Hydrodynamic stress induces anthraquinones production in Rubia tinctorum cell suspension cultures.

BUSTO, V.; GIULIETTI, AM.; RODRIGUEZ TALOU, J.

Barcelona, España

Congreso; 13th European Congress on Biotechnology.; 2007

Elicitation of plant cell cultures is seen as an effective strategy to enhance the production of many pharmaceutical compounds, secondary metabolites among them. These cultures can be elicited by traditional biotic elicitors, such as chitosan, arachidonic acid and pathogen cell walls. Abiotic elicitors have been used as well to induce secondary metabolite accumulation, which includes hydrodynamic stress. Rubia tinctorum cell suspension cultures produce anthraquinones, typical secondary metabolites, which are important in the pharmaceutical and food industries. In the present work, we investigated the effect of hydrodynamic stress on anthraquinones (AQs) production in Rubia tinctorum cell suspension cultures. A 1.5 L stirred tank bioreactor agitated by two six-bladed turbine impellers was used. To induce hydrodynamic stress rotational speed was set at 450 and 800 rpm. Control experiments were carried out in 125 mL shake flasks agitated at 100 rpm. Cell growth, extracellular pH, intracellular H2O2, AQs production and cell viability were evaluated. Loss of cell viability measured as Evans Blue uptake increased 200% and 100% after 24 hs of shear stress at 450 and 800 rpm respectively, compared to non-stressed cells. Despite of the decrease in cell viability after 24 hs of shear stress, regrowth assays showed that control and 24 hs stressed cells reached similar biomass concentration after 7 days of culture. AQs production increased 48% after 24 hs of hydrodynamic stress at 450 rpm. This value was 116% higher than non-stressed cells when stress was suspended for 7 days. However, after 24 hs at 800 rpm, AQs production increased 59% and this value did not change when stress was suspended. When cells were cultured during 13 days at 450 rpm, a decrease in biomass concentration (28%) was observed compared with control shake flask cultures, cells cultured at 800 rpm were severed injured and no growth was observed after 24 hs. AQs accumulation increased up to 230% compared with control after 10 days of shear stress at 450 rpm. The H2O2 concentration of stressed cells was higher than non-stressed cells after shear stress treatment at 450 and 800 rpm, showing that oxidative burst was trigged by the imposition of hydrodynamic stress. In this study it was found that R. tinctorum cells subjected to hydrodynamic stressed during 24 hs in a stirred tank recovered the ability to growth. Moreover, hydrodynamic stress elicit AQs production in R. tinctorum cell cultures and could be and alternative strategy to the traditional biotic elicitors.