WO1994026917A1

WO1994026917A1 - Polyester purification

Info

Publication number: WO1994026917A1
Application number: PCT/GB1994/000950
Authority: WO
Inventors: Timothy Hammond; John Mcdonald Liddell
Original assignee: Zeneca Limited
Priority date: 1993-05-07
Filing date: 1994-05-04
Publication date: 1994-11-24
Also published as: GB9309453D0; AU6579394A

Abstract

A method of purifying microbiologically produced polyester comprising removing microbial lipids and pigments from the polyester by washing with organic solvent characterised in that the washing with organic solvent is carried out after removal of non-polymer cell wall material from the polyester, and preferably after removal of the non-polymer cell wall material by enzyme treatment.

Description

POLYESTER PURIFICATION

THIS INVENTION relates to polyester purification and in particular to purification of microbiologically produced polyester giving polyester which is low in impurities and thereby less liable to discolouration at melt processing temperatures.

Methods of extraction of polyesters such as polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) have included the steps of harvesting the bacterial cells from the aqueous fermentation medium, e.g. by centrifugation, to give a mass of wet cells which are then contacted with acetone to effect drying and cell breakage. After removal of the acetone, the PHB (or PHBV) is extracted with a suitable solvent, e.g. pyridine (OSP 3036959) or a dichloromethane/ethanol mixture (USP 3044942). Such methods have the advantage that in addition to effecting drying and cell breakage, acetone also extracts lipids and pigments (if any) which would otherwise contaminate the product.

However, treatment of a mass of wet cells with acetone to effect drying and cell breakage is not economic on a large scale.

It is also possible to extract PHB or PHBV directly from the aqueous cell suspension produced by fermentation, preferably after some concentration, by contact with certain solvents such as chloroform, dichloromethane, or 1 ,2-dichloroethane, with, where necessary, a cell disruption step, e.g. milling, prior to contact with the solvent. However, the solvent and extraction conditions have to be selected with care to avoid undue uptake by the solvent of non-polyester material particularly lipids and pigments (if any) present in the bacterial cell. Not only does such non-polyester material contaminate the product and so present purification difficulties but also the co-extraction of lipids may tend to result in the formation of a relatively stable emulsion between the solvent and aqueous phases rendering separation thereof difficult. With such a direct extraction process, a separate lipid extraction step prior to contact with the PHB or PHBV extraction solvent is generally not practical as the solvents that extract lipids would need to be removed, together with the lipids, prior to contact with the PHB or PHBV extraction solvent and, because the more effective lipid solvents tend to be water miscible, such removal of the lipid solution presents practical difficulties.

EP 001 51 23 discloses a process in which the bacterial cells containing PHB are weakened by a drying process, the lipids and pigments are extracted with solvent and then PHB is extracted by contact with an extraction solvent, such as pyridine, cyclic carbonates and partially haiogenated hydrocarbons, such as chloroform, dichloromethane and 1 ,2-dichloroethane. In this process it is preferred to subject the cells to the lipid/pigment extraction step prior to extraction with the extraction solvent. The lipids and pigments may be extracted with acetone, methanol, ethanol, butanol, hexane, or petroleum ether.

EP 145233 discloses a process in which non-polymer cell material is soiubilised, preferably enzymatically, leaving the PHB undissolved and over several stages it is possible to obtain PHB products of progressively increasing purity. In all such processes it is very desirable to remove lipids and pigments from the microbially produced polyester because such materials cause yellowness of the final processed polymer and also give off a pungent odour.

Thus, according to the invention in its first aspect there is provided a method of purifying microbiologically produced polyester

(PHA) comprising removing microbial lipids and pigments from PHA by washing with organic solvent characterised in that the washing with organic solvent is carried out after removal of non-polymer cell wall material from PHA. The polyester (PHA) is especially a polyhydroxyalkanoate capable of a relatively high level of crystaliinity, for example over 30%, especially 50-90%, in the absence of plasticiser. Suitably such PHA is at least one having units of formula (I):

- O - C_m H_n - CO - (I) where m is in the range 1 -1 3 and n is 2m or (if m is at least 2) 2m-2.

Typically C_m H_n contains 2-5 carbon atoms in the polymer chain and the remainder (if any) in a side chain. In very suitable polyesters n is 2m and especially there are units with m = 3 and m = 4 copolymerised and with respectively a C. and C₂ side chain on the carbon next to oxygen in the chain. Particular polyesters contain a preponderance of m = 3 units, especially over 95 mol% of such units, the balance being units in which m = 4. The molecular weight Mw of the PHA is for example over 50000, especially over 100000, up to e.g. 2 x 10⁶. In PHAs having units with m = 3 and m = 4 there may be very small, typically fractional, percentages of units having higher values of m.

Especially preferred are polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-valerate (PHBV) and polyhydroxy-octanoate.

The PHA can be a product of fermentation, especially of a microbiological process in which a microorganism lays down PHA during normal growth or is caused to do so by cultivation in starvation of one or more nutrients necessary for cell multiplication. The microorganism may be wild or mutated or may have the necessary genetic material introduced into it. Alternatively the necessary genetic material may be harboured by an eukaryote, to effect the microbiological process.

Examples of suitable microbiological processes are the following: for Formula I material with m = 3 or m = partly 3, partly 4 : EP-A- 69497 (Alcaliqenes eutrophus) for Formula I materials with m = 3: US 4101 533 (A. eutrophus H-1 6)

EP-A-14401 7 (A. latus) : for Formula I material with m = 7-1 3:

EP-A-0392687 (various Pseudomonas).

The PHA is isolated from the fermentation product cells by removing cellular material leaving microscopic granules of PHA. A typical method for isolating PHA is to heat shock the fermentation cell mass which causes slight damage to the cell walls, followed by an enzyme treatment in which the cell walls and general cell debris are broken down and solubilised in aqueous solution. The isolated PHA is subjected to a number of aqueous and detergent washes. The PHA at this stage is still in aqueous suspension and to isolate it as, for example, PHA granules, it may be spray dried or thermally agglomerated according to the process described in PCT/GB 93/01465. The aqueous suspension will still contain some cell remains notably lipids and pigments which are carried through to the PHA granule or powder.

Therefore, in a preferred embodiment of the process of the present invention the lipids and pigments are removed from PHA by washing with organic solvent after non-polymer cell material has been removed by enzyme treatment.

Thus, according to a further aspect of the present invention there is provided a method of purifying a polyester comprises the steps of: (a) cultivating a microorganism in conditions in which it accumulates polyester in its cells; (b) enzymatically decomposing into water soluble products constituents of such cells other than such polyester;

(c) washing the resulting polyester with water; and

(d) drying the washed polyester: and is characterised by washing the granules after step (b) with a organic solvent.

The organic wash may follow (b) immediately, or follow the water wash in step (c). For operational convenience the polyester may be agglomerated into larger granules preparatory to the organic wash. For this reason the organic wash may preferably be applied to dried granules or powder from step (d). The drying step need not remove water to an extent greater than necessary for such agglomeration, since the organic wash removes remaining water. After the organic wash the granules are preferably dried to remove the organic solvent.

In a further aspect of the present invention there is provided a method for preparing polyester comprising cultivating a microorganism under conditions in which polyester is accumulated in its cells and subjecting the polyester to a purification method as disclosed herein. The organic solvent usually has a boiling point at atmospheric pressure which is less than 200°C, preferably less than 100°C. The solvent is preferably water-miscible, especially a water-miscible alcohol such as methanol, ethanol, n-propanol, isopropanol or tert-butanol; or the solvent may be acetone, ethylene glycol or other aliphatic diols and tetrahydrofuran alcohols. Any of the previous mentioned lipid extraction solvents could be used in mixture with each other or any other non-PHA- dissolving solvents, for example, diethyl ether. The solvent must not dissolve the PHA. One or more other organic solvents may be present, provided the mixture of organic solvents is still water miscible. It is not necessary that the solvent is water miscible if the solvent is used in the later stage of the process, i.e. after step (d). In this case, as there is no water present, the solvent could be water immiscible, for example, petroleum ether or hexane.

The solvents methanol, ethanol or a mixture of methanol and ethanol are particularly preferred solvents for the present invention. The purified polyester can' be used in any of the known methods and processes for making any of the known shaped articles, for example, fibres, films, sheets and vessels and is especially valuable when those articles have to meet high purity standards or to be minimally off-white or to be bright in colour, or to have a low odour The polyester purified according to the present invention is useful in all processing of the polyester which involves heat in the process, for example, extrusion, production of film, injection moulding, thermoforming, fibre spinning and blow moulding. The process of the present invention is particularly useful for providing polyester of a purity suitable for use in medical applications.

Another important use of the purified polyester is where multiple or lengthy high temperature heat treatments are required.

The invention is now particularly described with reference to the following example but is not limited thereby. EXAMPLE 1

Example 1 demonstrates removal of lipid and pigment impurities by using the yellowness of the final polymer as a measure of the efficiency of removal. Methanol Washing PHBV copolymer is prepared according to methods described hereinbefore. 250 kg of PHBV was charged to a Rosenmund pressure filter (stainless steel), the pressure filter was purged with nitrogen and 1 m³ methanol added. The entire system was heated to 30°C and the PHBV allowed to soak in the methanol at 30°C for 90 minutes. The methanol was filtered off under pressure until 1 -2cm of methanol was left above the polymer bed. The pressure was released and a further 1 m³ of methanol added and the PHBV was allowed to soak in the methanol without heating for 90 minutes. Then the methanol was filtered off as previously described and further 1 m³ of methanol was added and the

PHBV was allowed to soak in the methanol without heating for 90 minutes. The methanol is then discharged completely under pressure. The PHBV was then dried using a nitrogen blower system and a sample used in the next stage of the testing. Yellowness Index Measurement '

Preparation of samples :

1 .Og of polymer (unwashed or methanol washed) was placed in a mould between two pieces of Melinex^" film. The mould was heated to above Tm (approximately 170°C) to melt the polymer, left for 1 minute, then subjected to pressure at 10, 1 5 and 20 tons and sudden removal of this pressure at each level, in order to remove the air bubbles. The pressure was raised again to 20 tons and the sample held at this pressure. The pressure and heat was removed after 3.5 minutes from the start of the heating stage of the process. The mould was quickly placed into water at 60°C and left until the sample was completely crystallised before removing the film. The moulded sample was dried and trimmed. Samples which contained air bubbles, particulates or had not been completely melted were rejected. The sample was then conditioned at 23°C + /- 2°C for 40 hours in the dark. The sample thickness was measured and any which were not from

1 .3mm to 1 .5mm were rejected.

The yellowness of the sample was measured by a Minolta CR-300 tristimulus colorimeter. Three measurements per sample were taken and the mean value of yellowness was calculated. ^" = trademark. PHBV % YELLOWNESS

BATCH PHB PHV METHANOL NO NO. MOL % MOL % WASH METHANOL WASH

1 85 1 5 41 -44 54

2 94 6 48 84-94

3 94 6 59 76

Conclusion

In all tests the yellowness of the polymer was significantly reduced, particularly when the polymer contained 6% HV units. This is a particularly useful result as the high yellowness of the control sample indicated a very high level of impurities prior to washing.

Claims

1 Method of purifying microbiologically produced polyester comprising removing microbial lipids and pigments from the polyester by washing with organic solvent characterised in that the washing with organic solvent is carried out after removal of non-polymer cell wall material from the polyester.

2 Method according to claim 1 wherein the cell wall material is removed from the polyester by enzyme treatment.

3 Method according to claim 2 wherein the method of purifying the polyester comprises the steps of:

(a) cultivating a microorganism in conditions in which it accumulates polyester in its cells;

(b) enzymatically decomposing into water soluble products constituents of such cells other than such polyester;

(c) washing the resulting polyester with water; and

(d) drying the washed polyester: and is further characterised by washing the polyester after step (b) with a organic solvent.

4 Method according to claim 3 which is further characterised by washing the polyester after step (c).

5 Method according to claim 3 which is further characterised by washing the polyester after step (d).

6 Method according to any of claims 1 to 5 wherein the organic solvent has a boiling point less than 200°C at atmospheric pressure.

7 Method according to any of claims 1 to 6 wherein the organic solvent is selected from the group: methanol, ethanol, n-propanoi, isopropanol or tert-butanol, acetone, ethylene glycol, other aliphatic diols, petroleum ether, hexane and tetrahydrofuran alcohols, or a mixture of any of this group with each other or another solvent.

8 Method according to any of the preceding claims wherein the solvent is a water-miscible alcohol. Method according to claim 8 wherein the water-miscible alcohol is methanol or ethanol or a mixture of methanol and ethanol. Method of preparing a polyester comprising cultivating a microorganism under conditions in which polyester is accumulated in its cells and subjecting the polyester to a purification method as defined in any of claims 1 to 9.