This invention relates to dispersions of polyhydroxyalkanoates (PHA) in water. In our co-pending applications such as GB 9502522.7 filed 9 February 1995 we describe latices of PHA containing surfactant, generally of the conventional type consisting of a single hydrophobic group having a single hydrophilic group at or near one of its ends. Such latices are of great practical usefulness, but could desirably be improved in stability and preparative convenience. According to the invention in its first aspect a dispersion of PHA particles in water is characterized by steric stabilization.

Stabilization may be provided by the presence on the surface of such particles of at least one water-soluble copolymeric dispersant. Copolymeric dispersants are characterized by containing a plurality - at least 2 and typically at least 10 and up to e.g. several hundred - of repeating units, including units of two types: A PHA - compatible; and

B hydrophilic.

Type A units may be for example aliphatic hydrocarbon (for example as in addition polymers) or aromatic hydrocarbon or (in chain lengths sufficient to give water-insolubility in a corresponding polymer consisting of such units) polyoxyalkylene, especially poly- 1,2- propylene oxide or polyester of the head-to-tail or head-to-head/tail-to-tail types such as for example, 12-hydroxy stearic acid polycondensate or alkyd resin. Preferably type A units carry substituents such as esterified carboxy groups or esterified or etherified hydroxy groups or both, since these afford greater compatibility with the PHA. Particular examples of such substituents are disclosed below.

Type B units can be anionic, for example carboxylate, sulphate, sulphonate, phosphate or phosphonate; or cationic, for example ammonium, especially quaternary ammonium; or non¬ ionic, for example polyalkylene oxide especially polyethyleneoxide, or polyglycerol or sorbitan or glycoside or amine oxide. The dispersant may contain hydrophilic groups of more than one chemical composition of ionic category. Very suitably it is polyethyleneoxy, especially 10 to 100 ethylene oxide units long, as is typical of conventional water-soluble surfactants.

The dispersant may contain a minor proportion, for example under 20 mol percent, of units falling into neither type A nor type B.

The balance of type A and type B units should be such as to provide the water solubility, which typically is at least 1% w/w in water at 20°C. Preferably the type B units are in a minority by moles, for example less than one-third of the total units in the copolymer chain; correspondingly the water-soluble portion of the type B units, if polyethyleneoxy, should be sufficiently long. The HLB number (HLB signifies hydrophile-lipophile balance rating) of the dispersant is suitably in the range 10-15. Generally the dispersant is preferably from the class of non-ionic emulsifiers, especially when the PHA particles are non-crystalline to the extent described below.

The PHA is especially capable of a relatively high level of crystallinity, for example over 30%, especially 50-90%. Although so capable, it is preferably non-crystalline to the extent described below. It typically has units of formula 1 :

- O - C _m H _n - CO - where m is in the range 1-13 and n is 2m or (except when m is one) 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 m is 3 or 4, n is 2m and especially there are units with m = 3 and m = 4 copolymerized together with respectively a C, and C ₂ side chain on the carbon next to oxygen. Particular polyesters contain a preponderance of m = 3 units, especially with at least 70 mol % of such units, the balance being units in which m = 4. The molecular weight of the polymer is for example over 50000, especially over 100000, up to 2 x 10 . PHA of formula (1 ) containing only m = 3 units may be referred to as PHB; and PHA containing m = 3 and m = 4 units is the co-polymer polyhydroxy-butyrate-co-valerate (PHBV). PHBV preferably contains 4-25% of m = 4 units. Since the intended PHA product can be a blend of two or more PHAs differing in the value of m. a corresponding mixture of suspensions can be used in the process ofthe invention. A particular example contains: (a) PHA consisting essentially of Formula 1 units in which 2-5 mol % of units have m = 4, the rest = 3; and (b) PHA consisting essentially of Formula 1 units in which 5-30 mol % of units have m = 4, the rest m = 3. The proportions of the PHAs in such blends preferably give an average m = 4 content in the range 4-25 mol %.

The PHA may be the product of chemical synthesis but is more particularly the product of a microbiological process. In such a process the microorganism may lay down PHA during

normal growth or may be caused to do so by cultivation in the absence of one or more nutrients necessary for cell multiplication. The microorganism may be wild or mutated or may have had the necessary genetic material introduced into it. Alternatively the necessary genetic material may be harbored by a eukariote, to effect the microbiological process. PHA produced microbiologically is (R)-stereospecifιc.

Examples of suitable microbiological processes are the following: for Formula 1 materials with m = 3; or m = party 3, partly 4: EP-A-69497 (Alcaligenes eutrophus); for Formula 1 materials with m = 3; US 4101533 (A. eutrophus), EP-1-144017 (AJatus); for Formula 1 material with m = 7-13: EP-A-0392687 (various Pseudomonas).

The type A unit compatibility with the PHA when in its amorphous state appears to correspond to a solubility parameter closer to that of the PHA than that of conventional paraffin- chain surfactants. The balance may also afford substantial insolubility in water at over 80°C. It is believed that in such dispersants the hydrophilic groups are so in virtue of inteφenetration of polymeric water molecules with polyethylene oxide chains, but that this structure is 'melted out' at the higher temperature.

The particles can carry a part layer of material such as surfactant other than the dispersant. The liquid phase can contain surfactant additional to that absorbed on the particles. It may contain hydrotropic compounds such as water soluble monomeric or oligomeric compounds, for example glycols and polyols.

The invention provides processes of making the dispersion, in particular by any one of: a) shearing liquid-form PHA in a solution of the dispersant; b) dissolving PHA in a liquid of low water solubility, emulsifying the resulting solution in an aqueous solution of the dispersant, and removing the liquid. This is especially convenient when the liquid is volatile, since then it can be removed by evaporation or diffusion; and c) making PHA-containing microbiological biomass, solubilizing biomass, solubilizing non-PHA material and applying the dispersant to the resulting PHA particles. In any of the above processes a dispersion of PHA in a solution of a conventional surfactant can be treated with the dispersant to replace that surfactant by the dispersant.

SUBSTITUTE SHEET (RULE 26

In the dispersion the PHA particles are on average preferably under 30, especially under 20, especially under 1 , % w/w crystalline. It appears that each individual particle is either maximally or 0% crystalline: thus the percentage crystallinity is the weight proportion of maximally crystalline particles. It is believed that the effectiveness of the dispersant may be due to surface mixing or deeper mixing of its type A domains with non-crystalline PHA. Contacting PHA particles with the dispersant is at a temperature preferably over 5°C, for example 10-50°C.

In the above processes these levels of crystallinity apply to the particles the time of contacting with the dispersant. Thus for process (c) in particular the particles should be in the never-dried virgin state.

In a particular dispersant there may be present units carrying at least one oxygen-linked hydrocarbon group. The oxygen-links may be ester or ether. Examples of esters are: (a) those of the acids acrylic (as hereinafter defined), maleic, fumaric and itaconic, with C _g alcohols and phenols; (b) those of allyl alcohol or the notional vinyl alcohol with C ₈ carboxylic acids.

Examples of ether groups are those of allyl alcohol or the notional vinyl alcohol with C ₈ alcohols and phenols. Such alcohols and carboxylic acids can be straight-chain, branched or cyclic but, if substituted, do not include groups conferring water-solubility on the polymer in the proportion used. The above-mentioned esterifying alcohols and carboxylic acids and etherifying alcohols preferably each contain at least 2, preferably up to 9, carbon atoms.

Other type A units can be the residues of for example one or more of ethylene, propylene, styrene, vinyl halides, vinylidene halides, vinyl methyl ether, vinyl acetals, vinyl carbonate, acrylic (as hereinafter defined) nitrile or methyl ester and conjugated olefins. The term 'acrylic' is herein defined by the general formula:

-CH ₂ -C(R,) -

I C=O

OR ₂

where R] is hydrogen C ₂ alkyl (especially methyl), cycloalkyl, aryl, halogen or cyano and R ₂ is a C,. _|8 hydrocarbon group. The analogous definition of R[ applies to corresponding nitrile units if present.

In its second aspect the invention provides a process of making a PHA latex by the steps of:

(a) producing a biomass containing particles of PHA and non-PHA cell material (NPCM); (b) solubilizing the NPCM;

(c) suspending the PHA particles in an aqueous solution of a dispersant as herein defined. Step (b) can be carried out by methods involving, for example, one or more of the following: (i) heat shock

(ii) hydrolase solubilization of glycopeptides (iii) protease solubilization of proteins (iv) solubilization of nucleic acids (v) surfactant action on proteins (vi) oxidation by hypochlorite

(vii) oxidation by peroxide

Methods (vii) and (v), possibly following (i) and (iii) and/or (ii) and (iv) are preferred. If the dispersant is sufficiently soluble in water at the required operating temperature it can be used as the solubilizing surfactant in (v); otherwise a conventional surfactant can be used in the solubilization and thereafter replaced or supplemented by the dispersant.

In its third aspect the invention provides a process of producing dry PHA suitable for solvent processing or melt processing, by the steps of:

(a) producing a biomass of PHA-containing cells in water;

(b) solubilizing and removing at 50% w/w of NPCM by one or more of the methods specified for step (b) above;

(c) stabilizing an aqueous dispersion of the resulting impure PHA by means of the dispersant herein defined;

(d) concentrating the dispersion to e.g. at least 200 g/l of PHA;

(e) applying further steps of NPCM solubilization and removal to the so- concentrated dispersion.

Steps (d) and (e) may be carried out by separating a cake or pellet of PHA and redispersing it.

The dispersion containing the dispersant, whether concentrated or not, can be used directly for puφoses such as coating water-sensitive materials such as paper and board as described in our co-pending application above mentioned. Alternatively it can be converted to solid PHA, possibly via steps or particle agglomeration at over 80°C. EXAMPLE

A raw PHA latex was produced by the following steps:

1 growth of Alcaligenes eutrophus on glucose substrate in an aqueous medium containing sources of nitrogen and phosphate and usual other nutrients and trace elements until phosphate exhaustion; 2 accumulation of 76.6:23.4 mol percent PHBV by feeding further glucose and also sodium propionate and a trace of phosphate until the rate of accumulation became slow;

3 heat shock at 150°C;

4 treating with proteolytic enzyme to effect solubilization of NPCM. The raw latex was treated further as follows: (a) Samples of raw latex containing 134.3 g/kg of solids (90% PHA) and 38.6 g of solubles were formulated as follows:

A No further addition;

B 0.5% w/w of dispersant CG6 which is an acrylic graft copolymer formulation in water/propylene glycol containing 32% w/w of active agent of HLB number approximately 1 1 -12, available from Imperial

Chemical Industries PLC under the name HYPERMER (RTM) CG6.

C, D, E 0.99% 3.0%o, and 4.97% w/w of dispersant A respectively.

(These percentages are calculated on the PHA).

Each sample was centrifuged at 4300 φm for 30 min. The supernatant was discarded and the pellet resuspended in deionized water. The particle size distribution of the resulting suspension was then measured. The samples that did not flocculate were then centrifuged and resuspended again to determine if the dispersant was washed off the solids. The particle size distribution of the samples that did not flocculate are shown in

Table 1.

TABLE 1

1 st Resuspension 2nd Resuspension

Dispersant w/w w/w CG6 (% w/w)

10% 50% 90% 10% 50% 90% (μm) (μm) (μm) (μm) (μm) (μm)

0 00 2 34 58 46 502 77

0 50 0 64 2 92 48 84

0 99 0 58 1.52 20 13

3 00 0 53 1 1 1 3 16 0 60 3 00 50 96

4 97 0 53 1 09 2 77 0 58 1 81 33 44

Even 0.5% addition of dispersant had a significant effect on the redispersibihty of the centrifuge pellet The lowest dose that prevented flocculation completely was 3% When centrifuged and resuspended a second time, both the 3% and 5% pellets flocculated This is explained as follows ¹ when resuspended the first time the solubles become diluted 10 to 20 fold, reducing the dispersant concentration to between 0.15 and 0.5% The particle sizes obtained after the second centrifugation are similar to those from the 0 5% addition to the original material. This suggests that the dispersant would need "topping up" after each centrifuge wash, or that resuspension should be carried out in dispersant solution at suitably 0.5% w/w.

(b) Samples of raw latex containing 368 g/kg of solids, viscosity initially 8 to 15 mpas were formulated with 3% w/w of various surfactants and dispersants and tested in a Bohlin Rheometer system at 25°C, shear rate 1460 sec ^" for periods up to 1 hour. Their viscosities were observed to increase grossly at times as shown in Table 2. TABLE 2

Surfactant Type Viscosity Increase Time, ks

Aerosol OT Dioctyl sulphosuccinate 0 25

SDS Sodium dodecylsulphate 0 25

Nansa AS40 Sodium dodecylbenzene- 0 3, 0 6 sulphonate

Sarcozyl Sodium lauryl sarcosinate 0 3

Dowfax 3A 1 Sodium dodecyldiphenyl oxide 0 6 disulphonate

Surfactant Type Viscosity Increase Time, ks

No Addition 1.2

Triton 405 Octylphenoxy PEO, HLB 17 9 2 7 (slow inc from 1 5)

Synperonic PE/F 108 (80 % w/w polypropylene over 3 5

(oxide NW 3250 (20% w/w polyethylene

(oxide (total MW 50000 approx

CG6 Acrylic graft copolymer over 3 5

It is evident that most of the conventional surfactants provoke a viscosity increase at time much shorter than in their absence; but that the dispersants stabilize the viscosity.

(c) A sample of the raw latex was digested with hydrogen peroxide and surfactant Synperonic A20 (TTM) (C, ₃ alkyl 20 mols ethylene oxide, 19 g/l), at 80°C for lOh and allowed to cool. A test sample was evaluated for crystallinity by centrifuging with 40% w/v Nycodenz (RTM) at 15000 φm for 15 min. No crystalline fraction was observed. Three further samples were centrifuged without Nycodenz and the attempt made to resuspend the resulting solid pellets in water. Results are shown in Table 3. TABLE 3

CG6 Ccntπfug Ccntπfug pellet Supernatant Resuspendabi ty Crystalline/amorphous

% w/v e speed e mm φm

0 9000 10 very very clear very difficult to crystalline firm resuspend any pelleted mateπal

0 5 9000 10 firm clear difficult crystalline

0 5 5000 5 soft very cloudy very eas amoφhous ca (50% solids pelleted out)

It is evident that it is possible to resuspend amoφhous latex particles if dispersant CG6 is present, even at 0.5% w/v, provided centrifugation conditions are not too severe.

Optimization is likely to lead to conditions intermediate between the second and third runs in Table 4, possibly (see Table 1 ) at a higher CG6 concentration. EXAMPLE 2

A PHA latex was prepared by the following steps: 1 Powdered crystalline PHVB (76.6:23.4 by moles) was dissolved in chloroform to give a

5% w/w solution;

2 The solution was emulsified in an aqueous solution (1% w/v) of Sarkosyl (RTM) (sodium N-lauroyl sarcosinate).

3 The chloroform was removed by stirring in a current of nitrogen leaving a latex of solid amoφhous particles of average diameter 0.56 μm. The latex was concentrated to 6% w/v solids and freed of excess Sarkosyl by dia-filtration. It was then conentrated to 40% w/v solids by evaporation. Samples 2A (without CG6) and 2B (with CG6 5% w/v on the PHVB) were tested for shear ability in a Bohlin Rheometer system at 21°C, 3000 φm: The times to flocculation were:

2A: 500 sec 2B: over 3600 sec.

It is evident that the dispersant is as effective for emulsification-route latex as it is for virgin latex.