CN109504715A - A method of preparing polyhydroxyalkanoate (PHA) - Google Patents
A method of preparing polyhydroxyalkanoate (PHA) Download PDFInfo
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- CN109504715A CN109504715A CN201710840777.2A CN201710840777A CN109504715A CN 109504715 A CN109504715 A CN 109504715A CN 201710840777 A CN201710840777 A CN 201710840777A CN 109504715 A CN109504715 A CN 109504715A
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- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title claims abstract description 109
- 238000000034 method Methods 0.000 title claims abstract description 84
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims abstract description 83
- 238000000855 fermentation Methods 0.000 claims abstract description 55
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- 241000894006 Bacteria Species 0.000 claims abstract description 50
- 239000007788 liquid Substances 0.000 claims abstract description 49
- 238000005406 washing Methods 0.000 claims abstract description 39
- 230000008569 process Effects 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 239000000463 material Substances 0.000 claims abstract description 9
- 210000002421 cell wall Anatomy 0.000 claims abstract description 7
- 238000005336 cracking Methods 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 241000206596 Halomonas Species 0.000 claims description 25
- 238000001035 drying Methods 0.000 claims description 12
- 239000012141 concentrate Substances 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 9
- 239000003945 anionic surfactant Substances 0.000 claims description 7
- 229920001634 Copolyester Polymers 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 6
- 230000000996 additive effect Effects 0.000 claims description 6
- 229920000070 poly-3-hydroxybutyrate Polymers 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- REKYPYSUBKSCAT-UHFFFAOYSA-N 3-hydroxypentanoic acid Chemical compound CCC(O)CC(O)=O REKYPYSUBKSCAT-UHFFFAOYSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 claims description 4
- 238000001694 spray drying Methods 0.000 claims description 4
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 claims description 3
- 229940006015 4-hydroxybutyric acid Drugs 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 241000203069 Archaea Species 0.000 claims description 2
- 241000205035 Halobacteriaceae Species 0.000 claims description 2
- 241001135694 Halomonadaceae Species 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- AFENDNXGAFYKQO-UHFFFAOYSA-N 2-hydroxybutyric acid Chemical compound CCC(O)C(O)=O AFENDNXGAFYKQO-UHFFFAOYSA-N 0.000 claims 1
- 241000247079 Bacteroidales bacterium Species 0.000 claims 1
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 40
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 238000004519 manufacturing process Methods 0.000 description 16
- 239000012452 mother liquor Substances 0.000 description 14
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 12
- 239000005015 poly(hydroxybutyrate) Substances 0.000 description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 11
- 238000000605 extraction Methods 0.000 description 11
- 229910052760 oxygen Inorganic materials 0.000 description 11
- 239000001301 oxygen Substances 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000009423 ventilation Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000002028 Biomass Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000005265 energy consumption Methods 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 230000003519 ventilatory effect Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- -1 for example Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 238000004065 wastewater treatment Methods 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 2
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 2
- 238000005660 chlorination reaction Methods 0.000 description 2
- 229910017052 cobalt Inorganic materials 0.000 description 2
- 239000010941 cobalt Substances 0.000 description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229960002413 ferric citrate Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 2
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 2
- 238000011169 microbiological contamination Methods 0.000 description 2
- SPIFDSWFDKNERT-UHFFFAOYSA-N nickel;hydrate Chemical compound O.[Ni] SPIFDSWFDKNERT-UHFFFAOYSA-N 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011684 sodium molybdate Substances 0.000 description 2
- 235000015393 sodium molybdate Nutrition 0.000 description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 239000011686 zinc sulphate Substances 0.000 description 2
- 235000009529 zinc sulphate Nutrition 0.000 description 2
- JRHWHSJDIILJAT-UHFFFAOYSA-N 2-hydroxypentanoic acid Chemical compound CCCC(O)C(O)=O JRHWHSJDIILJAT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001037822 Bacillus bacterium Species 0.000 description 1
- 241000252867 Cupriavidus metallidurans Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000005259 measurement Methods 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
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 229920001013 poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Invention provides a kind of methods for preparing polyhydroxyalkanoate (PHA), method includes the following steps: (1) prepares Halophiles fermentation liquid and obtains concentration bacterium solution by being separated by solid-liquid separation；(2) the concentration bacterium solution obtained to step (1) is washed；(3) bacterium solution after the washing obtained to step (2) carries out cell wall breaking walls and cracking to obtain shell-broken liquid；(4) the non-PHA material in the shell-broken liquid that removal step (3) obtains, collects PHA；(5) PHA is purified；(6) dry PHA.This method process warm and, it is low in cost, it is low for equipment requirements, it can be achieved that large-scale industrial production.
Description
Technical field
The invention belongs to bioengineering downstreams to post-process field, and in particular, to a kind of to prepare polyhydroxyalkanoate
(PHA) method.
Background technique
Polyhydroxyalkanoate (polyhydroxyalkanoates, abbreviation PHA) be microorganism in other nutrition limitation and
A kind of shelf stability particulate matter synthesized under the conditions of carbon source is superfluous, is a kind of boiomacromolecule petchem.PHA has ordinary hot
The characteristic of thermoplastic plastic, but traditional petrochemical industry plastics are different from, PHA has good biocompatibility, degradability, pressure
Electrical property and optical activity have broad application prospects it in the fields such as industry, agricultural, medical and health, food, degradable
Agricultural mulching field and degradation plastic articles and medical domain, PHA have been shown as biodegradable material best at present
Reveal huge potentiality.
Traditional PHA production is with the Escherichia coli (E.coli) of the true oxygen bacillus (R.eutropha) of Roche, genetic modification
It is formed for production strain fermentation, that there are microbiological contamination probabilities is high for production, energy consumption is high, the substrate transformation rate is not high, downstream processing is at high cost etc.
Disadvantage causes cost to remain high for a long time, seriously hinders the large-scale application of PHA.
Using Halophiles as the PHA production technology that chassis bacterium is developed overcome traditional PHA production present in multiple problems, it is first
First Halophiles is grown under high alkali environment with high salt, and this growth conditions can inhibit the growth of most microorganisms, reaction
In production be exactly it is open type fermented without sterilizing, not only will not microbiological contamination, but also without sterilizing, greatly reduce energy consumption and management at
This.Secondly, by the engineering bacteria of synthetic biology technological development can inexpensive carbon source efficiently synthesize poly- 3-hydroxybutyrate and 3-
The multiple performances such as hydroxypentanoic acid copolyesters (PHBV), poly- 3-hydroxybutyrate and 4 hydroxybutyric acid copolyesters (P3HB4HB) are excellent
PHA product has further speeded up commercial applications speed.
For PHA as bacterium content, ingredient intracellular is considerably complicated, and extraction difficulty is quite big, in the very long course of PHA development
In, researchers have done a large amount of research to extraction.Existing extraction process include method of organic solvent extraction, mechanical crushing method,
Sodium hypochlorite-surfactant method, enzyme process etc..The shortcomings that organic solvent method is difficult solvent recovery, and production environment is dangerous, only suitable
Laboratory is closed to extract；Mechanical crushing method mainly includes supersonic wave wall breaking or high-pressure homogenization broken wall, and energy consumption is high, and amplification difficulty is big；
The shortcomings that sodium hypochlorite-surfactant method is that sodium hypochlorite irritation is strong, and working environment is poor, is destroyed seriously to PHA molecular weight,
And the wastewater treatment difficulty containing surfactant is big；The shortcomings that enzyme process is the enzyme that use a variety of valuableness, and cost is very high.
Therefore develop a kind of reaction condition it is mild, to equipment without particular/special requirement, raw material is cheap, is suitble to the side of industrialization amplification
Method can substantially reduce production cost, accelerate the commercial applications of PHA.The patent of invention of publication number CN02112208 discloses
A method of can effectively reduce PHA separation and Extraction cost, this method first carries out broken wall to bacterium with physical method, then uses lye
It adjusts pH to alkalinity, in the pretreatment fluid of alkalinity, is added anionic surfactant and flocculating agent, in separation and Extraction treatment fluid
Precipitating, is washed out drying, obtains finished product.This method reaction condition is mild, and equipment is simple, but is carried out brokenly using physical method
Wall, energy consumption is higher, and a large amount of surfactant is added, and wastewater treatment is complicated, and is washed with a large amount of clear water, sewage row
It is high-volume big.
The patent of invention of publication number CN98100266 discloses the method for separation and Extraction PHA out of microorganism a kind of, packet
It includes following steps: 1) handling thallus with the alkaline solution containing surfactant；2) it is separated by solid-liquid separation, separates most of non-
PHA ingredient；3) basic protein enzymatic treatment PHA is used；4) separation and Extraction PHA particle；5) it is dried to obtain PHA product.This method reaction
Mild condition, cost is relatively low, but alkali consumption is big, and surfactant additive amount is big, and needs to add alkali protease, extraly
Cost is increased, and only laboratory process, does not consider industrialization amplification.
A big advantage of Halophiles production PHA is extraction process simplicity, after fermentation liquid desalination, can use inside and outside osmotic pressure
Difference is diluted with water broken wall, a small amount of lye and surfactant can also be added at high temperature to realize quick broken wall, accelerate reaction speed
Rate improves product purity.
Summary of the invention
The present inventor, in conjunction with the self-characteristic of Halophiles (for example, Halomonas), develops on the basis of previous work
It is a kind of efficiently, low cost, the PHA extraction process of high-purity, which is suitble to a variety of PHA products, for example, PHB, PHBV,
P3HB4HB etc..1m of the present invention in laboratory 7.5L fermentation level and pilot scale3Fermentor and 5m3Fermentation level has all carried out largely
Experiment has very strong industrialization amplification value.
The object of the present invention is to provide a kind of method for preparing polyhydroxyalkanoate (PHA), this method is from Halophiles
In fermentation liquid high efficiency, low cost extraction PHA process, this method process warm and, it is low in cost, it is low for equipment requirements, can
Realize large-scale industrial production.
Therefore, invention provides a kind of method for preparing polyhydroxyalkanoate (PHA), this method includes following step
It is rapid:
(1) it prepares Halophiles fermentation liquid and obtains concentration bacterium solution by being separated by solid-liquid separation；
(2) the concentration bacterium solution obtained to step (1) is washed；
(3) bacterium solution after the washing obtained to step (2) carries out cell wall breaking walls and cracking to obtain shell-broken liquid；
(4) the non-PHA material in the shell-broken liquid that removal step (3) obtains, collects PHA；
(5) PHA is purified；
(6) dry PHA.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that the PHA can produce for various PHA
Product, for example, polyhydroxybutyrate (PHB), poly- 3-hydroxybutyrate and 3- hydroxypentanoic acid copolyesters (PHBV), poly- 3-hydroxybutyrate and 4-
Hydroxybutyric acid copolyesters (P3HB4HB) etc..
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that Halophiles described in step (1)
It can be the Halophiles of halophilic archaea (halobacteriaceae), Halomonas (halomonadaceae) etc..
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (1), the separation of solid and liquid
Centrifugal process perhaps filtration method or flocculence, preferably centrifugal process can be used.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (2), washing for bacterium solution is concentrated
The method of washing can be to add water to be sufficiently stirred washing to the concentration bacterium solution after separation of solid and liquid, and centrifugation removes impurity, wherein amount of water can be
It is concentrated 1-10 times of bacterium solution.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (3), somatic cells wall is broken
Wall cleavage method can comprise the following steps that
A. the Halomonas bacterium solution rehydration after washing concentrating creates hypotonic environment；
B. bacterium solution temperature is adjusted to most suitable broken wall temperature；
C. the pH of bacterium solution is adjusted to optimal pH；
D. anionic surfactant is added.
Preferably, in above-mentioned steps a, the volume after Halomonas bacterium solution rehydration after washing concentrating is concentration bacterium solution volume
1-10 times.
Preferably, in above-mentioned steps b, bacterium solution broken wall required temperature can be 30 DEG C -120 DEG C, preferably 40 DEG C -100 DEG C, more
Preferably 60 DEG C -90 DEG C.
Preferably, in above-mentioned steps c, optimal pH needed for bacterium solution broken wall can be 8.5-13.0, preferably 9.0-12.0, more
Preferably 9.5-11.5.
Preferably, in above-mentioned steps d, anionic surfactant needed for bacterium solution broken wall can be dodecyl sodium sulfate,
Abbreviation SDS.
Preferably, in above-mentioned steps d, anionic surfactant needed for bacterium solution broken wall (for example, SDS) additive amount can be
The 0%-5% (w/v) of broken wall bacterium solution volume, preferably 0.2%-1.0% (w/v).
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (3), needed for bacterium solution broken wall
Time can be 0.5h-2h, preferably 0.5h-1.5h.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (3), bacterium solution broken wall process
In can be used higher revolving speed (preferably 2000rpm-20000rpm) centrifugation, to promote the cracking of cell wall.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (4), the separation
The method of PHA and non-PHA material can be centrifugal process, filtration method or flocculence, preferably centrifugal process.The non-PHA material packet
It includes but is not limited only to, the impurity such as cell fragment, protein, nucleic acid, NaCl, SDS.Preferably, the centrifugal process use from
Scheming can be low speed centrifuge or supercentrifuge, preferably supercentrifuge, and the separating factor of centrifuge is 2000-
20000, preferably 4000-20000, preferably 5000-20000.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (5), the PHA's
Purification process can are as follows: water is added to the PHA concentrate for removing non-PHA material, washing is sufficiently stirred, is then centrifuged for removal washing
Liquid repeats this process 2-3 times, the purifying of PHA can be completed.In above-mentioned PHA purification process, PHA concentrate adds water washing mistake
Cheng Zhong, amount of water can be 1-10 times, preferably 1-5 times of concentrate volume.
In the method for preparing polyhydroxyalkanoate (PHA) of the invention, it is preferable that in step (6), the PHA is dry
Drying method can be for room temperature aeration-drying, heating aeration-drying, vacuum freeze drying, spray drying, the separation of solid and liquid with upstream
Effect is related.
PHA extraction process of the invention is not high to the PHA content requirement of thallus, and due to Halophiles itself is unique can be
The voluntarily physiological property of broken wall under hypotonic environment, the broken wall condition of thallus is compared with traditional broken wall condition, and broken wall pH is lower, table
The additive amount of face activating agent is lower, or even under some purposes, surfactant can be not added, and reduces wastewater treatment difficulty.
The method provided by the invention that PHA is extracted from Halomonas halomonas TD bacterial strain fermentation liquor is completed from reality
Room 7.5L fermentor is tested to 1m3Fermentor pilot plant test arrives 5m again3The a series of experiments of Showcase Production Line, practicability, science,
Industrialization amplification value has obtained sufficient verifying.
The present invention has the advantage that compared with existing purifying technique
(1) technique is that work is extracted in the industrialization in the world for the first time for Halophiles production polyhydroxyalkanoate exploitation
Skill is the core technology of blue aquatic organism technology, has important market value；
(2) the technique overall process does not use any organic solvent, and production environment is simple and safe；
(3) extract equipment is simple, and slightly transformation can put into production on the basis of existing fermentation plant；It is former needed for extracting
Expect it is cheap, it is from a wealth of sources.
Specific embodiment
Embodiment 1: PHA is extracted from Halomonas fermented liquid under laboratory condition
One, [deposit number: CGMCC No.4353, in patent application, (patent is public by Halomonas halomonas TD01
Announcement number: authorization in CN102120973B)] using glucose as carbon source through fermentation production PHB, ferment 48h, biomass 83g/L, PHB
Content 80%.
Fermentation medium:
Sodium chloride 40g/L, glucose 30g/L, corn starch 10g/L, urea 2g/L, magnesium sulfate 0.2g/L, biphosphate
Potassium 4.2g/L, microelement mother liquor I 10mL/L, microelement mother liquor II 1mL/L.
1L microelement mother liquor I: 5g ferric citrate and 2g CALCIUM CHLORIDE DIHYDRATE are mixed with 0.5mol/L aqueous hydrochloric acid solution
It closes, complements to 1 liter with 0.5mol/L aqueous hydrochloric acid solution until completely dissolved.
1L microelement mother liquor II: by white vitriol 100mg, tetrahydrate manganese chloride 30mg, boric acid 300mg, six water chlorinations
Cobalt 200mg/L, cupric sulfate pentahydrate 10mg, six water nickel chloride 20mg, sodium molybdate 30mg and 0.5mol/L combined, to
1 liter is complemented to 0.5mol/L aqueous hydrochloric acid solution after being completely dissolved.
Fermentation culture conditions:
Secondary seed solution is linked into the fermentation medium after above-mentioned optimization by 10% inoculum concentration, at the beginning of 7.5L fermentor
Begin dress liquid 3L, the unsterilised direct fermentation of fermentation system.37 DEG C of temperature of control, initial dissolved oxygen control pass through adjusting in 30%-50%
Revolving speed and ventilation control dissolved oxygen, maximum (top) speed 800rpm, Maximum Ventilatory Volume 1.5vvm, after revolving speed and ventilation reach maximum, no
Control dissolved oxygen；In fermentation process through control of additive raw material sugar concentration between 5-20g/L；Controlling fermentation pH with 5M NaOH is 8.5.
Extracting method:
1, the centrifugation and washing of thallus
Halomonas halomonas TD01 fermentation liquid 300mL is taken, is symmetrically placed in supercentrifuge after trim, item is centrifuged
Part are as follows: revolving speed 6000rpm, time 20min.After centrifugation, supernatant is carefully removed, then plus water restores volume, and stirring is equal
Continue to be centrifuged after even, centrifugal condition are as follows: 5000rpm, time 10min.After centrifugation, supernatant is discarded, thallus restores volume extremely
300mL, after mixing evenly, the bacterium solution after washing are poured into 500mL beaker, prepare broken wall.
2, the cell wall broken wall of bacterium solution
500mL beaker equipped with 300mL Halomonas halomonas TD01 bacterium solution is placed in 60 DEG C of band magnetic agitation water
In bath, after bacterium solution temperature rises to 60 DEG C, adjusting bacterium solution pH with 5M NaOH is 10.0, and 2g dodecyl sodium sulfate is added
(SDS), 60min is reacted.
3, the purifying of PHB
Broken wall after reaction, go unless PHA impurity, centrifugal condition are as follows: 4500rpm, 10min by centrifugation.After centrifugation,
Supernatant carefully is removed, adds water to restore volume and continues to be centrifuged after mixing evenly, washing centrifugally operated is repeated 2 times.Washing centrifugation
PHA and centrifuge tube adhesion afterwards is close, can discard supernatant completely, PHA solid content will not lose.
4, the drying of PHB
Centrifuge tube equipped with PHB sediment is placed in -80 DEG C of ultra low temperature freezers and is freezed 2 hours, it is cold to be subsequently placed in vacuum
It is lyophilized in lyophilizer, is uniformly mixed after PHB is ground after freeze-drying, measure PHB mass, content, molecular weight.Last products obtained therefrom is pure
Degree 98%, molecular weight 5 × 105Da, the rate of recovery 90%.
Two, Halomonas halomonas TD40 (strain discloses in following non-patent literature: Chen, X., Yin,
J.,Ye,J.,Zhang,H.,Che,X.,Ma,Y.,Li,M.,Wu,L-P.,Chen,G-Q.,Engineering Halomonas
bluephagenesis TD01 for Non-sterile Production of Poly(3-hydroxybutyrate-co-
4-hydroxybutyrate), (2017) Bioresource Technology, and by the limited public affairs of Beijing aquamaine microorganism science and technology
Department provides) P3HB4HB is produced using glucose and gamma-butyrolacton as carbon source through fermentation, ferment 48h, biomass 77g/L, P3HB4HB
Content 70%.
Fermentation medium:
Sodium chloride 60g/L, glucose 20g/L, corn starch 15g/L, urea 2g/L, magnesium sulfate 0.2g/L, biphosphate
Potassium 5g/L, microelement mother liquor I 10mL/L, microelement mother liquor II 2mL/L.
1L microelement mother liquor I: 5g ferric citrate and 2g CALCIUM CHLORIDE DIHYDRATE are mixed with 0.5mol/L aqueous hydrochloric acid solution
It closes, complements to 1 liter with 0.5mol/L aqueous hydrochloric acid solution until completely dissolved.
1L microelement mother liquor II: by white vitriol 100mg, tetrahydrate manganese chloride 30mg, boric acid 300mg, six water chlorinations
Cobalt 200mg/L, cupric sulfate pentahydrate 10mg, six water nickel chloride 20mg, sodium molybdate 30mg and 0.5mol/L combined, to
1 liter is complemented to 0.5mol/L aqueous hydrochloric acid solution after being completely dissolved.
Fermentation culture conditions:
Secondary seed solution is linked into the fermentation medium after above-mentioned optimization by 10% inoculum concentration, at the beginning of 7.5L fermentor
Begin dress liquid 3L, the unsterilised direct fermentation of fermentation system.37 DEG C of temperature of control, initial dissolved oxygen control pass through adjusting in 30%-50%
Revolving speed and ventilation control dissolved oxygen, maximum (top) speed 800rpm, Maximum Ventilatory Volume 1vvm, after revolving speed and ventilation reach maximum, do not control
Dissolved oxygen processed；Gamma-butyrolacton is added between 5-20g/L, in fermentation process to promote by control of additive raw material sugar concentration in fermentation process
Into the accumulation of 4HB；Controlling fermentation pH with 5M NaOH is 8.5.
Extracting method:
1, the centrifugation and washing of thallus
Halomonas halomonas TD40 fermentation liquid 300mL is taken, is symmetrically placed in supercentrifuge after trim, item is centrifuged
Part are as follows: revolving speed 6000rpm, time 20min.After centrifugation, supernatant is carefully removed, then plus water restores volume, and stirring is equal
Continue to be centrifuged after even, centrifugal condition are as follows: 5000rpm, time 10min.After centrifugation, supernatant is discarded, thallus restores volume extremely
300mL, after mixing evenly, the bacterium solution after washing are poured into 500ml beaker, prepare broken wall.
2, the cell wall broken wall of bacterium solution after Halomonas halomonas TD40 is washed
500mL beaker equipped with 300mL Halomonas halomonas TD40 bacterium solution is placed in 90 DEG C of band magnetic agitation water
In bath, after bacterium solution temperature rises to 90 DEG C, adjusting bacterium solution pH with 5M NaOH is 10.5, and 1.5g dodecyl sodium sulfate is added
(SDS), 90min is reacted.
3, the purifying of P3HB4HB
Broken wall after reaction, go unless PHA impurity, centrifugal condition are as follows: 4500rpm, 10min by centrifugation.After centrifugation,
Supernatant carefully is removed, adds water to restore volume and continues to be centrifuged after mixing evenly, washing centrifugally operated is repeated 2 times.Washing centrifugation
PHA and centrifuge tube adhesion afterwards is close, can discard supernatant completely, PHA solid content will not lose.
4, the drying of P3HB4HB
Centrifuge tube equipped with P3HB4HB sediment is placed in -80 DEG C of ultra low temperature freezers and is freezed 2 hours, is subsequently placed in true
It is lyophilized in vacuum freecing-dry machine, is uniformly mixed after PHB is ground after freeze-drying, measure PHB mass, content, molecular weight.Finally measure production
Product purity 95%, molecular weight 6.0 × 105Da, the rate of recovery 90%.
Embodiment 2: PHA is extracted from Halomonas fermentation liquid under pilot plant conditions
Fermentation process: using Halomonas halomonas TD40 as fermentation strain, using glucose and gamma-butyrolacton as carbon source
Fermenting and producing P3HB4HB, 1m3Ferment tank system is 700L, fermentation time 36h, biomass 89g/L, P3HB4HB content
65%.
Fermentation medium:
Sodium chloride 30g/L, glucose 30g/L, corn slurries 50mL/L, urea 2g/L, magnesium sulfate 0.8g/L, biphosphate
Potassium 4g/L, microelement mother liquor I 10mL/L, microelement mother liquor II 10mL/L.
Microelement mother liquor I and II preparation method is identical as 7.5L fermentation system in embodiment 1.
Fermentation culture conditions:
Secondary seed solution is linked into the fermentation medium after above-mentioned optimization by 10% inoculum concentration, 1000L fermentor
Initial dress liquid 600L, the unsterilised direct fermentation of fermentation system.37 DEG C of temperature, ventilatory capacity 1vvm, tank presses 0.05MPa, initial speed
100rpm, manually adjust the speed after dissolved oxygen is lower than 30%, maximum (top) speed 280rpm；Revolving speed and ventilation reach maximum, do not control molten
Oxygen.Concentration of glucose is controlled in fermentation process by way of fed-batch between 5-20g/L；By adding in fermentation process
Gamma-butyrolacton accumulates 4HB；It is 8.5 that 5M NaOH, which automatically adjusts pH,.
Extracting method:
1, the centrifugation of thallus
700 liters of Halomonas halomonasTD40 fermentation liquids are centrifuged with disc-type supercentrifuge, centrifuge speed
6000rpm, separating factor 8000, the specified treating capacity 500L/h of centrifuge.
2, the washing centrifugation of thallus
Concentration liquid after centrifugation is pumped into washing tank, adds water to 700 liters of graduation marks, and speed of agitator 150rpm fills
Divide and shake up, is then centrifuged with disc centrifuge, centrifugal condition is same as above.
3, Halomonas halomonas TD40 somatic cells wall broken wall
Concentration liquid after washing centrifugation is pumped into broken wall tank, and water is added to restore volume to 700L, speed of agitator
150rpm is sufficiently stirred evenly, and adjusting bacterium solution temperature is 80 DEG C, after temperature rises to 80 DEG C, is adjusted pH to 10.5 with 8M NaOH, is added 5kg
SDS, timing 2h, after broken wall, adjusting shell-broken liquid temperature is 30 DEG C, after temperature is down to 40 DEG C or less, with hydrochloric acid tune pH to 8.0
Below.
4, the separation of solid and liquid of shell-broken liquid
Shell-broken liquid is separated by solid-liquid separation using centrifugal process, and centrifuge used is disc centrifuge, and centrifuge treating capacity is
500L/h。
5, the washing purifying of PHA
The washing centrifugal method of PHA is roughly the same with the washing centrifugation of thallus, and the concentrate of every first-stage centrifugal, which is driven into, to be washed
It washs in tank, adds water to restore volume, stir, continue to be centrifuged, washing is centrifuged 2 PHA concentrates, uses tube centrifuge
Continue to be centrifuged, tube centrifuge revolving speed 20000rpm, separating factor 15000, PHA concentrate is after tube centrifuge is centrifuged in solid
Shape object state, it is similar to laboratory test effect.
6, the drying of PHA
The PHA block generated after tubular type centrifugation is placed in aeration-drying in 70 DEG C of baking ovens after being sufficiently crushed, completely dry
Measurement PHA yield, purity and molecular weight after dry.Final products obtained therefrom purity 90%, molecular weight 5.5 × 105Da, the rate of recovery
85%.
Embodiment 3: PHA is extracted from Halomonas fermentation liquid under industrialized condition
Fermentation process: using Halomonas halomonas TD40 as fermentation strain, using glucose and gamma-butyrolacton as carbon source
Fermenting and producing P3HB4HB, 5m3Ferment tank system is 4m3, fermentation time 36h, biomass 99g/L, P3HB4HB content
65%.
Fermentation medium:
Sodium chloride 30g/L, glucose 30g/L, corn slurries 30mL/L, urea 2g/L, magnesium sulfate 1.2g/L, biphosphate
Potassium 4g/L, microelement mother liquor I 10mL/L, microelement mother liquor II 10mL/L.
Microelement mother liquor I and II preparation method is identical as 7.5L fermentation system in embodiment 1.
Fermentation culture conditions:
Secondary seed solution is linked into the fermentation medium after above-mentioned optimization by 10% inoculum concentration, 5m3At the beginning of fermentor
Begin dress liquid 3m3, the unsterilised direct fermentation of fermentation system.37 DEG C of temperature, ventilatory capacity 180m3/ h, tank press 0.05MPa, initial speed
90rpm, manually adjust the speed after dissolved oxygen is lower than 30%, maximum (top) speed 170rpm；After revolving speed and ventilation reach maximum, do not control molten
Oxygen.Concentration of glucose is controlled in fermentation process by way of fed-batch between 5-20g/L；By adding in fermentation process
Gamma-butyrolacton accumulates 4HB；It is 8.5 that 8M NaOH, which automatically adjusts pH,.
Extracting method:
1, the centrifugation of thallus
4m3Halomonas halomonas TD40 fermentation liquid is centrifuged with disc-type supercentrifuge, centrifuge speed
5500rpm, separating factor 8000, the specified treating capacity 2m of centrifuge3/h。
2, the washing centrifugation of thallus
Concentration liquid after centrifugation is pumped into washing tank, adds water to 4m3Graduation mark, speed of agitator 80rpm, sufficiently shakes
It is even, it is then centrifuged with disc centrifuge, centrifugal condition is same as above.
3, Halomonas halomonas TD40 somatic cells wall broken wall
Concentration liquid after washing centrifugation is pumped into broken wall tank, and water is added to restore volume to 4m3, speed of agitator 80rpm,
Sufficiently stir evenly, adjusting bacterium solution temperature is 90 DEG C, after temperature rises to 90 DEG C, pH to 10.5 is adjusted with 8M NaOH, adds 30kg SDS,
Timing 1.5h, after broken wall, adjusting shell-broken liquid temperature is 30 DEG C, after temperature is down to 40 DEG C or less, with hydrochloric acid tune pH to 8.0 with
Under.
4, the separation of solid and liquid of shell-broken liquid
Shell-broken liquid is separated by solid-liquid separation using centrifugal process, and centrifuge used is disc centrifuge, and centrifuge treating capacity is
2m3/h。
5, the washing purifying of PHA
The washing centrifugal method of PHA is roughly the same with the washing centrifugation of thallus, and the concentrate of every first-stage centrifugal, which is driven into, to be washed
It washs in tank, adds water to restore volume, stir, continue to be centrifuged, washing centrifugation is repeated 3 times.
6, the drying of PHA
The PHA volume of the concentrated liquid that afterbody centrifugation generates is 800L spray dryer spray drying, spray dryer evaporation
Amount is 50kg/h, PHA yield, purity and molecular weight after measuring spray drying.Final products obtained therefrom purity 85%, molecular weight 7.0 ×
105Da, the rate of recovery 80%.
Claims (10)
1. a kind of method for preparing polyhydroxyalkanoate (PHA), method includes the following steps:
(1) it prepares Halophiles fermentation liquid and obtains concentration bacterium solution by being separated by solid-liquid separation；
(2) the concentration bacterium solution obtained to step (1) is washed；
(3) bacterium solution after the washing obtained to step (2) carries out cell wall breaking walls and cracking to obtain shell-broken liquid；
(4) the non-PHA material in the shell-broken liquid that removal step (3) obtains, collects PHA；
(5) PHA is purified；
(6) dry PHA.
2. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein the PHA is selected from poly- hydroxyl
Butyric acid (PHB), poly- 3-hydroxybutyrate and 3- hydroxypentanoic acid copolyesters (PHBV), poly- 3-hydroxybutyrate and 4 hydroxybutyric acid copolyesters
(P3HB4HB)。
3. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein thermophilic described in step (1)
Salt bacterium is selected from halophilic archaea (halobacteriaceae) and Halomonas (halomonadaceae)；And/or in step (1),
Described be separated by solid-liquid separation uses centrifugal process perhaps filtration method or flocculence, preferably centrifugal process.
4. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein in step (2), bacterium is concentrated
The washing methods of liquid is to add water that washing, centrifugation removal impurity, wherein amount of water is sufficiently stirred to the concentration bacterium solution after separation of solid and liquid
For 1-10 times that bacterium solution is concentrated.
5. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein in step (3), thallus is thin
Cell wall breaking walls and cracking method the following steps are included:
A. the Halomonas bacterium solution rehydration after washing concentrating creates hypotonic environment；
B. bacterium solution temperature is adjusted to most suitable broken wall temperature；
C. the pH of bacterium solution is adjusted to optimal pH；
D. anionic surfactant is added.
6. the method according to claim 5 for preparing polyhydroxyalkanoate (PHA), wherein in above-mentioned steps a, washing
The volume after Halomonas bacterium solution rehydration after concentration is 1-10 times that bacterium solution volume is concentrated；And/or in above-mentioned steps b, bacterium solution
Broken wall required temperature is 30 DEG C -120 DEG C, preferably 40 DEG C -100 DEG C, more preferably 60 DEG C -90 DEG C；And/or above-mentioned steps c
In, optimal pH needed for bacterium solution broken wall is 8.5-13.0, preferably 9.0-12.0, more preferably 9.5-11.5；And/or it is above-mentioned
In step d, anionic surfactant needed for bacterium solution broken wall is dodecyl sodium sulfate, it is preferable that in step d, bacterium solution is broken
(for example, dodecyl sodium sulfate) additive amount of anionic surfactant needed for wall is the 0%-5% (w/ of broken wall bacterium solution volume
V), more preferably 0.2%-1.0% (w/v).
7. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein in step (3), bacterium solution is broken
It is 0.5h-2h, preferably 0.5h-1.5h the time required to wall；And/or in step (3), used during bacterium solution broken wall
The centrifugation of 2000rpm-20000rpm revolving speed.
8. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein described in step (4)
The method for separating PHA and non-PHA material is centrifugal process, filtration method or flocculence, preferably centrifugal process, it is preferable that it is described from
The centrifuge that heart method uses is low speed centrifuge or supercentrifuge, more preferably supercentrifuge, the separating factor of centrifuge
For 2000-20000, preferably 4000-20000, more preferably 5000-20000.
9. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein described in step (5)
The purification process of PHA are as follows: water is added to the PHA concentrate for removing non-PHA material, washing is sufficiently stirred, is then centrifuged for removal and washes
Liquid is washed, repeats this process 2-3 times, the purifying of PHA can be completed, it is preferable that during PHA concentrate adds water washing, amount of water
For 1-10 times of concentrate volume, more preferably 1-5 times.
10. the method according to claim 1 for preparing polyhydroxyalkanoate (PHA), wherein described in step (6)
PHA drying means is selected from room temperature aeration-drying, heating aeration-drying, vacuum freeze drying, spray drying.
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| CN111349218A (en) * | 2020-04-29 | 2020-06-30 | 吉林中粮生化有限公司 | Method for separating polyhydroxyalkanoate and polyhydroxyalkanoate prepared by same |
| CN111348766A (en) * | 2020-04-29 | 2020-06-30 | 吉林中粮生化有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using membrane filtration and application of obtained fermentation waste liquor |
| CN111362445A (en) * | 2020-04-29 | 2020-07-03 | 吉林中粮生化有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using adsorbent and application of obtained fermentation waste liquor |
| CN111363126A (en) * | 2020-04-29 | 2020-07-03 | 南京钛净流体技术有限公司 | Ceramic membrane reactor and method for extracting polyhydroxyalkanoate by using ceramic membrane reactor |
| CN111348766B (en) * | 2020-04-29 | 2020-12-11 | 吉林中粮生化有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using membrane filtration and application of obtained fermentation waste liquor |
| CN111362445B (en) * | 2020-04-29 | 2021-01-05 | 吉林中粮生化有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using adsorbent and application of obtained fermentation waste liquor |
| CN111363126B (en) * | 2020-04-29 | 2021-02-02 | 南京钛净流体技术有限公司 | Ceramic membrane reactor and method for extracting polyhydroxyalkanoate by using ceramic membrane reactor |
| CN111349218B (en) * | 2020-04-29 | 2021-02-09 | 吉林中粮生化有限公司 | Method for separating polyhydroxyalkanoate and polyhydroxyalkanoate prepared by same |
| US11203663B2 (en) | 2020-04-29 | 2021-12-21 | Cofco (Jilin) Bio-Chemical Technology Co., Ltd | Method for separating PHA and PHA prepared therefrom |
| CN111647151A (en) * | 2020-06-10 | 2020-09-11 | 珠海麦得发生物科技股份有限公司 | Efficient full-automatic secondary PHA purification process |
| US11155483B1 (en) | 2020-06-30 | 2021-10-26 | Nutrition & Health Research Institute, COFCO Corporation | Method for efficiently producing PHA |
| CN112552500A (en) * | 2021-01-04 | 2021-03-26 | 珠海麦得发生物科技股份有限公司 | Method for removing endotoxin in PHAs by fermentation method |
| CN112552500B (en) * | 2021-01-04 | 2022-12-20 | 珠海麦得发生物科技股份有限公司 | Method for removing endotoxin in PHAs by fermentation method |
| CN112813112A (en) * | 2021-01-07 | 2021-05-18 | 上海碧州环保能源科技有限公司 | Non-methanation process with PHA production as guide |
| CN113354802B (en) * | 2021-05-26 | 2022-04-22 | 清华大学 | High-purity extraction method of polyhydroxyalkanoate |
| CN113354802A (en) * | 2021-05-26 | 2021-09-07 | 清华大学 | High-purity extraction method of polyhydroxyalkanoate |
| CN113801810A (en) * | 2021-08-13 | 2021-12-17 | 珠海麦得发生物科技股份有限公司 | Halomonas strain and application thereof |
| CN113801810B (en) * | 2021-08-13 | 2022-06-24 | 珠海麦得发生物科技股份有限公司 | Halomonas strain and application thereof |
| CN114308409A (en) * | 2021-11-19 | 2022-04-12 | 中粮生物科技股份有限公司 | Method and system for separating polyhydroxyalkanoate by using horizontal spiral centrifugal separation |
| CN114277067B (en) * | 2021-12-27 | 2023-08-04 | 常州柯纳生物科技有限公司 | Method for producing PHA material by utilizing bast fibers |
| CN114277067A (en) * | 2021-12-27 | 2022-04-05 | 常州柯纳生物科技有限公司 | Method for producing PHA material by using bast fiber |
| CN115058461A (en) * | 2022-06-20 | 2022-09-16 | 宁波天安生物材料有限公司 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor |
| CN115058461B (en) * | 2022-06-20 | 2024-05-28 | 宁波天安生物材料有限公司 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation broth |
| CN115717157A (en) * | 2022-09-08 | 2023-02-28 | 常州柯纳生物科技有限公司 | PHA extraction method and equipment |
| CN115807044A (en) * | 2022-11-09 | 2023-03-17 | 华南理工大学 | Method for efficiently extracting and purifying high-purity polyhydroxyalkanoate |
| CN115807044B (en) * | 2022-11-09 | 2023-10-13 | 华南理工大学 | Method for efficiently extracting and purifying high-purity polyhydroxyalkanoate |
| CN115786411A (en) * | 2023-01-09 | 2023-03-14 | 北京微构工场生物技术有限公司 | Method for extracting polyhydroxyalkanoate |
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