2.0 LITERATURE REVIEW
Biopolymers are polymers that are synthesized by populating beings to carry through biological maps for endurance. Microorganisms have the ability to synthesise biopolymers through enzymatic procedures to be manipulated for assorted applications in medical, pharmaceutical, and fabricating industries ( Chen, 2009 ) . Man-made polymers which are chemical-based polymers ( Khanna and Srivastava, 2005 ) , are present in the current market but lack features of degradability and are risky to the environment.
Plastics have become portion of the modern life style of world. The broad scope applications of plastics allow them to go critical constituents of many industries and are extensively used as packaging stuff. The dependance on plastics for coevalss to come, would present a risky menace to the environment as these conventional plastics are immune to debasement. The scientific discovery of biodegradable plastics offers a solution as an environment-friendly option ( Reddy et al. , 2003 ) .
Biodegradable plastics are derived from biopolymers with degradability features. However, non all biopolymers are degradable. Harmonizing the Khanna and Srivastava, 2005, biopolymers can be differentiated into 3 classs ; chemically synthesized polymers, starch-based biodegradable plastics every bit good as polyhydroxyalkanoates ( PHAs ) .
Among the three classs, merely PHAs are known to be a 100 per cent biodegradable into C dioxide and H2O in aerophilic conditions, and into methane in anaerobiotic conditions ( Khanna and Srivastava, 2005 ) . Starch-based biodegradable plastics are considered semi-degradable as they are starch-linked polythene fragments. The amylum associating the fragments can be degraded by dirt bugs, but debasement action is halted by the polythene fragments therefore go forthing the polythene fragments undegraded ( Reddy et al. , 2003 ) . Chemically synthesized polymers such as polyglycollic acid, polylactic acid, and polyvinyl intoxicant nevertheless, are chemically every bit good as physically different from the belongingss in plastics therefore can non be straight compared to plastics ( Khanna and Srivastava, 2005 ) .
2.3 Polyhydroxyalkanoates ( PHAs )
PHAs are biodegradable thermoplastics that exist as intracellular C and energy storage stuffs in bacterial cells. This intracellular accretion occurs in alimentary limited conditions with the presence of extra C beginning ( Anderson and Dawes, 1990 ) . These bacterial cells have the ability to roll up PHA up to 90 % of the cell dry weight without doing important consequence to the osmotic force per unit area in the cell ( Madison and Huisman, 1999 ) . This is due to the accretion of intracellular granules in polymerized indissoluble signifiers that neither affects the cell map nor cause escape of the polymer out of the cell. ( Madison and Huisman, 1999 ; Verlinden et al. , 2007 )
PHAs resemble polypropene in its efficiency of being processed into plastics therefore doing them widely applicable to a assortment of countries. As demonstrated in Figure 2.1, PHAs are additive 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 figure on the chemical construction determines the type of PHA it is, such as shown in Table 2.1 ( Loo and Sudesh, 2007 ) . The PHA that occurs of course in micro-organisms, is Poly ( 3-hydroxybutyrate ) , P ( 3HB ) or besides known as PHB ( Taguchi and Doi, 2004 ) .
The polymerizing enzyme, PHA synthase ( PhaC ) is the chief enzyme that determines the type of PHA being produced in the micro-organism. PHA synthases are classified based on their primary constructions and substrate specificity. Substrate specificity of the PHA synthase affects the figure of C atoms that polymerizes to hydroxyalkanoates ( Sudesh et al. , 2000 ) .
Thermoplastic ability, degradability, every bit good as its versatility to be able to be modified to obtain coveted belongingss are among the features of PHA that have caught the involvement of many scientists across the universe since its find by Lemoinge in 1926 ( Anderson and Dawes, 1990 ; Sudesh et al. , 2000 ) . These critical features prove to be an advantage to this polyester and has opened up many doors for assorted industrial applications ( Chen, 2009 ) . Other general characteristics of PHA include being able to be produced from renewable resources, nontoxic, possessing a high grade of polymerisation, indissoluble and extremely crystalline ( Steinbuchel and Fuchtenbusch, 1998 ) .
2.3.1 Formation of PHA inclusion
PHA inclusion in vivo is in the signifier of a nomadic formless 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 non happen unless treated with chemical dissolvers such as done in extraction methods, where the phospholipids and the proteins are so removed ( Stuart et al. , 1998 ) .
Figure 2.2 Conventional pulling showing the proteins located in the monolayer phosholipid membrane involved in the construction of PHA inclusion organic structure ( Sudesh et al. , 2000 )
The organisation of these cistrons and enzymes that are involved in PHB biogenesis are as demonstrated in Figure 2.3. Cardinal proteins such as the PHA synthase ( PhaC ) for the biogenesis of PHA every bit good as the intracellular PHA depolymerase ( PhaZ ) , which is involved in debasement of PHA have been identified ( Sudesh et al. , 2000 ) . phaCBA cistron bunch plays an of import function in biogenesis, katabolism and ordinance of P ( 3HB ) . It encodes for proteins PhaA ( B-ketothiolase ) , PhaB ( NADPH-oxidoreductase ) and PhaC ( PHB polymerase ) . These proteins catalyze the chief reactions in the P ( 3HB ) metabolic tract ( Luengo et al. , 2003 ) .
There are besides proteins called phasins ( PhaP ) which are non-enzymatic proteins which is a common happening at the interface of PHA granules. The measure of granules accumulated every bit good as their size were found to be influenced by these phasins ( Luengo et al. , 2003 ; Verlinden et al. , 2007 ) . The accretion of these low molecular weight PhaP were found to hold an influence in advancing PHA synthesis, therefore the look of these proteins are to be able to move 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 organic structures although Sudan Black B is unable to stain animal starch and polyphosphate ( Loo and Sudesh, 2007 ) .
Figure 2.3 Organization of cistrons and enzymes involved in biogenesis of PHB in C. necator H16 ( besides known as R. eutropha H16 ) ( Luengo et al. , 2003 )
2.3.2 PHA biosynthetic tract
There are three chief tracts that exists for the biogenesis of PHA ; Pathway I, Pathway II, and Pathway III. The type of tract in which PHA is synthesized is dependent on the C beginning being fed to the bacterial cell ( Tsuge, 2002 ) . Pathway I is one of the most common tracts that is popular among bacteriums such as C. necator in bring forthing P ( 3HB ) through the action of 3 chief biosynthetic enzymes. The PHA biosynthetic tract begins with PhaA which is the B-ketothiolase that acts by uniting two acetyl-coenzyme molecules to organize an acetoacetyl-CoA molecule. PhaB, the acetoacetyl-CoA reductase so 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 turning polymer ( Verlinden et al. , 2007 ; Sudesh et al. , 2000 ) . In fortunes where there is no alimentary restriction in bacterial growing, suppression of B-ketothiolase occurs by the free coenzyme-A from the TCA rhythm. However, when there is alimentary restriction with available C beginning supplied, acetyl-CoA is prevented from come ining the TCA rhythm, go forthing it to take the path of P ( 3HB ) production ( Verlinden et al. , 2007 ) .
Pathway II and III differs from Pathway I by using fatty acerb B-oxidation intermediates and fatty acerb biogenesis intermediates, severally, for the production of MCL- ( R ) -3HA monomers by specific enzymes to be used by PHA synthase to bring forth P ( 3HB ) ( Tsuge, 2002 ) . PhaJ protein generates MCL-3-HA-CoA from fatty acerb debasement ( B-oxidation ) tract while PhaG, the ( R ) -3-hydroxyacyl-acyl-carrier protein ( ACP ) -CoA transferase generates MCL-3-HA-CoA from the fatty acerb biogenesis tract ( Tsuge, 2002 ; Taguchi and Doi, 2004 ) . PhaJ and PhaG enzymes present act as metabolic links between the fatty acerb metamorphosis to the biogenesis of PHA ( Taguchi and Doi, 2004 ) .
Figure 2.4 Metabolic tracts that produces assorted hydroxyalkanoate ( HA ) monomers for PHA biogenesis. 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 belongingss of PHA
The type of PHA formed is based on its chemical construction that is dependent on the R groups that vary in concatenation length ( alkyl groups ) at R constellation at the C-3. The figure of Cs added at C-3 scopes from one C atom to every bit many as 14 C atoms ( Suriyanmongkol et al. , 2007 ) . As mentioned earlier, PHA synthase is the chief enzyme that maps with a polymerisation action. This polymerisation action is the key to the categorization of PHA produced by the micro-organism ( Sudesh et al. , 2000 ) .
The 3 classs 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 sum of C atoms happening in the molecule determines its categorization. Short-chain-length ( SCL ) monomers contain 3 to 5 C atoms and medium-chain-length ( MCL ) monomers with 6 to 14 C atoms ( Sudesh et al. , 2000 ) .
2.4.1 SCL- PHA
Short-chain-length PHA ( SCL-PHA ) are distinguished by their characteristic high thaw temperatures and are more brickle ( Madison and Huisman, 1999 ) . This is due to the fact that SCL-PHA such as P ( 3HB ) which is the PHA normally produced in wild-type bacteriums, ( Madison and Huisman, 1999 ) has a high grade of crystallinity ( Sudesh et al. , 2000 ; Suriyanmongkol et al. , 2007 ) . SCL-PHA is considered instead boring to treat due to the high thaw temperature it contains which is around 170 oC. This high thaw temperature AIDSs thermic decomposition of this PHA polymer ( Madison and Huisman, 1999 ) .
This category of PHA is known to demo lesser crystallinity compared to the SCL-PHA, therefore doing it less brickle with lower thaw temperature ( Madison and Huisman, 1999 ; Chen et al. , 2009 ) . It possesses hapless tensile strength as an elastomer ( Wu et al. , 2003 ) . An illustration of MCL-PHA are copolymers such as poly ( 3-hydroxybutyrate-co-3-hydroxyvalerate ) [ P ( 3HB-3HV ) ] . These features of the MCL-PHA is non near to the conventional plastics, nevertheless, they have the ability to be modified into rubber-based stuffs ( Suriyanmongkol et al. , 2007 ) .
2.4.3 SCL-MCL PHA
These distinguishable features of SCL-PHA monomers and MCL-PHA monomers hamper their application capablenesss. The combination of SCL-MCL copolymers of PHA nevertheless, have shown to be able to supply better features for a broader scope of applications ( Zhao and Chen, 2007 ) . For the PHA to incorporate 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 belongingss such as Young ‘s modulus of 3.5 GPa, and tensile strength of 43 MPa resembing that of polypropene ( PP ) , it is frequently compared to these conventional plastics. However, P ( 3HB ) is comparatively more brickle and stiff as they possess merely 5 % elongation to interrupt compared to the 400 % achieved by PP ( Sudesh et al. , 2000 ) . Co-feeding of assorted substrates to the cells is normally carried out to integrate other monomers to the bing homopolymer ( Madison and Huisman, 1999 ) . The incorporation of these other monomers alter the belongingss of stuffs produced which may heighten its desirable features therefore enabling a broad scope 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 construction 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 assistance of precursors such as valeric acid that is co-fed with glucose and even with valeric acid as exclusive C beginning ( Anderson and Dawes, 1990 ; Lee and Choi, 1999 ) . Propionic acid can besides be used to bring on the formation of the HV monomer but valeric acid precursors are known to give better consequences of the copolymer formation ( Bhubalan et al. , 2008 ) . These precursors have to be monitored in footings of concentration fed as they were toxic to the cells and may interrupt the cellular activities of the cell, therefore taking to cell decease ( Loo and Sudesh, 2007 ) . Unlike C.necator being able to bring forth this copolymer through the three-step P ( 3HB ) tract, there are other PHA bring forthing bacteriums bring forthing copolymer P ( 3HB-co-3HV ) through different tracts such as methylmalonyl-CoA tract and other tracts affecting B-oxidation and fatty acerb biogenesis 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 construction as demonstrated in Figure 2.6, possesses physical belongingss similar to common plastics such as PP and low-density polythene ( LDPE ) ( Loo and Sudesh, 2007 ) . The 3HHx constituent in the copolymer can significantly increase the snap and flexibleness 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 interrupt ability of the polymer up to 400 % ( Sudesh et al. , 2000 ) . C. necator was reported to be able to bring forth P ( 3HB-co-3HHx ) when even-numbered fatty acids ironss were used as C beginning while odd-numbered fatty acid ironss produced P ( 3HB-co-3HV ) ( Dennis et al. , 1998 ) . P ( 3HB-co-3HHx ) consists of both SCL-PHA monomers every bit good as MCL-PHA monomer ; 3HB and 3HHx severally, which are able to be of course produced by a few micro-organisms such as the Aeromonas caviae and A. hydrophila ( Loo and Sudesh, 2007 ) . As for mutation strains, Loo and colleagues discovered that C. necator harbouring PHA synthase cistron of A. caviae was able to bring forth around 87 % ( w/w ) of cell dry weight P ( 3HB-co-3HHx ) when it was fed with C beginning of thenar meats 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 harbouring the PHA synthase of A. caviae by feeding with palm meats oil every bit good as the Na valerate and Na propionate precursors ( Bhubalan et al. , 2008 ) . Park and colleagues were besides able to bring forth P ( 3HB-co-3HV-co-3HHx ) from genetically modified Escherichia coli incorporating A. hydrophila PHA biogenesis cistrons with eating of dodecanoic acid with another uneven C figure fatty acids ( Park et al. , 2001 ) . This terpolymer was found to be more flexible compared to its brickle homopolymer and copolymer opposite numbers ( Zhao and Chen, 2007 ) . The addition in elastic behavior exhibited by P ( 3HB-co-3HV-co-3HHx ) at room temperature is attributed to the lessening in glass passage temperature Tg ( Zhao and Chen, 2007 ) .
2.6 PHA-producing bacteriums
2.6.1 Cupriavidus necator
Cupriavidus necator antecedently referred to as Hydrogenous eutropha, Alcaligenes eutrophus, Wautersia eutropha and Ralstonia eutropha is a Gram-negative bacterium which is able to bring forth PHA ( Sudesh et al. , 2000 ; Jendrossek, 2009 ) . The C. necator is a popular pick for this intent as it has the ability to roll up high PHA content ( Byrom, 1992 ) every bit good as use a assortment of C substrates for its growing ( Anderson and Dawes, 1990 ; Fukui and Doi, 1998 ) . The PHA-biosynthetic operon nowadays in C. necator allows it to transport out its PHA-producing map even in recombinant strains ( Jendrossek, 2009 ) . It is known to synthesise PHA when its growing is limited by the deficiency of N, P, or O ( Anderson and Dawes, 1990 ) .
Cultivation techniques used for C. necator are two chief types, the one-step cultivation technique and the two-step cultivation technique. One-step cultivation technique utilizes the same medium to bring on cell growing ( Madison and Huisman, 1999 ) and PHA production while two-step cultivation technique involves two different mediums which are the rich medium and the alimentary modification medium ( Kichise et al. , 1999 ) . Rich medium would be foremost used to bring on growing of the bacterial cells, followed by a alimentary modification medium ( deficiency of Nitrogen beginning ) but supplied with sufficient C beginning to bring on the accretion of PHA ( Madison and Huisman, 1999 ) .
2.6.2 Chromobacterium violaceum
Other PHA manufacturers that have been studied and characterized include Chromobacterium violaceum. This Gram-negative proteobacteria has a distinguishable ability to bring forth violacein, a purple-coloured pigment under aerophilic conditions which is utile in antibiotic production every bit good as in cytotoxic activities ( Robert, 2003 ; Konzen et al. , 2006 ) .
C. violaceum is able to bring forth PHA polymers that comprises of 3HB and 3HV monomers every bit good as merely 3HV homopolymers by feeding on valerate ( Steinbuchel et al. , 1993 ) . Surveies have found that it was possible to insulate and clone the polyhydroxyalkanoate synthase cistron of C. violaceum to be expressed in another PHA bring forthing micro-organism such as C. necator ( Kolibachuk et al. , 1999 ) . The synthase cistron of C. violaceum is known to be able to integrate 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 besides been engineered to harbor the PHA synthase cistrons of other high bring forthing PHA strains to better PHA production in footings of content and composing every bit good as cell dry weight ( Madison and Huisman, 1999 ) . Recombinant strain of C. necator is manipulated by the incorporation of the PHA synthase cistron from another PHA manufacturer such as the C. violaceum. The phaC from C. violaceum is incorporated to work together with the bing phaA and phaB nowadays in the C. necator ( Kolibachuk et al. , 1999 ) . Kolibachuk and colleagues suggested the possibility of C. necator possessing extra enzymes, perchance an alternate ketoacyl-CoA reductase, that has different substrate-specificity to the phaC of C. violaceum ( Kolibachuk et al. , 1999 ) .
2.7 Carbon beginning
Different PHA-producing bacterial strains may necessitate different C beginnings for accretion of PHA ( Lee et al. , 1999 ; Reddy et al. , 2003 ) . Therefore, feeding of C. necator with suited alternate C beginnings that are cheaper and renewable are of import to cut down the cost of PHA production from bacterial cells as C beginning histories for 70 to 80 per cent of the entire disbursal of natural stuffs ( Cavalheiro et al. , 2009 ) . Examples of cheap renewable C beginning for PHA production include works oils, fatty acids, agricultural or nutrient industrial wastes, organic acids, every bit good as C dioxide ( Tsuge, 2002 ) .
Plant oils such as soybean oil, olive oil, maize oil and thenar oil have been identified as a C beginning that is expeditiously utilized by C. necator H16 to roll up up to 80 % of the cell dry weight ( Fukui and Doi, 1998 ) . Plant oils turn out to be an advantage in footings of output obtained every bit good every bit cost-efficiency as they were found to incorporate high C content per weight as compared to common sugar substrates used ( Akiyama et al. , 2003 ) . C. necator H16 was able to bring forth high cell dry weight every bit good as high PHA content up to 76 % ( w/w ) , while its recombinant PHB-4/pJRDEE32d13 was found to be able to bring forth PHA content up to 74 % ( w/w ) when fed with soybean oil ( Kahar et al. , 2004 ) . Palm oil has besides proven to be expeditiously utilized by bacterial cells to synthesise PHA of different monomer composings with the assistance of different precursors ( Lee et al. , 2008 ; Bhubalan et al. , 2008 ) .
The copiousness and renewability of soybean oil and thenar oil in nature allows their use in the mass production of PHA to be feasible ( Loo et al. , 2005 ) . Linoleic acid ; an unsaturated fatso acid, was found to be ill utilized by C. necator, by perchance suppressing the incorporation of the fatty acids into the cells. Kahar and colleagues suggested that palm oil was a much better pick of C beginning for the eating of C. necator for PHA production as palm oil contains lesser linoleic acid ( Kahar et al. , 2004 ) . Furthermore, palm oil as the universe ‘s taking comestible oil in footings of efficiency of production, supply and cost makes it a superior campaigner than the other comestible oils available ( Lam et al. , 2009 ) .
Normally studied sugars such as glucose and saccharose are normally utilized as a C beginning by bacterial cells in the agitation procedure of PHA production ( Tsuge, 2002 ) . There has been several efforts for large-scale production of PHA using these simple sugars as C beginnings for bacterial strains such as C. necator, A. latus, and E. coli ( Chen, 2009 ) . However, even with the comparatively low cost of sugars, bacterial cells fed on these sugars face the disadvantage of obtaining low output when compared to cheaper renewable C beginnings such as works oils ( Akiyama et al. , 2003 ) .
2.7.2 Palm oil
C. necator H16 every bit good as its recombinant strains were found to be able to bring forth PHA copolymers every bit good as terpolymers with different monomer composings with the eating of palm oil merchandises as C beginning with the assistance of precursors ( Lee et al. , 2008 ; Bhubalan et al. , 2008 ) . Crude palm meats oil ( CPKO ) and the rough thenar oil ( CPO ) are the chief merchandises of the oil thenar fruit. Malaysia, being one of the universe ‘s largest manufacturers of palm oil, produced a sum of 2.13 million metric tons of rough palm meat oil in the twelvemonth 2008 ( GOFB, 2009 ) . CPKO is the expeller that is pressed from the meat or the endosperm of the oil thenar fruit ( Lee et al. , 2008 ) . CPKO proves to be an efficient C beginning due to its low production cost, handiness, and the renewability of palm oil because unlike CPO, CPKO is barely utilised in nutrient industries, therefore conveying no inauspicious impact of big graduated table PHA production to handle oil-based nutrient merchandises sustainability ( Bhubalan et al. , 2008 ) . The chief difference between palm meats oil and thenar oil in fundamentally in their fatty acerb composing with palm meats oil incorporating saturated fatty acids while palm oil incorporating unsaturated fatty acids ( Loo et al. , 2005 ) .
2.7.3 Fatty Acids
Fatty acids are able to be utilized by C. necator to bring forth PHA under Nitrogen deficient conditions ( Chakraborty et al. , 2009 ) . The use nevertheless is dependent on the degree of concentration of the fatty acids and pH used, as the cells can digest a certain scope 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 incursion of undissociated lipotropic molecules of fatty acids. This disrupts the gradient of protons every bit good as the energy production and conveyance system affecting the cell membrane every bit good ( Yu et al. , 2002 ) . Lo and colleagues suggested that fatty acids incorporating individual dual bond activates the enzymatic reactions of PHA when several bacteriums strains were fed with glucose as C beginning and fatty acids were added as nutritionary addendums ( Lo et al. , 2005 ) .
2.8 Pathway inhibitor
Acrylic acid is known to be an inhibitor of the fatty acerb B-oxidation tract as it prevents the action of 3-ketoacyl-CoA thiolase that frees acetyl-CoA from 3-ketoacyl-CoA. This suppression leads to the build-up of fatty acerb B-oxidation intermediates ( Qi et al. , 1998 ) . The presence of acrylic acid during cultivation of PHA bring forthing cells together with C beginnings was found to change the monomer composing of co-polymer every bit good as the sum of polymer formed ( Steinbuchel et al. , 1998 ) . Acrylic acerb inhibits 3-ketoacyl-CoA thiolase action that catalyzes the concluding measure in B-oxidation that releases acetyl-CoA from 3-ketoacyl-CoA ( Qi et al. , 1998 ) .
2.9 Biodegradation of PHA
Due to the deficiency of degradability in conventional plastics that contribute to the high sums of solid waste, the biodegradability of PHA offers the solution that drives the uninterrupted surveies done on PHA production.
PHA has been found to be able to degrade in microbic active environments such as dirt ( Mergaert et al. , 1993 ) , lake H2O, and marine H2O ( Ohura et al. , 1999 ) and even in sewerage sludge ( Lee and Choi, 1999 ) . This happening is due to PHA degrading enzymes besides known as PHA depolymerase which are secreted by micro-organisms to hydrolyse non-water-soluble PHA into water-soluble signifiers as foods which can be utilized by these micro-organisms ( Sudesh et al. , 2000 ) . PHA depolymerase has been found to be a construction consisting spheres including catalytic sphere and a substrate-binding sphere to adhere the non-water-soluble PHA stuffs to the enzyme, where both spheres are bonded by a linker sphere ( Numata et al. , 2009 ) .
PHA biodegradation are influenced by factors such as the type of environment they are being degraded in, the microbic activity nowadays in the environment, every bit good as the belongingss of the PHA stuff ( Khanna and Srivastava, 2005 ; Lee and Choi, 1999 ) . The physical and chemical belongings of PHA stuff besides contribute to their debasement consequence such as molecular weight ( Mw ) , runing temperature ( Tm ) , crystallinity and snap ( Tokiwa and Calabia, 2004 ) . Li and colleagues suggested that enzymatic debasement of PHA is based on the length of side concatenation of PHA, which implies that the longer side ironss 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 debasement capableness 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 alone features of PHA let it to be utilized in a broad scope of applications. PHA application involves industries such as the packaging, medical, energy, stuff, fabric, all right chemical every bit good as bulk chemical industries ( Chen, 2009 ) .
The packaging industry which to a great extent depends on plastics as packaging stuff allows PHA to be marketed as a biodegradable plastic ( Madison and Huisman, 1999 ) . These attempts are aimed to cut down the solid waste stuffs generated from packaging. PHA produced can besides be diversified to organize coveted features such as through use of bacterial strains, feeding substrate every bit good as the use of inhibitors and precursors in certain phases of growing ( Steinbuchel, 2001 ) . Furthermore, new plastic belongingss can be produced by assorted composing mixtures of PHB and other polymers to change the physical belongingss and crystallinity of the plastic produced to accomplish assorted belongingss of plastic for a wider scope of packaging applications ( Zinn et al. , 2001 ) .
The chief application of PHA in the medical industry involves its use as a drug bearer and as medical implant stuff in tissue technology ( Chen, 2009 ) . PHA features emphasized for medical implant biomaterials include compatibility with cells, ability to back up cell growing, proliferate merely with cell adhesion for normal cell growing, conducts proper cell organisation and the ability to degrade one time no longer in usage ( Williams et al. , 1999 ; Zinn et al. , 2001 ) . Current stuffs being used as implant stuff such as Si are suspected to be able to be malignant and may do malignant neoplastic disease or bring forth toxic compounds ( Zinn et al. , 2001 ) . PHAs normally studied for the intent of bio-material medical implant include P ( 3HB ) , P ( 3HB-co-4HB ) , P ( 4HB ) , and P ( 3HO ) ( Chen, 2009 ) . A popular illustration is P ( 4HB ) , which is the stuff for merchandises 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 bearers involves the general mechanism that utilizes microspheres of SCL-PHA to let go of the drugs quickly with 90 per cent of release within the clip frame of 24 hours ( Zinn et al. , 2001 ) . PHAs used for this intent are manufactured to incorporate increased porousness every bit good as increased surface country to heighten degradability every bit good 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 atom size used every bit good as the type of drug loaded ( Pouton and Akhtar, 1996 ; Zinn et al. , 2001 ) . Besides biodegradability, biocompatibility of PHB every bit good as P ( 3HB-co-3HV ) co-polymers is besides a chief concern in drug bringing as they influence polymer debasement every bit good as the mode tissues respond to the foreign polymers introduced ( Pouton and Akhtar, 1996 ) .
Another popular field of PHA application is besides the energy industry which is the production of PHA-based biofuels. PHB is able to be acerb hydrolyzed to organize hydroxyalkanoate methyl esters such as ( R ) -3-hydroxybutyrate methyl ester ( 3HBME ) and medium concatenation length hydroxyalkanoate methyl ester ( 3HAME ) that are combustible ( Chen, 2009 ) . Blended fuels such as 3HAME-gasoline and 3HAME-diesel contribute better every bit fuels as they are unable to bring forth higher burning heat as pure gasolene or Diesel ( Chen, 2009 ) . This opens up a new avenue for PHA applications in the biofuel production.
2.11 Economicss of PHA production
The development of engineering has increased the use of natural resources available on this planet. Due to this, the depletion of natural resources such as fossil fuels are going a chief concern across the Earth. One such illustration is with the use of conventional plastics which are non readily degraded in the natural environment, therefore going an environmental pollutant. Recognizing the depletion of crude oil based merchandises from the crude oil crisis in the 1970s ( Sudesh and Iwata, 2008 ) , PHA has gained involvements of many, as a possible natural option for this intent. Commercialization of PHA is by and large hindered due to its high cost, US $ 16 per Kg as produced by C. necator ( Lee, 1996 ) which is 18 times higher every bit compared to its polypropylene opposite number ( Reddy et al. , 2003 ) . In order to bring forth PHA at a commercially feasible monetary value, which is around US $ 4 per Kg ( Lee, 1996 ) , there are several parametric quantities that have to be taken into consideration. These include cost of substrate, efficient runing procedure, high transition efficiency from substrate to give every bit good as methods in cut downing downstream recovery cost ( Reddy et al. , 2003 ) . Familial use of cistrons for heterologic and homologous looks every bit good as technology high production strains can besides be considered to better the output every bit good as alter the metabolic procedures to obtain desirable send merchandises ( Steinbuchel, 2001 ; Reddy et al. , 2003 ) . Further surveies in these countries can significantly cut down the production cost of PHA enabling them to be commercially feasible, in replacing of the current petrochemical-based plastic.