244 JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 94, No. 3, 244–250. 2002 Effect of Mixing Time on Taxoid Production Using Suspension Cultures of Taxus chinensis in a Centrifugal Impeller Bioreactor JIAN-JIANG ZHONG, 1 * ZHI-WEI PAN, 1 ZHEN-YU WANG, 1 JIANYONG WU, 2 FENG CHEN, 3 MUTSUMI TAKAGI, 4 AND TOSHIOMI YOSHIDA 4 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China, 1 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China, 2 Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China, 3 and International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan 4 Received 26 February 2002/Accepted 25 June 2002 The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active second- ary metabolite, were used as a model system for this investigation. The mixing time (t m ) and volu- metric oxygen transfer coefficient (k L a) in the bioreactor were characterized at various cell densi- ties and operating conditions. A constant t m of 5 s or 10 s was maintained during cultivation by ad- justing the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accu- mulation was significantly increased through culture elicitation using 100 mM methyl jasmonate (MJA). The lower Tc production at the longer t m of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing. [Key words: plant cell culture, mixing time, oxygen transfer, centrifugal impeller bioreactor, taxoid production, Taxus chinensis] The commercial production of the anticancer drug pacli- taxel using plant cell suspension cultures was recently achieved by the Samyang Genex (Taejon, Korea) and Phy- ton Catalytic (NY, USA) companies. Without doubt, this successful industrial application of plant cell cultures will trigger further research on the production of other plant-de- rived beneficial bioactive compounds. The main problems hindering the development of the large-scale cultivation of plant cells have been low productivity, cell line instability, and difficulty in scale-up. Because plant cells are much larger than most microbial cells and have a tendency to grow in aggregates with sizes ranging from about 40 mm to more than a few mm, they sed- iment easily in cultivation vessels. During the suspension cultivation of plant cells, a rapid increase in fluid viscosity occurs (1). Therefore, fluid mixing is one of the most criti- cal issues and we need to consider its interactions with fluid viscosity, sedimentation, oxygen transfer, shear stress, and culture kinetics (1). Adequate mixing at a relatively high cell density can be achieved by increasing agitation speeds and/or aeration rates in agitated bioreactors. On the other hand, shear stress caused by intense agitation or aeration may be detrimental to plant cells because of their shear-sen- sitive nature. In the scale-up and optimization of microbial processes, fluid mixing affected cell growth physiology and the pro- duction of primary metabolites such as amino acids (2–4). The conditions of poor mixing and non-homogeneous sub- strate distribution in bioreactors of industrial scale were simulated and studied in scaled-down reactors (2–4). The degree of mixing was also linked to plasmid stability in the cultivation of recombinant Escherichia coli strains (5). In algal cultures, mixing played an important role because it induced light/dark cycles in photobioreactors (6, 7). The effects of fluid flow and mixing were also investigated in plant root cultures (8–10) and plant micropropagation sys- tems (11). In all of these studies, mixing time was not con- trolled at a constant level during cultivation. As suggested in our recent work on suspension cultures of Panax notogin- seng cells (12) and related studies (e.g., 13–15), fluid mix- ing is an important factor in the process optimization and scale-up of plant cell suspension cultures for the efficient production of useful metabolites. There is a need to investi- gate the effect on plant cell physiology and metabolism of a constant mixing time in long-term agitated cultures. To the best of our knowledge, there are no reports to date on the effects of mixing time (t m ) on metabolite production in plant * Corresponding author. e-mail: jjzhong@ecust.edu.cn phone: +86-21-6425-2091 fax: +86-21-6425-3904