CA1223687A - 3-hydroxybutyrate polymers - Google Patents

Abstract

Abstract 3-hydroxybutyrate polymers High molecular weight copolymers containing 3-hydroxybutyrate residues, i.e. units of the formaula -O.CH(CH3).CH2.CO-and up to 50 mole % of residues of other hydroxy acids, viz units of the formula -0.CR1R2.(CR3R4)n.CO-where n is 0 or an integer and, if n = 1 and R3, H3, and R4 = H,R1 is not methyl.
The copolymers are made microbiologically: for part of the cultivation the micro-organism is under conditions such that polymer is accumulated, e.g. by limitation of a nutrient, e.g.
nitrogen source, required for growth but not polyester accumulation.
For at least part of this period of polymer accumation the substrate is an acid or a derivative thereof that gives the comonomer units.
Propionic acid, which gives polymers where n = 1, R2=R3=R4=H and R1 = C2H5, is the preferred acid.

Description

3~87 l 3 31910 ~ his in~ention relates to 3-hydroxybutyrate polymers hereinafter referred to as P~B~
P~B is accum~lated by ~arious micro org~nisms, princip-all~ bacte~ia, as an enexgy reserve material as ~ra~ule~ withinthe microbial cells.
PhB extracted ~rom such cells is a thermoplastic poly-ester of the repeat structure ~ OoCEI(CH~;) eCE2~CO ~
that rapidly c~ystallise~ to a relati~ely high level eOg. of the order of 7~/o or mo~e~ ~his c~tallisation behaviour is often disadvantageous when the polymer i~ to be used as, for exampleg a moulding material.
Wë'`'have'found'that the crystallisatio~ of P~B can be modified by incorporation of u~its of a dissimilar monomer into the pol~mer chai~. ~hus a minor proportion of como~omer units may be ;ntroduced Lnto the polymer chain by culti~ation of the micro~org~nism under certain conditions in t~e presence of oertain organic acids. One example of such an acid is propionic acid~
Although we do not wish to be bound by the following theory, it i9 thou~ht that the meta~olic pathwa~ leading to such copolymers is as follows, in which CoAS~ is unesterified Coe~zyme A, (Sa C~3.CO~SoCoA is the acetyl thioester of Coenzyme A a~d is more commonly termed acet~l CoA), and ~ Z ~ ~ 6 ~ ~

2 ~ 31910 ~TADP is nicotinamide adenine din~cleotide phosphate in the 02idîsed state. ~TADPH2 is reduced ~P.
It is believed that, in the biosynthesis of PEB by a micro~organism the first step is the synthesis of acetyl CoA~
This can be formed, for example, from Coenzyme A and acetate, or by the decarboXylation of pyruvate, which is a product of the glycolysis of carbohydrates, or which can be fo~med by decarbo.~y-lation of oxaloacetate, the latter being a member of the tricarb-ox~lic acid, ~CA, cycle9 otherwise know~ as the Krebs cycle.
10 ~hus with aoetate as the source o~ acetyl CoA, the PHB
is prod~ced by a metabolic pathway invol~ing the reactio~s:
1 CH CO ~~ + CoA S~ thiokina e > C~ CO S CoA OH-2. 2CE3.CO.S~CoA ~ CH3.CO.CH2COoS.CoA ~ CoA.SH

3- C~3~CO C 2~CO.S~CoA ~ ~TA3PH2 ~
CE3. CHOH~CE2~COoSoCOA + l~ADP
3-hydroxybutyryl CoA

4. CH30CHO~oCH2~CO~S~COA ~ Y--m-eraS ~ -O~CH(CH3).C~ .CQ- + CoASH
repeat unit in polymer ~hus reaction 4 adds a -O.CE(CH3).C~2.CO- u~it to a growing polymer chain.
Because of lack of specificity of the enzymes L~volved, with propionic acid, the corresponding pathway is thought to bes la. CE3,C~2.COO + CoA.Sa ~ CE3~CH2oCOoS~COA + OH
2~ 2a. C~3~CH2oCO~S.CoA + CH30COoS.COA ~ C~3~CH2.CO~CH2~COoS.CoA
+ Co~.SH
3aO CEI30CH2oCO~Cl~[2~COSoCOA ~ CE30GE2oCEOH~CE2~COISCOA
+ ~
4a. C~30CH2oCHOHoCH2~CO~SoCOA ~ -OoCH(C2H5)~C~2~CV- CoAO~E
i,eO xepeat u~its having a pendant ethyl group are introduced into the polymer chain: Ln contrast PH3 has pendant methyl groups.
Cert~in polJmers contalning 3-h~droxybut~rate units, i.e~
( 3) 2 u~its, together with othe~ ~nits have been described in the literature.
35 ~hus polymers exhibiting a~ infra-red band said to be ~L~72~7~

indicati~e of ethylenic unsatuxation are described by Davis in "Applied ~icrobiology" 12 (1964) pagss 301 - 304. These polymers which are said by Davis to be copolymers containilg 3-hydroxy~
butyrate U71its and 3-hydroxy-2-butenoate units, i.e. units of the

5 formula - OOC(CH3) - CLI.CO -were prepared by cultivating 7~ocardia on n-butane.
~ lso 7~Jallen et al describe in "Environmental Science a~d 17echnolo~sr" 6 (1972) pages 161 - 164 and 8 (1974) pages 576 - 579 10 a polymer melting at 97 - 100C (after repeated washing) isolated from activated sludges and containing 3-hydroxybutyrate units and 3-hydroxyvalexate units, iOe.
2 5) 2 units in the ratio of 1:5. The polymer thus contains only about 15 17% of 3-hydroxybutyrate ~its. l~archessault et al report in "II~P~C Macro ~710rence 1980 I~ternational Sgmposium on l~acromolecules Prepr~ts" 2 (1980) pages 272 - 275 a study of this polymer and confirmed that it oontained mainly 3-h~droxyvalerate units~
7~ited States Patenl; Specification 3275610 describes 20 the microbiological productio~ of polye~ters by cultivati~lg certain micro~organisms, especially ~ocardia salmonicolor, on carbox~rlic acids containing 4 carbon atoms. In ~xamples 2 and 3, whe~e the acids were 3-butenoic and 2-h~rdroxybutyric acids res-pectively, the polymers appear, from the quoted melting points of 25 the order of 178 - 184C, to be poly(3-hydro~rJbutyrate). In ~xample 1 however, wherein 2-methyI acrylic acid, i.e. methacrylic acid, was employed the polymer produced is unidentiIied but is described as having a meltin~ point of 215 - 220C and as being soluble in methyl ethyl ketone. In cGntrast thereto, copolgmers 30 in accordance with the present invention, containing predominanLtly 3-h~dxoxgbutgrate xesidues, have melting poirLts below 180C and are insoluble in cold methyl et~l ketoleO
When P~accumulating micro-or~a~is7ns are aerobically cultured on a ~uitable sub9trate, i.eO a source of energr and 3~ carbon, they reproduce until ons or moxe OI the essential 4 ~ ~1910 requirements for reproduc-tion is exhausted. ~his reproduction of the micro-organism is hereinafter referred to as growth. ~pon exhaustion of an essential ~rowth requirement, further growth occurs only to a ~ery limited extent, if a-t all, but, providing the substrate is not exhausted, PEB may be accum~lated by the micro-organism.
With some micro-organisms, even in the absence of a PHE-inducing constraint such as a limitation on one or more of the essential growth requirements9 PHB may also be accumulated ~hile growth of -the micxo-organism is taking place: however, except in the case of micro-org~nisms that produce P~B conætitutively, the amount of P~B so accumulated is ge~erally small and typically is less than about l~/o by weight of the cells produced. ~hus whe~
grown in batch culture, the micro organisms that do not produce P~B constitutively, will g~row, with little or no PEB accumulation, until one or more of the essential requirements for growth becomes exhausted, and then the micro-organism synthesises P~B. I~ order to produce copolymers it is therefore necessary to use the acid (or a derivative thereof) that is to fi ve rise to the copolymex 20 units other tha~ 3-hydro~ybutyrate units as at least part of the substrate present during the period when polymer is accumulated.
~he acid, or derivative thereof, that ~ives rise to the copolymer units other than 3-~ydroxybutyrate units is herein ter~ed the comonomer componen-t of-the substrate.
When the cultivation conditions are such that the polyester, e.g. PY~, is not being accumulated to any significant extent, the comonomer component of the substrate will often be metabolised by the micro-org~Lism by other pathways leading to e.g. acetyl Co~ or to a member of the ~CA cycle, and 30 copolymers will not be produced. Thus, as an~example, propionic acid ca~ be metabolised by micro-organisms, in the absence of a~y growth limitation, via propionyl CoA, with the incorporation of c~rbon dioxide to methyl malonyl CoA, a~d the~ce to succinate, a member of the ICA cycleO
Metabolism of the comonomer co~ponent of the substrate 3L ~T ;~ 3 7 3 31~10 by such other pathways may al~o occur when using micro-organisns that produce P~3 constitutively. Hence we prefer, even when using constitutive PHB-accumulating micro-organisms to cause the polymer to be accumulated by cultivation of the micro-organism under conditions T~herein the amount of one or more of the essential re-quirements ~or growth, but not P~3 accumulation, i3 limited. ~ven when cultivating the micro-organism under conditions where there is a rastrictio~ of an essential requireme~t for growth, 30 that polymer is accumulated by the micro~organism, some of the comonomer compo~ent of the substrate ma~ be metabolised by pathwa~s leading to acetyl CoA or a member of the TCA cycle. ~hi~ enables the micro-org ni~m to synthesise 3-hydroxybutyrate units for incorpor-ation into the copolymer as well as the dissimilar copolymer units, even if the comonomer component i~ the 301e substrate during the polymer accumulation stage. Al~o, as indicated by the metabolic pathway suggested above for the production of 3-hydrox~alerate units ~rom propionate, one qtep, reaction 2a, Lnvolves the reaction of propio~yl CoA with acetyl CoA0 ~ence if propionate is the sole substrate, some of the propionate is metabolised to acetyl CoA in order that 3~hydroxyvalerate u~it3 can be produced.
In addition to the metabolic pathways shown above, lead-ing to 3-hydroxyvalerate units in tho copolymer, other reactions can occur with various other materials a3 the comonomer component of the substrate.
For example it is possible that other hydroxy substituted carboxylic acids, if not metabolised by other routes leading to e.
acetyl or propio~yl CoA, could in some cases be inco~porated directly by the polymerase Into the polymer, e~g.
Ho~CRlR2~(CR3R4)n~Co4CoA~ OOCR R2~(CR3R4)n.Co- + CoAS~
where n is 0 or an integer and R , R , R , R4, which may be the same or di~erent, are selected from hydrocarbon radical3, such as alkyl, aralkyl, a~yl, or alkaryl radicals; halo- and hydroxy-sub~tituted hydroca^bon radicals; hydrox~y radicals; halogen atom~;

~2;236~37

6 ~ 31910 a~d hydrogen atoms. Of couxse where n = 1 and R = R3 = R4 =
hydrogen, i~ R is methyl the h~droxycarbo~ylic acid is 3-hydroxybutyric acid which of course will be directly assimil-ated to give 3-hydrox~butyrate unitsO
Preferably the groups R1, R2, R3 and R4 each contain less than 4 carbon atoms. Generally at least one of the grol~ps R , R2, R3 and R4 is hydroge~. n is prefera~ly 0, 1 o~ 20 Such hydroxy-carboxylic acids may be added as such (as part, or all, of the comonomer component of the substrate) or may by synthesised by the micro-organism from other comonomer compon-ent materials.
In particular we ha~e found that acids such as acrylic, 3-chloropropionic, and 3-hydroxypropionic acids may give rise to polymers containing 3-hydroxybutyra-te units and other units. Ia some cases all these other units are 3-hydroxyvalerate u~its while in other cases, the other units ~hich are hereinafter termed units ~ a~d which may occur in co~junction with 3~hydroxy~alerate units~ are characterised by proton a~d 13C nuclear magnetic reson-ance spectra exhibitin~, inter alia, (i) a p~oton ~MR triplet at 4.3 ppm, and (ii) 3C ~R peaks at 59~89 a~d 33.83 ppm (relative to a tetramethyl silane sta~dard).
From these ~MR data it is believed that these units are 3-~ydroxy-propionate units (i.e. n = l; R = R = R3 = R4 = ~).
~he copolymers may also contain 4-hydroxy~alerate units i.e. n = 2; R = C~3; R2 = R3 = R4 = H.

,. /
/

361!37

7 ~ 31910 3-~ydroxypropionate and/or 4-hydro~valerate urlits could result from the intermediates CH20HoCH2~COoS~COA and CH3OCEOHoCE2~CE2~COoSCOA~ ~he former col~d be formed f~om 3~
~ydroxypropionate and CoA.S~. In addition to being supplied as such, 3-hydroxypropionate could be formed by the h~dration o~
acrylate:
CH2 = CHoCOO + El20--~CHOE~C~I2oCOO
or by the hydrolysis of 3-chloropropionate CH2CloCE2oCOO + OH -- ~--~ C130H~CH2~COO + Cl 4-hydroxyvaleryl CoA could be derived from the condensation of acetyl CoA a~d acrylyl CoA fcllowed by reduction, i.e~
ClI3~COoSoCO~ CE2 = CE~COoS~COl~--~CE33~COoCH = CHoCOoSoCOA
+ CoA.S~
CE3~COoCH ~ CH~COoSoCOA ~ 2 ~AIP~ ~ C~3C~O~C~2CH2CO-S-CA
~ 2 ~AIP
Similarly 4-h~dro~yvaleryl CoA could be produced from 3-hydrox~-, or 3-chloro~propionate by reactions involving condensation with acetyl CoA, reduction, and dehy~ratio~ (with an intermediate hydrolysis step in the case of 3-chloropropionate)O
~y the process of the present invention it is therefore possible to obtain copolymers containing 3-hydrox~butyrate unitsO
I -O.C~(CE~).CE2oCO
together with units of the formula II -o.CR1R2.(CR3R4)n.Co-where n is 0 or an integer and R1, R2, R3, and R4 are each selected ~`rom hydrooarbon radicals, such as alk~l, aralkyl, aryl, or alkaxyl radicals, halo- and ~drox~ substituted h~drocarbon radicals;
hydroxy radicals; halogen atoms; a~d hydrogen atoms, provided that, ~here n is 1, if R2, R3 and R4 are each hydroge~ atoms, R is not methyl.
Preferably the groups R1, R2, R3 ~nd R4 each contai~ less than 4 carbon atomsO Ge~erally at least one of the groups R , RZ, R3 and R4 is hydrogen. Pre~erably ~ is 0, 1 or 2.
~he copolyme~s m~y contai~ more than one t~pe of unit IIo Preferably in at least some of the u~its II n = 1, ~2236~

8 3 31910 R2 = R3 = ~4 = H and R1 = ethyl.
~o be of any practical use as plastics materials, the polymers should have a wei~ht average molecular weight, (~w), above lO,000 eOg. as measured by gel permeation chromatography.
~he proportion of repeat units II in the copolymer is preferably between 0.1 and 50, particularly 1 to 40, mole percent of the total repeat units i~ the copolymer. In some cases the polymer produced by the micro-orOoanism may be a blend o~ a homo-polymer o~ repeat units I with a copolymer con-taining repeat units I and II. In this case the overall proportion of repeat units II Ln the polymer is pre~erably betwee~ 0.1 and 50 mole per-cent of the total repeat units. Most preferably the proportion of repeat u~its II in the poly~er i3 between 3 and 30 mole %.
In order to obtain a significant proportion of comonomer u~its II, the amount of combined carbon in the comonomer component of the substrate should be at least 2/O,preferably at least 10% by weight of the total combined carbon in the ~ubst~ate present duri~g the period when the cultivatio~ conditions are such that polymer is being accumulated by the micro organism.
Duri~g this period, we prefer that the comonomer compo~ent i8 the only carboxylic acid (or derivative thereof) present in the 3ubstxate, although in some case~ aceta~e may also be present.
According to the present invention we therefore provide a process for the production of a thermoplastic polyester wherei~
a micro-o~ganism that is capable of accumulati~g a polyester is cultivated in a~ aqueous medium on a water soluble assimilable carbon co~ai~ing substrate with at least part of the cultivation being co~ducted under conditions causing polyester to be accumNlated b~ the micro-organism characterised in that, at least ~uri~g part of the period when polyestex is being accumulated, the substrate comprises an organic acid, or deri~tati~e thereof, that is meta;bolis-able by said micro-orga~ism under said polyester accum~lating co~-ditions, to a polyester other than one composed solely of -O.CE(C~3).C~2oC0- repeat units, the amou~t of combL~ed oarbon in ~ ~ ~ 3 ~ ~ 7

9 3 31910 said acid, or derivative thereof, constituting at least 2% by weight of the total combined carbon in the substrate present throughout said period.
The concentration of the comonomer component in the aqueous medium should be above 0.05 g/l and will preferably be at a level between 0.1 and 5 g/l. It will therefore be appreciated that the wa-ter solubility of the comonomer component should be above 0.05 g/l a~d sufficient to provide the desired level of the comonomer component concentration at the rultivation

10 temperatureO
The comonomer component may be the acid itself or a salt, ester (including a lactone in the oase of a h~droxy substituted acid), anhydride, amide or halide.
As mentioned hereinbefore, it is preferred that at least part of the cultivation is conducted under conditions of limitation of an essential requirement for growth but not polyester accumul-ation. ~he most convelient growth requirement limitation is limit-ation of nitrogen. ~or this reason, where nitrogen limitation is employed, the substrate is preferably ritrogen free a~d so amides are less preferred substrates.
Acids that can be used to produce copolymers should be those that do not give rise only to repeat units I, ~hen the cultiv-ation is in the polymer accumulating stage. ~nsuitable acids there-fore i~clude acetic and 3-hydroxybutyric acids, members of the ~CA
cycle, and acid3 giviQg only acetyl Co~ and/or a member of the ~CA
cyole when the cultivation is Ln the polymer accumNlating stage.
Thus u~suitable aoids also include phosphoglyce~ic, pyruvic, citric, isocitric, o~-ketoglutaric, succinic, fumaric, maleic, m~lic, oxalacetic, oxalosuccinic, aco~itic, and methyl malonic, acids.
Amino acids are likewise unsuitableO Other acids that ha~e bee~
found not to give copol~mers include fo~mic, butyxic, phenyl acetic, benzoic, chloroacetic, 2-chloropropionic, 3-hydroxybutyric, 4~hydroxybutyric, 2-chlorobutyrio, 2-meth~lacrylic, 2,3-dimethyl-acrylic7 3,3-dimethylac~y1ic, lactic, glyo~ylic and glycolic acids~
Acids that ha~e bee~ fou~d to give copolymers include ~ 7 ~ 31910 propionic, }-hydro~propionic, 3-chloropropionic, 3-ethoxypropionic, 2-hydroxybutyric, isobutyric, and acrylic acids. Other acids that could be used include higher saturated carboxylic acids containi~g an odd number of carbon atoms, e,g. valeric and heptanoic acids;
pivalic acid; and substituted propenoic acids, eOgO 2- and 3-chloropropenoic acidsO
As mentioned hereinbefore, in some cases the micro-organism _ay perform further reactions on the acid: thus isobutyric acid would be expected to give repeat units II in which n is l, R =
R3 = R4 = H, and R1 is isopropylO In fact repeat units of the type II
wherein n is 1, R _ R3 = R4 = ~, and R1 is ethyl are fou~d indicat-ing that the micro-organism here substitutes hydrogen for a methyl group during the metabolic pathway to -the copolymer.
Prefe~red acids that can give rise to copol~mers are propionic, isobutyric, and acrylic acidsr Suitable derivatives of such acids include the alkali metal salts thereof and the lower alkyl esters (in which the alkyl group contains l to 4 carbon atoms).
Particularly suitable esters include methyl, ethyl, isopropyl, propyl, butyl and isobutyl prOpionates; methyl and eth~l isobuty-rates; and methyl and ethyl acr~lates~
Mixtures of copolymer forming acids (or derivatives thereof) may be used as the comonomer component of the substrateO
For example interesting results have bee~ obtained using a mixture of propionic and acrylic acids.
As indicated above, it is preferred, e~en whe~ using a micro-org2nism that produces P~B constitutively, to conduct the period of culti~ation of the m~cro-organism where polyester is being accumNlatea under conditions of limitation of a nutrient required for growth but not polyester accumulation.
Tn ad~ition to the substrate and oxygen (which is ge~er-ally supplied by injecting air into the aqueous medium in the fermenter), variou~ nutrient salta are requixed to enable the micro-organism to grow. mus sources of the following eleme~ts i~
assimilable form, nor~ally as water soluble salts, are generally rsquired: nitrogen, phosphorus, sulphur, potassium, sodium9 ~ 2 ~ 7 ll ~ 31910 magnesium, calcium, and iron, togethe~ with traces of elements such as manganese, zinc and copper. ~hile it _ay be possible to ind~ce polyester accumulation by restricting the ~upply of oxygen to the fermenter, it is preferred to restrict the amount of one or more of the nutrient salts. ~he most praotical elements to limit are nitrogen, phosphoxus, or, less preferably, magnesium, sulphur or potassium. Of these it is most preferred to restrict the amount of nitrogen (which is conveniently supplied as an ammonium salt)a ~he amount of assimilable nitrogen required is about 8 - 15% by weight of the desired weight of cells less accumulated polyester.
The fermentation is preferably conducted so that the dxy weight of the polyester-containing cells is at least 5 g per litre of aqueous medium. ~ence if, for example~ it i9 desired to produce lO g per litre of P~B-containing cells having a P~B
content of 4~/0 b~ weight, the amount of the essential nutrient fed to the fermenter that is used to limit the amount of cell growth must be that required to support the growth of 6 g per litre of cells containing no PEB: thus, if nitrogen is employed as the growth limiting nutrient, since the nitrogen content of P9B free bacterial cells is about 8 ~ 15% by weight, the amount of assimilable nitrogen required wot~d be between about 0O5 and O.9,g per litre, e~g. about o.6 to 1.2 g of ammonium ions per litre. ~
~he fermentation may be conducted under the conditions e.g~ pE, temperature, and degree of aeration (unless oxygen is utilised as the l;m;ting nutrient) conventionally used for the micro-orga~isma Likewise the amounts of nutrient salts (other than the growth limiting nutrient whose amount may be ds-te~mined following the considera-tions outlined hereinbefore) employed may be those normally used for growth of the micro or~anism~
~ he micro-organism is pxeferably grot~n to a certain desixed weight by culti~ation in the presence of ~ufficient of th0 nutrie~t required for ~rowth that is to be restricted Ln the pol~mer accumulation stage on a readily metabolisable substrate, such a a carbohydrate, and the~ cultivated under conditions of ~ ~ ~ 3 ~ ~ ~
12 ~ 31910 ~rowth requirement restriction to cause the pol~Jmer acc~mulation.
In some cases the substra-te for at least part, and in some cases all, of the growth stage may be the acid (or derivative thereof) that gives rise to the copolymer repeat units II in the polymer accumulation stage~
~ he fermentation may be performed as a batch ferme~tation in which case polymer accumulatlon will occur as the amount of the nutrient that is required for growth but not polymer acc~mLulation becomes depleted~ i~e~ e~hausted. Alte~natively t'ne fe~mentation may be conducted as a conti~uous pxocess wherein aqueous medium containing the bacterial cells is removed, continuously or inter-mittently9 from the fermentation vessel at a rate correspondin~ to the rate of addition of fresh aqueous medium and subs-trate thereto.
It is preferred that the amount of the nutrient that is restricted that is fed to the feDmentation vessel is such that the aqueous medium removed from the vessel contains little cr none of that nutrie~t, and the aqueou medium removed from the vessel is then fed to a second fermentation vessel7 operated either Ln batch or, preferably, continuous fashion wherein polymer accumulation is caused to take place by continuing the aerobic cultivatio~ with the addition of a fresh quantity of substrate comprising the comonomer component. I~hile additional quantities of substrate and nutrient salts may be added in this further fermentation step, since further growth is generally not desired, little or no further quantity of the nutrient utilised to limit growth should be added.
It will however be appreciated that the aqueous medium fed to the further fermenter or fe~menters from the first fermenter may contain some residual quantity of the limiting nutrient and/or the addition o~ a furthar small quantity thereof may be desirable for effician-t operation~
Alte~nati~ly the fermentation may be conducted as a single stage continuous pxocess. I~ order to achie~e polyester accumulatio~ by means of nutrient limitation the residence time of the medium in tha ~ermenter is made su~ficiently long to allow the micro-organism to grow and exhaust the limiting nutrient supplied ~L2~3~37 to the fe~m~nter and to allow the micro-organism then to accumNlate the polyester~
In either a batch process, or continuous processes as desclibed above, the acid (or derivative thereof) used to provide the copolymer repeat units II is used as part, or all~ of the substrate during the polymer accumulation stage occurring upon exhaustion of the nutrient required for growth. The comonomer component ma~ be used in admixture with a substrate, e.g. a carbo-hydrate, that will give repeat units I, or may be the ~ole substrate:
in the latter case sufficient of the comonomer componen-t will normally be metabolised by other pathways to acetyl CoA to provide the repeat units I and any acetyl Co~ required to produce the repeat units lI, eOgO if a pathway involving reaction 2a, is employed. ~owever, when the comonomer component is the sole sub-strate, the yield of polymer is often low~
The comonomex component may be present for o~ly partof the pol~Jmer accu~ulatio~ stage: for the rest of the polymer accumulation stage, which ma~ occur before and/or after the part of the polymer accumulation sta~e whexein the comonomer compo~ent is prese~t, a substra-te giving only repeat units I may be the sole sub~trate.
In some cases it may be possible to prevent the "normal"
metabolism of the como~omer component, io e. to acetyl CoA, by blocking enzyme required for that pathway and/or by using micro-organisms that lack the ability to synthesise the ~ecess ~J
enzymes. Eowever in order to obtai~ substantial yields of polymer a period of cultivatio~ under conditions of limitation, and prefer-ably depletion~ of a nutrient required for ~rowth is generally desirable~
~ ~he fe~mentatio~ is preferably conducted so that the amount of accumulated polyester comprises about 50 to 80% by weight of the bacterial cells.

~236~3~
1~ 3 ~1910 Micro-organisms that may be used include any poly (3_hydroxybuty~ate accumulating mic~o-organism~ that are capable of assimilating the acid (or derivative the~eo~) from which it is desired to producs the copolymers. ~he bacteria Alcali~enes eutro~hus (previously k~own as ~3~3~3~g~Q~ ) species, e~g. strain H 16 widely employ0d in academic studies of this species, see e.g. J General Microbiology (1979) 115 pages 185 -192, and which is available as A~CC strain 17699, and mutantsof strain H 16 such as mutants 11~7B, S301/C5, S501/C29 and $501jC41, which have been deposited, on 18 August 1980 with the National Collection of Industrial 3acteria, Torry Research Station9 Aberdeen, Scotla~d, under NCI3 Nos. 11600, 11599, 11597 and 11598 respectively, are particularly suitable. The ATeC number refers to the number designated by the American ~ype Culture Collection, 15 12301 Park Lawn Dri~e, Rockville, M~ryland 20852, U.S.A. As mentioned hereinbefore a carbohydrate is preferably used as the substrate during the growth stage. While ~s~_Y~ eutrophus strain H 16 (ATCC 17699) will not utilise glucose, certain mut-ants thereof, eOg. the aforesaid mutants 11/7B, S301/C5, S501/C29 and S501/c~l can utilise glucose. Carbohydrates~ particularly glucose, are the preferred substrates for the growth stage in view of the cost and the fact that the micro-orga~isms can grow efficiently thereon.
The polyester is produced as granules inside the micro-organism cellsO While the cells containing the polyester maythemselves be used as a moulding material, for example as des-cribed in ~SP 3,107,172, it is generally desirable to separate the polyester from the bacterial cells. This may be accomplished by subjecting the cells to a cell breakage step ~ollowed by ex-3 traction of the pol~ester with a suitable solventO Examples ofsuitable extraction processes are described in our ~uropean Patent Application 15123, published on September 3, 1980.
As mentioned hereinbefore the copolymers should have a weight average molecular weight (Mw) abo~e lO,CC0 as measured by gel permeation chromatography, if they are to be of any practical ~P"~

~ ~ ~ 3 ~ ~ ~

useO Preferably ~w is above 50,000, more preferably abGve 1001000 and in particular above 200,000.
The copolymers invariably have the D-configuration and eæhibit melting points below that of the 3-hydroxybutyr~te homopolymer.
The copolymers are of particular utility in the prepar-ation of melt-fabricated articles, where their reduced crystal-linity compared to 3~hydro~ybut~rate homopolymer is o~ten advantageous.
Of particular interest i9 the use of small amounts of the copolymers as high molecular weight processing aids for vinyl chloride polymers. For this application the amount of copolymer is preferabl3 0.5 to 10~ by weight of the vinyl chloride polymer.
For the best results in this application, we have found that the co-15 polymer ~hould be random: to obtain random copolym~rs, the como~omar component used -to produce the comoromer units II is preferably the sole substrate presP~lt, at least throughout the period of Cl~tiV-ation of the micro-organism under the conditions ofpolymer accumulat~o~
Copolymers are also of particular utility in the produc-tion of film by melt extrusion, preferably followed by rolling, e.g. passage through one or more pairs of rollsl to reduce the film thickness and induce some orientation, at a temperature be-tween the glass transition temperature (Tg~ and the melting point of the polymer.
The i~vention is illustrated by the following eæamples.
EXA
In the normal metabolism of propionate, -the latter is converted~ as described herei~before, to succinate which can give 3 rise to acetyl CoA by oxidation in -the ~CA cycle to oxaloacetic acid followed by decarboxylation. In the decarboxylation of oxaloacetic acid both terminal acid groups are removed as carbon dioxide~ Hence if propionate having the carbon atom of the carboxyl group radio labelled~ i.e.1 - 14C_ propionate, is sup-plied to the cells conversion to acetyl CoA will result in loss 36~7 of the radioactivity as 1 C0z. Any incorporation of 1 C intothe pol~Jmer must result from conversion of propionyl CoA into 3~hydroxyvaleryl CoA and subsequent polymerisation.
caligenes eutro~hus mutant NCI~ 11599 was grown by aerobic cultivation in a batch fermenter employing an aqueous medium A which contained sufficient assimilable nitrogen to sup-port a biomass free of accumulated polyester of 3.5 g 1 and glucose as the substrate. Medium A had the composition, per litre of deionised water:
(N~4)2 S04 2 g g 04~ o.8 g K2SOL~ O.L~5 g X3P04 (l.l M) 12 ml e 4 7~2 15 mg Trace element solution 24 ml The trace element solution had the following composition~ per litre of de ionised water CUS04O5E20 0.02 g ZnS04 6X20 0.1 g MnS04.4H20 0.1 g CaC12.2E~0 2.6 g When the biomass concentration reached 4.5 g 1 1, iOe. after the system became starved of assimilable nitrogen, 1 g l 1 of sodium propionate containing1-14C- propionate was added to the fermenter in addition to glucose and fermentation continued for 5 minutes.
The cells were then harvested by filtratîon and the polymer ex-tracted with chloroform. The labelled carbon was found almost exclusively in the chloroform solution indicating that the label-led terminal carbon atom had not been lost as carbon dioxide.
3 Eence at least some propionate had been incorporated into the polymer other than as acetyl CoA.
~e~e ~
~ 3~iL~2~ eutrophus mutant NCIB 11599 was ~ro~n by aerobic cultivation at pE 6.8 and 34 C in a 5 litre batch fer-menter containing 4000 ml of an aqueous medium B havin~ the ~L~2~ 37 17 ~ 31~10 composition, per litre of deionised water:
(NH4)2S4 4 g MgS04-7H20 o.8 g K2~04, -45 g 3 4 (1 1 M) 12 ml Fe~04.7H20 15 mg Trace element solution (as used in ~xample 1) 24 ml Glucose was fed to the fermenter at a rate of ~ g hr 1, ~he amount of assimilable nitrogen in medium B was sufficient to support 26 g of PHB~free cells.
After 40 hours the eells were harvested by centrifugation;
they were then freeze dried and the pol~mer extracted with chloroform.
EXAMP~æ 3 Exampla 2 was repeated except that when the cell weight reached 34 g, pr~pionic acid was fed to the fermenter instead of glucose at a rate of 2.8 g hr 1.

Example 3 was repeated except that feed of propionic acid was comDenced when the cell weight reached 39 g.

Ex~mple 3 was repeated except that the feed of propionic acid was commenced whe~ the cell weight reached 56 g.

Example 3 was repeated except when the cell weight reached 48 g a single addition of 12 g of propionic acid was made.

Ex~mple 2 was repeated except that medium ~A was used and propionic acid was fed at a rate of 4 g hr 1 instead of glucose 3 throughout the fermentation.
EX~PLE 8 Example 2 was repeated except that when the cell weight reached 38 g, a mixture of-glucose and propionic acid was fed to the fermenter~ instead of glucose, at a rate of 5.2 g hr gluc~
ose and 2.8 g hr 1 propionic acid.

~2~687 18 ~ 31910 Example o was repea-ted except that the mixture of glu-cose and propionic acid was fed at a rate of 6~8 g hr 1 glucose and 1.2 g hr 1 propionic acid, commencing when the cell weight reached 28 g.
In Examples 2 to 9 the propionic acid was added as a solution containing 400 g 1 1.
EXAMPI.E 10 Example 2 was repeated except that when the cell weight reached 28 g, isobutyric acid was fed to the fermentation ~essel at a rate of 2 g hr 1 in place of glucose. ~he isobutyric acid was added as a solution containing 150 g 1 1.
In Examples 3 - 6 and 8 _ 10 the fermentations were con-tinued until the ratio of the weight of acid fed to the fermenter 15 to the sum of the weight of glucose fed to the fexmenter after the cell weight had reached 26 gg i.e. when the system became nitrogen depleted, and the weight of acid fed to the fermenter, reached the ~alu0s set out in Table 1 E~AMPLE 11 Example 2 was repeated except that when the cell weight reached 26.4 g, 3-c'nloropropionic acid was fed to the fermenter instead of glucose at a rate of 4 g hr 1 for 5 hours.

Example 11 was repeated except that feed of 3-chloro 25 propionic acid was commenced when the cell weight reached 34.4 g.
~.~
Example 12 was repeated except that a single addition of 4 g of 3-chloropropionic was made when the cell weight reached 30 g and then glucose was fed at a rate of 6.8 g hr 1 for 7 hours.
3 In Examples 11 - 13 the 3-chloropropionic acid was added as a soluti~n containing 50 g 1 1 E~ample 2 was repeated except that when the cell weight reached 31 g, acrylic acid was fed to the fermenter at a rate of 4 g hr 1 for 5 hours instead of glucose. ~he acr~lic ~2;~
19 3 ~1910 acid ~la8 fed as a solution containing 100 ~ 1 1.
Table 1 ~ ~ ~ ~~ ~~ ~~~~ - Final Amount of .
Acid feed cell Polymer ~xample Acid ratio~ concentra~ion in cells.
(~) (~ (~ by weight) _ _ 2 none 0 20.0 7o 10 3 propionic 75 15~6 7o _ _ 4 propionic 50 13.3 60 propionic 33 16.0 7o . , . ~ ._ _ 15 6 propionic 4 13.0 63 7 propionic 100 6.4 55 _ _ , _ ~.
8 propionic 17 13.6 55 9 propionic 9-5 14.2 67 _ _ ~ _ isobutyric 66 13.0 5o 113-chloropropionic 61 7.4 25 _ ~_ __ 123~chloropropionic 33 4.5 20 25133-chloropropionic 6.5 9-3 35 __ _ _ - ._............... __ 1~ ~ ~G 6.0 ~5 * acid feed ratio is defined as the weigkt Or acid fed to the fermenter divided by the sum of the weight of glucose added after the cell dry weight reached 26 g and the weight ofacid fed to the fe~me~ter, said ratio being e~pressed as a peroentage~
The amount of comonomer units in the polymers of Examples 2 to 14 was determined (a) by metha~ol~9i3 and ga8 chromatograph~
and (b) by 13C nuclear magnetic resonance spectroscopy.

36~7 20 3 319~0 ~he molecular weights of the polymers were determined by gel pe~meation chromatography.
Chlorine analyses were also performed on the polymexs of ~xamples 2, 11, 12 and 130 ~he results are shown in Table 2.
In the table 3 - 3~ indicates 3~hydro~yvalerate units while the units ~ are those mentioned hereiabefore and which are believed to be ~-hydroxypxopionate units~
It is seen that little of the chlorine from 3~chloro-propionic acid is to be fouud i~ the polymerO It would therefore appear that during the metabolism o~ 3-chloropropionic acid, chlorine is replaced and the resultin~ product metabolised to ~ive the units ~ ~nd the 3~hydroxyvalerate unitsO However the chlorine content of the pol~mers o~ Examples 11 - 13 ma~ indicate that some of the chlorine is present as chlorine containing groups R in the units II, possibly as R = chloroethyl and R2 = R3 = R4 =
. = lo 23~

. _ _ _ ___ ._ .. ~
.~ ~ o ~L~ U~ U~
_ ~ ~ ~ , ., _ ~D _ _ ~ ~ ~ a:~ ~ ~o~ ~ ' a~ ~ ~ ~D
~1 ~ ~ ,--i 1`-~, r-i ~i rt N C~i C~i r-~ . ~i C`J
_.. _ . __ _ _ O ~ ~0 ~ 0 ad), ~ C~ d- co 1~ r~

._ _ ______ r : :: ~ _ _ ~i h ~ C`l 'I ~ ~ ~ ~ C\l l l l l i l l ~ ~ , .
C~J ~1 ~ g}~ . . .
__ ... _._ _ _ ___ _ _.
æ ~ co c~ ~o ~ o ~ ~1 ~ ~I~o o C-~ ~ C~l o ~
_ , _ ~ 1- .
H S~ ~ ~ !~ ~ l~i i~ i !~ ~ cq ~i ~ I Cli ,~L~ ''5i _~, _.._ _ ,_ ___ , .0---O` ,0 .
~ o o o C~ o o o ,1 . ,~ ,~, ' ,1 o ~ M ~ M M h ~ . ~ g' ~ M
~oi ~ P~ ~ P, f~ ):~ ~ ~,~ ~! ~o ~o I .
_.___, _ _ __ ._ __ 1-~ ~ r~
a~
~ C~J ~ e~ U~ ~O C~ C~ 1:~ O ~i - N ~ ~t ~i rl ~1 rl ~1 ~1 1 . __ _ _ _ _ _ ~ _ __ _ ~ 7 22 ~ 31910 ~ i~h resolution 13C N~R wa~ used to investi~ate the monomer sequences o~ the copolymers of Examples 3 ~ 10~ The signal derived from the carbon atom of the carbonyl group was found to occur at different chemical shifts depending upon its environment. Thus with polymers containing the units I and II(where n = 1, PL = C2H5, R2 _ R3 = ~4 - H) the possible ~equences are A.butyrate - butyrate7 i.eO
CIH3 C~3 ~0~ CH~C~I2o CO.CE.CH2.CO-lO ~.valerate-valerate, i.e.
IC2~5 ~C2H5 -O.CH.C~ CO.CE~CEI2.CO-C.butyrate - valerate, io e.

-O.C~C~2.CO,C~OC~2Co-~ MR examination of the polymers of Examples 2 - lO re-vealed three resonances occurring at 169.07, 169~25 and 169.44 ppm respectively. Following the work o~ M. Iida et al(~u~molecules _ , 1978, p 490) the resonance at 169.07 ppm can be assigned to the butyrate-bu-~yrate sequence A, a~d that at 169.44 ppm to the valerate valerate SeqUeQCe ~. ~y iD~err~lcr3 the si~al at 169025 must arise from the butyrate-valerate seque~ce C.
Qu2~titative anal~sis of the ~qR results of the co-polymer of example lO gave the followi~g results:
sequence A (butyrate-butyrate) 55%
sequenca B (valerate-valerate) 1~%
sequence C (butyrate-valerate) 31%
~hese res~lts clearly indicate that the polymer of Example 10 contains a substantial amount of a copolymer of u~its I and II (~here n = l, R - C2~ = R = H). HOWQ~r it is possible that some homopolymer of repeat unit I is also present.
All the polymers of Examples 2 - 14 had the D(-) con-figarationO
The melting beha~iours of the copolymers as extracted was first determined by differential scanning calorimetry (D~C) -~2~3~37 23 ~ 31910 using the Dupont 1090 system with computerised data analysis.
DSC was also performed on the samples after compression mould-ing at 190C and slow cooling 1~ the press in order to obtain a fully crystallised product. I~ each case the specimens were heated at 20C/min ~ air and the temperatures at the start (~s) and peak (~p) of the melting endotherm, together with its area, were noted. ~eating o~ the annealed sa~ple was continued to 200C a~d, after isothe~mlng for one minute to ensure complete melting, it was quenched in liquid nitrogen. ~he 3SC was then re-run in order to deter~ine the glass transition temperature (~g) of the amorphous phaseO ~inally the densities of the ~nnealed copolymers were measured by flotation in a density gradient.
The results are shown in ~able 3.
/
/

8~

__.__.. -- . ,~ .................. _ DSC on extracted polymer 3SC on an~ealed polymer Example _ I _ _ _ _ Density Ts C Tp C lArea ~g 1 ~g C Ts C Tp C ~raa g~cm~3 ~ g-1 _ _ _ ____ - -2140 _ 18~ _ 100 59 140 1~1 127 1~256 3 120 166 20 -1~9 140 171 18 10172 ... __ ~ .. ... _ . _ 4 120 170 50 008 140 182 44 1~174 120 5 _ _ __ _ 110 170 5 2.2 140 177 45 1.2~0 _ __ _ ~___ ~ ..
6 120 172 100 207 120 173 96 1.225 __ . . _ . . _ . .. _ _ _ 7 80132 34 oo4 80 132 4o 1.198 _ __ __ 120 _ _ _ - ~ 110166 6o _ 2.0 140 174 43 10199 9 _ 110156 99 4~o- 110 163 81 1.210 2010 5 120 3 ~2.0 130 172 26 1~13 `168 25 _ _

11 110170 57 5.0 120 180 73 .... _ , ~.. _ __ .~ . _ _ ~ _

12 110 177 86 4.1 120 173 86 lol82 . ~ .

13 1~0172 98 59 120 171 96 1.218 . _~ ~ . . _. . . . ~ . _ _ _ ~. . _ 25 14 -- 110 172 84 2.7 110 174 75 1.212 The wide meltin~ ra~ges of the copolymers indicated that the copolymers were o~ rather heterogeneous composition.
However significant randomisatio~ by ester interchange occurred on annealing si~ce the melting endotharms became much sharper and slightly reduc~ea in area. This is indicati~e that the polymers are not physical ble~ds of homopolymers but are gen~ine copoly~ers.
~ltiple DSC peaks were observed for the "a3 extracted"
polymers of Eæamples 3, 5, 8 and 10.
The area o~ the melting endothe~ms gives an i~dic~tion 1;2 23G87 of the degree of crystallinity. All the polymers of Examples 3 to 14 after annealing were significantly less crystalline than the control homopolymer of Example 2.

Alcaligenes eutrophus mutant NCI~ 11599 ~as grown by aerobic cultivation at pH 6.8 and 34C in a 5 litre batch fer-menter containing 4000 ml of an aqueous medium C which was the same as medium B except that the amount of a~monium sulphate was 5.2 g 1 1 which is sufficient to support 8.5 g 1 1 of P~B-free cells.
The substrate was glucose supplied at a rate of 5.5 g l ~
hr . Whe~ the cell concentration reached 7 g 1 1, propionic acid was fed at a rate of 1.58 g 1 1 hr 1 i~ addition to the glucose. The cells were harvested when the cell dry weight reached 15 g 1 1. ~he cell suspension was spray dried, lipids extracted by refluxing the dried cells with methanol, and the polymer then extracted by refluxing with chloroform. The polymer was reco~ered by precipit-ation by adding the chloroform solution to a methanol/water mixture.
~ he copolymer contained 20 mole % of repeat units II
where R1 was ethyl, R2, R3, R4 were each hydrogen, and n = 1.
The copolymer had a molecular weight of 350,000, and was insoluble in cold methyl ethyl ketone. When 2 g of the copolymer was re-fluxed with 100 ml of methyl ethyl ketone for 1 hour it all dis-soived: on cooling the solution a gelatinous mass was formed. In contrast less than 0.1 g of a 3-hydro~Jbutyrate homopolymer dissol~ed when 2 g of the homopolymer was refluxed with 100 ml of methyl ethyl ketone~ ldhen these solubility tests were repeated with ethanol in place of methyl ethyl ~etone, about 0.7 g of the copolymer, and less than 0.04 g of the homopolymer, had dissolved after refluxing for 1 hour.
3 The solubility of the copolymer in ethanol was also assessed at a lower concentration: thus 0.5 g of the copolymer was refluxed with 1 litre of ethanol for 1 hour. Less than 0.2 g of the copolymer dissol~red In contrast it is noted that the pol~mers described by 35 Wallen et al in 't~nvironmental Science and Technology" 8 (1974) ~ ~ ~ 3 ~ ~ ~

pages 576 - 579 were said to be soluble in hot ethanol.
E ~LE 16 Aqueous media D, E, and E were made up to the following compositions, per litre of deionised water:
Medium D
(~4)2S04 12 g g 4 7H2 102 g K2S0~ 1.5 g CaC12 0.12 g 4 7H2 0.1 g znso4~ 7H20 O ~ oo6 g MnS04 4X2 0.006 g CU!S~4 5~2 o.oOl5 g H2S04 (concentrated) 1 ml Medium E
H3P04 (1.1 M) 2.4 ml glucose 40 g Medium ~
H3P04 (1.1 M) 2~4 ml propionic acid 4 g A sterilised batch fermenter of nominal capacity 250 litres was filled to the 130 litre mark with a mixture of approximately equal volumes of media D and ~. A small sample of the medium in the fermenter was then analysed for nitrogen content. ~he fermenter was then inoculated with Alcaliæenes eutroE~s mutant NCI~ 11599 and fermenta~tion conducted aerobically at 34 C with automatic pH
control at 6.8 by addition of a sodium hydroxide solution.
~ he amount of assimilable nitrogen present in the fer-menter was sufficient to support the growth of the micro-organism 3 to only about lh2 kg of poly~er-free ~ells. When the cell weight reached about 1005 kæ feed of medium E was commenced at a rate of 6.5 litres~hour.
When the weight of cells reached appro~cimately 1700 g feed of medium E was stopped and feed of medium F commenced at a rate of o.5 litres/hour, and fermentation continued until about ~ ~ ~ 3 ~ ~ 7 27 3 31~10 2.6 kg of cells had been produced.
~ he cell suspension was then concentrated by centri-fugation to a concentration of about 60 g/litre and the pol~Jmer extracted therefrom by contacting 1 volume of the suspension with 2 volumes of 1,2-dichloroethane (DCE) in a Sil~erson mlxer at 20C
for 15 minutesO lhe DCE phase was separated from the aqueous phase, which contained the cell debris, and filtered. The polymer was precipitated by adding 1 volume of the filtered DCE phase to 4 volumes of a methanol/water mixture (4 volumes of methanol to 1 volume of water). The precipitated polymer was collected by filtration, washed with methanol, and dried in an o~en for 4 hours at 100C.
~ he polymer had a melting range, as determined by dif~
ferential scanning calorimetry~ of about 100 C to loO C wit'n a 5 peak in the melting endotherm at 168C.
EXAMP~E 17 The fermentation procedure of Example 16 was repeated except that the changeo~er from feeding medium E to feeding medium E;~ook place when the weight of cells was approximatsly 3.5 kg. ~he medium F was fed at a rate of 11~4 litres/hour for 4 hours and then reduced to 3.2 litres/hour and maintained at this le~el for a further 9 hours at which stage the weight of cells was about 3~9 kg.
In this example the amount of assimilable nitrogen present ln the fermenter was sufficient to support the ~rowth of the micro-organism to only about 1.5 kg of polymer-free cells.
The cell suspension was concentrated by centrifugation and then the polymer was extracted from the concentrated cell sus-pension by the procedure described in Example 15.
~XAPEIE 18 A 250 litre fermenter was charged and Lnoculated as in Example 16. ~he amount of assimilable nitrogen was sufficient to support growth of the mlcro-organisms only to about 1.9 kg of polymer-free cells. As in Example 16 fermentation was conducted aerobically at 34C at a p~ of 6.8.

~L~223~7 2~ 3 31910 When the cell weight reached about 1.0 kg feeds of medium E and of a medi~n G at rates of 8.7 litres/hour and 4.6 litres/hour respectively were commenced and continued until the cell weight reached about 3.9 kg.
Medium G had the composition, per litre of deionised water:
H~P04 (lol M) 1.2 ml propionic acid 20 g The cell suspension was concentrated by centriugation and then the pol~mer extracted from the concentrated cell suspen-sion by the procedure described in Example 15.

~ he procedure of Example 17 ~as repeated on a larger scale usirg a fe~nenter of nominal capacity lOC0 litres which was filled to the 500 litre mark with approximatel~ equal ~olumes of media D and E. In this ex2mple the feed of medium E was com-menced, at a rate of 25 litres/hour, when the weight of cells was about 4 kg and the feed of medium F l~as commenced~ at a rate of 37.5 litres/hour7 when the weight of the cells was about 8 kg.
The feeds of media E and ~ were continued until the weight of cells was about 10 kg. ~he amount of assimilable nitrogen pre-sent was sufficient to support the growth of the micro-organisTn to onl~ about 4.1 kg of pol~ner-free cells.

_ Example 19 was repeated except that the feed rate of medium F was 25 litres/hour and the fe~nentation was continued until the weight of the cells was about 11 kg. In this case the amount of assimilable nitrogen present was sufficient to support the growth of the micro-organism to only about 4 kg of polymer-3 free cells.
~ he polymers of Examples 16 ~ 20 were each copol~ners co~taining 3-hydroxybutyrate (~B) unit9 and ~hydroxy~aler-ate (~) units, and had weight average molecular weights abo~e 300,000. They each had the D(-) configuration.
~5 100 parts by weight of each of the polymers of E-~nples ~3~7 16 - 20, and of a 3-hydrox~butyrate homopolymer were slurried with about 10 parts by weight of chloroform and 1 part by weight of Steamic talc, and granulated at room temperature through a domestic mincer. The compositions were then dried to remove the chloroform and extruded at 190C and regranulated. ~he resulting granules were injection moulded at 185 C into test bars using a mould temperature of 60 C and a cooling time of 20 sec. The ten~ile properties were measured according to AS~M D 638-77a at a rate of 50 mm/min and the impact strength assessed by the Izod impact test according to ASTM D 256-78.
The results are shown in Table 4.
Table 4 .. ~_ HV/HB molar Izod Impact strength 15 Example ratio Modulus* Tensile Extension ~J/m) ~ (~P~) Strength to 3reak by GC bg NMR (MPa) (%) 1 mm notch unnotched ,__ , __ _ ~ _ 1618/82 20/80 1.47 25 10-31 66 463 2017 4/96 6/94 2.98 33 5-7 23 1L~o 18 8/92 7/93 2.10 31 14-19 106 408 19 1/99 4/96 2.70 35 8-14 56 191 4/96 4/96 2.48 35 8~15 23 1 L~o homo-25polymer 0/lpC 0/100 3.25 40 6-13 65 115 , _ . _ , . .. ~ _ _ __ * at 0.5% extension A PVC formulation was made by dry blending the following 3 ingredients at room temperature:
parts by_weight (i) vinyl chloride homopol~Jner (K62) 100 (ii) a complex tin thiooctyl stabiliser based on a di-N-dithioglgcollic acid ester 105 ~;2723~7 (iii) methyl methac~ylate/butadiene/
styrene PVC i pact modifier 8 (i~) wax (external lubricant) 0.8 (v) glyce~Jl monoester (inter~al lubricant) (vi) ~3 polymer (processing aid) 2 ~he HB polymer processin~ aids were a) a 3-hydroxybutyrate homo~olymer prepared by the procedure of Example 2 b) the copolymer of Example 7 (copolymer A) c) -the copolymer of E~ample 16 (copolymer 3) ~he processing aids were slurried with about l~/o by weight of chlorofo~m7 gxa~ulated at room temperature through a domestic mincer, dried, melt extruded at 190C, regra~ulated, and ground to a 5 partiole size below 150 ~ before incorporation i~to the PVC dry blend.
The dry blends were tested as follows:
l. 50 g of the mixture was poured into the miæing head of a 3rabender Plastograph maintained at 180C rotating at 18 rpm under a pressure ram loaded with a 5 kg weight. ~he time taken fox gelation to occur was measured.
2. ~he mixture was cold compres ed to fo~m a candle ~hich was then chaxged to an extrusio~ rheometer maintained at 170C and fitted with a die having a circular orifice of 1 mm diameter a~d 20 mm la~d le~gth. A~ter the cha~ge had heated to 170C, it was extruded at inoreasing rate~. ~he appearance of the extrudate was noted and the melt extensibilit~ assessed b~ attempting to draw the eætrudate away from the die.
~he results are shol~n in ~able 5.

~3Ç~37 31 ~ 31910 Processing aid Gelation tim8 Melt ~ ~ ~ _~1~ .
5 ~one 12 Severe sharkskin at low Poor extrusion rates;
ripple at higher rates homopolymer 9-5 Poor with a lot of Poor u~melted polymer clearly visible copolymer A 1.0 Excellent - very smooth Good copolymer ~ 1.5 Smooth, but occasional ~air unmelted particles . _ _ . .
~his example shows that the copolymers are superior to 3-hydroxybutyrate homopol~mer as a vinyl chloride polymer p~ocessing aid. ~he more random copolymer A was clearly superiox to the oopolymer ~.
~E~MPL~ 22 A medium E was made up to the fol.lowing composition:
(~H4~2S4 1 ~
E~2P04 2 g ~a~2~P04 3 g M~S04.7a20 0.2 g CaC12 0.01 g ~eS04.7E20 0.005 g M~S04.4~20 0.002 g 2 ~ 2 0.1 g (~2)2 C0 1.5 g deionised wate~ to 1 litre This medium had a pE of 7.
~ight 1 litre shake flasks each containing 500 ml of medium E, in which 0.5 g of methacrylio acid had been dissolved, were each inoculated with 5 ml of a tarter culture of ~ocardia ~almonicolor strain ~CC 19149 and incubated at 32C on a gyratory shaker.

~2~6~37 0.5 g of methacrylic acid was added to each ~lask at intervals OI 24, 48 and 72 hours after inoc~ation, and a final addition of 0.25 g of methacrylic acid was made 96 hours after i-noculation. After a total of 108 hours after inoculation the flasks were exami~ed: little or no growth of the microorganism was apparent in any of the flasks. The conte~ts of the flasks were combined and centrifu~ed to give a pellet of cells which was dried in an oven and weighedO ~he weight o~ the pellet was 2.81 g. ~he cell content of the inoculum was also dete~mined and ~0 found to be 69.75 g.l~ ence the total ~eight of cells added, as the inoculum, -to the flasks was 2.79 g.
It is concluded that, at the concent~ations o~ methacr~lic acid employed, this strai~ does not assimilate methacrJlic acid.

In these examples a r~ge of acids and derivatives there-of were scree~ed for their ability to give copolymers.
The technique employed was as follows:
~ o~r~hus mutant ~CIB 11599 was ~rown by aerobic cultiv-ation at pE 6.8 and 34C in a 5 litre batch fermenter containing 3500 to 4000 ml of an aqueous medium h~ing the composition, per litre of de-ionised water:
glucose 17 g (~4)2S4 4 g MgSO~.7~ 0 0.8 g H ~04(1-1 M) 12 ml ~eS04.7H20 15 mg ~race element solution (as used in Example 1) 36 ml ~he p~ was controlled at 6.8 by the automatic addition of a 9;1 v/v mixtures of 4 M potassium hydroxide and 4 M sodium hydroxide. ~his addition of a EO~ aOH mixture to co~trol pH also ser~ed to supply potassium and sodium to the cultivation medium.
~ he amount of ass;milable nitroge~ (provided by the 4 g/l of ammonium sulphate) was su~ficient to support only about 6.5 ~/1 of ~B polymer-free cellsO As about 16 ~/1 of glucose are required ~ 2 ~ 3 ~

to produce 6.5 ~/1 of HB polymer free cells, the amount of glucose presen-t was sufficient to provide a æmall carbon excess. ~y monitoring the residual glucose concentration and also the dis-solved oxygen tension trace, a clear m dication was obtaLned whan the system became star~ed of assimilable nitrogen. At this stage feed of the comonomer component, i.e~ the acid or derivative under study, was commenced and contirlued u~til a total of about 0.1 to 5 g/l of the comonomer componant had been added.
After completion of the addition of the comonomer com-ponent, the cells were har~ested by centrifugation. ~ sample ofthe centrifuged cells was freeze dried and the polymer was extxacted with chloroform. The polymer was analysed by a gas chxomatography/
mass spectroscopy (~CMS) technique (which in~olves a prelimi~ary transesterification step) or by nuclear magne-tic resonance spectros-copy (~MR).
The rasults are shown in table 6~ In this table the ~MRresults are quoted where possible as thesa are considered to be the more definitive ~ince the ~IMR technigue doeæ not involve a trans-esterification stepO
_ - /

/

23 Ei~77 TABLæ 6 . _--Comonomer component Example period over ~ ~nits II ~ound which comonomer total amount natureco~ponent added added (hr) (g/l) ~ ~ ~___ _ 23 propionic acid 5 5 3-BV
24 ethyl propionate 1.75 2053 ~V~
3-hydroxypropionic 1 13-HY and "A"
acid 26 3-hydxoxypropionic 16.5 3~43-~V and "A"
acid 27 2-chloropropionic ~5 . 0.3none acid 28 3~chloropropionic 0.5 0.33-EV and "~"
acid .
29 3-ethox~propionic 1 1 3-EV*
acid isobu~yric acid 5 5 3-EV
31 2-chlorobutyric 0.5 0.3~o~e acid 32 2-hydroxybutyric 005 1 3-HV
acid 33 4-hydroxybutyric 3 2 no~e acid 34 phenyl acetic acid 2 2 none acid ~ 305 003none 36 acrylic acid 5 3.53-EV and l'A7' 37 2-met~ylacrylic O.5 1 none acid _ _ _ ~ __ 6~7~

3 319~0 ~able 6 (cont'd) .,.
Comonomer component . . .~ _~ ___ 5 Example period over ~nits II found which comonomer total amound .. nature component added added (hr) (g/l) ~ _~ ____ --- - - - ................. .
38 2,3-dimethyl-acr~lic acid 03 5 1 none 39 3,3-dimet~yl-acrylic acid 1 1 none glyoxylic 5 2 none 41 glycolic 3 2 . none 42 lactic 5.5 3 none*
43 benzoic 24 5 no~e*
44 formic 4 2 none valeric 4 2.5 3-HV~
~ . __ ~ -- ~ . , ~ _ _ ~
~ Only GCMS analysis performed~ + polymer contai~ed about 33% 3-~V
units.
Similar results.were obtaLned ~hen E~amples 23-44 were repeated using an ~lternative cultivation regime wherein a mixture of glucose and the comonomer component was continuously added a~ter nitrog~n starvatio~
had been attained.
~a~
~ s ~ mutant ~C B 11599 was grown by con-tinuous aerobic cultivation at p~ 6.8 and 34C in a 5 litre fe~menter with a working volume of about 4 litres at a dilution ratio (recip-rocal of residence time) o~ 0.085 hr . ~he aqueous medium employedhad the followi~g composition, per litre o~ deionised water:
M~04.7E20 0.8g 2 4 0O45g 2S4 0.05g 35 ~3P04(1.1 M) 12 ~1 ~race element solution (as used i~ Example 1) 36 ml ~;223~7 36 3 31~1~

The ingredients set out in Table 7 were also continuously supplied to the fermenter ~_~
Amount added per Supplied as a solution Ingredient litre of medium containing, per litre in the fe~menter of deionised water Fe 5 mg ( 2 g FeS04.7E20 ( 1 ml conc~ ~ S04.
~ 850 mg 50 g il~40H
propionic acid 26 g 400 g propionic acid acrylic acid see ~able 3 40 g acr~lic acid ... , . _ ~he amount of nitroge~ was sufficient to support only 6.5 g/l of HB-polymer fxee cells.
pH was controlled at 6.8 by the automatic addition of a 9ol v/v mixture of 4 M potas~ium hydroxide and 4 M sodium hydroxide.
Fermentation was commenced usi~g propionic acid as the sole substrateO 3 days after steady state conditions had been achieved a sample of the aqueous cell suspension product was taken and then ac~ylic acid was added as a cosubstrate in increasing amounts. Prior to each Lncrease in acrylic acid conce~tration a sample of the aqueous cell suspension product was take~. Fsrment-ation under steady state conditio~s for at least 3 days was pex-fo~med at each acrylic acid concentration before taki~g each sample.
~ach sample of the aqueous cell suspension product was centrifuged to harvest the cells which were then freeze driedO ~he polymer was extracted from the cells with chloroform a~d then analysed by ~MR~
~he resul~s are shown in ~able 8.
., ~ ~;, ~ ~ ~ 3 ~ ~ ~

37 ~ 31910 ~ABLE 8 ~ . _ __ ~ , ~mount of acr~lic acid added, per Cell a~ Polymer content Example litre of weight (% by weight) ~MR analysis medium in g/l (mole %) the fermenter ~ _ (g) 3-~B 3-EV other ~ ....... ~ .. _ _ - _ __ 47 0025 11~8 45 7o 1911 (units A) 48 0.5 10.8 4 72 208 (units ~) 49 - 1.0 1005 38 81 19 O
15 5 2.0 9-3 3 90 10 O
5~- 4.5 7.1 ~ _ 89 11 O

~ 3~ 2 In this example ~ls~i læ~ eut~ mutant ~CIB 11599 is grown aerobically at 34C in under phosphorus, rathe~ than nitrog~n,limitation. ~ 5 litre fe~menter was charged with 3 litres of a~ aqueous medium containing, per litre of deionised watero (~E4)2S04 6.2 g E ~04(1.1 M) 1.5 ml Mgso~.7~2 0.8 g ~eS0~7 ~ 0 15 mg Trace element solution (a used i~ 3xample 1) 36 ml ~ermentation was controlled at p~ 6.8 automaticall~J by the addition of a 9:1 v/v mi~ture of 4 M EO~ aad 4 M ~TaOE. ~he amount of phosphorus was sufficient to support onl~ about 8 g/l of ~B polymer-free cells~
The farme~ter was L~oculated with a 48 hr shake flask culture and then 5 g/l of glucose was added. When all the glucose had bee~ utilised (at which 3tage the cell concentration wa~ about ~ ~ ~ 3 ~ ~ 7 205 g/l), propionic acid was added, as a solution containing 300 g/l propionic acid) at a rate of 0.8 g/l ~ for 54 hours.
The cells were then har~ested, the polymer extracted with chlorofo~m, and analysed by C~MSo ~he results were as ~ollows-Final cell concentration 20 g/l Polymer content 600/o mole % 3-~ 4/

In these E~amples two strains of ~ocardia salmonicolor were grown on glucose and then polymer accumulation Lnduced using various acids.
In each example a 250 ml shake flask was charg~d with 50 ml of an aqueous medium contai~i~g, per litre of deionised water glucose 10 g 2 o4 109 g ~2P4 1~56 g (~4)2 S4 1.8 g M~S04.7H20 0.2 g 3 2 0.001 g ~race element solution (as used in Example 1) 1 ml The pE of the aqueou~ medium was 7~0G The flask was ~noculated with the oxganism and grown, with g~ratory shaking, at 30C Por 24 hours. The resultant suspension was the~ centrifuged a~d the SUpernataQt a~ueous medium discardedO The centrifuged pellet was then resuspended in 50 ml of an a~ueous medium identic~l to that above except that the 10 g/l of glucose was replaced b~ 1 g~l o~
the acid and the 1.8 g/l of am~onium sulphate was omittedO ~he resuspe~ded cells were shaken ~or a ~urther 24 hours at 30C a~d then the cell suspe~sion centxi~uged~ m e centrifuged pelle-t o~
cells was washed twice with methanol and a~alysed for polymer content~ The polymer was ~nalysed by GCMSo ~he results are shot~n in Table 9.

~23~37 39 ~ 31910 E~ample = _- ; . - - - mole stx~in polymer content (% by weight) 3-HB 3-HV
__ ~ _ 53 ATCC 19149 isobutyxic 10 35 65 54 ATCC 19149 2-chloropxopionic 12 63 37 A~CC 21243 propionic 10 38 72 56 ATCC 21243 lsobutyxic 10 3o 7o 57 AqCC 21243 2-cli1oropropionic 12 38 62 . . _. , . . . _ PA/CG ~

14 June 1982

Claims (20)

1. Copolymers having a weight average molecular weight above 10,000 and containing repeat units I -0.CH(CH3).CH2.CO-and repeat units II -0.CR1R2.(CR3R4)n.C0-where n is 0 or an integer and R1, R2, R3, and R4 are each selected from hydrocarbon radicals; halo- and hydroxy- substituted hydro-carbon radicals; hydroxy radicals; halogen atoms; and hydrogen atoms, provided that, where n is 1 and R2, R3, and R4 are each hydro-gen atoms, R1 is not methyl, said repeat units II constituting 0.1 to 50 mole % of the total repeat units in said copolymer.

2. Copolymers according to claim 1 wherein n is 1.

3. Copolymers according to claim 1 wherein R1, R2, R3 and R4 each contain less than 4 carbon atoms.

4. Copolymers according to claim 1 wherein at least one of R1, R2, R3 and R4 is hydrogen.

5. Copolymers according to claim 4 wherein R2, R3 and R4 are each hydrogen.

6. Copolymers according to claim 5 containing repeat units I -0.CH(CH3).CH2.CO-and repeat units II -0.CH(C2H5).CH2.CO-

7. Copolymers according to claim 5 containing repeat units -0.CH(CH3).CH2CO- together with repeat units -0.CH2.CH2.CO- alone or in conjuction with repeat units -0.CH(C2H5)CH2.CO-.

8. Copolymers according to claim 1 containing 1 to 40 mol %
of repeat units II.

9. Copolymers having a weight average molecular weight above 10,000 and containing at least 50 mol % of repeat units -0.CH(CH3)CH2.CO-and being characterised by a triplet at 4.3 ppm as measured by proton nuclear magnetic resonance spectroscopy and peaks at 59.89 and 33.83 ppm (relative to a tetramethyl silane standard) measured by 13C
nuclear magnetic resonance spectroscopy.

10. Copolymers according to claim 1 having a weight average molecular weight above 200,000.

11. A process for the production of a thermoplastic polyester wherein a micro-organism that is capable of accumulating a polyester is cultivated in an aqueous medium on a water soluble assimilable carbon containing substrate with at least part of the cultivation being conducted under conditions causing polyester to be accumulated by the micro-organism, characterised in that, at least during part of the period when polyester is being accumulated, the substrate comprises an organic acid, or derivative thereof, that is metabolis-able by said micro-organism under said polyester accumulating con-ditions to a polyester other than one composed solely of -0.CH(CH3).CH2.CO- repeat units, the amount of combined carbon in said acid, or derivative thereof, constituting at least 2% by weight of the total combined carbon in the substrate present throughout said period.

12. A process according to claim 11 wherein the acid is selected from propionic, isobutyric, and acrylic acid.

13. A process according to claim 11 wherein the acid is the sole substrate for at least part of the period during which the cultivation of the micro-organism is conducted under conditions causing polyester to be accumulated.

14. A process according to claim 13 wherein the acid is the sole substrate throughout the cultivation of the micro-organism.

15. A process according to claim 11 wherein the micro-organism is grown using a carbohydrate as a substrate.

16. A process according to claim 15 wherein the carbohydrate is glucose.

17. A process according to claim 15 wherein, for at least past of the period when the cultivation is under conditions causing poly-ester accumulation, the substrate is a mixture of the acid and the carbohydrate.

18. A process according to claim 11 wherein the micro-organism is caused to accumulate polyester by cultivation under conditions of limitation of one or more of the essential requirements for microbial growth but not polyester accumulation.

19. A process according to claim 18 wherein the essential requirement for growth, but not polyester accumulation, that is limited, is the nitrogen source, or the phosphorus source.

20. A process according to claim 11 wherein the acid, or derivative thereof, that is metabolisable to a polyester other than one composed solely of -0.CH(CH3).CH2.CO- units, is the sole organic acid, or derivative thereof, present during the period when polyester is being accumulated.