J Soils Sediments DOI 10.1007/s11368-016-1554-7

SOILS, SEC 1 • SOIL ORGANIC MATTER DYNAMICS AND NUTRIENT CYCLING • RESEARCH ARTICLE

N2O production pathways relate to land use type in acidic soils in subtropical China Yi Zhang 1 & Wei Zhao 1,2 & Jinbo Zhang 1,3,4 & Zucong Cai 1,3,5

Received: 10 July 2016 / Accepted: 7 September 2016 # Springer-Verlag Berlin Heidelberg 2016

Electronic supplementary material The online version of this article (doi:10.1007/s11368-016-1554-7) contains supplementary material, which is available to authorized users.

WD, accounting for 44.6 % of N2O emissions, whereas heterotrophic nitrification contributed less than 2.7 % in all three agricultural soils, due to a lower organic C content and soil C/ N ratio. In contrast, denitrification dominated N2O production in agricultural soils, accounting for 54.5, 72.8 and 77.1 % in UA, TP and BP, respectively. Nitrate (NO3−) predominantly affected the contribution from denitrification in soils under different land use types. Autotrophic nitrification increased after the conversion of woodland to agricultural lands, peaking at 42.8 % in UA compared with only 21.5 % in WD, and was positively correlated with soil pH. Our data suggest that pH plays a great role in controlling N2O emissions through autotrophic nitrification following conversion of woodland to agricultural lands. Conclusions Our results demonstrate the variability in N2O production pathways in soils of different land use types. Soil pH, the quantity and quality of organic C and NO3− content primarily determined N2O emissions. These results will likely assist modelling and mitigation of N2O emissions from different land use types in subtropical acidic soils in China and elsewhere.

* Jinbo Zhang [email protected]

Keywords Agricultural soil . Land use . N2O production pathway . Subtropical China . Woodland soil

Abstract Purpose Agricultural practises impact soil properties and N transformation rate, and have a greater effect on N2O production pathways in agricultural soils compared with natural woodland soils. However, whether agricultural land use affects N2O production pathways in acidic soils in subtropical regions remains unknown. Materials and methods In this study, we collected natural woodland soil (WD) and three types of agricultural soils, namely upland agricultural (UA), tea plantation (TP) and bamboo plantation (BP) soils. We performed paired 15N-tracing experiment to investigate the effects of land use types on N2O production pathways in acidic soils in subtropical regions in China. Results and discussion The results revealed that heterotrophic nitrification is the dominant pathway of N2O production in Responsible editor: Zhihong Xu

1

2

School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China College of Environmental Science, Nanjing Industry University, Nanjing 210009, China

3

Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Nanjing, China

4

State Key Laboratory Cultivation Base of Geographical Environment Evolution, Nanjing, Jiangsu Province 210023, China

5

Key Laboratory of Virtual Geographical Environment (VGE), Ministry of Education, Nanjing Normal University, Nanjing, China

1 Introduction Nitrous oxide (N2O) is a potent long-lived greenhouse gas that contributes ~6 % to radiative forcing, and it is the single most important substance depleting the stratospheric ozone (Ravishankara et al. 2009; WMO 2015). N2O sources and sinks therefore attract considerable interest from climate scientists. Globally, soil is a major source and accounts for approximately 37 % of global N2O emissions (IPCC et al. 2013).

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Autotrophic nitrification of NH4+, heterotrophic nitrification of organic N, denitrification of NO3−, chemical nitrification of NO2− or NH2OH and dissimilatory nitrate reduction to ammonia (DNRA) are all believed to contribute to N2O production in soils (Van Cleemput and Baert 1984; Papen et al. 1989; Wrage et al. 2001; Laughlin and Stevens 2002; Wrage et al. 2005; Rütting et al. 2010; Müller et al. 2014). These pathways are closely associated with soil NH4+, organic N and NO3− pools, which are affected by soil properties, vegetation type and land use (Xu and Cai 2007; Zhang et al. 2015). Considering the aforementioned major N pools, Zhang et al. (2015) generalised these complex systems into three main pathways: autotrophic nitrification, heterotrophic nitrification and denitrification. These relate to NH4+, organic N and NO3− pools, respectively, and suggest N2O production is regulated by N transformation rates of the three processes, as well as the proportion of N lost as N2O-N via these processes, which are influenced by soil moisture, temperature, pH and other environmental factors (Koponen et al. 2006; Stehfest and Bouwman 2006; Butterbach-Bahl et al. 2013). Anaerobic denitrification is considered an important pathway for N2O production (Mosier et al. 1986; Friedl et al. 2016), especially in soil with a low O2 content (Firestone and Davidson 1989), and high N and labile C availability (Mulvaney et al. 1997; Azam et al. 2002; Morley and Baggs 2010). Soil pH is an important factor regulating the contribution of denitrification to N2O production (Cheng et al. 2015). During denitrification, N2O can be emitted either as an intermediate product, or as a terminal product, in neutral and alkaline soils. While N2O is more likely to be produced as the terminal product in acidic soils, fungi generally lack the nosZ gene product needed to further reduce N2O to N2, and may therefore play a more important role in N2O production than bacteria in acidic conditions (Firestone and Davidson 1989; Laughlin and Stevens 2002; Hu et al. 2015). The factors involved in N2O production via denitrification in acidic soils may therefore differ in acidic soils, compared with neutral and alkaline soils. Ammonia oxidation also has an important impact on N2O production. The activity of ammonia oxidising bacteria can be suppressed in acidic soils, and microbiological oxidation of NH4+ to NO3− by these organisms appears not to occur in soils at pH <4.5 (Weber and Gainey 1962). In contrast, heterotrophic nitrification (oxidation of organic N to NO3−) is perhaps more widespread in acidic soils (Kreitinger et al. 1985; Wood 1990; Huygens et al. 2008). Studies on heterotrophic nitrification and its contribution to N2O production have been carried out in acidic woodland soils (Zhang et al. 2011b; Stange et al. 2009) and grassland soils (Rütting et al. 2010; Müller et al. 2004). The results showed that heterotrophic nitrification is important in N2O production in acidic soils, accounting for 27–69 % in woodland soils (Zhang et al. 2011b; Stange et al. 2009) and 54–85 % in grassland soils (Rütting et al. 2010; Müller et al. 2004), and the

contribution of this process is positively affected by soil organic C content and C/N ratio (Zhang et al. 2015). Soils in subtropical zones in China are heavily weathered, characterised by a high redox potential, and are highly acidic (Qafoku et al. 2004; Xu and Cai 2007; Ding 2008). Humid subtropical woodland soils are reported to be the largest natural source of N2O globally (Davidson 1991; Zhao 2002; Xu and Cai 2007). Zhang et al. (2011b) reported that denitrification and heterotrophic nitrification are responsible for 53.5– 56.1 % and 27.4–41.8 % of N2O production, respectively, in subtropical acidic woodland soils, due to the low pH and high C/N ratio. Recent conversion of woodland to agricultural land has attempted to meet the increasing demands of agricultural production (Zhao 2002). During this process, soil properties and N transformation rates can be affected significantly (Zhang et al. 2013). For example, in acidic agricultural soils in China, conversion of woodland to upland agriculture is usually accompanied by the application of fertilisers and lime to improve crop production, which increases soil pH and nitrification rate (Xu and Cai 2007; Cai and Zhao 2009). Planting tea usually causes further soil acidification due to the physiological features of this plant species, and heavy application of fertilisers such as urea results in excessive soil nitrogen (Ruan et al. 2004; Cai and Zhao 2009), and a subsequent increase in heterotrophic nitrification (Zhu et al. 2014). N2O production is widely considered to increase in soil after conversion of woodland to agriculture, due to the agricultural management practises described above (Agustín et al. 2004; Lin et al. 2010; Petitjean et al. 2015). However, whether agricultural land use can affect N2O production pathways remains poorly understood, as do the factors influencing N2O production pathways following a change in land use, particularly in acidic soils in subtropical regions in China. Based on the current knowledge regarding soil N transformation, we made the following hypotheses: (1) heterotrophic nitrification is the dominant pathway of N2O production in woodland soils, but the contribution from autotrophic nitrification would increase after conversion to agricultural soils and (2) soil pH, and organic C and inorganic N content may primarily affect N2O production in soils of different land use types. The purpose of the present study was to evaluate the effects of land use types on the contribution from autotrophic nitrification, heterotrophic nitrification and denitrification to N2O production, and to explore the factors regulating N2O emissions in humid subtropical acidic soils in China.

2 Materials and methods 2.1 Soil samples Seventeen soil samples were collected in October 2015 from Jiangxi Province in the subtropical region of China. Six

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woodland soils (WD) were collected, along with 11 agricultural soils from five upland agricultural areas (UA), three tea plantations (TP) and three bamboo plantations (BP). All agricultural sites were established after clearing native woodland, and all received lime amendment at the beginning of establishment. Farmers generally applied an average of 200– 300 kg N ha − 1 year − 1 , 400 kg N ha − 1 year − 1 and 50 kg N ha−1 year−1 in UA, TP and BP, respectively, for 10 consecutive years immediately prior to data collection. Research areas had a typical subtropical climate, with a mean annual temperature of 18.4 °C and a mean annual precipitation of 1785 mm. At each site, four plots (~4 m × 4 m) were selected randomly, one soil sample was taken from each plot at a depth of 20 cm and four samples were pooled together and passed through a 2-mm sieve. Soil samples from each site were then divided into two parts, and half was stored at 4 °C for the incubation experiment, while the other half was airdried for measurement of soil properties such as pH, organic C and total N content. 2.2 Paired 15N-tracing experiment For each soil sample, two 15N-labelled treatments (i.e. 15Nlabelled NH4+ and 15N-labelled NO3−) were performed. Nitrogen solution was prepared as 15NH4NO3 (15N at 9.76 atom% excess) and NH415NO3 (15N at 9.74 atom% excess). For each soil sample, 24 flasks (250 ml) containing 20 g (oven-dry basis) fresh soil were prepared and 2 ml nitrogen solution was applied to a final concentration of 20 mg N kg−1 soil (oven-dry basis). Soils were incubated for 6 days at 60 % WHC and 25 °C. Gas samples from each treatment (three replicates) were collected at 6 h, and 2, 4 and 6 days after N application. Before each gas sampling, flasks were flushed with ambient air and resealed with stoppers for 6 h. A total of 37 ml of gas was collected from each flask and injected into two pre-evacuated vials (18.5 ml) to determine the N2O concentration (using an Agilent 7890 gas chromatograph) and isotopic composition (using a Thermo DELTA V PLUS mass spectrometer). After gas sampling, soil in each flask was extracted using 2 M KCl solution to determine the NH4+ and NO3− concentration and isotopic composition. After NH4+ and NO3− extraction, soil was washed using distilled water, oven-dried at 60 °C and the isotopic composition of the organic N was determined (Zhang et al. 2009).

(SKALAR). The isotopic composition was determined using a Delta V plus isotope mass spectrometer (Thermo) (Zhang et al. 2009). 2.4 Calculations In this study, we assumed that N2O originated from NH4+, NO3− and organic N pools via autotrophic nitrification, denitrification and heterotrophic nitrification, respectively. The contribution of each pathway to N2O emissions was determined using Eq. (1.1) and Eq. (1.2) (Rütting et al. 2010; Zhang et al. 2011). aN 20 ¼ dad þ na aa þ nh ah

ð1:1Þ

d þ na þ nh ¼ 1

ð1:2Þ

where aN2O is the 15N abundance of total N2O produced during incubation; d, na and nh are N2O emission fractions originating from the NO3− pool via denitrification, the NH4+ pool via autotrophic nitrification and the organic N pool via heterotrophic nitrification, respectively; ad, aa and ah are the 15N abundance of NO3−, NH4+ and organic N, respectively. Inserting 15N abundance values of N2O produced from NO3−, NH4+ and organic N in the paired 15NH4NO3 and NH415NO3 treatments into the equations allowed calculation of d, na and nh. N2O production rates originating from the NO3− pool via denitrification, the NH4+ pool via autotrophic nitrification and the organic N pool via heterotrophic nitrification were calculated using Eq. (2.1) – (2.3) as follows: N 2 Od ¼ d  N 2 Ot

ð2:1Þ

N 2 Oa ¼ na  N 2 Ot

ð2:2Þ

N 2 Oh ¼ nh  N 2 Ot

ð2:3Þ

where N2Ot is the total N2O emission rate from incubated soil. The net nitrification rate (mg kg−1 day) was calculated using Eq. (3). Net nitrification rate ¼ ðNO3 − final −NO3 − initial Þ=t

ð3Þ

where NO3−final is the NO3− concentration measured after 6 days, NO3− initial is the NO3− concentration measured at 6 h and t is the incubation time (days).

2.3 Soil property analysis

2.5 Statistical analysis

Soil total N and organic C were determined with a C/N element analyser (Sample preparation system, Europa EA-GSL). Soil pH was measured at a 1:2.5 (v/v) dry-soil to water ratio using a DMP-2 mv/pH detector (Quark Ltd., Nanjing, China) (Lu 2000). NH4+ and NO3− concentrations were measured using a SANplus ANALYZER plus continuous flow analyser

All statistical analyses were performed using SPSS software package 18.0 for windows. One-way ANOVA analysis and the least significant difference (LSD) method at a significance of p < 0.05 was used to compare differences in soil properties, and the source of N2O among the four different land use types. Data on the contributions of

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autotrophic nitrification, denitrification and heterotrophic nitrification to N2O production were transformed using arcsine prior to ANOVA and t test analysis. Redundancy analysis and correlation analysis were employed to investigate relationships between soil properties and N2O production pathways.

3 Results 3.1 Soil properties All soil pH values measured in the present study were less than 5, and the lowest pH was observed in WD (average = 4.5), while the highest (average = 4.9) was in UA (Table 1). Soil organic C (SOC) and total N in UA were significantly lower than those in WD, TP and BP (p < 0.05), whereas the C/N ratio in WD was significantly higher than in all three agricultural soils (p < 0.05; Table 1). There were no significant differences in C/N ratio among the three agricultural soils. The NH4+ concentration in UA was significantly lower than in WD, TP and BP (p < 0.05), but the NO3− concentration in TP and BP was significantly higher than in WD and UA (p < 0.05). Agricultural land use clearly affected the net nitrification rate in the studied soils (Table 1). Net nitrification in WD was significantly lower than in all three agricultural soils (p < 0.05), and was highest in TP (3.0 mg N kg−1 day), but was only 1.7 mg N kg−1 day in UA and BP.

3.2 N2O emission rate The average N 2 O emission rate was highest in TP (10.3 μg N kg −1 day) and considerably lower in UA (2.8 μg kg − 1 day), BP (2.1 μg kg − 1 day) and WD (1.4 μg kg−1 day; p < 0.05). There were no significant differences in N2O emission rate among UA, BP and WD (Fig. 1a). N2O emission was significantly correlated with soil NO3− concentration and net nitrification rates (Fig. 2a, b). Table 1

3.3 Sources of N2O The results revealed significant differences in the contribution of autotrophic nitrification, heterotrophic nitrification and denitrification to soil N2O production with different land use types (Fig. 1b, Fig. 3, and Figures in Supplementary Material). 15N enrichment of N2O was close to the natural abundance in the 15NH4+ treatment (Fig. 3a), suggesting autotrophic nitrification and oxidation of NH4+ via heterotrophic nitrification had minimal influence on N2O production in WD. Meanwhile, 15N enrichment of N2O was above the natural abundance in the 15NO3− treatment (Fig. 3b), suggesting denitrification played an important role in N2O production. However, denitrification alone could not explain N2O production in WD, and another N pool with a 15N abundance close to the natural abundance may be responsible for N2O production (Fig. 3a, b). In agricultural soils, 15N enrichment of N2O was significantly higher than the natural abundance, and close to the abundance of 15NO 3− in the 15 NO3− treatment (Fig. 3d, f, h), suggesting denitrification was largely responsible for N2O production, especially in TP (Fig. 3f) and BP (Fig. 3h). Autotrophic nitrification also appeared to have a significant effect on N2O production in agricultural soils, because 15N enrichment of N2O was higher than the natural abundance in the 15NH4+ treatments (Fig. 3c, e, g). Quantitative analysis showed that heterotrophic nitrification was the dominant N2O production pathway, accounting for 44.6 % of N 2 O emissions in WD (Fig. 1b). The relative contributions of autotrophic nitrification and denitrification to N2O production were 21.5 and 33.9 %, respectively, in WD. Meanwhile, denitrification was the dominant N2O production pathway in agricultural soils, and accounted for 72.8, 77.1 and 54.5 % of N2O production in TP, BP and UA, respectively. The contribution of heterotrophic nitrification to N2O production was minimal (less than 2.7 %) in all agricultural soils (Fig. 1b). The contribution of autotrophic nitrification ranged from 22.9 to 42.8 %, and was highest in UA, and significantly less in WD. Redundancy and

Soil properties and net nitrification rates in soil samples from different land use (average ± standard deviation) NO3− (mg N kg−1)

pH

SOC (g kg−1)

Total N (g N kg−1)

C/N ratio

NH4+ (mg N kg−1)

WD UA

4.5 ± 0.1b 4.9 ± 0.1a

32.1 ± 13.4a 8.1 ± 2.6b

1.5 ± 0.3a 0.9 ± 0.2b

21.2 ± 5.4a 8.5 ± 0.9b

7.1 ± 2.7a 0.3 ± 0.0b

4.3 ± 1.3b 7.9 ± 1.0b

0.1 ± 0.5c 1.7 ± 0.5b

TP BP

4.7 ± 0.1ab 4.8 ± 0.5ab

23.7 ± 2.2a 22.8 ± 9.2a

1.8 ± 0.4a 1.7 ± 0.7a

13.3 ± 1.5b 13.8 ± 0.3b

7.9 ± 4.4a 6.4 ± 3.7a

27.4 ± 8.8a 28.5 ± 11.1a

3.0 ± 0.5a 1.7 ± 1.3b

Soil type

Identical letters indicate no significant differences in average values WD woodland soil, UA upland agricultural soil, TP tea plantation soil, BP bamboo plantation soil, SOC soil organic C content

Net nitrification rate (mg N kg−1 day)

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12

100

Contribution to N2O (%)

N2Oa

8 6 4 2

( g N kg d )

12 a

-1

40

UA

TP

WD

BP

UA

BP

Our results showed that N2O production pathways are related to land use type in acidic soils in subtropical China. Heterotrophic nitrification was mainly responsible for N2O production in woodland soils (44.6 % of N2O), consistent with the results of Zhang et al. (2011b). Following conversion of woodland to agricultural soils, the contribution of heterotrophic nitrification to N2O production significantly decreased to less than 2.7 % (Fig. 1b), and the contribution from heterotrophic nitrification also decreased from 0.6 μg kg−1 day to less than 0.01 μg kg−1 day (Fig. 1a). Heterotrophic nitrification is known to play an important role in N2O production in acidic forest soils (Kreitinger et al. 1985; Killham 1990; Wood 1990; Huygens et al. 2007; Huygens et al. 2008; Zhang et al. 2011b; Stange et al. 2013), especially in forest soils with high SOC and C/N ratio (Zhang et al. 2011b). Our results also showed that the contribution of heterotrophic nitrification to N2O production was significantly correlated with SOC content and soil C/N ratio (Fig. 4; Fig. 5b, c). SOC rather than oxidation of reduced nitrogen compounds is the main energy source of 12 b

p<0.05

p<0.001

12 c

8

8

8

4

4

4

0 -1 0 1 2 3 4 Net nitrification rate -1 -1 (mg N kg d )

0

0

TP

4 Discussion

correlation analyses were applied to probe correlations between soil properties and N2O production pathways (Fig. 4). The results indicated that the contribution of autotrophic nitrification to N2O production was highly correlated with soil pH (p < 0.05), the contribution of heterotrophic nitrification was more closely related to soil organic C (p < 0.01) and C/N ratio (p < 0.01) and the contribution of denitrification to N2O was correlated with NO3− concentration (p < 0.01; Fig. 4). N2O production rates via denitrification (7.3 μg kg−1 day) and autotrophic nitrification (3.1 μg kg−1 day) in TP were significantly higher than those in UA, WD and BP (p < 0.01; Fig. 1a). The average N2O production rate via heterotrophic nitrification in WD was 0.6 μg kg−1 day, which was significantly higher than rates in all three agricultural soils (p < 0.01). N2O production from autotrophic nitrification was positively correlated with soil net nitrification (Fig. 5a), and production from heterotrophic nitrification was positively correlated with soil organic C and C/N ratio (Fig. 5b, c). N2O production rates from denitrification increased significantly with increasing NO3− concentration (Fig. 5d).

-1

60

0

WD

N2O average production rate

80

20

0

Fig. 2 Relationships between N2O average production rate and NO3− concentration (a), net nitrification rate (b) and the contribution of denitrification to N2O emissions (c). Cd is the contribution of denitrification to N2O emissions. Dashed curves correspond to the 95 % CI for linear regression

Denitrification Heterotrophic nitrification Autotrophic nitrification

b

N2Oh

-1

-1

(µg N k g d )

120

N2Od

a

10

N2O production rate

Fig. 1 N2O production rates (a) and production pathways (b) in soils from different land use. N2Oa, N2Oh and N2Od contributions to N2O emission rates via autotrophic nitrification, heterotrophic nitrification and denitrification, respectively. WD woodland soil, UA upland agricultural soil, TP tea plantation soil, BP bamboo plantation soil

0 10 20 30 40 50 -

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NO3 (mg N kg )

p<0.05

0 20 40 60 80 100 Cd (%)

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A to m % N e x c e s s

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NH4

NO3

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N2O

b

6

4

4 2

2

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9 c

6 d

6

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A to m % N e x c e s s

Fig. 3 15N enrichment of N2O, NH4+ and NO3− during incubation of soils from different land use. Only one representative for each land use type is included, and detailed results from all other soil samples are in the Electronic Supplementary Material. Figure 3a, c, e and g refer to the 15 NH4+ labelled treatment, and Fig. 3b, d, f and h refer to the 15 NO3− labelled treatment in woodland, upland agricultural, tea plantation and bamboo plantation soils, respectively. Symbols are means and error bars correspond to standard deviation

15

Atom % N excess

15

Atom % N excess

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heterotrophic microorganisms (Kuenen and Robertson 1994). Thus, abundant SOC is favourable for heterotrophic nitrification and N2O production via this process. Conversion of forest to agricultural soils, especially in upland sites, decreases SOC and C/N ratio, which in turn affects heterotrophic nitrification and N2O production via this process. However, Cai et al. (2010) and Chen et al. (2014) reported that heterotrophic nitrification may also play an important role in N2O production in agricultural soils in Northeast China. This could be explained by the high SOC concentration (87– 119 g kg−1) in these previous studies, which was much higher than that measured in soils in the present study (8.1– 32.1 g kg−1). This further supports our conclusion that SOC plays an important role in regulating the contribution of heterotrophic nitrification to N2O production.

6

7

-1

0

1 2 3 4 5 6 Incubation time (d)

7

In contrast to heterotrophic nitrification, the contribution of autotrophic nitrification to N2O production increased after conversion of woodland to agricultural soils, and was highest in upland agricultural soils and considerably lower in WD, consistent with our hypothesis (1) discussed above. Increasing soil pH by lime application, which is widely practised in subtropical China, is crucial for controlling the contribution of autotrophic nitrification to N2O production. NH3 levels are known to be very low in acidic soils (Wrage et al. 2005), which are not favourable for the growth and activity of ammonia-oxidising microbes (Hu et al. 2015). Increasing soil pH presumably favoured N2O production via autotrophic nitrification (Fig. 4), and was significantly higher in agricultural soils compared with woodland soils (Fig. 1a). In addition, N fertilisation can also stimulate the growth and activity of

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Fig. 4 Relationships between N2O production pathways and soil properties based on redundancy analysis. Ca is the contribution of autotrophic nitrification to N2O emissions, and Ch is the contribution of heterotrophic nitrification

ammonia-oxidising microbes and increase the contribution of autotrophic nitrification to N2O production via this process (Lu et al. 2012; Zhang et al. 2013), consistent with our observation of a correlation between N2O production via autotrophic nitrification and net nitrification rates (Fig. 5a). It was somewhat surprising that the contribution of denitrification to N2O production was above 50 % in all three agriculture soils under aerobic conditions. Cheng et al. (2015) reported that denitrification dominated N2O production in

1.5 -1

N2Oh ( g N kg d )

a

-1

-1

p<0.01

-1

N2Oa ( g N kg d )

4

2

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1.5

p<0.05 0.5 0.0

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N2Od ( g N kg d )

c

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20 30 40 -1 Organic C (g kg )

50

d

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-1

1.0

p<0.01

-1

-1

b

1.0

-0.5

0 2 4 -1 -1 Net nitrification rate (mg N kg d )

N 2 O h ( g N kg d )

Fig. 5 Relationships between N2O production rate via autotrophic nitrification and net nitrification rates (a), via heterotrophic nitrification and organic C concentration (b), via C:N ratio (c) and via denitrification and NO3− concentration (d). N2Oa N2O production rate via autotrophic nitrification, N2Oh N2O production rate via heterotrophic nitrification, N2Od N2O production rate via denitrification. Dashed curves correspond to the 95 % CI for linear regression

acidic soils, and a decrease in soil pH significantly increased the contribution of denitrification. There are two main explanations for why denitrification accounts for such a large proportion of N2O production under aerobic conditions in acidic soils. Fungi in acidic soils can perform both denitrification and O2 respiration simultaneously, unlike bacteria (Shoun et al. 1992; Laughlin and Stevens 2002; Firestone and Davidson 1989), and many fungi lack N2O reductase, and therefore produce N2O as a terminal product (Shoun et al. 1992, Wrage et al. 2001). An alternative explanation is that acidic soils favour chemodenitrification by which N2O is produced via the chemical decomposition of NH2OH or NO2− (Van Cleemput and Baert 1984; Medinets et al. 2015). Following conversion of woodland to agricultural soils, N2O production was increased, consistent with previous studies (Agustín et al. 2004; Lin et al. 2010; Petitjean et al. 2015). There was a significant positive correlation between N2O production and denitrification, suggesting this process plays the most important role in N2O emissions in aerobic conditions in subtropical acidic soils (Fig. 1c). Our results revealed a significant increase in the contribution of denitrification to N2O with increasing NO3− concentration (Fig. 4). It is widely accepted that nitrification is stimulated by the application of N fertiliser following conversion of woodland to agricultural soils (Zaman et al. 1999; Stockdale et al. 2002; Hall and Matson 2003; Lu et al. 2012), since this increases the amount of substrate available for denitrification. Moreover, higher net nitrification indicated enhanced microbial activity, which could stimulate greater consumption of O 2 and promote the

0.5 0.0

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p<0.01

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C:N ratio

30

35

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10 20 30 -1 NO3 (mg N kg )

40

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anaerobic conditions required for N2O production via denitrification (Lan et al. 2014). Thus, the contribution of denitrification to N2O production was significantly increased following conversion of woodland to agricultural soils.

5 Conclusions Agricultural land use significantly affected N2O production pathways in acidic soils in subtropical China. Heterotrophic nitrification was the dominant pathway of N2O production in woodland soils. However, after conversion of woodland to agricultural soils, the contribution from heterotrophic nitrification became negligible, mainly due a decrease in soil C/N ratio and organic C content. Denitrification dominated N2O production in agricultural soils under aerobic conditions. Our results showed that N2O production pathways can vary significantly in soils of different land use types, and soil pH, quantity and quality of organic C, and NO3− content are the main regulatory factors. These results are valuable for modelling and mitigation of N2O production in areas of different land use types in subtropical acidic soils in China. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (41571227), the B973^ Project (2014CB953803) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 164320H116).

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