- Email: [email protected]
Agricultural land use affects nitrate production and conservation in humid subtropical soils in China
Soil Biology & Biochemistry 62 (2013) 107e114
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Agricultural land use affects nitrate production and conservation in humid subtropical soils in China Jinbo Zhang a, b, Tongbin Zhu a, Tianzhu Meng a, Yanchen Zhang a, Jiajia Yang a, Wenyan Yang a, Christoph Müller c, d, Zucong Cai a, b, * a
School of Geography Sciences, Nanjing Normal University, Nanjing 210023, PR China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, PR China Department of Plant Ecology, Justus-Liebig University Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany d School of Biology and Environmental Science, University College Dublin, Belfield, Dublin4, Ireland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 November 2012 Received in revised form 6 March 2013 Accepted 7 March 2013 Available online 29 March 2013
To date, very few studies have been conducted to investigate the characteristics of gross nitrogen (N) transformations in subtropical agricultural soils. In this study, 12 natural woodland and 10 agricultural soils were collected to investigate the effects of land use on soil gross N transformations in the humid subtropical zones in China. The results showed that gross autotrophic nitrification rates (average 0.19 mg N kg1 d1) in the woodland soils were significantly lower than those determined in the agricultural soils (average 1.81 mg N kg1 d1) (p < 0.01). However, the NO 3 immobilization rates (average 0.10 mg N kg1 d1) in the agricultural soils were significantly lower than in the woodland soils (average 0.47 mg N kg1 d1) (p < 0.01). On average, 98% of the total NO 3 produced could be immobilized into organic-N in the woodland soils, while, it accounted for only 10% in the agricultural soils. These differences in gross N transformations resulted in the inorganic N being dominated by NHþ 4 in the woodland soils; however, NO 3 dominated the inorganic N in the agricultural soils. The risk of N leaching and runoff from soil sharply increases after woodland soils are converted to agricultural soils. Application of organic fertilizers with high C/N ratios to agricultural soils in subtropical regions to increase soil organic C content and the C/N ratio is expected to improve NO 3 immobilization capacity and reduce the risk of N leaching and runoff from soil. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Agricultural soils N transformation NO3immobilization capacity Nitrification
1. Introduction Nitrogen (N) is one of the key elements required for biomass growth. Previous investigations have suggested that in some natural ecosystems (e.g. forest ecosystems), in particular under conditions of high rainfall, most available N may be conserved successfully in soils through inherent soil N conservation mechanisms, e.g. i) low mineral N production and low NHþ 4 oxidation rates; ii) a combination of high mineral N production with efficient N immobilization; and iii) NO 3 retention via processes such as dissimilatory NO 3 reduction to ammonium (DNRA) (Huygens et al., 2007; Rütting and Müller, 2007, 2008). Zhang et al. (2011a,b) and Zhu et al. (2012) have reported that the autotrophic nitrification (i.e. NHþ 4 oxidation) rate was low, resulting in the inorganic N being
* Corresponding author. School of Geography Sciences, Nanjing Normal University, Nanjing 210023, PR China. Tel.: þ86 25 8589 1203; fax: þ86 25 8589 1745. E-mail address: [email protected] (Z. Cai). 0038-0717/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2013.03.006
dominated by NHþ 4 , and produced NO3 could be conserved effi ciently in soils by NO3 immobilization processes in the humid, acidic subtropical forest soils in China. Over the past 60 years, diverse land use patterns have been developed to meet an increasing demand for food, cash crops, and fiber, thus making the subtropical region of China important agriculturally. The arable area in the humid subtropical region of China is about 446,890 km2, which accounts for approximately 4% of the world’s subtropical arable land surface or 37% of China’s arable land (Zhao, 2002). Previous observations have suggested that the organic carbon content in subtropical agricultural soils in China is generally low due to intensive land use and poor management (Zhao et al., 1988). Land use and management practices, such as the use of inorganic N fertilizer, organic manure, and lime, could markedly influence the physical and chemical properties of the soil, which would in turn affect soil N cycling. Therefore, the characteristics of soil N transformations in agricultural soils would be expected to differ from those in forest soils. However, very few studies have been conducted to investigate the characteristics of
108
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
soil gross N transformations in subtropical and tropical agricultural soils (Booth et al., 2005). It is currently unknown whether the mechanisms for conserving inorganic N in humid subtropical soils (i.e. NO 3 immobilization and DNRA) could be maintained after woodlands are converted for agricultural use. Understanding soil gross N transformations in subtropical soils and how it is affected by land use would provide a scientific basis for rationally applying and appropriately assessing the environmental impacts of N fertilizers. The objectives of this study were to investigate the effects of land use on soil gross N transformations. Our hypothesis is that soil N conservation mechanisms in humid subtropical forest soils would be destroyed by intensive land use and practice management such as fertilization and liming. Large amounts of mineral N fertilization and liming could stimulate NO 3 production, while a low organic carbon content and C/N ratio could result in a decrease in the NO 3 immobilization capacity, inducing NO3 dominance in the inorganic N present in agricultural soils, thus increasing N losses through NO 3 leaching and runoff. Twelve natural woodland and 10 agricultural soils were collected from typical subtropical zones in China to test the above hypothesis. 2. Material and methods 2.1. Soil samples Twelve natural woodland soils and ten agricultural soils (one orange grove, three rice paddies, three peanut fields, and three maize fields) were collected from the typical subtropical zones in China. The annual precipitation is approximately 1,700 mm, and the mean annual temperature is 18.4e19.4 C. All agricultural sites were established after clearing the native woodland, and had received lime amendments at the beginning of establishment. The orange orchard was approximately 10 years old, and mineral N fertilizer was applied at a rate of approximately 80 kg N ha1 y1. The peanut sites were approximately 20 years old, and were fertilized with mineral N at a rate of approximately 200 kg N ha1 y1. Both the rice paddies and maize fields were established more than 100 years ago, and w300 kg N ha1 y1 N fertilizer had been applied in the most recent 10 years. The majority of crop residues were removed after harvest. At each site, soil samples were taken from three grids (approximately 4 m 4 m) that were randomly selected from a representative 100 m 100 m plot. From each grid, the O horizon, if present, was removed, and one sample was then taken from the mineral A horizon (0e20 cm). Three samples were pooled together, sieved (2 mm), homogenized and subsequently split into two subsamples. One sub-sample was stored at 4 C for the incubation studies. The other sub-sample was air-dried for analysis of soil properties. 2.2.
15
N tracing experiment
In the present investigation, the combination of an 15N tracing experiment and full process-based N cycle models was used to quantify simultaneously occurring gross N transformations in soil, mineralization of recalcitrant organic-N to NHþ 4 , mineralization of þ labile organic-N to NHþ 4 , immobilization of NH4 to labile organic-N, immobilization of NHþ 4 to recalcitrant organic-N, release of adsorþ bed NHþ 4 , adsorption of NH4 on cation exchange sites, oxidation of þ NH4 to NO3 (autotrophic nitrification), oxidation of recalcitrant organic-N to NO 3 (heterotrophic nitrification), immobilization of NO 3 to recalcitrant organic-N, and dissimilatory NO3 reduction to þ NH4 (DNRA) (Müller et al., 2007). The transformation rates were calculated using zero-order, first-order, or MichaeliseMenten kinetics. In the present study several model modifications, varying in
the number of considered N transformation, kinetic settings and considered N pools, were tested to find the model which best describes the measured N dynamics (Rütting et al., 2008). The final model was selected according to Aikaike’s Information Criterion (AIC) (Cox et al., 2006). Parameter optimization was carried out with the Markov chain Monte Carlo Metropolis algorithm (MCMCMA). The steps in model development and the optimization algorithm are described in detail by Müller et al. (2007) (Müller et al., 2007). The misfit function f(m) between the simulation output and observations (see Equation (3) in Müller et al., 2007) takes into account the variance of the individual observations. Analyses using this parameter optimization concept in previous studies have shown that the mineralization of two conceptual organic-N pools produced realistic NHþ 4 dynamics (Huygens et al., 2007; Müller et al., 2009). The MCMC-MA routine was used in the MatLab software package (Version 7.2, The MathWorks Inc.), which calls models that are separately set up in Simulink (Version 6.4, The MathWorks Inc.). Initial concentrations of the mineral N pools were determined according to Müller et al. (2004). Briefly, concentra tions of NHþ 4 and NO3 are estimated for time zero by backextrapolation to t ¼ 0. The difference between NHþ 4 applied and þ NHþ 4 determined was considered to be the amount of NH4 that was immediately adsorbed on NHþ exchange sites (NH ads). 4 4 There were two NH4NO3 treatments (each in three replicates): one contained 15N labeled ammonium (15NH4NO3) while the other 15 contained 15N labeled nitrate (NH15 N at 20 4 NO3) (labeled with atom percentage excess). For each soil, a series of 250-ml conical flasks was prepared with 30 g of fresh soil (oven-dry basis). Two ml of 15NH4NO3 or NH15 4 NO3 solution was added to each of the conical 1 1 flasks at a rate of 20 mg NHþ soil and 20 mg NO 4 eN kg 3 eN kg soil. The soil was adjusted to 60% water holding capacity (WHC) and incubated for 144 h at 25 C. The conical flasks were sealed with silicone rubber stoppers. The soils (three replicates for each 15N label treatment) were extracted at 0.5, 24, 72, and 144 h after fertilizer application for the determination of the concentration and isotopic composition of the NHþ 4 and NO3 . The detailed principle of 15 the model and the N incubation experiment were from Müller et al. (2004, 2007). 2.3. Analyses Soil properties were determined following the Soil AgroChemical Analysis procedures of Lu (2000). Soil pH was measured in a 1:2.5 (v/v) soil to water ratio using a DMP-2 mV/pH detector (Quark Ltd, Nanjing, China). Soil organic carbon (SOC) was analyzed by wet-digestion with H2SO4eK2Cr2O7, and total nitrogen was determined by semi-micro Kjeldahl digestion using Se, CuSO4 and K2SO4 as catalysts. Ammonium and NO 3 were extracted with 2 M KCl at a soil/solution ratio of 1:5 on a mechanical shaker for 60 min at 300 rpm at 25 C. The extracts were filtered through filter paper (Qualitative Filter Paper, BH92410262) and the concentrations of NHþ 4 and NO3 were determined with a continuous-flow analyzer (Skalar, Breda, Netherlands). For isotopic analysis, NHþ 4 and NO3 were separated by distillation with magnesium oxide and Devarda’s alloy (Feast and Dennis, 1996; Zhang et al., 2011a,b). In detail, a portion of the extract was steam-distilled with MgO to separate NHþ 4 on a steam distillation system; thereafter, the sample in the flask was distilled again after addition of Devarda’s alloy to separate NO 3 . The liberated NH3 was trapped using boric acid solution. The trapped N was acidified and converted to (NH4)2SO4 using 0.02 mol L1 H2SO4 solution. The H2SO4 solution containing NHþ 4 was then evaporated to dryness at 65 C in an oven and analyzed for 15N abundance. Before separating NHþ 4 and NO3 in the extract using the steam distillation system, the þ 1 recovery of NHþ 4 and NO3 in a standard solution (1 g NH4 eN L
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114 1 and 1 g NO 3 eN L ) was determined. The results showed that almost all NHþ eN in the solution could be recovered (>99%), and 4 þ the recovery of NO 3 was >95%. Isotopic composition of the NH4 and NO was determined using an automated C/N analyzer coupled 3 to an isotope ratio mass spectrometer (IRMS 20-22, SerCon, Crewe, UK).
2.4. Data analysis The ratio of autotrophic nitrification rate to mineralization rate (M ¼ MNrec þ MNlab) was defined as nitrification capacity (NC), indicating the capacity of the soil to transform available NHþ 4 to NO 3 . The ratio of total NO3 consumption rate (NO3 immobilization ) to total nitrification rate was defined as the capacity of the soil to retain NO 3 (NR). The net NO3 production rate ¼ the total nitrifi cation rate e the total NO3 consumption rate. 2.5. Statistical analyses and model setup The optimization procedure resulted in a probability density function (PDF) for each parameter, from which parameter averages and standard deviations were calculated (Müller et al., 2007). Each analysis run was carried out with three parallel sequences to identify adequate iteration numbers. Based on the kinetic settings and the final parameters, average N transformation rates were calculated over the whole period and expressed in units of mg N kg1 soil day1. Pearson correlation coefficient analysis was performed to explore the relationship between measured variables using the SPSS 17.0 software package for Windows. Soil characteristics and N transformations between woodland and agricultural soils were compared by t-tests using SPSS 17.0 software (where p < 0.05 was considered to indicate statistical significance). 3. Results 3.1. Soil properties The soil pH ranged from 4.12 to 4.65 (average 4.35) in the woodland soils, which was significantly lower than that in the agricultural soils (average 4.77) (p < 0.05). However, most pH values were still less than 5.0 in the agricultural soils (Fig. 1a). Soil organic C (SOC) content (average 32.68 g kg1, p < 0.05) and C/N ratio (average 16.09, p < 0.05) in the woodland soils was significantly higher than those in the agricultural soils (average values were 18.35 g kg1, and 10.32, respectively, for SOC, and C/ N) (Fig. 1b,c), although no significant difference in the total N content was observed (Fig. 1d). The inorganic N was dominated þ by NHþ 4 in the woodland soils (the average ratios of NH4 /NO3 was 7.54), however, NO3 dominated the inorganic N in the agricultural soils (the average ratios of NHþ 4 /NO3 were 0.24). The NO 3 and total inorganic N concentrations in the agricultural soils were much higher than those in the woodland soils (p < 0.05) (Fig. 1f, g). 3.2. Gross N transformation There was no significant difference in the gross mineralization rates and NHþ 4 immobilization rates between woodland and agricultural soils (Fig. 2a,b). There was also no significant relationship between gross mineralization rates and individually measured soil properties (Fig. 1, i.e. soil organic C, total N, pH, and C/N ratio). Gross autotrophic nitrification (NHþ 4 oxidation) rates ranged from 0 to 0.65 mg N kg1 d1 (average 0.19 mg N kg1 d1) in the woodland soils, which was significantly lower than that
109
determined for the agricultural soils (range, 0.80e 2.74 mg N kg1 d1, with an average of 1.81 mg N kg1 d1; p < 0.05) (Fig. 2c). The gross autotrophic nitrification rate increased linearly with the increase in soil pH (R2 ¼ 0.46, p < 0.01) (Fig. 3a). Nitrification capacity (NC, presented as the ratio of NHþ 4 oxidation to mineralization) in the woodland soils (0.04 0.04) was substantially lower than that of the agricultural soils (1.06 0.67; p < 0.05) (Fig. 2d). The NC was also exponentially correlated with soil pH (R2 ¼ 0.35, p < 0.01) (Fig. 3b). The average heterotrophic nitrification (oxidation of recalcitrant organic-N to NO 3 ) rate in the woodland soils was 0.49 0.36 mg N kg1 d1 (Fig. 2e). However, the heterotrophic nitrification rate was negligible (0.002 0.01 mg N kg1 d1) in the agricultural soils. Heterotrophic nitrification increased with the increase in soil C/N ratio (R2 ¼ 0.75, p < 0.001), except in soil 4, in which heterotrophic nitrification was negligible, despite the fact that the C/N ratio was high (20.26). Also, heterotrophic nitrification decreased exponentially with an increase in total mineralization in the woodland soils (p < 0.01). DNRA rates were low in all studied soils, and not significantly different between woodland and agricultural soils (Fig. 2f). The NO 3 immobilization rates were significantly different between woodland and agricultural soils (p < 0.05) (Fig. 2g). All agricultural soils, with the exception of soil 22, which exhibited a NO 3 immobilization rate of 0.63 0.25 mg N kg1 d1, had very low NO 3 immobilization capacity. In contrast, the studied woodland soils had significantly higher NO 3 immobilization capacities (average 0.47 mg N kg1 d1) than did the agricultural soils (p < 0.01). The NO 3 immobilization rate was positively correlated with the C/N ratio (p < 0.001) and negatively correlated with soil pH (p < 0.05) (Fig. 4). NO 3 retention ability (NR) (i.e. ratio of NO3 consumption [NO 3 immobilization and DNRA] to total nitrification rate), which ranged from 0.39 to 2.37 (average 0.98) in the woodland soils, was significantly higher than in the agricultural soils, where it ranged from 0.01 to 0.29 (average 0.1; p < 0.05) (Fig. 2h). The net nitrification rate (average 1.59 mg N kg1 d1) in the agricultural soils was significantly higher than in the woodland soils (average 0.12 mg N kg1 d1; p < 0.05) (Fig. 1g). Both the net nitrification rate, which was calculated as the total NO 3 production rate (autotrophic nitrification þ heterotrophic nitrification) minus the total NO 3 consumption rate (NO3 immobilization þ DNRA), and the gross nitrification rate could predict NO 3 concentrations well in the studied soils (Fig. 5). 4. Discussion The results of the present study show that the conversion of woodland soil to agricultural soil mainly affected nitrate production and immobilization, while there were no significant differences in the gross mineralization rates and NHþ 4 immobilization rates between woodland and agricultural soils (Fig. 6). After conversion of woodland soil to agricultural soil, the gross NHþ 4 oxidation rate and NC increased significantly, compared to woodland soils (p < 0.01), resulting in the inorganic N being dominated by NO 3 in the agricultural soils (Fig. 6). The change in soil nitrogen transformation characteristics in the agricultural soils could result in a change in inorganic N forms, and increase the risk of N leaching and runoff from soil, inducing negative environmental effects as compared to the woodland soils. 4.1. Nitrate as dominant form of inorganic N in the agricultural soils Generally, the rates of N cycling are high in humid subtropical and tropical forests ecosystems (Martinelli et al., 1999; Matson et al., 1999), so they have to develop a strong capacity for retaining inorganic N in soils (Huygens et al., 2007; Rütting et al., 2008;
110
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
Fig. 1. Box plot of soil properties in the present study. Identical letters indicate no statistical differences in the average values between groups.
Zhang et al., 2011a,b). The results of the present investigation show that autotrophic nitrification, i.e. ammonium oxidation, was very low in all studied woodland soils (average 0.19 mg N kg1 d1), and the NC was only 6%. As a result, ammonium dominated in the studied woodland soils. Under low soil pH (average 4.35), ammonia volatilization is almost completely suppressed. Although in subtropical regions, besides negatively charged colloids, highly weathered soils usually also contain positively charged sites on the colloids; NO 3 is lost much more easily via leaching and runoff than is ammonium (Qian and Cai, 2007). Therefore, maintaining inorganic ammonium dominance is one of the strategies for humid subtropical forest soils to retain inorganic N.
Both the ammonium oxidation rate and NC were correlated significantly with soil pH, suggesting that the increase in soil pH resulting from liming was one of the important factors affecting nitrification in the agricultural soils. These relationships highlight the importance of soil pH on the nitrification process (Weber and Gainey, 1962; Katyal et al., 1988; Zhao et al., 2007; Zhang et al., 2011a). In addition, N fertilization could be another important factor stimulating nitrification activity in the agricultural soils. Previous observations have found that autotrophic nitrification is often stimulated by N addition (Zaman et al., 1999; Stockdale et al., 2002; Hall and Matson, 2003; Zhang et al., 2012a; Lu et al., 2012). In agricultural soils, most ammonium oxidation is carried out by
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
8
a
8 b 6 4 2 0
0
2 .5 b
2 .1 1 .4 a
0 .7
A g ric u ltu ra l
(d )
b
2 .0 1 .5 1 .0 0 .5 a 0 .0 -0 .5
0 .0 W o o d la n d
W o o d la n d
A g ric u ltu ra l
(e )
0 .6
A g ric u ltu ra l
a
(f)
b a
0 .4
-1
-1
1 .0
D N R A (m g k g d )
H e te ro tro p h ic n itrific a tio n -1 -1 (m g k g d )
2
W o o d la n d
(c )
2 .8
1 .5
a
4
A g ric u ltu ra l
N itrific a tio n c a p a c ity
A u to tro p h ic n itrific a tio n -1 -1 (m g k g d )
3 .5
a 6
-2
W o o d la n d
0 .5 a 0 .0
-0 .5
0 .0
(g )
A g ric u ltu ra l
W o o d la n d
2 .5
b
0 .8 a 0 .6 0 .4
(h )
A g ric u ltu ra l
b
2 .0 1 .5 1 .0 0 .5
a
-
0 .2
N O 3 re te n tio n c a p a c ity
-
1 .0
0 .2
-0 .2
W o o d la n d
Im m o b iliz a tio n o f N O 3 -1 -1 (m g k g d )
(b )
+
(a )
Im m o b iliz a tio n o f N H 4 -1 -1 (m g k g d )
-1
-1
M in e ra liz a tio n (m g k g d )
10
111
0 .0
0 .0 -0 .5
-0 .2 W o o d la n d 1
A g ric u ltu ra l 1
W o o d la n d
A g ric u ltu ra l
Fig. 2. Box plot of gross N transformation rates (mg N kg d ) in studied woodland and agricultural soils (0e20 cm) estimated by the þ NO 3 reduction to NH4 . Identical letters indicate no statistical differences in the average values between groups.
autotrophic ammonia-oxidizing bacteria (AOB) (Barraclough and Puri, 1995). Previous studies have observed that long-term field mineral N fertilization can stimulate the growth of AOB in the field by more than 20-fold when compared to control soils (no N application) (Chu et al., 2008; Shen et al., 2008; Zhang et al., 2012b). In addition, recent studies have demonstrated that the Nitrosospira cluster 3-like AOB was highly enriched in fertilized agricultural soils (Chu et al., 2007; Shen et al., 2008). Thus, the observed stimulation of gross autotrophic nitrification in the agricultural soils was most likely driven by the increase in AOB population size and the changes in AOB composition due to long-term mineral N application. Further studies are needed to investigate the characteristics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in
15
N tracing model. DNRA is dissimilatory
the woodland and agricultural soils, and to clarify the mechanisms of agricultural use that stimulate the autotrophic nitrification process. 4.2. Decrease in NO 3 immobilization The results of our investigation show that NO 3 immobilization is a widespread process in the studied woodland soils in humid subtropical regions; however, in the agricultural soils, the NO 3 immobilization rate and NO 3 immobilization capacity were negligible. On average, the NO 3 immobilization rate into organic-N accounted for 98% of the total nitrification rate (autotrophic nitrification plus heterotrophic nitrification of organic-N) in the
112
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
-1
3
a
-1
y = 2 .2 2 x - 9 .1 7
-1 5
e
6 .7 7 9 6 x
2
2
2
b y = 3 10
R = 0 .4 6 p < 0 .0 1
N itrific a tio n c a p a c ity
A u to tro p h ic n itrific a tio n (m g k g d )
3
1
0
2
R = 0 .3 5 p < 0 .0 1
1
0
4 .0
4 .4
4 .8
5 .2
4 .0
4 .4
pH
4 .8
5 .2
pH
Fig. 3. Relationship between autotrophic nitrification (a), nitrification capacity (b) and soil pH. Nitrification capacity is presented as the ratio of NHþ 4 oxidation to mineralization.
and C/N ratio after conversion of woodland to agricultural soils, mainly due to low organic matter input and high mineral N fertilization, was an important factor which resulted in a decrease in NO 3 immobilization. Furthermore, previous studies have found that fungi may preferentially utilize NO 3 (Marzluf, 1997), when compared with bacteria. Thus, the observed differences in NO 3 immobilization between woodland soils and agricultural soils could be related to fungal activity. Soil conditions, such as soil organic matter content, composition (C/N) and pH, could influence soil microbial community structure, which, in turn, controlled soil N transformations. Generally, fungi are favored by acidic soils that are low in available nutrients, recalcitrant organic materials, and that have high C/N soil ratios (Kreitinger et al., 1985; Wood, 1990; Blagodatskaya and Anderson, 1998; Högberg et al., 2007). However, tillage and fertilization were usually considered to be reasons for a decline in fungal
woodland soils. In contrast, it accounted for only 10% in the agricultural soils. The difference in NO 3 immobilization ability could further explain why the NO 3 concentrations in the agricultural soils were much higher than in the woodland soils. Our results showed that the NO 3 immobilization rate increased with the increase in C/N ratio (p < 0.001, Fig. 4a), suggesting that quantity and quality of soil organic C could be the important factor affecting NO 3 immobilization (Recous et al., 1990; Johnson, 1992; Stark and Hart, 1997; Barrett and Burke, 2000; Burger and Jackson, 2003; Häbteselassie et al., 2006). Previous studies have reported that microbes needed to immobilize more inorganic N when they decompose organic matter with high C/N ratios (Sollins et al., 1984; Zak et al., 1994; Janssen, 1996). Therefore, the NO 3 immobilization rate could be high in the soils which had a high organic C content and high C/N ratio (Johnson, 1992; Stark and Hart, 1997; Burger and Jackson, 2003). The significant decrease of soil organic C content
-1
y = 0 .0 7 x - 0 .5 5
0 .8
b
-1
N O 3 im m o b iliz a tio n (m g k g d )
2
R = 0 .7 0 p < 0 .0 0 1
0 .6
0 .4
0 .2
0 .8
y = -0 .5 0 x + 2 .5 9 2
R = 0 .2 2 0 .6
p < 0 .0 5
0 .4
0 .2
-
-1
1 .0
a
-1
N O 3 - im m o b iliz a tio n (m g k g d )
1 .0
0 .0 4
8
12
16
20
24
C /N ra tio Fig. 4. Relationship between
0 .0 4 .0
4 .5
5 .0 pH
NO 3
immobilization and (a) C/N ratio, (b) Soil pH.
5 .5
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
a
Agricultural soil Immobilization
-
-1
NO3 concentration (mg kg )
80
y=19.1x + 3.4 2 R =0.59 p<0.001
60
0.10±0.19 Autotrophic Mineralization
40
NO 3DNRA
20
0.12±0.20
Immobilization 1.29±1.63 Heterotrophic nitrification
1
2 -1
80
0.002±0.01
3 -1
Net nitrification rate (mg kg d )
-1
1.81±0.53
NH 4+
SOM
0
-
nitrification
2.37±1.74
0
NO3 concentrition (mg kg )
113
Woodland soil Immobilization
b
0.47±0.27 Autotrophic
y = 21.7x - 7.4 2 R = 0.59 p<0.001
60
Mineralization
nitrification
3.67±2.15
0.19±0.21
NH 4+
SOM
40
NO 3DNRA 0.09±0.12
Immobilization 2.20±1.83
20
Heterotrophic nitrification 0.49±0.36
0 0
1
2
3 -1 -1
Gross nitrification rate (mg kg d ) Fig. 5. Relationship between net nitrification rate (a), gross nitrification rate (b) and soil nitrate concentration. Net nitrification rate was calculated as the total NO 3 production rate (autotrophic nitrification þ heterotrophic nitrification) minus the total NO3 consumption rate (NO3 immobilization þ DNRA).
biomass (Beare et al., 1997; Frey et al., 1999; Högberg et al., 2003; Joergensen and Wichern, 2008). Further studies are needed to investigate fungal activity and community structure in the woodland and agricultural soils in order to clarify the microbial mechanisms of NO 3 immobilization. Our results highlight the fact that changes in land use can significantly affect N transformation characteristics in the humid subtropical region. Nitrate production increased significantly; however, NO 3 immobilization almost disappeared, resulting in NO 3 dominance in the inorganic N in the agricultural soils as compared with woodland soils (Fig. 6). The risk of N leaching and runoff from soil sharply increased after woodland was converted to agricultural use, which is not only due to the application of N fertilizers, but also stimulation of nitrification and depression of NO 3 immobilization. Previous observations in a long-term fertilizing system (from 1990 to 2006) showed that the N fertilizer apparent recovery efficiency only ranged from 23.4% to 61.6%, with an average of 36.5%, in the subtropical region of China (Wu et al., 2008). As a result, the negative environmental effects of NO 3 leaching and runoff could be induced in the subtropical region. Application of organic fertilizer with a high C/N ratio to agricultural
Fig. 6. Nitrogen cycle in agricultural and woodland soils in the humid subtropical region of China. The thickness of the arrows represents the relative importance of each flux. The data in the figure are the means of gross rates of N transformation SD (mg kg1d1).
soils in the subtropical region to increase soil organic C content and C/N ratio is expected to improve NO 3 immobilization capacity and reduce the risk of N leaching and runoff from soil. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (41101209, 41222005, 40830531), the Natural Science Foundation of Jiangsu Province (BK2010611, SBK201220477), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Barraclough, D., Puri, G., 1995. The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification. Soil Biology and Biochemistry 27, 17e22. Barrett, J.E., Burke, I.C., 2000. Potential nitrogen immobilization in grassland soils across a soil organic matter gradient. Soil Biology and Biochemistry 32, 1707e1716. Beare, M.H., Hu, S., Coleman, D.C., Hendrix, P.F., 1997. Influences of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Applied Soil Ecology 5, 211e219. Blagodatskaya, E.V., Anderson, T.H., 1998. Interactive effects of pH and substrate quality on the fungal-to-bacterial ratio and qCO2 of microbial communities in forest soils. Soil Biology and Biochemistry 30, 1269e1274.
114
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
Booth, M.S., Stark, J.M., Rastetter, E.B., 2005. Controls on nitrogen cycling in terrestrial ecosystems: a synthetic analysis of literature data. Ecological Monographs 75, 139e157. Burger, M., Jackson, L.E., 2003. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biology and Biochemistry 35, 29e36. Chu, H.Y., Fujii, T., Morimoto, S., Lin, X.G., Yagi, K., Hu, J.L., Zhang, J.B., 2007. Community structure of ammonia oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil. Applied and Environmental Microbiology 73, 485e491. Chu, H.Y., Fujii, T., Morimoto, S., Lin, X.G., Yagi, K., 2008. Population size and specific nitrification potential of soil ammonia-oxidizing bacteria under long-term fertilizer management. Soil Biology and Biochemistry 40, 1960e1963. Cox, G.M., Gibbons, J.M., Wood, A.T.A., Craigon, J., Ramsden, S.J., Crout, N.M.J., 2006. Towards the systematic simplification of mechanistic models. Ecological Modelling 198, 240e246. Feast, N.A., Dennis, P.E., 1996. A comparison of methods for nitrogen isotope analysis of groundwater. Chemical Geology 129, 167e171. Frey, S.D., Elliott, E.T., Paustian, K., 1999. Bacterial and fungal abundance and biomass in conventional and no-tillage agroecosystems along two climatic gradients. Soil Biology & Biochemistry 31, 573e585. Häbteselassie, M.Y., Stark, J.M., Miller, B.E., Thacker, S.G., Norton, J.M., 2006. Gross nitrogen transformations in an agricultural soil after repeated dairy-waste application. Soil Science Society of American Journal 70, 1338e1348. Hall, S.J., Matson, P.A., 2003. Nutrient status of tropical rain forests influences soil N dynamics after N additions. Ecological Monographs 73, 107e129. Högberg, M.N., Baath, E., Nordgren, A., Arnebrant, K., Högberg, P., 2003. Contrasting effects of nitrogen availability on plant carbon supply to mycorrhizal fungi and saprotrophs e a hypothesis based on field observations in boreal forests. New Phytologist 160, 225e238. Högberg, M.N., Högberg, P., Myrold, D.D., 2007. Is microbial community composition in boreal forest soils determined by pH, C-to-N ratio, the trees, or all three? Oecologia 150, 590e601. Huygens, D., Rütting, T., Boeckx, P., Van Cleemput, O., Godoy, R., Müller, C., 2007. Soil nitrogen conservation mechanisms in a pristine south Chilean Nothofagus forest ecosystem. Soil Biology and Biochemistry 39, 2448e2458. Janssen, B.H., 1996. Nitrogen mineralization in relation to C/N ratio and decomposability of organic materials. Plant and Soil 181, 39e45. Joergensen, R.G., Wichern, F., 2008. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biology and Biochemistry 40, 2977e2991. Johnson, D.W., 1992. Nitrogen retention in forest soils. Journal of Environmental Quality 21, 1e12. Katyal, J.C., Cater, M.F., Viiek, P.L.G., 1988. Nitrification activity in submerged soil and its relation to denitrification loss. Biology and Fertility of Soils 7, 16e22. Kreitinger, J.P., Klein, T.M., Novick, N.J., Alexander, M., 1985. Nitrification and characteristics of nitrifying microorganisms in an acid forest soil. Soil Science Society of American Journal 49, 1407e1410. Lu, L., Han, W., Zhang, J., Wu, Y., Wang, B., Lin, X., Zhu, J., Cai, Z., Jia, Z., 2012. Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea. The ISME Journal 6, 1978e1984. Lu, R.K., 2000. Soil Agro-chemical Analyses. Agricultural Technical Press of China, Beijing (in Chinese). Martinelli, L.A., Piccolo, M.C., Townsend, A.R., Vitousek, P.M., Cuevas, E., McDowell, W., Robertson, G.P., Santos, O.C., Treseder, K., 1999. Nitrogen stable isotope composition of leaves and soil: tropical versus temperate forests. Biogeochemistry 46, 45e65. Marzluf, G.A., 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiology and Molecular Biology Reviews 61, 17e32. Matson, P.A., McDowell, W.H., Townsend, A.R., Vitousek, P.M., 1999. The globalization of N deposition: ecosystem consequences in tropical environments. Biogeochemistry 46, 67e83. Müller, C., Rütting, T., Kattge, J., Laughlin, R.J., Stevens, R.J., 2007. Estimation of parameters in complex 15N tracing models via Monte Carlo sampling. Soil Biology and Biochemistry 39, 715e726.
Müller, C., Stevens, R.J., Laughlin, R.J., 2004. A 15N tracing model to analyse N transformations in old grassland soil. Soil Biology and Biochemistry 36, 619e632. Müller, C., Rütting, T., Abbasi, M.K., Laughlin, R.J., Kammann, C., Clough, T.J., Sherlock, R.R., Kattge, J., Jäger, H.J., Watson, C.J., Stevens, R.J., 2009. Effect of elevated CO2 on soil N dynamics in a temperate grassland soil. Soil Biology & Biochemistry 41, 1996e2001. Qian, C., Cai, Z.C., 2007. Leaching of nitrogen from subtropical soils as affected by nitrification potential and base cations. Plant and Soil 300, 197e205. Recous, S., Mary, B., Faurie, G., 1990. Microbial immobilization of ammonium and nitrate in cultivated soils. Soil Biology and Biochemistry 22, 913e922. Rütting, T., Huygens, D., Müller, C., van Cleemput, O., Godoy, R., Boeckx, P., 2008. Functional role of DNRA and nitrite reduction in a pristine south Chilean Nothofagus forest. Biogeochemistry 90, 243e258. Rütting, T., Müller, C., 2007. 15N tracing models with a Monte Carlo optimization procedure provide new insights on gross N transformations in soils. Soil Biology and Biochemistry 39, 2351e2361. Shen, J.P., Zhang, L.M., Zhu, Y.G., Zhang, J.B., He, J.Z., 2008. Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam. Environmental Microbiology 10, 1601e1611. Sollins, P., Spycher, G., Glassman, C.A., 1984. Net nitrogen mineralisation from light and heavy-fraction forest soil organic matter. Soil Biology and Biochemistry 16, 31e37. Stark, J.M., Hart, S.C., 1997. High rates of nitrification and nitrate turnover in undisturbed coniferous forests. Nature 385, 61e64. Stockdale, E.A., Hatch, D.J., Murphy, D.V., Ledgard, S.F., Watson, C.J., 2002. Verifying the nitrification to immobilization ratio (N/I) as a key determinant of potential nitrate loss in grassland and arable soils. Agronomie 22, 831e838. Weber, D.F., Gainey, P.L., 1962. Relative sensitivity of nitrifying organisms to hydrogen ions in soils and solutions. Soil Science 94, 138e145. Wood, P.M., 1990. Autotrophic and heterotrophic mechanisms for ammonia oxidation. Soil Use and Management 6, 78e79. Wu, P., Liu, J., Zhou, Y., Xie, X., Shen, Q., Guo, S., 2008. Effects of different long term fertilizing systems on fertilizer use efficiency in red paddy soil. Plant Nutrition and Fertilizer Science 14, 277e283 (in Chinese with English abstract). Zak, D.R., Tilman, D., Parmenter, R.R., 1994. Plant production and soil microorganisms in late-successional ecosystems: a continental-scale study. Ecology 75, 2333e2347. Zaman, M., Di, H.J., Cameron, K.C., Frampton, C.M., 1999. Gross nitrogen mineralization and nitrification rates and their relationships to enzyme activities and the soil microbial biomass in soils treated with dairy shed effluent and ammonium fertilizer at different water potentials. Biology and Fertility of Soils 29, 178e186. Zhang, J.B., Zhu, T.B., Cai, Z.C., Müller, C., 2011a. Nitrogen cycling in forest soils across climate gradients in Eastern China. Plant and Soil 342, 419e432. Zhang, J.B., Müller, C., Zhu, T.B., Yi, C., Cai, Z.C., 2011b. Heterotrophic nitrification is the predominant NO 3 production mechanism in coniferous but not broad-leaf acid forest soil in subtropical China. Biology and Fertility of Soils 47, 533e542. Zhang, J.B., Zhu, T.B., Cai, Z.C., Qin, S., Müller, C., 2012a. Effects of long-term repeated mineral and organic fertilizer applications on soil nitrogen transformations. European Journal of Soil Science 63, 75e85. Zhang, J.B., Cai, Z.C., Yang, W.Y., Zhu, T.B., Yu, Y.J., Yan, X.Y., Jia, Z.J., 2012b. Long-term field fertilization affects soil nitrogen transformations in a rice-wheat-rotation cropping system. Journal of Plant Nutrition and Soil Science 175, 939e946. Zhao, Q.G., 2002. Material Cycling and Regulation in Red Soils of China. Science Press, Beijing (in Chinese). Zhao, Q.G., Xie, W.M., He, X.Y., Wang, M.Z., 1988. Red Soils in Jiangxi. Science and Technology Press of Jiangxi, Nanchang (in Chinese). Zhao, W., Cai, Z.C., Xu, Z.H., 2007. Does ammonium-based N addition influence nitrification and acidification in humid subtropical soils of China? Plant and Soil 297, 213e221. Zhu, T.B., Meng, T.Z., Zhang, J.B., Yin, Y.F., Cai, Z.C., Yang, W.Y., Zhong, W.H., 2012. Nitrogen mineralization, immobilization turnover, heterotrophic nitrification, and microbial groups in acid forest soils of subtropical China. Biology and Fertility of Soils. http://dx.doi.org/10.1007/s00374-012-0725-y.
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Agricultural land use affects nitrate production and conservation in humid subtropical soils in China Jinbo Zhang a, b, Tongbin Zhu a, Tianzhu Meng a, Yanchen Zhang a, Jiajia Yang a, Wenyan Yang a, Christoph Müller c, d, Zucong Cai a, b, * a
School of Geography Sciences, Nanjing Normal University, Nanjing 210023, PR China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, PR China Department of Plant Ecology, Justus-Liebig University Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany d School of Biology and Environmental Science, University College Dublin, Belfield, Dublin4, Ireland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 November 2012 Received in revised form 6 March 2013 Accepted 7 March 2013 Available online 29 March 2013
To date, very few studies have been conducted to investigate the characteristics of gross nitrogen (N) transformations in subtropical agricultural soils. In this study, 12 natural woodland and 10 agricultural soils were collected to investigate the effects of land use on soil gross N transformations in the humid subtropical zones in China. The results showed that gross autotrophic nitrification rates (average 0.19 mg N kg1 d1) in the woodland soils were significantly lower than those determined in the agricultural soils (average 1.81 mg N kg1 d1) (p < 0.01). However, the NO 3 immobilization rates (average 0.10 mg N kg1 d1) in the agricultural soils were significantly lower than in the woodland soils (average 0.47 mg N kg1 d1) (p < 0.01). On average, 98% of the total NO 3 produced could be immobilized into organic-N in the woodland soils, while, it accounted for only 10% in the agricultural soils. These differences in gross N transformations resulted in the inorganic N being dominated by NHþ 4 in the woodland soils; however, NO 3 dominated the inorganic N in the agricultural soils. The risk of N leaching and runoff from soil sharply increases after woodland soils are converted to agricultural soils. Application of organic fertilizers with high C/N ratios to agricultural soils in subtropical regions to increase soil organic C content and the C/N ratio is expected to improve NO 3 immobilization capacity and reduce the risk of N leaching and runoff from soil. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Agricultural soils N transformation NO3immobilization capacity Nitrification
1. Introduction Nitrogen (N) is one of the key elements required for biomass growth. Previous investigations have suggested that in some natural ecosystems (e.g. forest ecosystems), in particular under conditions of high rainfall, most available N may be conserved successfully in soils through inherent soil N conservation mechanisms, e.g. i) low mineral N production and low NHþ 4 oxidation rates; ii) a combination of high mineral N production with efficient N immobilization; and iii) NO 3 retention via processes such as dissimilatory NO 3 reduction to ammonium (DNRA) (Huygens et al., 2007; Rütting and Müller, 2007, 2008). Zhang et al. (2011a,b) and Zhu et al. (2012) have reported that the autotrophic nitrification (i.e. NHþ 4 oxidation) rate was low, resulting in the inorganic N being
* Corresponding author. School of Geography Sciences, Nanjing Normal University, Nanjing 210023, PR China. Tel.: þ86 25 8589 1203; fax: þ86 25 8589 1745. E-mail address: [email protected] (Z. Cai). 0038-0717/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2013.03.006
dominated by NHþ 4 , and produced NO3 could be conserved effi ciently in soils by NO3 immobilization processes in the humid, acidic subtropical forest soils in China. Over the past 60 years, diverse land use patterns have been developed to meet an increasing demand for food, cash crops, and fiber, thus making the subtropical region of China important agriculturally. The arable area in the humid subtropical region of China is about 446,890 km2, which accounts for approximately 4% of the world’s subtropical arable land surface or 37% of China’s arable land (Zhao, 2002). Previous observations have suggested that the organic carbon content in subtropical agricultural soils in China is generally low due to intensive land use and poor management (Zhao et al., 1988). Land use and management practices, such as the use of inorganic N fertilizer, organic manure, and lime, could markedly influence the physical and chemical properties of the soil, which would in turn affect soil N cycling. Therefore, the characteristics of soil N transformations in agricultural soils would be expected to differ from those in forest soils. However, very few studies have been conducted to investigate the characteristics of
108
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
soil gross N transformations in subtropical and tropical agricultural soils (Booth et al., 2005). It is currently unknown whether the mechanisms for conserving inorganic N in humid subtropical soils (i.e. NO 3 immobilization and DNRA) could be maintained after woodlands are converted for agricultural use. Understanding soil gross N transformations in subtropical soils and how it is affected by land use would provide a scientific basis for rationally applying and appropriately assessing the environmental impacts of N fertilizers. The objectives of this study were to investigate the effects of land use on soil gross N transformations. Our hypothesis is that soil N conservation mechanisms in humid subtropical forest soils would be destroyed by intensive land use and practice management such as fertilization and liming. Large amounts of mineral N fertilization and liming could stimulate NO 3 production, while a low organic carbon content and C/N ratio could result in a decrease in the NO 3 immobilization capacity, inducing NO3 dominance in the inorganic N present in agricultural soils, thus increasing N losses through NO 3 leaching and runoff. Twelve natural woodland and 10 agricultural soils were collected from typical subtropical zones in China to test the above hypothesis. 2. Material and methods 2.1. Soil samples Twelve natural woodland soils and ten agricultural soils (one orange grove, three rice paddies, three peanut fields, and three maize fields) were collected from the typical subtropical zones in China. The annual precipitation is approximately 1,700 mm, and the mean annual temperature is 18.4e19.4 C. All agricultural sites were established after clearing the native woodland, and had received lime amendments at the beginning of establishment. The orange orchard was approximately 10 years old, and mineral N fertilizer was applied at a rate of approximately 80 kg N ha1 y1. The peanut sites were approximately 20 years old, and were fertilized with mineral N at a rate of approximately 200 kg N ha1 y1. Both the rice paddies and maize fields were established more than 100 years ago, and w300 kg N ha1 y1 N fertilizer had been applied in the most recent 10 years. The majority of crop residues were removed after harvest. At each site, soil samples were taken from three grids (approximately 4 m 4 m) that were randomly selected from a representative 100 m 100 m plot. From each grid, the O horizon, if present, was removed, and one sample was then taken from the mineral A horizon (0e20 cm). Three samples were pooled together, sieved (2 mm), homogenized and subsequently split into two subsamples. One sub-sample was stored at 4 C for the incubation studies. The other sub-sample was air-dried for analysis of soil properties. 2.2.
15
N tracing experiment
In the present investigation, the combination of an 15N tracing experiment and full process-based N cycle models was used to quantify simultaneously occurring gross N transformations in soil, mineralization of recalcitrant organic-N to NHþ 4 , mineralization of þ labile organic-N to NHþ 4 , immobilization of NH4 to labile organic-N, immobilization of NHþ 4 to recalcitrant organic-N, release of adsorþ bed NHþ 4 , adsorption of NH4 on cation exchange sites, oxidation of þ NH4 to NO3 (autotrophic nitrification), oxidation of recalcitrant organic-N to NO 3 (heterotrophic nitrification), immobilization of NO 3 to recalcitrant organic-N, and dissimilatory NO3 reduction to þ NH4 (DNRA) (Müller et al., 2007). The transformation rates were calculated using zero-order, first-order, or MichaeliseMenten kinetics. In the present study several model modifications, varying in
the number of considered N transformation, kinetic settings and considered N pools, were tested to find the model which best describes the measured N dynamics (Rütting et al., 2008). The final model was selected according to Aikaike’s Information Criterion (AIC) (Cox et al., 2006). Parameter optimization was carried out with the Markov chain Monte Carlo Metropolis algorithm (MCMCMA). The steps in model development and the optimization algorithm are described in detail by Müller et al. (2007) (Müller et al., 2007). The misfit function f(m) between the simulation output and observations (see Equation (3) in Müller et al., 2007) takes into account the variance of the individual observations. Analyses using this parameter optimization concept in previous studies have shown that the mineralization of two conceptual organic-N pools produced realistic NHþ 4 dynamics (Huygens et al., 2007; Müller et al., 2009). The MCMC-MA routine was used in the MatLab software package (Version 7.2, The MathWorks Inc.), which calls models that are separately set up in Simulink (Version 6.4, The MathWorks Inc.). Initial concentrations of the mineral N pools were determined according to Müller et al. (2004). Briefly, concentra tions of NHþ 4 and NO3 are estimated for time zero by backextrapolation to t ¼ 0. The difference between NHþ 4 applied and þ NHþ 4 determined was considered to be the amount of NH4 that was immediately adsorbed on NHþ exchange sites (NH ads). 4 4 There were two NH4NO3 treatments (each in three replicates): one contained 15N labeled ammonium (15NH4NO3) while the other 15 contained 15N labeled nitrate (NH15 N at 20 4 NO3) (labeled with atom percentage excess). For each soil, a series of 250-ml conical flasks was prepared with 30 g of fresh soil (oven-dry basis). Two ml of 15NH4NO3 or NH15 4 NO3 solution was added to each of the conical 1 1 flasks at a rate of 20 mg NHþ soil and 20 mg NO 4 eN kg 3 eN kg soil. The soil was adjusted to 60% water holding capacity (WHC) and incubated for 144 h at 25 C. The conical flasks were sealed with silicone rubber stoppers. The soils (three replicates for each 15N label treatment) were extracted at 0.5, 24, 72, and 144 h after fertilizer application for the determination of the concentration and isotopic composition of the NHþ 4 and NO3 . The detailed principle of 15 the model and the N incubation experiment were from Müller et al. (2004, 2007). 2.3. Analyses Soil properties were determined following the Soil AgroChemical Analysis procedures of Lu (2000). Soil pH was measured in a 1:2.5 (v/v) soil to water ratio using a DMP-2 mV/pH detector (Quark Ltd, Nanjing, China). Soil organic carbon (SOC) was analyzed by wet-digestion with H2SO4eK2Cr2O7, and total nitrogen was determined by semi-micro Kjeldahl digestion using Se, CuSO4 and K2SO4 as catalysts. Ammonium and NO 3 were extracted with 2 M KCl at a soil/solution ratio of 1:5 on a mechanical shaker for 60 min at 300 rpm at 25 C. The extracts were filtered through filter paper (Qualitative Filter Paper, BH92410262) and the concentrations of NHþ 4 and NO3 were determined with a continuous-flow analyzer (Skalar, Breda, Netherlands). For isotopic analysis, NHþ 4 and NO3 were separated by distillation with magnesium oxide and Devarda’s alloy (Feast and Dennis, 1996; Zhang et al., 2011a,b). In detail, a portion of the extract was steam-distilled with MgO to separate NHþ 4 on a steam distillation system; thereafter, the sample in the flask was distilled again after addition of Devarda’s alloy to separate NO 3 . The liberated NH3 was trapped using boric acid solution. The trapped N was acidified and converted to (NH4)2SO4 using 0.02 mol L1 H2SO4 solution. The H2SO4 solution containing NHþ 4 was then evaporated to dryness at 65 C in an oven and analyzed for 15N abundance. Before separating NHþ 4 and NO3 in the extract using the steam distillation system, the þ 1 recovery of NHþ 4 and NO3 in a standard solution (1 g NH4 eN L
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114 1 and 1 g NO 3 eN L ) was determined. The results showed that almost all NHþ eN in the solution could be recovered (>99%), and 4 þ the recovery of NO 3 was >95%. Isotopic composition of the NH4 and NO was determined using an automated C/N analyzer coupled 3 to an isotope ratio mass spectrometer (IRMS 20-22, SerCon, Crewe, UK).
2.4. Data analysis The ratio of autotrophic nitrification rate to mineralization rate (M ¼ MNrec þ MNlab) was defined as nitrification capacity (NC), indicating the capacity of the soil to transform available NHþ 4 to NO 3 . The ratio of total NO3 consumption rate (NO3 immobilization ) to total nitrification rate was defined as the capacity of the soil to retain NO 3 (NR). The net NO3 production rate ¼ the total nitrifi cation rate e the total NO3 consumption rate. 2.5. Statistical analyses and model setup The optimization procedure resulted in a probability density function (PDF) for each parameter, from which parameter averages and standard deviations were calculated (Müller et al., 2007). Each analysis run was carried out with three parallel sequences to identify adequate iteration numbers. Based on the kinetic settings and the final parameters, average N transformation rates were calculated over the whole period and expressed in units of mg N kg1 soil day1. Pearson correlation coefficient analysis was performed to explore the relationship between measured variables using the SPSS 17.0 software package for Windows. Soil characteristics and N transformations between woodland and agricultural soils were compared by t-tests using SPSS 17.0 software (where p < 0.05 was considered to indicate statistical significance). 3. Results 3.1. Soil properties The soil pH ranged from 4.12 to 4.65 (average 4.35) in the woodland soils, which was significantly lower than that in the agricultural soils (average 4.77) (p < 0.05). However, most pH values were still less than 5.0 in the agricultural soils (Fig. 1a). Soil organic C (SOC) content (average 32.68 g kg1, p < 0.05) and C/N ratio (average 16.09, p < 0.05) in the woodland soils was significantly higher than those in the agricultural soils (average values were 18.35 g kg1, and 10.32, respectively, for SOC, and C/ N) (Fig. 1b,c), although no significant difference in the total N content was observed (Fig. 1d). The inorganic N was dominated þ by NHþ 4 in the woodland soils (the average ratios of NH4 /NO3 was 7.54), however, NO3 dominated the inorganic N in the agricultural soils (the average ratios of NHþ 4 /NO3 were 0.24). The NO 3 and total inorganic N concentrations in the agricultural soils were much higher than those in the woodland soils (p < 0.05) (Fig. 1f, g). 3.2. Gross N transformation There was no significant difference in the gross mineralization rates and NHþ 4 immobilization rates between woodland and agricultural soils (Fig. 2a,b). There was also no significant relationship between gross mineralization rates and individually measured soil properties (Fig. 1, i.e. soil organic C, total N, pH, and C/N ratio). Gross autotrophic nitrification (NHþ 4 oxidation) rates ranged from 0 to 0.65 mg N kg1 d1 (average 0.19 mg N kg1 d1) in the woodland soils, which was significantly lower than that
109
determined for the agricultural soils (range, 0.80e 2.74 mg N kg1 d1, with an average of 1.81 mg N kg1 d1; p < 0.05) (Fig. 2c). The gross autotrophic nitrification rate increased linearly with the increase in soil pH (R2 ¼ 0.46, p < 0.01) (Fig. 3a). Nitrification capacity (NC, presented as the ratio of NHþ 4 oxidation to mineralization) in the woodland soils (0.04 0.04) was substantially lower than that of the agricultural soils (1.06 0.67; p < 0.05) (Fig. 2d). The NC was also exponentially correlated with soil pH (R2 ¼ 0.35, p < 0.01) (Fig. 3b). The average heterotrophic nitrification (oxidation of recalcitrant organic-N to NO 3 ) rate in the woodland soils was 0.49 0.36 mg N kg1 d1 (Fig. 2e). However, the heterotrophic nitrification rate was negligible (0.002 0.01 mg N kg1 d1) in the agricultural soils. Heterotrophic nitrification increased with the increase in soil C/N ratio (R2 ¼ 0.75, p < 0.001), except in soil 4, in which heterotrophic nitrification was negligible, despite the fact that the C/N ratio was high (20.26). Also, heterotrophic nitrification decreased exponentially with an increase in total mineralization in the woodland soils (p < 0.01). DNRA rates were low in all studied soils, and not significantly different between woodland and agricultural soils (Fig. 2f). The NO 3 immobilization rates were significantly different between woodland and agricultural soils (p < 0.05) (Fig. 2g). All agricultural soils, with the exception of soil 22, which exhibited a NO 3 immobilization rate of 0.63 0.25 mg N kg1 d1, had very low NO 3 immobilization capacity. In contrast, the studied woodland soils had significantly higher NO 3 immobilization capacities (average 0.47 mg N kg1 d1) than did the agricultural soils (p < 0.01). The NO 3 immobilization rate was positively correlated with the C/N ratio (p < 0.001) and negatively correlated with soil pH (p < 0.05) (Fig. 4). NO 3 retention ability (NR) (i.e. ratio of NO3 consumption [NO 3 immobilization and DNRA] to total nitrification rate), which ranged from 0.39 to 2.37 (average 0.98) in the woodland soils, was significantly higher than in the agricultural soils, where it ranged from 0.01 to 0.29 (average 0.1; p < 0.05) (Fig. 2h). The net nitrification rate (average 1.59 mg N kg1 d1) in the agricultural soils was significantly higher than in the woodland soils (average 0.12 mg N kg1 d1; p < 0.05) (Fig. 1g). Both the net nitrification rate, which was calculated as the total NO 3 production rate (autotrophic nitrification þ heterotrophic nitrification) minus the total NO 3 consumption rate (NO3 immobilization þ DNRA), and the gross nitrification rate could predict NO 3 concentrations well in the studied soils (Fig. 5). 4. Discussion The results of the present study show that the conversion of woodland soil to agricultural soil mainly affected nitrate production and immobilization, while there were no significant differences in the gross mineralization rates and NHþ 4 immobilization rates between woodland and agricultural soils (Fig. 6). After conversion of woodland soil to agricultural soil, the gross NHþ 4 oxidation rate and NC increased significantly, compared to woodland soils (p < 0.01), resulting in the inorganic N being dominated by NO 3 in the agricultural soils (Fig. 6). The change in soil nitrogen transformation characteristics in the agricultural soils could result in a change in inorganic N forms, and increase the risk of N leaching and runoff from soil, inducing negative environmental effects as compared to the woodland soils. 4.1. Nitrate as dominant form of inorganic N in the agricultural soils Generally, the rates of N cycling are high in humid subtropical and tropical forests ecosystems (Martinelli et al., 1999; Matson et al., 1999), so they have to develop a strong capacity for retaining inorganic N in soils (Huygens et al., 2007; Rütting et al., 2008;
110
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
Fig. 1. Box plot of soil properties in the present study. Identical letters indicate no statistical differences in the average values between groups.
Zhang et al., 2011a,b). The results of the present investigation show that autotrophic nitrification, i.e. ammonium oxidation, was very low in all studied woodland soils (average 0.19 mg N kg1 d1), and the NC was only 6%. As a result, ammonium dominated in the studied woodland soils. Under low soil pH (average 4.35), ammonia volatilization is almost completely suppressed. Although in subtropical regions, besides negatively charged colloids, highly weathered soils usually also contain positively charged sites on the colloids; NO 3 is lost much more easily via leaching and runoff than is ammonium (Qian and Cai, 2007). Therefore, maintaining inorganic ammonium dominance is one of the strategies for humid subtropical forest soils to retain inorganic N.
Both the ammonium oxidation rate and NC were correlated significantly with soil pH, suggesting that the increase in soil pH resulting from liming was one of the important factors affecting nitrification in the agricultural soils. These relationships highlight the importance of soil pH on the nitrification process (Weber and Gainey, 1962; Katyal et al., 1988; Zhao et al., 2007; Zhang et al., 2011a). In addition, N fertilization could be another important factor stimulating nitrification activity in the agricultural soils. Previous observations have found that autotrophic nitrification is often stimulated by N addition (Zaman et al., 1999; Stockdale et al., 2002; Hall and Matson, 2003; Zhang et al., 2012a; Lu et al., 2012). In agricultural soils, most ammonium oxidation is carried out by
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
8
a
8 b 6 4 2 0
0
2 .5 b
2 .1 1 .4 a
0 .7
A g ric u ltu ra l
(d )
b
2 .0 1 .5 1 .0 0 .5 a 0 .0 -0 .5
0 .0 W o o d la n d
W o o d la n d
A g ric u ltu ra l
(e )
0 .6
A g ric u ltu ra l
a
(f)
b a
0 .4
-1
-1
1 .0
D N R A (m g k g d )
H e te ro tro p h ic n itrific a tio n -1 -1 (m g k g d )
2
W o o d la n d
(c )
2 .8
1 .5
a
4
A g ric u ltu ra l
N itrific a tio n c a p a c ity
A u to tro p h ic n itrific a tio n -1 -1 (m g k g d )
3 .5
a 6
-2
W o o d la n d
0 .5 a 0 .0
-0 .5
0 .0
(g )
A g ric u ltu ra l
W o o d la n d
2 .5
b
0 .8 a 0 .6 0 .4
(h )
A g ric u ltu ra l
b
2 .0 1 .5 1 .0 0 .5
a
-
0 .2
N O 3 re te n tio n c a p a c ity
-
1 .0
0 .2
-0 .2
W o o d la n d
Im m o b iliz a tio n o f N O 3 -1 -1 (m g k g d )
(b )
+
(a )
Im m o b iliz a tio n o f N H 4 -1 -1 (m g k g d )
-1
-1
M in e ra liz a tio n (m g k g d )
10
111
0 .0
0 .0 -0 .5
-0 .2 W o o d la n d 1
A g ric u ltu ra l 1
W o o d la n d
A g ric u ltu ra l
Fig. 2. Box plot of gross N transformation rates (mg N kg d ) in studied woodland and agricultural soils (0e20 cm) estimated by the þ NO 3 reduction to NH4 . Identical letters indicate no statistical differences in the average values between groups.
autotrophic ammonia-oxidizing bacteria (AOB) (Barraclough and Puri, 1995). Previous studies have observed that long-term field mineral N fertilization can stimulate the growth of AOB in the field by more than 20-fold when compared to control soils (no N application) (Chu et al., 2008; Shen et al., 2008; Zhang et al., 2012b). In addition, recent studies have demonstrated that the Nitrosospira cluster 3-like AOB was highly enriched in fertilized agricultural soils (Chu et al., 2007; Shen et al., 2008). Thus, the observed stimulation of gross autotrophic nitrification in the agricultural soils was most likely driven by the increase in AOB population size and the changes in AOB composition due to long-term mineral N application. Further studies are needed to investigate the characteristics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in
15
N tracing model. DNRA is dissimilatory
the woodland and agricultural soils, and to clarify the mechanisms of agricultural use that stimulate the autotrophic nitrification process. 4.2. Decrease in NO 3 immobilization The results of our investigation show that NO 3 immobilization is a widespread process in the studied woodland soils in humid subtropical regions; however, in the agricultural soils, the NO 3 immobilization rate and NO 3 immobilization capacity were negligible. On average, the NO 3 immobilization rate into organic-N accounted for 98% of the total nitrification rate (autotrophic nitrification plus heterotrophic nitrification of organic-N) in the
112
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
-1
3
a
-1
y = 2 .2 2 x - 9 .1 7
-1 5
e
6 .7 7 9 6 x
2
2
2
b y = 3 10
R = 0 .4 6 p < 0 .0 1
N itrific a tio n c a p a c ity
A u to tro p h ic n itrific a tio n (m g k g d )
3
1
0
2
R = 0 .3 5 p < 0 .0 1
1
0
4 .0
4 .4
4 .8
5 .2
4 .0
4 .4
pH
4 .8
5 .2
pH
Fig. 3. Relationship between autotrophic nitrification (a), nitrification capacity (b) and soil pH. Nitrification capacity is presented as the ratio of NHþ 4 oxidation to mineralization.
and C/N ratio after conversion of woodland to agricultural soils, mainly due to low organic matter input and high mineral N fertilization, was an important factor which resulted in a decrease in NO 3 immobilization. Furthermore, previous studies have found that fungi may preferentially utilize NO 3 (Marzluf, 1997), when compared with bacteria. Thus, the observed differences in NO 3 immobilization between woodland soils and agricultural soils could be related to fungal activity. Soil conditions, such as soil organic matter content, composition (C/N) and pH, could influence soil microbial community structure, which, in turn, controlled soil N transformations. Generally, fungi are favored by acidic soils that are low in available nutrients, recalcitrant organic materials, and that have high C/N soil ratios (Kreitinger et al., 1985; Wood, 1990; Blagodatskaya and Anderson, 1998; Högberg et al., 2007). However, tillage and fertilization were usually considered to be reasons for a decline in fungal
woodland soils. In contrast, it accounted for only 10% in the agricultural soils. The difference in NO 3 immobilization ability could further explain why the NO 3 concentrations in the agricultural soils were much higher than in the woodland soils. Our results showed that the NO 3 immobilization rate increased with the increase in C/N ratio (p < 0.001, Fig. 4a), suggesting that quantity and quality of soil organic C could be the important factor affecting NO 3 immobilization (Recous et al., 1990; Johnson, 1992; Stark and Hart, 1997; Barrett and Burke, 2000; Burger and Jackson, 2003; Häbteselassie et al., 2006). Previous studies have reported that microbes needed to immobilize more inorganic N when they decompose organic matter with high C/N ratios (Sollins et al., 1984; Zak et al., 1994; Janssen, 1996). Therefore, the NO 3 immobilization rate could be high in the soils which had a high organic C content and high C/N ratio (Johnson, 1992; Stark and Hart, 1997; Burger and Jackson, 2003). The significant decrease of soil organic C content
-1
y = 0 .0 7 x - 0 .5 5
0 .8
b
-1
N O 3 im m o b iliz a tio n (m g k g d )
2
R = 0 .7 0 p < 0 .0 0 1
0 .6
0 .4
0 .2
0 .8
y = -0 .5 0 x + 2 .5 9 2
R = 0 .2 2 0 .6
p < 0 .0 5
0 .4
0 .2
-
-1
1 .0
a
-1
N O 3 - im m o b iliz a tio n (m g k g d )
1 .0
0 .0 4
8
12
16
20
24
C /N ra tio Fig. 4. Relationship between
0 .0 4 .0
4 .5
5 .0 pH
NO 3
immobilization and (a) C/N ratio, (b) Soil pH.
5 .5
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
a
Agricultural soil Immobilization
-
-1
NO3 concentration (mg kg )
80
y=19.1x + 3.4 2 R =0.59 p<0.001
60
0.10±0.19 Autotrophic Mineralization
40
NO 3DNRA
20
0.12±0.20
Immobilization 1.29±1.63 Heterotrophic nitrification
1
2 -1
80
0.002±0.01
3 -1
Net nitrification rate (mg kg d )
-1
1.81±0.53
NH 4+
SOM
0
-
nitrification
2.37±1.74
0
NO3 concentrition (mg kg )
113
Woodland soil Immobilization
b
0.47±0.27 Autotrophic
y = 21.7x - 7.4 2 R = 0.59 p<0.001
60
Mineralization
nitrification
3.67±2.15
0.19±0.21
NH 4+
SOM
40
NO 3DNRA 0.09±0.12
Immobilization 2.20±1.83
20
Heterotrophic nitrification 0.49±0.36
0 0
1
2
3 -1 -1
Gross nitrification rate (mg kg d ) Fig. 5. Relationship between net nitrification rate (a), gross nitrification rate (b) and soil nitrate concentration. Net nitrification rate was calculated as the total NO 3 production rate (autotrophic nitrification þ heterotrophic nitrification) minus the total NO3 consumption rate (NO3 immobilization þ DNRA).
biomass (Beare et al., 1997; Frey et al., 1999; Högberg et al., 2003; Joergensen and Wichern, 2008). Further studies are needed to investigate fungal activity and community structure in the woodland and agricultural soils in order to clarify the microbial mechanisms of NO 3 immobilization. Our results highlight the fact that changes in land use can significantly affect N transformation characteristics in the humid subtropical region. Nitrate production increased significantly; however, NO 3 immobilization almost disappeared, resulting in NO 3 dominance in the inorganic N in the agricultural soils as compared with woodland soils (Fig. 6). The risk of N leaching and runoff from soil sharply increased after woodland was converted to agricultural use, which is not only due to the application of N fertilizers, but also stimulation of nitrification and depression of NO 3 immobilization. Previous observations in a long-term fertilizing system (from 1990 to 2006) showed that the N fertilizer apparent recovery efficiency only ranged from 23.4% to 61.6%, with an average of 36.5%, in the subtropical region of China (Wu et al., 2008). As a result, the negative environmental effects of NO 3 leaching and runoff could be induced in the subtropical region. Application of organic fertilizer with a high C/N ratio to agricultural
Fig. 6. Nitrogen cycle in agricultural and woodland soils in the humid subtropical region of China. The thickness of the arrows represents the relative importance of each flux. The data in the figure are the means of gross rates of N transformation SD (mg kg1d1).
soils in the subtropical region to increase soil organic C content and C/N ratio is expected to improve NO 3 immobilization capacity and reduce the risk of N leaching and runoff from soil. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (41101209, 41222005, 40830531), the Natural Science Foundation of Jiangsu Province (BK2010611, SBK201220477), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Barraclough, D., Puri, G., 1995. The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification. Soil Biology and Biochemistry 27, 17e22. Barrett, J.E., Burke, I.C., 2000. Potential nitrogen immobilization in grassland soils across a soil organic matter gradient. Soil Biology and Biochemistry 32, 1707e1716. Beare, M.H., Hu, S., Coleman, D.C., Hendrix, P.F., 1997. Influences of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Applied Soil Ecology 5, 211e219. Blagodatskaya, E.V., Anderson, T.H., 1998. Interactive effects of pH and substrate quality on the fungal-to-bacterial ratio and qCO2 of microbial communities in forest soils. Soil Biology and Biochemistry 30, 1269e1274.
114
J. Zhang et al. / Soil Biology & Biochemistry 62 (2013) 107e114
Booth, M.S., Stark, J.M., Rastetter, E.B., 2005. Controls on nitrogen cycling in terrestrial ecosystems: a synthetic analysis of literature data. Ecological Monographs 75, 139e157. Burger, M., Jackson, L.E., 2003. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biology and Biochemistry 35, 29e36. Chu, H.Y., Fujii, T., Morimoto, S., Lin, X.G., Yagi, K., Hu, J.L., Zhang, J.B., 2007. Community structure of ammonia oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil. Applied and Environmental Microbiology 73, 485e491. Chu, H.Y., Fujii, T., Morimoto, S., Lin, X.G., Yagi, K., 2008. Population size and specific nitrification potential of soil ammonia-oxidizing bacteria under long-term fertilizer management. Soil Biology and Biochemistry 40, 1960e1963. Cox, G.M., Gibbons, J.M., Wood, A.T.A., Craigon, J., Ramsden, S.J., Crout, N.M.J., 2006. Towards the systematic simplification of mechanistic models. Ecological Modelling 198, 240e246. Feast, N.A., Dennis, P.E., 1996. A comparison of methods for nitrogen isotope analysis of groundwater. Chemical Geology 129, 167e171. Frey, S.D., Elliott, E.T., Paustian, K., 1999. Bacterial and fungal abundance and biomass in conventional and no-tillage agroecosystems along two climatic gradients. Soil Biology & Biochemistry 31, 573e585. Häbteselassie, M.Y., Stark, J.M., Miller, B.E., Thacker, S.G., Norton, J.M., 2006. Gross nitrogen transformations in an agricultural soil after repeated dairy-waste application. Soil Science Society of American Journal 70, 1338e1348. Hall, S.J., Matson, P.A., 2003. Nutrient status of tropical rain forests influences soil N dynamics after N additions. Ecological Monographs 73, 107e129. Högberg, M.N., Baath, E., Nordgren, A., Arnebrant, K., Högberg, P., 2003. Contrasting effects of nitrogen availability on plant carbon supply to mycorrhizal fungi and saprotrophs e a hypothesis based on field observations in boreal forests. New Phytologist 160, 225e238. Högberg, M.N., Högberg, P., Myrold, D.D., 2007. Is microbial community composition in boreal forest soils determined by pH, C-to-N ratio, the trees, or all three? Oecologia 150, 590e601. Huygens, D., Rütting, T., Boeckx, P., Van Cleemput, O., Godoy, R., Müller, C., 2007. Soil nitrogen conservation mechanisms in a pristine south Chilean Nothofagus forest ecosystem. Soil Biology and Biochemistry 39, 2448e2458. Janssen, B.H., 1996. Nitrogen mineralization in relation to C/N ratio and decomposability of organic materials. Plant and Soil 181, 39e45. Joergensen, R.G., Wichern, F., 2008. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biology and Biochemistry 40, 2977e2991. Johnson, D.W., 1992. Nitrogen retention in forest soils. Journal of Environmental Quality 21, 1e12. Katyal, J.C., Cater, M.F., Viiek, P.L.G., 1988. Nitrification activity in submerged soil and its relation to denitrification loss. Biology and Fertility of Soils 7, 16e22. Kreitinger, J.P., Klein, T.M., Novick, N.J., Alexander, M., 1985. Nitrification and characteristics of nitrifying microorganisms in an acid forest soil. Soil Science Society of American Journal 49, 1407e1410. Lu, L., Han, W., Zhang, J., Wu, Y., Wang, B., Lin, X., Zhu, J., Cai, Z., Jia, Z., 2012. Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea. The ISME Journal 6, 1978e1984. Lu, R.K., 2000. Soil Agro-chemical Analyses. Agricultural Technical Press of China, Beijing (in Chinese). Martinelli, L.A., Piccolo, M.C., Townsend, A.R., Vitousek, P.M., Cuevas, E., McDowell, W., Robertson, G.P., Santos, O.C., Treseder, K., 1999. Nitrogen stable isotope composition of leaves and soil: tropical versus temperate forests. Biogeochemistry 46, 45e65. Marzluf, G.A., 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiology and Molecular Biology Reviews 61, 17e32. Matson, P.A., McDowell, W.H., Townsend, A.R., Vitousek, P.M., 1999. The globalization of N deposition: ecosystem consequences in tropical environments. Biogeochemistry 46, 67e83. Müller, C., Rütting, T., Kattge, J., Laughlin, R.J., Stevens, R.J., 2007. Estimation of parameters in complex 15N tracing models via Monte Carlo sampling. Soil Biology and Biochemistry 39, 715e726.
Müller, C., Stevens, R.J., Laughlin, R.J., 2004. A 15N tracing model to analyse N transformations in old grassland soil. Soil Biology and Biochemistry 36, 619e632. Müller, C., Rütting, T., Abbasi, M.K., Laughlin, R.J., Kammann, C., Clough, T.J., Sherlock, R.R., Kattge, J., Jäger, H.J., Watson, C.J., Stevens, R.J., 2009. Effect of elevated CO2 on soil N dynamics in a temperate grassland soil. Soil Biology & Biochemistry 41, 1996e2001. Qian, C., Cai, Z.C., 2007. Leaching of nitrogen from subtropical soils as affected by nitrification potential and base cations. Plant and Soil 300, 197e205. Recous, S., Mary, B., Faurie, G., 1990. Microbial immobilization of ammonium and nitrate in cultivated soils. Soil Biology and Biochemistry 22, 913e922. Rütting, T., Huygens, D., Müller, C., van Cleemput, O., Godoy, R., Boeckx, P., 2008. Functional role of DNRA and nitrite reduction in a pristine south Chilean Nothofagus forest. Biogeochemistry 90, 243e258. Rütting, T., Müller, C., 2007. 15N tracing models with a Monte Carlo optimization procedure provide new insights on gross N transformations in soils. Soil Biology and Biochemistry 39, 2351e2361. Shen, J.P., Zhang, L.M., Zhu, Y.G., Zhang, J.B., He, J.Z., 2008. Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam. Environmental Microbiology 10, 1601e1611. Sollins, P., Spycher, G., Glassman, C.A., 1984. Net nitrogen mineralisation from light and heavy-fraction forest soil organic matter. Soil Biology and Biochemistry 16, 31e37. Stark, J.M., Hart, S.C., 1997. High rates of nitrification and nitrate turnover in undisturbed coniferous forests. Nature 385, 61e64. Stockdale, E.A., Hatch, D.J., Murphy, D.V., Ledgard, S.F., Watson, C.J., 2002. Verifying the nitrification to immobilization ratio (N/I) as a key determinant of potential nitrate loss in grassland and arable soils. Agronomie 22, 831e838. Weber, D.F., Gainey, P.L., 1962. Relative sensitivity of nitrifying organisms to hydrogen ions in soils and solutions. Soil Science 94, 138e145. Wood, P.M., 1990. Autotrophic and heterotrophic mechanisms for ammonia oxidation. Soil Use and Management 6, 78e79. Wu, P., Liu, J., Zhou, Y., Xie, X., Shen, Q., Guo, S., 2008. Effects of different long term fertilizing systems on fertilizer use efficiency in red paddy soil. Plant Nutrition and Fertilizer Science 14, 277e283 (in Chinese with English abstract). Zak, D.R., Tilman, D., Parmenter, R.R., 1994. Plant production and soil microorganisms in late-successional ecosystems: a continental-scale study. Ecology 75, 2333e2347. Zaman, M., Di, H.J., Cameron, K.C., Frampton, C.M., 1999. Gross nitrogen mineralization and nitrification rates and their relationships to enzyme activities and the soil microbial biomass in soils treated with dairy shed effluent and ammonium fertilizer at different water potentials. Biology and Fertility of Soils 29, 178e186. Zhang, J.B., Zhu, T.B., Cai, Z.C., Müller, C., 2011a. Nitrogen cycling in forest soils across climate gradients in Eastern China. Plant and Soil 342, 419e432. Zhang, J.B., Müller, C., Zhu, T.B., Yi, C., Cai, Z.C., 2011b. Heterotrophic nitrification is the predominant NO 3 production mechanism in coniferous but not broad-leaf acid forest soil in subtropical China. Biology and Fertility of Soils 47, 533e542. Zhang, J.B., Zhu, T.B., Cai, Z.C., Qin, S., Müller, C., 2012a. Effects of long-term repeated mineral and organic fertilizer applications on soil nitrogen transformations. European Journal of Soil Science 63, 75e85. Zhang, J.B., Cai, Z.C., Yang, W.Y., Zhu, T.B., Yu, Y.J., Yan, X.Y., Jia, Z.J., 2012b. Long-term field fertilization affects soil nitrogen transformations in a rice-wheat-rotation cropping system. Journal of Plant Nutrition and Soil Science 175, 939e946. Zhao, Q.G., 2002. Material Cycling and Regulation in Red Soils of China. Science Press, Beijing (in Chinese). Zhao, Q.G., Xie, W.M., He, X.Y., Wang, M.Z., 1988. Red Soils in Jiangxi. Science and Technology Press of Jiangxi, Nanchang (in Chinese). Zhao, W., Cai, Z.C., Xu, Z.H., 2007. Does ammonium-based N addition influence nitrification and acidification in humid subtropical soils of China? Plant and Soil 297, 213e221. Zhu, T.B., Meng, T.Z., Zhang, J.B., Yin, Y.F., Cai, Z.C., Yang, W.Y., Zhong, W.H., 2012. Nitrogen mineralization, immobilization turnover, heterotrophic nitrification, and microbial groups in acid forest soils of subtropical China. Biology and Fertility of Soils. http://dx.doi.org/10.1007/s00374-012-0725-y.