Acetylene Reduction Assays (ARA)

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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N2 fixation (acetylene reduction)
Approach: acetylene reduction to ethylene (ARA)
Context: incubation, in situ
Spatial scale: point sample
Temporal scale: minutes to hours
Units: nmol N L-1 h-1 (after conversion from ethylene)
Community captured: bulk; single cell; slurries
Co-measurements: C2H2:N2 reduction ratio for calibration

Method Overview

Nitrogenase, the enzyme responsible for N₂ fixation, also reduces acetylene (C₂H₂) to ethylene (C₂H₄) with high affinity. The Acetylene Reduction Assay (ARA) exploits this by adding acetylene gas to sealed incubation vessels containing seawater samples. Ethylene produced during the incubation is measured by gas chromatography with flame ionisation detection (GC-FID) or photoionisation detection (GC-PID), and converted to an equivalent N₂ fixation rate using a theoretical stoichiometry (3 or 4 mol C₂H₄ per mol N₂, depending on whether the natural H₂-producing reaction is considered).

ARA is simpler and faster than ¹⁵N₂ methods, making it suitable for high-throughput screening or field assays where isotope-ratio mass spectrometry is unavailable.

Scale of measurement

Point sample; incubations of minutes to hours. Short incubation times reduce the risk of feedback effects of acetylene on the diazotroph community.

Data generated

Ethylene production rate, which is converted to an N₂ fixation rate using the molar conversion ratio. Results are reported in nmol N L^-1 h^-1 or nmol N cell^-1 h^-1 for single-cell assays.

Units & currency

Units are nmol N L^-1 h^-1 (after conversion from ethylene). The currency is nitrogen (ethylene is the primary measured currency; nitrogen is derived).

Sample size

Typical samples are 10–200 mL in volume.

Limitations

The theoretical C₂H₄:N₂ conversion ratio (3:1 or 4:1) is not stable across environmental conditions, organisms, and nutrient states, introducing a systematic uncertainty in the conversion. Acetylene inhibits natural H₂ production by nitrogenase, which affects the theoretical ratio. Dissolution of acetylene gas and potential bubble formation introduce variability across replicate bottles. Incubations longer than ~24 h can stress cells and alter the measured rate. ARA does not distinguish N₂ fixation from other acetylene reduction reactions.

Example Applications & Protocols

Classic examples

• Wilson et al. (2012) Comparative assessment of nitrogen fixation methodologies, conducted in the oligotrophic North Pacific Ocean

Common calculations/conversions

• N₂ fixation rate (nmol N L^-1 h^-1) = C₂H₄ production rate (nmol L^-1 h^-1) / conversion ratio (3 or 4).
• Empirical conversion ratios should be determined from parallel ¹⁵N₂ measurements in the same environment when possible.