Repeated fed-batch culture methods
Provided herein are methods of culturing a microorganism. The method includes providing a container comprising one or more microorganisms and medium, wherein the microorganisms and medium form a start volume. The microorganisms and medium are cultured until the culture reaches a threshold indicator, wherein culturing comprises feeding one or more carbon sources to the culture and wherein the culture is at a threshold volume when the threshold indicator is reached. The method also includes harvesting a portion of the threshold volume to leave a residual volume that is 40% or less of the start volume and adding fresh medium to the container in an amount to return the volume of the culture to the start volume.
1 . A method of producing oil comprising the steps of:
(a) providing a container comprising one or more oil-producing microorganisms and medium, wherein the oil-producing microorganisms and medium form a start volume;
(b) culturing the oil-producing microorganisms in the medium in the container under conditions that favor biomass production then lipid production until the oil-producing microorganisms in the medium completes fermentation and the medium reaches a threshold volume, wherein the threshold volume is greater than the start volume, and wherein culturing comprises feeding one or more carbon sources to the medium;
(c) harvesting a portion of the threshold volume of the medium from the container to leave a residual volume that is 10% to 40% of the start volume; and
(d) adding fresh medium to the container in an amount to return the volume of the medium to the start volume;
(e) isolating the oil from the harvested portion of the threshold volume, wherein the oil comprises fatty acids and the fatty acids comprise docosahexaenoic acid (DHA); and
(f) repeating steps (a) to (e) one or more times.
2 . The method of claim 1 , wherein the residual volume is 20% to 30% of the start volume.
3 . The method of claim 1 , wherein the residual volume is 30% to 40% of the start volume.
4 . The method of claim 1 , further comprising detecting in step (b) a volume of the medium, optical density (OD), dissolved oxygen (DO), cell concentration, carbon dioxide production rate, pH, time, concentration of nutrient in the medium, biomass productivity, oil productivity, or any combination thereof.
5 . The method of claim 4 , wherein concentration of nutrient in the medium is detected and wherein the nutrient is carbon or nitrogen.
6 . The method of claim 4 , wherein volume of the medium is detected.
7 . The method of claim 4 , wherein cell concentration is detected.
8 . The method of claim 1 , wherein the steps (a) to (e) are repeated more than two times.
9 . The method of claim 1 , wherein the steps (a) to (e) are repeated 3, 4, 5, 6, 7, 8, 9, or 10 times.
10 . The method of claim 1 , wherein the fatty acids further comprise alpha linolenic acid, arachidonic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and combinations thereof.
11 . The method of claim 1 , wherein the oil comprises triglycerides.
12 . The method of claim 1 , wherein the fatty acids further comprise palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-vaccenic acid (C18:1 (n-7)), docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinations thereof.
13 . The method of claim 1 , wherein the oil-producing microorganism is of the family Thraustochytriaceae.
14 . The method of claim 13 , wherein the oil-producing microorganism is of the genus Thraustochytriu m.
15 . The method of claim 14 , wherein the oil-producing microorganism is a microorganism deposited under ATCC Accession Number PTA-6245.
16 . The method of claim 1 , wherein the method results in an oil productivity from 40 to 50 g/L/d.
17 . The method of claim 1 , wherein the method results in a biomass productivity from 50 to 70 g/L/d.
18 . The method of claim 1 , wherein the method produces a biomass and a biomass productivity and wherein the biomass productivity increases after each repeat of steps (a) to (e).
19 . The method of claim 1 , wherein the method has an oil productivity and the oil productivity increases after each repeat of steps (a) to (e).