Carotenoids as a Protection Mechanism against Oxidative Stress in Haloferax mediterranei

Abstract

Haloarchaea are extremophilic microorganisms that in their natural ecosystem encounter several sources of oxidative stress. They have developed different strategies to cope with these harsh environmental conditions, among which bacterioruberin production is a very notable strategy. Bacterioruberin (BR) is a C₅₀ carotenoid synthesized in response to different types of stress. Previous works demonstrated that it shows interesting antioxidant properties with potential applications in biotechnology. In this study, Haloferax mediterranei strain R-4 was exposed to different concentrations of the oxidant compound H₂O₂ to evaluate the effect on carotenoid production focusing the attention on the synthesis of bacterioruberin. Hfx. mediterranei was able to grow in the presence of H₂O₂ from 1 mM to 25 mM. Cells produced between 16% and 78% (w/v) more carotenoids under the induced oxidative stress compared to control cultures. HPLC-MS analysis detected BR as the major identified carotenoid and confirmed the gradual increase of BR content as higher concentrations of hydrogen peroxide were added to the medium. These results shed some light on the biological role of bacterioruberin in haloarchaea, provide interesting information about the increase of the cellular pigmentation under oxidative stress conditions and will allow the optimization of the production of this pigment at large scale using these microbes as biofactories.

Keywords: H2O2; archaea; bacterioruberin; haloarchaea; hydrogen peroxide; oxidative stress.

Figure 1

(a) Growth curve of Hfx. Mediterranei cell cultures. Vertical dotted line indicates the time of the H₂O₂ addition. (b) Specific growth velocity (µ) of Hfx. Mediterranei during exponential phase. From 10 mM to 25 mM, there are two µ values since under these conditions, cells presented a diauxic growth with two exponential phases. Each experimental sample was compared to the control to evaluate statistical significance. *** p < 0.001. **** p < 0.0001.

Figure 2

Growth of preadapted cells vs. growth of non-preadapted cells. Preadapted and non-preadapted Hfx. Mediterranei cell cultures were grown under 5 mM (black) and 15 mM (grey) H₂O₂ exposure. Vertical dotted line indicates time of the H₂O₂ addition.

Figure 3

Visual comparison of the acetone extracts obtained from a selection of Hfx. Mediterranei cell cultures exposed to different H₂O₂ concentrations (0 mM ©), 5 mM, 10 mM, 15 mM, 20 mM, and 25 mM.

Figure 4

BR quantification (µg/mL) from extracts of Hfx. Mediterranei cell cultures exposed to different concentrations of H₂O₂. Each experimental sample was compared to the control to evaluate statistical significance. *** p < 0.001. **** p < 0.0001.

Figure 5

Relative percentage average of (a) BR and (b) MABR abundance and in Hfx. mediterranei cell cultures exposed to different concentrations of H₂O₂. Relative percentage was calculated by the sum of each percentage observed in the different peaks per sample in which BR and MABR were detected by its total m/z value: 740.9 and 722, respectively. Each experimental sample was compared to the control to evaluate statistical significance. ** p < 0.005, *** p < 0.001, **** p < 0.0001.