METHOD OF SEPARATING LIPIDS FROM A LYSED LIPIDS CONTAINING BIOMASS

FIELD OF INVENTION

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

BACKGROUND OF THE INVENTION

PUFAs (polyunsaturated fatty acids) containing lipids are of high interest in the feed, food and pharmaceutical industry. Due to overfishing there is a high need for alternative sources for PUFAs containing lipids besides fish oil. It turned out that besides certain yeast and algal strains in particular microalgal cells like those of the order Thraustochytriales are a very good source for PUFAs containing lipids.

But with respect to microbial organisms and in particular cells of the order Thraustochytriales, which produce the PUFAs containing lipids, the isolation of the oil from the cells turned out as a particular problem. The most effective way of isolating the oil was the use of organic solvents like hexane. But the use of organic solvents leads to hazardous operating conditions, requires the use of expensive explosion-proof equipment and requires the implementation of an expensive solvent recovery process to avoid pollution of the

environment.

In the attempt to avoid the use of organic solvents, as an effective alternative way for isolating the oil has turned out the salting-out of the oil with high amounts of sodium chloride. But the use of high amounts of sodium chloride leads to a delipidated biomass by-product which due to the high salt content cannot be utilized as a feed ingredient, so that the process is not very sustainable. Further, the high salt concentration leads to fast corrosion of the used steel equipment.

Thus, it was the object of the current invention to provide an improved and effective method for isolating a lipid, in particular a PUFAs containing lipid, from lipids containing cells, in particular of the order Thraustochytriales, and simultaneously avoiding not only the need of organic solvents, but further to improve and increase the recovery of PUFA oil from the microbial biomass. BRIEF SUMMARY OF THE INVENTION

The present invention is providing an improvement of a method for separating a

polyunsaturated fatty acids (PUFAs) containing lipid or oil from the debris of a biomass, said method comprises the following steps: i. Providing a suspension of a biomass comprising cells which contain a PUFAs

containing lipid; ii. Lysing the cells of the biomass; iii. Heating the suspension as obtained in step (b) to a temperature of 50°C to 100°C; iv. Keeping the temperature for at least one hour, preferably for at least two hours, more preferably for two to four hours; v. Harvesting of the PUFAs containing oil, wherein harvesting of the PUFAs containing oil comprises neutralization of the demulsified suspension and subsequent separation of the thus obtained oil containing light phase from the water, salts, residual oil and cell debris containing heavy phase; vi. Concentrating of the heavy phase to total dry matter of at least 25 wt.-%; vii. Heating and stirring the concentrated dry phase as obtained in step (vi) to a

temperature of at least 60°C for at least 10, preferably for at least 12, more preferably for 20 to 24 hours; viii. Harvesting of separated, additional PUFAs containing oil from step vii.

Inventors have surprisingly found that a 2 ^nd treatment of the heavy phase of step (v) which still contains a reasonable concentration of PUFAs results in an additional recovery of PUFA oils.

In a preferred embodiment of the invention, the heavy phase is concentrated in step (vi) to total dry matter of at least 30%, for example to a dry matter of at least 35 wt.-%.

Concentration of the suspension is preferably carried out in a forced circulation evaporator (for example available from GEA, Germany) to allow fast removal of the water.

In another preferred embodiment, the concentrated dry phase of step (vii) is heated to a temperature of 80°C for 24 hours. A second subject of the current invention is a method of separating a polyunsaturated fatty acids (PUFAs) containing lipid from the debris of a biomass, comprising the following steps before the treatment of the heavy phase according the steps vi, vii and viii: a) Providing a suspension of a biomass comprising cells which contain a PUFAs

containing lipid; b) Lysing the cells of the biomass; c) Heating the suspension as obtained in step (b) to a temperature of 50°C to 100°C, , while adjusting the pH to a value of > 9; d) Keeping the temperature and pH value in the ranges as depicted in (c) for at least 10 hours, preferably 15 to 40 hours, more preferably 20 to 36 hours.

A third subject of the current invention is a method of separating a polyunsaturated fatty acids (PUFAs) containing lipid from the debris of a biomass, comprising the following steps before the treatment of the heavy phase according the steps vi, vii and viii: a) Providing a suspension of a biomass comprising cells which contain a PUFAs

containing lipid; b) Lysing the cells of the biomass; c) Heating the suspension as obtained in step (b) to a temperature of of 80°C to 100°C, preferably 85°C to 95°C, more preferably about 90°C, while adjusting the pH to a value of 9.5 to 1 1.5, preferably 10.0 to 11.0, more preferably 10.3 to 10.7; d) Keeping the temperature and pH value in the ranges as depicted in (c) for at least 10 hours, preferably 15 to 40 hours, more preferably 20 to 36 hours.

The steps (c) and (d) lead to the separation of the oil containing light phase and the water, cell debris, salts and residual oil containing heavy phase, as obtained by lysing the cells of the biomass. This separation of the light and heavy phase is also called“demulsification” in the context of this application.

DETAILED DESCRIPTION OF THE INVENTION

A particular advantage of the method of the current invention is that it allows to additionally remove PUFA oil from the heavy phase in a simple and effective way as described above. In addition to that the method as such can be carried out without the use of any organic solvent, in particular without the use of any polar or non-polar organic solvent. Thus, in a preferred embodiment of the invention, no or only little amounts of organic solvents, in particular of polar or non-polar organic solvents, are used for isolating the PUFAs containing oil from the biomass. Typical organic solvents are hexane and ethanol.

Preferably, in the steps (b), (c) and (d) of the method the suspension is continuously mixed by using a stirrer and/or an agitator. In the method steps (c) and/or (d) preferably low shear agitation and/or axial-flow agitation is applied, in particular as disclosed in WO 2015/095694. Impellers suitable for agitating prior and during steps (c) and/or (d) include in particular straight blade impellers, Rushton blade impellers, axial flow impellers, radial flow impellers, concave blade disc impellers, high-efficiency impellers, propellers, paddles, turbines and combinations thereof.

Lysing of the cells of the biomass can be carried out by methods as known to those skilled in the art, in particular enzymatically, mechanically, physically, or chemically, or by applying combinations thereof.

Depending on the time of exposure and/or the degree of force applied, a composition comprising only lysed cells or a composition comprising a mixture of cell debris and intact cells may be obtained. The term“lysed lipids containing biomass” insofar relates to a suspension which contains water, cell debris and oil as set free by the cells of the biomass, but beyond that may also comprise further components, in particular salts, intact cells, further contents of the lysed cells as well as components of a fermentation medium, in particular nutrients. In a preferred embodiment of the invention, only small amounts of intact cells, in particular less than 20 %, preferably less than 10 %, more preferably less than 5 % (relating to the total number of intact cells as present before lysing the cells of the biomass) are present in the lysed biomass after the step of lysing the cells.

Lysing of the cells may be realized for example by utilizing a French cell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill, pebble mill, bead mill, high pressure grinding roll, vertical shaft impactor, industrial blender, high shear mixer, paddle mixer, and/or polytron homogenizer.

In a preferred embodiment of the invention, lysing of the cells comprises an enzymatic treatment of the cells by applying a cell-wall degrading enzyme.

According to the invention, the cell-wall degrading enzyme is preferably selected from proteases, cellulases (e.g., Cellustar CL (Dyadic), Fibrezyme G2000 (Dyadic), Celluclast (Novozymes), Fungamyl (Novozymes), Viscozyme L (Novozymes)), hemicellulases, chitinases, pectinases (e.g., Pectinex (Novozymes)), sucrases, maltases, lactases, alpha- glucosidases, beta-glucosidases, amylases (e.g., Alphastar Plus (Dyadic); Termamyl (Novozymes)), lysozymes, neuraminidases, galactosidases, alpha-mannosidases, glucuronidases, hyaluronidases, pullulanases, glucocerebrosidases, galactosylceramidases, acetylgalactosaminidases, fucosidases, hexosaminidases, iduronidases, maltases- glucoamylases, xylanases (e.g., Xylanase Plus (Dyadic), Pentopan (Novozymes)), beta- glucanases (e.g., Vinoflow Max (Novozymes), Brewzyme LP (Dyadic)), mannanases, and combinations thereof. The protease may be selected from serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases, glutamic acid proteases, alcalases (subtilisins), and combinations thereof. The chitinase may be a chitotriosidase. The pectinase may be selected from pectolyases, pectozymes,

polygalacturonases, and combinations thereof.

The adequate pH for utilizing the enzyme depends on the pH optimum of the enzyme.

In a preferred embodiment of the invention, an enzyme with a pH optimum of between 7.0 and 8.0, in particular of about 7.5, is used, so that the pH applied in this step is from 7.0 to 8.0, preferably from 7.3 to 7.7. A preferred enzyme which can be used in this pH range is an alcalase.

The enzyme is preferably added as a concentrated enzyme solution, preferably in an amount of 0.01 to 1.5 wt.-%, more preferably in an amount of 0.03 to 1.0 wt.-%, above all in an amount of 0.05 to 0.5 wt.-%, relating to the amount of concentrated enzyme solution as added in relation to the total amount of the suspension after addition of the concentrated enzyme solution.

In a very preferred embodiment of the invention, lysing of the cells is carried out as follows: a) Heating the suspension of (a) to a temperature of between 50°C and 70°C, preferably to a temperature of between 55°C and 65°C, and adding a cell wall-degrading enzyme to the fermentation broth, and adjusting an adequate pH value, if necessary, at which the enzyme is properly working; b) Keeping the temperature and pH in the ranges as depicted in (b) for at least one

hour, preferably for at least two hours, more preferably for two to four hours.

In step (a), the enzyme can be added before or after heating up the suspension and/or before or after adjusting the pH. In the same way heating up of the suspension can be carried out before or after adjusting the pH. - But in a preferred embodiment, the enzyme is added after heating up of the suspension and after adjusting the pH, if adjusting of the pH is necessary, at all. - In a very preferred embodiment all measures are carried out more or less simultaneously.

Preferably, in the steps (a) and (b) the suspension is continuously mixed by using a stirrer and/or an agitator.

In a further preferred embodiment of the invention, after lysing the cells of the biomass and before the demulsification step, the suspension is concentrated to a total dry matter content of 30 to 60 wt.-%, more preferably 35 to 55 wt.-%, in particular 40 to 50 wt.-%.

Concentration of the suspension is preferably carried out by evaporation of water at a temperature not higher than 100°C, preferably 70°C to 100°C, more preferably 80°C to 90°C, until a total dry matter content of 30 to 60 wt.-% more preferably 35 to 55 wt.-%, in particular 40 to 50 wt.-%, is reached.

Concentration of the suspension is preferably carried out in a forced circulation evaporator (for example available from GEA, Germany) to allow fast removal of the water.

In general, adjusting the pH value can be carried out according to the invention by using either bases or acids as known to those skilled in the art. Decreasing of the pH can be carried out in particular by using organic or inorganic acids like sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrochloric acid, hydrobromic acid, perchloric acid,

hypochlorous acid, chlorous acid, fluorosulfuric acid, hexafluorophosphoric acid, acetic acid, citric acid, formic acid, or combinations thereof. As a high content of chloride is desirably avoided, in a preferred embodiment of the invention no or only small amounts of hydrochloric acid are used in the process of the current invention. According to the invention, sulfuric acid is the preferred substance for decreasing the pH value. - Increasing of the pH can be carried out in particular by using organic or inorganic bases like hydroxides, in particular sodium hydroxide, lithium hydroxide, potassium hydroxide, and/or calcium hydroxide, carbonates, in particular sodium carbonate, potassium carbonate, or magnesium carbonate, and/or bicarbonates, in particular lithium bicarbonate, sodium bicarbonate, and/or potassium bicarbonate. Due to easiness of handling, the acids and bases are preferably used in liquid form, in particular as concentrated solutions. Thus, caustic soda is the preferred substance for increasing the pH value.

The method according to the invention preferably comprises as a further step the harvesting of the PUFAs containing lipid from the demulsified composition as obtained in step (iv). The harvesting of the PUFAs containing lipid preferably comprises neutralization of the demulsified suspension and subsequent separation of the thus obtained oil containing light phase from the water, salts, cell debris and residual oil containing heavy phase.

Neutralization of the demulsified composition is preferably realized by adding an acid, preferably sulfuric acid, to adjust a pH value of 6.5 to 8.5, preferably 7.0 to 8.0. Before starting separation of the light phase from the heavy phase the neutralized composition may be stirred at neutral pH from several minutes up to several hours.

Separation of the oil containing light phase from the water, salts and cell debris containing heavy phase is preferably realized by mechanical means and preferably at a temperature of 60-90°C, more preferably 70-80°C, and at a pH value of preferably 6-9, more preferably 7- 8.5.“Mechanical means” refers in particular to filtration and centrifugation methods as known to those skilled in the art.

Separation of additional oil from the heavy phase is preferably realized by standard means and preferably at a temperature of 60-90°C, more preferably at a temperature of 70-80°C. Concentration of the heavy phase is preferably carried out in a forced circulation evaporator (for example available from GEA, Germany) to allow fast removal of the water.“Mechanical means” refers in particular to concentration and centrifugation methods as known to those skilled in the art.

A particular advantage of the method of the current invention is that it allows to additionally remove PUFA oil from the heavy phase in a simple and effective way, wherein the additionally separated oil shows similar or identical properties as the PUFA containing oil of the light phase.

Therefore, the method of the current invention allows a very effective separation of the oil contained in the biomass from the cell-debris and other substances as contained in the fermentation broth. By using the method of the current invention preferably more than 90 wt.-%, in particular more than 95 wt.-% of the oil contained in the biomass can be separated from the biomass and isolated.

It turned out that the two oil fractions (F1 = oil containing light phase and F2 = oil extracted from the heavy phase) as obtained by applying the method of the current invention have both some advantageous characteristics over the PUFAs containing oils as disclosed in the state of the art so far. In particular both fractions exhibit very low oxidation values, a low content of free fatty acids and impurities, a very low viscosity and a very high flash point. Thus, a further subject of the current invention is an oil (F1 and/or F2) as obtained or as obtainable by a method according to the current invention.

A further subject of the current invention is therefore also a PUFAs containing oil (F1 and/or F2) exhibiting the following characteristics: a) a peroxide value of less than 0.5, preferably less than 0.3, in particular less than 0.15; b) an anisidine value of less than 15, preferably less than 10; c) preferably a content of free fatty acids of less than 1 wt.-%; d) preferably a content of moisture and impurities of less than 1 wt.-%, preferably less than 0.5 wt.-%; e) preferably a viscosity of less than 250 cps, more preferably of less than 200 cps, in particular of less than 160 cps; e) preferably a flash point of at least 350°C, more preferably of at least 400°C, in particular of at least 450°C; f) preferably a content of omega-3 fatty acids, in particular of DHA and EPA, of at least 35 wt.-%, preferably at least 40 or 45 wt.-%, above all at least 50 wt.-%; g) preferably DHA and EPA each in an amount of at least 8 wt.-%, preferably at least 10 wt.-%, above all at least 15 wt.-%; h) preferably an amount of organic solvents of less than 0.5 wt.-%, more preferably less than 0.1 wt.-%, in particular less than 0.05 wt.-%, above all less than 0.01 wt.-%; i) preferably an amount of chlorides of less than 0.1 wt.-%, more preferably less than 0.05 wt.-%, in particular less than 0.01 wt.-%.

The anisidine value (AV) is determined in accordance with AOCS Official Method Cd 18-90. The AV is a measure for secondary reaction products of the fatty acids, such as aldehydes and ketones, that occur during oxidation of the oil.

The peroxide value (PV) is determined in accordance with the AOCS Official Method CD 8- 53. The PV is a measure for primary reaction products, such as peroxide and

hydroperoxides, that occur during oxidation of the oil. According to the invention the PV is measured in meq/kg.

The PUFAs containing cells of the biomass are preferably microbial cells or plant cells.

Preferably, the cells are capable of producing the PUFAs due to a polyketide synthase system. The polyketide synthase system may be an endogenous one or, due to genetic engineering, an exogenous one.

Accordingly,“delipidated biomass” according to the invention refers to the residues of such a PUFAs containing cells comprising biomass, in particular as disclosed further below, after having been subjected to an oil isolation process, in particular as disclosed further before.

The plant cells may in particular be selected from cells of the families Brassicaceae, Elaeagnaceae and Fabaceae. The cells of the family Brassicaceae may be selected from the genus Brassica, in particular from oilseed rape, turnip rape and Indian mustard; the cells of the family Elaeagnaceae may be selected from the genus Elaeagnus, in particular from the species Oleae europaea; the cells of the family Fabaceae may be selected from the genus Glycine, in particular from the species Glycine max.

The microbial organisms which contain a PUFAs containing lipid are described extensively in the prior art. The cells used may, in this context, in particular be cells which already naturally produce PUFAs (polyunsaturated fatty acids); however, they may also be cells which, as the result of suitable genetic engineering methods or due to random mutagenesis, show an improved production of PUFAs or have been made capable of producing PUFAs, at all. The production of the PUFAs may be auxotrophic, mixotrophic or heterotrophic.

The biomass preferably comprises cells which produce PUFAs heterotrophically. The cells according to the invention are preferably selected from algae, fungi, particularly yeasts, bacteria, or protists. The cells are more preferably microbial algae or fungi.

Suitable cells of oil-producing yeasts are, in particular, strains of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

Suitable cells of oil-producing microalgae and algae-like microorganisms are, in particular, microorganisms selected from the phylum Stramenopiles (also called Heterokonta). The microorganisms of the phylum Stramenopiles may in particular be selected from the following groups of microorganisms: Hamatores, Proteromonads, Opalines, Developayella,

Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus,

Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes,

Raphidophytes, Synurids, Axodines (including Rhizochromulinales, Pedinellales,

Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, and

Chromulinales. Other preferred groups of microalgae include the members of the green algae and dinoflagellates, including members of the genus Crypthecodiurn.

The biomass according to the invention preferably comprises cells, and preferably consists essentially of such cells, of the taxon Labyrinthulomycetes (Labyrinthulea, net slime fungi, slime nets), in particular those from the family of Thraustochytriaceae. The family of the Thraustochytriaceae (Thraustochytrids) includes the genera Althomia, Aplanochytrium, Aurantiochytrium, Botryochytrium, Elnia, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium, and Ulkenia. The biomass particularly preferably comprises cells from the genera Aurantiochytrium, Oblongichytrium,

Schizochytrium, or Thraustochytrium, above all from the genus Schizochytrium. In accordance with the invention, the polyunsaturated fatty acid (PUFA) is preferably a highly-unsaturated fatty acid (HUFA).

The cells present in the biomass are preferably distinguished by the fact that they contain at least 20% by weight, preferably at least 30% by weight, in particular at least 35% by weight, of PUFAs, in each case based on cell dry matter.

According to the current invention, the term "lipid" includes phospholipids; free fatty acids; esters of fatty acids; triacylglycerols; sterols and sterol esters; carotenoids ; xanthophylls (e. g., oxycarotenoids) ; hydrocarbons ; isoprenoid-derived compounds and other lipids known to one of ordinary skill in the art. - The terms“lipid” and“oil” are used interchangeably according to the invention.

In a preferred embodiment, the majority of the lipids in this case is present in the form of triglycerides, with preferably at least 50% by weight, in particular at least 75% by weight and, in an especially preferred embodiment, at least 90% by weight of the lipids present in the cell being present in the form of triglycerides.

According to the invention, polyunsaturated fatty acids (PUFAs) are understood to mean fatty acids having at least two, particularly at least three, C-C double bonds. According to the invention, highly-unsaturated fatty acids (HUFAs) are preferred among the PUFAs.

According to the invention, HUFAs are understood to mean fatty acids having at least four C- C double bonds.

The PUFAs may be present in the cell in free form or in bound form. Examples of the presence in bound form are phospholipids and esters of the PUFAs, in particular monoacyl-, diacyl- and triacylglycerides. In a preferred embodiment, the majority of the PUFAs is present in the form of triglycerides, with preferably at least 50% by weight, in particular at least 75% by weight and, in an especially preferred embodiment, at least 90% by weight of the PUFAs present in the cell being present in the form of triglycerides.

Preferred PUFAs are omega-3 fatty acids and omega-6 fatty acids, with omega-3 fatty acids being especially preferred. Preferred omega-3 fatty acids here are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

In a very preferred embodiment of the current invention, cells, in particular a Schizochytrium strain, is employed which produces a significant amount of EPA and DHA, simultaneously, wherein DHA is preferably produced in an amount of at least 20 wt.-%, preferably in an amount of at least 30 wt.-%, in particular in an amount of 30 to 50 wt.-%, and EPA is produced in an amount of at least 5 wt.-%, preferably in an amount of at least 10 wt.-%, in particular in an amount of 10 to 20 wt.-% (in relation to the total amount of lipid as contained in the cells, respectively). DHA and EPA producing Schizochytrium strains can be obtained by consecutive mutagenesis followed by suitable selection of mutant strains which demonstrate superior EPA and DHA production and a specific EPA:DHA ratio. Any chemical or nonchemical (e.g. ultraviolet (UV) radiation) agent capable of inducing genetic change to the yeast cell can be used as the mutagen. These agents can be used alone or in

combination with one another, and the chemical agents can be used neat or with a solvent.

Preferred species of microorganisms of the genus Schizochytrium, which produce EPA and DHA simultaneously in significant amounts, as mentioned before, are deposited under ATCC Accession No. PTA-10208, PTA-10209, PTA-10210, or PTA-1021 1 , PTA-10212, PTA- 10213, PTA-10214, PTA-10215.

The suspension of biomass according to the present invention is preferably a fermentation broth, in particular a fermentation broth with a biomass density of at least 80 or 100 g/l, preferably at least 120 or 140 g/l, more preferably at least 160 or 180 g/l (calculated as dry- matter content). Thus, the suspension may be obtained by culturing and growing suitable cells in a fermentation medium under conditions whereby the PUFAs are produced by the microorganism.

Methods for producing the biomass, in particular a biomass which comprises cells containing lipids, in particular PUFAs, particularly of the order Thraustochytriales, are described in detail in the prior art (see e.g. WO91/07498, WO94/08467, WO97/37032, W097/36996,

W001/54510). As a rule, the production takes place by cells being cultured in a fermenter in the presence of a carbon source and of a nitrogen source, along with a number of additional substances like minerals that allow growth of the microorganisms and production of the PUFAs. In this context, biomass densities of more than 100 grams per litre and production rates of more than 0.5 gram of lipid per litre per hour may be attained. The process is preferably carried out in what is known as a fed-batch process, i.e. the carbon and nitrogen sources are fed in incrementally during the fermentation. When the desired biomass has been obtained, lipid production may be induced by various measures, for example by limiting the nitrogen source, the carbon source or the oxygen content or combinations of these.

In a preferred embodiment of the current invention, the cells are grown until they reach a biomass density of at least 80 or 100 g/l, more preferably at least 120 or 140 g/l, in particular at least 160 or 180 g/l (calculated as dry-matter content). Such processes are for example disclosed in US 7,732,170. Preferably, the cells are fermented in a medium with low salinity, in particular so as to avoid corrosion. This can be achieved by using chlorine-free sodium salts as the sodium source instead of sodium chloride, such as, for example, sodium sulphate, sodium carbonate, sodium hydrogen carbonate or soda ash. Preferably, chloride is used in the fermentation in amounts of less than 3 g/l, in particular less than 500 mg/I, especially preferably less than 100 mg/I.

Suitable carbon sources are both alcoholic and non-alcoholic carbon sources. Examples of alcoholic carbon sources are methanol, ethanol and isopropanol. Examples of non-alcoholic carbon sources are fructose, glucose, sucrose, molasses, starch and corn syrup.

Suitable nitrogen sources are both inorganic and organic nitrogen sources. Examples of inorganic nitrogen sources are nitrates and ammonium salts, in particular ammonium sulphate and ammonium hydroxide. Examples of organic nitrogen sources are amino acids, in particular glutamate, and urea.

In addition, inorganic or organic phosphorus compounds and/or known growth-stimulating substances such as, for example, yeast extract or corn steep liquor, may also be added so as to have a positive effect on the fermentation.

The cells are preferably fermented at a pH of 3 to 11 , in particular 4 to 10, and preferably at a temperature of at least 20°C, in particular 20 to 40°C, especially preferably at least 30°C. A typical fermentation process takes up to approximately 100 hours.

After the fermentation has ended, the cells may be pasteurized in order to kill the cells and to deactivate enzymes which might promote lipid degradation. The pasteurization is preferably effected by heating the biomass to a temperature of 50 to 121 °C, preferably 50 to 70°C, for a period of 5 to 80 minutes, in particular 20 to 60 minutes.

Likewise, after the fermentation is ended, antioxidants may be added in order to protect the PUFAs present in the biomass from oxidative degradation. Preferred antioxidants in this context are BHT, BHA, TBHA, ethoxyquin, beta-carotene, vitamin E, in particular tocopherol, and vitamin C. The antioxidant, if used, is preferably added in an amount of 0.001 to 0.1 wt- %, preferably in an amount of 0.002 to 0.05 wt.-%, relating to the total amount of the fermentation broth after addition of the antioxidant. EXAMPLE

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. The concentrated lysed cell mixture was transferred into a new vessel, heated up to 90°C under low shear agitation, while adjusting the pH to 10.5 by adding caustic soda. Low shear agitation was continued for about 30 hours, while keeping the temperature at 90°C and the pH above 9.0 by adding caustic soda.

After that the resulting demulsified mixture was neutralized by adding sulfuric acid to adjust a pH of 7.5. Phase separation into a light phase, containing the oil, and a heavy phase, containing water, cell-debris, residual oil and salts, was carried out mechanically by using a disc stack separator (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20).

After separation of the crude oil, the remaining cell debris were resuspended in the aqueous phase, concentrated and dried by spraygranulation.

Due to the efficient demulsification, more than 90 wt.-% of the oil could be separated from the biomass without the addition of organic solvents or sodium chloride.

The remaining heavy phase (Biomeal) containing total dry matter of“Biomeal” of 21 ,4wt.-% was concentrated to TDM of 31.1 wt.-%. Concentration of the suspension was carried out in a forced circulation evaporator (for example available from GEA, Germany) to allow fast removal of the water. After that, the suspension was stirred for 1 day at 80°C.

After centrifugation the oil containing phase has separated from the remaining solid phase.

Analytical results are shown in Figure 1.