METHOD FOR PRODUCING AND SEPARATING LIPIDS

Abstract

There are provided methods for the production of lipids such as sophorolipids. Also provided are apparatus for use in the production.

Claims

1. A method of producing lipids, wherein the method comprises: (a) performing a fermentation in a fermenter to produce a broth comprising lipid product; (b) transferring broth comprising lipid product from the fermenter to a separator; (c) allowing a lipid phase comprising lipid product to separate from other constituents of the broth in the separator; (d) returning broth having had lipid product separated therefrom from the separator to the fermenter; and (e) transferring lipid product from the separator.

2-30. (canceled)

31. The method according to claim 1, wherein the method comprises producing lipids selected from the group consisting of hydrocarbons, terpenoids, fats, oils, fatty acids and glycolipids, preferably terpenoids, fats, oils, fatty acids and glycolipids.

32. The method according to claim 1, wherein the lipid product is selected from sophorolipids, rhamnolipids and mannosylerythritol lipids.

33. The method according to claim 32, wherein a gravity separator is the only separator used within the method in step c).

34. The method according to claim 33, wherein the method comprises circulating broth between the fermenter and gravity separator, preferably continuously, over a period of at least 50 hours or over the duration of the production phase of the fermentation, wherein the method comprises transferring broth from the fermenter and returning broth to the fermenter such that the broth in the fermenter comprises at least 80% by weight, preferably at least 90% by weight, most preferably at least 95% by weight of the sum of the broth in the fermenter and gravity separator; and wherein the method comprises adding substrate to the fermenter over the duration of the production phase of the fermentation.

35. The method according to claim 1, wherein the method comprises maintaining the concentration of lipids in the broth in the fermenter below 80 g.Math.L.sup.1 to prevent bioreactor overflow and dissolved oxygen depletion.

36. The method according to claim 1, wherein the broth is agitated to prevent phase separation in the fermenter, and substantially no phase separation occurs in the fermenter.

37. The method according to claim 1, wherein the gravity separator comprises a separating chamber and is adapted such that, in use, the longitudinal axis of the chamber lies at between 10 degrees and 60 degrees to the horizontal.

38. The method according to claim 1, wherein step (c) comprises allowing separation of the broth by gravity in a gravity separator to provide a lipid phase comprising lipid product and a bulk broth comprising other constituents of the broth and wherein the bulk broth in the gravity separator comprises substrate and/or cells and wherein the method comprises separating the lipid phase comprising substantially no cells; and wherein the lipid phase comprises lipid product and water in a concentration of at least 80% by weight, preferably at least 90% by weight, most preferably at least 95% by weight.

39. The method according to claim 33, wherein a sophorolipid product is generated by controlling fermentation conditions, wherein the pH is between 2-5, a sugar is fed at a rate of at least 0.5 g.Math.L.sup.1.Math.h.sup.1, a vegetable oil is fed at a rate of at least 0.5 g.Math.L.sup.1.Math.h.sup.1 and a dissolved oxygen level of the broth in the fermenter is at least 20%.

40. A gravity separator adapted to separate a lipid phase from other constituents of a broth comprising lipids, wherein the gravity separator comprises: (I) a separating chamber in which, in use, said broth can be allowed to reside for a period of time such that a lipid phase comprising lipid product separates from other constituents of the broth; and wherein optionally the separating chamber is adapted such that, in use, a longitudinal axis of the chamber lies at between 10 degrees and 60 degrees to the horizontal; (II) a broth inlet to the separating chamber for transferring broth comprising lipid product into the separating chamber, in use; and (III) outlets from the separating chamber for: (i) transferring broth having had lipid product separated therefrom from the separating chamber, in use; and (ii) transferring lipid product from the separating chamber, in use.

41. The gravity separator according to claim 40, wherein the gravity separator comprises three outlets (III): (IIIa) a first outlet located toward the upper, in use, end of the separating chamber; (IIIb) a second outlet located toward the lower, in use, end of the separating chamber; and (IIIc) a third outlet located toward the lower, in use, end of the separating chamber.

42. The gravity separator according to claim 41, wherein outlets (IIIa), (IIIb) and (IIIc) are configured, to be selectively used as follows: (1) (IIIa) used for pressure relief, (IIIb) for transferring a lipid product from the separating chamber, (IIIc) for transferring broth having had a lipid product removed therefrom from the separating chamber; or (2) (IIIa) for transferring a lipid product from the separating chamber; (IIIb) for transferring broth having had a lipid product removed therefrom from the separating chamber; (IIIc) not used.

43. The gravity separator according to claim 40, wherein the gravity separator comprises: (I) a separating chamber in which, in use, said broth can be allowed to reside for a period of time such that a lipid phase comprising lipid product separates from other constituents of the broth; (II) a broth inlet which located toward a first end of the separating chamber for transferring broth comprising lipid product into the separating chamber, in use; (IIIA) a first outlet located toward a second end of the separating chamber; and (IIIB) a second outlet located toward a second end of the separating chamber; and wherein the broth inlet (II) comprises an opening to the separating chamber at a lower, in use, end of said separating chamber which lies on the longitudinal axis of said separating chamber; the first outlet (IIIA) comprises an opening to the separating chamber which lies in a side wall of said separating chamber in a region of said side wall towards an upper, in use, end of the separating chamber and wherein said opening to the separating chamber lies below the longitudinal axis of said separating chamber, in use; and the second outlet (IIIB) comprises an opening to the separating chamber which lies in a side wall of said separating chamber in a region of said side wall toward the upper, in use, end of the separating chamber and wherein said opening to the separating chamber lies above the longitudinal axis of said separating chamber, in use.

44. An apparatus for producing lipids, said apparatus comprising a fermenter having a fermentation chamber and the gravity separator according to claim 40, wherein the fermentation chamber and separating chamber of the gravity separator are in fluid communication such that, in use, broth comprising lipid product can be transferred from the fermentation chamber to the separating chamber and broth having had lipid product separated therefrom can be transferred from the separating chamber to the fermentation chamber; and wherein optionally the separating chamber is adapted such that, in use, the longitudinal axis of the chamber lies at between 10 degrees and 60 degrees to the horizontal.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0209] The present invention will now be illustrated by way of example with reference to the accompanying drawings in which:

[0210] FIG. 1 shows a separator;

[0211] FIG. 2 shows an apparatus comprising a fermenter and separator;

[0212] FIG. 3 shows the apparatus of FIG. 2 in an alternative configuration;

[0213] FIG. 4 is a graph showing feeding of substrate for fermentations;

[0214] FIG. 5 is a graph showing feeding of substrate for a fermentation;

[0215] FIG. 6 is a graph showing stirrer speed and dissolved oxygen;

[0216] FIG. 7 shows an alternative embodiment of a separator;

[0217] FIG. 8 is a graph showing concentrations and lipid production for a fermentation; and

[0218] FIG. 9 is a graph showing substrate feeding for the fermentation of FIG. 8.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0219] Example lipid productions were performed using a fermenter and separator to produce a broth comprising sophorolipids (lipid product) and to separate a sophorolipid phase from the broth (Examples 1 and 2). For comparison, a production of sophorolipids was performed without using a separator (Comparative Example 1C). A further example lipid production was performed using a fermenter and an alternative separator (Example 3).

[0220] Apparatus

[0221] For each of Examples 1 and 2 and Comparative Example 1C, the fermenter used was an Electrolab Fermac 320 fermentation system (Electrolab, UK) with a 2 l maximum working volume, H:D of 2, and an initial working volume of 1 l was used.

[0222] For Examples 1 and 2 the separator used was an in house built settling column illustrated in FIG. 1.

[0223] The separator 100 comprises a separating chamber 110. At the first, upper, in use, end 120 of the separator 100 the separating chamber 110 has a frusto conical section 111. The separating chamber 110 further comprises a cylindrical section 112 having a diameter of 50 mm and a length of 150 mm. The base 113 of the cylindrical section forms the lower, in use, end 130 of the separator.

[0224] The separator 100 comprises a broth inlet 140 and has an opening to the separating chamber 110 located on the longitudinal axis A-A of the separating chamber 110 at the first end 120 of the separator 100.

[0225] The separator comprises a first outlet 150, a second outlet 160 and a third outlet 170.

[0226] The first outlet 150 is located toward the first end 120 of the separator 100 and has an opening to the separating chamber 110 through a side wall 114 of the separating chamber 110. In use, the separator 100 is oriented such that the first outlet 150 lies above the longitudinal axis A-A of the separating chamber 110. Depending on the density of a lipid phase separated from a broth in the separator 100, in use, the first outlet 150 can be used to transfer a lipid product from the separator 100 or alternatively can be used for pressure relief.

[0227] The second outlet 160 is located toward the second end 130 of the separator 100 and has an opening to the separating chamber 110 through a side wall 114 of the separating chamber 110. In use, the separator 100 is oriented such that the second outlet 160 lies below the longitudinal axis A-A of the separating chamber 110. Depending on the density of a lipid phase separated from a broth in the separator 100, in use, the second outlet 160 can be used to transfer a lipid product from the separator 100 or alternatively can be used to transfer a broth having had lipid product separated therefrom from the separator 100.

[0228] The third outlet 170 is located toward the second end 130 of the separator 100 and has an opening to the separating chamber 110 through a side wall 114 of the separating chamber 110. In use, the separator 100 is oriented such that the second outlet 170 lies above the longitudinal axis A-A of the separating chamber 110. Depending on the density of a lipid phase separated from a broth in the separator 100, in use, the third outlet 170 can be used to transfer a broth having had lipid product separated therefrom from the separator 100 or alternatively can be redundant.

[0229] The third outlet 170 is positioned on the circumference of the cylinder 180 degrees from the second outlet 160 at the same distance from the end wall 113 of the separating chamber 110. The third outlet 170 is positioned on the circumference of the cylinder 0 degrees from the first outlet 150 but at the opposite end of the cylindrical section 112.

[0230] The separating chamber 110 is attached to a stand (not shown) which allows the angle of the longitudinal axis A-A of the separating chamber 110 relative to the horizontal to be varied. For Examples 1 and 2 the angle used was 30 degrees to the horizontal.

[0231] Apparatus 10 comprising a fermenter 200 and separator 100 configured as used for Example 1 is illustrated in FIG. 2. The separator was configured to separate a sophorolipid phase (lipid product phase) that was less dense than the broth.

[0232] The separator 100 comprises a separator according to FIG. 1.

[0233] The apparatus 10 is configured for the separator 100 to receive broth 300 comprising lipid product from the fermenter and to separate a lipid product phase 310 which is denser than a broth 300 from said broth to provide broth 320 having had the lipid product separated therefrom. The apparatus is further configured for the lipid product phase 310 to be transferred by a pump (not shown) and collected in a container 400 and the broth 320 having had the lipid product separated therefrom to be returned to the fermenter 200.

[0234] The separator 100 is angled such that the longitudinal axis A-A of the separating chamber 110 lies at 30 degrees to the horizontal. The first outlet 150 serves as an outlet for the lipid product phase 310, the second outlet 160 serves as an outlet for broth 320 having had the lipid product separated therefrom and the third outlet 170 is sealed with a stopper 180 and not used.

[0235] The fermenter 200 comprises a fermentation chamber 210 for holding broth 300. The fermenter 200 also comprises a stirrer 220 for agitating the broth. The fermenter also comprises an air sparger (not shown) for aerating the broth. The fermenter 200 further comprises a broth outlet 230 in fluid communication with a broth inlet 140 of the separator via a pump (not shown). In addition the fermenter comprises a broth inlet 240 in fluid communication via a pump (not shown) with the second outlet 160 of the separator 100.

[0236] Apparatus 10a comprising a fermenter 200 and separator 100 configured as used for Example 2 is illustrated in FIG. 3. The separator was configured to separate a sophorolipid phase (lipid product phase) that was denser than the broth.

[0237] The apparatus 10a is substantially the same as apparatus 10 and like parts are numbered accordingly. The main difference between the configuration of apparatus 10a and apparatus 10 is in the use of the outlets 150, 160, 170 and the fluid communication between the separator 100 and fermenter 200.

[0238] The separator 100 comprises a separator according to FIG. 1.

[0239] The apparatus 10a is configured for the separator 100 to receive broth 300 comprising lipid product from the fermenter and to separate a lipid product phase 310a which is less dense than a broth 300a from said broth to provide broth 320a having had the lipid product separated therefrom. The apparatus is further configured for the lipid product phase 310a to be transferred by a pump (not shown) and collected in a container 400 and for the broth 320a having had the lipid product separated therefrom to be returned to the fermenter 200.

[0240] The separator 100 is angled such that the longitudinal axis A-A of the separating chamber 110 lies at 30 degrees to the horizontal. The first outlet 150 is used as a pressure relief outlet and provided with an air filter 190, the second outlet 160 serves as an outlet for the lipid product phase 310a and the third outlet 170 serves as an outlet for the broth 320a having had the lipid product separated therefrom.

[0241] The fermenter 200 comprises a fermentation chamber 210 for holding broth 300a. The fermenter 200 also comprises a stirrer 220 for agitating the broth. The fermenter also comprises an air sparger (not shown) for aerating the broth. The fermenter 200 further comprises a broth outlet 230 in fluid communication with a broth inlet 140 of the separator via a pump (not shown). In addition the fermenter comprises a broth inlet 240 in fluid communication via a pump (not shown) with the third outlet 170 of the separator 100.

[0242] Fermentations

[0243] For each of Examples 1 and 2 and Comparative Example 1C, the sophorolipid productions were fed batch fermentations using C. bombicola ATCC 22214.

[0244] The substrates fed for the fermentations were glucose and rapeseed oil. The feeding rates of substrates were different for Example 1 and Example 2 so as to give a sophorolipid phase that separated to the top of the separator in Example 1 and to the bottom of the separator in Example 2. The substrates were fed at substantially the same rate for Comparative Example 1C as for Example 1. FIG. 4 shows the feeding of substrates for Example 1 and Comparative Example 10 and FIG. 5 shows the feeding of the substrates for Example 2.

[0245] For each of Examples 1 and 2 and Comparative Example 10 the growth medium for the fermentations, preculture and agar plates contained 6 g l.sup.1 yeast extract and 5 g l.sup.1 peptone. The initial concentration of glucose in all fermentations, preculture and agar plates was 100 g l.sup.1, with an initial rapeseed oil concentration of 50 g l.sup.1 in the fermenters, 100 g l.sup.1 in the preculture and 0 in the agar plates.

[0246] C. bombicola was first transferred from cryogenic storage (80 C.) onto agar plates, and incubated at 25 C. for 48 hours. Single colonies from these plates were then used to inoculate 50 ml of medium in 250 ml shake flasks, which were incubated at 25 C. and 200 rpm for 30 hours. This inoculum was diluted to an optical density at 600 nm of 20 with fresh media and used to inoculate the fermenter.

[0247] Fermentations were run at 25 C., and dissolved oxygen was controlled to 30% by varying the stirrer speed, whilst maintaining a constant aeration rate of 1 l min.sup.1. Fermenter pH was controlled to a value of 3.5 by the addition of 3M sodium hydroxide.

[0248] For Examples 1 and 2 separation was run depending on production rate with the separation operated on a plurality of occasions during fermentation with pauses between those occasions of operation. When separation was performed sophorolipid rich fermentation broth was continuously circulated from the fermenter, through the separator and back around to the fermenter, being pumped in 8 mm external diameter silicon tubing of 1 mm wall thickness, using peristaltic pumps. The flow rate of broth into and out of the separator was controlled to around 1 ml s.sup.1 giving a residence time in the separating chamber of 76 s.

[0249] In the separating chamber the sophorolipid phase separated out from the broth due to differences in relative density. In Example 1 the sophorolipid phase separated towards the top of the separating chamber and in Example 2 it separated towards the bottom.

[0250] For both Example 1 and Example 2, during the separation operations, initially broth was continuously circulated and the sophorolipid phase accumulated in the separating chamber. When the sophorolipid phase accumulating in the separating chamber reached 50% of the height of the settling chamber, which typically occurred after around three minutes of operation, the outlet pump was started to continuously remove the sophorolipid product phase at a rate controlled between 0.5 and 2 ml min.sup.1, depending on the accumulation or reduction of the sophorolipid phase in the settling vessel.

[0251] The separation was run periodically in both Examples 1 and 2, with the majority of the available sophorolipid phase separated, and run again when sufficient sophorolipid phase had accumulated.

[0252] In the illustrated Examples, whilst the separator is designed for continuous operation at the scale used separation occurs at a rate of 30-150 times the production rate. For this reason, the separator was run intermittently. Using larger scale apparatus (not shown) the broth can be continuously circulated from the fermenter, through the separator and back around to the fermenter.

[0253] Separation was carried out at 111, 184 and 261 hours in Example 1, and 71.5, 281, 355 and 376 hours in Example 2, and no separation of the sophorolipid phase was carried out in Comparative Example 1C.

[0254] Analytical Techniques

[0255] For all analyses, 5 ml of broth was removed from the fermenter at each sample time point. The sample was centrifuged at 5000 rpm for 5 minutes using a Sigma 6-16S centrifuge (Sigma laboratory centrifuges, Germany) and the glucose in the supernatant quantified using a TrueResult blood glucose monitor (Nipro Japan).

[0256] Residual oil and sophorolipid concentration were measured gravimetrically, with a hexane extraction first used to separate the residual oil and a triple ethyl acetate extraction used to separate the sophorolipid. The extracts were dried to constant weight in weighing dishes at ambient temperature for 30 h.

[0257] Cell growth was determined by both dry cell weight and optical density measurement. After solvent extraction, 8 ml distilled water was added to the remainder of the sample in the centrifuge tubes, which were then centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded and the resulting cell pellet was resuspended in 8 ml distilled water. This cell suspension was transferred to drying trays, which were dried to constant weight at 90 C. in a drying oven. Optical density was used as a proxy for dry cell weight when diluting the inoculum, at a wavelength of 600 nm.

[0258] The structure of the sophorolipids produced was determined using negative ionisation electrospray ionisation, using an Agilent 6520 QTOF mass spectrometer (Agilent, United States). Samples were prepared by dissolving sophorolipid extracts in ethyl acetate, and filtering using a 0.2 m filter. Flow injection analysis was used, at 0.3 ml min.sup.1, 50% acetonitrile, 0.1% formic acid, 49.9% water, with an injection volume of 2 l.

[0259] Results

[0260] The separation parameters achieved in Examples 1 and 2 are shown in Table 1 and the key metrics for the Examples 1 and 2 and Comparative Example 1C are shown in Table 2.

TABLE-US-00001 TABLE 1 Separation parameters Sophorolipid Sophorolipid Sophorolipid concentration concentration Recov- Time recovered in fermenter in extract Enrich- ery (h) (g) (g l.sup.1) (g l.sup.1) ment (%) Example 1 111 97.1 147.7 550.6 3.73 37 184 99.2 118.0 461.6 3.91 37 261 83.8 89.2 540.9 6.07 22 total 280.15 86 Example 2 71.5 16.8 103.7 582.9 5.62 21 281 79.5 168.3 654.1 3.89 41 355 59.2 109.2 616.9 5.66 48 376 45.5 106.3 638.7 6.01 43 total 201.0 57

TABLE-US-00002 TABLE 2 Key metrics Example 1 Yield substrate consumed (g g.sup.1) Yield substrate fed (g g.sup.1) 0.53 0.37 Productivity starting volume (g l.sup.1 h.sup.1) Productivity max volume (g l.sup.1 h.sup.1) 1.07 0.69 Comparative Example 1C Yield substrate consumed (g g.sup.1) Yield substrate fed (g g.sup.1) 0.43 0.33 Productivity starting volume (g l.sup.1 h.sup.1) Productivity max volume (g l.sup.1 h.sup.1) 1.07 0.62 Example 2 Yield substrate consumed (g g.sup.1) Yield substrate fed (g g.sup.1) 0.42 0.39 Productivity starting volume (g l.sup.1 h.sup.1) Productivity max volume (g l.sup.1 h.sup.1) 0.77 0.57

[0261] As can be seen from Table 1, the capacity to recover the majority of the sophorolipid from a fermentation broth during fermentation was demonstrated, whether the sophorolipid phase was less dense (Example 1) or denser (Example 2) than the fermentation broth.

[0262] Sophorolipid recovery in Example 1 using separation resulted in a higher productivity than was achieved by Comparative Example 1C without the separation due to the reduced maximum fermenter volume. As shown by FIG. 6 which shows the effect of adding collected sophorolipids back to the broth of Example 1, using separation also provides for a lower agitation requirement to maintain the desired dissolved oxygen level.

[0263] For both Example 1 and Example 2, the majority of the sophorolipid was removed from the fermentation broth. Sophorolipid recovery was significantly higher for separation from the surface of the broth (Example 1), than from the bottom (Example 2), at 86% compared to 57%, but this may be largely due to the lower separation time for the final separation in Example 2.

[0264] In Examples 1 and 2 which were performed on a laboratory scale, the separation had to be stopped as the layer of sophorolipids at the bottom/top of the settling column became too low, to prevent the media and cell phase being entrained in the product stream. It is likely that with an increased scale, recoveries may be improved as the minimum sophorolipid phase depth, which must be recycled back to the fermenter, would be similar irrespective of fermenter volume.

[0265] Almost no cells or oil were removed by the separation in Example 2. For Example 1, cell removal was negligible. Whilst 68 g of oil was removed in Example 1, it may be possible to substantially reduce or eliminate that by better control of the oil feeding rates to maintain a low oil concentration in the fermenter.

[0266] The enrichment varied significantly between extractions at different time points, from 3.73 to 6.07. This is thought to be largely due to the sophorolipid concentration present in the fermenter before the separation, as there was little variation of the concentration in the extract, of approximately 550 g l.sup.1. It is likely that with an increased fermenter volume, the system may operate at lower initial sophorolipid concentrations and so may give an improved enrichment.

[0267] The total sophorolipid produced was calculated by adding the mass of sophorolipid in the fermenter and the mass of sophorolipid extracted from the fermenter. Substrate feeding meant the volume was higher than the 1 l starting volume during much of the fermentations.

[0268] For Example 1, the productivity at the maximum volume was 0.69 g l.sup.1 h.sup.1, and for Comparative Example 1C it was 0.62 g l.sup.1 h.sup.1, showing an effective productivity increase of 11% when using separation. This was due to the decreased maximal volume reached when separation was used, at 1540 ml with separation rather than 1720 ml without separation. The corresponding productivity for Example 2 of 0.57 g l.sup.1 h.sup.1 is not directly comparable due to the differences in feeding rates between the fermentations.

[0269] It is likely that using separation in continuous mode from early in a fermentation may make it possible to control the fermentation volume to around 1.3 times the initial volume (which without control may rise to around 1.7 times the initial volume after 280 hours). This may amount to an effective productivity increase of over 30%.

[0270] Sophorolipid was first extracted at 71.5 hours in Example 2, when a sophorolipid phase could be observed to settle in a sample bottle within 2 minutes. Settling then became ineffective until 283 hours due to the high residual glucose concentrations caused by pulse glucose feeding. Whilst the settling or floating of the sophorolipid also depends on other factors, a glucose concentration of 50 g l.sup.1 tends to represent a threshold of settling or floating to the surface. It is likely better control of the feeding rate may have enabled the sophorolipid to be settled throughout the fermentation.

[0271] In Example 1, the glucose concentration initially rose, and remained above 50 g l.sup.1 for the majority of the fermentation, which coupled with the significant residual rapeseed oil concentrations after around 140 hours led to the sophorolipid rising to the surface of the fermenter and forming a mixed phase with the residual oil, when oil was present in significant quantities.

[0272] Hydrodynamic and Mass Transfer Effects

[0273] The sophorolipid extracts from Example 1 were pooled after the fermentation, and returned to the fermenter at 308.3 h, over a period of 12 minutes. FIG. 6 shows the dissolved oxygen level and stirrer speed at the end of Example 1, with a drop in dissolved oxygen upon addition of the viscous sophorolipid phase. The presence of this sophorolipid phase effectively reduced the Kla in the fermenter, resulting in an increase in stirrer speed to maintain the dissolved oxygen at the setpoint. A stirring rate increase of around 75 rpm was required to maintain the desired dissolved oxygen level when the sophorolipid phase produced over the whole fermentation was added which resulted in a greater than 40% increase in stirring power requirement.

Example 3

[0274] An alternative separator and fermentation conditions were used for Example 3. The separator used was an in house built settling column illustrated in FIG. 7.

[0275] The separator 1100 comprises a separating chamber 1110. At the first, lower, in use, end 1120 of the separator 1100 the separating chamber 1110 has a frusto conical section 1111. The separating chamber 1110 further comprises a cylindrical section 1112 having a diameter of 50 mm and a length of 150 mm. The base 1113 of the cylindrical section forms the upper, in use, end 1130 of the separator.

[0276] The separator 1100 comprises a broth inlet 1140 and has an opening to the separating chamber 1110 located on the longitudinal axis A-A of the separating chamber 1110 at the first end 1120 of the separator 1100. The inlet receives broth 300 comprising lipid product from a fermenter 200.

[0277] The separator comprises a first outlet (IIIA) shown as 1160 in FIG. 7 and a second outlet (IIIB) shown as 1170 in FIG. 7.

[0278] The first outlet 1160 is located toward the second end 1130 of the separator 1100 and has an opening to the separating chamber 1110 through a side wall 1114 of the separating chamber 1110. In use, the separator 1100 is oriented such that the first outlet 1160 lies below the longitudinal axis A-A of the separating chamber 1110. Depending on the density of a lipid phase separated from a broth in the separator 1100, in use, the first outlet 1160 can be used to transfer a lipid product from the separator 1100 or alternatively can be used to transfer a broth having had lipid product separated therefrom from the separator 1100. In Example 3 as illustrated by FIG. 7 the outlet 1160 was used to transfer a broth 1320 having had lipid product separated therefrom to the fermenter 200

[0279] The second outlet 1170 is located toward the second end 1130 of the separator 1100 and has an opening to the separating chamber 1110 through a side wall 1114 of the separating chamber 1110. In use, the separator 1100 is oriented such that the second outlet 1170 lies above the longitudinal axis A-A of the separating chamber 110. Depending on the density of a lipid phase separated from a broth in the separator 100, in use, the second outlet 1170 can be used to transfer a lipid product from the separator 1100 or alternatively can be used to transfer a broth having had lipid product separated therefrom from the separator 1100. In Example 3 as illustrated by FIG. 7 the outlet 1170 was used to transfer lipid product 1310 to a container 400.

[0280] The separating chamber 1110 is attached to a stand (not shown) which allows the angle of the longitudinal axis A-A of the separating chamber 1110 relative to the horizontal to be varied. For Example 3 the angle used was 30 degrees to the horizontal.

[0281] In Example 3, an increased media concentration, comprising 18 g l.sup.1 yeast extract and 15 g l.sup.1 peptone was used to achieve a high cell density and more rapid production. This necessitated higher feeding rates, as shown in FIG. 9. The stirring rate was fixed at 900 rpm, and the fermentation carried out in a 3 l volume Applikon bioreactor (fermenter), 1.5 l initial volume. The separator of FIG. 7 was used in Example 3 so as to achieve better separation of lipid product from the surface of the broth. All other parameters were as for Examples 1 and 2.

[0282] Higher cell densities resulted in a much faster sophorolipid production rate, and a total production of 689.8 g l.sup.1 sophorolipid was reached, The integrated separation system was necessary for extended running of the fermentation, to both prevent bioreactor overflow (which would have otherwise occurred around 165 h and 392 g l.sup.1 sophorolipid) and oxygen depletion, which dropped below the desired 30% set point within 100 hours and was corrected by separation.

[0283] This meant a productivity of 2.24 g l.sup.1 could be maintained for the whole fermentation, with no decrease in productivity apparent, other than at the point of additional nitrogen sources. These were added around 270 h, and resulted in a brief biomass growth period followed by continued production. It will be appreciated that this may be useful for periods of cell growth between long periods of sophorolipid production in continuous fermentation using this technique.

[0284] 79.2% of the sophorolipid produced during the fermentation was recovered, i.e. a total of 819 g.

[0285] FIG. 8 illustrates the concentrations of substrates namely glucose (illustrated by filled triangles) and oil (indicated by stars) and dry cell weight (indicated by open squares), within the bioreactor and the production of sophorolipid (with the concentration of sophorolipid indicated by filled circles and the total sophorolipid produced indicated by open circles).

[0286] FIG. 9 illustrates the substrate feeding rate for oil (indicated by dashed line) glucose (indicated by dotted line) and total (indicated by solid line).

[0287] In FIG. 8 the arrows with dotted lines show the times when integrated sophorolipid separation was performed and solid lines show the addition of 12 g yeast extract and 10 g peptone dissolved in 100 ml water to the bioreactor.

[0288] It will be appreciated that preferred embodiments of the present invention may provide improved methods of sophorolipid production and in particular may allow for improved productivity and lower energy requirements.

[0289] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0290] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0291] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0292] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.