GB2120671A - Separation process - Google Patents

Abstract

Hydroxybutyrate polymers are separated from solutions thereof by causing the solution to gel and then shearing the gel, and removing any expressed solvent, to give discrete gel particles free of any continuous liquid phase. Solvent is then evaporated from the discrete particles e.g. in a fluid bed drier.

Description

SPECiFICATION Separation process This invention relates to a separation process, and in particular to the separtion of 3hydroxybutyrate polymers from solution thereof.

Poly(3-hydroxybutyrate) is a thermoplastic polyester consisting of repeat units of the formula --CH(CH,) . CH,.CO.O-- which is accumulated by many micro-organisms, particularly bacteria, for example of the genera Alcaligenes, Athiorhodium, Azotobacter, Bacillus, Nocardia, Pseudomonas, Rhizobium, and Spirillium, as an energy reserve material.

The polymer is conveniently prepared by cultivating the micro-organism in an aqueous medium on a suitable substrate, such as a carbohydrate or methanol, as an energy and carbon source. The substrate must, of course, be one that is assimilable by the micro-organism. In order to promote accumulation of the polymer, at least part of the cultivation is preferably conducted under conditions wherein there is a limitation of a nutrient that is essential for growth of the micro-organism but which is not required for polymer accummulation. Examples of suitable processes are described in EP-A-1 5669 and 46344.

Polymers containing both 3-hydroxybutyrate units and other hydroxycarboxylic acid units, such as 3-hydroxyvalerate units, can also be produced microbiologically. Thus a microbilogically produced heteropolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate residues is described by Wallen et al in "Environmental Science and Technology" 8 (1974) 576-9. Also, as described in EP-A-52459 various copolymers can be produced by cultivating the micro-organism on certain substrates, such as propionic acid which gives rise to 3-hydroxyvalerate units in the copolymer.

Accordingly, in the present specification, by the term HB polymer we mean not only the homopolymer, poly(3-hydroxybutyrate), but also copolymers as described above, provided that the 3hydroxybutyrate residues form at least 40 mole %, and preferably at least 50, mole % of the polymer chain.

While cells containing the polymer can be used as such as a moulding material, for example as described in US-A-3 107172, it is generally desirable to separate the polymer from the remainder of the cell material.

During the processing of HB polymers, particularly during the extraction route employed to separate the polymer from the michoorganism cells, it is often necessary to separate the polymers from a solution.

In extraction processes the polymer is generally extracted from the micro-organism cells, usually after some preliminary treatment of the cells, with a solvent in which the HB polymer is soluble. For example, in EP--AA-15123 a 'wet' process is described wherein the aqueous cell suspension, in some cases after milling, is contacted with 1,2-dichloroethane, and the solvent phase, containing the dissolved HB polymer, is separated from the aqueous medium.Another process described in EP-A-1 5123 involves drying the cell suspension, e.g. by spray drying, prior to contact with the extraction solvent. EP--AA-15123 discloses that the polymer may be recovered from the solution in the extraction solvent by addition of the solution to a precipitant liquid, e.g. a water-methanol mixture where the extraction solvent is methyiene chloride, chloroform, or 1 2-dichlorethane, with which the extraction solvent is miscible and in which the polymer is insoluble.

Recovery and re-use of the solvents employed is desirable for economic operation of such processes. In such a precipitation process, solvent recovery is expensive since relatively large volumes of the precipitation liquid are required to ensure precipitation of the polymer in a form that is readily separable, e.g. by filtration. Also solvent recovery is often difficult: indeed the extraction solvent and precipitation liquid often forms azeotropic mixtures rendering solvent separation by simple distillation impractical.

An alternative procedure for separation of the polymer from the solvent involves direct evaporation of the solvent from the solution. In such a process it is generally necessary to agitate the solution in order to permit even and rapid evaporation: such agitation is relatively costly since the viscosity of the solution increases very rapidly as the solvent content decreases. Indeed we have found that solvent removal beyond a certain level is not practical by such a process using conventional thin film evaporators since a tough skin of polymer tends to build up preventing further removal of the solvent.

Therefore a process in which polymer can be separated from the solvent and solvent recovery is facilitated is to be desired.

In EP-A-2481 0 there is described a process wherein a gel formed from an HB polymer solution is subjected to a non-random deformation step, e.g. pressing: during the deformation step, solvent is expelled from the gel leaving the HB polymer in the form of what was termed therein, a 'parchment'.

We have now found that if such a gel is sheared, e.g. by stirring, i.e. a random deformation process, the gel can be broken into a particulate form from which the solvent can readily be removed to give discrete particles free of a continuous liquid phase.

Accordingly we provide a process for the separation of an HB polymer from a solution thereof comprising (a) causing said solution to gel, (b) shearing said gel and removing any solvent expressed therefrom until a mass of discrete gel particles that is free of a continuous liquid phase is produced, and thereafter (c) removing solvent from discrete particles by evaporation while agitating said particles.

Solid HB polymers separated from micro-organism cells by solvent extraction followed by precipitation are crystalline, or partly crystalline, materials, melting at about 1 800C (3-hydroxybutyrate homopolymer) or generally at somewhat lower temperatures in the case of copolymers. Freshly precipitated HB polymers are partially crystalline and crystallise further on heating to e.g. 1 000C or more. The difference in crystallinity is evident from the solubility characteristics of the polymer. For example freshly precipitated poly(3-hydroxybutyrate) is soluble in chloroform at room temperature whereas the same material after heating for 30 minutes at 1000C is not soluble in chloroform at room temperature.Methylene chloride will dissolve freshly precipitated poly(3-hydroxybutyrate) at room temperature only if the polymer has not been allowed to become warmer than about 60"C after precipitation. Freshly precipitated poly(3-hydroxybutyrate) is not soluble at room temperature in 1,2dichiorethane even if the precipitated polymer has not been ailowed to warm to above 400 C.

Chloroform, methylene chloride, and 1,2-chloroethane do however dissolve poly (3-hydroxybutyrate) at elevated temperatures, e.g. under reflux conditions. However poor solvents such as 1,2-chloroethane can be used to extract poly(3-hydroxybutyrate) from micro-organism cells even at relatively low temperatures, e.g. 10 to 400 C: in the state in which the polymer exists in the micro-organism cells, it is more readily dissolved by such solvents that after the polymer has been precipitated or otherwise separated from a solution thereof. This is presumably due to the configuration of the polymer in the micro-organism cells being different to that of polymer precipitated from a solution thereof.

In aforesaid EP-A-248 10 it was disclosed that gels can be formed from solution of HB polymers in Poor solvents, such as 1,2-dichloroethane, in which the freshly precipitated HB polymer that has not been heated to above 400C has a solubility of less than 0.1% by weight at temperatures below 250C.

By the term "gel" is meant a three-dimentional network of polymer chains in an environment of the "solvent" in which a significant number of the polymer chains contain at least three linking points along their length which are linked to other polymer chains. The polymer chains may be molecular in nature or may consist of fibrals made up of molecular chains. The gel used in the invention are so viscous that they are capable of supporting a stainless steel ball bearing 1.5 mm in diameter whilst the gel still contains solvent. The gels are also preferably coherent in that they can be picked up with forceps without the gel disintegrating to any substantial extent.While coherent gels which are able to support a stainless steel ball bearing of 1.5 mm diameter are readily obtained using concentrations of 1% or more by weight of the HB polymer, at lower concentrations the gel will readily support the ball bearing but it may not be possible to remove a coherent gel from its surrounding solvent using forceps.

The HB polymer solution to which the process of the present invention is applied preferably has an HB polymer content between 0.5 and 10% by weight. With more dilute solutions it is difficult to make a satisfactory gel that will break into particulate form in the shearing process, while more concentrated solutions may be too viscous to be readily filtered or otherwise handled prior to the shearing process.

The solutions may be caused to gel by a number of methods which may be used singly or in combination. For example gelation may be induced by allowing the solution to stand at a temperature between 1 00C and 400C, optionally with one or more of the following prelimin ry steps: (i) cooling the solution to below OOC (ii) shearing the solution, e.g. by stirring, and (iii) seeding the solution with a preformed gel of the HB polymer.

The time taken to produce a gel depends on, inter alia, the gelation inducing conditions and the concentration of the solution. Thus while a solution may gel simply by storage at room temperature for several days, gelation may be induced in an identical solution by cooling and/or stirring for an hour or two prior to leaving the solution to stand.

In order to produce a gel spontaneously on storage at room temperature the HB polymer concentration of the solution must generally be at least 1.5% by weight. For solutions of lower concentration gelation usually only occurs if an additional step, such as shearing or cooling, is applied.

The preferred methods of inducing gelation of the HB polymer are shearing and/or cooling, followed by standing at a temperature between 10"C and 400 C.

Other solvents that can be used to form gels include ketones such as acetone or methyl ethyl ketone and mixtures of a good solvent, such as chloroform, with a poor solvent such as methanol.

Thus, although HB polymers are not normally considered to be soluble in solvents such as acetone and methyl ethyl ketone, we have found that HB polymers can be extracted from micro-organism cells at elevated temperatures using such solvents (under superatmospheric pressure) and gel are readily formed on cooling to e.g. room temperature.

Likewise HB polymers can be extracted with a mixture of methanol and chloroform at elevated temperature and a gel formed by cooling. The ease of gel formation e.g. the degree of cooling necessary and/or the time taken for gel formation, depends on the proportion of methanol in the mixture and on the HB polymer content of the solution. Alternatively gels can be formed by addition of methanol to a hot solution of the HB polymer in chloroform and then cooling the mixture. Generally the solvent mixture should contain at least 30% by volume of methanol for gels to be produced.

During formation of the gel, some syneresis may occur. The separated solvent may be removed (for re-use if desired) prior to subjecting the gel to the shearing process. Likewise some solvent may evaporate from the gel simply on storage.

The gel is conveniently sheard by stirring, for example in a mixer of the Hobart type, or by extrusion through a die.

The amount of shearing that is employed should be such as to give discrete particles of gel with no continuous liquid phase.

When the gel is sheared initially some of the solvent may be expressed from the gel so as to produce a slurry of gel particles in a continuous liquid phase of the expressed solvent.

Expressed solvent may be removed by evaporation if shearing is continued during this evaporation step.

The expressed solvent can be removed by application of a vacuum during the shearing process and/or by passing a stream of gas, e.g. air, over or through the gel as it is being sheared so that the gas carries the evaporated solvent away from the sheared gel. The gas stream may be heated is desired to provide the heat of evaporation of the solvent. Alternatively, or additionally, the heat of evaporation may be provided by the heat generated by the shearing process.

Since gels tend to revert to a solution when heated to above a certain temperature, e.g. about 50-600C in the case of gels where the solvent is 1,2-dichloroethane, the shearing operation should be conducted below the temperature at which the gel reverts to a solution. Where higher than ambient temperatures can be tolerated without reversion of the gel to a solution, for economic operation the temperature of the gel may be maintained, during the shearing process, at above ambient temperature e.g. by means of a heated gas passed through the sheared gel and/or by means of a suitable heat exchanger e.g. water jacket on the vessel in which the shearing is conducted.Where the shearing operation is such that there is a risk that the heat generated during shearing could cause the temperature of the gel to rise to the level at which it reverts to a solution, such a heat exchanger may be used to cool the gel as necessary.

The removed evaporated solvent can be condensed and re-used as desired.

Alternatively the expressed solvent may be removed from the slurry mechanically e.g. by centrifuging, filtering, or decanting. Since compaction and cohesion of the gel particles may occur during such mechanical removal of expressed solvent, further shearing of the gel from which expressed solvent has been removed may be necessary to produce discrete gel particles free of a continuous liquid phase. Such further shearing may result in expression of further quantities of the solvent which has to be removed by evaporation, for example as described above, or by mechanical means as aforesaid, before discrete particles free from a continuous liquid phase are produced.

In general we have found that discrete gel particles free from a continuous liquid phase can be obtained when the amount of solvent that has been removed is such that the composition contains at least 40% by weight of the HB polymer.

The shearing process may be conducted in a batch operation with the discrete particles being discharged when the residual solvent has reached a desired level, or in a continuous manner with fress gel being continuously, or intermittently, fed to the shearing apparatus and the discrete particles having the desired residual solvent content removed as an equivalent rate.

It is normally desirable to reduce the solvent content of the particles to below 10, often to below 5, % by weight. This effected by evaporation of the solvent from the particles while agitating the latter.

The solvent may be evaporated simply by continuing shearing the discrete particles while evaporating the solvent therefrom as described above.

Alternatively the solvent may be evaporated from the discrete particles in a suitable low temperature particle drier, e.g. a fluid bed drier wherein a gas is passed through a bed of the discrete particles with sufficient velocity to effect fluidisation of the particles. In this case it may be desirable to employ discrete particles in the form of granules obtained by extension of the gel, without melting, through a suitable die.

To obtain such granules, sufficient solvent should be removed prior to the cold extrusion that discrete granular particles free from a continuous liquid phase are formed by the cold extrusion process.

However to assist such cold extrusion, the amount of solvent removed prior to granulation is preferably such that the composition contains 40 to 60% by weight of the HB polymer.

In addition to solvent removal by evaporation from agitated discrete particles, residual amounts of solvent may subsequently be evaporated from the composition by melt processing, with evaporation of the solvent from the melt, e.g. by the use of a vented extruder, of the discrete particles. the discrete particles may be in the cold extruded granular form produced as described above. However since the composition fed to such a melt processing step preferably contains less than 10% by weight of solvent, we prefer that the solvent content of the composition is reduced to below this level, by the techniques described above, prior to melt processing. Such a melt processing step mey be used directly to produce melt fabricated, e.g. extruded or moulded, articles of the HB polymer.

While the process of the present invention is of utility wherever it is desired to separate an HB polymer from a gel forming solution, it is of particular utility as part of a process of extracting the HB polymer from micro-organism cells in such a process, where the solvent is the extraction solvent, the solution is preferably filtered, prior to forming the gel, in order to remove micro-organism cell residues.

The recovered solvent may be re-used in the extraction process, after purification if necessary.

The invention is illustrated by the following examples. In Examples 1-5 the starting solutions were solutions of poly (3-hydroxybutyrate) in 1,2-dichloroethane. These solutions were prepared by the spray drying/methanol refiuxing/1,2-dichloroethane refluxing procedure described in EP-A-15123 from an aqueous suspension of poly(3-hydroxybutyrate)-containing cells of Alcallgenes eutrophus mutant NCIB 11 599 grown by aerobic batch fermentation on glucose in an aqueous medium containing a limited amount of assimilable nitrogen source. The starting solutions were separated from the cell residue by vacuum filtration.

EXAMPLE 1 This example shows how the time required for causing a poly(3-hydroxybutyrate) solution in 1,2dichloroethane to gel, simply by cooling, varies with the solution concentration. In this example, and in Examples 2 and 3, the starting solution contained 4.1 g of poly(3-hydroxybutyrate) per 100 ml of 1,2dichloroethane.

25 ml samples of solutions of various concentrations were prepared by (a) diluting the starting solution with fresh 1,2-dichloroethane or (b) by concentrating the starting solution by vacuum distillation of 1,2-dichloroethane from the starting solution. Test tubes containing the 25 ml samples were held for 1 hour at 370C to standardise their temperatures and then placed in a deep freeze at --39 OC. The tubes were inspected at 15 minute intervals and the degree of gelation noted. A control tube containing 25 ml of 1,2-dichloroethane was similarly treated and its temperature recorded at the 1 5 minute intervals to determine the cooling rate of the samples. The results are shown in Table 1.

TABLE 1

Sample Concentration of polymer 1 2 3 4 5 6 7 (g/1 00 ml solvent) 1.03 2.05 4.1 4.7 5.5 6.6 7.9 Cooling duration Temperature Degree of gelation (min) ( C) 0 37 - - - - - - - 15 -20 - - - - - - 30 -29 - - - x x xx xxx 45 -33 - - - x xxx xxx 60 -35 - - - xxx 75 -37 - - xx 90 -37.5 - x xxx 105 -38 - xxx 120 -39 xx 135 -39 xx 150 -39 xx 165 -39 xxx no gel x gelation starting; small pieces of gel present xx gelation approximately 50% complete xxx gelation complete; coherent gel, the tube can be inverted without the gel falling out.

EXAMPLE 2 15 litres of the starting solution was left overnight in a deep freeze at -390C. The solution formed a coherent gel that was allowed to warm to 200C and then placed in a Hobart mixer fitted with a 20 litre capacity bowl and an industrial "egg-whisk" type stirrer. The mixture, maintained at 200C by an external water bath, was stirred at 50 rpm. After a short time the gel had broken into pieces and some solvent has separated therefrom. The separated solvent was removed by filtration through muslin, and the gel particles then returned to the mixer bowl. The mixer was then fitted with a "dough-hook" stirrer and stirred at 90 rpm for 3 hours. During this time solvent evaporated from the gel particles to give about 600 g of a friable powder containing less than 5% by weight of residual solvent.

EXAMPLE 3 (comparative) When 1 5 litres of the starting solution were stirred, at 20CC without prior gelation, in a 20 litre bowl using a Hobart mixer fitted with an "egg-whisk" type stirrer, fibrils formed round the stirrer, thereby clogging it up. With the "dough-hook" stirrer, as the solvent evaporated, the solution became very viscous and difficult to stir. No separation of the solvent occurred. When the solution concentration reaches about 1 5% by weight it was so viscous that it formed large sticky balls that were not broken up by the stirrer.

Similar results were obtained when higher temperatures were used and also a film of the viscous solution formed on the vessel surfaces which gave very poor heat transfer to the remainder of the solution.

EXAMPLE 4 A starting solution containing about 5 g of poly(3-hydroxybutyrate) per 100 ml of 1,2dichloroethane was caused to gel by freezing for 10 hours at -200C followed by allowing the solution to thaw to room temperature. The gel was sheared by feeding to a screw fed Hobart food mixer fitted with a die having an orifice of 6 mm diameter, and extruded therethrough, at room temperature, at a rate of about 6 kg/hr. The product, which was a slurry of gel particles in expressed solvent was collected and subjected to vacuum filtration to remove the bulk of the expressed solvent. The resultant gel particles were then re-sheared and extruded, under the same conditions, and the product re-filtered to remove a further quantity of expressed solvent. The resultant gel product was coherent mass and had a polymer content of approximately 50% by weight.

This coherent gel mass was then again sheared and extruded, under the same conditions, to give a product in the form of approximately cylindrical granules of length about 12 mm and 6 mm diameter.

These granules wre free flowing and no continuous liquid phase was present. Solvent was then evaporated from the granules by passing air at room temperature through a bed of the granules at sufficient velocity to fluidise the granules. After 1 hour the residual solvent content of the polymer pellets was less than 1% by weight.

EXAMPLE 5 A sample of the gel obtained by the freezing/thawing cycle of Example 4 was stored at room temperature but exposed to the atmosphere. After 1 week the solvent content of the gel has reduced to 49% by weight On shearing and extrusion of this stored gel under the conditions employed in Example 4, free flowing granules free of any continuous liquid phase were produced in a single pass through the food mixer. Again, after drying the granules for 1 hour in a fluid bed drier as in Example 4, the residual solvent content was below 1 R6 by weight.

EXAMPLE 6 Spray dried cells of Alcaligenes eutrophus NCIB 11 599 containing 55% by weight of poly(3 hydroxybutyrate) were refluxed with methanol for 15 minutes to extract lipids and then the cells were dried at about 300C on trays in an air current.

3.5 g of the dried, lipid-extracted, cells were then refluxed with 35 ml of methyl ethyl ketone, under superatmospheric pressure, at 1 200C for 10 minutes. The resultant slurry was cooled to 700C and filtered to separate the cell residues. The resultant solution, which contained about 6.7% by weight of the polymer, was then cooled to room temperature. The solution gelled instantaneously as the liquid temperature reached 450C.

The solvent could be removed to give a solvent-free polymer powder by shearing the gel at room temperature to produce discrete gel particles while applying a vacuum to remove evaporated solvent.

Similar results were obtained using acetone in place of methyl ethyl ketone although, because of the lower boiiing point of acetone, the filtration had to be performed under superatmospheric pressure.

The filtered solution was cooled, while still under superatmospheric pressure, and gelation occurred instantaneously as the solution temperature reached about 600C EXAMPLE 7 Dried, lipid-extracted, cells were obtained by the procedure described in Example 6 from spray dried Alcaligenes eutrophus cells containing about 60% by weight of poly(3-hydroxybutyrate).

8 g of the lipid extracted cells were refluxed with 200 ml of an extraction solvent at atmospheric pressure for 30 minutes. The resultant slurry was filtered hot to remove the cell residues and then the resulting syrup was cooled. The procedure was repeated with various methanol/chloroform mixtures as the extraction solvent.

TABLE 2

Methanol content of Polymer content Gelation Run extraction solvent of syrup characteristics % by volume % by weight of syrup 7a O 1.6 Did not gel when maintained at -25 to -300C overnight 7b 30 1.9 7c 40 1.9 gelled when maintained at -25 to -300C overnight* 7d 45 1.5 gelled after 4 hours at 200C 7e 50 0.5 gelled after 2 hours at 200C 7f 100 0 * on warming to room temperature a very weak gel was produced. On storage for 2 days at room temperature a coherent gel was obtained.

The solvent could be removed from the gels or Runs 7c-7e to give a solvent-free polymer powder by shearing the gels at room temperature to produce discrete gel particles while applying a vacuum to remove evaporated solvent.

Claims (10)

1. A process for the separation of a 3-hydroxybutyrate polymer from a solution thereof comprising (i) causing said solution to gel, (ii) shearing said gel and removing any solvent expressed therefrom until a mass of discrete gel particles that is free of a continuous liquid phase is produced, and (iii) thereafter removing solvent from the discrete particles by evaporation while agitating said particles.

2. A process according to claim 1 wherein said solution contains 0.5 to 10% by weight of said 3 hydroxyb utyrate polymer.

3. A process according to claim 1 or claim 2 wherein expressed solvent is separated mechanically from said sheared gel and then the gel is again sheared to produce said mass of discrete gel particles.

4. A process according to any one of claims 1 to 3 wherein solvent is removed by evaporation from the gel while the latter is being sheared.

5. A process according to any one of claims 1 to 4 wherein the amount of solvent removed is such that said mass of discrete gel particles contains at least 40% by weight of said 3-hydroxybutyrate polymer.

6. A process according to claim 5 wherein said mass of discrete gel particles is obtained in granular form by extrusion of a gel containing at least 40% by weight of said 3-hydroxybutyrate polymer through a die without melting.

7. A process according to claim 6 wherein said gel contains 40 to 60% by weight of said 3hydroxybutyrate polymer.

8. A process according to claim 6 or claim 7 wherein solvent is evaporated from said granules by passing a gas through a bed of said granules.

9. A process according to any one of claims 1 to 7 wherein solvent is removed from the discrete gel particles by evaporation while said particles are being sheared.

10. A process according to claim 8 or claim 9 wherein solvent is evaporated from the discrete gel particles until the solvent content is less than 10% by weight.