Bioreactor design for scaleup of Catharanthus roseus hairy root cultures for production of indole alkaloids
Vani, Sundeep N.
Doctor of Philosophy
C. roseus is the source of the important anticancer indole alkaloids, vincristine and vinblastine. Hairy root cultures are an attractive alternative for the production of plant derived secondary metabolites. C. roseus hairy root cultures were established in our laboratory by A. rhizogenes mediated transformation of C. roseus seedlings prior to the beginning of this project. The objectives of this project were twofold: (i) Alkaloid analysis of C. roseus hairy root cultures, (ii) Scaleup of hairy root cultures from shake flasks to bioreactors. An extraction method was adapted for the rapid quantification of C. roseus hairy root extracts, and was used for quantifying alkaloid yields from five different root clones. Optimum analytical wavelengths were chosen for quantification after analyzing chromatograms at five different wavelengths using PDA detection. Extracts were analyzed using different analytical techniques--GC/MS, MS, HPLC-PDA and TLC--and several alkaloids were identified. The presence of ajmalicine, akuammine, apparicine, catharanthine, hoerhammericine, lochnericine, serpentine and tabersonine was confirmed. The presence of vindoline--an important precursor of vinblastine and vincristine--could not be confirmed although GC/MS indicated the presence of vindoline fragment ions. A novel method based on non-linear regression analysis was developed for online measurement of oxygen uptake rates. Thus, mass transfer rates could be adjusted in bioreactor cultures to provide sufficient oxygen. Three different bioreactor configurations--two recirculation reactors (60 mL, 1L) and a 500 mL spinner flask--were designed for scaleup studies. Growth, nutrient utilization and alkaloid productivities were measured. This is the first study where several biochemical parameters have been measured in multiple bioreactor configurations and found to be comparable--without exception--to shake flask cultures. Cultures were cultivated in different gas phase environments--'air' and '2.5% CO$\sb2$'--in each bioreactor configuration. This is the first study of the effect of CO$\sb2$ on hairy roots cultivated in liquid culture. A beneficial effect ($>$98% confidence) of CO$\sb2$ was observed on biomass accumulation. Possible metabolic importance of CO$\sb2$ on heterotrophic plant cultures is discussed.