Optimized Production Of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) From Cupriavidus Sp. Usmaa2-4 And Its Characterization
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Date
2015-10
Authors
KANNUSAMY, SHANTINI
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Abstract
Polyhydroxyalkanoate (PHA) is a biocompatible and biodegradable thermoplastic that has the potential to replace non-renewable resources. It is produced by microorganisms as intracellular energy and reserve material. P(3HB) homopolymer is stiff while copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was found flexible depending on the 3HV monomer composition. This study explores the potential of a local isolate, Cupriavidus sp. USMAA2-4, a Gram-negative bacterium to utilize the combination of oleic acid and 1-pentanol for the production of copolymer P(3HB-co-3HV). This local strain was found to have the potential for the production of various copolymers and the combination of this carbon source has never been reported. It was found that Cupriavidus sp. USMAA2-4 could produce 58 wt% of P(3HB-co-3HV) with 8 mol% 3-hydroxyvalerate (3HV) monomer by using 0.50 wt% C oleic acid and 0.06 wt% C 1-pentanol. The P(3HB-co-3HV) concentration obtained was 4.5 g/L with a growth of 3.4 g/L. Enhancement of P(3HB-co-3HV) production and growth were obtained through optimization of culture parameters by using the response surface methodology (RSM) in shake flasks. After optimization, the usage of 1-pentanol reduces 33%. The PHA content increased by 10.3% to 64 wt% and the PHA concentration increased 42% to 6.4 g/L. The 3HV monomer composition increased to 10 mol% with the growth of 3.5 g/L. Enhancement was also obtained through bioreactor optimization in batch fermentation using RSM whereby a significantly
higher growth of 9.8 g/L which represented a 180% increment and PHA concentration of 8.7 g/L which represented 36% increment. The productivity of the copolymer produced in the bioreactor increased by two fold to 0.16 g/L/h as compared to shake flask which was at 0.088 g/L/h. The productivity of PHA was improved by varying the frequency of feeding through the fed-batch culture. The production of copolymer by this local strain was found to be growth related; therefore method of feeding was used to increase the growth and copolymer production. The highest productivity (0.48 g/L/h) that represented 200% increment was obtained by feeding the carbon source and nitrogen source three times and also by considering the oxygen uptake rate (OUR) and oxygen transfer rate (OTR). A significantly higher P(3HB-co-3HV) concentration of 25.7 g/L and PHA content of 66 wt% were obtained. The 3HV monomer composition obtained was 24 mol% with the growth of 13.3 g/L. The P(3HB-co-3HV) copolymer produced were characterized. In general, the copolymer produced through three times feeding regimen exhibited greater flexibility (Young’s Modulus 187 MPa) and elongation at break (193 %) as compared to the other copolymers. The weight average molecular weight of the copolymer ranged between 233 to 849 kDa. As the frequency of feeding increases, the number of granules also increased which correlated with the polydispersity value obtained. In general, the fermentation strategy obtained in this study from shake flask to bioreactor can be used as a platform for the production of P(3HB-co-3HV) copolymer by the industries as it has resulted in the highest productivity of 0.48 g/L/h.
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Optimized Production Of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) , From Cupriavidus Sp. Usmaa2-4 And Its Characterization