APPLICABILITY OF WOOL COVALENT BONDED Bacillus circulans 25 CELLS FOR MILK-CLOTTING ENZYME PRODUCTION BY BATCH, REPEATED BATCH AND CONTINUOUS PROCESS

Abstract The production of milk-clotting enzyme (MCE) by free and immobilized Bacillus circulans 25 cells was investigated. The investigation evaluates cell immobilization through entrapment and covalent binding to different carriers. B. circulans 25 cells immobilized covalently via glutaraldehyde (cross-linker) to natural wool exhibited higher effectiveness factor (0.76) compared to other carriers. Immobilized cells produce maximum level of MCE and highest ratio of milk-clotting activity/proteolytic activity (MCA/PA) after 24 h using 0.4g wet weight cells/g wool. In batch operation, decreasing biomass loading to 0.2 g enhanced the MCA/PA ratio and specific productivity by 5.94 and 59.88%, respectively. Immobilized cells on natural wool as an effective and suitable carrier were able to produce the same level of MCA, productivity and MCA/PA ratio for 9 repeated cycles (216 h). Under continuous operation immobilized cells in a packed-bed bioreactor were able to keep producing MCA with MCA/PA ratio for about 7 days at the same level. Moreover, continuous operation demonstrates a very good productivity of 0.385 KU/L/h, which is higher than the other production systems by 7.0-11.7 fold. Immobilized B. circulans 25 cells proved to be fully capable of continuous MCE production in a packed-bed bioreactor.