Purification of rhamnolipids via extraction

Abstract

A process for purifying rhamnolipids via a liquid-liquid extraction process is disclosed. In the process, an acidic aqueous medium comprising acidic rhamnolipids is intimately mixed with water while maintaining the pH of the aqueous medium at an acidic pH, such as a pH of between 1.0 and 4.0. The water extracts impurities from the acidic rhamnolipids, resulting in purified rhamnolipids having a reduced amount of impurities, such as residual antifoam, as well as other impurities. The acidic aqueous medium may be heated to liquefy the rhamnolipids to facilitate extraction of the impurities. The liquidliquid extraction process can be conducted as a batch process or a continuous process.

Description

PURIFICATION OF RHAMNOLIPIDS VIA EXTRACTIONFIELD OF THE INVENTION

[0001] The present technology relates to a process for purifying rhamnolipids. In particular, the present technology relates to a liquid-liquid extraction process for extracting impurities from an acidic rhamnolipid solution.BACKGROUND OF THE INVENTION

[0002] There has been a recent trend to formulate products with ingredients that are based on renewable raw materials rather than fossil fuels. Such ingredients are considered “green” or “natural”, since they are derived from renewable and / or sustainable sources. As a result, they are more environmentally friendly than ingredients derived from fossil fuels.

[0003] Rhamnolipids are interface-active glycolipids produced by various bacterial species and are an example of a “green” ingredient, since they can be prepared by means of fermentation based on renewable raw materials. Rhamnolipids are of interest in a wide variety of technical applications, such as bioremediation and enhanced oil recovery (EOR), personal care and cosmetics, detergents and cleaners, and agriculture.

[0004] Although a great deal of research has been devoted in recent years to the production of rhamnolipids, they are typically available on the market only in small quantities and at high prices, due in part to cumbersome and expensive downstream processing to isolate and purify the rhamnolipids. A common isolation procedure involves acidifying the fermentation broth after fermentation forproducing the rhamnolipids is complete. Acidification causes the rhamnolipids to separate out of solution, resulting in a rhamnolipids dense layer that also includes solid cellular debris from the organism used in fermentation. The rhamnolipids are isolated from this cell debris by extracting them into an organic solvent, such as ethyl acetate. After stripping the ethyl acetate, a concentrated oily form of the product results. However, the solvent extraction process brings along any hydrophobic impurities with it, potentially including intact and partially hydrolyzed oils, long-chain free fatty acids, hydrophobic metabolites, and antifoams, such as silicones, used for foam control during the fermentation process. Additional processing is required to remove these hydrophobic impurities, particularly if the rhamnolipids are intended for applications with stringent purity requirements.

[0005] Rhamnolipids can also be extracted using solvent-free techniques. U.S. Patent No. 9,884,883 describes processes in which the liquid medium is aged to allow solid cellular waste to settle out, followed by separation of the solid waste from the liquid medium containing the rhamnolipids. The liquid medium is treated with an acid to form a solid, an aqueous phase and an oily phase. After removing the aqueous phase, the solid and oily phase are treated with a base to redissolve the rhamnolipids, and any solid waste is removed from the neutralized solution containing the rhamnolipids. However, impurities, such as antifoam, may still need to be removed from the rhamnolipid-containing solution.

[0006] There is a need in the art for an improved process for removing impurities from rhamnolipids. It would also be desirable to have a commercially viable process for purifying rhamnolipids with good yields and having low levels of impurities,including low levels of residual antifoam. The applicants have discovered an improved process for removing impurities from rhamnolipids that meets one or more of the above objectives, while also advancing UN Sustainability Goals (“SDG”). The process promotes sustainable industrialization by providing a more efficient rhamnolipid purification process. The purified rhamnolipids are advantageously bio-based, renewably sourced materials obtained from a bacterial fermentation process that generates biodegradable waste products that are less impactful on the environment. These benefits further SDG #9 (Industry, Innovation and Infrastructure) and SDG #12 (Responsible Consumption and Production).SUMMARY OF THE INVENTION

[0007] One aspect of the present technology is directed to a process for isolating and purifying rhamnolipids comprising (a) providing an acidic liquid comprising an acidic rhamnolipid phase comprising at least one rhamnolipid; and (b) mixing the acidic rhamnolipid phase with water while maintaining an acidic pH to thereby extract impurities from the acidic rhamnolipid phase into the water; and (c) removing the water from the acidic rhamnolipid phase. In some embodiments, the acidic liquid comprising an acidic rhamnolipid phase contains a solvent and is obtained by (i) treating an aqueous fermentation broth comprising at least one rhamnolipid with an acid to form an acidic medium; (ii) adding a solvent to the acidic medium to form an upper solvent layer comprising the at least one rhamnolipid and a lower aqueous layer; and (iii) removing the lower aqueous layer from the acidic medium to obtain the acidic liquid comprising the acidic rhamnolipid phase.

[0008] In some embodiments, the acidic liquid comprising an acidic rhamnolipid phase does not include a solvent and is obtained by (i) treating an aqueous fermentation broth comprising at least one rhamnolipid with an acid to form an acidic medium comprising an upper aqueous liquid phase and a lower rhamnolipid- containing phase comprising the at least one rhamnolipid; and (ii) removing the aqueous liquid phase from the acidic medium to obtain the acidic rhamnolipid phase. In some embodiments, after the aqueous phase is removed, the resulting acidic liquid comprising the acidic rhamnolipid phase is heated to a temperature in the range of 50°C to 100°C, preferably 50°C to 90°C, to liquefy the at least one rhamnolipid. In other embodiments, the acidic liquid comprising an acidic rhamnolipid phase is obtained by (i) treating an aqueous fermentation broth comprising at least one rhamnolipid with an acid while heating the aqueous fermentation broth to a temperature in the range of 50°C to 100°C, preferably 50°C to 90°C, to form an acidic medium comprising an upper aqueous liquid phase and a lower rhamnolipid-containing phase comprising at least one liquefied rhamnolipid; and (ii) removing the upper aqueous liquid phase from the acidic medium.

[0009] The resulting purified rhamnolipid phase can be further processed using, for example, solvent extraction techniques or solvent-free extraction techniques to obtain a purified rhamnolipid product.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a schematic diagram showing a countercurrent extraction system for removing impurities from a crude acidulated fermentation broth containing rhamnolipids.

[0011] Figure 2 is a graph illustrating the reduction in antifoam resulting from the water washing process of the present technology.

[0012] Figure 3 is a graph illustrating the increase in rhamnolipid concentration resulting from the water washing extraction process of the present technology.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] While the present technology will be described in connection with one or more preferred embodiments, it will be understood by those skilled in the art that the technology is not limited to only those particular embodiments. To the contrary, the presently described technology includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims.

[0014] As defined herein, a “rhamnolipid” is a glycolipid that has a lipid portion that includes one or more, typically linear, saturated or unsaturated P-hydroxy- carboxylic acid moieties and a saccharide portion of one or more units of rhamnose.

[0015] The saccharide portion and the lipid portion are linked via a p-glycosidic bond between the 1-OH group of a rhamnose moiety of the saccharide portion and the 3-OH group of a p-hydroxy-carboxylic acid of the lipid portion. Thus, the carboxylic acid of one carboxylic acid moiety defines the end of the rhamnolipid. Where more than one rhamnose-moiety is included in a rhamnolipid, each of the rhamnose moieties not linked to the lipid portion is linked to another rhamnosemoiety via a 1,4 p-glycosidic bond. In embodiments where two or more p-hydroxy- carboxylic acids are present in a rhamnolipid, the p-hydroxy-carboxylic acid moieties are selected independently from each other, p-hydroxy carboxylic acid moieties may in some embodiments be identical. In some embodiments, they are different from each other.

[0016] As defined herein, “an aqueous medium” is a composition that comprises at least 5% by weight rhamnolipids or salts of rhamnolipids in water. In a particular embodiment, it comprises the product of a fermentation reaction producing one or more rhamnolipids from at least one carbon source under fermentation conditions known in the art.

[0017] The term “fermentation broth” refers to the composition resulting from fermentation and bacterial growth.

[0018] The terms “liquefied rhamnolipids” and “liquefied or molten rhamnolipids” are used interchangeably and refer to an acidic rhamnolipid-containing phase that is heated to a temperature of about 50°C to about 100°C.

[0019] As used herein, “pH” refers to the value obtained after measuring at ambient temperature (20-25°C) using a calibrated electrode.

[0020] The process of the present technology enables the removal of impurities, such as residual antifoam, salts, and organic impurities from rhamnolipids. The rhamnolipids to be purified may be obtained from a rhamnolipid-producing microorganism as a result of fermentation. The term “rhamnolipid-producing microorganism” refers to any microorganism, such as bacteria, that has the capacityto synthesize / produce rhamnolipids under suitable conditions. Such microorganisms include, but are not limited to, bacteria of the phyla Pseudomonadota, Actinobacteria, Fimicutes, and Proteobacteria. In a particular embodiment, the rhamnolipid-producing microorganism is a bacterium of the Gamaproteobacteria class. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonadales order. In yet a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonadacae family. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonas genus, such as P. alcaligenes, P. aeruginosa, P. chlororaphis, P. clemancea, P. collierea, P. fluorescens, P. luteola, P. putida, P. stutzeri, and P. teessidea. In a particular embodiment, the rhamnolipid-producing microorganism is P. aeruginosa.

[0021] The rhamnolipid-producing microorganism is cultured under fermentation conditions that allow the production of rhamnolipids. Such conditions are known in the art and include providing a culture medium comprising a carbon source, such as vegetable oil, monosaccharides, disaccharides, or glycerol; a nitrogen source, such as ammonium sulfate, ammonium phosphate, urea, or yeast extract; and other nutritional sources such as mineral salts and vitamins.

[0022] After fermentation, the rhamnolipid-containing aqueous fermentation broth is recovered and further processed. In some embodiments, the further processing can include sterilization of the broth. Sterilization methods are known in the art and may be heat-based, chemical-based, ultraviolet light radiation-based, or gamma radiation-based. The heat-based treatment may be via moist heatsterilization, particularly autoclaving. A combination of sterilization methods may also be employed.

[0023] Waste from the fermentation broth may be removed or separated from the broth using separation procedures known in the art. These procedures include, but are not limited to, quiescent settling, batch or continuous centrifugation, or ultracentrifugation, which may be followed by decantation or filtration using procedures known in the art. The waste can be either a solid phase or a liquid phase, depending on where in the process the separation occurs.

[0024] The fermentation broth may be decolorized and / or deodorized. For example, decolorization and / or deodorization may be accomplished via peroxide treatment using hydrogen peroxide or organic (e.g. benzoyl peroxide or peroxyacetic acid) or inorganic peroxide (e.g., lithium peroxide, sodium peroxide, barium peroxide). Alternatively, sodium percarbonate or sodium chlorite could be used for decolorization and / or deodorization of the fermentation broth.

[0025] Further processing of the rhamnolipid-containing aqueous fermentation broth also includes treating the broth with an acid, or with CO2 under pressure, to obtain an acidic medium. Treatment with an acid or CO2 under pressure causes the rhamnolipids to separate out of solution, resulting in an acidic medium comprising a lower dense rhamnolipid-containing phase and an upper aqueous liquid phase. The acid added to the fermentation broth is in an amount sufficient to obtain an acidic medium having a pH in the range of about 1.0 to about 4.0, alternatively about 1 .5 to about 4.0, preferably about 2.0 to about 3.0. The acid can be an organic acid such as acetic acid, or a mineral acid. In some embodiments,the acid is a mineral acid, such as HCI, H2SO4, HNO3, or H3CIO4. The upper aqueous liquid phase is removed from the lower rhamnolipid-containing phase using standard separation techniques. In some embodiments, the remaining rhamnolipid-containing phase is heated to a temperature in the range of about 50 °C to about 100 °C, alternatively about 50 °C to about 90 °C. Heating the rhamnolipids causes a phase change in the rhamnolipids, resulting in a concentrated acidic liquid comprising liquefied rhamnolipids having a lowered viscosity. The acidic liquid may also include suspended solids.

[0026] In an alternative embodiment, heating to a temperature in the range of 50 °C to about 100 °C, alternatively about 50 °C to about 90 °C, can take place during acidulation of the aqueous fermentation broth, thereby creating a dense phase of liquefied or molten rhamnolipids. Suspended solids may also be present in the dense phase comprising the liquefied or molten rhamnolipids. The liquefied or molten rhamnolipids can be separated from the upper aqueous liquid phase using standard liquid / liquid separation techniques or a quiescent settling vessel, resulting in a concentrated acidic liquid comprising an acidic rhamnolipid phase.

[0027] In a further alternative embodiment, after acidulation, instead of separating the aqueous liquid phase from the rhamnolipid-containing phase, an organic solvent, such as ethyl acetate or chloroform, is added to the acidulated fermentation medium and the rhamnolipid-containing phase is extracted into the organic solvent, resulting in an aqueous phase and an acidic rhamnolipid phase. The aqueous phase is removed using standard separation techniques, leaving the acidic rhamnolipid phase in solvent.

[0028] The acidic rhamnolipid phase (either solvent-free or containing a solvent) is washed to remove impurities, including residual antifoam and inorganic salts, using the liquid-liquid extraction process of the present technology. The liquid-liquid extraction process comprises mixing the concentrated acidic liquid comprising the acidic rhamnolipid phase, or the acidic rhamnolipid phase in solvent, with a volume of water while maintaining the pH of the rhamnolipids at an acidic pH. Mixing of this acidic rhamnolipid phase with water, while simultaneously maintaining the pH of the rhamnolipid phase at an acidic pH, such as in the range of 1.0 to 4.0, alternatively 1.5 to 4.0, alternatively 2.0 to 3.0, extracts impurities from the rhamnolipid phase. The liquid-liquid extraction with water to remove impurities can be a batch process or a continuous process. Additional liquid-liquid extractions can be conducted to remove additional impurities from the acidic rhamnolipid phase.

[0029] The liquid-liquid extraction process to remove impurities can be conducted at a temperature in the range of about -5°C to about 100 °C, alternatively about 20 °C (ambient) to about 95 °C. Preferably, if the acidic rhamnolipid phase does not contain a solvent, the liquid-liquid extraction process is conducted at a temperature in the range of about 50°C to about 95 °C, alternatively about 50°C to about 85°C. The elevated temperature facilitates extraction of the impurities and allows enhanced impurity removal and rhamnolipid recovery. When the acidic rhamnolipid phase contains a solvent, the liquid-liquid extraction can be conducted at ambient temperature, or lower or higher than ambient temperature.

[0030] In some embodiments, impurities may be present in the acidic rhamnolipid phase that prevent the rhamnolipids from becoming completelyliquefied or molten after heating. In such cases, conducting one or more liquid-liquid extractions of the rhamnolipid phase with water at an acidic pH can remove sufficient impurities to allow the rhamnolipids to become completely liquefied, which can improve extraction of the impurities.

[0031] The liquid-liquid extraction process to remove impurities can be accomplished in a variety of ways over a wide range of pressures. Typically, the liquid-liquid extraction process is performed at ambient pressure, but higher pressures could also be used. Liquid-liquid extraction involves two basic elements: a mixer followed by a settler. A liquid-liquid extractor will mix a feed stream containing a solute with a solvent stream. The solvent in the solvent stream must be at least partially immiscible in the feed stream so that a phase separation will occur in the settler. The feed stream that exits the settler with a portion of the solute removed is referred to as the raffinate stream and the solvent stream that contains the portion of the removed solute is referred to as the extract stream. One or more mixers and one or more settlers can be used for the liquid-liquid extraction of impurities from the acidic rhamnolipid phase. The one or more mixers and one or more settlers can be arranged in series or in a counter current fashion. The series configuration typically uses more solvent per unit volume of solute removed.

[0032] Any type of mixing equipment that accomplishes intimate mixing of the water with the acidic rhamnolipid phase can qualify as a mixer in the liquid-liquid extraction process. Examples of the one or more mixers include, but are not limited to, pump(s), static mixer(s), agitator(s), moving sieve plate(s), etc. Combinations of different mixers may also be used. In some embodiments, the mixing equipmentmay be a mechanical mixer containing a rotor or impeller, with or without a stator, or an array of rotors and stators. In other embodiments, the mixing equipment may be a motionless or static mixer that allows for the continuous mixing of fluids within a pipeline.

[0033] Preferably, the water is at a pH in the range of 1.0 to 4.0, alternatively 1.5 to 4.0, alternatively 2.0 to 3.0, to aid in maintaining the pH of the rhamnolipids at an acidic pH. It is also beneficial to use heated water to maintain the rhamnolipids in a liquefied or molten state. Suitable temperatures for the water are in the range of about 50 °C to about 95 °C, alternatively about 50 °C to about 90°C, alternatively about 50 °C to about 85°C. The amount of water used for the liquid-liquid extraction is not critical but should be of a volume sufficient to extract impurities from the rhamnolipids. In one embodiment, the amount of acidic water mixed with the acidic liquid comprising the acidic rhamnolipid phase is in a volume ratio of 1 :1 . In other embodiments, the volume of water may be less than or greater than the volume of the acidic liquid comprising the acidic rhamnolipid phase. For example, the volume ratio of acidic water to acidic liquid comprising the acidic rhamnolipid phase could be 1 :4 to 4:1. The time for mixing may be about 15 minutes, although the mixing time could be shorter or longer. After mixing, the water and acidic liquid comprising the acidic rhamnolipid phase are separated in the one or more settlers.

[0034] Any type of settling equipment that allows separation of the acidic liquid comprising the acidic rhamnolipid phase from the water can qualify as a settler in the liquid-liquid extraction process. Examples of the one or more settlers include, but are not limited to, tank(s), column(s) or other volumetric space(s), or acombination thereof, that operate at any pressure and / or under any gravitational force. In some embodiments, the settler can be a static settler that allows separation of the acid liquid comprising the acidic rhamnolipid phase from the water, such as by quiescent settling or hydrocyclone separation. Alternatively, the settler can be a rotational settler, such as a centrifugal separator, or a continuous countercurrent extraction column or continuous countercurrent centrifuge. The rhamnolipid raffinate exiting or removed from the settler is reduced in impurities, while the spent water extract stream contains the removed impurities.

[0035] The liquid-liquid extraction can be repeated one or more times to increase the amount of impurities extracted from the acidic rhamnolipid phase. For example, the liquid-liquid extraction could be repeated one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, or more, if necessary to remove the impurities from the acidic rhamnolipid phase. However, in many cases, 3-4 liquid-liquid extractions will be sufficient to remove a substantial amount of impurities, and additional extractions may not remove an amount of impurities sufficient to justify the increased amount of wastewater generated by the additional extractions. For example, 3-4 liquid-liquid extractions can be sufficient to obtain rhamnolipids having a purity of at least 80% by weight, based on the total dried weight of the solids in the acidic rhamnolipid phase.

[0036] Figure 1 illustrates one way of accomplishing the liquid-liquid extraction process of the present technology. Referring to Figure 1 , a system 10 includes a countercurrent extraction column 16 for the liquid-liquid extraction of impurities from the acidic rhamnolipid phase. The system 10 includes a tank 12 for holding theacidic liquid comprising the acidic rhamnolipids to be purified, and a pump 14 that pumps the acidic liquid comprising the acidic rhamnolipid phase (feed stream) into the top of the countercurrent extraction column 16. The system 10 also includes a tank 20 for holding the acidic water for extracting the impurities from the acidic rhamnolipid phase, and a pump 22 that pumps the acidic water (solvent stream) into the bottom of the countercurrent extraction column 16. The countercurrent extraction column may include one or more stages 18, which enable intimate admixture of the acidic rhamnolipid phase and acidic water in the extraction column. Countercurrent extraction columns are commercially available from a number of different suppliers.

[0037] The amount of acidic water used as the solvent stream for the countercurrent extraction may be equal in volume to the amount of the rhamnolipid feed stream that is pumped into the extraction column. Alternatively, the volume of the acidic water solvent stream may be 0.5x, 1.5x, 2x, 2.5x, 3x, 3.5x or 4x the volume of the rhamnolipid feed stream. Preferably, the pH of the acidic water pumped into the extraction column is in the range of 1.0 to 4.0, alternatively 1.5 to 4.0, alternatively 2.0 to 3.0, to aid in maintaining the pH of the rhamnolipids at an acidic pH. The acidic water may also be heated to a temperature of about 50 °C to about 85 °C and / or the extraction column may be fitted with a heating jacket to ensure that the rhamnolipids remain in a molten or liquefied state during the countercurrent extraction.

[0038] The rhamnolipid feed stream and acidic water solvent are pumped simultaneously into the countercurrent extraction column. As depicted in Figure 1 ,the rhamnolipid feed stream flows through the extraction column from top to bottom while, simultaneously, the acidic water flows through the extraction column from bottom to top. As the respective fluids flow in countercurrent fashion through the extraction column 16, they become intimately mixed, allowing the impurities to be removed from the rhamnolipid feed stream and into the acidified water. The raffinate stream comprising purified rhamnolipids is withdrawn from the bottom of the extraction column, while the extract stream, comprising spent wash water and impurities, is withdrawn from the top of the extraction column.

[0039] One advantage of countercurrent extraction, whether performed in an extraction column or a different countercurrent extraction device, is that it minimizes the use of water for extracting impurities from the rhamnolipids. It may also reduce processing time, since multiple extractions may not be necessary with countercurrent extraction.

[0040] After the liquid-liquid extraction process, the resulting composition comprises from about 40% to about 55% by weight of purified rhamnolipids, and about 35% to about 50% by weight water, with a residual antifoam content of less than 0.5% by weight, preferably less than 0.35% by weight of the resulting rhamnolipid composition. The antifoam content in the resulting composition may range from about 0.01 % to less than 0.5% by weight of the composition, alternatively about 0.02% to about 0.35% by weight of the composition. The purified rhamnolipids have a purity of at least 80% by weight, based on the total weight of dried solids in the rhamnolipid composition. In some embodiments, the purity can be from 80% to about 99% by weight, alternatively 80% to about 95% by weight,alternatively 80% to about 90% by weight, based on the total weight of dried solids in the rhamnolipid composition.

[0041] The rhamnolipid composition may be further processed to provide a final aqueous composition comprising rhamnolipids. For example, the rhamnolipid composition can be neutralized with a base to a pH of about 7 to obtain a viscous solution of rhamnolipids in water. Suitable bases include, but are not limited to, NaOH, KOH, LiOH, or NaHCOs. The rhamnolipid composition can also be further processed by extracting the rhamnolipids into an organic solvent, such as ethyl acetate, followed by solvent stripping, dilution in water, and neutralization with a base to obtain the final aqueous composition comprising rhamnolipids. Since impurities, such as antifoams, have already been removed through the water washing process of the present technology, the rhamnolipids resulting from solvent extraction are substantially reduced in amounts of such impurities. If the liquidliquid extraction is performed on the acidic rhamnolipid phase containing a solvent, the resulting composition can be solvent stripped, diluted in water and neutralized with the base to obtain the final aqueous composition comprising rhamnolipids. If necessary, the final rhamnolipid composition can be treated with activated carbon and / or a polishing filter to remove haze and / or suspended solids or trace particulates.

[0042] Removal of antifoam, salts, and organic impurities through the process of the present technology produces several advantages in the final rhamnolipid composition: (1 ) increased rhamnolipid solution stability over a wide temperature range since the solution does not have impurities that can precipitate out of solution;(2) reduction of precipitation and haze in rhamnolipid formulations used with hard water; (3) increased clarity of neutralized rhamnolipid solutions; and (4) removal of active bleaching agents used for color reduction.EXAMPLES

[0043] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific embodiments of the present technology. By providing these examples, the inventors do not limit the scope and spirit of the present technology.Example 1

[0044] An initial draw (DFO) and first draw (DF1 ) of fermentation broth were withdrawn from culture media from culturing Pseudomonas aeruginosa bacteria (DFO + 1 run). The fermentation broth contained rhamnolipids produced by the P. aeruginosa bacteria, and also residual antifoam resulting from the use of a silicone anti-foam aqueous dispersion during fermentation. The fermentation broth was centrifuged, sterilized, and standard solid / liquid separation techniques were used to separate a clarified rhamnolipid rich solution from bacterial cell matter. An acid was then added to the clarified solution until the pH of the solution was about 2, resulting in an acidic medium. The acidic medium comprises rhamnolipids that have separated out of solution, forming a lower rhamnolipid-containing phase, and an upper aqueous liquid phase. The aqueous liquid phase was separated from the acidic medium using standard separation techniques. The remaining rhamnolipid- containing phase was measured and heated to a temperature of 60°C, forming anacidic dense liquefied rhamnolipid phase, also referred to as Acidulated Concentrated Clarified Broth (“ACCB”). Soft or distilled water was pH-adjusted to pH of 2.5 using dilute sulfuric acid. The acidic water solvent was subsequently measured out to an equal volume of the acidic liquefied rhamnolipid phase, and also heated to a temperature of 60°C. The two mixtures were combined and thoroughly mixed for at least 1 minute. Impurities transfer from the acidic liquefied rhamnolipid phase and into the acidic water solvent. The mixture was allowed to settle for at least 5 minutes while maintaining elevated temperature. The acidic liquefied rhamnolipid phase settles to the bottom while the spent wash water remains as a top layer. The top wash water phase was decanted out, leaving behind a purer acidic liquefied rhamnolipid phase. More spent wash water will exit the system compared to the acidic water charged to the system. Subsequent washes were completed following the same procedure, each using an equal volume ratio of acidic liquefied rhamnolipid phase to fresh heated acidic water. The amount of residual silicone-based antifoam remaining in the acidic liquefied rhamnolipid phase was measured prior to the first water washing, and then after each water wash. A total of ten water washes were used to purify the liquefied rhamnolipid phase. The final acidic liquefied rhamnolipid phase obtained after washing has significantly reduced impurities.Example 2

[0045] Example 1 was repeated except that a third draw (DF3) of fermentation broth was used in the washing procedure, and a different silicone antifoam, provided in neat form rather than as a dispersion, was used during the culturing(DF3 run). The final acidic liquefied rhamnolipid phase obtained after washing has significantly reduced impurities.

[0046] The amount of silicone-based antifoam in the acidic liquefied rhamnolipid phase (ACCB) prior to washing and after each wash for each of the DFO + 1 and DF3 runs is shown in Figure 2. The results in Figure 2 show that the water washing procedure is effective for removing the residual silicone-based antifoam remaining in the acidic liquefied rhamnolipid phase. The antifoam in the DF0+1 run was reduced from 0.36% to 0.035% by weight, and the antifoam in the DF3 run was reduced from 0.434% to 0.318% by weight.

[0047] The concentration of rhamnolipids in the acidic liquefied rhamnolipid phase for the DFO + 1 and DF3 was also measured prior to the first water washing, and then after each water wash. The concentration of rhamnolipids after each wash for the two runs is graphically illustrated in Figure 3. As shown in Figure 3, the water washing procedure substantially increased the purity of the acidic liquefied rhamnolipid phase. The purity of the rhamnolipids from the DF0+1 run increased from 64.4% to 86.2% by weight, based on total weight of dried solids in the DF0+1 run, and the purity of the rhamnolipids from the D3 run increased from 73.3% to 84.3% by weight, based on total weight of dried solids in the D3 run.

[0048] The present technology is now described in such full, clear and concise terms as to enable a person skilled in the art to which it pertains, to practice the same. It is to be understood that the foregoing describes preferred embodiments of the present technology and that modifications may be made therein without departing from the spirit or scope of the present technology as set forth in theappended claims. Further, the examples are provided to not be exhaustive but illustrative of several embodiments that fall within the scope of the claims.

Claims

Claims1 . A process for isolating and purifying rhamnolipids comprising:(a) providing an acidic liquid comprising an acidic rhamnolipid phase comprising at least one rhamnolipid;(b) mixing the acidic rhamnolipid phase with water while maintaining an acidic pH to thereby extract impurities from the acidic rhamnolipid phase into the water; and(c) removing the water from the acidic rhamnolipid phase.

2. The process of claim 1 , wherein the acidic liquid has a pH in the range of 1 .0 to 4.0, preferably 2.0 to 3.0.

3. The process of claim 1 or claim 2, wherein steps (b) and (c) are repeated one or more times to extract additional impurities.

4. The process of any one of claims 1-3, wherein the water is acidulated to a pH in the range of 1 .0 to 4.0, preferably 1 .5 to 4.0.

5. The process of any one of claims 1-4, wherein the water is heated to a temperature in the range of 50°C to 95°C.

6. The process of any one of claims 1-5, wherein the water is mixed with the acidic rhamnolipids in a 1 :1 volume ratio of water to acidic liquid comprising the acidic rhamnolipid phase.

7. The process of any one of claims 1-5, wherein the water is mixed with the acidic rhamnolipid phase in a volume ratio of 4:1 to 1 :4.

8. The process of any one of claims 1-7, wherein mixing of the acidic rhamnolipid phase with water is accomplished using a mechanical mixing device comprising at least one rotor or impeller and, optionally, at least one stator.

9. The process of any one of claims 1-7, wherein mixing of the acidic rhamnolipid phase with water is accomplished using a static mixer.

10. The process of any one of claims 1 -7, wherein mixing of the acidic rhamnolipid phase with water is accomplished using a countercurrent extraction column.11 . The process of any one of claims 1 -9, wherein the water is removed from the acidic rhamnolipid phase by using quiescent settling, hydrocyclone settling, or a centrifugal separator.

12. The process of claim 10, wherein the water is removed from the acidic rhamnolipid phase by using countercurrent extraction.

13. The process of any one of claims 1-12, wherein the impurities extracted from the acidic rhamnolipid phase include residual antifoam.

14. The process of claim 13, wherein the antifoam is a silicon-based antifoam.

15. The process of any one of claims 1 -14, wherein the acidic liquid comprising the acidic rhamnolipid phase is obtained by:(i) treating an aqueous fermentation broth comprising at least one rhamnolipid with an acid to form an acidic medium comprising an upper aqueous liquid phase and a lower rhamnolipid-containing phase comprising the at least one rhamnolipid; and(ii) removing the aqueous liquid phase from the acidic medium to obtain the acidic liquid comprising the acidic rhamnolipid phase.

16. The process of claim 15, wherein after step (i), the acidic liquid comprising the acidic rhamnolipid phase is heated to a temperature in the range of 50°C to 100°C, preferably 50°C to 90°C.

17. The process of claim 15, wherein the aqueous fermentation broth comprising at least one rhamnolipid is heated to a temperature in the range of 50°C to 100°C, preferably 50°C to 90°C, during treatment with the acid in step (i).

18. The process of claim 1 , wherein the acidic liquid comprising the acidic rhamnolipid phase is obtained by:(i) treating an aqueous fermentation broth comprising at least one rhamnolipid with an acid to form an acidic medium;(ii) adding a solvent to the acidic medium to form an upper solvent layer comprising the at least one rhamnolipid and a lower aqueous layer; and(iii) removing the lower aqueous layer from the acidic medium to obtain the acidic liquid comprising the acidic rhamnolipid phase.

19. The process of any one of claims 1 -18, wherein the acidic rhamnolipid phase from step (c) has a purity of at least 80% by weight, based on a total solids dry weight basis.

20. The process of any one of claims 13 or 14, wherein the acidic rhamnolipid phase after step (c) has a residual antifoam content of less than 0.35% by weight of the acidic rhamnolipid phase.

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