Hydroxy-terminated polyhydroxyalkanoates

BACKGROUND OF THE INVENTION
The present invention relates generally to biodegradable polymers. More particularly, it concerns methods for the bioproduction of novel hydroxy-terminated polyhydroxyalkanoate (PHA) polymer compositions, and their subsequent use in production of novel copolyesters, polyester carbonates, polyester ethers, polyester urethanes, polyester amides, polyester acetals and other elastomeric, thermoplastic and thermoset polymers and copolymers.
There has been considerable interest in recent years in the use of biodegradable polymers to address concerns over plastic waste accumulation. The potential worldwide market for biodegradable polymers is enormous (>10B lbs/yr). Some of the markets and applications most amenable to the use of such biopolymers range from single use applications, which include packaging, personal hygiene, garbage bags, and others where the biopolymers become soiled and are ideally suited for biodegradation through composting, to markets and applications in which the biopolymers can be recovered as clean materials, such as garment bags, shopping bags, grocery bags, etc. and are suitable for recycling, as well as composting, or biodegradation in landfills.
PHA biopolymers are thermoplastic polyesters produced by numerous microorganisms in response to nutrient limitation. The commercial potential for PHA's spans many industries, and is derived primarily from certain advantageous properties which distinguish PHA polymers from petrochemical-derived polymers, namely excellent biodegradability and natural renewability.
Widespread use and acceptance of PHA's, however, has been hindered by certain undesirable chemical and physical properties of these polymers. For example, PHA's are among the most thermosensitive of all commercially available polymers. As such, the rate of polymer degradation, as measured by a decrease in molecular weight, increases sharply with increasing temperatures in the range typically required for conventional melt-processing of PHA's into end-products such as films, coatings, fibers etc. An additional limitation of the potential utility of PHA polymers relates to the observation that some polymer characteristics, for example ductility, elongation, impact resistance, and flexibility, diminish over time. This rapid "aging" of certain PHA-derived products is unacceptable for many applications. Thus, the success of PHA as a viable alternative to both petrochemical-derived polymers and to non-PHA biodegradable polymers, will depend upon novel approaches to overcome the unique difficulties associated with PHA polymers and with products derived therefrom.
One approach which has the potential to provide new classes of PHA-containing polymers having unique and improved properties, is based on graft, random and block polymers and copolymers. In generating such polymers and copolymers, it is possible to vary the nature, length and mass fraction of the different polymer constituents which are present. In doing so, the morphology of the polymer, and therefore the resulting properties, may be manipulated to meet the requirements of a given application.
The production of copolymers with PHA is limited, however, by the dissimilar ends of a PHA polymer chain (i.e. a carboxy group and a hydroxy group, respectively, on the ends of each polymer molecule). In order for PHA to be useful in the production of copolymers, it is desired that the ends of a polymer molecule chain possess the same chemical groups, and that those groups be capable of forming covalent bonds with the ends of other polymer molecules either by direct reaction or by use of a coupling agent (e.g. a diisocyanate). The reactive end groups of hydroxy-terminated PHA, for example, would be well suited for the preparation of high MW block copolymers by various known chain extension approaches.
Synthetic PHA hydroxy-termination has been reported. Hirt et al. (Macromol. Chem. Phys. 197, 1609-1614 (1996)) describe a method for the preparation of HO-terminated PHB and PHB/HV by a transesterification procedure using ethylene glycol and commercial grade PHB and PHBV (Biopol) in the presence of a catalyst. When catalysts such as H.sub.3 PO.sub.4, ethylene glycolate, or tripropyl amine were used, some diol was formed but the major products were oligomers with carboxylic acid end groups and olefinic end groups, even when a tenfold excess of ethylene glycol was used. When dibutyltin-dilaurate was used as catalyst with a 10-fold excess of ethylene glycol, end-group hydroxyl incorporation was obtained. The oligomer had a hydroxyl end-group content of .about.97%, but the molecular weight (Mn) of the hydroxy-terminated PHA obtained by Hirt et. al. was only about 2,300. The ratio of the weight- to number-average molecular weights (Mw/Mn) of their oligomers was .about.2.
When one prepares hydroxy-terminated PHA by reaction of PHA with EG in the presence of a catalyst, a steady decline in MW to less than about 3,000 is observed as the content of hydroxyl end groups increases from about 50% to greater than 80%. Experiments performed in our laboratory confirm this effect. (the results of which are presented in Example 1 and in FIG. 1).
For many applications, higher MW hydroxy-terminated PHA would be preferred for use in the preparation of copolymers. In addition, it would be advantageous to have the ability to produce hydroxy-terminated PHA by a simple and inexpensive method that is easily amenable to large scale polymer production.
Shi et al (Macromolecules 29 10-17 (1996)) reported in vivo formation of a hybrid natural-synthetic di-block copolymer. They describe a method to prepare PHA-polyethylene glycol (PEG) diblock copolymers where the carboxylate terminus of PHA chains are covalently linked by an ester bond to PEG chain segments. Polymer production was carried out by cultivation of A. eutrophus in a nitrogen free medium containing 4-HBA and 4% w/v PEG (Mn.about.200). The unfractionated product was complex in that it was composed of at least three different component polymers of different repeat unit composition. The product was separated into an acetone insoluble fraction (43% w/w, Mn.about.130 k, Mw/Mn.about.3.42) and an acetone soluble fraction (57% w/w, Mn.about.37.4 k, Mw/Mn.about.2.52). The mole fraction content of 3HB, 3HV, 4HB, and PEG in the acetone insoluble and acetone soluble fractions were 95, 2, 3, 0.1 and 13, 2, to 84, 1.6, respectively. Thus the PEG segments were found primarily in the acetone soluble fraction. It is important to note that the acetone soluble fraction is high in 4HB while the acetone insoluble fraction is high in 3HB. This process to make the di-block copolymer is not very selective.
Shi et. al. (Macromolecules 29 7753-8 (1996)) later reported the use of PEG's to regulate MW during A. eutrophus cultivations. They used PEG's that ranged in MW from PEG-106 (diethylene glycol) up to PEG-10,000 (MW.about.10 k) and reported that PEG-106 was the most effective in that only 0.25% was required to reduce Mn of the PHA by 74%. By adding 10% PEG-106 to the medium, the decrease in Mn was from 455 k to 19.4 k. They also reported that the use of monomethoxy ether CH.sub.3 O-PEG-OH-350 and PEG-300 resulted in almost identical molecular weight reductions. However, the dimethoxy ether of tetraethylene glycol was not an effective agent for molecular weight reduction. Thus, the chain end functionality useful in the work was a hydroxyl group. Shi et al. also reported that their PHA products did not contain PEG terminal groups. They concluded that molecular weight reduction was not due to chain termination reactions by PEG, but likely due to interaction between PEG and the PHA production system, leading to an increased rate of chain termination by water relative to chain propagation reactions.
We have now discovered that by using simple, monomeric aliphatic diols or aliphatic polyols, that clean and selective reactions can be obtained by in vivo formation of PHA-diols at concentrations of aliphatic diols or aliphatic polyols in the culture medium that are not toxic to the PHA-producing microorganisms. The hydroxyterminated PHAs so produced have improved thermal stability and in addition can be useful in the production of shaped articles containing branched PHA.
SUMMARY OF THE INVENTION
According to one aspect of this invention, there is provided a method of producing a shaped article using a polymer at least partly made of a PHA modified to have an end group hydroxyl content greater than 50%. Such PHAs exhibit improved thermal stability and therefore undergo less degradation as a result of exposure to temperatures and other conditions that cause PHA degradation. The PHAs are well suited for the production of polymeric and copolymeric articles by extrusion, injection molding, coating, thermoforming, spinning, blow molding, calendering etc. The PHAs most useful in this aspect of the invention have an end group hydroxyl content greater than 70%, preferably greater than 80%, and more preferably greater than 90%.
In another aspect of the invention, there is provided a branched PHA composition produced by melt processing hydroxyterminated PHAs having greater than two terminal hydroxyl groups per polymer molecule, i.e. that are polyol terminated. The branched structure results from chain condensation reactions in a PHA melt between the carboxyl groups of at least two PHA molecules with the hydroxyl groups at the polyol terminated end of a polyol terminated PUA molecule.

BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 demonstrates the advantages achieved by use of the methods of present invention for production of hydroxy-terminated PHA. The solid line shows the sharp decline in molecular weight of PHA when reacted with EG in the presence of a catalyst. The individually displayed data points, on the other hand, represent results obtained in accordance with the present invention, wherein hydroxy-terminated PHA is produced by cultivating a PHA-producing microorganism in the presence of an aliphatic diol or an aliphatic polyol. If a HO-content in the end-groups greater than 80% is desired, the MW of the PHA-diol is typically less than 7000 when produced by catalytic conversion. In accordance with the present invention, in contrast, the MW of the hydroxy-terminated PHA ranges, in these examples, from about 20,000 to greater than 80,000 depending on the strain of bacteria used. If HO-content in the end groups of greater than 90% is desired, the MW of the PHA-diol from the catalytic conversion is typically less than 3500 (2500 at 92% HO-content). In contrast, the present invention can PHA compositions having greater than 90% HO-content in a MW range up to greater than 80,000. The MW's can be influenced by the bacterial strain utilized in the method. If a high MW is desired (e.g. greater than about 60,000), Ralstonia eutropha may be preferably used. If lower MW's are desired (e.g. .about.20,000), Comamonas testosteroni may be preferred. If intermediate molecular weights are desired, Alcaligenes latus may be preferred.
FIG. 2 illustrates the improved thermal stability as evidenced by reduced molecular weight loss upon isothermal heating at 200 deg.C. The improved stability is a result of the increase in end-group hydroxyl content of the hydroxyterminated PHB.
FIG. 3 illustrates the Mark-Houwink plots for unprocessed and processed PHB homopolymer. It demonstrates that both materials have the same relationship between the logarithmic intrinsic viscosities and molecular weights. Thus, the linear nature of the PHB chains does not change after processing.
FIG. 4 illustrates the Mark-Houwink plots for unprocessed PHB homopolymer and unprocessed pentaerythritol ethoxylate terminated PHB homopolymer. The results demonstrate that both polymers have similar hydrodynamic volumes and are hence linear in nature.
FIG. 5 shows the Mark-Houwink plots for the unprocessed and processed pentaerythritol ethoxylate terminated PHB homopolymer. These results demonstrate that across the entire range of molecular weights measured, the processed material has lower intrinsic viscosities when compared to unprocessed material having similar molecular weight, indicative of the formation of a branched structure when polyol terminated PHB is processed in the extruder.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The embodiments of the present invention relate broadly to novel approaches for the production of PHA-derived materials having wide-ranging properties that can serve to increase the versatility of PHA for numerous applications without sacrificing biodegradability.
PHA is a polymer made from repeating units having the following general structure: ##STR1## wherein R is H, alkyl, alkenyl, aralkyl, aryl, or cyclohexyl; a is 0-9; and n is an integer typically from 1-150,000. PHA can consist entirely of a single monomeric repeating unit, in which case it is referred to as a homopolymer. For example, poly-3-hydroxybutyrate (P3HB) homopolymer has repeating monomeric units wherein R=C1 alkyl and a=0. Poly-4-hydroxybutyrate has R=H and a=1. Copolymers, in contrast, contain two different types of monomeric units. PHBV, for example, is a copolymer containing polyhydroxybutyrate and polyhydroxyvalerate (R=C2 alkyl and a=0) units. P3HB/4HB copolymer contains units where a=0 and 1 and R=C1 alkyl and hydrogen, respectively. When three different types of repeating units are present the polymer is referred to as a terpolymer.
There is much interest in developing methods for the production of graft, random and block polymers and copolymers which contain PHA polymers in addition to other aliphatic moieties. In this way, it should be possible to generate polymers and copolymers with the necessary characteristics for a given application by altering the amounts and compositions of the PHA and of the other aliphatic moieties present. For the preparation of graft, block and random polymers and copolymers, it is often desirable to have available crystallizable polymers that have similar functional end-groups, and to have end-groups which are compatible with the end-groups of other macromers and monomers containing appropriate functionality. A method for the rapid and large scale production of end-functionalized PHA, therefore, would be useful in the preparation of new classes of graft, random and block copolymers with superior characteristics.
In a first embodiment of the present invention, there is provided a method of making hydroxy-terminated PHA in a PHA-producing microorganism by cultivating the microorganism in the presence of an aliphatic diol or an aliphatic polyol. Many of the aliphatic diols suitable for use in the present invention are represented by structures (1) and (2) below: ##STR2## where n=0-25; where ##STR3## or cyclohexyl; R.sub.2 =H or CH.sub.3 (CH.sub.2).sub.m --, where m=0-25;
R.sub.3 and R.sub.4 =H or CH.sub.3 (CH.sub.2).sub.p -- where p=0-25.
HOCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2)nOH (2)
where n=1-10.
Examples of preferred aliphatic diols include ethylene glycol (EG), propylene glycol (PG), 1,3-propanediol (1,3-PD), 1,2-butanediol, 1,3-butanediol, 2,3-butanediol, neopentyl glycol (NG), 1,4-butanediol (1,4-BD), 1,4-cyclohexyldimethanol (1,4CHDM), 1,6-hexanediol (1,6-HD), diethylene glycol (DEG), tetraethylene glycol (TEG), hydroquinone, and 1,4-benzenedimethanol for linear hydroxy-terminated PHA's.
Many of the aliphatic polyols for use in the present invention are represented by structure (3) below:
HOCH.sub.2 (R.sub.3).sub.n CH.sub.b 2 OH (3)
where n=1-12; ##STR4## R.sub.4 =H or CH.sub.3 (CH.sub.2).sub.m -- where m=0-25.
Examples of preferred aliphatic polyols include glycerol (GL), pentaerythritol (PAE), pentaerythritol ethoxylate, pentaerythritol propoxylate, pentaerythritol ethoxylate/propoxylate, glycerol propoxylate, and mono-, di-, and oligosacharrides including alditols such as sorbitol (SL) and mannitol (ML) for branched or star-shaped hydroxy-terminated PHA's. Also dianhydro derivatives of the aliphatic polyols, such as 1,4:3,6-dianhydrosorbitol or 1,4:3,6-dianhydroglucitol, could be used.
It has unexpectedly been found that PHA hydroxyl content can be increased by cultivating a PHA-producing microorganism in the presence of an aliphatic diol or polyol. Surprisingly, these compounds show little toxicity at concentrations in the culture medium (typically less than 5%) sufficient to generate PHA compositions having increased hydroxyl content (i.e. greater than 50%). Particularly preferred are those PHA compositions having a having a hydroxyl content greater than about 80 mol %, more preferably greater than 90%. However, PHA compositions having between 50 and 80 mol % hydroxyl end groups are also expected to be suitable for certain applications. Indeed, we have found that PHAs having an increased end group hydroxyl content greater than 50% display improved thermostability compared with PHAs in which the end group hydroxyl content has not been increased.
The PHA compositions so produced have molecular weights that can range up to greater than 100,000 while still having a high % HO-content. Furthermore, it is possible to vary the molecular weight of the hydroxy-terminated PHA by careful selection of the bacterial strain and diol/polyol utilized. For example, if a high MW is desired (e.g. greater than about 60,000), Ralstonia eutropha may be preferably used. If lower MW's are desired (e.g. .about.20,000), Comamonas testosteroni is preferred. If intermediate molecular weights are desired, Alcaligenes latus may be preferred. The selection of the aliphatic diol or polyol used in accordance with the present invention can also influence the MW of the hydroxy-terminated PHA obtained (see Example 21). Routine optimization of such experimental parameters can be readily determined by the skilled individual in this art.
It is well know in the art that PHA polymer is produced by diverse types of microorganisms. (see for example, Alistair et al., Micro. Rev. 54(4), 450-472 (1990)) For example, strains from bacterial genus Alcaligenes (e.g. eutrophus, latus), Bacillus (e.g. subtilis, megaterium), Pseudomonas (e.g. oleovorans, putida, fluorescent), Aeromonas (e.g. caviae, hydrophila), Azotobacter vinelandii, Comamonas testosteroni, Ralstonia eutropha and others, produce PHA. Several bacterial strains are demonstrated herein to be useful in the production of hydroxyterminated PHA, including Ralstonia eutropha, Alcaligenes latus, and Comamonas testosteroni. Based on the demonstrated applicability of the disclosed method to three distinct strains, it is anticipated that the methods provided herein can be applied to essentially any microorganism which naturally produces PHA, or which has been genetically manipulated in a manner so as to effect PHA production.
Upon culturing a PHA-producing microorganism in the presence of aliphatic diols or aliphatic polyols, the PHA chains apparently are modified by a transesterification reaction. Although the exact mechanism of incorporation is not known, it may involve the addition of the aliphatic diol or polyol to the growing PHA chain by replacing the Coenzyme A at the end of a growing chain, serving as a chain-growth terminating agent. Such a mechanism might be used to explain the truncation of MW as compared to PHA biosynthesis in the absence of EG, etc. Also, it is clear that higher MW's are produced than in the case of random attack by EG catalyzed by Sn catalysts. The end-groups obtained are either primary hydroxyl groups arising from transesterified ethylene glycol units, or secondary hydroxyl groups from normal end units of the PHA chain.
The PHA compositions produced according to the first embodiment of the present invention can be recovered from the PHA-producing microorganism by conventional methods. Typically, a solvent-based approach is utilized, wherein the cells are harvested, dried, and the PHA is extracted with a solvent capable of dissolving PHA from other bacterial components. However, methods suitable for the recovery of PHAs from microbial and other biomass sources are expected to also be suitable for the recovery of hydroxy-terminated PHA made in accordance with the present invention.
In a second embodiment of the present invention, there are provided hydroxy-terminated PHA compositions. The compositions are preferably produced by the method disclosed in the first embodiment of the present invention, wherein a PHA-producing microorganism is grown in the presence of an aliphatic diol or polyol. The structure of the hydroxy-terminated PHA can vary depending on cultivating conditions, for example the carbon source utilized, and which aliphatic diol or aliphatic polyol is used in the culture medium. The MW's of the hydroxy-terminated PHA compositions are greater than 3,000, and can range up to 100,000 or more. Thus, in accordance with the method of the present invention, it is possible to produce hydroxy-terminated PHA's having molecular weights much higher than was previously possible. In addition, the HO-content of the PHA compositions obtainable from the microorganisms is typically greater than 70%, preferably greater than 80%, and more preferably greater than 90%.
In one important embodiment, the PHA compositions comprise hydroxy-terminated PHA, wherein the compositions have an end group hydroxyl content greater than 90 mol % and a molecular weight greater than 3000. Further provided are PHA compositions wherein the molecular weight is greater than about 20,000 and the end group hydroxyl content is greater than 80%, or those where the molecular weight is greater than about 100,000 and the end group hydroxyl content is greater than about 70%. Although hydroxyl contents greater than 70 or 80% may be preferred for many applications, other applications may not necessarily require such levels. PHA compositions produced in accordance with the present invention which have greater than 50% hydroxyl content, therefore, are also expected to be useful in a variety of applications. In particular, such compositions have been found to have improved thermal stability compared to unmodified PHAs, and are therefore useful in the melt processing of polymeric and copolymeric articles.
These high MW PHA compositions are desirable for use in the production of polymer and copolymer compositions since many of the beneficial properties of high MW PHA's, such as hydrophobicity, biodegradability in soil and aqueous environments and biodegradability in humans and animals, are retained in a polymer or copolymer which contains the PHA.
Furthermore, polymers and copolymers containing the hydroxyterminated PHAs described herein have the benefit of being less thermosensitive than polymers and copolymers which have not been modified to contain an increased end-group hydroxyl content.
Hydroxy-terminated PHAs produced in accordance with this invention are useful in the production of biodegradable graft, block and random polymers and copolymers comprising hydroxy-terminated PHAs and other suitable polymers, oligomers or monomers. Therefore, several embodiments of the present invention relate to methods of using the hydroxy-terminated PHA of the present invention in the production of graft, block or random polymer and copolymer compositions, and the PHA-containing polymer and copolymer compositions produced therefrom. The molecular weight of the hydroxy-terminated PHA, by appropriate selection of the bacterial strain and aliphatic diol or polyol used, can be manipulated such that the properties required or desired for a given PHA-containing polymer or copolymer may be more readily achieved.
In a particularly important embodiment of the present invention, there is provided a method of using the hydroxy-terminated PHA of the present invention to produce a polymer or copolymer, wherein the PHA is reacted with a coupling agent. The polymer or copolymer so produced could be, for example, a block polymer or copolymer. Also provided are the polymer and copolymer compositions produced therefrom. Suitable coupling agents may include, for example, alkyl or aryl diisocyanate or triisocyanate, phosgene, alkyl or diaryl carbonate, a monomeric organic diacid, a monomeric organic diacid chloride, a monomeric organic diacid anhydride or a monomeric organic tetraacid dianhydride. Alternatively, the coupling agent can be an oligomer with end-groups that are reactive with hydroxy-terminated PHA, such as carboxy-terminated oligomeric polyesters or a isocyanate-terminated oligomeric polyol or polyester. This approach can be used, for example, to produce polyesters, copolyesters, polyester-carbonates, and polyester urethanes.
In a further embodiment of the present invention, there is provided a method of using the hydroxy-terminated PHA of the present invention to produce a polymer of copolymer, wherein the PHA is reacted with a coupling agent and with a different hydroxy-terminated moiety. The polymer so produced could be, for example, a block or random block polymer or copolymer. Also provided are the polymer and copolymer compositions produced therefrom. Suitable coupling agents may include, for example, alkyl or aryl diisocyanate or triisocyanate, phosgene, alkyl or diaryl carbonate, a monomeric organic diacid, a monomeric organic diacid chloride, a monomeric organic diacid anhydride or a monomeric organic tetraacid dianhydride. Alternatively, the coupling agent can be an oligomer with end-groups that are reactive with hydroxy-terminated PHA, such as a carboxy-terminated oligomeric polyester or polyamide, or a isocyanate-terminated oligomeric polyol, polyester or polyamide. The hydroxy-terminated moieties for use in this embodiment can include polyester diols such as polycaprolactone diol, polybutylene succinate diol, polybutylene succinate co-butylene adipate diol, polyethylene succinate diol, and similar aliphatic polymeric and copolymeric diols. Alternatively, the hydroxy-terminated moiety can be a polyether diol such as a polyethylene oxide-diol, polypropylene oxide-diol, or polyethylene oxide-propylene oxide diol. This approach can be used, for example, to produce polyesters, copolyesters, polyester carbonates, polyester urethanes, polyester ethers, polyester amides, copolyester ethers, polyester ether carbonates, and polyester ether urethanes.
In yet a further embodiment of the present invention, there is provided a method of using the hydroxy-terminated PHA of the present invention to produce a random or random block polymer or copolymer, wherein the PHA is reacted with a different polyester, copolyester, polyamide, or organic dicarboxylic acid in the presence of a catalyst. Also provided are the PHA-containing polymer or copolymer compositions produced therefrom. Suitable polyesters and copolyesters can include polyethylene terephthalate, polybutylene terephthalate, polycaprolactone, polyhydroxyalkanoates, polylactides, polyglycolides, polyethylene succinate, polybutylene succinate, polyethylene adipate, polyethylene succinate adipate, and other combinations of such polyesters or copolyesters. Suitable dicarboxylic acids may include, for example, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, azealic acid, terephthalic acid, phthalic acid, maleic acid, fumaric acid, and their anhydrides. Suitable polyamides may include for example nylon 6, nylon 66, and nylon 12. The catalyst can be, for example, Sn, Sb, or Ti catalysts. This approach can be used to produce polyesters, copolyesters, and polyester amides.
In yet a further embodiment of the present invention, there is provided a method of using the hydroxy-terminated PHA of the present invention to produce a block polymer or copolymer, comprising the steps of reacting the PHA with a reactive monomer. Also provided are the PHA-containing copolymer compositions produced therefrom. Where needed, catalysts and other reactants known in the art to facilitate the reaction, are used. The reactive monomer used in this embodiment can include, for example, alkyl epoxides such as ethylene oxide and propylene oxide, lactones such as caprolactone, butyrolactone, propiolactone, valerolactone, lactams such as caprolactam, and formaldehyde. This approach can be used to produce polyesters, copolyesters, polyester ethers, polyester amides, and polyester acetals.
In yet a further embodiment of the present invention, there is provided a method of using the hydroxy-terminated PHA of the present invention to produce thermoset resins, wherein the hydroxy-terminated PHA is first derivatized with a derivatizing agent, followed by curing the derivatized PHA with a curing agent. Also provided are the thermoset resin compositions produced therefrom. The curing agents suitable for use in this embodiment can include amide, amine, dicarboxylic acid or dicarboxylic acid anhydride based agents, in addition to other such agents well known in the art. Similarly, the derivatizing agent can be selected from those known in the art. One preferred derivatizing agent for use in this embodiment is epichlorohydrin, where the resulting derivatized PHA is a mono- or di-glycidyl PHA.
Suitable approaches for the production of the novel PHA-containing polymer and copolymer compositions described in the above embodiments are further illustrated below. A block copolymer of the present invention, for example, can comprise hydroxy-terminated PHA and hydroxy-terminated aliphatic polyesters and polyethers such as those selected from hydroxy-terminated polyoxyethylenes, polycaprolactone diol, polybutylene succinate diol, polyethylene succinate diol, and polybutylene succinate co-butylene adipate diol. The polymers and copolymers that can be made include, for example, copolyesters, polyester carbonates, polyester ethers, polyester urethanes, polyester amides, polyester acetals, and others.
Block copolyesters may be synthesized by the reaction of a hydroxy-terminated PHA with an organic diacid, an organic diacid chloride, an organic acid anhydride, an organic acid dianhydride, a tetracarboxylic acid dianhydride, etc. The example below illustrates the reaction of the PHA-diol with an organic diacid chloride.
xHO-(PHA)-OH+xClCO(CH.sub.2).sub.n COCl=x-[O-(PHA)-OCO(CH.sub.2).sub.n CO]-+2xHCl
n=0 to 25
Block copolyesters may also be made by reaction of the PHA-diol with lactones such as beta-butyrolactone, delta-valerolactone, beta-propiolactone, epsilon-caprolactone, etc. They can also be made by reaction of the PHA-diol with lactides and glycolides including L-lactide, L-glycolide, D,L-dilactide, etc. The lactones, lactides, and glycolides selected may be optically active or inactive, depending upon the application. Catalysts for the reactions are known in the art and can include compounds of Sn [e.g. Sn octanoate (see for example U.S. Pat. No. 5,321,088)], Ti [e.g. Ti(OBu).sub.4 (see for example JP 05 202173)], Zn [e.g. Et.sub.3 Zn, (see for example JP 05 132549)], and Al [e.g. Me.sub.3 Al or Et.sub.3 Al (see for example CA 111:120806)].
Block polyester-carbonates may be synthesized by the reaction of hydroxy-terminated PHA with phosgene or alkyl or aryl carbonate, [e.g. diphenyl carbonate (see for example EP 684,270)]. The example below illustrates the reaction of the PHA-diol with phosgene.
xHO-(PHA)-OH+xClCOCl=x-[O-(PHA)-OCO]-+2xHCl
Block polyester ethers can be made by reaction of the PHA-diol with an alkylene oxide such as ethylene oxide (EO) or propylene oxide (PO) or their mixtures [see for example EP 570,121]. The reaction with EO as illustrated below.
xHO-(PHA)-OH+y+zEO=H-[(EO)y-(PHA)x-O(EO)z-]H
Block polyester-urethanes may be synthesized by the reaction of hydroxy-terminated PHA with isocyanates, diisocyanates such as hexamethylene diisocyanate (HMDI), dicyclohexyl methane 4,4'-diisocyanate (CHMDI), isophorone diisocyanate (IPDI) and triisocyanates. The example below illustrates the reaction of the PHA-diol with HMDI.
x.HO-(PHA)-OH+xOCN(CH.sub.2).sub.6 NCO=x-[O-(PHA)-OCON(CH.sub.2).sub.6 NOC]-
Block polyester-amides may be synthesized by the catalyzed reaction of hydroxy-terminated PHA with lactams (see for example JP 04 283233), or by reaction of the diol with an organo diacid chloride and hexamethylene diamine (see for example, CA 118:81550). The example below illustrates the reaction of PHA-diol with beta-caprolactam (b-CL).
xHO-(PHA)-OH+nb-CL=-[O-(PHA)-O]x-[CO(CH.sub.2).sub.5 NH]n-
Polyester acetals can be made by reaction of the PHA-diol with formaldehyde followed by endcapping with acetic anhydride. Catalysts for the reaction include Sn compounds such as (Bu).sub.2 Sn(OCH.sub.3).sub.2 (see for example JP 06 65468) and quaternary ammonium compounds such as Bu.sub.4 N+Oac-(see for example JP 05 43638).
xHO-(PHA)-O+2xyCH.sub.2 O+Ac2O=xAc(OCH.sub.2)nO-(PHA)-O(CH.sub.2 O)mAc
Use of the hydroxy-terminated PHA compositions of the present invention for production of novel random polymers and copolymers is illustrated below. Random polymers made in this way can include copolyesters, polyester carbonates, polyester ethers, polyester urethanes, an polyester amides.
Random copolyesters may be synthesized by the reaction of the PHA-diol with PET in the presence of a Sn catalyst in DMSO solvent at greater than about 100.degree. C.
xHO-(PHA)-OH+y-(EG)-(TPA)=-(HA)x-(EG)y-(TPA)y-
Random block copolyester amides could also be synthesized by reaction of the PHA-diol with a nylon (e.g. nylon 12) in the presence of a catalyst [e.g. Zn(OAc).sub.2, (see for example CA 118:40168)] or by reaction of PHA-diol, a diacid such as dodecanedioic acid (DDDA) and caprolactam (CL) in the presence of a catalyst such as Sb.sub.2 O.sub.3 (see for example JP 04 283233) as shown in the example below.
xHO-(PHA)-OH+yDDDA+zCL=-[CL]z[O-(PHA)-O]x[DDDA]y-
Random block polyester-ethers may be synthesized by the reaction of the PHA-diol, a polyalkylene glycol and a diacid, diacid chloride, or a dianhydride. The example below illustrates the reaction of PHA-diol, polyethylene glycol and a diacid chloride.
xHO-(PHA)-OH+yClCORCOCl+zH-(OCH.sub.2 CH.sub.2)n-OH=-[O-(PHA)x-O(CORCO)yO(CH.sub.2 CH.sub.2 O)nz]-+2yHCl
The hydroxy-terminated PHA produced in accordance with the present invention can also be converted into biodegradable thermoset resins by selected, known reactions. For example, conversion of the diol with epichlorohydrin to PHA-diepoxide followed by a curing step can give a biodegradable epoxy resin, depending on selection of the curing agent and other components of the formulation.
xHO-(PHA)-OH+2xClepox=x[epox-O-(PHA)-O-epox]
x[epox-O-(PHA)-O-epox]+curing agent=cured resin
Other thermoset resins that can be made using PHA-diol as a starting material include polyurethanes and unsaturated polyesters.
Most PHAs are highly thermosensitive. As a result, PHAs typically undergo a drastic reduction in molecular weight when subjected to the conditions required of the melt processing operations that are used in commercial plastics production. This thermosensitivity and the resulting polymer degradation severely compromise the ability to produce PHA-derived products having commercially desired properties. Indeed, the well documented thermosensitivity of PHAs has contributed to their lack of acceptance as a viable biodegradable alternative to other polymer types.
In addition to the numerous applications described hereinabove involving the use of hydroxy-terminated PHAs in the production of novel polymers, copolymers, resins etc., we have found in another embodiment of this invention an unexpected use for PHAs having end-group hydroxyl contents greater than 50%. In particular, hydroxy-terminated PHA polymers and copolymers have been found to exhibit improved thermal stability, i.e, reduced thermosensitivity, compared with PHAs that have not been modified to contain increased end group hydroxyl contents. As used herein, improved thermal stability refers to a reduced tendency of the disclosed PHAs to undergo thermal mediated degradation as measured for example by the decrease in polymer molecular weight as a result of exposure to temperatures or other conditions, such as shear, that cause such degradation. Other measures of improved thermal stability can include reduced weight loss and increased energy of activation for thermal degradation.
Although not entirely understood, it is thought that there are competing reactions occurring in a PHA melt (N. Grassie, E. J. Murray, P. A. Holmes, Polymer Degradation and Stability 6 (1984), 127-134). In the first reaction shown below, condensation is occurring between the hydroxyl and carboxyl ends of the polymer molecules. Such reactions can continue until all avaliable hydroxyl groups are consumed and result in an overall increase in polymer molecular weight. Indeed, polymer molecular weight is observed to initially increase in a PHA melt as a result of these condensation reactions. However, over time, the molecular weight of the polymer in the melt rapidly declines to unacceptable levels. Presumably, this is a result of the random chain scissions of the polymer backbone, shown in the second reaction below, that are thought to take place concurrently with condensation reactions. One of the products of this scission reaction is a carboxyl group which, at least initially while hydroxyl groups are still available, participates in further condensation reactions. As a result of these reactions in a PHA melt, the avaliable hydroxyl groups become quickly consumed by condensation after which chain scission then dominates to degrade the polymer over time. These reactions are illustrated below. ##STR5##
We have found that PHAs having increased end group hydroxyl content display improved thermal stability, i.e., significantly less molecular weight reduction is observed upon exposure to melt conditions compared with unmodified PHAs under the same conditions. This appears to be a result of prolonging the condensation reaction by making available additional hydroxyl groups which can delay the degradative effects of the scission reaction.
PHAs having improved thermal stability are desirable for essentially any application in which the polymer is subjected to temperatures and/or other processing conditions which result in the degradation and molecular weight reduction of the polymer. They are particularly useful for producing shaped plastic polymeric and copolymeric articles by extrusion, injection molding, coating, thermoforming, fiber spinning, blow molding, calendering etc. Some such articles of interest include for example, polymeric pellets and granules, packaging films, non-woven fabrics, extruded nets, personal hygiene products, bottles and drinking vessels, agricultural and horticultural films and vessels, ostomy bags, coated products (such as paper, paperboard, non-woven fabrics etc.), slow release devices, adhesives, and many others.
The hydroxy-terminated PHAs used in the production of shaped articles can, if desired, be in the form of graft, block and random polymers and copolymers comprising hydroxy-terminated PHAs and other suitable polymers, oligomers or monomers.
Although it is typically preferred to use the hydroxy-terminated PHAs produced in accordance with this invention, this embodiment is not intended to be limited only to such polymers. In fact, hydroxy-terminated PHA produced by any known means will exhibit the improved thermal stability disclosed herein.
It may be preferred in this embodiment to use a hydroxy-terminated PHA produced using a polyol in accordance with this invention. Some of the PHA molecules so produced will have greater than two hydroxyl groups per PHA molecule. In this situation, the resulting polyol terminated polymer will have a greater number of hydroxyl groups per PHA molecule available for condensation, thereby providing for greater thermal stabilization. Furthermore, as a result of the condensation reaction occurring at multiple hydroxyl groups per molecule, a branched PHA structure results which can provide beneficial properties as a result of the improved extensional strength of branched material.
Therefore, this invention further provides branched PHA compositions, and shaped articles at least partly made therefrom, produced by melt-processing polyol terminated PHAs. The branched nature of a composition produced by melt processing polyol terminated PHA has been experimentally supported (see Example 20 and FIGS. 3,4 and 5). The resulting branched PHA is believed to have the following general structure: ##STR6## where: R=H, alkyl, alkenyl, aralkyl, aryl, cyclohexyl
R.sub.1 =H, alkyl, cyclohexyl, aralkyl, aryl, or
--(CH.sub.2 CH.sub.2 O)m-, --(CH.sub.2 CH(CH.sub.3)--O).sub.m -- where m=1-20;
n=1-150,000;
p=0-150,000;
q=0-150,000;
r=0-150,000;
wherein when p,q, r.gtoreq.1,
R.sub.2, R.sub.3, R.sub.4 =alkyl, cyclohexyl, aralkyl, aryl, or
--(CH.sub.2 CH.sub.2 O)m-, --(CH.sub.2 CH(CH.sub.3)--O).sub.m -- where m=1-20;
and wherein when p, q, r=0,
R.sub.1 =alkyl, cyclohexyl, aralkyl, aryl, H, or
--(CH.sub.2 CH.sub.2 O).sub.m --, --(CH.sub.2 CH(CH.sub.3)--O).sub.m -- where m=1-20, and
R.sub.2, R.sub.3, R.sub.4 =H or OH in which case the corresponding terminal hydroxyl group is removed from the structure.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent compositions and methods discovered by the inventor to finction effectively in the practice of the present invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1
Synthesis of Diol-Terminated Poly(3-hydroxybutyrate) by Transesterification With Ethylene Glycol
100 g poly(3-hydroxybutyrate) was dissolved in 300 ml diglyme at 140.degree. C. in a 4-necked flask equipped with a thermometer, overhead stirrer and reflux condensor, under nitrogen. To this 25 ml ethylene glycol was added and the mixture allowed to stir for about 5 minutes. Following this, 0.14 mL dibutyltin dilaurate was added via a syringe, and the reaction mixture was stirred for 7 hours. During the reaction, 0.14 mL dibutyltin dilaurate was added every 1.25 h (0h, 1.25 h, 2.5 h, 3.75 h, 5 h, 6.25 h). At the end of 7.5 h, the solution was immediately filtered hot, and the product precipitated by pouring it into approximately 10 times excess volume of cold distilled water. The product was separated by filtration under vacuum, washed with distilled water three times, and dried under vacuum at 80.degree. C. for 24 h. An NMR method was adapted from the literature (Spyros, A., et. al.; Macromolecules (1997), 30, 327-9) to determine the end groups of PHA's. Since phosphorus in both hydroxyl and carboxyl end groups are present in the same P-31 spectrum, a ratio can be calculated by simple integration of the peaks. Molecular weight was determined by GPC using polystyrene (PS) calibration.
Preparation of PHA-diol by reaction of PHB with EG.
______________________________________Preparation of PHA-diol by reaction of PHB with EG. HO-content Time, hours Mn % of endgroups______________________________________0 260 k 50 1.25 18 k 77 2.5 5.0 k 82 3.75 3.0 k 87 6.25 2.0 k 91 7.5 1.6 k 92______________________________________
EXAMPLE 2
Ralstonia eutropha strain TRON721 was grown in 250-ml shake flasks containing 50 ml of a minimal salt medium plus glucose (2%), Na-4-hydroxybutyrate (0.5%) and ethylene glycol (EG). Cells were grown at 30.degree. C. for 4 days. The MW of the PHA was measured by GPC using a PS standard. Content of hydroxyl end groups was determined by the NMR technique referenced in Comparative Example 1.
______________________________________ CDW PHA Mw EG (%) (mg/ml) Content (%) (.times. 1000) Mn (.times. 1000) mol %______________________________________ OH0 351 70.9 864 331 43 1 354 72.6 201 75 84 3 327 70.9 143 68 91 5 175 49.1 185 84 97______________________________________
At concentrations greater than about 3%, EG has a toxic effect on the bacteria as shown by a significant drop in CDW and PHA content.
EXAMPLE 3
Ralstonia eutropha strain TRON721 was grown in the same conditions as in Example 1, except the medium contained glucose (2%), Na-4-hydroxybutyrate (0.5%), Na-butyrate (0.1%) and ethylene glycol (2%). Cell dry weight (CDW) was 500 mg/flask and PHA content was 77.2%. This PHA contained 15.2% 4-hydroxybutyric acid and 84.8% 3-hydroxybutyric acid. The MW (Mn) as determined by GPC using a PS standard was 66,700 and the mole-% hydroxyl end groups was 94.6 by NMR analysis.
EXAMPLE 4
Ralstonia eutropha strain TRON721 was grown in 2-L shake flasks containing 1-L of a minimal salt medium with glucose (2%), Na-butyrate (0.1%) and ethylene glycol (2%). Cells were grown at 34.degree. C. for 3 days. The PHB production was 1.49 g/L with molecular weight by GPC using a PS standard Mw=298,000 and Mn=64,000. The mole-% hydroxyl end groups was 92.43% by NMR analysis.
EXAMPLE 5
Ralstonia eutropha strain TRON 721 was grown in 250-ml shake flasks containing 50 ml Luria-Bertani (LB) medium plus 2% oleic acid and different concentrations of ethylene glycol (EG). The culture flasks were incubated at 30.degree. C. for 3 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The MW of the PHB was measured by GPC using a polystyrene standard.
______________________________________ Mw EG (%) CDW (mg/ml) PHB Content (%) ( .times. 1000) Mn (.times. 1000)______________________________________0 12.6 70 3470 675 1 12.8 63.5 794 319 2 11.4 55 308 217 3 11.7 64 180 60______________________________________
EXAMPLE 6
Alcaligenes latus (ATCC 29713) was grown in 250 ml shake flasks containing 50 ml LB medium plus 2% sucrose and 3% ethylene glycol (EG). The culture flasks were incubated at 30.degree. C. for 3 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The MW of the PHB was measured by GPC using a polystyrene standard.
______________________________________ Mw EG (%) CDW (mg/ml) PHB Content (%) ( .times. 1000) Mn (.times. 1000)______________________________________0 9.7 67.6 698 341 3 5.5 47.6 69 31______________________________________
The content of hydroxyl endgroups of the polymer made with 3% EG added was 91.5% by NMR analysis.
EXAMPLE 7
Comamonas testosteroni was grown in 250-ml shake flasks containing 50 ml LB medium plus 2% oleic acid and different concentrations of ethylene glycol (EG). The culture flasks were incubated at 30.degree. C. for 3 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The MW of the PHB was measured by GPC using a polystyrene standard.
______________________________________ Mw EG (%) CDW (mg/ml) PHB Content (%) ( .times. 1000) Mn (.times. 1000)______________________________________0 3.3 23.5 247 96 1 5.7 33.6 59 37 2 4.3 29.5 34 19 3 4.3 32.4 40 22______________________________________
EXAMPLE 8
Comamonas testosteroni was grown in a 10-L fermenter (Braun Biostat B) containing 8.5 L of medium containing (g/L):
______________________________________K2HPO4 4.7 KH2PO4 1.1 MgSO4-7H2O 5 NaCl 5 Yeast extract 15 ferric citrate 0.01 EDTA 0.04 oleic acid 50 ml ethylene glycol 30 ml______________________________________
pH was adjusted to 6.5 with ammonia (28%). Agitation was 600-1200 rpm with dissolved oxygen minimum of 20%. Aeration was 5 L/minute. The culture was harvested after 65 hours and cells were extracted with chloroform at room temperature for 24 hours.
______________________________________CDW(g/L) PHB Content (%) Mw (.times. 1000) Mn (.times. 1000)______________________________________16.4 22 58.6 21.1______________________________________
The hydroxyl endgroup content was found to be 87% by NMR analysis.
EXAMPLE 9
Ralstonia eutropha strain TRON721 and Alcaligenes latus ATCC 29713 were grown in 250-ml shake flasks containing 50 ml LB medium plus 2% glucose (A.eutrophus) or 2% sucrose (A.latus) and with or without 1% neopentyl glycol (NG). The culture flasks were incubated at 30.degree. C. for 3 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The molecular weight of the PHB was measured by GPC using a polystyrene standard.
______________________________________NG CDW PHB Mw (%) (mg/ml) Content (%) (.times. 1000) Mn (.times. 1000)______________________________________R. eutropha 0 9.3 36 3759 661 1 9.5 29 3559 550 A. latus 0 7.8 58 614 249 1 3.2 24 396 182______________________________________
EXAMPLE 10
The experiments described in example 9 were repeated except that diethylene glycol was used.
______________________________________DEG CDW PHB Mw (%) (mg/ml) Content (%) (.times. 1000) Mn (.times. 1000)______________________________________R. eutropha 0 9.3 36 3759 661 1 9.1 45 735 243 A. latus 0 7.8 58 614 249 1 6.4 61 65 36______________________________________
EXAMPLE 11
The experiments described in example 9 were repeated except that tetraethylene glycol was used.
______________________________________TEG CDW PHB Mw (%) (mg/ml) Content (%) (.times. 1000) Mn (.times. 1000)______________________________________R. eutropha 0 9.3 36 3759 661 1 9.1 57 849 274 A. latus 0 7.8 58 614 249 1 7.9 52 128 84______________________________________
EXAMPLE 12
Ralstonia eutropha strain TRON 721 was grown in 250-ml shake flasks containing 50 ml LB medium plus 2% glucose and different concentrations of propylene glycol (PG). The culture flasks were incubated at 30.degree. C. for 4 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The MW of the PHB was measured by GPC using a PS standard.
______________________________________PG CDW PHB (%) (mg/ml) Content (%) Mw (.times. 1000) Mn (.times. 1000) % OH______________________________________1 10.7 57 346 131 80 2 11.0 54 272 120 78 3 10.2 49 227 97 80 4 11.1 47 187 69 92 5 11.1 56 180 63 87______________________________________
EXAMPLE 13
Ralstonia eutropha strain TRON 721 was grown in 250-ml shake flasks containing 50 ml LB medium plus 2% glucose and different concentrations of pentaerythritol (PAE). The culture flasks were incubated at 30.degree. C. for 4 days in a shaker (New Brunswick) orbiting at 250 rpm. Cells were harvested, dried, and extracted with chloroform at 100.degree. C. for 2 hours. The MW of the PHB was measured by GPC using a PS standard.
______________________________________PAE CDW PHB Mw (%) (mg/ml) Content (%) (.times. 1000) Mn (.times. 1000) % OH______________________________________0 8.7 57 1,362 420 51 0.25 10.4 46 778 254 79 0.5 9.3 52 1,269 493 77 1.0 8.4 51 1,308 406 (Not Determined) 2.0 9.1 51 1,535 728 67______________________________________
EXAMPLE 14
Synthesis of 50:50 Poly(3-hydroxybutyrate)-block-Poly(.epsilon.-caprolactone)Copolyesterurethane
4.5 g P3HB diol obtained as described in Example 8 (87% of end-groups determined to be hydroxyl, 13% carboxyl, Mn=19,300 by end-group analysis, Mn=21,100 by GPC relative to PS, 0.23 mmol) and 4.5 g polycaprolactone (PCL) diol (Aldrich, Mn=2000 reported by vendor, 2.2 mmol) were dissolved in 200 ml 1,2-dichloroethane in a 500 ml round-bottom flask. The solution was refluxed for about 20 hours via a soxhlet extractor with a thimble containing .about.15 g activated 4 .ANG. molecular sieves, to remove any residual water from the diols as a water/dichloroethane azeotrope. Following this, most of the dichloroethane was distilled off until a still stirrable, viscous solution remained. A nitrogen purge was maintained at all times. The distillation head was then removed and the flask fitted with an overhead stirrer and reflux condensor. A solution of 0.44 g hexamethylene diisocyanate (2.6 mmol) in 5 ml anhydrous 1,2-dichloroethane (EM Science, kept over molecular sieves for at least 24 h prior to use) was prepared, which was then slowly added via an addition funnel in two parts. Approximately half was added at the beginning of the reaction over a 2 h period, and the other half was added 12 h later, also over a 2 h period. A solution of 42 .mu.l of the catalyst, dibutyltin dilaurate, was prepared in 5 mL anhydrous 1,2-dichloroethane. 1.5 ml of this solution was added via a syringe at the beginning of the reaction. The reaction mixture was stirred at 79.degree. C. for about 110 hours. The product was recovered by precipitation in 1 L methanol, and further purified by redissolving in chloroform, reprecipitating in 1 L petroleum ether and drying in a vacuum oven at approximately 80.degree. C. for about 12 hours. Yield: 8.4 g. Molecular weight determination by GPC with PS calibration showed the product to have Mw=102,000 and Mn=52,000. Characterization by DSC of this polymer showed two endothermic peaks at 38.degree. C. and 161.degree. C. upon heating, and two exothermic peaks at 91.degree. C. and 4.degree. C. on cooling. The sample in tensile testing, showed 637% elongation at break and tensile strength of 1528 psi.
EXAMPLE 15
Synthesis of 75:25 Poly(3-hydroxybutyrate)-block-Poly(.epsilon.caprolactone)Copolyesterurethane
4.5 g P3HB diol obtained as described in Example 8 (87% of end-groups determined to be hydroxyl, 13% carboxyl, Mn=19,300 by end-group analysis, Mn=21,100 by GPC relative to PS, 0.23 mmol) and 1.5 g PCL diol (Aldrich, Mn=2000 reported by vendor, 0.75 mmol) were dissolved in 200 ml 1,2-dichloroethane in a 500 ml round-bottom flask. The solution was refluxed for about 18 h via a soxhlet extractor with a thimble containing .about.15 g activated 4 .ANG. molecular sieves, to remove any residual water from the diols as a water/dichloroethane azeotrope. Following this, most of the dichloroethane was distilled off until a still stirrable, viscous solution remained. A nitrogen purge was maintained at all times. The distillation head was then removed and the flask fitted with an overhead stirrer and reflux condensor. A solution of 0.17 g hexamethylene diisocyanate (1.03 mmol) in 5 ml anhydrous 1,2-dichloroethane (EM Science, kept over molecular sieves for at least 24 h prior to use) was prepared, which was then slowly added to the flask via an addition funnel. A solution of 28 .mu.l of the catalyst, dibutyltin dilaurate, was prepared in 5 mL anhydrous 1,2-dichloroethane. 1.5 ml of this solution was added via a syringe at the beginning of the reaction. The reaction mixture was stirred at 79.degree. C. for 141 hours. The product was then recovered by precipitation in 1 L methanol, and further purified by redissolving in chloroform, reprecipitating in 1 L petroleum ether and drying in a vacuum oven at 80.degree. C. for 12 hours. Yield: 5.6 g. Molecular weight determination by GPC with PS calibration showed the product to have Mw=59,900 and Mn=26,850.
EXAMPLE 16
A copolyester urethane was prepared with 25 g P3HB diol, 25 g PCL-diol and 4.4 g HMDI by essentially following the procedure of Example 14, with the exception that the P3HB-diol of Mn=2000 and 92% HO-endgroup content, was made by chemical transesterification of PHB and EG in the presence of a stannate catalyst, and the reaction time was approximately 24 hours. Yield: 42.3 g. Molecular weight determinations were made by GPC with PS calibration and showed the product to have Mw=74,950 and Mn=44,100. The final product in tensile testing, showed 101% elongation at break and tensile strength of 1105 psi.
EXAMPLE 17
Activation Energy of Thermal Degradation of PHA Samples by TGA (Thermogravimetric Analysis)
Hydroxy-terminated PHA samples in powder form were tested using the simultaneous DTA-TGA using Omnitherm STA 1500 Thermogravimetric Analyzer and analyzed using the accompanying software package. The instrument weight axis was calibrated using a calibration weight according to the instrument SOP. The TGA module measures weight loss as a function of temperature through the use of a null seeking balance. The procedure followed in the analysis was based on the ASTM Standard E 1641-94 ("Standard Test Method for Decomposition Kinetics by Thermogravimetry"). This procedure involved heating a series of four, 5.+-.1 mg, samples at different heating rates, specifically 1, 2, 5, and 10 K/min. The samples were heated from 25.degree. C. through their decomposition (from 230.degree. C. to 310.degree. C.). The temperature at which the sample lost a certain percentage of its weight was recorded for the four rates. A 10% loss was used for three of the samples but for two, better fits were generated for 5% and 15% weight losses. An Arrhenius plot--log (heating rate (K/min)) vs. 1/Temperature (K)--was obtained and the slope was given by least-squares fit. The slope was then used in the calculations contained in the standard to generate the activation energy (Ea).
The furnace was calibrated using a number of temperature stability readings at 2 degree intervals. A series of four 5.+-.1 mg samples were heated at different isothermal temperatures, specifically 170, 180 190 and 200.degree. C. The samples were heated from 25.degree. C. and maintained at the chosen isothermal temperature for 120 minutes. The weight lost over time was recorded. The results of these experiments are presented in the following table which shows the increased activation energy measured for the thermal degradation of hydroxy-terminated PHAs as compared to the control PHB sample. The percent residual weight after 120 minutes of isothermal heating also shows higher percentages of residue for the hydroxy-terminated PHAs as compared to the control, most probably due to the retardation of the random chain scission reaction and subsequent volatalization in the hydroxy-terminated PHAs. This is further illustrated by FIG. 2.
__________________________________________________________________________ Onset of thermal deg % residual wt. after 120 mins ofCarbon .degree. C. (5.degree. C./min) isothermal heatingSample Source Mw Mn % OH Ea (KJ/mol) 170.degree. C. 170.degree. C. 180.degree. C. 190.degree. C. 200.degree. C.__________________________________________________________________________PHB- glucose 877200 313500 40 98 256.76 99.5 90.6 58.9 13.1 G044 (control) 6025261 glu + pent. 137100 59000 82 272 281.17 100 100 98.9 73.7 ethoxylate 6025275 glu + EG 88000 27000 93 118 263.96 100 100 100 98.3 6025276 glu + PG 47000 16900 94 160 254.7 100 99.3 98.3 93.2__________________________________________________________________________
EXAMPLE 18
Molecular Weight Changes Under Isothermal Heating
Samples in powder form were divided into three portions of approximately 10 mg each in glass vials. The vials were then placed in a preheated oven at 170.degree. C., under a nitrogen purge, for intervals of 20, 40 and 100 minutes. At the specified intervals, the vials were withdrawn and allowed to cool down to room temperature in ambient atmosphere. These samples were then dissolved in chloroform at concentrations of approximately 2 mg/ml. Molecular weights were determined by Gel Permeation Chromatography (GPC) using a system with a Waters 410 differential refractometer, and a calibration curve of narrow polydispersity polystyrene standards. The weight and number average molecular weights of the samples tested this way are presented for each interval. As the results in the following table demonstrate, the hydroxy-terminated PHAs show minimal to no change in molecular weights after 100 minutes of isothermal heating at 170.degree. C., illustrating the prolonged condensation reaction between the available hydroxyl and carboxyl end-groups. In case of the pentaerythritol ethoxylate-terminated PHA, the weight average molecular weight of the polymer after 100 minutes is still measured to be marginally higher than the initial weight average molecular weight. In comparison, the PHB control shows a greater than 50% reduction in molecular weight.
__________________________________________________________________________Isothermal heating at 170.degree. C. carbon 0 mins 20 mins 40 mins 100 minsSample source Mw Mn Mw Mn Mw Mn Mw Mn__________________________________________________________________________PHB-G044 glucose 877200 313500 958500 269200 434200 139500 283200 101600 (control) 6025261 glu + pent. 137100 59000 161900 95200 186800 74500 155700 50200 ethoxylate 6025276 glu + PG 47000 16900 43200 17400 57500 15200 47500 17000__________________________________________________________________________
EXAMPLE 19
Production of Hydroxy-Terminated PHAs via Fed-Batch Fermentation
Ralstonia eutropha strain TRON 721 was grown in a minimal medium with 2% glucose in a shake flask (50-250 ml flask) overnight, and the whole culture was used to inoculate a Braun Biostat B, fermenter with a 2 L vessel. The medium at the start of fermentation consisted of the following:
______________________________________Glucose 20 g/L Diol/Polyol percentage specified Ammonium Sulfate 4 g/L MgSO.sub.4.7H.sub.2 O 2.2 g/L Citric acid 1.7 g/L Trace elements 8 ml Water 1 L______________________________________
(Trace elements: FeSO.sub.4.7H.sub.2 O, 10 g/L; ZnSO.sub.4.7H.sub.2 O, 2.25 g/L; CuSO.sub.4.5H.sub.2 O, 0.5 g/L; CaCl.sub.2.2H.sub.2 O, 2 g/L; H.sub.3 BO.sub.3, 0.1 g/L; (NH.sub.4).sub.6 Mo.sub.4 O.sub.24, 0.1 g/L; HCl, 10 ml)
After autoclaving, a filter sterilized solution of 3.5 g of KH.sub.2 PO.sub.4 in 30 ml water was added, and the pH was adjusted to 6.8 with ammonia. During the fermentation the pH was maintained at 6.8 with ammonia and 20% H.sub.2 SO.sub.4. The fermenter was maintained at 34.degree. C., and the medium agitated at 730-1200 rpm with dissolved oxygen maintained at a minimum of 20%. The aeration was at 4 L/min. When the glucose in the medium was exhausted (determined by YSI 2700 Select glucose analyzer, YSI, Inc. Yellow Springs, Ohio), feed consisting of 0.5 L water, 400 g glucose and the corresponding percentage of diol/polyol was introduced to the vessel. The feeding rate was varied depending on the rate of consumption of glucose, such that the glucose concentration was maintained at less than 2%. During the fermentation, foam was controlled by 50% SAG471 (OSI Specialties, Sistersville, W. Va.). The fermentation run was continued until the feed was exhausted. At the end of the run, the cells were harvested, dried and extracted for 2 h in refluxing chloroform. The polymer was recovered from the chloroform extract by precipitation into approximately 3-4 volumes of petroleum ether. Purification was carried out by redissolution and reprecipitation from chloroform into petroleum ether.
The following is a list of diols/polyols included in the feed during separate fermentation runs that were carried out as described above:
1% ethylene glycol
3% ethylene glycol
5% ethylene glycol
1% propylene glycol (1,2-propanediol)
3% propylene glycol
2% pentaerythritol ethoxylate (3/4 EO/OH)
Growth curves obtained from time course studies of these fermentations confirmed the accumulation of PHB at the levels of diol/polyol tested.
Another set of fermentations was performed essentially as described above, with the following modifications. The diol was not included in the initial medium. After 17 hours, when the glucose was exhausted in the medium and the cells had entered the stationary phase of growth, 10 mL of diol was added to the medium, and a continuous feed of 80% glucose and 5% diol in water was started. The fermentation was continued until the feeding was complete. The above procedure was used for two separate fermentations, one with 5% ethylene glycol and one with 5% propylene glycol. Growth curves of these runs confirmed that PHB accumulated over time.
EXAMPLE 20
Extrusion of Pentaerythritol Ethoxylate-Terminated PHB to Produce a Branched Polymer
PHB homopolymer (40% hydroxyl end-groups and 60% carboxyl end-groups; Mw=478,500 & Mn=168,100 based on universal calibration) and pentaerythritol ethoxylate terminated PHB (90% hydroxyl and 10% carboxyl end-groups; Mw=277,700 & Mn=100,000 based on universal calibration) were processed in a DACA.TM. Micro-compounder (DACA Instruments; Goleta, Calif.) by extrusion after circulating the melt in the extrusion chamber for 3 minutes at 170.degree. C. The resultant materials were collected and analyzed by gel permeation chormatography (GPC), along with the unprocessed materials. GPC was carried out on a Waters 150 CV system (Waters Corporation; Milford, Mass.) with a detection system consisting of a differential refractometer and single-capillary viscometer, and 3 mixed-bed PLGel columns with 10 .mu.m particle size (Polymer Laboratories Inc.; Amherst, Mass.), connected in series. The mobile phase was chloroform. Molecular weights and corresponding intrinsic viscosities were determined by injecting 300 .mu.L solutions with known concentrations of around 2 mg/mL in chloroform and analyzing them against a universal calibration curve generated by narrow-polydispersity polystyrene standards. Mark-Houwink plots were generated for these samples, and are represented in the following figures.
FIG. 3 shows the Mark-Houwink plots for unprocessed and processed PHB homopolymer. It demonstrates that both materials have the same relationship between the logarithmic intrinsic viscosities and molecular weights. This indicates that both polymers have similar hydrodynamic volumes (i.e. the volume the polymer chains occupy under identical conditions of solvent and temperature), as would be expected for linear polymers. Thus, the linear nature of the PHB chains does not change upon processing.
FIG. 4 shows the Mark-Houwink plots for unprocessed PHB homopolymer and unprocessed pentaerythritol ethoxylate terminated PHB homopolymer. The results demonstrate that both polymers have similar hydrodynamic volumes. Thus, it can be concluded that the pentaerythritol ethoxylate terminated PHB is also a linear polymer like the normal PHB homopolymer.
FIG. 5 shows the Mark-Houwink plots for the unprocessed and processed pentaerythritol ethoxylate terminated PHB homopolymer. These results demonstrate that across the entire range of molecular weights measured, the processed material has lower intrinsic viscosities when compared to unprocessed material having similar molecular weights. This indicates that the polymer chains in the processed polymer have lower hydrodynamic volumes compared to those of the unprocessed polymer. In other words, the chains of the processed polymer are more compact than those of the unprocessed polymer at the same molecular weights. This is indicative of the formation of a branched structure when the pentaerythritol ethoxylate terminated PHB is processed in the is extruder. This occurs as a result of the condensation of polymer carboxyl end-groups with the multiple polymer hydroxyl end-groups in the pentaerythritol ethoxylate terminated polymer, giving rise to branched structures.
The following table shows the weight and number average molecular weights of the processed and unprocessed samples:
______________________________________Sample Mw Mn______________________________________PHB, unprocessed 478,500 168,100 PHB, extruded at 170.degree. C. 229,500 88,500 Pentaerythritol ethoxylate 277,700 100,000 terminated PHB, unprocessed Pentaerythritol ethoxylate 215,900 89,200 terminated PHB, extruded at 170.degree. C.______________________________________
EXAMPLE 21
Effect of Diol/Polyol on Molecular Weight of Hydroxyterminated PHA
Ralstonia eutropha strain TRON was grown in 250-ml flask containing 50 ml of LB medium, 2% glucose, and a diol or polyol (concentration from 0.1 to 2%). Flasks were orbiting at 250 rpm in a New Bruswick controlled environment incubator shaker at 30 deg.C. for 3 days. Cells were harvested by centrifugation, and PHB was extracted with chloroform at 100 deg.C. for 2 hr. The PHB was isolated by precipitation in methanol, filtration, and drying under vacuum overnight. Molecular weights (Mn) were determined via GPC, using a standard calibration curve generated by narrow polystyrene standards. The molecular weight data of the polymers is summarized in the following table. These data show that the molecular weight of the hydroxyterminated PHAs produced in accordance with this invention can be influenced by the selection of diol/polyol.
______________________________________Diol/Polyol Conc., % Mn______________________________________Control 0 892 1,2-Butanediol 2 124 1,3-Butanediol 2 177 1,4-Butanediol 2 137 2,3-Butanediol 2 201 1,2,3-Butanetriol 2 207 Glycerol propoxylate 2 182 PPG425 2 423 PPG 725 2 535 PPG 1000 2 867 Glycerol 2 230 Hydroquinone 0.1 322 1,4-Benzenedimethanol 0.1 189 PET (Pentaerythritol) 2 728 PET ethoxylate 2 239 PET propoxylate 2 328 Sorbitol 2 415 Mannitol 1 524 PET 2 237 ethoxylate/propoxylate 1,2-hexanediol 0.5 106 1,6-hexanediol 0.5 78 2,5-hexanediol 0.5 248 1,3-cyclohexanediol 0.5 697 1,4cyclohexanediol 0.5 759 1,2-octanediol 0.1 120 1,8-octanediol 0.1 1406______________________________________
All of the methods and compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Number	Name	Date
5231148	Kleinke et al.	Jul 1993
5268422	Yalpani	Dec 1993
5545485	Hashitani et al.	Aug 1996
5550173	Hammond et al.	Aug 1996
5552515	Hubbs et al.	Sep 1996
5625030	Williams et al.	Apr 1997

Number	Date	Country
0 440 165	Aug 1991	EPX
2 272 904	Jun 1994	GBX
9707153	Feb 1997	WOX
9848028	Oct 1998	WOX

Hydroxy-terminated polyhydroxyalkanoates

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Hirt, Thomas D. et al. (1996) "Telechelic diols from poly[(R)-3-hydroxybutyric acid] and poly{[(R)-3-hydroxybutyric acid]-co-[(R)-3-hydroxyvaleric acid]}, " Macromol. Chem. Phys. 197:1609-1614.
Shi, Fengying et al. (1996) "Use of Poly(ethylene glycol)s To Regulate Poly(3-hydroxybutyrate) Molecular Weight dueing Alcaligenes eutrophus Cultivations," Macromolecules 29:7753-7758.
Grassie, N. et al. (1984) "The Thermal Degradation of Poly(-D)-.beta.-Hydroxybutyric Acid): Part 3--The Reachtion Mechanism," Polymer Degradation and Stability 6:127-134.