Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana

Abstract

In this work, we investigated the molecular basis of autotrophic vs. mixotrophic growth of Chlorella sorokiniana, one of the most productive microalgae species with high potential to produce biofuels, food and high value compounds. To increase biomass accumulation, photosynthetic microalgae are commonly cultivated in mixotrophic conditions, adding reduced carbon sources to the growth media. In the case of C. sorokiniana, the presence of acetate enhanced biomass, proteins, lipids and starch productivity when compared to autotrophic conditions. Despite decreased chlorophyll content, photosynthetic properties were essentially unaffected while differential gene expression profile revealed transcriptional regulation of several genes mainly involved in control of carbon flux. Interestingly, acetate assimilation caused upregulation of phosphoenolpyruvate carboxylase enzyme, enabling potential recovery of carbon atoms lost by acetate oxidation. The obtained results allowed to associate the increased productivity observed in mixotrophy in C. sorokiniana with a different gene regulation leading to a fine regulation of cell metabolism.

Figure 1

Growth curves, biomass productivity and accumulation of macromolecules in mixotrophy vs. autotrophy. Panel A: growth curves of C. sorokiniana growth in autotrophy vs. mixotrophy fitted with sigmoidal curves; Panel B: first derivate of growth curves reported in Panel A; Panel C: dry weight and average daily productivity; Panel D: relative protein, lipid and starch content per cell or volume of culture.

Figure 2

Photosynthetic properties of C. sorokiniana in autotrophy vs. mixotrophy. Panel A: PSII efficiency (Fv/Fm) variation during cultivation; Panel B: net oxygen evolution curves at different light intensities fitted with hyperbolic functions; Panel C: NPQ induction curves measured at 1500 µmol m⁻²s⁻¹. Panel D: proton motive force (PMF) induced by 940 µmol m⁻²s⁻¹ in autotrophic and mixotrophic cells. Chemical (ΔpH) and electric (ΔΨ) components of PMF are indicated.

Figure 3

Annotation of C. sorokoniana transcriptome. C. sorokiniana transcripts annotated by blast2Go were functionally grouped on the basis of Gene Onthology (GO) terms “cellular component”, “molecular function” and “biological processes”. The distribution of the different groups is reported on the basis of the node score associated to each group considering GO term with node score higher than 1%.

Figure 4

Heat map of selected genes differentially expressed in autotrophy vs. mixotrophy. The expression of genes involved in selected pathways modulated in autotrophy vs. mixotrophy is shown as log-transformed and mean normalized read counts for each sample analysed (blue, lower abundance; orange, higher abundance).

Figure 5

Western blot analysis of chlorophyll binding proteins. PSI, PSII, LHCII and PsbS accumulation in mixotrophy vs. autotrophy were investigated by immunoblot analysis. In the case of PSI and PSII their relative accumulation was investigated using antibody recognizing PsaA (subunit of PSI) and CP43 (subunit of PSII). Mitochondrial COX2 subunit was also quantified as a control. Samples were loaded on SDS-PAGE gels in different chlorophyll concentration reported in Panel A. Each immunoblotting analysis was performed loading samples from autotrophy and mixotrophy cultures on the same gel. PsaA, CP43 and LHCII immunoblotting were performed on the same filter cut between 30- and 40 KDa and between 50- and 60 KDa, while PSBS and COX2 immunoblotting analysis where performed on different filters. Quantification of resulting bands were performed by densitometry and normalized to the autotrophy case (Panel B). Significantly different data are indicated (n = 3; P < 0.05).

Figure 6

Model of metabolic pathways in autotrophy vs. mixotrophy in C. sorokiniana. Metabolic pathways are reported in yellow if not transcriptionally regulated, in red or blue if down or upregulated in mixotrophy respectively. PEP: phosphoenol-piruvate; RAF: RUBISCO accumulation factor; G3P: Glyceraldehyde 3-phosphate; PDK: pyruvate dehydrogenase kinase; PC: plastocianine; Fd: ferredoxin; FNR: Ferredoxin-NADP⁺ reductase; PPC: phosphoenolpyruvate carboxylase; ALDH: aldehyde dehydrogenase; GOX: glycolate oxidase; SHMT: serine hydroxymethyltransferase; AOX: alternative oxidase; PPDK: pyruvate-orthophosphate dikinase; AspAT: aspartate aminotransferase.