Gilles Curien
email: gilles.curien@cea.fr

Dagmar Lyska

Erika Guglielmino

Phillip Westhoff

Janina Janetzko

Marianne Tardif

Clément Hallopeau

Sabine Brugière

Davide Dal

Johan Decelle

Benoit Gallet

Denis Falconet

Michele Carone

Claire Remacle

Myriam Ferro

Andreas P M Weber

Giovanni Finazzi

Mixotrophic growth of the extremophile galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism

Keywords: Galdieria sulphuraria, mixotrophy, photorespiration, photosynthesis, red algae

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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words count for Results: 2048 words count for Discussion: 1059 5 figures (all in color) 1 supporting information file (with 12 figures) 3 supporting datasets

Introduction

The unicellular red alga Galdieria sulphuraria belongs to the Cyanidiophyceae, a class that includes five species often flourishing in different extreme environments [START_REF] Merola | Revision of Cyanidium caldarium. Three species of acidophilic algae[END_REF]Gross et al., 1998;Gross & Oesterhelt, 1999;[START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF]. From a phylogenetic perspective, plastids of the red algae gave rise to the complex plastids of, e.g., diatoms via secondary endosymbiosis [START_REF] Cho | Characterization of a non-thermophilic strain of the red algal genus Galdieria isolated from Soos (Czech Republic)[END_REF][START_REF] Bhattacharya | Photosynthetic eukaryotes unite: endosymbiosis connects the dots[END_REF]Yoon et al., 2004). Like other members of this class (Doemel & Brock, 1971;[START_REF] Reeb | The thermo-acidophilic Cyanidiophyceae (Cyanidiales)[END_REF] G. sulphuraria has an extremophile lifestyle, withstanding low pH (pH optimum at 2) and elevated temperatures (up to 56°C). It thrives in soils and forms biomats on rocks surrounding hot springs, fumaroles, or acid mining sites and even on burning coal spoil heaps [START_REF] Moreira | Characterization of 2 new thermoacidophilic microalgae -genome organization and comparison with Galdieria sulphuraria[END_REF]Gross et al., 1998;[START_REF] Castenholz | The Cyanidiales: Ecology, Biodiversity, and Biogeography[END_REF][START_REF] Barcyté | Burning coal spoil heaps as a new habitat for the extremophilic red alga Galdieria sulphuraria[END_REF]. Some mesophilic species have been isolated from environments with moderate temperatures and/ or a neutral pH [START_REF] Cho | Characterization of a non-thermophilic strain of the red algal genus Galdieria isolated from Soos (Czech Republic)[END_REF]Yoon et al., 2006;[START_REF] Azúa-Bustos | Ancient photosynthetic eukaryote biofilms in an Atacama desert coastal cave[END_REF][START_REF] Iovinella | Cryptic dispersal of Cyanidiophytina (Rhodophyta) in non-acidic environments from Turkey[END_REF]. Genome analysis [START_REF] Barbier | Comparative genomics of two closely related unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae[END_REF][START_REF] Schonknecht | Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote[END_REF]Rossoni et al., 2019a) has pinpointed a very high metabolic flexibility of this alga, which is confirmed by its ability to grow in photoautotrophy (exclusive use of light as an energy source) and heterotrophy (organic carbon respiration of more than 50 different substrates, [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF]). This capacity, along with the peculiar pH optimum for growth, allow G. sulphuraria to be cultivated in open ponds containing organic matter, overcoming other microorganisms, considered as contaminants in this case. Given these advantages for large-scale cultivation, G. sulphuraria is considered an emerging system for biotechnology applications [START_REF] Schmidt | Heterotrophic high cell-density fedbatch cultures of the phycocyanin-producing red alga Galdieria sulphuraria[END_REF][START_REF] Henkanatte-Gedera | Removal of dissolved organic carbon and nutrients from urban wastewaters by Galdieria sulphuraria: Laboratory to field scale demonstration[END_REF]Cizkova et al., 2019).

The relationship between photosynthesis and glycolysis/respiration in higher plants and microalgae is complex [START_REF] Avelange | Massspectrometric determination of O 2 and CO 2 gas exchange in illuminated higher-plant cells[END_REF][START_REF] Kromer | Mitochondrial oxidative phosphorylation participating in photosynthetic metabolism of a leaf cell[END_REF]Gemel & Randall, 1992;[START_REF] Pärnik | Decarboxylation of primary and end products of photosynthesis at different oxygen concentrations[END_REF][START_REF] Hoefnagel | Interdependence between chloroplasts and mitochondria in the light and the dark[END_REF][START_REF] Tcherkez | Respiratory metabolism of illuminated leaves depends on CO 2 and O 2 conditions[END_REF]. Photosynthesis/glycolysis/respiration interactions are prone to perturbation by mixotrophy, in which external organic carbon often interferes with carbon flow between chloroplasts, the cytosol and mitochondria. Several green microalgae are capable of mixotrophic growth (Combres et al., 1994;[START_REF] Wan | The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana[END_REF][START_REF] Johnson | Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii[END_REF][START_REF] Cecchin | Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana[END_REF]. While mixotrophy has always beneficial consequences on respiration, its effects on photosynthesis differ depending on the microalga considered: enhancement of photosynthesis was reported in one case (Ettlia oleoabundans, (Ferroni et al., 2018)), while in other algae, including the diatoms Phaeodactylum tricornutum [START_REF] Liu | Effects of organic carbon sources on growth, photosynthesis, and respiration of Phaeodactylum tricornutum[END_REF][START_REF] Villanova | Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum[END_REF] and Nannochloropsis (Fang et al., 2004;Xu et al., 2004) photosynthesis was unaffected. Decreased photosynthetic activity in mixotrophy has been reported in Chlorella vulgaris [START_REF] Martinez | Interactions between glucose and inorganic carbon metabolism in Chlorella vulgaris strain UAM-101[END_REF][START_REF] Cecchin | Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana[END_REF] and Chlamydomonas reinhardtii where the Carbon Concentrating Mechanism (CCM, [START_REF] Bogaert | Metabolic, physiological, and transcriptomics analysis of batch cultures of the green microalga Chlamydomonas grown on different acetate concentrations[END_REF]) and the light harvesting capacity [START_REF] Perrineau | Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii[END_REF] is decreased by acetate along with the enhancement of respiration. While it has been reported that G. sulphuraria 074G could grow in the simultaneous presence of light and a carbon source, heterotrophy seemed to prevail in these conditions, as no photosynthetic O 2 production could be measured in the presence of glucose [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF].

Here, we show instead that photosynthesis and carbon metabolism (glycolysis and respiration) operate simultaneously in Galdieria sulphuraria SAG21.92 (a close relative of the 074G strain) under mixotrophic conditions, provided that the temperature conditions are kept close to the ones experienced by this alga in its natural environment. We show that performances in mixotrophy, exemplified by photosynthetic activity and biomass production, actually exceed the sum of the heterotrophic and photoautotrophic yields under limiting inorganic carbon. This synergistic effect stems from a stimulation of photosynthesis by CO 2 of respiratory origin, which overcomes the limitation to the Calvin-Benson-Bassham cycle and suppresses photorespiration.

Limitation originates from the very low inorganic carbon concentration available in acidic conditions (pH 2) that constitute the alga's natural growth environment.

Notably, under these conditions, inorganic carbon is almost exclusively available as dissolved CO 2 (around 10 µM) while soluble bicarbonate is virtually absent.

Consistent with this hypothesis, the synergistic effect of 'light' and 'dark' energetic 6 metabolisms is sensitive to respiration inhibitors and is lost upon addition of exogenous CO 2 , which outcompetes endogenous CO 2 of respiratory origin in relieving inorganic carbon limitation of the Calvin-Benson-Bassham cycle. We conclude that mixotrophy constitutes an efficient mechanism to increase intracellular CO 2 concentration under physiological conditions, allowing G. sulphuraria to successfully exploit all the energy resources available for growth in its rather challenging environment.

Materials and Methods

Strains, growth and media composition

Galdieria sulphuraria SAG21.92 and 074G were obtained from the Culture Collection of Algae at Göttingen University (SAG) and were grown in sterile 2xGS modified Allen medium, pH 2.0, containing 20 mM of NaNO 3 , and 5 mM of inorganic phosphate (K 2 HPO 4 and KH 2 PO 4 in a 2/1 ratio [START_REF] Allen | Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte[END_REF]) at 42°C without or with organic substrates as indicated in the text. The concentration of organic substrates was selected on the basis of data reported in literature [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF]. More precisely, glucose was employed at the concentration of 25 mM, as in [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF]. The concentration of all the other organic compounds was adjusted to reach the same carbon atom concentration (150 mM). G. sulphuraria was grown either in 250 mL flasks (50 mL culture volume), in an incubator (Infors, Switzerland, continuous light, 30 µmol photons m -2 s -1 , 42°C, 100 rpm) or in a photobioreactor (Multicultivator, Photon System Instruments, Czech Republic). Inside the multicultivator, cells were provided with air or CO 2 -enriched air by active bubbling (see Methods S1).

Moreover, the incident light intensity was adjusted daily to maintain constant transmitted light through the culture (see Results). This 'luminostat' regime ensures maximal absorption of light without allowing a dark zone to develop inside the photobioreactor (Cuaresma et al., 2011). Growth was monitored daily by cell counting with a LUNA cell counter (Logos Biosystems, Inc. USA). Sorbitol consumption was measured using the D-Sorbitol/Xylitol assay kit (Megazyme).

Cell fresh weight and dry weight quantification

Cell pellet was resuspended in a small volume of water and centrifuged in preweighed Eppendorf tubes and pellet was weighed. For dry weight determination fresh cells pellets were dried for three days at 60°C, weighed and expressed as g.L -1 .

Clark electrode oxygen measurements

Oxygen exchanges in solution were measured with a Clark-type electrode (Hansatech Instruments, UK) at 42°C. Respiration and gross photosynthesis were quantified by measuring the slope of O 2 changes in the dark and under light exposure, respectively. Net photosynthesis was calculated assuming O 2 consumption by the mitochondrion in the light is identical to that in the dark (Net photosynthesis = v O2light +|v O2dark |).

Photophysiology measurements

Photosynthetic parameters were derived from quantification of chlorophyll fluorescence emission by cultures within the multicultivator. To this aim, we employed a custom-made fluorescence imaging system based on a previously published setup [START_REF] Johnson | A new setup for in vivo fluorescence imaging of photosynthetic activity[END_REF] modified as described in Methods S1. The photosynthetic electron transfer rate, ETR (a proxy of the carbon assimilation capacity [START_REF] Maxwell | Chlorophyll fluorescence -a practical guide[END_REF]) was calculated as (Fm'-Fs)/Fm'*PFD, where Fm' and Fs are the fluorescence intensities measured after exposure to a saturating pulse and in steady state, respectively, in light-acclimated cells and PFD (Photosynthetic Flux Density) is the incident light intensity, measured in µmol photons m -2 s -1 . The cells were allowed to reach steady state fluorescence emissions at each intensity (5-10 minutes of light exposure depending on the intensity) before increasing the photon flux.

Biochemistry and Proteomic analysis

Western blot analysis was performed on cells grown for 7 days in the indicated conditions. Cells (10 9 cells) were broken with a Precellys homogenizer (Bertin, France), through three cycles of 30 seconds at 10,000 rpm separated by a 30seconds interval. Total protein extracts were analyzed by immunoblotting with an anti-RuBisCO antibody (Agrisera, Sweden). An antibody against the β subunit of the ATPsynthase complex (Agrisera, Sweden) was used as a loading control. 10 µg of protein was loaded per well.

For proteomic analysis, algae were cultivated under the three conditions photoautotrophy, mixotrophy and heterotrophy in parallel in the same cultivator.

Three multicultivator experiments were carried out one week apart and constituted the three independent biological replicates. Cells were collected on day 7 (i.e. 4 days after addition of 25 mM D-sorbitol to mixotrophic and heterotrophic cultures).

Proteins from whole cell extracts (40 µg each) were solubilized in Laemmli buffer before being stacked in the top of a 4-12% NuPAGE gel (Life Technologies, US), stained with R-250 Coomassie blue (Bio-Rad, US) and in-gel digested using modified trypsin (sequencing grade, Promega, US) as previously described [START_REF] Bouchnak | Unraveling hidden components of the chloroplast envelope proteome: Opportunities and limits of better MS sensitivity[END_REF]. Resulting peptides were analyzed by online nanoLC-MS/MS (Ultimate 3,000 RSLCnano coupled to Q-Exactive HF, Thermo Fisher Scientific, US) using a 200-min gradient. Peptides and proteins were identified using Mascot (version 2.6.0, Matrix Science). Spectra were searched against Uniprot (G. sulphuraria taxonomy, July 2019 version, 7347 sequences) concomitantly with a home-made list of contaminants frequently observed in proteomics analyses (trypsins and keratins, 250 sequences).

The Proline software [START_REF] Bouyssie | Proline: an efficient and user-friendly software suite for large-scale proteomics[END_REF] was used to filter the results (conservation of rank 1 peptides, peptide identification FDR < 1% as calculated on peptide scores by employing the reverse database strategy, minimum peptide score of 25, and minimum of 1 specific peptide per identified protein group). Proline was then used to extract the MS1-based intensities values of protein groups from unique peptides. Proteins identified in the reverse and contaminant databases (i.e. trypsin or keratin), and proteins identified with only 1 peptide with a score <40 were further discarded from the list. Proteins identified in only one or two conditions were kept for analysis without statistical treatment. Proteins identified in all three conditions were submitted to statistical differential analysis using ProStaR [START_REF] Wieczorek | DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics[END_REF][START_REF] Wieczorek | Protein-level statistical analysis of quantitative label-free proteomics data with ProStaR[END_REF]. Detailed procedures are described in Methods S1.

Metabolite extraction and analysis

Cells grown in a multicultivator were harvested at day 5 by centrifugation (4°C, 5 min and 3,000 rcf), washed with ice-cold 0.9 % (w/v) NaCl, snap frozen in liquid nitrogen, and lyophilized overnight. Cells (5 x 10 8 ) were disrupted in a Mixer Mill (MM 400,Retsch GmbH,Haan,Germany) for 60 seconds at 30 Hz using metal beads and 500 µL of ice-cold chloroform and methanol (1:2.3 ratio; containing 5 µM Ribitol and 2,4-Dimethylphenylalanine (both Sigma-Aldrich, Munich, Germany) as internal standards) with another round of shaking in the Mixer Mill. After a 2 h incubation at -20°C, 400 µL of ice-cold deionized water (Milli Q, Merck Chemicals GmbH, Darmstadt, Germany) were added to induce phase separation. The samples were vortexed and centrifuged for 5 min at 4°C and 16,000 rcf. The aqueous phase was transferred to a new reaction tube and the organic phase was re-extracted with 400 µL of ice-cold deionized water. Aqueous phases of each sample were combined and lyophilized overnight. After resuspension in 500 µL of 50% methanol 50 µL were dried in a glass inlet for analysis by GC-MS and IC-MS.

For GC-MS analysis the samples were prepared and analyzed as described [START_REF] Gu | Identifying core features of adaptive metabolic mechanisms for chronic heat stress attenuation contributing to systems robustness[END_REF] and [START_REF] Shim | Loss of function of rice plastidic glycolate/glycerate translocator 1 impairs photorespiration and plant growth[END_REF]. Identification of metabolites was performed with MassHunter Qualitative (v b08.00, Agilent Technologies) by comparing spectra to the NIST14 Mass Spectral Library (https://www.nist.gov/srd/niststandard-reference-database-1a-v14) and to a quality control sample containing all target compounds. Peaks were integrated using MassHunter Quantitative (v b08.00, Agilent Technologies). For relative quantification, all metabolite peak areas were normalized to the peak area of the internal standard ribitol.

For IC-MS a combination of a Dionex ICS-6000 HPIC and a high field Thermo Scientific Q Exactive Plus quadrupole-Orbitrap mass spectrometer following the method described in [START_REF] Schwaiger | Anion-exchange chromatography coupled to high-resolution Mass Spectrometry: A powerful tool for merging targeted and non targeted metabolomics[END_REF] and in Methods S1. Data Analysis was conducted using Compound Discoverer (version 3.1, Thermo Scientific).

Setting parameters for the untargeted metabolomics workflow and peak annotation criteria are described in Methods S1.

Labelling experiments with 13 C-glucose

Cells were cultivated in 250 mL Erlenmeyer flasks (50 mL culture volume) for four days under continuous light at 60 µmol m -2 s -1 , 40 °C and ambient air (0.04% CO 2 ). U-13 C 6 -glucose (Cambridge Isotope Laboratories Inc, Tewksbury, Massachusetts) was added at day 4 in a final concentration of 25 mM and the irradiance was increased to 100 µmol m -2 s -1 either under ambient or elevated (2%) CO 2 conditions. 1-2.5 x10 8 cells were harvested 1, 4, 12, 24, 36, 48 and 60 hours after glucose addition as described above. Metabolites were extracted and measured by IC-MS as described above.

Data analysis was conducted with Compound Discoverer (version 3.1, Thermo Scientific) and the standard workflow for stable isotope labelling from Compound Discoverer was chosen. The default settings, which are 5 ppm mass tolerance, 30 % intensity tolerance and 0.1 % intensity threshold for isotope pattern matching were used and the maximum exchange rate was set to 95%.

Results

G. sulphuraria metabolises several organic substrates.

Previous work [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF] has suggested that G. sulphuraria is unable to grow mixotrophically in the presence of light plus organic carbon but rather alternates between heterotrophy (in the presence of an external source of organic carbon) and phototrophy (upon inorganic carbon consumption). This conclusion was based on experiments carried out at 25°C, i.e. a temperature that is far from the physiological optimum of this alga (above 40°C), and therefore decreases photosynthetic performances (Doemel & Brock, 1971;Ford, 1979;Rossoni et al., 2019b;Rossoni & Weber, 2019). Thus, we decided to reinvestigate the possible occurrence of mixotrophy under conditions that resemble natural growth conditions (42°C, pH 2.0) in which G. sulphuraria displays maximum photosynthetic capacity.

First, we sought compounds that could improve algal growth in presence of light.

We found that several hexoses, disaccharides and pentoses, but also some polyols and amino acids (L-alanine, L-glutamate) were able to boost G. sulphuraria's growth when compared to strict photoautotrophic conditions (Fig. S1). Conversely, some organic acids (malic and citric acid) and amino acids (Laspartate, L-leucine, L-valine, L-isoleucine, L-asparagine) had either no effect or led to a growth inhibition when compared to photoautotrophic conditions. Acetic acid was lethal to the cells. Compound concentrations were 25 mM, i.e., the same value employed in the study by Oesterhelt and colleagues. In the case of disaccharides, we reduced the concentration by a factor of two, to keep the overall carbon atoms concentration constant.

Based on these results, we focused on sorbitol, a compound that boosts growth in the dark without inducing a significant loss of photosynthetic pigments [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF]. We confirmed that sorbitol was able to sustain cell division in the dark (Fig. 1), but its effect on growth was largely enhanced by a concomitant exposure to light. The final dry weight and number of cells collected at the end of the exponential phase in the light plus carbon condition (orange symbols) exceeded the sum of cells obtained in the heterotrophic (black) plus the photoautotrophic (green) conditions (Fig. 1a-b). This finding indicates that G. sulphuraria is not only able to perform true mixotrophy under high temperature, dim light and presence of external organic carbon sources, but that this trophic mode is highly beneficial for its growth capacity. We obtained similar effects replacing sorbitol with a monosaccharide (glucose) and a disaccharide (saccharose) (Fig. S2).

Respiration boosts photosynthesis in mixotrophic Galdieria cells.

To further characterize the consequences of mixotrophy in G. sulphuraria, we transferred half of the cells grown in mixotrophy for five days (Fig. 1) to heterotrophic condition and monitored growth of the four samples (photoautotrophic: green, heterotrophic: black, mixotrophic: orange and mixotrophic transferred to heterotrophy: grey) for another five days. While the mixotrophic cells continued to display a higher growth capacity than the heterotrophic plus photoautotrophic ones, the mixotrophic cells transferred to the dark (grey) slowly reached the same cell density (Fig. 1a) and dry weight (Fig. 1b) as the heterotrophic culture, i.e., they lost the benefits provided by the simultaneous exposure to light and organic carbon within a few days. We conclude therefore that mixotrophy promotes a synergistic interaction between light and dark energy metabolisms, which slowly disappears when the light supply is halted.

In the experiments described above, photoautotrophic growth was most likely limited by the low light intensity (30 µmol photons m -2 s -1 ), which was kept constant during growth, and therefore rapidly became limiting for photosynthesis when the cell concentration was increased in the flasks. Gas diffusion could also be limiting in flasks. Therefore, we repeated experiments in a photobioreactor (Multicultivator, PSI, Czeck Republic, Fig. S3), where air bubbling ensured a more efficient gas delivery to the algae. Moreover, the intensity of the incident light was progressively increased in this setup to maintain a linear relationship between the cell number and the absorption. We reproduced the improvement of biomass productivity in mixotrophy with the photobioreactor (Fig. 2), where we could monitor biomass production and photosynthetic performances on actively growing cells using a custom-built fluorescence imaging setup to monitor photosynthesis via the electron transfer rate (ETR) parameter [START_REF] Maxwell | Chlorophyll fluorescence -a practical guide[END_REF] (Fig. S3).

The ETR in the photoautotrophic cultures was lower than in mixotrophic ones (Fig. 2a). The difference was larger during the first days of culturing (Fig. S4, days 4 and 5, i.e. the first and second day after sorbitol addition), and then photosynthesis progressively diminished in the mixotrophic cells (Fig. S4, days 6 and 7, i.e. the third and fourth day of mixotrophy), where we also observed a large variability in the photosynthetic capacity (Fig. S4,day 7). We ascribe this variability to a differential consumption of sorbitol, and therefore of the mixotrophic synergistic effect, in the various samples. Consistent with this hypothesis, enhancement of photosynthesis was re-established in mixotrophic cultures, provided that sorbitol, nearly exhausted after four days (Fig. S5, day 7) was added again to the growth medium (Fig. S6).

To explain the synergistic effect of mixotrophy, we reasoned that photosynthesis could be limited by the availability of inorganic carbon in photoautotrophic cells.

The latter is present only as CO 2 and at very low concentration (10 µM, (Gross et al., 1998)) at ambient air in G. sulphuraria's cultures, due to the acidic pH (pH 2).

Mixotrophy could alleviate this limitation by providing extra CO 2 of mitochondrial origin via enhanced respiration (Fig. 2c, Fig. S7). We tested this hypothesis by two approaches. First, we poisoned mixotrophic and photoautotrophic cultures with myxothiazol and SHAM, known inhibitors of the cyanide sensitive and insensitive respiratory pathways, respectively, and measured consequences on photosynthetic activity. Addition of these inhibitors completely abolished the enhancement of photosynthetic activity and of biomass productivity by mixotrophy (Fig. 2d-f). Next, we increased the CO 2 availability to the cells in mixotrophic cultures, to outcompete endogenous CO 2 of respiratory origin with an excess of exogenous inorganic carbon. As a prerequisite for this experiment, we calibrated the CO 2 requirement for optimum photosynthesis in our growth conditions (transmitted light of 10 µmol photons m -2 s -1 ). We found that photoautotrophic growth was increased by CO 2 up to a concentration of 2% CO 2 (Fig. S8a), the apparent affinity for CO 2 being 0.5 % ca. Upon addition of external CO 2 , the photosynthetic capacity in photoautotrophic conditions increased and the biomass produced in heterotrophic plus photoautotrophic conditions became equal to that observed in mixotrophy (Fig. 2g-i). Overall, these data indicate that the synergy between respiration and photosynthesis is lost when the respiration is inhibited or when the photosynthesis becomes saturated with externally supplied CO 2.

Moreover, we hypothesize that photorespiration should be decreased to very low rates at saturating CO 2 , possibly contributing to the gain in biomass productivity at high CO 2 concentrations. In these experiments, we also observed that the amount of biomass produced by phototrophic cells supplemented with air was the same irrespective of the light intensity employed (Fig S8b). Conversely, biomass production could be increased by increasing the light intensity in both phototrophic cells supplemented with 0.5% CO2 (Fig S8c) or in mixotrophic cells (Fig S8d).

These findings are fully consistent with the hypothesis that photosynthesis is CO 2 limited in air, and that this limitation can be alleviated by endogenous (mixotrophy) or exogenous CO 2 .

Metabolic acclimation to mixotrophy in G. sulphuraria.

The observation that mixotrophy enhances photosynthesis to a similar level as upon addition of external CO 2 to G. sulphuraria, suggests that mixotrophy behaves as a strategy to traffic CO 2 from the mitochondria to the plastid, allowing this alga to successfully exploit all the energy resources available for growth and to minimize energy loss through photorespiration.

To substantiate this hypothesis, we performed a complete survey of the metabolic changes between the three trophic lifestyles, and relate these changes to modifications in the cell proteome. We found that photosynthetic proteins (complexes of the electron transfer chain, enzymes of the Calvin-Benson-Bassham cycle and transporters involved in triose phosphate export) were downregulated in mixotrophic conditions compared to photoautotrophic conditions (Fig. 3), in agreement with previous suggestions [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF][START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF]. Conversely, respiratory protein levels remained relatively constant in the three conditions. This observation (photosynthetic activity is enhanced in mixotrophy despite the downregulation of the photosynthetic machinery) fully supports the conclusion that intracellular increase in CO 2 due to enhanced respiratory activity more than compensates for the decrease in Rubisco (see also Fig. S9).

In line with the hypothesis that mixotrophy channels CO 2 from respiration to photosynthesis, proteomic analysis indicates that all the enzymes involved in photorespiration (phosphoglycolate phosphatase, glycolate oxidase, serineglyoxylate aminotransferase, glycine decarboxylase, glycine/serine hydroxymethyltransferase, hydroxypyruvate reductase, glycerate kinase) were less abundant in mixotrophy. This is also true for Rubisco activase (Gasu_19410, dataset S1-2), an enzyme that was shown to be important under low CO 2 in Chlamydomonas [START_REF] Pollock | Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO 2 atmosphere[END_REF]. These findings are also corroborated by metabolite analysis (Fig. 4a). The amounts of the oxygenation product of Rubisco, 2-phosphoglycolate are highest under photoautotrophic conditions, lower in mixotrophic conditions, and very low in heterotrophic conditions. Glycine accumulates, likely due to a higher reduction potential inside the mitochondrial matrix under mixotrophic conditions, which will reduce the rate of oxidative decarboxylation of glycine by glycine decarboxylase. Consequently, the glycine to serine ratio was inverted compared to photoautotrophic conditions. The amounts of glycerate and 3-phosphoglycerate under mixotrophic conditions are mimicking those in heterotrophically grown cells (Fig. 4a).

Proteins involved in a putative C4-like carbon concentrating cycle (Rademacher et al., 2017) (Fig. 3, 4b) followed the same pattern as the photorespiratory enzymes.

Carbonic anhydrase and phosphoenolpyruvate carboxylase were more abundant under photoautotrophic conditions than under mixotrophic or heterotrophic conditions, and, in agreement with this finding, photoautotrophic cells also contained higher amounts of phosphoenolpyruvate (PEP) than mixotrophic or heterotrophic cells (Fig. 4b). The presence of this carbon concentrating cycle in photoautotrophic conditions could be supported by increased steady-state levels of phosphorylated C3 compounds, as suggested by the downregulation of pyruvate kinases in phototrophy (PEP consumption might be decreased) and strong induction of pyruvate ortho-phosphate dikinase (producing PEP). We assume that oxaloacetate (OAA) under photoautotrophic conditions is decarboxylated by PEP carboxykinase (PEPCK), and not after reduction into malate by malate dehydrogenase and decarboxylation by mitochondrial malic enzyme. This hypothesis is supported by the finding that mitochondrial malic enzyme (ME) is strongly decreased in photoautotrophy (Fig. 3) and the malate pool size is smaller under this condition than in mixo-or heterotrophy. We note that decarboxylation by PEPCK (as compared to ME) is energy conserving and directly yields PEP for a new round of carboxylation by PEP carboxylase.

While part of the increase in biomass production in mixotrophic conditions can be attributed to the repression of photorespiration, other benefits of the mixotrophic lifestyle may come from more carbon units being shuttled into anabolic pathways.

Indeed, we observed a general increase of metabolites of the oxidative pentose phosphate pathway (Ribulose-5P, Sedoheptulose-7P) and nucleoside triphosphates (ATP, GTP, UTP, CTP) as successor metabolites (Fig. 4c). The amounts do not differ between autotrophic and heterotrophic cultures but are clearly increased under mixotrophic conditions providing evidence for a higher flux of carbon at least into purine and pyrimidine synthesis.

Labelling experiments using 13 C-labelled glucose, a sugar with similar effects on cell growth and biomass production as sorbitol (Fig. S2), further support this notion.

We found a rapid incorporation of CO 2 from 13 C glucose into photosynthetic metabolites, such as Ribulose1,5-bisphosphate (RuBP), Sedoheptulose 1,7bisphosphate (SBP), and 2-phosphoglycolate (2-PG) (Fig. 5). While RuBP also occurs in the oxidative pentose phosphate pathway, SBP and 2-PG are metabolites solely formed in the Calvin-Benson-Bassham cycle and can only carry a label when 13 CO 2 released by respiration is fixed. Incorporation of the label by shuttling of carbon backbones from the cytosol into the chloroplast is unlikely since the solute transporters in the G. sulphuraria's chloroplast envelope do not favor the import of glycolytic intermediates under photosynthetic conditions [START_REF] Linka | Functional characterization of the plastidic phosphate translocator gene family from the thermo-acidophilic red alga Galdieria sulphuraria reveals specific adaptations of primary carbon partitioning in green plants and red Algae[END_REF]. The rate of incorporation of 13 C is much reduced when external CO 2 levels are increased to 2%. Interestingly, the tricarboxylic acid cycle intermediate succinate exhibits a similar pattern of delayed labelling under high CO 2 conditions when compared to ambient air, indicating a slowed down glucose usage. Thus, the delay of label incorporation into the Calvin-Benson-Bassham cycle intermediates can be traced back to lower levels of labelled respiratory CO 2 under CO 2 saturated conditions.

Discussion

At variance with a previous report [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF], G. sulphuraria cells are capable of a true mixotrophy, when transferred from strictly photoautotrophic conditions to a light and organic carbon regime, provided that the temperature conditions are kept close to the ones experienced by this alga in its natural environment. This is not only true for the G. sulphuraria SAG21.92 strain used here, but also for G. sulphuraria 074G, i.e., the strain used in the previous study by [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF] when tested in the presence of the same external organic carbon sources employed here (See Fig. S10). Acclimation of the strictly photoautotrophic Cyanidiophyceae Cyanidioschyzon merolae to suboptimal growth temperatures of 25°C, led to massive rearrangements of the photosynthetic apparatus and a lower photon-to-oxygen conversion rate when compared to cells grown at 42°C [START_REF] Nikolova | Temperature-induced remodeling of the photosynthetic machinery tunes photosynthesis in the thermophilic alga Cyanidioschyzon merolae[END_REF]. Also, cultivation of G. sulphuraria at 25°C led to a downregulation of transcripts encoding components of the photosynthetic machinery (Rossoni et al., 2019b). Therefore, cultivation at suboptimal temperatures may not be in favor of high photosynthetic rates and drive Cyanidiophyceae species capable of heterotrophic growth towards this trophic state.

Mixotrophy deeply alters the carbon metabolism of G. sulphuraria cells. Under photoautotrophic conditions, CO 2 is mainly concentrated by the PEPC/PEPCK driven CCM [START_REF] Rademacher | Transcriptional response of the extremophile red alga Cyanidioschyzon merolae to changes in CO 2 concentrations[END_REF]. This process is repressed in mixotrophy.

Moreover, enhanced respiration relieves the limitation of photosynthesis by inorganic carbon, which is at a low concentration (µmolar range) in the acid growth medium of this alga. We can quantify the extent of this process using the Clark electrode (as in Fig S7 ) in a closed configuration, to avoid gas exchanges with the atmosphere (representative traces in Fig S11a,c). In this case, the amount of O 2 produced by photosynthesis (32.3 ± 7.5 µM and 76.6 ± 2 µM in photoautrotrophy and mixotrophy, respectively) is commensurate with the amount of CO 2 available to Rubisco, i.e. the sum of respiratory CO 2 (22 ± 10.5 µM and 61 ± 1.5 µM respectively, assuming a 1/1 stoichiometry with consumed O 2 ) plus the small CO 2 amount present in the medium (10 µM ca at pH 2) (Fig. S11c,d). These estimates clearly indicate that respiration in mixotrophy sets the rate of photosynthesis under limited CO 2 conditions. The proximity between mitochondria and chloroplasts (highlighted in red and green, respectively, in Fig. S12) may favor this process, facilitating intracellular gas exchange between the two cell organelles, as already shown in the case of other microalgae [START_REF] Lavergne | Mitochondrial responses to intracellular pulses of photosynthetic oxygen[END_REF].

At the same time, increased intracellular CO 2 concentration is expected to lower the rate of photorespiration in mixotrophy. Although the Rubisco enzymes form Cyanidiophyceae show some of the highest carboxylation specificities reported to date [START_REF] Sugawara | Crystal structure of carboxylase reaction-oriented ribulose 1,5bisphosphate carboxylase oxygenase from a thermophilic red alga, Galdieria partita[END_REF], photorespiration is expected to proceed at high rates at the high temperatures and low CO 2 concentrations under which Galdieria grows.

Indeed, knockout of peroxisomal glycolate oxidase in the transformable Cyanidiales alga C. merolae demonstrated that a functional photorespiratory pathway is essential for survival of these algae under ambient CO 2 concentrations [START_REF] Rademacher | Photorespiratory glycolate oxidase is essential for the survival of the red alga Cyanidioschyzon merolae under ambient CO 2 conditions[END_REF]. The observed increase in the rate of photosynthesis and gain of biomass under high CO 2 and mixotrophic conditions (25 ± 6 %, when combining data from Fig. 12) is consistent with overcoming the expected loss of biomass gain due to photorespiration under photoautotrophic conditions.

In conclusion, by bypassing the possible metabolic antagonism between respiration and photosynthesis, G. sulphuraria can exploit the plethora of transporters encoded by its genome [START_REF] Schonknecht | Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote[END_REF]Rossoni et al., 2019a) and import organic carbon available in its environment and to boost CO 2 availability for photosynthesis. While this phenomenon certainly exists in other phototrophs (e.g. [START_REF] Rolland | Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas[END_REF], the capacity to enhance photosynthesis with respiratory CO 2 when the latter process is increased by exogenous carbon sources could be particularly relevant in G. sulphuraria. This alga thrives in an extreme environment, where growth is limited by low pH, high temperature and possibly light availability. Dissolved inorganic carbon is very low in this hot and acidic milieu (in fact its concentration inversely correlates with the pH of different collection sites in Yellowstone National Park) [START_REF] Boyd | Modeling the habitat range of phototrophs in Yellowstone National Park: toward the development of a comprehensive fitness landscape[END_REF][START_REF] Hamilton | Environmental constraints defining the distribution, composition, and evolution of chlorophototrophs in thermal features of Yellowstone National Park[END_REF] and its light-driven uptake is fairly low when compared to alkaline thermal habitats. Conversely, the concentration of dissolved organic carbon can be relatively high in the acidic hot springs (from 17 µM to 3 mM [START_REF] Nye | A novel PARAFAC model for continental hot springs reveals unique dissolved organic carbon compositions[END_REF]).

Values could become much higher when this alga proliferates in mats (Gross et al., 1998), where by-products of every group of microorganisms may serve as "food" for other groups.

Based on these considerations, it is tempting to speculate that while photosynthesis should allow cells to colonize new environments devoid of any organic carbon source, the peculiar division mode of G. sulphuraria (formation of endospores associated with the release in the media of the mother cell wall remnants), may favor mixotrophy on a longer time scale. In the acidic conditions where G. sulphuraria lives, this material is probably rapidly hydrolyzed, providing an organic carbon source to the algae, along with other external sources for dissolved organic carbon, like e.g. high-temperature acid-digested wood [START_REF] Nye | A novel PARAFAC model for continental hot springs reveals unique dissolved organic carbon compositions[END_REF]. Thanks to the abundance of transporters, this alga could outcompete other microorganisms such as fungi, which are also found in these extreme conditions, for growth. Thus, the coexistence of phototrophic, mixotrophic, and heterotrophic lifestyles thanks to the subtle compromise between the activity of the two energy-producing pathways (photosynthesis and respiration) would represent a key element for fitness and explain the success of G. sulphuraria to thrive in its extreme ecological niche. High fluctuations in the availability of dissolved organic and inorganic carbon, light, temperature, etc. in these environments possibly selected for the maintenance of a metabolic flexibility (Gross, 1999;Gross, Wolfgang et al., 2002;Ciniglia et al., 2004;[START_REF] Cho | Characterization of a non-thermophilic strain of the red algal genus Galdieria isolated from Soos (Czech Republic)[END_REF], which may have also allowed Cyanidiales to invade more moderate habitats (Yoon et al., 2006;[START_REF] Azúa-Bustos | Ancient photosynthetic eukaryote biofilms in an Atacama desert coastal cave[END_REF][START_REF] Castenholz | The Cyanidiales: Ecology, Biodiversity, and Biogeography[END_REF] and to disperse over long distances to geographically isolated extreme habitats (Rossoni et al., 2019a).

The same metabolic flexibility opens interesting perspectives to exploit this alga for biotechnology applications. 22 evolution in thermoacidophilic Galdieria (Rhodophyta) (vol 20, 112, 2020) Galdieria sulphuraria SAG21.92 cells and biomass production. Cells were inoculated at 3.5 10 6 cell per mL and grown in a photobioreactor in the light (transmitted light 10 µmol photons m -2 s -1 ) and air before D-sorbitol was added in the absence (heterotrophy, black) and in the presence of light (mixotrophy, orange). Light was increased every day to keep the transmitted light to a constant value of 10 µmol photons m -2 s -1 . Growth was followed at 42°C and pH 2. a, d, g, after five days of growth (i.e. 2 days after the addition of D-sorbitol), ETR was measured directly on cultures within the photobioreactor, to avoid possible temperature stress. Measurements were done in air, in the absence (a, b) and presence (d, e) of respiratory inhibitors (SHAM 1 mM and myxothiazol (10 µM), added 24 h before measurements), or in a CO 2 -enriched (0.5%) atmosphere (g, Methods S1

Microalgae and media composition

Galdieria sulphuraria SAG21.92 and 074G were obtained from the Culture

Collection of Algae at Göttingen University (SAG) and was grown in sterile 2xGS modified Allen medium, pH 2.0, containing 20 mM of NaNO3, and 5 mM of inorganic phosphate (K2HPO4 and KH2PO4 in a 2/1 ratio, [START_REF] Allen | Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte[END_REF])) at 42°C.

The medium and flasks were sterilized in an autoclave for 20 min at 121 °C in order to prevent any contamination. For the experiments with different organic carbon sources, the 2xGS modified Allen media was supplemented by adding filtersterilized concentrated organic substrate solutions as indicated in the text. The concentration of organic substrates was selected on the basis of data reported in literature [START_REF] Oesterhelt | Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria[END_REF]. G. sulphuraria was grown either in 250 mL flasks (50 mL culture volume) in an incubator (Infors, Switzerland, continuous light, 30 µmol photons m -2 s -1 , 42°C, 100 rpm) or in a multicultivator (Photon System Instruments, Czech Republic). For growth in the multicultivator, cells were provided with air or CO2-enriched air (see below) bubbled into the solution ensuring both aeration and mixing. Culture volume was 80 mL. Incident light intensity was adjusted daily to maintain constant transmitted light thus offering a constant light intensity to the cells (see Results and below). The fraction of CO2 in the feed air was regulated by a flow metering system (Photon System Instruments, Czech Republic). The total gas flow was 0.4 L/min for each culture.

Growth

Cultures used to inoculate flasks or multicultivator tubes were grown photoautotrophically in the air in flasks placed in the incubator. Adequate volumes of cultures at a density of 7 x 10 7 cells per mL were centrifuged and resuspended in a fresh medium to obtain an initial cell concentration of 1.5 x 10 6 cells per mL (in flasks) or 3.5 x 10 6 cells per mL (multicultivator). For organic substrates screening in flasks under mixotrophic conditions (see Fig. S1 & S2), substrate was added at a concentration of 150 mM carbon atom (e.g. 12.5 mM saccharose). For growth in the multicultivator cells were adapted for 3 days with an incident light of 60 µmol photons m -2 s -1 (transmitted light ~ 10 µmol photons. m 2 .s -1 ), before addition of organic carbon (150 mM C) for the mixotrophic and heterotrophic conditions. For the heterotrophic condition, light was switched-off and tubes were covered with aluminum foil to keep them in the dark. For experiments with the multicultivator, the incident light intensity was adjusted daily to maintain constant transmitted light though the culture (see Results). This 'luminostat' regime should ensure maximal absorption of light without allowing a dark zone to develop inside the multicultivator (Cuaresma et al., 2011) (see Supplementary Fig. S3). For most conditions, a transmitted light of 10 µmol photons m -2 s -1 was used. Tests were carried out in the range from 5 to 30 µmol photons m -2 s -1 (see Supplementary Fig. S8).

Experiments were either carried out in air or in air enriched with CO2 (0.5 to 4%, see Supplementary Fig. S8). Sorbitol consumption was measured using the D Sorbitol/Xylitol assay kit (Megazyme).

To monitor algal growth, samples were taken daily and growth was estimated using a LUNA™ cell counter (Logos Biosystems, Inc. USA).

Cell fresh weight and dry weight estimates

Cells were collected by centrifugation in 50 mL tubes, cell pellet was resuspended in a small volume of water and centrifuged in pre-weighted eppendorf tubes and pellet was weighted. For dry weight determination fresh cells pellets were exposed for three days at 60°C, weighted and expressed as g L -1 .

Clark electrode oxygen measurements

Net oxygen exchanges in solution were measured with a Clark-type electrode (Hansatech Instruments, UK) at 42°C. The electrode equilibrated for 12 h in distilled water was calibrated at 42°C in the air (100% of O2) and by bubbling argon (0% of O2). For oxygen exchanges measurements, an aliquot of the cell culture was collected immediately before the measurement (30 10 6 cells) and centrifuged at 42°C. The pellet was resuspended in 1 mL 2 x GS medium, pH 2.0, 42°C and introduced in the measure chamber thermostated at 42°C. The electrode was closed and measurement was first carried out in the dark for 10 minutes before light was switched on (320 µmol photons m -2 s -1 ). Respiration and gross photosynthesis were quantified by measuring the slope of oxygen changes in the dark and under light exposure. Net photosynthesis was calculated assuming O2 consumption by the mitochondrion in the light is identical to that in the dark (Net photosynthesis = vO2light +|vO2dark|).

Photophysiology measurements

Photosynthetic parameters were derived from quantification of chlorophyll fluorescence emission by cultures within the multicultivator. To this aim, we employed a custom-made fluorescence imaging system based on a previously published setup [START_REF] Johnson | A new setup for in vivo fluorescence imaging of photosynthetic activity[END_REF]. The system was modified by replacing the green LEDs providing actinic light with orange LEDs (emission peak 630 nm, Full

Width at Half Maximum: 40 nm), and the acquisition setup with a GigE µeye camera (IDS, Germany). Measuring light was provided by a LED peaking at 590 nm, to maximize excitation of the phycobiliprotein complexes of Galdieria. The photosynthetic electron transfer rate, ETRPSII, was calculated as the product of the light intensity times the photochemical yield in the light (Fm'-Fs)/Fm' x PFD, where Fm' and Fs are the fluorescence intensities measured after exposure to a saturating pulse and in steady state, respectively, in light-acclimated cells and PFD (Photosynthetic Flux Density) is the incident light intensity, measured in µmol photons m-2 s-1.(see [START_REF] Maxwell | Chlorophyll fluorescence -a practical guide[END_REF] for more details). Cells were allowed to reach steady state fluorescence emissions at every light ( 5-10 minutes of light exposure depending on the intensity) before increasing the photon flux.

Mass spectrometry-based proteomic analyses

Experimental design

Galdieria sulphuraria SAG21.92 was cultivated under the three conditions (photoautotrophy, mixotrophy and heterotrophy) in parallel in the same cultivator

Statistical treatment

The remaining list was considered for bioanalysis. Since there were many missing proteins in the photoautotrophy condition compared to the other two conditions, the list was divided into two sets. One set on which no statistical treatment was applied was constituted of proteins entirely missing in one condition (having no abundance values in all 3 replicates). The other set (proteins having at least 1 abundance value in all three conditions) was used for differential analysis using ProStaR [START_REF] Wieczorek | DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics[END_REF][START_REF] Wieczorek | Protein-level statistical analysis of quantitative label-free proteomics data with ProStaR[END_REF]. For this, the intensity

Metabolic analyses by IC-MS

The dried sample was reconstituted in 100 µL deionized water and 5 µL were injected via a Dionex AS-AP autosampler in push partial mode with a 10 μL loop.

The temperature of the autosampler was set to 4 °C to maintain sample stability.

Anion exchange chromatography was conducted on a Dionex IonPac AS11-HC column (2 mm × 250 mm, 4 μm particle size, Thermo Scientific) equipped with a Dionex IonPac AG11-HC guard column (2 mm × 50 mm, 4 μm, Thermo Scientific) at 30 °C. The mobile phase was established using an eluent generator with a potassium hydroxide cartridge to produce a potassium hydroxide gradient. The column flow rate was set to 380 µL min -1 with a starting KOH concentration of mM. The concentration was held for 3 min, then increased to 50% within 9 min followed by a steeper increase to 100% within 7 min. After 2 min of plateau the concentration dropped immediately back to 10 % for 8 min of equilibration. ). Growth rates were 0.5 ± 0.1 day -1 ; 1.2 ± 0.1 day -1 and 0.9 ±0.05 day -1 in photoautotrophic, mixotrophic and heterotrophic cultures, respectively. b, Sorbitol consumption in mixotrophic and heterotrophic cultures. Data from 3 biological replicates ± S.D.

  G. sulphuraria has already been employed in the fields of pigment/antioxidant production, bioremediation and bioenergy (Cizkova et al., 2019) and the possibility to exploit its lifestyle flexibility should be explored to cultivate this alga in organic matter-rich open ponds without contamination by other microorganisms.

Fig. 3 .

 3 Dataset S2: Compared proteomic analysis between photoautotrophic, mixotrophic and heterotrophic growth conditions. Dataset S3: Compared metabolomic analysis between photoautotrophic, mixotrophic and heterotrophic growth conditions.

Figure S1 :

 S1 Consequences of different substrates on G. sulphuraria growth in the light.

Figure S2 :

 S2 G. sulphuraria growth in photoautotrophic, mixotrophic and heterotrophic conditions driven by a polyol, an hexose and a disaccharide.

Figure S3 :

 S3 Experimental setup to expose cells to a constant photons/cell ratio.

Figure S4 :

 S4 In situ measurements of photosynthetic electron transfer rate (ETR) in photoautotrophic (light) and mixotrophic (light + 25 mM D-sorbitol) cells.

Figure S5 :

 S5 Enhancement of cell growth by mixotrophy and sorbitol consumption in G. sulphuraria.

Figure S6 :

 S6 Mixotrophy is restored in G. sulphuraria upon addition of a carbon source.

Figure S7 :

 S7 Respiration and net photosynthesis were measured every day in the three different growth conditions (photoautotrophy, mixotrophy and heterotrophy).

Figure S8 :

 S8 Biomass production as a function of transmitted light intensity and CO 2 concentration. FigureS9: Immunodetection of RuBisCO in phototrophic and mixotrophic cultures under ambient and enhanced CO 2 atmosphere.

Figure S10 :

 S10 Figure S10: Comparative analysis of phototrophic, mixotrophic and heterotrophic performances in G. sulphuraria 074 G and SAG21.92 species with D-sorbitol and D-glucose.Figure S11: Respiration fuels photosynthesis in photoautotrophic and mixotrophic G. sulphuraria cultures. Figure S12: Transmission electron microscopy of G. sulphuraria SAG21.92 grown 5 days under photoautotrophic, mixotrophic and heterotrophic conditions.

Figure S11 :Figure 1 :

 S111 Figure S10: Comparative analysis of phototrophic, mixotrophic and heterotrophic performances in G. sulphuraria 074 G and SAG21.92 species with D-sorbitol and D-glucose.Figure S11: Respiration fuels photosynthesis in photoautotrophic and mixotrophic G. sulphuraria cultures. Figure S12: Transmission electron microscopy of G. sulphuraria SAG21.92 grown 5 days under photoautotrophic, mixotrophic and heterotrophic conditions.

Figure 2 :

 2 Figure 2: In situ measurements of photosynthetic electron transfer rate (ETR) in photoautotrophic (light) and mixotrophic (light + 25 mM D-sorbitol)

  photosynthesis in the three examined conditions. ** indicate that at the 0.01 level the means of the two populations (mixotrophy on one side; heterotrophy+photoautotrophy on the other one) means are statistically different (Anova test).

Figure 3 :

 3 Figure 3: Synthesis of proteomic changes between phototrophic, mixotrophic and heterotrophic growth conditions of Galdieria sulphuraria SAG21.92. Plastid is indicated in green, cytosol in white, mitochondrion in orange and peroxisome in grey. Proteins are identified by their SwissProt accessions; boxes on top of protein names represent fold-changes (protein average abundance in one condition was compared with the average abundance of the other two conditions -left photoautotrophy, middle mixotrophy, right heterotrophy). Proteins displaying statistically significant changes (see methods) are highlighted in yellow. They include proteins involved in carbon concentrating mechanism (Pyruvate phosphate dikinase -M2XY57, carbonic anhydrase-M2XTP2, PEP carboxylase-M2XIX2), which are much more abundant in photoautotrophic conditions than in mixotrophic or heterotrophic conditions. Enzymes involved in photorespiration-dashed arrows-(Phosphoglycolate phosphatase-M2XAQ1, Glycolate oxidase-M2WRR8, Serine-glyoxylate aminotransferase-M2Y9J6, Glycine decarboxylase P proteins-M2X9U4, Glycine/serine hydroxymethyltransferase-M2XX08, Hydroxypyruvate reductase-M2XII5, Glycerate kinase-M2XRC5) follow the same pattern as CCM enzymes.

Figure 4 :

 4 Figure 4: Metabolic changes between phototrophic, mixotrophic and heterotrophic growth conditions of Galdieria sulphuraria SAG21.92. Nonphosphorylated metabolites were analyzed by GC-MS, phosphorylated metabolites were analyzed using IC-MS. Quantification of metabolites is provided in Dataset S3. Green bars: photoautotrophy; orange bars: mixotrophy; black bars: heterotrophy. a, Changes of metabolites involved in photorespiration. 2P-glycolate

Figure 5 :

 5 Figure 5: Incorporation of carbon derived from 13 C-labelled glucose into intermediates of the Calvin-Benson-Bassham (a) and tricarboxylic acid (b) cycles during mixotrophic cultivation of Galdieria sulphuraria SAG21.92 in two different CO 2 concentrations. Incorporation rates are displayed as average number of labelled carbon atoms in each molecule (average exchange). Error bars represent the standard deviation of biological quadruplicates. Light orange: cells grown in ambient air (0.02% CO 2 ). Dark orange cells grown in air supplied with 2% CO 2 . RuBP: Ribulose 1,5-bisphosphate; 3-PGA: 3-phosphoglycerate; 2-PG: 2phosphoglycolate; BPG: 1,3-bisphosphoglycerate; DHAP: Dihydroxyacetone phosphate; GAP: glyceraldehyde 3-phosphate; FBP: Fructose 1,6-bisphosphate; F6P: Fructose 6-phosphate; E4P: Erythrose 4-phosphate; Xu5P: Xylulose 5phosphate; SBP: Seduheptulose 1,7-bisphosphate; S7P: Seduheptulose 7phosphate; R5P: Ribose 5-phosphate; Ru5P: Ribulose 5-phosphate.

( 2 -

 2 3 tubes per condition). Three multicultivator experiments were carried out one week apart and constituted the biological replicates. Only one tube per condition and per cultivator was chosen having the closest ODs within the same condition. Cell breakage was performed on 10 9 cells with a Precellys homogenizer (Bertin, France). Protein digestion Each protein sample (40 μg) was stacked by a 1 cm-migration on the top of a NuPAGE 4-12% gel, (Invitrogen) before Coomassie blue staining (R250, Bio-Rad). Gel bands of concentrated proteins were manually excised and cut into pieces before being washed by 6 successive incubations of 15 min in 25 mM NH4HCO3 containing 50% (v/v) acetonitrile. Gel pieces were then dehydrated in 100% acetonitrile and incubated at 53 °C with 10 mM DTT in 25 mM NH4HCO3 for 45 min and in the dark with 55 mM iodoacetamide in 25 mM NH4HCO3 for 35 min. Alkylation was stopped by adding 10 mM DTT in 25 mM NH4HCO3 and mixing for 10 min. Gel pieces were then washed again by incubation in 25 mM NH4HCO3 before dehydration with 100% acetonitrile. Modified trypsin (Promega, sequencing grade) in 25 mM NH4HCO3 was added to the dehydrated gel pieces for an overnight incubation at 37 °C. Peptides were then extracted from gel pieces in three 15-min sequential extraction steps in 30 μl of 50% acetonitrile, 30 μl of 5% formic acid and finally 30 μl of 100% acetonitrile. The pooled supernatants were then vacuum-dried.MS/MS analysisThe dried extracted peptides were resuspended in acetonitrile 5%, trifluoroacetic acid 0.1% and analyzed via online nano-LC-MS/MS (nano-liquid chromatographytandem mass spectrometry; Ultimate 3000 RSLCnano and Q-Ex HF, Thermo Fischer Scientific, Thermo Scientific, Waltham, MA, USA). Peptide mixtures were desalted on line using a reverse phase precolumn (PepMap C18 Thermo Fisher Scientific) and resolved on a C18 column (ReproSil-Pur 120 C18-AQ 1.9 μm column, Dr. Maisch GmbH). The nanoLC method consisted in a 200-min gradient at a flow rate of 300 nL•min-1 ranging from 5.1% to 72.2% acetonitrile in 0.08% formic acid in 182 min. This gradient was operated as multi-steps optimized by GOAT software (V1.0.1) for Galdieria whole cell extracts. MS (mass spectrometry) and MS/MS data were acquired using the Xcalibur software (Thermo Fisher Scientific). The spray voltage was set at 2 kV and the heated capillary was adjusted to 270 °C. Survey full-scan MS spectra (m/z = 400-1600) were obtained in the Orbitrap with a resolution of 60,000 after accumulation of 10 6 ions (maximum filling time: 200 ms). The 20 most intense ions from the preview survey scan delivered by the Orbitrap were fragmented via collision-induced dissociation in the LTQ after accumulation of 1e5 ions (maximum filling time: 50 ms).Database searches and quantificationData were processed automatically using the Mascot Distiller software (version 2.7.1.0, Matrix Science). Peptides and proteins were identified using Mascot (version 2.6.0) through concomitant searches against Uniprot (Galdieria sulphuraria taxonomy, July 2019 version), classical contaminants database (homemade) and their corresponding reversed databases. Trypsin/P was chosen as the enzyme and three missed cleavages were allowed. Precursor and fragment mass error tolerance were set, respectively, to 10 ppm and 25 mmu. Peptide modifications allowed during the search were: carbamidomethylation (fixed), acetyl (protein N-terminal, variable) and methionine oxidation (variable). The Proline software ([START_REF] Bouyssie | Proline: an efficient and user-friendly software suite for large-scale proteomics[END_REF], http://proline.profiproteomics.fr) was used to filter the merged results: conservation of rank 1 peptide-spectrum match (PSM) with a minimal length of 7 and a minimal score of 25. PSM score filtering is then optimized to reach a False Discovery Rate (FDR) of PSM identification below 1% by employing the target decoy approach. A minimum of one specific peptide per identified protein group was set. Proline was then used to perform MS1-based label free quantification of the peptides and protein groups. The "Crossassignment" option was activated and allowed within replicates of the same condition only. Intensity values were extracted from specific peptides and used to infer protein abundances from the different samples. An ultimate filter was applied to discard non-relevant or doubtful identifications. This concerns proteins identified in the reverse and contaminant databases, proteins identified with only one peptide with a Mascot score inferior to 40 and few unquantified proteins. Proteins identified in the reverse and contaminant databases (i.e. trypsin or keratin), and proteins identified with only 1 peptide with a score <40 were further discarded from the list.

  values were log2 transformed and normalized by Variance Stabilizing Normalization (VSN) algorithm(Huber et al., 2002) with the « overall » option. The missing abundances were imputated according to the Structured Least Square Adaptative (SLSA) method. The Limma test was applied for comparison of one condition versus the other two combined. Due to huge differences in the matrixproteins identified and intensitiesin photoautotrophy compared to mixotrophy or heterotrophy, the strategy consisting of comparing one condition to the average of the other two (option "One_vs_All" in ProStaR) gave better discriminating results than comparisons of one condition to another one condition ("One_vs_One" option in ProStaR). Differentially recovered proteins were sorted out using a log2 foldchange (FC) cut-off of 1 and a p-value threshold (on the remaining proteins) that guarantees a Benjamini-Hochberg FDR of approximately 0.01%. The variations in abundance between the three cultivation conditions are important which made it difficult to objectively determine the latter threshold. We deliberately chose a very low FDR threshold to select a protein set of reasonable size in each comparison.

Figure S1 :

 S1 Figure S1: Consequences of different substrates on G. sulphuraria SAG21.92 growth in the light. Growth was measured in flasks at ambient CO2 in the presence of 25 mM reduced carbon source added from the beginning of the culture (day 0). Cultures were inoculated at a cell density of 1.5 10 6 photoautotrophic cells mL -1 . Growth of photoautotrophic cells is indicated in green, for comparison. Light intensity was 30 µmol photons m -2 s -1 . Experiments were carried out at 42°C with shaking at 100 rpm, pH 2. a, hexoses, b, pentoses, c, disaccharides, d, organic acids, e, amino acids, f, polyols. Inserts in panel d and e are zooms on the data.

Figure S2 :

 S2 Figure S2: G. sulphuraria SAG21.92 growth in photoautotrophic, mixotrophic and heterotrophic conditions driven by a polyol, an hexose and a disaccharide. Cells were grown in flasks, in photoautotrophic (light only, 30 µmol photons m -2 s -1 , green), mixotrophic (30 µmol photons. m -2 .s -1 plus a source of reduced carbon, orange) and heterotrophic (absence of light, presence of a source of reduced carbon, black) conditions at ambient CO2, 42°C and pH 2. a,b: D-sorbitol 25 mM; c,d: D-glucose 25 mM; e,f: saccharose 12.5 mM. b-d-f: Mixotrophic growth (orange bars) exceeds the sum of photoautotrophic (green bars) and heterotrophic (black bars) growth, highlighting the existence of a synergy under mixotrophic conditions. Cultures were started from photoautotrophic cells, which were diluted at 1.5 10 6 cells mL -1 for each condition. Experiments were carried out at 42°C with shaking at 100 rpm. b, d, f, Final cell concentration (day 8) in photoautotrophic, heterotrophic and mixotrophic conditions. Data from 3 biological replicates ± S.D. * indicate that at the 0.05 level the means of the two populations (mixotrophy on one side, heterotrophy+photoautotrophy on the other one) means are statistically different (Anova test). ** indicate that at the 0.01 level the means of the two populations (mixotrophy on one side, heterotrophy+photoautotrophy on the other one) means are statistically different (Anova test).

Figure S3 :

 S3 Figure S3: Experimental setup to expose cells to a constant photons/cell ratio. a, Schematic side view of the photobioreactor with the LED panel (yellow spots), water bath (blue) and culture tube (green). I0 and I represent the incident light (settings on the device) and the light transmitted through the device, respectively. I0 was progressively increased during cell growth, to keep a linear relation between cell density and absorption. b, Relation between absorbance and cell number under mixotrophic and photoautotrophic conditions for I = 10 µmol photons m -2 s -1 . The slopes differ for mixotrophic (orange) and photoautotrophic (dark green) cells possibly because the photosynthetic apparatus is downregulated in the presence of organic carbon. The lower amount of photosynthetic complexes reduces light absorption for the same amount of cell. c, Custom-made setup to evaluate photosynthetic performances of G. sulphuraria cells within the photobioreactor. The system is equipped with orange LEDs ( = 590 nm), to enhance absorption by the phycobiliprotein complexes of G. sulphuraria, and a near infrared camera to measure fluorescence emitted by the algae inside the photobioreactor (PBR tubes).

Figure S4 :

 S4 Figure S4: In situ measurements of photosynthetic electron transfer rate (ETR) in photoautotrophic (light) and mixotrophic (light + 25 mM D-sorbitol)G. sulphuraria SAG21.92 cells. Cells were inoculated at 3.5 x 10 6 cell mL -1 and grown in the light (transmitted light 10 µmol photons m -2 s -1 ) and air for three days before D-sorbitol was added (mixotrophy, orange) or not (autotrophy, green). Light was increased every day to keep the transmitted light to a constant value of 10 µmol photons m -2 s -1 . a-f, After three days of growth, ETR was followed every day directly on cultures within the photobioreactor, to avoid possible temperature stress. Data from 3 biological replicates ± S.D.

Figure S5 :

 S5 Figure S5: Enhancement of cell growth by mixotrophy and sorbitol consumption in G. sulphuraria SAG21.92. G. sulphuraria was inoculated at 3.5 10 6 cells mL -1 . At day 3, cultures were either continued without any addition (photoautotrophy, green), or supplemented with 25 mM D-sorbitol in the presence of light (mixotrophy, orange) or in the dark (heterotrophy, black). a, Growth curves (data from 12 biological replicates ± S.D.). Growth rates were 0.5 ± 0.1 day -1 ; 1.2 ± 0.1 day -1 and 0.9 ±0.05 day -1 in photoautotrophic, mixotrophic and heterotrophic cultures, respectively. b, Sorbitol consumption in mixotrophic and heterotrophic cultures. Data from 3 biological replicates ± S.D.

Figure S6 :

 S6 Figure S6: Mixotrophy is restored in G. sulphuraria SAG21.92 upon addition of a carbon source. a, Mixotrophic cells (orange) were let consuming sorbitol (day 7, i.e. 4 days after addition of D-sorbitol). b, Cells at day 7 were diluted to 3.5 x 10 6 cells mL -1 , and growth was resumed in the light (transmitted light 10 µmol photons m -2 s -1 ) without D-sorbitol (photoautotrophy, green) or in the presence of 25 mM D-sorbitol (orange). ETR measurements carried three days later (c) indicate that mixotrophic photosynthetic capacity was fully restored. Data from 3 biological replicates ± S.D.

Figure S7 :

 S7 Figure S7: Respiration and net photosynthesis of G. sulphuraria SAG21.92 were measured every day in the three different growth conditions (photoautotrophy, mixotrophy and heterotrophy). O2 consumption or production were measured with a Clark electrode at 42°C. Cells were centrifuged and resuspended at 3 x 10 7 cells in 1 mL of fresh 2xGS medium. Net photosynthesis corresponds to gross photosynthesis +|respiration|. Data from 3 biological replicates ± S.D.

Figure S8 :

 S8 Figure S8: Biomass production of G. sulphuraria SAG21.92 as a function of transmitted light intensity and CO2 concentration. a, Cells were grown in a photobioreactor at four different constant transmitted light intensities and different CO2 concentrations.During growth, the light intensity was increased to maintain a constant transmitted light (from 5 to 30 µmol photons m -2 s -1 ). Cells exposed to a given light ramp were used to inoculate the next experiment at higher CO2 concentration, while repeating the same light ramp. Progressive adaptation to higher CO2 concentration was chosen to avoid acidification of the cytosol in the presence of high CO2 concentration and to induce a progressive reduction in the accumulation of carbonic anhydrase concentration, which is expected to be high in low CO2 grown cells. After three days of adaptation, cells were grown for 4 additional days and collected at day 7, dried for three days at 60°C and weighted. Data from 3 biological replicates ± S.D. b: Focus on the light dependency of biomass production in phototrophic conditions in air. Same colour code as in panel a. Data from 3 biological replicates ± S.D. c, Focus on the light dependency of biomass production in phototrophic conditions supplemented with 0.5% CO2. Same colour code as in panel a. Data from 3 biological replicates ± S.D. d, Focus on the light dependency of biomass production in mixotrophic conditions (sorbitol 25 mM). Two light intensities were tested corresponding to a transmitted light of 10 and 30 µmol photons m -2 s -1 . Data from 3 biological replicates ± S.D .

Figure S9 :

 S9 Figure S9: Immunodetection of RuBisCO in phototrophic and mixotrophic cultures of G. sulphuraria SAG21.92 under ambient and enhanced CO2 atmosphere. Cells were grown in a photobioreactor in air ([CO2]= 0.04 %) or air supplemented with CO2. After 7 days of adaptation, cells were collected and broken with a Precellys homogeniser, through three cycles of 30 seconds at 10.000 rpm separated by a 30 seconds interval. Total protein extracts were analyzed by immunoblotting with anti-RuBisCO (AS03 037, Agrisera, Sweden).An antibody against the  subunit of the ATP synthase complex (AS05 085, Agrisera, Sweden) was used as a loading control. 10 µg of protein was loaded per well.

Figure S10 :

 S10 Figure S10: Comparative analysis of phototrophic (green), mixotrophic (orange) and heterotrophic (black) performances in G. sulphuraria 074 G and SAG21.92 species with D-sorbitol and D-glucose. G. sulphuraria 074 G and SAG21.92 species were grown in a photobioreactor as described in Fig. S3 & S4 under conditions similar to those described in Oesterhelt et al. (2007) in the presence of 2% CO2 but at 42°C. At day 3 cells were either placed in the dark (black) in the presence of 25 mM D-sorbitol or D-glucose or exposed to light (transmitted light 10 µmol photons m -2 s -1 ) in the absence of organic carbon (green) or in the presence of either 25 mM D-sorbitol or D-glucose as indicated in the graph. Cells were collected at day 7, dried and weighted. Growth performances of the 074G and SAG21.92 strains are very similar in the presence of D-glucose or D-sorbitol. Data from 3 biological replicates ± S.D.

Figure S11 :

 S11 Figure S11: Respiration fuels photosynthesis in photoautotrophic (green) and mixotrophic (orange) G. sulphuraria SAG21.92 cultures. a,c: representative experiment of O2 consumption or production measured in a closed system (Clark electrode) at 42°C. Photoautotrophic cells (a,b) or mixotrophic cells (c,d) were centrifuged and resuspended at 30 10 6 cells in 1 mL of fresh 2xGS medium. Stars indicate a dynamic equilibrium reached when all excess CO2 from the media (CO2 initially present plus CO2 produced by respiration) is fixed. Thus, photosynthesis becomes limited by CO2 production by respiration, leading to the compensation point. b (photoautotrophic cells), d (mixotrophic cells): the amount of O2 produced by photosynthesis (white) is commensurate with the amount of CO2 available to Rubisco, i.e. the sum of respiratory CO2 (evaluated from O2 consumption, assuming a 1/1 stoichiometry between CO2 released and consumed oxygen, black) plus the small CO2 amount initially present in the medium (10 µM ca at pH 2, grey), Data from 3 biological replicates ± S.D.

Figure S12 :

 S12 Figure S12: Transmission electron microscopy of G. sulphuraria SAG21.92 grown 5 days under photoautotrophic (a), mixotrophic (b) and heterotrophic (c) conditions. b, c show the presence of a huge floridean starch grain in the cytosol of the cells four days after addition of 25 mM D-sorbitol. Scale bars: a: 0.5 µm; b, c: 1 µm. In a, mitochondria and plastids are highlighted in red and green, respectively.

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Acknowledgments

GC, DL, AW, GF, MT, SB and MF acknowledge funds from the ANR (French Research Foundation) 'Momix' (Projet-ANR-17-CE05-0029). GC and GF also acknowledge support of the LabEX GRAL, ANR-10-LABX-49-01 financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). DDB acknowledged a PhD grant from CEA. J.D. was supported by the ATIP-Avenir program. CR and MC acknowledge the ARC grant (DARKMET proposal) for Concerted Research Actions (17/21-08), financed by the French Community of Belgium (Wallonia-Brussels Federation). This project received funding from the European Research Council: ERC Chloro-mito (grant no. 833184) to GC and GF. Metabolite analyses were supported by the CEPLAS Plant Metabolism and Metabolomics laboratory, which is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy -EXC-2048/1project ID 390686111. MT, SB and MF acknowledge support from the ANR-funded French national infrastructure in biology and health ProFI (Proteomics French Infrastructure; ANR-10-INBS-08). We acknowledge the excellent technical assistance by E. Klemp, K. Weber, and M. Graf for GC-MS and IC-MS measurements.

Author

Data availability

The original contributions presented in the study are included in the article and in the Supplementary information and supplementary dataset files. Further inquiries can be directed to the corresponding author Other supplementary materials for this manuscript include the following:

level by comparing MS 2 fragment spectra using mzCloud (ddMS2) (match factor 50) and MS 1 level ChemSpider (by formula or exact mass). Highest identification level via an in-house MS 2 spectral library and retention time was established using mzVault. QC-based batch normalization was performed with QC sample injection every 5 samples during the sequence. Differential analysis, determination of pvalues, adjusted p-values, ratios, fold change, were also calculated using Compound Discoverer.

Labelling experiments with 13 C-glucose

Cells were cultivated in 250 mL Erlenmeyer flasks (50 mL culture volume) for four days under continuous light at 60 µmol m-2 s-1, 40 °C and ambient air (0.04% CO2). U-13 C6-glucose (Cambridge Isotope Laboratories Inc, Tewksbury, Massachusetts) was added at day 4 in a final concentration of 25 mM and the irradiance was increased to 100 µmol m-2 s-1 either under ambient or elevated (2%) CO2 conditions.

1-2.5 x10 8 cells were harvested 1, 4, 12, 24, 36, 48 and 60 hours after glucose addition as described above. Metabolites were extracted and measured by IC-MS as described above.

Data analysis was conducted with Compound Discoverer (version 3.1, Thermo Scientific) and the standard workflow for stable isotope labelling from Compound Discoverer was chosen. The default settings, which are 5 ppm mass tolerance, 30 % intensity tolerance and 0.1 % intensity threshold for isotope pattern matching were used and the maximum exchange rate was set to 95%.

Electron microscopy sample preparation and observation

Cells were pelleted (1000g, 5 min, 4°C) and fixed in reduced osmium tetroxide.

Dataset S2 (separate file). Compared proteomic analysis between photoautotrophic, mixotrophic and heterotrophic growth conditions.

Proteomics analyses were performed on biological triplicates for each growth