Aarhus University Centre for Water Technology, Department of Biology, Section for Microbiology
Involved scientists: F. Steininger, T. Merl and K. Koren
Oxygenic photosynthesis was and is one of the most important metabolic processes in the biosphere as it is eliminating carbon dioxide from the atmosphere while producing oxygen1. Photosynthesis is predominantly responsible for producing and maintaining the oxygen content in the Earth’s atmosphere and supplies most of the energy necessary for life on Earth. Phototrophic organisms use photo-pigments, such as chlorophylls, carotenoids, etc. to harvest light and to convert light energy into chemical energy, i.e. generate ATP 2. In addition to that, organic matter is being produced by photosynthetic carbon dioxide fixation. This is an essential processes within the carbon cycle.1
The carbon cycle starts with the reduction of CO2 by using the energy of light and resulting in (CH2O)n and oxygen, where (CH2O)n characterises organic compounds. The inverse reaction from (CH2O)n to carbon dioxide and water is known as respiration. A higher degree of photosynthesis than respiration is therefore necessary in order to obtain organic matter.1
Oxygenic photosynthesis: CO2 + H2O --> (CH2O)n + O2
Respiration: (CH2O)n + O2 --> CO2 + H2O
Hence, oxygenic photosynthesis can be determined by measuring the O2 concentration and its change at different light intensities. Furthermore, assessing the pH is an indication of CO2 fixation as an increase in pH exhibits the decrease of carbon dioxide.
In here we describe how the combined O2, pH and temperature respiration vials can be used to study this important biochemical processes. We demonstrate the basic experimental setup and show exemplary data using green algae from the Tetraselmis genus as an example.
The algae from the Tetraselmis genus was cultured in the standard medium F/2 and maintained at room temperature at a 12 h day 12 h night rhythm providing ~300 µmol photons/(m²*s) during the day phase.
2.2 Sensor Calibration
The sensor vial with the integrated optical oxygen, pH and temperature sensors was calibrated, each sensor at a time. For the temperature calibration a 1-point calibration was performed by positioning an external Pt100 temperature sensor (provided by PyroScience) in close proximity to the temperature optode. The oxygen sensor was calibrated by 2-point calibration (de-oxygenated water and air saturated water). pH sensor calibration was performed by 2-point calibration (pH 4.01 and pH 10 buffers, provided by PyroScience).
2.3 Measurement Setup
The sensor vial was filled with algae solution and a magnetic stirring bar (Note: gas bubbles need to be avoided when filling the vial).The optical fibres were attached pointing on to the respective sensors, as can be seen in Figure 1 and the vial was placed on a magnetic stirrer.
The respiration vial was covered with a black cloth to exclude light for obtaining a darkness measurement (respiration). The cloth was removed, and a halogen lamp was positioned in front of the vessel and the light intensity was measured using a spectral light meter (Gigahertz-Optik MSC15), as shown in Figure 2. The distance of the lamp was then increased after each measurement to decrease the irradiance of the sample. The sample was exposed to each light intensity for 3 minutes, followed by 30 s of measuring. After the last light-measurement the vessel was again covered with a black cloth to measure the parameters during respiration of the algae.
3. Results and Discussion
In figure 3 the obtained raw data of the measurement can be seen. While the oxygen concentration decreases during dark phases (respiration) an overall increase is observed during light phases. The pH increases in the course of the experiment. This is in accordance with expectations, due to the CO2 fixation during photosynthesis. After about 80 minutes the vessel was again covered to exclude light, which resulted in a slight decrease in pH. As respiration is now the predominant process leading to the production of CO2 and therefore a decrease in pH. In blue the internal temperature compensation, measured by the optical temperature sensor, can be seen. Interestingly the temperature increase was more pronounced when the vial was covered rather than illuminated. A temperature increase of nearly 2 °C over the course of around 30 min is rather drastic and demonstrates the importance of internal temperature measurement.
Oxygen production is the change of oxygen concentration per time (d[O2]/dt). Therefore, the slopes of each oxygen measurement interval were plotted against the measured irradiance before each measurement (Figure 4). In general, the photosynthesis rate and therefore oxygen production increases with increasing light intensity until a plateau is reached, which corresponds to the irradiance during incubation of the algae culture.
The respiration vial with the combined O2, pH and temperature sensor is an ideal tool to study photosynthesis. The internal temperature compensation ensures stable readings and compensates well for fluctuations. By measuring both O2 and CO2 the product and one of the educts of photosynthesis are measured (at least indirect for CO2). This allows a better description of the system and the respective dynamics.
Sensor calibration and use is straightforward. The provided accessories (e.g. adapter rings) ensure a fast start-up and smooth operation.
Lars Borregaard Pedersen, Mette L. G. Nikolajsen and Ronny Mario Baaske are thanked for excellent technical support and for maintaining the culture.
1 Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H. & Stahl, D. A. Brock Biology of Microorganisms. (Pearson, 2015).
2Bryant, D. A. & Frigaard, N. U. Prokaryotic photosynthesis and phototrophy illuminated. Trends in Microbiology (2006).