Acetylene-block proxy for denitrifcation enzyme activity
| Denitrification enzyme activity (acetylene block) |
|---|
| Approach: acetylene inhibition of N2O reductase; GC measurement of N2O accumulation |
| Context: incubation, lab |
| Spatial scale: point sample |
| Temporal scale: minutes to hours |
| Units: nmol N2O-N L-1 h-1 |
| Community captured: bulk |
| Co-measurements: nitrate, oxygen, acetylene |
Method Overview
Acetylene (C2H2) at ~10 kPa partial pressure blocks the final step of denitrification, the reduction of N2O to N2 by N2O reductase (nosZ). In acetylene-amended, anoxic incubations supplied with saturating nitrate, denitrifiers reduce NO3- → NO2- → NO → N2O, but cannot proceed further to N2. The accumulating N2O is measured by gas chromatography (GC-FID or GC-ECD) and the rate of N2O accumulation provides an estimate of denitrification enzyme activity (DEA)[1]. Because nitrate is provided at saturating concentrations, the assay measures potential (Vmax) rather than in situ denitrification activity.
Scale of measurement
Point sample; short incubations (minutes to hours) under anoxic conditions with nitrate and acetylene amendments.
Data generated
N2O-N accumulation rates (nmol N2O-N L-1 h-1), which provide a proxy for potential denitrification enzyme activity. These rates overestimate in situ denitrification because they are measured at saturating nitrate and anoxia.
Units & currency
Units are nmol N2O-N L-1 h-1. The currency is N2O (nitrogen equivalent).
Sample size
Samples are typically 250 mL to 1 L in volume.
Repositories & databases
Limitations
Acetylene assumes complete blockage of N2O reductase, but incomplete inhibition can occur, particularly at low acetylene concentrations. Acetylene also inhibits nitrification (AMO) and can scavenge nitric oxide (NO), creating additional artifacts. The theoretical assumptions of complete anoxia and saturating nitrate may not reflect in situ conditions. The method measures potential activity, not actual in situ denitrification rates.
Example Applications & Protocols
Classic examples
- Balderston et al. (1976) Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus [1]
Recent applications
- Groffman et al. (2006) Methods for measuring denitrification: diverse approaches to a difficult problem [2]
Common calculations/conversions
- DEA (nmol N L-1 h-1) = Δ[N2O] / time; assuming equimolar conversion from NO3- via N2O.
References
- ↑ 1.0 1.1 Balderston, W. L., Sherr, B., & Payne, W. J. (1976). Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus. Applied and Environmental Microbiology, 31(4), 504–508. https://doi.org/10.1128/aem.31.4.504-508.1976
- ↑ Groffman, P. M., Altabet, M. A., Böhlke, J. K., Butterbach-Bahl, K., David, M. B., Firestone, M. K., Giblin, A. E., Kana, T. M., Nielsen, L. P., & Voytek, M. A. (2006). Methods for measuring denitrification: diverse approaches to a difficult problem. Ecological Applications, 16(6), 2091–2122. https://doi.org/10.1890/1051-0761(2006)016[2091:MFMDDA]2.0.CO;2