METHOD FOR PRODUCING A BIOMASS WITH AN INCREASED CONTENT OF POLYUNSATURATED FATTY ACIDS

Abstract

According to the invention, it has been found that when the amount of sulfate used in the culturing of PUFAs-producing cells is selected such that the sulfate concentration in the last phase of fermentation is always above zero, but at the same time always below the saturation limit of the cells, a biomass is obtained which has an increased proportion by mass of polyunsaturated fatty acids (PUFAs) in the final product, the process having at the same time a high product/substrate yield and space/time yield.

Claims

1.-15. (canceled)

16. A process for producing a biomass containing PUFAs comprising culturing PUFAs-producing cells in a fermentation medium, wherein the sulfate content of the fermentation medium is adjusted such that sulfate is always present in the last phase of fermentation but the sulfate concentration in the medium is always below the saturation limit of the cells.

17. The process of claim 16, wherein the culturing comprises a growth phase and an oil-production phase, and wherein sulfate is present in the fermentation medium during the entire period of the oil-production phase but the sulfate concentration is below the saturation limit of the cells during most of the oil-production phase.

18. The process of claim 17, wherein the sulfate concentration is below the saturation limit of the cells at least during the second half of the oil-production phase.

19. The process of claim 17, wherein the sulfate concentration is below the saturation limit of the cells at least during the last 70% of the oil-production phase.

20. The process of claim 17, wherein the sulfate concentration is below the saturation limit of the cells during the entire oil-production phase.

21. The process of claim 17, wherein the sulfate concentration is between 0.005 and 5 g of sulfate per kg of lipid-free biomass at least during the second half of the oil-production phase.

22. The process of claim 17, wherein the sulfate concentration is between 0.01 and 4 g of sulfate per kg of lipid-free biomass, at least during the last 70%, of the oil-production phase.

23. The process of claim 17, wherein the sulfate concentration is between 0.01 and 2 g of sulfate per kg of lipid-free biomass during the entire oil-production phase.

24. The process of claim 17, wherein the cells are cultured in the growth phase up to a biomass density of at least 50 g per litre of fermentation medium.

25. The process of claim 17, wherein the oil-production phase is introduced by limiting at least one limiting nutrient component.

26. The process of claim 17, wherein sulfate is continuously metered into the fermentation medium during most of the oil-production phase at a rate of 5 to 100 mg per kg of fermentation medium.

27. The process of claim 17, wherein, during the oil-production phase, in a period in which the sulfate concentration is below the saturation limit, sulfate is continuously metered into the fermentation medium in an amount giving a sulfate concentration below 0.05 g per kg of fermentation medium.

28. The process of claim 16, wherein sulfate is initially charged in the starting medium and the metered addition of sulfate is started once the sulfate concentration has fallen below 2 g per kg of fermentation medium.

29. The process of claim 16, wherein the sulfate concentration falls below 5 g of sulfate per kg of lipid-free biomass, just before the start of the oil-production phase, after a biomass density of at least 50 g per litre of fermentation medium has been reached, and remains below this concentration for the rest of the fermentation.

30. The process of claim 16, wherein the sulfate concentration is always below 0.5 g per kg of fermentation medium until the start of the metered addition of sulfate.

31. The process of claim 16, wherein the fermentation medium used has a chloride concentration of less than 1 g/l during the entire fermentation.

32. The process of claim 16, wherein the fermentation medium used has a chloride concentration of less than 250 mg/l during the entire fermentation.

33. The process of claim 16, wherein the PUFA-producing cells are of the taxon Labyrinthulomycetes (Labyrinthulea).

34. The process of claim 16, wherein the PUFAs are omega-3 fatty acids.

35. A biomass containing PUFAs and comprising a sulfate content of 12 to 20 g per kg of biomass and cells the family Thraustochytriaceae.

Description

EXEMPLARY EMBODIMENTS

Example 1

Producing Biomass by Fermentation of Aurantiochytrium limacinum SR21 in a Medium Having a High Sulfate Content and Subsequent Drying of the Biomass

[0169] The cells were cultured for about 70 h in a feed process using a steel fermenter having a fermenter volume of 2 litres with a total starting mass of about 700 g and an attained total final mass of about 1.5 kg. During the process, a glucose solution (570 g/kg glucose) was metered in (“fed-batch process”).

[0170] The composition of the starting medium was as follows:

[0171] Medium 1: 20 g/kg glucose; 4 g/kg yeast extract; 2 g/kg ammonium sulfate; 12 or 16 g/kg sodium sulfate; 2.46 g/kg magnesium sulfate (heptahydrate); 0.45 g/kg potassium chloride; 4.5 g/kg potassium dihydrogen phosphate; 0.1 g/kg thiamine (HCl); 5 g/kg trace element solution.

[0172] The composition of the trace element solution was as follows: 35 g/kg hydrochloric acid (37%); 1.86 g/kg manganese chloride (tetrahydrate); 1.82 g/kg zinc sulfate (heptahydrate); 0.818 g/kg sodium EDTA; 0.29 g/kg boric acid; 0.24 g/kg sodium molybdate (dihydrate); 4.58 g/kg calcium chloride (dihydrate); 17.33 g/kg iron sulfate (heptahydrate); 0.15 g/kg copper chloride (dihydrate). Culturing was carried out under the following conditions: Culture temperature 28° C.; aeration rate 0.5 vvm, stirrer speed 600-1950 rpm, control of pH in the growth phase at 4.5 using ammonia water (25% v/v). Fermentation was carried out up to a biomass density of 80 g/l before the oil-production phase was introduced by limitation of phosphate and nitrogen. Because of the high sulfate content in the starting medium until the end of fermentation, i.e. including until the end of the oil-production phase, the sulfate concentration is always above the saturation limit of the cells, which is about 5 g per kg of dry biomass. The biomasses obtained are hereinafter referred to as “Biomass 1” (12 g/kg sodium sulfate in the starting medium) and “Biomass 2” (16 g/kg sodium sulfate in the starting medium).

Example 2

Producing Biomass by Fermentation of Aurantiochytrium limacinum SR21 in a Medium Having a Low Sulfate Content and Subsequent Drying of the Biomass

[0173] The cells were cultured for about 70 h in a feed process using a steel fermenter having a fermenter volume of 2 litres with a total starting mass of 712 g and an attained total final mass of about 1.5 kg. During the process, a glucose solution (570 g/kg glucose) was metered in (“fed-batch process”).

[0174] The composition of the starting medium was as follows:

[0175] Medium 1: 20 g/kg glucose; 4 g/kg yeast extract; 0 g/kg ammonium sulfate; 2.46 g/kg magnesium sulfate (heptahydrate); 0.45 g/kg potassium chloride; 4.5 g/kg potassium dihydrogen phosphate; 0.1 g/kg thiamine (HCl); 5 g/kg trace element solution.

[0176] The composition of the trace element solution was as follows: 35 g/kg hydrochloric acid (37%); 1.86 g/kg manganese chloride (tetrahydrate); 1.82 g/kg zinc sulfate (heptahydrate); 0.818 g/kg sodium EDTA; 0.29 g/kg boric acid; 0.24 g/kg sodium molybdate (dihydrate); 4.58 g/kg calcium chloride (dihydrate); 17.33 g/kg iron sulfate (heptahydrate); 0.15 g/kg copper chloride (dihydrate). Culturing was carried out under the following conditions: Culture temperature 28° C.; aeration rate 0.5 wm, stirrer speed 600-1950 rpm, control of pH in the growth phase at 4.5 using ammonia water (25% v/v). Fermentation was carried out up to a biomass density of 80 g/l before the oil-production phase was introduced by limitation of phosphate and nitrogen. The sulfate concentration had already fallen below the detection limit of 0.05 g per kg of fermentation medium upon introduction of the oil-production phase, and the sulfate concentration was accordingly also below the detection limit during the entire oil-production phase. And since there was no metered addition of sulfate, the sulfate concentration fell to zero in the course of the oil-production phase. The biomass obtained is hereinafter referred to as “Biomass 3”.

Example 3

Producing Biomass by Fermentation of Aurantiochytrium limacinum SR21 in a Medium Having a Low Sulfate Content with Simultaneous Addition of Sulfate During the Fermentation and Subsequent Drying of the Biomass

[0177] The cells were cultured for about 70 h in a feed process using a steel fermenter having a fermenter volume of 2 litres with a total starting mass of 712 g and an attained total final mass of about 1.5 kg. During the process, a glucose solution (570 g/kg glucose) was metered in (“fed-batch process”), as were either sodium sulfate solutions of differing concentration (7.4; 14.8 or 22.2 g/kg sodium sulfate) or ammonium sulfate solutions of differing concentration (6.8; 13.6 or 20.4 g/kg ammonium sulfate) in six different batches at a constant feed rate of 3 g/h. The feed rates of the sulfate solutions were selected such that the sulfate content in the storage phase remains below the detection limit (0.05 g/l) for sulfate.

[0178] Furthermore, water with the same feed rate (3 g/h) was used in one batch as a negative control in order to rule out a “dilution effect”.

[0179] The composition of the starting medium was as follows:

[0180] Medium 1: 20 g/kg glucose; 4 g/kg yeast extract; 2 g/kg ammonium sulfate; 2.46 g/kg magnesium sulfate (heptahydrate); 0.45 g/kg potassium chloride; 4.5 g/kg potassium dihydrogen phosphate; 0.1 g/kg thiamine (HCI); 5 g/kg trace element solution.

[0181] The composition of the trace element solution was as follows: 35 g/kg hydrochloric acid (37%); 1.86 g/kg manganese chloride (tetrahydrate); 1.82 g/kg zinc sulfate (heptahydrate); 0.818 g/kg sodium EDTA; 0.29 g/kg boric acid; 0.24 g/kg sodium molybdate (dihydrate); 4.58 g/kg calcium chloride (dihydrate); 17.33 g/kg iron sulfate (heptahydrate); 0.15 g/kg copper chloride (dihydrate). Culturing was carried out under the following conditions: Culture temperature 28° C.; aeration rate 0.5 vvm, stirrer speed 600-1950 rpm, control of pH in the growth phase at 4.5 using ammonia water (25% v/v). Fermentation was carried out up to a biomass density of 50 g/l before the oil-production phase was introduced by limitation of phosphate and nitrogen. The sulfate concentration had already fallen below the detection limit of 0.05 g per kg of fermentation medium upon introduction of the oil-production phase, and the sulfate concentration was accordingly also below the detection limit during the entire oil-production phase. Because of the continuous metered addition of sulfate, the sulfate concentration in the medium is, however, prevented from falling to zero. Hereinafter, the biomasses with the sodium sulfate feed are referred to as “Biomasses 3, 4 and 5” and the biomasses with the ammonium sulfate feed are referred to as “Biomasses 6, 7 and 8”.

Example 4

Work-Up and Comparison of the Biomasses Obtained and of the Processes

[0182] After the culturing process, the fermentation broths were heated to 60° C. for 20 minutes in order to prevent further cellular activity.

[0183] This was followed by a two-stage drying of the biomass: Firstly, the fermentation broth was concentrated by evaporation to a dry mass of about 20% by weight. Thereafter, the concentrated fermentation broth was spray-dried using a Production Minor™ Spray Dryer (GEA NIRO) at an inlet temperature of the drying air of 340° C. Spray-drying thus gave a powder with more than 95% by weight of dry mass.

TABLE-US-00001 TABLE 1 Comparison of the biomasses obtained Sulfate content Yield [%] Product, Purity [%] Bio- [g/kg] (sulfate (g DHA per g total [g] (g DHA per g mass per kg biomass) dextrose) (g DHA) dry biomass) 1 29.7 7.8 44.2 15.7 2 32.5 7.3 45.4 15.4 3 8.4 7.1 35.5 20.1 4 12.4 8.3 52.5 21.2 5 13.6 8.7 53.9 22.5 6 15.0 8.5 52.1 21.5 7 12.2 8.3 51.8 21.3 8 13.9 8.6 55.8 21.7 9 15.4 8.4 51.9 21.3

[0184] Biomasses 1 and 2 are the biomasses obtained by fermentation at high sulfate content as per Example 1, Biomass 3 is the biomass obtained by fermentation at low sulfate content as per Example 2, whereas Biomasses 4 to 9 were obtained by fermentation at low sulfate content and with continuous feeding of sulfate at low metering rate as per Example 3.

[0185] It can be discerned that the fermentation processes which led to Biomasses 4 to 9 delivered a distinctly higher yield of DHA and also more product and a purer product than that process carried out at low sulfate content without continuous feeding of sulfate (biomass 3) or those processes carried out at high sulfate content (biomasses 1 and 2). The biomasses preferred according to the invention that were obtained by the processes as per Example 3 contained sulfate in an amount of 12 to 16 g per kg of biomass.