Perfusion culture of glycyrrhiza inflata suspension cells in a stir-tank bioreactor

Cell and tissue browning is a major impediment of plant culture in vitro. Glycyrrhiza inflata Batalin cells died of lethal browning on Day 9 when cultivated in batch mode. Perfusion culture in bioreactors has a high potential for overcoming plant browning in vitro. In the present study, a continuous perfusion-culture system of G. inflata suspension cells was established in a stir-tank bioreactor. The key component of the bioreactor is an annular gravitational settling zone, through which we achieved continuous separation of cells from the medium. We determined the cell-retention efficiency of the reactor by varying rates of agitation, perfusion and aeration. Complete cell retention was obtained under the following conditions: agitation 0-200 rpm, aeration 0-0.6 volume per volume per minute (vvm) and perfusion rate 0-1.0 day(-1). These results were also in good agreement with theoretical estimation. An orthogonal test, including three factors and three levels, showed the optimal culture parameters were as follows: agitation rate 150 rpm, aeration rate 0.3 vvm and perfusion rate 1.0 day(-1). Under these conditions, the perfusion-culture system in the bioreactor was operated smoothly, and the maximum cell concentration reached 26 +/- 1 g L-1 dry weight.