A Low-Cost Fertilizer Medium Supplemented with Urea for the Lutein Production of Chlorella sp. and the Ability of the Lutein to Protect Cells against Blue Light Irradiation

Abstract

This study aimed to investigate the use of organic fertilizers instead of modified f/2 medium for Chlorella sp. cultivation, and the extracted lutein of the microalga to protect mammal cells against blue-light irradiation. The biomass productivity and lutein content of Chlorella sp. cultured in 20 g/L fertilizer medium for 6 days were 1.04 g/L/d and 4.41 mg/g, respectively. These values are approximately 1.3- and 1.4-fold higher than those achieved with the modified f/2 medium, respectively. The cost of medium per gram of microalgal biomass reduced by about 97%. The microalgal lutein content was further increased to 6.03 mg/g in 20 g/L fertilizer medium when supplemented with 20 mM urea, and the cost of medium per gram lutein reduced by about 96%. When doses of ≥1 μM microalgal lutein were used to protect mammal NIH/3T3 cells, there was a significant reduction in the levels of reactive oxygen species (ROS) produced by the cells in the following blue-light irradiation treatments. The results show that microalgal lutein produced by fertilizers with urea supplements has the potential to develop anti-blue-light oxidation products and reduce the economic challenges of microalgal biomass applied to carbon biofixation and biofuel production.

Keywords: Chlorella; ROS; blue light; fertilizer; lutein; urea.

Figure 1

Growth profiles (a) and lutein contents (b) of the Chlorella sp. cultured in different fertilizer concentrations (10, 20, and 30 g/L), and compared to the cultures in modified f/2 medium as control. The initial microalgal biomass concentration was approximately 0.3 g/L. The culture was operated at 26 ± 1 °C with 300 μmol/m²/s of light intensity and with a 2% CO₂ aeration rate of 0.2 vvm for 7 days. The microalgal cells were sampled every 24 h for growth determinations and in a 6-day and a 7-day culture for lutein content determinations. Data were compared with a one-way ANOVA test to evaluate the differences between multiple groups on day 6. Different letters indicate significant differences between groups (p < 0.05). Statistical significance for each group on day 6 and day 7 is indicated by asterisks. Two-tailed paired Student t-test p-values indicate statistical significance (* p < 0.05).

Figure 2

Growth profiles (a) and lutein contents (b) of the Chlorella sp. cultured in 20 g/L fertilizer with the supplement of different nicotine concentrations (25, 50, 100, and 200 μM), and compared to the cultures without nicotine supplement. The initial microalgal biomass concentration was approximately 0.3 g/L. The culture was operated at 26 ± 1 °C with 300 μmol/m²/s of light intensity and with a 2% CO₂ aeration rate of 0.2 vvm for 7 days. The microalgal cells were sampled every 24 h for growth determinations and in a 6-day and a 7-day culture for lutein content determinations. Data were compared with a one-way ANOVA test to evaluate the differences between multiple groups on day 6. Different letters indicate significant differences between groups (p < 0.05). Statistical significance for each group on day 6 and day 7 is indicated by asterisks. Two-tailed paired Student t-test p-values indicate statistical significance (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Figure 3

Growth profiles (a) and lutein contents (b) of the Chlorella sp. cultured in 20 g/L fertilizer with the supplement of different NaNO₃ concentrations (5, 10, 20, 30, and 60 mM), and compared to the cultures without NaNO₃ supplement. The initial microalgal biomass concentration was approximately 0.3 g/L. The culture was operated at 26 ± 1 °C with 300 μmol/m²/s of light intensity and with a 2% CO₂ aeration rate of 0.2 vvm for 7 days. The microalgal cells were sampled every 24 h for growth determinations and in a 6-day and a 7-day culture for lutein content determinations. Data were compared with a one-way ANOVA test to evaluate the differences between multiple groups on day 6. Different letters indicate significant differences between groups (p < 0.05). Statistical significance for each group on day 6 and day 7 is indicated by asterisks. Two-tailed paired Student t-test p-values indicate statistical significance (* p < 0.05).

Figure 4

Growth profiles (a) and lutein contents (b) of the Chlorella sp. cultured in 20 g/L fertilizer with the supplement of different urea concentrations (5, 10, 20, 30, and 60 mM), and compared to the cultures without urea supplement. The initial microalgal biomass concentration was approximately 0.3 g/L. The culture was operated at 26 ± 1 °C with 300 μmol/m²/s of light intensity and with a 2% CO₂ aeration rate of 0.2 vvm for 7 days. The microalgal cells were sampled every 24 h for growth determinations and in a 6-day and a 7-day culture for lutein content determinations. Data were compared with a one-way ANOVA test to evaluate the differences between multiple groups on day 6. Different letters indicate significant differences between groups (p < 0.05). Statistical significance for each group on day 6 and day 7 is indicated by asterisks. Two-tailed paired Student t-test p-values indicate statistical significance (* p < 0.05).

Figure 5

Cell viability (a) and ROS production (b) of NIH/3T3 cells treated by blue light for the different irradiated time. NIH/3T3 cells were exposed to blue light with 300 μmol/m²/s of intensity for 3, 6, 12, 18, and 24 h, cell viability was detected using an MTT assay and the ROS production. The cell viability and ROS production of NIH/3T3 cells without exposure to blue light (0 h) were defined as the control (C; 100%). All values were expressed as the mean ± SD. Data were compared with a one-way ANOVA test to evaluate the differences among multiple groups. Different letters indicate significant differences between groups.

Figure 6

ROS production of NIH/3T3 cells treated with microalgal lutein for different time (a) and different doses (b) against the damage induced by blue-light irradiation. Before the cells were exposed to blue light with 300 μmol/m²/s of intensity for 12 h, NIH/3T3 cells were treated with 10 μM lutein for different time 0, 1, 3, 6, 12, and 24 h, and different doses of 0, 0.01, 0.1, 1, and 10 μM lutein for 6 h, individually. After exposure to blue light, the ROS production of NIH/3T3 cells was measured. The value of relative ROS production of NIH/3T3 cells without lutein supplement and no blue-light exposure was defined as control (C; 100%). All values were expressed as the mean ± SD. Data were compared with a one-way ANOVA test to evaluate the differences between multiple groups. Different letters indicate significant differences between groups.