# US 9,506,100 B2 · Production of astaxanthin and docosahexaenoic acid in mixotrophic mode using Schizochytrium > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,506,100 B2 - Document ID: US9506100B2 - Status: Granted - Kind: B2 - Application number: 14/385,294 - Filed: 2013-03-15 - Published: 2015-02-05 - Granted: 2016-11-29 - Earliest claimed: 2012-03-16 - Assignees: Fermentalg - Applicants: Fermentalg - Inventors: Khadidja Romari; Adeline Le Monnier; Cyril Rols; Cindy Merlet; Julien Pagliardini; Pierre Calleja; Claude Gudin - Web page: https://patentgrep.com/patent/US9506100B2 ## CPC - C12P 23/00: Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes (containing heterorings C12P17/00) - C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00) - C12N 1/125: Unicellular algae isolates - C12P 7/6434: Docosahexenoic acids [DHA] - C12P 7/6472: containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone - C12R 2001/89: Algae {; Processes using algae} - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract New strains of protists belonging to the Schizochytrium genus, allow high-yield production of lipids and carotenoids, in particular of astaxanthin and docosahexaenoic acid (DHA), in mixotrophic mode, and a method for selecting and culturing such strains, using a variable and/or discontinuous light source, in particular a flashing light. ## Claims ### Claim 1 A method of producing docosahexanoic acid (DHA) and astaxanthin in a culture of protists lacking chloroplasts of the genus Schizochytrium, comprising the following step: a) culturing of one or more strains of Schizochytrium over several generations in a culture medium containing an organic carbon-containing substrate, under conditions of alternation of illuminated phases and phases of darkness, wherein the illumination is provided in the form of flashes of light, each flash having an intensity between 5 μmol·m−2·s−1 and 1,000 μmol·m−2·s−1, said flashes of light occurring between 2 and 3,600 times per hour, and wherein said phases of darkness occupy more than half of the time during which said strains of Schizochytrium are cultured. ### Claim 2 (depends on 1) The method according to claim 1, wherein the culture is carried out in the presence of an organic carbon-containing substrate at a concentration 300 mM to 1.2 M, the organic carbon-containing substrate being at least one selected from the group consisting of saccharose, glycerol, glucose, cellulose derivatives, and a mixture thereof. ### Claim 3 (depends on 2) The method according to claim 2, wherein the concentration of the at least one organic carbon-containing substrate is from 600 mM to 900 mM. ### Claim 4 (depends on 1) The method according to claim 1, wherein the total light supply per hour in micromoles of photons is between 2,000 to 600,000. ### Claim 5 (depends on 1) The method according to claim 1, further comprising the following steps: b) recovering the thus cultured protists, and optionally c) recovering the DHA and astaxanthin from the recovered protists. ### Claim 6 (depends on 1) The method according to claim 1, wherein said protists of the genus Schizochytrium corresponds to the strain FCC 1104, deposited at the CCAP (Culture Collection of Algae and Protozoa), under number CCAP 4087/1. ### Claim 7 (depends on 1) The method according to claim 1, wherein said flashes of light have an intensity between 50 and 400 μmol·m−2·s−1. ### Claim 8 (depends on 1) The method according to claim 1, wherein said flashes of light have a duration of between 5 seconds and 10 minutes. ### Claim 9 (depends on 1) The method according to claim 1, wherein said flashes of light have a duration of between 20 seconds and 1 minute. ### Claim 10 (depends on 1) The method according to claim 1, wherein the number of flashes is between 10 and 150 times per hour. ### Claim 11 (depends on 1) The method according to claim 1, wherein the number of flashes is between 20 and 50 times per hour. ### Claim 12 (depends on 1) The method according to claim 1, wherein said at least one organic carbon-containing substrate is at least 300 mM of glucose and/or glycerol. ### Claim 13 (depends on 1) The method according to claim 1, wherein the cultured protists have a productivity (quantity of DHA and astaxanthin produced, per liter of culture, per hour) of at least 0.015 mg/L/h. ### Claim 14 (depends on 1) The method according to claim 1, wherein the cultured protists have a productivity (quantity of DHA and astaxanthin produced, per liter of culture, per hour) of more than 0.025 mg/L/h. ### Claim 15 (depends on 1) The method according to claim 1, wherein the cultured Schizochytrium produces DHA in an amount representing more than 40% of the total lipids contained in the Schizochytrium. ### Claim 16 (depends on 1) The method according to claim 1, wherein the cultured Schizochytrium produces DHA in an amount that represents more than 60% of the total lipids contained in the Schizochytrium. ### Claim 17 (depends on 1) The method according to claim 1, wherein the cultured Schizochytrium produces astaxanthin in an amount that represents more than 0.1% of the total weight of dry matter. ### Claim 18 (depends on 1) The method according to claim 1, wherein the cultured Schizochytrium produces astaxanthin in an amount that represents more than 0.2% of the total weight of dry matter.