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Title Nitrous and nitric oxide emissions from a cornfield and managed grassland : 11 years of continuous measurement withmanure and fertilizer applications, and land-use change

Author(s) Mukumbuta, Ikabongo; Shimizu, Mariko; Jin, Tao; Nagatake, Arata; Hata, Hiroshi; Kondo, Seiji; Kawai, Masahito;Hatano, Ryusuke

Citation Soil science and plant nutrition, 63(2), 185-199https://doi.org/10.1080/00380768.2017.1291265

Issue Date 2017-06

Doc URL http://hdl.handle.net/2115/70655

Rights This is an Accepted Manuscript of an article published by Taylor & Francis in Soil Science and Plant Nutrition on June2017, available online: http://www.tandfonline.com/10.1080/00380768.2017.1291265

Type article (author version)

File Information SSPN IKABONGO SSPN63(2).pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

https://eprints.lib.hokudai.ac.jp/dspace/about.en.jsp

For review

Nitrous and nitric oxide emissions from a cornfield and

managed grassland: 11 years of continuous measurement with manure and fertilizer applications, and land-use

change.

Journal: Soil Science and Plant Nutrition

Manuscript ID SSPN-16-116-F.R5

Manuscript Type: Full-length paper

Date Submitted by the Author: 26-Jan-2017

Complete List of Authors: Mukumbuta, Ikabongo; Hokkaido University, Soil Science Laboratory Shimizu, Mariko; Hokkaido University, Soil Science Laboratory Jin, Tao; Hokkaido University, Soil Science Laboratory Nagatake, Arata; Hokkaido University, Soil Science Laboratory Hata, Hiroshi; Hokkaido University, Field Science Center for Northern BIosphere Kondo, Seiji ; Hokkaido University, Field Science Center for Northern Biosphere

Kawai, Masahito; Hokkaido University, Field Science Center for Northern Biosphere HATANO, Ryusuke; Hokkaido University, Soil Science Laboratory

Keywords: global environment < Environment, soil biochemistry < Soil Biology

http://mc.manuscriptcentral.com/sspn

Soil Science and Plant Nutrition

For review

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Nitrous and nitric oxide emissions from a cornfield and managed grassland: 11 years of 1

continuous measurement with manure and fertilizer applications, and land-use change. 2

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Ikabongo Mukumbuta a*, Mariko Shimizu a, Tao Jin a, Arata Nagatake a, Hiroshi Hata b, Seiji 4

Kondo b, Masahito Kawai b, Ryusuke Hatano a 5

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a Soil Science Laboratory, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo, Hokkaido 7

060-8589, Japan. 8

b Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Hokkaido 9

060-0811, Japan. 10

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*Corresponding author email: [email protected]. 12

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Abstract 26

Changes in weather and management practices such as manure and fertilizer applications 27

have a major effect on nitrous oxide (N2O) and nitric oxide (NO) emissions from soils. N2O 28

and NO emissions exhibit high intra- and inter-annual fluctuations, which are also highly 29

influenced by land-use change. In this study we investigated how land-use change between 30

grassland and cornfield affects soil N2O and NO emissions using long-term field 31

measurements in a mollic andosol soil in Southern Hokkaido, Japan. Soil N2O and NO 32

emissions were monitored for 5 years in a 30-year old grassland (OG), which was then 33

ploughed and converted to a cornfield for 3 years and then converted back to grassland (new 34

grassland; NG) for another 3 years. We established four treatments plots; control, without 35

any nitrogen (N) input (CT plot), chemical fertilizer only (F plot), chemical fertilizer and 36

manure (MF plot), and manure only (M plot). 37

Changing land-use from OG to cornfield increased annual N2O emissions by 6-7 times, 38

while the change from cornfield to NG resulted in 0.3-0.6 times reduction in annual N2O 39

emissions. N2O emissions in the newly established grassland were 2-5 times higher than 40

those in the 30-year old grassland. Soil mineral N (NO3– and NH4

+) was higher in cornfield, 41

followed by NG and lowest in OG, while water extractable organic carbon (WEOC) did not 42

significantly change with changing land-use but tended to be higher in OG and NG than in 43

cornfield. The ratio of WEOC to soil NO3– was the most important explanatory variable for 44

differences in N2O emissions as land-use changed. High N input, surplus soil N, and 45

precipitation and low soil pH led to increased N2O emissions. N2O emissions in fertilizer 46

and/or manure-amended plots were 3-4, 2-5 and 1.4-2 times higher than those in the control 47

treatment in OG, cornfield and NG, respectively. NO emissions were largely influenced by 48

soil mineral N and N addition and showed less response to changing land-use. There were 49

high inter-annual variations in both NO and N2O emissions in all plots, including the control 50

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treatment, highlighting the need for long-term measurements when determining local 51

emission rates. 52

Keywords: N2O emission, grassland, cornfield, manure and fertilizer, land-use change. 53

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1. Introduction 76

Nitrous oxide (N2O) is an important greenhouse gas (IPCC 2007; UNEP 2013) and is 77

currently the most important substance emitted into the atmosphere causing the depletion of 78

the ozone layer (UNEP 2013), whereas nitric oxide (NO) is a highly reactive trace gas 79

important in atmospheric chemistry as it contributes to acid rain deposition and for its 80

regulation of photochemical production of ozone in the troposphere (Crutzen 1979; 81

Davidson et al. 1993; Eickenscheidt and Brumme 2013; Logan 1983). 82

The largest source of anthropogenic N2O emissions is agricultural soils, accounting for about 83

66% of gross anthropogenic emissions (UNEP 2013). N2O and NO emissions from soils and 84

agricultural systems are expected to increase further due to increased use of nitrogen (N) 85

fertilizers and manure to meet demand for increased food production (Ciais et al. 2013; FAO 86

2003; Mosier and Kroeze 2000; Smith et al. 2007; UNEP 2013; US-EPA 2006). Microbial 87

transformation of chemical N is an important source of both N2O and NO emission 88

(Medinets et al. 2015; Vitousek et al. 1997). While agricultural soils are not considered to be 89

the major source of NO globally, they are still very important sources especially when fossil 90

fuels are not considered (Bouwman et al. 2002). Tillage and manure application can increase 91

NO emission by up to 7 times and as high as 11% of applied fertilizer N can be emitted as 92

NO (Skiba et al. 1997). 93

It is generally accepted and widely reported that N2O and NO emission are increased by N 94

fertilization (Jin et al. 2010; Owen et al. 2015; Shimizu et al. 2013; Vanderzaag et al. 2011). 95

However, some studies have reported possible reductions in N2O emissions with improved 96

management of organic materials (Alluvione et al. 2010; Ryals and Silver 2012; UNEP 97

2013). Changing fertilizer type to those less susceptible to nitrification, timing of fertilization 98

and use of organic N sources could help mitigate NO emissions (Davidson et al. 1993; Skiba 99

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et al. 1997; Smith et al. 1997). There are still a lot of unknowns and high uncertainties in 100

estimating representative annual N2O and NO emissions from an individual site, as 101

emissions from the same site greatly vary year after year. 102

In grasslands, large amounts of N accumulate in plant biomass resulting in N rich organic 103

matter overtime (Davies et al. 2001; Shepherd et al. 2001; Velthof et al. 2010). When 104

grasslands are ploughed, there is increased soil available N, as this N is mineralized, 105

(Necpa lova et al. 2013; Whitehead et al. 1990) resulting in increased N losses through 106

leaching (Necpa lova et al. 2013; Whitehead et al. 1990) and N gas emissions (Oenema et 107

al. 2005; Smith et al. 2007; Smith and Conen 2004; UNEP 2013). Compared to grasslands, 108

croplands are ploughed annually, increasing the physical breakdown of soil structure and 109

organic matter, soil aeration and consequently leading to rapid microbial decomposition of 110

organic matter (Necpa lova et al. 2013; Ussiri and Lal 2009). Assessing how N2O and NO 111

emissions change when land use is changed back and forth between grassland and cropland 112

is important to fully understand the potential for mitigation of the emissions during the 113

transition from one land-use to the other. 114

Freezing and thawing can stimulate N2O and NO emissions (Burchill et al. 2014; Katayanagi 115

and Hatano 2012) by releasing carbon (C) and N through microbial lysis and through 116

physical entrapment and release during soil freezing and melting. However, the contribution 117

of winter and thawing periods to annual N2O and NO emissions, and its annual variation is 118

not well known. 119

Long-term field data on N2O and NO emissions is currently scarce (Tubiello et al. 2013). In 120

this study we report results of continuous monitoring of N2O emissions for 11 years 121

following manure and chemical fertilizer applications, combined with changing land-use. 122

While many studies have reported the effects of fertilizer, manure and land-use change on 123

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N2O emissions, very few, if any, have measured continuously the changes in the soil 124

properties and N2O emissions for as long as 11 years with changing land-use, covering a 125

permanent grassland, cornfield and a newly established grassland. The objectives were: (i) 126

To assess the effect of long-term manure and chemical fertilizer applications on N2O and NO 127

emissions; (ii) To investigate the effect of land-use change (from grassland to cornfield and 128

back) on N2O and NO emissions, (iii) to investigate the factors driving intra and inter-annual 129

variations in N2O and NO emissions, and (iv) to quantify the contribution of winter and 130

thawing periods to annual N2O and NO emissions. 131

2. Materials and methods 132

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2.1. Study site 134

This study was carried out at the Hokkaido University Shizunai experimental livestock farm 135

of the Field Science Center for Northern Biosphere in Shin-Hidaka city, Southern Hokkaido, 136

Japan (42°26’N, 142°29’E). The site is relatively cool in summer and cold in winter with 137

average annual air temperature and precipitation values of 8.1 ºC and 1252 mm respectively. 138

The soil surface is covered with snow from the end of December to the beginning of March. 139

The soil is derived from Tarumae (b) volcanic ash (Jin et al. 2010; Shimizu et al. 2010), and 140

is classified as Mollic Andosol (IUSS Working Group WRB 2006). 141

2.2. Field experimental designs and plot management 142

During the study period, land-use was an old grassland (OG) from 2005 to 2009, cornfield 143

(2010-2012) and newly established grassland (NG) (2013-2015). The old grassland had been 144

established more than 30 years prior to the beginning of this study in 2005. The dominant 145

grass species was reed canary grass (Phalaris arundinacea L.) and meadow foxtail 146

(Alopecurus pratensis L.) in OG, and timothy grass (Phleum pretense) in NG. 147

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The average amount of mineral fertilizer applied in OG before commencement of this study 148

was 133±36 kg N ha–1 year–1. From 1990 to 2004 the grassland was harvested for hay at least 149

twice a year. In September 2009 herbicide was applied and the field ploughed in December. 150

Three treatments plots namely; (i) control without N addition (CT plot), (ii) chemical N 151

fertilizer only (F plot), and (iii) Chemical N fertilizer and composted beef cattle manure (MF 152

plot) were set up in 2005. In 2011, a fourth plot with composted beef cattle manure only (M 153

plot) was added. Each plot was 5x5 m in size and all the treatment plots were replicated four 154

times and arranged as shown in Figure S1. The treatment plots for this study were set up 155

within a large 2-hectare field as shown in Figure S1 as previously described by Shimizu et al. 156

(2010). 157

Table 1 shows the timing of fertilizer and manure applications, and other management 158

practices. The type of chemical fertilizer was ammonium sulfate and the manure was 159

composited beef cattle manure with bedding litter (bark). The gross manure N and fertilizer 160

N application rates were as shown in Table 2. Lime was applied in all the plots from 2008 to 161

2015 at an average rate of 400 kg CaCO3 ha–1year–1. 162

2.3. Soil and weather measurements 163

164

Soil samples were collected at 5 cm depth during each sampling day from April to 165

November (non freezing period) in all treatment plots. Soil samples were sieved (2 mm 166

sieve) and extracted in deionized water or in 2 M KCl solution, and the extracts stored at 4°C 167

until analysis for dissolved nutrients after being filtered through 0.2 µm membrane filters. 168

From the water extracts; Soil NO3- concentrations were analyzed by ion chromatography 169

(Dionex QIC Analyzer; Dionex Japan, Osaka, Japan); soil pH was measured by using a 170

combined electrode pH meter (F-8 pH meter; Horiba, Kyoto, Japan); and water extractable 171

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organic carbon (WEOC) was measured using a total organic carbon (TOC) analyzer (TOC 172

5000A; Shimadzu, Japan). NH4+-N in the 2 M KCl extract solution was determined using the 173

indophenol- blue method (UV mini 1240; Shimadzu, Kyoto, Japan). 174

In the OG and NG, soil moisture was measured at 0–6 cm depth using the Frequency 175

Domain Reflectometry (FDR) method (DIK- 311A; Daiki, Saitama, Japan). Calibration 176

curves were made to calculate water–filled pore space (WFPS) from the FDR device reading 177

and percent total porosity (Jin et al. 2010; Linn and Doran 1984). In the cornfield, soil 178

moisture content was measured gravimetrically from soil samples collected at a depth of 0–5 179

cm. 180

Daily precipitation and air temperature were obtained from the nearest Automated 181

Meteorological Data Acquisition System (AMEDAS) station of the Japan Meteorological 182

Agency. Thermocouple thermometers (TR-52, T&D, Nagano, Japan) were permanently 183

installed in each plot to measure soil temperature at 5 cm depth at 30-minute intervals. On 184

each sampling day air temperature inside the chamber and soil temperature (5 cm depth) 185

were measured using a hand-held thermometer (CT220; CUSTOM, Tokyo, Japan). 186

2.4. Gas flux sampling and measurement 187

N2O and NO fluxes were measured using static closed chambers. The chambers were made 188

of stainless steel and were 20 cm in diameter and 25 cm in height in the cornfield, and 40 cm 189

wide and 30 cm high in OG and NG. Detailed information of the chambers was as reported 190

by Toma and Hatano (2007). The chambers were placed onto chamber bases, which were 191

installed permanently during the measurement period to a depth of 5 cm. Chamber bases 192

could not be used in winter, therefore chambers were inserted directly to 5 cm depth a day 193

before measurements. We did not remove the snow during winter measurements. After each 194

sampling the chambers were removed from the bases. 195

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Gas samples were taken between 8:00 am and 12:00 pm on each sampling day using a gas 196

tight syringe through a three-way valve fitted onto the chamber cover. The normal sampling 197

frequency was once or twice every fortnight, except in winter when sampling was conducted 198

once or twice every month. A more intensive sampling regime of every two to five days was 199

carried out after fertilization and other events that are known to stimulate gas flux. Gas 200

samples from the headspace of each chamber were collected into pre-vacuumed Tedlar bags 201

for NO analysis or a 20-mL vial bottle for N2O. Samples were taken at 0 and 30 minutes in 202

OG, 0 and 20 minutes in cornfield and 0, 15 and 30 minutes in NG after chamber closure. To 203

check the accuracy of flux calculated using only two headspace concentrations, we compared 204

the slope of the change of N2O concentration inside the chamber with time using the three 205

headspace concentrations (at 0, 15, and 30 min) and using two headspace concentrations (at 206

0 and 30 min) for all chambers in the 2013–2015 period (n=772). The results showed that the 207

slopes from the three and two headspace concentrations had a 1:1 linear relationship 208

(R2=0.9997). We then compared the slopes of three and two headspace concentrations when 209

N2O was low (below the median), high (above the median) and the whole data set, and there 210

was no significant difference among the three regression lines (F=0.0018, p=0.9981). This 211

result means flux from the two headspace concentrations could be used for treatment 212

comparisons (Stolk et al. 2009; De Klein and Harvey 2015; De Klein et al. 2003). 213

NO gas concentrations were analyzed in the laboratory within the same day of sampling 214

using a nitrogen oxides (NOx) analyzer (Model 265P; Kimoto Electric, Osaka, Japan). N2O 215

gas concentrations were analyzed within three months using a gas chromatograph fitted with 216

an electron capture detector (Model GC-14B; Shimadzu, Kyoto, Japan). NO and N2O 217

concentrations in the samples were calculated using calibration curves made by standard 218

gases. The concentrations of standards gases used were 0.3, 0.6 0.9, 2.8, 6.2, 9.3 and 30.9 219

ppm for N2O, and 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1 and 2 ppm for NO. 220

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221

The gas flux from the soil was calculated using the following linear regression equation 222

(Katayanagi and Hatano 2012). 223

F = ρ × V/A ×∆c/∆t × [273/(273 + T)] × α [Equation 1] 224

ｗhere F is the gas flux in µg m–2 hr–1; ρ is the density of each gas at standard conditions 225

(N2O = 1.97 × 106 mg m–3, and NO = 1.34 × 106 mg m–3); V is the volume of the chamber 226

(m3), A is the surface area of the chamber (m2); ∆c/∆t (10–6 m3 m–3 h–1) is the ratio of change 227

in gas concentration in the chamber during the sampling time; T is the air temperature inside 228

the chamber (°C); and α is ratio of molar mass of N of the molecular weight of each 229

respective gas. 230

Cumulative annual emissions were calculated by linear interpolation between sampling 231

events and numerical integration of underlying area using the trapezoid rule (Whittaker and 232

Robinson 1967; Ussiri et al. 2009). Winter period was defined as the period from Mid-233

December, when maximum soil temperature fell below 5oC, to the end of February when 234

maximum temperatures recorded reached 0 oC. The thawing period was defined as the period 235

when minimum daily temperatures reached 0 oC, to the time when soils were completely 236

melted (minimum soil temperatures ~5 oC) (Katayanagi and Hatano 2012; Kurganova et a. 237

2007). 238

2.5. Heterotrophic soil respiration and estimation of mineralized N 239

Heterotrophic respiration (RH) was measured as carbon dioxide (CO2) emission from bare 240

soil (plant and root excluded soil) as described by Limin et al. (2015). Bare plots were 241

established as described by Shimizu et al. (2009). Briefly, the aboveground plants and root 242

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were removed, and a root-proofing sheet (BKS9812; TOYOBO, Osaka, Japan) was 243

vertically inserted from soil surface until 30 cm depth to inhibit regrowth of roots. 244

RH was measured using the closed chamber method as described in section 2.4. RH was 245

measured in the CT plot from 2005 to 2009, and in all plots from 2010 to 2015. RH in CT 246

and F plots was regarded as heterotrophic respiration from soil organic matter decomposition 247

(RHs), while RH from manure amended plots included RHs and heterotrophic respiration 248

from manure decomposition (RHm). Therefore, RHm in MF was estimated by subtracting 249

the RH from F plot, while in M plot by subtracting the RH from CT plot. From 2005 to 2009, 250

RHm was calculated as the difference in total CO2 emissions in planted plots between MF 251

and F plots (Li et al. 2015; Shimizu et al. 2015). 252

The total mineralized N was calculated as the sum of soil organic matter N and manure N 253

mineralization. The mineralized N from soil organic matter and manure was calculated by 254

dividing RHs and RHm by the soil and manure C/N ratios, respectively. 255

2.6. Plant N uptake, total N input and soil surplus N 256

Net primary production (NPP) was measured as the net increase in plant biomass 257

(aboveground and belowground biomass) annually (Shimizu et al. 2015). 258

In grassland the plant biomass was collected four times in a year in April, June, August and 259

October as described by Shimizu et al. (2009). The aboveground biomass was manually 260

harvested by cutting all the plant biomass within a 0.5 m × 0.5 m quadrate. Two 261

aboveground samples were collected and averaged for each of the four treatment replicates 262

during each sampling event. The belowground biomass was measured by taking a soil block 263

(0.25 x 0.25 x 30 cm) at each of the 4 replications, from the same points where the 264

aboveground biomass was collected, and then manually separating the roots from the soil. 265

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In cornfield the plant biomass was collected once a year at the end of the growing season just 266

before harvesting. For each of the four treatment replications, corn plants within a 1.5 m x 1 267

m area were collected by uprooting them (by digging) to 30 cm depth to include all the roots 268

for each plant. 269

Plant roots were washed in water using a 0.5 mm sieve to completely remove the soil 270

particles and other debris. The plant samples were oven–dried at 70 ˚C for more than 72 271

hours and weighed. Each dried sample was analysed for total carbon (C) and N contents with 272

N/C analyzer (SUMIGRAPH NC–1000, Sumika Chemical Analysis Service, Ltd., Osaka, 273

Japan). 274

Surplus soil N was calculated as the difference between total N input (sum of soil and 275

manure mineralized N and chemical fertilizer N) and plant N uptake. 276

277

2.7. Data analysis 278

Statistical analysis was done using STATA-13 (Stata corporation, Texas, USA). Two-way 279

analysis of variance (ANOVA) was used to evaluate the differences in annual fluxes across 280

years and treatments within each land-use. One-way ANOVA was used to assess the 281

differences in annual N2O emissions and chemical properties among the land-uses for each 282

treatment. Annual N2O and NO data was natural log transformed [y = log (x + 1)] before 283

analysis of variance. The value of one was added to prevent generation of negative log 284

transformed values. 285

Pearson’s correlation test was used to test the relationship between weather and soil variables 286

with N2O fluxes and cumulative annual emissions. Step-wise single and multiple regression 287

analyses were used to explain the influence of soil and environmental variables on annual 288

N2O and NO emissions. 289

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290

3. Results 291

3.1 Soil and weather variables 292

Mean annual air temperatures were within long-term normal values for most of the years 293

during this study except for 2007, which recorded 0.7 ºC higher than the long-term average 294

value of 8.2 ºC. 2005 was the coolest as well as the driest year (8.0 ºC, 999 mm). 2009, 2010, 295

2011, and 2013 were wetter than average with annual precipitation at least 200 mm higher 296

than the 30-year average of 1252 mm. 297

Soil nitrate (NO3–) and ammonium (NH4

+) concentrations were significantly higher in 298

cornfield than grassland and higher in NG than OG (p<0.01) (Fig. 1). NO3– significantly 299

increased in 2010 after converting grassland to cornfield and decreased slightly in 2011 and 300

2012. In the first year after conversion from grassland to cornfield, NO3––N concentration in 301

control plot (without N addition) increased from an average of 1 mg kg–1 to 60 mg kg–1, but 302

decreased to 12 mg kg–1 by the third year of the cornfield (Fig. 1) and reduced further in the 303

new grassland. NH4+ concentration on the other hand did not increase in the first year of 304

cornfield but showed high values in 2012, the third year of cornfield. Soil NO3– and NH4

+ 305

concentrations were higher in chemical fertilizer amended plots (MF and F) compared to CT 306

and M plots in all three land-uses throughout the study period and always lowest in the 307

control treatment. Peaks of both soil NO3– and NH4

+ concentrations were observed following 308

chemical fertilizer applications in spring and short-lived peaks in NO3– concentrations were 309

sometimes observed after manure application. 310

Water extractable organic carbon (WEOC) did not change much with changing land-use but 311

tended to be higher in OG and NG compared to cornfield. Water extractable organic carbon 312

was significantly lower in 2010, the first year of conversion from grassland to cornfield, and 313

increased annually in the 3 years of cornfield. Water extractable organic carbon was higher 314

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in the manure-amended plots (MF and M) than the plots without manure application 315

(p<0.01). 316

The ratio of WEOC to NO3– was highest in OG, followed by NG and lowest in cornfield in 317

all the plots. 318

Soil pH was always lower in F plot compared to MF, M and CT plots (p<0.001). Soil pH in 319

MF and M (long term manure application) was higher than in CT plot. Soil pH in all plots 320

increased annually from 2008 due to liming. 321

322

3.2 Temporal variations of N2O fluxes 323

Nitrous oxide fluxes were very episodic and displayed high variations within and across 324

years throughout the study period (Fig 2). Intra-annual variations were highly influenced by 325

mean daily temperature and precipitation. 326

The timing when the highest fluxes were found was different depending on the land-use and 327

fertilizer application. In OG, the highest fluxes in MF plot; 275.5, 1290.6, 140.3, and 93.5 µg 328

N2O-N m–2hr–1 were found on May 20th, 11th, 29th, and 18th in 2005, 2006, 2007 and 2009 329

respectively. All these followed combined chemical fertilizer and manure applications in 330

spring, except in 2008 when the highest flux (71.7 µg N2O-N m–2hr–1) was found on July 14th 331

after the second fertilizer application. In F plot the highest fluxes; 313.8, 211.1, 206.8, 175.7 332

and 333.8 µg N2O-N m–2hr–1 were found after the second fertilization on 18th, 15th, 18th, 7th 333

July and 25th June in 2005, 2006, 2007, 2008 and 2009 respectively. This was despite the 334

lower N application rate in the second application compared to the first one in May. In the 335

control plot, highest fluxes in OG; 50.3, 66.2, 114.5, 22.8 and 30.4 µg N2O-N m–2hr–1 in 336

2005, 2006, 2007, 2008 and 2009 respectively were always found between July and August, 337

and were all preceded by cumulative precipitation of more than 40 mm within 7 days before 338

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sampling. In cornfield (2010–2012) the highest fluxes in all the treatment plots were found in 339

either June or July and were preceded by high precipitation. The highest fluxes in cornfield 340

ranged from 223.7 to 638.4 µg N2O-N m–2hr–1 in control plot, 822.1 to 2461.4 µg N2O-N m–341

2hr–1 in F plot, and 527.4 to 2223.5 µg N2O-N m–2hr–1 in MF plot. 342

In 2013, the first year of new grassland (but before it was well established), 343

disproportionately high fluxes (713.8, 871.0, 2260.9 and 1359.2 µg N2O–N m–2 hr–1 in CT, 344

F, MF and M plot respectively) were found on 18th September two days after very high 345

precipitation (97mm in one day) on 16th September. On June 5 and 20 in 2013, 54.3 and 40 346

mm rainfall was recorded and high fluxes were found for samples collected within 5 days. 347

Precipitation higher than 40 mm per day was recorded at least 6 times in 2014 and 2015 but 348

the fluxes were relatively low. 349

In all the plots winter N2O emissions were very low throughout the study. High fluxes during 350

the thawing period were found in all plots throughout the study period. 351

Nitrous oxide fluxes were highest in the cornfield, followed by NG and lowest in OG (Fig. 2 352

and Table 3). Nitrous oxide fluxes in chemical fertilizer-amended plots (F, MF) were higher 353

than those without chemical fertilizer application (p<0.01). The manure only plot tended to 354

have higher emissions than the control plot. 355

356

Inter-annual variations in cumulative N2O emissions were more pronounced in cornfield and 357

NG than OG (Table 3). Annual N2O emissions were lower in OG, followed by NG and 358

highest in cornfield (Table 3). Averaged over the entire study period for each land-use and 359

compared within each treatment, annual N2O emissions in cornfield were 6-7 times higher 360

than in the OG (p<0.001) and 1.5-3 times higher than NG (Table 3). The emissions in NG 361

were 2-5 times higher than those in OG (p<0.001). 362

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The emissions in the cornfield were highest in the first year after conversion from grassland 363

(2010) and lowest in the third year. After conversion from cornfield to NG, the annual 364

emissions reduced slightly in 2013 (first year of conversion), but by the second and third 365

year after conversion, the emissions in NG were significantly lower than in the cornfield. 366

Within each land-use type, there were significant differences in annual N2O emissions 367

among plots and among the years (p<0.01). 368

369

Contribution of winter and thawing periods to annual emissions 370

In grassland (both OG and NG) contributions of winter N2O emissions to annual emissions 371

ranged from 0–7% in all plots except for 2008 where winter emissions in CT plot accounted 372

for 25% and 2015 where cumulative winter emissions in F and CT plots were negative 373

(Table 4). In cornfield, winter emissions in CT and F plots contributed 2–18%, while in the 374

manure-amended plots, winter emissions contributed as high as 35% to the total annual 375

emissions. 376

The thawing period tended to have a higher contribution to annual emissions in the 377

unfertilized control treatments (Table 5). In 2014, the thawing period accounted for more 378

than 45% of total annual emissions in all plots. 379

380

3.3 Temporal variations of NO fluxes 381

Intra-annual variations of NO fluxes showed a similar trend with N2O fluxes. However, the 382

NO fluxes were very low throughout the study period with only the MF plot showing higher 383

values (Fig. 3). The highest NO fluxes were always found after fertilizer and manure 384

applications. 385

Annual NO emissions were higher in MF and F plots and lowest in the control plots 386

(p<0.05). Annual NO emissions ranged from 0.01-0.18, 0.03-0.65, -0.16-1.8 and -0.01-0.66 387

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kg N ha–1 in CT, F, MF and M plots respectively. There was no significant difference in 388

annual NO emissions between the grassland and cornfield. 389

During winter and thawing periods NO fluxes were generally low and varied widely. Winter 390

and thawing period NO fluxes in CT plot contributed more to annual emissions compared to 391

MF and F plots. The highest contributions of winter and thawing seasons to annual NO 392

emissions were 55% and 32% respectively in CT plot (Table S1, S2). 393

394

3.4 N2O-N/NO-N ratio 395

The ratio of N2O-N to NO-N (N2O-N/NO-N) is used an indicator of the dominant 396

mechanism of N2O production in the soil. If the ratio is less than 1, nitrification is the 397

mechanism of N2O production, if greater that 100 denitrification is the main mechanism 398

(Bouwman 1990). In OG, 2.4%, 79.2% and 18.4% of the N2O-N/NO-N ratio values were 399

less than 1, between 1-100 and greater than 100, respectively. In cornfield and NG, less than 400

1% (0.7% and 0.9%, respectively) of the N2O-N/NO-N values were less than 1. About 401

69.6% and 64.5 % of the N2O-N/NO-N values were between 1-100 and 29.7% and 34.5 were 402

greater than 100 in cornfield and NG, respectively. Nitrous oxide flux increased with 403

increasing N2O-N/NO-N values in all plots and land-uses combined. 404

405

3.5 Factors controlling N2O and NO emissions 406

Daily N2O fluxes were influenced by soil temperature, precipitation, soil pH, moisture 407

content, and N supply. In OG, the instantaneous N2O fluxes had significant positive 408

correlations with soil temperature (p<0.001), NO3– concentration (p<0.01) and NH4

+ 409

concentration (p<0.001) and non-significant negative correlations with WFPS, soil pH and 410

WEOC. In the cornfield, correlations were positive with NO3– (ns) and soil temperature 411

(p<0.001), and negative but non-significant with NH4+, WFPS, pH and WEOC. In NG, N2O 412

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correlated positively with soil temperature, NH4+ concentration and NO3

– (p<0.01), and 413

negatively with soil pH (p<0.05), WEOC (p<0.05) and WFPS (ns). 414

Annual N2O emissions, in all three land-uses, increased with total N input and surplus N in 415

the soil (p<0.05). Annual precipitation had a significant positive linear correlation with 416

annual N2O emission in cornfield and an exponential relationship in NG (Fig. 4). Soil pH 417

showed a negative correlation with annual N2O emission, but it was significant only in 418

cornfield (Fig. 5). However, the ratio of surplus N emitted as N2O (N2O-N/surplus N) had a 419

stronger negative correlation with soil pH in all three land-uses (Fig. 5). 420

The ratio of WEOC to soil NO3– (WEOC/ NO3

–) was the major driver of changing N2O 421

emission as the land-use changed (Fig. 6). The WEOC/ NO3– ratio explained 78% of changes 422

in annual N2O emission as land-use changed in the control plot (Table 6). 423

Nitric oxide fluxes only showed significant correlations with WFPS (negative) in all 424

treatments and with soil NO3– and NH4

+ (positive) in F and MF plots (p<0.05). N addition 425

was the one most important factor affecting annual NO emissions. 426

427

3.6 Heterotrophic soil respiration (RH), mineralized N, plant N uptake and surplus 428

N. 429

Total RH and total mineralized N were higher in manure-amended plots than F and CT plots, 430

and higher in cornfield than OG and NG (p<0.05) (Table S3). Plant N uptake in OG and 431

cornfield was not statistically different, but was higher than in NG (p<0.01) (Table S4). 432

Surplus soil N in cornfield was higher than in both OG and NG (p<0.01), and higher in NG 433

than OG (p<0.05) (Table S4). Chemical fertilization significantly increased plant N uptake 434

(p<0.05) 435

436

437

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4. Discussion 438

4.1 Temporal variation in N2O and NO emissions 439

The N2O fluxes in this study were highly variable and peak emissions occurred either after N 440

addition or after high rainfall. In OG and last two years of NG, all peak emissions occurred 441

after N addition, with less influence of rainfall. In cornfield and first year of NG rainfall had 442

a larger impact on peak emissions than N addition. These differences in the response of peak 443

N2O fluxes among the three land-uses were assumably due to differences in soil mineral N 444

content, aeration and redox conditions. High NO3– content in cornfield and in first year of 445

NG provided substrate for denitrifiers while high precipitation created favourable conditions 446

for denitrification. Occurrence of high rainfall when WEOC/NO3– ratio was high, in OG and 447

last two years of NG, would have favoured complete denitrification to N2 gas and hence less 448

N2O fluxes (Burchill et al. 2014; Iqbal et al. 2015). 449

450

In OG N2O peaks following N application were higher and lasted longer after the second 451

fertilization in summer compared to the first application in spring in F plot. In MF and M 452

plots the peaks were higher in spring when both manure and chemical fertilizer were applied. 453

Manure applications enhance microbial activity, which reduces soil O2 levels, creating 454

conditions that favour N2O emissions (Collins et al. 2011; Zhang et al. 2014), which could 455

explain the observed differences between F and manure plots. 456

The timing of peak NO fluxes were similar to those of N2O despite being much smaller in 457

magnitude (Fig. 3), which should be expected as both gases are mainly the products of 458

nitrification and denitrification processes and are driven by similar abiotic factors (Davidson 459

et al. 1993; Medinets et al. 2015; Yan et al. 2013; Skiba et al. 1997). Smaller peak NO fluxes 460

relative to N2O is in agreement with results reported by Wang et al. (2011), Yan et al. (2013) 461

and Zhu et al. (2013). In this study, the peak N2O-N fluxes were up to 200 times higher than 462

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peak NO-N fluxes which is significantly higher than those reported by Wang et al. (2011). 463

However, higher peak N2O than NO fluxes found in this study are contrary to results from 464

other studies (Akiyama and Tsuruta 2002; Akiyama et al. 2000; Smith et al. 1997) which 465

reported up to 20 times more NO-N than N2O-N. This contradiction among difference 466

studies could be due to differences in soil moisture and fertilizer types (Smith et al. 1997; 467

Akiyama et al. 2000). When WFPS is greater than 60%, denitrification, which produces 468

more N2O than NO, is predominant (Davidson et al. 1993; Smith et al. 1997) and diffusion 469

of NO is limited which allows further consumption of NO by denitrification (Skiba et al. 470

1997; Smith et al. 1997). The average WFPS value in this study was above 70%. 471

472

Few studies have reported long-term data of N2O and NO emissions. There was up to a 10-473

fold difference in inter-annual N2O emissions within each land-use and treatment in this 474

study. Differences in annual NO emissions were as high as 6 times. This high variation in 475

annual emissions emphasises the need for long-term studies to reduce uncertainties 476

associated with chamber flux measurements for individual sites. 477

478

4.2 Influence of N application on N2O and NO emissions 479

The N2O emissions in fertilizer and manure-amended plots were 3-4, 2-5 and 1.4-2 times 480

higher than in the control treatment in OG, cornfield and NG, respectively (Fig. 2 and Table 481

3). These results are similar to those of Mosier et al. (1991) who reported an increase of 2-3 482

times in N2O emission due to fertilization in native grassland and wheat prairies in the USA. 483

Several studies have reported increased N2O emission with manure and fertilizer applications 484

(Alluvione et al. 2010; Collins et al. 2011; Mu et al. 2006; Ryals and Silver 2012; Zhang et 485

al. 2014). Manure applications enhance microbial activity, which reduces soil O2 levels, 486

creating conditions that favour N2O emissions (Collins et al. 2011; Zhang et al. 2014). 487

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Our results indicate that soil organic matter mineralization and plant N uptake are important 488

parameters affecting N2O-N emissions as shown by significant positive relationship between 489

N2O emissions and surplus N and total N input. Therefore soil organic matter decomposition 490

and plant type should be included when evaluating the emission factors of different soils. 491

Chemical fertilizer and long-term manure application had a significant influence on soil 492

properties such as pH, mineral N content and organic carbon content (Fig. 1). Soil pH was 493

significantly decreased by chemical fertilizer application and increased by long-term manure 494

application. Manure application increased and maintained soil pH probably due to the high 495

pH of the manure (manure pH was around 7). The second reason is that manure increases the 496

buffering capacity of soils due to the presence of carboxyl and phenolic hydroxyl groups in 497

the manure (Whalen et al. 2000). The negative relationship between pH and N2O emission 498

(Fig. 5) suggests that under similar conditions, long-term manure could have benefits of 499

reducing N2O emissions indirectly by increasing soil pH, while the opposite is true for 500

chemical fertilizer. 501

Nitric oxide fluxes were stimulated just after fertilization similar to many published reports 502

(Akiyama and Tsuruta 2002; Bouwman et al. 2002; Cui et al. 2012; Skiba et al. 1997). 503

Although annual NO emissions were higher in inorganic N fertilized plots, regression 504

analysis showed a non-significant increase in annual NO emissions with increasing N input, 505

which disagrees with other studies (Cui et al. 2012; Yan et al. 2013) that have reported a 506

significant linear relation between annual NO emissions and fertilizer N input. One possible 507

explanation for this seemingly non-significant response of annual NO emissions N input is 508

that high moisture content in our site limited the diffusion of NO to the surface (Firestone 509

and Davidson 1989; Medinets et al. 2015; Skiba et al. 1997) which in turn increases the 510

likelihood of NO consumption in the soil by denitrification (Akiyama and Tsuruta 2003; 511

Aneja et al. 1996; Pilegaard 2013; Yao et al. 2010). 512

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513

4.3 Soil and environmental factors controlling N2O emissions 514

As expected, total N input and surplus N, NO3- and NH4

+ concentrations in the soil were 515

important controlling factors. In cornfield and NG in 2013, highest N2O fluxes were 516

recorded following rainfall higher than 40 mm in one day. Other factors such as tillage 517

(Chapin et. 2011; Li et al. 2015), oxygen availability (Firestone and Davidson 1989; Igbal et 518

al. 2014; Venterea et al. 2005) and precipitation (Koga et al. 2004) are more important when 519

inorganic N is not limiting in the soil, and hence were very important factors in cornfield. In 520

this study, the higher soil mineral N content (both NO3- and NH4

+) in cornfield and NG even 521

in the control treatment without any N addition, could have been due to enhanced 522

mineralization resulting from tillage (Shimizu et al. 2013). The higher heterotrophic 523

respiration values observed in cornfield and NG compared to OG supports this claim (Table 524

S3). 525

Effects of soil moisture and rainfall on N2O production have been reported by many studies 526

(Alluvione et al. 2010; Choudhary et al. 2001; Mosier et al. 1991; Sehy et al. 2003). High 527

N2O fluxes associated with high soil moisture were likely to have come primarily from 528

denitrification (Alluvione et al. 2010; Sehy et al. 2003; Shimizu et al. 2013). Precipitation 529

enhanced N2O emission due to stimulation of substrate diffusivity and microbial activity 530

with increased soil moisture content (Bateman and Baggs 2005; Kusa et al. 2002), reduced 531

oxygen diffusivity (Saggar et al. 2013) and the resulting increase in denitrification (Li et al. 532

2015; Saggar et al. 2013). A negative but non-significant correlation between annual N2O 533

emissions and precipitation in OG was found. In 2009, when the highest rainfall was 534

recorded in OG, N2O emissions were very low. This could be due to lower total N input 535

(Table 2) and surplus N (Table S4) and also lower NO3– and NH4

+ concentrations in OG 536

(Fig. 1). Another reason could be that high rainfall in grassland, given the limited drainage in 537

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our site and high available carbon relative to NO3– (Fig. 1), might have promoted complete 538

denitrification (Burchill et al. 2014; Iqbal et al. 2015). 539

The amount of surplus N emitted as N2O (N2O-N/surplus N) had a much stronger negative 540

correlation with soil pH than just N2O-N and pH in all three land-uses (Fig. 5). These results 541

suggest that it’s the excess (surplus) N in the soil that is much more influenced by soil 542

conditions and transformed to N2O. This is supported by a significant positive correlation 543

between N2O emissions and surplus N. A negative relationship between N2O and soil pH 544

has been reported by a number of studies (Clough et al. 2004; Pan et al. 2012). Increased 545

activity of N2O reductase enzyme relative to activities of NO3- and NO2

- reductase enzymes 546

at high pH may be the main reason for the low N2O at high pH (Pan et al. 2012). However, 547

this result is contrary to the increased cumulative N2O production with increasing pH in 548

grassland and forest soils in Canada reported by Cheng et al. (2013). 549

Multiple regression analysis showed that soil moisture and NH4+ concentration were the key 550

factors regulating NO fluxes, although NO fluxes showed strong positive correlation with 551

temperature and NO3– concentration as single factors. The negative correlation of NO with 552

WFPS is consistent with the reported impediment of the diffusion of NO at high moisture 553

content and thereby allowing NO consumption (Davidson et al. 1993; Medinets et al. 2015). 554

The fact that NH4+ showed a stronger controlling effect on NO than NO3

– agrees with reports 555

that nitrification was the major source of the NO fluxes (Cui et al. 2012). However, Skiba et 556

al. (1997) reported that denitrification produces more NO than nitrification but net release of 557

NO from denitrification is lower due to impediment of NO diffusivity and NO consumption 558

by denitrifiers. 559

560

561

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4.4 Importance of winter and thawing periods N2O and NO emissions. 562

Winter emissions contributed as high as 35% and 55% in N2O and NO emissions 563

respectively (Table 4, S3). Contribution of winter N2O emissions was higher when manure 564

was applied in autumn in cornfield compared to spring in grassland. Winter sampling was 565

done twice or once a month and therefore these values might have been underestimated. 566

However this study clearly shows that winter emissions contribute a significant amount to 567

annual emissions and this calls for more intensive sampling and inclusion of winter 568

emissions in annual budgets. 569

The two-months long thawing period (March to early May) contributed as high as 60% to 570

annual emissions in some years (Table 5, S2). In the control plots, thawing period emissions 571

were even more important compared to the other plots. The N2O emissions increased 572

following soil melting and as soil temperatures became warmer. The high fluxes in this period 573

could be due to high accumulation of N2O through denitrification during freezing period and the 574

physical release as the snow melts (Burchill et al. 2014) and low N2O reduction rate during 575

thawing (Katayanagi and Hatano 2012; Sehy et al. 2003). Peaks of N2O emissions in the 576

thawing period may also be due to enhanced mineralization of easily decomposable organic 577

substrates by increased microbial activity (Wu et al. 2010). 578

579

4.5 Effect of land-use type on N2O and NO emissions 580

In this study, the average annual N2O emissions in grassland (OG and NG) ranged from 0.4 581

to 4.9 kg N ha–1yr–1 except in 2013 in NG when emissions ranged from 5.8 to13.3 9 kg N ha–582

1yr–1. The high N2O emissions in NG in 2013 may have been due to ploughing twice, in May 583

and September and reseeding of the grass. Higher precipitation in 2013 just after ploughing 584

and seeding in spring may have further stimulated N2O emissions. The average annual N2O 585

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emissions in cornfield ranged from 3.6 to 22.9 kg N ha–1yr–1, and they were significantly 586

higher than values reported by Alluvione et al. (2010) in Italy of 3.9 to 8.7 and 3.9 kg N2O–587

N ha–1 and those of Chouldry et al. (2001) who found mean values of 2.3 to 3.4 kg N2O–N 588

ha–1yr–1 in a silt clay loam soil. Higher N input and precipitation in this study could explain 589

the observed differences in the N2O emissions. 590

Higher N2O emissions in cornfield compared to OG and NG were probably due to higher 591

soil NO3- concentrations (Fig. 1), higher heterotrophic soil respiration and consequently 592

higher mineralized N and higher surplus N. Furthermore, the perennial plants, in grassland, 593

were always in the field and hence capable of taking up available soil N, especially in spring. 594

In the cornfield on the other hand, there was no plant uptake of available N in early spring 595

and autumn, and yet manure was applied in autumn and chemical fertilizer at the time of 596

seeding. This lack of N uptake by plants in some periods, and hence lack of synchronisation 597

of plant uptake and soil N availability in some periods, combined with higher precipitation, 598

could have led to overall higher annual emissions in cornfield (FAO and IFA 2001; Iqbal et 599

al. 2014; UNEP 2013). Tillage activities which were conducted every year in the cornfield 600

further influenced heterotrophic soil respiration, N mineralization and hence N2O emissions. 601

Increasing N2O emissions due to tillage activities has been reported by several studies (Palm 602

et al. 2014; Ruan and Philip Robertson 2013; Yonemura et al. 2014). The cornfield 603

emissions were not significantly different with NG emissions of 2013 when tillage was 604

conducted. 605

606

Average N2O emissions over the whole study period were higher in NG than OG (Fig. 2 and 607

Table 2). This could be attributed to higher NO3- concentration (Fig. 1), lower plant N uptake 608

and as a result higher surplus N in NG compared to OG (Table S4). This means more applied 609

N in OG was taken up by the plant hence acting as a sink for N (Iqbal et al. 2014; 610

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Necpa lova et al. 2013; Velthof et al. 2010). In this study, tillage activities and very high 611

precipitation in 2013 in the NG may have played a part in the observed higher emissions. In 612

2014, the emissions were much lower in NG and by 2015 (3 years after establishment of new 613

conversion) N2O emissions in NG were not significantly different from those in OG. Our 614

results suggest that within 3 years after conversion from annual cropland to managed 615

grassland, significant reductions in N2O emissions could be achieved. 616

617

Soil NO3- concentrations in the cornfield and 2013 in NG were significantly higher than in 618

OG, while the WEOC did not differ significantly among the land-use types (Fig. 1). The 619

ratio of WEOC to NO3–-N was highest in OG and lowest in cornfield. High abundance of 620

NO3– relative to labile organic carbon favour N2O release over N2 (Chapin et al. 2011; 621

Firestone and Davidson 1989; Iqbal et al. 2014). This is because high NO3– (electron 622

acceptor) will lead to depletion of the relatively less abundant, electron donor (carbon) (Iqbal 623

et al. 2014) resulting in incomplete denitrification and accumulating higher amounts of N2O 624

in the soil. Lower NO3-, on the other hand may stimulate the reduction of N2O to N2 625

(Firestone and Davidson 1989; Iqbal et al. 2014). Our results in figure 6 are in agreement 626

with this interpretation. 627

628

Our study shows no significant differences in NO emissions among the three land-uses. This 629

finding is supported by Van Lent et al. (2015). Skiba et al. (1997) reviewed several papers 630

and found conflicting reports of land-use effect on NO emissions. However, other studies 631

have reported lower NO emissions in grassland compared to cornfield and attributed this to 632

greater N-use efficiency due to longer growing seasons in grasslands (Boumans et al. 2002). 633

634

635

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CONCLUSSION 636

Annual N2O emissions in cornfield were 6-7 times higher than in OG and 1.5-3 times higher 637

than in NG, and NG had 2-5 times higher N2O emissions than OG. Higher cornfield 638

emissions compared to grassland, and higher emissions in NG compared to OG were due to 639

higher available soil mineral N relative to labile soil organic carbon which could have led to 640

incomplete reduction of NO3- to N2, producing more N2O in the process. Lack of 641

synchronisation of N availability in the soil and plant N uptake may have further led to the 642

high emissions in the cornfield as well as in first year of NG. Within the first year of 643

converting grassland to cornfield N2O emissions increased by more than 500% and remained 644

high three years later, while after converting cornfield to new grassland emissions 645

significantly reduced within three years. Peaks of N2O flux following fertilization were 646

heavily influenced by land-use and interacted strongly with rainfall. Nitric oxide emissions 647

were more influenced by nitrogen addition than soil and weather variables. 648

649

Winter and thawing period N2O and NO emissions contributed significantly to annual 650

emissions, highlighting the need for high frequency of measurements in these periods. There 651

was up to a 10-fold difference in inter-annual N2O emissions within each land-use and 652

treatment in this study. Differences in annual NO emissions were as high as 6 times. This 653

high variation in annual emissions emphasises the need for long-term studies to reduce 654

uncertainties associated with chamber flux measurements for individual sites. 655

656

Acknowledgements 657

This study was partly supported by a research grant provided by the Projects; ‘Establishment 658

of good practices to mitigate Greenhouse Gas emissions from Japanese grasslands’ (FY 659

2004-2009) organized by the Japan Grassland Agriculture and Forage Seed Association 660

(GAFSA) and “Development of Mitigation Technologies to Climate Change in the 661

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Agriculture Sector (FY 2010-2014)” run by Ministry of Agriculture, Forestry and Fisheries 662

of Japan. The author thanks the staff and management of the Hokkaido University’s Shizunai 663

Livestock experimental farm for their assistance in field management activities. 664

665

666

References 667

Akiyama H and Tsuruta,H 2002. Effect of chemical fertilizer form on N2O, NO and NO2 668

fluxes from Andisol field. Nutr. Cycl. Agroecosyst, 63. 669

Akiyama H and Tsuruta H 2003. Nitrous oxide, nitric oxide, and nitrogen dioxide fluxes 670

from soils after manure and urea application. J. Environ. Qual. 3, 423–431. 671

Akiyama H, Tsuruta H and Watanabe T 2000. N2O and NO emissions from soils after the 672

application of different chemical fertilizers. Chemosphere - Global Change Science, 2, 673

313–320. 674

Alluvione F, Bertora C, Zavattaro L, Grignani C 2010: Nitrous Oxide and Carbon Dioxide 675

Emissions Following Green Manure and Compost Fertilization in Corn. Soil Sci. Soc. Am. 676

J. 74, 384–395. 677

Aneja PV, Robarge WP, Sullian LJ, Moore TC, Pierce TE, Geron C, Gay B 1996. Seasonal 678

variations in nitric oxide flux from agricultural soils in the Southeast United States. 679

Tellus, 48B, 626–640 680

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892

893

894

895

896

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Figure captions: 897

Fig. 1 Soil nitrate N, ammonium N and water extractable soil organic carbon (WEOC). CT is 898

control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure 899

plot; M is manure only plot. Dashed arrows indicate dates of manure application; full arrows 900

with open V shaped tip indicate dates of chemical fertilizer application; full arrows with 901

round top and normal closed tip indicate dates of ploughing. 902

903

Fig. 2 Daily precipitation and air temperature (a) and daily N2O flux. CT is control plot; F is 904

chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is manure 905

only plot. Dashed arrows indicate dates of manure application; full arrows with open V 906

shaped tip indicate dates of chemical fertilizer application; full arrows with round top and 907

normal closed tip indicate dates of ploughing. 908

909

Fig. 3 Daily NO flux. CT is control plot; F is chemical fertilizer plot; MF is combined 910

chemical fertilizer and manure plot; M is manure only plot. Dashed arrows indicate dates of 911

manure application; full arrows with open V shaped tip indicate dates of chemical fertilizer 912

application; full arrows with round top and normal closed tip indicate dates of ploughing. 913

914

Fig. 4 Relationship between annual N2O emission and annual precipitation in old grassland 915

(a), cornfield (b) and new grassland (c). CT is control plot; F is chemical fertilizer plot; MF 916

is combined chemical fertilizer and manure plot; M is manure only plot. 917

918

Fig. 5 Relationship between annual N2O emission and soil pH and ratio of annual nitrogen 919

emitted as N2O (N2O–N) to surplus nitrogen and soil pH in old-grassland (a,b), in cornfield 920

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(c,d) and in new-grassland (e,f). CT is control plot; F is chemical fertilizer plot; MF is 921

combined chemical fertilizer and manure plot; M is manure only plot. 922

923

Fig. 6 Relationship between annual N2O emission and the ratio of mean water extractable 924

organic carbon to mean soil NO3– (WEOC/NO3

-). Data in white symbols is in old grassland, 925

grey symbols in cornfield and black symbols in new grassland. CT is control plot; F is 926

chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is manure 927

only plot. 928

929

930

931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

955

956

957

958

959

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Table 1 Timing and kind of field management activities. 960

Land-use Management activity Time

OG Manure application May

Fertilizer application May and June/July

Harvesting June and August

Cornfield Tillage October/November (ploughing), May (harrowing and planting)

Manure application October/November

Fertilizer application May

Harvesting September/October

NG Tillage May 2013 (harrowing and planting), September 2013 (herbicide application, ploughing and re-planting)

Manure application October 2012, September 2013 and May 2015

Fertilizer application May 2013, May and July in 2014 and 2015

Harvesting September 2013, June and August 2014 and 2015

961

962

963

964

965

966

967

968

969

970

971

972

973

974

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41

Table 2 Manure and chemical fertilizer N application rates from 2005 to 2015 975

Land use Year Type F MF M

kg N ha–1

OG

2005 Manure N 0 253.7 – Fertilizer N 164 130 –




2009 Manure N 0 491.2 – Fertilizer N 91.4 0 –

Cornfield 2010 Manure N 0 559.0 – Fertilizer N 104 104 –

2011 Manure N 0 282.6 282.6 Fertilizer N 104 104 0

2012 Manure N 0 343.5 343.5 Fertilizer N 96.6 96.6 0

NG 2013 Manure N 0 448.4 448.4

Fertilizer N 40 40 0 2014 Manure N 0 0 0

Fertilizer N 150.2 47 0 2015 Manure N 0 165.4 165.4 Fertilizer N 103.8 56.9 0

CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; 976

M is manure only plot. OG is old grassland and NG is new grassland. 977

978

979

980

981

982

983

984

985

986

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Table 3 Annual N2O emissions (mean±sd) from 2005-2015 in unfertilized control plots 987

(CT), chemical fertilizer plot (F), manure and chemical fertilizer plot (MF) and manure plot 988

(M). 989

Land use Year CT F MF M

kg N2O-N ha–1 2005 0.7±0.4 2.8±0.7 3.6±1.2 – 2006 0.5±0.3 2.9±0.7 4.9±2.8 –

OG† 2007 0.7±0.5 1.5±0.5 2.2±0.7 – 2008 0.6±0.1 2.1±1.5 0.9±0.2 – 2009 0.4±0.1 1.2±0.7 1.4±0.4 – Average 0.6 2.1 2.6 –

2010 3.9±1.2 17.4±16.1 22.9±11.3 –

Cornfield 2011 5.8±2.3 13.6±8.7 14.3±2.2 11.7±2.3 2012 3.6±0.7 7.1±3.3 7.7±1.2 5.6±1.7 Average 4.4 12.7 14.9 8.7

2013 5.8±1.2 7.5±2.6 11.1±1.5 13.3±2.3 NG 2014 2.8±2.5 4.1±2.5 2.9±0.5 2.4±1.4

2015 1.2±0.2 2.0±0.6 2.3±1.2 1.1±0.4 Average 3.2 4.5 5.4 5.6

ANOVA

d.f. MS F p value

plot 4 117.31 4.29 0.0065

Land use 2 216.42 7.91 0.0015

990

OG is old grassland, NG is new grassland. 991

†Annual N2O emissions in old grassland were previously reported by Shimizu et al. (2013). 992

993

994

995

996

997

998

999

1000

1001

1002

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Table 4 Winter N2O emissions (kg N ha–1) and their contribution to total annual emissions in 1003

brackets (%) 1004

Year† CT F MF M 2005 0.02 (2.3) 0.02 (0.7) 0.01 (0.3) 2006 0.00 (0.0) 0.04 (1.4) 0.02 (0.5) 2007 0.04 (5.6) 0.07 (4.6) 0.04 (1.9) 2008 0.12 (24.8) 0.03 (1.6) 0.03 (3.6) 2009 -0.01 (-2.0) 0.02 (2.0) 0.07 (5.3) 2010 0.17 (4.3) 0.31 (1.8) 0.36 (1.5) 2011 0.49 (8.4) 0.69 (5.1) 3.65 (25.4) 3.65 (31) 2012 0.65 (17.9) 0.48 (6.8) 2.00 (26.0) 2.00 (35.8) 2013 0.14 (2.4) 0.32 (4.3) 0.32 (2.9) 0.45 (2.3) 2014 0.11 (5.1) 0.11 (2.6) 0.06 (-1.9) -0.05 (-2.3) 2015 -0.01 (-1.2) -0.28 (-13.6) 0.16 (6.9) 0.08 (7.2) CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; 1005

M is manure only plot. Winter period was defined as the period from Mid-December, when 1006

maximum soil temperature fell below 5oC, to the end of February when maximum temperatures 1007

recorded reached 0oC. 1008

† Winter N2O emissions were significantly higher in cornfield (2010-2012) than grassland (p<0.01) 1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

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Table 5 N2O emissions during the thawing period (kg N ha–1) and their contribution to total 1040

annual emissions in brackets (%) 1041

Year* CT F MF M 2005 0.04 (6) 0.04 (1) 0.03 (1) 2006 0.01 (2) 0.05 (2) 0.05 (1) 2007 0.14 (20) 0.18 (11) 0.12 (6) 2008 0.08 (18) 0.20 (12) 0.08 (10) 2009 0.03 (7) 0.05 (5) 0.03 (3) 2010 0.23 (5.8) 0.51 (2.9) 0.87 (3.8) 2011 2.1 (35) 0.68 (5.0) 0.71 (4.9) 0.71 (6.0) 2012 1.4 (38) 1.03 (14.) 1.43 (18.6) 1.57 (28.1) 2013 0.61 (10.6) 0.73 (9.7) 0.73 (6.6) 0.63 (3.2) 2014 1.32 (61) 1.92 (46.8) 1.75 (60.7) 1.63 (67.8) 2015 0.29 (25.0) 0.83 (40.2) 0.10 (4.5) 0.17 (15.4) CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; 1042

M is manure only plot. The thawing period was defined as the period when minimum daily 1043

temperatures reached 0oC(typically early march), to the time when soils were completely melted 1044

(minimum soil temperatures ~5oC) in early May. 1045

*N2O emissions during thawing were significantly lower in old grassland (2005-2009) than in corn 1046

and new grassland (p<0.01). 1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

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Table 6 Multiple and single linear regression models accounting for change in annual N2O 1080

emission with changing land-use in the unfertilized control plots (CT), chemical fertilizer 1081

plot (F) and manure and chemical fertilizer plot (MF). 1082

Treatment† Variable§ Coefficient SE p value Model R2 CT WEOC/NO3

– -0.006 0.002 0.018 0.78 F WEOC/NO3

– -0.018 0.002 0.001 0.93 Rainfall 0.011 0.001 0.004 pH -0.705 0.197 0.023 MF WEOC/NO3

– -0.024 0.008 0.016 0.55 †Annual N2O data were transformed using natural log transformation: In (N2O+1) 1083

§WEOC/NO3 is the ratio of the mean annual soil water extractable carbon to soil nitrate, Rainfall is 1084

total annual precipitation, soil pH is mean annual values, SE is standard error. 1085

1086

1087

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Fig. 1 Soil nitrate N, ammonium N and water extractable soil organic carbon (WEOC). CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is manure only plot. Dashed arrows indicate dates of manure application; full arrows with open V shaped tip indicate dates of chemical

fertilizer application; full arrows with round top and normal closed tip indicate dates of ploughing.

297x420mm (300 x 300 DPI)

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Fig. 2 Daily precipitation and air temperature (a) and daily N2O flux. CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is manure only plot. Dashed arrows

indicate dates of manure application; full arrows with open V shaped tip indicate dates of chemical fertilizer

application; full arrows with round top and normal closed tip indicate dates of ploughing.

297x420mm (300 x 300 DPI)

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Fig. 3 Daily NO flux. CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is manure only plot. Dashed arrows indicate dates of manure application; full arrows with open V shaped tip indicate dates of chemical fertilizer application; full arrows with round top and normal

closed tip indicate dates of ploughing.

297x420mm (300 x 300 DPI)

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Fig.4 Relationship between annual N2O emission and annual precipitation in old grassland (a), cornfield (b) and new grassland (c). CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and

manure plot; M is manure only plot.

297x420mm (300 x 300 DPI)

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Fig. 5 Relationship between annual N2O emission and soil pH and ratio of annual nitrogen emitted as N2O (N2O–N) to surplus nitrogen and soil pH in old-grassland (a,b), in cornfield (c,d) and in new-grassland (e,f).

CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer and manure plot; M is

manure only plot.

297x420mm (300 x 300 DPI)

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Fig. 6 Relationship between annual N2O emission and the ratio of mean water extractable organic carbon to mean soil NO3

–¬ (WEOC/NO3-). Data in white symbols is in old grassland, grey symbols in cornfield and

black symbols in new grassland. CT is control plot; F is chemical fertilizer plot; MF is combined chemical

fertilizer and manure plot; M is manure only plot.

297x420mm (300 x 300 DPI)

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For review

Nitrous and nitric oxide emissions from a cornfield and managed grassland: 11

years of continuous measurement with manure and fertilizer applications, and

land-use change.

Ikabongo Mukumbuta a1, Mariko Shimizu

a, Tao Jin

a, Arata Nagatake

a, Hiroshi Hata

b, Seiji Kondo

b, Masahito Kawai

b, Ryusuke Hatano

a

a Soil Science Laboratory, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo,

Hokkaido 060-8589, Japan.

b Field Science Center for Northern Biosphere, Hokkaido University, Sapporo,

Hokkaido 060-0811, Japan.

1Corresponding author email: [email protected].

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Figure S1: Layout of the treatment plots in the field experiment. The treatment plots

were located in a large experimental field (100 x 200 m) of the Hokkaido university

experimental farm. Each treatment plot was 5x5 m in size and was replicated 4 times

(as shown in figure below) for gas, soil and biomass sampling. M is manure only

treatment, MF is manure plus chemical fertilizer, F is chemical fertilizer only; and CT

is the control with neither manure nor inorganic fertilizer application. Manure and

chemical fertilizer in the treatment plots were applied by hand, but within one day

after the rest of the field was applied with manure or chemical fertilizer by farm

management.

5x5 m

M

F

MF F

M

CT

MF

M

F

MF

CT

F

M

CT

MF

Outside of experimental plots:

Manure and supplemental chemical

fertilizer applied by farm

management

Outside of experimental plots:

No manure, only chemical fertilizer

application by farm management

100m

100m ROAD

Drainage

ditch

CT

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Table S1 Winter NO emissions (g N ha–1) and their contribution to total annual

emissions in brackets (%)

Year CT F MF M

2005 5.8 (10) 10.0 (6) 7.1 (2)

2006 10.6 (7) 10.0 (4) 8.3 (2)

2007 12.3 (23) 40.0 (29) 16.0 (5)

2008 8.0 (55) 6.7 (17) 8.5 (15)

2009 -4.4 (-33) 20.5 (3) 1.0 (0.5)

2010 2.4 (6) 0.0 (0) 9.9 (3)

2011 4.4 (2) 2.0 (0.4) 46.7 (3) 46.7 (7)

2012 8.6 (9) 9.4 (2) 14.3 (2) 14.4 (11)

2013 0.0 (0) -3.2 (-3) -3.2 (-2) 7.0 (3)

2014 9.7 (28) 9.7 (14) -25.8 (-666) -25.8 (200)

2015 -1.2 (-6) -2.5 (--2) -3.6 (2.2) -1.9 (-8)

CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer

and manure plot; M is manure only plot

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Table S2 Thawing period NO emissions (g N ha–1) and their contribution to total

annual emissions in brackets (%)

Year CT F MF M

2005 6.1 (11) 6.3 (4) 7.1 (2)

2006 9.1 (6) 7.4 (3) 13.7 (3)

2007 6.0 (8) 1.8 (1) 4.8 (2)

2008 3.4 (23) 3.5 (9) 14.2 (25)

2009 -0.9 (-7) 1.0 (0.1) 1.5 (1)

2010 10.1 (25) 0.0 (0) 86.9 (27)

2011 60.9 (32) 20.1 (4) 30.3 (2) 30.3 (5)

2012 11.9 (13) 5.0 (1) 121.7 (16) 8.1 (6)

2013 0.8 (1) -0.9 (-1) -0.9 (0) 3.2 (1)

2014 10.3 (30.2) 7.9 (11.6) -17.2 (-442) -16.1 (130)

2015 0.3 (2) 7.1 (5) -0.9 (1) -1.9 (-8)


and manure plot; M is manure only plot

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For review

Table S3 Average annual heterotrophic soil respiration (RH; Mg C ha-1yr

-1) and

estimated total mineralized N (kg N ha-1yr

-1) from 2005-2015.

CT F MF M

OG 2005 RH 4.8±0.8 4.8±0.8 5.3±1.5 –

Mineralized N 444.4 444.4 464.6 –

2006 RH 4.6±0.7 4.6±0.7 6.4±1.2 –

Mineralized N 425.9 425.9 519.7 –

2007 RH 4.9±0.5 4.9±0.5 9.2±1.9 –

Mineralized N 453.7 453.7 638.3 –

2008 RH 4.0±1.1 4.0±1.1 5.1±2.7 –

Mineralized N 370.4 370.4 412.5 –

2009 RH 4.5±2.5 4.5±2.5 7.8±1.8 –

Mineralized N 416.7 416.7 599.0 –

Corn 2010 RH 6.8±0.8 6.9±1.1 10.2±0.7 –

Mineralized N 646.7 654.0 872.4 –

2011 RH 6.5±0.9 6.1±1.1 7.8±1.1 7.8±1.1

Mineralized N 612.8 574.3 627.9 653.4

2012 RH 6.8±1.2 4.9±0.3 8.8±0.6 10.3±0.5

Mineralized N 642.3 461.7 628.9 793.5

NG 2013 RH 4.4±0.3 4.8±0.3 9.4±0.8 9.2±0.9

Mineralized N 415.3 455.6 734.2 707.6

2014 RH 4.0±0.5 4.3±0.4 5.5±0.5 5.9±0.5

Mineralized N 376.4 406.7 475.0 484.3

2015 RH 5.0±0.9 5.0±0.9 6.8±1.0 7.0±1.4

Mineralized N 477.1 481.3 552.0 557.9

ANOVA RH

d.f. MS F p

Plot 4 15.55 14.03 <0.001

Land-use 2 12.88 11.62 <0.001


and manure plot; M is manure only plot. Mineralized N is sum of soil organic matter

and manure N mineralization.

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Table S4 Plant N uptake and surplus N (kg ha-1yr

-1).

CT F MF M

2005 Plant N uptake 106.3 231.3 185.3 –

2005 Surplus N 338.2 377.2 409.3 –

2006 Plant N uptake 106.3 194.0 178.3 –

2006 Surplus N 319.7 415.0 474.4 –

2007 Plant N uptake 116.9 179.0 146.9 –

2007 Surplus N 336.8 348.7 512.4 –

2008 Plant N uptake 85.0 130.5 118.9 –

2008 Surplus N 285.4 313.8 293.6 –

2009 Plant N uptake 81.5 145.5 139.9 –

2009 Surplus N 335.2 362.6 459.1 –

2010 Plant N uptake 54.8 106.5 159.0 _

2010 Surplus N 591.9 651.5 817.4 _

2011 Plant N uptake 57.3 117.8 193.6 95.2

2011 Surplus N 555.4 560.6 538.3 558.1

2012 Plant N uptake 86.5 109.9 218.5 113.5

2012 Surplus N 555.7 448.3 507.0 680.0

2013 Plant N uptake 35.2 49.3 75.2 59.4

2013 Surplus N 380.1 446.3 699.1 648.2

2014 Plant N uptake 63.2 108.6 109.7 80.7

2014 Surplus N 313.3 448.3 412.1 403.6

2015 Plant N uptake 43.8 145.3 62.6 66.3

2015 Surplus N 433.3 439.8 546.3 491.7

ANOVA Plant N uptake Surplus N

d.f. MS F MS F

Plot 4 9909 8.68** 21202 2.89*

Land-use 2 13043 11.43** 128953 17.6**


and manure plot; M is manure only plot. Surplus N was calculated as difference

between total N input (total mineralized N from soil organic matter and manure, and

chemical fertilizer N) and the plant N uptake. **p<0.01, *p<0.05

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