# US 2023/0265237 A1 · Solution and method for the oxidative lysis and conditioning of polyhydroxalkanoate producing cells

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- Number: US 2023/0265237 A1
- Document ID: US20230265237A1
- Status: Published application
- Kind: A1
- Application number: 18/004,854
- Filed: 2021-07-06
- Published: 2023-08-24
- Earliest claimed: 2020-07-13
- Assignees: Newlight Technologies, LLC
- Applicants: Newlight Technologies, Inc.
- Inventors: Anna Coragliotti; Garrett Sell; Kenton Kimmel
- Web page: https://patentgrep.com/patent/US20230265237A1

## CPC

- C08G 63/06: derived from hydroxycarboxylic acids
- C08G 63/89: Recovery of the polymer
- C08G 63/90: Purification; Drying
- C12N 1/06: Lysis of microorganisms
- C12P 7/625: Polyesters of hydroxy carboxylic acids
- B01D 11/0288: {Applications, solvents}
- C08G 2230/00: Compositions for preparing biodegradable polymers
- C08L 101/16: the macromolecular compounds being biodegradable
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or diesel
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present specification generally relates to a method for the oxidative conditioning of a microbial cell wall, and its eventual lysis. In particular, the specification pertains to an oxidative lysis solution and methodology, wherein the selective lysis solution is capable of conditioning and lysing the cell wall of polyhydroxyalkanoate-producing microbes, while having a limited effect on the degradation of the polyhydroxyalkanoate (PHA) biopolymer solids contained therein, thereby allowing for the further high yield recovery, and purification of high molecular weight PHA biopolymer solids.

## Claims

### Claim 1

A method for decreasing the production of foam when adding an oxidizing agent to a cellular biomass, comprising:
heating the cellular biomass to a temperature sufficient to deactivate any enzymes present in the cellular biomass that adversely interact with said oxidizing agent; and
adding said oxidizing agent to said heated cellular biomass forming a heated suspension to release the cellular content of said cellular biomass.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cellular biomass is heated to a temperature in the range of 80° C.-130° C.

### Claim 3 (depends on 1)

The method of claim 1, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (EO) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

### Claim 4 (depends on 3)

The method of claim 3, wherein said oxidizing agent comprises hydrogen peroxide (H2O2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na2O2), sodium perborate (Na2H4B2O8), sodium percarbonate (Na2H3CO6), and sodium persulfate (Na2S2O8); chlorite, chlorate, metachloro perbenzoic acid (C7H5ClO3) perchlorate, performic acid (CH2O3), peracetic acid (CH3CO3H), perchlorate (ClO4), chlorine dioxide (ClO2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

### Claim 5 (depends on 1)

The method of claim 1, wherein said heated suspension of cellular biomass in contact with said oxidizing agent contains a final concentration of 0.1%-10% of said oxidizing agent.

### Claim 6 (depends on 1)

The method of claim 1, wherein said cellular biomass comprises polyhydroxyalkanoate (PHA) containing microorganisms containing greater than 25% PHA by dry cell weight.

### Claim 7

(canceled)

### Claim 8 (depends on 6)

The method of claim 6, further comprising the step of separating and purifying said PHA from the cellular biomass and said cellular content.

### Claim 9 (depends on 8)

The method of claim 8, wherein said PHA has a molecular weight of about 100 kDa-3,000 kDa and a purity greater than 95.5%.

### Claim 10

A method for providing a source of polyhydroxyalkanoate (PHA) for further purification, comprising:
heating a suspension of microorganisms containing PHA;
exposing said heated suspension of PHA-containing microorganisms to an oxidizing agent; and
maintaining said heated suspension of PHA-containing microorganisms in contact with said oxidizing agent for a period of time and at a temperature sufficient to condition and lyse said microorganisms thereby releasing PHA and non-polymer cell mass (NPCM).

### Claim 11 (depends on 10)

The process of claim 10, wherein said heated suspension of PHA-containing microorganisms in contact with said oxidizing agent contains a final concentration of 0.1%-10% of said oxidizing agent.

### Claim 12

(canceled)

### Claim 13 (depends on 11)

The process of claim 11, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (EO) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

### Claim 14 (depends on 11)

The process of claim 11, wherein said oxidizing agent comprises hydrogen peroxide (H2O2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na2O2), sodium perborate (Na2H4B2O8), sodium percarbonate (Na2H3CO6), and sodium persulfate (Na2S2O8); chlorite, chlorate, metachloro perbenzoic acid (C7H5ClO3) perchlorate, performic acid (CH2O3), peracetic acid (CH3CO3H), perchlorate (ClO4), chlorine dioxide (ClO2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

### Claim at position 15

15-16. (canceled)

### Claim 17 (depends on 10)

The process of claim 10, wherein said temperature and time sufficient to condition and lyse said microorganisms and dissolve said NPCM is in the range of 80° C.-130° C. for greater than 1 hour.

### Claim at position 17

18-20. (canceled)

### Claim 21 (depends on 10)

The process of claim 10, wherein said polyhydroxyalkanoates (PHAs) is selected from the group consisting of poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO, or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), or other short- or medium-chain length, saturated or unsaturated PHAs; or polylactic acid (PLA); or their copolymers or any combinations thereof.

### Claim 22 (depends on 10)

The process of claim 10, wherein the suspension of microorganisms containing greater than 25% PHA by dry cell weight.

### Claim 23

An aqueous extraction process of polymers from a culture of microorganisms, comprising:
contacting a suspension of microorganisms with an oxidizing agent wherein said oxidizing agent has a final concentration of approximately 0.5%-6.0%;
maintaining said suspension of microorganisms in contact with said oxidizing agent at a temperature greater than about 87° C. for approximately 2.5 hours to lyse open said microorganisms freeing said polymer and dissolve said non-polymer cell mass.

### Claim 24 (depends on 23)

The process of claim 23, wherein said freed polymer has a molecular weight of about 100 kDa-3,000 kDa.

### Claim 25 (depends on 23)

The process of claim 23, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (EO) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

### Claim 26 (depends on 25)

The process of claim 25, wherein said oxidizing agent comprises hydrogen peroxide (H2O2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na2O2), sodium perborate (Na2H4B2O8), sodium percarbonate (Na2H3CO6), and sodium persulfate (Na2S2O8); chlorite, chlorate, metachloro perbenzoic acid (C7H5ClO3) perchlorate, performic acid (CH2O3), peracetic acid (CH3CO3H), perchlorate (ClO4), chlorine dioxide (ClO2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

### Claim at position 24

27-28. (canceled)

### Claim 29 (depends on 25)

The lysis solution of claim 25, wherein said non-gaseous compound comprises a 30-35% hydrogen peroxide solution having a final concentration of 0.4-4% volume/volume.