WO2013016558A1 - Procédés d'extraction et de purification de polyhydroxyalcanoates contenus dans des cellules bactériennes - Google Patents

Procédés d'extraction et de purification de polyhydroxyalcanoates contenus dans des cellules bactériennes Download PDF

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WO2013016558A1
WO2013016558A1 PCT/US2012/048374 US2012048374W WO2013016558A1 WO 2013016558 A1 WO2013016558 A1 WO 2013016558A1 US 2012048374 W US2012048374 W US 2012048374W WO 2013016558 A1 WO2013016558 A1 WO 2013016558A1
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Prior art keywords
pha
solvent
mixture
dichloromethane
alcohol
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Makoto N. MASUNO
Shawn Michael BROWNING
John Bissell
Ryan L. Smith
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Origin Materials Operating Inc
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Micromidas Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present disclosure generally relates to a process for producing polyhydroxyalkanoates (PHAs).
  • PHAs polyhydroxyalkanoates
  • the present disclosure relates to extracting and purifying PHAs from PHA-producing bacteria.
  • PHAs Polyhydroxyalkanoates
  • PHAs are linear polyester macromolecules composed of hydroxyl fatty acid monomer subunits.
  • PHBs polyhydroxybutyrate
  • PV polyhydroxyvalerate
  • PHAs are UV-stable, resistant to a wide range of temperatures, and have attractive barrier properties.
  • PHA-based plastics are completely biodegradable when placed in environments that foster decomposition, such as landfills, composting sites, or aquatic environments.
  • PHA-based plastics can degrade quickly without any harmful effects on sea life or the greater ocean environment from chemical residues or other pollutants.
  • PHAs are also biocompatible, gradually and harmlessly breaking down without inducing an inflammatory response in the body.
  • PHAs also have the potential to be useful for biomedical applications, such as medical sutures and tissue repair devices.
  • PHAs are produced by bacterial fermentation of sugars and lipids; however, extracting PHAs from these biological systems requires a robust mechanism of selectively isolating PHA from a mixture of amphiphilic compounds, including cell wall and other cellular structures and debris. As such, there exists a need in the art for a commercially viable process that can extract PHAs directly from PHA- containing bacterial cells.
  • the present disclosure addresses this need by providing a process for extracting PHAs from PHA-containing bacterial cells, and isolating the PHAs from cells.
  • 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; (b) adding dichloromethane to the PHA-containing bacterial cells to form a first mixture that includes dichloromethane, PHA and residual solids; (c) separating the PHA and the dichloromethane from the residual solids; (d) adding alcohol to the PHA and the dichloromethane to form a second mixture that includes PHA, dichloromethane, and alcohol; and (e) isolating the PHA from the second mixture.
  • PHA polyhydroxyalkanoate
  • the method further includes: after step (d), removing dichloromethane from the second mixture in an amount sufficient to achieve substantial solid- liquid phase separation.
  • step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with the PHA-producing bacteria to produce PHA- containing bacterial cells.
  • the process further includes converting the isolated PHA into one or more plastics.
  • the first mixture has a low viscosity.
  • step (c) is by pumping, filtering, or a combination thereof.
  • step (c) is performed by filtration.
  • step (c) is performed by using a coarse filter.
  • the process includes: (a) providing PHA-containing bacterial cells in a first vessel; (b) adding dichloromethane to the PHA-containing bacterial cells in the first vessel to form a first mixture that includes dichloromethane, PHA and residual solids; (c) separating the PHA and the dichloromethane from the residual solids by pumping, filtering, or pumping and filtering the PHA and the dichloromethane into a second vessel; (d) adding alcohol to the PHA and the dichloromethane in the second vessel to form a second mixture that includes PHA, dichloromethane, and alcohol; (e) removing dichloromethane from the second mixture in an amount sufficient to achieve substantial solid-liquid phase separation in the second vessel; and (f) isolating the PHA from the second mixture.
  • the process further includes sterilizing and/or disinfecting the PHA and the dichloromethane after separation from the residual solids and before addition of the alcohol.
  • the sterilization and/or disinfection employs ozonation.
  • the adding of alcohol to form the second mixture and the removing of at least a portion of the dichloromethane from the second mixture removes the off-color, reduces the odor, or a combination thereof from the isolated PHA.
  • the alcohol is Ci_6 alcohol. In one embodiment, the alcohol is methanol.
  • the weight/volume/volume ratio of PHA to dichloromethane to alcohol is about 1 to 10-25 to 10- 25. In certain embodiments, the weight/volume/volume ratio of PHA to dichloromethane to alcohol is 1:10:25; 1:20:20; 1:20:25; 1:25:20; or 1:25:25. In certain embodiments, the weight/volume/volume ratio of PHA to dichloromethane to methanol is about 1 to 10-25 to 10- 25. In certain embodiments, the weight/volume/volume ratio of PHA to dichloromethane to methanol is 1:10:25; 1:20:20; 1:20:25; 1:25:20; or 1:25:25.
  • the residual solids may include butyric acid, membrane lipids, fatty acids, one or more pyrrole compounds, or any combination thereof.
  • the process further includes heating the first mixture.
  • the first mixture is heated to a temperature between 30°C and 180°C.
  • the temperature is between 40°C and 120°C.
  • the temperature is about 100°C.
  • the temperature is about 40-50°C.
  • the isolated PHA has a recovery of at least 30%.
  • the isolated PHA has a recovery of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%.
  • the isolated PHA has a recovery between 50-100%, 75-95%, or 80-90%.
  • 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%.
  • the PHA includes polyhydroxybutyrate (PHB), polyhydroxy valerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof.
  • the PHA includes polyhydroxybutyrate (PHB), polyhydroxy valerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof.
  • the PHA is PHB.
  • the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0.
  • 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.
  • the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV.
  • 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.
  • Another aspect of the disclosure provides a process for isolating polyhydroxyalkanoate (PHA) from PHA-containing bacterial cells by: (a) providing PHA- containing bacterial cells; (b) adding a PHA-extracting solvent to the PHA-containing bacterial cells to form a first mixture that includes PHA-extracting solvent, PHA, and residual solids; (c) separating the PHA and the PHA-extracting solvent from the residual solids; (d) adding a PHA- purifying solvent to the PHA and the PHA-extracting solvent to form a second mixture that includes PHA, PHA-extracting solvent, and PHA-purifying solvent; and (e) isolating the PHA from the second mixture.
  • PHA polyhydroxyalkanoate
  • the method further includes: after step (d), removing PHA- extracting solvent from the second mixture in an amount sufficient to achieve substantial solid- liquid phase separation.
  • step (a) includes: providing a carbonaceous feedstock; providing PHA-producing bacteria; and contacting the carbonaceous feedstock with the PHA-producing bacteria to produce PHA- containing bacterial cells.
  • the process further includes converting the isolated PHA into one or more plastics.
  • the first mixture has a low viscosity.
  • step (c) is by pumping, filtering, or a combination thereof.
  • step (c) is performed by filtration.
  • step (c) is performed by using a coarse filter.
  • the process includes: (a) providing PHA-containing bacterial cells in a first vessel; (b) adding a PHA-extracting solvent to the PHA-containing bacterial cells in the first vessel to form a first mixture that includes PHA-extracting solvent, PHA and residual solids; (c) separating the PHA and the PHA-extracting solvent from the residual solids by pumping, filtering, or pumping and filtering the PHA and the PHA-extracting solvent into a second vessel; (d) adding a PHA-purifying solvent to the PHA and the PHA-extracting solvent in the second vessel to form a second mixture that includes PHA, PHA-extracting solvent, and PHA-purifying solvent; (e) removing PHA-extracting solvent from the second mixture in an amount sufficient to achieve substantial solid-liquid phase separation in the second vessel; and (f) isolating the PHA from the second mixture.
  • the PHA has a rate of dissolution of at least 3 mg/ PHA/min » g solvent in the PHA-extracting solvent.
  • the process further includes sterilizing and/or disinfecting the PHA and the PHA-extracting solvent after separation from the residual solids and before addition of the PHA-purifying solvent.
  • the sterilization and/or disinfection employs ozonation.
  • the adding of PHA-purifying solvent to form the second mixture and the removing of a portion of the PHA-extracting solvent from the second mixture removes the off-color, reduces the odor, or a combination thereof from the isolated PHA.
  • the process further includes heating the first mixture.
  • the first mixture is heated to a temperature between 30°C and 180°C. In other embodiments, the temperature is between 40°C and 120°C. In yet other embodiments, the temperature is about 100°C.
  • the PHA-extracting solvent is a halogenated solvent.
  • the PHA-extracting solvent is a chlorinated solvent, a fluorinated solvent, or any combination thereof.
  • the PHA-extracting solvent is a chlorinated solvent that may include chloroform, dichloromethane, dichloroethane, carbon tetrachloride, or any combination thereof.
  • the PHA-extracting solvent is dichloromethane.
  • the PHA-extracting solvent is a fluorinated solvent that may include hexafluoro isopropyl alcohol, fluoroform, difluorormethane, difluoroethane, carbon tetrafluoride, or any combination thereof.
  • the PHA-purifying solvent is a polar organic solvent.
  • the PHA- purifying solvent is alcohol.
  • the PHA-purifying solvent is Ci_6 alcohol.
  • the PHA-purifying solvent is methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, or any combination thereof.
  • the PHA-purifying solvent is methanol.
  • the boiling point of the PHA-extracting solvent is lower than the boiling point of the PHA-purifying solvent.
  • the PHA-extracting solvent may be cleanly separated from the resultant mixture, providing a clean precipitation (including in certain embodiments, crystallization) of the PHA from the PHA-purifying solvent.
  • the heat of vaporization of the PHA- extracting solvent is below 40 kJ/mol. When the heat of vaporization is lower, the separation may be more energetically efficient.
  • the weight/volume/volume ratio of PHA to PHA-extracting solvent to PHA-purifying solvent is about 1 to 10-25 to 10-25. In certain embodiments, the weight/volume/volume ratio of PHA to PHA-extracting solvent to PHA-purifying solvent is 1:10:25; 1:20:20; 1:20:25; 1:25:20; or 1:25:25.
  • the PHA may be suspended in the first mixture. In other embodiments, the PHA may be dissolved or partially dissolved in the first mixture. In yet other embodiments, the PHA may be suspended in the first mixture as a colloid. In some embodiments that may be combined with any of the preceding embodiments, the second mixture is a suspension.
  • the residual solids may include butyric acid, membrane lipids, fatty acids, one or more pyrrole compounds, or any combination thereof.
  • the process further includes: adding a second PHA-extracting solvent to the isolated PHA to form a third mixture that includes a second PHA, and second residual solids; separating the second PHA and the second PHA-extracting solvent from the second residual solids; adding a second PHA-purifying solvent to the second PHA and the second PHA- extracting solvent to form a fourth mixture that includes the second PHA, the second PHA- extracting solvent, and the second PHA-purifying solvent; removing the second PHA-extracting solvent from the fourth mixture in an amount sufficient to achieve substantial solid-liquid phase separation; and isolating the second PHA from the fourth mixture.
  • the second PHA-extracting solvent may be the same or different than the PHA-extracting solvent.
  • the second PHA-extracting solvent is a halogenated solvent.
  • the second PHA-extracting solvent is a chlorinated solvent, a fluorinated solvent, or any combination thereof.
  • the second PHA-extracting solvent is a chlorinated solvent that may include chloroform, dichloromethane, dichloroethane, carbon tetrachloride, or any combination thereof.
  • the second PHA-extracting solvent is dichloromethane.
  • the second PHA-extracting solvent is a fluorinated solvent that may include hexafluoro isopropyl alcohol, fluoroform, difluorormethane, difluoroethane, carbon tetrafluoride, or any combination thereof.
  • the second PHA-purifying solvent may be the same or different than the PHA-purifying solvent.
  • the second PHA-purifying solvent is a polar organic solvent.
  • the second PHA-purifying solvent is alcohol.
  • the second PHA-purifying solvent is Ci_6 alcohol.
  • the PHA-purifying solvent is methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, or any combination thereof.
  • the second PHA-purifying solvent is methanol.
  • the process further includes combining the PHA-extracting solvent removed in step (e) with second PHA-containing bacterial cells.
  • 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%.
  • 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%.
  • the PHA includes polyhydroxybutyrate (PHB), polyhydroxy valerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), polyhydroxydecanoate (PHD), polyhydroxybutyratevalerate (PHBV), or any combination thereof.
  • the PHA includes polyhydroxybutyrate (PHB), polyhydroxy valerate (PHV), polyhydroxybutyratevalerate (PHBV), or any combination thereof.
  • the PHA is PHB.
  • the PHA is a blend of PHB and PHV in a weight ratio of between 50:50 to 100:0.
  • 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.
  • the PHA is a blend of PHB and PHV, in which less than 35% by weight is PHV.
  • 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.
  • FIG. 1 depicts an exemplary process for extracting polyhydroxyalkanoates (PHAs) from PHA-containing bacterial cells using dichloromethane and purifying the extracted PHAs using methanol.
  • PHAs polyhydroxyalkanoates
  • process 100 is an exemplary embodiment for extracting and purifying PHA from PHA-containing bacterial cells by using dichloromethane as the PHA-extracting solvent and methanol as the PHA-purifying solvent.
  • dichloromethane as the PHA-extracting solvent
  • methanol as the PHA-purifying solvent.
  • other halogenated solvents or in certain embodiments, other chlorinated solvents
  • other alcohols or in certain embodiments, other alcohols, such as C 2 -6 alcohols
  • the PHA-containing bacterial cells in step 102 are provided from bacterial fermentation of sugars, fats, and fatty acids. PHA is produced inside the cells of the PHA- producing bacteria.
  • dichloromethane is added to form a mixture that includes the cells and PHA.
  • the mixture in step 104 is typically a heterogeneous mixture due to the presence of residual solids from the PHA-containing cells.
  • step 106 this mixture is heated to between 40°C and 50°C at 1 atm pressure.
  • step 108 the mixture is filtered using a coarse filter to separate the PHA from the residual solids (e.g. , cellular debris, residual butyric acid, membrane lipids, fatty acids, pyrrole compounds).
  • step 110 methanol is added to filtrate containing a mixture of PHA and dichloromethane. This mixture containing PHA, dichloromethane and methanol may be homogeneous or heterogeneous.
  • step 112 a portion of dichloromethane is removed from the filtrate by distillation. Once a portion of the dichloromethane is removed, solid-liquid phase separation is observed, and the PHA precipitates (more specifically, in certain instances, the PHA crystallizes).
  • step 114 the precipitated PHA (more specifically, in certain instances, the crystallized PHA) is isolated by solid- liquid separation (e.g. , filtration).
  • Process 100 includes optional step 116 to further purify the isolated PHA.
  • step 116 additional dichloromethane may be added to the isolated PHA, and steps 104 to 114 may be repeated. It should be understood that steps 104 to 114 may be repeated any number of times to purify the PHA.
  • step 112 may be omitted if the PHA-purifying solvent, such as methanol in process 100, is added in an amount and under conditions suitable to achieve solid-liquid phase separation.
  • an additional sterilization and/or disinfection step may be performed between steps 108 and 110, which may help with reducing the odor in the final PHA product.
  • Suitable methods may include, for example, ozonation, bleaching, pasteurization, irradiation, heat, filtration, antibiotics, chlorination, sulfur dioxide, electroporation, pH, or peroxides (e.g. , hydrogen peroxide, peracetic acid), or any combination of these methods.
  • the additional sterilization and/or disinfection step involves ozonation.
  • ozonation ozone can be bubbled through the PHA/dichloromethane mixture, before methanol is added.
  • additional steps may include recycling the PHA-extracting solvent (e.g. , dichloromethane) obtained from the distillation in step 112 for use in step 104.
  • the PHA-purifying solvent e.g. , methanol
  • process 100 may be recovered by distillation and/or purified by activated charcoal, and recycled for use in step 110 as the PHA-purifying solvent.
  • 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 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.
  • any methods known in the art may be used to provide the PHA-containing bacterial cells in process 100.
  • carbonaceous feedstock may be fermented to produce fatty acids (e.g. , butyrate, propionate, acetate, caproic acid, caprylic acid, capric acid, and lauric acid).
  • the fatty acids may be fermented by PHA-producing bacteria to produce PHAs.
  • bacteria suitable for PHA production may include Cupriavidus necator, Alcaligenes latus, Azotobacter, Comamonas, Pseudomonads, Burkholderia, and Delftia acidovorans.
  • Genetically- engineered organisms such as Cupriavidus, Escherichia coli, Klebsiella, and Delftia, may also be used to produce PHA.
  • 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 is produced inside the bacteria).
  • the PHA-containing bacterial cells may also be treated (e.g., by adding bleach or adjusting pH) before adding the PHA-extracting solvent, it should be understood that such a treatment is optional in the process described herein.
  • the PHA-extracting solvent used in the process described herein that is added to the PHA-containing bacterial cells includes any solvent system that may help with extracting the intracellular PHA from the PHA-containing bacterial cells.
  • the PHA- extracting solvent selected for the process described herein may have a boiling point that is lower than the boiling point of the PHA-purifying solvent by more than 10°C. This allows for more efficient distillation.
  • the heat of vaporization of the PHA-extracting solvent may also be less than 100 kJ/mol.
  • the PHA-extracting solvent may include one or more halogenated solvents.
  • the PHA-extracting solvent is a chlorinated solvent. Suitable chlorinated solvents may include, for example, chloroform, dichloromethane, dichloroethane, carbon tetrachloride, or any combination of these solvents. In certain embodiments, the chlorinated solvent is dichloromethane, dichloroethane, or any combination of these solvents. In other embodiments, the PHA-extracting solvent is a fluorinated solvent.
  • Suitable fluorinated solvents may include, for example, hexafluoro isopropyl alcohol, fluoroform, difluorormethane, difluoroethane, carbon tetrafluoride, or any combination of these solvents.
  • the PHA-extracting solvent is dichloromethane.
  • dichloromethane is used as the PHA-extracting solvent unexpectedly results in a mixture with a low viscosity.
  • the low viscosity of the mixture resulting from the addition of dichloromethane to the PHA-containing bacterial cells allows the mixture to be pumped and/or filtered more easily from one vessel to another compared to the use of other PHA-extracting solvents.
  • the low viscosity of the mixture offers a processing advantage, particularly when extracting PHA on a commercial scale.
  • One of skill would recognize the various methods and techniques known in the art to determine or measure viscosity.
  • viscosity can be qualitatively determined by observing the ease of movement of the liquid, mixture or slurry when poured. Viscosity can be measured using various types of viscometers and rheometers. Examples may include a glass capillary viscometer, a Zahn cup, a Stormer viscometer, a Ford viscosity cup, a vibrating viscometer, and an acoustic rheometer.
  • the solubility of PHA in the PHA-extracting solvent may vary depending on the type of solvent selected, and the amount of solvent added relative to the amount of PHA.
  • the PHA-extracting solvent may have a low solubility for PHA.
  • the PHA-extracting solvent when added to PHA, the PHA-extracting solvent may form a suspension with the PHA.
  • the PHA-extracting solvent may form a colloid with the PHA.
  • the PHA-extracting solvent may dissolve, or partially dissolve, the PHA.
  • dichloromethane is the PHA-extracting solvent added, dichloromethane may dissolve, or partially dissolve, at least a portion of the PHA.
  • the PHA may have a rate of dissolution of at least 3 mg/ PHA/min » g solvent in the PHA-extracting solvent.
  • PHA-extracting solvent may also be added in addition to the PHA-extracting solvent to help with lysing the PHA-containing bacterial cells.
  • ethyl acetate may also be used to help with extraction of PHA from the cells.
  • the mixture that includes the PHA-extracting solvent and PHA-containing bacterial cells may be heated to further improve extraction of the intracellular PHA from the cells.
  • the mixture may be heated to a temperature below the thermal degradation temperature of the PHA.
  • the mixture may also be heated to the boiling point or the reflux temperature of the PHA-extracting solvent.
  • the mixture is heated to a temperature 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.
  • the mixture that includes the dichloromethane and the PHA-containing bacterial cells may be heated to 40°C.
  • pressure may be increased to increase the temperature for heating.
  • the mixture is placed in a sealed vessel, the mixture is heated but not refluxed.
  • the residual solids separated from the PHA and the PHA-extracting solvent may include, for example, cellular debris that is insoluble in the PHA-extracting solvent, such as residual butyric acid, membrane fatty acids, and pyrrole compounds.
  • the residual solids may be separated from PHA-extracting solvent/PHA mixture by any solid/liquid separation methods known in the art. For example, gravity filtration, decanting, centrifugation, or screening may be used.
  • a coarse filter is typically used. For example, the coarse filters ranging from 1 micron to 10,000 microns are suitable for separating out the residual solids.
  • the PHA-purifying solvent helps with purifying the PHA solids extracted from the PHA-containing bacterial cells. It should be understood that any solvent system that has low PHA-solubility, but high solubility for impurities (e.g. , compounds causing the brown color and unpleasant odor) may be suitable for the process described herein. Additionally, the PHA- purifying solvent selected for the process described herein may, in certain embodiments, have a higher boiling point than the PHA-extracting solvent.
  • the PHA-purifying solvent is alcohol.
  • the PHA-purifying solvent is Ci_6 alcohol.
  • CW refers to the number carbons present.
  • Ci_6 alcohol has 1 to 6 carbons.
  • butanol is meant to include n- butanol, seobutanol, /so-butanol, and ieri-butanol;
  • propanol includes n-propanol and iso- propanol.
  • the PHA-purifying solvent is an alcohol selected from methanol, ethanol, propanol, butanol, isopropanol, and any combination of these solvents. In one embodiment, the PHA-purifying solvent is methanol.
  • the addition of the PHA-purifying solvent to the PHA and the PHA-extracting solvent may cause at least a portion of the PHA to precipitate.
  • precipitate or “precipitation” refers to a solid coming out of a solution, a partially-dissolved solution, or a homogenous mixture.
  • precipitate or “precipitation” also includes “crystallize” or “crystallization”, respectively.
  • the process may also include removing PHA-extracting solvent in an amount sufficient to achieve substantial solid-liquid phase separation.
  • Solid-liquid phase separation may occur when solids are observed to appear from the liquid phase.
  • solid- liquid phase separation may be the result of PHA precipitating (more specifically, in certain instances, crystallizing) from a solution, a partially-dissolved solution, or a homogenous mixture.
  • dichloromethane is the PHA-extracting solvent and methanol is the PHA-purifying solvent
  • removal of dichloromethane by distillation causes at least some of the PHA to precipitate (more specifically, in certain instances, crystallize).
  • the amount of dichloromethane removed may vary; for example, half or more of the dichloromethane that was initially added to the PHA-containing bacterial cells may be removed.
  • Any suitable techniques known in the art to remove a portion of the PHA-extracting solvent may be employed. For example, distillation may be used to remove a portion of the PHA-extracting solvent, which may also further increase PHA extraction yield.
  • the resulting PHA solids may be isolated by any solid/liquid separation methods known in the art ⁇ e.g. , gravity filtration, decanting, centrifugation, and screening).
  • the Isolated PHA The Isolated PHA
  • 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%.
  • GPC gel permeation chromatography
  • HPLC high-performance liquid chromatography
  • GCMS gas chromatography-mass spectrometry
  • GC-FID gas chromatography-flame ionization detector
  • Other techniques such as a crotonic acid assay may also be used to determine purity. It should be understood that the purity of the PHA may be increased by repeating the extraction process, as depicted in optional step 116 (FIG. 1).
  • the process described herein isolates PHA with a recovery of at least 30%.
  • the PHA 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%.
  • the recovery is between 30% and 99%.
  • the recovery is between 50% and 99%.
  • the recovery is between 90% and 99.9%.
  • the recovery is between 95% and 99%.
  • the term "recovery" or “extraction recovery” refers to the amount by weight of PHA extracted and purified from the PHA-containing bacterial cells relative to the amount by mass of PHA produced in the cells before extraction and purification.
  • the amount of PHA-extracting solvent relative to the amount of PHA-purifying solvent used in the extraction process may affect purity and yield of the isolated PHA.
  • the use of certain PHA-extracting solvents and PHA- purifying solvents may help to remove or reduce the off-color and/or odor from the isolated PHA.
  • the use of a dichloromethane/methanol solvent system may help to remove or reduce the off-color and/or odor from the isolated PHA.
  • the volume ratio of PHA-extracting solvent to PHA-purifying solvent used in the process is about 2:5.
  • the ratio of PHA-extracting solvent to PHA-purifying solvent used in the process is about 1 :2, 1 :3, or 1 :4.
  • the ratio of dichloromethane to methanol used in the process is about 2:5.
  • the amount of PHA-extracting solvent and PHA-purifying solvent used relative to the amount of PHA in the extraction process may also affect purity and yield of the isolated PHA.
  • the amount of PHA-extracting solvent and PHA-purifying solvent used relative to the amount of PHA may affect the amount of PHA that may be extracted from inside the PHA-containing bacterial cells, and may also affect the solubility of the impurities (e.g. , compounds causing the brown and/or other off-color(s) and unpleasant odor).
  • the weight/volume/volume ratio of PHA to PHA-extracting solvent to PHA- purifying solvent used in the process e.g. , steps 104 to 112 in FIG.
  • the weight/ volume/volume ratio of PHA to dichloromethane to methanol is 1 : 10:25 ; 1 :20:20; 1 :20:25; 1 :25 :20; or 1 :25 :25.
  • the ratio of PHA to PHA-extracting solvent to PHA-purifying solvent may also be expressed in other ways, such as by a volume/volume/volume ratio.
  • 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.
  • the PHA product includes a blend of PHB and PHV.
  • the weight ratio of PHB and PHV may be between about 50:50 to 100:0. In some embodiments, the weight 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.
  • 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.
  • 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.
  • Example 1 The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.
  • Example 1
  • a crotonic acid assay was used to determine the purity of the extracted PHA.
  • a 90.0mg of dried extracted PHA was transferred to a lOmL glass test tube.
  • 6mL of concentrated (95 -98 ) sulfuric acid was added to the test tube.
  • the sample was digested and reacted by heating at 100°C for 10-15 minute with periodic vortexing.
  • the sample was then cooled and diluted with concentrated sulfuric acid.
  • a 10-fold or 100-fold dilution was performed within a 1.5 ml eppendorff test tube to bring the sample within the sensitivity of the instrument.
  • the absorbance reading at 235nm was determined using the Shimatzu UV Spec.
  • CAAV crotonic acid absorbance value
  • PHA-containing bacterial cells such as Delftia sp. (Dsp) and Cupriavidus sp. (Csp) were dried in the oven until dry (12-24 hr) at about 107°C. Some of the cells were pretreated according to the protocol described in Table 1 below. In cell treatment A, bleach (5.25% NaOCl) was added to the cells. In cell treatment B, the pH of the cells was raised to pH 12 using 50% NaOH solution, followed by neutralization with 38% H2SO4. In cell treatment C, the cells were untreated.
  • Dsp Delftia sp.
  • Csp Cupriavidus sp.
  • a syrup (a concentrated thin stillage from corn ethanol process, 35% w/w solid) was obtained from a corn ethanol producer and 2 L of the syrup was fed into a 5-L fermentation reactor with 2 L of water. After raising the pH to 6.0 with 40 % sodium hydroxide solution, 1 L of a previously-fermented syrup was combined with the half-diluted syrup to inoculate additional fermentation bacteria. The stream was fermented for a week under anaerobic conditions in the reactor to generate volatile fatty acids (VFA). The pH was maintained at 6.0 with 40 % sodium hydroxide solution. The final VFA concentration, measured using Hach TNT 872 kit, was 29.7 g/L.
  • the fermentate containing suspended solid was sterilized by autoclaving in lL-bottles at 121°C for 30 minutes.
  • the autoclaved fermentate was then neutralized to pH at 7.0-7.2 with 20% sodium hydroxide solution (autoclaved).
  • 800 mL of the fermentate was combined with 3200 mL of autoclaved deionized water to make a 5-fold diluted fermentate.
  • Half of the diluted fermentate was transferred to autoclaved 450-mL centrifuge tubes and centrifuged at 8300 x g for 10 min to remove suspended solid.
  • the fermentate in which solid was removed (NSF) and the fermentate containing suspended solid (SF) were transferred (1425 mL each) into 2-L autoclaved baffled flasks.
  • the initial VFA concentrations were 5.63 g/L (NSF) and 6.18 g/L (SF).
  • the cultures in flasks were incubated on a shaker (190 rpm) at 30°C for 48 hours.
  • Optical densities at 600 nm were measured periodically along with solid contents, to monitor bacterial growth. Changes in PHA concentrations were monitored by converting PHB (the major component of PHA copolymer polyhydroxybutyrate-co-valerate (PHBV) synthesized by DEA01) to crotonic acid by heating ethanol-washed and dried cell pellet/solid mixtures in concentrated sulfuric acid at 100°C for 10-15 min and analyzing crotonic acid concentrations by HPLC (column: Sepax Carbomix H-NP5:8%, 7.8 x 50 mm; mobile phase: 18 mM sulfuric acid in water; flow rate: 1 mL/min; temperature: 60°C; sample injection: 7 ⁇ ; detection: UV at 210 nm; elution time: 2.32 min).
  • PHB the major component of PHA copolymer polyhydroxybutyrate-co-valerate (PHBV) synthesized by DEA01
  • HPLC columnumn: Sepax Carbomix
  • the final solid/cell weights (at 48 hours) of NSF and SF cultures were 1.63 and 12.6 g/L, with 27.9 % and 6.2 % PHB contents per solid (PHB concentrations were 455 mg/L (NSF) and 750 mg/L (SF), assuming 100 % conversion of PHB to crotonic acid), respectively.
  • a portion of the organic layer was placed in vials for analysis by GC (Agilent 6890 with FID detection; column: Agilent Innowax, 180 ⁇ ID x 20 m long x 0.18 um film thickness; injection: 2 ⁇ , 50: 1 split ratio; carrier gas: helium; flow rate: 1.1 mL/min; temperature program: ramped from an initial 35°C to 240°C at 60°C/min with a final hold for two minutes.
  • PHBV formed methyl- 3 -hydroxybutyrate and methyl-3-hydroxyvalerate, and the elution times were 2.54 and 2.75 min, respectively.
  • Quantitation was performed by comparing the peak areas of methyl-3- hydroxybutyrate and methyl-3-hydroxyvalerate from the digested polymer to peak areas from standards which contained approximately 30 mg of each of the two methyl esters and which were taken through the same process. To convert the observed quantities of methyl ester back into PHA there was a gravimetric factor required. The observed quantity of methyl-3- hydroxybutyrate multiplied by 86/118 provides the amount of PHB and the observed quantity of methyl- 3 -hydroxy valerate multiplied by 100/132 provides the amount of PHV from the sample.
  • NSF NSF
  • SF 35%

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Abstract

La présente invention concerne un procédé d'extraction et de purification de polyhydroxyalcanoates (PHA) contenus dans des cellules bactériennes, au moyen d'un système de solvants contenant un solvant d'extraction de PHA et un solvant de purification de PHA.
PCT/US2012/048374 2011-07-26 2012-07-26 Procédés d'extraction et de purification de polyhydroxyalcanoates contenus dans des cellules bactériennes Ceased WO2013016558A1 (fr)

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CN106687502A (zh) * 2014-06-03 2017-05-17 布拉格化学与技术大学 从产生聚羟基烷酸酯(pha)的微生物发酵的生物质和/或从包含至少一种产生聚羟基烷酸酯的作物的生物质分离聚羟基烷酸酯(pha)的方法
CN112608463A (zh) * 2020-11-25 2021-04-06 珠海麦得发生物科技股份有限公司 一种调控脂肪族聚酯的分子量的方法

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US3044942A (en) * 1960-09-27 1962-07-17 Grace W R & Co Process for preparing poly-beta-hydroxybutyric acid
EP0036699A1 (fr) * 1979-02-21 1981-09-30 Imperial Chemical Industries Plc Extraction de l'acide poly-bêta-hydroxybutyrique
EP0124309A2 (fr) * 1983-04-28 1984-11-07 Imperial Chemical Industries Plc Procédé d'extraction
EP0479043A1 (fr) * 1990-10-05 1992-04-08 PCD-Polymere Gesellschaft m.b.H. Procédé de production d'un polyhydroxyalcanoate à partir du matériel cellulaire d'un micro-organisme
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US3044942A (en) * 1960-09-27 1962-07-17 Grace W R & Co Process for preparing poly-beta-hydroxybutyric acid
EP0036699A1 (fr) * 1979-02-21 1981-09-30 Imperial Chemical Industries Plc Extraction de l'acide poly-bêta-hydroxybutyrique
EP0124309A2 (fr) * 1983-04-28 1984-11-07 Imperial Chemical Industries Plc Procédé d'extraction
EP0479043A1 (fr) * 1990-10-05 1992-04-08 PCD-Polymere Gesellschaft m.b.H. Procédé de production d'un polyhydroxyalcanoate à partir du matériel cellulaire d'un micro-organisme
WO1999051760A1 (fr) * 1998-04-08 1999-10-14 Metabolix, Inc. Procedes pour la separation et la purification des biopolymeres

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106687502A (zh) * 2014-06-03 2017-05-17 布拉格化学与技术大学 从产生聚羟基烷酸酯(pha)的微生物发酵的生物质和/或从包含至少一种产生聚羟基烷酸酯的作物的生物质分离聚羟基烷酸酯(pha)的方法
CZ307015B6 (cs) * 2014-06-03 2017-11-15 Nafigate Corporation, A.S. Způsob izolace polyhydroxyalkanoátů z biomasy fermentované mikroorganismy produkujícími polyhydroxyalkanoáty a/nebo z biomasy obsahující alespoň jednu plodinu produkující polyhydroxyalkanoáty
US9944748B2 (en) 2014-06-03 2018-04-17 Vysoka Skola Chemicko-Tech-Nologicka V Praze Method of isolation of polyhydroxyalkanoates (PHAs) from biomass fermented by microorganisms producing polyhydroxyalkanoates (PHAs) and/or from biomass containing at least one crop-plant producing polyhydroxyalkanoates
CN106687502B (zh) * 2014-06-03 2019-09-24 布拉格化学与技术大学 分离pha的方法
CN112608463A (zh) * 2020-11-25 2021-04-06 珠海麦得发生物科技股份有限公司 一种调控脂肪族聚酯的分子量的方法

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