Carboxymethyl Cellulase Production by Extremotolerant Bacteria in Low-Cost Media and Application in Enzymatic Saccharification of Stevia Biomass

Abstract

The aim of this study was to characterize novel cellulase-producing bacteria isolated from desertic environments in Tunisia, and then applied for cellulosic ethanol production. In the first instance, 27 alkali-thermo-halotolerant bacterial strains, belonging to Bacillaceae family, were screened on agar plates containing carboxymethyl cellulose as the sole carbon source. The selected high cellulase producing bacteria were identified as Bacillus mojavensis AG21, Bacillus vallismortis J77, Bacillus sonorensis 7.26 and Bacillus safiensis AJ49. Production of cellulolytic enzymes by selected bacteria was further tested in submerged cultures with Posidonia oceanica leaves, Peganum Harmala biomass, Stevia rebaudiana leaves (SRL), Olive cake, Spent coffee ground and peanut hulls as low-cost cellulosic substrates. Response surface methodology was applied to evaluate the effect of SRL concentration, NaCl concentration, cultivation pH and temperature on carboxymethyl cellulase production and to optimize enzyme yields by selected bacteria. Maximum cellulase production yields of about 1890, 1642, 1516 and 1455 IU/L were recorded for B. mojavensis AG21, B.vallismortis J77, B. sonorensis 7.26 and B. safiensis AJ49, respectively. Production of reducing sugars by hydrolysis of Stevia rebaudiana leaves with bacterial cellulases and commercially fungal cellulases was investigated and compared. The overall reducing sugar yields of B. mojavensis, B.vallismortis, B. sonorensis, B. safiensis, Aspergillus niger and Trichoderma reesei treated- SSR biomasses reached 3620 ± 181, 3040 ± 164, 3656 ± 164, 2654 ± 106, 2646 ± 145 and 2253 ± 123 mg/L respectively. The unique property of cellulase-producing extremotolerant bacteria, proves the potential candidature for current mainstream agricultural cellulosic biomass conversion into fuel ethanol.