VOI. 42 NO. 4 SCIENCE IN CHINA (Series B) ~ugust 1999 A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures ZHANG Wei (% X! p) * , Shintaro Furusaki2 and Chris Franco3 ( 1 . Department of Biochemical Engmeering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116012, China; 2. Department of Chemistry and Biotechnology , Graduate School of Engineering, The University of 'Tokyo, 7-3- 1 , Hongo , Bunkyo-ku , Tokyo 113, Japan; 3. Biotechnology , School of Medicine, Flinders University of South Australia, Bedford Park, SA 5042, Australia) Received July 2, 1998 Abstract A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspen- sion cultures of strawbeny cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawbeny cells were grown at 30T (the optimum temperature for cell gowth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthocyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature. was de- creased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influenced by the shift-time point but cell growth was not. Consequently, the maximum antho- cyanin content of 2.7 mg. g-fresh cell - ' was obtained on day 9 by a temperature-shift from 309: , after 3-d culture, to 159:. The highest anthocyanin yield of 318 mg. L-I on day 12 was achieved when the temperature was shifted from 30°C , after 5-d culture, to 20°C . For a global optimization of both the yield and productivity, the optimum anthocyanin yield and productivity of 272 mg- L- ' and 30.2 mg. L- ' . d- ' on day 9 were achieved by a two-stage culture with a tem- perature-shift from 309: after 3 d to 20°C . Keywords : strawberry cell, suspension cultures, anthocyanin, temperature, two-stage culture, temperahupshift. Significant improvement in the volumetric productivity and yield is a must for commercial-scale production of secondary metabolites by plant cell cultures. Since the volumetric productivity and yield are determined mainly by cell lines, culture conditions, process strategy and bioreactor configura- tions, different techniques ranging from environmental stress to genetic manipulation have been used to realize the improvernent"l . Of the environmental stresses that can be imposed upon the plant cells, culture temperature is considered to be the easiest for the manipulation of secondary metabolite pm- duction from the viewpoint of reactor design. It is known that temperature has significant and varied effects on cell growthr2' and secondary metabolite synthesis in plant cell culture^^^-*^ . The cultures therefore are usually carried out at the optimized constant temperature for different plant species. However, little research has been done on the advanced manipulation of temperature for further en- hancement of productivity. Zhang et al. have reported that the production of harringtonine and homo- harringtonine in Cephalotaxus fortunei callus culture could be enhanced by periodic temperature oscil- * Corresponding author. Mail address: Biotechnology, School of Medicine, Flinders University of South Australia, Bedford Park, SA 5042, Australia.