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Applied and Environmental Microbiology, May 2007, p. 3151-3158, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.01898-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

Nordic Center for Earth Evolution, Institute of Biology, University of Southern Denmark, DK-5230 Odense M,¹ National Environmental Research Institute, DK-8600 Silkeborg, Denmark²

Received 9 August 2006/ Accepted 8 March 2007

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 µM^–1, and there was 95% inhibition at 30 µM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 µM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 µM^–1; 95% inhibition at 70 µM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM^–1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.

Published ahead of print on 16 March 2007.