Screening and optimization of fermentation conditions for a high protease-producing strain derived from marine mangrove

To explore microbial resources in mangrove habitats with high protease-producing capacity, mud samples collected from the intertidal zone of Yintan Mangrove in Beihai, Guangxi, China, were used for strain isolation. Protease-producing strains were isolated and screened using the casein plate transparent zone method. Strain identification was carried out based on morphological characteristics, Gram staining, and 16S rRNA gene sequencing. The fermentation process for enzyme production was optimized through single-factor experiments combined with the Box-Behnken response surface methodology. A total of 456 protease-positive strains were isolated from the mud samples, and 20 strains with H/C ratio (hydrolysis zone diameter to colony diameter) > 4.0 were obtained after secondary screening. Among them, strain 298 exhibited the highest enzyme activity in liquid fermentation and was identified by 16S rRNA sequencing as Bacillus aerius . Single-factor experiments identified temperature, initial pH, and dosage of shrimp shell powder as three key factors affecting enzyme activity. The optimal fermentation conditions determined by response surface optimization were: dosage of shrimp shell powder 5.88 g/L, initial pH 8.66, and culture temperature 32.2°C. Under these conditions, the measured enzyme activity reached 639.5 U/mL, which was 57.9% higher than the basal medium under shake-flask conditions (404.75 U/mL). The relative error between the model-predicted value and the measured value was only 1.93%. This strain can directly utilize shrimp shells, an aquatic processing waste, as the fermentation substrate without the need for additional exogenous carbon or nitrogen sources, resulting in low fermentation costs. This study obtained a high-protease-producing strain of Bacillus aerius , providing a candidate strain and preliminary reference for laboratory-scale process development for the development of mangrove microbial resources, valorization of aquatic processing by-products, and industrial production of protease.