MXPA97001249A - Production of acidoshidroxialcanoi polymers - Google Patents

Abstract

The present invention relates to a process for recovering PHA from a suspension comprising PHA particles in a liquid, comprising the suspension at a temperature at which the PHA is melted and substantial coagulation of the PHA particles occurs forming with it at least partially a layer of molten PHA, and separating the PHA layer optionally after cooling it to form a solid

Description

PRODUCTION OF HYDROXYLCOLIC ACIDS POLYMERS This invention relates to the production of hydroxyalkanoic acid polymers. F? It is known, for example from European patent.com 69497 and 52460, produce polymers of hydroxyalkanoic acids (PHfl), especially polymers and copolymers of hydroxybutyric acid by microbial fermentation. The European country 145233 describes a procedure in which rcrocrogam s produced can be treated by heat and reagents in one or more steps to release PHfi particles contained therein by giving a suspension of the particles in an aqueous phase whose particles can be separated eg by centrifuging. It may be convenient to resuspend the particles in water to carry £ 5 out additional purification. According to this invention, a method of recovering PHfl from a suspension comprising particles of PHfl in a liquid comprises maintaining the suspension at a temperature at which the PHfl melts and occurs Substantial coagulation of PHfl particles thereby forming, in at least a least part, a meltblown PHfl layer of the liquid, and separating the PHfl layer from the liquor layer optionally after cooling to form a solid. If the procedure starts from a suspension of PHO When produced microbiologically, any of the following or a mixture thereof may be used: 1. Biomass intact as produced by fermentation, possibly concentrated or diluted; 2. Biomass of lysed cells originating from thermal or mechanical treatment of (1); 3. The treatment product of (D or (2) with a surfactant agent; 4. The treatment product of (1) or (2) with an enzyme hydrolase and / or protease; 1, 2, 3, or 4 with an oxidizing agent, preferably in the presence of < 1e a chelant and / or surfactant For each of such suspensions there may be a step of concentration or dilution or separation of soluble substances before the passage of melt separation For each of the suspensions made by particular surfactant treatment, it may be convenient to remove any excess surfactant before the melt separation step.The PHfl layer and the liquid layer can be formed by allowing the PHfl sedimente in a molten condition either normal gravity or ba or an increased gravitational field.The layers are suitably subjected to little or no turbulence during the sedimentation procedure. a to which the layers are formed should not be much higher than the melting point of the PHfl. It can for example be up to 25 ° C above said point of fusion. The temperature and residence time of the PHfl should be such that during the exposure period at that temperature the molecular weight of the PHfl is not reduced below 200,000 and preferably it is not reduced below 400,000 The PHfl is preferably a polymer or hydroxyl-butyl acid copolymer, for example a copolymer thereof with another hydroxyalkanoic acid, preferably having 3 10 carbon atoms, for example hydroxyl acid Lvalepco Conveniently, the liquid comprises water The layers can be formed in the presence of materials that are water soluble Such materials may include degradation products of cellular material other than PHfl of the microorganisms in which the PHO is formed, reagents used to degrade or solubilize said materials, for example proteases, surfactants or compounds of peroxide, for example hydrogen peroxide and decomposition products thereof and nutrients for microorganisms We are for example carbon nutrients for example glucose and inorganic nutrients. If desired, materials may be added to improve the formation of the layers for example by reducing the surface activity or other materials that tend to stabilize the suspension. If a component of the liquid ebul e at a temperature lower than the melting point of the PHfl, the procedure is suitably carried out under pressure to avoid turbulence. resulting from boiling. In general, the membranes used to form the layers are above 100 ° C, for example 140 to 200 ° C; If the liquid comprises water, the operation under pressure is usually convenient for the same. In a preferred form of the process, a suspension of PHfl containing microorganisms in water which may contain nu-pentes for the mycorrhizal smop is contacted with hydrogen peroxide in the presence of a complexing agent which may comprise acid et al. 11 in di ami a tet racetico or acid and ilendiarnma-tetra (rnet? Lenfosfómco) at a temperature sufficient to solubilize materials other than PHfl and kept at a temperature sufficient to allow a layer of PHfl and an aqueous layer to form; and separate the PHfl layer. This form of the invention is advantageous since it is not necessary, as in known processes, to introduce added proteases or surfactants and the hydrogen peroxide tends to be reduced by water. It will be appreciated that the PHfl layer may not include all of the PHfl present, and some loss of PHfl may be accepted due to this, or an additional separation step may be employed to reduce such losses. The separation can be improved using increased gravity and suitable centrifuges available commercially to separate liquid layers, and can be used in the invention. It will also be appreciated that the layer of PHfl may comprise some liquid and other matter other than PHfl. If the liquid is water, it is eliminated if the PHfl is treated in molten state; and little contamination has been found with material other than PHfl, but if desired, the PHfl layer can be washed additionally eg pass through or drops through fresh water and repeating the process of this invention to recover a second layer of PHfl in the molten state or by separating solid particles from the washing medium for example by filtration or centrifugation. The invention may comprise a further step of dividing the separated liquid PHfl with surfactant and water and thereby producing a suspension of PHfl in a water / surfactant phase. The process can be carried out by the steps of: (a) subjecting a suspension comprising PHfl and materials from, or derived from, living cells in water at a pressure and temperature at which the PHf1 and the water are both in the liquid until the molten PHfl separates as a lower layer; (b) separating the PHfl layer from the aqueous layer and mixing it while it is fused with a surfactant and water at a temperature and pressure such that the PHf1 and the water are both in the liquid state, thereby forming a fine dispersion of PHfl in water and solidifying the PHfl by cooling said d? sp > fine ers? To limit the decrease in molecular weight of PHfl at the relatively high temperatures involved, the separated PHfl layer should be cooled or resuspended and cooled as rapidly as possible. Suitable PHfls comprise units of formula L: -0 - CMHn - CO - where m is on the scale of 1-13 and n is 2rn or (when rn is at least 2) 2rn-2. Typically, CmHn contains 2-5 atoms of carbon in the polymer chain and the remainder (if it is the case) in a side chain. In very suitable PHfls rn ee 3 or 4, n is 2rn and especially there are units with rn = 3 and m = 4 copol abraded together with a side chain of Ci and C2 respectively on the carbon following oxygen. Particular PHfls contain a preponderance of n = 3 units, especially with at least 70 mole% of said units, the rest being units in which m = 4. The molecular weight of PHfl is for example over 50,000, especially over 100,000, per example up to 7 x 106. The PHfl of formula I containing only m-3 units is referred to as PHD; the PHfl that contains m - 3 and m = 4 units is the polydroxybutyrate-covalerate copolymer of PHBV. The PHBV preferably contains 4-20% of rn = 4 units. The PHfl can also be a mix of 2 or more different PHfls in the value of m. Correspondingly a mixture of starting suspensions is used. A particular example contains: a) PHfl consisting essentially of units of the formula T in which 2-5 mol% of units have rn = 4, the rest - 3; and b) PHfl consisting essentially of units of the formula I in which 5-30 mol% of units have rn-4, the remainder -3. The proportions in such a mixture are preferably such to give an average content rn-4 in the 4-20% scale. The PHf1 is preferably the product of a microbiological process in which the microorganism can be wild or mutated or it can have the necessary genetic material introduced into it. Alternatively, the necessary genetic material can be hosted by a eucalyptus, to carry out the pucrobiological process. The PHfl produced microbiologically is chiral (R) and stereospecific. Examples of suitable microbiological processes are the following: for material of formula I with m = 3 or m = partially 3, partially 4: EP-A-69497 (fllcaligenes eutrophus): for materials of formula I with rn-3; US 4101533 (fl eutrophus), EP-fl-144017 (fl Latus); for material of formula I with m = 7-13; EP-fl-0392687 (different P is dominated), The microbiological production of PHfl is preferably carried out in two stages: a) aerobic growth of microorganisms and b) aerobic fermentation of the resulting organisms in a medium that contains a source of carbon but is deficient in at least one nutrient for the growth. The nutrient defi ciently prefers phosphate. The surfactant may be cationic, ammonic, nonionic, zwitterionic, or contain hydrophilic groups of more than one type. The hydrophobic part of the surfactant preferably contains at least 8, especially 12-20 carbon atoms per hydrophilic group. It can be (almost) completely aromatic as in sulfonated naphthalenes and naphthylnanenes; or partially aromatic as alkylbenzenesulfonates or ethoxylates; or completely alifatico. Very conveniently, the surfactant contains a linear alkyl group. If the surfactant is cationic, preferably its hydrophilic part is quaternary ammonium, based for example on trialkylamine of C 1 -C 4. If it is unique, the hydrophilic group is typically sulfate, sulfonate, sulfonate, carboxylate, phosphate or phosphonate. If it is nonionic, it can be, for example, an ethoxylate, for example an alkyl ethoxylate containing from 7 to 16 carbon atoms and up to 100 ethoxylate units, or a block copolymer of ethylene oxide and propylene oxide or an alkylfeethoxylate. Suitable surface cationic surfactants include, typically co or chloride or bromide: dodecyl-, tetradecyl- and cetii-tmethylmomo, ceti-l-di-methyl- Ethyl omo, dodecyl-, tetradecyl- and hexadecyl-benzylidene or benzalcole, benzetomo, and 1 benzetomo and cetiipyridyme. Suitable ammonium surfactants typically include sodium salts: dodecyl sulfate, N-lauroyl sarcosmate, dioctylsul phosuccinate, cholate, deoxycholate, laurate, miptate, palmitate, and stearate. Suitable nonionic surfactants influence rnonopaipitate of sorbitan, alkyl glucoside and N-alkyl-ethoxylates. Preferred surfactants are cetyl-phenyl-1-ammonium bromide and deoxycholate, dodecylsulfate, N-lauroyl-sarcoside and sodium dioctyl-sulfosuccinate. The concentration of PHfl in the latex is typically 100 to 600, especially 200 to 500 g / 1- ?. The concentration of surfactant in the partition step is typically in the range of 0.25% to 10, especially 1 to 5% w / w in the polyester component of the mixture. Typical pressures and temperatures for different PHfls are as follows: Pressure Temperature ° C Homopolymer PHfl 106 pa 10 barias 180 97: 3 B: V 8 X 105 e barias 170 88:12 B: V 5.4 x IOS 5.4 barias 155 79:21 B: V 3.7 x lOSPa 3.7 barias 142 High-partition mixing may employ one or more of the following dispersion media, for example: nozzle (s) or fine spinners, possibly with vibration, possibly ultrasonic; ultrasonic agitation of a sub-zone or larger body of liquid; furnace in extreme space such as with SILVERSON or ULTRATURRAX; high pressure homogemzation such as with APV Manton-Gaulin, Ron e or Braun a Luebhe; homogesation by jet impact; plastic milling such as with HOBART, 8AKER-PERKTNS or UERNER-PFLEIDERER; grinding in ball mill or gravel mill; agitation with paddle, agitation with impeller-toothed. Which of these is used, depends on temperatures, pressures and viscosity of PHA and generally the convenience of the design. The conditions in the mixing zone are controlled according to the particle size and particle size distribution of the latex to be produced. Typically, the average particle size is on the scale of 0.05 to 5, especially 0.1 to 1.5. μm. The average particle sizes of 0.1 - 0.4, 0.4 - 0.6 and 0.8 - 1.1 μrn seem to be - especially suitable for particular applications. Preferably, the PHf1 in the particles is at least 96% pure, especially at least 98% w / w. Low in crystallinity, especially less than 30, particularly less than 20, for example less than 1 percent crystalline, as measured by density or amplitude of X-ray angle scattering (UflXS) is preferred. The percentages are by weight and it is believed which are represented by: Weight of crystalline PHA X 100 Total weight of PHA in the sample where each particle is either completely amorphous or crystalline in all practicable extent. If "desired, the molten PHA can be mixed, before step d), with a water-soluble liquid that dissolves it at a temperature of 100 °. C: this allows it to flow at a temperature below its melting point, and it can also make possible the use of more severe partition conditions, since the PHA is precipitated from the solution in step d). there is found propylene carbonate, 1,2-? ropanod? ol, C4-10 alkanols, C4-10 alkanol acetates, rnetii-ieobutil-ketone and cycloalkanones.

Co-solvents such as partially esterified glycols can be introduced in this step. The dispersion made according to the invention can be subjected, for example, to one or more of the following treatments: concentration or dilution; removal of excess surfactant, addition of an additional or different surfactant; addition of a thickener or stabilizer; addition of a pigment or co-solvent.

The dispersion, or latex, can be used as such, for example, as a coating for paper, polymer films, non-woven splints or food products. Alternatively it can be an intermediate to make dry PHA to be processed as a molten mixture or in solution. Such a route may be shorter than conventional routes and, in any case, should allow latex and dry PHA to be processed in a single stream process.

EXAMPLE 1 A strain of Al cali enes eutrophus is grown in a batch culture in an aqueous medium in a mixture of glucose and propionic acid under phosphorus limitation to yield a culture containing 176 g / 1 of cells containing 72.12% of a copolymer (3-hydroxybutyrate (HB) / 3-h? DrOx? Valerate (HB)), with a hydroxyvalerate content of 21% (the remainder of the polymer is hydroxybutyrate). A sample of the cells is first treated with heat at 150 ° C for 80 seconds at pH 6.5. This heat breaks down the cells which are then treated with a proteolytic enzyme (EC 3.4.21.14) at pH 8, 70 ° C for 2 hours. At the end of this period the polymer particles are separated by washing the solubilized cellular components by centrifugation and resuspension. The washed particles are resuspended in water, the temperature is raised to 80 ° C and the pH is adjusted to 7. It is added a hydrogen peroxide solution of 350 g / liters to give a final aqueous phase concentration of 16 g H2O2 per liter. The temperature and pH conditions were maintained for 12 hours. A sample of the resulting Lime po suspension is heated to 140 ° C in a sealed thick-walled glass tube immersed in an oil bath at 140 ° C for 10 minutes. The polymer particles in the hot suspension coagulate and form by cooling a solid mass of polymer at the base of the tube. After cooling the sample, the polymer plug is removed from the surrounding fluid and analyzed for residual polymer impurities. Analysis of the polymer product indicates that the residual nitrogen concentration is 550 pprn which correspond to a protein concentration of approximately 3500 pprn. The polymer product was thus considered poly (3-hydroxybutyrate / 3-hydroxyvalerate) 99.6% pure. Analysis of the polymer product that was separated by centrifugation before heat treatment at 140 ° C gave a residual nitrogen concentration of 1530 pprn. The molecular weight measurements carried out on the polymer indicate that before heating to 140 ° C, the molecular weight of the polymer was 620,000. After heating at 140 ° C for 10 minutes, the molecular weight was reduced to 540,000, but the product was still suitable for polymer applications.

EXAMPLE 2 A strain of Alcaligenes was developed < .jutrophus in cultivation per batch in an aqueous medium in a mixture of glucose and propionic acid under phosphorus limitation to yield a culture having 176 g / l of cells containing 72.2% of a copolymer of (3-hydroxybutyrate (HB) ) / 3-hydroxyvalerate (HV)) with a hydroxyvalerite content of 21% (the rest of the polymer is hydroxy butadiene). A sample of the cells was first heat treated at 150 ° C for 80 seconds at pH 6.5. This heat broke cells that were then treated with a proteolytic enzyme (EC 3.4.21.14) at pH 8, 70 ° C for 2 hours. At the end of this time, the pH of the suspension was adjusted to pH 7, the temperature was raised to 80 ° C and diethylene-pentaethylene-phenephonic diethyl acid was added to give a final concentration of 6mM. A solution of hydrogen peroxide (350 g / liter) is added to give a final aqueous phase concentration of 50 g / 11 tro. The temperature and pH conditions are maintained for 12 hours. A sample of the resulting polymer suspension is heated to 140 ° C in a sealed thick-walled glass tube, immersed in an oil bath at 140 ° C for 10 minutes. The polymer particles in the hot suspension clump together and form a solid polymer mass at the base of the tube. After cooling the sample, the polymer plug is removed from the surrounding fluid and analyzed for residual polymer impurities. Analysis of the polymer product indicates that the residual nitrogen concentration was 780 ppm, corresponding to a protein concentration of approximately 4,800 ppm. In this way, the polymer product is considered poly (3-hydroxybutyrate / 3-hydroxyvalerate) 99.5% pure. Analysis of the polymer product separated by centrifugation before heat treatment at 140 ° C gave a residual nitrogen concentration of 3,300 pprn. Measurements of molecular weight carried out on the polymer indicated that before heating to 140 ° C the molecular weight of the polymer was 640,000. After heating at 140 ° C for 10 minutes, the molecular weight was reduced to 560,000, but the product was still suitable for polymer applications.

EXAMPLE 3 The apparatus for this operation comprises a propeller extruder which ends in a spinner which supplies a one liter pressure vessel which includes a SILVERSON homogenizer. The pressure vessel is charged with 500 rnl of a 3.3% w / w solution of the surfactant sodium dodecylulfate, then closed and heated to 150 ° C. The barrel of the extruder it is heated to 150 ° C, its propeller is started and it is supplied with 400 g of PHBV (15-20 mol% V) as a powder. The liquid polymer comes out of the spinner and breaks into very tight particles through the oven; the particles remain in suspension with a latex contaminated by the surfactant. Then the pressure vessel is cooled and the latex is discharged. Instead of, or in addition to, the SILVERSON homogenizer, the liquid polymer mixture and the surfactant solution can be subjected to ultrasonic agitation or mechanical partitioning or multiple homogenization in successive steps or by recirculation.

EXAMPLE 4 A 1-liter pressure vessel equipped with a stirring system is charged with 600 ml of water, 20 ml of sodium dodecylsulfate and 400 g of pulverized PHBV (15-20 mol% V). The stirring is started and the container is closed and heated to 150 ° C, sufficient to melt the polymer. After 10 minutes, the vessel is allowed to cool, continuing the stirring at the same time. The product is discharged, a PHBV latex.

EXAMPLE 5 Example 2 is repeated, except that instead of adding polymer as a powder, a PHA suspension is used as described in copending application GB No. 941669.1.5. That is, a suspension of cells of Alcaligenes eutrophus developed in glucose + propionic acid under phosphorus limitation to give 176 g / 1 of cells containing 72.2% of PHBV (21 mol% V) is treated with heat at 150 ° C for 80 seconds at pH 6.5, treated with a proteolytic enzyme (EC 3.4.21.14) at pH 8.70 ° C for 2 hours, then released from solubilized NPCM by centrifugation and resuspension. The washed particles of PHBV are resuspended in water and at 80 ° C, pH 7, treated with hydrogen peroxide, 16 g / 1, for 12 hours.

Claims (15)

NOVELTY OF LR INVENTION CLAIMS

1. - A method of recovering PHfl from a suspension comprising PHA particles in a liquid, comprising maintaining the suspension at a temperature at which the PHA is melted and substantial coagulation of PHA particles occurs thereby partially forming a smaller layer of PHA melted, and separate the PHA layer optionally after cooling to form a solid.

2. A method according to claim 1, characterized in that the PHf1 is produced microbiologically.

3. A process according to claim 1 or 2, further characterized in that the PHf1 is a polymer or copolymer of hydroxybutyl acid.

4. A process according to any of the preceding claims, further characterized in that the PHf1 is a copolymer of hydroxybutyl acid and hydroxyvaleric acid.

5. A process according to any of the preceding claims, further characterized in that the suspension is maintained at a temperature of at most 25 ° C above the melting point of PHfl.

6.- A procedure in accordance with any of The above indications, further characterized because it is carried out in the presence of materials that are soluble in gua.

7. A process according to claim 6, further characterized in that the water-soluble materials include at least one of (a) degradation products of cellular material different from PHA of the microorganisms in which the PHA has been formed; (b) the agents used to degrade or solubilize such materials; and / or (c) nutrients for microorganisms.

8. A process according to any of the preceding claims, further characterized in that the liquid boils at a temperature below the melting point of the PHA and the process is carried out under pressure.

9.- A procedure in accordance with any of The preceding claims, further characterized in that the temperature is 140 to 200 ° C.

10. A method according to any of the preceding claims, further characterized in that the liquid is water.

11. A process according to any of the preceding claims, further characterized in that a suspension of PHA containing microorganisms in water makes contact with hydrogen peroxide in the presence of a complexing agent at a temperature sufficient to solubilize materials other than PHA and then it maintained at a sufficient temperature to allow a PHA layer and an aqueous layer to form, and separate the PHA layer.

12. The method according to any of the preceding claims, further characterized in that the separation of the layers is carried out using increased gravity.

13. A process according to any of the preceding claims further characterized in that the separated liquid PHA is divided with a surfactant and water thereby producing a suspension of the PHA in a water / surfactant phase.

14. A method according to claim 13, further characterized in that it comprises the steps of submitting a suspension comprising PHA and materials of, or derivatives of, living cells in water at a temperature and pressure at which the PHA and the The water is both in a liquid state until the molten PHA is separated as a lower layer, separating the PHA layer from the aqueous layer and mixing it while it is fused with a surfactant and water at a high partition rate and at a temperature and pressure such that the PHA and the water are both in liquid form thereby forming a fine dispersion of PHA in water and solidifying the PHA by cooling said fine dispersion.

15. A method according to any of the preceding claims, further characterized in that the molecular weight of the PHA is maintained above 200,000, restricting the exposure of PHA at high temperatures.