PRODUCTION OF POLYHYDROXYALKANOATES BY HALOPHILIC BACTERIA

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable plastic synthesized by a number of microbes as an intracellular carbon and energy reserves in response to excess carbon source and limitation of nitrogen and phosphorus. Polyhydroxybutyrate (PHB) is one of the major group of PHA, which has similar thermal and mechanical properties as petrochemical derived plastic such as polyethylene and polypropylene. It has been reported that more than 300 different microbes can produce PHB and its copolymer such as P(3HB-Co-3HV) & P(3HB-Co-4HB), by utilizing the different substrates. The leading microbes producing PHAs are Ralstonia eutropha, Bacillus megaterium, Pseudomonas oleovorans, Pseudomonas putida, Zobellella denitrificans, Cupriavidus necator, Halomonas compisalis, recombinant E. coli. Most researchers have concentrated on enhancing the thermal and mechanical properties of PHB by incorporating the copolymer such as 3HV, 4HB & 3HHx. Today, approximately 150 different monomer constituent of PHA have been reported. PHA and its copolymer are extensively used in packaging, tissue engineering, and drug delivery. The major drawback that hampers the large-scale production is the production cost. Various studies suggested that cost of carbon source is major contributor in PHA prodution. In this context, a low-cost by-product from agriculture and industry such as bagasse, molasses, cheese whey and crude glycerol is suggested as a promising carbon source to cut down the product cost. Different fermentation strategies have also been developed to improve the production yield, but the cost of PHA is still high as compared to conventional plastic. The present work is focused on screening of halophilic bacterial isolates, which can utilize renewable carbon source such as glycerol (crude glycerol), a by-product of the biodiesel industry and optimization of the fermentation medium for improving the PHA production. In this work eight halophilic isolates from Sambhar Lake, Rajasthan and two procured strain MCC 2171 & MCC 2172 from Microbial Collection Center (MCC), Pune, India were used for PHA production. On the basis of staining result, four out of eight halophilic isolates and two Halomonas sp. were found to be capable of PHA production. Next level of screening involved PHA quantification by crotonic acid assay and GC-MS analysis of PHA. The halophilic bacteria were first grown on PHA producing medium (HSM medium) supplemented with glucose (1%) as a carbon source, cell OD at 600 nm was recorded at regular interval and at stationary phase cell pellet was collected and used for PHA analysis. The result concluded halophilic isolates KBL, GSL3, GA, and KBD were capable to produce PHA copolymer P(3HB-co-3HV) with polymer yield of 0.84%, 0.51%, 0.36 & 0.87% respectively. The highest fraction of copolymer, 3HV (28 %) was recorded in ii halophilic isolate KBL. Halophilic bacteria along with Halomonas sp. MCC2171 & MCC2172 were grown on modified HSM medium, glycerol (2%) was supplemented in the medium. To check the possibilities of copolymer 4-methyl valeric acid was added as precursor. The result reveled that halophilic bacteria KBL, GSL3, GA, and KBD were capable to accumulate the copolymer P(3HBco- 3HV-co-3H4MV), a rarely produced PHA copolymer. The Halomonas sp. (MCC2171 & MCC2172) were found to accumulate PHB. The study concludes with the selection of KBL for PHA copolymer P(3HB-co-3HV) production and Halomonas sp. MCC2171, MCC2172 for PHB production. Different carbon sources such as sucrose, glycerol, molasses and vegetable oil along other medium components were optimized for production of PHA copolymer by halophilic bacteria KBL. On the basis of PHA yield of 1.57 % glycerol was suggested the best carbon source for PHA production. Further the effect of media components along with temperature and pH were studied for optimum PHA yield, the result concludes that at 3.0 M NaCl and 20 g/L MgSO4, 45 ºC of incubation temperature and 9.0 pH were desirable for optimum PHA yield and copolymer composition for halophilic bacteria KBL. The PHA yield of (2.63%) was further increased using Definitive Screening Design, with medium composition (in g/L) 80 glycerol, 4.5 yeast extract, 20 MgSO4 and 4.0 M NaCl. This low PHA yield is not up to the mark of commercialization. In next step of work Halomonas sp. MCC2171 was used for PHB production, the effect of media components along with temperature and pH were studied for optimizing the PHB production. The culture was cultivated on PM media, a nutrient-limited media reported in the literature, temperature and pH were set at 300C and 8.5 respectively. Glycerol was used as a carbon source, PHB was quantified by the GC-MS method. In recent year, the valorization of crude glycerol, a by-product of biodiesel industry has created significant interest. For selecting the crude glycerol as a carbon source in PHB production medium, Halomonas sp. MCC2171 was grown at high concentration of glycerol (50-150 g/L). Experiment was designed in such a way that simultaneous effects of glycerol and NaCl were considered for PHB production. It was found that at higher glycerol concentration, biomass of 11.12 g/L and PHB yield 55.6 % were recorded with 100 g/L glycerol and 45 g/L NaCl. In the next step, central composite design (CCD) was used to study the interaction between NaCl and glycerol and find optimum concentrations. The glycerol concentration and NaCl concentration range were selected from the previous study and varied in the range of 60-125 g/L and 20-40 g/L respectively. To keep the C/N ratio constant (90), the yeast extract concentration was varied based on glycerol iii concentration. From the result, it was observed that maximum biomass of 20.4 g/L was achieved at 92.5 g/L glycerol with 30 g/L of NaCl. Similarly, PHB production was also maximum (16.0 g/l) at glycerol and NaCl concentrations of 92.5 g/L and 30 g/L, respectively. CCD model was validated by five independent experiments with combination of glycerol concentration and NaCl concentration of 78/29, 82/29, 65/25, 92.5/20 &125/30, the experimental value was found close to theoretically predicted value. Optimum concentrations of glycerol and NaCl were numerically determined and found to be 81 g/L and 29.3 g/l. In next step, Box Behnken Design (BBD), a response surface methodology (RSM) was used to study the interaction between NaCl, yeast extract and glycerol. The result concluded the effect of yeast extract on biomass as well as PHB production. Further optimization of yeast extract concentration for PHB production was carried out by varying the concentration of yeast extract from 3 g/L to 18 g/L by keeping the glycerol and NaCl concentrations was fixed at 82±1 g/Land 30 g/l. Maximum biomass concentration of 53.92 g/L and PHB production of 32.5 g/L were achieved at 6 g/L of yeast extract concentration. The PHB production in controlled parameter was studied using a 3L bench top fermenter (Bioflo 110). Effect of pH & aeration were studied on 82±1 g/L glycerol, 29 g/L NaCl & 6.0 g/L yeast extract concentration. The result obtained in bioreactor indicates that maximum PHB production was achieved in batch II, in surface aeration condition as compare to the batch I, where aeration rate was higher. The specific growth rate of 0.65 h-1 and productivity of 0.27 g/L/h were achieved in uncontrolled condition (batch II), this suggest that low oxygen concentration favor growth as well as PHB production. The polymer was extracted and purified, and the characterization was done by FTIR, NMR (1H and 13C), SEM, TGA/DTA/DSC and XRD. The result was compared with data reported in the literature and the result was found in well agreement with the reported literature.