Influence of process parameters on the morphology and enzyme production of Trichoderma reesei

Abstract

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol as it represents a major part of the cost. A research project was initiated in order to understand the intimate relationship that exists between the key process parameters and the morphological characteristics of T. reesei during the course of fermentation and to improve the production of enzymes. Experiments were performed using three types of bioreactors with completely different mixing system: (1) stirred tank bioreactor (STB) with Rushton turbines, (2) reciprocating plate bioreactor (RPB) with six perforated plates, and (3) Couette flow bioreactor (CFB) with outer wall rotating. Fermentations normally followed the protocol that is currently used industrially. It consists of two phases: a growth phase to generate the necessary biomass and the secretion phase where the desired enzymes are produced. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology during the fermentation process. To evaluate the performance of STB and RPB in T. reesei fermentation, series of fed-batch experiments at different agitation speeds were conducted. Due to the limitation in evaluating the effect of shear in conventional bioreactors such as STB, a 5-L Couette flow bioreactor was designed and built to grow the microorganism in a uniform and defined shear conditions. Experiments in CFB were successfully performed in a continuous mode. Oxygen mass transfer coefficient is an important process parameter in fungal fermentation. Therefore, first a review of available techniques to estimate KLa was done. Two new techniques based on neural network, and varying agitation and/or flow rate are presented. Also, two new methods based on image analysis and DNA quantification are developed to estimate the biomass concentration during T. reesei fermentation in the presence of an insoluble substrate. The findings from this study provide new insight into the T. reesei fermentation. Also, the techniques developed can be implemented in large scale industrial bioreactors to better understand and monitor the T. reesei fermentation.