Effects of specific inhibitors on anammox and denitrification in marine sediments

Abstract

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 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. 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 microM-1; 95% inhibition at approximately 70 microM). 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 approximately 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.

FIG. 1.

Plots of concentrations of excess isotopically labeled N₂ (²⁹N₂ and ³⁰N₂) versus time for slurry incubations with ¹⁵NH₄⁺ plus ¹⁴NO₃⁻ (A), ¹⁵NO₃⁻ (B), and ¹⁵NO₃⁻ plus 220 μM acetylene (C).

FIG. 2.

Plots of production of excess ²⁹N₂ and ³⁰N₂ versus time for slurry incubations with ¹⁵NH₄⁺ plus ¹⁴NO₃⁻ (A), ¹⁵NO₃⁻ (B), and ¹⁵NO₃⁻ plus 3.3 mM methanol (C).

FIG. 3.

Rates of dinitrogen production by anammox and denitrification as a function of the concentration of acetylene. A and D indicate anammox and denitrification, respectively. The error bars indicate standard errors. The solid line represents the following function: anammox rate = 0.61 × e^{−0.10 × [acetylene]}. The dotted line represents the following function: denitrification rate = 6.47 × e^{−0.049 × [acetylene]}.

FIG. 4.

Concentrations of NO₃⁻ and NO₂⁻ in control incubation and incubation with 220 μM acetylene. The error bars indicate standard deviations. The data are data for examples of incubations amended with ¹⁵NO₃⁻. The same trends were observed in incubations with ¹⁵NH₄⁺ plus ¹⁴NO₃⁻.

FIG. 5.

Anammox and denitrification rates as a function of the methanol concentration. A and D indicate anammox and denitrification, respectively. The error bars indicate standard errors. The solid line represents the following function: anammox rate = 1.40 × e^{−0.79 × [methanol]}. The dashed line represents the following linear function: denitrification rate = 0.33C + 2.04 (R² = 0.99).

FIG. 6.

Net increases and decreases in NO₂⁻ and NH₄⁺ concentrations as a function of the methanol concentration. Rates were calculated from the slopes of linear regression lines of the concentrations of NO₂⁻ and NH₄⁺ versus time in incubations amended with ¹⁵NO₃⁻. The decreases and increases in nutrient concentrations over time are indicated by negative and positive rates, respectively. The error bars indicate standard errors.