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Extraction Process Europaisches Patentamt •J European Patent Office @ Publication number: 0124 309 A2 Office europeen des brevets D EUROPEAN PATENT APPLICATION g) Application number: 84302508.1 © Int. Cl.3: C 12 P 7/62, C 08 G 63/72 g) Date of filing: 12.04.84 © Priority: 28.04.83 GB 8311677 @ Applicant: IMPERIAL CHEMICAL INDUSTRIES PLC, Imperial Chemical House Millbank, London SW1P3JF (GB) @ Date of publication of application: 07.1 1.84 (72) Inventor: Stageman, John Frederick, 30 Linden Avenue, Bulletin 84/45 Darlington County Durham DL3 8PP (GB) @) Representative: Gratwick, Christopher et al, Imperial Chemical Industries PLC Legal Department: Patents Po @ Designated Contracting States: DEFRGBIT Box 6, Welwyn Garden City Herts, AL7 1 HD (GB) @ Extraction process. A continuous process for the extraction of 3-hydroxy- butyrate polymers from micro-organism cells wherein a non- solvent is added to the extracted syrup to permit separation of the polymer and, by distillation, the bulk of the extraction liquid, which may be a solvent/non-solvent mixture, is recov- ered for re-use. Preferably the non-solvent added to the syrup is also recovered in the distillation. This invention relates to an extraction process and in particular to the extraction of 3-hydroxybutyrate polymers from micro-organisms. Poly(3-hydroxybutyrate) is a thermoplastic polyester consisting of repeat units of the formula -CH(CH3) . CH2.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 assimil- able 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 accumulation. Examples of suitable processes are described in EP-A-15669 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 micro- biologically produced heteropolymer containing 3-hydroxybuty- rate and 3-hydroxyvalerate residues is described by Wallen et al "Environmental Science and Technology" 8 (1974) 576-9. Also, as described in EP-A-52459 and 69497 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-hydroxy- butyrate), but also copolymers as described above, provided that the 3-hydroxybutyrate residues form at least 40 mole %, and pre- ferably 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-3107172, it is generally desirable to separate the polymer from the re- mainder of the cell material. Extraction processes that have been proposed involve contacting the cells with an extraction liquid and then the extrac- tion liquid, containing the dissolved HB polymer, is separated from the cell residue. Extraction liquids that have been proposed include various partially chlorinated hydrocarbons such as chloro- form, 1,2-dichloroethane, and methylene chloride, and cyclic carbon- ates such as propylene carbonate, and certain mixtures, e.g. chloro- form/methanol (see US-A-3275610) and dichloromethane/ethanol (see US-A-3044942). In order to obtain good yields, generally the cells are subjected to a cell disruption step to render the cell walls permeable to the extraction solvent. Examples of cell disruption procedures include treatment with acetone (see US-A-3036959 and 3044942); ultrasonic vibration, grinding, French pressing, freez- ing/thawing cycles, and lysozyme treatment (see US-A-3275610); flocculation (see EP-A-46017); and milling, and spray or flash drying (see EP-A-15123). Spray drying is particularly suited to large scale operation. The extraction processes give a solution of the HB polymer in the extraction liquid. Hereinafter this solution is referred to as the syrup. Unless the syrup is to be used as such, e.g. for casting films, it is necessary to separate the HB polymer from the syrup. Simple evaporation of the extraction liquid from the syrup presents difficulties since the syrup vis- cosity rises rapidly as the extraction liquid is removed: syrups containing more than about 15% by weight of HB polymers are ex- tremely viscous and difficult to handle. Separation of the HB polymer by precipitation simply by cooling the syrup is generally not feasible when using the extraction liquids proposed heretofore except where the extraction liquid is a cyclic carbonate. However, cyclic carbonates are relatively expensive and are also high boiling: hence recovery and purification of the extraction liquid for re-use tends to be highly energy consuming. Cooling of syrups made using 1,2-dichloro- ethane as the extraction liquid can give rise to coherent gels from which the liquid be removed as described in EP-A-24810 and 58480 or GB-A-2120671. However the techniques necessary for obtaining satisfactory gels from 1,2-dichloroethane syrups tend to be time consuming and unsuitable for large scale operation. In alternative method of separating the HB polymer from the syrup is by precipitation by mixing the syrup with a liquid in which the HB polymer is insoluble but with which the extraction liquid is miscible. Petroleum ether, methanol, and methanol/water mixtures are examples of such precipitants. Again, recovery of the extraction liquid often presents serious difficulties. We have now found that with certain solvent/non-solvent combinations, extraction of the polymer, separation of the polymer from the syrup, and recovery of the extraction liquid for re-use is relatively simple. Accordingly the present invention provides a continuous, or cyclic batch, process for the extraction of an HB polymer from dried, solvent-permeable, HB polymer-containing micro-organism cells comprising: (a) contacting said cells with an extraction liquid to form a syrup containing HB polymer dissolved in the extraction liquid, said extraction liquid con- taining sufficient of a solvent for said polymer that said extraction liquid dissolves said polymer under the extraction conditions, (b) separating said syrup from the undissolved cell residue, (c) treating said syrup by adding thereto a sufficient quantity of a liquid composition, that is miscible with said syrup and that has a lower content of said solvent than said extraction liquid and com- prises an organic, non-solvent, liquid or a mixture thereof with said solvent, that said syrup is con- verted to a composition that can be mechanically separated into a solid polymer phase and a liquid phase, and effecting said mechanical separation, (d) distilling at least a proportion of said separated liquid phase to recover therefrom a first component consisting of said solvent or a mixture of said solvent and said non-solvent that is richer in said solvent than said liquid phase, and a second com- ponent consisting of said non-solvent or a mixture of said solvent and non-solvent that is richer in said non-solvent than said liquid phase, and (e) recycling at least a proportion of said recovered first component to the syrup forming stage, the proportion of said liquid phase that is subject to distillation and of said first component that is recycled being such that at least 8Q% by weight of said extraction liquid consists of said recycled first component. In the first stage of the process, the micro-organism cells that have been dried and rendered solvent-permeable, e.g. by spray drying, are contacted with an extraction liquid to form the syrup. For optimum extraction the cells are preferably dried so that their water content is less than 5% by weight. The extraction, or syrup-forming, stage may be conducted at elevated temperatures, preferably above 50°C, but is preferably conducted at a temperature below 125°C to minimise the risk of thermal degradation of the polymer. To minimise costs of recovery of the extraction liquid, the latter preferably has a boiling point, or boiling range, at atmospheric pressure below 125 C. The extraction should be conducted under conditions such that the cells are contacted with the extraction liquid with the latter in the liquid state, e.g. by refluxing. In some cases it may thus be desirable to operate the extraction step under superatmospheric pressure to maintain the presence of the extrac- tion liquid in the liquid state. The extraction liquid consists of a solvent for the polymer or a mixture of a solvent and a non-solvent. By the term solvent, we mean a liquid that will dissolve the polymer in the cells under the extraction conditions. Particularly suitable solvents include partially halogenated hydrocarbons such as dichloromethane, chloroform, and 1,2-dichloroethane. Where the extraction liquid contains a non-solvent, the proportion thereof should be such that, under the extraction conditions, the extraction liquid will dissolve the polymer in the cells. Preferably the extraction liquid contains at least 60% by weight of solvent. The amount of extraction liquid and the extraction conditions are preferably such that at least 75% by weight of the HB polymer in the micro-organism cells is extracted. The amount of extraction liquid employed will depend on the HB poly- mer content of the micro-organism cells, the proportion of this polymer that is extracted by the extraction liquid and the desired syrup concentration.
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