Scale-down approach: tools for the understanding of cellulase production for 2G-bioethanol

Abstract : 2G-ethanol production from lignocellulosic biomass requires transforming this biomass into fermentable sugars before performing alcoholic fermentation. This first step of biotransformation is accomplished by cellulases and represents the major technical and economic challenge for the process development. Cellulases are industrially produced by the filamentous fungus Trichoderma reesei, endowed with a strong secretory capacity. This fungus is strictly aerobic, it is influenced by the shear stress induced by strong agitation rate and its growth induces a viscosity increase as a consequence of the formation of three-dimensional structures of microorganisms (from micrometer to millimeter). These characteristics make the production process scale-up complex as strong links exist between morphological and physiological states of fungus and the process conditions. In this context, this work aims at developing a scale-down approach to better understand the answer of Trichoderma reesei to its culture conditions. Based on a multi-tools combination, this method involves the use of microscopy coupled to image analysis of T. reesei cells and the development of a specific method for viscosity measurement using original agitation devices. Both the growth of the fungus and its enzyme production capacity are taken into account as physiological parameters. They are characterized at the laboratory scale, at mixing time and shear stress values expected at the industrial scale. The cultures are carried out using fed-batch and chemostat modes in order to impose different constraints. The obtained results allow validating the methods at small scale and providing their main advantages and limitations.