PRODUÇÃO APERFEIÇOADA DE RAMNOLIPÍDEOS USANDO PELO MENOS DUAS FONTES DE CARBONO

Abstract

The present invention relates to a method for improving the yield of rhamnolipids comprising a culture medium containing a triglyceride-containing oil and a sweetener as a carbon source.

Description

Improved production of rhamnolipids using at least two carbon sources. TECHNICAL FIELD

[0001] The present invention relates to an improved method for producing rhamnolipids (RLs) comprising culturing a rhamnolipid-producing microorganism in a medium comprising at least two carbon sources, in particular, a triglyceride-containing oil and a sweetener. BACKGROUND

[0002] Due to growing environmental concerns, biosurfactants have gained much attention from the public and consumers. A primary focus after biosurfactants is rhamnolipids (RL) because they have high foaming, cleaning, dispersing, emulsifying abilities and low surface tensions [1,2]. Rhamnolipids are active interface glycolipids containing carbohydrates (rhamnose) and aliphatic acids (hydroxy fatty acids). They contain one (monorhamnosylipids or monorhamnolipids) or two rhamnose units (di-rhamnosylipids or di-rhamnolipids) and one or two (predominantly two) 3-hydroxy fatty acid residues. Rhamnolipids are predominantly produced through aerobic fermentation by Pseudomonas aeruginosa. Other Pseudomonas and E. coli species have also been reported to produce rhamnolipids, but their yields have a much lower titer and productivity than P. aeruginosa [3].

[0003] In order for rhamnolipids (RL) to compete with synthesized petroleum-based surfactants such as sodium laureth sulfate (SLES) and sodium lauryl sulfate (SLS or SDS), the cost of RL production must decrease significantly. Method optimization and fermentation performance are among the main cost drivers. A number of approaches including different Petition 870260062528, dated 06 / 25 / 2026, page 12 / 85 2 / 28 Feedstocks, genetically modified strains, and fermentation strategies have been implemented to increase RL titer productivity. Banat et al. [4] and Kaskatepe et al. [5] have extensive reviews on biosurfactant production using low-cost feedstock (i.e., waste stream from farms and various industries). Since rhamnolipids contain a rhamnose (sugar) half and a 3-hydroxy fatty acid tail, several researchers have tried using molasses as the sole carbon feedstock. None of them showed rhamnolipid concentrations > 6 g / L with molasses concentrations of 2-10% [6-9]. Vegetable oil, on the other hand, has been used to produce rhamnolipid at a higher concentration compared to molasses feedstock. No one has yet combined both feedstocks for RL production. A summary of fermentation performance for RL production with vegetable oil is shown in Table 1. Table 1: Fermentation performance of P. aeruginosa with different types of vegetable oil Carbon source Fermentation type RL concentration (g / L) Fermentation time (h) RL productivity (g / L / h) Reference Soybean oil Feed - batch 95 216 0.44

[10] Corn oil Batch 27 120 0.23

[11] Palm oil Batch 71 144 0.49

[12] Sunflower oil Batch 27 72 0.38

[11] Soybean oil Feed - batch 65 90 0.72

[13] * *pH control at 7-7.5 in the first 24 hours then at 6-6.5 after

[0004] Although US Patent No. 5,501,966

[10] claimed a batch feeding method producing RL as high as 112 g RL / L in 11 days (264 h) of fermentation and thus, the Petition 870260062528, dated 06 / 25 / 2026, page 13 / 85 3 / 28 The calculated RL productivity is only 0.42 g RL / L / h, which is considered low. Productivity (g RL / L / h) is a very important method parameter since it represents how quickly rhamnolipids can be produced from a certain fermentation volume. The higher the RL productivity, the cheaper the cost of RL production. SUMMARY

[0005] A means is provided for improving the production of by introducing an addition of a sweetener (e.g., an unrefined sweetener or sugar) to a medium containing oil or long-chain triglycerides (e.g., coconut oil or vegetable oil) or a combination of the two oils, thereby reducing the de novo synthesis of rhamnoses from fatty acids. This results in a shorter fermentation time and thus an improvement in RL productivity (g RL / L / h).

[0006] A semi-continuous method is also provided for producing a plurality of fermentations comprising one or more rhamnolipids (RL) comprising: (a) culturing a rhamnolipid-producing microorganism in a culture medium comprising at least two carbon sources, wherein at least one carbon source is a sweetener and at least one carbon source is an oil containing medium- or long-chain triglycerides, at least one nitrogen source, at least one phosphorus source, at least one magnesium source, at least one potassium source, at least one sulfur source, at least one chloride source, and at least one sodium source, for at least about 1 day and more particularly between about 1 day and about 4 days, even more particularly between about 1 day and about 3 days and even more particularly between about 1 day and about 2 days to obtain a first fermentation medium comprising one or Petition 870260062528, dated 06 / 25 / 2026, p. 14 / 85 4 / 28 plus rhamnolipids (RL) and one or more rhamnolipid-producing microorganisms, yielding RL at a rate of at least about 1.5 g RL / L / h, particularly about 1.7 g RL / L / h, more particularly at least about 1.8 RL / L / h, even more particularly yielding between about 1.8 g RL / L / h and about 3.0 g RL / L / h, even more particularly between about 1.8 g RL / L / h and about 2.7 g RL / L / h; (b) removal of at least about 70% of said first fermentation medium obtained in (a), which in a particular embodiment, occurs during agitation and while maintaining airflow, wherein in a particular embodiment, said airflow is maintained with oxygen-enriched air, in a vessel containing said fermentation medium;(c) replacing said first fermentation medium removed in (b) with culture medium having the composition shown in step (a) and (d) repeating steps (a)-(c) at least once to obtain a subsequent fermentation comprising rhamnolipids, wherein said steps (a)(c) are capable of being repeated for at least about 20 days and more particularly for at least about 30 days.;

[0007] In one embodiment, the method may further comprise the addition of a composition comprising one or more micronutrients at a concentration of 0.1–0.2% v / v of total fermentation volume per day. In yet another particular embodiment, at least about 40 g of RL / L are obtained using said method. In yet another particular embodiment, 50 g of RL / L are obtained; in an even more preferred embodiment, at least about 55 g of RL / L are obtained; in yet another particular embodiment, at least 60 g of RL / L are obtained; in an even more particular embodiment, at least about 65 g of RL / L are obtained; in yet another even more particular embodiment, at least about 70 g of RL / L are obtained; in yet another even more preferred embodiment, Petition 870260062528, dated 06 / 25 / 2026, p. 15 / 85 5 / 28 at least about 80 g of RL / L are obtained; in a still more particular embodiment, at least about 90 g of RL / L are obtained. In an even more particular embodiment, between about 40 g of RL / L and 110 g of RL / L are obtained.

[0008] A method is also provided for producing one or more rhamnolipids comprising culturing a rhamnolipid-producing microorganism in a culture medium comprising at least two carbon sources, wherein at least one carbon source is an unrefined sweetener and at least one carbon source is a vegetable oil, at least one nitrogen source, at least one phosphorus source, at least one magnesium source, at least one potassium source, at least one sulfur source, at least one chloride source, at least one sodium source and optionally at least one emulsifier for at least about 1 day yielding a titer of at least about 40 g RL / L, more particularly at least about 50 g RL / L; even more particularly, at least about 55 g RL / L; even more particularly, at least about 60 g RL / L, even more particularly, at least about 70 g RL / L;Even more particularly, at least about 80 g of RL / L; even more particularly, at least about 90 g of RL / L are obtained or alternatively between about 40 g of RL / L to about 110 g of RL / L / h or at a rate of at least about 1.5 g of RL / L / h. The method may further comprise isolating said rhamnolipid(s) from said rhamnolipid-containing fermentation medium. In a particular embodiment, the culture medium is micronutrient-free. This culture medium can be used in a semi-continuous fermentation, particularly the semi-continuous method shown above, as well as batch and feed-batch fermentations. Rhamnolipids can be isolated and; Petition 870260062528, dated 06 / 25 / 2026, p. 16 / 85 6 / 28 purified using methods known in the art (see, for example, US Patent No. 9,884,882 and patent application ser. No. 1,561,1045, filed June 1, 2017). DEFINITIONS

[0009] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other established or intervening value in that established range is encompassed within the invention. The upper and lower limits of those smaller ranges may be independently included in the smaller ranges and are also covered within the invention, subject to any limit specifically excluded in the established range. Where the established range includes one or both limits, ranges excluding any or both of those excluded limits are also included in the invention.

[00010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[00011] All publications and patents cited in this disclosure are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to precede such disclosure by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with this descriptive report, the descriptive report shall supersede any such material.

[00012] It should be noted that as used here and in Petition 870260062528, dated 06 / 25 / 2026, p. 17 / 85 7 / 28 claims bet, the singular forms “a”, “and” and “the” include plural references unless the context clearly dictates otherwise.

[00013] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or will be able to determine using no more than routine experimentation, many equivalents for the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. Throughout this descriptive report and the claims that follow, unless the context requires otherwise, the word “comprises,” and variations such as “comprises” and “comprising,” shall be understood to imply the inclusion of a given whole or step or group of wholes or steps but not the exclusion of any other whole or step or group of wholes or steps. Thus, the terms “comprising,” “including,” “containing,” “having,” etc., should be read expansively or open-ended and without limitation.When used here, the term "comprising" can be replaced by the term "containing" or sometimes, when used here, by the term "having".

[00014] As defined herein, a "sweetener" is a substance that sweetens an edible product.

[00015] As defined herein, an unrefined sweetener is a sweetener containing water and sugar as a byproduct of sugar processing but has not undergone the refining method. It may be extracted directly from including but not limited to sap, roots (e.g., potatoes, sweet potatoes, beets, particularly sugar beets), nectars, flowers, leaves, fruits, cane, trees, stems.

[00016] As defined herein, a refined sweetener is a sweetener Petition 870260062528, dated 06 / 25 / 2026, page 18 / 85 8 / 28 which underwent a refining method using methods known in the art.

[00017] As defined herein, an emulsifier is a type of surfactant typically used to keep emulsions (metastable mixtures of immiscible fluids) well dispersed. Emulsifiers typically have a hydrophobic (water-repelling) and a hydrophilic (water-loving) half. In an emulsion involving an oil and water, emulsifiers will surround the oil with their hydrophobic half oriented toward the oil, thus forming a protective layer so that the oil molecules cannot coalesce. This action helps to keep the dispersed phase in small particles and preserves the emulsion. Emulsifiers can be anionic, non-ionic, or cationic.

[00018] As defined herein, a medium-chain triglyceride contains between fatty acids having an aliphatic tail of 6-12 carbon atoms.

[00019] As defined herein, a long-chain triglyceride contains fatty acids having an aliphatic tail of more than 13 carbon atoms.

[00020] As defined herein, a “vegetable oil” contains mixtures of triglycerides derived from a plant or part thereof.

[00021] As defined herein, a "rhamnolipid" refers to a glycolipid that has a lipid portion that includes one or more saturated or unsaturated β-hydroxycarboxylic acid moieties, typically linear, and a saccharide portion of one or more rhamnose units. The saccharide portion and the lipid portion are linked via a β-glycosidic bond between the 1-OH group of a rhamnose moiety of the saccharide portion and the 3-OH group of a β-hydroxycarboxylic acid moiety of the lipid portion. Thus, the carboxylic group of a carboxylic acid moiety defines the rhamnolipid end. Where more than one Petition 870260062528, dated 06 / 25 / 2026, page 19 / 85 9 / 28 rhamnose moieties are included in a rhamnolipid; each of the rhamnose moieties not attached to the lipid portion is linked to another rhamnose moiety via a 1,4-ε-glycosidic linkage. In embodiments where two or more β-hydroxycarboxylic acids are present in a rhamnolipid, the β-hydroxycarboxylic acid moieties are selected independently of each other. β-hydroxycarboxylic acid moieties of a respective plurality of β-hydroxycarboxylic acid moieties in some embodiments may be identical. In some embodiments, they are different from each other.

[00022] As defined herein, a “micronutrient composition” is a composition comprising a micronutrient present in an amount of not more than about 20 mg / L.

[00023] The terms “culture medium” and “fermentation medium” are synonymous and are used interchangeably. BRIEF DESCRIPTION OF THE FIGURES

[00024] Figure 1 shows fermentation pH trends with and without changes in molasses addition. DETAILED DESCRIPTION

[00025] An improved method for producing rhamnolipids is provided here. In a particular embodiment, the rhamnolipid may have structure (I). Petition 870260062528, dated 06 / 25 / 2026, p. 20 / 85 10 / 28 where m = 2, 1 or 0, in particular 1 or 0, n = 1 or 0, or in particular 1, R1 and R2 = independently of each other an identical or different organic radical with 2 to 24, preferably 5 to 13, carbon atoms, in particular optionally branched alkyl radical, optionally substituted, in particular substituted with hydroxyl, optionally unsaturated, and particularly optionally mono-, di- or tri-unsaturated, preferably one selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl, and tridecenyl and (CI3 / 4)OCI3 / 43, where o = 1 to 23, preferably 4 to 12.

[00026] Both the main chain and the branches may contain heteroatoms such as N, O, S, Se, or Si, or a carbon atom may be substituted by one of these heteroatoms. An aliphatic half may be substituted or unsubstituted with one or more functional groups. Substituents may be any functional groups, such as, but not limited to, amino, amido, carbonyl, carboxyl, hydroxyl, nitro, thio, and sulfonyl. Microorganism producing rhamnolipid

[00027] As noted above, the method involves culturing a microorganism that produces rhamnolipid. A microorganism Petition 870260062528, dated 06 / 25 / 2026, p. 21 / 85 11 / 28 producing rhamnolipid can be a host cell producing rhamnolipids. A recombinant host cell producing rhamnolipids can be a host cell, such as a bacterial cell expressing an Rh1A gene or its ortholog and / or an Rh1B gene or its ortholog, and / or an Rh1C gene or its ortholog, and / or an Rh1R gene or its ortholog, and / or an Rh11 gene or its ortholog, and / or an Rh1G gene or its ortholog and others.

[00028] Alternatively, a "rhamnolipid-producing microorganism" can be any microorganism, such as bacteria, that has the ability to synthesize / produce rhamnolipids under appropriate conditions, including but not limited to bacteria of the phyla Actinobacteria, Fimicutes, and Proteobacteria. In one particular embodiment, the rhamnolipid-producing microorganism is a bacterium of the class Gammaproteobacteria. In another embodiment, the rhamnolipid-producing microorganism is a bacterium of the order Pseudomonadales. In yet another embodiment, the rhamnolipid-producing microorganism is a bacterium of the family Pseudomonadaceae. In yet another embodiment, the rhamnolipid-producing microorganism is a bacterium of the genus Pseudomonas, such as P. alcaligenes, P. aeruginosa, P. chlororaphis, P. clemancea, P. collierea, P. fluorescens, P. luteola, P. putida, P. stutzeri, and P. teessidea. In one embodiment, the microorganism producing rhamnolipid is P. aeruginosa. Culture medium (fermentation)

[00029] The rhamnolipid-containing microorganism is cultivated in a culture medium (also referred to as fermentation). Said culture medium comprises at least two carbon sources, at least one nitrogen source, at least one phosphorus source, at least one sulfur source, at least one sodium source, at least one magnesium source, at least one potassium source, by Petition 870260062528, dated 06 / 25 / 2026, page 22 / 85 12 / 28 minus one source of sulfur and at least one source of chloride.

[00030] The carbon source, in a particular embodiment, may be a sweetener and an oil containing one or more medium-chain and / or long-chain triglycerides (also referred to herein as medium-chain triglyceride oil and long-chain triglyceride oil, respectively). In a more particular embodiment, each sweetener may be present in an amount of about 0.1% to about 2% weight / volume and / or each oil may be present in an amount of about 3% to about 15% weight / weight, particularly between about 4% to about 10% weight / weight, and more particularly between about 6% and about 12% weight / weight.

[00031] The sweetener may be a refined or unrefined sweetener. Examples of refined sweeteners may include, but are not limited to, sucrose (table sugar) and stevia. The unrefined sweetener may be derived from the processing of sugar and / or sap, one or more roots, fruit, one or more seeds, one or more nectars, one or more flowers, one or more leaves, one or more trees, one or more stems, and / or one or more animals. In a more particular embodiment, said unrefined sweetener used may be at least one of molasses, barley or rice malt syrup, nectar, yacon syrup, sugar beet syrup, corn syrup, sorghum syrup, maple syrup, palm sugar, or sweetener derived from potatoes or sweet potatoes. In a more particular embodiment, the unrefined sweetener is molasses.In another embodiment, the carbon source may still comprise a monosaccharide, for example, glucose, a disaccharide, for example, sucrose, a sugar alcohol, for example, glycerol, a long-chain alkane, for example, n-hexadecane, a fatty acid such as caprylic acid (also called octanoic acid), or mixtures thereof, organic acids (for example, lactic acid, acetic acid, etc.). Petition 870260062528, dated 06 / 25 / 2026, page 23 / 85 13 / 28 citric acid, propionic acid), alcohols (e.g., ethanol) and mixtures thereof.

[00032] In a particular embodiment, the oil is a medium-chain triglyceride-containing oil that may be commercially available medium-chain triglyceride oil, which may contain a mixture of coconut oil, palm oil and / or other medium-chain triglycerides (e.g., containing caprylic acid), coconut oil or palm oil. The long-chain triglyceride may be soybean oil, canola oil, sunflower oil, safflower oil, peanut oil, hemp seed oil, jatropha oil, gourd oil, flaxseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil. In one embodiment, the long-chain triglyceride contains fatty acids having an aliphatic tail of more than 13 carbon atoms; in a particular embodiment, it contains fatty acids having an aliphatic tail of between 13-21 carbon atoms.

[00033] In a particular embodiment, the oil may be a vegetable oil. The vegetable oil may be soybean oil, safflower oil, peanut oil, hemp seed oil, canola oil, jatropha oil, gourd oil, linseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grapeseed oil, walnut oil, wheat germ oil, or a combination of vegetable oils.

[00034] In a more particular embodiment, the long-chain triglyceride may be a vegetable oil and the sweetener may be an unrefined sweetener.

[00035] In another particular embodiment, the medium- or long-chain triglyceride may be a vegetable oil where said vegetable oil is corn oil, canola oil or soybean oil or a medium-chain triglyceride where the medium-chain triglyceride is Petition 870260062528, dated 06 / 25 / 2026, p. 24 / 85 14 / 28 coconut oil and the sweetener is an unrefined sweetener which can be molasses, beet sugar syrup or sorghum syrup.

[00036] In one particular embodiment, the culture medium may comprise at least three carbon sources, wherein at least two of the carbon sources are sweeteners and at least one carbon source is an oil containing medium- or long-chain triglycerides. In an even more particular embodiment, at least two of the carbon sources are unrefined sweeteners and at least one carbon source is an oil containing medium-chain triglycerides, for example, coconut oil.

[00037] In another particular embodiment, the culture medium comprises at least four carbon sources, wherein at least two of the carbon sources are oils containing medium- or long-chain triglycerides and at least two of the carbon sources are sweeteners. In a more particular embodiment, at least one of the carbon sources is an oil containing medium-chain triglycerides (e.g., coconut oil), one of the carbon sources is an oil containing long-chain triglycerides (e.g., vegetable oil such as canola oil), and at least two of the carbon sources are unrefined sweeteners (e.g., molasses, sorghum syrup, sugar beet syrup).

[00038] The nitrogen source may be ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, and corn infusion liquor. In one particular embodiment, the nitrogen source is NaNOa. In yet another embodiment, nitrogen may be present in the amount of about 5-20 g / L.

[00039] The source of phosphorus may, in a particular embodiment, be H3PO4 or K2HPO4. In yet another particular embodiment, said phosphorus is present in the amount of about 1-15 g / L.

[00040] The magnesium ion, in a particular embodiment, can Petition 870260062528, dated 06 / 25 / 2026, p. 25 / 85 15 / 28 being MgSO4*7H2O and / or MgCb. In one particular embodiment, magnesium is present in the amount of approximately 0.2-2 g / L.

[00041] Potassium can be KCl and / or KOH. In one particular embodiment, potassium is present in an amount of about 0.1 to about 2 g / L.

[00042] Sodium can be NaCl, NaNO3, and NaOH. In one particular embodiment, said sodium ion is present in the amount of about 1-15 g / L.

[00043] Chloride can be KCl and NaCl. In a particular embodiment, said chloride ion is present in the amount of about 0.1 - 1 g / L.

[00044] Sulfur can be H2SO4. In a particular embodiment, said sulfur ion is present in the amount of about 0.1-1 g / L.

[00045] The sources of sulfur and chloride can be derived from the aqueous layer discharge stream, also referred to as the aqueous liquid phase or aqueous phase of an acid-treated clarified fermentation broth obtainable using procedures described in US patent 9,884,883. In a specific embodiment, rhamnolipids precipitate from solution from an acid-treated clarified fermentation broth and form a solid phase and an oily liquid phase at the bottom, and an aqueous liquid phase is generated at the top of the vessel used for this step. The aqueous liquid phase is removed using procedures known in the art and in a specific embodiment using methods shown above (e.g., filtration, or centrifugation, or deposition combined with decantation).The aqueous layer mentioned above is a source of sulfur or chloride (depending on the type of acid used during this pH adjustment of approximately 1.5 to 2.5, preferably approximately 2.05 to approximately 2.15) and is a source of micronutrients.

[00046] The cultural environment can still encompass a Petition 870260062528, dated 06 / 25 / 2026, p. 26 / 85 16 / 28 Emulsifier. In one particular embodiment, the emulsifier may include, but is not limited to, gum arabic, guar gum, and rhamnolipids. In yet another particular embodiment, the ratio of emulsifier to carbon source in said culture medium is between about 0.1% and about 20% w / w. In yet another particular embodiment, said emulsifier may be present in an amount of about 0.1-2% by weight.

[00047] In a particular embodiment, the culture or fermentation medium is sterilized using methods known in the art. These methods may be filtration-based, heat-based, chemical-based, or ultraviolet light radiation-based. In a particular embodiment, the heat-based treatment may be via wet thermal sterilization, particularly autoclaving.

[00048] In one embodiment, the culture medium (e.g., fermentation medium) can be sterilized by one of the above procedures. In another embodiment, the fermentation media can be sterilized by more than one of the above procedures, and these sterilizations can be in any order. It can be sterilized in fermentation during the first fermentation cycle but can be sterilized in another vessel in subsequent cycles. Micronutrient composition

[00049] As noted above, said method may further comprise the addition of a micronutrient solution or composition. Said micronutrient may be a trace of Fe, Mn, Zn, Cu, or Na. In a particular embodiment, said micronutrient is a salt of Fe, Mn, Zn, Na, or Cu. In a more particular embodiment, said micronutrient composition comprises salts of Fe, Mn, Zn, Na, and Cu. The composition may be sterilized by filtration.

[00050] In particular forms, said Cu salt is at least Petition 870260062528, dated 06 / 25 / 2026, p. 27 / 85 17 / 28 one of CuCli2*2H2O and CuSO4*5H2O and may be present in the amount of about 0.5-3 g / L of micronutrient solution; said Mn salt is at least one of MnSO4*H2O and MnCl2*4H2O and may be present in the amount of about 0.1-2 g / L of micronutrient solution; said Zn salt is ZnSO4*7H2O or ZnCh and may be present in the amount of about 0.5-3 g / L of micronutrient solution; said Fe salt is at least one of FeCl3*6H2O or FeSC”4 and may be present in the amount of approximately 0.1-1 g / L of micronutrient solution; said sodium salt is Na3C6HsO7^2H2O and may be present in the amount of approximately 1-5 g / L of micronutrient solution. EXAMPLES Example 1: Semi-continuous fermentation of 6% rhamnolipid soybean oil with added non-sulfurized sugarcane molasses and gum arabic as an emulsifier.

[00051] Rhamnolipid fermentation is carried out in a 10 L fermenter vessel (Labfors 5, Infors HT, Switzerland) with a working volume of 7.5 L. The fermentation media contain emulsified oil and nutrient solution in a deionized water (DI) balance. First, 1.5 L of 8% emulsified soybean oil with 0.8% gum arabic used as an emulsifier is prepared using a kitchen mixer. With the addition of molasses (sulfur-free cane honey molasses, Golden Barrel, USA), molasses is added to the emulsified oil at 1%, 0.5% or 0.25% w / v before sterilization in an autoclave at 1212°C for 50 minutes. After cooling to 37°C, a sterile filtered nutrient solution at 0.2 microns containing 9.69 g / L of 85% H3PO4, 5.21 g / L of NaOH, 1 g / L of MgSO4*7H2O, 1 g / L of KCl, and 15 g / L of NaNO3 is added. All chemical compounds are at least 99% pure except for the 85% H3PO4. H2SO4 is used to adjust the pH of the fermentation media to 6.3 before inoculation. Petition 870260062528, dated 06 / 25 / 2026, page 28 / 85 18 / 28 with R4 2.5% culture obtained from Example 3 of US Patent Application No. 1561 1045, filed June 1, 2017.

[00052] Fermentation is conducted at 37°C, a 0.14 vvm air feed rate, and a 300-650 rpm stirring speed to maintain dissolved oxygen (DO) at least 15%. When the stirring speed reaches 650 rpm but the DO percentage is still below 15%, pure oxygen is added along with air to maintain a constant total gas flow rate (0.14 vvm). Approximately 20% of a microtrace element composition prepared according to Example 2 listed in US patent application 15 / 146 508, published as US 2016 0326561, is continuously added to the fermenter at 80 mL / day using a peristaltic pump. A silicon-based antifoaming agent (Snapsil FD30, BRB, Netherlands) is automatically added to reduce foaming during fermentation. Fermentation occurs without pH control unless the pH exceeds 7.9. At this point, 25% H2SO4 is automatically added to control the pH at 7.9.

[00053] After fermentation is complete, approximately 77% of the fermentation broth (5.8 L) is removed while maintaining a % DO at 15% (i.e., agitation and gas feed still exist) using a pump. 5.8 L of newly sterilized 8% emulsified oil culture media prepared in a separate container as mentioned in the first paragraph of this example are fed into the fermenter as a new feed stock. This method, called "Remove and Fill (DF)," is shown in US patent application 15 / 146 508, published as US2016 0326561. The first 77% fermentation broth removed from the fermenter after inoculation is referred to as batch DF0. Subsequently, the next fermentation broth being removed from the fermenter after DF0 is called DF1, and so on. Petition 870260062528, dated 06 / 25 / 2026, page 29 / 85 19 / 28

[00054] A pH trend over the course of fermentation for DF1 (no molasses), DF5, 6 and 7 (0.5% molasses addition) shown in Figure 1 demonstrates a 3-phase pattern of pH changes. First, the pH increases rapidly at the beginning of fermentation. Second, the pH remains stable or decreases slightly before reaching phase 3, in which the pH increases again. Phase 2 is shortened with the addition of molasses. In phase 3, the pH increases rapidly along with an increase in % DO while agitation and airflow remain constant, indicating that fermentation is complete. A clear supernatant without an oil layer on top is obtained after the removed fermentation broth is centrifuged at 9500 rpm for 10 minutes or 14000 rpm for 5 minutes.The clear RL supernatant obtained from each DF is then sterilized and centrifuged again to obtain clarified broth (CB) which is filtered to 0.2 micron before being diluted with DI water at least 100-200 times depending on the starting concentration of the material. The diluted samples are then injected into HPLC-ELSD (methodology details shown in Example 2) for rhamnolipid quantification.

[00055] The fermentation results with various concentrations of added molasses are shown in Table 2. Fermentation was run continuously for 18 days using this "remove and fill" method, generating over 65 L of fermentation broth using a 10 L fermentation vessel without shutting it off. The results in Table 2 clearly show that the addition of molasses shortens the fermentation time, mainly during the 2nd pH change phase (Figure 1), yielding higher RL productivities compared to those without molasses, regardless of molasses concentration. This may also be due to an increase in bacterial cell mass shown in the g CDW / L column (g of dry cell weight / L). Petition 870260062528, dated 06 / 25 / 2026, page 30 / 85 20 / 28 Table 2: Fermentation performance of RL with 6% soybean oil with and without molasses DF# % of molasses addition Fermentation time (h) RL (g / L) RL productivity (g / L / h) g CDW / L % of soybean oil consumption DFO* 0% 76 75 1.0 16 90% DF1 0% 50 67 1.3 17 92% DF2 1% 38 72 1.9 32 96% DF3 1% 34 69 2.0 37 94% DF4 1% 36 69 1.9 37 95% DF5 0.50% 28 59 2.1 36 94% DF6 0.50% 28 60 2.2 31 93% DF7 0.50% 28 63 2.2 36 94% DF8 0.25% 33 63 1.9 28 91% DF9 0.25% 34 62 1.8 25 93% DF10 0.25% 34 65 1.9 22 95% * 8% soybean oil was used. Example 2: Quantification and structure of rhamnolipid analyses

[00056] An Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system equipped with an 1290 Infinity evaporative light scattering detector (ELSD) and a Pinnacle DB C18 reversed-phase column (100 x 2.1 mm, 3 micron part #9414312) by Restek is used to quantify the rhamnolipid concentration in the samples. The column temperature is maintained constant at 40°C. The sample injection volume is 25 microliters. The mobile phase contains an equal volume of 5mM ammonium acetate and acetonitrile at 25 mL / minute. The nebulized and evaporator temperatures are at 40°C with 1.7 SLM of nitrogen. The RL concentration is calculated using the dilution factor and the known concentration of the standards (i.e., the calibration curves of pure di-rhamnolipids and pure mono-rhamnolipids) obtained in-house using thin-layer chromatography.

[00057] The structure of rhamnolipids is analyzed using a Waters Corporation 2695 separation module connected to a Petition 870260062528, dated 06 / 25 / 2026, p. 31 / 85 21 / 28 Waters ZQ2000 single quadrupole mass spectrometer with electrospray ionization (LC / MS). The MS column is the same as that used in the HPLC setup. Injection volume is 5 microliters. Mobile phases consist of 5mM ammonium acetate (A) and acetonitrile (B). The flow rate is 0.2 mL / minute, holding A = 60% (B = 40%) for 2 minutes, then gradient to 100% B in 15 minutes, where it is maintained for the remainder of the LC treatment. Samples are kept at 4°C and the column temperature is maintained constant at 40°C. The LC / MS conditions for rhamnolipid detection are listed in Table 3 below. Table 3: LC / MS conditions Parameter Capillary Fixation (kV) 3.2 Cone (V) Per Ion Extractor (V) 5 Source Temp. (°C) 100 Desolvation Temp. (°C) 300 Desolvation Gas (L hr) 250 Cone Gas (L hr) 50 Example 3: Semi-continuous fermentation of RL from soybean oil 7.8% and unsulfurated sugarcane honey molasses 0.5% with rhamnolipids as an emulsifier

[00058] The fermentation conditions, media compositions, and nutrient are the same as shown in Example 1 except that purified rhamnolipid produced from Example 1 is used as an emulsifier. The feed stock is 7.8% soybean oil with 0.5% unsulfurated cane molasses. The purified sulfurized rhamnolipid is added to the culture media as an emulsifier at the beginning with the culture media freshly sterilized. Petition 870260062528, dated 06 / 25 / 2026, page 32 / 85 22 / 28

[00059] The rhamnolipid (RL) concentration and productivity are shown in Table 4. Since RL is added to the media at the beginning as an emulsifier at 0.5% for DFO and 0.1% for DF1DF3, those amounts are subtracted and the actual RL concentration produced from the fermentation is reported as adjusted RL (g / L). Table 4: Fermentation performance of RL with 7.8% soybean oil and 0.5% molasses Batch % of RL as an emulsifier Adjusted RL (g / L) Fermentation (h) RL Productivity (g / L / h) % of C in oil to C in RL DFO 0.5% 78 44 1.8 80% DF1 0.1% 90 35 2.6 92% DF2 0.1% 93 34 2.7 95% DF3 0.1% 80 33 2.4 82%

[00060] It is worth noting that fermentation will be longer for DFO since the microorganism needs time to adjust to the new environment from the stirring flask containing LB broth to the fermenter containing soybean oil. The overall RL productivity of DFO shown is lower than that obtained from DF1+. This is also an advantage of the semi-continuous fermentation method since RL productivity and fermentation method efficiency increase after the first inoculation (DFO). Batch fermentation methods will suffer from this delay every time a new batch starts since fermentation has to start from the beginning (i.e., new inoculation for each batch).

[00061] % carbon conversion is calculated based on the amount of carbon contained in soybean oil converted to carbon in rhamnolipids. LC / MS results showed that the rhamnolipid samples predominantly contain mono- and diramnose with C10-C10 and C10-C12 tails. Based on this result, the carbon conversion calculated from soybean oil to Petition 870260062528, dated 06 / 25 / 2026, page 33 / 85 23 / 28 rhamnolipid production is greater than 80%. Example 4: Semi-continuous fermentation of RL from corn oil. 8.8% with 0.5% non-sulfurized cane molasses

[00062] The fermentation conditions, media, and nutrient compositions are the same as shown in Example 1 except that purified rhamnolipid produced from Example 1 is used as an emulsifier and 7.5 mL of trace elements are added daily. The carbon feedstock is 8.8% corn oil with 0.5% non-sulfurized cane molasses, and purified rhamnolipid is added to culture media as an emulsifier at the beginning of DFO only at 0.1%. No rhamnolipid is added as an emulsifier for DF1-DF5. Table 5: Fermentation performance of RL with 8.8% corn oil and 0.5% molasses Batch RL adjusted (g / L) Fermentation time (h) RL productivity (g / L / h) % mono RL DF1 106 46 2.3 55% DF2 93 45 2.1 54% DF3 106 51 2.1 58% DF4* 82 40 2.1 53% DF5 97 49 2.0 60% *7.8% corn oil is used.

[00063] The RL productivity obtained from corn oil fermentation is as good as that of soybean oil. RL productivity is in the range of 2-2.3 g RL / L / h. Example 5: Shake-flask experiments with sugar beet and sorghum syrups at various concentrations.

[00064] The shaker flask experiment is performed at 37°C, 250 rpm using a MaxQ 8000 Stackable Orbital Shaker (Thermo Petition 870260062528, dated 06 / 25 / 2026, page 34 / 85 24 / 28 Scientific) in 250 mL Pyrex Erlenmeyer baffle flasks. Each flask contained 40 mL of culture medium containing 8% soybean oil with a nutrient composition identical to that described in Example 1 but without trace elements. The baffle flasks are autoclaved at 121 °C for 20 minutes and cooled to room temperature before inoculation at 2.5% v / v with P. aeruginosa culture. Samples are collected at 68, 92, and 116 h using sterile pipettes. The samples are centrifuged at 14,000 rpm for 5 minutes to obtain a clear supernatant (no oil layer) which is then sterilized and filtered to 0.2 micron before dilution for RL concentration analysis using FIPLC / ELSD.

[00065] The sample without a clear supernatant (i.e., with an oil layer on top) is represented as “no CB”, meaning that it was not subjected to HPLC due to a very high concentration of oil in the sample. The results shown in Table 6 clearly show that rhamnolipid production is also improved by the addition of sugar beet syrup and sorghum syrup. Table 6: Rhamnolipid concentrations with sugar beet and sorghum syrups Bottle (h) No additives Beetroot syrup Sugarcane syrup Sorghum syrup 0.50% 1% 1.50% 0.50% 1% 1.50% 68 None CB None CB None CB None CB None CB None CB 74 92 None CB 72 84 80 None CB 90 103 116 56 71 77 80 88 85 91 Example 6: Batch fermentation of RL with 8% soybean oil and 0.5% non-sulfurized sugarcane molasses.

[00066] The fermentation conditions, media and nutrient compositions are the same as shown in Example 4 except that this is a batch fermentation meaning that fermentation is initiated with inoculation R4 (time = 0) and once fermentation is Petition 870260062528, dated 06 / 25 / 2026, page 35 / 85 25 / 28 completed, fermentation is stopped and cleaned. The carbon feedstock is 8% soybean oil with 0.5% non-sulfurized cane molasses. Purified rhamnolipid is added to the culture media as an emulsifier at 0.1% with the culture media freshly sterilized.

[00067] Fermentation takes 44 hours to complete. The rhamnolipid (RL) concentration is obtained at 88 g / L in 44 hours, thus the RL productivity is 1.9 g / L / h compared to 1 g / L / h obtained in DF0 shown in Example 1 without the addition of molasses. Example 7: Semi-continuous fermentation of RL from coconut oil 8% and 0.5% unsulfurized sugarcane honey molasses with rhamnolipids as an emulsifier.

[00068] The fermentation conditions, media, and nutrient compositions are the same as shown in Example 3 except that the carbon feedstock is 8% coconut oil. No rhamnolipid is added as an emulsifier for DF1-DF4 since it is generated from DF0. The fermentation time is consistent at 32-36 h with the addition of 0.5% molasses. Table 8: Fermentation performance with 8% coconut oil and 0.5% molasses Batch RL (g / L) Fermentation Time (h) RL Productivity (g / L / h) % Mono RL DF0 68 38 1.8 66% DF1 75 33 2.2 63% DF2 74 33 2.3 64% DF3 74 32 2.3 62% DF4 75 36 2.1 61% Example 8: Fermentation of 8% RL coconut oil with a combination of sugar additives.

[00069] The fermentation conditions, media and nutrient compositions are the same as shown in Example 7 except that the sugar additives are molasses from non-cane honey. Petition 870260062528, dated 06 / 25 / 2026, page 36 / 85 26 / 28 sulfur compounds, sorghum syrup and sugar beet syrup. Table 9: Fermentation performance of RL with 8% coconut oil and various sugar additives RL Sugar (g / L) Fermentation Time (h) RL Productivity (g / L / h) % Mono RL No sugar 91 88 1.0 57% 0.25% Molasses + 0.25% Sorghum 79 33 2.4 63% 0.25% Molasses + 0.25% Sugar beet 80 35 2.3 60% 0.25% Sugar beet + 0.25% Sorghum 82 57 1.4 56% 0.5% Sugar beet + 0.5% Sorghum 81 41 2.0 59% Example 9: RL fermentation of medium and long chain triglyceride oils with 0.5% non-sulfurized sugarcane molasses

[00070] The fermentation conditions, media and nutrient compositions are the same as shown in Example 7, except that 4% coconut oil and 4% canola oil, representing medium- and long-chain triglyceride oils respectively, are used as a feed stock with 0.5% molasses. Fermentation is completed in 32 hours with a RL concentration of 88 g / L and thus the RL productivity is 2.8 g / L / h. References 1. Müller, MM, et al., Rhamnolipids—Next generation surfactants? Journal of Biotechnology, 2012. 162(4): p. 366-380. 2. Sekhon Randhawa, KK and PKSM Rahman, Rhamnolipid Biosurfactants—Past, Present, and Future Global Market Scenario. Frontiers in Microbiology, 2014. 5: p. 454. 3. Wittgens, A., et al., Growth-independent rhamnolipid production from glucose using nonpathogenic Pseudomonas putida KT2440. Microbial Cell Factories, 2011. 10(1): p. 1-18. Petition 870260062528, dated 06 / 25 / 2026, p. 37 / 85 27 / 28 4. Banat, I.M., et al., Cost effective technologies and renewable substrates for biosurfactants’ production. Frontiers in Microbiology, 2014. 5: p. 697. 5. Kaskatepe, B. and S. Yildiz, Rhamnolipid Biosurfactants Produced by Pseudomonas Species. Brazilian Archives of Biology and Technology, 2016. 59. 6. Desai, R.M.P.a.A.J., Biosurfactant production by Pseudomonas aeruginosa GS3 from molasses. Letters in Applied Microbiology, 1997. 25: p. 91-94. 7. Onbasli D., A.B., Biosurfactant production in sugar beet molasses by some Pseudomonas spp. J Environ Biol. , 2009. 30(1): p. 161-163. 8. Gudina, E.J., et al., Valorization of agro-industrial wastes towards the production of rhamnolipids. Bioresource Technology, 2016. 212: p. 144-150. 9. Rashedi, H., et al., Environmental importance of rhamnolipid production from molasses as a carbon source. International Journal of Environmental Science & Technology, 2005. 2(1): p. 59-62. 10. Giani, C., et al. Pseudomonas aeruginosa and its use in a process for the biotechnological preparation of L-rhamnose. US5501966 A, 1996. 11. Li, A.-h., et al., Rhamnolipid Production by Pseudomonas Aeruginosa GIM 32 Using Different Substrates Including Molasses Distillery Wastewater. Applied Biochemistry and Biotechnology, 2011. 163(5): p. 600-611. 12. Gong, Z., Y. Peng, and Q. Wang, Rhamnolipid production, characterization and fermentation scale-up by Pseudomonas aeruginosa with plant oils. Biotechnology Letters, 2015. 37(10): p. 2033-2038. Petição 870260062528, de 25 / 06 / 2026, pág. 38 / 85 28 / 28 13. Zhu, L., et al., Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fed-batch fermentation process. Bioresource Technology, 2012. 117: p. 208-213.

Claims

1. Semi-continuous method for producing a plurality of fermentations comprising one or more rhamnolipids (RL), said method characterized by comprising: (a) culturing a microorganism producing rhamnolipid in a culture medium, wherein said one or more rhamnolipids is / are obtained at a rate of at least 1.7 g RL / L / hr and said culture medium comprises: i. at least two carbon sources, wherein a first carbon source is an unrefined sweetener selected from the group consisting of molasses, rice or barley malt syrup, nectar, yacon syrup, beet syrup and sorghum syrup and a second carbon source is a vegetable oil containing medium or long chain triglycerides, wherein the unrefined sweetener in the culture medium is in an amount between 0.1% and 2.0% by weight, and the vegetable oil in the culture medium is in an amount between 3% and 15% by weight; ii. at least one source of nitrogen; iii. at least one source of phosphorus; iv.at least one source of magnesium; v. at least one source of potassium; vi. at least one source of sulfur; vii. at least one source of chloride; viii. at least one source of sodium; ix. and optionally in the presence of an emulsifier, wherein said rhamnolipid-producing microorganism is cultivated in said culture medium for at least 1 day to obtain a first fermentation medium comprising one or more rhamnolipids and the rhamnolipid-producing microorganism; (b) removal of at least 70% of said first medium from Petition 870260062528, dated 06 / 25 / 2026, page.40 / 85 2 / 4 fermentation obtained in step (a); (c) replacement of said first fermentation medium removed in (b) with culture medium having the composition shown in step (a); (d) repetition of steps (a)-(c) at least once to obtain a subsequent fermentation medium comprising one or more rhamnolipids and the rhamnolipid-producing microorganism, wherein said method yields a rhamnolipid titer of at least 60 g RL / L.

2. Method according to claim 1, characterized in that said rhamnolipid-producing microorganism is cultivated in step (a) for 1 to 4 days.

3. Method according to any one of claims 1 or 2, characterized by further comprising adding a micronutrient composition at a concentration of not more than 20 mg / L to said culture medium in step (a) at 0.1% v / v of total fermentation volume per day, wherein said micronutrient composition comprises iron, manganese, zinc, copper, sodium or a combination thereof.

4. Method according to any one of claims 1 to 3, characterized in that said fermentation medium is removed in step (b) during agitation and while maintaining airflow, wherein optionally the airflow is maintained with oxygen-enriched air.

5. Method according to any one of claims 1 to 4, characterized in that there is no sedimentation step between steps (a) and (b).

6. Method for producing one or more rhamnolipids, said method characterized by comprising: (a) culturing a microorganism producing rhamnolipid in a culture medium comprising at least two sources of carbon, wherein a first carbon source is an unrefined sweetener selected from the group consisting of molasses, rice or barley malt syrup, nectar, yacon syrup, beet syrup and sorghum syrup and wherein a second carbon source is a vegetable oil containing medium-chain or long-chain triglycerides, wherein the unrefined sweetener in the culture medium is in an amount between 0.1% and 2.0% by weight, and the vegetable oil in the culture medium is in an amount between 3% and 15% by weight;at least one nitrogen source, at least one phosphorus source, at least one magnesium source, at least one potassium source, at least one sulfur source, at least one chloride source, at least one sodium source and optionally an emulsifier, for at least 1 day, wherein the culture is carried out using the semi-continuous fermentation method as defined in claim 1, wherein said one or more rhamnolipids is / are obtained at a rate of at least 1.7 g RL / L / h; and wherein said method yields a rhamnolipid titer of at least 60 g / L; and (b) optionally isolating said one or more rhamnolipids from said culture medium.

7. Method according to any one of claims 1 or 6, characterized in that said rhamnolipid-producing microorganism is a microorganism of the genus Pseudomonas.

8. Method according to any one of claims 1 or 6, characterized in that said vegetable oil is at least one of soybean oil, safflower oil, peanut oil, hemp seed oil, jatropha oil, coconut fat, gourd oil, linseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grape seed oil, walnut oil, wheat germ oil, coconut oil or medium-chain triglyceride oil.

9. A method according to any one of claims 1 or 6, characterized in that said rhamnolipid-producing microorganism is cultivated in a culture medium selected from the group consisting of: (a) a culture medium comprising at least three carbon sources, wherein at least two of the carbon sources are unrefined sweeteners and at least one carbon source is a vegetable oil containing medium- or long-chain triglycerides; (b) a culture medium comprising at least three carbon sources, wherein at least two of the carbon sources are unrefined sweeteners and at least one carbon source is a vegetable oil containing medium-chain triglycerides; (c) a culture medium comprising at least four carbon sources, wherein at least two of the carbon sources are vegetable oils containing medium- or long-chain triglycerides and at least two carbon sources are unrefined sweeteners;and (d) a culture medium comprising at least four carbon sources, wherein at least one of the carbon sources is a vegetable oil containing medium-chain triglycerides, one of the carbon sources is a vegetable oil containing long-chain triglycerides and at least two of the carbon sources are unrefined sweeteners.

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