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N2 fixation estimates in real-time by cavity ring-down laser absorption spectroscopy

Cassar, N, J-P Bellenger, RB Jackson, J Karr, BA Barnett
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Real-time estimates of nitrogen fixation are needed to close the global nitrogen budget and remain a critical gap in both laboratory and field experiments. The most common currency for estimating N2 fixation is acetylene reduction to ethylene. We present a new method for continuous real-time measurements of ethylene production: Acetylene Reduction Assays by Cavity ring down laser Absorption Spectroscopy (ARACAS). In ARACAS, air in the headspace of an incubation chamber is circulated with a diaphragm pump through a cavity ring-down ethylene spectrometer and back to the incubation chamber. This paper describes the new approach and its many benefits compared to the conventional detection of ethylene by flame ionization detector gas chromatography. First, the detection of acetylene reduction to ethylene is non-intrusive and chemically non-destructive, allowing for real-time measurements of nitrogenase activity. Second, the measurements are made instantaneously and continuously at ppb levels, allowing for observation of real-time kinetics on time intervals as short as a few seconds. Third, the instrument can be automated for long time periods of measurement. Finally, our technique will be widely accessible by the research community as it can be readily adapted to most existing acetylene reduction protocols and is based on a modestly priced, commercially available instrument. We illustrate its use for measuring N2 fixation using two species, the diazotrophic bacterium Azotobacter vinelandiiand the lichen Peltigera praetextata. We also discuss potential limitations of the approach, primarily the implications of potential leaks in analyzers, as well as future improvements.

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