CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species

Abstract

Microalgae represent a potential solution to reduce CO₂ emission exploiting their photosynthetic activity. Here, the physiologic and metabolic responses at the base of CO₂ assimilation were investigated in conditions of high or low CO₂ availability in two of the most promising algae species for industrial cultivation, Chlorella sorokiniana and Chlorella vulgaris. In both species, high CO₂ availability increased biomass accumulation with specific increase of triacylglycerols in C. vulgaris and polar lipids and proteins in C. sorokiniana. Moreover, high CO₂ availability caused only in C. vulgaris a reduced NAD(P)H/NADP⁺ ratio and reduced mitochondrial respiration, suggesting a CO₂ dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria. Several rearrangements of the photosynthetic machinery were observed in both species, differing from those described for the model organism Chlamydomonas reinhardtii, where adaptation to carbon availability is mainly controlled by the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. Acclimation strategies to cope with a fluctuating inorganic carbon supply are thus diverse among green microalgae, and these results suggest new biotechnological strategies to boost CO₂ fixation.

Keywords: Chlorophyta; carbon assimilation; chlorella; lipids; microalgae; photosynthesis; respiration; triacylglycerols.

FIGURE 1

Growth curve and biomass productivity in AIR versus CO₂. Growth curve and biomass productivity are reported for C. sorokiniana in panel a, c, e and for C. vulgaris in panel b, d, f in AIR condition (⁓0.04% CO₂) compared to CO₂ condition (3% CO₂). (a, b): growth curve obtained measuring OD at 720 nm fitted with sigmoidal function. (c, d): first derivate of growth curves reported in panels a and b. (e, f) Dry weight (g/L), average and maximum daily productivity (g/L day⁻¹) obtained harvesting the biomass at the end of the growth curve. Data are means of four replicates and error bars represent standard deviation [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Starch, lipids and protein content in AIR versus CO₂. Relative starch, protein and lipid content per dry weight in C. sorokiniana (Panel a) and C. vulgaris (Panel b) in AIR versus CO₂ condition. Data are means of three biological replicates with standard deviation shown. Significantly different values, respectively, for starch, protein and lipids content per dry weight in CO₂ versus AIR or in C. vulgaris versus C. sorokiniana are indicated with different letters (p < .05, n = 3) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Lipid composition and fatty acids profile in AIR versus CO₂ conditions. Lipids composition of C. sorokiniana (panel a–c) and C. vulgaris (panel d–f) cells grown in AIR or CO₂ conditions. Panel a and d: lipid composition in AIR versus CO₂ condition in terms of phospholipids, galactolipids, DGTS and triacylglycerol (TAG). Panel b and e: Fatty acids profile obtained by gas chromatography. Panel c and f: Polar lipid profile obtained by thin layer chromatography. Data are means of three biological replicates with standard deviation shown. Significantly different values in CO₂ versus AIR are indicated by * (p < .05). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DGTS, diacylglycerol N,N,N‐trimethylhomoserine [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Chlorophyll content and PSII maximum yield in AIR versus CO₂ conditions. (a) Chlorophyll content per cell, (b) chlorophyll a/b ratio and (c) PSII maximum quantum yield expressed as F_v/fm = (F_m − F₀)/F_m in C. sorokiniana (grey colour) and C. vulgaris (red colour) in AIR (full colour) or CO₂ (dash colour) condition. Data are means of three biological replicates with standard deviation shown. Significantly, different values in CO₂ versus AIR are indicated by * (p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Analysis of PSI, PSII and LHCII content by immunoblots, P700 activity and functional PSII antenna size. (a, b) Immunoblot analysis of PSI (α‐PsaA antibody), PSII (α‐CP43 antibody) and LHCII (α‐LHCII antibody). Loading was performed on a chlorophyll basis: total μg of chlorophylls loaded in each lane is reported on the top of Panel a and b. (c, d) PSI/PSII (c) and LHCII/PSII (d) ratios calculated by densitometry of immunoblot signals for C. sorokiniana (panel a, grey colour) and C. vulgaris (panel b, red colour) in AIR (full colour) or CO₂ (dash colour) condition. (e) Maximal P700 oxidation on a chlorophyll basis in C. sorokiniana (left, grey colour) and C. vulgaris (right, red colour) in AIR (full colour) or CO₂ (dash colour) normalized to AIR condition. (f) Functional antenna size of the photosystem II (1/τ_2/3) normalized to AIR condition in C. sorokiniana (grey colour) and C. vulgaris (red colour). Data are means of three biological replicates with standard deviation shown. Significant different values in CO₂ versus AIR are indicated by ** (p < .01) and by * (p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

State transitions in AIR versus CO₂. State transition analysis by 77 K fluorescence emission spectra in state 1 (S1, red lines) or state 2 (S2, blue lines) conditions in C. sorokiniana (a–c) and C. vulgaris (d–f) in AIR (A, D) or CO₂ (b, e). S1 was induced by shaking vigorously cells in a low light (⁓5 μmol m² s⁻¹) with 10 μm of DCMU for at least 15 min to oxidize the plastoquinone pool while S2 was induced by adding 250 μm sodium azide to inhibit mitochondrial respiration and to reduce the plastoquinone pool as described in Fleischmann et al. (1999). Black lines are related to cells harvested in the different growing conditions (AIR or CO₂) and dark adapted for 1 min before freezing at 77 K. maximum capacities for state transitions were then quantified from the maximum fluorescence emission at 720 nm as (F_S2 − F_S1)/F_S2. Data reported are means of three biological replicates with standard deviation shown. Significant difference in CO₂ versus AIR are indicated with different letters (a, b, or c, p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 7

ATPase content and electrochromic shift in AIR versus CO₂. Immunoblot analysis of ATPase content (atpC subunit antibodies) and ECS measurements in C. sorokiniana and C. vulgaris in AIR (solid line,) or CO₂ (dashed line) condition. ECS results in presence of DCMU are also reported with open symbol. Data are means of three biological replicates with standard deviation shown. Significantly, different values in CO₂ versus AIR are indicated by * (p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 8

NAD(P)H formation rate in AIR versus CO₂. Light dependent rate of NAD(P)H formation upon exposure to light (300 μmol photons m⁻² s⁻¹) for 120 s in C. sorokiniana (grey colour) and C. vulgaris (red colour) in AIR (full colour) or CO₂ (dash colour) condition. The data reported were calculated from the slope of the NAD(P)H fluorescence emission curve upon exposure to actinic light. Data are means of three biological replicates with standard deviation shown. Significantly, different values in CO₂ versus AIR are indicated by * (p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 9

Dark respiration in C. sorokiniana and C. vulgaris in AIR versus CO₂ conditions. The relative contribution of cytochrome (filled bars) and alternative respiration (empty bars) was reported normalized to cell content. Data are means of three biological replicates with standard deviation shown. Significant difference in CO₂ versus AIR are indicated by * (p < .05) [Colour figure can be viewed at wileyonlinelibrary.com]