The ability to synthesize biopolymers

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2.0 LITERATURE REVIEW

2.1 Biopolymers

Biopolymers are polymers that are synthesized by living organisms to fulfil biological functions for survival. Microorganisms have the ability to synthesize biopolymers through enzymatic processes to be manipulated for various applications in medical, pharmaceutical, and manufacturing industries (Chen, 2009). Synthetic polymers which are chemical-based polymers (Khanna and Srivastava, 2005), are present in the current market but lack characteristics of degradability and are hazardous to the environment.

2.2 Bioplastics

     Plastics have become part of the modern lifestyle of mankind. The wide range applications of plastics allow them to become vital components of many industries and are extensively used as packaging material. The dependence on plastics for generations to come, would pose a hazardous threat to the environment as these conventional plastics are resistant to degradation. The scientific breakthrough of biodegradable plastics offers a solution as an environment-friendly alternative (Reddy et al., 2003).

Biodegradable plastics are derived from biopolymers with degradability characteristics. However, not all biopolymers are degradable. According the Khanna and Srivastava, 2005, biopolymers can be differentiated into 3 categories; chemically synthesized polymers, starch-based biodegradable plastics as well as polyhydroxyalkanoates (PHAs).

Among the three categories, only PHAs are known to be a hundred per cent biodegradable into carbon dioxide and water in aerobic conditions, and into methane in anaerobic conditions (Khanna and Srivastava, 2005). Starch-based biodegradable plastics are considered semi-degradable as they are starch-linked polyethylene fragments. The starch linking the fragments can be degraded by soil microbes, but degradation action is halted by the polyethylene fragments thus leaving the polyethylene fragments undegraded (Reddy et al., 2003). Chemically synthesized polymers such as polyglycollic acid, polylactic acid, and polyvinyl alcohol however, are chemically as well as physically different from the properties in plastics thus cannot be directly compared to plastics (Khanna and Srivastava, 2005).

2.3 Polyhydroxyalkanoates (PHAs)

PHAs are biodegradable thermoplastics that exist as intracellular carbon and energy storage materials in bacterial cells. This intracellular accumulation occurs in nutrient limited conditions with the presence of excess carbon source (Anderson and Dawes, 1990). These bacterial cells have the ability to accumulate PHA up to 90% of the cell dry weight without causing significant effect to the osmotic pressure in the cell (Madison and Huisman, 1999). This is due to the accumulation of intracellular granules in polymerized insoluble forms that neither affects the cell function nor cause leakage of the polymer out of the cell. (Madison and Huisman, 1999; Verlinden et al., 2007)

PHAs resemble polypropylene in its efficiency of being processed into plastics thus making them widely applicable to a variety of areas. As demonstrated in Figure 2.1, PHAs are linear polyesters made up of 3-hydroxyalkanoates (3HAs) that has an alkyl group positioned at the C-3 (Taguchi and Doi, 2004). The R and x number on the chemical structure determines the type of PHA it is, such as shown in Table 2.1 (Loo and Sudesh, 2007). The PHA that occurs naturally in microorganisms, is Poly(3-hydroxybutyrate), P(3HB) or also known as PHB (Taguchi and Doi, 2004).

The polymerizing enzyme, PHA synthase (PhaC) is the main enzyme that determines the type of PHA being produced in the microorganism. PHA synthases are classified based on their primary structures and substrate specificity. Substrate specificity of the PHA synthase affects the number of carbon atoms that polymerizes to hydroxyalkanoates (Sudesh et al., 2000).

Thermoplastic ability, degradability, as well as its versatility to be able to be modified to obtain desired properties are among the characteristics of PHA that have caught the interest of many scientists across the world since its discovery by Lemoinge in 1926 (Anderson and Dawes, 1990; Sudesh et al., 2000). These vital characteristics prove to be an advantage to this polyester and has opened up many doors for various industrial applications (Chen, 2009). Other general features of PHA include being able to be produced from renewable resources, nontoxic, possessing a high degree of polymerization, insoluble and highly crystalline (Steinbuchel and Fuchtenbusch, 1998).

2.3.1 Formation of PHA inclusion

PHA inclusion in vivo is in the form of a mobile amorphous polymer (Sudesh et al., 2000). The phospholipids and the proteins coat the granules accumulated in the cells such as depicited in Figure 2.2 below (Verlinden et al., 2007). Crystallization of PHA inclusions does not occur unless treated with chemical solvents such as done in extraction methods, where the phospholipids and the proteins are then removed (Stuart et al., 1998).

Figure 2.2 Schematic drawing demonstrating the proteins located in the monolayer phosholipid membrane involved in the structure of PHA inclusion body (Sudesh et al., 2000)

The organization of these genes and enzymes that are involved in PHB biosynthesis are as demonstrated in Figure 2.3. Key proteins such as the PHA synthase (PhaC) for the biosynthesis of PHA as well as the intracellular PHA depolymerase (PhaZ), which is involved in degradation of PHA have been identified (Sudesh et al., 2000). phaCBA gene cluster plays an important role in biosynthesis, catabolism and regulation of P(3HB). It encodes for proteins PhaA (B-ketothiolase), PhaB (NADPH-oxidoreductase) and PhaC (PHB polymerase). These proteins catalyze the main reactions in the P(3HB) metabolic pathway (Luengo et al., 2003).

There are also proteins called phasins (PhaP) which are non-enzymatic proteins which is a common occurrence at the interface of PHA granules. The quantity of granules accumulated as well as their size were found to be influenced by these phasins (Luengo et al., 2003; Verlinden et al., 2007). The accumulation of these low molecular weight PhaP were found to have an influence in promoting PHA synthesis, thus the expression of these proteins are to be able to act as a marker for the production of intracellular PHA (York et al., 2001). PHA granules can be observed through staining with Sudan Black B or Nile Blue A. Both dyes are able to stain lipid bodies although Sudan Black B is unable to stain glycogen and polyphosphate (Loo and Sudesh, 2007).

Figure 2.3 Organization of genes and enzymes involved in biosynthesis of PHB in C. necator H16 (also known as R. eutropha H16) (Luengo et al., 2003)

2.3.2 PHA biosynthetic pathway

There are three main pathways that exists for the biosynthesis of PHA; Pathway I, Pathway II, and Pathway III. The type of pathway in which PHA is synthesized is dependant on the carbon source being fed to the bacterial cell (Tsuge, 2002). Pathway I is one of the most common pathways that is popular among bacteria such as C. necator in producing P(3HB) through the action of 3 main biosynthetic enzymes. The PHA biosynthetic pathway begins with PhaA which is the B-ketothiolase that acts by combining two acetyl-coenzyme molecules to form an acetoacetyl-CoA molecule. PhaB, the acetoacetyl-CoA reductase then reduces the acetoacetyl-CoA stereospecifically by NADH to (R)-3-hydroxybutyryl-CoA. Subsequently, PHA synthase (PhaC) incorporates (R)-3-hydroxybutyryl-CoA to P(3HB) into a growing polymer (Verlinden et al., 2007; Sudesh et al., 2000). In circumstances where there is no nutrient limitation in bacterial growth, inhibition of B-ketothiolase occurs by the free coenzyme-A from the TCA cycle. However, when there is nutrient limitation with available carbon source supplied, acetyl-CoA is prevented from entering the TCA cycle, leaving it to take the route of P(3HB) production (Verlinden et al., 2007).

Pathway II and III differs from Pathway I by utilizing fatty acid B-oxidation intermediates and fatty acid biosynthesis intermediates, respectively, for the production of MCL-(R)-3HA monomers by specific enzymes to be used by PHA synthase to produce P(3HB) (Tsuge, 2002). PhaJ protein generates MCL-3-HA-CoA from fatty acid degradation (B-oxidation) pathway while PhaG, the (R)-3-hydroxyacyl-acyl-carrier protein (ACP)-CoA transferase generates MCL-3-HA-CoA from the fatty acid biosynthesis pathway (Tsuge, 2002; Taguchi and Doi, 2004). PhaJ and PhaG enzymes present act as metabolic links between the fatty acid metabolism to the biosynthesis of PHA (Taguchi and Doi, 2004).

Figure 2.4 Metabolic pathways that produces various hydroxyalkanoate (HA) monomers for PHA biosynthesis. PhaA, 3-Ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3-hydroxyacyl-ACP-CoA transferase; PhaJ, (R)-specific enoyl-CoA hydratase; FabG, 3-ketoacyl-ACP reductase (adapted from Tsuge, 2002).

2.4 Types and properties of PHA

The type of PHA formed is based on its chemical structure that is dependent on the R groups that vary in chain length (alkyl groups) at R configuration at the C-3. The number of carbons added at C-3 ranges from one carbon atom to as many as 14 carbon atoms (Suriyanmongkol et al., 2007). As mentioned earlier, PHA synthase is the main enzyme that functions with a polymerization action. This polymerization action is the key to the classification of PHA produced by the microorganism (Sudesh et al., 2000).

     The 3 categories of PHA produced are short-chain-length (SCL) monomers, medium-chain-length (MCL) monomers and PHA with both short-chain-length and medium-chain-length (SCL-MCL) polymers (Madison and Huisman, 1999; Sudesh et al., 2000). The amount of carbon atoms occuring in the molecule determines its classification. Short-chain-length (SCL) monomers contain 3 to 5 carbon atoms and medium-chain-length (MCL) monomers with 6 to 14 carbon atoms (Sudesh et al., 2000).

2.4.1 SCL- PHA

Short-chain-length PHA (SCL-PHA) are distinguished by their characteristic high melting temperatures and are more brittle (Madison and Huisman, 1999). This is due to the fact that SCL-PHA such as P(3HB) which is the PHA commonly produced in wild-type bacteria, (Madison and Huisman, 1999) has a high degree of crystallinity (Sudesh et al., 2000; Suriyanmongkol et al., 2007). SCL-PHA is considered rather tedious to process due to the high melting temperature it contains which is around 170 oC. This high melting temperature aids thermal decomposition of this PHA polymer (Madison and Huisman, 1999).

2.4.2 MCL-PHA

This class of PHA is known to show lesser crystallinity compared to the SCL-PHA, thus making it less brittle with lower melting temperature (Madison and Huisman, 1999; Chen et al., 2009). It possesses poor tensile strength as an elastomer (Wu et al., 2003). An example of MCL-PHA are copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-3HV)]. These characteristics of the MCL-PHA is not close to the conventional plastics, however, they have the ability to be modified into rubber-based materials (Suriyanmongkol et al., 2007).

2.4.3 SCL-MCL PHA

These distinct characteristics of SCL-PHA monomers and MCL-PHA monomers hamper their application capabilities. The combination of SCL-MCL copolymers of PHA however, have shown to be able to provide better characteristics for a broader range of applications (Zhao and Chen, 2007). For the PHA to contain SCL-MCL copolymers with both the monomers covalently linked, it requires PHA synthase that exhibits substrate specificity which is a combination of both monomers' synthases (Steinbuchel and Lutke-Eversloh, 2003).

2.5 Established polymers

2.5.1 Poly(3-hydroxybutyrate) [P(3HB)]

P(3HB) is the well-studied PHA that is able to be accumulated by bacterial cells (Madison and Huisman, 1999). With its mechanical properties such as Young's modulus of 3.5 GPa, and tensile strength of 43 MPa resembing that of polypropylene (PP), it is often compared to these conventional plastics. However, P(3HB) is relatively more brittle and stiff as they possess only 5% elongation to break compared to the 400% achieved by PP (Sudesh et al., 2000). Co-feeding of various substrates to the cells is usually carried out to incorporate other monomers to the existing homopolymer (Madison and Huisman, 1999). The incorporation of these other monomers alter the properties of materials produced which may enhance its desirable characteristics thus enabling a wide range of applications (Anderson and Dawes, 1990; Sudesh et al., 2000).

2.5.2 Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]

Figure 2.5 illustrates the chemical structure of P(3HB-co-3HV). It is a copolymer that is able to be produced by C. necator. The HV monomer is formed with the aid of precursors such as valeric acid that is co-fed with glucose and even with valeric acid as sole carbon source (Anderson and Dawes, 1990; Lee and Choi, 1999). Propionic acid can also be used to induce the formation of the HV monomer but valeric acid precursors are known to give better results of the copolymer formation (Bhubalan et al., 2008). These precursors have to be monitored in terms of concentration fed as they were toxic to the cells and may disrupt the cellular activities of the cell, thus leading to cell death (Loo and Sudesh, 2007). Unlike C.necator being able to produce this copolymer through the three-step P(3HB) pathway, there are other PHA producing bacteria producing copolymer P(3HB-co-3HV) through different pathways such as methylmalonyl-CoA pathway and other pathways involving B-oxidation and fatty acid biosynthesis intermediates (Madison and Huisman, 1999).

2.5.3 Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)]

P(3HB-co-3HHx) copolymer, with its chemical structure as demonstrated in Figure 2.6, possesses physical properties similar to common plastics such as PP and low-density polyethylene (LDPE) (Loo and Sudesh, 2007). The 3HHx component in the copolymer can significantly increase the elasticity and flexibility of the PHA polymer (Doi et al., 1995). It was reported that the incorporation of 10% mol of 3HHx to the P(3HB) homopolymer was able to increase the elongation to break ability of the polymer up to 400 % (Sudesh et al., 2000). C. necator was reported to be able to produce P(3HB-co-3HHx) when even-numbered fatty acids chains were used as carbon source while odd-numbered fatty acid chains produced P(3HB-co-3HV) (Dennis et al., 1998). P(3HB-co-3HHx) consists of both SCL-PHA monomers as well as MCL-PHA monomer; 3HB and 3HHx respectively, which are able to be naturally produced by a few microorganisms such as the Aeromonas caviae and A. hydrophila (Loo and Sudesh, 2007). As for mutant strains, Loo and co-workers discovered that C. necator harboring PHA synthase gene of A. caviae was able to produce around 87 % (w/w) of cell dry weight P(3HB-co-3HHx) when it was fed with carbon source of palm kernel oil (Loo et al., 2005)

2.5.4 Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) [P(3HB-co-3HV-co-3HHx)]

The P(3HB-co-3HV-co-3HHx) terpolymer has been proven to be able to be produced by a recombinant strain of C. necator harboring the PHA synthase of A. caviae by feeding with palm kernel oil as well as the sodium valerate and sodium propionate precursors (Bhubalan et al., 2008). Park and co-workers were also able to produce P(3HB-co-3HV-co-3HHx) from genetically modified Escherichia coli containing A. hydrophila PHA biosynthesis genes with feeding of dodecanoic acid with another odd carbon number fatty acids (Park et al., 2001). This terpolymer was found to be more flexible compared to its brittle homopolymer and copolymer counterparts (Zhao and Chen, 2007). The increase in elastic behaviour exhibited by P(3HB-co-3HV-co-3HHx) at room temperature is attributed to the decrease in glass transition temperature Tg (Zhao and Chen, 2007).

2.6 PHA-producing bacteria

2.6.1 Cupriavidus necator

Cupriavidus necator previously referred to as Hydrogenous eutropha, Alcaligenes eutrophus, Wautersia eutropha and Ralstonia eutropha is a Gram-negative bacteria which is able to produce PHA (Sudesh et al., 2000; Jendrossek, 2009). The C. necator is a popular choice for this purpose as it has the ability to accumulate high PHA content (Byrom, 1992) as well as utilize a variety of carbon substrates for its growth (Anderson and Dawes, 1990; Fukui and Doi, 1998). The PHA-biosynthetic operon present in C. necator allows it to carry out its PHA-producing function even in recombinant strains (Jendrossek, 2009). It is known to synthesize PHA when its growth is limited by the lack of N, P, or O (Anderson and Dawes, 1990).

Cultivation techniques used for C. necator are two main types, the one-step cultivation technique and the two-step cultivation technique. One-step cultivation technique utilizes the same medium to induce cell growth (Madison and Huisman, 1999) and PHA production while two-step cultivation technique involves two different mediums which are the rich medium and the nutrient limiting medium (Kichise et al., 1999). Rich medium would be first used to induce growth of the bacterial cells, followed by a nutrient limiting medium (lack of Nitrogen source) but supplied with sufficient carbon source to induce the accumulation of PHA (Madison and Huisman, 1999).

2.6.2 Chromobacterium violaceum

Other PHA producers that have been studied and characterized include Chromobacterium violaceum. This gram-negative proteobacteria has a distinct ability to produce violacein, a purple-coloured pigment under aerobic conditions which is useful in antibiotic production as well as in cytotoxic activities (Robert, 2003; Konzen et al., 2006).

C. violaceum is able to produce PHA polymers that comprises of 3HB and 3HV monomers as well as just 3HV homopolymers by feeding on valerate (Steinbuchel et al., 1993). Studies have found that it was possible to isolate and clone the polyhydroxyalkanoate synthase gene of C. violaceum to be expressed in another PHA producing microorganism such as C. necator (Kolibachuk et al., 1999). The synthase gene of C. violaceum is known to be able to incorporate higher fractions of 3HV monomer in the PHA accumulated in the cell (Kolibachuk et al., 1999).

2.6.3 Recombinant strains of C. necator

Recombinant strains of C. necator have also been engineered to harbour the PHA synthase genes of other high producing PHA strains to improve PHA production in terms of content and composition as well as cell dry weight (Madison and Huisman, 1999). Recombinant strain of C. necator is manipulated by the incorporation of the PHA synthase gene from another PHA producer such as the C. violaceum. The phaC from C. violaceum is incorporated to function together with the existing phaA and phaB present in the C. necator (Kolibachuk et al., 1999). Kolibachuk and co-workers suggested the possibility of C. necator possessing additional enzymes, possibly an alternate ketoacyl-CoA reductase, that has different substrate-specificity to the phaC of C. violaceum (Kolibachuk et al., 1999).

2.7 Carbon source

Different PHA-producing bacterial strains may require different carbon sources for accumulation of PHA (Lee et al., 1999; Reddy et al., 2003). Therefore, feeding of C. necator with suitable alternative carbon sources that are cheaper and renewable are important to reduce the cost of PHA production from bacterial cells as carbon source accounts for 70 to 80 per cent of the total expense of raw materials (Cavalheiro et al., 2009). Examples of inexpensive renewable carbon source for PHA production include plant oils, fatty acids, agricultural or food industrial wastes, organic acids, as well as carbon dioxide (Tsuge, 2002).

Plant oils such as soybean oil, olive oil, corn oil and palm oil have been identified as a carbon source that is efficiently utilized by C. necator H16 to accumulate up to 80% of the cell dry weight (Fukui and Doi, 1998). Plant oils prove to be an advantage in terms of yield obtained as well as cost-efficiency as they were found to contain high carbon content per weight as compared to common sugar substrates used (Akiyama et al., 2003). C. necator H16 was able to produce high cell dry weight as well as high PHA content up to 76% (w/w), while its recombinant PHB-4/pJRDEE32d13 was found to be able to produce PHA content up to 74% (w/w) when fed with soybean oil (Kahar et al., 2004). Palm oil has also proven to be efficiently utilized by bacterial cells to synthesize PHA of different monomer compositions with the aid of different precursors (Lee et al., 2008; Bhubalan et al., 2008).

The abundance and renewability of soybean oil and palm oil in nature allows their usage in the mass production of PHA to be viable (Loo et al., 2005). Linoleic acid; an unsaturated fatty acid, was found to be poorly utilized by C. necator, by possibly inhibiting the incorporation of the fatty acids into the cells. Kahar and co-workers suggested that palm oil was a much better choice of carbon source for the feeding of C. necator for PHA production as palm oil contains lesser linoleic acid (Kahar et al., 2004). Furthermore, palm oil as the world's leading edible oil in terms of efficiency of production, supply and cost makes it a superior candidate than the other edible oils available (Lam et al., 2009).

2.7.1 Sugars

Commonly studied sugars such as glucose and sucrose are usually utilized as a carbon source by bacterial cells in the fermentation process of PHA production (Tsuge, 2002). There has been several attempts for large-scale production of PHA utilizing these simple sugars as carbon sources for bacterial strains such as C. necator, A. latus, and E. coli (Chen, 2009). However, even with the relatively low cost of sugars, bacterial cells fed on these sugars face the disadvantage of obtaining low yield when compared to cheaper renewable carbon sources such as plant oils (Akiyama et al., 2003).

2.7.2 Palm oil

C. necator H16 as well as its recombinant strains were found to be able to produce PHA copolymers as well as terpolymers with different monomer compositions with the feeding of palm oil products as carbon source with the aid of precursors (Lee et al., 2008; Bhubalan et al., 2008). Crude palm kernel oil (CPKO) and the crude palm oil (CPO) are the main products of the oil palm fruit. Malaysia, being one of the world's largest producers of palm oil, produced a total of 2.13 million tonnes of crude palm kernel oil in the year 2008 (GOFB, 2009). CPKO is the expeller that is pressed from the kernel or the endosperm of the oil palm fruit (Lee et al., 2008). CPKO proves to be an efficient carbon source due to its low production cost, availability, and the renewability of palm oil because unlike CPO, CPKO is hardly utilized in food industries, thus bringing no adverse impact of large scale PHA production to palm oil-based food products sustainability (Bhubalan et al., 2008). The main difference between palm kernel oil and palm oil in basically in their fatty acid composition with palm kernel oil containing saturated fatty acids while palm oil containing unsaturated fatty acids (Loo et al., 2005).

2.7.3 Fatty Acids

Fatty acids are able to be utilized by C. necator to produce PHA under Nitrogen deficient conditions (Chakraborty et al., 2009). The utilization however is dependent on the level of concentration of the fatty acids and pH used, as the cells can tolerate a certain range before being toxic to the cells (Yu et al., 2002; Chakraborty et al., 2009). Toxicity of volatile fatty acids is due to cytoplasm acidification which occurs through penetration of undissociated lipophilic molecules of fatty acids. This disrupts the gradient of protons as well as the energy production and transport system involving the cell membrane as well (Yu et al., 2002). Lo and co-workers suggested that fatty acids containing single double bond activates the enzymatic reactions of PHA when several bacteria strains were fed with glucose as carbon source and fatty acids were added as nutritional supplements (Lo et al., 2005).

2.8 Pathway inhibitor

Acrylic acid is known to be an inhibitor of the fatty acid B-oxidation pathway as it prevents the action of 3-ketoacyl-CoA thiolase that frees acetyl-CoA from 3-ketoacyl-CoA. This inhibition leads to the build-up of fatty acid B-oxidation intermediates (Qi et al., 1998). The presence of acrylic acid during cultivation of PHA producing cells together with carbon sources was found to alter the monomer composition of co-polymer as well as the amount of polymer formed (Steinbuchel et al., 1998). Acrylic acid inhibits 3-ketoacyl-CoA thiolase action that catalyzes the final step in B-oxidation that releases acetyl-CoA from 3-ketoacyl-CoA (Qi et al., 1998).

2.9 Biodegradation of PHA

Due to the lack of degradability in conventional plastics that contribute to the high amounts of solid waste, the biodegradability of PHA offers the solution that drives the continuous studies done on PHA production.

PHA has been found to be able to degrade in microbial active environments such as soil (Mergaert et al., 1993), lake water, and marine water (Ohura et al., 1999) and even in sewage sludge (Lee and Choi, 1999). This occurrence is due to PHA degrading enzymes also known as PHA depolymerase which are secreted by microorganisms to hydrolyze water-insoluble PHA into water-soluble forms as nutrients which can be utilized by these microorganisms (Sudesh et al., 2000). PHA depolymerase has been found to be a structure comprising domains including catalytic domain and a substrate-binding domain to bind the water-insoluble PHA materials to the enzyme, where both domains are bonded by a linker domain (Numata et al., 2009).

PHA biodegradation are influenced by factors such as the type of environment they are being degraded in, the microbial activity present in the environment, as well as the properties of the PHA material (Khanna and Srivastava, 2005; Lee and Choi, 1999). The physical and chemical property of PHA material also contribute to their degradation effect such as molecular weight (Mw), melting temperature (Tm), crystallinity and elasticity (Tokiwa and Calabia, 2004). Li and co-workers suggested that enzymatic degradation of PHA is based on the length of side chain of PHA, which implies that the longer side chains provide better degradability (Li et al., 2007). Furthermore, copolymers of PHA have been found to degrade better compared to homopolymers (Mergaert et al., 1993). This higher degradation capability is attributed to the surface morphology of co-polymers which combines low crystallinity and porous surface (Sridewi et al., 2006; Wang et al., 2004).

2.10 Applications of PHA

The unique characteristics of PHA allow it to be utilized in a wide range of applications. PHA application involves industries such as the packaging, medical, energy, material, textile, fine chemical as well as bulk chemical industries (Chen, 2009).

The packaging industry which heavily depends on plastics as packaging material allows PHA to be marketed as a biodegradable plastic (Madison and Huisman, 1999). These efforts are aimed to reduce the solid waste materials generated from packaging. PHA produced can also be diversified to form desired characteristics such as through manipulation of bacterial strains, feeding substrate as well as the usage of inhibitors and precursors in certain stages of growth (Steinbuchel, 2001). Furthermore, new plastic properties can be produced by various composition mixtures of PHB and other polymers to alter the physical properties and crystallinity of the plastic produced to achieve various properties of plastic for a wider range of packaging applications (Zinn et al., 2001).

The main application of PHA in the medical industry involves its usage as a drug carrier and as medical implant material in tissue engineering (Chen, 2009). PHA characteristics emphasized for medical implant biomaterials include compatibility with cells, ability to support cell growth, proliferate only with cell adhesion for normal cell growth, conducts proper cell organization and the ability to degrade once no longer in use (Williams et al., 1999; Zinn et al., 2001). Current materials being used as implant material such as silicon are suspected to be able to be malignant and may cause cancer or produce toxic compounds (Zinn et al., 2001). PHAs commonly studied for the purpose of bio-material medical implant include P(3HB), P(3HB-co-4HB), P(4HB), and P(3HO) (Chen, 2009). A popular example is P(4HB), which is the material for products of medical implant, has been reported to be approved and marketed under the name PHA4400 by Tepha Inc. (Cambridge, MA) (Martin and Williams, 2003).

PHA as drug carriers involves the general mechanism that utilizes microspheres of SCL-PHA to release the drugs rapidly with 90 per cent of release within the time frame of 24 hours (Zinn et al., 2001). PHAs used for this purpose are manufactured to contain increased porosity as well as increased surface area to enhance degradability as well as the rate of drug diffusion through the pores (Chen and Wu, 2005). The rate of drug release was found to be dependent on the particle size used as well as the type of drug loaded (Pouton and Akhtar, 1996; Zinn et al., 2001). Besides biodegradability, biocompatibility of PHB as well as P(3HB-co-3HV) co-polymers is also a main concern in drug delivery as they influence polymer degradation as well as the manner tissues react to the foreign polymers introduced (Pouton and Akhtar, 1996).

Another popular field of PHA application is also the energy industry which is the production of PHA-based biofuels. PHB is able to be acid hydrolyzed to form hydroxyalkanoate methyl esters such as (R)-3-hydroxybutyrate methyl ester (3HBME) and medium chain length hydroxyalkanoate methyl ester (3HAME) that are combustible (Chen, 2009). Blended fuels such as 3HAME-gasoline and 3HAME-diesel contribute better as fuels as they are unable to produce higher combustion heat as pure gasoline or diesel (Chen, 2009). This opens up a new avenue for PHA applications in the biofuel production.

2.11 Economics of PHA production

The development of technology has increased the usage of natural resources available on this planet. Due to this, the depletion of natural resources such as fossil fuels are becoming a main concern across the globe. One such example is with the usage of conventional plastics which are not readily degraded in the natural environment, thus becoming an environmental pollutant. Realizing the depletion of petroleum based products from the petroleum crisis in the 1970s (Sudesh and Iwata, 2008), PHA has gained interests of many, as a possible natural alternative for this purpose. Commercialization of PHA is generally hindered due to its high cost, US$ 16 per Kg as produced by C. necator (Lee, 1996) which is 18 times higher as compared to its polypropylene counterpart (Reddy et al., 2003). In order to produce PHA at a commercially viable price, which is around US$ 4 per Kg (Lee, 1996), there are several parameters that have to be taken into consideration. These include cost of substrate, efficient operating process, high conversion efficiency from substrate to yield as well as methods in reducing downstream recovery cost (Reddy et al., 2003). Genetic manipulation of genes for heterologous and homologous expressions as well as engineering high production strains can also be considered to improve the yield as well as alter the metabolic processeses to obtain desirable send products (Steinbuchel, 2001; Reddy et al., 2003). Further studies in these areas can significantly reduce the production cost of PHA enabling them to be commercially viable, in replacement of the current petrochemical-based plastic.

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