PROCESS FOR THE FERMENTATIVE PRODUCTION OF A BIOSURFACTANT

Abstract

A process for the fermentative production of a biosurfactant brings a microorganism into contact with a medium that includes a mixture of saccharides. The mixture of saccharides includes glucose and at least one further saccharide which may be fructose, isomaltose, maltose, maltulose, or panose. The microorganism and the medium are provided under conditions where the microorganism is capable of synthesizing the biosurfactant.

Claims

1. A process for the fermentative production of a biosurfactant comprising: bringing a microorganism into contact with a medium comprising a mixture of saccharides consisting of glucose and at least one further saccharide selected from the group consisting of fructose, isomaltose, maltose, maltulose, and panose, under conditions where the microorganism is capable of synthesizing the biosurfactant.

2. The process according to claim 1, wherein said mixture of saccharides accounts for at least 85 wt. %, of all saccharides dissolved in the medium.

3. The process according to claim 1, wherein said mixture of saccharides accounts for at least 85 wt. % of all utilizable carbon sources present in the medium.

4. The process according to claim 1 wherein said mixture of saccharides comprises glucose in an amount of from 85.0 wt. % to 99.5 wt. %, and a sum of the further saccharides is contained in an amount of from 15.0 wt. % to 0.5 wt. %, wherein the percentages by weight refer to said mixture of saccharides contained in the medium.

5. The process according to claim 1, wherein the mixture of saccharides comprises fructose in an amount of from 0.1 wt. % to 10.0 wt. %, and wherein the percentage by weight refers to said mixture of saccharides contained in the medium.

6. The process according to claim 1, wherein the mixture of saccharides comprises isomaltose in an amount of from 0.1 wt. % to 4.0 wt. %, and wherein the percentage by weight refers to said mixture of saccharides contained in the medium.

7. The process according to claim 1, wherein the mixture of saccharides comprises maltose in an amount of from 0.1 wt. % to 8.0 wt. %, and wherein the percentage by weight refers to said mixture of saccharides contained in the medium.

8. The process according to claim 1, wherein the mixture of saccharides comprises maltulose in an amount of from 0.1 wt. % to 2.0 wt. %, and wherein the percentage by weight refers to said mixture of saccharides contained in the medium.

9. The process according to claim 1, wherein the mixture of saccharides comprises panose in an amount of from 0.1 wt. % to 3.0 wt. %, and wherein the percentage by weight refers to said mixture of saccharides contained in the medium.

10. The process according to claim 1, wherein the biosurfactant is selected from the group consisting of rhamnolipids, sophorolipids and glucolipids.

11. The process according to claim 1, wherein the biosurfactant is selected from the group consisting of rhamnolipids and the microorganism is selected from the group consisting of Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli and Burkholderia thailandensis, or the biosurfactant is selected from the group consisting of sophorolipids and the microorganism is selected from the group consisting of Starmerella bombicola, Candida bogoriensis, Candida batistae, Candida apicola and Wickerhamiella domericqiae, or the biosurfactant is selected from the group consisting of glucolipids and the microorganism is selected from the group consisting of Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli and Burkholderia thailandensis.

12. The process according to claim 2, wherein said mixture of saccharides accounts for at least 95 wt. % of all saccharides dissolved in the medium.

13. The process according to claim 3, wherein said mixture of saccharides accounts for at least 95 wt. % of all utilizable carbon sources present in the medium.

14. The process according to claim 4, wherein said mixture of saccharides comprises glucose in an amount of from 93.0 wt. % to 97.0 wt. %, and the sum of the further saccharides is contained in an amount of from 7.0 wt. % to 3.0 wt. %.

15. The process according to claim 5, wherein the mixture of saccharides comprises fructose in an amount of from 1.5 wt. % to 6.0 wt. %.

16. The process according to claim 6, wherein the mixture of saccharides comprises isomaltose in an amount of from 1.0 wt. % to 2.0 wt. %.

17. The process according to claim 7, wherein the mixture of saccharides comprises maltose in an amount of from 2.0 wt. % to 5.0 wt. %.

18. The process according to claim 8, wherein the mixture of saccharides comprises maltulose in an amount of from 0.4 wt. % to 1.0 wt. %.

19. The process according to claim 9, wherein the mixture of saccharides comprises panose in an amount of from 0.7 wt. % to 1.5 wt. %.

Description

DESCRIPTION OF THE INVENTION

[0011] It was found that, surprisingly, by using a well-defined mixture of saccharides, the problem underlying the invention could be solved.

[0012] The present invention therefore provides a process for the fermentative production of a biosurfactant as described in claim 1.

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

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

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

[0016] Another advantage is that the reduced viscosity and defoamer consumption lead to lower energy consumption of the mechanical agitation during fermentation

[0017] An additional advantage is that final product colour (darkening) is reduced

[0018] The instant invention thus provides a process for the fermentative production of a biosurfactant comprising the step of [0019] A) bringing a microorganism into contact with a medium containing a mixture of saccharides consisting of [0020] glucose and at least one further saccharide selected from the group of fructose, isomaltose, maltose, maltulose and panose, [0021] under conditions where the microorganism is capable of synthesizing the biosurfactant.

[0022] Within the context of the present invention, biosurfactants are understood as meaning all glycolipids produced by fermentation. The term biosurfactant also covers glycolipids that are chemically or enzymatically modified after fermentation, as long as structurally a glycolipid remains.

[0023] In the context of the present invention, the terms surfactant is understood to mean organic substances having interface-active properties that have the ability to reduce the surface tension of water at 20 C. and at a concentration of 0.5% by weight based on the overall composition to below 45 mN/m. Surface tension is determined by the DuNoy ring method at 20 C.

[0024] Where average values are stated hereinbelow, then, unless stated otherwise, these are number-averaged average values.

[0025] Unless stated otherwise, percentages are data in percent by weight.

[0026] Wherever measurement values are stated hereinbelow, then, unless stated otherwise, these have been determined at a temperature of 25 C. and a pressure of 1013 mbar.

[0027] A preferred process according to the instant invention is characterized in, that said mixture of saccharides accounts for at least 85 wt. %, preferably for at least 90 wt. %, even more preferably for at least 95 wt. %, of all saccharides dissolved in the medium.

[0028] Other saccharides that may be dissolved in the medium and are not part of said mixture in the process of the instant invention can be one or more selected from the group of lactose, trehalose, maltotriose, raffinose, saccharose.

[0029] It is preferred in the instant invention, if the process is characterized in, that said mixture of saccharides accounts for at least 85 wt. %, preferably for at least 90 wt. %, even more preferably for at least 95 wt. %, of all utilizable carbon source present in the medium.

[0030] Utilizable carbon that can be present in the medium as described above may be for example other carbohydrates than 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 process according to the instant invention is characterized in, that in said mixture of saccharides [0032] glucose is contained in an amount of [0033] from 85.0 wt. % to 99.5 wt. %, preferably from 90.0 wt. % to 98.0 wt. %, more preferably from 93.0 wt. % to 97.0 wt. %, and [0034] the sum of the further saccharides is contained in an amount of [0035] from 0.5 wt. % to 15.0 wt. %, preferably from 2.0 wt. % to 10.0 wt. %, more preferably from 3.0 wt. % to 7.0 wt. %, [0036] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0037] Preferably, the process according to the instant invention is characterized in, that in the mixture of saccharides [0038] fructose is contained in an amount of [0039] from 0.1 wt. % to 10.0 wt. %, preferably from 0.5 wt. % to 8.0 wt. %, more preferably from 1.5 wt. % to 6.0 wt. %, [0040] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0041] Preferably, the process according to the instant invention is characterized in, that in the mixture of saccharides [0042] isomaltose is contained in an amount of [0043] from 0.1 wt. % to 4.0 wt. %, preferably from 0.5 wt. % to 3.0 wt. %, more preferably from 1.0 wt. % to 2.0 wt. %, [0044] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0045] Preferably, the process according to the instant invention is characterized in, that in the mixture of saccharides [0046] maltose is contained in an amount of [0047] from 0.1 wt. % to 8.0 wt. %, preferably from 1.0 wt. % to 6.0 wt. %, more preferably from 2.0 wt. % to 5.0 wt. %, [0048] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0049] Preferably, the process according to the instant invention is characterized in, that in the mixture of saccharides [0050] maltulose is contained in an amount of [0051] from 0.1 wt. % to 2.0 wt. %, preferably from 0.3 wt. % to 1.5 wt. %, more preferably from 0.4 wt. % to 1.0 wt. %, [0052] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0053] Preferably, the process according to the instant invention is characterized in, that in the mixture of saccharides [0054] panose is contained in an amount of [0055] from 0.1 wt. % to 3.0 wt. %, preferably from 0.5 wt. % to 2.0 wt. %, more preferably from 0.7 wt. % to 1.5 wt. %, [0056] wherein the percentage by weight refer to said mixture of saccharides contained in the medium.

[0057] Preferably the process according to the instant invention is characterized in, that the biosurfactant present in the medium is higher than 50 g/l, preferably from 60 g/l to 200 g/l, more preferably from 80 g/l to 180 g/l, during at least some period of time of the process according to the instant invention.

[0058] Preferably, the process according to the instant invention is characterized in, that the biosurfactant is selected from the group of rhamnolipids, sophorolipids and glucolipids.

[0059] The term rhamnolipids in the context of the present invention preferably is understood to mean particularly compounds of the general formula (I) and salts thereof,

##STR00001## [0060] where [0061] mRL=2, 1 or 0, [0062] nRL=1 or 0, [0063] R.sup.1RL and R.sup.2L=mutually independently, identical or different, organic residues having 2 to 24, preferably 5 to 13 carbon atoms, in particular optionally branched, optionally substituted, particularly hydroxy-substituted, optionally unsaturated, in particular optionally mono-, bi- or tri-unsaturated alkyl residues, preferably those selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH.sub.2).sub.oCH.sub.3 where o=1 to 23, preferably 4 to 12.

[0064] If nRL=1, the glycosidic bond between the two rhamnose units is preferably in the -configuration. The optically active carbon atoms of the fatty acids are preferably present as R-enantiomers (e.g. (R)-3-{(R)-3-[2-O-(-L-rhamnopyranosyl)--L-rhamnopyranosyl]oxydecanoyl}oxydecanoate).

[0065] The term di-rhamnolipid in the context of the present invention is understood to mean compounds of the general formula (I) or salts thereof, where nRL=1.

[0066] The term mono-rhamnolipid in the context of the present invention is understood to mean compounds of the general formula (I) or salts thereof, where nRL=0.

[0067] Distinct rhamnolipids are abbreviated according to the following nomenclature:

[0068] diRL-CXCY are understood to mean di-rhamnolipids of the general formula (I), in which one of the residues R.sup.1RL and R.sup.2L=(CH.sub.2).sub.oCH.sub.3 where o=X-4 and the remaining residue R.sup.1 or R.sup.2=(CH.sub.2).sub.oCH.sub.3 where o=Y-4.

[0069] monoRL-CXCY are understood to mean mono-rhamnolipids of the general formula (I), in which one of the residues R.sup.1RL and R.sup.2L=(CH.sub.2).sub.oCH.sub.3 where o=X-4 and the remaining residue R.sup.1RL or R.sup.2RL=(CH.sub.2).sub.oCH.sub.3 where o=Y-4.

[0070] The nomenclature used therefore does not distinguish between CXCY and CYCX.

[0071] For rhamnolipids where mRL=0, monoRL-CX or diRL-CX is used accordingly.

If one of the abovementioned indices X and/or Y is provided with :Z, this signifies that the respective residue R.sup.1RL and/or R.sup.2RL is equal to an unbranched, unsubstituted hydrocarbon residue having X-3 or Y-3 carbon atoms having Z double bonds.

[0072] In the context of the present invention, the term sophorolipids preferably is understood as meaning compounds of the general formulae (IIa) and (IIb) and salts thereof

##STR00002## [0073] where [0074] R.sup.1SL=H or COCH.sub.3, [0075] R.sup.2SL=H or COCH.sub.3, [0076] R.sup.3SL=a divalent organic moiety which comprises 6 to 32 carbon atoms and which is unsubstituted or substituted by hydroxyl functions, is unbranched and optionally comprises one to three double or triple bonds, [0077] R.sup.4SL=H, CH.sub.3 or a monovalent organic radical which comprises 2 to 10 carbon atoms and which is unsubstituted or substituted by hydroxyl functions, which is unbranched and which optionally comprises one to three double or triple bonds, and [0078] nSL=1 or 0.

[0079] Sophorolipids may be produced in accordance with the invention in their acid form or their lactone form.

[0080] Preferred processes according to the instant invention produce a sophorolipid in which the ratio by weight of lactone form to acid form is in the range of 20:80 to 80:20, especially preferably in the ranges of 30:70 to 40:60.

[0081] To determine the content of sophorolipids in the acid or lactone form in a formulation, refer to EP14111111B, page 8, paragraph [0053].

[0082] In connection with the present invention, the term glucolipids preferably is understood as meaning compounds of the general formula (III) and salts thereof,

##STR00003## [0083] where [0084] mGL=1 or 0, [0085] R.sup.1GL and R.sup.2GL=independently of one another identical or different organic radical having 2 to 24 carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy-substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated, alkyl radical, preferably one selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH.sub.2).sub.oCH.sub.3 where o=1 to 23, preferably 4 to 12.

[0086] Distinct glucolipids are abbreviated according to the following nomenclature:

[0087] GL-CXCY is understood as meaning glucolipids of the general formula (III) in which one of the radicals R.sup.1GL and R.sup.2GL=(CH.sub.2).sub.oCH.sub.3 where o=X-4 and the remaining radical R.sup.1GL or R.sup.2GL=(CH.sub.2).sub.oCH.sub.3 where o=Y-4.

[0088] The nomenclature used thus does not differentiate between CXCY and CYCX.

[0089] If one of the aforementioned indices X and/or Y is provided with :Z, then this means that the respective radical R.sup.1GL and/or R.sup.2GL=an unbranched, unsubstituted hydrocarbon radical with X-3 or Y-3 carbon atoms having Z double bonds.

[0090] Preferably the process according to the instant 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 rubidaea SNAU02, Escherichia coli and Burkholderia thailandensis.

[0091] Preferably the process according to the instant invention is characterized in, that the biosurfactant is selected from rhamnolipids and that the concentration of all rhamnolipids present in the 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 some period of time of the process according to the instant invention.

[0092] Methodparameters and microorganisms suited for preparing rhamnolipids are disclosed, for example, in EP2786743 and EP2787065.

[0093] Fermentation of Pseudomonas, especially Pseudomonas aeruginosa, which are preferably non genetically modified cells, a technology already disclosed in the eighties, as documented e.g. in EP0282942 and DE4127908 can be conducted just as described within the scope of the instant invention by using the special mixture of saccharides. Pseudomonas aeruginosa cells which have been improved for higher rhamnolipid titres by genetical modification can also be used in the context of the instant invention; such cells have for example been disclosed by Lei at al. in Biotechnol Lett. 2020 June; 42(6):997-1002.

[0094] Preferably the process according to the instant invention is characterized in, that the biosurfactant is selected from sophorolipids and the microorganism is selected from the group of Starmerella bombicola, Candida bogoiensis, Candida magnoliae, Candida babstae, Candida apicola or Wickerhamiella domericqiae.

[0095] If in the process of the instant invention the biosurfactant is selected from sophorolipids and the microorganism is selected from yeast, then mixture of saccharides preferably accounts for at least 25 wt. %, preferably for at least 45 wt. %, even more preferably for at least 70 wt. %, of all utilizable carbon source present in the medium. Other utilizable carbon that is preferably present in the medium in this embodiment of the instant invention 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.

[0096] Preferably the process according to the instant invention is characterized in, that the biosurfactant is selected from sophorolipids and that the concentration of all sophorolipids present in the 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 some period of time of the process according to the instant invention.

[0097] Preferably the process according to the instant 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, Serrdia rubidaea, preferably strain ATCC 27593, and Burkholderia thailandensis.

[0098] Preferably the process according to the instant invention is characterized in, that the biosurfactant is selected from glucolipids and that the concentration of all glucolipids present in the medium is higher than 50 g/l, preferably from 50 g/l to 120 g/l, more preferably from 60 g/l to 100 g/l, during at least some period of time of the process according to the instant invention.

[0099] Methodparameters and microorganisms suited for preparing of glucolipids are disclosed, for example in WO2019154970.

[0100] Preferably the process according to the instant invention is characterized in, that it comprises the step of B) purifying the biosurfactant, preferably by separating it from the microorganism and/or at least parts of the medium.

[0101] The examples adduced hereinafter describe the present invention by way of example, without any intention that the invention, the scope of application of which is apparent from the entirety of the description and the claims, be restricted to the embodiments specified in the examples.

EXAMPLES

Example 1: Production of Rhamnolipids with Pseudomonas on Glucose and Fructose

[0102] A fermentation is carried out using a Pseudomonas putida strain pBBR1MCS2-Plac-rhIABC-T-Pfac-rhIC-T, the preparation of which is described in US2014296188, comprising the rhamnolipid biosynthesis genes RhIA, RhIB and RhIC. The preculture is carried out in a shaking flask as described in WO2012013554A1. For the main culture, a mineral medium M9 was likewise employed. This medium consists of 2% (w/v) glucose, 0.3% (w/v) KH.sub.2PO.sub.4, 0.679% Na.sub.2HPO.sub.4, 0.05% (w/v) NaCl, 0.2% (w/v) NH.sub.4Cl, 0.049% (w/v) MgSO.sub.47 H.sub.2O and 0.1% (v/v) of a trace element solution. This consists of 1.78% (w/v) FeSO.sub.47 H.sub.2O, 0.191% (w/v) MnCl.sub.27 H.sub.2O, 3.65% (w/v) HCl, 0.187% (w/v) ZnSO.sub.47 H.sub.2O, 0.084% (v/v) Na EDTA2H.sub.2O, 0.03% (v/v) H.sub.3BO.sub.3, 0.025% (w/v) Na.sub.2MoO.sub.42 H.sub.2O and 0.47% (w/v) CaCl.sub.2)2H.sub.2O. The pH of the medium is adjusted to 7.4 using NH.sub.4OH and the medium is subsequently sterilized by means of an autoclave (121 C., 20 min).

[0103] The fermentation is conducted in a 2 litre fermenter in a carbon-limited manner via a glucose feed input. The glucose feed input takes place by reference to the dissolved oxygen signal. The dissolved oxygen was regulated at 20% saturation via the stirrer speed. The pH is regulated to 7 via a pH electrode and addition of NH.sub.4OH. To prevent and measure the foaming of the fermentation broth, the defoamer DOW Corning 1500 was added as required. The fermentation was conducted over 4 days.

[0104] Furthermore, a medium is prepared and used, where the 2% (w/v) glucose are substituted by 1.98% (w/v) glucose and 0.02% (w/v) fructose, which is called M9*F.

[0105] Furthermore, a rhamnolipid producing wildtype strain, P. aeruginosa PAO1, is used.

TABLE-US-00001 Relative defoamer amount used Strain M9 M9*F strain pBBR1MCS2-Plac- 100 92 rhlABC-T-Ptac-rhlC-T PAO1 100 93

[0106] The results show, that the substitution of parts of the glucose by fructose reduces foaming and defoamer consumption, whereas the overall rhamnolipid yield remains the same.

Example 2: Production of Rhamnolipids with Pseudomonas on Glucose and Maltose

[0107] Example 1 is repeated, but a medium is prepared and used, where the 2% (w/v) glucose are substituted by 1.96% (w/v) glucose and 0.04% (w/v) maltose, which is called M9*M. Furthermore, a rhamnolipid producing wildtype strain, P. aeruginosa PAO1, is used.

TABLE-US-00002 Relative defoamer amount used Strain M9 M9*M strain pBBR1MCS2-Plac- 100 98 rhlABC-T-Ptac-rhlC-T PAO1 100 99

[0108] The results show, that the substitution of parts of the glucose by maltose reduces foaming and defoamer consumption, whereas the overall rhamnolipid yield remains the same, but to a lesser extend than substitution by fructose.

[0109] After separating off the cells of the fermentations described above by means of centrifugation at 10 000 g, the fermentation broth is adjusted to a pH of 3.1 by adding concentrated H.sub.2SO.sub.4. By centrifugation again at 500 g, a paste-like solid concentrate is obtained with a rhamnolipid fraction of 45% by weight and a viscosity of >10 000 mPas.

[0110] The relative colour was determined via the Platinum-Cobalt Scale:

TABLE-US-00003 Relative colour Strain M9 M9*M strain pBBR1MCS2-Plac- 100 83 rhlABC-T-Ptac-rhlC-T PAO1 100 89

[0111] The results show, that the substitution of parts of the glucose by maltose results in a purer rhamnolipid composition.

Example 3: Production of Rhamnolipids with Pseudomonas on Glucose and Maltose and Fructose

[0112] Example 1 is repeated, but a medium is prepared and used, where the 2% (w/v) glucose are substituted by 1.96% (w/v) glucose and 0.03% (w/v) maltose and 0.01% (w/v) fructose, which is called M9*FM.

[0113] Furthermore, a rhamnolipid producing wildtype strain, P. aeruginosa PAO1, is used.

TABLE-US-00004 Relative defoamer amount used Strain M9 M9*FM strain pBBR1MCS2-Plac- 100 90 rhlABC-T-Ptac-rhlC-T PAO1 100 91

[0114] The results show, that the substitution of parts of the glucose by maltose and fructose reduces foaming and defoamer consumption in a synergistic way, whereas the overall rhamnolipid yield remains the same.

[0115] After separating off the cells of the fermentations described above by means of centrifugation at 10 000 g, the fermentation broth is adjusted to a pH of 3.1 by adding concentrated H.sub.2SO.sub.4. By centrifugation again at 500 g, a paste-like solid concentrate is obtained with a rhamnolipid fraction of 45% by weight and a viscosity of >10 000 mPas.

[0116] The relative colour was determined via the Platinum-Cobalt Scale:

TABLE-US-00005 Relative colour Strain MF9 M9*FM strain pBBR1MCS2-Plac- 100 85 rhlABC-T-Ptac-rhlC-T PAO1 100 87

[0117] The results show, that the substitution of parts of the glucose by maltose results in a purer rhamnolipid composition.

Example 4: Production of Rhamnolipids with Pseudomonas on Glucose and Isomaltose

[0118] Example 2 is repeated, but a medium is prepared and used, where the 2% (w/v) glucose are substituted by 1.96% (w/v) glucose and 0.04% isomaltose, which is called M9*l.

[0119] The relative colour was determined via the Platinum-Cobalt Scale:

TABLE-US-00006 Relative colour Strain M9 M9*I strain pBBR1MCS2-Plac- 100 92 rhlABC-T-Ptac-rhlC-T PAO1 100 95

[0120] The results show, that the substitution of parts of the glucose by isomaltose results in a purer rhamnolipid composition.

Example 5: Production of Rhamnolipids with E. coli on Glucose and Panose or Glucose and Maltulose

[0121] Production of rhamnolipids is carried out as described in example 10 of EP2598646 in recombinant E. coli M110 pBBR1MCS-2::ABC cells.

[0122] CMP medium is used. This consists of 2% (w/v) glucose, 0.007% (w/v) KH.sub.2PO.sub.4, 0.11% Na.sub.2HPO.sub.42 H.sub.2O, 0.2% (w/v) NaNO.sub.3, 0.04% (w/v) MgSO.sub.4H.sub.2O, 0.01% (w/v) CaCl.sub.2)2H.sub.2O and 0.2% (v/v) of a trace element solution. This consists of 0.2% (w/v) FeSO.sub.47 H.sub.2O, 0.15% (w/v) MnSO.sub.4H.sub.2O and 0.06% (w/v) (NH.sub.4)MO.sub.7O.sub.244 H.sub.2O. The pH of the medium is adjusted to 6.7 using NaOH.

[0123] Additionally medium is prepared and used, where the 2% (w/v) glucose are substituted by 1.98% (w/v) glucose and 0.02% panose, which is called CMP*P. and where the 2% (w/v) glucose are substituted by 1.99% (w/v) glucose and 0.01% maltulose, which is called CMP*M.

[0124] The viscosities of the final fermentation broth is measured using a Rheometer (Anton Haak) with concentrical cylinders geometry at a constant shear rate of 100 1/s.

TABLE-US-00007 Relative viscosity Strain CMP CMP*P CMP*M E. coli W3110 pBBR1MCS-2::ABC 100 93 94

[0125] The results show, that the substitution of parts of the glucose by panose or maltulose results in lower fermentation broth viscosities. This is an advantage, as less energy needs to be introduced into the vessel during fermentation.

Example 6: Production of Glycolipids with Pseudomonas on Glucose and Fructose

[0126] Production of glucolipids is carried out as described in example 2 of WO2019154970 in recombinant P. putida BS-PP-368 cells in a 1 litre Dasgip fermenter.

[0127] Additionally, the pure 500 g/L glucose feed (G) is replaced by a 485 g/L glucose plus 15 g/l fructose (G*F).

TABLE-US-00008 Relative defoamer amount used Strain G G*F P. putida BS-PP-368 100 94

[0128] The results show, that the substitution of parts of the glucose by fructose reduces foaming and defoamer consumption, whereas the overall glucolipid yield remains the same.

Example 7: Production of Glucolipids with Pseudomonas on Glucose and Maltose

[0129] Production of glucolipids is carried out as described in example 2 of WO2019154970 in recombinant P. putida BS-PP-368 cells in a 1 litre Dasgip fermenter.

[0130] Additionally, the pure 500 g/L glucose feed (G) is replaced by a 490 g/L glucose plus 10 g/l maltose (G*M).

[0131] 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 addition of concentrated H.sub.2SO.sub.4.

[0132] After a second centrifugation at 5.000 g for 20 minutes the aqueous upper phase is separated off and the remaining lower phase is a concentrate, which had a content of more than 50 wt.-% of glucolipids.

[0133] The relative colour is determined via the Platinum-Cobalt Scale:

TABLE-US-00009 Relative colour Strain G G*M P. putida BS-PP-368 100 92

[0134] The results show, that the substitution of parts of the glucose by maltose results in a purer rhamnolipid composition.

Example 8: Production of Glucolipids with Pseudomonas on Glucose and Panose or on Glucose and Maltose or Glucose and Panose and Maltose

[0135] Production of glucolipids is carried out as described in example 7.

[0136] Additionally, the pure 500 g/L glucose feed (G) is replaced by a 496 g/L glucose plus 4 g/l panose feed(G*P), by a 496 g/L glucose plus 4 g/l maltose (G*M) feed and by a 496 g/L glucose plus 2 g/l maltose plus 2 g/l panose (G*MP) feed.

[0137] The viscosities of the final fermentation broth is measured using a Rheometer with concentrical cylinders geometry at constant a shear rate of 100 1/s.

TABLE-US-00010 Relative viscosities Strain G G*P G*M G*MP P. putida BS-PP-368 100 88 86 84

[0138] The results show, that the substitution of parts of the glucose by panose or maltose results in lower fermentation broth viscosities and that panose and maltose exert a synergistic effect. This is helpful, as less energy needs to be introduced into the vessel during fermentation.

Example 9: Production of Sophorolipids with S. Bombicola on Glucose and Fructose

[0139] Sophorolipids are produced as described in example 12 of WO2011061032 in the wildtype strain S. bombicola ATCC 22214 and the recombinant strain S. bombicola ATCC 22214 sbg3-hyg. The medium used for producing the sophorolipids SL is composed of 0.1% KH.sub.2PO.sub.4, 0.5% MgSO.sub.47 H.sub.2O, 0.01% FeCl.sub.3, 0.01% NaCl, 0.4% yeast extract, 0.1% urea, 10.5% rapeseed oil and 10% glucose. The pH is brought to 4.5.

[0140] Furthermore, a medium is prepared and used, where the 10% (w/v) glucose are substituted by 9.9% (w/v) glucose and 0.1% fructose, which is called SL*F.

[0141] The relative foam height in the flasks as well as the sophorolipid concentration is determined

TABLE-US-00011 Relative foam height Strain M9 M9*F S. bombicola ATCC 22214 100 82 S. bombicola ATCC 22214 100 67 sbg3-hyg

[0142] The results show, that the substitution of parts of the glucose by fructose prevents foaming, whereas the overall sophorolipid yield remains the same.

Example 10: Production of Sophorolipids with S. bombicola on Glucose and Isomaltose or Glucose and Maltose

[0143] Sophorolipids are produced according to example 2 of Example 2 WO2021236904 (M).

[0144] A second analogous experiment is performed with the glucose being substituted by a 99:1 mixture of glucose and isomaltose (M*I).

[0145] A second analogous experiment is performed with the glucose being substituted by a 99:1 mixture of glucose and maltose (M*M).

[0146] The relative colour was determined via the Platinum-Cobalt Scale:

TABLE-US-00012 Relative colour Strain M M*I M*M S. bombicola ATCC 100 96 95 22214

[0147] The results show, that the substitution of parts of the glucose by isomaltose or maltose results in a purer sophorolipid composition.