Plant Metabolism Department, Rheinland-Pfälzische Technische Universität
Kaiserslautern-Landau
Ayla Atila and Haim Treves

Chlorella ohadii is a microalga isolated from the biological sand crusts in the southern deserts of Israel, one of the harshest environments on earth. This alga exhibits a multiphasic growth pattern under both extreme 3000 µmol photons m⁻² s⁻¹ (EIL) and low 100 µmol photons m⁻² s⁻¹ (LL) illumination levels. Previous studies suggest that shifts in metabolic processes drive the transitions between the growth phases described here. However, the precise mechanisms distinguishing these phases, especially in terms of photosynthetic fluxes, remain poorly characterized. We therefore aimed to identify differences in in vivo photosynthetic rates between the phases using a highly sensitive system.

Methods

O₂ evolution rate was measured using FireSting optical oxygen meter (PyroScience FireSting,
OXROB3 oxygen probe), Each measurement was conducted under both in vivo light, i.e. under
incident light, considering the shadowing in the vessel and saturated maximum light. For each
measurement, 5 mL of the culture was sampled from a bioreactor, aerated by shaking in a 50
mL tube, and then transferred to a 5 mL quartz cuvette connected to an O₂ probe. To account
for light respiration, O₂ exchange was also measured using Membrane Inlet Mass
Spectrometer (MIMS). The O₂ metabolic chamber was fully controlled for temperature and
illumination (FIG S1).

FIG 1. Growth curves of C. ohadii cell cultures grown in photobioreactors. Cell density (O.D._720nm, green),
pH (purple) and Oxygen level (blue) are shown along the different growth phases

Results

Using the FireSting optical oxygen meter with robust oxygen probe, we were able to extract
in vivo O₂ evolution rate from C. ohadii different growth phases. Different light intensities and
growth phases influenced oxygen dynamics in this alga. At lower light intensities, respiration
exceeds photosynthesis, resulting in a net consumption of oxygen, whereas higher light
intensities tend to promote positive net photosynthesis (FIG 2). The transition between
growth phases also plays a role, with cultures adjusting their metabolic activity over time.
Notably, in phase 1, both cultures grown at LL and EIL show significant variations in
consumption rates, with higher consumption rate than in phase 2. Additionally, in phase 2,
algae grown in LL exhibit oxygen consumption, while those grown EIL in P2 demonstrate net
oxygen production.

FIG 2: Oxygen concentration of C. ohadii under various growth and lighting conditions and phases (n=3).
The Y-axis represents oxygen concentration (O₂ [µM]), and the X-axis shows time (minutes). A and B display
data for cultures grown at 100 and 3000 µmol photons m⁻² s⁻¹ during phase 1 (P1), respectively, while C
and D show corresponding data for phase 2 (P2). Each condition involved two cycles: 4 minutes of darkness
followed by 5 minutes of in vivo light exposure in the first cycle, and 4 minutes of darkness followed by 5
minutes of maximum light in the second cycle (n=3).

Conclusion

This study provides important insights into the metabolic responses of C. ohadii under
varying light intensities and growth phases, particularly in relation to oxygen dynamics.
Furthermore, O₂ exchange rates measured with the FireSting optical oxygen meter were
in qualitative agreement with MIMS data, yet the former provided a far more sensitive
and robust readout.

Related Products from PyroScience

• FireSting-O2 4-channel oxygen meter (FSO2-C4)
• OXROB3 robust oxygen probe
• OXVIAL4 sensor vial with ADVIAL4T adapter kit