Kinetics of growth and alkaloid production of Cephalotaxus harringtonia plant cell cultures

Paul John Westgate, Purdue University

Abstract

Cephalotaxus harringtonia plant-cell cultures have been examined to characterize growth kinetics. The requirement for an undefined media supplement (coconut water) was eliminated by maintaining high cell concentrations in semicontinuous and batch growth. Sucrose fed to batch cultured cells was completely inverted and a diauxic growth pattern was observed corresponding to first glucose and then fructose uptake. Specific growth rates were highest during glucose uptake, but cell yields were comparable for the two sugars. In contrast, batch studies with glucose or fructose as the sole carbon source indicate that, under these conditions, cell yields were significantly lower with fructose but specific growth rates were comparable for the two sugars. Since periods of substrate-limited batch growth are too short to make an accurate estimate of the Monod saturation constant, other forms of operation have been investigated to accomplish substrate limited growth. Continuous cultures are infeasible on a laboratory scale as a result of special properties of the plant cells. Semicontinuous growth is shown to require too frequent sampling to be a practical alternative. However, fed-batch operation, consisting of intermittent removal from a culture that is fed continuously, eliminates these problems. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed with a multichannel syringe pump. Results indicate that not only are values of the saturation constant estimated from batch culture much too large, but Monod's kinetics cannot adequately describe growth under these conditions. Monod's equation using internal glucose concentration gives a better, but not perfect correlation. Alkaloid production by the cultures has been studied using an HPLC procedure developed for this purpose. Cells grown in the defined media exhibited very low production of desired alkaloids. In the undefined media, however, quantities of harringtonine were produced that were comparable to those reported in callus culture. Treatment with a fungal elicitor during the early stages of growth resulted in a substantial increase in the total harringtonine present in the cells and media.

Degree

Ph.D.

Advisors

Emery, Purdue University.

Subject Area

Chemical engineering|Plant propagation

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