CHAPTER 1
Polyhydroxyalkanoates (PHAs), their Blends, Composites and Nanocomposites: State of the Art, New Challenges and Opportunities
VISAKH P. M.
1.1 Polyhydroxyalkanoates: Structure, Properties and Sources
Polyhydroxyalkanoates are biopolyesters with various side chains and fatty acids with hydroxyl groups at the 4- or 5-position. They consist of (R)-3-hydroxy fatty acids. There are three types of polyhydroxyalkanoates: (a) short chain length hydroxyalkanoic acids (PHASCL) with an alkyl side chain, which are produced by Ralstonia eutropha and many other bacteria. PHASCL contain 3–5 carbon atoms, for example poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB); (b) medium chain length hydroxyalkanoic acids (PHAMCL) with alkyl side chains that are produced by Pseudomonas oleovorans and other Pseudomonas sensu stricto. PHAMCL contain 6–14 carbon atoms and (c) long chain length (PHALCL) obtained from long chain fatty acids, which contain more than 14 carbon atoms The monomer composition, macromolecular structure and physical chemical properties of PHAs vary, depending on the producer organism as well as on the carbon source used for the growth. PHAs containing double bonds can also be produced by recombinant Methylobacterium extorquens strains when fed unsaturated fatty acids. These PHAs comprise PHASCL and PHAMCL. One reason for choosing amethylotrophic microorganism for such a purpose was that an important portion of the production process would use methanol. In spite of PHB being considered an environmentally friendly polymer with similar material properties to polypropylene (PP), it has not been used on a large scale to replace conventional polymers because it presents some drawbacks in its mechanical properties. Considering polymer mechanical properties, it is important to consider three basic properties when comparing the usefulness of a polymer for a given commodity application. It is hard to process PHB due to its high melting temperature of approximately 170 1C, which is very near to its degradation temperature. Therefore, a solution to these drawbacks could be the copolymerization of 3-HB with other monomers that confer less stiffness and tougher properties (which bestow greater flexibility and lessen breakage) and to reduce the melting point.
The monomer composition of PHA has considerable effects on its physical properties.
The PHA structure can effectively be controlled by adjusting the carbon substrates to achieve desired monomer contents, by engineering metabolic pathways in the hosts or by feeding the culture with carbon substrates containing functional side chains that in a second step can suffer chemical modifications. PHAs, such as PHB and poly(3-hydroxybutyrate-co-3 -hydroxyvalerate (PHBV), are brittle, which is related to their high crystalline degree and they may lack the superior mechanical properties required for biomedical and packaging applications. These properties are a consequence of PHA's chemical structure. Therefore, since these different types of PHA have various structural and physical chemical properties, they should be classified according to their properties and modified in order to be easy to use for target applications. Different approaches were explored for the production of PHASCL by the Cupriavidus genus. The main studies were: the utilization of noble versus waste carbon sources and the utilization of a limiting factor to trigger PHA production versus the operation under "nutrient-sufficient" conditions. Thus, Chen et al. (2011) observed that a smaller C–N ratio was more favorable for PHA accumulation in a culture of Gamma proteobacterium in 72 hours, whereas a higher C–N ratio was more favorable for PHA accumulation in longer cultures of up to 150 hours of cultivation.9 Concerning activated sludge systems, Moralejo-Garate et al. (2013) have shown that the presence of ammonia during the PHA accumulation step was not damaging for PHA production.
4-Hydroxybutyrate (4-HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3 -propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442, which are capable of transforming 1,4 -butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for the production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. In this respect, attention has been given to producing PHAs bearing terminal double bonds in their side chains. The reason behind the preference for unsaturated PHAs can be found in the possibility to establish high-yield production processes. Alkylic substrates containing double bonds are cheaper and generally exhibit less toxicity compared to substrates with reactive functional groups. At the same time, the unsaturated side chains of PHAs are susceptible to chemical modifications. Starting from alkene function, PHAs have been modified to exhibit functionalities en route towards new PHA-based biomaterials. The mechanical properties of PHAs are directly correlated with their structure and crystallinity. An increase in the variety of side chains within one polymer chain of PHAMCL can modify its ability to crystallize and as a consequence there are some distinct differences in the crystallinity of PHAMCL. Obtaining a low crystallinity is possibly done once the polymers have large and irregular pendant side groups attached. These groups inhibit the close packing of the polymeric chains in a regular three-dimensional fashion to form a crystalline array.
The physical and material properties of PHAs are greatly influenced by their monomer composition and chemical structure i.e. the length of the pendant groups that extend from the polymer backbone, the chemical nature of the pendant groups and the distance between the ester linkages in the polymer. The variety of bacterial PHA that can be directly produced by fermentation is extraordinary large with more than 150 different hydroxyalkanoic acids, and even mercaptoalkanoic acid, that are known constituents of these PHAs. Depending on the subunit compositions and substrate specificities of the enzymes, PHA synthases are generally classified into four groups: class I, II, III, and IV.
1.2 Recovery of Polyhydroxyalkanoates
The recovery system may affect the amount of product recovered, the convenience of the subsequent purification steps and the quality of the final product. Cell separation from the fermentation broth is the preliminary step of the recovery method. In order to recover the PHA granules, it is necessary to rupture the bacterial cell and remove the protein layer that coats the PHA granules. Alternatively, the PHA has to be selectively dissolved in a suitable solvent. Generally, two methods are usually utilized for the recovery and purification of PHAs from cell biomass, which include PHA solubilization or non-polymer cellular material (NPCM) dissolution. The majority of the PHA recovery method is performed using a solvent extraction process mainly by chloroform and methanol. Modifying the cell wall's permeability and then PHA dissolution in the solvent are the mechanisms for PHA extraction. The separation of PHA from the solvent is carried out using solvent evaporation or polymer precipitation in a non-solvent material. PHA is very viscous and the removal of cell debris is difficult. Without considering solvent recycling, the large amount of solvent required for PHA extraction is costly. Consequently, PHA solubilizing into an immiscible solvent in water was explored at high temperature (above 120 °C). Then, cold water is added to extract the PHA, although the solvent can be recycled many times before being distilled.
A new PHA recovery process was developed to obtain the benefits of both digestion methods using sodium hypochlorite and chloroform in a solvent extraction. The combined method creates three separate phases, which include hypochlorite solution at the upper phase, NPCM and undisrupted cells at the middle phase and a chloroform phase containing PHA. The polymer is then recovered by precipitation in a non-solvent and filtration. The molecular weight reduction due to polymer degradation is significantly reduced using this process.
A few mechanical methods have also been developed to supplement these systems or as independent systems, which are widely used to recover intracellular PHA. This field involves either solid shear (e.g. bead milling, extrusion of frozen cells) or liquid shear (e.g. high pressure homogenization). Combinations of methods, such as chemical and physical processes, can sometimes produce acceptable results whilst one method alone fails. It has been reported that chemical pretreatments increase the sensitivity of bacteria to disruption. They allow equal disruption to be obtained at lower operating pressures or fewer passes during the physical process. The success of PHA as a viable option to petrochemical-based plastics will depend upon the design and performance of efficient and selective means of PHA production and recovery. Thus, further investigations on mixed cultures, recombinant microbial strains, cheap carbon substrates and efficient fermentations has allowed the production of significant quantities of PHAs, which can significantly decrease the production cost. A commercial recovery system with a simple, efficient and economical procedure will probably focus on a non-solvent extraction-based recovery amongst a variety of PHA recovery methods. In addition, the tolerance of the final product to the conditions employed is an important criterion for the selection of a PHA recovery process and the PHA properties have to be considered for the development of downstream processes. If the PHA molecular weight is too low, the transition temperatures and the mechanical properties will usually decrease, which is not suitable for any useful commercial applications. Hence, the challenge in the recovery process should be the maintenance of the original molecular weights, while not compromising the degree of purity for various applications. However, severe degradation of polymer molecular weight was reported during PHA extraction of C. necator using a sodium hypochlorite treatment. It is necessary to characterize and compare the extracted and non-extracted polymer properties to assess the feasibility of the developed recovery methods on PHA extraction and address the possible market demand and intended applications. The selection of suitable PHA extraction methods depends on several process parameters such as concentration of chemicals, reaction time, recovery temperature and pH. Basically, the impact of process parameters on the effectiveness of PHA extraction procedures have been studied and proven, but there is a limitation of concrete data on the effect of external factors on PHA recovery.
1.3 Blends of Polyhydroxyalkanoates
Various PHA blends have been developed to improve the performance and to offset the high price of PHAs. The blending of PHAs will offer more scope to expand their range of applications. The P(3HB)/PLA blend is one of the most studied blends, which exhibits mechanical properties that are intermediate between the individual components. Although PLA and P(3HB) are biodegradable polymers synthesized from renewable resources, their potential applications are hampered due to brittleness and formation of very large spherulites. Zhao and co-workers (2013) reported the preparation of a P(3HB-co-3HV)/PLA blend using a co-rotating twin-screw extruder. The melt mixing was carried out above the glass transition temperature (Tg) of amorphous polymer. The reason for using a twin-screw extruder was to ensure that all the specimens undergo the same thermal–mechanical history. Blending of PCL and P(3HB) offers a good option to improve the performance of both homopolymers. They had prepared a blend of P(3HB)/PCL by melting the mixture in an internal mixer with compositions of PCL varying from 0 to 30 wt% to study the miscibility, morphology and physical– chemical properties of these systems.
Cellulose derivatives also have attracted much interest for their compatibility with P(3HB). Ethyl-cellulose (EtC) is also a biomaterial like P(3HB) that is approved by the FDA (Food and Drug Administration) and is widely used as a blood coagulant, in coatings for pharmaceutical tablets and matrices for poorly soluble drugs. Zhang and co-workers (1997) had investigated the miscibility, thermal behaviour and morphological structure of P(3HB) with ethyl cellulose (EtC) blends. A P(3HB)/starch blend was prepared either by a conventional solvent casting method or by melt processing methods, such as injection molding and compression molding after compounding. Two types of maize starch, Starch 1 (containing 70% amylose) and Starch 2 (containing 72% amylopectin), were blended with P(3HB) using a melt compounding method at a ratio of 70 : 30 wt% and characterised in terms of their morphology, structure, thermal, rheological and mechanical properties. Ikejima and co-workers (1999) had prepared P(3HB)/chitosan blend films in order to investigate the effect of deacetylation on the crystallisation behaviour of P(3HB). Chitosan is a copolysaccharide with a high degree of deacetylation. A solvent-casting method was employed to prepare the P(3HB)/chitosan blend films. P(3HB) and chitosan were dissolved separately in HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) before blending.
Zembouai et al. (2013) reported that the degradation of each polymer occurred separately and it was found that PLA was more thermally stable than P(3HB-co-3HV) copolymer. They also reported that the decomposition temperature, Td, for all the blends was between PLA and the P(3HB-co-3HV) copolymer. Therefore, the thermal stability of the P(3HB-co-3HV)/PLA blends could be improved by increasing the amount of PLA. The Tm for the blend was found to be in between these two polymers. Miscibility of any polymer can be determined by evaluating the Tg. A single Tg indicates the miscibility of the polymer. A study by Nanda et al. (2011) reported one Tg which corresponded to PLA and the Tg for P(3HB-co-3HV) copolymer was poorly observed. They also reported that when the content of P(3HB-co-3HV) copolymer was increased, a reduction in Tg from 60 °C to 45 °C was observed. The same observation was reported by Richards et al. (2008)33 and Modi et al. (2012). Chee and co-workers (2002) had performed viscometric studies on polymer-blend solutions of P(3HB) with PCL because it is a powerful method to assess the miscibility of the components in an amorphous state. Viscometric analysis demonstrated that P(3HB) was immiscible with PCl. Phase behaviour and crystallisation kinetics for the binary blend P(3HB)/ cellulose propionate (CP) had been studied by Maekawa and co-workers (1999).A strong dependence of the measure Tg on composition was detected at high levels of CP. The Flory–Fox equation is one of the best equations to describe the dependence of Tg on composition in miscible blend systems. Wang and co-workers (2003) studied the miscibility, crystallisation behaviour, tensile properties and environmental biodegradability of P(3HB)/cellulose acetate butyrate (CAB) blends. Ismail et al. (2010) studied the effect of starch content in a P(3HB) film matrix on its degree of swelling in water. Swelling in water and degradability are the most important characteristic for biodegradable materials. Polymer films were degraded by surface absorption of moisture and microorganisms.
1.4 Nanocomposites of Polyhydroxyalkanoates
Ten et al. produced evenly exfoliated nanocomposites of PHBV/cellulose nanowhiskers, with a well-defined distribution of nanofillers within the polymer matrix. Zhijiang et al. reported an improvement in the mechanical properties of a PHB nanocomposite made of bacterial cellulose nanofibrils that was prepared by a solution-casting method. In addition, they found the nanocomposite to show better biocompatibility and mechanical properties than pure PHB based on cell-adhesion analysis using Chinese hamster lung (CHL) fibroblast cells and stress strain tests, respectively. Ten et al. studied the isothermal crystallization kinetics of poly(3-hydroxybutyrate-co-3 -hydroxyvalerate) (PHBV) nanocomposites containing cellulose nanowhiskers (CNWs). The researchers studied the effects of the CNWs concentration and temperature on the crystallization rate and crystallinity of PHBV nanocomposites. Mook Choi et al. for the first time described the production of an intercalated PHBHV-clay nanocomposite using melt extrusion in a Brabender mixer at 165 °C, 50 rpm agitation rate for 15 min. The same melt extrusion was employed by Maiti et al. to produce a well dispersed PHB/ layered silicate nanocomposite. Zhang et al. prepared nanocomposites of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)/layered silicates and PHBHHx/expanded graphite. For the nanofillers used in the two nanocomposites, at a lower nanofiller content the researchers observed an exfoliated morphology with good dispersion. Gorrasi et al. obtained exfoliated nanocomposites of a poly-6-hydroxyhexanaote matrix through in situ intercalative ring opening polymerization of caprolactone with modified montmorillonite in the presence of a dibutyltin dimethoxide catalyst. Reports on the application of in situ intercalative polymerization for the production of bacterial PHA nanocomposites, especially those containing a medium-chain-length poly-3-hydroxyalkanoate matrix, are virtually nonexistent. Zhang et al. suggested that the dispersion of the polymeric side chain into the nanofiller's layered spaces results in exfoliation. The characterization of the state of nanoparticle dispersion allows for the interpretation of the preceding morphologies, and the structural characterization relies heavily on techniques such as X-ray diffraction (XRD), wide angle X-ray diffraction (WAXD), simultaneous small angle X-ray scattering (SAXS) and electron microscopy (transmission, TEM, or scanning, SEM). Zhijiang et al. employed the use of field emission scanning electron microscopy (FESEM) to qualitatively characterize a PHA/bacterial cellulose nanocomposite. Zhang et al. reported the use of nanocomposite particles based on a PHA/folate ligand which carries doxorubicin as an anticancer therapy. The researchers reported that the membrane showed an excellent selectivity with relatively high permeation flux towards water compared to existing commercial membranes. Shape memory nanocomposite polymers have the ability to revert back to their original shape after being deformed thus expanding their applications to such areas as dry adhesion, microfluidics, biosensors and tissue engineering. Ishida et al. reported the synthesis of a shape memory polymer nanocomposite derived from poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) composited with nanofillers of silsesquioxane (POSS). A nanocomposite membrane based on a PHB-functionalized multi-walled carbon nanotubes/chitosan matrix was used to efficiently pervaporate a mixture of water and 1,4-dioxane without the use of an entrainer. The researchers reported that the membrane showed an excellent selectivity with relatively high permeation flux towards water compared to existing commercial membranes.
