Enhanced production of lipids by limitation of at least two limiting nutrient sources

Abstract

The present invention is directed to a method of enhanced production of lipids by limitation of at least two limiting nutrient sources during the fermentation of the lipids producing cells.

Claims

1. A method of producing lipids comprising polyunsaturated fatty acids by the fermentation of a microorganism of the genus Schizochytrium or the genus Aurantiochytrium, said fermentation comprising: a) a biomass concentration increasing phase, in which a carbon source and at least two limiting nutrient sources are added to fermentation medium containing said microorganism to increase the biomass concentration of said fermentation medium to at least 50 g/L; and b) a lipid production phase in which the addition of at least two of said limiting nutrient sources is stopped or reduced while supply of the carbon source is maintained; wherein one of the two limiting nutrient sources is a source of phosphate and the other is a source of nitrogen.

2. The method of claim 1, wherein by stopping or reducing the addition of at least the two limiting nutrient sources, the amounts of said limiting nutrient sources fall to concentrations where the microorganism is in deprivation of said limiting nutrient sources.

3. The method of claim 2, wherein the microorganism is in deprivation of both of said at least two limiting nutrient sources for at least 30% of the time of the lipid production phase.

4. The method of claim 1, wherein the concentration of both of said at least two limiting nutrient sources falls below 0.01 mol/l in the course of the fermentation.

5. The method of claim 4, wherein the concentrations of said at least two limiting nutrient sources fall below 0.01 mol/l within 40 hours after initiation of the lipid production phase.

6. The method of claim 1, wherein the concentrations of said at least two limiting nutrient sources are below 0.01 mol/l, at least during the final 40% of the time of the lipid production phase.

7. The method of claim 1, wherein the concentrations of said at least two limiting nutrient sources are below 0.01 mol/l, at least during the last 20 hours of the fermentation.

8. The method of claim 1, wherein the nitrogen source is selected from the group consisting of: ammonia, urea, a nitrate, a nitrite, an amino acid, an inorganic ammonium salt, and mixtures thereof.

9. The method of claim 8, wherein the phosphate source is selected from the group consisting of: phosphoric acid; an inorganic phosphate salt, a hydrogen phosphate salt; a dihydrogen phosphate salt, and mixtures thereof.

10. The method of claim 9, wherein said nitrogen source comprises an inorganic ammonium salt, ammonia, or mixtures thereof.

11. The method of claim 9, wherein the phosphate source comprises phosphoric acid, inorganic phosphate salts, or mixtures thereof.

12. The method of claim 9, wherein, in the lipid production phase, the nitrogen source is reduced to below a concentration of 5.6×10.sup.−3 mol/l and the phosphate source is reduced to below a concentration of 5.4×10.sup.−3 mol/l within a time window of not more than 40 hours.

13. The method of claim 9, wherein, in the lipid production phase, the amount of the nitrogen source and the phosphate source is reduced to zero and/or below the detection limit of those compounds within a time window of not more than 40 hours.

14. The method of claim 9, wherein the concentration of the nitrogen source is always below 5.6×10.sup.−3 mol/l and the concentration of the phosphate source(s) is always below 5.4×10.sup.−3 mol/l during at least the last 75% of the time of the lipid production phase.

15. The method of claim 1, wherein, during the lipid production phase, the supply of the at least two limiting nutrient sources is stopped.

16. The method of claim 1, wherein the biomass concentration increasing phase is carried out until a biomass concentration of at least 80 g/L is reached.

17. A method of producing lipids comprising polyunsaturated fatty acids by the fermentation of a microorganism of the genus Schizochytrium or Aurantiochytrium, said fermentation comprising: a) a biomass concentration increasing phase, in which a carbon source and at least two limiting nutrient sources are added to fermentation medium containing said microorganism to increase the biomass concentration of said fermentation medium to at least 50 g/L; and b) a lipid production phase in which the addition of at least two of said limiting nutrient sources is stopped or reduced while supply of the carbon source is maintained; wherein: one of the two limiting nutrient sources is a source of phosphate is selected from the group consisting of: phosphoric acid; an inorganic phosphate salt, a hydrogen phosphate salt; a dihydrogen phosphate salt, and mixtures thereof; the other is a source of nitrogen selected from the group consisting of: ammonia, urea, a nitrate, a nitrite, an amino acid, an inorganic ammonium salt, and mixtures thereof; and the carbon source is selected from the group consisting of: methanol, ethanol, isopropanol, glycerol, fructose, glucose, sucrose, molasses, starch, cane sugar; beet sugar, hydroxy fatty acids, triacylglycerides, diglycerides, monoacylglycerides and mixtures thereof.

Description

EXAMPLES

Example 1: Production of Lipid Containing Biomass of Schizochytrium Sp ATCC PTA-9695 by Single Limitation of Ammonia at Transition into the Lipid Production Phase of the Main Fermentation

(1) Cultures were inoculated with 10% (wt/wt) of a seed fermentation of ATCC PTA-9695 and cells were cultivated in total for about 192 hours in 10 L fermenters with a start mass of 7.5 liters and a final mass of 10 liters. During the fermentation a dextrose solution of 85% (wt/vol) was fed to maintain a glucose concentration of about 50 g/l in the fermentation broth. In the biomass formation phase (about 60 hours of the fermentation time) the ammonia concentration in the broth was maintained in a range between 0.2 and 0.4 g/l. At certain intervals KH.sub.2PO.sub.4 solution was added to keep the phosphate concentration between 0.5 and 2 g/l during the whole time of the fermentation. The pH setpoint was maintained by titration with ammonia water into the fermenter. After about 60 hours of fermentation it was decided to initiate the transition into the oil production phase by stopping the titration of ammonia water, leading to complete exhaustion of the ammonia from the broth, and instead switching to titration with NaOH, while KH.sub.2PO.sub.4 titration was maintained. Particularly, the phosphate was kept during the oil production phase at a concentration of about 0.5 g/l in one run and at a concentration of about 1.5 g/l in another run. The dissolved oxygen (DO) level was maintained at 20% saturation or higher in the broth during the whole fermentation. The DO was controlled by stirrer speed.

(2) The start medium of the main-fermenter had the following composition:

(3) TABLE-US-00001 Compound Formula Concentration Optional ranges Sodium NaSO.sub.4 g/L 8.8 0-25, 2-20, sulfate or 3-10 Sodium NaCl g/L 0.625 0-25, 0.1-10, chloride or 0.5-5 Potassium KCl g/L 1 0-5, 0.25-3, chloride or 0.5-2 Magnesium MgSO.sub.4 * g/L 5 0-10, 2-8, or 3-6 sulfate 7H.sub.2O Ammonium (NH.sub.4+) g/L 0.42 0-10, 0.25-5, sulfate 2SO.sub.4 or 0.05-3 Calcium CaC.sub.12 * g/L 0.29 0.1-5, 0.15-3, chloride 2H.sub.2O or 0.2-1 Yeast Extract g/L 1 0-20, 0.1-10, or 0.5-5 Mono- KH.sub.2PO.sub.4 g/L 1.765 0.1-10, 0.5-5, potassium or 1-3 phosphate Post autoclave (Metals) Citric acid C.sub.6H.sub.8O.sub.7 * mg/L 46.82 0.1-5000, 10-3000, H.sub.2O or 40-2500 Ferrous (II)+ FeSO.sub.4 * mg/L 10.3 0.1-100, 1-50, sulfate 7H.sub.2O or 5-25 Manganese MnCl.sub.2 * mg/L 3.1 0.1-100, 1-50, chloride 4H.sub.2O or 2-25 Zink sulfate ZnSO.sub.4 * mg/L 9.3 0.01-100, 1-50, 7H.sub.2O or 2-25 Sodium Na.sub.2MoO.sub.4 * mg/L 0.04 0-1, 0.001-0.1, molybdate 2H.sub.2O or 0.01-0.1 Copper CuSO.sub.4 * mg/L 2.07 00.1-100, 0.5-50, sulfate 5H.sub.2O or 1-25 Nickel NiSO.sub.4 * mg/L 2.07 0.1-100, 0.5-50, sulfate 6H.sub.2O or 1-25 Post Autoclave (Vitamins) Thiamin* C.sub.12H.sub.18 mg/L 9.75 0.1-100, 1-50, HCL Cl.sub.2N.sub.4OS or 5-25 Calcium C.sub.18H.sub.32 mg/L 3.33 0.1-100, 0.1-50, D(+)- Ca.sub.14N.sub.2O.sub.10 or 1-10 pantothenate Biotin 1% C.sub.10H.sub.16 mg/L 0.00358 0.1-100, 0.1-50, D(+) N.sub.2O.sub.3S or 1-10 Post autoclave (Carbon) Dextrose C.sub.6H.sub.12O.sub.6 g/L 30 5-150, 10-100, or 20-50

(4) TABLE-US-00002 Target Nitrogen Nitrogen Feed Feed Addition Optional ranges NH4OH (28-30% w/w) 23.6 mL/L 0-150, 10-100, or 15-50
Cultivation conditions were as follows:

(5) TABLE-US-00003 Parameter Value Optional ranges Remark Temperature 22.5° C. 18-30; 20-27; 22-25 Dissolved ≥20% 10-100; 15-75; 20-60 Oxygen (DO) pH 7.0 6.5-7.5; 6.7-7.3; 6.8-7 Controlled by addition of ammonia water or NaOH

(6) After a fermentation time of 192 h in total, the fermentation process was stopped by heating the broth to 60° C. for 20 minutes, which stopped the metabolism of the cells. Subsequently, the total amount of oil as produced by the cells was determined. It turned out that stopping the nitrogen supply, while simultaneously maintaining the phosphate supply at concentrations of about 500 mg/l or about 1500 m/l lead to final total fat content in the cells of 47 and 43%, respectively.

Example 2: Production of Lipid Containing Biomass of Schizochytrium Sp ATCC PTA-9695 by Single Limitation of PO4 at Transition into the Lipid Production Phase of the Main Fermentation

(7) Cultures were inoculated with 10% (wt/wt) of seed fermentation of ATCC PTA 9695 and cells were cultivated in total for about 192 hours in fermenters with a start mass of 7.5 liters and a final mass of 10 liters. During the fermentation a dextrose solution of 85% (wt/vol) was fed to maintain a glucose concentration of about 50 g/l in the fermentation broth. In the biomass formation phase (about 60 hours of the fermentation time) phosphate concentration in the broth was maintained between 500 and 2000 mg/l by adding KH.sub.2PO.sub.4 solution at certain intervals. To initiate the oil production phase, the PO4 concentration was allowed to continuously drop until it was completely exhausted from the broth. The ammonia concentration was maintained during the whole fermentation, and in particular during the oil production phase, in range between 0.2-0.4 g/l. The pH setpoint was maintained by titration with ammonia water into the fermenter or by titration of a combination of ammonia water and NaOH. The dissolved oxygen (DO) level was maintained at 20% saturation or higher in the broth during the whole fermentation. The DO was controlled by stirrer speed.

(8) The start medium of the main-fermenter had the following composition:

(9) TABLE-US-00004 Compound Formula Concentration Ranges Sodium NaSO.sub.4 g/L 8.8 0-25, 2-20, or 3-10 sulfate Sodium NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 chloride Potassium KCl g/L 1 0-5, 0.25-3, or 0.5-2 chloride Magnesium MgSO.sub.4 * g/L 5 0-10, 2-8, or 3-6 sulfate 7H.sub.2O Ammonium (NH.sub.4+) g/L 0.42 0-10, 0.25-5, sulfate 2SO.sub.4 or 0.05-3 Calcium CaC.sub.l2 * g/L 0.29 0.1-5, 0.15-3, or 0.2-1 chloride 2H.sub.2O Yeast g/L 1 0-20, 0.1-10, or 0.5-5 Extract Mono- KH.sub.2PO.sub.4 g/L 1.765 0.1-10, 0.5-5, or 1-3 potassium phosphate Post autoclave (Metals) Citric acid C.sub.6H.sub.8O.sub.7 * mg/L 46.82 0.1-5000, 10-3000, H.sub.2O or 40-2500 Ferrous (II)+ FeSO.sub.4 * mg/L 10.3 0.1-100, 1-50, sulfate 7H.sub.2O or 5-25 Manganese MnCl.sub.2 * mg/L 3.1 0.1-100, 1-50, chloride 4H.sub.2O or 2-25 Zink sulfate ZnSO.sub.4 * mg/L 9.3 0.01-100, 1-50, 7H.sub.2O or 2-25 Sodium Na.sub.2MoO.sub.4 * mg/L 0.04 0-1, 0.001-0.1, molybdate 2H.sub.2O or 0.01-0.1 Copper CuSO.sub.4 * mg/L 2.07 00.1-100, sulfate 5H.sub.2O 0.5-50, or 1-25 Nickel NiSO.sub.4 * mg/L 2.07 0.1-100, 0.5-50, sulfate 6H.sub.2O or 1-25 Post Autoclave (Vitamins) Thiamin* C.sub.12H.sub.18Cl.sub.2 mg/L 9.75 0.1-100, 1-50, HCL N.sub.4OS or 5-25 Calcium C.sub.18H.sub.32Ca.sub.14 mg/L 3.33 0.1-100, 0.1-50, D(+)- or 1-10 pantothenate N.sub.2O.sub.10 Biotin 1% C.sub.10H.sub.16 mg/L 0.00358 0.1-100, 0.1-50, D(+) N.sub.2O.sub.3S or 1-10 Post autoclave (Carbon) Dextrose C.sub.6H.sub.12O.sub.6 g/L 30 5-150, 10-100, or 20-50

(10) TABLE-US-00005 Target Phosphate Phosphate Feed Feed Addition Range KH.sub.2PO.sub.4 Solution 40 mL/L 5-150, 15-100, (8.75% w/w) or 20-75
Cultivation conditions were as follows:

(11) TABLE-US-00006 Parameter Value Optional ranges Remark Temperature 22.5° C. 18-30; 20-27; 22-25 Dissolved ≥20% 10-100; 15-75; Oxygen (DO) 20-60 pH 7.0 6.5-7.5; 6.7-7.3; Controlled by 6.8-7 addition of ammonia water and/or NaOH

(12) After a fermentation time of 192 h in total, the fermentation process was stopped by heating the broth to 60° C. for 20 minutes, which stopped the metabolism of the cells. Subsequently, the total amount of oil as produced by the cells was determined. It turned out that stopping the phosphate supply, while simultaneously maintaining the ammonia supply at concentrations between 0.2 and 0.4 g/l lead to a final total fat content in the cells of between 50 and 60%.

Example 3: Production of Lipid Containing Biomass of Schizochytrium Sp. ATCC PTA-9695 by Simultaneous Limitation of Ammonia and Phosphate at Transition into the Lipid Production Phase of the Main Fermentation

(13) This fermentation was run essentially under the same conditions as described in “Example 1” with KH.sub.2PO.sub.4 solution added only in the biomass formation phase of the fermentation to keep the phosphate concentration between 500 and 2000 mg/l. After the biomass formation phase the phosphate concentration was allowed to continuously drop without further addition of KH.sub.2PO.sub.4 solution. By removing addition of the KH.sub.2PO.sub.4 solution, the culture run into simultaneous limitation of ammonia and phosphate. It turned out that stopping both the phosphate supply and the ammonia supply, delivered the best yield of final total fat in the cells, namely an amount of total fat of about 70%.