Method of isolating lipids from a lipids containing biomass with aid of hydrophobic silica

Abstract

The current invention relates to a method of isolating lipids from a lipids containing biomass with aid of hydrophobic silica and to an oil and to a delipidated biomass as obtained by such a method.

Claims

1. A method of isolating lipids from a lipids containing biomass, comprising the following steps: a) providing a biomass suspension, wherein the biomass comprises cells containing an average of at least 10 wt.-% of lipids; b) adding hydrophobic silica to the suspension; c) heating the suspension thus obtained to a temperature of at least 50° C. and then incubating the suspension for at least 20 minutes; d) separating an oil containing light phase from a water, salts, cell debris and residual oil containing aqueous phase.

2. The method of claim 1, wherein the hydrophobic silica is added to the suspension to a final concentration of between 0.005 and 0.25 wt.-%.

3. The method of claim 2, wherein the hydrophobic silica is added to the suspension so as to adjust the final concentration to be between 0.015 to 0.10 wt.-% of hydrophobic silica.

4. The method of claim 1, wherein besides silica, at least one surfactant is added to the suspension to a final concentration of surfactant of between 0.1 and 5.0 wt.-%.

5. The method of claim 4, wherein besides silica, at least one surfactant is added to the suspension in an amount so as to adjust the final concentration of surfactant to be between 0.25 and 3.0 wt.-%.

6. The method of claim 1, wherein the biomass suspension of step a) has a total dry matter content of between 20 and 60 wt.-%.

7. The method of claim 6, wherein hydrophobic silica is at a final concentration of between 0.005 and 0.25 wt.-%, and surfactant is at a final concentration of between 0.1 and 5.0 wt.-%.

8. The method of claim 1, wherein the suspension is heated in step c) to a temperature of 50 to 100° C.

9. The method of claim 8, wherein the suspension is heated in step c) to a temperature of 65 to 90° C.

10. The method of claim 1, wherein the suspension is incubated in step c) for 20 minutes to 30 hours.

11. The method of claim 1, wherein the suspension is incubated in step c) for one hour to 24 hours.

12. The method of claim 1, further comprising, as an additional step, lysing the cells of the biomass, wherein lysing is carried out before adding hydrophobic silica.

13. The method of claim 1, wherein the biomass suspension is a fermentation broth and the cells are of the genus Schizochytrium.

14. Oil comprising hydrophobic silica in an amount of 5 ppm (w/w) to 10 wt.-%, and wherein the oil comprises PUFAs in an amount of at least 10 wt.-%.

15. The oil of claim 14, wherein the oil comprises hydrophobic silica in an amount of 0.5 wt.-% to 2.5 wt.-%, and wherein the oil comprises PUFAs in an amount of 45 to 60 wt.-%.

16. The oil of claim 14, wherein the oil comprises fatty acid esters, in an amount of at least 50 wt.-%, and free fatty acids (FFA) in an amount of less than 1 wt.-%.

17. The oil of claim 16, wherein the oil comprises fatty acid esters, in an amount of at least 85 wt.-%, and free fatty acids (FFA) in an amount of less 0.8 wt.-%.

18. A PUFAs containing delipidated biomass comprising: 10 to 25 wt.-% of ashes; less than 0.1 wt.-% of a non-polar organic solvent; and less than 0.1 wt.-%, of chloride; wherein the biomass contains cells and/or cell debris of the genus Schizochytrium.

19. The PUFAs containing delipidated biomass of claim 18, wherein the PUFAs containing delipidated biomass comprises 15-20 wt.-% of ashes, less than 0.05 wt.-%, of a non-polar organic solvent and less than less than 0.05 wt.-%, of chloride.

20. The PUFAs containing delipidated biomass of claim 4, wherein the biomass comprises a mixture of DHA and EPA in a ratio of about 3:2 to about 4:1, and wherein the content of DHA is at least 10 wt.-% of the total amount of lipid.

Description

WORKING EXAMPLES

Example 1: Preparation of the Lysed And Concentrated Fermentation Broth

(1) An unwashed cell broth containing microbial cells (Schizochytrium sp.) at a biomass density of over 100 g/l was heated to 60° C. in an agitated vessel. After heating up the suspension, the pH was adjusted to 7.5 by using caustic soda (50 wt.-% NaOH solution), before an alcalase (Alcalase® 2.4 FG (Novozymes)) was added in liquid form in an amount of 0.5 wt.-% (by weight broth). Stirring was continued for 3 hours at 60° C. After that, the lysed cell mixture was transferred into a forced circulation evaporator (obtained from GEA, Germany) and heated to a temperature of 85° C. The mixture was concentrated in the forced circulation evaporator, until a total dry matter content of about 35 wt.-% was reached.

Example 2: Demulsification Using a Mixture of Hydrophobic Silica and Surfactants

(2) To a sample of 1000 g of the lysed and concentrated fermentation broth as obtained according to example 1 were added 5 g of a demulsifier mixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-% of a hydrophobic silica selected from Sipernat® D10, Sipernat® D17 and Aerosil® 202 R (Evonik Industries, Germany).—As negative control 5 g of the mixture of Tween 80 and Span 80 was used without addition of hydrophobic silica.—The demulsifier mixture was prepared by thoroughly mixing the components before applying the mixture. The suspension thus obtained, after addition of the demulsifier mixture, was heated in an agitated vessel to a temperature of 90° C. Samples of 100 ml were taken after 1 and 22 hours, respectively, and oil was subsequently separated from the aqueous phase by centrifugation. The results are disclosed in the following table.

(3) TABLE-US-00001 TABLE 1 Demulsification using a mixture of hydrophobic silica and surfactants Hydrophobic Yield after Yield after 22 FFA after 22 surfactant one hour hours hours [wt.-%] Sipernat D10 83.5% 86.8% 0.60 Sipernat D17 83.2% 92.1% 0.60 Aerosil 202 R 86.3% 87.0% 0.55 none No free oil No free oil —
As can be seen a high yield of oil can be obtained already after one hour of incubation, but the yield is higher after 22 hours of incubation.

Example 3: Demulsification Using Different Amounts of Demulsifier Mixture

(4) To samples of 1000 g of the lysed and concentrated fermentation broth as obtained according to example 1 were added 1, 3, 5 or 8 g of a demulsifyer mixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-% Sipernat® D17 (Evonik Industries, Germany), corresponding to a final amount of hydrophobic silica in the suspension of 0.005, 0.015, 0.025 and 0.040 wt.-%, respectively.

(5) The suspensions thus obtained were heated in an agitated vessel to a temperature of 90° C. Samples of 100 ml were taken after 24 hours, respectively, and oil was subsequently separated from the aqueous phase by centrifugation.

(6) The results are disclosed in the following table.

(7) TABLE-US-00002 TABLE 2 Demulsification using a mixture of hydrophobic silica and surfactants Amount of Amount of hydrophobic demulsifyer silica in aqeous Yield after FFA after 24 mixture suspension 24 hours hours [wt.-%] 0.1 wt.-% 0.005 wt.-% 87.4% 0.60 0.3 wt.-% 0.015 wt.-% 87.1% 0.70 0.5 wt.-% 0.025 wt.-% 89.0% 0.70 0.8 wt.-% 0.040 wt.-% 86.6% 0.75
As can be seen, an efficient demulsification takes place already by adding an amount of demulsifier mixture of 1 g per kg suspension, corresponding to an amount of hydrophobic silica in the suspension of 0.005 wt.-%.

Example 4: Demulsification at Different Incubation Times

(8) To a sample of 1000 g of the lysed and concentrated fermentation broth as obtained according to example 1 were added 5 g of a demulsifier mixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-% Sipernat® D17 (Evonik Industries, Germany), corresponding to a final amount of hydrophobic silica in the suspension of 0.025 wt.-%.

(9) The suspension thus obtained was heated in an agitated vessel to a temperature of 90° C. Samples of 100 ml were taken after 2, 4, 6 and 24 hours, respectively, and oil was subsequently separated from the aqueous phase by centrifugation.

(10) The results are disclosed in the following table.

(11) TABLE-US-00003 TABLE 3 Demulsification using a mixture of hydrophobic silica and surfactants Incubation time [hours] Yield FFA [wt.-%] 2 86.7% 0.36 4 84.6% 0.38 6 88.4% 0.41 24 89.0% 0.70
As can be seen, and in accordance with the results as disclosed in table 1, an efficient demulsification takes already place with short incubation times, but better yields are achieved with incubation times of more than 5 hours.—The advantage of relatively short incubation times is the very low amount of free fatty acids (FFA) in the final product.

Example 5: Demulsification at Different Temperatures

(12) To four samples of 1000 g of the lysed and concentrated fermentation broth as obtained according to example 1 were added 5 g of a demulsifier mixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-% Sipernat® D17 (Evonik Industries, Germany), respectively, corresponding to a final amount of hydrophobic silica in the suspensions of 0.025 wt.-%.

(13) The samples such obtained were heated in agitated vessels for 24 hours at different temperatures of 30, 50, 70 and 90° C., respectively. Samples of 100 ml were taken after 24 hours, respectively, and oil was subsequently separated from the aqueous phase by centrifugation.

(14) The results are disclosed in the following table.

(15) TABLE-US-00004 TABLE 4 Demulsification at different temperatures Incubation temperature [° C.] Yield FFA [wt.-%] 30 77.8% 0.37 50 72.0% 0.38 70 85.0% 0.40 90 89.0% 0.70
As can be seen, demulsification takes place already at very low temperatures, but good yields are obtained at temperatures above 50° C.—Like for low incubation times, lower FFA values are also obtained at incubation at lower temperatures.

Example 6: Variation of the Amount and Nature of Surfactants in the Demulsifier Mixture

(16) To samples of 1000 g of the lysed and concentrated fermentation broth as obtained according to example 1 were added different amounts of a demulsifier mixture containing the hydrophobic silica Sipernat® D17 (Evonik Industries, Germany) and optionally different amounts of surfactants.

(17) The samples such obtained were heated in agitated vessels at 90° C. Samples of 100 ml were taken after 24 hours, respectively, and oil was subsequently separated from the aqueous phase by centrifugation.

(18) The different compositions of the demulsifier mixture and amounts as added are disclosed in the following table, as well as the yields of oil as obtained:

(19) TABLE-US-00005 TABLE 5 Composition and employed amounts of demulsifier mixtures Comp. Surfactant 1 Surfactant 2 Hydrophobic silica No. [wt.-%] [wt.-%] [wt.-%] 1 — — 100 Sipernat D17 2 45.5 Tween 80 45.5 Span 80 9.0 Sipernat D17 3 48.4 Tween 80 48.4 Span 80 3.2 Sipernat D17 4 47.5 Tween 80 47.5 Span 80 5.0 Sipernat D17 5 71.4 Tween 80 23.8 Span 80 4.8 Sipernat D17 6 23.8 Tween 80 71.4 Span 80 4.8 Sipernat D17 7 47.6 Brij 98 47.6 Brij 92 4.8 Sipernat D17

(20) TABLE-US-00006 TABLE 6 Influence of surfactants on the oil yield Comp. Amount Amount of hydrophobic FFA No. added [g] silica in aq. suspension Yield [wt.-%] 1 0.24 0.024 wt.-% 84.2% 0.70 2 2.5 0.023 wt.-% 86.4% 0.75 3 7.5 0.024 wt.-% 90.2% 0.75 4 5 0.025 wt.-% 89.0% 0.70 5 5 0.024 wt.-% 88.6% 0.70 6 5 0.024 wt.-% 86.5% 0.75 7 5 0.024 wt.-% 87.7% 0.60
As can be seen, efficient demulsification does take place with different surfactants and even without using surfactants. But surfactants obviously have a positive effect on the yield.