Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria

Abstract

Ammonia (NH3)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH3 oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤ 20 μM concentrations of 1-alkynes C8 and C9. In contrast, NH3 oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C8 (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH4(+)-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH4(+)-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH4(+)-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.

Fig 1

(A and B) Response of NO₂⁻ production to C₂ to C₉ 1-alkynes by N. europaea (A) and N. maritimus (B) cultures. N. europaea and N. maritimus were incubated with 5 μM and 20 μM (C_aq) each alkyne, respectively, in the presence of 1 mM NH₄⁺. (C and D) Effects of C₂ to C₉ alkynes on net NO₂⁻ plus NO₃⁻ production by slurries of Corvallis cropped (AOB-dominated) soil (C) and Corvallis pasture (AOA-dominated) soil (D) incubated with 2 and 4 μM (apparent C_aq; see the information in the supplemental material) each alkyne, respectively, in the presence of 1 mM NH₄⁺. See Materials and Methods for further experimental details. NO₂⁻ (A and B) or NO₂⁻ plus NO₃⁻ (C and D) production in the presence of each alkyne was compared with that of a no-inhibitor control (Con). Error bars represent the SD of the mean (n = 3).

Fig 2

Sensitivity of NO₂⁻ production by N. europaea (A), N. multiformis (B), and N. maritimus (C) to different octyne (Oct) concentrations. N. europaea and N. multiformis cultures were suspended in 30 mM TES buffer (pH 7.2) with 1 mM NH₄⁺. N. maritimus cells were suspended in SCM medium with 1 mM NH₄⁺ (pH 7.5). The loss of linearity of NO₂⁻ production by N. europaea and N. multiformis after 5 h was likely due to a low affinity for NH₄⁺. See Materials and Methods for further experimental details. Controls contained no octyne. Error bars represent the SD of the mean (n = 3).

Fig 3

Comparison of the effects of alkyne (acetylene [+Ace], octyne [+Oct]), and no-alkyne ([NA]) treatments on net NO₂⁻ plus NO₃⁻ accumulation and on AOA and AOB amoA gene copy numbers during incubations of CC soil amended with no NH₄⁺ (A and D), low NH₄⁺ (B and E), or high NH₄⁺ (C and F) (0, 2, and 20 μmol NH₄⁺/g soil, respectively). (A, B, and C) NO₂⁻ plus NO₃⁻ accumulation over the time course of the experiment. Error bars represent the SD of the mean NO₂⁻ plus NO₃⁻ concentration (n = 3). d, day. (D, E, and F) AOB and AOA amoA gene copy numbers/g soil. Error bars represent the SD of the average number of copies of the amoA gene/g soil from triplicate qPCRs for each treatment (n = 3). *, NO₂⁻ plus NO₃⁻ accumulation was significantly different in the no-alkyne and octyne treatments within an NH₄⁺ treatment (P < 0.001). Different lowercase letters indicate that NO₂⁻ plus NO₃⁻ accumulation was significantly different in the no-alkyne treatment between different NH₄⁺ levels (P < 0.001). There was no significant difference in the rate of NO₂⁻ plus NO₃⁻ accumulation in the octyne treatment between different NH₄⁺ levels.

Fig 4

Correlation among three methods used to assess the relative contributions of AOA and AOB to soil slurry NPs of diverse Oregon soils. (A) Correlation between the rates of net NO₂⁻ plus NO₃⁻ production that were resistant to octyne (NP_oct) versus resistant to 100 μM ATU (NP_atu). (B) Correlation between the fractions of the RNPs that were insensitive to bacterial protein synthesis inhibitors (RNP_ab/RNP) versus the fractions of activity that were resistant to octyne (NP_oct/NP). Open and closed symbols represent cropped and noncropped soils, respectively. PG, Pendleton grassland; MR, Madras rangeland; KW, Klamath woodlot; CP, Corvallis pasture; PC, Pendleton cropped; MC, Madras cropped; KC, Klamath cropped; and CC, Corvallis cropped. See Table S1 in the supplemental material for site locations and soil characteristics.