Production of alkaline serine protease subtilisin Carlsberg by Bacillus licheniformis on complex medium in a stirred tank reactor

Abstract

Bacillus licheniformis (DSM 641) was cultivated on complex medium in batch and fed-batch operations in a 20-l working volume stirred tank reactor. The medium composition (maltose, glucose, sucrose, fructose, ammonia, phosphate) and O₂ and CO₂ in the off-gas were monitored on-line; pH, pO₂, turbidity, culture fluorescence were monitored in situ; optical density, concentrations of sugars, amino acids, phosphate, proteins, DNA, protease activity and total solids content were monitored off-line. Problems of on-line sampling, cell concentration monitoring, and culture fluorescence measurements and the influence of medium components on the enzyme productivity are discussed. Close relationships between variations of pH, pO₂, O₂ transfer rate and CO₂ production rate on the one hand and cell mass and fluorescence intensity on the other were demonstrated in batch and in fed-batch cultures. Using suitable cultivation conditions, alkaline protease with high volume activity [15300 units (U)/ml] and specific activity (510 U/mg) was produced. By replacing the complex medium with a semisynthetic one, the volumetric activity was reduced by a factor of ten (to 1650 U/ml), but the specific productivity by a factor of only two (to 210 U/ml).