] In addition to overcoming antagonistic surfactant incompatibility and promoting the viability, growth, and biological activity of beneficial microorganisms, the additives of the present technology unexpectedly improve the dispersibility and stability of beneficial microorganisms in water-thin aqueous compositions and can also prevent or minimize spore aggregation. This additional benefit can be obtained regardless of whether an incompatible surfactant is present in the aqueous composition.

[0041] The compositions of this technology may contain optional components depending on the end use of the composition. Such other optional components include additional surfactants, hydrotropes or other solubilizers for obtaining and maintaining a clear, single-phase concentrated composition or a ready-to-use composition, additional carbohydrates other than those mentioned above, builders, pH adjusters, electrolytes to enhance the cleaning power of surfactants, enzymes to enhance cleaning properties, fragrances for various attractive scents, dyes for desirable colors, preservatives, and other functional components. Preferably, any optional component is compatible with beneficial microorganisms.

[0042] Surfactants that have been found to be compatible with beneficial microorganisms and may be included in the composition as additional surfactants include, but are not limited to, castor oil ethoxylate, alpha-olefin sulfonates, e.g., BIO-TERGE® AS-40 of C14-16 olefin sulfonates, polyoxyethylene sorbitan monooleate, e.g., those sold under the trademarks TWEEN® and SPAN®, certain alkyl (alkenyl) dimethylamides, e.g., STEPOSOL MET-10U of unsaturated C10 N,N-dimethylamide, blends of alkyl ether sulfate sodium with alkylamidopropyl betaine and / or alkyl betaine, and combinations thereof. In some embodiments, it has been found beneficial to combine castor oil ethoxylate with additives to improve the ability of the additive to overcome incompatibility between the surfactant and beneficial live microorganisms.

[0043] Suitable hydrotropes for use in the compositions of this technology include sodium xylenesulfonate, cumenesulfonate, amphoteric dipropionate salts, and combinations thereof. Suitable carbohydrates include cellulose, maltodextrin, fiber, amylose, amylopectin, glycogen, starch, or combinations thereof. Sodium gluconate and sodium citrate dihydrate are builders that have been found to be compatible with beneficial microorganisms.

[0044] To enhance the cleaning properties, enzymes may be included in the composition of this technology. Suitable enzymes include proteases, amylases, and lipases. Preservatives may be included in the composition of this technology, provided that the amount of preservative does not affect the viability, growth, and / or biological activity of beneficial microorganisms at the dilution ratio of the composition used. Examples of preservatives that may be used include, but are not limited to, phenoxyethanol, isothiazolinone and / or benzisothiazolinone, benzyl alcohol, fatty acids and organic acids such as benzoic acid or benzoates with appropriate pH adjustment, or combinations thereof.

[0045] The compositions of this technology can be used in combination with beneficial microorganisms for a variety of end uses. In particular, the compositions of this technology are intended to be used in any end use that utilizes beneficial microorganisms and inhibitory surfactants. Such end uses include microorganism-enhanced detergents, including detergents for hard and soft surfaces; bio-insecticides and bio-fungicides for agricultural use; personal care formulations; bioremediation; wastewater treatment; fermentation; probiotics; animal health; aquaculture; water recycling; and food applications.

[0046] As an example, the compositions of this technology can be combined with microbial spores to form a microbially enhanced cleaning composition. When the composition is used to clean a surface, the spores germinate and digest dirt that is often inaccessible during initial cleaning, such as grout, flooring, and dirt embedded in porous surfaces such as countertops. The surfactant in the cleaning composition provides initial cleaning of the surface, while the additives in the composition enable the survival and proliferation of microbial spores, resulting in enhanced cleaning. It is assumed that the microbially enhanced cleaning compositions using the additives described herein can be formulated without microbial spores, and the microbial spores can be added to the composition at the time of use. Alternatively, the microbially enhanced composition may be formulated to include microbial spores together with the surfactant and additives.

[0047] Microbial-enhanced cleaning compositions may be formulated, for example, as ready-to-use products or concentrated dilution products. Concentrated products may be up to 1000 times the ready-to-use component levels. Whether in ready-to-use form or concentrated dilution, the final usable concentrations of the components are equivalent. The final usable concentration consists of approximately 0.05% to 1% by weight of surfactant active ingredients, approximately 0.05% to 1% by weight of total additives, and approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 8 Another option is approximately 1 × 10 5 ~Approx. 1×10 8 It may contain beneficial microorganisms in CFU / g. The dilution concentrate contains approximately 5.0% to 20% by weight of surfactant active ingredients, approximately 5.0% to 25% by weight of total additives, or as an alternative, approximately 5.0% to 20% by weight of total additives, and approximately 1 × 10⁻¹⁶ 7 ~Approx. 1×10 10 It may contain beneficial microorganisms in CFU / g. In some embodiments, dilution compositions are preferred as cost-saving and expense-reducing options, as they reduce packaging and transport costs. In some embodiments, concentrates may be packaged as liquids or sprays that can be diluted to working concentrations on-site and used immediately.

[0048] The diluent for diluting the concentrated form of the composition may be any diluent system known in the art. Suitable diluents include, but are not limited to, water, glycols (preferably propylene glycol), alcohols (e.g., isopropanol, ethanol, methanol), other polar solvents known in the art, and mixtures thereof. Water is a preferred diluent for the techniques described in this invention and may be deionized water, hard water, soft water, distilled water, tap water, or a combination thereof.

[0049] As another example of end-use, the compositions of this technology can be combined with microorganisms to enable the use of inhibitory surfactants in agricultural biocontrol formulations, biofertilizer formulations, biostimulant formulations, bioinsecticide formulations, and biofungicide formulations. In agriculture, microorganisms, typically of the spore type, and typically of the Bacillus genus, are applied to plants and / or fields to suppress the growth of pathogenic organisms, including fungi, and help crops grow more healthily. Many agricultural applications rely on surfactants to effectively wet the leaf surface for the efficient use of components in formulations containing microorganisms such as Bacillus. However, these agricultural surfactants can adversely affect the viability, growth, and / or bioactivity of microorganisms, both in formulations and at concentrations suitable for end-use. The additives in the compositions of this technology can overcome the adverse effects of surfactants, thereby enabling the use of these surfactants in agricultural bioinsecticide and biofungicide formulations.

[0050] For agricultural applications, the compositions of this technology are typically formulated as concentrates and diluted before use at dilution ratios ranging from 1:2.5 to 1:3200, 1:100 to 1:1000 as an alternative, and 1:256 to 1:512 as an alternative, preferably at a dilution ratio of 1:400. The concentrates consist of about 1.0% to about 20% by weight of surfactant active ingredients, about 1.0% to about 20% by weight of total additives, and about 1 × 10⁻⁶ 6 ~Approx. 5×10 10 CFU / g, another option is approximately 1 × 10 7 ~Approx. 1×10 10 It may contain beneficial microorganisms in CFU / g.

[0051] As a further example of end-use, the compositions of this technology can be used in environmental remediation applications that allow beneficial microorganisms to remove contaminants from soil. Beneficial microorganisms, such as Bacillus species, can be used to decompose chemicals and contaminants in the soil through the production of enzymes and acids. Surfactants can be used in combination with bacteria to emulsify contaminants, particularly hydrocarbons, and to increase their bioavailability to the bacteria. Rhamnolipid, in particular, is an attractive biosurfactant for use in soil remediation applications due to its favorable biodegradability and low toxicity. However, rhamnolipid also has antimicrobial properties and has been found to adversely affect the viability, growth, and / or biological activity of bacteria at surfactant concentrations considerably lower than those useful for soil remediation. The additives in the compositions of this technology can overcome the adverse effects that rhamnolipid has on beneficial microorganisms, thereby enabling the use of rhamnolipid together with beneficial microorganisms in soil remediation applications.

[0052] The soil remediation using rhamnolipid and additives of this technology can be achieved by applying an aqueous composition containing rhamnolipid to the soil to be remediated, and by applying an additive containing (a)(i) a blend of divalent metal salts and L-amino acids, and (ii) optionally carbohydrates, (b) beneficial live microbial spores, and (c) a carrier to the soil to be remediated. The aqueous composition contains rhamnolipid in an active ingredient amount of about 0.1% to about 25% by weight, as an alternative, about 1% to about 20% by weight, and as an alternative, about 5% to about 10% by weight, and can be applied to the soil in amounts ranging from about 1g to about 200g per kg of soil, as an alternative, about 2g to about 150g per kg of soil, and as an alternative, about 5g to about 100g per kg of soil. The composition containing the additive is a blend of divalent metal salts and L-amino acids, based on the weight of the composition, in amounts of approximately 0.1% to approximately 25% by weight, as an alternative option, approximately 1% to approximately 20% by weight, as an alternative option, approximately 5% to approximately 10% by weight, approximately 1 × 10 6 ~Approx. 1×10 10The composition contains CFU / g of microbial spores and a carrier in an amount totaling 100% by weight of the composition. If carbohydrates are included as an additive, the amount of carbohydrates is preferably less than or equal to the total weight of divalent metal salts and L-amino acids in the composition. The relative amounts by weight of divalent metal salts and L-amino acids in the composition may be 1:1 or more in weight ratio of L-amino acids to divalent metal salts if no carbohydrates are present, and 1:1 or more in weight ratio of divalent metal salts to L-amino acids if carbohydrates are present. In some embodiments, the L-amino acid includes L-alanine, the divalent metal salt includes magnesium chloride or its hydrate, and the carbohydrate, if present, includes glucose. Bioremediation may be carried out using various species of bacteria, fungi, or algae, including Bacillus species in spore form, or combinations thereof. The carrier may be any carrier that is compatible with microbial spores and suitable for soil remediation. Water is a suitable carrier for compositions containing additives. The amount of composition applied to the soil may range from approximately 1 g to 200 g per kg of soil, or alternatively, from approximately 2 g to 150 g per kg of soil, or alternatively, from approximately 5 g to 100 g per kg of soil.

[0053] This technology also includes a method for overcoming surfactant incompatibility between beneficial microorganisms and surfactants, which adversely affects the viability, growth, or biological activity of beneficial microorganisms. The method involves combining the additives described herein with a surfactant exhibiting antagonistic surfactant incompatibility and beneficial microorganisms, wherein the amount of the additive is 0.2:1 to 1:1 by weight ratio of additive (L-amino acids, divalent metal salts, and / or monovalent salts) to surfactant, and the additive overcomes antagonistic surfactant incompatibility and promotes the viability, growth, and biological activity of the beneficial microorganisms. In one embodiment of the method, the additive is combined and mixed with an aqueous composition containing the incompatible surfactant, and then, as a final step, beneficial microorganisms are added to the aqueous composition. The beneficial microorganisms may be added to the aqueous composition at the site of application, or may be pre-mixed with the aqueous composition to form the final product. Alternatively, the beneficial microorganisms may be added to the aqueous composition containing the incompatible surfactant in the absence of the additive, in which case the additive is added at a later stage. The absence of additives during the initial mixing of the surfactant and beneficial microorganisms ensures that microbial activation does not occur too quickly. Adding additives to the composition activates the microorganisms at that point, consuming the soil on and within the surface to act beneficially on crops or provide other beneficial properties.

[0054] The pH of the composition may be adjusted to 6-8 if necessary, depending on the selection of the unsuitable surfactant. Standard mixing equipment may be used to combine the unsuitable surfactant, additives, and beneficial microorganisms. Before mixing, the efficiency of mixing and the dispersibility of the beneficial microorganisms can be improved by dispersing some of the surfactants and additives used in the entire composition.

[0055] Those skilled in the art will recognize that modifications can be made to the present invention without departing from the spirit or scope of the invention. The present invention is further described by the following examples, but these examples should not be construed as limiting the spirit or scope of the invention to the specific procedures or compositions described therein.

[0056] The pH of the composition was determined at room temperature (20-25°C) using a calibration electrode. In Examples 1-3 below, bacterial spore germination, growth, and biological activity were evaluated using a stained extra virgin olive oil (EVOO) digestion test, which is a restriction nutrient suspension test similar to the resazurin method. Since EVOO is a food source for vegetative cells, thinly stained EVOO added to a test sample containing bacterial spores can serve as an indicator of biological activity. Spores that have germinated and grown into fully functional vegetative cells secrete enzymes that can digest EVOO, so a decrease in the amount of oil over time indicates that the spores have germinated and grown into vegetative cells that digest the oil. For the digestion test, 100 g of each test composition was added to the test surfactant, different amounts of additives, and 1 × 10⁶ spores from a commercially available source. 8The test compositions were prepared by combining CFU / g Bacillus species mixes. Each test composition also contained 0.20g of a 33g / L nutrient mix (30g / L triptych soy broth BD211825 and 3g / L yeast extract BD212750, pH 7.1-7.4). The test compositions were mixed and then stored at 37°C for 2-3 days to condition the spores. After conditioning, 1g of the test composition was placed in a scintillation vial, and one drop (approximately 15mg) of lightly stained (Sudan III, 62.5ppm) EVOO was added to the test sample in the scintillation vial without mixing. The initial oil droplets were photographed digitally and then photographed again after being gently swirled on an orbital shaker set to 1-2 on a scale of 0-10 for 4 days. The oil droplet size is determined by transferring the captured images to a computer and measuring the major axis of each oil droplet using Tracker.jar line profile analysis (https: / / physlets.org / tracker / ), thereby determining the diameter length in pixels. The oil droplet sizes of the initial and post-digestion test samples are compared. A decrease in measured droplet diameter indicates bacterial spore germination and growth, as well as EVOO digestion, which indicates that the additives in the test composition have overcome antagonistic surfactant incompatibility. An increase in measured droplet diameter is interpreted as a combination of a change in surface tension due to the surfactant and the absence or minimal digestion of EVOO by bacteria.

[0057] Using Stat-Ease 360 ​​software, sample sets for each experimental design were created, the test results were modeled from the obtained data, and statistical results for the optimal model were generated. Subsequently, optimization plots were created using this model and the numerical optimization function of Stat-Ease 360. The optimization included contour plots of the "desirability" of component levels against a defined design space. These plots visually represent the complex relationships between surfactant components and additive components, showing areas where the desired performance is high and areas where it is low or zero. [Examples]

[0058] Example 1: Formulation containing rhamnolipid and additives Test compositions containing rhamnolipid surfactant and different amounts of additives were prepared as described above to evaluate the ability of additives to overcome antagonistic incompatibility between rhamnolipid surfactant and bacterial spores. The additives used were L-alanine, magnesium chloride hexahydrate, potassium nitrate, and glucose. The rhamnolipid surfactant was a mixture of mono-rhamnolipid and di-rhamnolipid in a weight ratio of di-rhamnolipid to mono-rhamnolipid in the range of 55:45 to 56.5:43.5. The mixture contained RhaRha-C10-C10 in amounts ranging from 36% to 38% by weight, and RhaRha-C10-C10 in amounts ranging from 35% to 37% by weight, based on the total weight of rhamnolipid in the mixture.

[0059] Samples of each test composition were tested for EVOO digestion using the test procedure described above. Using the EVOO digestion data, Stat-Ease 360 ​​optimized contour plots were created for the test compositions with and without glucose as one of the additives. The contour plots for the test compositions with and without glucose as an additive are shown in Figures 1 and 2, respectively. The optimized plots show contours indicating high-desirability regions and zero-desirability regions corresponding to different amounts of different additives. In the contour plot shown in Figure 1, the high-desirability region is along the left side of the plot, with a wider area in the lower left corner, while the low-desirability region is the dark area on the right side of the plot. The high-desirability region occurs with L-alanine or a blend of L-alanine and magnesium chloride, and may also contain small amounts of potassium nitrate. As shown in Figure 2, adding glucose to the additive changes the desirability region. In the contour plot shown in Figure 2, the high-desirability region is in the lower left corner of the plot. These high-desirability regions indicate that when glucose is present, the additives can contain greater amounts of magnesium chloride and potassium nitrate than when glucose is absent. The clear region from high desirability to zero desirability indicates that the amount and ratio of specific additives are important in order to overcome the antagonistic effects that rhamnolipid surfactants have on beneficial microorganisms.

[0060] Example 2: Formulation containing alkyl sarcosinate and additives Test compositions containing alkyl sarcosinate surfactants and different amounts of additives were prepared as described above to evaluate the ability of the additives to overcome the antagonistic incompatibility between alkyl sarcosinate surfactants and bacterial spores. The additives used were L-alanine, potassium nitrate, and glucose. Magnesium chloride hexahydrate was not used in this example because magnesium chloride and alkyl sarcosinate may coprecipitate. The alkyl sarcosinate used was MAPROSYL® 30-B, a sodium lauroyl sarcosinate available from Stepan Company, Northfield, Illinois. Samples of each test composition were tested for EVOO digestion using the test procedure described above, and contour plots were created from the test data. Stat-Ease 360 ​​optimized contour plots for the test compositions are shown in Figure 3. The optimized plots show contours indicating high-desirability regions and zero-desirability regions corresponding to different amounts of different additives. In the contour plots shown in Figure 3, the high-desirability region is the dark area on the right side of the plot. These regions of high desirability arise with potassium nitrate and / or L-alanine as additives, but small amounts of glucose may also be included. The distinct regions from high desirability to zero desirability indicate that the amount and ratio of specific additives are crucial in overcoming the antagonistic effects that alkyl sarcosinate surfactants have on beneficial microorganisms.

[0061] Example 3: Formulation containing alpha-sulfonated alkyl ester and additives Test compositions containing alpha-sulfonated alkyl esters and varying amounts of additives were prepared as described above to evaluate the ability of the additives to overcome the antagonistic incompatibility between the alpha-sulfonated alkyl ester surfactant and bacterial spores. The additives used were L-alanine, magnesium chloride hexahydrate, potassium nitrate, and glucose. The alpha-sulfonated alkyl ester surfactant was ALPHA-STEP® PC-48, which is sodium methyl-2-sulfolaurate and disodium methyl-2-sulfolaurate, available from Stepan Company, Northfield, Illinois.

[0062] Samples of each test composition were tested for EVOO digestion using the test procedure described above, and contour plots were created from the test results. Stat-Ease 360 ​​optimized contour plots for the test compositions with and without glucose as one of the additives are shown in Figures 4 and 5, respectively. The optimized plots show contours indicating high-desirability and zero-desirability regions corresponding to different amounts of different additives. In the contour plot shown in Figure 4, the high-desirability region is along the right side of the plot, and the dark area in the lower left corner of the plot indicates the zero-desirability region. The high-desirability region occurs with potassium nitrate and / or L-alanine as additives, and the zero-desirability region occurs with magnesium chloride as additives. As shown in Figure 5, the presence of glucose in the additives changes the desirability region. When glucose is present in the additive, the high-desirability region is located in the lower right corner of the plot, occurring when the majority of the additive is potassium nitrate, while the low-desirability to zero-desirability region occurs when L-alanine and magnesium chloride are present in the additive. The distinct region from high-desirability to zero-desirability indicates that the amount and ratio of specific additives are important in overcoming the antagonistic effects that alpha-sulfonated alkyl ester surfactants have against beneficial microorganisms.

[0063] In Examples 4 to 7 below, bacterial spore germination and growth were visually evaluated using an unstained EVOO test. For the test, each test composition was prepared with 0.2% by weight of the active ingredient surfactant, 0.2% by weight of the total additives, and 1 × 10⁶ spores from a commercially available source. 7 The test formulations were prepared by adding 0.1% by weight of unstained EVOO to a test jar containing an aqueous solution of a Bacillus species mix at CFU / g. The test additives used in each formulation contained 0.09% by weight of L-alanine, 0.09% by weight of potassium nitrate, and 0.02% by weight of magnesium chloride hexahydrate. Comparative compositions containing surfactants and bacterial spores, but without additives, were also prepared. The control compositions, both with and without additives, contained bacterial spores but did not contain surfactants. All test compositions, comparative compositions, and control compositions contained 2% by weight of a 3.3 g / L nutrient mix. The pH was adjusted to pH 7 using L-lactic acid or dilute NaOH, if necessary. The compositions were initially photographed, followed by repeated photography after shaking for 4 days and 5-7 days in an orbital mixer set to setting "5". The initial and aged compositions were visually compared to determine the differences.

[0064] Example 4: Formulation containing alcohol ethoxylate and additives Test formulations were prepared containing 0.2% by weight of various alcohol ethoxylate surfactants in the medium HLB range (HLB=12-14) and the aforementioned additive composition, namely 0.09% by weight of L-alanine, 0.09% by weight of potassium nitrate, and 0.02% by weight of magnesium chloride hexahydrate. The alcohol ethoxylate surfactants tested were MAKON® UD-7, a 7-mol ethoxylated branched undecyl alcohol; MAKON® DA-6, a 6-mol ethoxylated decyl alcohol; and BIO-SOFT® N1-7, a 7-mol ethoxylated semilinear undecyl alcohol, all of which were available from Stepan Company, Northfield, Illinois. For each surfactant, a comparative formulation without additives was also prepared. The formulations were evaluated for bacterial spore germination and growth using the unstained EVOO procedure described above.

[0065] Figure 6 shows photographs of test composition jars with and without additives after shaking on an orbital mixer for 7 days. Also shown are photographs of control composition jars containing bacterial spores without surfactants, with and without additives. The turbidity of the test jars indicates biological activity; greater turbidity indicates higher biological activity. The control compositions are shown in the leftmost pair in Figure 6, with the jar on the left containing only spores and the jar on the right containing spores in addition to the additive. The comparison of the control jars demonstrates that, even in the absence of surfactants, the additive provides benefits such as improved dispersion of bacterial spores and elimination of aggregation.

[0066] For surfactant-containing formulations without additives (jars on the left in each set), all were inhibitory to biological activity, and turbidity was attributable solely to the initial population of Bacillus species. The turbidity levels of these jars were 1 × 10⁻⁶. 7CFU / g is a good visual measure for Bacillus species. When additives are included (right-hand jar in each set), surfactant inhibition is overcome, resulting in good bioactivity and stronger visual turbidity. It is important to note that turbidity due to bioactivity can be distinguished from turbidity due to surfactants based on the timing of its appearance. Turbidity due to surfactants appears almost immediately after formulation, while turbidity due to bioactivity takes at least 12 hours, more typically 48-96 hours, to appear and can continue to progress over time. The appearance of turbidity is a visual indicator that the additive in the test formulation has overcome surfactant incompatibility.

[0067] Example 5: Formulation containing a low-HLB surfactant and additives Test formulations were prepared containing various low-HLB surfactants (HLB=5-10) in 0.2% by weight of active ingredient, as well as the aforementioned additive composition, namely 0.09% by weight of L-alanine, 0.09% by weight of potassium nitrate, and 0.02% by weight of magnesium chloride hexahydrate. The low-HLB surfactants tested were BIO-SOFT® N23-3, a C12-13 semilinear alcohol ethoxylate with 3 moles of ethylene oxide; MAKON® UD-5, a 5-mol-ethoxylated branched undecyl alcohol; STEPAN-MILD® GCC, a glyceryl caprylate / caprate; and MAKON® DA-4, a branched 4-mol-ethoxylated decyl alcohol, all of which were available from Stepan Company, Northfield, Illinois. The initial turbidity of the test formulation was partially removed by adding 1.0% by weight of castor oil ethoxylate containing 36 moles of ethylene oxide to the test formulation. The formulation was evaluated for bacterial spore germination and growth using the unstained EVOO procedure described above.

[0068] Figure 7 shows photographs of the test jars with and without additives (left jars in each set) after shaking on an orbital mixer for 5 days. The photographs also show a control composition jar (far left set) containing bacterial spores with and without additives (right jars) and without surfactants. The increase in turbidity due to biological activity is evident in the photographs of the control jars and the surfactant test jars with additives. The surfactant test jar without additives showed strong inhibition of Bacillus species, exhibiting visual turbidity similar to that of the initial Bacillus population only, and was significantly clearer. The presence of additives in the test formulations is beneficial because these low-HLB surfactants help overcome the inhibitory effect on Bacillus spores that would otherwise be present.

[0069] Example 6: Formulation containing a dispersant and additives Test formulations were prepared containing various dispersants in 0.2 wt% of the active ingredient, as well as the aforementioned additive compositions, namely 0.09 wt% L-alanine, 0.09 wt% potassium nitrate, and 0.02 wt% magnesium chloride hexahydrate. Dispersants are a specific class of surfactants that can separate particles from each other and assist in the physical stability of the composition. The dispersants tested were STEP-FLOW® 26F, an EO / PO block copolymer; STEPFAC® 8181 PT3K, a phosphate ester salt; and STEPWET® DOS 60 ROE, a sodium dioctyl sulfosuccinate containing rapeseed oil methyl ester, all of which were available from Stepan Company, Northfield, Illinois. The formulations were evaluated for bacterial spore germination and growth using the unstained EVOO procedure described above.

[0070] Figure 8 is a photograph showing the test jars with and without additives (right jar in each set) after shaking on an orbital mixer for 7 days. Both the EO / PO block copolymer STEP-FLOW® 26F and the phosphate ester salt STEPFAC® 8181 PT3K strongly inhibit biological activity, as indicated by the lack of turbidity in the control formulation without additives. However, the test formulations containing additives show significant turbidity, suggesting biological activity. The additives in the test formulations help overcome the inhibitory effect on Bacillus spores that would otherwise be present due to these dispersants. For DOS 60 ROE, which is sodium dioctyl sulfosuccinate containing methyl rapeseed oil, 0.8 wt% of sodium xylenesulfonate, STEPANATE® SXS, was added as a hydrotrope to the initial formulation composition to achieve sufficient clarity for monitoring biological activity. The slightly stronger visual turbidity in this formulation without additives may simply be due to the methyl ester not being completely solubilized.

[0071] Example 7: Formulation containing hydroxysultaine and additives A test formulation was prepared containing 0.2% by weight of the active ingredient hydroxysultaine surfactant, and the aforementioned additive composition, namely 0.09% by weight of L-alanine, 0.09% by weight of potassium nitrate, and 0.02% by weight of magnesium chloride hexahydrate. The hydroxysultaine was AMPHOSOL® CS-50, a cocoamidopropyl hydroxysultaine available from Stepan Company, Northfield, Illinois. The formulation was evaluated for bacterial spore germination and growth using the unstained EVOO procedure described above.

[0072] Figure 9 is a photograph showing the test jars with and without the additive (right jar) after shaking on an orbital mixer for 7 days. The control jar is on the far left. AMPHOSOL® CS-50, which is cocoamidopropyl hydroxysultaine, inhibits biological activity, as indicated by the absence of turbidity in the control formulation without the additive. However, the test formulation containing the additive shows turbidity, suggesting biological activity. The additive in the test formulation helps hydroxysultaine overcome the inhibitory effect on Bacillus spores that would otherwise be present.

[0073] Example 8: Digestion and stability evaluation of EVOO The alpha-sulfonated alkyl ester test composition of Example 3 was used for this stability evaluation because, unexpectedly, even when the test composition was diluted with water at the dilution ratio used, some of the spores in the test composition remained completely dispersed in the composition even after standing for one week. The physical stability of the test composition was evaluated by photographing the test composition in the parent sample jar (from which the sample of Example 3 was taken) with the sample jar viewed from above, and quantifying the degree of precipitation by Tracker.jar image analysis. A contour plot was created combining the digestion results of Example 3 and the physical stability test results of this example to identify compositions exhibiting both good biological activity and good spore stability. The contour plot is shown in Figure 10. Regions of higher desirability are shown along the right side of the plot, and regions of zero desirability are the dark areas shown in the lower left corner of the plot. Regions of higher desirability that exhibit both good biological activity and good stability are produced with potassium nitrate and L-alanine as additives, but tend to be higher in amounts of L-alanine.

[0074] Example 9: Comparative formulation containing alpha-sulfonated alkyl ester and additives A comparative study was conducted using compositions containing alpha-sulfonated alkyl esters and different amounts of additives. According to the contour plots in Figure 4, the region of high desirability is along the right side of the plot, occurring when potassium nitrate and / or L-alanine are additives and magnesium chloride hexahydrate is minimal or absent, while the region of low desirability is in the lower left corner of the plot, occurring when the additive composition contains more magnesium chloride hexahydrate. Five comparative compositions were prepared as described in Examples 1-3, using 0.10 wt% ALPHA-STEP(registered trademark) PC-48 as the alpha-sulfonated alkyl ester surfactant and additives containing 0.07 wt% MgCl2-6H2O + 0.015 wt% L-alanine + 0.015 wt% KNO3. Five test compositions were prepared in the same manner, except that the additives included 0.045% by weight of L-alanine, 0.045% by weight of KNO3, and 0.01% by weight of MgCl2-6H2O. Bacterial spore germination and growth were visually evaluated for Examples 4-7 using the unstained EVOO test described above.

[0075] Figure 11 shows the results after 7 days on an orbital shaker at room temperature. The five jars on the left containing the comparative composition show minimal turbidity, suggesting minimal biological activity. The comparative composition demonstrates the inhibitory effect of the alpha-sulfonated alkyl ester surfactant, and that additive compositions containing higher amounts of MgCl2-6H2O cannot overcome it. The five jars on the right containing the test composition show that additives containing higher amounts of potassium nitrate and L-alanine, along with minimal amounts of MgCl2-6H2O, can restore biological activity, as demonstrated by the visible turbidity of the aqueous phase in these jars. These results indicate that the amount and ratio of specific additives are important in overcoming the antagonistic effect that alpha-sulfonated alkyl ester surfactants have on beneficial microorganisms.

[0076] Example 10: Formulation comparing potassium salts as an alternative additive option. In this example, potassium salts are compared as alternative options to determine whether salts other than KNO3 can be used in the additive composition. Repeated samples of aqueous test formulations were prepared using an additive composition containing 0.2 wt% of MAKON® DA-6 as the surfactant, along with 0.09 wt% L-alanine, 0.09 wt% potassium salt, and 0.02 wt% magnesium chloride hexahydrate. The potassium salts tested were KNO3, KCl, and K2SO4. Comparative compositions containing the surfactant and bacterial spores, but without the additive, were also prepared. The control compositions, with and without the additive, contained bacterial spores but did not contain the surfactant to confirm the viability of the spores. KNO3 was used as the potassium salt in the control composition containing the additive. All test compositions, comparative compositions, and control compositions contained 2 wt% of a 3.3 g / L nutrient mix. The pH was adjusted to pH 7 using L-lactic acid or dilute NaOH, if necessary. Bacterial spore germination and growth were visually evaluated for Examples 4-7 using the unstained EVOO test described above. The compositions were photographed after 7 days of shaking in an orbital mixer set to "5" at room temperature.

[0077] Figure 12 shows the results after 7 days on an orbital shaker. The jar on the far left of the photograph is a control formulation without additives or surfactants, and the second jar from the left is a control formulation containing additives but without surfactants. Figure 12 shows that in the absence of additives, the spores in the leftmost control jar aggregate and are not well dispersed. As seen in the second control jar from the left, the presence of additives promotes stronger biological activity and maintains excellent spore dispersibility. The strong inhibitory effect of MAKON® DA-6 is observed in the third jar from the left, which shows minimal biological activity in the absence of additives. The remaining jars in the photograph contained test formulations with additives. The set of three jars on the far right contained KNO3, KCl, and K2SO4 as potassium salts, in that order. The set of three jars immediately to the left of the set of three jars on the far right is a set of repeat samples, demonstrating the repeatability of the test. Each potassium salt helps overcome the inhibitory effect of the MAKON® DA-6 surfactant and restore bioactivity, as demonstrated by the visible turbidity of the aqueous phase in these jars. These results indicate that potassium salts other than KNO3 can be used in the additive composition, and that potassium sulfate is the most effective at promoting bioactivity.

[0078] Example 11: Formulation containing magnesium salt as an alternative option In this example, magnesium salts are compared as alternative options to determine whether salts other than MgCl2 can be used in the additive composition. Repeated samples of the test formulation were prepared using an additive composition containing 0.2 wt% rhamnolipid as the active ingredient, along with 0.03 wt% L-alanine, 0.03 wt% potassium nitrate, and 0.14 wt% magnesium salt. The magnesium salts tested were MgCl2-6H2O, MgNO3, MgSO4, and Mg(citrate) (dibasic). Control compositions were also prepared, one containing bacterial spores and additives but no surfactant, and the other containing surfactant and bacterial spores but no additive. Both the test and control compositions also contained 2 wt% of a 3.3 g / L nutrient mix. The pH was adjusted to pH 7 using L-lactic acid or dilute NaOH if necessary. Bacterial spore germination and growth were visually evaluated using the unstained EVOO test described above for Examples 4-7. The composition was photographed after 7 days of shaking in an orbital mixer set to "5" at room temperature.

[0079] Figure 13 shows the results after 7 days on an orbital shaker. The jar on the far left of the photograph is a control formulation with an additive composition based on MgCl2-6H2O and does not contain the rhamnolipid surfactant. The second jar from the left is a control formulation containing the rhamnolipid surfactant but no additives. The control formulations demonstrate spore viability and the inhibitory effect of the rhamnolipid surfactant. The remaining jars in the photograph contained test formulations containing the rhamnolipid surfactant and additives containing various Mg salts. The jars from left to right next to the control jars contain a set of two formulations, each formulation containing MgCl2-6H2O, MgSO4, Mg(NO3)2, or dibasic Mg(citrate) as magnesium salts in that order. Each magnesium salt helps overcome the inhibitory effect of the rhamnolipid surfactant and restore bioactivity, as demonstrated by the visible turbidity of the aqueous phase in these jars. These results indicate that magnesium salts other than MgCl2-6H2O can be used in additive compositions.

[0080] Example 12: Formulation containing a low-foaming nonionic alkoxylate and additives An important class of nonionic alkoxylates is the group known as alkylene oxide-containing alcohol alkoxylates. Alkylene oxides include not only ethylene oxide (EO) but also propylene oxide (PO), butylene oxide (BO), or even pentylene oxide (PTO). These alkoxylates can provide special grease cleaning with low foaming properties when the alkyl chain is slightly elongated by alkoxylation. When more PO is used, the HLB decreases and foaming decreases, making the surfactant useful for low-foaming mechanical cleaning. Test formulations were prepared containing various low-foaming nonionic alkoxylated surfactants in an active ingredient amount of 0.2 wt%, as well as additive compositions containing 0.09 wt% L-alanine, 0.09 wt% potassium nitrate, and 0.02 wt% magnesium chloride hexahydrate. The low-foaming nonionic alkoxylated surfactants tested were MAKON® NF-180 (proprietary alkoxylated polymer), MAKON® NF-12 (C10-12 alcohol alkoxylate), DA-1PO-8EO (isodecyl-1PO-8EO), and C42 MEA Amide-7EO-4PO (C12-C14 monoethanolamide-7EO-4PO). All materials under the trade name MAKON® are available from Stepan Company, Northfield, Illinois, while the other two were experimental materials. The surfactants were solubilized in aqueous formulations by adding 1.0% by weight of castor oil ethoxylate (TOXIMUL® 8240) containing 36 moles of ethylene oxide to the test formulations. Comparative compositions containing the surfactant, 1.0% by weight of the TOXIMUL® 8240 solubilizer, and bacterial spores, without additives, were also prepared. Control compositions were prepared, one containing bacterial spores but no surfactants or additives, and the other containing spores and additives but no surfactants. In this example, the control jar also contained 1.0 wt% TOXIMUL 8240 solubilizer. All test compositions, comparative compositions, and control compositions contained 2 wt% of a 3.3 g / L nutrient mix. The pH was adjusted to pH 7 using L-lactic acid or dilute NaOH if necessary.The germination and proliferation of bacterial spores were visually evaluated for Examples 4-7 using the unstained EVOO test described above.

[0081] Figure 14 is a photograph of the results after shaking on an orbital mixer for 7 days. Each alkoxylate is shown in a set, with the jar on the left of each set containing the respective alkoxylate without additives, and the jar on the right of each set containing additives. The photograph also shows a control composition jar (far left set) containing bacterial spores, with and without additives (right jar), and without surfactants, containing only 1% by weight of TOXIMUL® 8240 solubilizer. The test formulation containing MAKON® NF-180 is next to the control set, followed by MAKON® NF-12, then a formulation containing DA-1PO-8EO, and on the far right, a formulation containing C42 MEA Amide-7EO-4PO. Most of these alkoxylates do not inhibit the growth of Bacillus species as much as the alcohol alkoxylates tested in Example 4, as is evident from the turbidity due to biological activity present even in the test composition jars without additives. The formulation containing DA-1PO-8EO without additives showed the most inhibitory signs. All test formulations showed increased biological activity in the presence of additives. Additives can be beneficial even when conditions are not so inhibitory. Also, from the photographs, the amount of biological activity (turbidity) appears to be higher than typical, which suggests that TOXIMUL® 8240 may promote further germination and initial growth of Bacillus microorganisms.

[0082] Example 13: Use of rhamnolipid and additives for bioremediation (hypothetical example) Prepare 10 liters of a 10% solution of the active ingredient rhamnolipid in freshwater. This solution is sprayed onto a 1-metric-ton pile of soil containing Bacillus spores. Simultaneously, a separate 1-metric-ton pile of untreated soil is set aside for analysis. 1 kg of ammonium chloride (nitrogen source) is added to both soil piles. 10 liters of the additive composition is also added to the treated soil. The additive composition contains 10% by weight of a mixture of L-alanine and magnesium chloride hexahydrate in a 1:1 weight ratio in water. Soil samples are taken from both piles before treatment, and 20, 40, and 70 days after treatment. Total petroleum hydrocarbons (TPH) in all samples are measured using flame ionization detection gas chromatography (GC / FID system compliant with EPA 8015C). The treated soil shows lower TPH than the untreated soil.

[0083] Example 14: Crude oil digestion In this example, the ability of a spore-form Bacillus species mix to germinate, grow, and digest West Texas Intermediate (WTI) crude oil in an aqueous composition containing rhamnolipid and additives was investigated. Repeated samples were prepared by combining a stock suspension of 44.6 g DI water, 0.2 g L-alanine, 0.2 g MgCl2-6H2O, 2.0 g rhamnolipid, 0.5 g Toximul 8240, 2.0 g 33 g / L nutrient mix, and 0.5 g 10^9 CFU / g spore-form Bacillus species mix, and adding 2.0 g WTI crude oil to the mixture ("all at once" samples). The samples were mixed on the same stirring plate at 500 rpm for 15-30 minutes, followed by phase separation and then photographic imaging. The samples were then stirred for 3 days, followed by phase separation and further imaging.

[0084] A second set of replicate samples was prepared with the same components and quantities ("sequential addition" samples), except that water, rhamnolipid, TOXIMUL® 8240, and WTI crude oil were pre-emulsified, followed by the sequential addition of additives, nutrients, and a mix of Bacillus species in spore form. The second set of samples was prepared by mixing water, rhamnolipid, TOXIMUL® 8240, and WTI crude oil for 15-30 minutes. After phase separation, the samples were photographed. The samples were stirred for 3 days, and the WTI crude oil was pre-emulsified with rhamnolipid and TOXIMUL® 8240. After phase separation, the samples were photographed again. Next, additives, nutrients, and a mix of Bacillus species in spore form were added to the samples, and the samples were stirred for 4 days. After phase separation, the samples were photographed.

[0085] The results indicate that in both "all at once" and "sequential addition" samples, the spore-form Bacillus species mix was able to germinate, grow, and digest WTI crude oil. The results also show that pre-emulsifying rhamnolipid, TOXIMUL® 8240, and WTI crude oil, followed by the addition of additives, nutrients, and Bacillus species, can improve the digestion of WTI crude oil.

[0086] Example 15: Formulation containing alkyl ether sulfate A test formulation was prepared containing an alkyl ether sulfate (STEOL® 23-2s.70 CP, sodium laureth sulfate, ethoxylate 2 mol, available from Stepan Company) as a surfactant. While alkyl ether sulfates are suitable surfactants for use in agricultural compositions, they are incompatible with beneficial microorganisms at concentrations typically used in agricultural formulations. One difficulty in formulating agricultural compositions is the frequent requirement to include organic materials review institute (OMRI) compliant components. Potassium nitrate, magnesium chloride, and L-alanine, used as additives in many of the above examples, are not designated as OMRI compliant. Therefore, in this example, an alternative additive containing a compliant component was used. This alternative additive contained a mixture of magnesium nitrate and potassium chloride salts, but did not contain L-amino acids.

[0087] 7.5% by weight of alkyl ether sulfate surfactant as an active ingredient, 10.0% by weight of Bacillus spore blend (5 x 10 9 Two concentrates of the test formulation were prepared, each containing a mixture of 0.75% by weight potassium chloride and 0.75% by weight magnesium nitrate, and water to make up 100% of the total volume. 1.5% by weight of castor oil 36EO alkoxylate (TOXIMUL® 8240) (100% active ingredient) was added to one of the test formulations. Two comparative concentrates were also prepared. One comparative composition contained the same amount of surfactant and spores, but no additives or TOXIMUL® 8240, while the other comparative composition contained the same amount of surfactant, spores, and TOXIMUL® 8240, but no additives. A total of 20 g of each concentrate was prepared.

[0088] For testing, each concentrated composition was mixed with 1% by weight of 33g / L nutrients (30g / L tripty soy broth + 3g / L yeast extract), 0.2% by weight of extra virgin olive oil (EVOO), and water to a total weight of 50.0g in a 4-ounce glass jar, and 2.0% by weight (1 × 10 8 The solution was diluted to CFU / g. The jar was mixed in an orbital shaker for 15-30 minutes, photographed initially, then mixed in an orbital shaker for 7 days, and photographed again.

[0089] The imaging results showed that the comparative compositions (without additives and TOXIMUL® 8240, and without additives) were free of turbidity after 7 days, suggesting that alkyl ether sulfate inhibits the biological activity of spores. The lack of turbidity in the comparative composition with only TOXIMUL® 8240 added to 7.5 wt% alkyl ether sulfate is surprising because different results were obtained for low-foaming alkoxylate surfactants when TOXIMUL® 8240 was used in the test formulation and control formulation of Example 12. The results of Example 12 showed that the amount of turbidity observed in the composition of Example 12 was greater than that of the typical state, suggesting that the TOXIMUL® 8240 solubilizer may have more strongly promoted the germination and initial growth of Bacillus microorganisms.

[0090] The photographic results in this example also show that the test composition containing only the additive and not TOXIMUL® 8240 showed no turbidity after 7 days, suggesting that the specific combination of salts used as the additive was insufficient to overcome the incompatibility between alkyl ether sulfate and Bacillus spores. The test composition containing both the additive and TOXIMUL® 8240 showed increased turbidity after 7 days, suggesting that the combination of the additive and the TOXIMUL® 8240 solubilizer can overcome the incompatibility between alkyl ether sulfate surfactant and spores.

[0091] Example 16: Formulation containing alkylbenzene sulfonate Alkylbenzenesulfonates are another type of surfactant suitable for use in agricultural compositions, but at concentrations typically used in agricultural formulations, they are still incompatible with beneficial microorganisms. To evaluate the ability of different amounts of additive components to overcome the incompatibility between alkylbenzenesulfonate surfactants and microbial spores, four different test formulations were prepared. The test formulations contained calcium alkylbenzenesulfonate (NINATE® 100L, available from Stepan Company) as the surfactant, and the following as different additive components: (1) 1.0 wt% L-alanine + KCl and Mg(NO3)2, each 0.25 wt%; (2) 1.0 wt% KCl and L-alanine and Mg(NO3)2, each 0.25 wt%; (3) 1.0 wt% Mg(NO3)2 and L-alanine and KCl, each 0.25 wt%; and (4) L-alanine, Mg(NO3)2, and KCl, each 0.5 wt%.

[0092] 7.5% by weight of the active ingredient calcium alkylbenzene sulfonate surfactant, 10.0% by weight of the Bacillus spore blend (5 × 10 9 Four concentrates of the test formulation were prepared, each containing 1.5% by weight of the corresponding additive mixture (CFU / g), and water to make up 100% of the total. The total weight of each concentrate was 20 g. Two control concentrates were also prepared. The positive control concentrate contained 10% by weight of the Bacillus spore blend and water, while the negative control concentrate contained 10% by weight of the Bacillus spore blend, 7.5% by weight of the surfactant active ingredient, and water.

[0093] For testing, each concentrated composition was mixed with 1% by weight of 33g / L nutrients (30g / L tripty soy broth + 3g / L yeast extract), 0.2% by weight of extra virgin olive oil (EVOO), and water to a total weight of 50.0g in a 4-ounce glass jar, and 2.0% by weight (1 × 10 8The solution was diluted to CFU / g. The jar was mixed in an orbital shaker for 15-30 minutes, photographed initially, then mixed in an orbital shaker for 7 days, and photographed again.

[0094] The imaging results showed varying initial turbidity in the sample jars, partly due to the solubility of dispersed EVOO, spores, and the surfactant itself. After 7 days, the imaging results showed an increase in turbidity in the positive control jar, suggesting spore viability, and a decrease in turbidity in the negative control jar, demonstrating incompatibility between the surfactant and spores. The imaging results also showed a certain degree of turbidity increase after 7 days for each test composition containing either L-alanine or Mg(NO3)2 as the main additive component, and for test compositions containing equal amounts of the additive component. This increase in turbidity indicates that these particular additive mixtures can overcome, at least to some extent, the antagonistic effect that the alkylbenzene sulfonate calcium surfactant has on beneficial microorganisms.

[0095] The embodiments and examples described herein are illustrative and do not limit the technology described in this invention. The scope of the technology described herein is the entire scope defined or implied by the claims. In addition, any references listed in the detailed description section of this application are incorporated herein by reference in their entirety unless otherwise noted.

[0096] Further embodiments of the present invention are described in the following numbered paragraphs. Paragraph 1. A composition for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein the surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms, and the composition comprises: (a) at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; (b) an additive comprising: (i) at least one L-amino acid in combination with at least one inorganic divalent metal salt or monovalent salt; and (ii) optionally glucose, malt A composition comprising: (c) an additive comprising one or more carbohydrates including sucrose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan; and (i) water in an amount totaling 100% by weight of the composition, wherein the additive is present in the composition in an amount such that the weight ratio of additive component (i) to surfactant in the composition is in the range of 0.2:1 to 1:1, and when the composition is combined with beneficial live microorganisms, the additive overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial live microorganisms, thereby promoting the viability, growth, and biological activity of the beneficial microorganisms.

[0097] Paragraph 2. A method for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein the surfactant incompatibility adversely affects the viability, growth, or biological activity of the beneficial living microorganisms, the method comprising: (a) providing an aqueous composition comprising at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of the beneficial living microorganisms; and (b) combining an additive with at least one surfactant in the aqueous composition, wherein the additive comprises (i) at least one L-amino acid combined with at least one inorganic divalent metal salt or monovalent salt; and (ii) optionally, a glucose A method comprising: adding an additive in an amount such that the weight ratio of additive component (i) to surfactant is 0.2:1 to 1:1, comprising one or more carbohydrates including sucrose, maltose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan; and combining beneficial live microorganisms with at least one surfactant in an aqueous composition either before the additive is combined with at least one surfactant or after the additive is combined with at least one surfactant, wherein the additive overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial live microorganisms and promotes the viability, growth, and biological activity of the beneficial microorganisms.

[0098] Paragraph 3. Embodiments of paragraph 1 or 2, further comprising one or more additional components selected from the group consisting of castor oil alkoxylate, alpha-olefin sulfonate, polyoxyethylene sorbitan monooleate, sodium gluconate, alkenyldimethylamide, a blend of sodium alkyl ether sulfate and alkylamidopropyl betaine and / or alkyl betaine, and combinations thereof.

[0099] Paragraph 4. Embodiments according to any one of paragraphs 1 to 3, further comprising a hydrotrope, preferably sodium xylenesulfonate, sodium cumenesulfonate, amphoteric dipropionate, or a combination thereof.

[0100] Paragraph 5. An embodiment according to any one of paragraphs 1 to 4, further comprising one or more additional carbohydrates selected from the group consisting of cellulose, maltodextrin, fiber, amylose, amylopectin, glycogen, and starch.

[0101] Paragraph 6. An embodiment according to any one of paragraphs 1 to 5, further comprising at least one enzyme, preferably one or more proteases, amylases, lipases, or combinations thereof.

[0102] Paragraph 7. An embodiment according to any one of paragraphs 1 to 6, wherein the surfactant (a) is one or more rhamnolipids, and the additive contains L-alanine and MgCl2 in a weight ratio of 1:1 or more, or the additive contains L-alanine and MgCl2 in a weight ratio of 1:1 or more MgCl2:L-alanine in combination with an amount of glucose not exceeding the weight of (MgCl2 + L-alanine) in the composition.

[0103] Paragraph 8. An embodiment according to any one of paragraphs 1 to 6, wherein the surfactant (a) is one or more types of rhamnolipids, and the additive comprises a combination of L-alanine, MgCl2, and KNO3 in a weight ratio of 4:1 or greater (L-alanine + MgCl2):KNO3, or the additive comprises a combination of L-alanine, MgCl2, and KNO3 in combination with glucose in an amount not exceeding the weight of (L-alanine + MgCl2 + KNO3) in the composition, in a weight ratio of 1:1 or greater MgCl2:(L-alanine + KNO3).

[0104] Paragraph 9. Embodiments of Paragraph 7 or 8, wherein the composition comprises 5% to 10% by weight of rhamnolipid and 5% to 10% by weight of additives, or 0.05% to 1.0% by weight of rhamnolipid and 0.05% to 1.0% by weight of additives, based on the weight of active ingredients.

[0105] Paragraph 10. An embodiment according to any one of paragraphs 1 to 6, wherein the surfactant (a) is an alkyl sarcosinate, and the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 1:1 or greater (KNO3 + L-alanine):glucose, or the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 2:1 or greater KNO3:(L-alanine + glucose).

[0106] Paragraph 11. Embodiments of Paragraph 10, wherein the alkyl sarcosinate is sodium lauroyl sarcosinate, and the composition comprises 5% to 20% by weight of sodium lauroyl sarcosinate and 5% to 20% by weight of an additive, or the composition comprises 0.05% to 1.0% by weight of sodium lauroyl sarcosinate and 0.05% to 1.0% by weight of an additive, by weight.

[0107] Paragraph 12. An embodiment according to any one of paragraphs 1 to 6, wherein the surfactant (a) is a sulfonated alkyl ester, and the additive comprises a combination of L-alanine, MgCl2, and KNO3 in a weight ratio of 3:1 or greater (L-alanine + KNO3):MgCl2, or the additive comprises a combination of L-alanine, MgCl2, and KNO3 in combination with an amount of glucose not exceeding the weight of (L-alanine + MgCl2 + KNO3) in the composition, in a weight ratio of 1:1 or greater KNO3:(L-alanine + MgCl2).

[0108] Paragraph 13. The embodiment described in Paragraph 12, wherein the composition comprises 0.05% to 1.0% by weight of a sulfonated alkyl ester and 0.05% to 1.0% by weight of an additive.

[0109] Paragraph 14. Spores of beneficial living microorganisms such as one or more species of fungi, yeasts, or bacterial spores, preferably bacterial spores, more preferably Bacillus amyloricephasiens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus filmus, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus megatherium, Bacillus methylotropus, An embodiment according to any one of paragraphs 1 to 13, further comprising spores of one or more Bacillus species, such as Bacillus mycoides, Bacillus pasteuli, Bacillus polyfermenticus, Bacillus polymixa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thiaminolyticus, or Bacillus thuringiensis.

[0110] Paragraph 15. Spores, 1 × 10 7 ~1 × 10 10 Preferably 1 × 10 9 ~5×10 9 This is the amount in CFU / g, or 1 × 10⁻⁶ 5 ~1 × 10 9 The embodiment described in paragraph 14, which is the quantity of the embodiment described in paragraph 14.

[0111] Paragraph 16. An embodiment according to any one of paragraphs 1 to 13, wherein the bacteria, fungi, or yeast are beneficial living microorganisms. Paragraph 17. An embodiment according to any one of paragraphs 1 to 16, further comprising at least one preservative selected from the group consisting of phenoxyethanol, methylisothiazolinone, benzylisothiazolinone, benzyl alcohol, fatty acids, benzoic acid, and combinations thereof.

[0112] Paragraph 18. An embodiment according to any one of paragraphs 2 to 15 or 17, wherein beneficial living microorganisms comprise microbial spores, the microbial spores are added to an aqueous composition in the absence of an additive to maintain the microbial spores in an inactive state; and then an additive is added to the aqueous composition to activate the microbial spores.

[0113] Paragraph 19. A method for remediating the environment of soil, comprising the steps of: applying an aqueous composition containing 0.1% to 25% by weight of rhamnolipid as an active ingredient to soil to be remediated; and applying a composition to soil to be remediated, wherein the composition comprises (a) (i) a blend of L-alanine and MgCl2 in a weight ratio of 1:1 or more L-alanine:MgCl2, based on the weight of the composition, in an amount of 0.1% to 25% by weight; and (ii) an additive comprising, optionally, at least one carbohydrate; and (b) 1 × 10 6 ~1 × 10 10 (c) a carrier in an amount of beneficial live microbial spores in CFU / g, and (a) an amount of carrier totaling 100% by weight of the composition, or the composition is an additive comprising (a) an additive in an amount of L-alanine and MgCl2 in a weight ratio of 1:1 or greater, from 0.1% to 25% by weight based on the weight of the composition, (ii) an amount of glucose not exceeding the weight of (L-alanine + MgCl2) in the additive composition, (b) 1 × 10 6 ~1 × 10 10 A method comprising the steps of (c) a beneficial amount of live microbial spores in CFU / g, and (c) a carrier in an amount totaling 100% by weight of the composition.

[0114] Paragraph 20. The embodiment described in Paragraph 19, wherein an aqueous composition containing rhamnolipid and a composition containing additives are applied to the soil in amounts of about 1 to about 200 g / kg of soil, each. Paragraph 21. A microbially enhanced cleaning composition comprising: (a) at least one surfactant in an amount of about 0.05% to 1.0% by weight of the active ingredient that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms, wherein the surfactant comprises one or more of alkyl sulfates, alkyl sulfonates, alpha-sulfonated alkyl esters, alkyl sarcosinates, alkyl glutamates, alkyl ether sulfates, alkyl betaines, alkylamidopropyl betaines, alkylamine oxides, alkylamine alkoxylates, quaternated alkylamine alkoxylates, sulfonated alkyl esters, alkyl sulfoacetates, alcohol alkoxylates, EO / PO block copolymers, phosphate ester salts, sulfosuccinates, monoglycerides and / or diglycerides, hydroxysultaine, or rhamnolipids; and (b) at least one surfactant in an amount of about 0.05% to 1.0% by weight of the active ingredient.An additive that overcomes antagonistic surfactant incompatibility between at least one surfactant and beneficial living microorganisms, in a quantity of 0% by weight, and promotes the viability, growth, and biological activity of beneficial microorganisms, wherein the additive comprises (i) at least one L-amino acid selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, glutamic acid, serine, arginine, cysteine, glutamine, glycine, proline, and tyrosine; and (ii) at least one inorganic divalent metal salt or at least one monovalent salt or a combination thereof, wherein the inorganic divalent metal salt is magnesium chloride, calcium chloride, manganese chloride, iron chloride, copper chloride, zinc chloride, cobalt chloride, magnesium nitrate, magnesium sulfate, calcium sulfate, and magnesium sulfate. (iii) an additive comprising: (iii) an inorganic divalent metal salt or at least one monovalent salt or a combination thereof, wherein the monovalent salt is potassium nitrate (KNO3), sodium nitrate, potassium iodide, potassium chloride, potassium manganese oxide, potassium sulfate, sodium bicarbonate, sodium sulfate, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium citrate, potassium citrate, or ammonium citrate, or a combination thereof; (iii) an additive comprising one or more carbohydrates, optionally including glucose, maltose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan; (c) 1 × 10. 4 ~Approx. 1×10 8 A microbially enhanced cleaning composition comprising (d) microbial spores in an amount of CFU / g, and (f) water in an amount totaling 100% by weight of the composition.

[0115] Paragraph 22. The embodiment according to Paragraph 21, wherein the surfactant (a) is one or more types of rhamnolipids, and the additive contains L-alanine and MgCl2 in a weight ratio of 1:1 or more, or the additive contains L-alanine and MgCl2 in a weight ratio of 1:1 or more MgCl2:L-alanine in combination with an amount of glucose not exceeding the weight of (MgCl2 + L-alanine) in the composition.

[0116] Paragraph 23. The embodiment described in Paragraph 21, wherein the surfactant (a) is one or more types of rhamnolipids, and the additive contains a combination of L-alanine, MgCl2, and KNO3 in a weight ratio of 4:1 or more (L-alanine + MgCl2):KNO3, or the additive contains a combination of L-alanine, MgCl2, and KNO3 in a weight ratio of 1:1 or more MgCl2:(L-alanine + KNO3) in combination with glucose in an amount not exceeding the weight of (L-alanine + MgCl2 + KNO3) in the composition.

[0117] Paragraph 24. The embodiment described in Paragraph 21, wherein the surfactant (a) is an alkyl sarcosinate, and the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 1:1 or greater (KNO3 + L-alanine):glucose, or the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 2:1 or greater KNO3:(L-alanine + glucose).

[0118] Paragraph 25. The embodiment described in Paragraph 21, wherein the surfactant (a) is an alkyl sarcosinate, and the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 1:1 or greater (KNO3 + L-alanine):glucose, or the additive comprises a combination of L-alanine, KNO3, and glucose in a weight ratio of 2:1 or greater KNO3:(L-alanine + glucose).

[0119] Paragraph 26. The spores are one or more species of fungal spores, yeast spores, or bacterial spores, preferably bacterial spores, more preferably Bacillus amyloricephasiensis, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus filmus, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus megatherium, Bacillus metirotropis, Bacillus An embodiment according to any one of paragraphs 21-25, which is a spore of one or more Bacillus species, such as Bacillus mycoides, Bacillus pasteuli, Bacillus polyfermenticus, Bacillus polymixa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thiaminolyticus, or Bacillus thuringiensis.

[0120] Paragraph 27. An embodiment according to any one of paragraphs 21 to 26, further comprising one or more additional components selected from the group consisting of castor oil alkoxylate, alpha-olefin sulfonate, polyoxyethylene sorbitan monooleate, sodium gluconate, alkenyldimethylamide, a blend of sodium alkyl ether sulfate and alkylamidopropyl betaine and / or alkyl betaine, and combinations thereof.

[0121] Paragraph 28. A composition for overcoming antagonistic surfactant incompatibility between alkyl ether sulfate surfactants and beneficial living microorganisms, wherein surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms, wherein the composition comprises (a) an alkyl ether sulfate surfactant in the weight of the active ingredient, in an amount of 5% to 20% by weight, which exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; and (b) an additive in an amount of 1% to 10% by weight, which is (i) potassium nitrate (KNO3), sodium nitrate, potassium iodide, potassium chloride, potassium manganese oxide, potassium sulfate, sodium bicarbonate, sodium sulfate, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium citrate, potassium citrate, or ammonium citrate. A composition comprising: (ii) an additive comprising: (ii) at least one monovalent salt comprising: magnesium chloride, calcium chloride, manganese chloride, iron chloride, copper chloride, zinc chloride, cobalt chloride, magnesium nitrate, magnesium sulfate, calcium sulfate, manganese sulfate, iron sulfate, copper sulfate, zinc sulfate, cobalt sulfate, magnesium citrate, calcium citrate, any of the above hydrates, or any of the above combinations; (c) alkoxylated castor oil; and (d) water in an amount totaling 100% by weight of the composition, wherein when the composition is combined with beneficial live microorganisms, the combination of the additive and alkoxylated castor oil overcomes antagonistic surfactant incompatibility between alkyl ether sulfate surfactants and beneficial live microorganisms, and promotes the viability, growth, and biological activity of beneficial microorganisms.

[0122] This document describes the present technology in sufficient, clear, and concise language so that those skilled in the art in which the technology pertains can implement it. It should be understood that the above describes preferred embodiments of the technology, and that modifications may be made without departing from the spirit or scope of the technology as set forth in the attached claims. Furthermore, the examples are provided not exhaustively, but to illustrate some embodiments that fall within the scope of the claims.

Claims

1. A composition for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein the surfactant incompatibility adversely affects the viability, growth, or biological activity of the beneficial living microorganisms, and the composition is (a) at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of the beneficial living microorganisms; (b) Additives, (i) At least one L-amino acid in combination with at least one inorganic divalent metal salt or monovalent salt in an amount that results in a weight ratio of additive component (i) to surfactant of 0.2:1 to 1:1; and (ii) If desired, at least one type of carbohydrate, Additives, including; (c) an amount of water totaling 100% by weight of the composition, A composition comprising, when the composition is combined with the beneficial live microorganism, the additive in the composition overcomes the antagonistic surfactant incompatibility between the at least one surfactant and the beneficial live microorganism, thereby promoting the viability, growth, and biological activity of the beneficial microorganism.

2. The composition according to claim 1, wherein the surfactant (a) is one or more of the following: alkyl sulfate, alkyl sulfonate, alpha-sulfonated alkyl ester, alkyl sarcosinate, alkyl glutamate, alkyl ether sulfate, alkyl betaine, alkylamidopropyl betaine, alkylamine oxide, alkylamine alkoxylate, quaternized alkylamine alkoxylate, sulfonated alkyl ester, alkyl sulfoacetate, alcohol alkoxylate, EO / PO block copolymer, phosphate ester salt, sulfosuccinate, monoglyceride and / or diglyceride, hydroxysultaine, or rhamnolipid.

3. The composition according to claim 1 or 2, wherein the L-amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, glutamic acid, serine, arginine, cysteine, glutamine, glycine, proline, and tyrosine.

4. The composition according to any one of claims 1 to 3, wherein the inorganic divalent metal salt is selected from the group consisting of magnesium chloride, calcium chloride, manganese chloride, iron chloride, copper chloride, zinc chloride, cobalt chloride, magnesium nitrate, magnesium sulfate, calcium sulfate, manganese sulfate, iron sulfate, copper sulfate, zinc sulfate, cobalt sulfate, magnesium citrate, calcium citrate, any of the above hydrates, and any combination thereof.

5. The aforementioned monovalent salt is potassium nitrate (KNO ３ The composition according to any one of claims 1 to 4, which is sodium nitrate, potassium iodide, potassium chloride, potassium manganese oxide, potassium sulfate, sodium bicarbonate, sodium sulfate, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium citrate, potassium citrate, or ammonium citrate, or a combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the carbohydrate is one or more of glucose, maltose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan.

7. The composition according to any one of claims 1 to 6, wherein the surfactant (a) is one or more types of rhamnolipids, and the additive comprises the L-amino acid, the divalent metal salt, and optionally the carbohydrate.

8. The composition according to any one of claims 1 to 6, wherein the surfactant (a) is one or more types of rhamnolipids, and the additive comprises the L-amino acid, the divalent metal salt, the monovalent salt, and optionally the carbohydrate.

9. The composition according to claim 7 or 8, comprising 5% to 10% by weight of rhamnolipid and 5% to 10% by weight of additives.

10. The composition according to claim 7 or 8, comprising 0.05% to 1.0% by weight of rhamnolipid and 0.05% to 1.0% by weight of an additive.

11. The composition according to any one of claims 1 to 6, wherein the surfactant (a) is an alkyl sarcosinate, and the additive comprises the L-amino acid, the monovalent salt, and the carbohydrate.

12. The composition according to claim 11, wherein the alkyl sarcosinate in the composition is present in an amount of 5% to 20% by weight of the active ingredient, and the additive is present in the composition in an amount of 5% to 20% by weight.

13. The composition according to claim 11, wherein the alkyl sarcosinate in the composition is in an amount of 0.05% to 1.0% by weight of the active ingredient, and the additive is in an amount of 0.05% to 1.0% by weight.

14. The composition according to any one of claims 1 to 6, wherein the surfactant (a) is a sulfonated alkyl ester, and the additive comprises the L-amino acid, the monovalent salt, the divalent metal salt, and optionally the carbohydrate.

15. The composition according to claim 14, comprising 0.05% to 1.0% by weight of a sulfonated methyl ester and 0.05% to 1.0% of an additive by weight of the active ingredient.

16. The composition according to any one of claims 1 to 15, further comprising spores of beneficial living microorganisms.

17. The aforementioned spores are 1 × 10 ７ ~1 x 10 １０ Preferably 1 x 10 ９ ~5 x 10 ９ The amount is CFU / g, or 1 × 10⁻⁶ ５ ~1 x 10 ９ The composition according to claim 16, in an amount of [amount].

18. A method for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial living microorganisms, wherein the surfactant incompatibility adversely affects the viability, growth, or biological activity of the beneficial living microorganisms, and the method is (a) A step of providing an aqueous composition comprising at least one surfactant that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of the beneficial living microorganisms; (b) A step of combining an additive with the aqueous composition, wherein the additive is (i) At least one L-amino acid in combination with at least one inorganic divalent metal salt or monovalent salt in an amount that results in a weight ratio of additive component (i) to surfactant of 0.2:1 to 1:1; and (ii) If desired, at least one type of carbohydrate, Processes including; (c) A step of combining the beneficial live microorganisms with the aqueous composition before or after step (b), A method comprising the additive, wherein the additive overcomes the antagonistic surfactant incompatibility between the at least one surfactant and the beneficial living microorganism, and promotes the viability, growth, and biological activity of the beneficial microorganism.

19. A method for remediating the soil environment, (a) A step of applying an aqueous composition containing 0.1% to 25% by weight of rhamnolipid as an active ingredient to the soil to be remediated; (b) A step of applying the composition to the soil to be repaired, wherein the composition comprises: (i) a blend of an L-amino acid and a divalent metal salt in an amount of 0.1% to 25% by weight based on the composition, and optionally at least one carbohydrate in an amount not exceeding the weight of the blend of the L-amino acid and the divalent metal salt in the composition; (ii) beneficial viable microbial spores in an amount of 1×10 ６ to 1×10 １０ CFU / g; and (iii) a carrier in an amount such that the total is 100% by weight of the composition. Methods that include...

20. A microbially enhanced cleaning composition, (a) at least one surfactant in an amount of about 0.05% to 1.0% by weight of the active ingredient that exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of the beneficial living microorganisms; (b) An additive in an amount of about 0.05% to 1.0% by weight that overcomes the antagonistic surfactant incompatibility between the at least one surfactant and the beneficial living microorganism, and promotes the viability, growth, and biological activity of the beneficial microorganism, wherein the additive is (i) at least one L-amino acid; (ii) at least one inorganic divalent metal salt or at least one monovalent salt or a combination thereof; (iii) If desired, at least one type of carbohydrate, Additives, including; (c) 1 x 10 ４ ~Approx. 1×10 ８ Microbial spores in amounts of CFU / g; (d) an amount of water totaling 100% by weight of the composition, A composition containing the following:

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