Processes for the fermentative production of biosurfactants.

Abstract

The present invention relates to a fermentation process for the production of a biosurfactant.

Description

[Technical Field]

[0001] The present invention relates to a fermentation process for the production of biosurfactants. [Background technology]

[0002] As reported in the literature (Varjani and Upasani, Bioresource Technology 232(2017):389-397; Chong and Li, Microb Cell Fact (2017)16-137), the increase in global surfactant production is estimated to exceed 24 million tons per year by 2020 (Gudina et al, Biore Technol. 2016;212:144-50). The urgent need to replace non-biodegradable, petroleum-derived surfactants, which can lead to environmental problems, is driven primarily by the increasing demand for biosurfactants, particularly rhamnolipids, as environmentally friendly alternatives (Dobler et al, New Biotechnol. (2016) 33:123-35; Muller et al, J Biotechnol. (2012) 162:366-80; Dusane et al, Biotechnol Genetic Eng Rev. Harding SE editor. (2010) 27:159-184).

[0003] As stated by Chong and Li (2017), the most attractive feature of biosurfactants is that they are easily biodegradable and have low toxic effects on the environment, while possessing properties similar to synthetic surfactants.

[0004] Fermentative production of biosurfactants remains extremely challenging (Geys et al., Current Opinion in Biotechnology (2014) 30:66-72). Fermentative production is dictated by the nature of the product itself, its raw materials, and the production process. Biosurfactants are primarily valued in end-use products for their foaming properties, which are essential for generating stable foam during product application. In contrast, excessive foaming is a problem that must be minimized and, if possible, avoided in fermentative production processes (Bator et al., Frontiers in Bioengineering and Biotechnology (2020) 8:Article 899). In this regard, most fermentation processes are characterized by increasing product titer as well as vigorous agitation and aeration of the fermentation broth, which promotes foam formation in the headspace of the bioreactor. This poses a significant challenge for successful commercial processes because these stable foams accumulate and increase in the headspace over time and product titer. If not properly removed or tightly controlled, they can clog pipes, filters, and measuring devices, as well as effectively reduce the available reaction volume of the fermenter. Furthermore, accumulation of microorganisms typically occurs at the gas-liquid interface of the foam. This has the powerful consequence of removing biocatalysts from the liquid fermentation broth in the fermenter and reducing biosurfactant production, as reported for rhamnolipids (Blesken et al., Frontiers in Bioengineering and Biotechnology (2020) 8, Article 572892). For example, in the case of rhamnolipids, it is customary to use antifoam agents during the fermentation process (Beuker et al., AMB Express (2016) 6:124), but this is hindered by the large amounts of antifoam agents required and the associated additional costs, as well as increased difficulties in downstream processing (DSP) (Ochsner et al., Adv Biochem Eng Biotechnol (1996) 53:89-118).

[0005] Regarding the raw materials used in biosurfactant production, as reported in detail by Geys et al. (Current Opinion in Biotechnology (2014) 30:66-72), Chong and Li (Microb Cell Fact (2017) 16:137), and Liu et al. (Biotechnology and Bioengineering (2018) 115:796-814), glucose, glycerol, and some oils are primarily used as carbon sources. In the specific case of rhamnolipid production using Pseudomonas species, titers have been reported to be 0.3 g / L–40 g / L using glucose, 0.6 g / L–30 g / L using glycerol, and significantly up to 150 g / L using edible vegetable oils such as sunflower oil, corn oil, coconut oil, palm oil, soybean oil, and olive oil.

[0006] Vegetable oils, as renewable resources, are a well-suited substrate for the production of biotensides and have the added advantage of acting as anti-foaming agents during the fermentation process; however, downstream processing of the fermentation broth presents a problem in that the remaining oil and by-products are difficult and expensive to remove to obtain the final purified biosurfactant (Heyd et al, Anal Bioanal Chem (2008) 391:1579-1590; Varjani et al, Int. J. Innovative Res. Sci. Eng. Technol. 3(2):9205-9213; Chong and Li Microb Cell Fact (2017) 16:137).

[0007] As addressed by Tan and Li (Microb Cell Fact (2018) 17:89), the biggest challenges when sugars or sugar-containing waste materials are used for biosurfactant production remain low yields and excessive foaming during fermentation. Nevertheless, because glucose-based processes are widely established at industrial scale, alternatives that overcome the limitations primarily caused by extreme foaming during fermentation have been intensively investigated in recent years, for example, by using recyclable in situ liquid-liquid extraction solvents for the foam-free synthesis of rhamnolipids in two-stage fermentation (Demling et al., Green Chem (2020) 22:8495-8510) or by applying process-integrated foam fractionation columns to separate biosurfactants from the culture medium and bacterial cells (Blesken et al., Frontiers in Bioengineering and Biotechnology (2020) 8, Article 572892). Although glucose was used as the main C source in both cases, and the foam fractionation method was supported by the deletion of genes encoding cell surface structures (hydrophobic proteins) present on the producing microorganism Pseudomonas putida KT2440, neither process alternative delivered rhamnolipids with titers above 40 g / L under 100 h of operation.

[0008] Specifically, it has been reported that the foam stability of rhamnolipids produced by fermentation increases significantly with both agitation and increased product concentration. The foam remained very stable over a 30-minute half-life. While the primary product of rhamnolipids contributes significantly to the extreme foaming during the fermentation process, other products, such as cells, have a more limited effect. This explains why extreme foaming occurs in the late stages of fermentation when rhamnolipid-rich solutions are mechanically agitated (Long et al., Journal of Surfactants and Detergents (2016), 19(4):833-840).

[0009] The object of the present invention is to improve the process for the fermentative production of biosurfactants in that it is more manageable with respect to excessive foaming, fermentation broth viscosity, and / or coloration of the final product. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Varjani and Upasani, Bioresource Technology 232(2017):389-397 [Non-patent document 2] Chong and Li, Microb Cell Fact(2017)16-137 [Non-patent document 3] Gudina et al,Biores Technol.2016;212:144-50 [Non-patent document 4] Dobler et al, New Biotechnol.(2016)33:123-35 [Non-Patent Document 5] Muller et al, J Biotechnol. (2012) 162:366-80 [Non-patent document 6] Dusane et al,Biotechnol Genetic Eng Rev.Harding SE editor.(2010)27:159-184 [Non-Patent Document 7] Geys et al,Current Opinion in Biotechnology(2014)30:66-72 [Non-patent document 8] Bator et al,Frontiers in Bioengineering and Biotechnology(2020)8:Article 899 [Non-Patent Document 9] Blesken et al.,Frontiers in Bioengineering and Biotechnology(2020)8,Article 572892 [Non-Patent Document 10] Beuker et al.,AMB Express(2016)6:124 [Non-Patent Document 11] Ochsner et al., Adv Biochem Eng Biotechnol (1996) 53:89-118 [Non-Patent Document 12] Liu et al,Biotechnology and Bioengineering(2018)115:796-814 [Non-Patent Document 13] Heyd et al, Anal Bioanal Chem (2008) 391:1579-1590 [Non-Patent Document 14] Varjani et al,Int.J.Innovative Res.Sci.Eng.Technol.3(2):9205-9213 [Non-Patent Document 15] Tan and Li, Microb Cell Fact (2018) 17:89 [Non-Patent Document 16] Demling et al,Green Chem(2020)22:8495-8510 [Non-Patent Document 17] Long et al, Journal of Surfactants and Detergents (2016), 19(4):833-840

[0011] Summary of the Invention Surprisingly, it has been found that the problem underlying the present invention can be solved by using a well-defined mixture of sugars.

[0012] Therefore, the present invention provides a process for the fermentative production of biosurfactants according to claim 1.

[0013] One advantage of the present invention is that high titers can be reached.

[0014] Another advantage of the present invention is that the viscosity of the culture medium decreases during fermentation.

[0015] A further advantage is that antifoam consumption is reduced (compared to cultivation with a single sugar), i.e. foam formation is reduced.

[0016] Another advantage is that the reduction in viscosity and antifoam consumption results in lower energy consumption of mechanical agitation during fermentation.

[0017] An additional benefit is that the color (darkening) of the final product is reduced.

[0018] Thus, the present invention provides a process for the fermentative production of a biosurfactant, comprising: A) glucose and at least one additional sugar selected from the group consisting of fructose, isomaltose, maltose, maltulose, and panose; contacting the microorganism with a medium containing a mixture of sugars consisting of: The present invention provides a process including:

[0019] Within the context of the present invention, "biosurfactants" is understood to mean all glycolipids produced by fermentation. The term "biosurfactants" also encompasses glycolipids that are chemically or enzymatically modified after fermentation, as long as the glycolipid remains structurally intact.

[0020] In the context of the present invention, the term "surfactant" is understood to mean an organic substance having surface-active properties capable of reducing the surface tension of water to less than 45 mN / m at 20°C and at a concentration of 0.5% by weight based on the total composition. The surface tension is determined by the DuNouil ring method at 20°C.

[0021] Where average values ​​are specified below, these are number-averaged average values ​​unless otherwise specified.

[0022] Unless otherwise stated, percentages are given as percent by weight.

[0023] Wherever measurements are stated below, these are determined at a temperature of 25° C. and a pressure of 1013 mbar, unless otherwise specified.

[0024] A preferred process according to the invention is characterized in that said mixture of sugars represents at least 85% by weight, preferably at least 90% by weight, even more preferably at least 95% by weight of all sugars dissolved in the medium.

[0025] Other sugars that may be dissolved in the medium but are not part of the mixture in the process of the invention may be one or more selected from the group: lactose, trehalose, maltotriose, raffinose, sucrose.

[0026] In accordance with the present invention, a process is preferred, characterized in that the mixture of sugars accounts for at least 85% by weight, preferably at least 90% by weight, and even more preferably at least 95% by weight of all available carbon sources present in the medium.

[0027] The available carbon that may be present in the above-described medium may be, for example, carbohydrates other than those listed above, particularly sugars, and / or lipophilic carbon sources such as fats, oils, partial glycerides, fatty acids, fatty alcohols, long-chain saturated carbohydrates, or unsaturated hydrocarbons.

[0028] Preferably, the process according to the invention comprises in said mixture of sugars: Glucose is It is contained in an amount of 85.0 wt % to 99.5 wt %, preferably 90.0 wt % to 98.0 wt %, more preferably 93.0 wt % to 97.0 wt %, The total amount of further sugars is It is contained in an amount of 0.5% by weight to 15.0% by weight, preferably 2.0% by weight to 10.0% by weight, more preferably 3.0% by weight to 7.0% by weight, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0029] Preferably, the process according to the invention comprises in the mixture of sugars: Fructose is It is contained in an amount of 0.1% by weight to 10.0% by weight, preferably 0.5% by weight to 8.0% by weight, and more preferably 1.5% by weight to 6.0% by weight, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0030] Preferably, the process according to the invention comprises in the mixture of sugars: Isomaltose is It is contained in an amount of 0.1% by weight to 4.0% by weight, preferably 0.5% by weight to 3.0% by weight, and more preferably 1.0% by weight to 2.0% by weight, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0031] Preferably, the process according to the invention comprises in the mixture of sugars: Maltose is It is contained in an amount of 0.1% by weight to 8.0% by weight, preferably 1.0% by weight to 6.0% by weight, and more preferably 2.0% by weight to 5.0% by weight, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0032] Preferably, the process according to the invention comprises in the mixture of sugars: Maltulose is It is contained in an amount of 0.1 wt % to 2.0 wt %, preferably 0.3 wt % to 1.5 wt %, more preferably 0.4 wt % to 1.0 wt %, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0033] Preferably, the process according to the invention comprises in the mixture of sugars: Panose, It is contained in an amount of 0.1% by weight to 3.0% by weight, preferably 0.5% by weight to 2.0% by weight, more preferably 0.7% by weight to 1.5% by weight, The weight percentage refers to the mixture of sugars contained in the medium. It is characterized by:

[0034] Preferably, the process according to the invention is characterized in that the biosurfactant present in the culture medium is greater than 50 g / l, preferably between 60 g / l and 200 g / l, more preferably between 80 g / l and 180 g / l, during at least part of the process according to the invention.

[0035] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from the group of rhamnolipids, sophorolipids and glucolipids.

[0036] The term "rhamnolipids" in the context of the present invention preferably refers specifically to lipids of the general formula (I) [ka] and salts thereof, in which mRL=2, 1, or 0; nRL=1 or 0, R 1RL and R 2RL = identical or different organic residues, independent of one another, having 2 to 24, preferably 5 to 13, carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or tri-unsaturated alkyl residues, preferably those selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl, and (CH) o-CH3 (wherein o=1 to 23, preferably 4 to 12).

[0037] When nRL=1, the glycosidic bond between the two rhamnose units is preferably in the α-configuration. The optically active carbon atom of the fatty acid is preferably present as the R-enantiomer (e.g., (R)-3-{(R)-3-[2-O-(α-L-rhamnopyranosyl)-α-L-rhamnopyranosyl]oxydecanoyl}oxydecanoate).

[0038] The term "di-rhamnolipid" in the context of the present invention is understood to mean a compound of general formula (I) or a salt thereof, in which nRL=1.

[0039] The term "monorhamnolipid" in the context of the present invention is understood to mean a compound of general formula (I) or a salt thereof, in which nRL=0.

[0040] The different rhamnolipids are abbreviated according to the following nomenclature: "diRL-CXCY" is understood to mean a dirhamnolipid of general formula (I), in which the residues R 1RL and R 2RL One of these = (CH2) o -CH3, where o=X-4 and the remaining residues R 1 or R 2 =(CH2) o -CH3, where o=Y-4.

[0041] "monoRL-CXCY" is understood to mean a monorhamnolipid of general formula (I), in which the residues R 1RL and R 2RL One of these = (CH2) o -CH3, where o=X-4 and the remaining residues R 1RL or R 2RL =(CH2) o -CH3, where o=Y-4.

[0042] Therefore, the nomenclature used does not distinguish between "CXCY" and "CYCX".

[0043] For rhamnolipids where mRL=0, monoRL-CX or diRL-CX are used as appropriate.

[0044] If one of the above indices X and / or Y is given a "Z", this means that the respective residue R 1RL and / or R 2RL is equivalent to an unbranched, unsubstituted hydrocarbon residue having X-3 or Y-3 carbon atoms bearing a Z double bond.

[0045] In the context of the present invention, the term "sophorolipid" preferably refers to a compound of the general formula (IIa) and (IIb) [ka] [ka] and salts thereof, in which R 1SL =H or CO-CH3, R 2SL =H or CO-CH3, R 3SL = a divalent organic moiety containing 6 to 32 carbon atoms, unsubstituted or substituted with hydroxyl functional groups, unbranched, and optionally containing 1 to 3 double or triple bonds; R 4SL =H, CH3, or a monovalent organic radical containing 2 to 10 carbon atoms, unsubstituted or substituted with a hydroxyl functional group, unbranched, and optionally containing 1 to 3 double or triple bonds; nSL=1 or 0.

[0046] Sophorolipids can be produced in accordance with the present invention in their acidic or lactone form.

[0047] A preferred process according to the present invention produces sophorolipids in which the weight ratio of lactone to acidic forms is in the range of 20:80 to 80:20, particularly preferably in the range of 30:70 to 40:60.

[0048] To determine the content of acidic or lactone sophorolipids in a formulation, see EP 1411111, page 8, paragraph

[0053] .

[0049] In the context of the present invention, the term "glucolipid" preferably refers to a glycolipid of the general formula (III) [ka] and salts thereof, in which mGL=1 or 0, R 1GL and R 2GL = identical or different organic residues having 2 to 24 carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di-, or tri-unsaturated alkyl radicals, preferably those selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl, and tridecenyl, and (CH) o -CH3 (wherein o=1 to 23, preferably 4 to 12).

[0050] The different glucolipids are abbreviated according to the following nomenclature: "GL-CXCY" is understood to mean a glucolipid of general formula (III), in which the radical R 1GL and R 2GL One of these = (CH2) o -CH3, where o=X-4 and the remaining radical R 1GL or R 2GL =(CH2) o -CH3, where o=Y-4.

[0051] For this reason, the nomenclature used does not distinguish between "CXCY" and "CYCX".

[0052] If "Z" is given for one of the above indices X and / or Y, this is the same as the respective radical R 1GL and / or R 2GL It means an unbranched, unsubstituted hydrocarbon radical having X-3 or Y-3 carbon atoms with a ═Z double bond.

[0053] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from rhamnolipids and the microorganism is selected from the group of Pseudomonas putida, Pseudomonas aeruginosa, Serratia rubidae SNAU02, Escherichia coli, and Burkholderia thailandensis.

[0054] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from rhamnolipids and that the concentration of all rhamnolipids present in the culture medium is greater than 80 g / l, preferably between 80 g / l and 180 g / l, more preferably between 100 g / l and 160 g / l, during at least part of the process according to the invention.

[0055] Suitable process parameters and microorganisms for the preparation of rhamnolipids are disclosed, for example, in EP 2786743 and EP 2787065.

[0056] Fermentation of Pseudomonas, particularly Pseudomonas aeruginosa, preferably non-genetically modified cells, is a technique that was disclosed as early as the 1980s, as documented, for example, in EP 0 282 942 and DE 4 127 908, and can be carried out as described within the scope of the present invention by using special sugar mixtures. Pseudomonas aeruginosa cells that have been genetically modified to produce higher rhamnolipid titers can also be used in the context of the present invention; such cells are disclosed, for example, by Lei et al. in Biotechnol Lett. 2020 Jun;42(6):997-1002.

[0057] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from sophorolipids and the microorganism is selected from the group Starmerella bombicola, Candida bogoriensis, Candida magnolia, Candida batista, Candida apicola or Wiccahamierella domericae.

[0058] In the process of the present invention, when the biosurfactant is selected from sophorolipids and the microorganism is selected from yeast, the mixture of sugars preferably accounts for at least 25% by weight, preferably at least 45% by weight, and even more preferably at least 70% by weight of all available carbon sources present in the medium. In this embodiment of the present invention, other available carbon sources preferably present in the medium are selected from fats, oils, partial glycerides of fatty acids, fatty acids, fatty alcohols, and long-chain, preferably C8 to C32, saturated or unsaturated hydrocarbons.

[0059] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from sophorolipids and the concentration of all sophorolipids present in the culture medium is greater than 80 g / l, preferably between 80 g / l and 180 g / l, more preferably between 100 g / l and 160 g / l, during at least part of the process according to the invention.

[0060] Preferably, the process according to the invention is characterized in that the biosurfactant is selected from glucolipids and the microorganism is selected from the group of Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli, Serratia rubidae, preferably strain ATCC 27593, and Burkholderia thailandensis.

[0061] Preferably, the process according to the invention is characterized in that the biosurfactants are selected from glucolipids and that the concentration of all glucolipids present in the culture medium is greater than 50 g / l, preferably between 50 g / l and 120 g / l, more preferably between 60 g / l and 100 g / l, during at least part of the process according to the invention.

[0062] Suitable process parameters and microorganisms for the preparation of glucolipids are disclosed, for example, in WO2019154970.

[0063] Preferably, the process according to the invention is characterized in that it comprises a step B) of purifying the biosurfactant, preferably by separating it from the microorganism and / or from at least part of the culture medium.

[0064] The examples presented below are intended to illustrate the present invention, the scope of which is clear from the entire specification and claims, and are not intended to be limited to the embodiments embodied in the examples. [Example]

[0065] Example 1: Rhamnolipid production using Pseudomonas on glucose and fructose Fermentation was performed using the Pseudomonas putida strain pBBR1MCS2-Plac-rhlABC-T-Ptac-rhlC-T, whose preparation is described in U.S. Patent Application Publication No. 2014296168 and contains the rhamnolipid biosynthetic genes RhIA, RhIB, and RhIC. Preliminary cultures were performed in shake flasks as described in WO 2012013554. For the main cultures, mineral medium M9 was similarly employed. This medium consisted of 2% (w / v) glucose, 0.3% (w / v) KH2PO4, 0.679% Na2HPO4, 0.05% (w / v) NaCl, 0.2% (w / v) NH4Cl, 0.049% (w / v) MgSO4 × 7 H2O, and 0.1% (v / v) trace element solution. It consists of 1.78% (w / v) FeSO4 x 7 HO, 0.191% (w / v) MnCl2 x 7 HO, 3.65% (w / v) HCl, 0.187% (w / v) ZnSO4 x 7 HO, 0.084% (v / v) NaEDTA x 2 HO, 0.03% (v / v) H3BO3, 0.025% (w / v) Na2MoO4 x 2 HO, and 0.47% (w / v) CaCl2 x 2 HO. The pH of the medium is adjusted to 7.4 with NH4OH, and the medium is then sterilized by autoclaving (121 °C, 20 min).

[0066] Fermentations are carried out in 2-liter fermentors with a carbon-limited glucose feed input. The glucose feed input is referenced to the dissolved oxygen signal. Dissolved oxygen is controlled at 20% saturation by the agitator speed. The pH is controlled at 7 by a pH electrode and the addition of NH4OH. Antifoam DOW Corning 1500 was added as needed to prevent and measure foaming of the fermentation broth. Fermentations were carried out for 4 days.

[0067] Additionally, 2% (w / v) glucose was replaced with 1.98% (w / v) glucose and 0.02% (w / v) fructose, M9 * A medium called F is prepared and used.

[0068] Furthermore, we use Pseudomonas aeruginosa PAO1, a wild-type strain that produces rhamnolipids. [Table 1]

[0069] The results show that replacing part of the glucose with fructose reduces foaming and antifoam consumption, while the overall rhamnolipid yield remains the same.

[0070] Example 2: Rhamnolipid production using Pseudomonas on glucose and maltose Example 1 was repeated, but 2% (w / v) glucose was replaced with 1.96% (w / v) glucose and 0.04% (w / v) maltose, M9 * A medium called M is prepared and used.

[0071] Furthermore, we use Pseudomonas aeruginosa PAO1, a wild-type strain that produces rhamnolipids. [Table 2]

[0072] The results show that partial replacement of glucose with maltose reduces foaming and antifoam consumption, while the overall rhamnolipid yield remains the same, but to a lesser extent than with fructose replacement.

[0073] After separating the cells of the above fermentation by centrifugation at 10,000 g, the fermentation broth is adjusted to pH 3.1 by adding concentrated H2SO4. After centrifugation again at 500 g, a pasty solid concentrate is obtained with a rhamnolipid fraction of 45% by weight and a viscosity of more than 10,000 mPas.

[0074] Relative color was judged by the platinum-cobalt scale. [Table 3]

[0075] The results indicate that replacing some of the glucose with maltose results in a purer rhamnolipid composition.

[0076] Example 3: Rhamnolipid production using Pseudomonas on glucose, maltose, and fructose Example 1 was repeated, but the 2% (w / v) glucose was replaced with 1.96% (w / v) glucose, 0.03% (w / v) maltose, and 0.01% (w / v) fructose. * A medium called FM is prepared and used.

[0077] Furthermore, we use Pseudomonas aeruginosa PAO1, a wild-type strain that produces rhamnolipids. [Table 4]

[0078] The results show that replacing part of the glucose with maltose and fructose synergistically reduces foaming and antifoam consumption, while the overall rhamnolipid yield remains the same.

[0079] After separating the cells of the above fermentation by centrifugation at 10,000 g, the fermentation broth is adjusted to pH 3.1 by adding concentrated H2SO4. After centrifugation again at 500 g, a pasty solid concentrate is obtained with a rhamnolipid fraction of 45% by weight and a viscosity of more than 10,000 mPas.

[0080] Relative color was judged by the platinum-cobalt scale. [Table 5]

[0081] The results indicate that replacing some of the glucose with maltose results in a purer rhamnolipid composition.

[0082] Example 4: Rhamnolipid production using Pseudomonas on glucose and isomaltose Example 2 was repeated, but 2% (w / v) glucose was replaced with 1.96% (w / v) glucose and 0.04% isomaltose. * A medium called I is prepared and used.

[0083] Relative color was judged by the platinum-cobalt scale. [Table 6]

[0084] The results indicate that replacing part of the glucose with isomaltose results in a purer rhamnolipid composition.

[0085] Example 5: Rhamnolipid production using Escherichia coli with glucose and panose, or glucose and maltose Rhamnolipid production is carried out in recombinant Escherichia coli W3110 pBBR1MCS-2::ABC cells as described in Example 10 of EP 2598646.

[0086] Use CMP medium, which consists of 2% (w / v) glucose, 0.007% (w / v) KH2PO4, 0.11% Na2HPO4 x 2 H2O, 0.2% (w / v) NaNO3, 0.04% (w / v) MgSO4 x 7 H2O, 0.01% (w / v) CaCl2 x 2 H2O, and 0.2% (v / v) trace element solution, which consists of 0.2% (w / v) FeSO4 x 7 H2O, 0.15% (w / v) MnSO4 x 7 H2O, and 0.06% (w / v) (NH4)MO7O. 24 x 4 HO. Adjust the pH of the medium to 6.7 using NaOH.

[0087] Additionally, CMP, in which 2% (w / v) glucose was replaced with 1.98% (w / v) glucose and 0.02% panose, * P, and CMP, in which 2% (w / v) glucose was replaced with 1.99% (w / v) glucose and 0.01% maltulose. * A medium called M is prepared and used.

[0088] The viscosity of the final fermentation broth is measured using a rheometer (Anton Haak) with a concentric cylindrical geometry at a constant shear rate of 100 1 / s. [Table 7]

[0089] The results show that replacing part of the glucose with panose or maltulose reduces the viscosity of the fermentation broth, which is advantageous because less energy needs to be introduced into the vessel during fermentation.

[0090] Example 6: Glucolipid production using Pseudomonas on glucose and fructose Glucolipid production is performed on recombinant Pseudomonas putida BS-PP-368 cells in a 1 liter Dasgip fermentor as described in Example 2 of WO2019154970.

[0091] Additionally, a pure 500g / L glucose feed ("G") was replaced with 485g / L glucose + 15g / L fructose (G * F). [Table 8]

[0092] The results show that replacing part of the glucose with fructose reduces foaming and antifoam consumption, while the overall glucolipid yield remains the same.

[0093] Example 7: Glucolipid production using Pseudomonas on glucose and maltose Glucolipid production is performed on recombinant Pseudomonas putida BS-PP-368 cells in a 1 liter Dasgip fermentor as described in Example 2 of WO2019154970.

[0094] Additionally, a pure 500g / L glucose feed ("G") was replaced with 490g / L glucose + 10g / L maltose (G * M).

[0095] The cells are separated by centrifugation at 10,000 g for 20 minutes. The fermentation broth is separated as the supernatant and adjusted to pH 3.1 by the addition of concentrated H2SO4.

[0096] After a second centrifugation at 5000 g for 20 minutes, the aqueous upper phase is separated and the remaining lower phase is a concentrate containing more than 50% by weight of glucolipids.

[0097] Relative color is judged by the platinum-cobalt scale. [Table 9]

[0098] The results indicate that replacing some of the glucose with maltose results in a purer glucolipid composition.

[0099] Example 8: Glucolipid production using Pseudomonas for glucose and panose, or glucose and maltose, or glucose, panose, and maltose Glucolipid production is carried out as described in Example 7.

[0100] Additionally, a pure 500 g / L glucose feed ("G") was replaced with a 496 g / L glucose + 4 g / L panose feed (G * P), 496 g / L glucose + 4 g / L maltose (G *M) and 496 g / L glucose + 2 g / L maltose + 2 g / L panose (G * MP) supply.

[0101] The viscosity of the final fermentation broth is measured using a rheometer with concentric cylindrical geometry at a constant shear rate of 100 1 / s. [Table 10]

[0102] The results show that replacing part of the glucose with panose or maltose reduces the viscosity of the fermentation broth, and panose and maltose have a synergistic effect, which is beneficial because less energy needs to be introduced into the vessel during fermentation.

[0103] Example 9: Production of sophorolipids using Starmerella bombicola on glucose and fructose Sophorolipids were produced for the wild-type strain Starmerella bombicola ATCC 22214 and the recombinant strain Starmerella bombicola ATCC 22214 sbg3-hyg as described in Example 12 of WO 2011061032. The medium used to produce sophorolipid SL consisted of 0.1% KH2PO4, 0.5% MgSO4 x 7 H2O, 0.01% FeCl3, 0.01% NaCl, 0.4% yeast extract, 0.1% urea, 10.5% rapeseed oil, and 10% glucose, with a pH of 4.5.

[0104] Additionally, 10% (w / v) glucose was replaced with 9.9% (w / v) glucose and 0.1% fructose, SL * A medium called F is prepared and used.

[0105] The relative foam height in the flask as well as the sophorolipid concentration are determined. [Table 11]

[0106] The results show that replacing part of the glucose with fructose prevents foaming, while the total sophorolipid yield remains the same.

[0107] Example 10: Production of sophorolipids using Starmerella bombicola on glucose and isomaltose, or glucose and maltose Sophorolipids are produced according to Example 2 of WO2021236904(M).

[0108] Another similar experiment was performed by mixing glucose with a 99:1 mixture of glucose and isomaltose (M * I) and replace it with

[0109] Another similar experiment was performed by mixing glucose with a 99:1 mixture of glucose and maltose (M * M) and replace it with

[0110] Relative color was judged by the platinum-cobalt scale. [Table 12]

[0111] The results indicate that replacing part of the glucose with isomaltose or maltose results in a purer sophorolipid composition.

Claims

1. A process for the fermentation production of biosurfactants, A) Glucose and at least one further sugar selected from the group consisting of fructose, isomaltose, maltose, maltulose, and panose. A step of contacting microorganisms with a culture medium containing a mixture of sugars, under conditions that enable the microorganisms to synthesize the biosurfactant. A process that includes this.

2. The process according to claim 1, characterized in that the mixture of sugars constitutes at least 85% by weight of all sugars dissolved in the culture medium.

3. The process according to claim 1 or 2, characterized in that the mixture of sugars accounts for at least 85% by weight of all available carbon sources present in the culture medium.

4. In the mixture of the aforementioned sugars, Glucose, It is contained in an amount of 85.0% to 99.5% by weight. The totality of the further sugars is It is contained in an amount of 15.0% to 0.5% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

5. In the mixture of the aforementioned sugars, Fructose, It is contained in an amount of 0.1% to 10.0% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

6. In the mixture of the aforementioned sugars, Isomaltose, It is contained in an amount of 0.1% to 4.0% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

7. In the mixture of the aforementioned sugars, Maltose, It is contained in an amount of 0.1% to 8.0% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

8. In the mixture of the aforementioned sugars, Martz loin, It is contained in an amount of 0.1% to 2.0% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

9. In the mixture of the aforementioned sugars, Panos, It is contained in an amount of 0.1% to 3.0% by weight. The weight percentage refers to the mixture of sugars contained in the culture medium. The process according to claim 1 or 2, characterized in that

10. The process according to claim 1 or 2, characterized in that the biosurfactant is selected from the group consisting of rhamnolipids, sophorolipids, and glucolipids.

11. The biosurfactant is selected from rhamnolipids, and the microorganism is selected from the group Pseudomonas putida, Pseudomonas erginosa, Escherichia coli, and Burkholderia tyrandensis, The biosurfactant is selected from sophorolipids, and the microorganism is selected from the group of Starmerella bombicola, Candida bogoriensis, Candida batistae, Candida apicola, or Wickelhamiera domericae, or The biosurfactant is selected from glucolipids, and the microorganism is selected from the group Pseudomonas putida, Pseudomonas erginosa, Escherichia coli, and Burkholderia tylandensis. The process according to claim 1 or 2, characterized in that