Inhanced shikonin production from lithospermum erythrorhizon cell cultures by in situ extraction

Abstract

Plant cell cultures of lithospermum erythrorhizon were performed to produce plant derived secondary metabolizes such as shikonin. L. erythrorhizon was cultured in 250 mL shake flasks. Dry cell concentration of 13 g/L and fresh cell concentration of 390 g/L were obtained after 210 hours. Specific growth rate of 0.26 $day^{-1}$ was obtained, and it was about 20 times lower than that of microbial cells. The slower growth rate lowers cellular productivity and makes the operation difficult. Owing to the slow growth rate, specific oxygen uptake rate was 7.25 mg/g.h which is much lower than that of microbial cells (Chap. 2). Plant cell immobilization effect on secondary metabolite production was studied. Plant eclls cells were immonilized in a dual hollow fiber bioreactor to maintain high cell density and to operate continuously. The dual hollow fiber bioreactor consists of outer silicone membrane for oxygen transport and an inner isotopic polypropylene hollow fiber for liquid nutrient transport. its performance was examined by culturing. L erythorhizon cells. The cells have grown well and the dry biomass density of the cells was 325 g/L of void volume for cell growth, which was highest among the methods for plant cell culture ever reported. Volumetric productivity of phenolics as a rough indication of plant cell secondary metabolizes was 221 mg/L.day at a medium flow rate of 0.3 mL/h, which corresponds to a 58 fold increase over that of shake flask culture. the specific productivity of the cells in the reactor was 0.68 mg/g cell.day, which was 2 times higher than that of batch culture (Chap.3). Effects of cell immobilization and in sits extraction on shikonin production were studied. In sits extraction and cell immobilization in calcium alginate beads enhanced shikonin production, respectively, and they greatly increased shikonin production when they were employed together. This simultaneous treatment increased cellular shikonin productivity and volumetric productivi...