1 Introduction

Nitrification is an important nitrogen transformation process in soils, which converts a relatively immobile form of nitrogen, ammonium (NH4 +), into a mobile form (nitrate, NO3 −) (Joye and Hollibaugh 1995). Nitrate is subjected to losses by leaching and denitrification when oxygen is limited. Nitrification and denitrification can also lead to the emission of potent greenhouse gas, nitrous oxide (N2O). The application of nitrification inhibitors is one possible way to reduce nitrogen losses and thereby increase nitrogen fertilizer efficiency for ammonium-based fertilizers. Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) are responsible for the first and rate-limiting step of autotrophic nitrification. Many studies have found that nitrification inhibitors blocked nitrification by stopping AOA or AOB growing (Di et al. 2009, 2010; Gubry‐Rangin et al. 2010).

3,4-Dimethylpyrazole phosphate (DMPP) is a widely used nitrification inhibitor which in some cases has been very effective in reducing nitrification at low application rates of 0.5–1.0 kg active compound ha–1 (Zerulla et al. 2001; Barth et al. 2008). However, its efficacy at inhibiting nitrification varies markedly with soils (Barth et al. 2001; Shi et al. 2011), temperature, and moisture (Chen et al. 2010). Acetylene (C2H2) is another effective inhibitor of nitrification (Bremner and Blackmer 1978). Moreover, C2H2 is bacteriostatic (Juliette et al. 1993) and commonly works on autotrophic nitrification at a low concentration (e.g., 10 Pa) (De Boer and Kowalchuk 2001). Previous studies have shown that C2H2 is very effective in inhibiting nitrification in acid soils (Hynes and Knowles 1982; Gubry‐Rangin et al. 2010), but the interactions between C2H2 and soil properties are rarely reported. Therefore, it is essential to determine what causes the inconsistent efficacy of nitrification inhibitors. Soil pH is believed to be a key factor that affects biological processes in soils (Šimek and Cooper 2002) by affecting the chemical form, concentration, and availability of substrates (Kemmitt et al. 2006) and influencing bacterial diversity and community structure on a global scale (Fierer and Jackson 2006). It has been reported that soil pH was the main factor driving the community changes of AOA and AOB in a series of soil pH gradient plots (He et al. 2007; Nicol et al. 2008; Shen et al. 2008). However, the relationship between soil pH and the effectiveness of nitrification inhibitors has rarely been studied. There are several studies on the responses of AOA and AOB to the addition of nitrification inhibitors (Shen et al. 2008; Di et al. 2009, 2010; Gubry‐Rangin et al. 2010; Zhang et al. 2011), though their findings are often contradictory. A comprehensive study is needed to determine the effects of nitrification inhibitors on nitrification and ammonia oxidizer population in different soils.

The objectives of this study were (i) to investigate the efficacy of two types of nitrification inhibitors (DMPP and C2H2) to inhibit nitrification in three contrasting soil types of different pH from Australia and (ii) to determine the effects of DMPP and C2H2 on ammonia oxidizers in these soils.

2 Materials and methods

Surface soil samples (0–10 cm) of three soil types from different regions and industries and with different pH values were collected from Clare (sugarcane, pH 7.0), Queensland (19.78°S, 147.23°E), Tamworth (pasture, pH 8.0), New South Wales (31.09°S, 150.93°E), and Hamilton (cropping, pH 4.6), Victoria (38.32°S, 142.07°E), air-dried, and ground to pass through a 2-mm sieve prior to analysis. Details of selected soil properties are shown in Table 1. The soils were stored at 4 °C prior to incubation experiments.

Table 1 Properties of the surface soil (0–10 cm) collected at field sites
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Laboratory incubation experiments were conducted in the dark. Sixty grams of air-dried soil from each of the three soil types was placed in capped 500-ml vials at 25 °C and 60 % water filled pore space (WFPS). Samples were pre-wetted and incubated for 3 weeks under 25 °C and just below 60 % WFPS to equilibrate the soil before the application of treatments. Ammonium chloride (NH4Cl) was applied to all soils at the rate of 100 μg N g−1 soil. The treatments applied were as follows: control (NH4Cl), DMPP (3.37 ml of 29 % DMPP per g NH4Cl), and C2H2 (1 % of the headspace in the vials). The vials were aerated every 3 days when water content and C2H2 were replenished.

Triplicate samples were extracted for NH4 + and NO3 − analyses on days 0, 7, 14, 21, and 28 with 2 M KCl (soil-to-solution ratio 1:5) by shaking for 1 h. The soil extracts were filtered through Whatman number 42 filter papers and analyzed for NH4 + and NO3 − using a segmented flow analyzer (Skalar SAN++).

The amoA gene copy numbers were quantified from triplicate samples on day 28 using real-time polymerase chain reaction (PCR) with two different primer sets to target the AOA (Francis et al. 2005) and AOB (Rotthauwe et al. 1997). Each archaeal amoA real-time PCR reaction was performed in a 20-μl volume containing 10 μl SensiFAST (Bio-Rad Laboratories, USA), 0.5 μM of each primer, and 2 μl of 10-fold dilution DNA template (1–10 ng). Amplification conditions were as follows: 95 °C for 3 min, 40 cycles of 5 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C. Each bacterial amoA real-time PCR reaction was performed in a 10-μl volume containing 5 μl iTaq Universal SYBR GREEN Supermix (Bio-Rad Laboratories, USA), 0.6 μM of each primer, and 2 μl of 10-fold dilution DNA template (1–10 ng). Amplification conditions were the same as the AOA QPCR assay. A known copy number of plasmid DNA for AOA or AOB was used to create a standard curve. For all assays, PCR efficiency was 90–100 % and r 2 was 0.96–0.99.

Data were analyzed using SPSS 19, and means were compared using one-way ANOVA between treatments to test the variance with a level of significance of P < 0.05.

3 Results

The inhibition of nitrate production by C2H2 and DMPP varied with soil type. Nitrate concentrations in the three control soils increased gradually during the incubation, with more NO3 − being produced in the alkaline soil than the other two soils (Fig. 1b, d, f). The addition of C2H2 completely blocked the production of NO3 − in all soils (Fig. 1b, d, f). DMPP completely inhibited the production of NO3 − in the neutral soil (Fig. 1b) and markedly slowed its formation in the other two soils (Fig. 1d, f and Table 2). In the first 7 days, the NH4 + concentrations in the control soils decreased from 105, 106, and 113 mg N kg−1 soil to 60, 28, and 90 mg N kg−1 soil for the neutral, alkaline, and acid soils, respectively. After that, the rate at which the NH4 + concentrations decreased was slower, and overall, more NH4 + was lost from the alkaline soil and less from the acid soil (Fig. 1a, c, e). In the inhibitor treatments, a slight decrease in NH4 + concentration was observed during the first 7 days, but after that, the concentrations were generally increased. The largest increase occurred in the acid soil treated with C2H2 (Fig. 1a, c, and e).

Fig. 1
figure 1

Ammonium and nitrate concentrations in a neutral soil (a, b), alkaline soil (c, d), and acid soil (e, f) incubated at 25 °C and 60 % WFPS. Error bars indicate standard errors of three replicates

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Table 2 Mean nitrification rate and inhibition by DMPP and C2H2 during incubation at 25 °C and 60 % WFPS for 28 days
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AOA and AOB populations changed with incubation time. After incubation of the soils with NH4Cl (100 μg N g−1 soil) for 28 days, the AOA amoA gene copy numbers in the control soils ranged from 5.2 to 39 × 108 g−1 dry soil. The AOA population in the acid soil was greater (39 × 108 g−1 dry soil) than that that in the alkaline soil (18 × 108 g−1of dry soil) and neutral soil (5.2 × 108 g−1of dry soil) (P < 0.05) (Fig. 2). The AOB population was smaller than that of the AOA in all three soils (Fig. 2). There were more AOB amoA gene copy numbers in the alkaline soil (3.7 × 108 g−1 dry soil) than in the acid soil (2 × 108 g−1 dry soil) and neutral soil (0.4 × 108 g−1dry soil). In the acid control soil, the ratio of AOA to AOB was highest at 19.5 followed by neutral control soil at 13 and alkaline control soil at 4.9.

Fig. 2
figure 2

AOA and AOB amoA gene copy numbers at day 28 in neutral soil (a), alkaline soil (b), and acid soil (c). The number above the bar indicates the ratio of AOA to AOB. Error bars indicate standard errors of three replicates

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The inhibition of AOA and AOB populations by C2H2 and DMPP varied with soil type. The addition of DMPP and C2H2 significantly reduced the AOA population in all soils, but the effect on the neutral soil was much greater than that on the acid and alkaline soils. Acetylene significantly inhibited AOB population in the neutral soil (P < 0.05) and slightly blocked AOB growth in alkaline soil (Fig. 2). However, the AOB population in the acid soil was not affected by the application of the nitrification inhibitors (Fig. 2). The reduction in the ratio of AOA to AOB was greatest (from 19.5 to 2.1) with C2H2 in the acid soil and least (from 4.9 to 3.4) with DMPP in the alkaline soil.

4 Discussion

Our study indicated that application of DMPP and C2H2 reduced nitrification for all three soil types, but to different extents. Acetylene was much more effective than DMPP in inhibiting nitrification in all three soil types. DMPP performed better in the neutral soil than the other two soils. There was no effect on the rate of NH4 + immobilization after inhibitor addition (Fig. 1), although the inhibitors blocked transformation of NH4 + to NO3 − supporting previous reports by Chalk (1990) and Crawford and Chalk (1993). In our study, soil pH might be a key factor influencing the effectiveness of DMPP and C2H2 on nitrification; however, their effectiveness might also have been affected by other soil properties such as soil texture (Barth et al. 2001). A multiple regression including more soil physico-chemical properties is therefore necessary. Although C2H2 completely inhibited nitrification, we still measured N2O emission (data not reported) which we hypothesize must have originated from denitrification of the original NO3 − or heterotrophic nitrification.

In the neutral soil, the two inhibitors significantly suppressed both AOA and AOB and decreased NO3 − content (by 57–85 %). In contrast, the effect of these inhibitors on nitrification differed in the other two soil types. This difference may be attributed to soil pH or other properties such as organic matter content (Table 1). It has also been shown that the relative abundance of these organisms is affected by NH4 + concentration (Di et al. 2009; Martens-Habbena et al. 2009; Verhamme et al. 2011), pH (Nicol et al. 2008; Hu et al. 2013), soil type (Girvan et al. 2003; Suzuki et al. 2009), and nutrient content (Di et al. 2009; Erguder et al. 2009). In the alkaline soil, both DMPP and C2H2 halved the AOA abundance and decreased AOB gene copy numbers by 27 and 35 %, respectively. Our results differ from the study conducted by Kleineidam et al. (2011) who observed that DMPP only reduced AOB but not AOA abundance in an acid soil 8 weeks after fertilizer application. He et al. (2007) found that AOB and AOA population sizes were the lowest in the N treatment with the lowest soil pH and that soil pH was significantly correlated with the abundance of AOB and AOA. However, this study found no clear relationship between soil pH and AOB abundance. Over the incubation time, we measured soil pH at each sample time and found there was no obvious difference between day 0 (Table 1) and day 28.

In the acid soil, both inhibitors inhibited only AOA but not AOB indicating that nitrification in acid soil was mainly associated with the dynamics of the AOA populations rather than with that of AOB. DMPP was less effective in lowering AOA, indicating AOA may be more sensitive to C2H2 than DMPP in acid soil. Offre et al. (2009) demonstrated a similar result in which AOA was inhibited by C2H2 in acid soil. It has been reported that soil environmental factors can determine the ecological niche of AOA and AOB (Girvan et al. 2003; Suzuki et al. 2009). Compared to AOB, AOA is better adapted to low NH4 availability (Martens-Habbena et al. 2009) and low pH (Nicol et al. 2008). Studies have shown that AOA were more abundant in unfertilized agricultural soils with low NH4 + content (Offre et al. 2009), while more AOB were found in fertilized soils or grazed pastures receiving additional N from animal excrement (Di et al. 2009; Jia and Conrad 2009). Soil heterogeneity and physiological differences between AOA and AOB may explain why they can coexist in the same soil despite competing for NH4 +. AOA and AOB have different niches and may therefore respond differently to inhibitors having specific targets.

5 Conclusions

Acetylene and DMPP effectively inhibited nitrification in all three soil types. C2H2 provided better inhibition than DMPP in our study, and DMPP was most effective when applied to neutral soil than alkaline and acid soils. AOA were significantly inhibited by C2H2 and DMPP in all soils; however, AOB were significantly inhibited by both inhibitors in neutral soil, slightly inhibited in alkaline soil, and was not affected in acid soil. Therefore, we propose that AOA might play a more important role than AOB in autotrophic nitrification in alkaline and acid soils in Australia. DMPP and C2H2 were effective in inhibiting both AOA and AOB in neutral soil.