Method of isolating lipids from a lipids containing biomass

Abstract

The current invention relates to a method of isolating polyunsaturated fatty acids containing lipids from a lipids containing biomass. The method involves providing a suspension of cells with a PUFAs-containing lipid; optionally lysing the cells; concentrating the suspension; adding base equivalents to the suspension based on the total dry matter present; feeding a continuous reactor with the suspension; and separating the lipids-containing light phase from the water and cell debris-containing heavy phase.

Claims

1. A method of isolating a polyunsaturated fatty acids (PUFAs) containing lipid from a biomass, comprising the following steps: a) providing a suspension of a biomass comprising cells which contain a PUFAs containing lipid; b) optionally lysing the cells of the biomass; c) determining the total dry matter content (TDM) of the suspension and if the suspension is determined to have a TDM lower than 20 wt-%, then concentrating the suspension to a TDM of 20 to 60 wt. % prior to step d); d) adding a total of 7.5 to 25 moles of base equivalent to 10 kg of total dry matter in the suspension from step c); e) feeding a continuous reactor with the suspension from step d) at a hydrodynamic residence time of 2 to 36 hours at a temperature of 20° C. to 100° C.; f) separating a light phase containing lipids from a heavy phase containing water and cell debris.

2. The method of claim 1, wherein, in step d), 12 to 17 moles of base equivalent are added to 10 kg of total dry matter in the suspension and, in step e), the hydrodynamic residence time is 2 to 24 hours at a temperature of 70 to 90° C.

3. The method of claim 1, wherein in step d), base equivalents are added to the suspension to adjust the pH of the suspension to a value of 8 to 11.5.

4. The method of claim 1, wherein the suspension is mixed with the base equivalents in a static mixer, and wherein the static mixer forms part of the continuous reactor or is located just before the continuous reactor.

5. The method of claim 1, wherein the base as added in step (d) is a hydroxide and/or carbonate and/or a bicarbonate.

6. The method of claim 5, wherein: a) the hydroxide is selected from the group consisting of: sodium hydroxide; lithium hydroxide; potassium hydroxide; and/or calcium hydroxide; b) the carbonate is selected from the group consisting of: sodium carbonate; potassium carbonate; and/or magnesium carbonate; and/or c) the bicarbonate is selected from the group consisting of: lithium bicarbonate; sodium bicarbonate; and potassium bicarbonate.

7. The method of claim 1, wherein the base as added in step (d) is sodium hydroxide provided as an aqueous solution.

8. The method of claim 1, wherein the suspension leaving the continuous reactor has, or adjusted to have, a pH value of between 5.5 to 8.5 before the separation of the lipids containing light phase from the water and cell debris containing heavy phase is carried out.

9. The method of claim 1, wherein the continuous reactor of step (e) is a column reactor, a tube or a plug-flow reactor.

10. The method of claim 1, wherein separating the lipids containing light phase from the water and cell debris containing heavy phase according to step (f) is carried out by centrifugation or filtration.

11. The method of claim 1, wherein concentration of the suspension in step (b) is carried out by evaporation of water at a temperature not higher than 100° C.

12. The method of claim 1, wherein, in step (c), the suspension is concentrated to a TDM of 30-55% if the suspension is determined to have a TDM lower than 30%.

13. The method of claim 1, wherein cells are lysed using either no salt or salt at less than 0.1 g/l fermentation broth.

14. The method of claim 1, wherein cells are lysed using either no organic solvent or organic solvent in an amount of less than 0.1 g/l fermentation broth.

15. The method of claim 1, wherein cells are lysed enzymatically, mechanically, chemically and/or physically.

16. The method of claim 1, wherein steps (c) to (f) are carried out without prior lysing of the cells of the biomass.

17. The method of claim 1, wherein the suspension is provided as a fermentation broth.

18. The method of claim 17, wherein the fermentation broth has a biomass density of at least 100 g/l.

19. The method of claim 1, wherein the cells which contain a PUFAs containing lipid are selected from algae, fungi, protists, bacteria, microalgae, plant cells, and mixtures thereof.

20. The method of claim 1, wherein the cells which contain a PUFAs containing lipid are from the family Thraustochytrids.

Description

WORKING EXAMPLES

Example 1: Preparation of the Suspension for Use in the Demulsification Tests

(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 30 wt.-% was reached.

Example 2: Influence of the Amount of Added Base Equivalents on the Liberation of the Oil

(2) To test the significance of the amount of added base equivalents on the efficiency of the oil liberation from the biomass, the effect of addition of different amounts of caustic soda to the biomass with respect to the liberation of oil was tested. The ratio of base equivalents added to total dry matter is depicted in table 1 as well as the amount of oil as set free by the addition of the caustic soda. All experiments were carried out with one liter of enzymatically treated and subsequently concentrated fermentation broth using a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The total dry matter of the samples was 30 wt.-%. Demulsification was carried out for 24 hours at a temperature of 80° C. The suspension was stirred with 300 rpm. Caustic soda was added at the beginning as one shot. After 24 hours, the demulsified compositions were neutralized to pH 7.5. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(3) TABLE-US-00001 TABLE 1 Influence of the amount of added NaOH on the amount of liberated oil Added amount of NaOH 10 12.5 15 22.5 [mol/10 kg TDM] Added amount of NaOH [wt.-%/TDM] 4 5 6 9 Yield [wt.-%] 90.8 91.9 93.4 87.7

(4) The results show that even with quite low amounts of added base equivalents already very good yields of liberated oil can be realized without addition of organic solvents or salts like sodium chloride. Further it becomes clear that there is a ratio of added base equivalent to total dry matter, where a maximized yield can be realized. Further increasing the amount of base equivalents beyond that ratio do not increase the yield, but leads to even worse results in comparison to smaller amounts of added base equivalents. Best results were obtained with an amount of 12.5 and 15 moles NaOH per 10 kg of TDM.

Example 3: Influence of the Amount of Added Base Equivalents on the Liberation of the Oil

(5) To further test the significance of the amount of added base equivalents on the efficiency of the oil liberation from the biomass, the effect of addition of different amounts of caustic soda to the biomass with respect to the liberation of oil was tested. The ratio of base equivalents added to total dry matter is depicted in table 2 as well as the amount of oil as set free by the addition of the caustic soda. All experiments were carried out with one liter of enzymatically treated and subsequently concentrated fermentation broth using a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The total dry matter of the samples was 30.5 wt.-%. Demulsification was carried out for 24 hours at a temperature of 80° C. The suspension was stirred with 300 rpm. Caustic soda was added stepwise in three shots to keep the pH low. After 24 hours the demulsified compositions were neutralized to pH 7.5. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(6) TABLE-US-00002 TABLE 2 Influence of the amount of added NaOH on the amount of liberated oil Added amount of NaOH 12.5 17.5 20 22.5 [mol/10 kg TDM] Added amount of NaOH 5 7 8 9 [wt.-%/TDM] Yield [wt.-%] 93.9 92.3 92.6 84.5

(7) The results show that even with quite low amounts of added base equivalents already very good yields of liberated oil can be realized without addition of organic solvents or salts like sodium chloride. Further it becomes clear that there is a ratio of added base equivalent to total dry matter, where a maximized yield can be realized. Further increasing the amount of base equivalents beyond that ratio do not increase the yield, but leads to even worse results in comparison to smaller amounts of added base equivalents. Best results were obtained with an amount of 12.5 to 20 moles NaOH per 10 kg of TDM.

Example 4: Influence of the Amount of Added Base Equivalents on the Liberation of the Oil

(8) To test the significance of the amount of added base equivalents on the efficiency of the oil liberation from the biomass, the effect of addition of different amounts of caustic soda to the biomass with respect to the liberation of oil was tested. The ratio of base equivalents added to total dry matter is depicted in table 3 as well as the amount of oil as set free by the addition of the caustic soda. All experiments were carried out with one liter of enzymatically treated and subsequently concentrated fermentation broth using a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The total dry matter of the samples was 30 wt.-%. Demulsification was carried out for 24 hours at a temperature of 80° C. The suspension was stirred with 300 rpm. Caustic soda was added continuously to avoid high pH values. After 24 hours the demulsified compositions were neutralized to pH 7.5. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(9) TABLE-US-00003 TABLE 3 Influence of the amount of added NaOH on the amount of liberated oil Added amount of NaOH 10 12.5 15 17.5 20 [mol/10 kg TDM] Added amount of NaOH 4 5 6 7 8 [wt.-%/TDM] Yield [wt.-%] 92.5 94.0 95.2 95.2 93.8

(10) The results show that even with quite low amounts of added base equivalents already very good yields of liberated oil can be realized without addition of organic solvents or salts like sodium chloride. Further it becomes clear that there is a ratio of added base equivalent to total dry matter, where a maximized yield can be realized. Further increasing the amount of base equivalents beyond that ratio do not increase the yield, but leads to even worse results in comparison to smaller amounts of added base equivalents. Best results were obtained with an amount of 12.5 to 17.5 moles NaOH per 10 kg of TDM.

Example 5: Influence of the Amount of Added Base Equivalents on the Liberation of the Oil

(11) To test the significance of the amount of added base equivalents on the efficiency of the oil liberation from the biomass, the effect of addition of different amounts of caustic soda to the biomass with respect to the liberation of oil was tested. The ratio of base equivalents added to total dry matter is depicted in table 4 as well as the amount of oil as set free by the addition of the caustic soda. All experiments were carried out with one liter of enzymatically treated and subsequently concentrated fermentation broth using a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The total dry matter of the samples was 35.5 wt.-%. Demulsification was carried out for 24 hours at a temperature of 80° C. The suspension was stirred with 300 rpm. Caustic soda was added continuously to avoid high pH values. After 24 hours the demulsified compositions were neutralized to pH 7.5. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(12) TABLE-US-00004 TABLE 4 Influence of the amount of added NaOH on the amount of liberated oil Added amount of NaOH 10 12.5 15 17.5 20 [mol/10 kg TDM] Added amount of NaOH 4 5 6 7 8 [wt.-%/TDM] Yield [wt.-%] 88.5 91.8 91.0 89.0 87.0

(13) The results show that even with quite low amounts of added base equivalents already very good yields of liberated oil can be realized without addition of organic solvents or salts like sodium chloride. Further it becomes clear that there is a ratio of added base equivalent to total dry matter, where a maximized yield can be realized. Further increasing the amount of base equivalents beyond that ratio do not increase the yield, but leads to even worse results in comparison to smaller amounts of added base equivalents. Best results were obtained with an amount of 12.5 to 15 moles NaOH per 10 kg of TDM.

Example 6: Influence of Temperature, Total Dry Matter, Amount of Caustic and Stirring Speed on the Liberation of the Oil

(14) To test the influence and interdependence of temperature, total dry matter content (TDM), amount of base euqivalents and stirring speed on the liberation of the oil, tests were carried out with enzymatically treated fermentation broths which were concentrated by forced circulation evaporation to a total dry matter content of either 25, 30 or 35 wt.-%. Tests were carried out in a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The volume of the concentrated suspensions as used in the tests was 1 L for each sample. In the tests the total amount of base equivalent added was varied from 5 to 7 wt.-%, while the demulsification was carried out either at 70, 80 or 90° C. As base equivalent NaOH was added in liquid form (20 wt.-% NaOH solution) in one shot at the beginning of the demulsification step. Demulsification took place at a stirrer speed of either 100, 550 or 1000 rpm for 24 hours. After 24 hours the resulting compositions were neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(15) TABLE-US-00005 TABLE 5 Influence of temperature, TDM and stirring speed on the yield of liberated oil Temperature [° C.] 70 70 90 90 70 70 90 90 80 80 TDM [wt.-%] 25 35 25 35 25 35 25 35 30 30 Amount of NaOH 5 7 7 5 7 5 5 7 6 6 [wt.-% per TDM] Amount of NaOH 12.5 17.5 17.5 12.5 17.5 12.5 12.5 17.5 15 15 [mol/10 kg TDM] Stirrer speed [rpm] 100 100 100 100 1000 1000 1000 1000 550 550 Yield [wt.-%] 90.3 96.2 82.9 95.5 76.6 93.1 93.1 90.0 94.9 93.9

(16) As can be seen with a TDM of 35 wt.-% always very good results could be realized, i.e. an oil yield of at least 90 wt.-%, even at a rather high amount of base equivalents and a rather low temperature. On the contrary, at a TDM of only 25 wt.-%, the results might get significantly worse, when the amount of base equivalent is quite high, in particular when the temperature is quite low.

Example 7: Influence of Temperature on the Liberation of the Oil

(17) To test the significance of the temperature on the liberation of the oil, tests were carried out with enzymatically treated fermentation broths which were concentrated by forced circulation evaporation to a total dry matter content of 33 wt.-%. Tests were carried out in a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The volume of the concentrated suspensions as used in the tests was 1 L for each sample. In each test the same total amount of base equivalent (6 wt.-% NaOH per TDM, i.e. 15 moles NaOH per 10 kg TDM) was added, while the demulsification was carried out either at 40, 50 or 90° C. The base equivalent was added in liquid form (20 wt.-% NaOH solution) in one shot at the beginning of the demulsification step. Demulsification took place at a stirrer speed of 300 rpm for 24 hours. After 24 hours the resulting composition was neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(18) TABLE-US-00006 TABLE 6 Influence of the temperature on the yield of liberated oil Temperature [° C.] 40 50 90 Yield [wt.-%] 85.4 87.6 93.0

(19) It turned out that even at low temperatures as like 40° C. or 50° C. a very efficient liberation of oil can be realized, when the appropriate amount of base equivalents is added.

Example 8: Demulsification at Very Low Temperatures

(20) To further test the significance of the temperature on the liberation of the oil, tests were carried out with enzymatically treated fermentation broths which were concentrated by forced circulation evaporation to a total dry matter content of 33.5 wt.-%. Tests were carried out in a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The volume of the used concentrated suspensions was 1 L for each sample. In each test the same total amount of base equivalent (6 wt.-% NaOH per TDM, i.e. 15 moles NaOH per 10 kg TDM) was added, while the demulsification was carried out either at 30° C. or 40° C. The base equivalent was added in liquid form (20 wt.-% NaOH solution) in one shot at the beginning of the demulsification step. Demulsification took place at a stirrer speed of 300 rpm for 24 hours. After 24 hours the resulting composition was neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(21) TABLE-US-00007 TABLE 7 Influence of the temperature on the yield of liberated oil Temperature [° C.] 30 40 Yield [wt.-%] 85.8 84.6

(22) It turned out that even at temperatures as low as 30° C. an efficient liberation of oil can be realized, when the appropriate amount of base equivalents is added.

Example 9: Influence of Demulsification Time on the Liberation of Oil

(23) To test the significance of the exposure time on the liberation of the oil, tests were carried out with enzymatically treated fermentation broths which were concentrated by forced circulation evaporation to a total dry matter content of 36.2 wt.-%. Tests were carried out in a stirring vessel. The volume of the used concentrated suspensions was 300 L for each sample. In each test the same total amount of base equivalent (6 wt.-% NaOH per TDM, i.e. 15 moles NaOH per 10 kg TDM) was added and the temperature was kept at 80° C. The base equivalent was added stepwise in liquid form (20 wt.-% NaOH solution) in the course of the exposure, so that the pH of the suspension never exceeded pH 9.5. The exposure times were varied from 4 to 23 hours. After the incubation the resulting composition was neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(24) TABLE-US-00008 TABLE 8 Influence of the time of demulsification on the yield of liberated oil Exposure time [h] 4 9 13 23 Yield [wt.-%] 90.5 92.0 92.0 91.3

(25) It turned out that surprisingly almost the same amount of oil could be isolated at exposure times varying from 4 to 23 hours. That means that quite short exposure times already lead to very good yields, so that longer, time and energy consuming incubation times can be avoided.

Example 10: Use of Ca(OH).SUB.2 .as Base in the Demulsification

(26) Enzymatically treated fermentation broths were concentrated by forced circulation evaporation to a total dry matter content of 34 wt.-%. Tests were carried out in a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The volume of the used concentrated suspensions was one liter for each sample. The demulsification was carried out at a temperature of 80° C. and at an exposure time of 24 hours. The suspensions were stirred with 300 rpm. Either 10.6 moles base equivalents or 14.0 moles base equivalents of Ca(OH).sub.2 per 10 kg total dry matter were added to the suspension. Ca(OH).sub.2 was added stepwise in suspended form (20 wt.-% Ca(OH).sub.2 in water) in the course of the exposure, so that the pH value of the suspension never exceeded pH 9.5. After the incubation the resulting composition was neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(27) TABLE-US-00009 TABLE 9 Influence of the amount of added Ca(OH).sub.2 on the amount of liberated oil Added amount of Ca(OH).sub.2 10.6 14.0 [mol/10 kg TDM] Added amount of Ca(OH).sub.2 [wt.-%/TDM] 3.9 5.2 Added amount of Ca(OH).sub.2 [g] 13.3 17.8 Yield [wt.-%] 90.2 90.1

(28) As can be seen, increasing the amount of Ca(OH).sub.2 from 10.6 to 14.0 moles per 10 kg total dry matter has no influence on the yield of liberated oil. Addition of the same amount of base equivalents leads to similar good results as in case of NaOH.

Example 11: Demulsification without Enzymatic Treatment of the Cells

(29) To test the significance of the enzymatic treatment of the cells on the liberation of the oil, tests were carried out with fermentation broths which after pasteurisation were not treated enzymatically, but directly concentrated by forced circulation evaporation to a total dry matter content of 32.8 wt.-%. Tests were carried out in a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). The volume of the used concentrated suspensions was 1 L for each sample. In each test the same total amount of base equivalent (6 wt.-% NaOH per TDM, i.e. 15 moles NaOH per 10 kg TDM) was added, and the demulsification was carried out at 90° C. for 24 hours at a stirrer speed of 300 rpm. The base equivalent was added in liquid form (20 wt.-% NaOH solution) in one shot at the beginning of the demulsification step. After 24 hours the resulting composition was neutralized by adding sulfuric acid. After neutralization, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

(30) It turned out that surprisingly even without prior enzymatic treatment of the cells quite good oil yields of about 80% could be realized, when an appropriate amount of total dry matter is provided and an appropriate amount of base equivalents is applied during demulsification.

Example 12: Continuous Demulsification Using a Heated Column Reactor

(31) For carrying out the continuous demulsification process in lab scale, the following equipment was provided: a) a feed tank with concentrated lysed broth as obtained according to example 1 having a TDM content of about 35 wt.-%. The feed tank was heated to 90° C. b) a NaOH tank, containing an aqueous solution of NaOH (20 wt.-% NaOH water). c) a column reactor with a heat jacket and a capacity of 0.3 liter, where glass beads are located on the inlet of the column reactor to simulate a static mixer and wherein the column reactor is heated to 90° C.

(32) The experiment was carried out as follows: the concentrated lysed broth is fed into the column reactor with a feed stream of 1.25 g/min. The NaOH solution is fed into the plastic tube carrying the broth feed shortly before entering the glass beads at the inlet of the column reactor with a feed stream of 0.13 g/min, so that the broth and the base are thoroughly mixed before entering the column reactor, resulting in a feed stream of 1.38 g/min, corresponding to a volume stream of 1.25 ml/min and a hydrodynamic residence time of 4 hours.

(33) After leaving the column reactor, samples are taken and neutralized to pH 6.5 by adding diluted sulfuric acid (20 wt.-% sulfuric acid in water) at different points in time. The neutralized suspension is subsequently centrifuged to separate the oil containing light phase from the aqueous phase and then the amount of isolated oil is determined.

(34) TABLE-US-00010 TABLE 10 Yield of isolated oil at different point in time applying a hydrodynamic residence time of 4 hours Process time [h] 4 20 27 44 51 68 75 Yield [wt.-%] 84.8 88.9 84.7 89.7 87.9 90.5 85.7

(35) The experimental data show that with a hydrodynamic residence time of 4 hours a quite efficient oil yield (in average 88 wt.-%) can be realized constantly over time, when the demulsification is carried out in a continuous flow reactor.