Haloferax mediterranei Cells as C50 Carotenoid Factories

Abstract

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL^-1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL^-1 of carotenoids for a culture with turbidity 14.0.

Keywords: C/N ratio; Haloferax mediterranei; bacterioruberin; carotenoids; osmotic stress.

Figure 1

Time course of the growth of Hfx. mediterranei and the cellular content of carotenoids. Cells were grown in a complex medium. At the indicated times, turbidity (-●-) and carotenoids production (bars) were determined. Error bars show the standard deviation of replicates. More details are provided in the Materials and Methods section.

Figure 2

Effect of glucose and/or yeast extract starvation on the growth of Hfx. mediterranei. Cells growing in complex medium (OD₆₀₀ of approximately 6.0) were harvested, washed, and resuspended in complex medium (-○-), lacking glucose (-Δ-), lacking yeast extract (-□-), or lacking both (-◇-). The turbidity was determined at the indicated times. Error bars show the standard deviation of replicates. More details are provided in the Materials and Methods section.

Figure 3

Effect of glucose and/or yeast extract starvation on the carotenoids production by Hfx. mediterranei. Samples of cultures corresponding to days 1, 2, and 3, under nutrient deficiency (see Figure 2), were used as the sources of carotenoids, which were extracted and quantified. The data correspond to control culture (dark bars), lacking yeast extract (grey bars), lacking glucose (white bars), or lacking both yeast extract and glucose (dashed bars). Error bars show the standard deviation of replicates. More details of experimental procedures are provided in the Materials and Methods section. This yield was obtained from a two-step procedure over seven days and was slightly lower than that obtained in the one-stage culture. Cultures lacking yeast extract showed a 1.77-fold higher carotenoid content than the control, while those lacking in both yeast extract and glucose increased the carotenoid content by 1.23-fold only.

Figure 4

Effect of inorganic salts on the growth of Hfx. mediterranei and the yield of bacterioruberin (BR). The cells were grown in a complex medium, except that the concentration of inorganic salts (SW = salts in water) was as indicated. (A) Turbidity was measured using aliquots of each culture. (B) BR was measured at the stationary phase of growth (turbidity 8.0–9.0). Error bars show the standard deviation of replicates. Other conditions are described in the Materials and Methods section.