Impaired reduction of N2O to N2 in acid soils is due to a posttranscriptional interference with the expression of nosZ

Abstract

Accumulating empirical evidence over the last 60 years has shown that the reduction of N2O to N2 is impaired by low soil pH, suggesting that liming of acid soils may reduce N2O emissions. This option has not gained much momentum in global change research, however, possibly due to limited understanding of why low pH interferes with N2O reductase. We hypothesized that the reason is that denitrifying organisms in soils are unable to assemble functional N2O reductase (N2OR) at low pH, as shown to be the case for the model strain Paracoccus denitrificans. We tested this by experiments with bacteria extracted from soils by density gradient centrifugation. The soils were sampled from a long-term liming experiment (soil pH 4.0, 6.1, and 8.0). The cells were incubated (stirred batches, He atmosphere) at pH levels ranging from 5.7 to 7.6, while gas kinetics (NO, N2O, and N2) and abundances of relevant denitrification genes (nirS, nirK, and nosZ) and their transcripts were monitored. Cells from the most acidic soil (pH 4.0) were unable to reduce N2O at any pH. These results warrant a closer inspection of denitrification communities of very acidic soils. Cells from the neutral soils were unable to produce functional N2OR at pH values of ≤6.1, despite significant transcription of the nosZ gene. The N2OR expressed successfully at pH 7.0, however, was functional over the entire pH range tested (5.7 to 7.6). These observations lend strong support to our hypothesis: low soil pH diminishes/prevents reduction of N2O, primarily by precluding a successful assembly of functional N2O reductase.

Importance: Impaired N2O reduction in acid soils was first observed ~60 years ago, and the phenomenon has been rediscovered several times since then. The practical implication would be that the emissions of N2O from cropped soils could be controlled by soil pH management, but this option has largely been ignored till now. One reason for this could be that the mechanisms involved have remained obscure. Here, we provide compelling evidence that the primary reason is that low pH interferes with the making of the enzyme N2O reductase rather than the function of the enzyme if properly assembled. The implications are important for understanding how pH controls the kinetics of N2O and N2 production by denitrification. The improved understanding provides credibility for soil pH management as a way to mitigate N2O emissions.

FIG 1

Outline of the oxic/anoxic incubations of cells extracted from soils with different pH (pH_s 4.0, 6.1, and 8.0). The cells were incubated in minimal media with different pH (pH_m 5.7, 6.1, and 7.6) at 15°C.

FIG 2

Kinetics of NO, N₂O, and N₂ production by bacteria from soils with different pH levels (pH_s 4.0, 6.1, and 8.0) during anoxic incubation in medium with three different pH levels (pH_m 5.7, 6.1, and 7.6). The figure shows the measured NO (red circle), N₂O (green square), and N₂ (blue triangle), all as µmol vial⁻¹ (note the different scales). The production of N₂ was below the detection limit (~150 nmol vial⁻¹) for all treatments except pH_s 6.1 and pH_s 8.0 at pH_m 7.6. The pH was measured at the end of the incubation and was found to be identical to the initial pH except for a slight increase (<0.2 pH units) in the treatments with high denitrification rates.

FIG 3

Transient accumulation of N₂O and NO by bacteria extracted from soils with different pH (pHs 4.0, 6.1, and 8.0), depending on the pH in the medium (pH_m). Panel a shows the N₂O index plotted against pH_m (each point is the index for a single vial). The index is a measure of the relative amount of N₂O, cumulating in equation 1 in the text. Panel b shows the corresponding index for NO (see equation 2 in the text for more details).

FIG 4

Copy numbers of the nirS, nirK, and nosZ genes (a to c) and the corresponding transcripts (d to f) throughout the 100 h of anoxic incubation of cells extracted from soil in which the pH (pH_s) was 6.1 and incubated in media of different pH (pH_m 5.7, 6.1, and 7.6) (see Fig. 1). Results are shown in a separate plot for each pH level of the medium. Quantitative PCR was performed in triplicates for each sample, and average values are shown (average coefficients of variation were 32% for DNA and 40% for cDNA). Time = 0 is the time of oxygen removal.

FIG 5

Average transcription ratios for nirS and nosZ genes (mRNA copies/DNA copies) during the first 40 h of anoxic incubation. The values are based on data presented in Fig. 4. Standard deviations are shown as vertical lines.

FIG 6

Transcription of nirS and nosZ during denitrification at pH_m 7.6, by bacteria extracted from soil with pH_s 4.0. The number of transcripts per vial is shown on the left y axis, and the accumulated N₂O production is shown on the right y axis. N₂ production was below the detection limit during the entire incubation.

FIG 7

Effect of pH_m (pH level of the medium) on the metabolism of a denitrifying community with functional N₂O reductase was expressed at pH 7.0. The cells (extracted from soil with pH 6.1) were allowed to express denitrification at pH 7.0 and then transferred to vials with pH_m 5.7 to 7.6. (A) Rate of oxic respiration measured in vials with oxic headspace; (B) rate of N₂O reduction in vials with anoxic headspace containing N₂O (and NO₃⁻-free medium). All rates are averages from the first 5 h of incubation (i.e., after transfer to the different pH_m levels), and standard deviations are shown as vertical bars (3 replicate vials for panel A, n = 6 for panel B).