High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance

Abstract

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photobioreactors. However, wild-type strains show biological limitations making algal bioproducts expensive compared to those extracted from other feedstocks. Such constraints include inhomogeneous light distribution due to high optical density of the culture, and photoinhibition of the surface-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobioreactors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent.

Keywords: biomass; carotenoids; chloroplast; microalgae; norflurazon; photooxidative stress; photoprotection.

Figure 1

Characterization of C. vulgaris norflurazon-resistant (NFR) strains of Chlorella vulgaris. (A) PSII functional antenna size. Variable Chl fluorescence was induced with a green light (15 μmol photons m⁻² s⁻¹), on dark-adapted cells of WT and NFR (about 1 × 10⁷ cells mL⁻¹) in BG-11 medium supplemented with 50 μM DCMU. Data are expressed as mean ± SD, n = 10. The reciprocal of time needed for reaching two-thirds of the fluorescence rise (T_2/3) was taken as a measure of the PSII functional antenna size (see Table 1). (B,C) Immunoblotting used for the quantification of photosynthetic subunits. (upper) Immunotitration was performed with antibodies directed against individual gene products: LHCII, the major light harvesting complex of PSII; the PSII core subunit PsbC (CP43); the PSI core subunit (PsaA). The amounts of Chls (panel B) and of cells (panel C) loaded for each lane are indicated. (lower) In each table, significantly different values (ANOVA followed by Tukey’s post-hoc test at a significance level of p < 0.05), within the same row, are marked with different letters. (D) Light-saturation curves of photosynthesis, measured in cultures grown in BG-11 minimal medium. Data are expressed as mean ± SD, n = 4.

Figure 2

Photooxidation of C. vulgaris WT and NRF mutant genotypes under photooxidative stress. (A) Cell suspensions of WT and mutant strains were treated with 1400 µmol photons m⁻² s⁻¹ at 20 °C, and kinetics of malondialdehyde (MDA) formation were followed. MDA is an index of membrane lipid peroxidation, and was quantified by HPLC as thiobarbituric reactive substances. Slopes of the linear fit (proportional to the rate of MDA release) were 0.0195 ± 0.0056 (WT), 0.0029 ± 0.0024 (NFR-3), 0.0034 ± 0.0031 (NFR-13). (B) Cell suspensions were treated with strong white light (14,000 µmol photons m⁻² s⁻¹, 20 °C) and the amount of Chl was evaluated by measuring the absorption area in the region 600–750 nm. See Materials and Methods for details. Symbols and error bars show means ± SD, n = 4. Values marked with the same letters are not significantly different from each other within the same time point (ANOVA followed by Tukey’s post-hoc test at a significance level of p < 0.05).

Figure 3

Growth curves of WT and mutant strains. Growth of WT and NRF mutant lines was performed under photoautotrophic conditions, in 1-L cylinders, illuminated with 1400 µmol photons m⁻² s⁻¹, 25 °C. Cultures were maintained in a semi-batch system fed with air/CO₂ mix; CO₂ supply was modulated in order to keep the pH of the medium always between 6.8 and 7.2. Symbols and error bars show means ± SD, n = 5. Values marked with the same letters are not significantly different from each other within the same time point (ANOVA followed by Tukey’s post-hoc test at a significance level of p < 0.05).

Figure 4

Analysis of Chl fluorescence during photosynthesis under EL. (A) Chl fluorescence was monitored in cultures dark-adapted for 2 h. Cell suspensions were illuminated for 25 min and the non-photochemical quenching (NPQ) was determined at the end of light treatment (i.e., during steady-state photosynthesis). (B) PSII quantum yield recovery after EL treatment was quantified on WT and NFR strains by measuring F_v/F_m recovery in low light (20 μmol photons m⁻² s⁻¹, 24 °C, light yellow bar) upon 3 h of EL treatment (1800 μmol photons m⁻² s⁻¹, 24 °C, yellow bar). (Inset) kinetics of F_v/F_m were zeroed at the end of the EL treatment and normalized to the maximum F_v/F_m during low light recovery. Data are expressed as mean ± SD, n = 4.