Domestication of the green alga Chlorella sorokiniana: reduction of antenna size improves light-use efficiency in a photobioreactor

Abstract

Background: The utilization of biomass from microalgae for biofuel production is one of the key elements for the development of a sustainable and secure energy supply. Among the different microalgae, Chlorella species are of interest because of their high productivity, high lipid content, and resistance to the high light conditions typical of photobioreactors. However, the economic feasibility of growing algae at an industrial scale is yet to be realized, in part because of biological constraints that limit biomass yield. A key issue is the inefficient use of light due to uneven light distribution, and the dissipation of excess absorbed light as heat. The successful implementation of biofuel production facilities requires the development of algal strains with enhanced light use efficiency in photobioreactors. Such domestication strategies include decreasing the absorption cross section in order to enhance light penetration, increasing the size of metabolic sinks per chlorophyll and minimizing feedback energy dissipation.

Results: In this work we applied random mutagenesis and phenotypic selection to the thermotolerant, fast-growing Chlorella species, C. sorokiniana. Truncated antenna mutants (TAMs) were selected that exhibited a lower fluorescence yield than the wild-type (WT) strain. Six putatively interesting mutants were selected by high throughput fluorescence video imaging, two of which, TAM-2 and TAM-4, were found to have approximately half the chlorophyll content per cell and LHCII complement per PSII with respect to the WT. In batch culture, TAM-2 showed an increased photon use efficiency, yielding a higher Pmax at saturating irradiances with respect to the WT. Cultivation of TAM-2 in both laboratory-scale and outdoor photobioreactors showed higher productivity than WT, with a 30% higher biomass yield in dense cell suspensions typical of industrial photobioreactors.

Conclusions: These results suggest that generation of mutants with low chlorophyll content can significantly improve the light-to-biomass conversion efficiency of C. sorokiniana under mass culture conditions. However, owing to the lack of sexual reproduction in this species, the presence of additional mutations might affect growth rate, suggesting that selection should include evaluation of multiple independent mutants for each desired phenotype.

Keywords: Antenna size; Biofuel; Biomass; Chlorella sorokiniana; Light-use efficiency; Photobioreactor; Photosynthesis.

Figure 1

Growth and fluorescence analysis of six putative truncated antenna mutants (TAM1-6) of Chlorella sorokiniana . Culture samples were spotted onto minimal medium (upper panels: phototrophic growth) or acetate-containing medium (lower panels: mixotrophic growth), grown in continuous light (50 μmol photons m^-2 s^-1) for seven days, and then dark-adapted for pulse amplitude modulation (PAM) fluorescence analysis. False color images reveal that all six mutants have lower fluorescence emissions compared to the wild ype (WT), with TAM-2 and TAM-4 the most pronounced.

Figure 2

Polypeptide composition of thylakoid membranes from wild-type, TAM-2, and TAM-4 mutants. (a) Thylakoid pigment-protein complexes were separated by nondenaturing Deriphat PAGE upon solubilization with 1% β-DM. Thylakoids corresponding to 25 mg of chlorophylls were loaded in each lane. The composition of each band is indicated. (b) Immunoblotting used for the quantification of photosynthetic subunits in the wild-type (WT) and TAM thylakoids. Immunoblot analysis 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). Thylakoids corresponding to 0.25, 0.5, and 1 μg of Chls were loaded for each sample. All samples were loaded on the same SDS-PAGE slab gel. (c) Results of the immunotitration of thylakoid proteins. Data of PSII antenna subunits (left panel) and PSI core subunit (right panel) were normalized to the PSII core amount (PsbB content) and expressed as a percentage of the corresponding wild-type content ± SD. Significantly different values from wild type are marked with an asterisk.

Figure 3

Functional antenna size of PSII and PSI measured in wild-type and mutants TAM-2 and TAM-4. (a) Variable Chl fluorescence was induced with a green light of 15 μmol photons m^-2 s^-1, on dark-adapted cells (about 1.0 · 10⁷ cells/ml) in BG-11 medium supplemented with 50 μM DCMU. The reciprocal of time corresponding to two-thirds of the fluorescence rise (T_2/3) was taken as a measure of the PSII functional antenna size. (b) The kinetics of P700 oxidation (ΔAbs at 705 nm) were measured on thylakoid suspension (75 μg Chl/ml) treated with 50 μM 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) and 1 mM methylviologen, upon illumination with a 10-s pulse of red actinic light (λ =630 nm, 560 μmol photons m^-2 s^-1). Data are expressed as mean ± SD, n =7.

Figure 4

Light-saturation curves of photosynthesis. Curves were obtained with the C. sorokiniana wild-type and the TAM-2 (a) and TAM-4 (b) mutants. The light-saturated P_max was 1.5-fold higher in the TAM-2 mutant than in the wild type, suggesting a greater productivity on a per-Chl basis. In the TAM-4 mutant, the light-saturated O₂ yield was not significantly higher than that for the wild type. Data are expressed as mean ± SD, n =4.

Figure 5

Growth curves of wild-type, TAM-2, and TAM-4 mutants under autotrophic conditions. Wild-type was grown with TAM-2 (a) or TAM-4 (b) and the cell content was measured once a day. All experiments were performed in 1-L cylinders, illuminated with 450 μmol photons m^-2 s^-1, 25°C. Growths were performed in a semi-batch system fed with air/CO₂ mix; the CO₂ supply was modulated in order to keep the pH of the medium always below 7.1.

Figure 6

Growth curves of wild-type, TAM-2, and TAM-4 mutants in outdoor photobioreactor. (a) Time-dependent course of cell concentration. All experiments were performed in 7-L hanging bag reactors, fed with air/CO₂ mix modulated in order to keep the pH of the medium always below 7.1. Two runs with nine reactors, each operated in parallel, were prepared. Data are expressed as mean ± SD, n =9. (b) Picture of the outdoor plant with 7-L reactors, showing growth stage of WT and TAM after two (left), four (center), and eight (right) days from initial inoculum.