Preparation and Properties of a Novel Class of Polyhydroxyalkanoate

Jan 12, 2005 - PHA copolymers with different mcl-3HA types and contents can be made either by bacterial fermentation or by chemical synthesis. The inc...
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Biomacromolecules 2005, 6, 580-586

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Preparation and Properties of a Novel Class of Polyhydroxyalkanoate Copolymers† Isao Noda,*,‡ Phillip R. Green,§ Michael M. Satkowski,‡ and Lee A. Schechtman| The Procter & Gamble Company, 8611 Beckett Road, West Chester, Ohio 45069, The Procter & Gamble Company, 11530 Reed Hartman Highway, Cincinnati, Ohio 45241, and The Procter & Gamble Company, P.O. Box 538707, Cincinnati, Ohio 45253-8707 Received August 31, 2004; Revised Manuscript Received December 17, 2004

Polyhydroxyalkanoates (PHAs) are biodegradable aliphatic polyesters, known to be produced by many common microorganisms. Nodax is a recently introduced family of PHA copolymers comprising 3-hydroxybutyrate units and a relatively small amount of other medium chain length 3-hydroxyalkanoate (mcl-3HA) comonomers with side groups of at least three carbon units or more. There are several different grades of copolymers available, depending on the average molecular weight, average mcl-3HA content within the copolymer, and side group chain length of the chosen mcl-3HA unit. PHA copolymers with different mcl-3HA types and contents can be made either by bacterial fermentation or by chemical synthesis. The incorporation of mcl-3HA units into PHAs effectively lowers the crystallinity and Tm in a manner similar to the effect of R-olefins in linear low-density polyethylene. The Tm can be lowered well below the thermal decomposition temperature of PHAs to make this material much easier to process. The reduced crystallinity provides the ductility and toughness required for many practical applications. The mcl-3HA content regulates the Tm and crystallinity of copolymer almost independently of the branch size, as long as more than three carbons are present in the side group. On the other hand, the side group chain length of the mcl-3HA has a profound effect on the flexibility of copolymer. Introduction Polyhydroxyalkanoates (PHAs) are biodegradable aliphatic polyesters, known to be produced by many common microorganisms. These microbes accumulate PHAs as an energy storage mechanism in a manner similar to lipid accumulation in higher organisms.1 There are now over 100 different types of known basic building blocks for PHA polymers reported.2 Poly(3-hydroxybutyrate) (PHB) homopolymer and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) are the most well-known types of PHAs.3-5 Copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate and elastomeric PHA comprising predominantly 3-hydroxyoctanoate have also been studied.6,7 The properties of PHA copolymers depend strongly on the type, content, and distribution of comonomer units comprising the polymer chains, as well as the average molecular weight and molecular weight distribution. While PHAs have been investigated by various researchers, the successful commercial utilization of PHAs has been surprisingly slow. The production cost of PHAs by conventional fermentation processes has been high, making the entry of this material into the commodity plastics market difficult. * To whom correspondence should be addressed. Tel. +1-513-634-8949; fax +1-513-634-9342; e-mail [email protected]. † This paper was presented at the ISBP 2004 (International Symposium on Biological Polyesters), held in Beijing, China, August 22-28, 2004. ‡ The Procter & Gamble Company, 8611 Beckett Rd., West Chester, OH 45069. § The Procter & Gamble Company, 11530 Reed Hartman Hwy., Cincinnati, OH 45241. | The Procter & Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707.

Limited availability of this class of materials also did not contribute favorably toward the establishment of a robust cost structure and product development. Most importantly, the physical properties of earlier commercial PHAs, like PHBV copolymers, were inadequate for many of the applications envisioned for the replacement of commodity plastics. Because of the remarkable stereo-regularity of the perfectly isotactic chain configuration created by the bio-catalyzed polymerization process, PHB homopolymer exhibits an unusually high degree of crystallinity. The high crystallinity results in a rather hard and brittle material that is not very useful for many applications. The melt temperature (Tm) of PHB is also high (>170 °C), relative to the region of its thermal decomposition temperature, making PHB much more difficult to manipulate using melt processing equipment used for conventional plastics. It was hoped that the incorporation of a comonomer unit, 3-hydroxyvalerate (3HV), could control the excessively high Tm and crystallinity limitations. Unfortunately, the desired effect of 3HV incorporation to regulate the crystallinity and Tm was surprisingly limited because of the isodimorphism phenomenon,4 where 3HV units can be easily included in the crystal lattice of 3-hydroxybutyrate (3HB) units and vice versa without the anticipated disruption of crystallinity. An alternative structure of PHA copolymers, therefore, had to be designed to overcome this limitation. Nodax PHA Copolymers. Nodax is a family of recently introduced PHA copolymers comprising 3HB units and a relatively small amount of medium chain length 3-hydroxyalkanoate (mcl-3HA) comonomer units, which are different from 3HV. These mcl-3HAs are chosen to be larger than 3HV, having the copolymer side groups of at least three

10.1021/bm049472m CCC: $30.25 © 2005 American Chemical Society Published on Web 01/12/2005

Preparation and Properties of PHA Copolymers

carbon units or more.8-11 Within the Nodax family, there are several different grades of copolymers available, depending on the average molecular weight, the average mcl-3HA content within the copolymer, and the side group chain length of the chosen mcl-3HA unit. The simplest form of this class of copolymer is the PHBHx copolymer comprising 3HB and 3-hydroxyhexanoate (3HHx) units. Other 3HA units, such as 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), are also available to form various copolymers comprising 3HB and one or more mcl-3HAs. The unique molecular structure of Nodax copolymers provides a set of useful properties not achieved by more traditional PHA polymers, like PHB homopolymer or PHBV copolymers.11-13 These copolymers exhibit high toughness and ductility, as well as convenient thermal property ranges similar to those of polyethylene. The incorporation of mcl3HA units effectively lowers the crystallinity and Tm in a manner similar to the effect of R-olefins in linear low-density polyethylene. The Tm can be lowered well below the thermal decomposition temperature of PHAs to make this material much easier to process. The reduced crystallinity provides the ductility and toughness required for many practical applications. The mcl-3HA content regulates the Tm and crystallinity of copolymer almost independently of the mcl size, as long as more than three carbons are present in the side group. On the other hand, the side group chain length of mcl-3HA has a profound effect on the flexibility of the copolymer.11-13 Another noticeable advantage of PHA copolymers is that the material undergoes total biodegradation not only in aerobic but also in anaerobic environments.12-14 The copolymers also show robust ambient hydrolytic stability, digestibility in hot alkaline solution, superb oxygen and odor barrier performance, excellent surface property for printing and adhesion, and compatibility with many other materials, including other degradable polymers, like poly(lactic acid).12,13 By selecting different mcl-3HA structures and contents, wide ranges of PHA copolymers can be provided for applications. Currently, the developmental scale production of Nodax PHA copolymer is carried out by the bacterial fermentation of various carbon feeds derived from renewable resources. In addition to the conventional biosynthesis route, we can also prepare a broad range of PHA copolymers with different mcl-3HA types and contents by using the in vitro chiral chemical synthesis technique based on ring-opening polymerization.15 In this report, we will discuss the preparation of Nodax copolymers based on both fermentation biosynthesis and chemical polymerization to explore the full range and potential of this class of polymeric material. We then present some of the important physical properties of this family of PHA copolymers. Experimental Section Biosynthesis. PHA copolymers comprising 3HB and 3HHx were produced by fermentation of wild-type Aeromonas hydrophila using lauric acid as described,16 and a strain of Ralstonia eutropha genetically modified to express the synthase gene from Pseudomonas fluorescens GK-1317 was used to produce the longer chain 3-hydroxybutyrate-

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co-3-hydroxyalkanoate copolymers. R. eutropha were grown overnight in Luria-Bertani broth, and 1 mL was used to inoculate 250 mL in shake flasks containing, per liter, 9.0 g of Na2PO4‚12H2O, 1.5 g of KH2PO4, 2.0 g of nutrient broth, 0.2 g of MgSO4‚7H2O, 0.02 g of CaCl2‚2H2O, 0.0012 g of FeNH4(SO4)2, and 10 mL/L of a trace element solution containing, per liter, 500 mg ethylenediaminetetraacetic acid, 200 mg of FeSO4‚7H2O, 10 mg of ZnSO4‚7H2O, 3 mg of MnCl2‚4H2O, 30 mg of H3BO3, 20 mg of CoCl2‚6H2O, 1 mg of CuCl2‚2H2O, 2 mg of NiCl2‚6H2O, and 3 mg of Na2MoO4‚2H2O. The cultures included 5 g/L fructose plus varied amounts of the sodium salts of fatty acids and were grown at 30 °C and 200 rpm for 72 h. Cells were centrifuged, washed once with 0.9% NaCl, and freeze-dried. Extraction and Fractionation of PHA. Dried cells from large shake flasks were extracted into 20 mL refluxing chloroform per gram cells for 4 h. The extract was vacuumfiltered through two sheets of Whatman #4 filter paper, and the cell retentate was washed once by passing 100 mL of hot chloroform through it under a vacuum. The extract was flash evaporated to lower the volume of chloroform, or, for smaller samples, dried under a stream of nitrogen. Concentrated chloroform extracted PHA was added to 10× volume of ethyl ether/hexane (3:1 v/v) with constant stirring. The precipitated PHA was centrifuged and washed once with the ethyl ether/hexane or, for larger volumes, vacuum filtered through two sheets of Whatman #4 filter paper. The PHA was vacuum-dried overnight and weighed. Further fractionation of the PHA copolymers was accomplished by refluxing the chloroform extracted PHA in ethyl acetate (about 50 mL/g PHA) for 4 h. PHB and PHA with very low mcl-3HA comonomer composition remained precipitated and were removed from the hot ethyl acetate by vacuum filtration as described above. The filtered hot ethyl acetate was allowed to cool at room temperature while being stirred with a magnetic stirrer. When cool, PHA with an average of about 4% mcl-3HA comonomer content precipitated and could be collected by vacuum filtration or centrifugation. Hexane was then added at 1.5× volume of ethyl acetate while stirring, and the precipitated PHA with 5-15% comonomer was recovered by centrifugation or filtration. PHA with 15-25% comonomer could be recovered similarly by adding more hexane until it was 3× volume versus ethyl acetate. Chemical Synthesis. Materials. [R]-β-Butyrolactone (BL) was prepared by the chiral reduction of diketene18 or by a multistep synthesis similar to a published procedure.19 This latter multistep procedure was also used to make the longer side-chain β-lactones. BL, [R]-3-propyl-β-propiolactone (3HxL), and [R]-3-pentyl-β-propiolactone (3-OL) were distilled from CaH2, flash chromatographed down an alumina column with pentane as the eluent, redistilled either once or twice (fractional or spinning band column), and stored over activated alumina or activated molecular sieves at least 24 h prior to use. The [R]-3-pentadecyl-β-propiolactone (3-OdL) was recrystallized from hexane. Diethylzinc (1.1 M toluene solution) was used as received. Anhydrous toluene was purchased from Aldrich and typically contained 13 ppm water as determined by Karl Fischer titration, and 2,4-

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dimethyl-2,4-pentanediol (Aldrich) was dried over activated 3A molecular sieves prior to use. Preparation of Difunctional Initiator (EZPD). The initiator was prepared by the addition of dry, argon-sparged 2,4dimethyl-2,4-pentanediol (0.78 mL, 0.7197 g, 5.44 mmol) dropwise from a syringe to 9.67 mL (8.8486 g) of 1.1 M (15 wt %) diethylzinc in toluene (11.0 mmol diethylzinc) contained in a dry, septum-capped, argon-purged flask. The ethane evolved was removed through a needle attached to a bubbler with or without an argon purge. The reaction mixture may warm depending on the rate of addition. Occasionally, a small amount of solid will form. This solid can be dissolved by the addition of a small amount of dry toluene (1 mL) or gentle warming of the solution to 30-35 °C. The cloudy initiator mixture can be used as-is, if it is stirred or shaken to suspend the solid. Polymerization. PHB homopolymer and copolymers were all prepared via similar procedures. All manipulations of dry monomer and initiator were carried out in oven-dried, then flamed-out, septa-capped glassware under dry argon with transfers made with a syringe or a cannula. Butyrolactone was combined with 3-alkyl-β-propiolactone in the desired molar ratio and diluted with dry toluene to give a 15% monomer solution. The monomer solution was run through a short column of activated alumina directly into an argonfilled, flame-dried reaction flask. The initiator was added via syringe, and the mixture was heated with an oil bath to 60-65 °C for the designated time. Afterward, chloroform was added to obtain a solution of reasonable viscosity, and the polymer was recovered by precipitation into an etherhexane mixture (3:1 v/v). The solid was dried under a vacuum (20-40 °C). Characterization. Molecular Weight Determination. Molecular weights were determined through standard size exclusion chromatography (SEC) techniques done at room temperature. Three Waters Ultrastyragel linear columns (one 50 × 7.8 mm and two 300 × 7.8 mm) in series where used with chloroform (1 mL/min) as the eluent. Calibration was performed with narrow molecular weight polystyrene standards. Previously we had found that polystyrene in chloroform calibration resulted in molecular weights that closely approximated the actual PHA molecular weight determined by light scattering. Molecular weights determined with polystyrene standards were only 5-10% higher than those determined with light scattering.15 All molecular weights given here are based on the polystyrene-calibrated SEC. NMR. Proton spectra were collected on a Varian UNITYplus spectrometer at 300 MHz using standard parameters in CDCl3. The data were analyzed using NUTS-2D software (Acorn NMR). The comonomer content (mol %) was calculated by comparing the side-chain methyl resonance integral for the comonomer at 0.95 ppm to the total backbone methine resonance integral at 5.3 ppm. Additional Characterization. A standard set of polymer characterization procedures, including thermal analysis, X-ray diffraction, and mechanical and rheological tests, were carried out. The details of test methods have been provided elsewhere.11 Thermal analysis (melting temperature and glass transition temperature) were conducted on a Mettler dif-

Noda et al.

ferential scanning calorimeter (DSC). A nominal melting point determination (a practical “processing” melt temperature, a melt temperature that could be expected at reasonable storage times and temperatures) was obtained by first exposing all the samples to uniform thermal history by melting 1 g of sample at 170 °C (185 °C for comonomer content less than 5 mol %) for 1 min in a Carver press under low force (250 kg) and cooling the sample to room temperature by placing the sample between 5-kg Al blocks at room temperature. The samples were kept at room temperature for at least 10 days. The melting temperature was taken as the highest temperature of the peak of the endotherm scanning at 10 °C/min. The glass transition temperature (Tg) was obtained by melting the samples, rapidly quenching to liquid nitrogen temperatures (as fast as the DSC would allow), and heating the sample at 10 °C/ min. The Tg reported is the midpoint of the transition. Crystallinity was determined by wide-angle X-ray diffraction. The method of Ruland was employed.20 The general shape of amorphous halos was easily deduced from samples with low crystallinity. Samples of nominally 0.5-mm-thick were measured with Cu KR radiation in transmission geometry. Samples were corrected for background scattering, absorption, and Compton scattering. Tensile mechanical measurements were conducted on PHA films. Films of copolymer samples were made by melting the material between the Teflon sheets in a Carver press at 20 °C above the melt temperature. Pressure on the sheets was adjusted to produce films of approximately 0.1-mm thickness. The films were then identically cooled to room temperature by placing the molds between large (5 kg) aluminum plates and allowing the films to cool to room temperature. The films were cut into a dog-bone shape with a 6.43-mm width in the necking region. The films were analyzed using the Instron model 1122 tensile tester for stress-strain measurements. The Instron tensile test conditions used a 19.05-mm gauge length and a crosshead speed of 5.08 mm/min. The flexural modulus was measured dynamically with a TA Instruments Q800 dynamic mechanical analyzer. Samples were measured under a dual cantilever geometry with sample dimensions of 35 mm × 12.5 mm × 1.5 mm. Samples were prepared by first compression molding the materials into 1.5mm-thick sheets at a temperature 10 °C above the offset melt temperature, where the offset melt temperature was determined by differential scanning calorimetry and is the lowest temperature at which the crystalline phase is completely melted. After cooling and crystallization, test bars were then machined out from the polymer sheets and allowed to equilibrate for 1 week at room temperature. Samples were measured through a frequency sweep from 0.1 to 100 Hz at 27 °C under a controlled deformation amplitude of 0.1%. The flexural modulus was calculated using a standard TA Instruments algorithm and reported at the 1 Hz frequency. Results and Discussion PHAs Produced by Fermentation. The use of biocatalysts, such as enzymes, microorganisms, and even higher

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Preparation and Properties of PHA Copolymers Scheme 1

organisms, to convert non-petroleum-based feedstocks (e.g., fats, oils, sugars, and polysaccharides) to novel and functional polymers is now gaining a strong interest. As opposed to the use of conventional industrial chemical synthesis (which relies primarily on petroleum derivatives as raw materials), such biosynthetic approaches are attractive because of the potential utilization of abundant and renewable bio-based raw materials and the possibility of finding unique bio-catalyzed conversion processes to efficiently produce desirable end products. Furthermore, in the long run, bio-catalytic production of polymers may offer an intriguing opportunity for establishing the so-called sustainable supply of various functional materials.21 With the needs defined above in mind, the production of plastics by biological means has been investigated. The biosynthesis of PHA copolymers involves parallel enzymatic production of 3-hydroxybutyryl CoA and 3-hydroxyacyl CoA units via fatty acid biosynthesis and fatty acid oxidation followed by enzymatic copolymerization. The key metabolic pathways utilized in the production of the PHA copolymers used for these studies are shown in Scheme 1.12 Two units of acetyl CoA form acetoacetyl CoA catalyzed by the β-ketothioloase coded by the phaA gene.22 Acetoacetyl-CoA is then converted by the β-ketoreductase to 3-hydroxybutyryl CoA, coded by the phaB gene.22 Parallel to these steps are the other metabolic pathways leading to the long chain 3-hydroxyacyl CoA units. These include fatty acid biosynthesis coupled to PHA synthesis via the phaG gene product 3-hydroxyacyl-ACP-CoA transacylase23 and fatty acid oxidation facilitated by the phaJ gene product R specific enoyl-CoA reductase.24 Finally, the copolymerization of 3HB CoA and 3HA CoA with phaC PHA synthase results in the production of PHA copolymers. A series of PHA copolymers were produced in R. eutropha by expressing the P. fluorescens GK13 phaC2 gene in the PHB-4 mutant strain DSM.17,25 R. eutropha was grown for 3 days in 1-L volumes of medium with 5 g/L fructose and up to 0.5 g/L fatty acid. The overall PHA yield was 1030% by dry weight. Production of mcl-3HA comonomers required the presence of a fatty acid co-feed. The choice of fatty acid affected the mcl composition (Table 1). Feeding octanoate yielded PHA with 3HO as the primary comonomer with small amounts of 3HHx and 3HD. Feeding decanoate yielded PHA with 3HD and lesser amounts of 3HO. Feeding

Table 1. Gas Chromatographic Analysis of PHAa % total fatty acid feed % 3HB % 3HHx % 3HO % 3HD % 3HDD mcl-3HA C8, 0.25 g/L C8, 0.35 g/L C8, 0.45 g/L C8, 0.50 g/L C10, 0.10 g/L C10, 0.15 g/L C10, 0.20 g/L C10, 0.25 g/L C12, 0.10 g/L C12, 0.15 g/L C12, 0.20 g/L C12, 0.25 g/L C12, 0.30 g/L C14, 0.10 g/L C14, 0.15 g/L C14, 0.20 g/L C14, 0.25 g/L C14, 0.30 g/L C16, 0.20 g/L C16, 0.25 g/L C16, 0.30 g/L C16, 0.35 g/L

83.6 59.8 29.5 27.6 93.9 87.7 82.5 60.6 93.2 80.6 53.0 28.2 20.6 93.3 86.5 81.6 61.2 45.3 87.6 78.2 71.2 48.5

Tr Tr 4.6 5.1 Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr

12.4 35.3 60.3 67.1 Tr 5.1 6.2 14.1 Tr 4.8 10.3 16.2 17.0 Tr 5.8 7.9 12.3 17.5 5.8 10.7 13.6 19.7

3.9 4.7 5.5 Tr 5.8 7.0 11.1 25.1 6.5 7.7 19.6 29.2 32.4 6.4 7.5 10.3 16.7 23.4 6.4 10.9 15.0 21.8

Tr Tr Tr Tr Tr Tr Tr Tr Tr 6.8 16.4 26.3 29.9 Tr Tr Tr 9.7 13.7 Tr Tr Tr 9.9

16.4 40.2 70.5 72.4 6.1 12.3 17.5 39.4 6.8 19.4 47 71.8 79.4 6.7 13.5 18.4 38.8 54.7 12.4 21.8 28.8 51.5

a Gas chromatographic analysis of PHA extracted from bacteria grown in the presence of 5 g/L fructose plus 0.1-0.5 g/L sodium octanoate (C8), sodium decanoate (C10), sodium laurate (C12), sodium myristate (C14), and sodium palmitate (C16). Methyl esters of hydroxyalkanoates (HAC#) were fractionated and measured. Data are peak integrations. Tr ) trace.

C12-C16 fatty acids yielded PHA with 3HD and lesser amounts of 3HO and 3HDD (3-hydroxydodecanoate). PHA isolated from R. eutropha grown on 5 g/L fructose + 0.5 g/L sodium palmitate was fractionated into different comonomer compositions by ethyl acetate solubilization, cooling, and hexane precipitation. Material with about 4.5% comonomer precipitated when the ethyl acetate extract was cooled to 4 °C and comprised 68.5% of the total PHA. PHA of 14.5% average mcl-3HA comonomer content was precipitated by treating the filtered cooled ethyl acetate extract with 1.5× hexane (v/v) and was 17% of the total PHA. Treating the filter extract with more hexane to bring it to 3× hexane/ethyl acetate (v/v) precipitated 14.5% of the recovered PHA and was comprised of 22% comonomer material. PHA with higher levels of comonomer remained soluble. PHAs Prepared by Chemical Synthesis. As opposed to biological synthesis via fermentation, chemical synthesis allows one to more easily and predictably accommodate variations in molecular weight, comonomer type, and comonomer content if pure monomers are available. The

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Scheme 2

approach is especially attractive for the testing of new classes of PHAs, where microbes capable of biosynthesizing such PHAs have not yet been discovered. Indeed, early research on Nodax copolymers was often conducted on the basis of synthetic PHAs.8,10,14,15 PHA copolymerization typically involves ring-opening polymerization of β-lactones as shown in Scheme 2. A new, soluble difunctional zinc initiator was used to prepare the isotactic PHA copolymers used in this study. Although the literature contains numerous reports on initiators used to polymerize β-lactones,26-34 only the studies on aluminoxanes,26 distannoxanes,30 and alkylzinc alkoxide34,35 report the preparation of PHA with molecular weights >30 000. Because the desirable mechanical properties of isotactic PHA polymers are not realized until the weight-average molecular weights (Mw) are 500 000 or greater,11,16 an initiator that produces high molecular weight, isotactic copolymer is needed. The use of ethylzinc isopropoxide with highly purified and highly enantiomerically (enantiomeric excess > 90%) enriched monomers allowed us to consistently prepare isotactic PHA polymers and copolymers with Mw up to several hundred thousand.34b,c However, we could not consistently obtain polymer with Mw of 500 000 or greater. The soluble, difunctional initiator derived from 2,4-dimethyl2,4-pentanediol and diethylzinc (EZDP) enabled us to meet the Mw requirement.11,15 Synthetic PHA is expected to differ only slightly from the equivalent naturally derived PHA. The differences are due to the synthetic copolymers not being perfectly isotactic. This is a result of the starting 3-alkyl-β-propiolactones being of only 92-95% stereo-purity. Similarly, low levels of stereoimperfections in PHB homopolymer lead to a 10 °C drop in Tm for synthetic PHB made with EZPD initiator compared to the Tm of bacterial PHB.11 Data on the homo- and copolymerizations of BL initiated with EZPD are given in Table 2. The EZPD initiator works well in solution polymerization in toluene. It also can be used for solution polymerization in tetrahydrofuran (THF) if the copolymer produced is

Figure 1. Plot of Mn and polydispersity versus time for the solution polymerization of racemic β-butyrolactone in toluene at 100 °C using a Zn/BL mole ratio of 0.001.

soluble in THF. Bulk polymerizations have also been performed with EZPD. In all cases the yields are good. Figure 1 contains data on the homopolymerization of racemic BL. It clearly shows that the molecular weight increases with time, and the polydispersities of the resultant polymers are around 1.5. Copolymerization was typically run at 60-65 °C because the 3-alkyl-β-lactones tend to be unstable at elevated temperatures. The longer the alkyl group, the more thermally labile the lactones become, decomposing to carbon dioxide and terminal alkene. Therefore, one must balance the polymerization temperature and reaction time with the thermal stability of the β-lactone monomers. Incorporation of the comonomers is good, and even at lower conversions (30-50%) the comonomer incorporation is typically >50% of the feed ratio and often almost the same. Physical Properties. Figure 2 shows the changes in melt temperature (Tm) of various PHA copolymers with different levels of 3HA comonomer contents. The abbreviations PHBO, PHBD, and PHBDD stand respectively for copolymers of 3HB with 3HO, 3HD, and 3HDD. The melt temperature of Nodax PHAs is significantly depressed by the incorporation of mcl-3HAs with side groups having at least three carbon units, such as 3HHx or 3HO. In contrast, PHBV copolymers with short ethyl side groups (3HV) do not depress Tm much, even at a relatively high level of 3HV incorporation. Figure 2 shows that it is difficult to bring down the Tm of PHBV well below 150 °C. This result has a

Table 2. Polymerization of BL with [R]-3-Alkyl-β-propiolactones Using EZPD comonomer level (mol %) feed polymer

Mn (× 10-3)

Mw (× 10-3)

Mw/Mn

333 380 116

505 572 165

1.52 1.51 1.43

6 11 7

521 410 308

765 665 509

1.47 1.62 1.65

9 25

8 24

437 234

643 297

1.47 1.29

10 5

10 5

333 318

530 490

1.59 1.54

temperature (°C)

time (h)

Zn/monomer ratio

yield (%)

65 65 100

48 65 7

0.0003 0.0003 0.001

91 88 97

3-HxL 3-HxL 3-HxL

60 65 65

65 65 67

0.0002 0.0002 0.0004

90 93 97

7 11 7

3-OL 3-OL

65 65

70 64

0.0002 0.0004

96 89

3-OdL 3-OdL

60 65

88 44

0.0002 0.0003

91 84

comonomer

Preparation and Properties of PHA Copolymers

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Figure 2. Melt temperature of PHA copolymers. Symbols stand for PHB and PHBV (9), PHBHx (b), PHBO ([), and PHBD (2).

Figure 4. Glass transition temperature of PHA copolymers measured by the DSC. Symbols stand for PHB and PHBV (9), PHBHx (b), PHBO ([), and PHBD (2). The straight line is for copolymers with 8 mol % 3HA, and the broken line is for copolymers with 12 mol % 3HA.

Figure 3. Crystallinity of PHA copolymers. Symbols stand for PHB and PHBV (9), PHBHx (b), PHBO ([), and PHBOd (2).

profound implication in the ease of melt processing for different PHAs. PHAs have low to moderate thermal stability because of their propensity to undergo a random chain scission reaction at an elevated temperature by a β-elimination mechanism. Typical PHAs suffer substantial thermal decomposition at a temperature above 170 °C.36,37 This temperature is near the melt temperatures of PHB or PHBV. As one would prefer to keep the melt process temperature of PHAs well below their thermal decomposition temperature, the depression of the Tm of PHA copolymer to below 150 °C obviously becomes a major advantage compared to that of PHB homopolymer or PHBV. The susceptibility of PHA copolymers to thermal decomposition may be used as an advantage. The molecular weight of PHAs can be regulated by the judicious choice of the process temperature to intentionally reduce the molecular weight. Typical intended applications for PHAs call for the molecular weight to range from 500 000 to 700 000. Because biologically produced PHA copolymers can have initial weight-averaged molecular weights well over 1 000 000, the ability to bring down the molecular weight at will to a target range by controlled thermal decomposition provides additional design space for the material. The crystallinity of PHA copolymers is also greatly affected by the type of comonomer as shown in Figure 3. The incorporation of 3HV units provides very little change in the crystallinity, so PHBV copolymers remain somewhat brittle and fragile even if a large amount of 3HV units are incorporated. On the other hand, a small amount of other

Figure 5. Flexural modulus of PHAs measured by the dynamic cantilever bending.

mcl-3HA units can effectively depress the crystallinity to make PHAs more ductile and tough. Indeed, the incorporation of mcl-3HA units, as low as 5 mol %, can make the mechanical properties of PHAs comparable to those of ductile polyethylene and greatly expand the potential utility of these materials as general purpose plastics. Further incorporation of larger mcl-3HA comonomer units makes PHAs even more soft and flexible. Figure 4 shows the glass-to-rubber transition temperatures (Tg) of PHA copolymers with various 3HA monomer types, estimated by DSC measurement. The effect of the length of the PHA side group branches is apparent. The Tg of PHA with longer side groups clearly shows the pronounced depression, indicating the enhanced local segmental mobility of polymer chains in the presence of longer side groups. The effect of the mcl-3HA content also is evident. The higher the mcl-3HA content, the lower the Tg of the PHA. Striking differences were observed for the flexibility of the different PHA copolymers. Figure 5 shows the flexural modulus of PHA copolymers measured by dynamic cantilever bending at a frequency of 1 Hz. The content of 3HA comonomer units was kept to about 8 mol % for most of the samples, except for the PHBD (7 mol %) and PHB homopolymer. The extension of the PHA side groups by even

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Knapmeyer

for

physical

property

measurements.

References and Notes

Figure 6. Young’s modulus of PHAs.

a few carbon units dramatically increased the flexibility of the samples. Similar results were obtained for Young’s modulus obtained by tensile measurements. Figure 6 shows the steady decrease of Young’s modulus as the side group length is increased. Conclusion We have investigated a new and promising family of PHA copolymers. Nodax PHA copolymers, comprising predominantly 3HB repeat units with additional mcl-3HA comonomer units with their side groups made of at least three carbons, can be prepared either by bacterial fermentation or chiral chemical synthesis based on ring-opening polymerization. PHA copolymers with different types and contents of mcl-3HA can be produced. The incorporation of mcl-3HA units with more than three carbon units reduces the regularity of the polymer chain structure to depress the melt temperature and crystallinity. The higher the incorporation level of the mcl-3HA, the more pronounced is the reduction of Tm and crystallinity. Interestingly, the incorporation of the smaller 3HV unit, instead of mcl-3HA, has much less of an effect on the Tm depression or the crystallinity reduction. The depression of Tm is beneficial, because the material can be processed at a temperature much lower than the thermal decomposition temperature of typical PHAs. The reduction of crystallinity imparts the desired ductility to the material compared to the excessively crystalline and brittle PHB homopolymer. The incorporation of mcl-3HA comonomers also seems to increase the local segmental mobility of PHA copolymers, indicated by the depression of their glass transition temperatures. This effect is more pronounced for longer side branches. The flexibility of PHA copolymers is greatly enhanced by the incorporation of mcl-3HA with longer side branches. Both the flexural modulus and Young’s modulus were dramatically reduced by incorporating long side branches. Thus, Nodax PHA copolymers, especially those comprising long side groups, have superior ductility and toughness, as well as ease of processing compared to conventional PHAs, like PHBV copolymers or PHB homopolymer. Acknowledgment. We thank J. J. Kemper for contributions to the synthetic work, and J. T. Grothaus and J. T.

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