CA2239871A1 - Polyester particles - Google Patents

Abstract

This invention relates to a process for producing polyhydroxyalkanoate (PHA) that overcomes a problem encountered when seeking to separate PHA particles without centrifugation. The process comprises producing a biomass containing PHA particles and non-PHA cell material (NPCM), solubilising the NPCM, oxidizing the resulting product mixture to further solubilise NPCM, adding a water soluble surfactant to produce a suspension, and heating the suspension below the melting point of the PHA to agglomerate the PHA particles to a suitable weight average diameter. In one emdobiment, the surfactant is a monomeric water soluble surfactant. In another embodiment, the weight average particle diameter is 200 µm to 500 µm.

Description

W O 97122654 PCT~US96/20016 POLYESTER PARTICLES

This invention relates to polyester particles and in particular to a process for producing such particles by agglomer~tinp finer particles laid down in microbiological cells. Such polyester is hereinafter referred to as PHA.
GB application 9215791.6 filed 24 July 1992 and published as WO 94/02622 describes a process of agglomerating PHA particles in suspension in water optionally co~ inp at least partly chemically degraded non-PHA microbial cell matter (NPCM) by m~int:~ining the suspension at a relatively high l~ cl~ , for example over 100~C, but at least 30~C below the o peak melting point of the PHA (as determined by differential sc~nninp calorimetry). The Examples show operation at 130~C for 30 min, 126~C for 2 min or (in a continuous process) at 125~C for a residence time of 1 min. It is indicated, and has been found in practice that, before the high t~ln~ dLule treatment it is desirable to separate the particles resulting from the preceAing chemical degradation step (çhemi(~l includes enzymatic and/or heating in water), re-lS suspend them in a second liquid medium. Such separation and re-suspension are, however, inconvenient, since they involve centrifugation of very fine particles.
GB application 9307674.3 filed 14 April 1993 and published as WO 94/24302 discloses recovery of PHA particles by solubilising NPCM with an oxidizing agent in the presence of a chel~ting agent. The effect of the chelating agent is that the oxidizing tre~tm~nt can be applied to a PHA suspension that has not been subjected to separation and re-suspension and thus contains heavy metal ions present as trace elements in the microbiological ferment~tion and which now would catalyze decomposition of the oxidizing agent if not chelated.
It has been found, however, that the agglomeration process of WO 94/02622 does not readily take place when applied to a starting suspension that has not undergone the separation and re-suspension, for example a suspension made by the process of WO 94/24302. The mech~ni~m of this effect is believed to be that degraded NPCM includes a component that is not decomposed by hydrogen peroxide, yet has surface activity sufficient to stabilize small PHA
particles against agglomeration.
It has now been found that addition of a surfactant to a suspension of PHA particles in such a solution of NPCM solubilisation products promotes high temperature agglomeration of such particles.

W O 97/22654 PCT/US96~0016 According to the invention a process for producing R-stereospecific PHA comprises rnicrobiological ferment~tion, harvesting PHA particles by solubilising NPCM andagglomerating them by heating the aqueous suspension thereof, and is characterized by the succession of steps:
5 (a) partly solubilising NPCM by the action of for example one or more of proteolytic enzyme, hydrolase lysozyme, homogenization and heat-shock;
(b) reacting the product of (a) oxidatively in conditions effecting further solubilisation of NPCM;
(c) providing for the presence of a water soluble surfactant preferably a monomeric water soluble llrf~f t~nt, and (d) agglomerating the suspended particles produced in (c) by heating the suspension at a temperature below the melting point of the PHA as measured by differential sc~nning calorimetry (DSC).
A preferred subsequent step (e) is defined below.
In this process step (a) can be carried out by methods well established in industry and research, for example as described in EP-A-145233. Step (b) can be applied to the whole product of step (a) or to that product after subjection to minor procedures such as concentration but short of separation and re-suspension.
Oxidative step (b) is preferably carried out by the action of a peroxide (especially hydrogen peroxide); preferably in presence of a chelator. Suitably the process of WO 94/24302 is used. Typically the PHA content is in the range 60-250 g/l. The pH is suitably in the range 5 to 9 and the tclllpel~Lulc in the range 60-180~C. Suitable chelators are ethylene ~ mine tetra-acetic acid, nitrilo triacetic acid, citric acid and diethyl~n~ mine penta-methylenephosphonic acid.
2s By monomeric water soluble surfactant is meant a compound co~ lg in its molecule a small number of hyclrophobic groups which are C6+ aliphatic hydrocarbon chains (especially I
or 2 such chains) and a small number of hydrophilic groups (especially 1 or 2 such groups). The said chains and groups may be connected by direct links or through for example at least one of oxygen, ester, arnide or aromatic hydrocarbon. The hydrophilic group(s) may be:

anionic, for example, carboxylate, sulphonate, sulphate, phosphonate or phosphate; or cationic, for example qn~"~.y arnrnonium; or non-ionic, for example polyalkyleneoxy, poly glycerol, glycoside or arnine oxide; or a combination of two or more of these. Such surfactants are generally characterized by micelle s formation in aqueous solution and they decrease the surface tension of water markedly. It may be that their hydrophobic groups are capable of wetting the surface of the PHA granules but not of penetrating such granules. Such wetting may be less favored as the granules agglomerate and crystallize.
It appears that s--rf~et~nf~ having a negative temperature coefficient of water solubility o are preferable. Good results have been obtained using non-ionic surf~rt~nt~, especially those having a C1o 20 hydrophobic group and 6 to 100 ethylene oxide units.
The process is especially applicable to suspension initially free of such cllrf~ct~nt~
especially those forrned by subjecting a fermentation biomass at tempcldLul~s in the range 100-200~C and/or to the action of proteolytic enzymes, in each case without addition of surfactant.
However, it is also usable in processes in which the biomass has already undergone tre~tment in presence of a surfactant ("first surfactant"): then steps (b) and (c) at least partly overlap. In such processes the same surfactant may suffice for the agglomeration step, or the content of surfactant may be sllhst~ntiçllly increased or a different surfactant added. Usually the first surfactant is not removed or inactivated and replaced by another; but this is not excluded. The surfactant can, in 20 general, thus be introduced at any convenient stage, provided that it is present in step (d).
The content of ~ulra;lal L required depends on the detailed operating conditions, in particular:
extent of prior decomposition of NPCM;
concentration of solubilised NPCM (which in turn depends on the content of PHA in the 2s microorganism cells);
type of surfactant and balance of hydrophobic and hydrophilic groups;
extent of further NPCM (adsorbed or solubilised) decomposition;
extent of agglomeration required;
time available;

W 097/22654 PCT~US96/20016 temperature. r Typically 0.1 to 10, especially 0.5 to 7, % w/w on the PHA is used.
Step (d) can be carried out in presence of polymer processing additives such as pigments, nucleants and plasticisers, such that co-agglomeration takes place.
The temperature in step (d) is by 30-80, preferably 40-70, ~C lower than the melting point of the PHA as measured by DSC. Typically the temperature is over 100~C andagglomeration is carried out under superatmospheric pressure. It will be appreciated that the te~ dlul~ is stated in terms of the DSC melting point of the PHA because the PHA particles at the time of agglomeration are in transition between the amorphous state and the crystalline state, o so that their melting point cannot be known.
The PHA is especially capable of a relatively high level of crystallinity, for exarnple over 30%, especially 50-90%. It typically has units of formula 1:
- O - CmHn - CO -where m is in the range 1-13 and n is 2m or (except when m is unity) 2m-2. Typically CmHn contains 2-5 carbon atoms in the polymer chain and the rem~in-lPr (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 Cl and C2 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 eg 2 x 106.
PHA of formula (1) cont~ining only m = 3 units may be referred to as PE~B, and PHA
c~ lg 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 intçnflPtl PHA product can be a blend of two or more PHAs differing in the value of m, a corresponding mixture of fermf~nt~tion products or suspensions can be used in the process of the invention. A particular example contains:
(a) PHA con~i~ting ç~Pnti~l~y of Forrnula 1 units in which 2-5 mol % of units have m = 4, the rest m = 3; and W O 97/226~4 PCT~US96/20016 (b) PHA consi~tin~ 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 are preferably such as given an average m = 4 content in the range 4-25 mol %.
s In the initial microbiological 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 mllt~ted or may have had the necessary genetic material introduced into it. Alternatively the necessary genetic material may be harboured by a eukariote, to effect the microbiological process.
o Examples of suitable microbiological processes are the following:
for Formula l materials with m = 3, or m = partly 3, partly 4: EP-A-69497 (Alcali~enes eukophus~;
for Formula l materials with m = 3: US 4101533 (A. eutrophus), EP-A-144017 (A. Iatus), for Formula 1 material with m = 7-13: EP-A-0392687 (various Pseudomonas).
Whereas the starting PHA particles are typically of weight average diameter in the range 0.1 to 5 llm, the process of the invention typically increases this to at least S0, preferably 100-5000, for example 200-500, ,~Lm. Their porosity is then typically at least 0.6, especially 0.7 to 0.8, by volume.
As a result the filtration rate of the suspension is typically 100 to 10000 times greater 20 than that of the starting suspension.
After the agglomeration step the agglomer~t~s may be separated from the aqueous phase of the suspension by for example ~lec~nt~tion, filtration or centrifugation. In any such method there may be one or more steps of resuspension, washing and re-s~dLion, to ensure more complete removal of solubilised NPCM and surfactant from the agglomer~tPs. It is an 2s advantage if using an agglomeration step that such separation and washing can be effected by ~nt~tion and/or filtration, without the expense of enh~n-~e-1-gravity m~-~hin~ry such as centrifuge. Thus one or more steps of washing by ~lec~nt~tion and/or filtration preferably co~ ule step (e) of the process hereinbefore defin~

WO 97/22654 PCT~US96/20016 If the agglomerates are to be washed, the washing liquid is usual~y water. However, other liquids may be used, for example alcohols (especially meth~nol) to remove liquid components of NPCM and less polar licluids (such as ethers, esters and hydrocarbons). The process is capable of producing PHA of better colour, as measured by yellowness index, as a result of low occlusion of impurities by the agglomerates.
Usually the separated and washed agglomer~tes are dried. As a result of agglomeration they consist substantially of cr,vstalline PHA. They are suitable for uses involving melting, such as:
Injection moulding, injection blow moulding, con~l~;s~ion moulding and casting (which o usually do not involve post-shaping mechanical treatment c~ncin~ substantial cryct~lli7~tion);
film casting, fiber spinning, each of which commonly is followed by stretching to increase crystallinity towards the m~xinlu... possible;
fluidized bed coating, as described in WO 93/10308.
For any of these processes the agglomerates may be used as such (especially if they are large enough to afford good die-fill and avoid serious dusting) or may be extruded to granular feed.
Another potentially valuable use of the agglomerates is as carriers for biochemically active m~teriz~l~ such as human medicines, animal mefiiCinec and agrochemicals. Such a component may be introduced during the agglomeration step or into the separated agglomerates taking advantage of their porosity. Depending on its chemis~y, it may itself act as the surfactant or part of it or may form water-insoluble complex with a surfactant. As a result of the relatively short time of the agglomeration step, a wide variety of such components can be used with minor risk of decomposition.

2s A ferment~tion biomass was forrned by growing Alcaligenes eutrophus on a nutrient me~ m co~ .i.,g glucose as carbon source, then ~<cl~m~ tin~ PHA by feeding glucose and propionic acid under phosphate limitation.
The biomass, co"1~; " ;. ,~ l 70g/litre of cells of 70% w/w polyester content (B:V = 92:8 by moles), was heat shocked at 1 50~C for 1.5 min, then cooled to 20~C and digested with proteolytic enzyme. Sarnples of the product were treated by addition of chelator DEQUEST, (RTM) (diethylene triamine pent~mcthylenephosphonic acid), hydrogen peroxide, the surfactant Synperonic A-11 water soluble Cl3 alkyl ethoxylate (11 EO) and silicone antifoam and stirred at 80~C for up to 15h. Test sarnples were removed at 7h and 15h, transferred to a glass tube, sealed, and heated at 140~C for 30 min. This test procedure correlates with steam-injected continuous agglomeration as described in Example 3 of our co-pending application WO
94/02622: if a PHA layer separates at the bottom of the tube, the particles of the dispersion would agglomerate at 125~C, 0.5 min, to filterable particles. (The s~ ct~nt can be added at any time up to the te~ er~ re increase for agglomeration). Results are shown in Table 1.

SampleNo A-ll Anti-foam Agglomerationat 140~C Final % w/w on PHA% w/w on PHA Yellowness 7h 1 Sh Index 0.5 Slight Good 26.4 2 5 0.25 Slight Good 26.4 3 5 0.05 Slight Good 26.6 4 3 0.25 None Good 26.0 Control None None 31.1 In the presence of A-ll the reaction time was shortened, agglomeration increased and the yellowness index (measured by m~tching the density scale in a colour meter) improved. A-l 1 at 5% was more effective than at 3% and all anti-foam levels inhibited foaming.

Similar results were obtained using the su~ t~nt~ Synperonic A-7 (C~3 alkyl 7 EO) and Synperonic A50 (Cl3 alkyl 50 EO), except that using A50 a concentration of 1% was found to ~be sufficient.

In a repeat of Exarnple I in an approximately 1 m3 scale the data shown in Table 2 were recorded for a PHBV (95:5 mol %).

TA~BLE 2 Suspended I SurfactantH2O2 35% w/wAgglomeration solids g/l w/w on used ml/g time, h solids PHA
131.40 none 2.82 20 154.07 1% ~.013 10 PHBV of 95% w/w purity 2 Synperonic A50 (C,3 alkyl + 50 mols ethylene oxide) s It is evident that, as well as shortening the agglomeration time, the surfactant also usefully decreases the conservation of hydrogen peroxide.

Claims

1. A process for producing polyhydroxyalkanoate (PHA), comprising:
producing a biomass containing particles of PHA and non-PHA cell material (NPCM); solubilising the NPCM to produce a product mixture;
oxidizing the product mixture under conditions to effect further solubilisation of NPCM; adding a water soluble surfactant to produce a suspension; and heating the suspension at a temperature below the melting point of the PHA
to agglomerate the PHA particles.

2. The process of Claim 1 wherein the surfactant is a monomeric water soluble surfactant.

3. The process of Claim 1 wherein the surfactant is non-ionic and has a hydrophobic group containing 10 to 20 carbon atoms, and 6 to 100 ethylene oxide units.

4. The process of Claim 1 wherein said solubilising is carried out by one or both of heat shock or protease solubilisation of proteins.

5. The process of Claim 1 wherein said oxidizing comprises adding hydrogen peroxide.

6. The process of Claim 1 wherein said oxidizing is conducted in the presence of a chelator.

7. The process of Claim 1 wherein the amount of surfactant is between 0.1 and 10.0% (w/w).

8. The process of Claim 1 wherein the amount of surfactant is between 0.5 and 7.0% (w/w).

9. The process of Claim 1 wherein the temperature of said heating is 30°C to 80°C below the melting point of the PHA.

10. The process of Claim 1 wherein the temperature of said heating is 40°C to 70°C below the melting point of the PHA.

10. The process of Claim 1 wherein the temperature of said heating is at least 100°C.

11. The process of Claim 1 wherein the agglomerated PHA particles have a weight average diameter of 50 µm to 5000 µm 13. The process of Claim 1 wherein the agglomerated PHA particles have a weight average diameter of 200 µm to 500 µm.

14. The process of Claim 1 wherein the PHA consists of repeating units of.
- O - CmHn - CO -wherein m is between 1 and 13 and n is 2m or 2m-2.

15. The process of Claim 1 wherein the PHA is polyhydroxy-butyrate-co-valerate is copolymer.

16. The process of Claim 1 further comprising separating the agglomerated PHA
particles from the suspension.

17. The process of Claim 16 wherein said separating comprises decanting the suspension.

18. The process of Claim 16 wherein said separating comprises filtering the suspension.