Proteolytic Bacillus sp. Isolation and Identification from Tannery Alkaline Baths

The application of microbial alkaline proteases holds significant potential for eco-sustainable industrial processes by reducing chemical usage and lowering the costs of effluent treatment. In the search for novel proteases with industrial relevance, several microbial strains were isolated from alkaline baths of the Portuguese tannery agroindustry. The most promising protease-producing strains were selected for identification and further study. Two isolates demonstrated the highest proteolytic activity, reaching 0.51 ± 0.01 U mL^-1 and 0.70 ± 0.01 U mL^-1 after 7.5 h of submerged cultivation in nutrient broth. Based on API biochemical tests, molecular biology techniques, and GC-FAME analysis of membrane lipids, the isolates were identified as <i>Bacillus subtilis</i> and incorporated into INIAV's collection of industrial microbial cultures as <i>B. subtilis</i> CCMI 1253 (BMR2) and <i>B. subtilis</i> CCMI 1254 (BMR1). The most promising protease producer, <i>B. subtilis</i> CCMI 1253 (BMR2), exhibited a maximum specific growth rate of 0.88 ± 0.10 h^-1. The proteases produced exhibited good extracellular proteolytic activity, with adaptability to industrial conditions, indicating their suitability for agroindustry applications such as leather making, detergent formulations and the treatment of effluents and protein residues. The results support the potential of microbial proteases as valuable tools in the bioeconomy and green chemistry.