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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
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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
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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
133
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
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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
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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
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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
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964
965
966
967
968
969
970
971
972
973
974
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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 –
2006 Manure N 0 310.2 – Fertilizer N 183 133 –
2007 Manure N 0 331.4 – Fertilizer N 74 21 –
2008 Manure N 0 308.1 – Fertilizer N 74 0 –
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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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)
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 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
CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer
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**
CT is control plot; F is chemical fertilizer plot; MF is combined chemical fertilizer
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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