PHYTASE PRODUCTION BY SOLID-STATE FERMENTATION AND BIOREACTOR DESIGN

Abstract

Solid-State Fermentation (SSF) is the process of cultivation of microorganisms on moist solid substrates or inert carriers that take place in the absence or near absence of a free aqueous phase. Use of solid organic wastes for the production of large quantity of biologically active secondary metabolites, single cell proteins, enzymes, industrial chemicals, biofuel, food, phenolics, feed, and pharmaceutical products has made SSF technology as an attractive alternative to submerged fermentation (SMF). Over the last few decades, the technique with its broad application and operational advantage over SMF has led to improvement in bioreactor design, operation, and scale-up strategies. However, the true industrial potential of SSF technology has not been fully realized which is due to the lack of suitable bioreactors designs which can operate aseptically at high substrate bed loading, facilitate efficient heat transfer and mitigate heterogeneity of heat and mass. In this thesis, design and the operation of a novel, intermittently mixed, modular in nature, SSF bioreactor system is proposed. The bioreactor performance was experimentally validated with a heat transfer design equation during the production of phytase, by Rhizopus oryzae MTCC 1987, using wheat bran and linseed oil cake (1:1) as the main substrate. Phytase is an important enzyme in the animal feed industry. It catalyzes the hydrolysis of phytate; an anti-nutrient compound present in cereal and grains, that forms complexes with phosphorus and other nutrients, thereby, making them unavailable for absorption. Phytase produced by R. oryzae in the present study is thermo-tolerant and acid stable. This is highly preferred for the enzyme to withstand the temperature of the pelleting process and to retain activity in the acidic environment of organism‘s digestive tract. The Bioreactor system is modular in nature wherein multiple modules may be stacked vertically around a central pipe. The central pipe not only facilitates the supply of conditioned air into the module but also acts as the shaft of the mixing apparatus. The bioreactor operates at high substrate bed loading (59.2%, v/v) while providing strict aseptic condition; such that all the fermentation operations can be performed in a single module in a highly contained fashion. 6 Intense heat buildup and heterogeneity of heat and mass, across the substrate bed, constitute major challenges for scale-up of SSF process. To address these issues, a design equation describing heat transfer in the axial and radial direction was experimentally validated during phytase production in the proposed bioreactor at different operating conditions of inlet air flow rate and relative humidity (RH). The experimentally obtained bed temperature profiles were well predicted by the design equation (error < 5%). An inlet air flow rate of 4 L min-1 at 80% RH was optimum for phytase production. Sensitivity analysis of the system‘s transport and kinetic parameters showed that R. oryzae specific growth rate constant, maximum biomass concentration, and metabolic heat yield, and substrate density and thermal conductivity had a significant effect on critical bed height. Among the parameters, the effect of specific growth rate constant was most profound. Phytase production was examined in a 0.5 L mini-bioreactor system using optimized synthetic media, in submerged fermentation (SMF) conditions. Phytase yield in SMF was 15.9 times less than SSF. Water activity in the substrate bed was optimized by varying RH of the inlet air stream. Desorption Isotherm of the substrate was used to correlate water activity and bed moisture content. A bed moisture content of 0.55 mL g-dry-solid-1 corresponding to bed water activity ≥0.95 was optimum for phytase production. Mixing constitutes a critical design parameter in SSF bioreactor and its effect on heat and water transport, and microbial growth in substrate bed can significantly influence overall productivity. Effects of mixing events were studied on phytase production in the proposed SSF bioreactor. A critical mixing phase was identified, in the absence of which fungal growth led to the onset of heat accumulation and subsequent bed drying. At this critical phase, the tensile strength required to disengage R. oryzae hyphal bonds was experimentally estimated (600 N m-2) and related to the mixing intensity in the bioreactor that gave an optimum working value of 15 rpm. Effect of mixing time on bioreactor performance was also investigated where a 3 min mixing duration, at every six hours, increased the biomass and phytase productivity to 2.2- and 4.5-fold, respectively than packed bed bioreactor (PBR) operation. The proposed bioreactor system with intermittent mixing gave better performance than PBR and tray bioreactor, concerning maximum bed temperature, axial bed temperature and biomass gradient, average bed moisture content, biomass, and phytase productivity. 7 Direct measurement of cell biomass is difficult in an SSF process involving filamentous fungi since the mycelium and the solid substrate are often inseparable. However, respiratory data is rich in information for real-time monitoring of microbial biomass production. In this regard, a correlation was obtained between oxygen uptake rate (OUR) and biomass concentration (X) of Rhizopus oryzae during phytase production in the proposed SSF bioreactor. To obtain the correlation, various models describing sigmoidal growth were tested, namely the logistic, Gompertz, Stannard, and Schnute models. Regression analysis of experimental results, at different operating conditions of inlet air flow rate and relative humidity, suggested that OUR and X were correlated well by the logistic model (R2 > 0.90). To corroborate the use of respiratory data for on-line measurement of metabolic activity, OUR was related to metabolic heat generation rate (Rq), and the logistic model was found to satisfactorily correlate Rq and X as well. The model parameter, YQ/X, when substituted into the heat transfer design equation, along with the values of other parameters and operating variables, gave reliable estimates of bed temperature. The correlations developed in the present study, between respiratory activity and biomass concentration may be extended on to other SSF processes for further validation. Effect of periodic air pressure pulsation (APP) was studied on bed temperature and moisture content, and on biomass and phytase production in a different fermentation setup for SSF. Air pressure amplitude of 60 mBar for 1 min duration, at a frequency of 10 min, during the log phase of R. oryzae increased the cell biomass and phytase productivity by 1.43 and 1.90 times than packed bed operation. Scale-up of phytase production, using design equations, in the proposed SSF bioreactor with APP shall be taken up in future studies.