Isolation and characterization of proteolytic bacteria strains from poultry waste-contaminated soil

Soil contaminated with poultry waste may harbor probiotic bacteria with beneficial traits. This study aims to isolate, characterize, and evaluate proteolytic probiotic bacteria from such soil for potential use in health and biotechnology applications. Soil samples were aseptically collected from five spots at a farmland in Enugu State and serially diluted using sterile normal saline. Isolates were cultured and screened for proteolytic activity using casein agar. Biochemical characterization and Gram staining were performed. Genomic DNA was extracted with ZR Miniprep™ kit, and 16S rRNA and ITS genes were amplified via PCR, visualized with gel electrophoresis, and sequenced. Six bacterial isolates were successfully obtained and identified morphologically and biochemically as members of the Bacillus and LactoBacillus/LactiplantiBacillus genera. All isolates were Gram-positive rods. Bacillus strains displayed dry, rough, opaque colonies, while LactoBacillus and LactiplantiBacillus formed moist, smooth, round colonies. Biochemical tests revealed that Bacillus isolates were catalase-, oxidase-, and citrate-positive, whereas LactoBacillus strains were generally catalase- and oxidase-negative. Sugar fermentation tests showed that all isolates fermented glucose, fructose, maltose, lactose, and mannitol, with lactic acid bacteria producing acid and gas, consistent with heterofermentative metabolism. Molecular identification via 16S rRNA confirmed species identity, and gel electrophoresis revealed bands at ~1500 bp. Proteolytic screening demonstrated that Bacillus subtilis strain NBT-15 and LactoBacillus plantarum strain ML05 exhibited the highest casein hydrolysis zones (28 mm), indicating strong protease production. The presence of robust protease-producing strains from both genera underscores the potential of poultry waste soil as a reservoir for industrially significant microorganisms. These isolates hold promise for applications in probiotic formulation, food fermentation, enzyme production, and biotechnological innovation.