Materials and reagents

Inorganic ¹³C (Sigma-Aldrich, catalog number: 364592)
N₂ gas (Oxygen and Argon Works Ltd.)
O₂ gas (Oxygen and Argon Works Ltd.)
¹²CO₂ gas (Oxygen and Argon Works Ltd.)
¹³CO₂ (isotopic purity 99-atom percentage) gas (Sigma-Aldrich, catalog number: 364592-10L-EU)
Lime soda (Merck Millipore, catalog number: 1068395000)
Chlamydomonas reinhardtii strain CC-124 (UTEX Culture Collection of Algae)
Chlorella sorokiniana UTEX 1663 (UTEX Culture Collection of Algae)
Chlorella ohadii
HEPES (Sigma-Aldrich, catalog number: 54457)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 655104)
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C4901)
Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: 213330)
Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: 230391)
Dipotassium phosphate (K₂HPO₄) (Sigma-Aldrich, catalog number: P3786)
Monopotassium phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: PX1562)
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)
Boric acid (BO₃H₃) (Sigma-Aldrich, catalog number: B0394)
Zinc sulphate heptahydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: 221376)
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sigma-Aldrich, catalog number: 221279)
Iron(II) sulphate heptahydrate (FeSO₄·7H₂O) (Sigma-Aldrich, catalog number: 215422)
Cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) (Sigma-Aldrich, catalog number: 255599)
Copper sulphate pentahydrate (CuSO₄·5H₂O) (Sigma-Aldrich, catalog number: 209198)
Ammonium molybdate tetrahydrate (Mn₇O₂₄(NH₄)₆·4H₂) (Sigma-Aldrich, catalog number: 09878)
Methanol (Sigma-Aldrich, catalog number: 34860)
Chloroform (Sigma-Aldrich, catalog number: 650498)
tert-Butyl methyl ether (Sigma-Aldrich, catalog number: 650560)
Liquid nitrogen (N₂)
Dry ice
Ethanol, technical
HP (HEPES-phosphate) medium (pH 7.2) (see Recipes)
1. Nutrient stock (see Recipes)
2. Phosphate buffer 1,000× (see Recipes)
3. Hutner’s solution (see Recipes)
MTBE (methyl tert-butyl ether) solution (see Recipes)

Equipment

Gas (mass-flow) controllers calibrated for N₂, O₂, and CO₂ (Brooks Instruments, model: 5850S) and smart control software (Brooks Instruments)
20 mL syringes
Syringe pump (KF Technology, model: NE-1600 Multi-Channel Syringe Pump)
Transparent tubing (Vici Jour, catalog number: R-T-4007-M10)
Luer adapters (Upchurch Scientific, Union Polypropylene, UPP series)
1. Flangeless ferrule (Upchurch Scientific, catalog number: UPP-200N)
2. Union assembly (Upchurch Scientific, catalog number: UPP-360)
3. Luer adapter (Upchurch Scientific, catalog number: UPP-658)
Gas washing bottle (Robu Glasfilter-Geräte, catalog number: 41101)
Flat glass photobioreactors (PSI, model: FMT-150)
Cool-white LED array (PSI, model: 3500-D)
Centrifuge (Eppendorf, model: 5418 R)
LC-MS (Thermo Fisher Scientific, model: TSQ Quantum Ultra with Excalibur 2.07 SP1 and TSQ Quantum 1.4 software)
GC-MS (LECO Instruments GmbH, model: Pegasus III TOF-MS)
Gas chromatograph (Agilent Technologies, model: 6890N24)
Humidifier:
1. Laboratory bottles (DURAN, Sigma-Aldrich, catalog number: Z305197)
2. Head with filter-disc for GL 45 (ROBU Glasfilter-Geraete GmbH, catalog number: ISO 4793-80)
50 mL centrifuge tubes (Sarstedt, catalog number: 62.547.004)
Lyophilizer (Martin Christ, model: Alpha 2-4 LSCbasic)
Microfluidic mixer (Manufactured based on original design from Sivashankar et al., 2016)
Light meter (LI-COR, model: LI-250A)
Air pump (Sera precision, model: air 275 R plus, catalog number: 08814)
Autoclave (Witeg, model: WAC-60 230 V)
Filter 0.22 μm (Sigma-Aldrich, catalog number: SLMP025SS)
Water bath (GFL, model: 1002)
Oxygen sensor (Mettler Toledo, model: InPro 6800)
pH sensor (Mettler Toledo, model: InPro 3250)
Upright microscope (Nikon, model: Eclipse E200)
SpeedVac concentrator (Thermo Fisher Scientific, model: SPD210)
Gas analyser (LI-COR, model: LI850)

Procedure

Algal cultures
Grow algal cultures in flat glass bioreactor vessels, as specified in the instructions below. Further details associated with this technique are described in Treves et al. (2017).
1. Prepare HP medium (see Recipes) for the bioreactors.
2. Autoclave (121 °C, saturated steam, 30 min) each bioreactor cuvette (see https://photo-bio-reactors.com/products/photobioreactors/#details) with 1 L of HP medium and both oxygen and pH sensors connected.
3. On a sterile bench, connect 0.22 μm filters to all air inlets/outlets of the system and add 10 mL of axenic inoculum into the bioreactor medium, to reach an initial cell density of OD_735nm = 0.02. The optical density of the culture is measured by the bioreactor following activation (see https://photo-bio-reactors.com/products/photobioreactors/#details).
4. Initiate the bioreactors at the optimal or target temperature defined for each strain and irradiate the cells at light regimes (in μmol photons m^-2·s^-1) optimised and designed for the study goals. For reliable estimation of in vivo fluxes, illumination levels used for labelling should be set to reflect the penetrating light levels within the bioreactor, as measured using an integrating sphere inside the running culture in the bioreactors.
5. Supply air for bubbling using an air-pump at approximately 1 L/min and monitor pH, dissolved oxygen concentration, and optical densities (OD) at 680 and 735 nm every 1–5 min, through integral bioreactor sensors (https://photo-bio-reactors.com/products/photobioreactors/#details).
6. Perform independent biological replicates of three separate bioreactor runs for each alga or condition.
7. Determine the weight of organic material within the samples through the measurement of ash-free dry weight per volume for each culture, as previously described in Klassen et al. (2015).
  Note: Assess and validate the axenicity of the cultures with light microscopy and Luria-Bertani plating/incubation.
¹³CO₂-labelling and sampling procedures
Two setups are used to provide inorganic ¹³C to algal liquid cultures (Figure 1).

Figure 1. Simplified labelling procedure scheme. Flowchart presenting the main steps of rapid (left) and slow (right) labelling procedures.
1. Very short pulses (up to 40 s) and rapid labelling (time points 5, 10, 20, and 40 s):
  1. Fill a glass bottle with 400 mL of fresh HP medium (pH 7.2).
  2. Place the glass bottle in a temperature-controlled water bath kept at the growth temperature of each algal culture, preferably in a separate room to avoid ¹³CO₂ contamination in the bioreactors’ running cultures prior to mixing.
  3. Bubble with the synthetic gas mixture (78% N₂, 21% O₂, and 400 ppm ¹³CO₂) for 30 min to reach < 1% residual ¹²CO₂ levels (see Figure 2B). Run exhaust flow through lime soda pellets via gas washing bottle to avoid ¹³CO₂ cross contamination of running cultures.
    
    Figure 2. Kinetics of ¹³CO₂ supply via bubbling (A) through algal cultures or (B) HP medium or double-distilled water (DDW). Monitoring of ¹²CO₂ levels at the pulse, as measured on the exhaust of algal cultures/medium/DDW using a gas analyser. (A) Different algal cultures, grown to a density corresponding to T0 for each alga and condition, were bubbled with natural air. Thereafter, the natural air was rapidly changed (red arrow) to ¹³C-based synthetic labelled air mixture (see Procedure). Reciprocally, approximately 20 min after the first change, bubbling is rapidly replaced again for natural air (green arrow).
  4. Wash a 20 mL syringe three times and fill it with the bubbled solution (keep contact with air to a minimum).
  5. Load the syringe to a syringe pump.
  6. Connect the syringe with transparent tubing by screwing the luer to the screw tip of the syringe.
  7. Turn on the light source above the syringe pump and bioreactor and adjust to illumination levels measured in step A4.
  8. Connect transparent tubing (with length adjusted to pulse time, see Notes) between the mixer and the cooled 50 mL tubes (see Figure 3A).
    
    Figure 3. Rapid algal labelling system scheme. (A) Fresh medium bubbled with ¹³CO₂-based synthetic air mixture (see Procedure) is mixed with fresh algal sample withdrawn from the bioreactor under controlled LED illumination. (B) Solutions are rapidly (< 1 s) mixed in a tailor-made transparent chip and injected through transparent tubing into 70% methanol solution kept at -70 °C. Pulse length (5–40 s) is controlled via tubing length.
  9. Withdraw a fresh algal sample into a 20 mL syringe using a transparent tube under the light. Load the syringe to the pump while avoiding any shading throughout the process.
  10. Connect the syringe to transparent tubing by screwing the luer to the screw tip of the syringe (Figure 3A).
  11. Operate the pump manually to push the liquids in both syringes and remove residual air from the tubes to the microfluidic mixer. Program the syringe pump to the required volume in each of the four tube lengths (see Section B, step 1h, above). Push the cultures and fresh media through the transparent mixer and tubing and collect the 1:1 mixture to a 70% methanol solution in 50 mL tubes cooled to -70 °C. Tubes are cooled in an ethanol bath precooled with dry ice (see scheme in Figure 3A). Inject the mixed sample into the cooled 70% methanol to reach a 1:2 ratio (e.g., 15 mL of sample into 30 mL of 70% methanol) (see Notes).
  12. Keep the cooled 50 mL tubes at -70 °C until centrifugation (3,200× g, 3 min, -9 °C).
  13. Discard the supernatant (see Notes) and freeze the pellet immediately in liquid nitrogen.
  14. Repeat the same procedure for the non-labelled samples for each pulse time series replica by mixing the same algal culture with fresh HP medium pre-bubbled with ambient air at the same temperature as in Section B, step 1b.
  15. Following rapid freezing, samples may be stored in a -80 °C freezer until extraction.
  16. Resuspend the frozen pellets in ice-cold methanol/chloroform (5:1, v/v).
  17. Perform four freeze-thaw cycles (see Notes) of resuspended cells and perform metabolite extraction following the methanol/chloroform procedure (see Mettler et al., 2014).
    Notes:
    1. Synthetic gas mixture is mixed using mass flow controllers as described previously (Szecowka et al., 2013).
    2. Light is provided by an upper-positioned cool-white LED array through the entire route from sampling at the bioreactor to the quenching tube, with intensities corresponding to the penetrating light for each culture and avoiding shading.
    3. Pulse duration is implemented by fitting the length of the transparent tubing downstream to the mixer to spray the culture/¹³CO₂ solution mixture into the quenching tube at desired timings.
    4. Metabolites level within the supernatant (see section B, step 1m) should be assessed by LC-MS/MS and should ideally be up to approximately 1% that of equivalent pellets.
    5. Depending on the cell wall rigidity of the algal strains, the number of freeze-thaw cycles should be adjusted to the point where all chlorophyll migrates to the chloroform phase.
    6. Microfluidic mixer (Figure 3B) may be produced by 3D printing, according to the detailed design provided in Sivashankar et al., 2016.
2. Long ¹³C incubation times (15–300 min) and slow labelling:
  Introduce inorganic ¹³C via direct bubbling of the cultures with the gas mixture used for rapid labelling.
  1. Bubble algal cultures with ¹³CO₂ gas mixture (approximately 1 L/min) through a humidifier as done for the rapid labelling. Switch the gas mixture from natural air to the same synthetic ¹³C-labelled air mixture (T0). (Residual ¹²CO₂ in the cultures should be below 2% within a few minutes of pulse. See Figure 2A). Run exhaust flow from labelled culture through lime soda pellets via gas washing bottle to avoid ¹³CO₂ cross contamination of other running cultures.
  2. Collect samples into 50 mL Falcon tubes before switching (T0) and at 15, 30, 60, 120, 180, and 300 min of bubbling with ¹³C synthetic gas mixture.
  3. Centrifuge immediately (3,200× g, 3 min, -9 °C), remove supernatant, and freeze pellets in liquid nitrogen.
  4. Resuspend frozen pellets in precooled (-20 °C) MTBE solution.
  5. Perform four freeze-thaw cycles of resuspended pellets, followed by a two-phase MTBE extraction to analyse metabolites, protein, and starch, as described in Jüppner et al. (2017).
    i. Dry the upper MTBE phase, containing lipids, in a SpeedVac concentrator and store at -80 °C to analyse lipid content and ¹³C labelling.
    ii. Dry the lower phase, containing the polar and semipolar metabolites, in a SpeedVac concentrator and store at -80 °C for metabolite profiling.
    iii. Store the solid pellets containing the precipitated protein and starch at -20 °C for further analysis of labelling into starch and protein as previously described (Bradford, 1976; Arrivault et al., 2009; Ishihara et al., 2015; Fernandez et al., 2017).
    Notes:
    1. Slow labelling is required, since longer incubation times would lead to Ci depletion in the closed microfluidic system.
    2. Measured residual ¹²CO₂ in the cultures reaches below 2% within a few minutes of pulse (Figure 2A) and H¹²CO₃- is rapidly equilibrated by the cells as previously reported for low CO₂-acclimated algae (Tchernov et al., 2003) and demonstrated here by the absence of a slow ¹²CO₂ decay phase in the cultures (Figure 2A and 3B).
    3. In step 2c, centrifugation is necessary as some metabolites can accumulate in the medium over time, thus leading to labelling patterns in the complete suspension not reflecting that in the cells.

Data analysis

Data analysis, peak annotation, and quality control (QC) procedures are thoroughly described in Arrivault et al. (2009) and Ma et al. (2014). Briefly, data analysis includes metabolite peak annotation and correction, followed by correction of raw data for the abundance of stable isotopes using the Corrector software tool (https://www.mpimp-golm.mpg.de/19405/Corrector_package_V1_91.zip), analysis of calibration curves for each metabolite and correction for signal decay along each run using periodic injection of standard mix solution; relative isotopomer abundance (m + n) for each metabolite is calculated as in Szecowka et al. (2013).

Recipes

All recipes regarding the preparation of the HP medium and associated stocks are available in Harris (1989).

HP medium, pH 7.2 (for 1 L)
HEPES 1.19 g
Nutrient stock 25 mL
Phosphate buffer 1,000× 1 mL
Hutner’s solution 1 mL
Make up to 1 L with DDW, set pH at 7.2 using NaOH, and autoclave.
Store in a glass bottle at room temperature (solution is not light sensitive).
Note: Following autoclave, add 25 mL of CaCl₂ 18 mM.
Nutrient stock (for 1 L)
NH₄Cl 8.57 g
MgSO₄·7H₂O 4 g
Make up to 1 L with DDW and autoclave.
Store in a glass bottle at room temperature (solution is not light sensitive).
Phosphate buffer 1,000× (pH = 7) (for 1 L)
K₂HPO₄ 106 g
KH₂PO₄ 53 g
Make up to 1 L with DDW and autoclave.
Store in a glass bottle at room temperature (solution is not light sensitive).
Hutner’s solution, pH = 6.5–6.8 (for 1 L)
EDTA 50 g
Bo₃H₃ 11.4 g
ZnSO₄·7H₂O 22 g
MnCl₂·4H₂O 5.06 g
FeSO₄·7H₂O 4.99 g
CoCl₂·6H₂O 1.61 g
CuSO₄·5H₂O 1.57 g
Mn₇O₂₄(NH₄)·4H₂O 1.1 g
Store in a glass bottle at 4 °C and wrap the glass bottle in aluminium foil (solution is light sensitive).
MTBE solution
Methanol/methyl tert‐butyl‐ether (1/3).
Store in a glass bottle at 4 °C (solution is not light sensitive).

Acknowledgments

We thank Dr. Stephanie Arrivault from MPI-MP for providing support for the optimization of algal harvest and extraction procedures through iterative LC-MS/MS measurements. We thank Prof. Dr. Mark Stitt for constructive discussions regarding experimental setup and controls design. We thank Dr. Hirofumi Ishihara for his support of integrating the labelling mixer with existing ¹³C-labelling gas mixer setup. This work was supported by grant 1697/22 from the Israeli Science Foundation and by the Alon fund from the Israel Council of Higher Education. This protocol was adapted from Treves et al. (2022).

Competing interests

The authors declare no competing interests.

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