Enhanced Production of Artemisinin by Hairy Root Cultivation of Artemisia annua in a Modified Stirred Tank Reactor

Abstract

Artemisinin is an important drug commonly used in the treatment of malaria as a combination therapy. It is primarily produced by a plant Artemisia annua, however, its supply from plant is significantly lower than its huge demand and therefore alternative in vitro production routes are sought. Hairy root cultivation could be one such alternative production protocol. Agrobacterium rhizogenes was used to induce hairy roots of A. annua. Statistical optimization of media was thereafter attempted to maximize the biomass/artemisinin production. The growth and product formation kinetics and the significant role of O2 in hairy root propagation were established in optimized media. Mass cultivation of hairy roots was, thereafter, attempted in a modified 3-L Stirred Tank Bioreactor (Applikon Dependable Instruments, The Netherlands) using optimized culture conditions. The reactor was suitably modified to obtain profuse growth of hairy roots by segregating and protecting the growing roots from the agitator rotation in the reactor using a perforated Teflon disk. It was possible to produce 18 g biomass L(-1) (on dry weight basis) and 4.63 mg L(-1) of artemisinin in 28 days, which increased to 10.33 mg L(-1) by the addition of elicitor methyl jasmonate.

Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors

“…However, there was no statistically significant difference in terms of biomass growth yield in gas-and liquid-phase bioreactors. The biomass growth yield per unit limiting substrate was found to be 0.67 g/g of sucrose consumed in modified NMB, as compared to biomass yield of 0.64 g/g sucrose consumed in modified stirred tank bioreactor and 0.65 g/g in shake flask cultivation (Patra and Srivastava 2014a). Initial phosphate concentration in the medium has little effect on artemisinin content and nitrate concentration remains non-limiting till stationary phase in A. annua hairy root cultivation (Patra and Srivastava 2015).…”

“…The biomass (23.02 ± 2.61 g/l) and artemisinin concentrations of 25.78 mg/l (1.12 mg/g DW) reported in this investigation are significantly higher than the literature results for batch cultivation of hairy root cultivation in bioreactors. The production of artemisinin in liquid-phase reactor is indicated to be growth associated (Patra and Srivastava 2014a). Liquid-phase reactors have been reported to promote higher biomass concentration (Kim et al 2001).…”

“…This reactor featured segregation of roots from the agitator of reactor by a perforated Teflon disc. This arrangement ensured adequate nutrient availability to growing roots without any mechanical shearing (Patra and Srivastava 2014a).…”

“…Growth of plant cells and hairy root culture has been attempted to some extent and implemented in liquid-phase submerged reactors like stirred tank reactors (STR), convective flow reactor, bubble column reactor, airlift reactor, rotating drum bioreactor, centrifugal impeller bioreactor, spin filter bioreactor, etc. (Panda et al 1989;Patra and Srivastava 2014a;Prakash and Srivastava 2007;Srivastava et al 2008). Most of these bioreactor configurations were found to be suitable for enhancement of biomass growth.…”

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

“…At present, A. annua is the sole commercial source of artemisinin, but the content of artemisinin in its flowers and leaves is just 0.05%-1.00% of the dry weight depending on the species (Duke et al, 1994;Delabays et al, 2001;Abdin et al, 2003;Shen et al, 2012;Patra and Srivastava, 2014). Because of its low concentration, numerous efforts have been made to improve artemisinin production to facilitate the commercialization of A. annua.…”