CN115058461A - Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor - Google Patents
Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor Download PDFInfo
- Publication number
- CN115058461A CN115058461A CN202210697420.4A CN202210697420A CN115058461A CN 115058461 A CN115058461 A CN 115058461A CN 202210697420 A CN202210697420 A CN 202210697420A CN 115058461 A CN115058461 A CN 115058461A
- Authority
- CN
- China
- Prior art keywords
- polyhydroxyalkanoate
- purifying
- liquid
- directly separating
- sodium salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 60
- 229920000903 polyhydroxyalkanoate Polymers 0.000 title claims abstract description 60
- 238000000855 fermentation Methods 0.000 title claims abstract description 43
- 230000004151 fermentation Effects 0.000 title claims abstract description 43
- 239000007788 liquid Substances 0.000 claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 159000000000 sodium salts Chemical class 0.000 claims abstract description 28
- 238000003756 stirring Methods 0.000 claims abstract description 21
- 239000003945 anionic surfactant Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000002244 precipitate Substances 0.000 claims abstract description 14
- 238000005406 washing Methods 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 239000003513 alkali Substances 0.000 claims abstract description 9
- 210000002421 cell wall Anatomy 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 230000007935 neutral effect Effects 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 238000000926 separation method Methods 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 7
- 238000003828 vacuum filtration Methods 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- 229910001415 sodium ion Inorganic materials 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 239000000047 product Substances 0.000 abstract description 24
- 230000008569 process Effects 0.000 abstract description 21
- 239000004094 surface-active agent Substances 0.000 abstract description 10
- 238000009776 industrial production Methods 0.000 abstract description 6
- 238000000605 extraction Methods 0.000 description 14
- 238000001556 precipitation Methods 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 12
- 238000001816 cooling Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 241000252867 Cupriavidus metallidurans Species 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 5
- 239000005015 poly(hydroxybutyrate) Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241001052560 Thallis Species 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- 241000588986 Alcaligenes Species 0.000 description 2
- 241000589151 Azotobacter Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108091005658 Basic proteases Proteins 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000190967 Rhodospirillum Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229920000229 biodegradable polyester Polymers 0.000 description 1
- 239000004622 biodegradable polyester Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 101710108497 p-hydroxybenzoate hydroxylase Proteins 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
Images
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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/05—Alcaligenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/065—Azotobacter
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
Abstract
The invention discloses a method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor, belonging to the technical field of downstream post-treatment in bioengineering. The method comprises the following steps: (1) taking fermentation liquor of polyhydroxyalkanoate production bacteria, and performing cell wall breaking treatment to obtain wall-broken liquid; (2) adjusting the pH value of the wall breaking liquid to be alkaline by using alkali liquor; (3) adding sodium salt and anionic surfactant into the alkaline wall-breaking liquid, and stirring for reaction, wherein the order of adding the sodium salt and adding the anionic surfactant can be interchanged; (4) separating and collecting the precipitate in the reaction solution, washing the precipitate to be neutral by water, dehydrating and drying to obtain the polyhydroxyalkanoate. The invention adopts the process means of sodium salt and surfactant, the product is separated and purified under mild condition, the molecular shear is small, and the obtained product has the characteristics of high purity, high molecular weight and stable product quality. The method has the advantages of simple process, high production efficiency and low cost, and can realize large-scale industrial production.
Description
Technical Field
The invention relates to the technical field of downstream post-treatment in bioengineering, in particular to a method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor.
Background
Poly-beta-Hydroxyalkanoates (PHA) are a class of biopolyesters that are accumulated intracellularly by microorganisms under unbalanced growth conditions, and are typically represented by Polyhydroxybutyrate (PHB) and its copolymers (e.g., PHBV, PHBH, etc.), which have physical properties similar to polypropylene. Because of the characteristics of biodegradability, biocompatibility, edibility, piezoelectricity, nonlinear optical activity and the like which are not possessed by common petrochemical resins, the special biodegradable polyester resin has wide application prospects in the fields of industry, agriculture, medical treatment, sanitation, food, electronics and the like. However, to date, PHA has not been widely used on a large scale, primarily because the cost is still significantly higher than petrochemical resins. The production cost of PHA mainly includes raw material cost and separation and purification cost. The raw material cost depends on the production efficiency of the strains and the adopted fermentation process, and the separation and purification cost mainly depends on the adopted process.
The current extraction process is to separate the cell from the fermentation liquor by a high-speed centrifuge, and then purify PHA in the separated wet thallus, and the method generally adopts organic solvent extraction, chemical reagent method, surfactant + enzyme method and the like. These methods are either costly or highly polluting and difficult to achieve in industrial production.
The patent application of the invention with publication number CN1800235A discloses a method for extracting high-purity PHAs (polyhydroxyalkanoates) from wet thalli, which comprises the steps of firstly concentrating fermentation liquor to collect thalli, dissolving macromolecules by adopting an organic solvent, separating residues, concentrating organic phase and separating to obtain PHA. The method has the advantages of large usage amount of organic solvent, high safety requirement of equipment, difficult solvent recovery, and organic solvent leakage risk, is only suitable for extraction test in a laboratory, and is not suitable for industrial production.
The invention patent of publication No. CN1070534C discloses a method for separating and extracting PHA from bacterial cells, comprising the following steps: 1) treating the cells with an alkaline solution containing a surfactant; 2) solid-liquid separation, most non-PHA components are separated; 3) treating the PHA with an alkaline protease; 4) separating and extracting PHA grains; 5) drying to obtain PHA product. The method has mild reaction conditions and low cost, but has large alkali consumption, large addition amount of the surfactant, and needs to add the alkaline protease, thereby additionally increasing the cost, and is only a laboratory process without considering the industrial amplification.
The invention patent publication No. CN109504715A discloses a method for producing Polyhydroxyalkanoate (PHA), which comprises the steps of: (1) preparing halophilic bacteria fermentation liquor and obtaining concentrated bacterial liquid through solid-liquid separation; (2) washing the concentrated bacterial liquid obtained in the step (1); (3) performing cell wall breaking and cracking on the washed bacterial liquid obtained in the step (2) to obtain a wall-broken liquid; (4) removing non-PHA substances in the wall-broken liquid obtained in the step (3), and collecting PHA; (5) purifying the PHA; (6) the PHA is dried. The method has the advantages that the steps of bacterial liquid concentration, wall breaking, non-PHA substance removal, washing and the like are all required to be subjected to high-speed centrifugation at 2000-20000rpm and repeated centrifugation, the equipment investment cost is high, the method is only suitable for laboratory research and is not suitable for industrial production; the wall breaking process needs high-speed centrifugation under the conditions of high temperature, alkalinity and surfactant, not only can generate a large amount of foams and is easy to lose control in the processing process, but also under the high-temperature alkaline environment, PHA is degraded due to the shearing action of alkali, the molecular weight of PHA is reduced sharply, and the application field of the separated PHA product is greatly limited.
Disclosure of Invention
The invention aims to provide a process for separating and purifying polyhydroxyalkanoate, which has the advantages of simple process, high production efficiency, stable product quality and low cost and can realize large-scale industrial production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor comprises the following steps:
(1) taking fermentation liquor of polyhydroxyalkanoate production bacteria, and performing cell wall breaking treatment to obtain wall breaking liquid;
(2) adjusting the pH value of the wall breaking liquid to be alkaline by using alkali liquor;
(3) adding sodium salt and anionic surfactant into the alkaline wall-breaking liquid, and stirring for reaction, wherein the order of adding the sodium salt and adding the anionic surfactant can be interchanged;
(4) and (3) performing solid-liquid separation, collecting the precipitate, washing the precipitate to be neutral with water, dehydrating and drying to obtain the polyhydroxyalkanoate.
In the step (1), the invention directly carries out cell wall breaking treatment on the fermentation liquor containing the polyhydroxyalkanoate producing bacteria without separating the bacteria, and releases the intracellular substances of the bacteria.
In the method, the cell wall breaking can adopt a mechanical wall breaking method or a chemical wall breaking method. Preferably, the mechanical wall breaking method adopts an ultrasonic method or a high-pressure homogenization method.
Preferably, the ultrasonic treatment conditions are as follows: the power is 200-1000W, and the time is 10-30 min.
Preferably, the high-pressure homogenization conditions are as follows: the pressure is 50-100 MPa, more preferably 60-80 MPa, and the wall is broken for 2 times.
In the step (2), the pH value of the wall breaking liquid is adjusted to 8-12, so that mycoprotein can be solubilized, part of phospholipid molecules can be hydrolyzed, and the reaction of combining a surfactant and a non-PHA (polyhydroxyalkanoate) substance can be synergistically promoted.
The alkaline substance of the alkali liquor is NaOH and Na 2 CO 3 、NaHCO 3 Or ammonia. Preferably, the first and second liquid crystal materials are,and adjusting the pH value of the wall breaking liquid to 9-11.
In the step (3), sodium salt is added into the alkaline wall-breaking liquid for reaction and precipitation, and an anionic surfactant is added as a detergent for stirring to dissolve non-PHA substances such as cell fragments and the like in the wall-breaking liquid. In the process, sodium salt is added to react and separate out, so that the hydrolysis of alkali liquor on the polyhydroxyalkanoate is prevented; on the other hand, the high-salt environment changes the surface charge of the cell, reduces the stability of the cell membrane, is beneficial to the insertion of an anionic surfactant into the cell membrane and the wrapping of a phospholipid bilayer structure of the cell membrane, so that the polyhydroxyalkanoate is separated from cell fragments and released into a reaction solution.
The sodium salt can be selected from sodium chloride, sodium sulfate or sodium carbonate. Preferably, the sodium salt is sodium chloride.
The concentration of sodium ions is 0.3-1 mol/L per liter of fermentation liquid. The dosage of the sodium salt is 1-10% (W/V) of the fermentation liquor. Research shows that with the increase of the sodium salt dosage, the reaction precipitation can be promoted, the reaction precipitation time is shortened, and the reactant agglomeration effect is good. The concentration of sodium ions exceeds 1mol/L (fermentation liquor), and the synergy is not obvious. Preferably, NaCl is taken as an example, and the dosage is 2-6 g/100mL of fermentation liquor.
Preferably, the anionic surfactant is sodium lauryl sulfate.
Preferably, 1 to 5g of the anionic surfactant is added per 100mL of the fermentation broth.
Research shows that different sodium salts, anionic surfactant dosage, reaction temperature, stirring speed and other factors can cause the difference of reaction precipitation time. Therefore, one principle is to mix the raw materials for extraction reaction uniformly at the first time, and after mixing uniformly, the stirring speed is properly reduced, which is beneficial to the precipitation of the product. The reaction time is based on the complete precipitation of the product, and the stirring speed is adjusted according to specific conditions.
Preferably, the reaction time is 1-15 min after the sodium salt is added. And adding an anionic surfactant, and reacting for 1-5 min.
In order to ensure full reaction, sodium salt and anionic surfactant are respectively dissolved in water to prepare solution, and then the solution is added into a reaction system.
In the step (3), the reaction temperature is controlled to be not lower than 10 ℃. The research shows that the high-concentration SDS can be solidified under the condition of low temperature, and is not beneficial to the reaction. If the temperature is too high, the product is separated out too fast, the solid matters are agglomerated and loose, the particles are fine and soft, the solid-liquid separation is not facilitated, and the reaction temperature is preferably 10-50 ℃. Taking NaCl as an example, the reaction temperature is 10-35 ℃. More preferably, the reaction temperature is 15-25 ℃.
And (4) carrying out solid-liquid separation on the reaction solution, collecting the precipitate, and repeating the water washing step for multiple times to purify the PHA.
Preferably, the solid-liquid separation method can be vacuum filtration, centrifugation and filter pressing. The vacuum filtration conditions are as follows: the mesh number of the filter cloth is 100-200 meshes, and the vacuum gauge pressure is less than-0.05 MPa; the centrifugation conditions were: the separation factor is more than or equal to 600.
The method for purifying PHA comprises the following steps: adding water into the precipitate after solid-liquid separation, fully stirring and washing, then removing the washing liquid, and repeating the process for a plurality of times until the pH value of the solution after washing is neutral.
The dehydration method is the same as the solid-liquid separation method, namely vacuum filtration, centrifugation or filter pressing.
The drying method comprises heating, ventilating and drying, and vacuum freeze drying.
The invention is suitable for a wide range of processing objects, and can be applied to the separation and purification of fermentation liquor of various bacteria containing polyhydroxyalkanoate, variant strains or genetic engineering bacteria and the like. Suitable bacterial species include, but are not limited to: alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Azotobacter (Azotobacter), Rhodospirillum (Rhodospirillum), Methylotrophs (Methylotrophs) and Bacillus (Bacillus).
The invention has the following beneficial effects:
(1) the separation and purification process provided by the invention is used for directly separating and purifying the polyhydroxyalkanoate from the fermentation liquor without separating thalli in advance, avoids complicated and energy-consuming processes such as using a high-speed centrifuge and the like, saves investment and reduces cost investment.
(2) The invention adopts the process means of sodium salt and surfactant, the product is separated and purified under mild condition, the molecular shear is small, and the obtained product has the characteristics of high purity, high molecular weight and stable product quality. In the invention, sodium salt is added after the pH value of the wall breaking liquid is adjusted to be alkaline, and the PHA product is protected from the degradation of alkali liquor under the salting-out action, so that the sharp reduction of the molecular weight is avoided; and simultaneously, the sodium salt and the surfactant act cooperatively to help improve the separation efficiency of the PHA and non-PHA substances.
(3) The PHA extraction process has low requirements on PHA content of thalli, and has the advantages of simple process, high production efficiency, stable product quality and low cost, and can realize large-scale industrial production.
Drawings
FIG. 1 is a diagram of the reaction process and the suction filtration cleaning process after adding sodium salt and surfactant to the wall-breaking liquid in example 1, wherein (A) is the initial stage of reaction-completion of charging, (B) is the middle stage of reaction-preliminary precipitation, (C) is the final stage of reaction-complete precipitation, (D) is complete precipitation of mother liquor, (E) is post-treatment of slurry-4 times of filtration cleaning, and (F) solid is collected.
Detailed Description
The present invention is further illustrated by the following specific examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The Alcaligenes eutrophus bacteria and the Eubacterium rosenbergii (Ralstonia eutropha) used in the examples are conventional polyhydroxyalkanoate-producing bacteria and can be obtained from commercial sources. The fermentation culture is carried out by adopting the conventional fermentation culture conditions in the field.
Example 1
Taking 100mL of fermentation liquor of Alcaligenes eutrophus bacteria, 145g/L of dry cell weight (the PHB content is 70.5%), breaking the cell wall by ultrasonic waves (300W, 10s over/10 s stop for 30 minutes), cooling to 35 ℃, adjusting the pH to 11 by using 30% NaOH solution, adding 10g of NaCl (firstly dissolved by about 40mL of water), stirring for 1 minute, adding 2.4g of sodium dodecyl sulfate (firstly dissolved by about 25mL of water), continuing stirring for 2 minutes, carrying out vacuum filtration on a filter flask (the vacuum surface is less than or equal to-0.05 MPa), washing the precipitate by using water until the pH is neutral, carrying out suction filtration and dehydration, and placing in an oven at 90 ℃ to dry to constant weight. The reaction and suction filtration processes are shown in figure 1.
The product detection method refers to the national standard GB/T30293-2013 biological preparation of polyhydroxyalkanoate, and the product obtained in the embodiment has the purity of 99.1 percent and the viscosity-average molecular weight of 5.5 multiplied by 10 5 Da, extraction yield is 89%.
Comparative example 1
The comparative example explores optimization of process parameter conditions, takes the same processing object in the example 1, namely the wall-broken liquid of the Alcaligenes eutrophus bacteria fermentation liquid, fixes other preparation conditions, only changes a single factor, and compares the performance of the purified products under different conditions.
1. The reaction parameters and results of the different sodium salt tests are shown in table 1.
TABLE 1
Remarking: the reaction time is related to the pH, temperature and surfactant level. The molecular weight is related to the initial molecular weight of the product in the fermentation liquor, and the extracted product is generally about 5-10 ten thousand lower than the initial molecular weight.
As can be seen from Table 1, the effect of precipitating sodium chloride is the best when the sodium ion strength is the same, and sodium sulfate and sodium carbonate are slightly inferior.
2. Different reaction pH test
The pH of the wall breaking liquid is respectively adjusted to 6, 7, 8, 9, 10, 11, 12, 13 and 14, and the results show that:
when the pH is less than 8, the reaction reagent is added, the precipitation amount is small, the particles are fine and loose, the subsequent separation is not facilitated, and the extraction yield is low;
when the pH value is more than or equal to 8 and less than or equal to 12, the reaction precipitation amount is large, precipitates can be agglomerated, the particles are thick, the separation and the cleaning are facilitated, and the extraction yield is high;
when the pH value is more than 12, the reaction precipitation amount is large, precipitates can be agglomerated, the particles are thick, but the requirement of the subsequent cleaning process is high, the mother liquor needs to be removed quickly, otherwise, polyhydroxyalkanoate molecules are easily sheared by strong alkalinity, and the molecular weight is easily relatively low.
3. Different amounts of sodium salt
Different sodium salt dosage and different reaction effects exist, for example, NaCl is taken as an example, when the concentration of sodium ions reaches 1mol/L (fermentation liquor), the reaction precipitation time is obviously shortened, the reaction is basically complete, the sodium salt dosage is increased, and the reaction effect is not obviously changed; but reducing the amount of sodium salt prolongs the reaction time, makes the precipitated precipitate looser, is not beneficial to subsequent separation and cleaning, and has less precipitated amount, incomplete reaction and low extraction yield when the concentration of sodium ions is lower than 0.3mol/L (fermentation liquor).
4. Reaction temperature
The temperature of the wall breaking liquid is controlled to be 10, 15, 20, 25, 30, 35, 40, 45 and 50 ℃, and the result shows that with the increase of the reaction temperature, the product precipitation time is shortened, the apparent agglomeration is complete, but the particles are fine and soft, which is not beneficial to the subsequent separation, the cleaning loss is large, and the yield is not high; the temperature is properly reduced, the precipitates separated out by the reaction are properly agglomerated, and the particles are coarse, so that the separation and the cleaning are facilitated. From the test case of NaCl, the reaction can be carried out under the condition of 10-35 ℃, but the optimal reaction temperature is between 15-25 ℃.
Example 2
Taking the fermentation broth of the same treated object in example 1, namely Alcaligenes eutrophus bacteria, with the dry cell weight of 145g/L (wherein the PHB content is 70.5%), cooling to 20 ℃, breaking the walls under 80MPa and high pressure, stirring the wall-breaking broth in a storage tank with a cooling coil to cool to 20 ℃, and repeating the process of breaking the walls once again. Taking 100mL of wall breaking liquid, adjusting pH to 11 by using 30% NaOH solution, adding 6g of NaCl (dissolved by about 25m of water firstly) and stirring for 1 minute, adding 2.4g of sodium dodecyl sulfate (dissolved by about 25mL of water firstly) and continuing stirring for 2 minutes, carrying out vacuum filtration in a filter flask (the vacuum surface is less than or equal to-0.05 MPa), washing the precipitate by using water until the pH is neutral, carrying out vacuum filtration and dehydration, and placing in an oven at 90 ℃ and drying to constant weight.
The purity of the obtained product is 98.9 percent, and the viscosity average molecular weight is 5.3 multiplied by 10 5 Da, extraction yield is 88%.
Example 3
Taking fermentation liquor of Alcaligenes eutrophus bacteria, cooling the cell dry weight to 20 ℃ and breaking the wall under 80MPa high pressure, wherein the cell dry weight is 189g/L (the PHBV content is 81%), stirring the wall breaking liquid in a storage tank with a cooling coil and cooling to 20 ℃, and repeating the process of breaking the wall once again. Taking 100L of the wall-broken liquid, adjusting pH to 9 with 30% NaOH solution, adding 3.5kg NaCl, stirring for 2 min, adding 2.0kg sodium dodecyl sulfate, stirring for 3 min, centrifuging with a three-foot centrifuge (separation factor 600), washing the filter cake with water until the pH of the filtrate is neutral, dehydrating, and drying in an oven at 70 deg.C to constant weight.
The purity of the obtained product is 99.3 percent, and the viscosity average molecular weight is 5.8 multiplied by 10 5 Da, extraction yield 91 percent.
Example 4
Fermenting with fermentation broth of Eutrophobacter rolfsii (Ralstonia eutropha) with dry cell weight of 195g/L (wherein PHBV content is 82%), cooling to 20 deg.C, breaking cell wall under 80MPa, stirring the wall-broken liquid in a storage tank with cooling coil, cooling to 20 deg.C, and repeating wall-breaking process once. Taking 100L of the wall-broken liquid, adjusting pH to 11 with 30% NaOH solution, adding 5kg of sodium dodecyl sulfate, stirring for 1 minute, adding 2.5kg of NaCl, continuously stirring for 5 minutes, centrifuging with a three-foot centrifuge, washing the filter cake with water until the pH of the filtrate is neutral, dehydrating, and drying in an oven at 70 deg.C to constant weight.
The purity of the obtained product is 99.3 percent, and the viscosity average molecular weight is 5.9 multiplied by 10 5 Da, extraction yield is 92%.
Comparative example 1
Taking the fermentation broth of the same treated object in example 1, namely Alcaligenes eutrophus bacteria, with the dry cell weight of 145g/L (wherein the PHB content is 70.5%), cooling to 20 ℃, breaking the walls under 80MPa and high pressure, stirring the wall-breaking broth in a storage tank with a cooling coil to cool to 20 ℃, and repeating the process of breaking the walls once again. Taking 100mL of wall-broken liquid, adjusting the pH value to 11 by using 30% NaOH solution, adding 0.5g of sodium dodecyl sulfate and 5g of sodium polyacrylate, adjusting the reaction temperature to 70 ℃, and stirring for reacting for 30 minutes. Then vacuum filtering in a filter flask (vacuum surface is less than or equal to-0.05 MPa), washing the filter cake with water until the pH of the filtrate is neutral, dehydrating, and placing in an oven at 90 ℃ to dry to constant weight.
The obtained product has a purity of 93.5% and a viscosity average molecular weight of 4.5 × 10 5 Da, extraction yield is 82%.
The test data show that the whole extraction reaction time is obviously shortened by adding the sodium salt, the purity and the molecular weight of the product are obviously higher than those of a comparative example, and the extraction yield is also greatly improved.
Claims (10)
1. A method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor is characterized by comprising the following steps:
(1) taking fermentation liquor of polyhydroxyalkanoate production bacteria, and performing cell wall breaking treatment to obtain wall breaking liquid;
(2) adjusting the pH value of the wall breaking liquid to be alkaline by using alkali liquor;
(3) adding sodium salt and anionic surfactant into the alkaline wall-breaking liquid, and stirring for reaction, wherein the order of adding the sodium salt and adding the anionic surfactant can be interchanged;
(4) and (3) performing solid-liquid separation, collecting the precipitate, washing the precipitate to be neutral with water, dehydrating and drying to obtain the polyhydroxyalkanoate.
2. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation broth as claimed in claim 1, wherein in step (1), the cell wall breaking is performed by ultrasonic method or high pressure homogenization method.
3. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation broth as claimed in claim 1, wherein in the step (2), the pH value of the wall-broken liquid is adjusted to 8-12; the alkaline substance of the alkali liquor is NaOH and Na 2 CO 3 、NaHCO 3 Or ammonia.
4. The method for directly separating and purifying polyhydroxyalkanoate from a fermentation broth as claimed in claim 1, wherein in step (3), the sodium salt is sodium chloride, sodium sulfate or sodium carbonate; the concentration of sodium ions is 0.3-1 mol/L per liter of fermentation liquid.
5. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation liquor as claimed in claim 1 or 4, wherein the reaction time after the sodium salt is added is 1-15 min.
6. The method for directly separating and purifying polyhydroxyalkanoate from a fermentation broth as claimed in claim 1, wherein, in the step (3), the anionic surfactant is sodium dodecyl sulfate; adding 1-5 g of anionic surfactant into every 100mL of fermentation liquor.
7. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation broth as claimed in claim 1 or 6, wherein the reaction time after adding the anionic surfactant is 1-5 min.
8. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation broth as claimed in claim 1, wherein, in the step (3), the reaction temperature is controlled to not lower than 10 ℃.
9. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation broth as claimed in claim 8, wherein the reaction temperature is 15-25 ℃.
10. The method for directly separating and purifying polyhydroxyalkanoate from the fermentation liquor as claimed in claim 1, wherein in the step (4), the solid-liquid separation method is vacuum filtration, centrifugation and filter pressing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210697420.4A CN115058461A (en) | 2022-06-20 | 2022-06-20 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210697420.4A CN115058461A (en) | 2022-06-20 | 2022-06-20 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115058461A true CN115058461A (en) | 2022-09-16 |
Family
ID=83201842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210697420.4A Pending CN115058461A (en) | 2022-06-20 | 2022-06-20 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115058461A (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07303490A (en) * | 1994-05-13 | 1995-11-21 | Repsol Quimica Sa | Method of extracting polyhydroxyalkanoate |
| KR20010009719A (en) * | 1999-07-13 | 2001-02-05 | 성재갑 | Method of separation polyhydroxyalkanoates from microorganism |
| CN1328160A (en) * | 2000-06-13 | 2001-12-26 | 中国科学院微生物研究所 | One-stop separation method for extracting polyhydroxyl phytanate directly from fermented liquid |
| CN1464063A (en) * | 2002-06-25 | 2003-12-31 | 宁波天安生物材料有限公司 | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria fermentation liquor |
| CN1800235A (en) * | 2005-10-19 | 2006-07-12 | 深圳市奥贝尔科技有限公司 | Method for extracting high purity PHAs (Polyhydroxyalkanoates) from wet thallus |
| US20070072276A1 (en) * | 2003-12-19 | 2007-03-29 | Xuejun Chen | Method for separating, extracting and purifying poly-beta-hydroxyalkanoates (phas) directly from bacterial fermentation broth |
| CN103431193A (en) * | 2013-08-01 | 2013-12-11 | 宁波天安生物材料有限公司 | Novel feed containing PHA cell |
| CN109504715A (en) * | 2017-09-15 | 2019-03-22 | 北京蓝晶微生物科技有限公司 | A method of preparing polyhydroxyalkanoate (PHA) |
| CN111393625A (en) * | 2020-06-03 | 2020-07-10 | 中粮营养健康研究院有限公司 | Method and system for extracting polyhydroxyalkanoate by utilizing ultrasound under combination of lysozyme and SDS |
| CN111500650A (en) * | 2020-06-30 | 2020-08-07 | 中粮营养健康研究院有限公司 | Method for efficiently producing PHA |
| CN112430557A (en) * | 2020-12-18 | 2021-03-02 | 河北大学 | high-PHA-yield Kabayeri strain, screening method and PHA production method thereof |
| WO2022000322A1 (en) * | 2020-06-30 | 2022-01-06 | 北京阿迈特医疗器械有限公司 | Developing composite material and preparation method therefor and use thereof, and implantable and interventional medical instrument and preparation method therefor |
-
2022
- 2022-06-20 CN CN202210697420.4A patent/CN115058461A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07303490A (en) * | 1994-05-13 | 1995-11-21 | Repsol Quimica Sa | Method of extracting polyhydroxyalkanoate |
| KR20010009719A (en) * | 1999-07-13 | 2001-02-05 | 성재갑 | Method of separation polyhydroxyalkanoates from microorganism |
| CN1328160A (en) * | 2000-06-13 | 2001-12-26 | 中国科学院微生物研究所 | One-stop separation method for extracting polyhydroxyl phytanate directly from fermented liquid |
| CN1464063A (en) * | 2002-06-25 | 2003-12-31 | 宁波天安生物材料有限公司 | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria fermentation liquor |
| US20070072276A1 (en) * | 2003-12-19 | 2007-03-29 | Xuejun Chen | Method for separating, extracting and purifying poly-beta-hydroxyalkanoates (phas) directly from bacterial fermentation broth |
| CN1800235A (en) * | 2005-10-19 | 2006-07-12 | 深圳市奥贝尔科技有限公司 | Method for extracting high purity PHAs (Polyhydroxyalkanoates) from wet thallus |
| CN103431193A (en) * | 2013-08-01 | 2013-12-11 | 宁波天安生物材料有限公司 | Novel feed containing PHA cell |
| CN109504715A (en) * | 2017-09-15 | 2019-03-22 | 北京蓝晶微生物科技有限公司 | A method of preparing polyhydroxyalkanoate (PHA) |
| CN111393625A (en) * | 2020-06-03 | 2020-07-10 | 中粮营养健康研究院有限公司 | Method and system for extracting polyhydroxyalkanoate by utilizing ultrasound under combination of lysozyme and SDS |
| CN111500650A (en) * | 2020-06-30 | 2020-08-07 | 中粮营养健康研究院有限公司 | Method for efficiently producing PHA |
| WO2022000322A1 (en) * | 2020-06-30 | 2022-01-06 | 北京阿迈特医疗器械有限公司 | Developing composite material and preparation method therefor and use thereof, and implantable and interventional medical instrument and preparation method therefor |
| CN112430557A (en) * | 2020-12-18 | 2021-03-02 | 河北大学 | high-PHA-yield Kabayeri strain, screening method and PHA production method thereof |
Non-Patent Citations (4)
| Title |
|---|
| BOWEN XIONG等: "Chemical digestion method to promote activated sludge cell wall breaking and optimize the polyhydroxyalkanoate (PHA) extraction process", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》, vol. 240, 7 April 2023 (2023-04-07) * |
| YUNG-HUN YANG等: "Improved detergent-based recovery of polyhydroxyalkanoates (PHAs)", 《BIOTECHNOLOGY LETTERS 》, vol. 33, 5 January 2011 (2011-01-05), pages 937 - 942 * |
| 尹芬等: "可降解包装材料聚羟基烷酸酯合成工艺及影响因素的研究进展", 《包装工程》, vol. 40, no. 3, 31 December 2019 (2019-12-31), pages 72 - 79 * |
| 陈柳清: "极端嗜盐菌XMQ19菌株发酵生产聚羟基烷酸酯（PHA）的提取工艺研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》, 15 March 2009 (2009-03-15), pages 018 - 99 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0015123B1 (en) | A process for the extraction of poly-3-hydroxy-butyric acid from microbial cells | |
| KR101130191B1 (en) | Process for recovering polyhydroxialkanoates (phas) from cellular biomass | |
| CN111019108B (en) | Method for extracting and purifying polyhydroxyalkanoate | |
| CN109504715A (en) | A method of preparing polyhydroxyalkanoate (PHA) | |
| AU2005257068A1 (en) | Production of polylactic acid (PLA) from renewable feedstocks | |
| EP1705250B9 (en) | A method for separating, extracting and purifying poly-beta-hydroxyalkanoates (phas) directly from bacterial fermented broth | |
| CN112409635B (en) | Method for separating, recycling and utilizing waste polyester-cotton blended fabric | |
| WO2010013174A2 (en) | Process for the co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by the fermentation of the yeast pichia pastoris | |
| JP2022514256A (en) | Production of polyhydroxyalkanoates from pulp and paper waste streams | |
| EP1554391B1 (en) | Separation of biomass from lactic acid containing fermentation products by means of flocculation | |
| JPH10158303A (en) | Alkali solution or gelled product of fine fibrous cellulose | |
| CN115058461A (en) | Method for directly separating and purifying polyhydroxyalkanoate from fermentation liquor | |
| CN116144046B (en) | Preparation method of polyhydroxyalkanoate aggregate | |
| CN115368486B (en) | Ternary eutectic solvent and application thereof in procambarus clarkia shell chitin extraction | |
| CN111547916A (en) | Salt and nitrate separation method for nitrate-rich wastewater | |
| JP2008054541A (en) | Method for separating and purifying 3-hydroxyalkanoic acid copolymer produced by fermentation | |
| JPS63198991A (en) | Separation and purification of poly-beta-hydroxy lactic acid | |
| CN1257982C (en) | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria fermentation liquor | |
| CN106554273B (en) | Method for purifying long-chain dicarboxylic acid in fermentation liquor | |
| US20050159593A1 (en) | Method for deproteinization of chitosan | |
| WO2024003698A1 (en) | Process for recovering and purifying polyhydroxyalkanoates from a fermentation broth | |
| CN115594883B (en) | Method for separating and utilizing waste polyester-cotton blended fabric based on acidic hydrogen peroxide solution | |
| CN117361699A (en) | Treatment method of cell wall-broken wastewater and application of treatment method in halophilic bacteria fermentation | |
| CN117187311A (en) | PHA extraction method | |
| JP2009207380A (en) | Method for purifying polyhydroxyalkanoic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |