US20010006802A1 - Methods for separation and purification of biopolymers - Google Patents
Methods for separation and purification of biopolymers Download PDFInfo
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- US20010006802A1 US20010006802A1 US09/288,832 US28883299A US2001006802A1 US 20010006802 A1 US20010006802 A1 US 20010006802A1 US 28883299 A US28883299 A US 28883299A US 2001006802 A1 US2001006802 A1 US 2001006802A1
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- pha
- ozone
- biomass
- poly
- hydroxybutyrate
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- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000000746 purification Methods 0.000 title abstract description 14
- 238000000926 separation method Methods 0.000 title abstract description 5
- 229920001222 biopolymer Polymers 0.000 title 1
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims abstract description 110
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 claims abstract description 109
- 208000037534 Progressive hemifacial atrophy Diseases 0.000 claims abstract description 74
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims abstract description 71
- 238000011282 treatment Methods 0.000 claims abstract description 43
- 239000002028 Biomass Substances 0.000 claims abstract description 37
- 239000004816 latex Substances 0.000 claims abstract description 23
- 229920000126 latex Polymers 0.000 claims abstract description 23
- 239000002002 slurry Substances 0.000 claims abstract description 13
- 239000000725 suspension Substances 0.000 claims abstract description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 19
- 239000003960 organic solvent Substances 0.000 claims description 12
- 229920000070 poly-3-hydroxybutyrate Polymers 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000000354 decomposition reaction Methods 0.000 claims description 5
- 230000000813 microbial effect Effects 0.000 claims description 5
- 239000004094 surface-active agent Substances 0.000 claims description 5
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 claims description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
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- 239000003125 aqueous solvent Substances 0.000 claims description 3
- 239000002270 dispersing agent Substances 0.000 claims description 3
- 239000003995 emulsifying agent Substances 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- ALRHLSYJTWAHJZ-UHFFFAOYSA-M 3-hydroxypropionate Chemical compound OCCC([O-])=O ALRHLSYJTWAHJZ-UHFFFAOYSA-M 0.000 claims description 2
- PHOJOSOUIAQEDH-UHFFFAOYSA-N 5-hydroxypentanoic acid Chemical compound OCCCCC(O)=O PHOJOSOUIAQEDH-UHFFFAOYSA-N 0.000 claims description 2
- IWHLYPDWHHPVAA-UHFFFAOYSA-M 6-hydroxyhexanoate Chemical compound OCCCCCC([O-])=O IWHLYPDWHHPVAA-UHFFFAOYSA-M 0.000 claims description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims description 2
- 229930188620 butyrolactone Natural products 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims description 2
- 239000013538 functional additive Substances 0.000 claims description 2
- 229920002791 poly-4-hydroxybutyrate Polymers 0.000 claims description 2
- 238000007669 thermal treatment Methods 0.000 claims description 2
- 150000004072 triols Chemical class 0.000 claims description 2
- 238000012017 passive hemagglutination assay Methods 0.000 claims 22
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims 2
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical group ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 claims 1
- 239000003139 biocide Substances 0.000 claims 1
- 238000000502 dialysis Methods 0.000 claims 1
- 150000001451 organic peroxides Chemical class 0.000 claims 1
- 238000000638 solvent extraction Methods 0.000 claims 1
- 150000003627 tricarboxylic acid derivatives Chemical class 0.000 claims 1
- UBOXGVDOUJQMTN-UHFFFAOYSA-N trichloroethylene Natural products ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 claims 1
- 239000004034 viscosity adjusting agent Substances 0.000 claims 1
- 229920000642 polymer Polymers 0.000 abstract description 33
- 238000000605 extraction Methods 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 6
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- 210000004027 cell Anatomy 0.000 description 13
- 239000007787 solid Substances 0.000 description 12
- 239000012535 impurity Substances 0.000 description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 238000011035 continuous diafiltration Methods 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- 150000002978 peroxides Chemical class 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 235000019645 odor Nutrition 0.000 description 8
- 238000006385 ozonation reaction Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 6
- 238000013019 agitation Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012467 final product Substances 0.000 description 5
- 239000004922 lacquer Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 102000016943 Muramidase Human genes 0.000 description 4
- 108010014251 Muramidase Proteins 0.000 description 4
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 4
- 229960000274 lysozyme Drugs 0.000 description 4
- 239000004325 lysozyme Substances 0.000 description 4
- 235000010335 lysozyme Nutrition 0.000 description 4
- 229960003330 pentetic acid Drugs 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 3
- 238000004061 bleaching Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000004332 deodorization Methods 0.000 description 3
- 239000006260 foam Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005063 solubilization Methods 0.000 description 3
- 230000007928 solubilization Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229920001410 Microfiber Polymers 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000589776 Pseudomonas putida Species 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 210000000991 chicken egg Anatomy 0.000 description 2
- 229910001882 dioxygen Inorganic materials 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000005188 flotation Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000009897 hydrogen peroxide bleaching Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003658 microfiber Substances 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
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- 229920000728 polyester Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011085 pressure filtration Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- -1 tricarboxylic acid esters Chemical class 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- NDPLAKGOSZHTPH-SSDOTTSWSA-N (R)-3-hydroxyoctanoic acid Chemical compound CCCCC[C@@H](O)CC(O)=O NDPLAKGOSZHTPH-SSDOTTSWSA-N 0.000 description 1
- WHBMMWSBFZVSSR-UHFFFAOYSA-M 3-hydroxybutyrate Chemical compound CC(O)CC([O-])=O WHBMMWSBFZVSSR-UHFFFAOYSA-M 0.000 description 1
- NDPLAKGOSZHTPH-UHFFFAOYSA-N 3-hydroxyoctanoic acid Chemical compound CCCCCC(O)CC(O)=O NDPLAKGOSZHTPH-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000589781 Pseudomonas oleovorans Species 0.000 description 1
- WHBMMWSBFZVSSR-UHFFFAOYSA-N R3HBA Natural products CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 229920006125 amorphous polymer Polymers 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000011968 cross flow microfiltration Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
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- 230000009089 cytolysis Effects 0.000 description 1
- 230000001877 deodorizing effect Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 239000004088 foaming agent Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000001465 metallisation Methods 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000002926 oxygen Chemical class 0.000 description 1
- 229940097156 peroxyl Drugs 0.000 description 1
- 229920000307 polymer substrate Polymers 0.000 description 1
- 239000012255 powdered metal Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
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- 230000009261 transgenic effect Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/88—Post-polymerisation treatment
- C08G63/90—Purification; Drying
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/88—Post-polymerisation treatment
- C08G63/89—Recovery of the polymer
Definitions
- the present invention is generally in the field of separation processes for polymers, and more specifically to purification of polymers derived from biological sources.
- PHAs Polyhydroxyalkanoates
- thermoplastic polyesters which can be produced from bacteria or plants (Williams & Peoples, CHEMTECH 26:33-44 (1996)). These polymers can be recovered from the biological systems (the biomass) by organic solvent processes, aqueous processes, or a combination of both organic solvent/aqueous processing. Examples of known organic solvent recovery processes are described in U.S. Pat. No. 4,310,684 and U.S. Pat. No. 4,705,604 to Vanlautem et al. (extraction of PHB from microbes with chlorinated solvents); U.S. Pat. No. 4,968,611 to Traussnig et al.
- EP 0 145 233 discloses aqueous methods for purifying a microbiological 3-hydroxybutyrate polymer wherein the cells are heat-treated at temperatures above 80° C. and then digested with enzymes, surfactants, and/or hydrogen peroxide.
- U.S. Pat. No. 5,110,980 to Ramsey et al. describes the use of hypochlorite for dissolving non-PHA biomass.
- U.S. Pat. No. 5,691,174 to Liddell et al. discloses a method for purifying microbial polyesters using the combination of hydrogen peroxide with a chelating agent.
- PCT WO 94/02541 describes additives to mask odor arising from the PHAs produced, for example, as described in EP 0 145 233 and U.S. Pat. No. 5,691,174 to Liddell et al. It would be advantageous to eliminate the odor associated with PHAs recovered from biomass, particularly if odor removal could be performed during the recovery process, thereby eliminating the need for odor-masking additives in the PHA resin.
- Methods are provided for the recovery and purification of polyhydroxyalkanoates (PHAs) from PHA-containing plant and microbial biomass, wherein the methods include contacting the biomass or partially purified PHA with ozone in at least one step of a purification process.
- Ozone has the beneficial effects of (a) bleaching, (b) deodorization, and (c) solubilization of impurities, facilitating their removal from aqueous polymer suspensions or latexes.
- the ozone treatment may be used alone or in combination with other treatment, extraction, and separation processes, and is especially suitable for the treatment of PHA-containing latexes, slurries, suspensions, and organic solvent solutions.
- the ozone contacting step advantageously can be conducted over a wide range of temperatures, including processing temperatures, for example between about 1 and 40° C., which are lower than processing temperatures used in known methods.
- Treatment with ozone of PHA-containing biomass, partially purified PHA, or solvent-extracted PHA yields an enhanced level of polymer purity suitable for coating and other applications.
- the ozone treatment also has the added advantage that the resulting PHA polymer or polymer latex is essentially odor-free.
- PHAs polyhydroxyalkanoates
- the methods include treating the biomass or partially purified PHA with ozone, in at least one step of a purification process.
- Ozone treatment can be beneficially applied to PHAs from any source, including PHAs extracted using organic solvents (e.g., by treating an organic solvent solution of the PHAs), in order to further purify and/or deodorize the PHAs.
- PHAs can be produced in a number of biological systems including bacteria and genetically engineered plant crops. In bacterial systems, the PHAs are accumulated intracellularly as granular inclusion bodies. PHA also can be produced in genetically engineered plant crops. Methods for constructing such crops are described, for example, in U.S. Pat. Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; U.S. Pat. No. 5,502,273 to Bright et al.; U.S. Pat. No. 5,534,432 to Peoples and Sinskey; U.S. Pat. No. 5,602,321 to John; U.S. Pat. No.
- the PHA-containing biomass derived from bacteria or plants which is to be used in the methods described herein typically is in the form of a polymer slurry, latex, or solution.
- the polymer slurry, latex, or solution preferably has a solids content between about 1 and 90% by weight, and more preferably between about 5 and 50% by weight.
- PHAs include poly-3-hydroxybutyrate (PHB), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV, marketed as BIOPOLTM by Monsanto), poly-3-hydroxybutyrate-co-4-hydroxybutyratepoly-3-hydroxypropionate, poly-3-hydroxybutyrate-co-3-hydroxypropionate, poly-4-hydroxybutyrate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate, poly-5-hydroxyvalerate, and poly-6-hydroxyhexanoate.
- activated oxygen is an allotropic form of oxygen, and is one of the strongest known oxidants. It may be generated by any convenient method, such as corona discharge or UV irradiation, applied to an air, oxygen, or oxygen
- ozone is applied to the PHA-containing biomass or solution in an oxygen (O 2 ) stream containing between about 1 and 10%, and more preferably between about 2 and 5%, ozone.
- O 2 oxygen
- Ozone advantageously is active at ambient temperatures and can be generated economically in situ from air or oxygen.
- the ozone preferably is generated at the PHA extraction site.
- the PHA-containing biomass (of microbial, plant, or other origin) is treated with ozone for purposes of bleaching, sterilization, solubilization/oxidation of impurities, and/or deodorization.
- the ozone-containing gas stream may be introduced in the polymer-containing fluid phase by sparging, nozzle-injection, or other means selected to provide efficient mass transfer of the ozone.
- the treatment with ozone is effective at any stage of a PHA isolation process for PHAs, which may comprise one or more treatment and/or separation steps.
- ozone may be used individually or in combination with other operations such as thermal treatment, enzyme treatment (e.g., nuclease, protease, or glycosidase), mechanical treatment (e.g., homogenizer or Microfluidizer), chemical treatment (e.g., surfactant, hypochlorite, or peroxide), centrifugation, filtration, and/or flotation.
- enzyme treatment e.g., nuclease, protease, or glycosidase
- mechanical treatment e.g., homogenizer or Microfluidizer
- chemical treatment e.g., surfactant, hypochlorite, or peroxide
- centrifugation filtration, and/or flotation.
- Ozone has a strong bleaching and deodorizing effect at any point of such a treatment process; however, for maximum efficacy
- Ozone treatment has a strong whitening and deodorization effect when applied at any of various stages of a PHA isolation process.
- the ozone can be applied (a) to a whole cell aqueous slurry, (b) to a crude polymer aqueous slurry following homogenization, lysozyme/protease treatment, and washing, (c) to a crude polymer aqueous slurry following homogenization, lysozyme/protease treatment, washing, hydrogen peroxide bleaching, and washing, and (d) to an organic solvent solution containing PHA generated, for example, using a process described in U.S. Pat. No. 4,310,684 and U.S. Pat. No.
- Ozone treatment can be performed in the presence of various functional additives useful for the stabilization and/or purification of PHA latexes and slurries.
- additives include surfactants, detergents, emulsifiers, dispersants, anti- or de-foaming agents, pH control agents, and chelators.
- the pH of the suspension generally is not vital for successful ozone treatment. Nonetheless, a pH approximately neutral is desirable to maintain a stable polymer suspension and/or to precipitation of soluble impurities.
- Foam formation may be a side effect of ozone treatment, due to the introduction of a largely insoluble gas stream into the polymer suspension. Since foaming can be disadvantageous for the production of stable, uniform latexes, it typically is necessary to add emulsifiers/dispersants and antifoaming agents immediately prior to ozone treatment.
- the ozone is applied in as concentrated a form as practical (e.g., a stream of having greater than 1% O 3 in oxygen/air), in order to minimize foam formation from the inert carrier gas.
- ozone treatment is applied in combination with hydrogen peroxide bleaching of PHA-containing biomass.
- ozone preferentially targets impurities containing unsaturated and/or aromatic rings, whereas hydrogen peroxide reacts via a peroxyl anion with electrophilic species. The effect is to render the impurities more highly water soluble (and hence easier to separate from the polymer suspensions by filtration or centrifugation) and less highly colored or odoriferous.
- ozone when applied prior to a peroxide-based treatment of the polymer suspension, usefully prolongs the lifetime of the peroxide in solution and increases the efficacy of the peroxide treatment.
- the quantity of ozone to utilize depends on the desired effect upon the final product, as well as the form of the PHA-containing biomass which is treated.
- a water-based slurry, suspension, or latex may require a relatively high dose of ozone for essentially complete decoloration, for example, between about 0.02 and 0.1 g ozone per gram of PHA-containing biomass.
- a relatively pure homogeneous solution of PHA in an organic solvent may be treated to reduce odor and color with a more modest dose, for example between about 0.002 and 0.01 g ozone per gram of solute.
- Slurries or latexes of PHA treated with ozone may be useful for any of the various applications for which PHAs are proposed, such as biodegradable objects; food coatings and additives; cathode-ray tube (CRT) lacquers; ceramic and powdered metal binders; biomedical microspheres, implants, and devices; and melt processed articles, such as described in U.S. Pat. No. 5,525,658 to Liddell et al.
- CTR cathode-ray tube
- a particularly suitable application for ozone-treated PHA latex is as a CRT lacquer.
- the lacquer is used to form a smooth substrate over the surface of the CRT phosphor particles allowing the subsequent deposition of a reflective aluminum layer, followed by thermal volatilization of the polymer substrate (PCT WO 96/17369).
- This application relies upon the ability of pure PHAs to decompose thermally and volatilize at temperatures of approximately 300° C.
- a high level of purification is necessary to use PHA latexes in this application, as cellular residues such as protein and nucleic acid may leave a charred residue on the CRT panel after baking out, resulting in poor picture quality.
- the usefulness of a PHA latex for application as a CRT lacquer may be judged in part from an ash analysis conducted at 450° C. in air atmosphere, conditions which approximate those used in CRT metallization.
- Ozone-treated latexes, and especially those treated with the combination of ozone followed by hydrogen peroxide, have exceptionally low ash contents upon baking out and are well suited to CRT lacquer use.
- compositions and methods of preparation and use thereof described herein are further described by the following non-limiting examples.
- the lysate was digested with (a) chicken egg lysozyme (Sigma Chemical Co., 0.2 g/L, pH 7.0, 45° C., 1 hr, 20 mM EDTA added); (b) ALCALASETM (Novo Nordisk, 1 mL/L, pH 7.5, 50° C., 2 hr); and (c) FLAVOURZYMETM (Novo Nordisk, 1 g/L, pH 7.2, 50° C., 8 hr). ALCALASETM and FLAVOURZYMETM digestions were conducted in the presence of 0.5% BRIJTM 721 (ICI Surfactants). Following FLAVOURZYMETM treatment, the PHA sample was washed by constant volume diafiltration with water containing 0.25% BRIJTM 721 and then concentrated to a volume of 16 L.
- chicken egg lysozyme Sigma Chemical Co., 0.2 g/L, pH 7.0, 45° C., 1 hr, 20 mM EDTA
- the washed latex was then further treated with hydrogen peroxide, 3% wt/vol, at 80° C., with the addition of 0.5% BRIJTM 721 and 0.01% DTPA (diethylenetriamine pentaacetic acid), until essentially all peroxide was exhausted (15 hr).
- BRIJTM 721 and 0.01% DTPA diethylenetriamine pentaacetic acid
- the product was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJTM 721.
- the final product from washing contained 0.8% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- Example 3 The product derived as in Example 3 was sparged with an ozone-containing oxygen stream (4 standard cubic feet per hour, 3-4% ozone) for 8 hr at room temperature, pH 7, with vigorous agitation. Following ozonation, the latex was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJTM 721. The final product from washing contained 0.9% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- ozone-containing oxygen stream 4 standard cubic feet per hour, 3-4% ozone
- the lysate was digested with chicken egg lysozyme (0.2 g/L, pH 7.0, 45° C., 1 hr, 20 mM EDTA added), followed by ALCALASETM (1 mL/L, pH 7.5, 50° C., 10 hr, 0.3% BRIJTM 721 added). Following ALCALASETM treatment, the sample was washed by constant volume diafiltration with water containing 0.1% BRIJTM 721.
- BRIJTM 721 was added to 0.2% wt/vol, and then the latex was sparged with an ozone-containing oxygen stream (4 standard cubic feet per hour, 3-4% ozone) for 10 hr at room temperature, pH 7, with vigorous agitation. Following ozonation, the latex was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.1% BRIJTM 721.
- the washed latex was then further treated with hydrogen peroxide, 3% wt/vol, at 80°C., with the addition of 0.15% BRIJTM 721 and 0.01% DTPA, until essentially all peroxide was exhausted (18 hr).
- the product was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.1% BRIJTM 721 (9 vol.) followed by water (2 vol.), and then concentrated to a solids content of 10.7%.
- the final product contained 0.05% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- the extract (26% wt/vol solids) then was sparged with an ozone-containing oxygen gas stream (8 standard cubic feet per hour, ca. 2% ozone) for 15 min at room temperature with agitation.
- a portion of ozonated extract (0.6 L) then was passed through a column of silica gel (ca. 60 g), and the colorless eluate (26% wt/vol solids) was collected (A 273 0.063, 1:9 dilution in hexane).
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Abstract
Description
- Priority is claimed to U.S. provisional application Ser. No. 60/081,112, filed Apr. 8, 1998.
- The present invention is generally in the field of separation processes for polymers, and more specifically to purification of polymers derived from biological sources.
- Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters which can be produced from bacteria or plants (Williams & Peoples,CHEMTECH 26:33-44 (1996)). These polymers can be recovered from the biological systems (the biomass) by organic solvent processes, aqueous processes, or a combination of both organic solvent/aqueous processing. Examples of known organic solvent recovery processes are described in U.S. Pat. No. 4,310,684 and U.S. Pat. No. 4,705,604 to Vanlautem et al. (extraction of PHB from microbes with chlorinated solvents); U.S. Pat. No. 4,968,611 to Traussnig et al. (use of diols, acetalized triols, di- or tricarboxylic acid esters or butyrolactone to extract poly-3-hydroxybutyrate (PHB) and its copolymers from microbes); U.S. Pat. No. 5,213,976 to Blauhut et al. (process for extracting PHB from microbial cells using methylene chloride followed by precipitation of the PHB in water); PCT WO 97/15681; PCT WO 93/11656 (use of acetone to extract poly-3-hydroxyoctanoate polymer from Pseudomonas oleovorans); PCT WO 96/06179 and PCT WO 97/15681 (solvent methods for recovering PHAs from transgenic plant crops); and U.S. Pat. No. 5,821,299 to Noda (the use of solvent/partial non-solvent mixtures for extracting PHAs from biomass). Typically, in each of these prior art processes, some of the biomass components are co-extracted with the PHA, which can cause the PHA product to be discolored and/or to have an unpleasant odor.
- In some cases, it is useful to extract the PHAs from biomass using aqueous processing techniques, in which the polymer remains in a microparticulate state and the non-PHA biomass is solubilized through mechanical, chemical, and/or enzymatic treatments. The PHA particles then are separated from the solubilized material using centrifugation, filtration, flotation, or other known methods.
- EP 0 145 233, for example, discloses aqueous methods for purifying a microbiological 3-hydroxybutyrate polymer wherein the cells are heat-treated at temperatures above 80° C. and then digested with enzymes, surfactants, and/or hydrogen peroxide. U.S. Pat. No. 5,110,980 to Ramsey et al. describes the use of hypochlorite for dissolving non-PHA biomass. U.S. Pat. No. 5,691,174 to Liddell et al. discloses a method for purifying microbial polyesters using the combination of hydrogen peroxide with a chelating agent. The disadvantage of these methods, however, is that the peroxide treatment must be carried out at high temperatures, e.g., 80-180° C., which requires extensive heating and cooling of the product and, in some cases, requires high pressure equipment. Furthermore, the hydrogen peroxide frequently is found to be unstable in the presence of high levels of cellular biomass, which results in the hydrogen peroxide decomposing nonproductively to water and oxygen and generating voluminous foam. Another drawback is that the prolonged high temperatures and hydrogen peroxide also can cause a decrease in the molecular weight of the polymers, and in some cases can promote crystallization of the polymers, which is undesirable for the production of an amorphous polymer latex.
- As discussed above, PHAs derived from biomass can have unpleasant odors. PCT WO 94/02541 describes additives to mask odor arising from the PHAs produced, for example, as described in EP 0 145 233 and U.S. Pat. No. 5,691,174 to Liddell et al. It would be advantageous to eliminate the odor associated with PHAs recovered from biomass, particularly if odor removal could be performed during the recovery process, thereby eliminating the need for odor-masking additives in the PHA resin.
- It is therefore an object of the present invention to provide improved methods of purifying polyhydroxyalkanoates derived from biomass.
- It is a further object of the present invention to enhance extraction of polyhydroxyalkanoates from biomass containing polyhydroxyalkanoates using organic solvent systems, aqueous solvent systems, or combinations thereof.
- It is another object of the present invention to provide extraction methods which reduce the exposure of the PHA polymers to conditions which can decrease the molecular weight of the polymers.
- It is a further object of the present invention to eliminate odors in the process of recovering polyhydroxyalkanoates from biomass.
- Methods are provided for the recovery and purification of polyhydroxyalkanoates (PHAs) from PHA-containing plant and microbial biomass, wherein the methods include contacting the biomass or partially purified PHA with ozone in at least one step of a purification process. Ozone has the beneficial effects of (a) bleaching, (b) deodorization, and (c) solubilization of impurities, facilitating their removal from aqueous polymer suspensions or latexes. The ozone treatment may be used alone or in combination with other treatment, extraction, and separation processes, and is especially suitable for the treatment of PHA-containing latexes, slurries, suspensions, and organic solvent solutions. The ozone contacting step advantageously can be conducted over a wide range of temperatures, including processing temperatures, for example between about 1 and 40° C., which are lower than processing temperatures used in known methods. Treatment with ozone of PHA-containing biomass, partially purified PHA, or solvent-extracted PHA yields an enhanced level of polymer purity suitable for coating and other applications. The ozone treatment also has the added advantage that the resulting PHA polymer or polymer latex is essentially odor-free.
- Improved methods for the recovery of polyhydroxyalkanoates (PHAs) from biomass including PHAs have been developed. The methods include treating the biomass or partially purified PHA with ozone, in at least one step of a purification process. Ozone treatment can be beneficially applied to PHAs from any source, including PHAs extracted using organic solvents (e.g., by treating an organic solvent solution of the PHAs), in order to further purify and/or deodorize the PHAs.
- Polyhydroxyalkanoates
- PHAs can be produced in a number of biological systems including bacteria and genetically engineered plant crops. In bacterial systems, the PHAs are accumulated intracellularly as granular inclusion bodies. PHA also can be produced in genetically engineered plant crops. Methods for constructing such crops are described, for example, in U.S. Pat. Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; U.S. Pat. No. 5,502,273 to Bright et al.; U.S. Pat. No. 5,534,432 to Peoples and Sinskey; U.S. Pat. No. 5,602,321 to John; U.S. Pat. No. 5,610,041 to Somerville et al.; PCT WO 91/00917; PCT WO 92/19747; PCT WO 93/02187; PCT WO 93/02194; PCT WO 94/12014, Poirier et al.,Science 256:520-23 (1992); van der Leij & Witholt, Can. J Microbiol. 41(supp.):222-38 (1995); Nawrath & Poirier, The International Symposium on Bacterial Polyhydroxyalkanoates, (Eggink et al., eds.) Davos Switzerland (Aug. 18-23, 1996); and Williams & Peoples, CHEMTECH 26: 38-44 (1996). Methods for recovering PHAs from plant biomass are described, for example in PCT WO 97/15681, PCT WO 97/07239, and PCT WO 97/07229.
- The PHA-containing biomass derived from bacteria or plants which is to be used in the methods described herein typically is in the form of a polymer slurry, latex, or solution. The polymer slurry, latex, or solution preferably has a solids content between about 1 and 90% by weight, and more preferably between about 5 and 50% by weight.
- The ozone purification methods described herein similarly are useful for purification of other PHAs, regardless of source organism or comonomer composition. Representative PHAs include poly-3-hydroxybutyrate (PHB), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV, marketed as BIOPOL™ by Monsanto), poly-3-hydroxybutyrate-co-4-hydroxybutyratepoly-3-hydroxypropionate, poly-3-hydroxybutyrate-co-3-hydroxypropionate, poly-4-hydroxybutyrate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate, poly-5-hydroxyvalerate, and poly-6-hydroxyhexanoate.
- Ozone
- Ozone (O3), also referred to as “activated oxygen”, is an allotropic form of oxygen, and is one of the strongest known oxidants. It may be generated by any convenient method, such as corona discharge or UV irradiation, applied to an air, oxygen, or oxygen-enriched air stream, as described for example in U.S. Pat. No. 5,855,856 to Karlson and U.S. Pat. No. 5,766,560 to Cole (corona discharge) and in U.S. Pat. No. 4,517,084 to Pincon, U.S. Pat. No. 4,329,212, U.S. Pat. No. 4,427,636 to Obenshain, U.S. Pat. No. 4,317,044 to Vaseen (UV irradiation).
- Preferably, ozone is applied to the PHA-containing biomass or solution in an oxygen (O2) stream containing between about 1 and 10%, and more preferably between about 2 and 5%, ozone.
- Ozone advantageously is active at ambient temperatures and can be generated economically in situ from air or oxygen. The ozone preferably is generated at the PHA extraction site.
- PHA Purification Methods Using Ozone
- In a preferred embodiment, the PHA-containing biomass (of microbial, plant, or other origin) is treated with ozone for purposes of bleaching, sterilization, solubilization/oxidation of impurities, and/or deodorization. The ozone-containing gas stream may be introduced in the polymer-containing fluid phase by sparging, nozzle-injection, or other means selected to provide efficient mass transfer of the ozone.
- The treatment with ozone is effective at any stage of a PHA isolation process for PHAs, which may comprise one or more treatment and/or separation steps. In an aqueous process, ozone may be used individually or in combination with other operations such as thermal treatment, enzyme treatment (e.g., nuclease, protease, or glycosidase), mechanical treatment (e.g., homogenizer or Microfluidizer), chemical treatment (e.g., surfactant, hypochlorite, or peroxide), centrifugation, filtration, and/or flotation. Ozone has a strong bleaching and deodorizing effect at any point of such a treatment process; however, for maximum efficacy, it may be desirable for the ozone treatment to be effected subsequent to lysis and partial removal of the non-PHA biomass.
- Ozone treatment has a strong whitening and deodorization effect when applied at any of various stages of a PHA isolation process. For example, the ozone can be applied (a) to a whole cell aqueous slurry, (b) to a crude polymer aqueous slurry following homogenization, lysozyme/protease treatment, and washing, (c) to a crude polymer aqueous slurry following homogenization, lysozyme/protease treatment, washing, hydrogen peroxide bleaching, and washing, and (d) to an organic solvent solution containing PHA generated, for example, using a process described in U.S. Pat. No. 4,310,684 and U.S. Pat. No. 4,705,604 to Vanlautem et al.; U.S. Pat. No. 4,968,611 to Traussnig et al; U.S. Pat. No. 5,213,976 to Blauhut et al.; U.S. Pat. No. 5,821,299 to Noda; PCT WO 93/11656; PCT WO 96/06179; or PCT WO 97/15681. Following ozone treatment, the polymer containing slurry can be purified by microfiltration with water washing. The ozone effects significant solubilization of non-PHA material, as evidenced by a decrease in ash content. (PHA polymers volatilize cleanly at approximately 300°C., whereas other cellular contents, such as DNA and protein, produce ash near this temperature).
- Ozone treatment can be performed in the presence of various functional additives useful for the stabilization and/or purification of PHA latexes and slurries. Examples of these additives include surfactants, detergents, emulsifiers, dispersants, anti- or de-foaming agents, pH control agents, and chelators. For aqueous polymer suspensions, the pH of the suspension generally is not vital for successful ozone treatment. Nonetheless, a pH approximately neutral is desirable to maintain a stable polymer suspension and/or to precipitation of soluble impurities.
- Foam formation may be a side effect of ozone treatment, due to the introduction of a largely insoluble gas stream into the polymer suspension. Since foaming can be disadvantageous for the production of stable, uniform latexes, it typically is necessary to add emulsifiers/dispersants and antifoaming agents immediately prior to ozone treatment. Preferably, the ozone is applied in as concentrated a form as practical (e.g., a stream of having greater than 1% O3 in oxygen/air), in order to minimize foam formation from the inert carrier gas.
- In a preferred embodiment, ozone treatment is applied in combination with hydrogen peroxide bleaching of PHA-containing biomass. Without being bound by the theory, it appears that ozone preferentially targets impurities containing unsaturated and/or aromatic rings, whereas hydrogen peroxide reacts via a peroxyl anion with electrophilic species. The effect is to render the impurities more highly water soluble (and hence easier to separate from the polymer suspensions by filtration or centrifugation) and less highly colored or odoriferous. Additionally, ozone, when applied prior to a peroxide-based treatment of the polymer suspension, usefully prolongs the lifetime of the peroxide in solution and increases the efficacy of the peroxide treatment.
- The quantity of ozone to utilize depends on the desired effect upon the final product, as well as the form of the PHA-containing biomass which is treated. For example, a water-based slurry, suspension, or latex may require a relatively high dose of ozone for essentially complete decoloration, for example, between about 0.02 and 0.1 g ozone per gram of PHA-containing biomass. A relatively pure homogeneous solution of PHA in an organic solvent, however, may be treated to reduce odor and color with a more modest dose, for example between about 0.002 and 0.01 g ozone per gram of solute.
- Applications Using the Purified PHAs
- Improving PHA recovery and purification methods is highly advantageous, as PHAs can be processed for use in an enormous variety of applications. It is evident that ozone may be especially useful for those applications where high purity, sterility, low odor, or low ash residue are important. In some cases, the purified latex or slurry will itself be useful as a product, while in other cases it may be advantageous to isolate the ozonated polymer in dry form for shaping and processing.
- Slurries or latexes of PHA treated with ozone may be useful for any of the various applications for which PHAs are proposed, such as biodegradable objects; food coatings and additives; cathode-ray tube (CRT) lacquers; ceramic and powdered metal binders; biomedical microspheres, implants, and devices; and melt processed articles, such as described in U.S. Pat. No. 5,525,658 to Liddell et al.
- A particularly suitable application for ozone-treated PHA latex is as a CRT lacquer. The lacquer is used to form a smooth substrate over the surface of the CRT phosphor particles allowing the subsequent deposition of a reflective aluminum layer, followed by thermal volatilization of the polymer substrate (PCT WO 96/17369). This application relies upon the ability of pure PHAs to decompose thermally and volatilize at temperatures of approximately 300° C. A high level of purification is necessary to use PHA latexes in this application, as cellular residues such as protein and nucleic acid may leave a charred residue on the CRT panel after baking out, resulting in poor picture quality. The usefulness of a PHA latex for application as a CRT lacquer may be judged in part from an ash analysis conducted at 450° C. in air atmosphere, conditions which approximate those used in CRT metallization. Ozone-treated latexes, and especially those treated with the combination of ozone followed by hydrogen peroxide, have exceptionally low ash contents upon baking out and are well suited to CRT lacquer use.
- The compositions and methods of preparation and use thereof described herein are further described by the following non-limiting examples.
- Cells ofPseudomonas putida were fermented to a density of 76 g/L by growth on octanoic acid in minimal mineral salts medium. Under these conditions, the cells typically accumulate between 30 and 60% PHA on a cell dry weight basis, with R-3-hydroxyoctanoic acid as the principal comonomer. The sample, consisting of 48 L, was washed by constant volume diafiltration with deionized water, using a ceramic cross-flow microfiltration apparatus (Niro Inc.). Commercial nuclease then was added (BENZONASE™, available from American International Chemical, 10 μL/L of cells), the pH was adjusted to 10 with ammonia, and the cells were disrupted by twice passing the sample through a Microfluidizer M-110EH (Microfluidics International Corp.) at an operating pressure of 15,000 psi. The lysate was digested with (a) chicken egg lysozyme (Sigma Chemical Co., 0.2 g/L, pH 7.0, 45° C., 1 hr, 20 mM EDTA added); (b) ALCALASE™ (Novo Nordisk, 1 mL/L, pH 7.5, 50° C., 2 hr); and (c) FLAVOURZYME™ (Novo Nordisk, 1 g/L, pH 7.2, 50° C., 8 hr). ALCALASE™ and FLAVOURZYME™ digestions were conducted in the presence of 0.5% BRIJ™ 721 (ICI Surfactants). Following FLAVOURZYME™ treatment, the PHA sample was washed by constant volume diafiltration with water containing 0.25% BRIJ™ 721 and then concentrated to a volume of 16 L.
- Half of the PHA crude latex from Example 1 (8 L) was diluted 1:1 with deionized water. Then, BRIJ™721 was added to 0.5% wt/vol, and the latex was sparged with an ozone-containing oxygen stream (4 standard cubic feet per hour, 3-4% ozone) for 8 hr at room temperature, pH 7, with vigorous agitation. Following ozonation, the latex was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJ™ 721.
- The washed latex was then further treated with hydrogen peroxide, 3% wt/vol, at 80° C., with the addition of 0.5% BRIJ™ 721 and 0.01% DTPA (diethylenetriamine pentaacetic acid), until essentially all peroxide was exhausted (15 hr). The product was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJ™ 721. The final product from washing contained 0.8% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- Half of the PHA crude latex from Example 1 (8 L) was diluted 1:1 with deionized water. BRIJ™ 721 (0.5%) and DTPA (0.01%) were added, and the latex was then treated with hydrogen peroxide, 3% wt/vol, at 80° C., until essentially all peroxide was exhausted (7 hr). The product was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJ™ 721. The final product from washing contained 4.6% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- The product derived as in Example 3 was sparged with an ozone-containing oxygen stream (4 standard cubic feet per hour, 3-4% ozone) for 8 hr at room temperature, pH 7, with vigorous agitation. Following ozonation, the latex was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.25% BRIJ™ 721. The final product from washing contained 0.9% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- Cells ofPseudomona sputida were fermented to density of 80 g/L by growth on octanoic acid in minimal mineral salts medium. The sample, consisting of 46 L, was washed by constant volume diafiltration with deionized water and concentrated to a volume of 20 L. Commercial nuclease then was added (BENZONASE™, 10 μL/L of cells), the pH was adjusted to 10 with ammonia, and the cells were disrupted by twice passing the sample through a Microfluidizer M-110EH (Microfluidics International Corp.) at an operating pressure of 15,000 psi. The lysate was digested with chicken egg lysozyme (0.2 g/L, pH 7.0, 45° C., 1 hr, 20 mM EDTA added), followed by ALCALASE™ (1 mL/L, pH 7.5, 50° C., 10 hr, 0.3% BRIJ™ 721 added). Following ALCALASE™ treatment, the sample was washed by constant volume diafiltration with water containing 0.1% BRIJ™ 721.
- BRIJ™ 721 was added to 0.2% wt/vol, and then the latex was sparged with an ozone-containing oxygen stream (4 standard cubic feet per hour, 3-4% ozone) for 10 hr at room temperature, pH 7, with vigorous agitation. Following ozonation, the latex was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.1% BRIJ™ 721.
- The washed latex was then further treated with hydrogen peroxide, 3% wt/vol, at 80°C., with the addition of 0.15% BRIJ™ 721 and 0.01% DTPA, until essentially all peroxide was exhausted (18 hr). The product was washed to remove solubilized impurities by constant volume diafiltration with water containing 0.1% BRIJ™ 721 (9 vol.) followed by water (2 vol.), and then concentrated to a solids content of 10.7%. The final product contained 0.05% ash (wt/wt of solids basis) following polymer decomposition at 450°C. in air.
- Dry samples (0.2-1.0 g) were heated in air to 450°C. at a rate of 10° C./min, in quartz or aluminum vessels. The furnace temperature was maintained at 450°C. for a period of 45 min, after which the samples were allowed to cool and the unvolatilized residue weighed. Liquid samples containing water (solids content 0.2-1.0 g) were heated in air to 150°C. at a rate of 10° C./min, and then maintained at 150°C. for 30 min to evaporate the water. The temperature then was increased to 450°C. at a rate of 10°C./min, and maintained at 450°C. for a period of 45 min. The samples then were allowed to cool. The unvolatilized residue measured and reported as a percentage of the weight of solids present in the original sample.
- Cells ofP. putida fermented, as described above, on octanoic acid were collected by centrifugation and freeze-dried to give a solid mass. The mass (440 g) was pulverized in a mill and placed in the thimble of a Soxhlet extractor. The dried cells were extracted in two batches for a total of 16 hr with n-hexane (2 L), yielding an amber extract (A273=0.457, 1:9 dilution in hexane) which contained 26% (wt/vol) solids. The extract was clarified by pressure filtration through a 2 μm glass microfiber filter. The extract (26% wt/vol solids) then was sparged with an ozone-containing oxygen gas stream (8 standard cubic feet per hour, ca. 2% ozone) for 15 min at room temperature with agitation. The resulting product was a clear, virtually colorless liquid (A273=0.152, 1:9 dilution in hexane). A portion of ozonated extract (0.6 L) then was passed through a column of silica gel (ca. 60 g), and the colorless eluate (26% wt/vol solids) was collected (A2730.063, 1:9 dilution in hexane).
- Solutions of poly-3-hydroxybutyrate (PHB) (Fluka) and poly-3-hydroxybutyrate-co-14%-3-hydroxyvalerate (PHBV) (Aldrich) were prepared at 5% (wt/vol) in dichloromethane. The solutions were clarified by pressure filtration through a 2.6-μm glass microfiber filter. Each solution (95 mL) was then sparged with an ozone-containing oxygen gas stream (8 standard cubic feet per hour, ca. 2% ozone) for 6 min at room temperature with agitation. The products were clear, virtually colorless solutions. The PHB solution after ozonation showed an A273=0.714 (1:9 dilution in dichloromethane) compared to A273=0.904 (1:9 dilution in dichloromethane) prior to ozonation. Similarly, the PHBV solution after ozonation showed an A273=0.599 (1:9 dilution in dichloromethane) compared to A273=1.029 (1:9 dilution in dichloromethane) prior to ozonation.
- Modifications and variations of the present invention will be obvious to those of skill in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.
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1999
- 1999-04-08 DE DE69942060T patent/DE69942060D1/en not_active Expired - Lifetime
- 1999-04-08 MX MXPA00009754A patent/MXPA00009754A/en active IP Right Grant
- 1999-04-08 WO PCT/US1999/007689 patent/WO1999051760A1/en active IP Right Grant
- 1999-04-08 JP JP2000542471A patent/JP2002510747A/en active Pending
- 1999-04-08 AT AT99916498T patent/ATE458822T1/en not_active IP Right Cessation
- 1999-04-08 ES ES99916498T patent/ES2339725T3/en not_active Expired - Lifetime
- 1999-04-08 AU AU34808/99A patent/AU757682B2/en not_active Ceased
- 1999-04-08 US US09/288,832 patent/US6368836B2/en not_active Expired - Lifetime
- 1999-04-08 EP EP99916498A patent/EP1070135B1/en not_active Expired - Lifetime
- 1999-04-08 CA CA002327086A patent/CA2327086A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8450448B2 (en) | 2004-08-31 | 2013-05-28 | Riken | Thermostable biopolyester |
CN114409886A (en) * | 2022-01-26 | 2022-04-29 | 珠海麦得发生物科技股份有限公司 | Purification method of polyhydroxy fatty acid ester |
Also Published As
Publication number | Publication date |
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JP2002510747A (en) | 2002-04-09 |
ES2339725T3 (en) | 2010-05-24 |
AU757682B2 (en) | 2003-02-27 |
AU3480899A (en) | 1999-10-25 |
US6368836B2 (en) | 2002-04-09 |
WO1999051760A1 (en) | 1999-10-14 |
ATE458822T1 (en) | 2010-03-15 |
EP1070135B1 (en) | 2010-02-24 |
DE69942060D1 (en) | 2010-04-08 |
CA2327086A1 (en) | 1999-10-14 |
MXPA00009754A (en) | 2002-05-08 |
EP1070135A1 (en) | 2001-01-24 |
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