Effects of Fe oxide on N transformations in subtropical acid soils.

Subtropical ecosystems are often characterized by high N cycling rates, but net nitrification rates are often low in subtropical acid soils. NO3(-)-N immobilization into organic N may be a contributing factor to understand the observed low net nitrification rates in these acid soils. The effects of Fe oxide and organic matter on soil N transformations were evaluated using a (15)N tracing study. Soil net nitrification was low for highly acidic yellow soil (Ferralsols), but gross ammonia oxidation was 7 times higher than net nitrification. In weakly acidic purple soil (Cambisols), net nitrification was 8 times higher than in Ferralsols. The addition of 5% Fe oxide to Cambisols, reduced the net nitrification rate to a negative rate, while NO3(-)-N immobilization rate increased 8 fold. NO3(-)-N immobilization was also observed in Ferralsols which contained high Fe oxides levels. A possible mechanism for these reactions could be stimulation of NO3(-)-N immobilization by Fe oxide which promoted the abiotic formation of nitrogenous polymers, suggesting that the absence of net nitrification in some highly acid soils may be due to high rates of NO3(-)-N immobilization caused by high Fe oxide content rather than a low pH.

Soils in subtropical and tropical forests are generally characterized by rapid N cycling rates and high n class="Chemical">N availability123. However, a few studies indicated that nitrification seems to be absent or a minor process in subtropical acid soils4567. This interpretation was supported by two observations: one is that NO3− concentrations are often consistently low in these soils, and that net nitrification rates are low or even negative during incubation assays of soil samples6789. However, this does not necessarily mean that the nitrification does not occur in these subtropical acid soils. Alternatively, it is possible that NO3−-N produced in these soils during nitrification (ammonia plus organic N oxidation) is immobilized through both abiotic and biotic processes as for observed in coniferous forest soils10. For example, high rates of NO3−-N immobilization into organic N can be responsible for N enrichment in subtropical acid forest soils3 which could protect the soil N from leaching. Furthermore, NO3−-N can be converted readily to other inorganic forms through denitrification or dissimilatory NO3− reduction to NH4+ (DRNA). Previous studies also suggested that N immobilization can be controlled by the concentration of available C11 and available inorganic N1213. Nugroho et al.14 observed that net nitrification rates in acid soils can vary about three fold at similar soil pH but with different organic matter content. Of course, both biotic and abiotic processes may be significant or one process may dominate, depending on the soil conditions. However, the causes of variation of N immobilization between soil types are not well known15.

Subtropical and tropical acid soils are often characterized by high levels of n class="Chemical">Fe oxides. It has been hypothesized that Fe may play a key role in regulating NO3− immobilization in acid forest soils16. Likewise, the inhibition of nitrification by high amounts of iron from pyrite (FeS2) can also occur17. Shindo and Huang18 found that Mn oxide promoted the abiotic formation of nitrogenous polymers. Since Fe and Mn oxides have similar chemical properties, it is possible that Fe oxide can also promote organic N formation from NO3− Therefore, Fe oxide may be a primary player in N transformations in subtropical acid soils and may help to explain the low NO3− concentrations often observed. Here, we hypothesized that nitrification does occur in subtropical acid soils, and NO3−-N produced during the nitrification process is immobilized quickly by Fe oxide. A weakly acidic purple soil (Cambisols, Chromic) and a highly acidic yellow soil (Ferralsols, Xanthic) were selected to test this hypothesis. Both soils formed from the same parent material in the same climate but had different Fe oxide content. The effects of Fe oxide or organic substances addition on N transformations in these soils were estimated using a 15N tracing method and N transformation model19.

Results

Effects of Fe oxide or organic substances addition on NH4+-N and NO3−-N concentrations during incubation

Half an hour after 50 mg kg−1 15NH4NO3 spiking, n class="Chemical">NH4+-N concentrations for the Ferralsols control averaged 43.1 ± 1.02 mg N kg−1. Organic substance or Fe oxide addition did affect NH4+-N concentrations significantly. During the 7 days incubation period, NH4+-N concentrations did not change significantly for the Ferralsols control and its two treatments (Figure 1A). However, NH4+-N dynamics for the Cambisols differed from Ferralsols, as Fe oxide and organic substances addition caused higher significant effects for Cambisols than Ferralsols. At half an hour, after 50 mg kg−1 15NH4NO3 was introduced, NH4+-N concentrations decreased rapidly for the Cambisols control, and further declined during the 7-day incubation (p < 0.05). A similar trend was observed when organic substances were added to Cambisols. However, Fe oxide addition did not change NH4+-N concentrations throughout the 7-day incubation (Figure 1B).

Figure 1

Effects of Fe oxide or organic substances addition on NH4+-N and NO3−-N dynamics during 7-day's by 15N tracing.

(NH4+-N concentration was measured following the addition of 50 mg kg−1 15NH4NO3; NO3−-N concentration was measured following the addition of 50 mg kg−1 NH4 15NO3). Error bars represent standard deviation, n = 3.

Half an hour after the addition of 50 mg kg−1 NH4 n class="Chemical">15NO3, NO3−-N concentrations for the Ferralsols control averaged 9.89 ± 0.36 mg NO3−-N kg−1. Addition of organic substances or Fe oxide did not affect NO3−-N concentrations. During the 7 days incubation period, NO3−-N concentrations did not change significantly, and did not respond to Fe oxide or organic substances addition in the Ferralsols control (Figure 1C). Conversely, NO3−-N dynamics in the Cambisols differed were significantly from the one in the Ferralsols. Half an hour, after 50 mg kg−1 NH4 15NO3 application, NO3−-N concentrations for the Cambisols control averaged 10.5 ± 0.20 mg N kg−1. NO3−-N concentrations increased significantly for the Cambisols control and organic substances treatment through time, but decreased significantly in the Fe oxide treatment during the 7 days incubation (Figure 1D).

Effects of Fe oxide or organic substances addition on N transformation rates

Our results clearly showed that soil net nitrification rates, gross autotrophic nitrification (ammonia oxidation) rates, NH4+ immobilization rates and NO3− immobilization rates were significantly affected by Fe oxide addition.

The net nitrification rate for the Ferralsols averaged 0.05 ± 0.01 mg N kg−1 soil day−1, which was significantly lower than for Cambisols (0.41 ± 0.04 mg N kg−1 soil day−1), and the organic substances treatment showed a non-significant decreasing trend in nitrification rates (p > 0.05). However, Fe oxide addition decreased the net nitrification rate significantly from 0.41 ± 0.04 to 0.28 ± 0.03 mg N kg−1 soil day−1 for Cambisols but not for Ferralsols.

The gross ammonia oxidation rates were 0.21 ± 0.03 mg n class="Chemical">N kg−1 day−1 and 0.74 ± 0.05 mg N kg−1 day−1 for the Ferralsols control and Cambisols respectively (p < 0.05) (Figure 2B). Furthermore, Fe oxide addition significantly decreased the gross NH4 oxidation rate in Cambisols, whereas the organic substances amendment did not change gross ammonia oxidation (p > 0.05). Neither Fe oxide nor organic substances amendments changed gross NH3 oxidation rates in the Ferralsol (Figure 2A).

Figure 2

Effects of Fe oxide or organic substances addition on gross heterotrophic nitrification and gross ammonia oxidation in Ferralsols and Cambisols estimated by the 15N tracing.

Error bars represent standard deviation, n = 3.

Fe oxide or organic substances amendments did not change heterotrophic nitrification rates for the n class="Chemical">Ferralsols (Figure 2C). However, the Fe oxide treatment increased the heterotrophic nitrification rate for Cambisols (Figure 2D).

NO3− immobilization rates for n class="Chemical">Cambisols were not significantly affected by organic substances addition (Figure 3D) but they significantly increased by Fe oxide addition. NO3− immobilization was also observed for the Ferralsol treatments, including the control and both Fe oxide and organic substances treatments. However, Fe and organic substances amendments did not significantly change NO3− immobilization rates for Ferralsols compared to the unamended control (Figure 3C).

Figure 3

Effects of Fe oxide or organic substances addition on the immobilization of NO3− and NH4+ (total of two immobilization rates including immobilization into recalcitrant and immobilization into labile organic N) (mg kg−1 d−1) in Ferralsols and Cambisols estimated by the 15N tracing model.

Error bars represent standard deviation, n = 3.

Gross NH4+ immobilization was four times higher for n class="Chemical">Cambisols than Ferralsols (Figure 3A, B). Hematite addition significantly decreased the NH4+ immobilization rate for Cambisols, and the organic substances treatment also showed a decreasing trend but the difference was not significant (p > 0.05). NH4+ immobilization rates for Ferralsols did not change in response to Fe oxide and organic substances addition.

Gross N mineralization rates did not difn class="Chemical">fer significantly between Ferralsols and Cambisols in the Fe oxide and organic substance treatment (p > 0.05), although Fe oxide addition tended to decrease gross N mineralization for both soils (Figure 4).

Figure 4

Effects of Fe oxide or organic substances addition on gross mineralization (mineralization of recalcitrant organic N and labile organic N to NH4+) (mg kg−1 d−1) in Ferralsols and Cambisols estimated by the 15N tracing model.

Error bars represent standard deviations, n = 3.

Ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies

Abundance of AOB and AOA was estimated by quantifying their respective amoA gene copy numbers after pre-incubation (Figure 5). The organic substances treatment tended to decrease AOB abundance for both Ferralsols and n class="Chemical">Cambisols but results were not significant (p > 0.05). However, Fe oxide addition significantly decreased AOB amoA gene copy numbers by about 50% for Cambisols. For Ferralsols, Fe oxide addition also tended to decrease AOB abundance, but not significantly. The abundance of AOB was significantly higher for Cambisols than Ferralsols, but the difference was not significant after 5% Fe oxide addition.

Figure 5

Effects of Fe oxide or organic substances addition on abundance of ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies.

Lowercase letters indicate statistically significant differences (p < 0.05). Error bars represent standard deviations, n = 3.

AOA amoA gene copy numbers were significantly higher for Cambisols than n class="Chemical">Ferralsols (Figure 5). Fe oxide addition decreased AOA amoA gene copy numbers for Cambisols to a similar level in Ferralsols. For Ferralsols, Fe oxide addition also did not change AOA abundance significantly. Similar to AOB, AOA abundance showed a non-significant decreasing trend after the organic substances addition for both Ferralsols and Cambisols (p > 0.05).

Discussion

While net nitrification was low for the highly acidic Ferralsols, gross autotrophic nitrification rates were about three times higher than net nitrification rates. The net nitrification rate was 8 times higher for weakly acidic n class="Chemical">Cambisols than highly acidic Ferralsols. However, the net nitrification rate decreased to a negative value for the weakly acidic Cambisols after Fe oxide addition. Results clearly showed that soil net nitrification rate was significantly inhibited by Fe oxide addition. However, only the evaluation of the associated individual gross N rates sheds light whether the oxidation to NO3− or immobilization of NO3− was responsible for this result.

Nitrification could be n class="Disease">retarded by high amounts of Fe from pyrite (FeS2). Blaise17 reported that nitrification was inhibited by 40.3% with the addition of 10000 mg pyrite kg−1 soil at the end of a 30-day incubation. However, they were unable to identify the exact mechanism by which pyrite inhibits nitrification/immobilization. Furthermore, postulated that the effect of pyrite could be due to the toxic action of any one or a combination of the following: (1) sulphides20; (2) the oxidized forms of the sulphides, or (3) presence of Fe2+ ions21. Compared to their results, a stronger inhibition of net nitrification by 50000 mg hematite kg−1 soil was observed in the present study. However, the mechanisms they postulated for pyrite inhibition of nitrification did not seem to be applicable for the present study since S or sulphides were not involved. Therefore, other mechanisms must be responsible for ferric iron inhibition of nitrification.

The toxicity of n class="Chemical">Fe to nitrifying microorganisms may partly contribute to the depression of nitrification. A pioneer study by Meiklejohn22 indicated that small amounts of Fe (0.1–0.6 mg L−1) stimulated growth of nitrifying bacteria and increased the oxidation of NH3 to NO2−, whereas high concentrations of Fe (>112 mg L−1) was toxic to nitrifying bacteria. The present results clearly demonstrated that both amoA genes for the ammonia monooxygenase (AMO) of AOB and AOA decreased two or three times after Fe oxide addition to Cambisols, and the soil gross ammonia nitrification rate also decreased. Higher amoA gene copy numbers of AOB and AOA were observed for Cambisols than for Ferralsols. Since Fe oxide content of Ferralsols (50.1 mg kg−1 soil) was significantly higher than for Cambisols (16.6 mg kg−1 soil), AOB and AOA were likely inhibited by high Fe oxide content. However, Fe toxicity to AOB and AOA cannot explain the decrease of gross ammonia nitrification completely, because the average activity was estimated around 40 fmol NH3 cell−1 day−1 for AOA and AOB2324. The gene copy numbers of AOB and AOA after Fe oxide addition still have potential nitrification up to 20 mg N kg−1 soil day−1.

The gross NO3−-n class="Chemical">N immobilization rate for Cambisols was 8 times higher in the 5% Fe oxide compared to the control treatment. In general immobilization of NO3− is assumed to be assimilated into microbial biomass. However, higher NO3−-N immobilization induced by Fe oxide in the present soils can not be explained by the assimilation of NO3−-N into microbial biomass. This was supported by the fact that NH4, rather than NO3, is the preferred form of N for assimilation by soil microorganisms252627. Further evidence was that the gene abundance of nitrifying microorganisms decreased after Fe oxide addition (Figure 5). Results from upland tropical forest soils in America also showed that microbial biomass 15N did not increase after NO3− addition28. Positive charges on the soil surface may also increase NO3−-N immobilization under field conditions293031. Anion exchange capacity (AEC) increased significantly after Fe oxide addition (Table 1). However, it cannot explain why NO3−-N concentrations kept decreasing with time after Fe oxide addition because most of the NO3−-N absorbed together with cations should have been released through ion exchange by the extractant (2 M KCl).

Table 1

Selected soil properties

Treatments	Texture	Parent material	pH	SOM	TN	CEC	AEC	Total Fe	Free Fe oxide	C/N
				(g/kg)	(g/kg)	(cmol/kg)	(cmol/kg)	(g/kg)	(g/kg)
Ferralsols	Sandy loam	Purple sandstone	4.2	16.0 ± 0.34b	1.96 ± 0.01a	7.50 ± 0.86b	1.91 ± 0.23b	50.1 ± 4.33a	4.47 ± 0.47b	4.73
Ferralsols + 5% hematite			4.0	16.1 ± 0.69b	1.86 ± 0.06a	7.30 ± 0.77b	3.77 ± 0.17a		6.37 ± 0.84a	5.02
Ferralsols + 5% organic substances			4.1	20.9 ± 1.19a	1.85 ± 0.11a	7.37 ± 3.00b	2.24 ± 0.15b		4.68 ± 0.86b	6.55
Cambisols	Sandy loam	Purple sandstone	5.8	14.6 ± 1.62b	0.96 ± 0.10b	35.9 ± 2.20a	1.04 ± 0.47c	16.6 ± 1.21b	1.52 ± 0.10c	8.82
Cambisols + 5% hematite			5.4	14.3 ± 0.69b	0.81 ± 0.04b	33.3 ± 0.73a	1.93 ± 0.35b		5.84 ± 0.68ab	10.2
Cambisols + 5% organic substances			5.6	19.1 ± 1.39a	0.82 ± 0.09b	32.5 ± 3.29a	1.38 ± 0.22c		1.60 ± 0.23c	13.5

aSOM reprents soil organic matter; TN reprents total nitrogen; CEC reprents cation exchange capacity; AEC reprents anion exchange capacity.

bValues behind ± represent standard deviation.

cMean values in a same column not followed by the same letter are different, p < 5%.

A potential mechanism of abiotic immobilization of NO3− has been postulated, and the authors suggested that n class="Chemical">Fe plays a key role in NO3−-N immobilization by promoting organic N formation from inorganic N16. Shindo and Huang found that Mn oxide significantly promoted the abiotic formation of nitrogenous polymers in the pH range 4 to 8 within 24 hours18. Since Fe and Mn oxides have similar chemical properties, it is possible that Fe oxide also functions similarly and can thus promote organic N formation. The mechanism for this organic N formation may explain the N dynamics for the two soils evaluated in our study. In a recent study, Zhang et al. reported that the gross autotrophic nitrification ranged from 0 to 0.5 μg N g−1d−1 and the NO3−-N immobilization rate into organic N ranged from 0.11 to 1.71 μg N g−1d−1 in a subtropical acid forest soil3. Their results indicated high rates of NO3−-N immobilization into organic N were responsible for the N enrichment in humid subtropical forest soils which contained large amounts of Fe and Mn oxides. A higher net nitrification rate occurred for weakly acidic Cambisols than for highly acidic Ferralsols in our study, and the NO3−-N immobilization was higher for Ferralsols than for Cambisols. The Fe oxide content of Ferralsols (50.1 g kg−1 soil) was higher than for Cambisols (16.6 g kg−1 soil), which implied that the absence of nitrification in some highly acid soils may due to high amounts of NO3−-N immobilization stimulated by high Fe oxide concentrations.

Soil net nitrification, gross NH4+ oxidation, total gross n class="Chemical">NH4+ mineralization and NO3−-N immobilization did not change significantly by organic substances addition for both soils. The insignificant response of N transformations by C/N ratio or organic substances addition may be due to the short incubation period which was not enough to highlight possible effects.

Cambisols and n class="Chemical">Ferralsols formed from the same parent material in the same climate, but differing in pH. While net nitrification occurred for the slightly acidic Cambisols, no obvious net nitrification was observed for highly acidic Ferralsols. The soil net nitrification rate strongly decreased to a negative value after Fe oxide addition to the slightly acidic Cambisols. However, significant autotrophic nitrification was observed using 15N tracers for both soils. Fe oxide greatly increased NO3−-N immobilization, probably by promoting the abiotic formation of nitrogenous polymers. This suggests that the absence of net nitrification in some highly acid soils may due to high amounts of NO3−-N immobilization caused by the high Fe oxide content rather than the low pH.

Methods

Site description and soil sampling

The yellow soil samples (Ferralsols, Xanthic) were collected from a pine forest soil derived from a sandstone parent material in Jingyun Mountain, Chongqing, China (29°83′n class="Chemical">N,106°39′E). In this region, the annual mean temperature is 19.6°C and annual rainfall is 1611 mm. Middle subtropical evergreen broad-leaved forests characterize the vegetation of this area. Purple soils (Cambisols, Chromic) were collected at a nearby hill. The Ferralsols samples were highly weathered and experienced a long developing process. To the contrary, Cambisols were slightly weathered and have not experienced a long developing process because of soil erosion. Selected soil properties for two soils and each treatment were listed in Table 1.

Four field replicates were taken at each site to a 0–20 cm depth in October, 2012, with each replicate being composed of seven individual soil cores (diameter was 5.5 cm), which were pooled and homogenized to reduce heterogeneity. Samples for each of the four replicates per site were were air-dried and separated into two parts. One was ground to pass a 2-mm sieve and used to make subsamples for incubation, another was ground to pass a 1-mm sieve and used for chemical analyses.

Preparation of Fe oxide and organic substances treatments

The precipitates of hematite were prepared from n class="Chemical">ferric chlorite of at least 98% purity, and solutions containing 100 g of FeCl3·6H2O/L were prepared. Ferric oxides were precipitated by the addition of NH3 to the FeCl3 solution until pH 7.0. The resultant suspension was thoroughly dialyzed for 24 hours at which time no further Cl− and Fe3+ was detected in the external solution, followed by aging for 48 hours in a 160°C oven32. Obtained hematite was checked by X-ray powder diffractometer (XRD) (see Supplementary figure 1). The organic substances were collected from the organic layer of the Ferralsols, and sieved to pass a sieve <2 mm to remove coarse roots, mixed thoroughly and then air dried (20°C) for 3 days before use.

Subsamples were obtained by adding 5% hematite or 5% organic substances into n class="Chemical">Ferralsols or Cambisols, respectively. Six subsamples were obtained as follows: Ferralsols + 5% hematite, Ferralsols + 5% humic substances, Cambisols + 5% hematite and Cambisols + 5% humic substances. Subsamples were air-dried, and separated into two parts. One was ground to pass a 2-mm sieve and stored at 4°C for 2 months before use, another was ground to pass a 1-mm sieve and used for chemical analyses.

Physical and chemical analyses

Soil pH was measured in a soil to water ratio of 1:2.5 (v/v) by a n class="Chemical">DMP-2 mV/pH detector (Quark Ltd, Nanjing, China). Total N (TN) and soil organic matter (SOM) contents were determined by a Macro Elemental Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Total soil Fe was extracted with HNO3–HF–HClO4, and free Fe oxides were extracted with Na2S2O4–Na3C6H5O7–NaHCO3. Fe content was measured by atomic absorption spectrophotometry with a graphite furnace (GFAAS) using a model Z-8200 spectrophotometer. Soil cation exchange capacity (CEC) and anion exchange capacity (AEC) were determined after extraction with 0.0215 mol (NH4NO3) L−1 solution using the method described by Gillman & Sumpter33.

Experimental design and 15N addition

The soil subsamples were adjusted to 60% water holding capacity (WHC) and pre-incubated for 7 days at 25°C. We employed a combination of n class="Chemical">15N tracing experiments and full process-based N cycle models to quantify process-specific and pool-specific N transformation rates, which is the standard method for the quantification of N dynamics in soils1934353637. For all samples, 15N tracing studies were carried out under controlled conditions. For the 15N tracing experiments, we employed two NH4NO3 treatments (each with three replications). In the first, NH4 (15NH4NO3) was labeled with 15N at 20 atom% excess, and in the second, NO3 (NH4 15NO3) was labeled. For each soil sample, a series of 250-ml conical flasks was prepared, each containing 30 g of fresh soil. 2 ml of 15NH4NO3 or NH4 15NO3 solution was added to each conical flask at a rate of 7.14 μmol N g−1 soil (50 μg NH4+-N g−1 soil and 50 μg NO3−-N g−1 soil). The soil was adjusted to 60% water-holding capacity (WHC) and incubated for 7 days at 25°C. All bottles were covered with polyethylene film punctured with needle holes to maintain aerobic conditions. The soils (three replications for each treatment) were extracted at 0.5, 24, 72, 120 and 168 hours after fertilizer application to determine the concentrations and isotopic composition of NH4+ and NO3−.

For isotopic analysis, NH4+ and n class="Gene">NO3− were separated by distillation with Mg oxide and Devarda's alloy3839 (see Supplementary information). The isotopic composition of NH4+ and NO3− were measured using an automated C/N analyzer coupled to an isotope ratio mass spectrometer (Europa Scientific Integra, UK). Simultaneous gross N transformations in soil were quantified using a process-based 15N tracing model19(see Supplementary information).

DNA extraction & quantitative PCR assay

Right after pre-incubation, 4 replicates of each treatment were randomly selected to extract DNA and amoA genes were analyzed by quantitative PCR (qPCR). The Dn class="Chemical">NA was extracted for three sub-samples from 0.50 g of soil with the FastDNA Spin Kit for soil (MP Biomedicals, United States), according to the protocol of the manufacturer. The quality and quantity of the DNA extracts were determined with a spectrophotometer (Nanodrop, PeqLab, Germany), and were pooled and stored at −20°C until use. Quantitative PCR of amoA genes was performed to estimate the abundance of the ammonia-oxidizing bacterial and archaeal communities, respectively (see Supplementary information).

Statistical analyses

Data (measured or calculated) were subjected to one-way ANOVA and mean values were separated using Duncan's n class="Chemical">New Multiple Range Test at p < 0.05. All statistical analyses were performed by SPSS statistical package.

Author Contributions

X.J. wrote the manuscript and carried out data analysis; X.X. and J.Z. prepared the experimental set-up and scientific protocols. X.X. and S.L. prepared the figures. C.M. provides the model for calculation and help in discussion. T.Z. did the calculation and data analysis. Z.C. help in discussion and data analysis. A.W. helped with discussion and language checking. All authors reviewed the manuscript.