# US 9,040,267 B2 · Polyhydroxyalkanoate production method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,267 B2 - Document ID: US9040267B2 - Status: Granted - Kind: B2 - Application number: 14/003,751 - Filed: 2012-03-08 - Published: 2013-12-19 - Granted: 2015-05-26 - Earliest claimed: 2011-03-08 - Assignees: Newlight Technologies, LLC - Applicants: Markus Herrema - Inventors: Markus Herrema - Web page: https://patentgrep.com/patent/US9040267B2 ## CPC - C12P 7/625: Polyesters of hydroxy carboxylic acids - C12P 39/00: Processes involving microorganisms of different genera in the same process, simultaneously - 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)} ## Abstract Provided are processes for the production and high efficiency processing of polyhydroxyalkanoates (PHA) from carbon sources comprising carbon-containing gases or materials. ## Claims ### Claim 1 A method for enhancing polyhydroxyalkanoate (PHA) generation in a methanotrophic culture, the method comprising: (a) contacting a culture of methanotrophic microorganisms with a medium comprising copper, one or more additional nutrients, and a carbon-containing gas that can be metabolized by said culture; (b) incubating said culture in said medium to cause growth of said culture; and (c) inducing a selection pressure in said culture to cause said culture to generate PHA by: (i) reducing the concentration of copper in said medium to enable production of soluble methane monooxygenase (sMMO) in species of methanotrophic microorganisms capable of producing sMMO and particulate methane monooxygenase (pMMO) in species of methanotrophic microorganisms capable of producing pMMO; (ii) reducing the concentration of said one or more nutrient in said medium to cause said culture to generate PHA from said carbon-containing gas, (iii) returning the culture resulting from step c (ii) to the growth conditions of step (b); and (iv) repeating steps (i), (ii) and (iii) at least two times, to generate a culture of microorganisms that metabolize said carbon-containing gas to produce PHA. ### Claim 2 (depends on 1) The method of claim 1, wherein the concentration of said copper in said medium is reduced to a concentration less than 0.001 mg/L. ### Claim 3 (depends on 1) The method of claim 1, wherein the concentration of said copper in said medium is reduced to be less than 0.5 micromolar. ### Claim 4 (depends on 1) The method of claim 1, wherein the concentration of said copper in said medium is reduced to be less than 0.1 mg per dry weight gram of said microorganisms. ### Claim 5 (depends on 1) The method of claim 1, wherein the concentration of said copper in said medium is reduced to be less than 100 mg/L. ### Claim 6 (depends on 1) The method of claim 1, wherein the concentration of said copper in said medium is reduced to be less than 1 mg/L. ### Claim 7 (depends on 1) The method of claim 1, wherein said reduction of said nutrient comprises a depletion of nitrogen from said media by about 50% or more. ### Claim 8 (depends on 1) The method of claim 1, wherein said carbon-containing gas comprises methane. ### Claim 9 (depends on 1) The method of claim 1, wherein said carbon-containing gas comprises carbon dioxide. ### Claim 10 (depends on 1) The method of claim 1, wherein said transformed culture produces PHA at a concentration of at least 70% of the dry biomass weight of said microorganisms. ### Claim 11 A method for enhancing particulate methane monooxygenase (pMMO)-based polyhydroxyalkanoate (PHA) production comprising (a) contacting a culture of methanotrophic microorganisms with a medium comprising copper and nitrogen; (b) incubating said culture in said medium to cause growth of said culture and contacting said culture with a carbon-containing gas that can be metabolized by said culture; and (c) transforming said culture into a culture that generates PHA by: (i) reducing the concentration of copper in said medium to enable production of soluble methane monooxygenase (sMMO) in species of methanotrophic microorganisms capable of producing sMMO and particulate methane monooxygenase (pMMO) in species of methanotrophic microorganisms capable of producing pMMO, (ii) reducing the concentration of nitrogen in said medium to cause said culture to generate PHA from said carbon-containing gas, (iii) returning the culture to the growth conditions of step (b); and (iv) repeating steps (i), (ii) and (iii) at least two times, wherein said repetitions selectively favor growth of microorganisms that metabolize said carbon-containing gas. ### Claim 12 (depends on 11) The method of claim 11, wherein said concentration of copper in said medium is reduced to a concentration less than about 0.001 mg/L. ### Claim 13 (depends on 11) The method of claim 11, wherein said concentration of nitrogen in said medium is reduced by about 50%. ### Claim 14 (depends on 11) The method of claim 11, wherein said microorganisms having higher intracellular concentrations of PHA comprise PHA at a concentration of at least about 70% of the dry biomass weight of the microorganisms. ### Claim 15 (depends on 11) The method of claim 11, further comprising contacting the culture with one or more additional carbon-containing materials selected from the group consisting of one or more methanol, acetone, formaldehyde, propane, and ethane.