How it works

In contrast with typical oxygen-based measurements of algal photosynthesis, where photosynthesis is derived from the change in O₂ concentration over time, the LI-6800 is an open, flow-through, steady-state gas exchange system where CO₂ and O₂ concentrations are constant during the measurement.

A differential CO₂ measurement

In the 6800-18 Aquatic Chamber, the carbon assimilation rate is determined from the mass balance of an air stream before and after it interacts with a liquid sample. The CO₂ and water vapor concentrations of the air stream are measured by a pair of high-precision infrared gas analyzers (IRGA). Assimilation is calculated from the concentration differences and flow rate:

Fundamentally, this mass balance gives the flux of CO₂ between the liquid sample and the cuvette headspace. The flux is coupled to the true biological carbon assimilation rate by mass transfer at the air-liquid interface and the kinetics of the carbonate system for the aquatic sample.

When normalized to cell density, mass, or chlorophyll content, the aquatic chamber provides measurements as µmol CO₂ cell^-1 s^-1, µmol CO₂ mg^-1 s^-1, and µmol CO₂ µg^-1 s^-1, respectively.

• The water vapor difference between the incoming and outgoing airstreams is minimized by a patent-pending method.
• Water vapor concentrations are included in the carbon assimilation calculation to account for volumetric dilution.

Rapid equilibration of gas-phase CO₂ and CO₂ in solution through aeration of liquid samples

A carefully controlled aeration scheme ensures that the mass transfer coefficient is not limiting and that the measured flux represents the biological assimilation rate of the sample. To keep the carbonate system at a steady state during measurements, carbonic anhydrase (CA) is added to the sample media for rapid hydration of CO₂ and interconversion with bicarbonate (HCO₃^–) .

Steady state sample conditions

The LI-6800 precisely controls environmental conditions of the sample, including the CO₂ concentration in headspace air and the light environment according to your preferences.

• The CO₂ concentration that the sample is subject to is stable during the measurement. The instrument maintains the CO₂ concentration with a mixer/scrubber system, preventing either CO₂ or O₂ from accumulating in the aquatic sample or headspace.
• The instrument controls light in the chamber – total light, as well as proportions of blue, red, and far-red – enabling a wide variety of light response measurements.
• With an external recirculating water bath, the sample temperature can be maintained at setpoints from >0 °C to 50 °C. Sample temperature is measured and recorded with the dataset.

These features enable time-series data collection on a sample while environmental conditions are kept stable. This ensures that the measured biological response is in the context of known, steady-state conditions.

Chlorophyll a fluorescence

Simultaneous measurements of CO₂ gas exchange and PAM chlorophyll a fluorescence of an aquatic sample provide a more complete impression of photosynthetic processes than either technique alone. CO₂ exchange measurements are indicators of the photosynthetic interaction between algae and dissolved inorganic carbon in solution. Chlorophyll a fluorescence is an indicator of the light reactions of the organisms.

When combined, they reveal more about the photochemistry of algae than either technique alone.

Example measurements

From basic irradiance response measurements to complex multifactor experiments, the aquatic chamber can be used to evaluate biological responses to different environmental conditions.

Algal photosynthetic response to irradiance at constant CO₂

Figure 3 shows assimilation measurements on Chlorella at ambient oxygen (21%) and low oxygen (0.5%) in response to light (Q). The CO₂ concentration entering the chamber was held constant at 400 µmol mol^-1 by the LI-6800 system. Air with 21% oxygen was ambient; low-oxygen air was from a tank of 0.5% oxygen balanced in air. Chamber temperature was held constant at 25 °C using an external water bath. Cells were measured in a saltwater media at 17 ppt salinity.

When O₂ concentrations are reduced in solution, the likelihood of oxygenation of RuBP by RuBisCO—the first step of photorespiration—is reduced, and the overall efficiency of carbon assimilation relative to energy capture increases. There is little impact on the efficiency of PS_II, shown here by the near identical behavior of Φ_PSII. Photorespiration impacts the strength of electron sinks such that 1-qL may be expected to decrease some with an increase in photorespiration (Figure 4).

Active regulation of a portion of NPQ related to cyclic electron flow balances the energetic products of photochemistry, ATP, and NADPH—so as photorespiration is suppressed at low oxygen, down regulation of NPQ is observed (Figure 5).

Algal photosynthetic response to CO₂ at constant O₂ and light intensity

The photosynthetic response to CO₂ was measured on Monoraphidium at ambient O₂ concentration and 700 µmol photons m^-2 s^-1 light intensity. The CO₂ concentration entering the chamber was controlled at different setpoints during these measurements. Chamber temperature was held constant at 25 °C using an external water bath. Cells were measured in a freshwater media buffered to pH 7.0 with a TRIS buffer.

The photosynthetic response to CO₂ is non-linear in nature (Figure 6). At low concentrations, CO₂ is limiting, and assimilation changes proportionally to the concentration.

LI-6800 Aquatic Chamber with 9968-338 Sample Adapter Kit

Sargassum photosynthetic response to irradiance at constant CO₂

With the sample adapter, the aquatic chamber can be used to measure the photosynthetic response of samples that must remain in humid air, as well as samples that are too large to fit into the upper chamber opening, such as coral. Figure 8 shows the irradiance response of Sargassum as measured with the aquatic chamber.

Figure 8.Sargassum photosynthetic response measurements, including assimilation, PhiPSII, and non-photochemical quenching plotted against irradiance (Q). Values are means of three biological replicate curves. Error bars are standard error

LI-COR 6800-18 Aquatic Chamber – Sigmatech Inc. Philippines