Process for the production of lipids from biomass

Abstract

Process for the production of lipids from biomass including at least one polysaccharide, which comprises fermentation of a biomass hydrolysate using an oleaginous microorganism and separation of oleaginous cellular biomass comprising lipids. An aqueous fraction obtained by reverse osmosis of a spent fermentation broth is recycled as a feed for growth of the oleaginous microorganism. The lipids thus obtained can be advantageously used in the production of biodiesel or green diesel which can be used as such, or in a mixture with other automotive fuels.

Claims

1. A process for the production of lipids from biomass including at least one polysaccharide, comprising: subjecting said biomass including at least one polysaccharide to hydrolysis obtaining a mixture comprising a first solid phase and a first aqueous phase; separating said first aqueous phase from said mixture: preparing an inoculum comprising at least one oleaginous microorganism in a first fermentation device obtaining a first fermentation broth; feeding said first aqueous phase and said first fermentation broth to a second fermentation device obtaining a second fermentation broth; subjecting said second fermentation broth to separation obtaining an aqueous suspension of oleaginous cellular biomass comprising lipids and a second aqueous phase; subjecting said second aqueous phase to reverse osmosis obtaining a permeate and a retentate; feeding said retentate to said first fermentation device or to said second fermentation device.

2. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said polysaccharide is selected from cellulose, hemicellulose, or mixtures thereof.

3. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said biomass including at least one polysaccharide is a lignocellulosic biomass.

4. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said biomass including at least one polysaccharide is subjected to a preliminary grinding process before being subjected to said hydrolysis.

5. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said first aqueous phase comprises; an amount of glucose higher than or equal to 100 g/l up to the solubility limit of the glucose in said first aqueous phase; from 0 g/l to 200 g/l of xylose; from 0 g/l to 20 g/l of arabinose; from 0 g/l to 20 g/l of mannose; from 0 g/l to 10 g/l of galactose; from 0 g/l to 8 g/l of acetic acid; from 0 g/l to 0.7 g/l of furfural; and from 0 g/l to 2.5 g/l of 5-hydroxymethylfurfural.

6. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said retentate is an aqueous solution comprising an amount of sugars higher than or equal to 40 g/l.

7. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein, in said first fermentation device, the fermentation is carried out at a temperature ranging from 20? C. to 40? C., for a time ranging from 10 hours to 36 hours, at a pH ranging from 4.5 to 7.

8. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein, in said second fermentation device, the fermentation is carried out at a temperature ranging from 20? C. to 40? C., for a time ranging from 2 days to 10 days, at a pH ranging from 4.5 to 7.

9. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said oleaginous microorganism is selected from the following yeasts: Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula graminis, Lypomices starkeyi, Lypomices lipofer, Trigonopsis variabilts, Candida kefyr, Candida curvata, Candida lipolytica, Torulopsis sp., Pichia stipitis, Trichosporon cacaoliposimilis, Rhodosporidium sp., Cryptococcus curvatus.

10. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein the fermentation in said second fermentation device is carried out in one or more steps in a mode selected from the group consisting of batch, semi-continuous, continuous, and combinations thereof.

11. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said reverse osmosis is carried out in the presence of at least one polymeric membrane selected from polymeric membranes generally used for desalination.

12. The process for the production of lipids from biomass including at least one polysaccharide according to claim 11, wherein said polymeric membrane has a maximum operating temperature ranging from 15? C. to 90? C., a maximum operating pressure ranging from 5 bar to 80 bar, a nominal molecular weight cut-off ranging from 30 daltons to 200 daltons, a maximum operating pH ranging from 1 to 13.

13. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said reverse osmosis is carried out at a temperature ranging from 5? C. to 90? C.

14. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said reverse osmosis is carried out applying a pressure at the retentate side ranging from 5 bar to 80 bar.

15. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said reverse osmosis is carried out operating at a specific flow-rate ranging from 5 kg/(m.sup.2?h) to 80 kg/(m.sup.2?h).

16. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein, before being subjected to reverse osmosis, said second aqueous phase, is subjected to microfiltration or ultrafiltration.

17. The process for the production of lipids from biomass including at least one polysaccharide according to claim 1, wherein said process comprises adding corn steep liquor or corn steep solid to said second fermentation device in an amount ranging from 2 g/l to 20 g/l.

Description

(1) The present invention will now be illustrated in greater detail by means of an embodiment with reference to FIG. 1 provided hereunder.

(2) FIG. 1 schematizes an embodiment of the process object of the present invention. For this purpose, the biomass including at least one polysaccharide (e.g., lignocellulosic biomass previously ground) is subjected to hydrolysis (operating according to one of the methods known in the art indicated above) obtaining a mixture comprising a first aqueous phase and a first solid phase including lignin.

(3) Said mixture is subjected to filtration or centrifugation (not represented in FIG. 1) obtaining a first solid phase and a first aqueous phase.

(4) In the meantime, an inoculum is prepared in a first fermentation device using an oleaginous microorganism (e.g., Rhodotorula graminis, Trichosporon cacaoliposimilis) obtaining a first fermentation broth: it should be noted that, as mentioned above, the first time that the process object of the present invention is carried out, the aqueous solution comprising a quantity of sugars higher than or equal to 40 g/l, preferably ranging from 45 g/l to 60 g/l, i.e. the retentate, will be substituted by an aliquot of the first aqueous phase obtained from the hydrolysis of the biomass including at least one polysaccharide, optionally diluted so as to have the desired quantity of sugars (indicated in FIG. 1 with the dashed line).

(5) Said first aqueous phase and said first fermentation broth are fed to a second fermentation device in the presence of an oleaginous microorganism (e.g., Rhodotorula graminis, Trichosporon cacaoliposimilis) obtaining a second fermentation broth.

(6) Said second fermentation broth is subjected to separation (e.g., by means of centrifugation) obtaining an aqueous suspension of oleaginous cellular biomass and a second aqueous phase.

(7) According to an embodiment of the present invention, at least a part of said second fermentation broth can be subjected to microfiltration obtaining an aqueous phase (permeateP) which is joined to said second aqueous phase and sent to reverse osmosis and an aqueous suspension of oleaginous cellular biomass (retentateR) which is sent to said second fermentation device (indicated in FIG. 1 with the dashed line).

(8) Said aqueous suspension of oleaginous cellular biomass is subjected to cell lysis (operating according to one of the methods described above), extraction in the presence of a solvent and subsequent evaporation of the solvent obtaining lipids.

(9) Said second aqueous phase is subjected to reverse osmosis (e.g., using a polymeric membrane comprising polyamide) obtaining a retentate (i.e. aqueous solution comprising a quantity of sugars higher than or equal to 40 g/l) which is fed to said first fermentation device and a permeate (i.e. water having a TOC lower than or equal to 300 mg/1).

(10) Alternatively, said retentate can be fed to said second fermentation device (indicated in FIG. 1 with the dashed line).

(11) Some illustrative and non-limiting examples are provided hereunder for a better understanding of the present invention and for its practical embodiment.

EXAMPLE 1

(12) Composition of the Lignocellulosic Hydrolyzate

(13) The lignocellulosic hydrolyzate (i.e. first aqueous phase) used in the following examples had the following composition: glucose (126 g/l), xylose (87.1 g/l), arabinose (7.5 g/l), mannose (2.9 g/l), galactose (6.5 g/l), acetic acid (4.9 g/l), furfural (0.46 g/l), 5-hydroxymethylfurfural (1.85 g/l), for a total sugar content equal to 230 g/l.

(14) The content of furfural and of 5-hydroxymethylfurfural was determined by means of High Performance Liquid Chromatography (HPLC) using a LichroCART Purospher RP-18 end-capped column (240 mm?mm; 5 ?m) of Merck, equipped with a photodiode UV detector, with a flow of 0.8 ml/min, temperature 40? C., and with phosphoric acid mobile phase at 0.05% in water (eluent A) and acetonitrile+phosphoric acid at 0.05% in water, in a ratio of 90/10 vol/vol (eluent B), using the elution gradient indicated in Table 1.

(15) TABLE-US-00001 TABLE 1 Time Eluent A Eluent B (min) (%) (%) 0 100 0 4 94 6 30 85 15

(16) The sugar content was determined by means of ion-exchange chromatography (HPAE-PAD), using a Dionex chromatograph, equipped with a Carbopac PA100 column, with a gradient of sodium hydroxide and sodium acetate as counter-ion.

(17) The quantitative determination of the organic acids, i.e. acetic acid, was carried out by means of a DIONEX BIOLC 4000 ion chromatograph coupled with a Pulsed Electrochemical Detector (PED), an Ice-AS1 chromatographic column (diameter: 9 mm; length: 250 mm), an AMMS-ICE (Anion MicroMembrane Suppressor), injection volume 50 ?l, elution in isocratic mode with heptafluorobutyric acid 0.4 mM as eluent.

EXAMPLE 2

(18) Preparation of Inoculum with Hydrolyzate (Rhodotorula Graminis)

(19) As the retentate obtained from the reverse osmosis according to the process, object of the present invention, was not yet available, the inoculum (i.e. first fermentation broth) was prepared using part of the lignocellulosic hydrolyzate (i.e. first aqueous phase) described in Example 1.

(20) For this purpose, 19.4 ml of said lignocellulosic hydrolyzate (i.e. first aqueous phase) suitably diluted with water (80.6 ml) so as to have a final concentration of sugars equal to 44.5 g/l, were introduced into a 500 ml flask, equipped with a magnetic stirrer, to which 10 g/l of yeast extract and g/l of peptone were subsequently added: the pH of the mixture obtained was brought to 6 by the addition of a few drops of potassium hydroxide (KOH) 2.5 M. The mixture obtained was sterilized in an autoclave at 80? C., for 45 minutes.

(21) At the end of the sterilization, the mixture obtained was brought to room temperature (25? C.) and inoculated with cells of Rhodotorula graminis DBVPG 4620 which were left to grow, for 24 hours, at 30? C., under stirring (200 rpm), until a first fermentation broth was obtained, having a concentration of oleaginous cellular biomass equal to 13.32 g/l (dry weight).

EXAMPLE 3

(22) Fermentation of Rhodotorula graminis (Feed-Batch Mode)

(23) The fermentation test with cells of Rhodotorula graminis DBVPG 4620 was carried out in feed-batch mode in a 20 liter fermenter, operating under the following conditions: 2.6 l of lignocellulosic hydrolyzate (i.e. first aqueous phase) described in Example 1, suitably diluted with water so as to have an initial sugar concentration equal 100 g/l; 1.0 g/l of yeast extract; 2.5 g/l of corn steep solid; 2.5 g/l of (NH.sub.4).sub.2SO.sub.4; 1 g/l of KH.sub.2PO; 0.05 g/l of MgSO.sub.4.7H.sub.2O; 0.01 g/l of NaCl; 0.01 g/l of CaCl.sub.2.2H.sub.2O; air fed: flow equal to 1 l/min; operating pH equal to 6, maintained by the addition, when necessary, of a few drops of a solution of potassium hydroxide (KOH) 5 M and phosphoric acid (H.sub.3PO.sub.4) 10% (v/v); stirring equal to 600 rpm-900 rpm, modulated with the air flow so as to maintain the concentration of dissolved oxygen (DO.sub.2) above 30%; initial volume: 6 liters; inoculum of Rhodotorula graminis DBVPG 4620 (i.e. first fermentation broth) obtained as described in Example 2, diluted at 10% (v/v) with the culture medium used for the fermentation, in order to start the fermentation with a concentration of oleaginous cellular biomass equal to 1.3 g/l (dry weight).

(24) The fermentation was carried out batchwise for the first 36 hours obtaining a fermentation broth having a concentration of oleaginous cellular biomass equal to 29.03 g/l (dry weight) and a residual concentration of sugars equal to 38.5 g/l.

(25) Additions were subsequently made to the fermentation broth, operating in feed-batch mode of lignocellulosic hydrolyzate (i.e. first aqueous phase) described in Example 1, suitably diluted with water so as to have a total concentration of sugars equal to 199.71 g/1: in particular, three additions were made, two of 1 liter each, after 36 hours and after 54 hours of fermentation respectively, and one of 2 liters after 44 hours of fermentation.

(26) At the end of the fermentation, after 122 hours, a second fermentation broth was obtained, having a concentration of oleaginous cellular biomass equal to 62.62 g/l (dry weight) and a content of total lipids equal to 60% by weight with respect to the dry weight of said oleaginous cellular biomass.

(27) Said second fermentation broth was subjected to separation by means of centrifugation at 7000 rpm, for 20 minutes obtaining 2.1 kg of oleaginous cellular biomass [626 g (dry weight)?concentration equal to 30% by weight with respect to the total quantity of oleaginous cellular biomass obtained] having a content of total lipids equal to 60% by weight with respect to the dry weight of said oleaginous cellular biomass and 7.9 l of exhausted fermentation water (i.e. second aqueous phase) having a content of non-metabolized sugars equal to 15.15 g/l.

(28) The total lipid content was determined using the total lipids-sulpho-phospho vanillin kit, operating as described above. The sugar content was determined operating as described in Example 1.

(29) A yield of oleaginous cellular biomass was reached with respect to the substrate consumed (Y.sub.X/S=g of biomass obtained per g of substrate consumed) equal to 0.41 g/g, and a yield of lipids with respect to the substrate consumed (Y.sub.L/S=g of lipids obtained per g of substrate consumed) equal to 0.25 g/g.

EXAMPLE 4

(30) Preparation of Inoculum with Lignocellulosic Hydrolyzate (Trichosporon cacaoliposimilis)

(31) As the retentate obtained from the reverse osmosis according to the process object of the present invention, was not yet available, the inoculum was prepared using part of the lignocellulosic hydrolyzate (i.e. first aqueous phase) described in Example 1.

(32) For this purpose, 19.4 ml of said lignocellulosic hydrolyzate (i.e. first aqueous phase) suitably diluted with water (80.6 ml) so as to have a final concentration of sugars equal to 44.5 g/l, were introduced into a 500 ml flask, equipped with a magnetic stirrer, to which 10 g/l of yeast extract and g/l of peptone were subsequently added: the pH of the mixture obtained was brought to 6 by the addition of a few drops of potassium hydroxide (KOH) 2.5 M. The mixture obtained was sterilized in an autoclave at 80? C., for 45 minutes.

(33) At the end of the sterilization, the mixture obtained was brought to room temperature (25? C.) and inoculated with cells of Trichosporon cacaoliposimilis ATCC 20509 which were left to grow, for 24 hours, at 30? C., under stirring (200 rpm), until a first fermentation broth was obtained, having a concentration of oleaginous cellular biomass equal to 15.7 g/l (dry weight).

EXAMPLE 5

(34) Fermentation of Trichosporon cacaoliposimilis (Feed-Batch Mode)

(35) The fermentation test with cells of Trichosporon cacaoliposimilis ATCC 20509 was carried out in feed-batch mode in a 20 liter fermenter, operating under the following conditions: 2.6 l of lignocellulosic hydrolyzate described in Example 1, suitably diluted with water so as to have an initial sugar concentration equal 100 g/l; 1.0 g/l of yeast extract; 2.5 g/l of corn steep solid; 2.5 g/l of (NH.sub.4).sub.2SO.sub.4; 1 g/l of KH.sub.2PO; 0.05 g/l of MgSO.sub.4.7H.sub.2O; 0.01 g/l of NaCl; 0.01 g/l of CaCl.sub.2.2H.sub.2O; air fed: flow equal to 1 l/min; operating pH equal to 6, maintained by the addition, when necessary, of a few drops of a solution of potassium hydroxide (KOH) 5 M and phosphoric acid (H.sub.3PO.sub.4) 10% (v/v); stirring equal to 600 rpm-900 rpm, modulated with the air flow so as to maintain the concentration of dissolved oxygen (DO.sub.2) above 30%; initial volume: 6 liters; inoculum of Trichosporon cacaoliposimilis ATCC 20509 (i.e. first fermentation broth) obtained as described in Example 4, diluted at 10% (v/v) with the culture medium used for the fermentation, in order to start the fermentation with a concentration of oleaginous cellular biomass equal to 1.6 g/l (dry weight).

(36) The fermentation was carried out batchwise for the first 30 hours obtaining a fermentation broth having a concentration of oleaginous cellular biomass equal to 24.1 g/l (dry weight) and a residual concentration of sugars equal to 38.5 g/l.

(37) Additions were subsequently made to the fermentation broth, operating in feed-batch mode of the lignocellulosic hydrolyzate (i.e. first aqueous phase) obtained as described in Example 4, suitably diluted with water so as to have a concentration of sugars equal to 199.71 g/1: in particular, four additions were made, three of 2 liters each, after 30 hours, after 40 hours and after 53 hours of fermentation respectively, and one of 3 liters after 47 hours of fermentation.

(38) At the end of the fermentation, after 60 hours, a second fermentation broth was obtained, having a concentration of oleaginous cellular biomass equal to 77.12 g/l (dry weight) and a content of total lipids equal to 54% by weight with respect to the dry weight of said oleaginous cellular biomass.

(39) Said second fermentation broth was subjected to separation by means of centrifugation at 7000 rpm, for minutes obtaining 3.9 kg of oleaginous cellular biomass [1157 g (dry weight)?concentration equal to 30% by weight with respect to the total quantity of oleaginous cellular biomass obtained] having a content of total lipids equal to 54% by weight with respect to the dry weight of said oleaginous cellular biomass and 11 l of exhausted fermentation water (i.e. second aqueous phase) having a content of non-metabolized sugars equal to 2.8 g/l.

(40) The total lipid content was determined using the total lipids-sulpho-phospho vanillin kit, operating as described above. The sugar content was determined operating as described in Example 1.

(41) A yield of oleaginous cellular biomass was reached with respect to the substrate consumed (Y.sub.X/S=g of biomass obtained per g of substrate consumed) equal to 0.42 g/g, and a yield of lipids with respect to the substrate consumed (Y.sub.L/S=g of lipids obtained per g of substrate consumed) equal to 0.21 g/g.

EXAMPLE 6

(42) Concentration of Fermentation Water by Means of Reverse Osmosis

(43) The test was carried out using an apparatus for tests with flat membranes, consisting of a cylindrical steel container, on whose base the polymeric membrane was assembled on a filtering porous septum, forming the support for the membrane. The container, equipped with stirring, can be pressurized with air or inert gas up to a pressure of 35 bar. The permeate filtered through the membrane and was collected in an underlying container, whereas the retentate remained inside the container.

(44) The membrane BW30 of Dow Chemical was used for the concentration of the fermentation water, which is a thin film composite membrane (TFC) based on polyamide, having the following characteristics: nominal molecular weight cut-off (MWCO)=50 Daltons; operating pH=2-11; maximum operating temperature=70? C.; maximum operating pressure=68 bar.

(45) For this purpose, at the end of the fermentation, the second fermentation broth, obtained as described in Example 3, was subjected, as indicated above, to centrifugation at 7000 rpm, for 20 minutes obtaining 2.1 kg of oleaginous cellular biomass and 7.9 l of exhausted fermentation water (i.e. second aqueous phase).

(46) 450 ml of said exhausted fermentation water (i.e. second aqueous phase) were subjected to filtration on a Millipore GF/C filter made of fibreglass, having a porosity of 0.22 ?m, and were charged into the apparatus for reverse osmosis described above, under stirring at 500 rpm, at an initial pressure of 15 bar, progressively increased up to 35 bar: the test was carried out at room temperature (25? C.)

(47) The exhausted fermentation water (i.e. second aqueous phase) had the following composition: total sugars: 15.15 g/l; furfural: 458 ppm; 5-hydroxymethylfurfural: 711 ppm; conductivity: 10.5 mS/cm; TOC: 14300 ppm; pH: 5.92.

(48) At the end of the reverse osmosis, 120 ml of retentate were recovered, having the following composition: total sugars: 47.2 g/l; furfural: 873 ppm; 5-hydroxymethylfurfural: 1915 ppm; conductivity: 19 mS/cm; TOC: 52000 ppm; pH: 5.85.

(49) During the reverse osmosis, a permeate was also recovered. After collecting 225 ml of permeate, equal to a 50% recovery (225 ml of permeate with respect to 450 ml of initial feed), a further 103.5 ml of permeate, were collected, bringing the overall recovery to 73%. The permeate relating to the recovery fraction between 50% and 73%, had the following composition: total sugars: 0.1063 g/l; furfural: 376 ppm; 5-hydroxymethylfurfural: 343 ppm; conductivity: 0.30 mS/cm; TOC: 140 ppm; pH: 5.5.

(50) From the above data, it can be deduced that the reverse osmosis allows a concentration factor equal to 3.1 to be obtained for the sugars, 1.9 for the furfural and 2.7 for the 5-hydroxymethylfurfural, with rejection indexes of 0.996 for the sugars, 0.395 for the furfural and 0.670 for the 5-hydroxymethylfurfural, respectively. Said data clearly indicate that the sugars are completely withheld by the membrane and concentrated, whereas the furfural and 5-hydroxymethylfurfural are withheld less and are partly distributed in the permeate. Furthermore, the permeate has a TOC equal to 140 ppm, which corresponds to 560 ppm of COD (COD=TOC?4): the value slightly exceeds the sewage discharge limit without treatment (i.e. COD equal to 500 ppm) and consequently a significant reduction in the treatment costs and water disposal costs is obtained, should it be eliminated. Alternatively, as specified above, said water can be used as process water within the process object of the present invention (for example, as washing or diluting water).

(51) The content of sugars, furfural and 5-hydroxymethylfurfural was determined, operating as described in Example 1. The TOC was determined operating as described above.

EXAMPLE 7

(52) Preparation of Inoculum with Retentate (Rhodotorula Graminis)

(53) The retentate obtained as described in Example 6 was used for the preparation of an inoculum (i.e. first fermentation broth).

(54) For this purpose, 100 ml of retentate were introduced into a 500 ml flask, equipped with stirring, to which 10 g/l of yeast extract and 10 g/l of peptone were subsequently added: the pH of the mixture obtained was maintained at 5.85 by the addition of a few drops of potassium hydroxide (KOH) 2.5 M. The mixture obtained was sterilized by filtration on Millipore Stericap? Plus 0.2 ?m filters.

(55) The sterilized mixture was inoculated with cells of Rhodotorula graminis DBVPG 4620 which were left to grow, for 24 hours, at 30? C., under stirring (200 rpm), obtaining a first fermentation broth having a concentration of oleaginous cellular biomass equal to 8.21 g/l (dry weight).

EXAMPLE 8

(56) Fermentation of Rhodotorula graminis (Feed-Batch Mode)] Using the Inoculum Prepared with the Retentate

(57) The fermentation test with cells of Rhodotorula graminis DBVPG 4620 was carried out in feed-batch mode in a 20 liter fermenter, operating under the following conditions: 2.6 l of lignocellulosic hydrolyzate (i.e. first aqueous phase) described in Example 1, suitably diluted with water so as to have an initial sugar concentration equal 100 g/l; 1.0 g/l of yeast extract; 2.5 g/l of corn steep solid; 2.5 g/l of (NH.sub.4).sub.2SO.sub.4; 1 g/l of KH.sub.2PO; 0.05 g/l of MgSO.sub.4.7H.sub.2O; 0.01 g/l of NaCl; 0.01 g/l of CaCl.sub.2.2H.sub.2O; air fed: flow equal to 1 l/min; operating pH equal to 6, maintained by the addition, when necessary, of a few drops of a solution of potassium hydroxide (KOH) 5 M and phosphoric acid (H.sub.3PO.sub.4) 10% (v/v); stirring equal to 600 rpm-900 rpm, modulated with the air flow so as to maintain the concentration of dissolved oxygen (DO.sub.2) above 30%; initial volume: 6 liters; inoculum of Rhodotorula graminis DBVPG 4620 (i.e. first fermentation broth) obtained as described in Example 6, diluted at 10% (v/v) with the culture medium used for the fermentation, in order to start the fermentation with a concentration of oleaginous cellular biomass equal to 1.4 g/l (dry weight).

(58) The fermentation was carried out batchwise for the first 24 hours obtaining a fermentation broth having a concentration of oleaginous cellular biomass equal to 12.07 g/l (dry weight) and a residual concentration of sugars equal to 23.5 g/l.

(59) Additions were subsequently made to the fermentation broth, operating in feed-batch mode of the hydrolyzate obtained as described in Example 1, suitably diluted with water so as to have a total concentration of sugars equal to 199.71 g/1: in particular, three additions were made, two of 1 liter each after 24 hours and after 34 hours of fermentation respectively, and one of 4 liters after 44 hours of fermentation.

(60) At the end of the fermentation, after 144 hours, a second fermentation broth was obtained, having a concentration of oleaginous cellular biomass equal to 57.14 g/l (dry weight) and a content of total lipids equal to 54% by weight with respect to the dry weight of said oleaginous cellular biomass.

(61) Said second fermentation broth was subjected to separation by means of centrifugation at 7000 rpm, for minutes obtaining 2.3 kg of oleaginous cellular biomass [686 g (dry weight)?concentration equal to 30% by weight with respect to the total quantity of oleaginous cellular biomass obtained] having a content of total lipids equal to 54% by weight with respect to the dry weight of said oleaginous cellular biomass and 9.7 l of exhausted fermentation water (i.e. second aqueous phase) having a content of non-metabolized sugars equal to 15.15 g/l.

(62) The total lipid content was determined using the total lipids-sulpho-phospho vanillin kit, operating as described above. The sugar content was determined operating as described in Example 1.

(63) A yield of oleaginous cellular biomass was reached with respect to the substrate consumed (Y.sub.X/S=g of biomass obtained per g of substrate consumed) equal to 0.48 g/g, and a yield of lipids with respect to the substrate consumed (Y.sub.L/S=g of lipids obtained per g of substrate consumed) equal to 0.26 g/g.

(64) If the retentate obtained as described in Example 6 were not used, the actual fermentation yields, considering the total sugars, would be Y.sub.x/s 0.43 g/g and Y.sub.L/S 0.23 g/g.

EXAMPLE 9

(65) Recovery of Lipids by Means of Cell Lysis (Thermal Treatment)

(66) For this purpose, at the end of the fermentation, 1180 ml of the second fermentation broth obtained as described in Example 8, having a concentration of oleaginous cellular biomass equal to 57.14 g/l (dry weight), were subjected to centrifugation at 7000 rpm, for 20 minutes, obtaining 200 ml of an aqueous suspension of oleaginous cellular biomass having a concentration of oleaginous cellular biomass equal to 336 g/l (dry weight) and 980 ml of exhausted fermentation water (i.e. second aqueous phase).

(67) The 200 ml of said aqueous suspension were introduced into an 0.5 l autoclave (Parr stirred reactor model PA 4575 A) which was brought to a temperature of 140? C., an autogenous pressure of 4.9 bar, with a stirring of 450 rpm, and kept under these conditions for 2 hours. After this period, the exhausted oleaginous cellular biomass was discharged and sent to the extraction process (Example 12).

EXAMPLE 10

(68) Recovery of Lipids by Means of Cell Lysis (Mechanical Treatment)

(69) For this purpose, at the end of the fermentation, 6 of the second fermentation broth obtained as described in Example 8, having a concentration of oleaginous cellular biomass equal to 57.14 g/l (dry weight), were subjected to centrifugation at 7000 rpm, for 20 minutes, obtaining 1 l of an aqueous suspension of oleaginous cellular biomass having a concentration of oleaginous cellular biomass equal to 336 g/l (dry weight) and 5 l of exhausted fermentation water (i.e. second aqueous phase).

(70) The 1 l of said aqueous suspension were pumped into a homogenizer (Mod. NS3006L of Gea NiroSoavi) at a homogenization pressure of 1500 bar, at room temperature and at a flow-rate of about 15 l/h.

(71) At the end of the treatment, the exhausted oleaginous cellular biomass was discharged and sent to the extraction process (Example 12).

EXAMPLE 11

(72) Recovery of Lipids by Means of Cell Lysis (Microwave Treatment)

(73) For this purpose, at the end of the fermentation, 1180 ml of the second fermentation broth obtained as described in Example 8, having a concentration of oleaginous cellular biomass equal to 57.14 g/l (dry weight), were subjected to centrifugation at 7000 rpm, for 20 minutes, obtaining 200 ml of an aqueous suspension of oleaginous cellular biomass having a concentration of oleaginous cellular biomass equal to 336 g/l (dry weight) and 980 ml of exhausted fermentation water (i.e. second aqueous phase).

(74) The 200 ml of said aqueous suspension were introduced into a 300 ml glass flask equipped with a cooler and a magnetic anchor and were brought to a temperature of 100? C. by means of a microwave device (Mod. MicroSYNTH of Milestone). The temperature was kept constant for 20 minutes at atmospheric pressure.

(75) At the end of the treatment, the exhausted oleaginous cellular biomass was discharged and sent to the extraction process (Example 12).

EXAMPLE 12

(76) Solvent Extraction

(77) In order to recover the lipids contained in the oleaginous cellular biomass obtained after the treatments described in Examples 9, 10 and 11, various extraction tests were carried out, using different types of solvents or mixtures thereof.

(78) For this purpose, 200 ml of the aqueous suspension of exhausted oleaginous cellular biomass, obtained as described in Example 9, Example 10, or Example 11, were used in the various tests.

(79) Said aqueous suspension was subjected to two extraction cycles, of 2 hours each, at the boiling point of the solvent or of the mixture of solvents used, in a reflux extractor, in the presence of a volume of solvent or of mixture of solvents equal to twice the volume of said aqueous suspension.

(80) The lipids were obtained after separating the organic phase containing the solvent and lipids from said aqueous suspension containing the exhausted oleaginous cellular biomass, and subjecting said organic phase to distillation of the solvent, which is recycled to the extraction.

(81) The solvents and the mixtures of solvents used, the treatments to which the oleaginous cellular biomass was subjected (Examples 9-11cell lysis), the extraction temperatures and the extraction yields, are indicated in Table 2.

(82) TABLE-US-00002 TABLE 2 Extrac- Extraction tion Treatment tem- yield* Treatment conditions Extraction solvent perature (%) Thermal autoclave, hexane/ 60? C. 98% 140? C., 2 hours iso-propanol (3:2; v/v) Thermal autoclave, ethyl acetate 72? C. 95% 140? C., 2 hours Thermal autoclave, iso-octane 82? C. 74% 140? C., 2 hours Thermal autoclave, xylene 93? C. 87% 140? C., 2 hours Thermal autoclave, ethyl tert-butyl 68? C. 83% 140? C., 2 hours ether Thermal autoclave, methyl iso-butyl 90? C. 97% 140? C., 2 hours ketone Thermal autoclave, iso-octane + 10% 70? C. 82% 140? C., 2 hours ethanol Mechanical homogenizer, ethyl acetate 72? C. 71% 1500 bar, 15 l/h Microwave microwave ethyl acetate 72? C. 80% reactor, 100? C., 20 min. *the extraction yield (%) is indicated, of lipids obtained from the extraction, with respect to the total quantity of lipids present in the oleaginous cellular biomass (dry) obtained after fermentation determined using the total lipids-sulpho-phospho vanillin kit, operating as described above.