N 2 O fluxes and related processes of denitrification in acidified soil

: 18 In North China, high levels of N fertilizer and irrigation water are used in fields, which cause 19 considerable N 2 O fluxes via several pathways, especially anaerobic denitrification. Anaerobic 20 denitrification is regarded as an important microbial process for N 2 O production in soils with a low 21 O 2 level and high N and labile C availability (the typical soil conditions caused by high levels of N 22 fertilizer and irrigation water in the field). We conducted an anaerobic incubation experiment to 23 determine the impact of soil acidification (with a series of soil pH levels, pH 6.2, pH 7.1, and pH 8.7) on N 2 O source partitioning with the addition of KNO 3 and glucose. Natural abundance isotope 25 techniques and gas inhibitor technique were applied to analyze the N 2 O flux derived from fungal 26 denitrification and bacterial denitrification and its isotopocule characteristics emitted from soils 27 after the addition of NO - 3 and glucose. A mapping approach was used to obtain further insight into 28 the N 2 O production processes. Our findings confirmed that soil pH strongly controlled the N 2 O 29 production and reduction rates of denitrification. Soil acidification significantly increased N 2 O 30 emissions varied from 0.76 mg N kg -1 for natural soil (pH 8.7), to 1.88 mg N kg -1 for pH 7.1, and to 31 2.35 mg N kg -1 for pH 6.2, and had a blockage effect on the reduction of N 2 O to N 2 . The addition 32 of carbon sources promoted complete denitrification. We assumed a higher contribution of fungal 33 denitrification to N 2 O production compared to total N 2 O emission associated with acidified soil. A 34 promotion of the contribution of fungal denitrification-derived N 2 O was indeed observed with 35 decreasing pH, increasing from 0.28 mg N kg -1 for pH 8.7 to 0.94 mg N kg -1 for pH 6.2. The addition 36 of glucose further increased the contribution of fungal denitrification to N 2 O production from 0.99 37 mg N kg -1 for pH 8.7 to 3.66 mg N kg -1 for pH 6.2. The mapping approach provided rational results 38 for correcting N 2 O reduction compared with the acetylene inhibition method. The results calculated 39 by both methods indicated a reasonably large contribution of fungal denitrification to total N 2 O 40 production in acidified soils. 41


Introduction 44
As a trace gas in the atmosphere, N2O is the third most important anthropogenic greenhouse (1) 144 where f is the emission rate of N2O, μg kg -1 h -1 ; ρ is the N2O density (1.25 kg m -3 ), at 273 K and 101 145 kPa; V is the effective headspace volume of a jar, 0.25 L; ∆C/∆t is the variation of N2O concentration 146 per unit time (nL L -1 h -1 ); m is the mass of dry soil, 40×10 -3 kg; T is the air temperature during 147 incubation, 25℃; 24 is the 24 hours of one day, and used for the translation gaseous emission rate 148 of per hour to per day. 149 The dual isotope and isotopocules of N2O (δ 15 N bulk , δ 15 N α and δ 18 O) were measured by the 150 IRMS with Delta V Plus-Precon, with the precision of 0.5‰ for δ 15 N bulk , 0.9‰ for δ 15 N α , and 0.6‰ 151 for δ 18 O. 152 δX =(R sample /R standard -1) ×1000 (2) 153 where X is 15 N bulk , 15 N α or 18 O. The standard reference gases were used for calibration.

Soil analysis 159
Soil pH was measured in suspension of soil (1:2.5, soil: 0.01 M CaCl2) using a Thermo Orion 160 8 pH meter (Mettler Toledo, China pH). Soil water content was determined after weight loss for 24 h 161 drying in 105°C. The NO -3 -N and NH + 4 -N content were determined by extracting soil with 2 M KCl 162 solution (1:5, soil: KCl). The extract was analyzed for the concentration of NO -3 -N and NH + 4 -N using 163 a Lachat Flow-Injection Auto-analyzer (Lachat Instruments, USA). 164

Identification of N2O production processes 165
In this study, the contribution of fungal denitrification (fBD) and bacterial denitrification (fFD; 166 fBD =1 -fFD) to total N2O production were calculated by the two end-member mixing model 167 (equation (5) and (6); Sutka et al., 2003) using two different approaches. In this model, SP values 168 are used to investigate the respective contribution of the two pathways, one from lower SP group 169 and one from higher SP group, which are selected as the end members. Four cases should be 170 considered depending on this model: (i) bacterial nitrification and bacterial denitrification, (ii) 171 bacterial nitrification and nitrifier denitrification, (iii) fungal denitrification and bacterial 172 denitrification, (iv) fungal denitrification and nitrifier denitrification. This experiment was 173 conducted under anaerobic environment, for which denitrification was the only process in soil. 174 Therefore, the two end-members are fungal denitrification (fFD) and bacterial denitrification (fBD). 175 Isotopic endmembers for SP values of produced N2O were assumed to be 35.5‰ (SPFD) for fungal 176 denitrification and -5‰ (SPBD) for bacterial denitrification (Sutka et al., 2008;Rohe et al., 2014a). 177 The reduction of N2O to N2 can cause an enrichment in SP values, thus moving the values 180 closer to those of fungal denitrification. If N2O reduction process is not blocked, SP values for fungal 181 and bacterial denitrification could be confounded.

9
In this study, two methods (i.e. acetylene inhibition approach and mixing approach) were 183 chosen to eliminate this confusion. 184

Acetylene inhibition method 185
In this method, the reduction of N2O to N2 can be inhibited by the addition of 10 kPa (10 vol%) 186 C2H2. Therefore, N2O emissions are the total (N2O+N2) emissions for treatments with C2H2 and the 187 net N2O production after the reduction of N2O for treatments free of C2H2. The SP values obtained 188 from groups with 10 kPa C2H2 which are not influenced by N2O reduction are able to distinguish 189 fungal and bacterial denitrification calculated by the two end-member mixing model (equation  before reducing to N2. If the SP0 value was lower than the measured SP value of N2O, the calculated 214 value (SP0) was used, since N2O reduction could not be negligible. 215 From the calculated SP0 values (or SP), the fraction of N2O derived from fungal denitrification 216 (fFD) or bacterial denitrification (fBD) can be estimated using the two end-member mixing model. 217 Results derived from this method are the calculated results (calculated fFD; Figure.

Effect of soil acidification on inorganic nitrogen 227
To obtain different pH levels, soils at natural pH (pH 8.7) were preincubated for a period of 228 two weeks after acidification. Data for all the pH groups are presented in Table 1. With H2SO4 added, 229 significant differences among pH groups were observed and varied from 0.9 to 2.5 units (p < 0.0001). 230 Moreover, there were also obvious changes in both NH + 4 and NO -3 concentrations. The average NH 231 + 4 -N content at pH 6.2 was significantly different from that in natural soil (pH 8.7; p < 0.01) and 232 increased 2.1 times by the end of preincubation (Table 1) in conjunction with a slight decrease of 233 the NO -3 -N content (5.9 mg N kg -1 ). 234

N2O and N2 fluxes 235
Compared to the groups free of C2H2, the addition of C2H2 significantly increased N2O 236 production in all treatments (p < 0.05; Fig. 2). With substrate and carbon source consumption, both 237 the N2O production and reduction rates generally deceased with incubation progress in all groups 238 (Fig. 2). Soil pH strongly controls the N2O production and reduction rates of denitrification. Both 239 soil N2O and (N2O+N2) cumulative emissions in acidic soils increased significantly compared to 240 soils of natural pH (p < 0.0001). No significant difference was observed between soils of pH 7.1 241 and pH 8.7, but higher values occurred in soils of pH 7.1. 242 The total N2O production determined in soils with C2H2 was significantly larger (p < 0.001) in 243 the presence of glucose, varying among 5.85 mg N kg -1 d -1 for pH 8.7, 8.89 mg N kg -1 d -1 for pH 244 7.1 and 12.67 mg N kg -1 d -1 for pH 6.2, compared to groups in the absence of glucose, varying 245 among 0.89 mg N kg -1 d -1 for pH 8.7, 2.14 mg N kg -1 d -1 for pH 7.1 and 2.93 mg N kg -1 d -1 for pH 246 6.2 ( Fig. 2; Table 2). The differences in N2O production among soils with different pH values were 247 all statistically significant under the same glucose conditions (p < 0.001).
12 Net N2O production in the absence of C2H2 varied between 0.76 and 2.35 mg N kg -1 d -1 (Fig.  249 2; Table 2), and was significantly increased after adding glucose (p < 0.001) with a variation between 250 2.76 and 8.44 mg N kg -1 d -1 . The N2O reduction rate, calculated based on the comparison between 251 the groups with and without C2H2, varied from 0.12 to 4.23 mg N kg -1 d -1 and was significantly 252 higher in the presence of glucose, showing a higher time-weighted mean value for a lower pH, 253 although higher values among all the pH groups were not observed in the first 3 days. Significant 254 differences of N2O reduction rate between pH 6.2 and 7.1 and between pH 6.2 and pH 8.7 were 255 observed in the absence of glucose (p < 0.05), but the addition of glucose decreased the differences 256 (p = 0.240 between pH 6.2 and 7.1; p = 0.054 between pH 6.2 and pH 8.7). This C-effect was also 257 reflected in the production ratio (N2O/(N2+N2O)), which was lower for all pH levels in the presence 258 of glucose varying from 0.47 for pH 8.7 to 0.67 for pH 6.2 compared to the groups free of glucose 259 varying from 0.80 for pH 6.2 to 0.88 for pH 8.7.  (Table 2). Significant differences were found among different 266 soil pH values (P < 0.001). In the absence of C2H2, the δ 15 N bulk values of natural soils were 267 significantly higher than that in the presence of C2H2 (P < 0.05), and this statistical significance was 268 not observed in soils after acidification. The δ 15 N bulk of produced N2O in soils with glucose varied 269 between -43.72‰ and -27.67‰ and was significantly higher in more acidic soils (p < 0.001). 270 13 The δ 18 O values showed no evident changes for pH 6.2 with incubation time, decreased in the 271 first three days and then increased for the other two soils (Fig. 3). The δ 18 O values of produced N2O 272 in soils with C2H2 varied from 16.83‰ to 20.64‰ (Table 2)  The SP values in groups with C2H2 varied between 4.90‰ and 21.73‰, with a wider variation 281 for a lower pH (Table 2; Fig. 3), and were higher for groups free of glucose compared to groups 282 with glucose in the early stage of the incubation. The highest SP values in groups free of glucose 283 were observed before day 5, with a general decline after day 5. Conversely, the lowest SP values in 284 the presence of glucose were observed before day 5 except for pH 7.1, which was observed on day 285 9. The addition of glucose increased the SP values on and after day 5 compared to the groups free 286 of glucose, and the differences between groups with and without glucose were higher for a lower 287 pH, varying among 2.89‰ for pH 8.7, 3.66‰ for pH 7.1 and 7.33‰ for pH 6.2. The higher average 288 SP values were followed by lower pH conditions. 289

The relationship between isotopocules and gaseous nitrogen fluxes 290
Pearson correlation analysis were used to identify isotopocules of N2O linked to N2O and N2 291 flux (Table 3). For all groups, the δ 15 N bulk , δ 18 O and SP values of N2O were all significantly 292 14 correlated with N2O flux, with positive correlations between δ 15 N bulk and N2O and between SP and 293 N2O (p < 0.01) and negative correlations between δ 18 O and N2O (p < 0.01) in the absence of glucose. 294 Additionally, a higher correlation coefficient was found between δ 15 N bulk and N2O at pH 8.7, a 295 negative correlation was found between SP and N2O in all soils, and a positive correlation was found 296 between δ 18 O and N2O at pH 6.2 after adding glucose. In the absence of glucose, no significant 297 correlation was observed between N2 flux and isotopocules of N2O at pH 8.7 and pH 7.1 except for 298 δ 18 O and N2 at pH 7.1. Positive correlations were found between δ 15 N bulk and N2 and between SP 299 and N2 (p < 0.01), and a negative correlation was found between δ 18 O and N2 at pH 6.2 that was 300 consistent with the groups after the addition of glucose. For soils at pH 8.7 and pH 7.1, positive 301 correlations were observed between δ 15 N bulk and N2, followed by negative correlations between δ 18 O 302 and N2 and between SP and N2. 303

Source partitioning 304
The mapping approach based on δ 18 O/SP plots for soil-derived N2O was used for source 305 partitioning (Fig. 1). δ 18 O values were highest in natural soils with glucose, coupled with the SP 306 values. These individuals were outside the vector that is interpretable by mixing N2O from fungal 307 denitrification and bacterial denitrification as source processes. This shift to higher values could be 308 interpreted by the incorporation of mixed production from fungal and bacterial denitrification and 309 the partial reduction of N2O to N2 during bacterial denitrification. Such a shift was only observed in 310 individuals from natural soil, where no acid was added. Individuals in the absence of glucose were 311 more concentrated, with a narrow range of 16.53‰ to 28.11‰ for δ 18 O and of 7.61‰ to 22.93‰ 312 for SP, compared to groups after adding glucose (18.48‰ to 34.28‰ for δ 18 O and 6.80‰ to 25.73‰ 313 for SP).

15
Based on the endmember signatures, fFD calculated from Equation (5) ranged from 23% to 76% 315 (Table S1). The highest SP values were measured in individuals at pH 6.2 with glucose, and closer 316 SP values were observed from samples in groups without carbon sources, especially natural soils. 317 Using the mean values for the SP and δ 18 O values of fungal denitrification and bacterial 318 denitrification (i.e., center values of endmember areas in Fig. 1), the contribution of fungal 319 denitrification to N2O production was 45% for pH 8.7, 48% for pH 7.1 and 49% for pH 6.2 and was 320 lower for pH 8.7 (37%) and pH 7.1 (43%) and higher for pH 6.2 (57%) after adding carbon sources. 321 From the calculated SP (or SP0) values estimated by the isotopocule map (calculated SP values) 322 and the measured SP values directly measured from the groups with C2H2 (measured SP values), the 323 contribution of fungal denitrification-derived N2O based on the above two approaches was 324 calculated by using the end-members mixing model (the calculated and measured fFD were estimated 325 by the above calculated and measured SP values, respectively). Hence, we compared the calculated 326 and measured fFD and found good agreement between them with a significant fit to the 1:1 line (Fig.  327   4), especially for the group in the absence of glucose (R 2 = 0.81). The mean absolute difference 328 between the measured and calculated fFD was 0.11 in the absence of glucose and 0.04 in the presence 329 of glucose. The mean relative error in the determination of the contribution of fungal denitrification 330 to N2O production was 28% in the absence of glucose and 9% in the presence of glucose. A better 331 fit was not obtained when the calculated and measured fFD for individual pH values were determined 332 and applied separately. From the correlation tested among different pH levels, we found that only 333 for acidified soils free of glucose (R 2 = 0.77 for pH 6.2; R 2 = 0.43 for pH 7.1) were the measured 334 and calculated fFD correlated. In the presence of glucose, although no correlation was observed for 335 individual pH values, a slightly significant fit to the 1:1 line was obtained when soils of different 336 pH values were determined and applied together (R 2 = 0.40), which indicated that these fFD values 337 were associated with the incubation conditions. 338 339

Discussion 340
It is well established that soil pH is a main controlling factor for N2O production/reduction 341 through regulating N mineralization, nitrification and denitrification processes (Xiao et al., 2013;342 Qu et al., 2014), especially in anaerobic environments. Modifying soil pH on site is difficult due to 343 the high buffering capacity of most soils. Nevertheless, soils differing in natural pH also have 344 differences in many other properties. Hence, the influence of soil pH is often conducted by 345 modulating the pH by liming, which requires repeated lime applications and is a gradual and typical 346 method that can take years (Adams and Adams, 1983; Nicol et al., 2008). In this study, natural soil 347 (pH 8.7) was significantly modified by the application of H2SO4 with a short-term preincubation. 348 This process was carried out under highly controlled conditions of pH and carbon sources (glucose), 349 adopted homogenized and sieved soil free of plants and animals, imposed conditions optimal for 350 denitrification in the anaerobic incubation, and examined N2O and (N2O+N2) emissions in response 351 to pH amendments, and distinguished the fraction of N2O derived from fungal or bacterial 352 denitrification by using an isotopocule mixing approach. 353

Effect of soil acidification on inorganic nitrogen 354
Acidification of soil was accompanied by changes in other soil properties (Table 1) resulting 355 from changes in soil function. Therefore, the significantly higher NH + 4 content (p < 0.01) coupled 356 with the decrease in NO -3 content in acidic soil during soil preincubation was partially associated 357 with a process of producing NH + 4 , i.e., DNRA. DNRA is the process by which NO -3 can be directly 358 17 and rapidly converted into NH + 4 in soils under similar conditions (i.e., low redox potential, available 359 NO -3 and labile C) for denitrification (Zumft, 1997;Silver et al., 2001). In soils of pH 6.2, the 360 addition of glucose made a gradual promotion of NH + 4 contents and changed the reduction of NO -3 , 361 moving this process closer to denitrification and farther from DNRA compared to the groups free 362 of glucose. This influence caused by the addition of glucose may be due to the promotion of the 363 N2O reduction process (Weier et al., 1993;Azam et al., 2002). Soil acidification resulted in this C 364 effect, which did not occur in neutral and alkaline soils. 365

N2O and N2 fluxes 366
The results showed that soil acidification was associated with both increasing denitrification 367 rates and the N2O/(N2O+N2) product ratios in the presence of glucose, in good agreement with 368 previous findings (Scholefield et al., 1997;Hanaki et al., 1992). The influence of pH may be indirect 369 -the availability of organic carbon and nitrogen mineralization and the microbial community are 370 regulated by the shift in soil pH, further leading to changes in the denitrifying component. A previous 371 study reported that the optimum pH for bacterial denitrification was from 7.0 to 8.0 (Sahrawat and 372 Keeney, 1986). Therefore, slightly acidic soil may have a partial blockage effect on bacterial 373 denitrifiers, whose significantly higher N2O production may be attributed to fungal denitrification it can be assumed that fungal denitrifiers are capable of higher N2O production with more substrates 378 due to the partial blockage of bacterial denitrifiers by acidic environments. Furthermore, a lower 379 soil pH increased the N2O/(N2O+N2) product ratio due to the lower tolerance of N2O reductase to 380