Measurement of N2, N2O, NO, and CO2 Emissions from Soil with the

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Measurement of N2, N2O, NO, and CO2 Emissions from Soil with the Gas-Flow-Soil-Core Technique Rui Wang,†,‡ Georg Willibald,† Qi Feng,‡ Xunhua Zheng,‡ Tingting Liao,‡ Nicolas Br€uggemann,†,§ and Klaus Butterbach-Bahl*,† †

Karlsruhe Institute of Technology, Institute for Meteorology and Climate Research (IMK-IFU), 82467 Garmisch-Partenkirchen, Germany ‡ Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing 100029, China

bS Supporting Information ABSTRACT: Here we describe a newly designed system with three stand-alone working incubation vessels for simultaneous measurements of N2, N2O, NO, and CO2 emissions from soil. Due to the use of a new micro thermal conductivity detector and the redesign of vessels and gas sampling a so-far unmatched sensitivity (0.23 μg N2
N h
1 kg
1 ds or 8.1 μg N2
N m
2 h
1) for detecting N2 gas emissions and repeatability of experiments could be achieved. We further tested different incubation methods to improve the quantification of N2 emission via denitrification following the initialization of soil anaerobiosis. The best results with regard to the establishment of a full N balance (i.e., the changes in mineral N content being offset by simultaneous emission of N gases) were obtained when the anaerobic soil incubation at 25 °C was preceded by soil gas exchange under aerobic conditions at a lower incubation temperature. The ratios of N and C gas emission changed very dynamically following the initialization of anaerobiosis. For soil NO3
contents of 50 mg N kg
1 dry soil (ds) and dissolved organic carbon (DOC) concentrations of approximately 300 mg C kg
1 ds, the cumulative emissions of N2, N2O, and NO were 24.3 ( 0.1, 12.6 ( 0.4, and 10.1 ( 0.3 mg N kg
1 ds, respectively. Thus, N gas emissions accounted (on average) for 46.2% (N2), 24.0% (N2O), and 19.2% (NO) of the observed changes in soil NO3
. The maximum N2 emission reached 1200 μg N h
1 kg
1 ds, whereas the peak emissions of N2O and NO were lower by a factor of 2
3. The overall N2:N2O and NO:N2O molar ratios were 1.6
10.0 and 1.6
2.3, respectively. The measurement system provides a reliable tool for studying denitrification in soil because it offers insights into the dynamics and magnitude of gaseous N emissions due to denitrification under various incubation conditions.

’ INTRODUCTION In the last century, humans have dramatically increased the amount of reactive nitrogen in the biosphere, thereby accelerating global nitrogen cycling by a factor of 2.1 The increasing availability of nitrogen has been a major factor ensuring global food production and security.2 The observed accelerated nitrogen cycling has a number of hazardous environmental consequences, such as eutrophication, acidification, and air pollution.3
5 Once reactive nitrogen is created (through fertilizer production, combustion processes, and biological dinitrogen (N2) fixation), it may remain in the global environment for days to centuries, thereby cascading in different forms downstream and downwind through the biosphere, hydrosphere, and atmosphere. The major pathway by which reactive nitrogen is inactivated is its conversion to the chemically stable N2 form via classical denitrification.3,6 Denitrification, a facultative anaerobic process,7 is the reduction of nitrate (NO3
) to nitrite (NO2
), to nitric oxide (NO), to nitrous oxide (N2O), and finally to N2. Denitrification is associated with the production of N2, N2O, and NO in soil, sediments, and bodies of water.8 The latter two r 2011 American Chemical Society

gases are primarily or secondarily active greenhouse gases affecting atmospheric chemistry and global warming.9 N trace gas emissions from soil and the importance of denitrification as a driving force of N2O and NO emission have received significant research attention, but data on N2 emission by denitrification are scarce3 due to the fundamental difficulty of quantifying the dominant end product of denitrification—N2—given its high background concentration in the environment.3,7 A number of methods have been used to obtain better insight into the factors controlling denitrification and to quantify N2 losses from soil. These approaches include (a) acetylene (C2H2) inhibition,10 (b) 15N tracers,11,12 (c) gas-flow-soil methods for direct N2 quantification,13
17 and (d) mass balances.18 Specifically gas-flow-soil methods have been combined in the recent

Received: November 3, 2010 Accepted: June 2, 2011 Revised: March 16, 2011 Published: June 16, 2011 6066

dx.doi.org/10.1021/es1036578 | Environ. Sci. Technol. 2011, 45, 6066–6072

Environmental Science & Technology past with analyses of isotopic fingerprints of N2O19 and/or transcription rates of denitrification enzymes.20 The gas-flow-soil-core technique is based on the replacement of the soil0 s atmosphere by an N2-free atmosphere, thus allowing direct measurement of N2 emission from the soil due to denitrification. The method was introduced in the 1970s21,22 to measure N2O and N2 during denitrification in small repacked soil samples under anaerobic conditions of a helium/argon atmosphere.23,24 The technique was further developed to allow the measurement of N2 emitted by denitrification under aerobic conditions.13
17 Shortcomings are mainly associated with (a) the time needed to establish an N2 free atmosphere, (b) the sensitivity for detecting N2 fluxes, and (c) the gas-tightness of the incubation vessel.7 Furthermore, depending on soil texture, soil moisture content, and volume of the soil samples, the time needed for a complete replacement of the soil atmosphere may have taken several days.7,15 Here we describe innovations to speed up the gas exchange process by alternating overpressure and negative pressure. In most studies a standard thermal conductivity detector (TCD) detector was used for N2 detection13,14 with the detection limit being rather high, translating to a soil N2 flux of 50 g N2
N ha
1 d
1.14 Therefore, studies were concentrated on highly fertilized soils13,14 or sediments with high denitrification rates.25 Recently, several groups started to use pulse-discharged helium ionization detectors for N2 detection. This allowed lowering the detection limit for N2 fluxes to approximately 2.4 g N2
N ha
1 d
1.15,16 Here we used a newly available microTCD for N2 measurements and tested its sensitivity for detecting denitrification N2 fluxes. Minimizing N2-diffusion into measuring vessels is crucial to ensure that measured N2 increases are indeed resulting from denitrification. In previous studies, the high detection limit for N2 may be attributed to N2 diffusion into incubation vessels or the gas sampling system.14 Similarly, measurement of very low concentration of N2 (10
50 ppmv) tended to be variable due to leakage.17 The system shown here is characterized by high gas tightness, ensured by respective leakage tests. A remaining problem is the choice of incubation conditions during the phase of soil gas exchange, since during this phase N2 emissions cannot be measured due to a too high background concentration. To overcome this problem, we tested the suitability of varying incubation conditions (anaerobic versus aerobic and high incubation temperature [25 °C] versus low temperature [2 °C]) with regard to their effects on total N and C gas production by agricultural soils. In summary, in this study, we describe a system based on the gas-flow-soil-core technique for simultaneously measuring all the gaseous N products of denitrification (N2, N2O, and NO) as well as soil CO2 emission. We further developed and tested this system to minimize the effects of the gas exchange process on the N2 emission processes. Finally, we used the system to analyze the temporal dynamics of N gas and CO2 emission following the initiation of anaerobiosis in soil.

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experimental treatment in Shanxi province, China (34.56° N, 110.43° E).26 General soil properties are given in Table S3. Following sampling (June 2009), the fresh soil (16% gravimetric water content) was sieved (2 mm), and stones, roots, and crop residues were removed. Subsequently, the soil was air-dried and stored at 25 °C (Experiments 1 and 2) or at 4 °C (Experiments 3a, b). Five days before the start of experiments (Experiment 3b: 16 days), the air-dried soil was remoistured with deionized water to values of 25% gravimetric water content (ca. 45% water filled pore space (WFPS)). Preincubation water content was held stable by adding respective amounts of water following daily weighing. For the measurements the soil was repacked into a cylindrical stainless steel ring (diameter 6 cm, height 4 cm) at a bulk density of approximately 1.07 g cm
3. Immediately before the start of the incubation the soil was additionally amended with 20 ml of a solution of nitrate and glucose, increasing soil moisture content from 25% to 30%. In Experiments 1 and 2 the initial soil NO3
concentration was set at 100 mg N kg
1 dry soil (ds) and initial glucose concentration was set at 300 mg C kg
1 ds. For Experiment 3 we set the soil NO3
content at 50 mg N kg
1 ds while keeping the glucose concentration at 300 mg C kg
1 ds in order to investigate the effect of a lowered N:C ratio on N and C gas production. Experimental Design. Following the complete replacement of the soil atmosphere by an N2-free gas, N2 emissions can be measured. Therefore, the correct incubation conditions during the gas exchange period for soil air replacement are essential to allow a full assessment of gaseous N losses. Three different conditions were examined to evaluate their suitability for quantifying N2 emissions from soil under initialized anaerobiosis (Table S4). Experiment 1: gas exchange and consecutive measurements under anaerobic conditions at 25 °C. Experiment 2: gas exchange under aerobic conditions at 25 °C followed by measurements of N2 emission under anaerobic condition at 25 °C. Experiment 3a and 3b: gas exchange under aerobic conditions at 2 °C, followed by an anaerobic incubation period at 2 °C and subsequent incubation at 25 °C. Please note that irrespective of the incubation conditions, N2O, NO, and CO2 emissions were also measured during the gas exchange period. All experiments were performed in triplicate using three independent incubation vessels. Gas Analysis. N2, N2O, and CO2 were detected by an Agilent gas chromatograph (Agilent Technologies Inc., Wilmington, DE) equipped with a micro-TCD, an electron capture detector, and a flame ionization detector, respectively. NO concentrations were measured with a NO-NO2-NOx chemiluminescent analyzer (42i Thermo Environmental Instruments Inc., USA). For more details see the Supporting Information. Calculation of Gas Fluxes. Sampling of the headspace involved dilution of the gas phase due to the injection of a gas mixture into the vessel over a 4-min period at a rate of 20 mL min
1. Therefore, the measured concentrations of gases were corrected for this gas dilution (for details see Supporting Information): 

C i ¼ Cm i


’ MATERIALS AND METHODS A detailed description of the principles and methods of the gas-flow-soil-core measurement system is presented in the Supporting Information. Soil Properties and Pretreatments. To test the system, we used arable soil from a winter wheat
summer maize rotation

i

∑1 ebi 3 ðe4a
1Þ when ðCmi > CinÞ

bi ¼ lnðjCm i
Cin jÞ þ a¼
6067

ð1Þ

vin t V head 3

vin V head

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Figure 1. Time course of gaseous N and CO2 emission from soil during an incubation experiment under anaerobic conditions at 25 °C (Experiment 1). The data shown are means ( standard error of three replicates.

where t is sampling time during headspace flushing (min); i is series number of measurement for determining a flux (i = 1, 2, ..., 5); C*i is corrected concentration of N2, N2O, NO, and CO2 considering dilution effects (ppmv); Cm is measured gas concentration of N2, N2O, NO, and CO2 (ppmv); Cin is inlet gas concentration during sampling (ppmv); Vhead is headspace volume (mL); vin is inlet gas flow rate during sampling (mL min
1); and bi is a constant, determined by the Cm i . Following recalculation of the headspace concentrations, the emissions of N2, N2O, NO, and CO2 were determined from the temporal linear change of the mixing ratios over time. 

F ¼

V head 3 ðΔCi
ΔCL Þ 3 M 273 3 273 þ T Mds 3 MV 3 103

ð2Þ

where F is N2, N2O, NO, or CO2 emission (μg N or C h
1 kg
1 ds); ΔCi* is change in N2, N2O, NO, or CO2 concentration (ppmv h
1); ΔCL is inherent leakage rate of the system (for N2 only, 0.4 ppmv N2 h
1); M is weight of pure N or C per mole in N2, N2O, NO, or CO2 (28, 28, 14, and 12 g mol
1, respectively); Mds is dry weight of the soil (kg); MV is molar volume of the gas at 273 K and 1013 hPa (L mol
1); and T is incubation temperature (°C). The system0 s inherent leakage rate for N2 (ΔCL) was determined with empty vessels and with vessels filled with sterilized soil (double autoclaved). Both approaches yielded a constant N2 leakage rate of 0.4 ppmv h
1 (Figure S4). Soil Analysis. The soil samples were analyzed for water content, mineral N (as NO3
and ammonium (NH4þ)), DOC, and soil microbial biomass C and N (SMBC/N) at the start and end of each experiment (Supporting Information).

’ RESULTS Experiment 1: Anaerobic Gas Exchange
Anaerobic Incubation. In this experiment, anaerobic conditions were estab-

lished at time zero by purging with pure He. Shortly after the establishment of anaerobiosis, emission of both N2O and NO dramatically increased (Figure 1). NO emission peaked at approximately 5 h, whereas N2O emission peaked somewhat later at approximately 25 h. The peak N2O emission was 400 μg N h
1 kg
1 ds, which was approximately four times higher than the peak NO emission (100 μg N h
1 kg
1 ds). N2 emission

could only be measured following complete gas exchange (50 h). However, at this time N2 emission was already 500 μg N h
1 kg
1 ds, while those of N2O and NO were 130 μg N h
1 kg
1 ds at the end of the experiment. Soil respiration showed a strong decline after the induction of anaerobiosis, with respiratory fluxes peaking at the same time as N2O emission (Figure 1). Thereafter, respiration declined and stayed constant at 200 mg C h
1 kg
1 ds until the end of the experiment. Soil NO3
levels decreased from 124 mg N kg
1 ds at the start of the experiment to 20 mg N kg
1 ds at the end, whereas soil NH4þ levels increased from 4.6 to 12.3 mg N kg
1 ds (Table 1). Experiment 2: Aerobic Gas Exchange
Anaerobic Incubation. N2 emission under aerobic incubation conditions, i.e., following soil gas exchange, was close to the detection limit and as low as 1 μg N h
1 kg
1 ds. Moreover, N2O and NO emissions were low at 40 times higher than that of NO. N2 emission declined steadily following the peak emission, but remained at approximately 120 μg N h
1 kg
1 ds from hours 200 to 450. Only after this period N2 emission declined further and finally approached zero (approximately 600 h). Cumulative N2 emissions were 65.0 ( 0.8 mg N kg
1 ds, representing 65% of the measured change in soil NO3
concentration (Table 1). Soil respiration was high at the start of the experiment but decreased steadily during the aerobic gas exchange period to values of approximately 200 μg C h
1 kg
1 ds. The initialization of anaerobiosis was accompanied by increased CO2 emission, and peak CO2 emission (54 h) followed those of N2O and NO (48 h). Experiment 3: Aerobic Gas Exchange at Lowered Incubation Temperature
Anaerobic Incubation. In Experiment 2 the chosen incubation temperature of 25 °C had already fueled high rates of soil respiration at the start of the experiment. During this phase, easily degradable carbon substrates may have been consumed, which can affect denitrification during anaerobic incubation. Therefore, the aerobic gas exchange was conducted at 2 °C instead of 25 °C. Thereafter, we changed to anaerobic conditions (2 °C) before finally setting the incubation temperature to 25 °C (Figure 3). N2 emission was close to zero under 2 °C aerobic incubation conditions. Following the initialization of anaerobiosis—still at 2 °C—N2 emission increased to 80 μg N h
1 kg
1 ds. Peak emissions of N gases were observed following the increase in the incubation temperature from 2 to 25 °C. As in Experiment 2, N2O and NO emissions peaked markedly before N2 emission. The peak N2 emission was 1200 μg N h
1 kg
1 ds, whereas the peak emissions of N2O and NO were lower by a factor of approximately 2
3. Cumulative N2, N2O, and NO emissions were 24.3 ( 0.1, 12.6 ( 0.4, and 10.1 ( 0.3 mg N kg
1 ds, accounting for 46%, 24%, and 19% of the measured changes in the soil NO3
pool, respectively (Table 1). Figure 3 shows that the results could be accurately reproduced with regard to the timing and magnitude of N gas fluxes, even though the time period between the two experiments was >2 weeks. N Gas Emissions and N-Balance. Table 1 provides overviews of the cumulative emissions of N gases and of the changes in soil inorganic N pools, SMBN, DOC, and SMBC during the individual experiments. SMBN did not significantly change in any 6068

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Table 1. Cumulative N Gas and CO2 Emissions and Changes in Soil Inorganic N, Soil Microbial Biomass Nitrogen (SMBN), and Carbon (SMBC) during the Entire Incubation Periods in Experiments 2, 3a, and 3ba Cumulative emissions Experiment

NO3




NH4þ

SMBN

DOC

SMBC

N2

N2O

NO

CO2

RR(%)

t0

123.8 ( 5.4

4.6 ( 0.5

n.a.

228.9 ( 5.8

n.a.

t1

19.9 ( 2.0

12.3 ( 0.3

n.a.

75.3 ( 0.7

n.a.

20.6 ( 1.1

9.5 ( 0.5

1.6 ( 0.04

21.5 ( 0.3

30.5

2

t0 t1

101.8 ( 2.8 1.2 ( 0.4

7.9 ( 0.3 6.1 ( 0.7

n.a. n.a.

265.2 ( 3.7 66.0 ( 1.4

n.a. n.a.

65.0 ( 0.8

1.7 ( 0.1

0.27 ( 0.01

56.0 ( 2.5

66.5

3a

t0

52.7 ( 0.4

4.5 ( 0.9

59.3 ( 1.3

339.2 ( 3.2

309.7 ( 88.0 24.3 ( 0.1

12.6 ( 0.4

10.1 ( 0.3

84.2 ( 13.0

89.4

3b

t0

22.5 ( 0.01 12.4 ( 0.3

10.0 ( 0.1

80.3 ( 8.7

91.3

1

t1 t1

0.2 ( 0.02 49.4 ( 0.7 0.2 ( 0.004

14.2 ( 0.5

58.1 ( 1.2

191.8 ( 14.9

315.2 ( 95.8

0.2 ( 0.02

53.2 ( 1.5

296.7 ( 3.6

262.3 ( 80.3

9.0 ( 0.9

52.3 ( 2.5

128.9 ( 6.9

296.2 ( 84.9

The data are means ( standard error for three replicate observations (3 vessels) with units of mg N or C kg
1dry soil. RR is the nitrogen recovery rate, which is the sum of the cumulative N gas emissions (N2, N2O, and NO) expressed as a percentage of the change in the soil NO3
pool. t0 and t1 are the time at the start of incubation and at the end of incubation, respectively. n.a.: not available. a

of the experiments. However, significant changes in the soil NO3
levels (p < 0.01) between the start and end of the incubation period were observed in Experiments 2 and 3, wherein the NO3
pool decreased from approximately 100 or 50 mg N kg
1 ds to values close to zero. In view of uncertainties in the measurements, the calculated average recovery rate of NO3
in gaseous products of about 90% demonstrates that the system is suitable for studying the dynamics of total N gas emission in soil.

’ DISCUSSION Evaluation of the Measuring System. Measurements of N2 production by denitrification in soils remain challenging. Here the measurement problem was addressed by exchanging the soil atmosphere with an N2-free gas mixture, which lowered the background N2 concentration to a few ppmv. The principles of this technique—the gas-flow-soil-core technique—had been previously introduced21,22 and further developed for soil.14
16 In this work, the technique was further refined with regard to (a) testing and use of a new micro-TCD for online N2 detection; (b) simultaneous detection of NO, N2O, N2, and CO2 emissions using a revised and updated instrumentation setup; (c) optimization of the incubation conditions for a full accounting of gaseous N losses; and (d) assessment of the repeatability of the denitrification measurements. The use of the new micro GC-TCD (Agilent Technologies) has the advantage that the small 200-nL internal volume eliminates peak broadening, thus providing better data quality and easier quantitation. In conjunction with a narrow-bore capillary column and the micromachined injector system one can achieve a measuring precision of 0.2 ppmv. The cross-sensitivity of the micro-TCD for N2 and O2 could be solved here by optimizing analytical conditions (see Supporting Information). A redesign of the measuring system, with automated headspace gas sampling and automated calibration of the TCD, resulted in a detection limit of 8.1 μg N m
2 h
1 (or 0.23 μg N2
N h
1 kg
1 ds) for soil N2 emission (Table S2). This is approximately 20% better than was described earlier15 for a comparable system and 4-fold better than was described for a gas-flow-soil-core system in Rothamsted Research, North Wyke.16 This enables the use of the system for measuring N2 production also in N limited soils, e.g., in soils from natural ecosystems. Moreover, the detection limits for N2O and CO2 emission are 1 order of magnitude better than

Figure 2. Dynamics of gaseous N and CO2 soil emissions in Experiment 2. Here, an initial phase of aerobic incubation (0
40 h) was followed by a phase where the headspace of the vessel was purged with an O2-free atmosphere for the rest of the incubation period. Incubation temperature: 25 °C. The data shown are the means of three replicates ( standard error.

those of the UK system,16 but similar with regard to N2O in another study.15 Simultaneous measurements of NO emission have not been previously reported. One shortcoming of the gas-flow-soil-core system is the relatively long time needed to complete the soil gas exchange process. With the new setup, using alternating periods of negative pressure and pressurized purging, the soil gas exchange could be completed within 25 h (Figure S5) or two times faster than described earlier.15,16,27 However, during these 25 h, quantifying N2 emission is not possible. This is problematic if the gas exchange for soil air replacement is performed under anaerobic conditions because cumulative losses of N2 will be severely underestimated. This problem can be solved if the soil gas exchange is done under aerobic conditions first while setting the soil temperature to around 2
4 °C to reduce soil microbial activity during the gas exchange period. Such an experimental design allows to successfully calculate total N balances as shown here. In our experiments, changes in soil N pools could be explained by the production of N gases, with nitrogen recovery rates ranging from 67 to 91%. In view of the 6069

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Figure 3. N- and C-gas emission dynamics and reproducibility of the results (a and b are independent experiments) in Experiment 3. The data shown are the means of three replicates. The vertical bars indicate the standard error.

uncertainties in the quantification of soil NO3
levels and gaseous N products, this is an encouraging result. Moreover, the accuracy with which N and C gas emission measurements could be repeated is remarkable. In Experiments 3a and 3b, even though conducted approximately two weeks apart, the temporal dynamics, emission levels, and timing of peak N and C gas emissions were reproducible (Figure 3, Table 1). This was favored by using sieved and previously dried soil, but allowed us to demonstrate the precision of our approach. Timing and Magnitude of N and C Gas Emissions. Denitrification is an enzymatic reaction controlled by the availability of substrates and certain environmental factors (such as temperature, moisture, O2, and pH).28 Following the initialization of anaerobiosis N gas emissions peaked in the following time sequence: NO e N2O < N2. This sequence and the nearly concomitant emission of NO and N2O are in agreement with our present understanding of N gas emission during denitrification and reflect the sequential expression of the enzymes of the denitrification chain following anaerobiosis29
34 as triggered by O2 deficiency and the availability of nitrogen oxides as electron acceptors. In Experiment 3, the observed maximum rates of N2 emission in the soil (which was enriched in nitrate (50 mg N kg
1 ds) and glucose (300 mg C kg
1 ds)) was 1200 μg N2
N h
1 kg
1 ds. This rate is well within the reported range of N2 emissions from clay silt loam soil taken from an arable site in Belgium, which was

enriched with 100 mg KNO3
N kg
1 ds and 950 mg glucose-C kg
1 ds and incubated under anaerobic conditions.35,36 N2 emissions of up to 1300 μg N2
N kg
1 h
1 were also reported for grassland soils amended with approximately the same amounts of glucose and NO3
-N as in our experiments.36 Molar Ratios between Cumulative N and C Gas Emissions. The molar ratios of the N and C gas emissions are summarized in Table S5 and are depicted in Figures 1
3. The N2:N2O ratios were highly variable but widened with the length of the anaerobic incubation period. Under strictly anaerobic conditions, N2 increasingly becomes the dominant N gas emitted (Figure 2). The ratios of N2:N2O emissions for the anaerobic incubation period at 25 °C are in the range of 1.6
1.7 for Experiments 3a/b and approximately 39 in Experiment 2. For temperate forest soils, N2:N2O ratios of approximately 7 for spruce forest soils and 1.9 for soils taken from a beech site have been reported.15 Cumulative N2O fluxes from an arable soil in Belgium exceeded cumulative N2 emission for several days during anaerobic incubation at 25 °C.37 Higher cumulative N2O than N2 emission was also observed in measurements of nitrate- and glucoseamended grassland soils.16 Much wider N2:N2O ratios in the range of 5
200 have been reported for arable soils in Uzbekistan38 and for beech forest soils in southern Germany.27 The few examples given here indicate that N2:N2O ratios can vary significantly, and the main drivers of this variability are the 6070

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Environmental Science & Technology soil nitrate concentration, the availability of easily degradable C substrates, the redox potential, soil moisture, and soil pH.27,39,40 Generally, the largest N2:N2O ratios are expected if available C is high, whereas at high soil nitrate concentrations and low concentrations of easy degradable C the conversion of N2O to N2 is inhibited and N2O may be the sole end product.40,41 This can also been seen in Experiment 2, where soil nitrate levels decreased from approximately 100 mg NO3
-N kg
1 ds at the start of the experiment to nearly zero at the end. N2O emission was in the same range as N2 emission only for the first 20 h after the initialization of anaerobiosis, whereas toward the end of the experiment N2 was the sole product of denitrification. The NO/N2O emission ratio has been used as an indicator of the relative contributions of nitrification versus denitrification to observe NO and N2O emissions. Emission ratios >1 have typically been associated with active populations of nitrifiers and soil conditions favorable for nitrification, whereas emission ratios 1 may be misleading.43 Even though the soil NO3
content at the start of Experiment 2 was two times higher than in Experiments 3a and b, the maximum N2, N2O, and NO emissions in Experiment 2 were significantly lower than those in Experiment 3 (Figures 2 and 3). This was most likely due to the greater availability of easily degradable carbon in Experiment 3, in which the DOC concentration was approximately 20% higher (Table 1). This interpretation is consistent with earlier findings.47 The slight increase in soil NH4þ concentrations is most likely due to anaerobic mineralization of SOM48 or dissimilatory nitrate reduction to ammonium.49 In our study, CO2 emission was closely related to denitrification activity. For an experiment with clayey silt loam amended with nitrate and glucose at optimal soil moisture and incubated anaerobically at 25 °C, the reported molar CO2 to N gas emission ratios were 0.9
2.7.35 In our experiments, the cumulative CO2 to N gas emission ratios were in the range of 0.5
3.0 (Table S5). When glucose is the only C substrate and NO3
is the only electron acceptor, reduction to N2O would produce 1.0 mol CO2 (mol NO3
reduced)
1 and reduction to N2 would result in a CO2/ NO3
molar ratio of 1.25.35 Thus, high CO2 to N gas emission ratios indicate that CO2 emissions may originate from microbial processes other than denitrification (which are not accompanied by N gas emission), e.g., alcohol fermentation, microbial iron reduction, and/or microbial sulfate reduction.50

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed descriptions of principles and methods of the gas-flow-soil-core measurement system and additional supporting figures and tables. This information is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Tel: þ49-8821-183136; e-mail: [email protected]. Present Addresses §

Forschungszentrum J€ulich GmbH, Institute of Bio- and Geosciences, IBG-3 (Agrosphere), 52425 J€ulich, Germany

’ ACKNOWLEDGMENT This work was funded by the German Research Foundation (DFG, FG536, MAGIM), the National Natural Science Foundation of China (40805061, 41021004), and the NitroEurope project. Technical assistance from Meike Sauerwein, Guangren Liu, Yinghong Wang, Yang Sun, and Dongsheng Ji is acknowledged. ’ REFERENCES (1) Gruber, N.; Galloway, J. N. An earth-system perspective of the global nitrogen cycle. Nature 2008, 451 (7176), 293–296. (2) Erisman, J. W.; Sutton, M. A.; Galloway, J.; Klimont, Z.; Winiwarter, W. How a century of ammonia synthesis changed the world. Nat. Geosci. 2008, 1 (10), 636–639. (3) Davidson, E. A.; Seitzinger, S. The enigma of progress in denitrification research. Ecol. Appl. 2006, 16 (6), 2057–2063. (4) Galloway, J. N.; Aber, J. D.; Erisman, J. W.; Seitzinger, S. P.; Howarth, R. W.; Cowling, E. B.; Cosby, B. J. The nitrogen cascade. Bioscience 2003, 53 (4), 341–356. (5) Rockstr€om, J.; et al. A safe operating space for humanity. Nature 2009, 461 (7263), 472–475. (6) Galloway, J. N.; Townsend, A. R.; Erisman, J. W.; Bekunda, M.; Cai, Z. C.; Freney, J. R.; Martinelli, L. A.; Seitzinger, S. P.; Sutton, M. A. Transformation of the nitrogen cycle: Recent trends, questions, and potential solutions. Science 2008, 320 (5878), 889–892. (7) Groffman, P. M.; Altabet, M. A.; B€ohlke, J. K.; Butterbach-Bahl, K.; David, M. B.; Firestone, M. K.; Giblin, A. E.; Kana, T. M.; Nielsen, L. P.; Voytek, M. A. Methods for measuring denitrification: Diverse approaches to a difficult problem. Ecol. Appl. 2006, 16 (6), 2091–2122. (8) Seitzinger, S.; Harrison, J. A.; Bohlke, J. K.; Bouwman, A. F.; Lowrance, R.; Peterson, B.; Tobias, C.; Van Drecht, G. Denitrification across landscapes and waterscapes: A synthesis. Ecol. Appl. 2006, 16 (6), 2064–2090. (9) Fowler, D.; et al. Atmospheric composition change: Ecosystemsatmosphere interactions. Atmos. Environ. 2009, 43 (33), 5193–5267. (10) Yoshinari, T.; Hynes, R.; Knowles, R. Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem. 1977, 9 (3), 177–183. (11) Mathieu, O.; Lev^eque, J.; Henault, C.; Milloux, M. J.; Bizouard, F.; Andreux, F. Emissions and spatial variability of N2O, N2 and nitrous oxide mole fraction at the field scale, revealed with 15N isotopic techniques. Soil Biol. Biochem. 2006, 38 (5), 941–951. (12) Russow, R.; Sich, I.; Neue, H. U. The formation of the trace gases NO and N2O in soils by the coupled processes of nitrification and denitrification: Results of kinetic 15N tracer investigations. Chemosphere 2000, 2 (3
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