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WO2025168637A1 - Procédé de production de rhamnolipides à partir de paille de maïs - Google Patents

Procédé de production de rhamnolipides à partir de paille de maïs

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Publication number
WO2025168637A1
WO2025168637A1 PCT/EP2025/052978 EP2025052978W WO2025168637A1 WO 2025168637 A1 WO2025168637 A1 WO 2025168637A1 EP 2025052978 W EP2025052978 W EP 2025052978W WO 2025168637 A1 WO2025168637 A1 WO 2025168637A1
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rhamnolipids
fatty acids
chaff
production
medium
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Adriana BAVA
Fabrizio Beltrametti
Mentore VACCARI
Chantal VIOLA
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Lamberti SpA
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Lamberti SpA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the present invention relates to a process for producing biosurfactants, and, in particular, rhamnolipids, by fermentation.
  • the process is characterized by the use, as one of the carbon sources used by microorganisms which can produce biosurfactants, of corn chaff.
  • Corn chaff is obtained by the mechanical shelling process to which corncobs are subjected after harvesting. This process responds to the comprehensive need of finding effective uses for some agricultural wastes generated by the transformation of cereals, currently not served by an adequate management chain.
  • the process enables the production of completely biobased surfactants, i.e. of surfactants obtained from renewable, biological raw materials and completely vegetal sources.
  • the production chains of the agricultural sector generate by-products and waste.
  • the quantities of the latter are often marginally considered by the official statistics of the sector, causing underestimations and uneven data, which do not provide a clear and exhaustive picture of the situation.
  • the latest available data on the production of vegetable waste of agricultural origin refers to 1997, the year in which only the Italian production exceeded 20 million t/year of dry matter, of which about 13 million t/year from cereal production. This corresponds to 2-4% of the product obtained, which is mainly sent to anaerobic digestion plants for producing biogas.
  • biosurfactants are in all their chemical-physical characteristics, surfactants, i.e. amphipathic molecules, able to accumulate at the interface between two immiscible phases because of their both hydrophilic and hydrophobic nature.
  • Biosurfactants reduce the surface and interfacial tension (IFT) in liquids with different phases of matter, such as gas, liquid and solid, thus increasing the miscibility of substances that are not compatible with each other, typically giving rise to stable foams and oil and water emulsions.
  • IFT interfacial tension
  • biosurfactants excellent and completely biobased alternatives to conventional synthetic surfactants in any domestic and industrial field, for example in detergents, textiles, paints, polymers, pharmaceuticals, agrochemicals, paper, personal care products, and in the oil and gas industry and remediation field.
  • biosurfactants in general is still small compared to that of synthetic surfactants, mainly due to higher production costs.
  • Several techniques and approaches have been adopted worldwide to reduce the costs of biosurfactant production and make it more efficient.
  • the use of cheaper substrates, optimized culture conditions in processes carried out in bioreactors, cost effective recovery processes and strain improvements, all of them have been investigated to improve biosurfactant productivity.
  • Biosurfactants are produced by bacteria, yeasts and filamentous fungi. At present, members of the genera Pseudomonas, Bacillus, Rhodococcus and Candida are the most widely implicated in the industrial production of these biomolecules. Biosurfactants are generally classified into glycolipids, fatty acid phospholipids, lipopeptides and lipoproteins, polymeric surfactants and particulate surfactants.
  • rhamnolipids represent a valid alternative to ethoxylated surfactants, responding to the increasing demand for natural, preferably vegetable-based, ingredients that are sustainably sourced, and which have no impact on the environment once released post-use.
  • Rhamnolipids are long known glycolipids and were described as early as in 1946. They have two moieties: the rhamnose moiety (also known as glycan part) and the lipid moiety (also known as aglycone part) linked via glycosidic linkage.
  • the glycan moiety is hydrophilic and made of one or two rhamnose units; the lipid moiety is hydrophobic and comprises one or more saturated/unsaturated [3-hydroxy fatty acids chains of C8-C24 length, linked together by an ester bond.
  • rhamnolipids can lower the surface tension of water to around 50 mN/m with a CMC of around 20 mg/L, as reported in Biomolecules 2019, 9, 885 by Shreve, G.S. and Makula, R.
  • Rhamnolipids are widely studied for their surfactant properties, but also for their antibacterial, antifungal and antiviral activities and have been widely used in the fields of bioremediation and biodegradation.
  • Rhamnolipids are also used in various biotechnological and industrial applications, such as the synthesis and stabilization of nanoparticles, the preparation of microemulsions; they are also used as anticaking agents and as a source of rhamnose.
  • rhamnolipids are usually performed with natural isolates of bacteria of the genus Pseudomonas a nd Burkholderia (US 4,933,281, US 2011/0306569, US 7,202,063), but cases of use of recombinant strains have also been reported (US 9,854,799; US 10,174,353; US 2014/0235561; US 2017/0096695; US 2013/0130319).
  • the Gram-negative bacterium Pseudomonas aeruginosa (PA) is the best studied rhamnolipid producer.
  • rhamnolipids Besides the microorganism properties, the production strategies for rhamnolipids rely on the use of different carbon sources (US 11,142,782, WO2016179249), usually a mixture of carbohydrates and lipids with salts that provide nitrogen, phosphorous, magnesium and other elements. Furthermore, several studies were performed in the production of rhamnolipids from food and/or agricultural waste.
  • US 2021/0079436 relates to a method for preparing rhamnolipids using anaerobic digestate prepared by anaerobic digestion of organic waste, preferably from food waste.
  • CN 104498566 describes the preparation of rhamnolipids by the semi-solid fermentation in the presence of agricultural waste comprising rapeseed meal, cottonseed meal, bran, straw, peanut shell, and rice husk.
  • CN 106801075 provides a production method of rhamnolipid based on the synchronous treatment of manioc waste with a cellulase and Pseudomonas aeruginosa (synchronous saccharification and fermentation).
  • US 2022/0364128 provides a production process that makes use of lignocellulosic substrates, such as cornstalks, in a method for preparing biosurfactants.
  • WO 2014/039940 describes a combination of bacteria that can generate rhamnolipids when grown on hemicellulose, cellulose, or lignin that have been produced because of agricultural activity; examples include, but are not limited to, lawn trimmings, food waste, corn stover, and forestry residues.
  • Lignocellulosic waste from the cereal supply chain is an excellent growth substrate for various kinds of microorganisms; they are rich in complex sugars, which can be used by numerous microorganisms as a source of nourishment. As reported in the literature, several microorganisms possess an enzymatic set suitable for the degradation of lignocellulosic components.
  • the object of the present invention is the development of an efficient process for producing rhamnolipids by fermentation, starting from a specific lignocellulosic waste biomass that can be used even without preliminary chemical or enzymatic degradation pretreatment.
  • this object may be achieved by using corn chaff as such, or mechanically shredded corn chaff, as culturing nutrient in a fermentation process for obtaining rhamnolipids.
  • Fig. 1 graph of the growth curves of Pseudomonas aeruginosa on corn chaff and other agricultural wastes.
  • Fig. 2 photographic reproduction of the result of the qualitative evaluation of the production of biosurfactants, by Pseudomonas aeruginosa grown on corn chaff, by oil displacement activity test (ODA).
  • Fig. 3 thin layer chromatography of Rhamnolipids obtained after the growth of Pseudomonas aeruginosa on corn chaff (A and B are replicas of the same sample, standard on the right).
  • Fig. 4 graph of the Rhamnolipids (RLs) production in Examples AA (on the right) and BB (on the left).
  • Fig. 5 graph of the production of Rhamnolipids (RLs) in fermentation medium inoculated at different incubation times of the seed culture.
  • Fig. 6 graph of rhamnolipids (RLs) production decrease after the addition of a- amylase to the fermentative medium
  • the process for producing rhamnolipids by fermentation comprises the steps of 1) inserting a rhamnolipid-producing microorganism into a culture medium suitable for the growth (seed inoculum); 2) inoculating a fermentation medium with the seed inoculum, the fermentation medium containing i) corn chaff, ii) at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol or any mixtures thereof; 3) culturing the rhamnolipid producing microorganism in the fermentation medium, to obtain a fermentation medium containing one or more rhamnolipids and the rhamnolipid producing microorganism; 4) isolating the rhamnolipids.
  • the microorganism is more preferably Pseudomonas aeruginosa or a Burkholderia, most preferably it is Pseudomonas aeruginosa.
  • Pseudomonas aeruginosa is a Gram negative, facultatively aerobic, opportunistic pathogenic bacterium capable of growing through aerobic respiration and through anaerobic respiration using nitrate as the final electron acceptor.
  • Suitable strain to be used in this method is any of those intrinsically able to produce rhamnolipids and any recombinant strains carrying heterologous genes to synthesize rhamnolipids.
  • There are no specific limitations in the preparation of the seed inoculum having the seed the role of supplying enough bacteria, in a suitable physiological state to allow growth in the production medium (or fermentation medium) which is used in the process.
  • the inoculum is grown on Luria Bertani medium (LB medium) and dosed in the fermentation medium at 1-5% by weight of the fermentation medium used for production.
  • the fermentation medium typically contains, by weight percentage (wt%), 1-20 % of corn chaff, 1-20 % of at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol or any mixtures thereof, and preferably 0.1-1 wt% of a nitrogen source, 0.05-1 wt% of a phosphorous source, and 0.1-1 wt% of other essential elements sources which can typically, without limitation, being supplied in the form of trace element solutions as described in (Sharma et al. 3 Biotech (2016) 8(1), 20; Sun et al. Biotechnol progress, 2021, 37(4), e3155), the balance being substantially water.
  • the characterising carbon source of the process is thus made of corn chaff mixed with at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids and/or glycerol.
  • Chaff is the dry, scaly protective casing of the seeds of cereal grains or similar plant material.
  • Corn chaff is mainly made of the beeswings, the usually brownish or reddish outermost material that holds the grains and is released from the corn cob during shelling and of a part of chaff that remains on the corn cob after the grains have been removed.
  • the corn chaff that is useful for the process (herein also called "loose corn chaff”) is made of the beeswings together with few fragments of grain that are gathered during shelling in small pieces together with the beeswings.
  • Loose corn chaff is a light weight small sized material (typically max. 5-10 mm 2 , 0.1-1 mm thick with a density of 100-500 g/L, preferably 200-400 g/L).
  • the corn chaff that is used in the process of the invention may be untreated, i.e. it may be, and it is preferably used, as such in the process without chemical or enzymatic treatments, optionally only shredded to a smaller size.
  • the untreated corn chaff preferably passes through a 20-mesh sieve or more preferably through a 40-mesh sieve.
  • cereal waste mainly consists of a lignocellulosic material that is normally difficult to be degraded by microorganisms (especially due to the macromorphology of the material). For this reason, these materials are not known to be ideal fermentation substrates unless after suitable treatments. Numerous chemical, enzymatic and/or thermal treatments have been described for the preparation of the lignocellulosic material suitable for microbial digestion.
  • untreated corn chaff can be used as such in the process of the present invention. It has also been found that using in the process untreated corn chaff shredded with a cereal mill further improves rhamnolipids yield.
  • shredding is by far to be preferred to chemical, enzymatic or thermal treatments due to its simplicity, eco-compatibility and to the possibility of being carried out directly at the source in the cereal treatment plants.
  • more drastic treatments such as maceration in the presence of strong acids at high temperatures did not show any advantages compared to what was obtained with shredding.
  • Suitable fatty acids that may be included in the fermentation medium are C8-C22 alkyl or alkenyl, possibly hydroxyl substituted, preferably linear, carboxylic acids.
  • C12-C18 alkyl or alkenyl linear carboxylic acids are preferred among fatty acids.
  • Mixtures of such fatty acids, conveniently found in natural vegetable oils, can be used.
  • Suitable mono-, di- or triglycerides of fatty acids that may be included in the fermentation medium are the mono-, di- and triglycerides of the above-mentioned fatty acids and mixtures thereof.
  • Natural triglycerides of the above fatty acids i.e. vegetable oils, such as soybean oil, olive oil, palm oil, coconut oil, castor oil, sunflower oil, rapeseed oil, and the like are the preferred component of the fermentation medium, especially in combination with glycerol.
  • Suitable components are also waste vegetable oils ("waste cooking oils”) that beside triglycerides also contain minor portions of monoglycerides, diglycerides and variable quantities of fatty acids (5-20 wt%).
  • Suitable alkyl esters of fatty acids that may be included in the fermentation medium are the alkyl esters, such as the methyl esters, of the above-mentioned fatty acids or of mixtures thereof.
  • the fermentation medium may contain glycerol.
  • the carbon source in the fermentation medium may essentially consist of (or consists of) corn chaff, vegetable oil or waste cooking oils and glycerol.
  • a nitrogen source at least a phosphorous source and other essential elements sources are added.
  • the nitrogen source may be sodium nitrate, when present typically accounting for
  • the fermentation medium is typically a pourable viscous aqueous fluid in which the corn chaff is dispersed.
  • the fermentation medium can eventually change its viscosity
  • the fermentation medium contains from 10 to 200 g/L of corn chaff and from 20 to 60 g/L of at least one of triglycerides of fatty acids and/or glycerol.
  • the rhamnolipid producing microorganism is typically fermented for from about 1 day to about 14 days.
  • the fermentation medium may also be a pasty, non-pourable medium, provided that enough aeration of it is dispensed with a suitable mixing apparatus or other means.
  • the average dissolved oxygen level in the fermentation medium is preferably from 5 to 50% during the culturing phase.
  • the fermentation medium has pH from 2.5 to 8.0, preferably from 3.0 to 7.0, more preferably from 3.5 to 6.0 and temperature from about 15°C to about 70°C, preferably from 20 to 50°C, more preferably from 25 to 40°C.
  • the culturing stage may be performed in batch or with continuous or stepwise addition (fed-batch) of nutrients (carbon source and other nutrients) of the fermentation medium.
  • the batch culture is performed by adding in one step all the nutrients, sterilizing the medium and performing the fermentation as described above.
  • the continuous fermentation is performed by adding some volume of the nutrients to the medium in a continuous manner and by removing some volume of the fermentation culture at the same time; stepwise addition (fed-batch) is performed by adding some of the nutrients to the fermentation culture according to specifical signals given by the fermentation parameters.
  • the recovery of the rhamnolipids may be made according to any methodology known in the art.
  • the corn chaff and some other agricultural waste supplied as the only nutrient source (concentration 100 g/L) were used, suspended in 100 mL of ultrapure water in 500 mL flasks with baffle, sterilized in autoclave at 123°C for 20 minutes:
  • the control is a common medium for the production of rhamnolipids, i.e. 20g/L soybean meal.
  • the growth of Pseudomonas aeruginosa was performed and evaluated according to the following protocol.
  • the strain was revitalized in 9 cm Petri dishes containing the selected agarized- medium by seeding the culture stored at -80°C (working cell bank, WCB) using a sterile loop. The prepared plates were then incubated at 24-28°C for 24-48 hours. The cultivation was then continued in 500 mL baffled-flasks, containing 100 mL of vegetative medium (Luria Bertani Broth, LB), sterilized in an autoclave at 121°C for 20 minutes.
  • vegetative medium Lia Bertani Broth, LB
  • the inoculation of the vegetative medium is carried out by suspending a loop of bacterial cells in Luria Bertani broth, taking a volume (1 to 5 ml) of the above suspension, and placing it in 500 ml baffled-flasks, containing 100 mL of vegetative medium.
  • the volume of the bacterial cell suspension is set in a way to obtain an OD600 (measured in LB medium as the reference) in the range 0.1-0.3 in the 500 ml flask.
  • the inoculation is carried out directly from the WCB into the vegetative medium according to the OD600 parameters set above.
  • the cultures are placed in the 500 mL baffled-flasks at 28 °C and stirred at 200-250 rpm.
  • Physiological solution (0.9% w/w), was used to prepare the dilutions of microorganisms and to carry out the viable counts.
  • Physiological solution was prepared by dissolving 9 g/L of sodium chloride (NaCI) in deionized water (H2O). Once dissolved, the solution was sterilized in an autoclave at a temperature of 121°C for 20 minutes.
  • the viable Petri dish plate count allowed to evaluate the concentration of viable cells expressed as Colony Forming Units per unit volume (CFU/mL). This method is used, as an indication of vitality, in all the phases of the fermentation in which a passage from one medium to another is involved, or as an indication of the growth of a microorganism on the substrate under analysis.
  • CFU/mL Colony Forming Units per unit volume
  • serial dilutions are made starting from 1 mL of broth culture (either vegetative culture or fermentative culture according to the experimental requirements), with subsequent plating on agar medium; 100 pL of the diluted suspension described above, are deposited on the plate, and are then spread over until the liquid is dried.
  • the rhamnolipid productivity of Pseudomonas aeruginosa was qualitatively evaluated by the oil displacement activity test (ODA) and analysis on thin layer chromatography, and subsequently confirmed and quantified by means of emulsification index ( E 124%) (Cooper and Goldenberg. 1987, Appl. Environ.
  • the oil dispersion test in water, or oil displacement assay represents a rapid and effective qualitative method for evaluating the surfactant activity of the molecule under investigation. It also offers the advantage of being able to allow testing of many samples in a short time.
  • the assay is commonly performed in Petri dishes with a diameter of 5 cm, to which 30 pL of light crude oil, 3 mL of demineralised water and finally 3 pL of the sample to be analysed have been added.
  • the diameter of the halo obtained on the oil deposited on the surface of water, after dropping the sample represents a qualitative indication of the surfactant power of the substance under examination.
  • This assay developed by Morikawa in 2000, (BBA-Molecular and Cell Biology of Lipids. 1488:211), exploits the ability of biosurfactants to create circular areas in which the fluid is displaced once added to an apolar liquid such as oil. The size of these zones can be correlated to the activity of the biosurfactant.
  • the ODA test result is considered positive if oil displacement is observed for a sample after growth on the tested substrates.
  • the ODA test made possible to identify the production of surfactant substances in the culture broths of Pseudomonas aeruginosa that have grown only on corn chaff (Fig. 2 shows ODA test on aliquots taken from the supernatant coming from culture broths of P. aeruginosa; the test turns positive 72 hours of growth of the strain in the suitable medium). Oat and emmer chaff and pea peel did not show positive results in this test (no biosurfactants production).
  • Negative controls were performed, using as sample an aliquot of supernatant from abiotic media, prepared with the same method.
  • the positive results obtained with corn chaff were confirmed by TLC (thin layer chromatography) (Fig. 3, where A and B are replicas of the same sample and on the right is the rhamnolipid standard).
  • a mixture of chloroform (CHCI3), methanol (MeOH) and ultrapure water (H2O) in a ratio of 65:15:1 is used as the mobile phase.
  • the development of the TLC takes place using the orcinol reagent.
  • the orcinol reagent is prepared by dissolving 0.2 g of orcinol in 89.6 mL of deionized water (H2O) and 10 mL of 96% sulfuric acid (H2SO4). The sulfuric acid is added slowly to limit the exothermic reaction.
  • the produced rhamnolipids were compared with a standard of rhamnolipids from Sigma-Aldrich.
  • the production of surfactant substances was also evaluated by determining the emulsification index; the test shows, in a simple and immediate way, the surfaceactive capacity of the compounds present in the culture broths.
  • the emulsification index (El 24%) represents a parameter for determining the emulsifying power of a surfactant molecule.
  • the protocol is reported in the literature (Cooper and Goldenberg 1987, Appl. Environ. Microbiol. 53: 224-229). In detail, the measurement was performed by mixing equivalent volumes, equal to 2 mL, of the solution (eventually after dilution) containing the biosurfactant under analysis and n-hexadecane or light crude oil. The mixture was stirred with the aid of a vortex mixer for exactly 2 minutes and then incubated at 25°C for 24 hours.
  • the emulsification index (expressed as a percentage) is calculated as the ratio between the height of the emulsified phase and the total height of the liquid column.
  • the test was replicated using light crude oil instead of n-hexadecane.
  • the result of the test showed an emulsification index of 90%, with an emulsifying index of 75% in the abiotic broth.
  • ODA oil displacement activity
  • chromatographic analyses were performed with the aid of thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the samples to which soybean oil and waste oil were added were analysed.
  • a very well-defined spot is visible at the rhamnolipids (both monorhamnolipids and di-rhamnolipids) retention time. Comparison was performed by use of a rhamnolipid standard (Sigma-Aldrich).
  • rhamnolipids produced by Pseudomonas aeruginosa in Examples AA and BB were determined by titration of rhamnose using an HPLC method:
  • the amount of RLs (g/L) is calculated by multiplying the amount of rhamnose (titrated on HPLC), for a correction factor that takes into account the sugar content in the mono- and di-RL molecule (Mohammad et al., 2011, Biosurfactants, Microbiology Monographs 20).
  • the media components were dissolved in ultrapure water and dispensed into 500 mL baffled-flasks and sterilized at 123 °C for 20 minutes.
  • Tests were performed also with the TLC (thin layer chromatography) method, to verify the actual presence of rhamnolipids; the trials had a positive outcome for all the mixtures tested (excluding the negative controls), showing two spots corresponding to mono-rhamnolipids and di-rhamnolipids.
  • the growth in vegetative medium is recognized as a fundamental step for optimizing the yield of the production of secondary metabolites; a growth step in vegetative medium was therefore verified to increase the production of rhamnolipids.
  • Microbial growth was confirmed by viable count at the time of transfer to evaluate the accuracy of the optical density measurement, which also includes non-viable cells.
  • Fig. 5 reports the production of rhamnolipids in P. aeruginosa cultures on BCS388 medium inoculated from vegetative cultures grown for different incubation times (exponential, late exponential-stationary phase of growth).
  • a-amylase In order to make the culture medium more fluid and to make available in the early stages of microbial growth a greater quantity of glucose, the enzyme a-amylase was used; a-amylase is able to hydrolyse the a-1,4 glycosidic bonds inside starch molecules. This gives low molecular weight molecules such as glucose, maltose and maltotriose.
  • the enzyme was added to the BCS388 medium a few minutes before sterilization in the autoclave; in fact, the enzyme is activated by the high temperatures reached, making the procedure standardisable.
  • a-amylase 50 pL and 100 pL (12.5 and 25 U/L of medium) were tested; a seed culture was prepared in vegetative medium (as described above) before transfer to the amylase-treated medium.
  • Fig. 6 shows the maximum production of rhamnolipids obtained under these conditions at 144 hours of fermentation in medium containing a-amylase (HPLC titration). The production of rhamnolipids seems to be reduced by the pretreatment with increasing amounts of the enzyme.

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Abstract

Procédé de production de rhamnolipides par fermentation comprenant l'utilisation d'un microorganisme producteur de rhamnolipides dans un milieu de fermentation contenant de la paille de maïs et au moins un composé choisi parmi les acides gras, les mono -, di- ou triglycérides d'acides gras, les esters alkyliques d'acides gras, le glycérol et tout mélange de ceux-ci, répondant au besoin global de trouver des utilisations efficaces pour certains déchets agricoles générés par la transformation de céréales et permettant la production de tensioactifs obtenus à partir de matières premières biologiques renouvelables et de sources totalement végétales.
PCT/EP2025/052978 2024-02-07 2025-02-05 Procédé de production de rhamnolipides à partir de paille de maïs Pending WO2025168637A1 (fr)

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Citations (13)

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US4933281A (en) 1987-03-17 1990-06-12 The University Of Iowa Research Foundation Method for producing rhamnose
US7202063B1 (en) 2004-09-01 2007-04-10 United States Of America As Represented By The Secretary Of Agriculture Processes for the production of rhamnolipids
US20110306569A1 (en) 2010-06-11 2011-12-15 Oregon State University Rhamnolipid biosurfactant from pseudomonas aeruginosa strain ny3 and methods of use
US20130130319A1 (en) 2010-07-28 2013-05-23 Evonik Goldschmidt Gmbh Cells and methods for producing rhamnolipids
WO2014039940A1 (fr) 2012-09-10 2014-03-13 Logos Technologies Llc Cellules et méthodes de production de rhamnolipides
US20140235561A1 (en) 2011-09-21 2014-08-21 Technische Universitaet Dortmund Means and methods for rhamnolipid production
CN104498566A (zh) 2014-12-17 2015-04-08 江南大学 一种半固态发酵法制备鼠李糖脂的方法及其应用
WO2016179249A1 (fr) 2015-05-05 2016-11-10 Logos Technologies, Llc Procédé semi-continu de production de rhamnolipides à rendement et titre élevés
US20170096695A1 (en) 2014-05-26 2017-04-06 Evonik Degussa Gmbh Methods of Producing Rhamnolipids
CN106801075A (zh) 2017-02-22 2017-06-06 北京林业大学 一种鼠李糖脂的生产方法
US20210079436A1 (en) 2019-09-13 2021-03-18 Hong Kong Baptist University Method for preparing rhamnolipids
US11142782B2 (en) 2017-07-31 2021-10-12 Stepan Company Enhanced production of rhamnolipids using at least two carbon sources
US20220364128A1 (en) 2021-05-12 2022-11-17 Mikros Biochem Methods of preparing biosurfactants using carbon dioxide and/or lignocellulose as substrate

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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