Investigating the structure–property relationship of bacterial PHA block copolymers
Introduction
Conversion of bio-renewable resources, such as starches, sugars, and oils, into useful commodity products is an important aim for a sustainable economy. Cost of production, physical properties of the product, safety, and other characteristics are among the aspects that must be considered for these new classes of materials. One class of materials that is currently under extensive investigation is polyhydroxyalkanoates (PHAs). These polyesters can be biologically synthesized, are generally biodegradable, biocompatible, and are synthesized from bio-renewable feedstocks. Further, biological systems have reportedly been shown to incorporate over 150 different repeat units (Steinbüchel and Valentin, 1995). Among these are two general classes: the short-chain-length (SCL) and medium-chain-length (MCL) repeat units. These classes are defined by the overall length of the aliphatic chain in the monomer unit. Repeat units less than 5 carbons in overall length are part of the SCL class, while repeat units having 6–12 carbons are in the MCL class. Most of these repeat units are of the 3-hydrxoyacyl variety, but units with hydroxyl groups at the fourth or fifth carbon have also shown to be incorporated into the polymer (Doi et al., 1990, Haywood et al., 1991, Liu and Steinbüchel, 2000).
Although several products have emerged which focus on specific secondary properties of PHAs (such as biocompatibility) (Williams et al., 1999, Zinn et al., 2001, Zhao et al., 2002), perhaps the biggest impact for the successful generation of a sustainable economy will be through the expansion of these materials into the commodity plastics market (Steinbüchel and Fuchtenbusch, 1998). Several studies have been completed to determine properties of poly(3-R-hydroxybutyrate) (PHB) homopolymer and PHA random copolymers consisting of SCL and/or MCL repeat units (Barham and Organ, 1994, Choi et al., 1999, Tanaka et al., 2004, Fischer et al., 2004, Furuhashi et al., 2004).
It is important to consider three basic mechanical properties when comparing the usefulness of a polymer for a given commodity application. The elongation at failure is a measurement of toughness and reflects the total deformation that the polymer can withstand before fracture. The Young's modulus is a measure of stiffness that considers the slope of the stress response to deformation at very low strains. Steeper slopes at low deformations are indicative of stiffer materials. Finally, the ultimate tensile strength is a measure for the maximum strength of the material prior to the onset of plastic deformation. This value is obtained from the maximum of the initial peak in the stress versus strain diagram. While the modulus (stiffness) and ultimate tensile strength (overall strength) of PHB and related copolymers identified in the previously mentioned studies are suitable for many commodity-type uses, a major setback in the commercialization of these materials relates to the extremely brittle character of these polymers. Under purely extensional stress, PHB and PHBV materials have been shown to fracture to less than 10% elongation (Steinbüchel, 2002). The brittle nature of these PHA polymers limits the process and end-use applications of the material. It is necessary to craft PHA polymers such that a suitable combination of desirable characteristics is achieved.
Crystallization phenomena have been studied in conjunction with some of the aforementioned polymer systems. Specifically, PHBV random copolymer has been studied and compared to the crystallization of PHB homopolymer. For PHBV containing low quantities of 3HV repeat unit (less than 25%), the orthorhombic crystal structure of PHB dominates because this structure is able to withstand the slight swelling caused by the infrequent addition of an extra carbon in the side chain of the monomer. However, at higher 3HV compositions, PHBV random copolymer forms an expanded crystal structure, whereby the spacing of the (1 1 0) plane of the original PHB lattice is increased. Further, the (0 0 2) plane in the PHB crystal is replaced by a (2 1 1) plane in the 3HV enriched copolymer. These differences have been attributed to steric hindrances resulting from the extra side chain carbon in the 3HV structure (Bluhm et al., 1986, Kunioka et al., 1989, Scandola et al., 1992). Since the isodimorphism (two different crystal structures) of PHBV manifests itself with changes in the local concentration of valerate in the polymer chain, block copolymer microstructure should lead to local microphase separation and eventually to development of distinct properties when compared to PHA random copolymers.
While investigating the compositional space of random copolymers synthesized by bacterial PHA producers is important, it is also necessary to evaluate potential advantages of increasing the microstructure within biological PHA systems. For example, block copolymers are polymer chains that contain thermodynamically different regions (or “blocks”) of polymer that are covalently linked together (Bates and Fredrickson, 1990). Examples of linear block copolymers include A–B diblock, A–B–C triblock, and (A–B)n repeating diblock, etc. These arrangements of polymer microstructure prevent large phase separations that would normally arise from comparable polymer blends. Instead, the polymers are limited to microphase separations. These new morphologies can lead to additional physical properties that cannot be obtained by applying random copolymerization, polymer blending, or filler-adding techniques (Bates, 1991). As an example, an empirical comparison was conducted on the effects of the number of blocks on the mechanical properties of block copolymers. It had been found that by merely increasing the number of blocks from two to three to five, the elastomeric properties of the polymer system would be enhanced. It is argued that the primary effect of block copolymer architecture (the covalent bonding of two distinct microphases) can be multiplied through the covalent linkage of several microphase domains (Hermel et al., 2003).
The development of bacterial PHA block copolymer synthesis offers the opportunity for complex microstructures for biologically produced PHAs. Substrate addition strategies to synthesize PHA block copolymers in Cupriavidus necator were developed using polymerization kinetics modeled by both computer and mathematical models (Mantzaris et al., 2001, Kelley et al., 2001, Mantzaris et al., 2002). Later, the ability to monitor the culture environment, and thus infer the status of cell physiology, allowed for the controlled and repeatable implementation of the developed synthesis schemes (Pederson and Srienc, 2004, Pederson et al., 2006). Quantitative analysis of the accuracy and precision of the population balance models cannot be determined using presently available methods because of several factors. The high degree of biological-based diversity ensures that there is a widely varying population of polymer chains synthesized in the bacteria. Also, while distinct physical and mechanical properties are observed for the portfolio of random copolymers, complications arise when using scattering techniques due to the structural and compositional similarity between the two blocks used in this repeating diblock architecture (PHB and PHBV). Although quantitative analysis is not possible, previous work has been completed to qualitatively show the presence of block copolymers using a combination of fractionation, calorimetry, rheological, and nuclear magnetic resonance techniques (Pederson et al., 2006).
It becomes necessary, therefore, to compare the progress in the compositional space to the subsequent progress within the property space of biological PHAs. This paper specifically compares block copolymers to similar random copolymers, both synthesized in vivo from monomers within the short-chain-length class of PHA repeat units. The differences in mechanical properties between the random copolymers and block copolymers are analyzed as the properties change over time.
Section snippets
Polymer synthesis and film casting
PHA random copolymer and block copolymer were synthesized with C. necator as reported previously (Pederson et al., 2006). As before, polymer was extracted from freeze-dried cell mass using chloroform at reflux. Approximately 0.1 mm thick and 5 cm diameter films were cast using 1,2,4-trichlorobenzene as the solvent (Sigma–Aldrich, St. Louis, MO). Two PHBV random copolymer samples, RC1 and RC2, were used as controls. These films contain 8 and 29% overall 3 HV content, respectively. The controls were
Chemical and molecular weight characterization
Films of PHA random copolymer and block copolymer were made by solvent casting. The chemical composition of each film as measured by gas chromatography as well as the theoretical microstructure composition as calculated using population balance models presented elsewhere (Mantzaris et al., 2001, Mantzaris et al., 2002) are presented in Table 1. Also included are the values (number average molecular weight, MN, and the polydispersity index, PDI) describing the molecular weight distribution for
Conclusions
It has been shown here that the introduction of block copolymer-based microstructure to PHAs synthesized in vivo result it long term properties that differ from the properties obtained by simpler PHA random copolymer microstructure. Over time, the block copolymer systems are able to withstand the effects of aging that lead to the brittle character of the random copolymer samples. While crystallization continues to occur in the block copolymer samples, the effects on the elongation at failure
Acknowledgements
The authors would like to thank Jack Lewis, Professor of Orthopedic Surgery at the University of Minnesota, for the equipment used to complete the tensile testing trials. Portions of this work were completed at the Minnesota Characterization Facility, which receives partial support by the NSF through the NNIN program. This research was supported by the 3M Science and Technology Fellowship and by the University of Minnesota's Initiative for Renewable Energy and the Environment (IREE).
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