Enhanced production of lipids by limitation of at least two limiting nutrient sources
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.
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 −3 mol/l and the phosphate source is reduced to below a concentration of 5.4×10 −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 −3 mol/l and the concentration of the phosphate source(s) is always below 5.4×10 −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.