Method of separating lipids from a lysed lipids containing biomass

Abstract

The current invention relates to a method of separating polyunsaturated fatty acids containing lipids from a lipids containing biomass by using acetone.

Claims

1. A method of separating polyunsaturated fatty acids (PUFAs) containing lipid from the debris of a biomass, comprising the steps: a) providing a suspension of a biomass comprising cells which contain a PUFAs containing lipid; b) lysing the cells of the biomass; c) adding acetone to the suspension obtained in step (b) until a final amount of between 25 and 47.5 wt.-% of acetone is reached; d) thoroughly mixing the suspension obtained in step (c); e) separating an oil containing PUFAs and acetone-containing light phase obtained in step (d) from a water, acetone, salt and cell debris containing heavy phase.

2. The method of claim 1, wherein acetone is added to the suspension of biomass in step (c) until a final amount of between 27.5 and 45.0, wt.-% of acetone is reached.

3. The method of claim 1, wherein mixing of the suspension in step (d) is carried out by shaking, stirring and/or vortexing.

4. The method of claim 1, wherein lysing of the cells of the biomass is carried out enzymatically, mechanically, chemically and/or physically.

5. The method of claim 4, wherein lysing of the cells of the biomass comprises an enzymatic treatment of the cells with a cell wall degrading enzyme.

6. The method of claim 5, wherein lysing of the cells of the biomass is carried by a method comprising: a) heating the suspension of biomass to a temperature of between 50° C. and 70° C., adding a cell wall-degrading enzyme to the fermentation broth, and, if necessary, adjusting the pH to a value at which the enzyme is active; b) maintaining the temperature and pH in the ranges of paragraph a) for at least one hour.

7. The method of claim 1, wherein after lysing the cells, the suspension is concentrated to a total dry matter content of 30 to 60 wt-%.

8. The method of claim 1, wherein steps (c) to (e) are carried out at a temperature of 10 to 50° C.

9. The method of claim 1, wherein, before addition of acetone in step (c), the pH of the suspension is adjusted to an acidic pH.

10. The method of claim 9, wherein before addition of acetone in step (c), the pH is adjusted to a an acidic pH of 2.5 to 6.8.

11. The method of claim 1, wherein separation of the oil and acetone-containing light phase from the water, acetone, salt and cell debris containing heavy phase is realized by mechanical means.

12. The method of claim 11, wherein separation of the oil and acetone-containing light phase from the water, acetone, salt and cell debris containing heavy phase by mechanical means takes place at a pH of 5.5 to 8.5.

13. The method of claim 1, further comprising separating the acetone from the PUFAs containing oil.

14. The method of claim 1, wherein the suspension has a biomass density of at least 80 g/l.

15. The method of claim 1, wherein the suspension has a biomass density of at least 140 g/l.

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

17. The method of claim 16, wherein the cells which contain a PUFAs containing lipid are microalgae selected from the phylum Stramanopiles.

18. The method of claim 17, wherein the cells which contain a PUFAs containing lipid are from the family Thraustochytrids.

19. The method of claim 18, wherein the cells which contain a PUFAs containing lipid are microalgae of the genus Schizochytrium.

20. The method of claim 5, wherein the cell-wall degrading enzyme is selected from the group consisting of: a protease, cellulase, hemicellulase, chitinase, pectinase, sucrase, maltase, lactase, alpha-glucosidase, beta-glucosidase, amylase, lysozyme, neuraminidase, galactosidase, alpha-mannosidase, glucuronidase, hyaluronidase, pullulanase, glucocerebrosidase, galactosylceramidase, acetylgalactosaminidase, fucosidase, hexosaminidase, iduronidase, maltase-glucoamylase, beta-glucanase, mannanase, and combinations thereof.

Description

WORKING EXAMPLES

Example 1

(1) An unwashed cell broth containing microbial cells (Schizochytrium sp.) at a biomass density of over 100 g/I 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.

(2) Fractions of the concentrated lysed cell mixture were then taken and a specific pH value was adjusted by either using NaOH or H.sub.2SO.sub.4, resulting in aliquots with a pH value of 3.1, 5.6, 8.1 and 10.4.

(3) Subsequently aliquots of those fractions were mixed with different amounts of acetone which were added to those aliquots at room temperature. After addition of acetone, the resulting suspensions were thoroughly mixed by using a vortex. After mixing, phase separation was carried out by using a centrifuge.

(4) After centrifugation, it was first determined whether an oil containing phase was obtainable. If an oil containing phase was obtained, then the amount of oil as contained in this phase in comparison to the total amount of oil as contained in the biomass at the beginning was determined. The results are disclosed in the following tables.

(5) TABLE-US-00001 TABLE 1 Acetone extraction at a pH of 3.1 Acetone [wt.-%] 27.5 30 32.5 35 37.5 40 42.5 45 47.5 Lysed broth [g] 29.0 28.2 27.3 26.0 25.0 24.2 23.1 22.1 21.2 Acetone [g] 11.1 12.5 13.2 14.1 15.4 16.6 17.2 18.6 19.2 Isolated oil [wt.-%] 88.3 84.8 75.2 81.0 74.3 72.1 78.1 60.4 61.3

(6) TABLE-US-00002 TABLE 2 Acetone extraction at a pH of 5.6 Acetone [wt.-%] 27.5 30 32.5 35 37.5 40 42.5 45 47.5 Lysed broth [g] 29.1 28.0 27.0 26.2 25.1 24.1 23.1 22.0 21.0 Acetone [g] 11.1 12.2 13.3 14.1 15.1 16.1 17.1 18.0 19.2 Isolated oil [wt.-%] 73.3 74.7 65.6 73.7 71.2 60.9 70.6 64.6 34.7

(7) TABLE-US-00003 TABLE 3 Acetone extraction at a pH of 8.1 Acetone [wt.-%] 25 27.5 30 32.5 37.5 40 42.5 45 47.5 Lysed broth [g] 30.1 29.2 28.1 27.3 25.0 24.2 23.0 22.0 21.0 Acetone [g] 10.1 11.3 12.2 13.4 15.0 16.2 17.0 18.2 19.8 Isolated oil [wt.-%] 54.8 61.3 67.2 52.1 61.3 61.3 41.0 45.2 36.0

(8) TABLE-US-00004 TABLE 4 Acetone extraction at a pH of 10.4 Acetone [wt.-%] 25 27.5 30 35 37.5 40 42.5 45 47.5 Lysed broth [g] 30.0 29.0 28.0 26.1 25.0 24.1 23.0 22.0 21.0 Acetone [g] 10.3 11.0 12.3 14.4 15.1 16.1 17.1 18.1 19.0 Isolated oil [wt.-%] 68.1 62.1 51.5 62.3 47.3 71.0 57.2 62.5 76.0

(9) As can be learnt from the table, acetone turned out to be a good means for isolating the oil from the biomass, if the amount of acetone was in the range of between 25.0 and 47.5 wt.-%, calculated on basis of the final suspension as obtained after addition of acetone. —If acetone was in that range, then an oil containing phase was observed on top of the centrifuged suspension, which contained besides oil also small amounts of acetone and water. —In case that the amount of acetone was either higher than 47.5 wt.-% or lower then 25.0 wt.-%, no phase separation could be observed.

(10) Further it turned out that oil isolation seem to work better at acidic pH values.

(11) After separation of the oil containing phase, the residual water and acetone can easily be removed by evaporation.