L'induzione di stress ossidativo aumenta la produzione di astaxantina in Haematococcus pluvialis

Astaxanthin is an important algal ketocarotenoid that is employed to improve human health and as a pigment for fish feeding in aquaculture. Therefore, its production has consistent economic and scientific interests. The biological activity of astaxanthin is related to its strong antioxidant activity that was reported to prevent age-related diseases. The best source of biologically active astaxanthin is the fresh water green microalga Haematococcus pluvialis that accumulates significant amounts of astaxanthin esters in response to stressed growth conditions. Although several studies have investigated the optimal environmental factors that improve ketocarotenoid production, few papers reported growth conditions compatible with an industrial scale up, whereas a relevant number of patents is available. In this thesis I evaluated the effects of oxidative stress on carotenoid accumulation in H. pluvialis by time-course experiments in order to look for scalable methods for industrial production. The microalga was treated with increasing concentrations of hydrogen peroxide (0, 1 and 10 µM) in mixotrophic batch cultures with medium containing sodium acetate. The experiments were performed under different light intensities (PPFD: 0, 20 and 80 µmol m-2 s-1). Spectrophotometric assays were used for chlorophyll quantification and to asses the hydrogen peroxide (H2O2) content in the mixotrophic medium. Qualitative screening of accumulated carotenoids and chlorophylls was performed by Thin Layer Chromatography (TLC) and the presence of astaxanthin esters was confirmed upon saponification of extracts. Chemical identification and quantification of accumulated carotenoids were carried out by HPLC-DAD with a C30 reverse phase column. After 7 days, the treated cultures with 1 and 10 μM of H2O2 showed a significant increase of astaxanthin esters at both 20 and 80 µmol m-2 s-1 with respect to control and a significant reduction of other carotenoids. On the base of our results, the best condition for astaxanthin accumulation was the treatment with 10 M of H2O2 at 80 mol m-2 s-2 PPFD. In conclusion the oxidative stress resulted a valid and economic stress factor able to induce a fast and significant accumulation of the bioactive astaxanthin esters in H. pluvialis in mixotrofic condition. This occurred at the highest light intensity (80 PPFD) used. However, these PPFD values were well below the standard light requirements for astaxanthin production experienced in other works or in industrial plants, indicating that the use of H2O2 might potentially induce energy saving and cost reduction.