Valorization of Agro-Industrial By-Products for Enhanced Amylase Production by Bacillu tropicus: A Sustainable Biotechnological Approach

Abstract

The increasing need for industrial enzymes, coupled with the growing need for sustainable waste management, has made it necessary to explore cost-effective bioprocessing strategies. In this study the possibility of using different agro-industrial wastes as alternative substrates for the production of amylase using a novel bacterial isolate from the sugarcane rhizosphere was explored. Bacterial strains were first tested on starch agar medium and the presence of amylolytic activity was verified by flooding with iodine. The isolate with the best morphology, biochemical and molecular characteristics was selected as the most promising. Gram staining showed that the strain was Gram-positive, rod-shaped, and 16S rRNA gene sequencing with universal primers (27F and 1492R) followed by Sanger sequencing and phylogenetic analysis confirmed the strain as Bacillu tropicus (Accession No. OQ674805). Wheat bran, rice polish, rice husk and soluble starch were used as substrates for amylase production by submerged fermentation. The optimization of the process was performed with the one factor at a time method. The amylase activity was the highest (299.068 IU/min/mL) when the substrate used was rice polish after incubation for 72 hours at 35°C and pH 7. Peptone (146.289 IU/min/mL) and urea (142.049 IU/min/mL) were the two nitrogen sources that greatly supported the production of enzymes. It is interesting to note that the addition of MnCl2 caused a dramatic increase in amylase activity to 508.26 IU/min/mL, indicating a metal-dependent catalytic mechanism. The process is also industrially feasible as 345.982 IU/min/mL was obtained after 72 hours of fermentation in a 2 L stirred bioreactor. The enzyme was found to be most active at moderately thermophilic conditions and neutral pH, which are desirable for several industrial applications such as food processing, detergents, textiles, and biofuels. This study proposes B. tropicus as a potential microbial producer of amylase with renewable agro-industrial residues, which is also cost-effective and sustainable, in line with circular bioeconomy principles.