Produção de celulases por Aspergillus fumigatus através da fermentação em estado sólido e recuperação e purificação por extração micelar em duas fases aquosas

Abstract

Threats to sustainability stemming from industrial development, energy dependency and the exponential demand for food production have led to the growing interest of alternative and renewable sources of energy such as plant biomass. In this scenario, the viability of ethanol from biomass (second generation ethanol (2G)), which does not generate competition for arable land, such as sugarcane bagasse, coconut fiber and the cashew peduncle. Enzymatic hydrolysis is an important step in the production of 2G ethanol. However, the producing costs of cellulases is still a bottleneck of the process. Thus, the aim of this work was to contribute in the reduction of cellulase production costs using residues from Brazilian industry, e.g. sugarcane bagasse, coconut fiber and cashew peduncle fiber, for the purpose of cellulases production by solid state fermentation (SSF) through an experimental planning using the Aspergillus fumigatus as microorganism. Initially, the three lignocellulosic fibers were characterized and used to measure the potential of cellulase production by SSF. Then, alkaline pre-treatments (NaOH and Ca(OH)2) were applied to the residue that presented cellulolytic activity (CMCase and FPase) and the total protein content. The enzymatic extract produced was used for the cellulose nanocrystals production (NNC). Recovery and purification of the cellulases were performed using the Two Phase Aqueous Micellar System (ATPMS). The characterization results showed the following composition: sugarcane bagasse (39.25% ± 5.49 cellulose, 25.20% ± 1.13 hemicellulose and 18.82% ± 0.01 lignin) , coconut fiber (36.23 ± 0.09 cellulose, 27.79 ± 0.37 hemicellulose and 30.23 ± 0.12 lignin); and cashew peduncle fiber (21.02% ± 0.31 cellulose, 11.50% ± 1.13 hemicellulose and 45.84% ± 1.28 lignin). Under optimized conditions, the activities of FPase (0.64 IU/g) and CMCase (4.28 IU/ g) and total proteins of 0.28 mg / mL were obtained. The CMCase activity was stable at 60 °C showing a relative activity of 70%, whereas FPase showed a stability of 30% at 50 °C. The pretreated of sugarcane bagasse using NaOH had a cellulose content of 56.63% ± 0.10; hemicellulose content of 19.23% ± 1.69 and lignin content of 9.92% ± 1.19. The produced of nanocrystals presented the best shape and size after 48h of hydrolysis, with a circular shape. The use of ATPMS allowed the recovery and the purification of the enzyme with partition coefficient (K) of 9.33 for the enzyme CMCase and of 5.00 for the enzyme FPase both at 60 °C. The purification factor for CMCase was 10.89 and for the FPase of 0.65. In the present study, the solid state fermentation conditions were optimized for the production of cellulases by A. fumigatus, the enzymes were applied in the enzymatic hydrolysis for the generation of cellulose nanocrystals successfully, and the purification technique applied in the cellulases can be considered a promising alternative.