Method for fermentative production of biosurfactants

Abstract

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

Description

Field of the Invention

[0001] The present invention relates to a fermentation method for producing biosurfactant. Prior art

[0002] As reported in the literature (Varjani and Upasani, Bioresource Technology 232 (2017): 389-397; Chong and Li, Microb Cell Fact (2017) 16-137), by 2020, the annual production of surfactants worldwide is expected to exceed 24 million tons (Gudina et al., Biores Technol. 2016; 212: 144-50). The desire to replace non-biodegradable petroleum-derived surfactants that may cause environmental problems can be said to be the main driving force for the increased demand for biosurfactants as environmentally friendly alternatives, especially rhamnolipids (Dobler et al., New Biotechnol, (2016) 33: 123-35; Müller et al., J Biotechnol, (2012) 162: 366–80; Dusane et al., Biotechnol Genetic Eng Rev, Harding SE ed., (2010) 27: 159-184).

[0003] As stated by Chong and Li (2017), the most attractive features of biosurfactants are that they are easily biodegradable and cause less toxic impact on the environment while having similar properties to synthetic surfactants.

[0004] Nevertheless, the fermentation production of biosurfactants is still quite challenging (Geys et al., Current Opinion in Biotechnology (2014) 30: 66-72). This is determined by the properties of the product itself, its raw materials and production methods. Biosurfactants are highly valued in final commercial products, mainly because of their foaming properties, which have stable foam during product application, which is an essential property. On the contrary, in fermentation production methods, excessive foaming is a problem that should be minimized and avoided as much as possible (Bator et al., Frontiers in Bioengineering and Biotechnology (2020) 8: Article 899). In this regard, most fermentation methods are characterized by increasing product titer, as well as vigorous stirring and aeration of the fermentation broth, which promotes the formation of foam in the headspace of the bioreactor. This is the main challenge of commercial success methods, because such stable foams will accumulate and increase in the headspace with the increase of the aforementioned processing time and product titer, and if not properly removed or strictly controlled, it can not only cause clogging of pipes, filters and measuring devices, but also actually reduce the available reaction volume of the fermenter. In addition, the accumulation of microorganisms usually occurs at the gas-liquid interface of the foam. This leads to the removal of the biocatalyst from the liquid fermentation broth of the fermenter, and its serious (strong) consequence is that the biosurfactant output is reduced, as reported for rhamnolipids (Blesken et al., Frontiers in Bioengineering and Biotechnology (2010) 8, Article 572892). Therefore, it is common practice to use defoamers in fermentation methods, such as for rhamnolipids (Beuker et al., AMB Express (2016) 6: 124), but due to the large amount of defoamers required and the additional associated costs, and the increase in the difficulty of downstream processing (DSP) is hindered (Ochsner et al., Adv Biochem Eng Biotechnol (1996) 53: 89-118).

[0005] As for the raw materials used in the production of biosurfactants, mainly glucose, glycerol and various oils are used as C-sources, 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). In the particular case of rhamnolipid production by Pseudomonas, titers ranging from 0.3 g / L to 40 g / L were reported when glucose was used, from 0.6 g / L to 30 g / L when glycerol was used, and up to a significant 150 g / L when edible vegetable oils such as sunflower seed, corn, coconut, palm, soybean and olive oil were used.

[0006] Although plant oils are highly suitable as substrates for biotensides production as a renewable resource and have the added benefit of acting as defoamers in fermentation processes, problems arise during downstream processing of the fermentation broth, where residual oil and byproducts are difficult and costly to remove, making it impossible to obtain a purified final 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 described by Tan and Li (Microb Cell Fact (2018) 17:89), when sugars or sugar-containing wastes are used for biosurfactant production, the biggest challenges of fermentation-based production remain low yields and excessive over-foaming. Nevertheless, since glucose-based methods have been widely established on an industrial scale, alternative approaches to overcome the limitations caused mainly by excessive foaming in fermentation methods have recently been intensively investigated, such as the use of recyclable in situ liquid-liquid extraction solvents for the foam-free synthesis of rhamnolipids in two-phase fermentation (Demling et al., Green Chem (2020) 22:8495–8510), or the separation of biosurfactants from culture medium and bacterial cells by applying process-integrated foam fractionators (Blesken et al., Frontiers in Bioengineering and Biotechnology (2020) 8, Article 572892). In both cases, glucose was used as the main C-source and although foam fractionation was supported by deletion of genes encoding cell surface structures (hydrophobins) present on the production microorganism P. putida KT2440, none of the alternative methods produced rhamnolipid titers above 40 g / L at 100 h of operation.

[0008] In particular, the significant improvement in foam stability of rhamnolipids produced by fermentation is reported to be affected by both stirring and increased product concentration. The overflowing foam maintains an ultra-high stability of more than 30 minutes in terms of half-life. The main product of rhamnolipids is largely responsible for severe foaming in the fermentation process, while the effects caused by other products such as cells are much more limited. This explains why severe foaming occurs in the late fermentation stage when a rhamnolipid-rich solution is mechanically stirred (Long et al., Journal of Surfactants and Detergents (2016), 19 (4): 833-840).

[0009] The object of the present invention is to enhance a process for the fermentative production of biosurfactants to make them easier to handle with respect to excessive foaming, broth viscosity and / or coloration of the final product.

[0010] Description of the invention

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

[0012] The present invention therefore provides a method for the fermentative production of biosurfactants as claimed in claim 1 .

[0013] An advantage of the present invention is that high titers can be achieved.

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

[0015] Another advantage is the reduced consumption of antifoaming agents (compared to incubation with simple sugars), ie reduced foam formation.

[0016] Another advantage is that the reduced viscosity and antifoam consumption lead to lower energy consumption for mechanical agitation during the fermentation process.

[0017] An additional advantage is the reduction in final product color (darkening).

[0018] The present invention therefore provides a method for the fermentative production of biosurfactants comprising the steps of

[0019] A) Under conditions where microorganisms are able to synthesize biosurfactants,

[0020] The microorganism is contacted with a medium containing a sugar mixture consisting of

[0021] Glucose and at least one other sugar selected from the group consisting of fructose, isomaltose, maltose, maltulose and panose.

[0022] In the context of the present invention, "biosurfactants" are understood to mean all glycolipids produced by fermentation. The term "biosurfactant" also encompasses glycolipids that have been chemically or enzymatically modified after fermentation, as long as the glycolipid is retained structurally.

[0023] In the context of the present invention, the term "surfactant" is understood to mean an organic substance with interface-active properties which is capable of reducing the surface tension of water to below 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 Du Noüy ring method at 20° C.

[0024] Where average values ​​are stated hereinafter, these are number average values ​​unless otherwise stated.

[0025] Unless otherwise stated, the percentages are expressed as percent by weight.

[0026] Wherever measured values ​​are stated hereinafter, unless otherwise stated, these have been determined at a temperature of 25° C. and a pressure of 1013 mbar.

[0027] A preferred method according to the invention is characterized in that the sugar mixture 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 culture medium.

[0028] Other sugars that may be dissolved in the culture medium and are not part of the mixture in the method of the present invention may be one or more selected from lactose, trehalose, maltotriose, raffinose, sucrose.

[0029] In the present invention it is preferred if the process is characterized in that the sugar mixture represents at least 85% by weight, preferably at least 90% by weight, even more preferably at least 95% by weight of all available carbon sources present in the culture medium.

[0030] As mentioned above, the available carbon that may be present in the culture medium may be, for example, other carbohydrates than those listed above, in particular sugars, and / or lipophilic carbon sources such as fats, oils, partial glycerides, fatty acids, fatty alcohols, long chain saturated or unsaturated hydrocarbons.

[0031] Preferably, the method according to the invention is characterized in that the content of glucose in the sugar mixture is

[0032] 85.0 wt % to 99.5 wt %, preferably 90.0 wt % to 98.0 wt %, more preferably 93.0 wt % to 97.0 wt %, and

[0033] The total content of other sugars is

[0034] 0.5 wt % to 15.0 wt %, preferably 2.0 wt % to 10.0 wt %, more preferably 3.0 wt % to 7.0 wt %,

[0035] The weight percentages are based on the sugar mixture contained in the culture medium.

[0036] Preferably, the method according to the invention is characterized in that in the sugar mixture

[0037] The fructose content is

[0038] 0.1 wt % to 10.0 wt %, preferably 0.5 wt % to 8.0 wt %, more preferably 1.5 wt % to 6.0 wt %,

[0039] The weight percentages are based on the sugar mixture contained in the culture medium.

[0040] Preferably, the method according to the invention is characterized in that the content of isomaltose in the sugar mixture is

[0041] 0.1 wt % to 4.0 wt %, preferably 0.5 wt % to 3.0 wt %, more preferably 1.0 wt % to 2.0 wt %,

[0042] The weight percentages are based on the sugar mixture contained in the culture medium.

[0043] Preferably, the method according to the invention is characterized in that the content of maltose in the sugar mixture is

[0044] 0.1 wt % to 8.0 wt %, preferably 1.0 wt % to 6.0 wt %, more preferably 2.0 wt % to 5.0 wt %,

[0045] The weight percentages are based on the sugar mixture contained in the culture medium.

[0046] Preferably, the method according to the invention is characterized in that the content of maltulose in the sugar mixture is

[0047] 0.1 wt % to 2.0 wt %, preferably 0.3 wt % to 1.5 wt %, more preferably 0.4 wt % to 1.0 wt %,

[0048] The weight percentages are based on the sugar mixture contained in the culture medium.

[0049] Preferably, the method according to the invention is characterized in that in the sugar mixture

[0050] The content of panose is

[0051] 0.1 wt % to 3.0 wt %, preferably 0.5 wt % to 2.0 wt %, more preferably 0.7 wt % to 1.5 wt %,

[0052] The weight percentages are based on the sugar mixture contained in the culture medium.

[0053] Preferably, the method according to the invention is characterized in that, during at least some period of the method according to the invention, the biosurfactant present in the culture medium is above 50 g / l, preferably from 60 g / l to 200 g / l, more preferably from 80 g / l to 180 g / l.

[0054] Preferably, the method according to the invention is characterized in that the biosurfactant is selected from rhamnolipids, sophorolipids and glycolipids.

[0055] The term "rhamnolipid" in the context of the present invention is preferably understood to mean in particular compounds of the general formula (I) and their salts,

[0056]

[0057] in

[0058] mRL=2, 1 or 0,

[0059] nRL=1 or 0,

[0060] R 1RL and R 2RL= independently of one another are identical or different organic residues 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 selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH 2 ) o -CH 3 Those of the group consisting of wherein o=1 to 23, preferably 4 to 12.

[0061] If 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).

[0062] The term "diarhamnolipid" is understood in the context of the present invention to mean a compound of the general formula (I) or a salt thereof in which nRL=1.

[0063] The term "monorhamnolipid" is understood in the context of the present invention to mean a compound of the general formula (I) or a salt thereof in which nRL=0.

[0064] The different rhamnolipids are abbreviated according to the following nomenclature:

[0065] "DiRL-CXCY" is understood to mean a dirhamnolipid of the general formula (I) in which the residue R 1RL and R 2RL One = (CH 2 ) o -CH 3 , where o = X-4, and the remaining residue R 1 or R 2 =(CH 2 ) o -CH 3 , where o=Y-4.

[0066] "Mono RL-CXCY" is understood to mean a monorhamnolipid of the general formula (I) in which the residue R 1RL and R 2RL One = (CH 2 ) o -CH 3 , where o = X-4, and the remaining residue R 1RL or R 2RL =(CH 2 ) o -CH 3 , where o=Y-4.

[0067] Therefore, the nomenclature used does not distinguish between "CXCY" and "CYCX".

[0068] For rhamnolipids where mRL=0, mono-RL-CX or di-RL-CX was used accordingly.

[0069] If one of the above labels X and / or Y is accompanied by ":Z", this means that the respective residue R 1RL and / or R 2RL Equivalent to an unbranched, unsubstituted hydrocarbon residue having X-3 or Y-3 carbon atoms with Z double bonds.

[0070] In the context of the present invention, the term "sophorolipid" is preferably understood to mean compounds of the general formula (IIa) and (IIb) and their salts

[0071]

[0072]

[0073] in

[0074] R 1SL =H or CO-CH 3 ,

[0075] R 2SL =H or CO-CH 3 ,

[0076] R 3SL = a divalent organic moiety comprising 6 to 32 carbon atoms and which is unsubstituted or substituted by hydroxyl functions, is unbranched and optionally comprises 1 to 3 double or triple bonds,

[0077] R 4SL =H、CH 3 or a monovalent organic radical which contains from 2 to 10 carbon atoms and which is unsubstituted or substituted by a hydroxyl function, is unbranched and which optionally contains from 1 to 3 double or triple bonds, and

[0078] nSL=1 or 0.

[0079] Sophorolipids can be produced according to the invention in their acid form or in their lactone form.

[0080] A preferred method according to the invention produces sophorolipids wherein the weight ratio of the lactone form to the acid form is in the range of 20:80 to 80:20, particularly preferably in the range of 30:70 to 40:60.

[0081] To determine the content of sophorolipids in the acid or lactone form in a preparation, reference is made to EP 1 41 1 1 1 B1 , page 8, paragraph

[0053] .

[0082] In connection with the present invention, the term "glycolipid" is preferably understood to mean compounds of the general formula (III) and their salts,

[0083]

[0084] in

[0085] mGL=1 or 0,

[0086] R 1GL and R 2GL = independently of one another are identical or different organic radicals 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 selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH 2 ) o -CH 3 A group consisting of wherein o=1 to 23, preferably 4 to 12.

[0087] The different glycolipids are abbreviated according to the following nomenclature:

[0088] "GL-CXCY" is understood to mean a glycolipid of the general formula (III) in which the radical R 1GL and R 2GL One = (CH 2 ) o -CH 3 , where o = X-4, and the remaining groups R 1GL or R 2GL =(CH 2 ) o -CH 3 , where o=Y-4.

[0089] Therefore, the nomenclature used does not distinguish between "CXCY" and "CYCX".

[0090] If one of the above symbols X and / or Y carries ":Z", this means that the respective radical R 1GL and / or R 2GL = an unbranched, unsubstituted hydrocarbon radical having X-3 or Y-3 carbon atoms with Z double bonds.

[0091] Preferably, the process according to the invention is characterized in that the biosurfactant is chosen from rhamnolipids and the microorganism is chosen from Pseudomonas putida, Pseudomonas aeruginosa, Serratia rubidaea SNAU02, Escherichia coli and Burkholderia thailandensis.

[0092] 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 during at least a period of time according to the process according to the invention is higher than 80 g / l, preferably between 80 g / l and 180 g / l, more preferably between 100 g / l and 160 g / l.

[0093] For example, EP2786743 and EP2787065 disclose suitable process parameters and microorganisms for preparing rhamnolipids.

[0094] Fermentation of Pseudomonas, especially Pseudomonas aeruginosa, which is preferably non-transgenic cells, is a technology that has been disclosed in the 1980s, as described in, for example, EP0282942 and DE4127908, and can be carried out within the scope of the present invention by using a special sugar mixture as described. Pseudomonas aeruginosa cells that have been improved by genetic modification for higher rhamnolipid titers can also be used in the context of the present invention; for example, Lei et al. disclose such cells in Biotechnol Lett. 2020 Jun; 42 (6): 997-1002.

[0095] Preferably, the process according to the invention is characterized in that the biosurfactant is chosen from sophorolipids and the microorganism is chosen from Starmerella bombicola, Candida bogoriensis, Candida magnoliae, Candida batistae, Candida apicola or Wickerhamiella domericqiae.

[0096] If in the method of the present invention, the biosurfactant is selected from sophorolipids and the microorganism is selected from yeast, the sugar mixture preferably accounts for at least 25% by weight of all available carbon sources present in the culture medium, preferably at least 45% by weight, even more preferably at least 70% by weight. In this embodiment of the invention, it is preferred that other available carbon present in the culture medium is selected from fats, oils, partial glycerides of fatty acids, fatty acids, fatty alcohols and long-chain, preferably C8 to C32 saturated or unsaturated hydrocarbons.

[0097] Preferably, the method according to the invention is characterized in that the biosurfactant is selected from sophorolipids and that the concentration of all sophorolipids present in the culture medium is higher than 80 g / l, preferably from 80 g / l to 180 g / l, more preferably from 100 g / l to 160 g / l during at least a period of time according to the method according to the invention.

[0098] Preferably, the method according to the invention is characterized in that the biosurfactant is chosen from glycolipids and the microorganism is chosen from Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli, Serratia rubrum (preferably strain ATCC 27593) and Burkholderia thailandensis.

[0099] Preferably, the method according to the invention is characterized in that the biosurfactant is selected from glycolipids and that the concentration of all glycolipids present in the culture medium during at least a period of time according to the method according to the invention is higher than 50 g / l, preferably between 50 g / l and 120 g / l, more preferably between 60 g / l and 100 g / l.

[0100] For example, WO2019154970 discloses method parameters and microorganisms suitable for preparing glycolipids.

[0101] Preferably, the method according to the invention is characterized in that it comprises a step B) purifying the biosurfactant, preferably by separating it from the microorganism and / or at least part of the culture medium.

[0102] The examples cited below illustrate the invention by way of example, without any intention of limiting the invention to the embodiments specified in the examples, the scope of application of the invention being apparent from the entire description and claims. Example

[0103] Example 1: Production of rhamnolipids on glucose and fructose using Pseudomonas

[0104] Fermentation was performed using the Pseudomonas putida strain pBBR1MCS2-Plac-rhlABC-T-Ptac-rhlC-T, the preparation of which is described in US2014296168, including the rhamnolipid biosynthesis genes RhIA, RhIB and RhIC. Pre-culture was performed in shake flasks as described in WO2012013554A1. For the main culture, mineral medium M9 was also used. The medium consisted of 2% (w / v) glucose, 0.3% (w / v) KH 2 PO 4 , 0.679% Na 2 HPO 4 , 0.05% (w / v) NaCl, 0.2% (w / v) NH 4 Cl, 0.049% (w / v) MgSO 4 x 7H 2 O and 0.1% (v / v) trace element solution. It consists of 1.78% (w / v) FeSO 4 x7h 2 O, 0.191% (w / v) MnCl 2 x7h 2 O, 3.65% (w / v) HCl, 0.187% (w / v) ZnSO 4 x 7H 2 O, 0.084% (v / v) Na EDTA x 2H 2 O, 0.03% (v / v) H 3 BO 3 、0.025%(w / v)Na 2 MoO 4 x 2H 2 O and 0.47% (w / v) CaCl 2 x 2H 2 O. 4 The pH value of the medium was adjusted to 7.4 with 4% HCl and then sterilized by autoclaving (121° C., 20 min).

[0105] Fermentation was performed in a 2-liter fermenter in a carbon-limited manner via glucose feed input. The glucose feed input was referenced to the dissolved oxygen signal. Dissolved oxygen was adjusted to 20% saturation via the stirrer speed. 4 The addition of OH adjusted the pH to 7. To prevent and measure foaming of the fermentation broth, an antifoaming agent DOW Corning 1500 was added as needed. The fermentation was carried out for 4 days.

[0106] In addition, a medium in which 2% (w / v) glucose was replaced by 1.98% (w / v) glucose and 0.02% (w / v) fructose was prepared and used, which was called M9*F.

[0107] In addition, the wild-type strain P. aeruginosa (P. aeruginosa) PAO1 producing rhamnolipids was used.

[0108]

[0109] The results showed that partial replacement of glucose with fructose reduced the consumption of foaming and antifoaming agents, while the total rhamnolipid production remained unchanged.

[0110] Example 2: Production of rhamnolipids on glucose and maltose using Pseudomonas

[0111] Example 1 was repeated, but a medium was prepared and used in which 2% (w / v) glucose was replaced by 1.96% (w / v) glucose and 0.04% (w / v) maltose, which was called M9*M.

[0112] In addition, the wild-type strain Pseudomonas aeruginosa PAO1 producing rhamnolipids was used.

[0113]

[0114] Results showed that partial replacement of glucose with maltose reduced the consumption of foaming and antifoaming agents, while total rhamnolipid production remained unchanged, but to a lesser extent than replacement with fructose.

[0115] After the fermented cells were separated by centrifugation at 10 000 g, concentrated H 2 SO 4 The pH of the fermentation broth was adjusted to 3.1. By further centrifugation at 500 g, a pasty solid concentrate was obtained with a rhamnolipid fraction of 45% by weight and a viscosity of >10 000 mPas.

[0116] Relative color is determined via the platinum-cobalt scale:

[0117]

[0118] The results showed that replacing part of the glucose with maltose resulted in a purer rhamnolipid composition.

[0119] Example 3: Production of rhamnolipids on glucose, maltose and fructose using Pseudomonas

[0120] Example 1 was repeated, but a medium was prepared and used in which 2% (w / v) glucose was replaced by 1.96% (w / v) glucose and 0.03% (w / v) maltose and 0.01% (w / v) fructose, which was called M9*FM.

[0121] In addition, the wild-type strain Pseudomonas aeruginosa PAO1 producing rhamnolipids was used.

[0122]

[0123]

[0124] The results showed that partial replacement of glucose with maltose and fructose synergistically reduced the consumption of foaming and antifoaming agents, while the total rhamnolipid production remained unchanged.

[0125] After the fermented cells were separated by centrifugation at 10 000 g, concentrated H 2 SO 4 The pH of the fermentation broth was adjusted to 3.1. By further centrifugation at 500 g, a pasty solid concentrate was obtained with a rhamnolipid fraction of 45% by weight and a viscosity of >10 000 mPas.

[0126] Relative color is determined via the platinum-cobalt scale:

[0127]

[0128] The results showed that replacing part of the glucose with maltose resulted in a purer rhamnolipid composition.

[0129] Example 4: Production of rhamnolipids on glucose and isomaltose using Pseudomonas

[0130] Example 2 was repeated, but a medium was prepared and used in which 2% (w / v) glucose was replaced by 1.96% (w / v) glucose and 0.04% isomaltose, which was designated M9*I.

[0131] Relative color is determined via the platinum-cobalt scale:

[0132]

[0133] The results showed that replacing part of the glucose with isomaltose resulted in a purer rhamnolipid composition.

[0134] Example 5: Production of rhamnolipids using E. coli on glucose and panose or glucose and maltulose

[0135] The production of rhamnolipids was performed as described in Example 10 of EP2598646 in recombinant E. coli W3110 pBBR1MCS-2::ABC cells.

[0136] CMP medium was used. This medium consisted of 2% (w / v) glucose, 0.007% (w / v) KH 2 PO 4 、0.11%Na 2 HPO 4 x 2H 2 O, 0.2% (w / v) NaNO 3 、0.04%(w / v)MgSO 4 x H 2 O, 0.01% (w / v) CaCl 2 x 2H 2 O and 0.2% (v / v) trace element solution. It consists of 0.2% (w / v) FeSO 4 x 7H 2 O, 0.15% (w / v) MnSO 4 x H 2 O and 0.06% (w / v) (NH 4 )MO 7 O 24 x 4H 2 O composition. The pH value of the medium was adjusted to 6.7 using NaOH.

[0137] Additionally, culture media were prepared and used in which 2% (w / v) glucose was replaced by 1.98% (w / v) glucose and 0.02% panose, which was designated as CMP*P; and in which 2% (w / v) glucose was replaced by 1.99% (w / v) glucose and 0.01% maltulose, which was designated as CMP*M.

[0138] The viscosity of the final fermentation broth was measured using a rheometer with concentric cylinder geometry (Anton Haak) at a constant shear rate of 100 1 / s.

[0139]

[0140] The results show that replacing part of the glucose with panose or maltulose results in lower fermentation broth viscosity. This is an advantage because less energy needs to be introduced into the vessel during the fermentation process.

[0141] Example 6: Production of glycolipids on glucose and fructose using Pseudomonas

[0142] The production of glycolipids was carried out as described in Example 2 of WO2019154970 in recombinant Pseudomonas putida BS-PP-368 cells in a 1 liter Dasgip fermenter.

[0143] In addition, the pure 500 g / L glucose feed ("G") was replaced by 485 g / L glucose plus 15 g / L fructose (G*F).

[0144]

[0145] The results showed that replacing part of the glucose with fructose could reduce the consumption of foaming and antifoaming agents, while the total glycolipid production remained unchanged.

[0146] Example 7: Production of glycolipids on glucose and maltose using Pseudomonas

[0147] The production of glycolipids was carried out as described in Example 2 of WO2019154970 in recombinant Pseudomonas putida BS-PP-368 cells in a 1 liter Dasgip fermenter.

[0148] In addition, the pure 500 g / L glucose feed ("G") was replaced by 490 g / L glucose plus 10 g / L maltose (G*M).

[0149] The cells were separated by centrifugation at 10.000 g for 20 min. The fermentation broth was separated into supernatant and the supernatant was separated by adding concentrated H 2 SO 4 The pH was adjusted to 3.1.

[0150] After a second centrifugation at 5.000 g for 20 minutes, the aqueous upper phase was separated, while the remaining lower phase was a concentrate with a glycolipid content of more than 50% by weight.

[0151] Relative color is determined via the platinum-cobalt scale:

[0152]

[0153] The results showed that replacing part of the glucose with maltose resulted in a purer rhamnolipid composition.

[0154] Example 8: Production of glycolipids by Pseudomonas on glucose and panose or glucose and maltose or glucose and panose and maltose

[0155] Production of glycolipids was performed as described in Example 7.

[0156] In addition, the pure 500 g / L glucose feed ("G") was replaced by a 496 g / L glucose plus 4 g / L panose feed (G*P), a 496 g / L glucose plus 4 g / L maltose (G*M) feed, and a 496 g / L glucose plus 2 g / L maltose plus 2 g / L panose (G*CP) feed.

[0157] The viscosity of the final fermentation broth was measured using a rheometer with concentric cylinder geometry at a constant shear rate of 100 1 / s.

[0158]

[0159] The results showed that replacing part of the glucose with panose or maltose resulted in a reduction in the viscosity of the fermentation broth, and that panose and maltose acted synergistically. This is helpful because less energy needs to be introduced into the vessel during the fermentation process.

[0160] Example 9: Production of sophorolipids on glucose and fructose using S. bombicola

[0161] The method for producing sophorolipids is as described in Example 12 of WO2011061032, and sophorolipids are produced in wild-type strain Candida albicans ATCC 22214 and recombinant strain Candida albicans ATCC 22214sbg3-hyg. The culture medium for producing sophorolipids SL consists of 0.1% KH 2 PO 4 、0.5% MgSO 4 x 7H 2 O, 0.01% FeCl 3 , 0.01% NaCl, 0.4% yeast extract, 0.1% urea, 10.5% rapeseed oil and 10% glucose. The pH value was adjusted to 4.5.

[0162] In addition, a medium in which 10% (w / v) of glucose was replaced by 9.9% (w / w) of glucose and 0.1% of fructose was prepared and used, which was called SL*F.

[0163] The relative foam height and the sophorolipid concentration in the flask were determined.

[0164]

[0165] The results showed that partial replacement of glucose by fructose prevented foaming, while the total sophorolipid production remained unchanged.

[0166] Example 10: Production of sophorolipids using Candida albicans on glucose and isomaltose or glucose and maltose

[0167] Sophorolipids were prepared according to Example 2 of Example 2 WO2021236904 (M).

[0168] A second similar experiment was performed using a 99:1 mixture of glucose and isomaltose (M*I) instead of glucose.

[0169] A second similar experiment was performed using a 99:1 mixture of glucose and maltose (M*M) instead of glucose.

[0170] Relative color is determined via the platinum-cobalt scale:

[0171]

[0172] The results showed that replacing part of the glucose with isomaltose or maltose resulted in a purer sophorolipid composition.

Claims

1. A method for the fermentation production of biosurfactants comprising the steps of A) Under conditions where microorganisms are able to synthesize biosurfactants, The microorganism is contacted with a medium containing a sugar mixture consisting of Glucose and at least one other sugar selected from the group consisting of fructose, isomaltose, maltose, maltulose and panose.

2. The method according to claim 1, characterized in that The sugar mixture accounts for 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 culture medium.

3. The method according to claim 1 or 2, characterized in that: The sugar mixture comprises at least 85%, preferably at least 90%, even more preferably at least 95% by weight of all available carbon sources present in the culture medium.

4. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of glucose is 85.0 wt% to 99.5 wt%, preferably 90.0 wt% to 98.0 wt%, more preferably 93.0 wt% to 97.0 wt%, and The total content of other sugars is 15.0 wt% to 0.5 wt%, preferably 10.0 wt% to 2.0 wt%, more preferably 7.0 wt% to 3.0 wt%, The weight percentages are based on the sugar mixture contained in the culture medium.

5. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of fructose is 0.1 wt% to 10.0 wt%, preferably 0.5 wt% to 8.0 wt%, more preferably 1.5 wt% to 6.0 wt%, The weight percentages are based on the sugar mixture contained in the culture medium.

6. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of isomaltose is 0.1 wt% to 4.0 wt%, preferably 0.5 wt% to 3.0 wt%, more preferably 1.0 wt% to 2.0 wt%, The weight percentages are based on the sugar mixture contained in the culture medium.

7. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of maltose is 0.1 wt% to 8.0 wt%, preferably 1.0 wt% to 6.0 wt%, more preferably 2.0 wt% to 5.0 wt%, The weight percentages are based on the sugar mixture contained in the culture medium.

8. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of maltulose is 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 percentages are based on the sugar mixture contained in the culture medium.

9. The method according to any one of the preceding claims, characterized in that In the sugar mixture The content of panose is 0.1 wt% to 3.0 wt%, preferably 0.5 wt% to 2.0 wt%, more preferably 0.7 wt% to 1.5 wt%, The weight percentages are based on the sugar mixture contained in the culture medium.

10. The method according to any one of the preceding claims, characterized in that The biosurfactant is selected from rhamnolipids, sophorolipids and glycolipids.

11. The method according to any one of the preceding claims, characterized in that The biosurfactant is selected from rhamnolipids, and the microorganism is selected from Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli and Burkholderia thailandensis, or The biosurfactant is selected from sophorolipids, and the microorganism is selected from Candida albicans, Candida bogoria, Candida pasta, Candida mellifera, or Candida pseudoviciliformis, or The biosurfactant is selected from glycolipids, and the microorganism is selected from Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli and Burkholderia thailandensis.

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