KINETIC MODELING AND EXPERIMENTAL STUDIES ON PRODUCTION OF BIOPOLYMERS USING MICROORGANISMS

Abstract

For the last 50 years, plastics derived from petroleum have been used extensively, because of their versatility, outstanding technical properties and relatively low price. It is well understood that fossil fuel-based synthetic plastics have high environmental impact and creates land-filing problems due to their non biodegradable nature. Biologically derived thermoplastics namely, polylactic acid (PLA) and polyhydroxyalkanoates (PHAs) represent potentially sustainable replacement to synthetic thermoplastics. Polylactic acid is produced via polymerization of lactic acid. Lactic acid can be produced by fermentation of sugar containing substrates. While, polyhydroxyalkanoates can be produced intracellular^ in a variety of bacteria cultivated on various sugar containing raw materials. Few of the hindrances in the successful commercialization of these biopolymers are high cost and poor productivities as compared to petroleum based plastics. Several factors contributing to higher manufacturing cost and poor productivities are use of pure and expensive substrates, insufficient process modelling and optimization studies etc. In this work, a kinetic model has been developed for the batch fermentation of sugarcane molasses for lactic acid production by Enterococcus faecalis RKY1 strain. Parameters for the model have been determined based on the experimental data available in the literature using an evolutionary optimization technique, genetic algorithm. The values of important kinetic parameters obtained are maximum specific growth rate (//max), 1-6 h*1; growth associated constant for lactic acid production (or), 0.26 g/g; substrate inhibition constant for growth of biomass (Kix), 167 g/l; substrate inhibition constant for substrate consumption and for lactic acid production (Kss), 303.17 g/l; product inhibition constants on biomass growth (Kpx), 17.074 g/l; product inhibition constants for sugar consumption (Kps), 29.1664 g/l; product inhibition constants for lactic acid production (Kpp), 29.1664 g/l; product inhibition constants on biomass growth (Kpx), 17.074 g/l; maximum specific lactic acid production rate (c/p,max), 3 g/(gh); maximum specific sugar i utilization rate (gs,max), 3.33 g/(gh); death rate coefficient (/cd), 0.0032 h"1. It has been observed that the growth of biomass and lactic acid production are affected by lactic acid inhibition. However, effects of substrate limitation and substrate inhibition have been found to be relatively small. The simulation results obtained are closely matching with experimental values. Effect of various operating parameters like pH on kinetic parameters was also studied. It has been found that the growth of biomass and lactic acid production are strongly influenced by extreme values of pH. Optimum pH is found to be 7. A kinetic model has also been developed for PHB production process based on batch fermentation of sugar using Alcaligenes latus strain. Parameters of the kinetic model have been determined using genetic algorithm as described for kinetic modelling of lactic acid production process. The values of important kinetic parameters are maximum specific growth rate (jjm), 0.15 h"1, saturation constant for nitrogen (Ksi), 0.021 g/l; saturation constant for sugar (KS2), 2 g/l; inhibition parameter value for ammonium ion concentration (Smi), 0.726 g/l, inhibition parameter value for sugar concentration (Sm2) 27.9 g/l; growth associated constant for PHB production (K2), 0.2806 g/g; non-growth associated constant for PHB production {K-j), 0.5548 g/(g h); product inhibition constant (Kp), 0.8387 l/g etc. It has been observed that the growth of biomass is strongly dependent on substrate inhibition, while the effect of substrate limitation is found to be relatively small. The PHB accumulation is found to be growth associated as well as non-growth associated. When compared with batch experimental data, the model provides good predictions for growth of biomass, sugar consumption and PHB production with few exceptions. The kinetic model was also validated against fed batch culture experimental data indicates that the developed model can be implemented for optimization, design and model-predictive control studies. The experimental production of poly(3-hydroxybutyrate) by A. latus bacteria has been studied using sugarcane molasses as a cheap renewable raw material. Shake-flask experiments were carried out with molasses as the sole source of nutrients to find out PHB producing capacity of molasses. Three levels of concentration of molasses studied are 4.85%, 6.3% and 8.2%, out of which molasses concentration of 6.3% gave best results with 8.6 g/l dry cell weight concentration, 58.2% PHB content and PHB productivity of 0.133 g/(lh). This shows that sugarcane molasses as the fermentation medium could fulfil the nutrients requirement for biomass growth and PHB production to some extent. Effects of supplementation of additives like peptone, yeast extract, beef extract and oleic acid to molasses (6.3%) on PHB production were also evaluated. 0.2% yeast extract gave the best results with 9.75 g/l dry cell weight concentration and 63.5% PHB content. However, oleic acid affected PHB production adversely. Addition of peptone resulted in the decrease of cell dry weight to 7.94 g/l and increase in PHB production to 64%. Beef extract could increase dry cell weight concentration to 9.25 g/l with negligible change in PHB content. In order to achieve higher PHB production, inorganic nutrients were added in varying compositions to molasses (6.3%). Significant improvement in PHB production resulted with the addition of phosphates to molasses. It is observed that molasses alone could fulfil nitrogen requirement for cell growth and PHB production without addition of any external nitrogen source. Highest PHB content of 72% has been obtained with supplementation of 0.5 g/l KH2P04 and 8 g/l Na2HP04-12H20 to molasses. Addition of other nutrients with phosphates resulted in PHB productivity of 0.212 g/(lh) and PHB production yield on sugar (yP/S2) of 0.342. Thus, it is clearly demonstrated that A. latus MTCC 2309 strain can produce good yield of PHB using sugarcane molasses as a substrate. Kinetic modelling of experimental results obtained for PHB production using molasses has been attempted but could not fit an unstructured kinetic model to the batch kinetic data with a reasonable error. Therefore, some more logical modelling studies are required for modelling of PHB production on complex substrate. Characterization of the extracted polymer from A. latus cultivated on molasses has been done using FTIR, NMR and thermogravimetry analysis. It is found from characterization that A. latus bacteria accumulates polymer exclusively in the form of PHB. Properties of obtained PHB are similar to the properties of PHB reported in the literature. In summary, kinetic models for microbial production of lactic acid and PHB have been developed. These models can be useful for optimization and modelpredicted control studies leading to improvement in the process performance. iii Experimental studies were carried out on the production of PHB using sugarcane molasses as the renewable agro-industrial raw material. PHB production process based on sugarcane molasses can be scaled up and have good potential for commercialization by coupling PHB production process with any cane-sugar producing industry.