Note: Descriptions are shown in the official language in which they were submitted.
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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.
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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:
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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;
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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
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(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~
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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.
EXAMPLE 1
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
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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.
EXAMPLE 2
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.
EXAMPLE 3
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 %).
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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.
