STUDIES ON THE KINETICS OF GADEN TYPE I FERMENTATION USING IMMOBILIZED WHOLE CELLS IN DIFFERENT BIOREACTORS

Abstract

There is an increasing interest in the practical applications of immobilized microbial cell systems for the production of biochemicals. The emphasis on the work in the immobilization of enzymes during the last two and half decades has resulted in the development of new immobilization techniques, many of which, are equally applicable to cells. This has provided an impetus to research activities in the area of immobilized whole cells with a view to improve the reactor productivity and to facilitate the repeated usage of cells while avoiding wash out. Intraparticle diffusion limitations are very important while working with immobilized whole cell systems. Considerable efforts have been made by researchers to use the theory of reaction and diffusion in porous media to immobilized enzymes and whole cell systems. Large number of model equations using different reaction rate functions and boundary conditions for different matrix shapes are available in literature. However as pointed out by Karel et al. (1985) a detailed experimental verification of the theory of reaction coupled with diffusion is not available in literature. Moreover, the kinetic parameters are determined under free cell conditions which are subsequently used in the studies of immobilized cell systems. Present study has been undertaken to develop unstructured models for three bioreactor configurations viz. batch, continuously stirred tank and column flow using immobilized whole iii cells using inhibition-free substrate-limiting Monod kinetics for the Gaden type I fermentations. Based on the model equations developed, experimental methodologies for the determination of kinetic parameters and effectiveness factor have been devised. Model equations have been developed in which a new effectiveness factor concept has been introduced and th e reaction contribution in liquid space as well as immobilization matrix have been included for batch, continuously stirred tank and column flow bioreactors. For the batch reactors, separate models have been developed for the exponential growth phase and the steady state growth phase. The resultant equations for immobilized whole cell systems could be reduced to equation for free cells as derived by Gates and Marlar (1968). These equations have been used to formulate experimental methodology the determination of kinetic parameters i.e. maximum specific growth rate, pmax and Monod constant, Km and the effectiveness factor, n from batch cultures of immobilized whole cells for exponential and steady state growth phases. Steady state growth phase modelling has been carried out for CSTR and used for developing experimental methodology for the determination of kinetic parameters and effectiveness factor. The expression for the dilution rate for maximum productivity, Dmax ' for a CSTF* has also been developed. Immobilized cell packed column flow reactor has also been modelled incorporating mixing effects. This generalized equation is reducible to free cell system equation as studied by IV Chen et al. (1972) and Todt et al. (1977). Mixed collocation methods as applied earlier by Fan et al. (1971) and Chen et al. (1972) to chemical and biochemical reactors have been employed for the solution of the nonlinear differential equation having split type boundary conditions. An experimental method for the determination of kinetic parameters and effectiveness factor from column reactors packed with immobilized cells has also been developed based on collocation methods. In order to test and verify the validity of the developed models, ethanol fermentation was selected as the suitable example for the Gaden type I fermentations. Saccharomyces cerevisiae cells were immobilized in calcium alginate and growing synthetic medium was employed throughout the experimental studies. For batch reactors the growth pattern of Saccharomyces cerevisiae cells immobilized in calcium alginate matrix as represented by cell loading, was investigated. Two distinct phases of growth-exponential and steady state have been observed as has also been reported by Wada et al. (1980). The theoretical models have been used to illustrate the determination of kinetic parameters i.e. umax and Km and effectiveness factor, n from batch reactor containing immobilized yeast cells. The effect of initial cell loading/concentration in gel beads, initial substrate concentration and gel bead size on kinetic parameters and effectiveness factor has also been investigated in detail. In the range of parametric values, cell loading , initial substrate concentration and gel bead size have no effect on kinetic parameters. However, the effectiveness factor showed strong functional dependence on these parameters^ With the increase in initial cell loading/concentration and gel bead size, effectiveness factor decreased parabolically. However with the increase in initial substrate concentration, the effectiveness factor increased. The kinetic parameters as determined from Gates and Marlar (1968) method for free cell systems have been compared with the values obtained for these parameters using the present methodology. The results show very good agreement. Experiments were also conducted in a CSTR to study the effect of inlet substrate concentration and gel bead size on the effectiveness factor, kinetic parameters and ethanol productivity. Dilution rates at which maximum ethanol productivity were found to occur are compared with the theoretically predicted values. The effect of inlet substrate concentration and gel bead diameter on kinetic parameters and effectiveness factor is qualitatively similar to those obtained for batch reactor experiments. For the column flow reactor using immobilized cells, parametric sensitivity with respect to Bodenstein number,, Monod constant, effective immobilized cell concentration and Damkohler number has been investigated. The Bodenstein numbers for the column flow reactor were determined by tracer technique using bovine albumin as a tracer. The effect of inlet substrate concentration and gel bead diameter on effectiveness factor and kinetic parameters has been experimentally investigated. The experimental data of Melick et al. (1987) have also been used to test the proposed model. It has been found that the proposed model represents their experimental data within ± 11% error in vi comparison to their own model which gives upto ± 63 % variations of predicted values with the data. The effectiveness factors as obtained from the present investigation using three types of reactors have been compared using Andrews' (1988) structured model. The effect of cell loading/concentration, initial/inlet substrate concentration and gel bead size on effectiveness factor has been found to follow similar pattern as predicted by Andrews (1988) model. However, the Andrews model predictions for n are higher than those actually determined using the methodology developed in the present studies. The higher values of n as predicted from Andrews model stems from the fact that Andrews model does not incorporate the effect of cell inactivation in the immobilization process itself and the matrix specificity. On the basis of the proposed theoretical models and experimental investigations it is concluded that the quantitative assessment of intraparticle mass transfer resistance in the immobilized whole cell matrices for Gaden type I fermentations could be made using batch, continuously stirred tank or column flow bioreactors. Additionally the kinetic parameters could also be determined from the presented methodolog ies.