WO2024118749A1 - Methods for producing bio-derivatized linear sophorolipids - Google Patents

Abstract

Materials and methods are provided for producing bio-derivatized sophorolipids (SLP) via yeast fermentation, wherein SLP molecules are transformed in situ into derivative molecules through selective modification of nutrients and feedstock. The final percentage of linear-type SLP with respect to total SLP produced is at least 70% or greater, through a single fermentation process, thereby eliminating the need for additional downstream processing or chemical reactions.

Description

METHODS FOR PRODUCING BIO-DERIVATIZED LINEAR SOPHOROLIPIDS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 63/428,460, filed November 29, 2022, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

From hard surface cleaners to laundry detergents, consumers are searching for naturally- derived products that they perceive as safer for themselves and the environment. Laundry liquid formulations typically contain around 12 to 40% surfactants, which are primarily alkyl ether sulfates (AES) and alkyl sulfonates. Personal care and household dishwashing formulations often contain linear alkyl sulfonates (LAS), linear alkylbenzene sulfonates (LABS) and linear AES, which are produced by sulfation/sulfonation of the corresponding alkane, alcohol or its ethoxylate with sulfur trioxide or chlorosulfonic acid, followed by neutralization. These common surfactants do not meet the growing consumer demand for green products, particularly for formulations that are sulfate-free or majority bio-based.

One promising group of surface-active substances that could meet this need are biosurfactants, a structurally diverse group of surface-active substances produced by microorganisms. All biosurfactants are amphiphiles. They consist of two parts: a polar (hydrophilic) moiety and non-polar (hydrophobic) group. Due to their amphiphilic structure, biosurfactants can, for example, increase the surface area of hydrophobic water-insoluble substances, increase the water bioavailability of such substances, and change the properties of bacterial cell surfaces.

Biosurfactants can also reduce the interfacial tension between water and oil and, therefore, lower the hydrostatic pressure required to move entrapped liquid to overcome the capillary effect. Biosurfactants accumulate at interfaces, thus reducing interfacial tension and leading to the formation of aggregated micellar structures in solution. The formation of micelles provides a physical mechanism to mobilize, for example, oil in a moving aqueous phase. The ability of biosurfactants to form pores and destabilize biological membranes also permits their use as antibacterial, antifungal, and hemolytic agents to, for example, control pest and/or microbial growth.

Like chemical surfactants, the properties of biosurfactants can be measured by hydrophile- lipophile balance (HLB). HLB is the balance of the size and strength of the hydrophilic and lipophilic moieties of a surface-active molecule. Specific HLB values are required for a stable emulsion to be formed. In water/oil and oil/water emulsions, the polar moiety of the surface-active molecule orients towards the water, and the non-polar group orients towards the oil, thus lowering the interfacial tension between the oil and water phases. HLB values range from 0 to about 20, with lower HLB (e.g., 10 or less) being more oil- soluble and suitable for water-in-oil emulsions, and higher HLB (e.g., 10 or more) being more water-soluble and suitable for oil-in-water emulsions.

There are multiple types of biosurfactants, including low molecular weight glycolipids, lipopeptides, flavolipids and phospholipids, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

Glycolipids, in particular, are biosurfactants comprising a carbohydrate and at least one fatty acid. Glycolipids include, for example, rhamnolipids (RLP), rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates, trehalose monomycolates, mannosylerythritol lipids (MEL), cellobiose lipids, ustilagic acids and/or sophorolipids (SLP).

SLP comprise a sophorose consisting of two glucose molecules, linked to a fatty acid by a glycosidic ether bond. SLP are categorized into two general forms: the lactonic form, where the carboxyl group in the fatty acid side chain and the sophorose moiety form a cyclic ester bond; and the acidic form, or linear form, where the ester bond is hydrolyzed. In addition to these forms, there exists a number of derivatives characterized by the presence or absence of double bonds in the fatty acid side chain, the length of the carbon chain, the position of the glycosidic ether bond, the presence or absence of acetyl groups introduced to the hydroxyl groups of the sugar moiety, and other structural parameters.

Lactonic and linear sophorolipids have different functional properties. For example, linear SLP are highly water soluble due to their free carboxylic acid groups. Altering the ratio of linear to lactonic SLP alters, e.g., emulsion droplet size, viscosity alteration properties, foaming and surface/interfacial tension reduction properties.

Fermentation of yeast cells in a culture substrate including a sugar and fatty acids with carbon chains of differing length is typically how SLP are produced. The yeast Starmerella (Candida) bombicola is one of the most widely recognized producers of SLP. Typically, the yeast produces both lactonic and linear SLP during fermentation, with about 60-70% of the SLP comprising lactonic forms, and the remainder comprising linear forms. The lactonic form is generated as a result of an enzyme produced by the yeast, lactone esterase, which catalyzes the esterification of the linear form in aqueous environments.

Thus, production of SLP using yeast fermentation generally results in a range of molecules with a distribution of structures. Additionally, because of the nature of biological processes, it is difficult to standardize the exact concentration of certain forms of SLP that can be extracted from a yeast culture, making it challenging and costly to produce products containing a single form of SLP from a cultivation batch, particularly the linear form. To produce a product containing a majority linear SLP involves either post-fermentation processing by alkali-catalyzed hydrolysis and ring opening of lactonic compounds or the use of engineered (genetically modified) yeast strains in which the lactone esterase enzyme is suppressed to avoid formation of lactonic moieties. Hydrolysis of the lactone ring usually results in de- acetylation of the linear SLP, which is not always desired.

SLP have potential to be use as a substitute for chemical surface active agents, and/or as a co-surfactant to reduce the negative effects of chemical surfactants, in a wide range of industries. SLP can be used in, for example, food preservation, biomedicine, cosmetics, bioremediation, remediation of heavy metals, and making various personal care and household cleaning products. SLP can also be applicable to the petroleum industry in, for example, drilling, cement slurries, fracturing, enhanced oil recovery, scale formation prevention, acidization, demulsification of crude fluids, corrosion inhibition, reduced oil viscosity, cleaning of equipment, waterflooding, and/or foam and steam flooding. Furthermore, in agriculture and livestock production, SLP can be used as, for example, soil amendments, broad spectrum biopesticides, antiviral, antifungal and antibacterial agents, and/or additives to animal feed to enhance nutrient absorption.

While current methods of producing SLP products containing a majority linear SLP may be sufficient for small scale production of SLP, for example, in research settings, these methods are not ideal for industrial applications or when the use of GMOs is undesirable. Therefore, improved methods are needed for production of linear sophorolipids that are suitable for industrial scale applications.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides materials and methods for producing compositions comprising sophorolipids (SLP). More specifically, the subject invention provides methods for the production of multi-functional SLP compositions comprising, in some embodiments, a mixture of SLP molecules, wherein the composition can be modified to exhibit one or more functional characteristics based on the desired use by altering the structures and ratios of different SLP molecules.

In certain embodiments, the subject invention provides materials and methods for producing bio-derivatized sophorolipids (SLP) via yeast fermentation, wherein SLP molecules are transformed in situ into derivative molecules through selective modification of nutrients and feedstock. Advantageously, the subject methods can be designed to increase the final percentage of linear-type SLP to, for example, at least 70%, at least 80%, or at least 90%, or greater, with respect to total SLP produced, through a single fermentation process, thereby eliminating or reducing the need for additional downstream processing or chemical reactions. Furthermore, the subject methods can decrease the fermentation time for achieving a desired volume of SLP compared with traditional fermentation methods, thereby increasing overall productivity and reducing the manufacturing footprint.

In certain embodiments, the methods of the subject invention comprise creating a bio- tailored fermentation medium and cultivating a sophorolipid-producing yeast in the fermentation medium to produce a yeast culture. The yeast culture comprises liquid broth, yeast cells, and a mixture of linear-type and lactonic SLP.

The bio-tailored fermentation medium comprises a source of fatty acids and/or triglycerides, and optionally, further comprises, or lacks, a particular component, wherein the presence or lack of the component alters the metabolic pathway through which sophorolipids are produced. The result of fermentation is thus altered from what is achieved through traditional fermentation parameters.

In certain embodiments, the bio-tailored fermentation medium comprises a bio-based component that alters the activity of the lactone esterase enzyme, which is responsible for catalyzing the intramolecular esterification (lactonization) of linear SLP to produce lactonic SLP. In some embodiments, the bio-based component is a mono-, di- or poly-alcohol, which can bind to and esterify the carboxyl group of a linear SLP fatty acid chain, thereby blocking intramolecular esterification of the sophorose moiety.

In certain embodiments, the use of an alcohol component in the bio-tailored fermentation medium eliminates the need for traditional carbohydrate sources, e.g., sugars such as glucose. This improves the sustainability, cost-effectiveness and efficiency of the production method through reduction in total raw materials. In some embodiments, a sugar can be included, although preferably sourced from a local supplier, e.g., within 100 miles of the fermentation facility.

The addition of an alcohol, such as, e.g., glycerol, to the bio-tailored fermentation medium shifts the traditional production of predominantly lactonic SLP to predominantly linear-type SLP at surprisingly and advantageously high ratios. For example, in certain embodiments, the yeast culture comprises at least 70%, at least 80% or at least 90% linear-type SLP with respect to the total amount of SLP produced. In certain embodiments, the use of an alcohol component also reduces the fermentation time from, e.g., 90-120 hours down to 70-80 hours, when compared with use of a traditional carbohydrate source.

Other examples of bio-based components for use in the bio-tailored fermentation medium include but are not limited to C2-C10 alkyl chain alcohols, including mono-alcohols, e.g., ethanol, methanol, propanol, butanol, isopropanol; diols, e.g., ethylene glycol, propylene glycol, butylene glycol, cyclohexane- 1,2-diol; and polyols, e.g., glycerine, polyethylene glycol, polypropylene glycol, polytetrahydrofuran, castor oil, sorbitol, mannitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and isomalt.

In certain embodiments, the method can further comprise collecting, extracting, isolating and/or purifying the SLP from the yeast culture. In some embodiments, the bio-derivatized linear SLP are separated from the lactonic SLP, while in other embodiments, the SLP mixture is left as a mixture.

In preferred embodiments, the sophorolipid-producing yeast is Starmerella bombicola, or another member of the Starmerella and/or Candida clades. For example, S. bombicola strain ATCC 22214 can be used according to the subject methods. GMO yeasts are not required to achieve the desired increased linear-type SLP ratio in the SLP mixture; however, the subject methods are not limited to non-GMO microorganisms.

In certain embodiments, in addition to the surprising increase in the ratio of linear-type SLP molecules and decrease in fermentation time, the subject methods also result in the production of novel bio-derivatized linear SLP molecules with surprisingly similar, or even improved, properties compared with conventionally-produced linear SLP, e.g., critical micelle concentration (CMC), surface tension reduction, interfacial tension reduction (FIGS. 1-3), wettability alteration (FIG. 4), and foaming (FIGS. 5-6). Thus, with fewer material inputs, fewer chemical reactions outside of standard fermentation, and no requirement for the use of GMO organisms, the subject methods allow for the production of bio-based SLP products with comparable or improved properties to non- derivatized SLP products.

In certain embodiments, the novel bio-derivatized linear-type SLP molecules of the subject invention are linear SLP alcohols having the following General Formula (A):

wherein R = a mono-, di-, or polyol. In a certain specific embodiment, R = glycerol.

Advantageously, the fermentation processes described herein eliminate the need for GMO microorganisms, as well as the costly and tedious downstream processing of lactonic SLP to produce linear SLP. Furthermore, the methods do not require complex or expensive equipment, but rather can be implemented using standard fermentation materials. Even further, the molecules produced according to the subject invention can be useful for replacing and/or reducing the amounts of chemical surfactants used in applications such as large scale industrial and agriculture uses, cosmetics, household products, health, medical and pharmaceutical fields, and oil and gas recovery.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B depict a comparison of a glycerol ester SLP composition according to an embodiment of the subject invention with a FermaSH (0-20% lactonic, 80-100% linear SLP obtained via hydrolysis) with regard to dynamic surface tension reduction (A) and interfacial tension reduction (B).

Figures 2A-2B depict a comparison of dynamic (A) and static (B) surface tension between a glycerol ester SLP composition according to an embodiment of the subject invention, FermaSL (53- 71% lactonic, 30-48% linear SLP)) and FermaSH.

Figure 3 depicts static surface tension of common laundry surfactants. SLES = sodium salt of lauryl ether sulfate; LABS = linear alkyl benzene sulfonate; SLABS = sodium linear alkyl benzene sulfonate.

Figure 4 depicts contact angle data comparing wettability of a glycerol ester SLP composition according to an embodiment of the subject invention and various other surfactants. C10-APG = alkyl poly glucoside with C10 chain.

Figure 5 depicts a sparge foam test comparison between a glycerol ester SLP composition according to an embodiment of the subject invention, FermaSL and FermaSH.

Figures 6A-6B depict results of a Ross-Miles foam test comparing foam volume over time between FermaSH and a glycerol ester SLP composition according to an embodiment of the subject invention (A) and the appearance of the foam for both SLP compositions (B).

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods for producing bio-derivatized sophorolipids (SLP) via yeast fermentation, wherein SLP molecules are transformed in situ into derivative molecules through selective modification of nutrients and feedstock. Advantageously, the subject methods can be designed to increase the final percentage of linear-type SLP to, for example, at least 70%, 80% or 90% of the total SLP produced, through a single fermentation process, thereby eliminating the need for additional downstream processing or chemical reactions.

Sophorolipids are glycolipid biosurfactants produced by, for example, various yeasts of the Starmerella clade. SLP consist of a disaccharide sophorose linked to long chain hydroxy fatty acids. They can comprise a partially acetylated 2-O-P-D-glucopyranosyl-D-glucopyranose unit attached p- glycosidically to 17-L-hydroxyoctadecanoic or 17-L-hydroxy-A9-octadecenoic acid. The hydroxy fatty acid can have, for example, 1 1 to 20 carbon atoms, and may contain one or more unsaturated bonds. Furthermore, the sophorose residue can be acetylated on the 6- and/or 6’-position(s). The fatty acid carboxyl group can be free (acidic or linear form) or internally esterified at the 4"-position (lactonic form). In most cases, fermentation of SLP results in a mixture of hydrophobic (water- insoluble) SLP, including, e.g., lactonic SLP, mono-acetylated linear SLP and di-acetylated linear SLP, and hydrophilic (water-soluble) SLP, including, e.g., non-acetylated linear SLP.

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

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

where R¹ and R¹' independently represent saturated hydrocarbon chains or single or multiple, in particular single, unsaturated hydrocarbon chains having 8 to 20, in particular 12 to 18 carbon atoms, more preferably 14 to 18 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups; R² and R² independently represent a hydrogen atom or a saturated alkyl functional group or a single or multiple, in particular single, unsaturated alkyl functional group having 1 to 9 carbon atoms, more preferably 1 to 4 carbon atoms, which can be linear or branched and can comprise one or more hydroxy groups; and R³, R^3', R⁴and R^4' independently represent a hydrogen atom or -COCH₃. R⁵ is typically -OH; however, the subject invention provides bio-derivatized linear-type SLP wherein R⁵ is, for example, an alcohol group. See General Formula (A).

Due to the structure and composition of SLP, these biosurfactants have excellent surface and interfacial tension reduction properties, as well as other beneficial biochemical properties, which can be useful in applications such as large scale industrial and agriculture uses, cosmetics, household products, health, medical and pharmaceutical fields, and oil and gas recovery.

Selected Definitions

In some embodiments, the subject invention provides microbe-based compositions, meaning a composition that comprises components that were produced as the result of the growth of microorganisms or other cell cultures. Thus, the microbe-based composition may comprise the microbes themselves and/or by-products of microbial growth. The microbes may be in a vegetative state, in spore form, in mycelial form, in any other form of propagule, or a mixture of these. The microbes may be planktonic or in a biofilm form, or a mixture of both. The by-products of growth may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. The microbes may be intact or lysed. The microbes may be present in or removed from the composition. The microbes can be present, with broth in which they were grown, in the microbe-based composition. The cells may be present at, for example, a concentration of at least 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10 ¹¹, 1 x 10¹², or more CFU per milliliter of the composition.

The subject invention further provides microbe-based products, which are products that are to be applied in practice to achieve a desired result. The microbe-based product can be simply a microbe-based composition harvested from the microbe cultivation process. Alternatively, the microbe-based product may comprise further ingredients that have been added. These additional ingredients can include, for example, stabilizers, buffers, carriers, such as water, salt solutions, or any other appropriate carrier, added nutrients to support further microbial growth, non-nutrient growth enhancers and/or agents that facilitate tracking of the microbes and/or the composition in the environment to which it is applied. The microbe-based product may also comprise mixtures of microbe-based compositions. The microbe-based product may also comprise one or more components of a microbe-based composition that have been processed in some way such as, but not limited to, filtering, centrifugation, lysing, drying, purification and the like.

As used herein, an “alcohol” is a compound comprising at least one hydroxyl functional group (-OH) bound to a saturated carbon atom. Within the definition of alcohol are “diols," which comprise two hydroxyl functional groups, and “polyols,” which are alcohols comprising more than one hydroxyl function group. As used herein, the term “alkyl” refers to straight chain or branched hydrocarbon groups. Suitable alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl. The term alkyl may be prefixed by a specified number of carbon atoms to indicate the number of carbon atoms or a range of numbers of carbon atoms that may be present in the alkyl group such as C1 -C10 alkyl, C1 -C20 alkyl, and C10-C20 alkyl. For example, C1 -C3 alkyl refers to methyl, ethyl, propyl and isopropyl.

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

As used herein, “harvested” refers to removing some or all of a microbe-based composition from a growth vessel.

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

In certain embodiments, purified compounds are at least 60% by weight the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 98%, by weight the compound of interest. For example, a purified compound is one that is preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

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

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

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

As used herein, “surfactant” means a compound that lowers the surface tension (or interfacial tension) between two liquids, between a liquid and a gas, or between a liquid and a solid. Surfactants act as, e.g., detergents, wetting agents, emulsifiers, foaming agents, and/or dispersants. A “biosurfactant” is a surface-active substance produced by a living cell and/or using naturally- derived sources.

The transitional term “comprising,” which is synonymous with “including,” or “containing,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Use of the term “comprising” contemplates other embodiments that “consist” or “consist essentially of’ the recited components).

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “and” and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All references cited herein are hereby incorporated by reference in their entirety. Fermentation Methods

In preferred embodiments, the subject invention provides methods of producing sophorolipidic compositions by cultivating a sophorolipid-producing yeast using submerged fermentation. The methods can be scaled up or down in size. Most notably, the methods can be scaled to an industrial scale, i.e., a scale that is suitable for use in supplying biosurfactants in amounts to meet the demand for commercial applications, for example, formulation of compositions for personal care, home care, agriculture and enhanced oil recovery.

Production of sophorolipids traditionally results in a mixture of different, but structurally similar, molecules. Traditional fermentation yields about a 60-80% lactonic SLP and 20-40% linear SLP, although this ratio can vary depending on set parameters of the fermentation cycle and carbon sources.

In certain embodiments, the subject invention provides materials and methods for the production of multi-functional SLP compositions comprising a mixture of SLP molecules, wherein the composition can be modified to exhibit one or more functional characteristics based on the desired use by altering the structures and ratios of different SLP molecules.

In certain preferred embodiments, the subject invention provides fermentation processes for producing SLP, wherein the fermentation parameters are altered in such a way that the amount of linear-type SLP produced is, for example, at least 70%, 75%, 80%, 85%, 90% or more (with respect to total SLP). The methods of the subject invention comprise cultivating a sophorolipid-producing yeast in a bio-tailored fermentation medium to produce a yeast culture. The yeast culture comprises liquid broth, yeast cells, and a mixture of linear-type and lactonic SLP.

In certain embodiments, the method comprises filling a fermentation reactor with the bio- tailored fermentation medium; inoculating the reactor with the sophorolipid-producing yeast; and cultivating the yeast under conditions favorable for production of SLP.

As used herein “fermentation” refers to growth or cultivation of cells under controlled conditions. The growth could be aerobic or anaerobic. Unless the context requires otherwise, the phrase is intended to encompass both the growth phase and product biosynthesis phase of the process. As used herein, a “broth,” “culture broth,” or “fermentation broth” refers to a culture medium comprising at least nutrients. If the broth is referred to after a fermentation process, the broth may comprise microbial growth byproducts, microbial cells and/or cellular components as well.

The microbe growth vessel used according to the subject invention can be any fermenter or cultivation reactor for industrial use. As used herein, the term “reactor,” “bioreactor,” “fermentation reactor” or “fermentation vessel” includes a fermentation device consisting of one or more vessels and/or towers or piping arrangements. Examples of such reactor includes, but are not limited to, the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact. In some embodiments, the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, when referring to the addition of substrate to the bioreactor or fermentation reaction, it should be understood to include addition to either or both of these reactors where appropriate.

In a further embodiment, the vessel may also be able to monitor the growth of microorganisms inside the vessel (e.g., measurement of cell number and growth phases). Alternatively, samples may be taken from the vessel for enumeration, purity measurements, SLP concentration, and/or visible oil level monitoring. For example, in one embodiment, sampling can occur every 12-24 hours.

The microbial inoculant according to the subject methods preferably comprises cells and/or propagules of the desired microorganism, which can be prepared using any known fermentation method. The inoculant can be pre-mixed with water and/or a liquid medium, if desired.

The microorganisms utilized according to the subject invention may be natural, or genetically modified microorganisms. For example, the microorganisms may be transformed with specific genes to exhibit specific characteristics. The microorganisms may also be mutants of a desired strain. As used herein, “mutant” means a strain, genetic variant or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion) as compared to the reference microorganism. Procedures for making mutants are well known in the microbiological art. For example, UV mutagenesis and nitrosoguanidine are used extensively toward this end.

In preferred embodiments, the microorganism is any yeast or fungus. Examples of yeast and fungus species suitable for use according to the current invention, include, but are not limited to, Acaulospora, Aspergillus, Aureobasidium (e.g., A. pullulans). Blakeslea, Candida (e.g., C. albicans, C. apicola), Cryptococcus, Debaryomyces (e.g., D. hansenii), Entomophthora, Fusarium, Hanseniaspora (e.g., H. uvarum), Hansenula, Issatchenkia, Kluyveromyces, Mortierella, Mucor (e.g., M. piriformis), Meyerozyma (e.g., M. guilliermondii), Penicillium, Phythium, Phycomyces, Pichia (e.g., P. anomala, P. guilliermondii, P. occidentalis, P. kudriavzevii), Pseudozyma (e.g., P. aphid is), Rhizopus, Saccharomyces (.S' cerevisiae, S. boulardii sequela, S. torula), Starmerella (e.g., .S', bombicola), Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei, T. harzianum, T. virens), Ustilago (e.g., U. maydis), Wickerhamomyces (e.g., W. anomalus), Williopsis, and Zygosaccharomyces (e.g., Z. bailii). In preferred embodiments, microorganism is a Starmerella spp. yeast and/or Candida spp. yeast, e.g., Starmerella (Candida) bombicola, Candida apicola, Candida batistae, Candida floricola, Candida riodocensis, Candida stellate and/or Candida kuoi. In a specific embodiment, the microorganism is Starmerella bombicola, e.g., strain ATCC 22214. GMO yeasts are not required to achieve the surprising increase in linear SLP percentage in the produced SLP mixture; however, the subject methods are not limited to non-GMO microorganisms.

Preferably, the subject methods utilize a liquid fermentation medium, or liquid growth medium. Traditional fermentation methods for SLP production utilize a nitrogen source for cellular growth, a hydrophilic carbon source for the hydrophilic sophorose ring formation, and a hydrophobic carbon source to provide the hydrophobic fatty acid chain. The biosynthesis begins with the hydroxylation of fatty acids. If the medium contains triglycerides, an extracellular lipase hydrolyzes them to fatty acids, which are consumed by the cells.

Thus, in preferred embodiments, the bio-tailored fermentation medium comprises purified fatty acids and/or triglycerides, and/or sources of fatty acids and/or triglycerides, such as, for example, vegetable oil, soybean oil, rice bran oil, olive oil, corn oil, sunflower oil, sesame oil, coconut oil, madhuca oil, linseed oil, canola oil, pecan oil, peanut oil, macadamia oil, grapeseed oil, poppyseed oil, palm oil, palm kernel oil, safflower oil, cocoa butter, mutton tallow, beef tallow, lard, and fatty acid alkyl esters.

In certain embodiments, the bio-tailored fermentation medium comprises a source of oleic acid, such as, for example, high oleic soybean oil, high oleic sunflower oil, high oleic canola oil, olive oil, pecan oil, peanut oil, macadamia oil, grapeseed oil, sesame oil, poppyseed oil, pure oleic acid, madhuca oil, oleic acid alkyl esters, and/or triglycerides of oleic acid.

In contrast to traditional fermentation, the bio-tailored fermentation medium preferably further comprises, or lacks, a particular component, wherein the presence or lack of the component alters the metabolic pathway through which sophorolipids are produced. The result of fermentation is thus altered from what is achieved through traditional fermentation parameters.

In certain embodiments, the bio-tailored fermentation medium comprises a bio-based component that alters the activity of the lactone esterase enzyme, which is responsible for catalyzing the intramolecular esterification (lactonization) of linear SLP to produce lactonic SLP. In some embodiments, the bio-based component is an alcohol, diol or polyol, which can bind to and esterify the carboxyl group of a linear SLP fatty acid chain, thereby blocking intramolecular esterification of hydroxyl groups on the sophorose ring.

In certain embodiments, the use of an alcohol component in the bio-tailored fermentation medium reduces and/or eliminates the need for traditional carbon sources, e.g., sugars such as glucose. This improves the sustainability, cost-effectiveness and efficiency of the production method through reduction in total raw materials. In some embodiments, a sugar or other carbon source can be included, although preferably sourced from a local supplier, e.g., within 100 miles of the fermentation facility. The traditional carbon source can be, for example, a carbohydrate, such as glucose, dextrose, sucrose, lactose, fructose, trehalose, mannose, molasses and/or maltose; or and organic acid such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid.

In certain embodiments, the bio-tailored fermentation medium comprises from 0 to 500 g/L, 0.01 to 400 g/L, 0.1 to 300 g/L, 0.5 to 200 g/L, 1.0 to 100 g/L, 2.0 to 75 g/L, 3.0 to 50 g/L, 3.5 to 25 g/L, 4.0 to 20 g/L, 4.5 to 15 g/L, or 5.0 to 10 g/L of the traditional carbon source. In certain preferred embodiments, the bio-tailored fermentation comprises less than 200 g/L, less than 150 g/L, less than 100 g/L, less than 50 g/L, less than 25 g/L, less than 10 g/L, less than 5 g/L, or less than 1 g/L of the traditional carbon source.

The addition of a bio-based alcohol component, such as, e.g., glycerol, to the bio-tailored fermentation medium can be used to shift the traditional production of predominantly lactonic SLP to predominantly linear-type SLP at surprisingly and advantageously high ratios. For example, in certain embodiments, the yeast culture comprises at least 70%, at least 80% or at least 90% linear- type SLP with respect to the total amount of SLP produced.

In certain embodiments, the use of an alcohol component also reduces the fermentation time from, e.g., 90-120 hours down to 70-80 hours, or preferably, around 72-76 hours, when compared with use of a traditional carbohydrate source. Thus, the subject invention provides methods for reducing the time required for producing linear-type SLP.

While glycerol or other alcohols can be a natural by-product of yeast metabolization of triglycerides, the subject invention employs the positive addition of an alcohol component in excess of an amount that would be produced naturally. In other words, without the “forced metabolism” and associated chemical transformation resulting from the human influence of adding non-natural amounts of glycerol to the yeast’s fermentation environment, the ratio of linear-type SLP to lactonic SLP would remain lactonic-predominant, and the derivatized SLP would not be produced in useful quantities.

Advantageously, glycerol is readily available as a waste product of, for example, biodiesel production. Other examples of bio-based alcohol components useful in the bio-tailored fermentation medium include but are not limited to mono-alcohols, e.g., ethanol, methanol, propanol, butanol, isopropanol, hexanol, octanol; diols, e.g., ethylene glycol, propylene glycol, butylene glycol, cyclohexane- 1,2-diol; and polyols, e.g., glycerine, polyethylene glycol, polypropylene glycol, polytetrahydrofuran, castor oil, sorbitol, mannitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and isomalt. In certain embodiments, the alcohols are C2-C 10 alcohols. In preferred embodiments, the amount of the alcohol component in the bio-tailored fermentation medium is from 0.1 to 500 g/L, 0.5 to 400 g/L, 1.0 to 300 g/L, 1.5 to 200 g/L, 2.0 to 100 g/L, 2.5 to 75 g/L, 3.0 to 50 g/L, 3.5 to 25 g/L, 4.0 to 20 g/L, 4.5 to 15 g/L, or 5.0 to 10 g/L.

In some embodiments, both the alcohol component and a traditional carbon source, e.g., glucose or dextrose, are present in the bio-tailored fermentation medium. Depending on the ratio of the alcohol to the traditional carbon source, the ratio of linear-type and lactonic SLP can be customized. For example, a 1 : 1 ratio of alcohol to traditional carbon source can produce a SLP mixture comprising from 40 to 60% linear-type SLP and 40 to 60% lactonic SLP, with respect to total SLP. As the concentration of the alcohol increases and the concentration of the traditional carbon source decreases, the percentage of linear-type SLP with respect to total SLP increases. Similarly, as the concentration of alcohol decreases and the concentration of traditional carbon source increases, the percentage of lactonic SLP with respect to total SLP increases.

In certain embodiments, the bio-tailored fermentation medium comprises a bio-based component that increases the yeast metabolic rate and SLP production rate. In some embodiments, the bio-based component is magnesium sulfate (MgSO4), which can reduce the time required for fermentation of a given amount of SLP and/or can increase yeast efficiency with regard to metabolizing the alcohol component. Thus, the result can be a reduction in the amount of the alcohol component required to produce a given amount of SLP in one fermentation cycle.

In one embodiment, the liquid growth medium comprises a nitrogen source. The nitrogen source can be, for example, yeast extract, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride. These nitrogen sources may be used independently or in a combination of two or more.

In one embodiment, one or more inorganic salts may also be included in the liquid growth medium. Inorganic salts can include, for example, potassium dihydrogen phosphate, monopotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate, calcium nitrate, magnesium sulfate, sodium phosphate, sodium chloride, and/or sodium carbonate. These inorganic salts may be used independently or in a combination of two or more.

In one embodiment, growth factors and trace nutrients for microorganisms are included in the medium. This is particularly preferred when growing microbes that are incapable of producing all of the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. Furthermore, sources of vitamins, essential amino acids, proteins and microelements can be included, for example, com flour, peptone, yeast extract, potato extract, beef extract, soybean extract, banana peel extract, and the like, or in purified forms. Amino acids such as, for example, those useful for biosynthesis of proteins, can also be included.

In some embodiments, the method for cultivation may further comprise adding additional acids and/or antimicrobials in the liquid medium before and/or during the cultivation process. Antimicrobial agents or antibiotics (e.g., streptomycin, oxytetracycline) are used for protecting the culture against contamination. In some embodiments, however, the metabolites produced by the yeast culture provide sufficient antimicrobial effects to prevent contamination of the culture.

In one embodiment, prior to inoculation, the components of the liquid culture medium can optionally be sterilized. In one embodiment, sterilization of the liquid growth medium can be achieved by placing the components of the liquid culture medium in water at a temperature of about 85-100°C. In one embodiment, sterilization can be achieved by dissolving the components in 1 to 3% hydrogen peroxide in a ratio of 1 :3 (w/v).

In one embodiment, the equipment used for cultivation is sterile. The cultivation equipment such as the reactor/vessel may be separated from, but connected to, a sterilizing unit, e.g., an autoclave. The cultivation equipment may also have a sterilizing unit that sterilizes in situ before starting the inoculation. Gaskets, openings, tubing and other equipment parts can be sprayed with, for example, isopropyl alcohol. Air can be sterilized by methods know in the art. For example, the ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the medium may be pasteurized or, optionally, no heat at all added, where the use of pH and/or low water activity may be exploited to control unwanted microbial growth.

The method of cultivation can further provide oxygenation to the growing culture. One embodiment utilizes slow motion of air to remove low-oxygen containing air and introduce oxygenated air. The oxygenated air may be ambient air supplemented daily through mechanisms including impellers for mechanical agitation of the liquid, and air spargers for supplying bubbles of gas to the liquid for dissolution of oxygen into the liquid. In certain embodiments, dissolved oxygen (DO) levels are maintained at about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, or about 50% of air saturation.

The pH of the culture should be suitable for the microorganism of interest. In some embodiments, the pH is about 2.0 to about 7.0, about 3.0 to about 5.5, about 3.25 to about 4.0, or about 3.5. Buffers, and pH regulators, such as carbonates and phosphates, may be used to stabilize pH near a preferred value. In certain embodiments, a base solution is used to adjust the pH of the culture to a favorable level, for example, a 15% to 30%, or a 20% to 25% NaOH solution. The base solution can be included in the growth medium and/or it can be fed into the fermentation reactor during cultivation to adjust the pH as needed.

In one embodiment, the method of cultivation is carried out at about 5° to about 100° C, about 15° to about 60° C, about 20° to about 45° C, about 22° to about 35 °C, or about 24° to about 28°C. In one embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to changing temperatures.

According to the subject methods, the microorganisms can be cultivated in the fermentation system for a time period sufficient to achieve a desired effect, e.g., production of a desired amount of cell biomass or a desired amount of one or more microbial growth by-products. The microbial growth by-product(s) produced by microorganisms may be retained in the microorganisms and/or secreted into the growth medium. The biomass content may be, for example from 5 g/1 to 180 g/1 or more, from 10 g/1 to 150 g/1, or from 20 g/1 to 100 g/1.

In certain embodiments, fermentation of the yeast culture occurs for about 36 to 150 hours, or about 100 to about 125 hours, or about 120 hours. In some embodiments, the fermentation cycle is ended once the alcohol component in the medium has been consumed (e.g., to a level of 0% to 0.5%). In some embodiments, the end of the fermentation cycle is determined to be a time point when the microorganisms have begun to consume trace amounts of SLP.

In certain embodiments, the method can further comprise collecting, extracting, isolating and/or purifying the SLP from the yeast culture. In some embodiments, the bio-derivatized linear SLP are separated from the lactonic SLP, while in other embodiments, the SLP mixture is left as a mixture.

Separation, isolation, extraction and purification can be performed using methods known in the art and as described in, e.g., WO2021/127339A1 (incorporated herein by reference). Advantageously, the fermentation processes described in the subject invention eliminate the need for GMO microorganisms, as well as the costly and tedious downstream processing of lactonic SLP to produce linear SLP. Furthermore, the methods do not require complex or expensive equipment, but rather can be implemented using standard fermentation materials. Bio-derivatized Linear-type Sophorolipids

In certain embodiments, in addition to the surprising increase in the ratio of linear-type SLP molecules, the subject methods also result in the production of novel bio-derivatized linear SLP molecules with surprisingly similar and/or improved properties compared with conventionally- produced SLP, e.g., critical micelle concentration (CMC), wettability alteration, and foaming. Thus, with fewer material inputs, fewer chemical reactions outside of standard fermentation, and no requirement for the use of GMO organisms, the subject methods allow for the production of bio- based SLP products with comparable and/or improved properties to non-derivatized SLP products. Without the use of harsh downstream processing, e.g., alkaline hydrolysis of lactonic SLP, the method can also reduce the de-acetylation of SLP, thus producing greater mono- and di-acetylated linear-type SLP. The bio-derivatized SLP produced are linear-type SLP in which the carboxylic end of the fatty acid tail is replaced with an alcohol ester. Utilizing an alcohol, such as glycerol, and canola oil as the hydrophilic and hydrophobic carbon sources, in addition to nitrogen sources, the SLP produced inside the yeast cell comprises a carboxylic acid chain occupied by the alcohol chain. This prevents the lactone esterase from acting on the molecule and prevents the ring closure found in the lactonic form of SLP.

In certain embodiments, the novel bio-derivatized linear-type SLP molecules of the subject invention are linear SLP alcohols have the following General Formula (A):

wherein R = an alcohol group such as, for example,

ethanol; methanol; heptanol; butanol; propanol; isopropanol; pentanol;

hexanol; octanol; nonanol; or decanol.

In certain embodiments, the subject invention provides compositions produced according to the subject methods, the compositions comprising a bio-derivatized linear SLP alcohol produced according to the subject fermentation methods. In certain embodiments, the composition also comprises some lactonic SLP, e.g., 30% or less of the SLP is lactonic.

In certain embodiments, the composition comprises a purified bio-derivatized linear SLP alcohol produced according to the subject methods.

Combined with the characteristics of low toxicity and biodegradability, SLP are advantageous for use in many settings including, for example, improved bioremediation, mining, and oil and gas production; waste disposal and treatment; enhanced health of livestock and other animals; food additives, such as preservatives and/or emulsifiers; cosmetic additives; and enhanced health and productivity of plants.

In particular, linear SLP, or SLP products comprising 70% or greater linear SLP, are hydrophilic (water-soluble), salt-tolerant, can be useful for foaming, cleansing, producing oil-in- water emulsions, and are stable at wide pH ranges. Thus, they are particularly suitable for personal care products such as shampoos and hand soaps.

Further components can be added to the sophorolipidic compositions as needed for a particular use. The additives can be, for example, carriers (e.g., water), solvents, organic and/or inorganic acids, essential oils, botanical extracts, cross-linking agents, chelators, fatty acids, alcohols, pH adjusting agents, reducing agents, buffers, enzymes, dyes, colorants, fragrances, preservatives, emulsifiers, foaming agents, bleaching agents, polymers, thickeners and/or viscosity modifiers, tracking agents, pesticides, herbicides, animal feed, food products and other ingredients specific for an intended use.

In certain embodiments, the methods of the subject invention can be carried out in such a way that minimal-to-zero waste products are produced, thereby reducing the amount of fermentation waste being drained into sewage and wastewater systems, and/or being disposed of in landfills. Furthermore, this can be achieved while increasing the overall linear SLP production from a single fermentation cycle.

The yeast cell biomass collected from the yeast culture after removal and purification of the SLP would typically be inactivated and disposed of. However, the subject methods can further comprise collecting the cell biomass and using it, in live or inactive form, for a variety of purposes, including but not limited to, as a soil amendment, a livestock feed supplement, an oil well treatment, and/or a skincare product. The cell biomass can be used directly, or it can be mixed with additives specific for the intended use.

Cultivation of microbial biosurfactants according to the prior art is a complex, time and resource consuming, process that requires multiple stages. Advantageously, the methods of the subject invention do not require complicated equipment or high energy consumption, and thus reduce the capital and labor costs of producing microorganisms and their metabolites on a large scale. Additionally, the methods and equipment of the subject invention reduce the capital and labor costs of producing highly useful linear sophorolipids.

Uses

In a specific embodiment, the novel molecules of the present invention can be useful for laundry formulations suitable for sensitive skin and eyes. Advantageously, the bio-derivatized SLP can be useful for reducing the amounts of chemical surfactants used in laundry formulations, while reducing, or even treating, skin and/or eye irritation caused by these surfactants. In certain specific embodiments, the bio-derivatized SLP can be used as an additive for improving the performance and/or reducing the dosage rate of chelating agents in formulations such as, for example, laundry detergents and other household and personal care goods. As used herein, “chelating agents,” or “chelators” are active complex ion-forming agents capable of removing a metal ion from a system by forming a complex so that the metal ion, for example, cannot readily participate in or catalyze oxygen radical formation.

Examples of chelating agents include, but are not limited to, dimercaptosuccinic acid (DMSA), 2,3 -dimercaptopropanesulfonic acid (DMPS), alpha lipoic acid (ALA), thiamine tetrahydrofurfuryl disulfide (TTFD), penicillamine, ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium citrate and citric acid. In certain embodiments, the chelating agent is a derivative of a chelating agent, for example, disodium EDTA. In one embodiment, a mixture of chelators is used. The total concentration of chelating agents can be, for example, about 0.1 g/L to about 50 g/L, about 1 .0 g/L to about 25 g/L, or about 5 g/L to about 10 g/L.

In certain specific embodiments, the bio-derivatized SLP can be used for improving irrigation of soil. Advantageously, the compositions and methods of the subject invention can be formulated as environmentally-friendly, non-toxic and cost-effective solutions to the growing problems of, for example, water shortages, water-use inefficiency, declining soil health, nutrient leaching and runoff, and soil-borne greenhouse gas emissions.

In certain embodiments, the compositions and methods of the subject invention can be useful for any of the following exemplary benefits: a) improving the dispersion, percolation and/or retention of water and nutrients throughout the layers of soil, thereby improving water and nutrient uptake by plant roots and reducing water and fertilizer usage requirements; b) improving the circulation of water and nutrients within plant vasculature, even in colder climates; c) increasing the capture of atmospheric moisture into soil; d) decreasing and/or preventing soil compaction, thereby improving water, nutrient, root and microbial movement throughout the soil; e) reducing the pooling of water on and in soil, thereby reducing evaporation, runoff and waterlogging; and f) increasing soil organic content (SOC) and reducing soil-borne greenhouse gas emissions.

The bio-derivatized SLP can serve as an irrigation additive, which, in some embodiments, can be enhanced by the presence of residual glycerol in the fermentation medium comprising the bio-derivatized SLP.

In certain embodiments, both the bio-derivatized SLP and the glycerol are active ingredients that serve as wetting agents, which, when contacted with water in soil and/or the atmosphere, enhance the watering efficiency for plants, including crops, turf and ornamentals.

The glycerol preferably works in synergy with the bio-derivatized SLP to reduce the surface tension of water in soil and facilitate water transport through dry and/or poorly draining soils. Furthermore, in certain embodiments, the bio-derivatized SLP improves the emulsification of glycerol in the water for even distribution during application in the field.

Glycerol can help draw atmospheric moisture into the soil and can also serve as a food source for beneficial soil microorganisms. Advantageously, glycerol is readily available as a waste product of, for example, biodiesel production; thus, the invention can provide an effective inter- industry solution for waste management.

Other examples of bio-based alcohol components that can be used according to the subject invention include but are not limited to mono-alcohols, e.g., ethanol, methanol, propanol, butanol, isopropanol, hexanol, octanol; diols, e.g., ethylene glycol, propylene glycol, butylene glycol, cyclohexane- 1,2-diol; and polyols, e.g., glycerine, polyethylene glycol, polypropylene glycol, polytetrahydrofuran, castor oil, sorbitol, mannitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and isomalt. In certain embodiments, the alcohols are C2-C10 alcohols.

In certain embodiments, the bio-derivatized SLP and/or glycerol irrigation additive can comprise additional substances, such as, for example, carriers, pH adjusters, pesticides, herbicides, fertilizers, microbial inoculants, mineral sources, plant seeds, dyes, stabilizers, emulsifiers, prebiotics and/or polymers.

In certain embodiments, the subject invention provides methods of irrigating a soil, the methods comprising combining an amount of the bio-derivatized SLP and/or glycerol-containing irrigation additive according to the subject invention with an aqueous fluid to create a treated irrigation fluid, and administering the treated irrigation fluid to the soil. In certain embodiments, the bio-derivatized SLP and/or glycerol-containing irrigation additive is applied with the aqueous fluid continuously throughout irrigation. Advantageously, the subject methods improve soil health, soil hydrology, and, consequently, lead to improved plant health.

In some embodiments, the method comprises applying the bio-derivatized SLP and/or glycerol-containing irrigation additive to soil, followed by applying an aqueous irrigation fluid to the soil or allowing rainwater to activate the irrigation additive upon contact therewith.

In certain embodiments, the bio-derivatized SLP and/or glycerol-containing irrigation additive is applied to a soil to reduce the surface and/or interfacial tension between the water and soil particles, thereby providing one or more of: improved dispersion, penetration and/or percolation of water and nutrients into the soil during irrigation; loosening of hard or compacted soils; and increased soil porosity and aeration. Advantageously, this can increase the space for root growth, air movement, and water holding capacity within the soil.

In certain embodiments, the bio-derivatized SLP is particularly helpful for reducing surface and/or interfacial tension between water and soil, as well as increasing porosity of compacted soils, due to its nano-micelle size. The ultra-small micelle size of the surface active agent allows water to penetrate into micro- and nano-sized pores in hard and tightly packed soils, thereby loosening the pores and allowing for increased air, nutrient and water flow. This can further help prevent root rot caused by excessive water, which can cause overgrowth of detrimental fungi in the soil and on roots.

In certain embodiments, the bio-derivatized SLP and/or glycerol-containing irrigation additive lowers the surface and/or interfacial tension between water and root cells, thereby facilitating enhanced transport of water and nutrients into, and throughout, plant vascular systems.

Advantageously, the method can reduce the water usage requirements for achieving a desired level of irrigation by at least 15%, at least 20%, at least 25%, or at least 30% compared with irrigating without the bio-derivatized SLP and/or glycerol-containing irrigation additive.

EXAMPLES

A greater understanding of the present invention and of its many advantages may be had from the following examples, given by way of illustration. The following examples are illustrative of some of the methods, applications, embodiments and variants of the present invention. They are not to be considered as limiting the invention. Numerous changes and modifications can be made with respect to the invention.

EXAMPLE 1 - CULTIVATION OF STARMERELLA BOMBICOLA AND BIO- DERIV ATIZATION OF SLP

Traditional fermentation of SLP utilizes glucose as a growth source. The standard fermentation uses high aeration to achieve high cell growth and SLP production. The resulting mixture of SLP is predominantly lactonic, ranging from 60 - 80% lactonic SLP and 40 - 20% linear SLP. To obtain a product having >80% of linear SLP, hydrolysis of the lactone ring is required and usually results in non-acetylated linear SLP.

The subject invention provides novel fermentation methods in which the lactone esterification is blocked in situ to yield new forms of bio-derivatized linear SLP. Thus, a larger ratio of linear-type SLP retaining acetyl groups is obtained, without the addition of harsh chemicals typically used in post-fermentation hydrolyzation of lactonic SLP. Bio-derivatization Fermentation Parameters:

Parameters are adjusted at the beginning of the stationary phase of the fermentation cycle to obtain the bio-derivatized linear SLP.

After about 4-7 days of cultivation, or if the yeast begins to consume the SLP, the batch is ready for harvesting.

EXAMPLE 2 - ADDITIONAL BIO-TAILORED FERMENTATION MEDIUM

While optimizing the production of the new bio-derivatized liner SLP, it was determined that magnesium plays a major role in the metabolism and uptake of the glycerol by Starmerella bombicola ATCC 22214. This is based on the expected glycerol pathway used by this strain, the glycerol-3-phosphate pathway. Magnesium acts as the cofactor for the presumed rate limiting reaction performed by the GUT1 glycerol kinase. By increasing the magnesium concentration in the media; the rate of consumption and utilization efficiency can be increased. Furthermore, the dosage rate of glycerol can be reduced.

EXAMPLE 3 - EXEMPLARY PROPERTIES OF GLYCEROL ESTER SLP

EXAMPLE 4 - COMPARISON OF GLYCEROL ESTER SLP WITH OTHER SURFACTANTS

The ring/plate method was used to measure static surface tension and critical micelle concentration (CMC) of the bio-derivatized linear SLP (“SLPBD”) produced according to the subject invention. The maximum bubble pressure method was used to calculate the dynamic surface tension. Dynamic measurements are taken between 22-24°C with 20 second time intervals. This reflects the surface tension with respect to a chosen bubble interval time. FIGS. 1-3.

The SLPBD exhibits low CMC, at 30 ppm (equivalent to FermaSL (53-71% lactonic, 30- 48% linear SLP)), when compared to linear alkyl benzene sulfonate (LABS), sodium salt of lauryl ether sulfate (SLES), alkyl poly glucoside with C10 chain (C10-APG) and FermaSH (0-20% lactonic, 80-100% linear SLP). Table 7.

Additionally, when applied at 250 ppm to a polycarbonate surface, the SLPBD is comparable or slightly better at wettability alteration (contact angle of 59.9°) than SLES (60.8°), APG (63.2°) and FermaSH (61.2°), but not as effective as LABS (33.8°) and C12-C15-Pareth- 7(linear alcohol ethoxylate) (37.1°). FIG. 4. Furthermore, in sparge tests at 500 ppm in DI water (22°C), the SLPBD were comparable with FermaSH at pH 5.8 for foaming but show more stable foam at higher concentrations when tested using a Ross-Miles Foam test. At pH 4.6, SLPBD exhibited very similar foaming behavior to FermaSL. FIGS. 5-6B. EXAMPLE 5 - LAUNDRY DETERGENT FORMULATION

In one embodiment, the bio-derivatized linear SLP are formulated into a laundry detergent suitable for sensitive skin and eyes. Preferably the pH is within a range of about 8.0 to about 12.5, more preferably about 8.0 to about 1 1 .0, which is suitable for cleaning stains and other soils from fabrics.

Claims

CLAIMS We claim:

1. A method for producing a sophorolipid (SLP) composition, the method comprising: cultivating a sophorolipid-producing yeast in a bio-tailored fermentation medium to produce a yeast culture, said yeast culture comprising liquid fermentation broth, yeast cells and a mixture of bio-derivatized linear SLP alcohols and lactonic SLP, said mixture of bio-derivatized linear SLP alcohols and lactonic SLP comprising at least 70% bio-derivatized linear SLP alcohols, wherein the bio-tailored fermentation medium comprises one or more sources of fatty acids and/or triglycerides and an alcohol component, and wherein the alcohol component is present in the bio-tailored fermentation medium at an amount that is greater than an amount produced by the yeast as a by-product of metabolism.

2. The method of claim 1, wherein the yeast is Starmerella bombicola.

3. The method of claim 1, wherein the alcohol component is ethanol, methanol, heptanol, butanol, propanol, isopropanol, pentanol, hexanol, octanol, nonanol, decanol, ethylene glycol, propylene glycol, butylene glycol, glycerol, cyclohexane- 1,2-diol, polyethylene glycol, polypropylene glycol, polytetrahydrofuran, castor oil, sorbitol, mannitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, or isomalt.

4. The method of claim 1, wherein the bio-derivatized linear SLP has the following General Formula (A):

wherein R = an alcohol group selected from the group consisting of:

; . i l l

; ethanol; methanol; heptanol; butanol; propanol; isopropanol; pentanol; hexanol; octanol; nonanol; and decanol.

5. The method of claim 1, wherein the bio-tailored fermentation medium does not comprise a traditional carbon source.

6. The method of claim 1, wherein the bio-tailored fermentation medium comprises less than 10 g/L of glucose or dextrose.

7. The method of claim 1, further comprising isolating the SLP mixture from the yeast culture.

8. The method of claim 7, further comprising isolating the bio-derivatized linear SLP alcohol from the lactonic SLP.

9. The method of claim 8, further comprising purifying the bio-derivatized linear SLP alcohol.

10. The method of claim 7, further comprising formulating the SLP mixture into a product by adding one or more carriers, solvents, surfactants, co-surfactants, organic and/or inorganic acids, essential oils, botanical extracts, cross-linking agents, chelators, fatty acids, alcohols, pH adjusting agents, reducing agents, buffers, enzymes, dyes, colorants, fragrances, preservatives, emulsifiers, foaming agents, bleaching agents, polymers, thickeners and/or viscosity modifiers, tracking agents, pesticides, herbicides, animal feed, food products and other ingredients specific for an intended use.

11. A composition comprising a bio-derivatized linear SLP and a lactonic SLP, wherein the percentage of bio-derivatized linear SLP is at least 70% with respect to total SLP.

12. The composition of claim 11, wherein the percentage of bio-derivatized linear SLP is at least 80% with respect to total SLP.

13. The composition of claim 11, wherein the percentage of bio-derivatized linear SLP is at least 90% with respect to total SLP.

14. The composition of claim 11, wherein the bio-derivatized linear SLP alcohol has the following General Formula (A):

wherein R = an alcohol group selected from the group consisting of:

; ethanol; methanol; heptanol; butanol; propanol; isopropanol; pentanol; hexanol; octanol; nonanol; and decanol.

15. A bio-derivatized linear SLP having the following General Formula (A):

wherein R = an alcohol group selected from the group consisting of:

ethanol; methanol; heptanol; butanol; propanol; isopropanol; pentanol; hexanol; octanol; nonanol; and decanol.

16. The bio-derivatized SLP of claim 15, wherein

17. A method for producing a sophorolipid (SLP) composition, the method comprising: cultivating a sophorolipid-producing yeast in a bio-tailored fermentation medium to produce a yeast culture, said yeast culture comprising liquid fermentation broth, yeast cells and a mixture of bio- derivatized linear SLP alcohols and lactonic SLP, said mixture comprising a customized ratio of bio- derivatized linear SLP alcohols to lactonic SLP, wherein the bio-tailored fermentation medium comprises one or more sources of fatty acids and/or triglycerides, an alcohol component, and a carbohydrate, and wherein the alcohol component is present in the bio-tailored fermentation medium at an amount that is greater than an amount produced by the yeast as a by-product of metabolism.

18. The method of claim 17, wherein the yeast is Starmerella bombicola.

19. The method of claim 17, wherein the alcohol component is ethanol, methanol, heptanol, butanol, propanol, isopropanol, pentanol, hexanol, octanol, nonanol, decanol, ethylene glycol, propylene glycol, butylene glycol, glycerol, cyclohexane- 1,2-diol, polyethylene glycol, polypropylene glycol, polytetrahydrofuran, castor oil, sorbitol, mannitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, or isomalt.

20. The method of claim 17, wherein the bio-derivatized linear SLP has the following General

Formula (A):

wherein R = an alcohol group selected from the group consisting of:

; ethanol; methanol; heptanol; butanol; propanol; isopropanol; pentanol; hexanol; octanol; nonanol; and decanol.

21. The method of claim 17, wherein the carbohydrate is glucose or dextrose.

22. The method of claim 17, wherein the amount of the alcohol component is greater than the amount of the carbohydrate, and wherein the mixture of bio-derivatized linear SLP alcohols to lactonic SLP comprises greater than 50% bio-derivatized linear SLP with respect to total SLP,

23. The method of claim 17, wherein the amount of the carbohydrate component is greater than the amount of the alcohol component, and wherein the mixture of bio-derivatized linear SLP alcohols to lactonic SLP comprises a greater than 50% lactonic SLP with respect to total SLP,

24. The method of claim 17, further comprising isolating the SLP mixture from the yeast culture.

25. The method of claim 24, further comprising formulating the SLP mixture into a product by adding one or more carriers, solvents, surfactants, co-surfactants, organic and/or inorganic acids, essential oils, botanical extracts, cross-linking agents, chelators, fatty acids, alcohols, pH adjusting agents, reducing agents, buffers, enzymes, dyes, colorants, fragrances, preservatives, emulsifiers, foaming agents, bleaching agents, polymers, thickeners and/or viscosity modifiers, tracking agents, pesticides, herbicides, animal feed, food products and other ingredients specific for an intended use.

26. The method of claim 25, comprising formulating the SLP mixture into a product comprising EDTA or disodium EDTA.

27. A method for reducing a fermentation time period required for producing an amount of a sophorolipid, the method comprising, cultivating a sophorolipid-producing yeast in a bio-tailored fermentation medium to produce a yeast culture, said yeast culture comprising liquid fermentation broth, yeast cells and a mixture of bio-derivatized linear SLP alcohols and lactonic SLP, said mixture comprising a customized ratio of bio-derivatized linear SLP alcohols to lactonic SLP, wherein the bio-tailored fermentation medium comprises one or more sources of fatty acids and/or triglycerides, an alcohol component, and a carbohydrate, wherein the alcohol component is present in the bio-tailored fermentation medium at an amount that is greater than an amount produced by the yeast as a by-product of metabolism, and wherein the fermentation period is reduced from 90-120 hours to 70-80 hours.

28. The method of claim 27, wherein the bio-tailored fermentation medium further comprises MgSO4.