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

Gilles Curien, Dagmar Lyska, Erika Guglielmino, Phillip Westhoff, Janina Janetzko, Marianne Tardif, Clément Hallopeau, Sabine Brugière, Davide Dal Bo, Johan Decelle, et al.

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To cite this version:

Gilles Curien, Dagmar Lyska, Erika Guglielmino, Phillip Westhoff, Janina Janetzko, et al. Mixotrophic growth of the extremophile galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism. New Phytologist, 2021, 10.1111/nph.17359. hal-03193983

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MIXOTROPHIC GROWTH OF THE EXTREMOPHILE GALDIERIA

SULPHURARIA REVEALS THE FLEXIBILITY OF ITS CARBON

ASSIMILATION METABOLISM

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Gilles Curien¹*, Dagmar Lyska², Erika Guglielmino¹, Phillip Westhoff², Janina

Janetzko², Marianne Tardif³, Clément Hallopeau¹, Sabine Brugière³, Davide Dal

Bo¹, Johan Decelle¹, Benoit Gallet⁴, Denis Falconet¹, Michele Carone⁵, Claire

Remacle⁵, Myriam Ferro³, Andreas P.M. Weber², Giovanni Finazzi¹

¹Laboratoire de Physiologie Cellulaire et Végétale. Univ. Grenoble Alpes, CNRS,
CEA, INRAe, 38054 Grenoble Cedex 9, France

²Institute of Plant Biochemistry, Cluster of Excellence on Plant Sciences
(CEPLAS), Heinrich Heine University, 40225 Düsseldorf, Germany

³EdyP Laboratoire Biologie à Grande Echelle. Univ. Grenoble Alpes, CEA, Inserm,
BGE U1038, 38054 Grenoble Cedex 9, France

⁴Institut de Biologie Structurale, Univ. Grenoble Alpes, CNRS, CEA, 71 Avenue
des Martyrs, 38044 Grenoble, France

⁵Genetics and Physiology of microalgae, InBios/Phytosystems Research Unit,
University of Liege, 4000, Belgium

*Corresponding author: Gilles Curien, phone: +33 4 38782509, email:

gilles.curien@cea.fr

ORCID numbers:

Gilles Curien: 0000-0002-5361-2399

Dagmar Lyska: 0000-0002-9175-6334

Marianne Tardif: 0000-0003-4438-8281

Clément Hallopeau : 0000-0002-0692-2407

Sabine Brugière : 0000-0002-0757-0968

Johann Decelle: 0000-0002-4343-8358

Benoit Gallet: 0000-0001-8758-7681

Denis Falconet: 0000-0001-8182-1182

Michel Carone: 0000-0002-5561-0792

Claire Remacle: 0000-0002-5016-9547

Myriam Ferro: 0000-0002-4222-6847

Andreas P.M. Weber: 0000-0003-0970-4672

Giovanni Finazzi: 0000-0003-0597-7075

total words count: 5225

words count for Introduction: 755.

words count for Material and methods: 1363
39 words count for Results: 2048

40 words count for Discussion: 1059

41 5 figures (all in color)

42 1 supporting information file (with 12 figures)

43 3 supporting datasets

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## 48 Summary

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* *Galdieria sulphuraria* is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that *G. sulphuraria* cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon.

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* Here, we show that *G. sulphuraria* SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO₂ supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO₂.

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* Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO₂ concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Rubisco and decreasing photorespiration.

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* We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

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74 red algae

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## Introduction

The unicellular red alga *Galdieria sulphuraria* belongs to the Cyanidiophyceae, a class that includes five species often flourishing in different extreme environments (Merola *et al.*, 1981; Gross *et al.*, 1998; Gross & Oesterhelt, 1999; Oesterhelt *et al.*, 2007). From a phylogenetic perspective, plastids of the red algae gave rise to the complex plastids of, e.g., diatoms via secondary endosymbiosis (Yoon *et al.*, 2002; Bhattacharya *et al.*, 2003; Yoon *et al.*, 2004). Like other members of this class (Doemel & Brock, 1971; Reeb & Bhattacharya, 2010) *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 (Moreira *et al.*, 1994; Gross *et al.*, 1998; Castenholz & McDermott, 2010; Barcyté *et al.*, 2018). Some mesophilic species have been isolated from environments with moderate temperatures and/ or a neutral pH (Gross, W. *et al.*, 2002; Yoon *et al.*, 2006; Azúa-Bustos *et al.*, 2009; lovinella *et al.*, 2018). Genome analysis (Barbier *et al.*, 2005; Schonknecht *et al.*, 2013; 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, (Gross & Schnarrenberger, 1995)). 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 (Schmidt *et al.*, 2005; Henkanatte-Gedera *et al.*, 2017; Cizkova *et al.*, 2019).

The relationship between photosynthesis and glycolysis/respiration in higher plants and microalgae is complex (Avelange *et al.*, 1988; Kromer *et al.*, 1988; Gemel & Randall, 1992; Pärnik & Keerberg, 1995; Hoefnagel *et al.*, 1998; Tcherkez *et al.*, 2008). 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
109 green microalgae are capable of mixotrophic growth (Combres et al., 1994; Wan
110 et al., 2011; Johnson & Alric, 2012; Cecchin et al., 2018). While mixotrophy has
111 always beneficial consequences on respiration, its effects on photosynthesis differ
112 depending on the microalga considered: enhancement of photosynthesis was
113 reported in one case (Ettlia oleoabundans, (Ferroni et al., 2018)), while in other
114 algae, including the diatoms Phaeodactylum tricornutum (Liu et al., 2009; Villanova
115 et al., 2017) and Nannochloropsis (Fang et al., 2004; Xu et al., 2004)
116 photosynthesis was unaffected. Decreased photosynthetic activity in mixotrophy
117 has been reported in Chlorella vulgaris (Martinez & Orus, 1991; Cecchin et al.,
118 2018) and Chlamydomonas reinhardtii where the Carbon Concentrating
119 Mechanism (CCM, (Bogaert et al., 2019)) and the light harvesting capacity
120 (Perrineau et al., 2014) is decreased by acetate along with the enhancement of
121 respiration. While it has been reported that G. sulphuraria 074G could grow in the
122 simultaneous presence of light and a carbon source, heterotrophy seemed to
123 prevail in these conditions, as no photosynthetic O₂ production could be measured
124 in the presence of glucose (Oesterhelt et al., 2007).

125 Here, we show instead that photosynthesis and carbon metabolism (glycolysis and
126 respiration) operate simultaneously in *Galdieria sulphuraria* SAG21.92 (a close
127 relative of the 074G strain) under mixotrophic conditions, provided that the
128 temperature conditions are kept close to the ones experienced by this alga in its
129 natural environment. We show that performances in mixotrophy, exemplified by
130 photosynthetic activity and biomass production, actually exceed the sum of the
131 heterotrophic and photoautotrophic yields under limiting inorganic carbon. This
132 synergistic effect stems from a stimulation of photosynthesis by CO₂ of respiratory
133 origin, which overcomes the limitation to the Calvin-Benson-Bassham cycle and
134 suppresses photorespiration.

135 Limitation originates from the very low inorganic carbon concentration available in
136 acidic conditions (pH 2) that constitute the alga's natural growth environment.
137 Notably, under these conditions, inorganic carbon is almost exclusively available
138 as dissolved CO₂ (around 10 µM) while soluble bicarbonate is virtually absent.
139 Consistent with this hypothesis, the synergistic effect of 'light' and 'dark' energetic
metabolisms is sensitive to respiration inhibitors and is lost upon addition of exogenous CO₂, which outcompetes endogenous CO₂ 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 CO₂ concentration under physiological conditions, allowing G. *sulphuraria* to successfully exploit all the energy resources available for growth in its rather challenging environment.

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## 148 Materials and Methods

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150 *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 NaNO₃, and 5 mM of inorganic phosphate (K₂HPO₄ and KH₂PO₄ in a 2/1 ratio (Allen, 1959)) 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 (Oesterhelt et al., 2007). More precisely, glucose was employed at the concentration of 25 mM, as in (Oesterhelt et al., 2007). 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 CO₂-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).
172 **Cell fresh weight and dry weight quantification**

173 Cell pellet was resuspended in a small volume of water and centrifuged in pre-
174 weighed Eppendorf tubes and pellet was weighed. For dry weight determination
175 fresh cells pellets were dried for three days at 60°C, weighed and expressed as
g.L⁻¹.
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178 **Clark electrode oxygen measurements**

179 Oxygen exchanges in solution were measured with a Clark-type electrode
180 (Hansatech Instruments, UK) at 42°C. Respiration and gross photosynthesis were
181 quantified by measuring the slope of O₂ changes in the dark and under light
182 exposure, respectively. Net photosynthesis was calculated assuming O₂
183 consumption by the mitochondrion in the light is identical to that in the dark (Net
184 photosynthesis = vO₂light + |vO₂dark|).

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186 **Photophysiology measurements**

187 Photosynthetic parameters were derived from quantification of chlorophyll
188 fluorescence emission by cultures within the multicultivator. To this aim, we
189 employed a custom-made fluorescence imaging system based on a previously
190 published setup (Johnson et al., 2009) modified as described in Methods S1. The
191 photosynthetic electron transfer rate, ETR (a proxy of the carbon assimilation
192 capacity (Maxwell & Johnson, 2000)) was calculated as (Fm'-Fs)/Fm'*PFD, where
193 Fm' and Fs are the fluorescence intensities measured after exposure to a
194 saturating pulse and in steady state, respectively, in light-acclimated cells and PFD
195 (Photosynthetic Flux Density) is the incident light intensity, measured in µmol
196 photons m⁻² s⁻¹. The cells were allowed to reach steady state fluorescence
197 emissions at each intensity (5-10 minutes of light exposure depending on the
198 intensity) before increasing the photon flux.
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200 **Biochemistry and Proteomic analysis**

201 Western blot analysis was performed on cells grown for 7 days in the indicated
202 conditions. Cells (10⁹ cells) were broken with a Precellys homogenizer (Bertin,
France), through three cycles of 30 seconds at 10,000 rpm separated by a 30-seconds 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 (Bouchnak et al., 2019). 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 (Bouyssie et al., 2020) 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 (Wieczorek et al., 2017; Wieczorek et al., 2019). 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 ($5 \times 10^8$) 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 (Gu et al., 2012) and (Shim et al., 2020). 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/nist-standard-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 (Schwaiger et al., 2017) and in Methods S1. Data Analysis
265 was conducted using Compound Discoverer (version 3.1, Thermo Scientific).

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

268

269 Labelling experiments with ¹³C-glucose

270 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).

271 U-¹³C₆-glucose (Cambridge Isotope Laboratories Inc, Tewksbury, Massachusetts)
272 was added at day 4 in a final concentration of 25 mM and the irradiance was
273 increased to 100 µmol m⁻² s⁻¹ either under ambient or elevated (2%) CO₂
274 conditions.
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276 1-2.5 x10⁸ cells were harvested 1, 4, 12, 24, 36, 48 and 60 hours after glucose
277 addition as described above. Metabolites were extracted and measured by IC-MS
278 as described above.

279 Data analysis was conducted with Compound Discoverer (version 3.1, Thermo
280 Scientific) and the standard workflow for stable isotope labelling from Compound
281 Discoverer was chosen. The default settings, which are 5 ppm mass tolerance, 30
282 % intensity tolerance and 0.1 % intensity threshold for isotope pattern matching
283 were used and the maximum exchange rate was set to 95%.

284

285

286 Results

287

288 G. sulphuraria metabolises several organic substrates.

289 Previous work (Oesterhelt et al., 2007) has suggested that *G. sulphuraria* is unable
to grow mixotrophically in the presence of light plus organic carbon but rather
290 alternates between heterotrophy (in the presence of an external source of organic
291 carbon) and phototrophy (upon inorganic carbon consumption). This conclusion
292 was based on experiments carried out at 25°C, i.e. a temperature that is far from
293 the physiological optimum of this alga (above 40°C), and therefore decreases
294 photosynthetic performances (Doemel & Brock, 1971; Ford, 1979; Rossoni et al.,
295 2019b; Rossoni & Weber, 2019). Thus, we decided to reinvestigate the possible
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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 (L-aspartate, 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 (Gross & Schnarrenberger, 1995). 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 (Maxwell & Johnson, 2000) (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 CO₂ 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 CO₂ 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 CO₂ availability to the cells in mixotrophic cultures, to outcompete endogenous CO₂ of respiratory origin with an excess of exogenous inorganic carbon. As a prerequisite for this experiment, we calibrated the CO₂ 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 CO₂ up to a concentration of 2% CO₂ (Fig. S8a), the apparent affinity for CO₂ being 0.5 % ca. Upon addition of external CO₂, 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 CO₂. Moreover, we hypothesize that photorespiration should be decreased to very low rates at saturating CO₂, possibly contributing to the gain in biomass productivity at high CO₂ 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
390 cells supplemented with 0.5% CO2 (Fig S8c) or in mixotrophic cells (Fig S8d).

391 These findings are fully consistent with the hypothesis that photosynthesis is CO2

392 limited in air, and that this limitation can be alleviated by endogenous (mixotrophy)

393 or exogenous CO2.

394

395 **Metabolic acclimation to mixotrophy in G. sulphuraria.**

396 The observation that mixotrophy enhances photosynthesis to a similar level as

397 upon addition of external CO2 to G. sulphuraria, suggests that mixotrophy behaves

398 as a strategy to traffic CO2 from the mitochondria to the plastid, allowing this alga

399 to successfully exploit all the energy resources available for growth and to

400 minimize energy loss through photorespiration.

401 To substantiate this hypothesis, we performed a complete survey of the metabolic

402 changes between the three trophic lifestyles, and relate these changes to

403 modifications in the cell proteome. We found that photosynthetic proteins

404 (complexes of the electron transfer chain, enzymes of the Calvin-Benson-

405 Bassham cycle and transporters involved in triose phosphate export) were

406 downregulated in mixotrophic conditions compared to photoautotrophic conditions

407 (Fig. 3), in agreement with previous suggestions (Gross & Schnarrenberger, 1995;

408 Oesterhelt et al., 2007). Conversely, respiratory protein levels remained relatively

409 constant in the three conditions. This observation (photosynthetic activity is

410 enhanced in mixotrophy despite the downregulation of the photosynthetic

411 machinery) fully supports the conclusion that intracellular increase in CO2 due to

412 enhanced respiratory activity more than compensates for the decrease in Rubisco

413 (see also Fig. S9).

414 In line with the hypothesis that mixotrophy channels CO2 from respiration to

415 photosynthesis, proteomic analysis indicates that all the enzymes involved in

416 photorespiration (phosphoglycolate phosphatase, glycolate oxidase, serine-

417 glyoxylate aminotransferase, glycine decarboxylase, glycine/serine

418 hydroxymethyltransferase, hydroxypyruvate reductase, glycerate kinase) were

419 less abundant in mixotrophy. This is also true for Rubisco activase (Gasu_19410,

420 dataset S1-2), an enzyme that was shown to be important under low CO2 in
421 *Chlamydomonas* (Pollock *et al.*, 2003). These findings are also corroborated by 422 metabolite analysis (Fig. 4a). The amounts of the oxygenation product of Rubisco, 423 2-phosphoglycolate are highest under photoautotrophic conditions, lower in 424 mixotrophic conditions, and very low in heterotrophic conditions. Glycine 425 accumulates, likely due to a higher reduction potential inside the mitochondrial 426 matrix under mixotrophic conditions, which will reduce the rate of oxidative 427 decarboxylation of glycine by glycine decarboxylase. Consequently, the glycine to 428 serine ratio was inverted compared to photoautotrophic conditions. The amounts 429 of glycerate and 3-phosphoglycerate under mixotrophic conditions are mimicking 430 those in heterotrophically grown cells (Fig. 4a).

431 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. 432 Carbonic anhydrase and phosphoenolpyruvate carboxylase were more abundant 433 under photoautotrophic conditions than under mixotrophic or heterotrophic 434 conditions, and, in agreement with this finding, photoautotrophic cells also 435 contained higher amounts of phosphoenolpyruvate (PEP) than mixotrophic or 436 heterotrophic cells (Fig. 4b). The presence of this carbon concentrating cycle in 437 photoautotrophic conditions could be supported by increased steady-state levels 438 of phosphorylated C3 compounds, as suggested by the downregulation of 439 pyruvate kinases in phototrophy (PEP consumption might be decreased) and 440 strong induction of pyruvate ortho-phosphate dikinase (producing PEP). We 441 assume that oxaloacetate (OAA) under photoautotrophic conditions is 442 decarboxylated by PEP carboxykinase (PEPCK), and not after reduction into 443 malate by malate dehydrogenase and decarboxylation by mitochondrial malic 444 enzyme. This hypothesis is supported by the finding that mitochondrial malic 445 enzyme (ME) is strongly decreased in photoautotrophy (Fig. 3) and the malate pool 446 size is smaller under this condition than in mixo- or heterotrophy. We note that 447 decarboxylation by PEPCK (as compared to ME) is energy conserving and directly 448 yields PEP for a new round of carboxylation by PEP carboxylase. 449

450 While part of the increase in biomass production in mixotrophic conditions can be 451 attributed to the repression of photorespiration, other benefits of the mixotrophic
lifestyle may come from more carbon units being shuttled into anabolic pathways. 452

Indeed, we observed a general increase of metabolites of the oxidative pentose 453
phosphate pathway (Ribulose-5P, Sedoheptulose-7P) and nucleoside 454
triphosphates (ATP, GTP, UTP, CTP) as successor metabolites (Fig. 4c). The 455
amounts do not differ between autotrophic and heterotrophic cultures but are 456
clearly increased under mixotrophic conditions providing evidence for a higher flux 457
of carbon at least into purine and pyrimidine synthesis. 458

Labelling experiments using ¹³C-labelled glucose, a sugar with similar effects on 459
cell growth and biomass production as sorbitol (Fig. S2), further support this notion. 460

We found a rapid incorporation of CO₂ from ¹³C glucose into photosynthetic 461
metabolites, such as Ribulose1,5-bisphosphate (RuBP), Sedoheptulose 1,7- 462
bisphosphate (SBP), and 2-phosphoglycolate (2-PG) (Fig. 5). While RuBP also 463
occurs in the oxidative pentose phosphate pathway, SBP and 2-PG are 464
metabolites solely formed in the Calvin-Benson-Bassham cycle and can only carry 465
a label when ¹³CO₂ released by respiration is fixed. Incorporation of the label by 466
shuttling of carbon backbones from the cytosol into the chloroplast is unlikely since 467
the solute transporters in the *G. sulphuraria*'s chloroplast envelope do not favor 468
the import of glycolytic intermediates under photosynthetic conditions (Linka et al., 469
2008). The rate of incorporation of ¹³C is much reduced when external CO₂ levels 470
are increased to 2%. Interestingly, the tricarboxylic acid cycle intermediate 471
succinate exhibits a similar pattern of delayed labelling under high CO₂ conditions 472
when compared to ambient air, indicating a slowed down glucose usage. Thus, the 473
delay of label incorporation into the Calvin-Benson-Bassham cycle intermediates 474
can be traced back to lower levels of labelled respiratory CO₂ under CO₂ saturated 475
conditions. 476

477

Discussion
***

479

At variance with a previous report (Oesterhelt et al., 2007), *G. sulphuraria* cells are 480
capable of a true mixotrophy, when transferred from strictly photoautotrophic 481
conditions to a light and organic carbon regime, provided that the temperature 482
conditions are kept close to the ones experienced by this alga in its natural 483
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 Oesterhelt *et al.*, (2007) 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 (Nikolova *et al.*, 2017). 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, CO₂ is mainly concentrated by the PEPC/PEPCK driven CCM (Rademacher *et al.*, 2017). 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 O₂ produced by photosynthesis (32.3 ± 7.5 µM and 76.6 ± 2 µM in photoautotrophy and mixotrophy, respectively) is commensurate with the amount of CO₂ available to Rubisco, i.e. the sum of respiratory CO₂ (22 ± 10.5 µM and 61 ± 1.5 µM respectively, assuming a 1/1 stoichiometry with consumed O₂) plus the small CO₂ 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 CO₂ 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 (Lavergne, 1989).
At the same time, increased intracellular CO₂ 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 (Sugawara et al., 1999), photorespiration is expected to proceed at high rates at the high temperatures and low CO₂ 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 CO₂ concentrations (Rademacher et al., 2016). The observed increase in the rate of photosynthesis and gain of biomass under high CO₂ and mixotrophic conditions (25 ± 6 %, when combining data from Fig. 1-2) 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 (Schonknecht et al., 2013; Rossoni et al., 2019a) and import organic carbon available in its environment and to boost CO₂ availability for photosynthesis. While this phenomenon certainly exists in other phototrophs (e.g. Rolland et al., 1997), the capacity to enhance photosynthesis with respiratory CO₂ 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) (Boyd et al., 2012; Hamilton et al., 2012) 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 (Nye et al., 2020)). 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 (Nye *et al.*, 2020). 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; Cho *et al.*, 2020), which may have also allowed Cyanidiales to invade more moderate habitats (Yoon *et al.*, 2006; Azúa-Bustos *et al.*, 2009; Castenholz & McDermott, 2010) 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. *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.

572

573

574

575
576 Acknowledgments

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

595

596

597 Author Contributions

598 G.C., D.L., A.P.M.W. and G.F. designed research; G.C., D.L., E.G., P.W., J.J.,
599 C.H., S.B., D.D.B., J.D., B.G., D.F., M.C. and G.F. performed research; G.C.,
600 D.L., P.W., M.T., C.R., M.F., A.P.M.W. and G.F. analyzed data; G.C., D.L.,
601 A.P.M.W and G.F. wrote the paper.

602

603 Data availability

604 The original contributions presented in the study are included in the article and in
605 the Supplementary information and supplementary dataset files. Further inquiries
606 can be directed to the corresponding author

607

608
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853 **Dataset S1:** Proteins involved in photosynthesis, central metabolism and respiration were selected from the complete proteomic dataset (see Supplementary Dataset S2) and used to build Fig. 3.
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857 **Dataset S2:** Compared proteomic analysis between photoautotrophic, mixotrophic
858 and heterotrophic growth conditions.

859
860 **Dataset S3:** Compared metabolomic analysis between photoautotrophic,
861 mixotrophic and heterotrophic growth conditions.

862
863 **Figure S1:** Consequences of different substrates on *G. sulphuraria* growth in the light.

864
865 **Figure S2:** *G. sulphuraria* growth in photoautotrophic, mixotrophic and heterotrophic conditions driven by a polyol, an hexose and a disaccharide.

866
867 **Figure S3:** Experimental setup to expose cells to a constant photons/cell ratio.

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

869
870 **Figure S5:** Enhancement of cell growth by mixotrophy and sorbitol consumption
871 in *G. sulphuraria*.

872 **Figure S6:** Mixotrophy is restored in *G. sulphuraria* upon addition of a carbon source.

873
874 **Figure S7:** Respiration and net photosynthesis were measured every day in the three different growth conditions (photoautotrophy, mixotrophy and heterotrophy).

875
876 **Figure S8:** Biomass production as a function of transmitted light intensity and CO₂ concentration.

877
878 **Figure S9:** Immunodetection of RuBisCO in phototrophic and mixotrophic cultures under ambient and enhanced CO₂ atmosphere.
880 **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.

881
882
883 **Figure S11:** Respiration fuels photosynthesis in photoautotrophic and mixotrophic *G. sulphuraria* cultures.

884
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886
887 **Figure S12:** Transmission electron microscopy of *G. sulphuraria* SAG21.92 grown 5 days under photoautotrophic, mixotrophic and heterotrophic conditions.

888 **Methods S1:** Microalgae and media composition, growth, cell fresh weight and dry weight estimates, Clark electrode oxygen and photophysiology measurements,
889
890 mass spectrometry-based proteomic analyses, metabolic analyses by IC-MS,
891 electron microscopy sample preparation and observation.

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**Figure 1: Growth enhancement of *Galdieria sulphuraria* SAG21.92 by reduced carbon sources is light dependent.** *a*, Growth curves. Data from 3 biological replicates ± S.D. Error bars are shown when larger than the symbol size. *G. sulphuraria* was grown in flasks at ambient CO₂ in photoautotrophic (light only, 30 µmol photons m⁻² s⁻¹, green), mixotrophic (30 µmol photons m⁻² s⁻¹ plus D-sorbitol 25 mM, orange) and heterotrophic (absence of light, presence of D-sorbitol 25 mM, black). The initial cell concentration was 1.5 × 10⁶ cells per mL. At day 5, the mixotrophic culture was split in two parts, and light was switched off in one culture, (grey). Growth was carried out at 42°C with shaking at 100 rpm, pH 2. **b**, Dry weight estimated at day 9. Mixotrophic biomass (orange bars) exceeds the sum of photoautotrophic (green bars) and heterotrophic (black bars) biomass, highlighting the existence of a synergy under mixotrophic conditions. Data from 3 biological replicates ± S.D. ** 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). The concentration of inorganic nitrogen was 20 mM, while that of inorganic phosphate was 5 mM.

**Figure 2: In situ measurements of photosynthetic electron transfer rate (ETR) in photoautotrophic (light) and mixotrophic (light + 25 mM D-sorbitol) *Galdieria sulphuraria* SAG21.92 cells and biomass production.** Cells were inoculated at 3.5 × 10⁶ 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 CO₂-enriched (0.5%) atmosphere (g, h). a, b, g photosynthetic electron transfer: data from 3 biological replicates ± S.D. b, e, h, Biomass production in photoautotrophic (green, data from 12 biological replicates ± S.D.), heterotrophic (black, data from 8 biological replicates ± S.D.) and mixotrophic (orange, data from 8 biological replicates ± S.D.) conditions, respectively. Cells were collected after 7 days of growth (i.e. 4 days after addition of D-sorbitol). c, f, i, Sketches representing possible CO₂ sources for 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).

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**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.
Conversely, pyruvate kinases (especially M2WVY6) are strongly repressed under phototrophic condition, possibly to maintain a high PEP-oxaloacetate (OAA) pool for efficient fluxes in the carbon concentration cycle. Enzymes involved in photosynthesis are reduced under mixotrophic condition compared to photoautotrophic condition and strongly reduced under heterotrophic conditions. Mitochondrial respiratory proteins involved in the Krebs cycle or in ATP production are virtually not affected with the exception of fumarase and malic enzyme strongly reduced under phototrophic condition. Only representative proteins of the different complexes (e.g. photosynthesis, respiration) are represented. A more complete list of proteins can be found in Dataset S1. The complete set of proteomic data is available in Dataset S2.

**Figure 4: Metabolic changes between phototrophic, mixotrophic and heterotrophic growth conditions of Galdieria sulphuraria SAG21.92.** Non-phosphorylated 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 as a photorespiration-specific metabolite is boxed in red. Glycolate, glycine, serine and glycerate are transported between cellular compartments as indicated by dashed arrows. PGLP: phosphoglycolate phosphatase; GOX: glycolate oxidase; GGAT: glutamate:glyoxylate aminotransferase; GDC: glycine decarboxylase complex; SHMT: serine hydroxymethyltransferase; SGAT: serine:glyoxylate aminotransferase; HPR: hydroxypyruvate reductase; GLYK: glycerate kinase. **b**, Metabolites involved in a putative C4-type CCM. CA: Carbonic anhydrase; PPC: phosphoenolpyruvate carboxylase; PEPCK: phosphoenolpyruvate carboxykinase; PPDK: pyruvate phosphate dikinase; PEP: phosphoenolpyruvate. **c**, Metabolic changes of intermediates of upper glycolytic pathways (EMP, ED, PPP) and purine/ pyrimidine metabolism. The pentose-5P (ribulose-5P, xylulose-5P, ribose-5P, marked with an asterisk) could not be distinguished and are plotted in a single boxed graph. SDH: sorbitol dehydrogenase; FRK: fructokinase; GPI: glucose-6P
isomerase; G6PD: glucose-6P dehydrogenase; GND: 6-phosphogluconate dehydrogenase; EBB: phosphogluconate dehydratase; EDA: KDPG aldolase; PFK: 6-phosphofructokinase; FBPase: fructose-1,6P bisphosphatase; FBA: fructose bisphosphate aldolase; TIM: triosephosphate isomerase; RPE: ribulose-5P epimerase; RPI: ribulose-5P isomerase; TKT: transketolase; TAL: transaldolase. The full list of metabolite changes can be found in Dataset S3. Y axes in the graphs correspond to normalized peak areas and error bars represent the standard deviation of biological quadruplicates.

<caption>**Figure 5: Incorporation of carbon derived from ¹³C-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 CO₂ 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% CO₂). Dark orange cells grown in air supplied with 2% CO₂.</caption>

RuBP: Ribulose 1,5-bisphosphate; 3-PGA: 3-phosphoglycerate; 2-PG: 2-phosphoglycolate; 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 5-phosphate; SBP: Seduheptulose 1,7-bisphosphate; S7P: Seduheptulose 7-phosphate; R5P: Ribose 5-phosphate; Ru5P: Ribulose 5-phosphate.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>
a **Photorespiration**

<figure><img src="image_7.png" /></figure>

b **C4-like CCM**

<figure><img src="image_8.png" /></figure>

c **Upper glycolysis**

<figure><img src="image_9.png" /><figcaption>Figure 4</figcaption></figure>
a

<figure><img src="image_10.png" /></figure>

b

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>Figure 5</figcaption></figure>
MIXOTROPHIC GROWTH OF THE EXTREMOPHILE GALDIERIA
SULPHURARIA REVEALS THE FLEXIBILITY OF ITS CARBON
ASSIMILATION METABOLISM

Gilles Curien¹*, Dagmar Lyska², Erika Guglielmino¹, Phillip Westhoff², Janina Janetzko², Marianne Tardif³, Clément Hallopeau¹, Sabine Brugière³, Davide Dal Bo¹, Johan Decelle¹, Benoit Gallet⁴, Denis Falconet¹, Michele Carone⁵, Claire Remacle⁵, Myriam Ferro³, Andreas P.M. Weber², Giovanni Finazzi¹

¹Laboratoire de Physiologie Cellulaire et Végétale. Univ. Grenoble Alpes, CNRS, CEA, INRA, 38054 Grenoble Cedex 9, France

²Institute of Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany

³EdyP Laboratoire Biologie à Grande Echelle. Univ. Grenoble Alpes, CEA, Inserm, BGE U1038, 38054 Grenoble Cedex 9, France

⁴Institut de Biologie Structurale, Univ. Grenoble Alpes, CNRS, CEA, 71 Avenue des Martyrs, 38044 Grenoble, France

⁵Genetics and Physiology of microalgae, InBios/Phytosystems Research Unit, University of Liege, Belgium

* Corresponding author: Gilles Curien. phone: +33 4 38782509, email: gilles.curien@cea.fr
article acceptance date: 18 March 2021.

**This file includes:**

Methods S1

Figures S1 to S12

Legends for Datasets S1 to S3

**Other supplementary materials for this manuscript include the following:**

Datasets S1 to S3
# 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, (Allen, 1959)) 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 filter-sterilized concentrated organic substrate solutions as indicated in the text. The concentration of organic substrates was selected on the basis of data reported in literature (Oesterhelt *et al.*, 2007). *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 10⁷ cells per mL were centrifuged and resuspended in a fresh medium to obtain an initial cell concentration of 1.5 x 10⁶ cells per mL (in flasks) or 3.5 x 10⁶ 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. m².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 (30 10⁶ 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 = V̇O₂light + |V̇O₂dark|).

## 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 (Johnson *et al.*, 2009). 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, ETR<sub>PSII</sub>, 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 (Maxwell & Johnson, 2000) 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 (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 10⁹ 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 analysis

The dried extracted peptides were resuspended in acetonitrile 5%, trifluoroacetic acid 0.1% and analyzed via online nano-LC-MS/MS (nano-liquid chromatography-tandem 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 10⁶ 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 quantification

Data 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 ((Bouyssie et al., 2020), 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 “Cross-assignment” 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.
## 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 (Wieczorek *et al.*, 2017; Wieczorek *et al.*, 2019). For this, the intensity values were log2 transformed and normalized by Variance Stabilizing Normalization (VSN) algorithm (Huber *et al.*, 2002) with the « overall » option. The missing abundances were imputed according to the Structured Least Square Adaptive (SLSA) method. The Limma test was applied for comparison of one condition versus the other two combined. Due to huge differences in the matrix – proteins identified and intensities – in 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 fold-change (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.

## 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 10 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. To prevent the high amounts of salt from entering the ESI source a Dionex ADRS 600, 2 mm suppressor was used in dynamic mode at a temperature of 15 °C. Spray stability was achieved with a make up consisting of methanol with 10 mM acetic acid delivered with 150 µL min⁻¹ by an AXP Pump. The electro spray was achieved in the ESI source using the following parameters: sheath gas 30, auxiliary gas 15, sweep gas 0, spray voltage - 2.8 kV, capillary temperature 300 °C, S-Lens RF level 45, and auxiliary gas heater 380 °C. For the untargeted approach the mass spectrometer operated in a combination of full mass scan and a data-dependent Top5 MS2 (ddMS2) experiment. The full scan (60-800 m/z) was conducted with a resolution of 140.000 and an automatic gain control (AGC) target of 10⁶ ions with a maximum injection time (IT) of 500 ms. The Top5 ddMS2 experiment was carried out with a resolution of 17.500 and an AGC target of 10⁵ and a maximum IT of 50 ms. The stepped collision energy was used with the steps (15,25,35) to create an average of NCE 25.

**Untargeted data analysis** was conducted using Compound Discoverer (version 3.1, Thermo Scientific) using the “untargeted Metabolomics workflow”. In this workflow automatic retention time alignment is performed in a window of 2 min within 5 ppm mass accuracy as well as unknown compound detection, and compound grouping across all samples. Elemental compositions are predicted based on accurate mass and chemical background is subtracted by using blank extraction samples. Levels of peak annotation are indicated in the supplementary Dataset S3. Lowest level of annotation is based on accurate mass (3 ppm mass accuracy) on MS¹ level and allows to evaluate potential sum compositions and comparisons with databases such as Chemspider (http://www.chemspider.com) with ranking by the mzLogic algorithm. The next level of identification is via MS²
level by comparing MS² fragment spectra using mzCloud (ddMS2) (match factor 50) and MS¹ level ChemSpider (by formula or exact mass). Highest identification level via an in-house MS² 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 p-values, adjusted p-values, ratios, fold change, were also calculated using Compound Discoverer.

## Labelling experiments with ¹³C-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-¹³C₆-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 x10⁸ 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. To this end, cell pellets were resuspended in 0.1 M phosphate buffer (pH 7.4) and 2.5% glutaraldehyde and incubated overnight at 4°C. Cells were then pelleted and washed five times in 0.1 M phosphate buffer. Cells were fixed by a 1h incubation on ice in 500 µL 0.1 M phosphate buffer containing 1% osmium and 1.5% ferricyanide potassium red before they were pelleted and washed five times with
0.1 M phosphate buffer. Pellets were resuspended in 0.1 M phosphate buffer containing 0.1% tannic acid and incubated for 30 min in the dark at room temperature. Again, cells were pelleted and washed five times with 0.1 M phosphate buffer. The samples were dehydrated in ascending sequences of ethanol and infiltrated with ethanol/Epon resin mixture. Finally, the cells were embedded in Epon. Ultrathin sections (70 nm) were prepared with a diamond knife on a PowerTome ultramicrotome (RMC products) and collected on nickel grids. Ultrathin sections were examined on a Philips CM120 transmission electron microscope operating at 80 kV.
<figure><img src="image_13.png" /><figcaption>**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 1.5 10⁶ 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.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**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 1.5 10⁶ 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).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**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). $I_0$ and $I$ represent the incident light (settings on the device) and the light transmitted through the device, respectively. $I_0$ 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 down-regulated 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 ($λ = 590$ 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).</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**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 10⁶ 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.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**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.</figcaption></figure>
<figure><img src="image_18.png" /></figure>

**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 10⁶ 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><img src="image_19.png" /></figure>

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 10⁷ cells in 1 mL of fresh 2xGS medium. Net photosynthesis corresponds to gross photosynthesis +|respiration|. Data from 3 biological replicates ± S.D.
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

**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><img src="image_24.png" /></figure>

**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 β 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><img src="image_25.png" /></figure>

**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><img src="image_26.png" /><figcaption>**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 30 10⁶ 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.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>**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.</figcaption></figure>
**Dataset S1 (separate file). Proteins involved in photosynthesis, central metabolism and respiration were selected from the complete proteomic dataset (see Supplementary Dataset S2) and used to build Fig. 3.** Proteins function and predicted localizations were checked manually (column "Manual annotation"). Cells highlighted in yellow in this column indicate proteins with statistically significant changes in abundance (see Threshold described in Supplementary Dataset S1). Blue-color and orange-color scales indicate decrease and increase in abundance, respectively (See Legend sheet).

**Dataset S2 (separate file). Compared proteomic analysis between photoautotrophic, mixotrophic and heterotrophic growth conditions.** Proteomics analyses were performed on biological triplicates for each growth condition. Quantification was obtained from extracting the MS1 intensity. Merging of results and filtering on both identification and quantification criteria as described in the Supporting Information resulted into 2100 proteins ("protein sets", sheet **Dataset S2-ALL**). These 2100 proteins were further divided into 694 proteins entirely missing in at least 1 condition (sheet **Dataset S2-MissingInOneCond.**) and 1406 proteins that have been quantified in all three conditions (in at least 1 replicate, sheet **Dataset S2-SeenInAllConditions**). This latter subset was submitted to differential analysis. Highlighted in blue: imputed values (in Dataset S2-SeenInAllConditions) that were assigned to partially missing abundance values (i.e. within one condition, in Dataset S2-ALL).

**Dataset S3 (separate file). Compared metabolomic analysis between photoautotrophic, mixotrophic and heterotrophic growth conditions.**

**GC-MS sheet:** In the table 32 compounds are listed with their retention time and chosen quantifier ion fragment. Compound identification was based on retention time and fragment pattern match with an in-house library or the NIST library. Peak areas of each compound were normalized to the internal standard and transformed to readable values by a multiplicator. The p-value for the sample group was calculated using the multiple comparison function from PRISM software based on
the Students t-test. **IC-MS sheet:** In the table 31 compounds are listed with their retention times and calculated molecular weights. Compound identification was based on 5 different sources of which the in-house database created with mzVault was the preferred source of identification. Batch normalization was carried out with pooled QC samples frequently measured during the batch. The p-value for the sample group was calculated in Compound Discoverer by running the Tukey HSD test (posthoc) after an analysis of variance (ANOVA) test and correction was performed by using the Benjamini-Hochberg algorithm for the false discovery rate.

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