Polyhydroxyalkanoates (PHAs), also referred to as bioplastics or biopolyesters have been biosynthesized through metabolic transformation of various carbon sources and composed of (R)-3-hydroxy fatty acids (Allen et al., 2010). These biopolyesters have been produced by many bacteria and some archaea, recombinant bacterial strains and recombinant eukaryotes. In native PHA producing microorganisms, these polyesters have been produced as intracellular water-insoluble inclusions and functioned as carbon storage polymers and energy reserves. Many PHA biopolyesters have also showed interesting properties, such as biodegradability and a wide array of uses, similar to petroleum based commodity plastics (Sudesh et al., 2000). Properties of PHAs have been defined by the number of carbon atoms in the individual monomer units and the physical structure of monomers followed by their incorporation into biopolymer chains by microbial enzymes (Jain et al., 2010).
Though several pathways for PHA biosynthesis have been suggested, but out of them, only three pathways were well established. Pathway I was the most well characterised and studied pathway for the biosynthesis of short chain length PHAs in Cupriavidus necator, while Pathways II and III were reported to utilized by bacteria such as Pseudomonas aeruginosa for biosynthesis of medium chain length PHAs (Tan et al., 2014). Biosynthesis of the most common short chain length PHA, i.e. polyhydroxybutyrate (PHB), has been carried out by three key enzymes: i) Î²-ketothiolase (encoded by gene PhaA), ii) acetoacetyl-CoA reductase (encoded by gene PhaB) and iii) PHA synthase (encoded by gene PhaC) (Jain et al., 2014a). Acetyl coenzyme-A as a staring molecule has been undergone condensation by PhaA and reduced to precursor molecule (R)-3-hydroxybutyryl-CoA by PhaB and finally polymerised into PHA by PhaC. PHA synthase has been found covalently attached to the core of the PHA granule and considered as an important tag for anchoring proteins on the surface of PHAs. In native producers of PHB, phasins (PhaP) has been contained up to 5% of the intracellular proteins and known as the most abundant granule-associated protein (Mezzina et al., 2014). Pathway II and III have been found to utilize intermediates generated from the fatty acid Î²-oxidation pathway, from related and non-related carbon sources. In these pathways, various Î²-oxidation metabolic intermediate compounds such as alkanes, alkenes and alkanoates have been converted into (R)-3-hydroxyacyl-CoA thioesters. Non-related carbon sources like sucrose, glycerol, glucose or gluconate have been catabolized to acetyl coenzyme-A (Rehm, 2007 and Lu et al., 2009). Irrespective to type of PHA biosynthesized, an associated pathway of PHA degradation catalysed by PHA depolymerase, dehydrogenase, dimer hydrolase and 3-hydroxybutyrate was also reported (Sudesh et al., 2000).
Biosynthesis of PHA has well studied over the past two decades and been concluded that the synthesis of PHA biopolymers has been influenced by several factors such as bacterial strain producing the PHA polymer, the carbon source on which microbial cells have been grown, how that carbon has been metabolized in the cells, the types and role of enzymes directly or indirectly involved (Amara et al., 2011). Commercial fermentation in industries also documented that rate of PHA biosynthesis have been known to differ under chemically mild conditions such as temperature, atmospheric pressure, pH and carbon source (Jain et al., 2015). All these observation has triggered to develop the first hypothesis of the present investigation. It was hypothesized that changes in physical environment and nutrition sources have been responsible for evolution of alternative pathways. These alternative pathways have worked as limiting factor either to increase or decrease rate of PHA biosynthesis.
To check the first hypothesis, in the present study, biosynthesis of PHA was studied with different soil microorganisms in the fermentation utilizing different waste carbon sources (such as apple pomace hydrolysate, waste frying oil and agricultural waste) at varying physical parameter such as pH, temperature and agitation. The present study has been justified in two ways: i) to confirm to role of physical parameters and carbon sources in triggering alternative pathways and, ii) to provide a ready data of PHA biosynthesis utilizing different carbon sources as raw material to the industry. Nowadays bioplastics have become very important for the industry and society as an emerging option for the replacement of synthetic plastics because of their eco-friendly nature (Jin et al., 2014). The technical substitution possibility of bioplastics for the replacement of conventional plastics has been estimated more than 85%. Among different bioplastics, PHA has received intensive attention because of a negligible environmental impact and thermoplastic properties (Heinrich et al., 2015).
The conventional plastics being used today were developed almost a century ago. Since their invention, plastics were exclusively derived from non-renewable fossil fuels and the industry was driven by two main factors: cost efficiency and utility (Li et al., 2014). Because of uniquely flexible material properties of plastics that has seen them occupy a huge range of functions, from packing materials to complex biomedical components (Jain et al., 2010). But their durability has raised concerns about their end-of-life disposal (because of their non-degradable properties). Non-degradable plastics have accounted for huge accumulation in our landfills, natural environments and oceans. Problems related to the management of plastic waste on the mother earth have been increasing day-by-day (Sudesh et al., 2000).
Biodegradable plastics were developed to be fully sustainable (Dinjaski et al., 2015). Their development was a responsible and holistic approach that involved a greater emphasis on the renewable sources from which they had been derived and ultimate fate after their complete utilization. Being main substrate for the production of PHAs, carbon sources have known to account major part of raw material cost (Amulya et al., 2015). The present work was an attempt to check not only the role of wastes carbon sources as precursor of alternative pathways in increased/deceased biosynthesis of biopolymers and also to check the feasibility of waste carbon substrate in the replacement of the standard carbon source, i.e. glucose in the fermentation. This approach has helped to over the conundrum of cost-effectiveness, which led to an effective commercial biopolymer production.
It has been well known that protein engineering combined with metabolic pathway manipulation can lead to the synthesis of PHA biopolymers of desired quality (Han et al., 2009). Importantly the genome sequence of Cupriavidus necator (formally known as Ralstonia eutropha) has revealed a number of potential ketothiolases, reductases and homologues for many other genes involved in PHA biosynthesis (Lykidis et al., 2010). This available information has triggered to develop another hypothesis, that the thorough understanding of genes homologues involved in polymer biosynthesis and function of their encoded proteins can provide strategies and clues to optimize the production of tailor-made PHA polymers (Park et al., 2012). To establish the hypothesis different genes homologues (responsible for encoding various enzymes and proteins) involved directly or indirectly in PHA biosynthesis and degradation and their functions were studied. Data available in well-established data base such as NCBI was also cross checked.
Notwithstanding to the presence of all the required characteristics similar to some petroleum-based polymers, PHAs have not escaped from the limitations and drawbacks (Mousavioun, 2011). Both hypotheses have not only brought an opportunity to get many insights about genetic metabolism of biopolymers, but also about their commercial feasibility, durability and ultimate fate of PHA in the environment. Other than the cost, the major drawback for PHA production was its lack of comparable properties, to the extent of adequate commercialization (PeÃ±a et al., 2014). The mechanical properties and biocompatibility of PHA were improved by blending (modifying the surface of PHA). FDA approved, cellulose based blending agents were sensibly employed for a meaningful pursuit of stable and commercially viable bioplastics. Physical & mechanical properties and degradation of produced PHA was checked as per ASTM standard.
To summarize hypothesises, goal of the study has been established and discussed below.
2. STUDY GOAL AND SPECIFIC OBJECTIVES
In the present study, soil bacteria were isolated and tested for their potential of polymer biosynthesis and compared with Cupriavidus necator (bacterial strain used in industry). One of the focuses of work was to investigate the utilization of different wastes material such as apple pomace hydrolysate, waste frying oil and agricultural waste as the alternative carbon sources for the biosynthesis of cost effective bioplastic. Other important aspect investigated was to study genes, encoding different enzymes and proteins involved in the biosynthesis and degradation pathways of biopolymer. The enzymes and proteins studied were Î²-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, PHB synthase, PHA depolymerase, dimer hydrolase and 3-hydroxybutyrate dehydrogenase, phasins and regulatory proteins. The physical & mechanical properties of produced biopolymer were also studied and improved by utilization of blending agents, keeping in view at biodegradability and non-toxicity.
To fulfil the main goals of the study following RDC approved objectives were established:
â¢ Biosynthesis (organisms oriented) of bioplastics.
â¢ Identification, expression of microbial genes and function of the encoded proteins involved in the biosynthesis of bioplastics.
â¢ Testing of samples on three point test for the quality of bioplastics
â¢ Biodegradation pattern of bioplastics
2.1 ADDITIONAL OBJECTIVES
â¢ Testing of the thermal stability
â¢ Testing of the toxicity
Objectives were achieved by;
o Isolation of bacteria and comparison of isolates with known Cupriavidus necator MTCC 1285 strain for production of bioplastics
o Optimization of the waste based medium and fermentation conditions for biopolymer production
o Preparation of the bioplastic film by taking different concentrations of blending agent (ethyl cellulose and cellulose acetate butyrate)
o Study of reputed public database to identify various genes sequences for encoded proteins involved in PHA biosynthesis and degradation pathways in Cupriavidus necator
o To establish similarity using multiple sequence alignment
o To check the motifs in gene sequences to identify expression and functional relations among the encoded proteins
o Homology modelling of proteins encoding genes and their structure analysis
o Testing of tensile strength
o Testing for flexural strength
o Testing of thermal stability
o Testing of biodegradation pattern
o Testing of toxicity
3. NOTEWORTHY CONTRIBUTION IN THE FIELD
In the present study, classical microbiology (such as isolation of microorganisms, characterization, microbial fermentation), chemical processing and genetic information of microorganism involved in biopolymer production have been brought together to decipher the PHA metabolism and commercial opportunities.
The study was significant; (i) to characterize the genes and encoded proteins that have provided useful information for metabolic engineering, (ii) to improve the basic understanding of the fermentation based PHA biosynthesis and related parameters, (iii) to understand properties of biopolymer, (iv) to develop reliable material characteristics through blending of bioplastic and, (v) to support large-scale implementation of renewable materials.
In general, the present work has provided a strategy to increase productivities for industrial-scale operations.