CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/511,962, filed July 26, 2011, which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure generally relates to a process for producing polyhydroxyalkanoates (PHAs). In particular, the present disclosure relates to extracting PHAs from PHA-containing bacterial cells.

BACKGROUND

[0003] Polyhydroxyalkanoates (PHAs) are linear polyester macromolecules composed of hydroxyl fatty acid monomer subunits. One of the most common forms of PHAs produced is a copolymer of polyhydroxybutyrate (PHB) and polyhydroxy valerate (PHV), which has properties very similar to polypropylene currently used in many containers, household items, and automotive parts. PHAs are typically UV-stable, resistant to a wide range of temperatures, and have attractive barrier properties. Unlike petroleum-based plastics that may take centuries to degrade, PHA-based plastics may biodegrade when placed in environments that foster decomposition, such as landfills, composting sites, or aquatic environments. Furthermore, PHA- based plastics may degrade quickly without any harmful effects on sea life or the greater ocean environment from chemical residues or other pollutants. In addition to these properties, PHAs may also be biocompatible, gradually and harmlessly breaking down without inducing an inflammatory response in the body. As such, PHAs also have the potential to be useful for biomedical applications, such as medical sutures and tissue repair devices.

[0004] These favorable properties of PHAs provide incentives to develop efficient ways of producing PHAs using biological systems. Currently, PHAs are produced by bacterial fermentation of sugar and lipids; however, extracting PHAs from these biological systems may require excising intracellular PHA granules. Current extraction methods rely on a limited number of solvents that may dissolve PHA. These solvents are typically hazardous, highly regulated, toxic, expensive, or feature poor dissolution kinetics. Furthermore, drying steps may be needed, and processing difficulties may arise from the high viscosity of polymer solutions. As such, there exists a need in the art for a commercially viable process that can extract PHAs directly from PHA-containing bacterial cells.

BRIEF SUMMARY

[0005] The present disclosure addresses this need by providing a process for extracting PHAs from PHA-containing bacterial cells by lysing the cells and isolating the PHAs from the lysed cell debris.

[0006] One aspect of the present disclosure provides a process for isolating polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells by: (a) providing PHA- containing bacterial cells suspended in a liquid; (b) contacting the PHA-containing bacterial cells suspended in the liquid with a base, a surfactant, or a combination thereof to form lysed cells; (c) adding a PHA anti-solvent to the lysed cells, wherein the PHA anti-solvent and the liquid releases PHA from the lysed cells; and (d) isolating the PHA from the lysed cells of step (c).

[0007] In some embodiments, the liquid may include water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, or any combination thereof. In certain embodiments, the liquid may include water.

[0008] In some embodiments that may be combined with any of the preceding embodiments, the base is ammonia, methyl amine, n-bromolithium, pyridine, acetylacetone, calcium hydroxide, bromine hydroxide, calcium oxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, alanine, or any combination thereof. In one embodiment, the base is sodium hydroxide.

[0009] In some embodiments that may be combined with any of the preceding embodiments, the surfactant is ammonium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, Triton X 100, Triton X 114, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), deoxycholate (DOC), nonylphenol ethoxylate (NP-40), cetyltrimethylammonium bromide (CTAB), octyl thio glucosides, docusates, perfluorooctanoic acid (PFOA), perfluorooctanoate (PFO), sodium stearate, sodium dodecyl sulfate, or any combination thereof. In one embodiment, the surfactant is sodium dodecyl sulfate. [0010] In some embodiments, the PHA-containing bacterial cells are contacted with a base and a surfactant. In some embodiments, the base and the surfactant are present in a weight ratio of about 0.1-10 to 1. In one embodiment, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate. In certain embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1-10 to 1. In some embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1-1 to 1 or 0.5-1 to 1. In other embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1 to 1, 0.2 to 1, 0.3 to 1, 0.4 to 1, 0.5 to 1, 0.6 to 1, 0.7 to 1, 0.8 to 1, 0.9 to 1, 1 to 1, 2 to 1, 5 to 1, or 10 to 1.

[0011] In some embodiments, the base, the surfactant, and the liquid are present in a weight/ weight/ volume ratio of about 0.1-10 to 1 to 100. In certain embodiments where the liquid includes water, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide, the sodium dodecyl sulfate and the water from the PHA-containing cell suspension are present in a weight/weight/volume ratio of about 0.1-10 to 1 to 100. In another embodiment where the liquid includes water, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide, the sodium dodecyl sulfate and the water from the PHA-containing cell suspension are present in a weight/weight/volume ratio of about 0.1 to 1 to 100, 0.2 to 1 to 100, 0.3 to 1 to 100, 0.4 to 1 to 100, 0.5 to 1 to 100, 0.6 to 1 to 100, 0.7 to 1 to 100, 0.8 to 1 to 100, 0.9 to 1 to 100, 1 to 1 to 100, 2 to 1 to 100, 5 to 1 to 100, or 10 to 1 to 100. One skilled in the art would recognize that the ratio of base to surfactant to water may also be expressed as a weight/weight/weight ratio.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the PHA-containing bacterial cells may be further lysed before adding the PHA anti-solvent. The PHA-containing bacterial cells may be further lysed by employing a lysing solvent, a lysing enzyme, a bacteriophage, a beta-lactam antiobiotic, bleach, mechanical shear, pressure change, freeze and thaw, dessication, or any combination thereof. Lysing solvents may include, for example, methanol, acetonitrile, acetone, acetic acid, ethanol, dioxane, tetrahydrofuran, hexane, heptane, ethyl acetate, toluene, chloroform, dichloromethane, dichloroethane, butyl acetate, xylene, or any combination thereof. Lysing enzymes may include, for example, lysozyme, lysostaphin, zymolase, cellulose, mutanolysin, glycanases, proteases, mannose, or any combination thereof.

[0013] In some embodiments that may be combined with any of the preceding embodiments, the PHA anti-solvent may include methanol, ethanol, propanol (e.g. , n-propanol, isopropanol), butanol (e.g. n-butanol, isobutanol), acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetic acid, formic acid, hexane, heptane, toluene, butyl acetate, xylene, water, or any combination thereof. In one embodiment, the PHA anti-solvent is methanol.

[0014] In some embodiments, the volume ratio of the PHA anti-solvent added to the liquid in the PHA-containing suspension is about 1-20 to 1. In other embodiments, the volume ratio of the PHA anti-solvent added to the liquid in the PHA-containing suspension is between about 1- 10 to 1. In certain embodiments, the volume ratio of PHA anti- solvent to the liquid is about 1 to 1, 1.5 to 1, 2.5 to 1, 3 to 1, 4 to 1, 5 to 1, 6 to 1, 10 to 1, 15 to 1, or 20 to 1.

[0015] In some embodiments, the isolated PHA has a recovery of at least 30%. In other embodiments, the isolated PHA has a recovery of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a recovery between 50-100%, 75-95%, or 80-90%.

[0016] In some embodiments, the isolated PHA has a purity of at least 70%. In other embodiments, the isolated PHA has a purity of at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a purity of at least 70- 100%, 80- 100%, 90-100%, 70-95%, 80-95%, or 90-95%. In yet another embodiment, the isolated PHA has a purity of about 100%.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In certain embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In one embodiment, the PHA is PHB. In other embodiments, the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0. In yet other embodiments, the weight ratio of PHB to PHV in the PHA product is 50:50, 70:30, 80:20, 86: 14, 85: 15, or 90: 10. In some embodiments, the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV. In yet certain embodiments, the PHA is a blend of PHB and PHV, in which less than 3%, between 15-25%, or between 30- 35% by weight is PHV.

[0018] In some embodiments that may be combined with any of the preceding embodiments, step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with PHA-producing bacteria to produce PHA-containing bacterial cells.

[0019] Another aspect of the present disclosure provides a process for isolating polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells by: (a) providing PHA- containing bacterial cells suspended in a liquid; (b) contacting the PHA-containing bacterial cells suspended in the liquid with a base, a surfactant, or a combination thereof to form lysed cells; (c) adding methanol to the lysed cells, wherein the methanol and the liquid releases PHA from the lysed cells; and (d) isolating the PHA from the lysed cells of step (c).

[0020] In some embodiments, the liquid may include water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, or any combination thereof. In certain embodiments, the liquid may include water.

[0021] In some embodiments that may be combined with any of the preceding embodiments, the base is ammonia, methyl amine, n-bromolithium, pyridine, acetylacetone, calcium hydroxide, bromine hydroxide, calcium oxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, alanine, or any combination thereof. In one embodiment, the base is sodium hydroxide.

[0022] In some embodiments that may be combined with any of the preceding embodiments, the surfactant is ammonium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, Triton X 100, Triton X 114, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), deoxycholate (DOC), nonylphenol ethoxylate (NP-40), cetyltrimethylammonium bromide (CTAB), octyl thio glucosides, docusates, perfluorooctanoic acid (PFOA), perfluorooctanoate (PFO), sodium stearate, sodium dodecyl sulfate, or any combination thereof. In one embodiment, the surfactant is sodium dodecyl sulfate. [0023] In some embodiments, the PHA-containing bacterial cells are contacted with a base and a surfactant. In some embodiments, the base and the surfactant are present in a weight ratio of about 0.1- 10 to 1. In one embodiment, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate. In certain embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1- 10 to 1. In some embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1- 1 to 1 or 0.5- 1 to 1. In other embodiments where the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1 to 1, 0.2 to 1, 0.3 to 1, 0.4 to 1 , 0.5 to 1, 0.6 to 1, 0.7 to 1, 0.8 to 1, 0.9 to 1, 1 to 1, 2 to 1, 5 to 1, or 10 to 1.

[0024] In other embodiments where the liquid includes water, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide, the sodium dodecyl sulfate and the water from the PHA-containing cell suspension are present in a weight/weight/volume ratio of about 0.1- 10 to 1 to 100. In another embodiment where the liquid includes water, the base is sodium hydroxide and the surfactant is sodium dodecyl sulfate, the sodium hydroxide, the sodium dodecyl sulfate and the water from the PHA-containing cell suspension are present in a weight/weight/volume ratio of about 0.1 to 1 to 100, 0.2 to 1 to 100, 0.3 to 1 to 100, 0.4 to 1 to 100, 0.5 to 1 to 100, 0.6 to 1 to 100, 0.7 to 1 to 100, 0.8 to 1 to 100, 0.9 to 1 to 100, 1 to 1 to 100, 2 to 1 to 100, 5 to 1 to 100, or 10 to 1 to 100. One skilled in the art would recognize that the ratio of base to surfactant to water may also be expressed as a weight/weight/weight ratio.

[0025] In some embodiments where the liquid includes water, the methanol and the water in the PHA-containing suspension may be present in a volume ratio of about 1-20 to 1. In other embodiments, the volume ratio of methanol added to the water in the PHA-containing suspension is between about 1- 10 to 1. In certain embodiments, the methanol and the water are present in a volume ratio of about 1 to 1, 1.5 to 1, 2.5 to 1, 3 to 1, 4 to 1, 5 to 1, 6 to 1, 10 to 1, 15 to 1, or 20 to 1.

[0026] In some embodiments that may be combined with any of the preceding embodiments, the process further includes adding one or more additional extraction solvents that may include ethanol, propanol (e.g. , n-propanol, isopropanol), butanol (e.g. n-butanol, isobutanol), acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetic acid, formic acid, hexane, heptane, toluene, butyl acetate, xylene, water, or any combination of these solvents. In certain embodiments, the one or more additional extraction solvents is one, two, three, or four additional extraction solvents.

[0027] In some embodiments that may be combined with any of the preceding embodiments, the PHA-containing bacterial cells may be further lysed before adding the methanol. The PHA- containing bacterial cells may be further lysed by employing a lysing solvent, a lysing enzyme, a bacteriophage, a beta-lactam antiobiotic, bleach, mechanical shear, pressure change, freeze and thaw, dessication, or any combination thereof.

[0028] In some embodiments, the isolated PHA has a recovery of at least 30%. In other embodiments, the isolated PHA has a recovery of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a recovery between 50-100%, 75-95%, or 80-90%.

[0029] In some embodiments, the isolated PHA has a purity of at least 70%. In other embodiments, the isolated PHA has a purity of at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a purity of at least 70-100%, 80-100%, 90-100%, 70-95%, 80-95%, or 90-95%. In yet another embodiment, the isolated PHA has a purity of about 100%.

[0030] In some embodiments that may be combined with any of the preceding embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In certain embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In one embodiment, the PHA is PHB. In other embodiments, the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0. In yet other embodiments, the weight ratio of PHB to PHV in the PHA product is 50:50, 70:30, 80:20, 86: 14, 85: 15, or 90: 10. In some embodiments, the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV. In yet certain embodiments, the PHA is a blend of PHB and PHV, in which less than 3%, between 15-25%, or between 30- 35% by weight is PHV.

[0031] In some embodiments that may be combined with any of the preceding embodiments, step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with PHA-producing bacteria to produce PHA-containing cells.

[0032] Yet another aspect of the present disclosure provides a process for isolating polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells by: (a) providing PHA- containing bacterial cells suspended in a liquid; (b) lysing the PHA-containing bacterial cells suspended in the liquid; (c) adding one or more extraction solvents to the lysed cells, wherein the one or more extraction solvents and the liquid releases PHA from the lysed cells; and (d) isolating the PHA from the lysed cells of step (c). In some embodiments, the liquid is water.

[0033] In some embodiments, the liquid may include water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, or any combination thereof. In certain embodiments, the liquid may include water.

[0034] In some embodiments that may be combined with any of the preceding embodiments, the lysing may employ a base, a surfactant, a lysing solvent, a lysing enzyme, a bacteriophage, a beta-lactam antiobiotic, bleach, mechanical shear, pressure change, freeze and thaw, dessication, or any combination thereof. Suitable bases may include, for example, ammonia, methyl amine, n-bromolithium, pyridine, acetylacetone, calcium hydroxide, bromine hydroxide, calcium oxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, alanine, or any combination thereof. In one embodiment, the base is sodium hydroxide. Suitable surfactants may include, for example, ammonium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, Triton X 100, Triton X 114, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), deoxycholate (DOC), nonylphenol ethoxylate (NP-40), cetyltrimethylammonium bromide (CTAB), octyl thio glucosides, docusates, perfluorooctanoic acid (PFOA), perfluorooctanoate (PFO), sodium stearate, sodium dodecyl sulfate, or any combination thereof. In one embodiment, the surfactant is sodium dodecyl sulfate. Suitable lysing solvents may include, for example, methanol, acetonitrile, acetone, acetic acid, ethanol, dioxane, tetrahydrofuran, hexane, heptane, ethyl acetate, toluene, chloroform, dichloromethane, dichloroethane, butyl acetate, xylene, or any combination thereof. Suitable lysing enzymes may include, for example, lysozyme, lysostaphin, zymolase, cellulose, mutanolysin, glycanases, proteases, mannose, or any combination thereof.

[0035] In some embodiments that may be combined with any of the preceding embodiments, the one or more extraction solvents may include methanol, ethanol, propanol (e.g. , n-propanol, isopropanol), butanol (e.g. n-butanol, isobutanol), acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetic acid, formic acid, hexane, heptane, toluene, butyl acetate, xylene, water, or any combination of these solvents. In one embodiment, the one or more extraction solvents is methanol.

[0036] In some embodiments, the isolated PHA has a recovery of at least 30%. In other embodiments, the isolated PHA has a recovery of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a recovery between 50-100%, 75-95%, or 80-90%.

[0037] In some embodiments, the isolated PHA has a purity of at least 70%. In other embodiments, the isolated PHA has a purity of at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a purity of at least 70- 100%, 80- 100%, 90-100%, 70-95%, 80-95%, or 90-95%. In yet another embodiment, the isolated PHA has a purity of about 100%.

[0038] In some embodiments that may be combined with any of the preceding embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In certain embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In one embodiment, the PHA is PHB. In other embodiments, the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0. In yet other embodiments, the weight ratio of PHB to PHV in the PHA product is 50:50, 70:30, 80:20, 86: 14, 85: 15, or 90: 10. In some embodiments, the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV. In yet certain embodiments, the PHA is a blend of PHB and PHV, in which less than 3%, between 15-25%, or between 30- 35% by weight is PHV.

[0039] In some embodiments that may be combined with any of the preceding embodiments, step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with PHA-producing bacteria to produce PHA-containing bacterial cells. [0040] In yet another aspect, provided is a method for isolating polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells, by: (a) providing PHA-containing bacterial cells suspended in a liquid comprising water; (b) contacting the PHA-containing bacterial cells suspended in the liquid with sodium hydroxide and sodium dodecyl sulfate to form lysed cells; (c) adding alcohol to the lysed cells, wherein the methanol and the liquid releases PHA from the lysed cells; and (d) isolating the PHA from the lysed cells of step (c).

[0041] In some embodiments, the alcohol is Ci_6 alcohol. In one embodiment, the alcohol is methanol.

[0042] In some embodiments that may be combined with any of the preceding embodiments, the sodium hydroxide and the sodium dodecyl sulfate are present in a weight ratio of about 0.1- 10 to 1. In other embodiments, the sodium hydroxide, the sodium dodecyl sulfate and the liquid are present in a weight/weight/volume ratio of about 0.1-10 to 1 to 100. In yet other embodiments, the volume ratio of the alcohol to the liquid is about 1-20 to 1. In yet other embodiments, the volume ratio of the methanol to the liquid is about 1-20 to 1.

[0043] In some embodiments that may be combined with any of the preceding embodiments, the isolated PHA has a recovery of at least 30%. In other embodiments, the isolated PHA has a recovery of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a recovery between 50-100%, 75-95%, or 80-90%.

[0044] In some embodiments that may be combined with any of the preceding embodiments, the isolated PHA has a purity of at least 70%. In other embodiments, the isolated PHA has a purity of at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In yet other embodiments, the isolated PHA has a purity of at least 70-100%, 80-100%, 90-100%, 70-95%, 80-95%, or 90- 95%. In yet another embodiment, the isolated PHA has a purity of about 100%.

[0045] In some embodiments that may be combined with any of the preceding embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In certain embodiments, the PHA includes polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof. In one embodiment, the PHA is PHB. In other embodiments, the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0. In yet other embodiments, the weight ratio of PHB to PHV in the PHA product is 50:50, 70:30, 80:20, 86: 14, 85: 15, or 90: 10. In some embodiments, the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV. In yet certain embodiments, the PHA is a blend of PHB and PHV, in which less than 3%, between 15-25%, or between 30- 35% by weight is PHV.

[0046] In some embodiments that may be combined with any of the preceding embodiments, step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with PHA-producing bacteria to produce PHA-containing bacterial cells.

DESCRIPTION OF THE FIGURES

[0047] The present application can be understood by reference to the following description taken in conjunction with the accompanying figure, in which like parts may be referred to by like numerals:

[0048] FIG. 1 depicts an exemplary process for extracting polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells by lysing the bacterial cells using sodium hydroxide and sodium dodecyl sulfate, and extracting the PHA using a methanol/water solvent system.

DETAILED DESCRIPTION

[0049] The following description sets forth numerous exemplary configurations, processes, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

[0050] The following description relates to a process for extracting PHA from PHA- containing bacterial cells. With reference to FIG. 1, process 100 is an exemplary embodiment for extracting PHA from PHA-containing bacterial cells by using a methanol/water solvent system. The PHA-containing bacterial cells in step 102 are Gram-negative bacteria that have produced intracellular PHA by fermenting sugars, fats, and fatty acids. In step 104, the PHA- containing bacterial cells are lysed by adding sodium hydroxide (i. e. , a base) and sodium dodecyl sulfate (i. e. , a surfactant). In step 106, methanol is added to the lysed cells to form a solvent system with the water from the PHA-containing cell suspension in step 102, in which the volume ratio of methanol to water is about 6 to 1. The methanol used in the methanol/water extraction system helps purify the PHA by dissolving or partially dissolving some of the impurities (e.g. , lipids, proteins, DNA, RNA, and other cell debris). After adding methanol, solid PHA granules are suspended in the mixture with the lysed cells. In step 108, the solid PHA is isolated by filtration. Process 100 includes optional step 110 to further purify the isolated PHA. In step 110, more methanol is added to re-suspend the isolated PHA, and the solid PHA is collected again by filtration. It should be understood that steps 106 and 108 may be repeated any number of times to purify the PHA.

[0051] It should be noted, however, that one or more steps may be omitted or added from process 100. For example, in other embodiments of the process described herein, process 100 may also include additional lysing steps (e.g. , bleaching, sonication, ross mixing, bead milling, microfluidization, and other shearing processes) before or after contacting the PHA-containing bacterial cells a base and/or a surfactant. In yet other embodiments, step 104 involving the use of base and surfactant may be omitted if there is already sufficient lysis. In yet other embodiments, process 100 may include a polishing step after isolating the solid PHA by filtration. The polishing step may include, for example, dissolving the PHA into a solvent that has high PHA solubility, washing the PHA with other solvents that have low PHA solubility), and heating, reacting, or modifying the system in a way that will help remove impurities.

[0052] As used herein, the term "about" refers to an approximation of a stated value within an acceptable range. Preferably, the range is +/- 10% of the stated value.

The PHA-Containing Bacterial Cells

[0053] The PHA-containing bacterial cells used in exemplary process 100 are provided from bacterial fermentation of carbonaceous feedstock containing sugars, fats, and fatty acids. The carbonaceous feedstock can be any material that contains carbon, and can serve as a source for producing PHAs. Such materials may include one or more of animal manure, municipal sewage and wastewater, pulp waste, waste from food processing plants (e.g. , tomato paste production waste), agricultural waste, restaurant waste, yard waste, forest waste, other plant-based materials, biodiesel transesterification waste products (e.g. , glycerol), ethanol fermentation waste products (e.g. , thin stillage from corn or cane sugar), other fermentation or industrial process waste, or any combination of these materials.

[0054] Any methods known in the art may be used to provide the PHA-containing bacterial cells in process 100. For example, carbonaceous feedstock may be fermented to produce fatty acids (e.g. , butyrate, propionate, acetate, caproic acid, caprylic acid, capric acid, and lauric acid). Then, the fatty acids may be fermented by PHA-producing bacteria to produce PHA. In some embodiments, the bacteria are Gram-negative bacteria. Examples of bacteria suitable for PHA production may include Cupriavidus necator, Alcaligenes latus, Azotobacter, Comamonas, Pseudomonads, Burkholderia, and Delflia acidovorans. Genetically-engineered organisms, such as Cupriavidus, Escherichia coli, Klebsiella, and Delflia, may also be used to produce PHA. Examples of bacteria suitable for PHA production may include Cupriavidus necator, Alcaligenes latus, Azotobacter, Comamonas, Pseudomonads, Burkholderia, and Delflia acidovorans. Genetically-engineered organisms, such as Cupriavidus, Escherichia coli, Klebsiella, and Delflia, may also be used to produce PHA.

[0055] It should be understood that PHA may be produced inside the bacteria, or released by the bacteria into the surrounding medium. The process described herein, however, is directed to PHA-containing bacterial cells with intracellular PHA (i. e. , PHA produced inside the bacteria).

[0056] The PHA-containing bacterial cells in process 100 may be provided as a mixture of PHA-containing bacterial cells suspended in water or any other suitable liquid (e.g., methanol, ethanol, propanol, butanol, acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, or any combination thereof). In some embodiments, the PHA- containing bacterial cells are first isolated from the cell broth after fermentation to produce PHA before re-suspending the PHA-containing bacterial cells in water or any other suitable liquid. In other embodiments, the PHA-containing bacterial cells are already suspended in the cell broth after fermentation to produce PHA, and PHA is directly extracted from the cell broth suspension using the process described herein. In yet other embodiments, the PHA-containing bacterial cells are suspended in the cell broth after fermentation to produce PHA, and at least a portion of the cell broth liquid is removed before PHA is directly extracted from the cell broth suspension using the process described herein.

[0057] The volume of water or other suitable liquid used to suspend the PHA-containing bacterial cells may vary depending on cell-density of the suspension, and/or viscosity of the suspension. In some embodiments, the density of the PHA mixture is between 0.05-0.25 g/mL. In other embodiments, the density of the PHA mixture is between 0.05 to 0.2 g/mL. In certain embodiments, the density of the PHA mixture is about 0.1 g/mL. Lysine the PHA- Containing Bacteria Cells

[0058] With reference to FIG. 1, sodium hydroxide and sodium dodecyl sulfate are used as the base and the surfactant, respectively, for lysing the PHA-containing bacterial cells. "Lyse" or "lysing" refers to breaking or partially breaking down the walls of the PHA-containing bacterial cells, thereby making the PHA more available for isolation.

[0059] While process 100 employs sodium hydroxide, other suitable bases may include, for example, ammonia, methyl amine, n-bromolithium, pyridine, acetylacetone, calcium hydroxide, bromine hydroxide, calcium oxide, magnesium hydroxide, potassium hydroxide, and alanine. Any combination of the bases described above may also be used.

[0060] While process 100 employs sodium dodecyl sulfate, other suitable surfactants may include, for example, ammonium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, Triton X 100, Triton X 114, CHAPS, DOC, NP-40, CTAB, octyl thio glucosides, docusates, perfluorooctanoic acid, perfluorooctanoate, and sodium stearate. Any combination of the surfactants described above may also be used. In some embodiments, the surfactant may be a cationic surfactant, an anionic surfactant, a non-ionic surfactant, or any combination thereof.

[0061] Furthermore, it should be understood that the base used may depend on the surfactant used. While process 100 employs sodium hydroxide and sodium dodecyl sulfate as the base/surfactant pair used for lysing cells, other base/surfactant combinations may be suitable.

[0062] The amount of base and surfactant used in process 100 may depend on the density of PHA mixture provided. The amount of base and surfactant used may affect the efficiency of lysis, and thus the PHA recovery yield. In certain embodiments where the base is sodium hydroxide, the surfactant is sodium dodecyl sulfate, and water is used in the PHA mixture, the ratio of sodium hydroxide (by weight) to sodium dodecyl sulfate (by weight) to water (by volume) is about 0.8 to 1 to 100. In other embodiments, the ratio of sodium hydroxide (by weight) to sodium dodecyl sulfate (by weight) to water (by volume) is about 0.1-10 to 1 to 100. In certain embodiments, the ratio of sodium hydroxide (by weight) to sodium dodecyl sulfate (by weight) to water (by volume) is 0.1 to 1 to 100, 0.2 to 1 to 100, 0.3 to 1 to 100, 0.4 to 1 to 100, 0.5 to 1 to 100, 0.6 to 1 to 100, 0.7 to 1 to 100, 0.8 to 1 to 100, 0.9 to 1 to 100, 1 to 1 to 100, 2 to 1 to 100, 3 to 1 to 100, 4 to 1 to 100, 5 to 1 to 100, 6 to 1 to 100, 7 to 1 to 100, 8 to 1 to 100, 9 to 1 to 100, or 10 to 1 to 100. [0063] While FIG. 1 depicts using a base and a surfactant to lyse the PHA-containing bacterial cells, it should be understood that the PHA-containing bacterial cells may be lysed by any methods known in the art. In some embodiments, the PHA-containing bacterial cells may be lysed by contacting the bacteria with a base or an acid. In other embodiments, a lysis buffer may be used. The surfactant may optionally be present. In one embodiment, a base and a surfactant are used. Other suitable methods to lyse the PHA-containing bacterial cells may include, for example, a lysing solvent, a lysing enzyme, a bacteriophage, a beta-lactam antiobiotic, bleach, mechanical shear, pressure change, freeze and thaw, dessication, sonication, or any combination of these methods. Lysing solvents may include, for example, methanol, acetonitrile, acetone, acetic acid, ethanol, dioxane, tetrahydrofuran, hexane, heptanes, ethyl acetate, toluene, chloroform, dichloromethane, dichloroethane, butyl acetate, xylene, or any combination of these solvents. Lysing enzymes may include, for example, lysozyme, lysostaphin, zymolase, cellulose, mutanolysin, glycanases, proteases, mannose, or any combination of these enzymes.

[0064] In other embodiments, lysing the PHA-containing bacterial cells may further include heating the cell broth containing the PHA-containing bacterial cells to a temperature below the thermal degradation temperature of the PHA. In some embodiments, the temperature is between 50°C and 180°C. In other embodiments, the temperature is between 80°C and 120°C. In yet other embodiments, the temperature is about 100°C. Depending on the solvent or solvent system used for the extraction process described herein, the pressure may need to be elevated. For example, to achieve a temperature above about 80°C when methanol and water are used as the extraction solvent system, the pressure may need to be increased. Furthermore, it should be understood that the pressure may affect the efficiency of lysis, and thus the PHA recovery yield.

The Extraction Solvent System

[0065] The PHA extraction solvent system includes the liquid that suspends the PHA- containing bacterial cells and the one or more extraction solvents that are used to isolate the PHA. The one or more extraction solvents form a mixture with the liquid suspending the PHA- containing bacterial cells. This mixture helps in releasing PHA from the lysed cell debris. The mixture may solubilize or partially solubilize the lysed cell debris and other residual materials, such as residual acetic, butyric, propionic, and valeric acid, membrane lipids, other fatty acids, pyrrole compounds and the surfactant(s). The mixture may also cause at least a portion of the PHA produced to dissociate from lysed cells. [0066] The one or more extraction solvents may include solvents that have low PHA- solubility, but can solubilize the lysed cell debris and other residual materials. In some embodiments, the one or more extraction solvents may be a "PHA anti-solvent", which refers to any solvent in which PHA is poorly dissolved. In some embodiments, PHA is insoluble in the PHA anti-solvent, or less than 5%, less than 1%, or less than 0.1% of PHA by weight is soluble in the PHA anti-solvent.

[0067] In certain embodiments, the extraction solvents are PHA anti-solvents that form a suspension with the PHA. The PHA is generally insoluble in the extraction solvent system, whereas the cellular debris and other residual materials are generally soluble in the extraction solvent system.

[0068] The one or more extraction solvents may be miscible with water. Suitable extraction solvents may include, for example, methanol, ethanol, propanol (e.g. , n-propanol, isopropanol), butanol (e.g. n-butanol, isobutanol), acetone, acetonitrile, ethyl acetate, diethyl ether, chloroform, dichloromethane, dichloroethane, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetic acid, formic acid, hexane, heptane, toluene, butyl acetate, xylene, water, or any combination of these solvents.

[0069] In certain embodiments, the liquid suspending the PHA-containing bacterial cells includes water and the extraction solvent is Ci_6 alcohol. As used herein, "Ci_6" refers to the number carbons present. Thus, Ci_6 alcohol has 1 to 6 carbons. When an alcohol having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, "butanol" is meant to include n- butanol, seobutanol, /sobutanol, and ieri-butanol; "propanol" includes n-propanol and isopropanol. In certain embodiments, the extraction solvent is an alcohol selected from methanol, ethanol, propanol, butanol, isopropanol, and any combination of these solvents. In one embodiment, the extraction solvent is methanol.

[0070] It should be recognized that the extraction solvent system used in the process described herein may depend on the constituents found in the carbonaceous feedstock. For example, the extraction solvent system may vary depending on the presence of high molecular weight fatty acids, insoluble salts, or other impurities that may be insoluble in the solvent system. [0071] In addition to the liquid used to suspend the PHA-containing bacterial cells, the number of extraction solvents used may vary. In some embodiments, one, two, three, or four solvents may be used. The ratio of the one or more extraction solvents to the liquid present in the PHA-containing suspension may affect the purity of the isolated PHA. In some embodiments where the liquid includes water and the extraction solvent is methanol, the methanol and the water are present in a volume ratio of about 1-20 to 1. In other embodiments, the methanol and water are present in a volume ratio of about 1- 10 to 1. In certain embodiments, the methanol and the water are present in a volume ratio of about 1 to 1, 1.5 to 1 , 2.5 to 1, 3 to 1 , 4 to 1, 5 to 1, 6 to 1, 10 to 1, 15 to 1, or 20 to 1.

[0072] It should be understood that a ratio of the one or more extraction solvents to the liquid present in the PHA-containing suspension that achieves optimal removal of the non-PHA components is used in the process described herein. For example, the optimal removal of non- PHA components may be achieved using a methanol concentration that solvates most of the lipids, but allows water to solvate the water-soluble components (e.g. , salts, fatty acids, sugars).

[0073] The PHA solids may be isolated from the solution containing the cellular debris and other residual materials by any solid/liquid separation methods known in the art (e.g. , gravity filtration, decanting, centrifugation, or screening).

The Isolated PHA

[0074] The isolated PHA extracted by the process described herein has a purity of at least 70%. In some embodiments, the isolated PHA has a purity of at least 80%, 85%, 90%, 95%, or 99%. In other embodiments, the isolated PHA has a purity between 80% and 95%. In yet other embodiments, the isolated PHA has a purity between 85% and 90%. One skilled in the art would recognize the different techniques that may be used to determine purity, such as GPC, HPLC, GCMS, or GC-FID. It should be understood that the purity of the PHA may be increased by repeating the extraction process as depicted in optional step 114 (FIG. 1), and improving the effectiveness of lysing the PHA-containing bacterial cells.

[0075] The process described herein isolates PHAs with a recovery of at least 30%. In some embodiments, the PHAs are extracted from PHA-containing bacterial cells with a yield of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9%. In other embodiments, the recovery is between 30% and 99%. In yet other embodiments, the recovery is between 50% and 99%. In yet other embodiments, the recovery is between 90% and 99.9%. In yet other embodiments, the recovery is between 95% and 99%. As used herein, the term "recovery" or "extraction recovery" refers to the amount by weight of PHAs extracted from the PHA-containing bacterial cells relative to the amount by mass of PHAs produced in the cells before extraction.

[0076] The PHA extracted from the process described herein may be blend of polymers and/or co-polymers including, for example, polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate, and polyhydroxybutyratevalerate (PHBV). The blend of polymers and/or co-polymers may include straight-chained or branched PHAs that may be substituted with different functional groups. It should be understood that the fermentation bacteria, the operating conditions (e.g. , pH), and the feed constituents may affect the blend of polymers and/or co-polymers inside the PHA-producing bacteria.

[0077] In some embodiments, the PHA product includes a blend of PHB and PHV. The ratio of PHB and PHV may be between about 50:50 to 100:0. In some embodiments, the ratio of PHB to PHV in the PHA product may be about 50:50, 70:30, 80:20, 86: 14, 85: 15, 90: 10, or 100:0. In some embodiments, the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV. In yet certain embodiments, the PHA is a blend of PHB and PHV, in which less than 3%, between 15-25%, or between 30-35% by weight is PHV.

[0078] Furthermore, the isolated PHA may be further processed and converted into one or more plastics. The biocompatible nature of PHA enables PHA-based plastics to be used in variety of biological applications, including medical sutures, tissue repair devices, or other biomedical uses.

EXAMPLES

[0079] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

Example 1

Providing a mixture of PHA-containing bacterial cells suspended in water

[0080] This Example describes the production of a PHA mixture of PHA-containing bacterial cells suspended in water, in which the PHA was produced inside Gram-negative bacteria. [0081] A 4L-reactor equipped with oxygen diffusion membranes, airlocks, and simethicone drip was used to grow Delftia Acidovorans bacteria to produce PHA. The reactor system was prepared by boiling the feedstock, followed by cooling it to room temperature. The feedstock was then added to a previously sterilized 4L-reactor containing a solution of 7.50g sodium acetate, 8.78g sodium propionate, 3g nutrient broth, and 0.75g potassium phosphate in 3L of distilled water. The bacteria were incubated overnight at 25°C, pH 7, 30% dissolved oxygen.

[0082] Once carrying capacity was reached, 300mL 5% hypochlorite solution (commercially available bleach) was added to the reactor and mixed thoroughly for 10 minutes. Two lOmL samples of the bleached cell broth were removed from the reactor and set aside in test tubes for GC analysis. The test tubes containing the bleached cell broth were centrifuged for 10 minutes at 8,000g in a VWR Clinical 100 centrifuge. The supernatant was decanted, washed with lOmL distilled water, and centrifuged for 10 minutes at 8,000g. The supernatant was decanted again, and a sampled was prepared for GC analysis.

[0083] The cell broth was then centrifuged for 10 minutes at 8,000g in a Sorvall RC 5B Plus Centrifuge and 25 °C. The supernatant was decanted, and a cell pellet containing PHA remained. The cell pellet was diluted in a 200mL beaker with lOOmL of distilled water to form a mixture of PHA-containing bacterial cells suspended in water.

Example 2

Extraction of PHA by using a methanol/water solvent system

[0084] This Example demonstrates the extraction of PHA from the mixture of PHA- containing bacterial cells suspended in water obtained from Example 1. The PHA-containing bacterial cells are lysed, and then the PHA is extracted from the lysed debris using a methanol/water solvent system.

[0085] To the mixture of PHA-containing bacterial cells suspended in water, lg (1.0% w/w to water) sodium dodecyl sulfate (SDS) was added. The mixture was then sonicated for 10 minutes at 20kHz and 50 amplitude in a Misonix S-4000 ultrasonic processor. After sonication, the mixture was transferred to an ice bath and 0.8g (0.8% w/w to water) NaOH was added, and mixed thoroughly for 10 minutes. The pH of the mixture was adjusted back to pH 7 using 12. IN HC1 (about 1.7mL), and then sonicated for 10 minutes at 20 kHz and 50 amplitude.

[0086] 250mL methanol was added to the mixture containing PHA and lysed cell debris, and mixed thoroughly for 10 minutes. After methanol addition, the PHA was suspended in the methanol layer. This suspension was centrifuged for 10 minutes at 8,000g, and the methanol layer was decanted. The PHA pellet was obtained and washed with 300mL methanol. The resulting suspension was centrifuged for 10 minutes at 8,000g, and the methanol was decanted. A PHA pellet was obtained, and dried for 2 hours at 80°C in an aluminum weigh boat in the oven.

[0087] Two samples of about 0.0200g each were removed from the PHA pellet, and were prepared for GC analysis.

[0088] In this example, a solvent system with a volume ratio of 2.5:1 methanol to water was used. It should be understood, however, that other ratios of methanol to water may be used, and varying the methanol/water ratio may affect the purity and yield of the PHA extracted. The purity of the PHA extracted in this Example is summarized in Table 2 below. It should be recognized, however, that the output purity readings may be above 100% since the response factors for 3-hydroxybutyrate and 3 -hydroxy valerate have not yet been identified, leading to large error bounds on the output values. Table 1 below notes the purity, error, and the PHV content of the two PHA samples produced from Example 1 above, and extracted using the methanol/water system described in this example.

Table 1. Purity and PHV content of PHA derived from bacteria and extraction using methanol/water solvent system

- Γ ^■ ..1 Π Ι■ % Ft! V

Sample 2 108.0+] 4.9 14.5

[0089] With reference to Table 2, the extracted PHA was observed to be at least 100% pure with ± 14% error. In addition, the extraction was observed to work well for multiple types of PHAs, including PHB and PHV.

[0090] The PHA recovery was also determined by first analyzing the amount of PHA initially in the reactor. The GC preparation hydrolysis solution can lyse and hydrolyze the entrained PHA granules, and offers a reading on the amount of PHA in a reactor system prior to processing. The initial PHA content of the reactor was determined from the two samples taken prior to extraction, and is summarized in Table 2 below. Table 2. Reactor content of PHA

PHA in Siiiivpto (gi PHA in R«i«or ig) ¾ PHV Average PHA in

Reactor < g>

[0091] After using cell lysis and the methanol/water extraction system, 0.88g of PHA was obtained. This recovery led to an 11.1% loss of PHA due to extraction. PHA loss may have occurred due to incomplete cell lysis, decomposition of PHA due to lysis conditions (e.g. , extreme pH and sonication), loss of PHA due to some minimal solubility in the methanol-water solution, or incomplete remove of PHA from the methanol-water solution due to Brownian suspension effects. The amount of PHA lost in these steps may be reduced by optimizing the lysis procedure and by centrifuging at a higher speed to force separation of the PHA from the methanol solution.