Continuous fermentation of glycolipids

Abstract

Continuous fermentation of glycolipids including rhamnolipids, mannosylerythritol lipids, and sophorolipids using a series of tanks under positive pressure in which different stages of fermentation occur in different tanks. The continuous flow through system is compatible with a sufficient period of dwell time in each stage of fermentation.

Description

CONTINUOUS FERMENTATION OF GLYCOLIPIDSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of the earlier filing of U.S. Provisional Patent Application No. 63 / 662,270, filed on June 20, 2024, which is incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to the continuous fermentation of glycolipids.BACKGROUND OF THE DISCLOSURE

[0003] Surfactants are molecules with hydrophobic and hydrophilic moieties and are used to lessen surface and interfacial tension. They are widely employed in a variety of industries as detergents and cleaning agents, emulsifiers, wetting agents, foaming agents, antistatic additives, and dispersants and have a global market valued at 40.5 billion per year with an estimated growth of 3.6% per year (Market Forecast for Global Surfactants, Freedonia Market Research 2021 ). However, most common surfactants are made from petroleum products and many of them bioaccumulate. Given the negative environmental impact of petroleum-based surfactants, there have been attempts to find replacements.

[0004] Microbial biosurfactants are biodegradable and non-toxic. Each microbial biosurfactant has a different biosynthetic pathway impacted by the nutritional environmental conditions provided during the growth stage. Generally, microbial biosurfactants such as glycolipids are secondary metabolites produced by the batch fermentation of microbes such as bacteria, yeast, and filamentous fungi. In batch fermentation, all stages of fermentation take place in a single tank and each batch yields a single harvest.

[0005] Batch fermentation has a number of drawbacks including frequent cleaning, sanitizing, and filing of fermenters. Further, foam production during batch fermentation requires 30% to 50% headspace in the tank, reducing yield. As fermentation systems are generally designed to handle liquids and not foams, heavy foaming may lead to a reduction in product, productivity, and purity. The low product yield and the requirement for sophisticated production processes make glycolipids expensive to produce using current systems (Sarubbo L.A., Da M., Silva G.C., Jose I., Durval B., Gercyane K., Bezerra O., Ribeiro B.G., Silva LA., Twigg M.S., et al. Biosurfactants: Production, properties, applications, trends, and general perspectives. Biochem. Eng. J. 2022;181 :108377).SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides a continuous fermentation process, and devices / vessels and systems for carrying out the process. In some aspects, the disclosure provides a method for manufacturing glycolipids using a plurality of sequentially connected tanks, each tank used for one stage of the fermentation process.

[0007] Exemplary glycolipids that can be produced using the methods described herein include rhamnolipids, mannosylerythritol lipids, and sophorolipids. Rhamnolipids are glycolipid biosurfactants that contain rhamnose as the sugar moiety linked to p-hydroxylated fatty acid chains. Mannosylerythritol lipids (MELs) and sophorolipids are glycolipids produced by a variety of microbes including yeast, bacterial, and fungal strains that exhibit excellent interfacial and biochemical properties. While any strains that produce glycolipids may be used, in exemplary embodiments, the glycolipid is a rhamnolipid (RL) such as 3-[3-[(2R,3R,4R,5R,6S)-4,5-dihydroxy- 6-methyl-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2- yl]oxydecanoyloxy]decanoic acid produced by the Pseudomonas genus, mannosylerythritol lipids (MEL) 4-0-0-d-mannopyranosyl-meso-erythritol lipids) produced using the Ustilago genus, Moesziomyces genus, or the Pseudozyma genus, or a Sopholipids (SL) (such as 17-[2-O-(6-O- Acetyl-beta-D-glucopyranosyl)-6-0-acetyl-beta-D-glucopyranosyloxy]-9-octadecenoic acid) produced using the Candida genus. In some aspects, the microbes (microbe culture) are added to the first tank in a series of tanks a single time while feedstock is added to the first tank continuously, episodically, or periodically. Exemplary species for use with the system described herein for the production of glycolipids include Pseudomonas aeruginosa, Pseudomonas putida, Moesziomyces antarcticus (Pseudozyma antarctica or Candida antarctica), Moesziomyces aphids (Pseudozyma aphids) and Ustilago maydis, though other glycolipid producing Pseudomonas, Ustilago, Candida, Moesziomyces, and Pseudozyma species as would be understood by one or ordinary skill in the art may also be used.

[0008] Feedstock as used herein may include a combination of one or more of water, any generally used nutrient broth such as lysogeny broth or beef broth, one or more inorganic salts (sodium chloride, magnesium sulfate, potassium phosphate, and the like), yeast extract, beef extract, tryptone, proteins (yeast powder, peptone, and the like), and sugars (glucose, maltose and the like). The various proportions of the different components of the feedstock may be altered depending on the particular microbe being used and the needs of the continuous flow fermentation system. In some aspects, the feedstock contains a combination of phosphate buffer, magnesium sulfate (MgSC ), sodium nitrate (NaNOs), yeast extract, calcium chloride (CaCh), and water. In some aspects, the feedstock may additionally include vegetable oil. Vegetable oil asused herein includes vegetable fats that are liquid at room temperature such as soybean oil, grape seed oil, canola oil, olive oil, palm oil, and rice bran oil. The components of the feedstock may be added to a first tank as a single mixture or in the form of one or more of the individual components. In some aspects, the feedstock is sterilized and / or pressurized prior to being added to a first tank of the continuous fermentation system. The various tanks in the continuous fermentation system may be maintained at the same or different temperatures.

[0009] After a sufficient dwell time, a portion of the first tank is moved to a second tank for a second stage of fermentation. After a sufficient dwell time in the second tank, a portion of the second tank is moved to a third tank for a third stage of fermentation though additional tanks may also be used. In some aspects, foam generated by the various stages of the fermentation tank is passed from each tank to the next prior to being collected in an optional fourth tank for additional processing. In some aspects, air is bubbled through each tank to encourage the generation of foam. Foam collected in the optional fourth tank may be processed through an evaporation column to remove water and sterilize the produced glycolipids.

[0010] To the accomplishment of the foregoing and related ends, certain illustrative aspects of the system are described herein in connection with the following description and the attached drawings. The features, functions, and advantages that have been discussed can be achieved independently in various embodiments of the present disclosure or may be combined in yet other embodiments, further details of which can be seen with reference to the following description and drawings. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of any subject matter described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic of a continuous fermentation system according to an embodiment.

[0012] FIG. 2 is a schematic of a continuous fermentation system according to an embodiment.

[0013] FIG. 3 is a schematic of a continuous fermentation system according to an embodiment.

[0014] FIG. 4 is a schematic of a continuous fermentation system according to an embodiment.

[0015] FIG. 5 is a collecting tank for extracting oil-based glycolipids according to an embodiment.

[0016] FIG. 6 is a collecting tank for extracting aqueous glycolipids according to an embodiment.

[0017] FIG. 7 illustrates a process for continuous fermentation according to an embodiment.DETAILED DESCRIPTION

[0018] Various implementations of the present disclosure will be described in detail with reference to the drawings, wherein like reference numerals present like parts and assemblies throughout the several views. Additionally, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible implementations.

[0019] Biosurfactants such as glycolipids have higher biodegradability, lower toxicity, greater thermostability, and higher tolerance in extreme conditions than petroleum-based surfactants and can be produced from renewable substrates. However, they have been challenging to produce on a commercial scale (Varvaresou & lakovou, Biosurfactants in cosmetics and biopharmaceuticals. Lett. Appl. Microbiol. 61 :214-223, 2015. doi: 10.1 111 / lam.12440; Vivek et al., Opportunities and challenges in omics approaches for biosurfactant production and feasibility of site remediation: Strategies and advancements, Environmental Technology & Innovation, 25:102132, 2022 ISSN 2352-1864, doi.org / 10.1016 / j.eti.2O21 .102132).

[0020] Glycolipids are components of cellular membranes with a carbohydrate attached by a glycosidic (covalent) bond. Exemplary glycolipids include rhamnolipids, mannosylerythritol lipids, and sophorolipids. Rhamnolipids are glycolipid biosurfactants that contain rhamnose as the sugar moiety linked to [3-hydroxylated fatty acid chains. Mannosylerythritol lipids (MELs) and sophorolipids are glycolipids produced by a variety of microbes including yeast, bacterial, and fungal strains that exhibit excellent interfacial and biochemical properties. While any strains that produce glycolipids may be used, in exemplary embodiments, the glycolipid is a rhamnolipid (RL) such as 3-[3-[(2R,3R,4R,5R,6S)-4,5-dihydroxy-6-methyl-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6- methyloxan-2-yl]oxyoxan-2-yl]oxydecanoyloxy]decanoic acid produced by the Pseudomonas genus, mannosylerythritol lipids (MEL) 4-O-p-d-mannopyranosyl-meso-erythritol lipids) produced using the Ustilago genus, or a Sopholipids (SL) (such as 17-[2-O-(6-O-Acetyl-beta-D- glucopyranosyl)-6-0-acetyl-beta-D-glucopyranosyloxy]-9-octadecenoic acid) produced using the Candida genus.

[0021] One method of producing glycolipids is fermentation. Fermentation generally has four stages: a lag stage, an exponential growth stage, a stable stage when the key nutrients are already used, and a stationary or production stage. During the lag stage, microbes and feedstock are combined, the microbes acclimate to the environment, and nutrients and oxygen are absorbed. Once the microbes have adjusted, they progress to the exponential growth stage in which the cell count starts to increase exponentially and large amounts of CO2 and foam are produced. Once most of the nutrients have been consumed and waste products begin to buildup, the microbes enter the production stage. During the production stage, the growth of the microbes slows and the production of secondary metabolites such as glycolipids occurs.

[0022] Traditional glycolipid fermentation uses a batch system in which the feedstock and microbes are added to a fermentation tank at the same time and left to ferment for 7-14 days in the single fermentation tank. Once activity in the tank slows or stops, fermentation is viewed as completed and the microbes and the liquid containing the glycolipids are removed and the liquid is filtered to capture the glycolipids. The fermentation tank is then cleaned, and the process is restarted.

[0023] During aerobic fermentation, there is a tendency for the liquid to generate foam. In large scale fermentation, foam may cause overflow, contamination, and dangerous or inefficient use of the reactor, leading to decreased yield. Conventional fermentation systems attempt to reduce foam by increasing back-pressure, decreasing agitation, decreasing aeration, adding antifoam or defoamer agent(s), altering the pH to 7.5-8, or selecting microbes with low foam production. However, many of these measures are not conducive to cell growth and can decrease glycolipid yield. Further, as batch fermentation takes place in a single tank, the different stages cannot be separated, and if there is an issue with one stage, the entire process is contaminated. The cleaning requirements between each batch may also result in delays and interruptions in production.

[0024] Continuous flow fermentation for the production of glycolipids, as described herein, allows each fermentation stage to be carried out in isolation from the other stages. The system as described herein is unidirectional, flowing from one end of the system to the other, further decreasing the possibility of cross-contamination between stages. If there is a problem (such as contamination) in the fermentation being carried out in any one tank in the system, that tank may be isolated and removed, allowing for the other tanks to continue the process while the contaminated tank is cleaned and re-attached.

[0025] In some aspects, the use of the described continuous flow process allows for the optimization of each stage of fermentation, simplifying the condition adjustments such as heat and pH of different stages. In some aspects, the system described herein reduces the risk of contamination as sterile air aerates the tanks, creating a positive pressure in the system and reducing the likelihood of the introduction of substances from outside of the tanks. Embodiments of the system described herein additionally allow for foam to be collected from the various tanks without opening the system, avoiding overflow and increasing production efficiency.

[0026] FIG. 1 depicts a continuous flow glycolipid fermentation system 100 which encourages foam production by bubbling air through the tanks. The various tanks shown in FIG. 1 may be thesame or different sizes depending on the needs of the continuous fermentation system. In some aspects, one or more tanks may include a stirring apparatus. In some aspects, each of the first tank 108, second tank 110, and third tank 112 may be encased in an insulating jacket (not shown), allowing for consistent temperature maintenance at the appropriate temperature for each stage of fermentation. In some aspects, the insulating jacket may additionally provide heating or cooling. The temperature of each of the first tank 108, the second tank 1 10, and third tank 1 12 may be the same or different.

[0027] After an initial setup, the system flows from container 102 to production tank 1 12 as shown by arrow 128. In some aspects, there may be an optional fourth tank for collecting the resulting foam. In batch fermentation, the absolute amount of microbe growth is proportional to the nutrients available. Thus, when the nutrients are consumed, the microbe growth ends. The continuous flow glycolipid fermentation system 100 allows for repeated infusions of feedstock from container 102 to the first tank 108, allowing the microbes in the first tank 108 to continue growing. By continuously, periodically, or episodically moving portions of the contents of each tank to subsequent tank(s), the system allows for multiple stages of fermentation to take place at the same time, creating a continuous fermentation system. Each stage of fermentation may take place over the same, or different periods of time such that the mixture in the first tank 108 is in the tank for a first period of time, the mixture is in the second tank 1 10 for a second period of time, and the mixture is in the third tank 112 for a third period of time. The first, second, and third periods of time are of such a length as needed to complete a single stage of fermentation and may be the same or different periods of time. In some aspects, the first, second, and third periods of time are each two days.

[0028] Feedstock as used herein may include one or more of water, any generally used nutrient broth such as lysogeny broth or beef broth, inorganic salts (sodium chloride, magnesium sulfate, potassium phosphate, and the like), yeast extract, beef extract, tryptone, proteins (yeast powder, peptone, and the like), and sugars (glucose, maltose and the like). The various proportions of the different components of the feedstock may be altered depending on the particular microbe being used and the needs of the continuous flow fermentation system. In some aspects, the feedstock may contain a combination of water, phosphate buffer, magnesium sulfate (MgSC ), sodium nitrate (NaNOs), yeast extract, and calcium chloride (CaCI2). In some aspects, the feedstock may additionally include vegetable oil. Vegetable oil as used herein includes vegetable fats that are liquid at room temperature such as soybean oil, grape seed oil, canola oil, olive oil, palm oil, and rice bran oil.

[0029] In some aspects, the feedstock flows from container 102 through pipe 150 to a pressurization unit 106 prior to being added to the first tank 108 via pipe 144. The pressurization unit may include a pressurized oven to heat the feedstock to the desired temperature. In some aspects, the pressure may be between 10 and 70 psi, including 20 psi, 30 psi, 40 psi, 60 psi, or 70 psi or any fraction thereof. In some aspects, the temperature may be between 100°C and 200°C, for example 105°C, 120°C, 130°C, 150°C,154°C, 180°C, or any fraction thereof. In some aspects, the feedstock may be heated to create superheated water. For example, the feedstock may be subjected to ultra-high temperature processing. Different infusions of feedstock may be heated for different lengths of time depending on the temperature being utilized. For example, when the feedstock is heated at a lower temperature, it may be heated for a longer period of time in an effort to kill any wild microbes. While any appropriate temperature and pressure may be used, in some aspects the feedstock is heated to, for example, 150°C and 50 psi. In some aspects, the sterilized feedstock is then cooled to room temperature before adding it to the first tank 108 via pipe 144.

[0030] The microbes are present or are added to the first tank 108, for example, through inlet 104, and combined with the feedstock to form a first mixture. The first mixture undergoes the lag and growth stage of fermentation in the first tank 108 and is aerated via air inlet 130 to increase foaming. Foam produced during this stage of fermentation is allowed to freely flow through tubing 120 into a second tank 110.

[0031] After a first time period, a portion of the first mixture is pumped via pipel 38 to a second tank 110 where vegetable oil is added from container 103 to form a second mixture. Enough oil is added so that the oil is 1% to 5%, for example 3% or any fraction thereof, of the second mixture. In some aspects, the oil flows through pipe 148 to pressurization unit 106 to be heated prior to being added to the second tank 1 10. While any desired temperature may be used, in some aspects the oil is heated to such a temperature and for a length of time that would result in sterilization of the oil. For example, it may be heated to a temperature between 100°C and 200°C, for example, 105°C, 120°C, 130°C, 150°C,154°C, 180°C, or any fraction thereof prior to adding it to the second tank 110. In some aspects, different types of oil may require different temperatures for different lengths of time in order to be sterilized. For example, fresh oil may be heated to a lower temperature such as 105°C for 45 minutes, whereas recycled oil may be heated to a higher temperature. The oil flows from the pressurization unit 106 via pipe 146 to the second tank 110 to form a second mixture for the stable stage of fermentation.

[0032] After a second period of time, a portion of the second mixture is pumped via pipe 140 into a third tank 112 for the production and collection stage. While varying flow rates from one tank tothe next may be used depending on the microbe and / or stage of fermentation, in some aspects the feedstock / microbe mixture is pumped at a rate of 10L / minute. Such a rate may be adjusted by calculating the rate at which 1 / 3 of the tank would be transferred from the first tank 108 to the second tank 110 or the second tank 110 to the third tank 112 over twenty minutes. In some aspects, the mixture may be pumped at a rate of 4.2% per hour. The mixture may be pumped from one tank to the next continuously, episodically, or periodically. In some aspects, the mixture is pumped once per day, twice per day, every other day, every three days, or weekly. In some aspects, air may be introduced into each tank via air inlet 130, air inlet 132, and air inlet 134, respectively. Sterile air added through air inlet 130, air inlet 132, and air inlet 134 respectively aerates the continuous fermentation, increases foaming, and maintains a positive pressure in the system. In some aspects, 30% of the total volume of a tank is air, with sufficient air added to the tank to maintain the volume during the continuous fermentation process.

[0033] After a third period of time, liquid and foam in the third tank 112, which contains the glycolipids, may be run through a condenser (not shown), for example, an evaporation column such as a bubble evaporation column, one or more times to evaporate water and sterilize the foam. In some aspects, the condenser is heated, sterilizing the foam / liquid. In some aspects, the liquid containing the glycolipid is run through the condenser until about 40%, 50%, 60%, 70%, 80% of the water is removed. In some aspects, the liquid is run through the condenser until the solution is clear and odor free. Running the foam / liquid through the condenser increases the concentration of the glycolipids in the foam / liquid at least two fold, three fold, four fold or more from the starting material.

[0034] The continuous glycolipid fermentation system 200 as shown in FIG. 2 includes a sterilization unit 205 and a separate collecting tank 214 including an air inlet 236. The continuous glycolipid fermentation system 200 further includes a feedstock container 202 similar to container 102, an oil container 203 similar to container 103, a pressurization unit 206 similar to pressurization unit 106, a first tank 208 similar to first tank 108, a second tank 210 similar to second tank 110, a third tank 212 similar to third tank 1 12, an additional storage tank 216 and various pipes and pumps such as pipe 248 similar to pipe 148, pipe 244 similar to pipe 144, pipe 246 similar to pipe 146, tubing 220 similar to tubing 120, tubing 222 similar to tubing 122, pipe 238 similar to pipe 138, pipe 240 similar to pipe 140, air inlet 230 similar to air inlet 130, air inlet 232 similar to air inlet 132, and air inlet 234 similar to air inlet 134.

[0035] Similarly to FIG. 1 , feedstock is introduced into the continuous glycolipid fermentation system 200 from container 202. The various stages of fermentation progress from the first tank 208 through to the storage tank 216 as shown by arrow 228. In the continuous fermentationsystem shown in FIG. 2, the feedstock may be sterilized prior to introduction into the first tank 208. For example, the feedstock may flow through a pipe 250 or other connection mechanism to a sterilization unit 205.

[0036] Sterilization unit 205 may provide any type of sterilization including moist heat sterilization, dry heat sterilization, gas sterilization, or various types of radiation, including ultraviolet radiation, infrared radiation, x-rays, gamma radiation, and high-velocity electrons. In some aspects, the sterilization method uses ultraviolet radiation. After sterilization, the sterilized feedstock may flow through pipe 242 to a pressurization unit 206. The pressurization unit may include a pressurized oven to heat the feedstock to the desired temperature. In some aspects, the feedstock may be pressurized to between 10 and 70 psi, including 20 psi, 30 psi, 40 psi, 60 psi, 70 psi, or any fraction thereof. In some aspects, the feedstock may be heated to between 100°C and 200°C, for example 105°C, 120°C, 130°C, 150°C,154°C, 180°C, or any fraction thereof. In some aspects, the feedstock may be heated to create superheated water. For example, the feedstock may be subjected to ultra-high-temperature processing. The feedstock may be heated for different lengths of time depending on the temperature being utilized. For example, when the feedstock is heated at a lower temperature, it may be heated for a longer period of time in an effort to kill any wild microbes. While any appropriate temperature and pressure may be used, in some aspects the feedstock is heated to 150°C and 50 psi. In some aspects, the sterilized feedstock is then cooled to room temperature before adding it to the first tank 208 via pipe 244.

[0037] The microbes of interest may be added to the first tank 208 to be combined with the feedstock via an inlet such as inlet 204 to form a first mixture. While the desired microbes may be added to the first tank 208 multiple times, in some aspects, the desired microbes are only added a single time during the initial setup. While any desired cell concentration may be used, in some aspects, the cell concentration is 1012- 1013cells per liter. The continuous, episodic, or periodic inflow of feedstock from container 202 through pipe 250 to pressurization unit 206 and from the pressurization unit 206 through pipe 244 to first tank 208 allows the microbes to continue growing without subsequent microbe inoculations, allowing for continuous fermentation. In some aspects, the first mixture in the first tank may be maintained at a temperature of 34°C to 37°C and stirred continuously using a mechanical stirrer. The mixture may be stirred at a rate of 50 to 1000 rpm or any fraction therefore, for example, 300 to 500 rpm.

[0038] After a first period of time, when sufficient growth and volume have been achieved, a portion of the first mixture is pumped from the first tank 208 to the second tank 210 via pipe 238. The mixture(s) may be pumped from one tank to the next continuously, episodically, or periodically. In some aspects, the mixture is pumped once per day, twice per day, every otherday, every three days, or weekly. In some aspects, the flow rate may be calculated as a fraction of the total fluid volume used and the fermentation time for each tank. For example, the total fluid volume may be divided by the fermentation time, the number of hours in the day, and the number of minutes in an hour to calculate the injection rate per minute. In some instances, the total fluid volume may be divided by seven days, with two days spent in each of the growth stage, the stable stage, and the production stage and the last day spent in the collecting tank, though the time spent in each tank may be shorter or longer. The resulting flow rate would be, for example (272 liters / 7 days)(1000mL / liter)(1 day / 24 hours)(1 hour / 60 minutes) or 27mL / min, though other flow rates may also be used. In some aspects, the flow rate may be such that 4.2% of a tank is pumped per hour. The transfer rate between first tank 208, second tank 210, and third tank 212 may be the same or different. In some aspects, foaming may be encouraged by aeration via air inlet 230 and any foam that is generated during the growth stage is moved to the second tank 210 via tubing 220.

[0039] The transferred first mixture then enters the stable stage in the second tank 210. In some aspects, the contents of the second tank 210 may be maintained at a lower temperature than the contents of the first tank 208. For example, the second tank may be maintained at a temperature of 30°C-34°C. Vegetable oil is added to the second tank 210 from container 203 to the first mixture to form a second mixture. Vegetable oil as used herein includes one or more vegetable fats that are liquid at room temperature such as soybean oil grape seed oil, canola oil, olive oil, palm oil, and rice bran oil. Enough oil is added so that the oil is 1 % to 5%, for example, 3% or any fraction thereof, of the second mixture. In some aspects, the oil flows from oil container 203 through pipe 248 to pressurization unit 206 to be heated. While any desired heat may be used, in some aspects the oil is heated to 150°C prior to adding it to the second tank 210. The heated oil then flows from the pressurization unit 206 via pipe 246 to the second tank 210.

[0040] In the second tank 210, the second mixture enters the stable stage of fermentation. In the stable stage, most of the nutrients from the feedstock have been consumed and secondary metabolites have been converted. Foam created in the second stage is allowed to flow into the third tank 212 via tubing 222. After a second period of time, the mixture from the stable stage is pumped into the third tank 1 12 where it enters the production stage. The third tank 212 may be maintained at a temperature between 30°C-34°C depending on the microbe. In some aspects, it is maintained at 30°C.

[0041] After a third period of time, the foam from the fermentation is collected in the collecting tank 214 via tubing 224. As the system provides continuous fermentation, in some aspects, the final stage of fermentation may not be completed in the third tank 212, and additional fermentationmay take place in the collecting tank 214. The foam in the collecting tank 214, which contains the glycolipids, may be run through a condenser such as condenser 226, for example, an evaporation column such as a bubble evaporation column, one or more times to evaporate water and sterilize the foam. In some aspects, the condenser 226 is heated, sterilizing the foam / liquid. In some aspects, the liquid containing the glycolipid is run through the condenser until about 40%, 50%, 60%, 70%, 80% of the water is removed. In some aspects, the liquid is run through the condenser until the solution is clear and odor free.

[0042] FIG. 3 depicts a continuous glycolipid fermentation system 300 with a plurality of containers for feedstock sources as shown by container 302a, container 302b, container 302c, and container 302d. Continuous glycolipid fermentation system 300 includes an oil container 303 similar to oil container 103, a pressurization unit 306 similar to pressurization unit 106, an inlet 304 for microbe deposition similar to inlet 104, a first tank 308 similar to first tank 108, a second tank 310 similar to second tank 110, a third tank 312 similar to third tank 112, a collection tank 314 similar to collection tank 214, a storage tank 316 similar to storage tank 216, a pipe 338 similar to pipe 238, a pipe 340 similar to pipe 240, tubing 320 similar to tubing 120, tubing 322 similar to tubing 122, tubing 324 similar to tubing 224, air inlet 330 similar to air inlet 130, air inlet 332 similar to air inlet 132, air inlet 334 similar to air inlet 134, air inlet 336 similar to air inlet 236.

[0043] As shown in FIG. 3, elements of feedstock are added from one or more containers such as container 302a, container 302b, container 302c, and container 302d. Each container may include one or more components of the feedstock. For example, container 302a may contain phosphate buffer, container 302b may contain magnesium sulfate, and container 302c may contain yeast extract, sodium nitrate and calcium chloride, though other combinations and components of feedstock may also be used including water, lysogeny broth, inorganic salts, beef extract, tryptone, proteins, sugars and the like. In some aspects, each of container 302a, container 302b, and container 302c may also contain water. In some aspects, container 302d may contain vegetable oil which is combined with the feedstock prior to pressurization and sterilization, that is, the oil from 302d is pumped through pipe 344 into first tank 308 and additional oil is added from container 303 through pipe 346 to second tank 310. The proportions of the feedstock components may be adjusted depending on the needs of the microbes. In an exemplary embodiment, the combined fermentation components may be used to form a fermentation medium containing vegetable oil 1 -8%, NaNO30.6%, K2HPO40.34%, KH2PO40.34%, CaCI20.012%, MgSO40.024%, and yeast extract 0.12% with the remainder water. In some aspects, fermentation conditions such as pH and temperature may also be adjusted to optimize glycolipid production.

[0044] As shown in FIG. 3, in some aspects, the feedstock may be heated and pressurized in pressurization unit 306 before being introduced via pipe 344 into a first tank 308. The pressurization unit may include a pressurized oven to heat the feedstock to the desired temperature. In some aspects, the pressure may be between 10 and 70 psi, including 20 psi, 30 psi, 40 psi, 60 psi, or 70 psi or any fraction thereof. In some aspects, the temperature may be between 100°C and 200°C, for example 105°C, 120°C, 130°C, 150°C, 154°C, 180°C, or any fraction thereof. In some aspects, the feedstock may be heated to create superheated water. For example, the feedstock may be subjected to ultra-high temperature processing. While any appropriate temperature and pressure may be used, in some aspects, the feedstock is heated to 150°C and 50 psi. The feedstock may be heated for different lengths of time depending on the temperature being utilized. For example, when the feedstock is heated at a lower temperature, it may be heated for a longer period of time in an effort to kill any wild microbes. In some aspects, the sterilized feedstock may be cooled to room temperature before adding it to the first tank 308 via pipe 344. While not shown, continuous glycolipid fermentation system 300 may additionally include a sterilization unit similar to sterilization unit 205.

[0045] During the initial set up, microbes may be added to the first tank 308 through inlet 304. While any desired cell concentration may be used, in some aspects, the cell concentration is 1012- 1013cells per liter. The mixture in the first tank 308 may be maintained at a temperature of 34°C to 37°C and stirred continuously using a mechanical stirrer. The mixture may be stirred at a rate of 50 to 1000 rpm or any fraction therefore, for example, 300 to 500 rpm. The first tank 308 is used for the growth stage of fermentation. During the growth stage, the microbes grow exponentially. In continuous fermentation, additional feedstock may be added continuously, episodically, or periodically via pipe 344 to the first tank 308 at a rate such that the nutrients in 308 are never depleted, allowing the microbes to continue to grow and avoiding nutrient depletion in the first tank 308.

[0046] After the initial set up, the contents of each tank may be transferred to a subsequent tank continuously, episodically, or periodically. In some aspects, the transfer occurs at a rate that allows sufficient dwell time in each tank for each stage of the fermentation process. For example, the dwell time in the first tank may be about two days. After two days a portion of the contents of the first tank 308 may flow to the second tank 310 and more feedstock may be added to the first tank 308, allowing for continuous growth of the remaining microbes in the first tank 308. Thus, different portions of each tank may have been dwelling for different amounts of time. In some aspects, each portion of the mixture may dwell in the first, second, and third tanks for two days in each tank. In some aspects 2% to 6%, for example, 4.17%, of the mixture is pumped to the nexttank every hour. In other aspects, a portion of each tank may be pumped into the next tank continuously, episodically, or periodically. Oil may be added to the second tank 310 via pipe 346. In some aspects, the oil from container 303 may be heated by pressurization unit 306 prior to being added to the second tank 310. In some aspects, the contents of the second tank 310 may be maintained at a lower temperature than the contents of the first tank 308. For example, the second tank may be maintained at a temperature of 30-34°C. After a second period of time, a portion of the resulting liquid is pumped through pipe 340 into the third tank 312 with any foam produced during the secondary stage flowing from the second tank 310 to the third tank 312 via tubing 322.

[0047] In the third tank 312, the mixture may be allowed to settle for up to fourteen days while the foam progresses to collection tank 314 via tubing 324. The foam in the collection tank 314 is condensed and sterilized through the evaporation column 326 and the foam, which contains the bulk of the glycolipids, is collected in the storage tank 328. While any size or relative proportion of tanks may be used, in some aspects, the third tank 312 contains the largest volume.

[0048] In some aspects, as shown in FIG. 4, there may be additional tanks for the various stages of fermentation. For example, the stable stage may take place in second tank 410 and third tank 411 and the production stage may take place in fourth tank 412 and fifth tank 413. The use of additional tanks increases the amount of glycolipids that may be produced and allows for additional manipulation of the dwell time and conditions for each stage of the fermentation process.

[0049] The continuous glycolipid fermentation system 400 of FIG. 4 depicts a flow from feedstock container 402a, feedstock container 402b, feedstock container 402c, and feedstock container 402d to a storage tank 416 as shown by arrow 428. The system 400 includes a sterilization unit 405 similar to sterilization unit 205, an oil container 403 similar to container 103, a pressurization unit 406 similar to pressurization unit 106, a first tank 408 similar to first tank 108, a second tank 410 similar to second tank 1 10, a fourth tank 412 similar to third tank 112, a collection tank 414 similar to collection tank 214, with and various pipes, and pumps such as pipe 448 similar to pipe 148, pipe 444 similar to pipe 144, pipe 446 similar to pipe 146, tubing 420 similar to tubing 120, tubing 422 similar to tubing 122, pipe 438 similar to pipe 138, pipe 440 similar to pipe 140, air inlet 430 similar to air inlet 230, air inlet 432 similar to air inlet 232, air inlet 434 similar to air inlet 234, and air inlet 436 similar to air inlet 236. The additional tanks 41 1 and 413 may have air inlets 433 and 435 respectively. Second tank 410 is connected to tank 411 via pipe 439 and fourth tank 412 is connected to fifth tank 413 via pipe 441 .

[0050] As shown in FIG. 4, elements of feedstock are added from one or more containers such as container 402a, container 402b, container 402c, and optionally 402d via a plurality of pipes 450. Each container may include one or more components of the feedstock. For example, container 402a may contain phosphate buffer, container 402b may contain magnesium sulfate, and container 402c may contain yeast extract, sodium nitrate and calcium chloride though other combinations and components of feedstock may also be used including lysogeny broth, inorganic salts, beef extract, tryptone, proteins, sugars and the like. In some aspects, container 402a, container 402b, container 402c may also contain water. In some aspects, container 402d may contain vegetable oil which is combined with the feedstock prior to pressurization and sterilization, that is, the oil from 402d flows through pipe 444 and additional oil is added from container 403 through pipe 446 to second tank 410.

[0051] The proportions of the feedstock components, the pH, and the temperature may be adjusted depending on the needs of the microbes. In an exemplary embodiment, the feedstock may contain vegetable oil 1 -8%, NaNOs 0.6%, K2HPO40.34%, KH2PC>40.34%, CaCI20.012%, MgSC 0.024%, and yeast extract 0.12% with the remainder of the feedstock as water. In some aspects, the feedstock may be sterilized prior to introduction into the continuous glycolipid fermentation system. For example, the feedstock may flow through a pipe 440 or other connection mechanism to a sterilization unit 405. The flow rate may be calculated as a fraction of the total fluid volume used and the fermentation time for each tank. For example, the total fluid volume may be divided by the fermentation time, the number of hours in the day and the number of minutes in an hour to calculate the injection rate per minute. In some instances, the total fluid volume may be divided by seven days, with two days spent in each of the growth stage, the stable stage, and the production stage and an additional day spent in the collection tank, though the amount of time spent in each tank may be shorter or longer. The resulting flow rate would be, for example, (272 liters / 7 days)(1000mL / liter)(1 day / 24 hours)(1 hour / 60 minutes) or about 27 mL / min, though other flow rates may also be used. In some aspects, once the system is up and running, that is, there is a mixture in each tank, 4.2% of each tank may be transferred per hour. Sterilization unit 405 may provide any type of sterilization including moist heat sterilization, dry heat sterilization, gas sterilization or various types of radiation including ultraviolet radiation, infrared radiation, x-rays, gamma radiation and high-velocity electrons. In some aspects, the sterilization method uses ultraviolet radiation.

[0052] After sterilization, the feedstock may flow through pipe 442 to a pressurization unit 406. The pressurization unit may include a pressurized oven to heat the feedstock to the desired temperature. In some aspects, the temperature may be between 100°C and 200°C, for example105°C, 120°C, 130°C, 150°C,154°C, 180°C, or any fraction thereof. In some aspects, the feedstock may be heated to create superheated water. For example, the feedstock may be subjected to ultra-high temperature processing. While any appropriate temperature and pressure may be used, in some aspects, the feedstock is heated to 150°C and 50 psi. The feedstock may be heated for different lengths of time depending on the temperature being utilized. For example, when the feedstock is heated at a lower temperature, it may be heated for a longer period of time in an effort to kill any wild microbes. In some aspects, the sterilized feedstock is then cooled to room temperature before adding it to the first tank 408 via pipe 444.

[0053] The desired microbes may be added to the feedstock in the first tank 408 via an inlet such as inlet 404 to form a first mixture. While any desired cell concentration may be used, in some aspects, the cell concentration is 1012- 1013cells per liter. The desired microbes may be added to the first tank 408 one or more times as needed to keep the system running. In some aspects, the continued inflow of feedstock from containers 402a, 402b, 402c, and 402d allows the microbes to continue growing in the continuous fermentation process without subsequent microbe inoculations.

[0054] During the initial set up, the microbes in the first mixture may enter an initial lag stage in first tank 408 in which the microbes acclimatize to the environment. In some aspects, the lag stage is carried out at a higher temperature than the rest of the fermentation. For example, the first mixture in the first tank may be maintained at a temperature of 34°C to 37°C and stirred continuously using a mechanical stirrer. The mixture may be stirred at a rate of 50 to 1000 rpm or any fraction thereof, for example 300 to 500 rpm.

[0055] After the lag stage of the initial setup and during the continuous fermentation, the microbes enter the exponential growth stage. The exponential growth stage generally takes 1 -2 days. In a standard batch fermentation system, once the microbes have consumed the available nutrients and oxygen levels have decreased, the growth stage ends. In the continuous flow system described herein, feedstock ingredients are added continuously, episodically, or periodically from containers 402a, 402b, 402c, and optionally 402d allowing the microbes to continue growing exponentially in the first tank 408.

[0056] After sufficient growth and volume has been achieved, a portion of the first mixture is pumped from the first tank 408 to the second tank 410 via pipe 438. This rate of transfer continues during the continuous fermentation. A portion of the first mixture may be pumped from one tank to the next continuously, episodically, or periodically. In some aspects, a portion of the mixture is pumped once per day, twice per day, every other day, every three days, or weekly. While varying flow rates may be used depending on the microbe, in some aspects, the feedstock / microbemixture is pumped at a rate of 10L / minute. Such a rate may be adjusted by calculating the rate at which 1 / 3 of the tank would be transferred from the first tank 408 to the second tank 410, the second tank 410 to the third tank 411 , from the third tank 41 1 to the fourth tank 412, and the fourth tank 412 to the fifth tank 413 over twenty minutes. In some aspects, 3% of the total volume of the tank may be transferred per minute. In other aspects, 4.2% of a tank may be transferred per hour. The transfer rate between the tanks may be the same or different. Any foam that is generated during the growth stage is moved to the second tank 410 via tubing 420.

[0057] The transferred first mixture then enters the stable stage in second tank 410. In some aspects, the contents of the second tank 410 may be maintained at a lower temperature than the contents of the first tank 408. For example, the second tank may be maintained at a temperature of 30°C-34°C. In the second tank 410, vegetable oil is added from oil container 403 to the first mixture to form a second mixture. Vegetable oil as used herein includes vegetable fats that are liquid at room temperature such as soybean oil grape seed oil, canola oil, olive oil, palm oil, and rice bran oil. Enough oil is added so that the oil is 1% to 5%, for example 3% or any fraction thereof, of the second mixture. In some aspects, the oil flows through pipe 448 to pressurization unit 406. While any desired heat may be used, in some aspects the oil is heated to 150°C prior to adding it to the second tank 410. to such a temperature and for a length of time that would result in sterilization. For example, it may be heated to a temperature between 100°C and 200°C, for example 105°C, 120°C, 130°C, 150°C,154°C, 180°C, or any fraction thereof prior to adding it to the second tank 410. In some aspects, different types of oil may require different temperatures for different lengths of time. For example, fresh oil would be heated to a lower temperature such as 105°C for 45 minutes, whereas recycled oil would be heated to a higher temperature. The oil flows from the pressurization unit 406 via pipe 446 to the second tank 410.

[0058] In the second tank 410, the first mixture enters the stable stage of fermentation. In the stable stage, most of the nutrients from the feedstock have been consumed and secondary metabolites have been converted. In some aspects, the stable stage may take place in second tank 410 and third tank 41 1 with excess foam being transferred via tubing 422 and tubing 423 respectively. Primary rhamnolipid production may occur during the stable or stationary stage. The mixture from the stable stage is then pumped into the fourth tank 412 where it enters the production stage. The fourth tank 412 may be maintained at a temperature between 30°C-34°C depending on the microbe. In some aspects, it is maintained at 30°C. In some aspects, the production stage may take place in two tanks, for example, fourth tank 412 and fifth tank 413 with excess foam flowing through tubing 425.

[0059] After the production stage, the foam is collected in the collection tank 414 via tubing 424. As the system provides continuous fermentation, in some aspects, the final stage of fermentation may not be completed in the fifth tank 413, and additional fermentation may take place in the collecting tank 414. The liquid in the collecting tank 414, which contains the glycolipids, may be run through a condenser such as condenser 426, for example, an evaporation column, one or more times to evaporate water from the liquid. In some aspects, the condenser 426 is heated, sterilizing the foam / liquid before depositing the concentrated liquid in the storage tank 416. In some aspects, the liquid containing the glycolipid is run through the condenser until about 40%, 50%, 60%, 70%, 80% of the water is removed. In some aspects, the liquid is run through the condenser until the solution is clear and odor free.

[0060] Glycolipids produced in the continuous fermentation system may be water soluble or oil soluble. As shown in FIG. 5, in the tank 514 similar to collection tank 214 or collection tank 314, the mixture including foam is input through tubing 524 similar to tubing 424 and settles into a series of layers with oil-based glycolipids such as MEL-A, B, C and SL settling to the bottom layer 530, a microbe layer 532 forming on top of the glycolipid layer, an aqueous phase 534 forming on top of the glycolipid layer, and residual oil separating to the top at 536. If the glycolipid is waterbased such as MEL-D and RL, microbes will settle in a bottom layer 632 with an aqueous layer 634 on top of the microbe and any residual oil 636 floating on top of the aqueous layer 634 in a tank 614, similar to collection tank 314, as shown in FIG. 6 which has received the foam and / or liquid through tubing 624, similar to tubing 224.

[0061] FIG. 7 depicts a process for continuous fermentation with feedstock in one or more containers being formed at 702. The feedstock may be optionally sterilized at 704, using for example, UV sterilization, prior to being heated and pressurized at 706. Once the heated and pressurized feedstock has been allowed to cool, the feedstock is transferred to a first tank at 708. The system loops, creating and adding more feedstock to the first tank continuously, episodically, or periodically. Microbes are added to the first tank at 710 and combined with the feedstock. After a first period of time, a portion of the microbe / feedstock mixture is transferred to a second tank at 712. Vegetable oil is heated at 714 and then transferred to the second tank and mixed with the microbe / feedstock mixture at 716. After a second period of time, a portion of the contents of the second tank is pumped to the third tank at 718. After a third period of time foam and liquid from the third tank is processed through an evaporation column at 722. The glycolipids are extracted from the foam / liquids for further processing at 724.

[0062] The Exemplary Embodiments and Example(s) below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize inlight of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Exemplary Embodiments.

[0063] 1 . A method of continuously manufacturing glycolipids including: adding a feedstock to a first tank; adding a quantity of a microbe culture to the feedstock in the first tank to form a first mixture for a first stage of fermentation; stirring the first mixture for a first time period; allowing foam generated in the first tank from the first stage of fermentation to flow to a second tank; pumping a portion of the first mixture to a second tank and combining the portion of the first mixture with an amount of vegetable oil to form a second mixture, stirring the second mixture for a second time period for a second stage of fermentation, wherein foam generated in the second tank from the second stage of fermentation flows from the second tank to a third tank; pumping a portion of the second mixture from the second tank to a third tank for a third stage of fermentation; bubbling the second mixture in the third tank into a condenser; and collecting sterilized foam from the condenser.

[0064] 2. The method of embodiment 1 , wherein the amount of vegetable oil introduced into the second tank is between 1% to 3% of a total volume of the first mixture.

[0065] 3. The method of embodiment 1 or 2, further including maintaining the first mixture of the first tank at a temperature that is greater than a temperature of the second mixture of the second tank.

[0066] 4. The method of any of embodiments 1 to 3, wherein the first mixture is maintained at a temperature of 34° to 37 °C.

[0067] 5. The method of any of embodiments 1 to 4, wherein the second mixture maintained at a temperature of 30° to 34 °C.

[0068] 6. The method of any of embodiment 1 to 5, wherein the feedstock is pressurized prior to being added to the first tank.

[0069] 7. The method of embodiment 6, wherein the feedstock is sterilized prior to being pressurized.

[0070] 8. The method of embodiment 7, wherein the sterilization is ultraviolet sterilization.

[0071] 9. The method of any of embodiments 1 to 8, wherein the feedstock is added to the first tank a plurality of times.

[0072] 10. The method of any of embodiments 1 to 9, wherein the first mixture is pumped at a rate of 4.2% of total volume of the first tank per hour.

[0073] 11 . The method of any of embodiments 1 to 10, wherein the condenser concentrates at least one glycolipid.

[0074] 12. The method of embodiment 11 , wherein the glycolipid is an oil-based glycolipid.

[0075] 13. The method of embodiment 1 1 , wherein the glycolipid is a water-based glycolipid.

[0076] 14. The method of embodiment 11 , wherein the glycolipid is a rhamnolipid (RL), a mannosylerythritol lipid (MEL), or a sophorolipid (SL).

[0077] 15. The method of any of embodiments 1 to 14, wherein the condenser includes an evaporation column.

[0078] 16. The method of embodiment 15, wherein the evaporation column includes a bubble column evaporator.

[0079] 17. The method of any of embodiments 1 to 16, wherein the microbe culture is Pseudomonas, Ustilago, or Candida.

[0080] 18. The method of embodiment 17, wherein the microbe culture is selected from Pseudomonas aeruginosa, Pseudomonas putida, Moesziomyces antarcticus, Moesziomyces aphids, and Ustilago maydis.

[0081] 19. A system for continuously manufacturing glycolipids through microbial fermentation, the system including: a first tank, wherein the first tank contains a first mixture including microbes and feedstock; a second tank operably connected to the first tank at at least two points; a third tank operably connected to the second tank at at least two points; a fourth tank, wherein a top of the third tank is operably connected to a bottom of the fourth tank; and a condenser, wherein the condenser connects the third tank to a storage tank.

[0082] 20. The system of embodiment 19, wherein the condenser is an evaporation column.

[0083] 21 . The system of embodiment 20, wherein the evaporation column is a bubble column evaporator.

[0084] 22. Use of the system of any of embodiments 19 to 21 to produce a glycolipid preparation.

[0085] 23. The system of claim 22, wherein the microbes are selected from Pseudomonas, Ustilago, Moesziomyces, or Candida.

[0086] 24. The system of claim 23, wherein the microbes are selected from Pseudomonas aeruginosa, Pseudomonas putida, Moesziomyces antarcticus, Moesziomyces aphids, and Ustilago maydis.

[0087] 25. The system of claim 23, wherein the glycolipid is an oil-based glycolipid.

[0088] 26. The system of claim 23, wherein the glycolipid is a water-based glycolipid.

[0089] 27. The system of claim 23, wherein the glycolipid is a rhamnolipid (RL), a mannosylerythritol lipid (MEL), or a sophorolipid (SL).Closing Paragraphs

[0090] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the production of glycolipids.

[0091] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0092] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in their respective testing measurements.

[0093] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0094] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0095] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0096] Furthermore, numerous references have been made to patents, printed publications, journal articles, other written text, and web site content throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching(s), as of the filing date of the firstapplication in the priority chain in which the specific reference was included. For instance, with regard to chemical compounds, nucleic acid, and amino acids sequences referenced herein that are available in a public database, the information in the database entry is incorporated herein by reference as of the date of an application in the priority chain in which the database identifier for that compound or sequence was first included in the text.

[0097] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0098] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0099] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11 th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8thEdition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

LISTING OF CLAIMSWhat is claimed is:1 . A method of continuously manufacturing glycolipids comprising: adding a feedstock to a first tank; adding a quantity of a microbe culture to the feedstock in the first tank to form a first mixture for a first stage of fermentation; stirring the first mixture for a first time period; allowing foam generated in the first tank from the first stage of fermentation to flow to a second tank; pumping a portion of the first mixture to a second tank and combining the portion of the first mixture with an amount of vegetable oil to form a second mixture, stirring the second mixture for a second time period for a second stage of fermentation, wherein foam generated in the second tank from the second stage of fermentation flows from the second tank to a third tank; pumping a portion of the second mixture from the second tank to a third tank for a third stage of fermentation; bubbling the second mixture in the third tank into a condenser; and collecting sterilized foam from the condenser.

2. The method of claim 1 , wherein the amount of vegetable oil introduced into the second tank is between 1 % to 3% of a total volume of the first mixture.

3. The method of claim 1 , further comprising maintaining the first mixture of the first tank at a temperature that is greater than a temperature of the second mixture of the second tank.

4. The method of claim 3, wherein the first mixture is maintained at a temperature of 34°C to 37°C.

5. The method of claim 3, wherein the second mixture maintained at a temperature of 30°C to 34 °C.

6. The method of claim 1 , wherein the feedstock is pressurized prior to being added to the first tank.

7. The method of claim 6, wherein the feedstock is sterilized prior to being pressurized.

8. The method of claim 7, wherein the sterilization is ultraviolet sterilization.

9. The method of claim 3, wherein the feedstock is added to the first tank a plurality of times.

10. The method of claim 9, wherein the first mixture is pumped at a rate of 4.2% of total volume of the first tank per hour.11 . The method of claim 1 , wherein the condenser concentrates at least one glycolipid.

12. The method of claim 1 1 , wherein the glycolipid is an oil-based glycolipid.

13. The method of claim 1 1 , wherein the glycolipid is a water-based glycolipid.

14. The method of claim 11 , wherein the glycolipid is a rhamnolipid (RL), a mannosylerythritol lipid (MEL), or a sophorolipid (SL).

15. The method of claim 1 1 , wherein the condenser comprises an evaporation column.

16. The method of claim 15, wherein the evaporation column comprises a bubble column evaporator.

17. The method of claim 1 , wherein the microbe culture is Pseudomonas, Ustilago, Moesziomyces, or Candida.

18. The method of claim 17, wherein the microbe culture is selected from Pseudomonas aeruginosa, Pseudomonas putida , Moesziomyces antarcticus, Moesziomyces aphids, and Ustilago maydis.

19. A system for continuously manufacturing glycolipids through microbial fermentation, the system comprising: a first tank, wherein the first tank contains a first mixture comprising microbes and feedstock;a second tank operably connected to the first tank at at least two points; a third tank operably connected to the second tank at at least two points; a fourth tank, wherein a top of the third tank is operably connected to a bottom of the fourth tank; and a condenser, wherein the condenser connects the third tank to a storage tank.

20. The system of claim 19, wherein the condenser is an evaporation column.21 . The system of claim 20, wherein the evaporation column is a bubble column evaporator.

22. Use of the system of claim 19 to produce a glycolipid preparation.

23. The system of claim 22, wherein the microbes are selected from Pseudomonas, Ustilago, Moesziomyces, or Candida.

24. The system of claim 23, wherein the microbes are selected from Pseudomonas aeruginosa, Pseudomonas putida, Moesziomyces antarcticus, Moesziomyces aphids, and Ustilago maydis.

25. The system of claim 23, wherein the glycolipid is an oil-based glycolipid.

26. The system of claim 23, wherein the glycolipid is a water-based glycolipid.

27. The system of claim 23, wherein the glycolipid is a rhamnolipid (RL), a mannosylerythritol lipid (MEL), or a sophorolipid (SL).

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