Inhibition of methane oxidation by nitrogenous fertilizers in a paddy soil

Abstract

Nitrogenous fertilizers are generally thought to have an important role in regulating methane oxidation. In this study, the effect of ammonium on methane oxidation activity was investigated in a paddy soil using urea at concentrations of 0, 50, 100, 200, and 400 μg N per gram dry weight soil (N/g.d.w.s) and ammonium sulfate at concentrations of 0, 50, and 200 μg N/g.d.w.s. The results of this study demonstrate that urea concentrations of 200 μg N/g.d.w.s. and above significantly inhibit methane oxidation activity, whereas no statistically significant difference was observed in methane oxidation activity among soil microcosms with urea concentrations of less than 200 μg N/g.d.w.s after incubation for 27 days. Similar results were obtained in a sense that methane oxidation activity was inhibited only when the ammonium sulfate concentration was 200 μg N/g.d.w.s in soil microcosms in this study. Phylogenetic analysis of pmoA genes showed that nitrogen fertilization resulted in apparent changes in the community composition of methane-oxidizing bacteria (MOB). Type I MOB displayed an increased abundance in soil microcosms amended with nitrogenous fertilizers, whereas type II MOB dominated the native soil. Furthermore, although no statistically significant relationship was observed between pmoA gene and amoA gene abundances, methane oxidation activity was significantly negatively correlated with nitrification activity in the presence of urea or ammonium sulfate. Our results indicate that the methane oxidation activity in paddy soils might be inhibited when the concentration of ammonium fertilizers is high and that the interactions between ammonia and methane oxidizers need to be further investigated.

Keywords: methane oxidation; nitrification activity; nitrogenous fertilizers; paddy soil; particulate methane monooxygenase gene pmoa.

Figure 1

Changes in the CH₄ concentrations in the headspaces of soil microcosms amended with urea (A) or ammonium sulfate (B) over an incubation course of 27 days as well as changes in the concentrations of NH⁺₄-N in the soil microcosms (C,D). The designations CK-1, U-50, U-100, U-200 and U-400 represent the soil microcosms that received no fertilization, 50, 100, 200, and 400 μg urea-N/g d.w.s., respectively. The designations CK-2, AS-50, and AS-200 represent the soil microcosms treated with no fertilization, 50 and 200 μg ammonium sulfate-N/g d.w.s., respectively. The arrows indicate the repeated addition of CH₄, i.e., an initial concentration of ~ 5,000 ppm CH₄ in the headspace was re-established after an incubation period of approximately three days. Error bars represent the standard deviation of the duplicate microcosms, and the same letter above the columns refers to no statistically significant difference among the treatments (P > 0.05).

Figure 2

Changes in the concentrations of NO⁻₃ and NO⁻₃-N (A) the abundances of the pmoA genes of the methane-oxidizing bacteria (B) and the amoA genes of the Archaea (C) and Bacteria (D) in the soil microcosms amended with urea. The designations are the same as in Figure 1. The error bars represent the standard deviation of the duplicate microcosms, and the same letter above the columns refers to no statistically significant difference among the treatments (P > 0.05).

Figure 3

Changes in the concentration of NO⁻₃ and NO⁻₃-N (A) the abundances of the pmoA genes of the methane-oxidizing bacteria (B) and the amoA genes of the Archaea (C) and Bacteria (D) in the soil microcosms amended with ammonium sulfate. The designations are the same as in Figure 1. The error bars represent the standard deviation of the duplicate microcosms, and the same letter above the columns refers to no statistically significant difference among the treatments (P > 0.05).

Figure 4

Neighbor-joining tree showing the relationships of the pmoA genes retrieved from the clone library in this study to those in GenBank. The scale bar indicates 5 changes per 100 nucleotide positions. Bootstrap values (>40%) are indicated at the branch points. • represents the native soil (day 0), and ▲ and ■ represent the ammonium sulfate-and urea-amended soil microcosms, respectively.

Figure 5

DGGE fingerprints of the bacterial amoA gene in the soil microcosms amended with urea (A) and ammonium sulfate (B). The arrows indicate DGGE bands excised for sequencing. Zero represents the original soil sample, and all other designations are the same as in Figure 1. R1 and R2 represent duplicate microcosms.

Figure 6

DGGE fingerprints of the archaeal amoA gene in the soil microcosms amended with urea (A) and ammonium sulfate (B). The arrows indicate DGGE bands excised for sequencing. Zero represents the original soil sample, and all other designations are the same as those in Figure 1. R1 and R2 represent duplicate microcosms.

Figure 7

Neighbor-joining tree showing the relationships of the bacterial amoA genes retrieved from the DGGE bands (bold) in this study to those in the GenBank. The scale bar indicates 5 changes per 100 nucleotide acid positions. Bootstrap values (>40%) are indicated at branch points.

Figure 8

Neighbor-joining tree showing the relationships of the archaeal amoA genes retrieved from the DGGE bands (bold) in this study to those in the GenBank. The scale bar indicates 5 changes per 100 nucleotide acid positions. Bootstrap values (>40%) are indicated at branch points.

Figure A1

Relationships among the concentration of CH₄ remained in the headspace, the amount of NO⁻₂ and NO⁻₃-N and the copy number of pmoA genes in the soil microcosms amended with urea and ammonium sulfate after 27 days of incubation. The black line is the linear regression line between two parameters. The P values for panel (A) 0.001, (B) 0.055, and (C) 0.064.