Optimization and Scale-Up of Thiarubrine a Production From Hairy Root Cultures of Ambrosia Artemisiifolia.
Date of Award
Doctor of Philosophy (PhD)
Martin A. Hjortso
Thiarubrine A (TA), a plant secondary metabolite synthesized by members of the family Asteraceae possesses antifungal and antiviral properties and is a potential pharmaceutical. The objective of the work was the optimization and scale-up of a bioprocess for the production of TA using hairy root cultures of Ambrosia artemisiifolia. At the inception of the work an established hairy root culture of the model system was used as inoculum in reactor experiments for the development of a protocol for the quantification of TA and in subsequent shake flask experiments to determine optimum culture conditions. TA accumulation closely paralleled growth and then decreased indicating TA degradation. Growth and TA accumulation decreased on increasing the temperature from 25$\sp\circ$C to 30$\sp\circ$C. No TA accumulation was observed in cultures exposed to light and this inhibitory effect was reversible. Elicitation, biotic and abiotic was studied as TA yield enhancement strategy using a statistical method. Biotic elicitation was done with Protomyces gravidus, a pathogen and Botrytis cinereae, a non pathogen of the model system and abiotic elicitation using vanadyl sulfate solution. In all the three cases stimulation of TA accumulation was observed. To obtain highly productive clones the root cultures were disorganized by the addition of phytohormones which facilitated somaclonal variation to provide a pool of variability. Hairy root clones were regenerated from the disorganized phase and a clone with thrice the productivity of the parent clone was achieved. Using the optimal culture conditions obtained, the cultures were scaled-up to a nine liter air sparged reactor with the inoculum concentration of root tips as the basis. Volumetric productivity of TA was of the same order of magnitude in the shake flask and the reactor. The results show that the inoculum concentration of root tips is a key parameter describing root growth and secondary metabolite accumulation at low biomass densities. Experiments performed in a shake flask indicated that manipulation of the inoculum concentration of root tips provides a way of manipulating the kinetics of root growth and TA accumulation early in the run.
Bhagwath, Santosh G., "Optimization and Scale-Up of Thiarubrine a Production From Hairy Root Cultures of Ambrosia Artemisiifolia." (1997). LSU Historical Dissertations and Theses. 6467.