Nanocellulose isolation using a thermostable endoglucanase-rich cocktail from Myceliophthora thermophila cultivated in a multilayer packed-bed bioreactor

The isolation of nanocellulose materials by means of enzymatic hydrolysis offers advantages in terms of their properties and operational conditions, but there is still a lack of enzymatic cocktails specifically designed for this application. The present work investigates the use of a thermostable endoglucanase-rich enzymatic cocktail from Myceliophthora thermophila for nanocellulose production. An initial set of experiments was conducted to determine the optimal conditions for enzyme production under solid-state cultivation in small-scale polypropylene plastic bags. A full factorial experimental design was used as a statistical tool to evaluate the effects of the solid substrate and moisture content on the production of endoglucanase and beta-glucosidase. Solid-state cultivations in a multilayer packed-bed bioreactor were then carried out under the selected conditions to obtain a larger volume of an enzymatic extract with high endoglucanase selectivity, given by the ratio of endoglucanase (203 +/- 6 U/g-substrate) to beta-glucosidase activity (1.6 +/- 0.1 U/g-substrate). The enzymatic hydrolysis of eucalyptus cellulose pulp at different temperatures, with this endoglucanase-rich cocktail at a loading of 10 mg-protein/g-pulp, resulted in higher glucose release (11 +/- 2 g-glucose/L) at 60 degrees C, with 61 +/- 9% cellulose conversion and 16.2% nanocellulose yield. The isolated nanomaterial with average diameter of 36.65 nm and length of 156.63 nm, presented a crystallinity index of 77.4% and thermal stability temperature of 316 degrees C. These results show the potential to isolate nanocellulose using a thermostable endoglucanase-rich enzymatic cocktail produced locally by solid-state cultivation in a packed-bed bioreactor, in accordance to the biorefinery concept.