Staal et al. 2001

Staal M, te Lintel-Hekkert S, Harren F, Stal L. 2001. Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring. Environmental Microbiology 3(5): 343-351.

These authors present two methods for measuring nitrogenase activity in cyanobacteria, both based on continuous on-line measurement of ethylene produced by the reduction of acetylene by nitrogenase. One method relies on a gas chromatograph to detect ethylene, the other on a not-yet-commercially available laser system. Nitrogenase normally reduces N2 to NH3, but will also reduce other triple bonds such as that between the carbon atoms in acetylene, hence this measurement assay was developed in the late 1960s. The nitrogenase enzyme is inhibited by oxygen, but is very energy-intensive when reducing N2, thus cyanobacteria may fix Nitrogen in a manner dependent upon but separated from photosynthesis in either (at night) time or space (specialized cells).

Previous ethylene-based methods were based on incubations of cells in air-tight containers, for incubation periods sufficient to saturate nitrogenase with acetylene and accumulate sufficient ethylene for detection. Changes in O2 and CO2 concentrations during these hours-long incubations introduce conflating variables; O2 is depleted and CO2 accumulates in the dark, vice-versa in the light. CO2 concentration affects pH, while O2 inhibits nitrogenase and indirectly relates to available energy. In addition, long incubations will fail to detect any event occuring on a frequency shorter than the incubation time, such that processes occurring on time scales of seconds to minutes will not be registered. Finally, saturation of nitrogenase with acetylene eventually leads to nitrogen starvation and the synthesis of more nitrogenase.

In contrast, on-line methods involve the continuous flow of gas over the sample. This can be used to measure gas flux only when the system reaches a steady state (however, see the discussion of steady-state and non-steady-state modes in gas sampling chambers in Davidson et al., 2002). For nitrogenase-ethylene, this steady state may be reached as quickly as 1 minute under ideal, high-surface-area conditions. In addition, while O2 and CO2 concentrations are controlled during on-line measurement, ethylene cannot accumulate, thus only very low concentrations will be present.

Nitrogenase activity was higher under light-saturation conditions than in the dark, but became inhibitory with longer incubation times. Changes in nitrogenase activity with time and light level probably relate to both energy limitation in the dark and oxygen inhibition in the light. Changes relating to growth, internal rythms, or energy depletion only occurred after very long incubations, such as 24 hours. I am not certain how long-term energy depletion is distinct from short term energy limitation in the dark.

This paper suggests it may be possible for us to measure nitrogenase activity with acetylene and ethylene using the Gasmet FTIR system and its chambers.