Process for the extraction of lipids from algal biomass

Abstract

A process for the extraction of lipids from algal biomass comprising: —producing an aqueous suspension of algal biomass; —adding to said aqueous suspension of algal biomass at least one organic solvent immiscible or substantially immiscible with water obtaining an organic-aqueous mixture; —subjecting said organic-aqueous mixture to evaporation of water and lipid extraction, operating at a temperature such to obtain the substantial complete removal of the water from said organic-aqueous mixture, obtaining: (i) an organic phase comprising lipids and said organic solvent; (ii) a semi-solid phase comprising a residue of said algal biomass.

Claims

1. A process for the extraction of lipid from algal biomass, said process consisting of: (a) producing an aqueous suspension of algal biomass; (b) adding to said aqueous suspension of algal biomass at least one organic solvent immiscible or substantially immiscible with water, to obtain an organic-aqueous mixture; (c) subjecting said organic-aqueous mixture to evaporation of water and lipid extraction, said evaporation of water and lipid extraction being carried out simultaneously, operating at a temperature such to obtain the substantial complete removal of the water from said organic-aqueous mixture, to obtain: (i) an organic phase comprising lipid and said organic solvent; and (ii) a semi-solid phase comprising a residue of said algal biomass, wherein a lipid extraction yield ranges from 62.9% to 88.8%.

2. The process according to claim 1, wherein the aqueous suspension of algal biomass is derived from the growth of unicellular algae (microalgae), carried out on wastewaters coming from industrial wastewaters.

3. The process according to claim 1 or 2, wherein, the aqueous suspension of algal biomass is subjected to thickening to obtain a higher concentration of dry matter in said algal biomass.

4. The process according to claim 1, wherein the aqueous suspension of algal biomass has a concentration of dry matter ranging from 2% by weight to 40% by weight with respect to the total weight of the aqueous suspension of algal biomass.

5. The process according to claim 4, wherein the aqueous suspension of algal biomass has a concentration of dry matter ranging from 4% by weight to 25% by weight with respect to the total weight of the aqueous suspension of algal biomass.

6. The process according to claim 1, wherein the organic solvent immiscible with water is one or more members selected from the group consisting of an aliphatic hydrocarbon having a boiling point higher than 100° C.; an aromatic hydrocarbon; a refinery cut comprising: (a) a mixture of aliphatic hydrocarbons having a boiling point higher than 100° C., (b) a mixture of aromatic hydrocarbons, or (c) a mixture of aliphatic and aromatic hydrocarbons.

7. The process according to claim 6, wherein the organic solvent immiscible in water is selected from the group consisting of n-octane, xylene, and a mixture thereof.

8. The process according to claim 1, wherein the organic solvent substantially immiscible with water is selected from the group consisting of an ester; a ketone having a boiling point higher than 100° C.; and a mixture thereof.

9. The process according to claim 8, wherein the organic solvent substantially immiscible with water is ethyl acetate.

10. The process according to claim 1, wherein the ratio between the concentration of dry matter in the aqueous suspension of algal biomass and the volume of organic solvent is from 1:1 to 1:80.

11. The process according to claim 10, wherein the ratio between the concentration of dry matter in the aqueous suspension of algal biomass and the volume of organic solvent is from 1:3 to 1:70.

12. The process according to claim 1, wherein the evaporation of water and the lipid extraction are carried out: (1) under atmospheric pressure, at 100° C., if the boiling temperature of said organic solvent is higher than 100° C.; or, (2) at the boiling temperature of the low boiling azeotrope, if said organic solvent forms a low boiling azeotrope with water.

13. The process according to claim 1, wherein the evaporation of water and lipid extraction are carried out for a time ranging from 30 minutes to 5 hours.

14. The process according to claim 13, wherein the evaporation of water and lipid extraction are carried out for a time ranging from 1.5 hours to 3.5 hours.

15. The process according to claim 1, wherein the organic phase (i) comprising lipid and organic solvent is subjected to evaporation in order to recover the organic solvent which is re-used in the above process and the lipid is extracted.

16. The process according to claim 15, wherein the lipid is subjected to esterification in the presence of an alcohol having from 1 to 4 carbon atoms and of an acid catalyst in order to produce glycerol and alkyl esters.

17. The process according to claim 15, wherein the lipid is subjected to hydrogenation/deoxygenation in presence of hydrogen and a catalyst, to obtain green diesel.

18. The process according to claim 1, wherein said organic phase (i) comprising lipid and organic solvent is directly subjected to esterification, or to hydrogenation/deoxygenation.

19. The process according to claim 1, wherein said semi-solid phase (ii) comprising a residue of the algal biomass is subjected to pyrolysis in order to obtain pyrolytic oils.

20. The process according to claim 1, wherein said semi-solid phase (ii) comprising a residue of the algal biomass, after removal of the organic solvent, is subjected to anaerobic digestion by micro-organisms in the absence of oxygen in order to obtain biogas.

Description

EXAMPLE 1

Preparation of the Algal Biomass

(1) In the following examples 2-7 the algal strain Scenedesmus sp., of internal collection, which normally grows in fresh water, was used. The cultivation process adopted is described hereunder.

(2) The inoculum to be introduced into the growth tank described hereunder, was prepared as follows: a sample of monoalgal culture previously preserved at −85° C. in a 10% glycerine solution, was defrosted leaving it at room temperature, it was then subjected to centrifugation to remove the supernatant, obtaining a cellular paste; the cellular paste thus obtained was inoculated into three 250 ml flasks containing 50 ml of solution comprising nutrients, obtaining an algal culture; said algal culture was grown in an illuminated climatic chamber at a constant temperature of 30° C., in the presence of CO.sub.2 at 0.5% in air; after about a week, the flask reached concentration of 0.3 g/l, this culture was used as inoculum for three 1 liter flasks containing 500 ml of solution comprising nutrients and placed in the climatic chamber; after 2 days the culture had a concentration of 0.5 g/l and this culture was used as inoculum for a laboratory growth tank having a volume of 35 liters.

(3) The inoculum, prepared as described above, was grown in the culture medium M4N, described in literature for the cultivation of microalgae. The growth conditions were the following:

(4) Water: drinkable;

(5) KNO.sub.3: 5.0 g/l;

(6) KH.sub.2PO.sub.4: 1.25 g/1;

(7) CaCl.sub.2: 0.01 g/l;

(8) FeSO.sub.4.7H.sub.2O: 0.003 g/1;

(9) MgSO.sub.4.7H.sub.2O: 2.5 g/l;

(10) Microelements: 1 ml/l of the following solution: 2.86 g of H.sub.3BO.sub.3, 1.81 g of MnCl.sub.2.4H.sub.2O, 80 mg of CuSO.sub.4.5H.sub.2O, 220 mg of ZnSO.sub.4.7H.sub.2O, 210 mg of Na.sub.2MoO.sub.4, 25 g of FeSO.sub.4.7H.sub.2O, 33.5 g of EDTA and 1 drop of concentrated H.sub.2SO.sub.4 per liter;

(11) operating pH: 7.8.

(12) Inoculum tank: 10% by volume of the above culture in M4N medium.

(13) The tank was illuminated from the outside by means of 17,500 Lux tungsten lamps and was maintained at 28° C. by means of thermostat-regulated water circulation. The tank was also fed with a mixture of air and CO.sub.2 at 10% in air, at a flow-rate of 200 liters/hour, under a pH control (pH set point 7.0).

(14) Culture Medium Tank (Optimized M4N):

(15) Water: drinkable;

(16) KNO.sub.3: 1.5 g/l;

(17) KH.sub.2PO.sub.4: 1.25 g/l;

(18) K.sub.2HPO.sub.4: 0.1 g/l;

(19) CaCl.sub.2: 0.01 g/l;

(20) FeSO.sub.4.7H.sub.2O: 0.003 g/l;

(21) MgSO.sub.4.7H.sub.2O: 1.5 g/l;

(22) Microelements: 1 ml/l of the following solution: 2.86 g of H.sub.3BO.sub.3, 1.81 g of MnCl.sub.2.4H.sub.2O, 80 mg of CuSO.sub.4.5H.sub.2O, 220 mg of ZnSO.sub.4.7H.sub.2O, 210 mg of Na.sub.2MoO.sub.4, 25 g of FeSO.sub.4.7H.sub.2O, 33.5 g of EDTA and 1 drop of concentrated H.sub.2SO.sub.4 per liter;

(23) operating pH: 7.0.

(24) When shifts of ±0.2 units were observed with respect to pH 7.0, the CO.sub.2 flow was manually modified.

(25) The composition indicated above was obtained by modifying the typical culture medium M4N described in literature for the cultivation of microalgae. In particular, the modifications carried out are: reduction of the KNO.sub.3 content from 5.0 g/l to 1.5 g/1; addition of K.sub.2HPO.sub.4 in a quantity of 0.1 g/l; reduction of the content of MgSO.sub.4.7H.sub.2O from 2.5 g/l to 1.5 g/l; reduction in the operating pH from 7.8 to 7.0.

(26) Three samples of tank culture were taken and subjected to optical density measurements, at a wave-length of 750 nm, by means of a Varian C 900 spectrophotometer in order to be able to follow the algal growth trend.

(27) In addition to this measurement, dry weight measurements were carried out to determine the effective concentration reached by the cultures. Table 1 summarizes the results obtained (triplicate values).

(28) TABLE-US-00001 TABLE 1 Dry Time Optical density (750 nm) weight (hrs) 0 3 5 20.5 24 29 44.5 48 52 (g/l) 0.415 0.500 0.570 1.550 1.840 2.200 2.850 3.180 3.350 0.835 0.670 0.760 0.830 1.500 1.550 1.720 2.300 2.410 2.540 0.925 0.510 0.570 0.620 1.330 1.250 1.460 1.900 1.980 2.060 0.580

(29) The algal biomass obtained was centrifuged in a disk centrifuge of the Alfa Laval type obtaining an aqueous suspension of algal biomass having a volume of about 2 liters (concentration of dry matter 10 g/liter).

(30) The aqueous suspension of algal biomass was then subjected to filtration under vacuum until a concentration of dry matter was obtained, varying from 5% by weight to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass.

(31) The samples obtained were preserved in a refrigerator before being subjected to lipid extraction.

EXAMPLE 2

Lipid Extraction (Concentration of Dry Matter Equal to 5%, Solvent n-Octane)

(32) 50 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 100 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 5% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(33) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature, for 3 hours.

(34) After 3 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(35) The organic phase, about 50 ml comprising n-octane and lipids, was recovered by removing it directly from the flask.

(36) The semisolid phase remaining in the flask was washed with n-octane (50 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(37) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids by modifying the spectrophotometric method described by Marsch J. B. and Weistein D. B. in “Journal of Lipid Research” (1996), Vol. 7, pages 574-576.

(38) For this purpose, 2 mg of starting algal biomass or of residue of dried algal biomass, were suspended in a ml test-tube, in 4.5 ml of a chloroform:methanol solution in a ratio of 1:2 (v/v). The test-tube was placed in an ultrasound bath (NEY Ultrasonik 104H) for 15 minutes, and subsequently in an ice bath for 5 minutes. 1.5 ml of chloroform and 1.5 ml of water were then added, obtaining an aqueous phase comprising methanol and an organic phase comprising chloroform and the extracted lipids. Said organic phase was transferred to a 10 ml test-tube and the chloroform was evaporated operating under a nitrogen flow.

(39) After evaporation of the chloroform, 2 ml of concentrated sulphuric acid were added. The test-tube was heated to 200° C., left at this temperature for 15 minutes, and rapidly brought back to room temperature (25° C.) by placing the test-tube in an ice bath. After 5 minutes in ice, 3 ml of deionized water were added, the test-tube was stirred and subsequently placed in ice for a further 5 minutes.

(40) The sample thus obtained was subjected to optical density measurement, at a wave-length equal to 375 nm using a Philips spectrophotometer mod. PU8625. The concentration of the lipids is obtained by interpolation taking as reference the values of the standard obtained by dissolving palmitin in chloroform at different concentrations: 100 μg/ml, 200 μg/ml and 300 μg/ml.

(41) The results obtained are indicated in Table 2.

EXAMPLE 3

Lipid Extraction (Concentration of Dry Matter Equal to 10%, Solvent n-Octane)

(42) 50 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 10% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(43) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature, for 2.5 hours.

(44) After 2.5 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(45) The organic phase, about 50 ml, comprising n-octane and lipids, was recovered by removing it directly from the flask.

(46) The semisolid phase remaining in the flask was washed with n-octane (50 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(47) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 2.

EXAMPLE 4

Lipid Extraction (Concentration of Dry Matter Equal to 20%, Solvent n-Octane)

(48) 100 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(49) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature, for 3 hours.

(50) After 3 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(51) The organic phase, about 100 ml, comprising n-octane and lipids, was recovered by removing it directly from the flask.

(52) The semisolid phase remaining in the flask was washed with n-octane (100 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(53) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 2.

EXAMPLE 5

Lipid Extraction (Concentration of Dry Matter Equal to 10%, Solvent Xylene)

(54) 50 ml of xylene were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 10% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(55) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 92° C. (boiling point of the low-boiling azeotrope) and was kept, under stirring, at this temperature, for 3 hours.

(56) After 3 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(57) The organic phase, about 50 ml, comprising xylene and lipids, was recovered by removing it directly from the flask.

(58) The semisolid phase remaining in the flask was washed with xylene (50 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(59) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 2.

EXAMPLE 6

Lipid Extraction (Concentration of Dry Matter Equal to 20%, Solvent Xylene)

(60) 100 ml of xylene were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(61) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 92° C. (boiling point of the low-boiling azeotrope) and was kept, under stirring, at this temperature, for 2 hours.

(62) After 2 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(63) The organic phase, about 100 ml, comprising xylene and lipids, was recovered by removing it directly from the flask.

(64) The semisolid phase remaining in the flask was washed with xylene (100 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(65) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 2.

EXAMPLE 7

Lipid Extraction (Concentration of Dry Matter Equal to 20%, Solvent Ethyl Acetate)

(66) 100 ml of ethyl acetate were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Scenedesmus sp. obtained as described in Example 1, having a concentration of dry matter equal to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(67) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 70° C. (boiling point of the low-boiling azeotrope) and was kept, under stirring, at this temperature, for 2.5 hours.

(68) After 2.5 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(69) The organic phase, about 100 ml, comprising ethyl acetate and lipids, was recovered by removing it directly from the flask.

(70) The semisolid phase remaining in the flask was washed with ethyl acetate (100 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(71) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 2.

(72) TABLE-US-00002 TABLE 2 B** LIPID A* TOTAL EXTRACTION TOTAL LIPIDS LIPIDS YIELD EXAMPLE (%) (%) (%) 2 22.4 6.7 70.1 3 22.4 8.3 62.9 4 22.4 8.2 63.4 5 22.4 5.3 76.3 6 22.4 5.0 77.7 7 22.4 2.5 88.8 A*: starting algal biomass; B**: residue of dried algal biomass after lipid extraction

EXAMPLE 8

Preparation of the Algal Biomass

(73) In the following examples 9-11, the algal strain Chlorella sp., of internal collection, which normally grows in salt water, was used. The cultivation process adopted is described hereunder.

(74) The inoculum to be introduced into the growth tank described hereunder, was prepared as follows: a sample of monoalgal culture previously preserved at −85° C. in a 10% glycerine solution, was defrosted leaving it at room temperature, it was then subjected to centrifugation to remove the supernatant, obtaining a cellular paste; the cellular paste thus obtained was inoculated into three 250 ml flasks containing 50 ml of solution comprising nutrients, obtaining an algal culture; said algal culture was grown in an illuminated climatic chamber at a constant temperature of 30° C., in the presence of CO.sub.2 at 0.5% in air; after about a week, the flask reached a concentration of 0.3 g/l, this culture was used as inoculum for three 1 liter flasks containing 500 ml of solution comprising nutrients and placed in the climatic chamber; after 2 days the culture had a concentration of 0.5 g/l and this culture was used as inoculum for a laboratory growth tank having a volume of 35 liters.

(75) The inoculum, prepared as described above, was grown in the culture medium F/2, described in literature for the cultivation of microalgae. The growth conditions were the following:

(76) Water: drinkable;

(77) Seawater salts: 33 g/l

(78) NaNO.sub.3: 600 mg/1;

(79) NaH.sub.2PO.sub.4.H.sub.2O: 45 mg/l;

(80) FeCl.sub.3.6H.sub.2O: 6.3 mg/l;

(81) Na.sub.4EDTA: 8.72 mg/l;

(82) Vitamins: 0.2 mg/l of thiamine-HCl, 1.0 μg/l of biotin, 1.0 μg/l of vitamin B12;

(83) Microelements: 0.0196 mg/l of CuSO.sub.4.5H.sub.2O, 0.044 mg/l of ZnSO.sub.4.7H.sub.2O, 0.020 mg/l of CoCl.sub.2.6H.sub.2O, 0.360 mg/l of MnCl.sub.2.4H.sub.2O, 0.0126 mg/l of Na.sub.2MoO.sub.4.2H.sub.2O;

(84) Operating pH: 7.8.

(85) Inoculum tank: 10% by volume of the above culture in F/2 medium.

(86) The tank was illuminated from the outside by means of 17,500 Lux tungsten lamps and was maintained at 28° C. by means of thermostat-regulated water circulation. The tank was also fed with a mixture of air and CO.sub.2 at 10% in air, at a flow-rate of 200 liters/hour, under a pH control (pH set point 7.0).

(87) F2 culture medium tank: the growth conditions are the same as those indicated for the growth of the inoculum.

(88) When shifts of +0.2 units were observed with respect to pH 7.0, the CO.sub.2 flow was manually modified.

(89) The algal biomass obtained was centrifuged in a disk centrifuge of the Alfa Laval type obtaining an aqueous suspension of algal biomass having a volume of about 2 liters (concentration of dry matter 10 g/liter).

(90) The aqueous suspension of algal biomass was then subjected to filtration under vacuum until a concentration of dry matter was obtained, varying from 5% by weight to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass.

(91) The samples obtained were preserved in a refrigerator before being subjected to lipid extraction.

EXAMPLE 9

Lipid Extraction (Concentration of Dry Matter Equal to 5%, Solvent n-Octane)

(92) 50 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 100 g of an aqueous suspension of algal biomass of algae of the strain Chlorella sp. obtained as described in Example 8, having a concentration of dry matter equal to 5% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(93) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature for 3 hours.

(94) After 3 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(95) The organic phase, about 50 ml, comprising n-octane and lipids, was recovered by removing it directly from the flask.

(96) The semisolid phase remaining in the flask was washed with n-octane (50 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(97) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 3.

EXAMPLE 10

Lipid Extraction (Concentration of Dry Matter Equal to 10%, Solvent n-Octane)

(98) 50 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Chlorella sp. obtained as described in Example 8, having a concentration of dry matter equal to 10% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(99) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature, for 2.5 hours.

(100) After 2.5 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(101) The organic phase, about 50 ml, comprising n-octane and lipids, was recovered by removing it directly from the flask.

(102) The semisolid phase remaining in the flask was washed with n-octane (50 ml) and dried in a thermostat-regulated oven at 80° C., obtaining a residue of dried algal biomass.

(103) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 3.

EXAMPLE 11

Lipid Extraction (Concentration of Dry Matter Equal to 20%, Solvent n-Octane)

(104) 100 ml of n-octane were added to a three-necked 500 ml flask, equipped with a Marcusson Dean-Stark distillation apparatus, containing 50 g of an aqueous suspension of algal biomass of algae of the strain Chlorella sp. obtained as described in Example 8, having a concentration of dry matter equal to 20% by weight with respect to the total weight of the aqueous suspension of algal biomass (ratio concentration of dry matter in the aqueous suspension of algal biomass:volume solvent equal to 1:10).

(105) The flask, maintained at atmospheric pressure, was brought by means of a heating jacket to 100° C. and was kept, under stirring, at this temperature, for 3 hours.

(106) After 3 hours, the stirring was stopped, the flask was brought back to room temperature (25° C.) and the two phases were left to separate by gravity.

(107) The organic phase, about 100 ml, comprising n-octane and lipids, was recovered by removing it directly from the flask.

(108) The semisolid phase remaining in the flask was washed with n-octane (100 ml) and dried in a thermostat-regulated oven at 100° C., obtaining a residue of dried algal biomass.

(109) The starting algal biomass, as well as the residue of dried algal biomass, were subjected to determination of the total lipids operating as described in Example 2: the results obtained are indicated in Table 3.

(110) TABLE-US-00003 TABLE 3 A* B** LIPID TOTAL TOTAL EXTRACTION LIPIDS LIPIDS YIELD EXAMPLE (%) (%) (%) 9 22.3 6.2 72.2 10 22.3 8.2 63.2 11 22.3 7.9 64.6 A*: starting algal biomass; B**: residue of dried algal biomass after lipid extraction