Nitrous oxide production and consumption by denitrification in a grassland: Effects of grazing and hydrology

Science of the Total Environment 532 (2015) 702–710

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Nitrous oxide production and consumption by denitrification in a grassland: Effects of grazing and hydrology Jing Hu, Kanika S. Inglett, Mark W. Clark, Patrick W. Inglett, K. Ramesh Reddy ⁎ Wetland Biogeochemistry Laboratory, Soil and Water Science Department, University of Florida, Gainesville, FL, USA

H I G H L I G H T S • • • • •

We quantified N2O production and consumption potentials along a hydrologic gradient. N2O production and consumption rates followed Gaussian functions of WFPS. NO− 3 was a key factor limiting rates of N2O production and consumption. Wetlands within agroecosystems could reduce both NO− 3 leaching and N2O emissions. Transient areas between wetland and upland could be hot spots of N2O emissions.

a r t i c l e

i n f o

Article history: Received 20 August 2014 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online xxxx Editor: D. Barcelo Keywords: Agroecosystem Wetland Upland Nitrate Glucose Water-filled pore space

a b s t r a c t Denitrification is generally recognized as a major mechanism contributing to nitrous oxide (N2O) production, and is the only known biological process for N2O consumption. Understanding factors controlling N2O production and consumption during denitrification will provide insights into N2O emission variability, and potentially predict capacity of soils to serve as sinks or sources of N2O. This study investigated the effects of hydrology and grazing on N2O production and consumption in a grassland based agricultural watershed. A batch incubation study was conducted on soils (0–10 cm) collected along a hydrological gradient representing isolated wetland (Center), transient zone (Edge) and pasture upland (Upland), from both grazed and ungrazed areas. Production and consumption potentials of N2O were quantified on soils under four treatments, including (i) ambient condition, − and amended with (ii) NO− 3 , (iii) glucose-C, and (iv) NO3 + glucose-C. The impacts of grazing on N2O production and consumption were not observed. Soils in hydrologically distinct zones responded differently to N2O production and consumption. Under ambient conditions, both production and consumption rates of Edge soils were higher than those observed for Center and Upland soils. Results of amended incubations suggested NO− 3 was a key factor limiting N2O production and consumption rates in all hydrological zones. Over 5-d incubation with NO− 3 amendment, cumulative production and consumption of N2O for Center soils were 1.6 and 3.3 times higher than Edge soils, and 3.6 and 7.6 times higher than Upland soils, respectively. However, cumulative N2O net production for Edge soils was the highest, with 2 to 3 times higher than Upland and Center soils. Our results suggest that the transient areas between wetland and upland are likely to be “hot spots” of N2O emissions in this ecosystem. Wetlands within agricultural landscapes can potentially function to reduce both NO− 3 leaching and N2O emissions. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In the context of environmental pollution and global climate change, denitrification receives much of the attention because of its control on nitrate (NO− 3 ) removal from soils, and involvement in nitrous oxide (N2O) emissions to the atmosphere (Bouwman et al., 2013; Saggar et al., 2013). Denitrification has been recognized as a major process for ⁎ Corresponding author at: Soil and Water Science Department, 2181 McCarty Hall, PO Box 110290, University of Florida, Gainesville, FL 32611, USA. E-mail address: krr@ufl.edu (K. Ramesh Reddy).

http://dx.doi.org/10.1016/j.scitotenv.2015.06.036 0048-9697/© 2015 Elsevier B.V. All rights reserved.

N2O production, and the only biological mechanism for N2O consumption in soils (Firestone and Davidson, 1989; Pérez et al., 2006). Nitrous oxide, an intermediate of denitrification, is produced by reducing NO− 3 or nitrite (NO− 2 ) through a series of steps catalyzed by intracellular enzymes, i.e., nitrate reductase (Nar), nitrite reductase (Nir), and nitric oxide reductase (Nor) (Saggar et al., 2013). A portion of the N2O formed during denitrification may be emitted to the atmosphere before being consumed, a step of reducing N2O to dinitrogen gas (N2) catalyzed by nitrous oxide reductase (Nos) (Saggar et al., 2013). Therefore, the environmental impacts of denitrification depend not only on the capability of NO− 3 removal (equivalent to N2O production), but also on the

J. Hu et al. / Science of the Total Environment 532 (2015) 702–710

partitioning of its end-products into N2O and N2 (N2O consumption/ production ratio). Understanding the biogeochemical controls regulating processes of N2O production and consumption during denitrification is vital for evaluating the reduction of NO− 3 loads to adjacent water bodies, and essential for predicting N2O emissions and developing effective mitigation technologies. Denitrification rates in soils exhibit a high degree of variability both spatially and temporally due to variations in environmental factors (Bouwman et al., 2013; Saggar et al., 2009). Several studies have examined the regulators of the N2O:N2 ratio, such as NO− 3 concentration (Senbayram et al., 2012; Weier et al., 1993; Zaman et al., 2008), availability of organic carbon (C) (Jahangir et al., 2012; Murray et al., 2004), oxygen (O2) (Morley and Baggs, 2010), pH (Stevens et al., 1998; Liu et al., 2010), soil water content (Weier et al., 1993; Liu et al., 2007), and microbial community composition (Cavigelli and Robertson, 2000, 2001). These factors interactively regulate denitrification through a hierarchical system (Beauchamp, 1997). Active denitrifying microorganisms, NO− 3 , and available organic C are considered as the “proximal factors” which directly affect denitrification and consequently regulate N2O production and consumption (Beauchamp, 1997). However, how the “proximal factors” interact and regulate N2O production and consumption under various environmental conditions of soils are still not well understood (Saggar et al., 2013). Grasslands cover approximately 25% of the earth's surface (Saggar et al., 2009), and are a major source of nitrogen (N) to the atmosphere and waters (Murray et al., 2004). In grasslands, grazing and hydrological conditions could be two environmental drivers of importance. Grazing has been reported to alter the availability of NO− 3 and organic C in soils and thus N2O production (Luo et al., 1999a; Patra et al., 2005; Xu et al., 2008). Grazing can have marked influences on soil microbial community composition, abundance, and activity (Le Roux et al., 2008; Patra et al., 2005; Yang et al., 2013). Wetlands within agricultural landscapes act as water storage systems (Moreno et al., 2007). The hydrological gradient that extends from a wetland to the adjacent upland could significantly influence microbial communities (Balasooriya et al., 2008; Yu and Ehrenfeld, 2010) and biogeochemical processes associated with organic C and NO− 3 (Bruland and Richardson, 2004; Jordan et al., 2007).

703

In this work, a laboratory incubation study was conducted to compare the potential production and consumption of N2O during denitrification between grazed and ungrazed areas along a hydrologic gradient. Specific objectives of this study were: (1) to quantify the potential of N2O production and consumption under grazed and ungrazed areas; (2) to quantify the potential of N2O production and consumption under varying hydrological conditions; (3) to identify how grazing and hydrology affect N2O production and consumption through their effects on the “proximal factors”. 2. Materials and methods 2.1. Site description and soil sampling The study site is located on a cow-calf ranch, Larson Dixie, north of Lake Okeechobee in south-central Florida (Fig. 1). The size of the isolated wetland chosen for this study was approximately 1 ha and the surrounding uplands were cow-calf pasture lands with low stocking density of approximately 1 head ha−1 (Cheesman et al., 2010; Dunne et al., 2010). This site was stratified into three zones according to distinct hydrological conditions and characteristic vegetation: isolated wetland center zone (Center), transient edge zone (Edge), and pasture upland zone (Upland). Center had open water present during wetland flooding with a hydroperiod of around 230 d y−1 (Cheesman et al., 2010). Vegetation composition in Center included Pontedaria cordata var. lancifolia (Muhl.) Torr., Bacopa monnieri (L.) Pennell, Panicum hemitomon Schult., Polygonum sp., and Ludwigia repens Forst. Edge had evidence of inundation, but the hydroperiod in Edge (approximately 90 d y−1) was much shorter than Center (Cheesman et al., 2010). Typical vegetation species at the Edge site were Juncus effuses L., Eleocharis baldwinii (Torr) Chapm, Paspalum acuminatum Raddi, and Hydrochloa caroliniensis Beauv. The Upland site (hydroperiod of approximately 2 d y− 1) was dominated by forage grass Paspalum notatum Flugge. Soils of the study site are classified as siliceous, hyper-thermic Spodic Psammaquents (Cheesman et al., 2010). Six transects extending from the center of the wetland to surrounding pasture uplands were set up for the study. Exclosure fences were installed outside of three transects

(a)

Upland

Edge

Center

(b)

Fig. 1. Study site. (a) Location of Larson Dixie in the four priority basins of Okeechobee Basin. White dash line indicates isolated wetland. (b) Study site showing three zones: Center, Edge, and Upland. White dash line indicates the boundary of zones.

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in February 2011 to exclude cattle grazing. Intact soil cores (0–10 cm) were taken from all six transects in January 2012. Along each transect, one location was randomly selected for quadrat sampling within each hydrological zone. Two intact soil cores (7 cm in diameter) were collected within each quadrate and amalgamated into one composite sample. A total of 18 composite soil samples were placed in airtight plastic bags and transported on ice to the Wetland Biogeochemistry Laboratory, University of Florida, where soil samples were stored at 4 °C until further analyses. 2.2. Soil properties Soil samples were weighed and homogenized in airtight plastic bags. Roots and recognizable organic fragments greater than 2 mm in diameter were removed from soils. Soil moisture content was determined from mass loss following oven drying at 70 °C for 72 h (Reddy et al., 2013). Soil pH was measured in a 1:2 (soil:water) suspension after equilibration for 30 min (Reddy et al., 2013). Soil bulk density (BD) was calculated as the dry weight of soil (dried for 72 h at 70 °C) per known soil volume (Reddy et al., 2013). Soil water-filled pore space (WFPS) was determined by Eq. (1) WFPS ¼ moisture content ð%Þ  BD=ð1−BD=PDÞ

ð1Þ

where PD (particle density) of 2.65 g cm−3 was used (Linn and Doran, 1984). Soil organic matter content was estimated by the loss-onignition (LOI) method after combustion of soil at 550 °C in a muffle furnace for 4 h (Wright et al., 2008). Total phosphorous (TP) was determined by dissolving the ash obtained after combustion in 6 M HCl, followed by an analysis of P using ascorbic acid colorimetric method (Method 365.1, USEPA, 1983). Total carbon (TC) and total nitrogen (TN) were analyzed on oven dried and ground subsamples with Thermo Flash EA 1112 elemental analyzer (CE Elantech Inc., USA). Fresh soil subsamples were extracted by 2 M KCl and the extraction was analyzed col− orimetrically for NH+ 4 (Method 350.1, USEPA, 1983) and NO3 (Method 353.2; USEPA, 1983). Extractable organic C (ext-OC) was determined from 0.5 M K2SO4 extraction of chloroform fumigated subsamples (White and Reddy, 2001) with a total organic C analyzer, TOC-5050A (Shimadzu, Norcross, Ga.). Microbial biomass C (MBC) was determined by the fumigation-extraction method (Vance et al., 1987), and a combined extraction efficiency factor kEC = 0.37 was applied to MBC calculation (Sparling et al., 1990). 2.3. N2O production and consumption assay Laboratory slurry incubations were established to determine N2O production and consumption potentials using the acetylene inhibition method (Tiedje et al., 1989). Briefly, two fresh subsamples of each soil (approximately 2.5 g equivalent oven dry weight) were placed in 60 mL serum bottles, and were made into slurries by adding 5 mL distilled de-ionized (DDI) water. All serum bottles were closed with gray butyl stoppers and sealed with aluminum crimps. Soil slurries were purged with N2 gas to create anaerobic conditions. One out of two serum bottles with the same soil sample was randomly chosen, and approximately 10% of headspace gas was replaced with acetylene (C2H2) gas to inhibit N2O reduction activity (Tiedje et al., 1989). All the serum bottles were incubated in the dark at room temperature (23 ± 1 °C) for 5 days. The incubation time of 5 days was employed because C2H2 could become a C source during longer incubation periods (Hill et al., 2004; Dodla et al., 2008). Headspace gas in serum bottles was sampled periodically, and analyzed for N2O on a gas chromatograph (Shimadzu GC-14-A, Shimadzu Scientific Instruments, Columbia, MD) equipped with a Poropak Q column and a 63Ni electron capture detector (ECD). The gas chromatograph used P5 gas (5% methane and 95% argon) as the carrier gas (30 mL min−1), and was operated at temperatures of 70 °C, 120 °C and 300 °C for column, injector and detector, respectively.

This set of incubation bottles without NO− 3 and glucose-C amendment was considered as an ambient treatment (AMB). Three other treatments −1 , NIT), with addition of 5 mL nitrate solution (56 mg KNO− 3 –N L glucose solution (288 mg C6H12O6–C L−1, GLU), and glucose + nitrate −1 and 288 mg C6H12O6–C L−1, GAN), resolution (56 mg KNO− 3 –N L spectively, instead of DDI water were also included in this study. Cumulative N2O production was determined by monitoring the total amount of N2O accumulated in the set of incubation bottles with presence of 10% C2H2 (Eq. (2)), and the cumulative net production was determined in the set of incubation bottles without C2H2 (Eq. (3)). The difference between production and net production was cumulative N2O consumption (Eq. (4)). Cumulative N2 O production ¼ N2 Owith




ð2Þ

acetylene

Cumulative N2 O net production ¼ N2 Owithout Cumulative N2 O consumption ¼ N2 Owith


acetylene


acetylene

– N2 Owithout

ð3Þ
acetylene

:

ð4Þ Initial N2O production rate, consumption rate and net production rate were calculated from the change in cumulative production, consumption and net production in the first 6 h of incubation using the linear regression. 2.4. Statistical analysis Statistical analysis was performed using SAS 9.2 (SAS Institute Inc., Cary, NC), and JMP v.8 statistical software (SAS Institute Inc., Cary, NC). A two-way ANOVA was applied to test the effects of hydrological zone, grazing and their interaction on soil biogeochemical properties. A split-plot linear model (Proc MIXED) was developed to test the effects of hydrological zone, grazing, treatment, and their interactions on rates of N2O production, consumption and net production. A split-plot repeated measures linear model (Proc GLIMMIX) was developed to test mean differences of cumulative N2O production, consumption and net production with consideration of 4 factors of interest (hydrological zone, grazing, treatment, and time) and their interactions. A heterogeneous first-order autoregressive structure was used to model the correlation among observations taken from the same soil sample under one treatment over time. Tukey's test was conducted for multiple comparisons. Pearson's Product correlations were calculated to determine correlation coefficients between variables. Stepwise multiple regression was employed to select the essential variables to predict N2O production and consumption. P values of 0.1 and 0.05 were used as entry and staying values, respectively, in the stepwise selection method (Majchrzak et al., 2001). Principal component analysis (PCA) was performed on data including soil biogeochemical properties, and initial rates of N2O production, consumption and net production under ambient condition. 3. Results 3.1. Soil biogeochemical properties A two-way ANOVA revealed that hydrological zones had significant effects (P b 0.05) on most of the soil biogeochemical properties, including soil moisture, pH, and LOI, and also had marginally effects on soil NO− 3 content (P = 0.053). In contrast, significant effects of grazing, and the interaction of grazing and hydrological zones were only observed on TP and MBC (Table 1). Soil moisture and WFPS decreased in the order of Center, Edge and Upland soils (Table 2), while pH of Center soils was significantly higher (P b 0.01) than Edge and Upland soils. Significant differences (P b 0.01) in LOI values indicated the soil organic matter content for Center soils

J. Hu et al. / Science of the Total Environment 532 (2015) 702–710 Table 1 Summary of effects of hydrological zones, grazing, and their interaction on soil biogeochemical parameters. * — P b 0.05, ** — P b 0.01, *** — P b 0.001, and NS — no significant difference.

Moisture WFPS pH BD LOI TC TN TP ext-OC MBC NO− 3 NH+ 4

Zone

Grazing

Zone × Grazing

*** *** ** NS ** ** ** ** NS * NS (P = 0.053) **

NS NS NS NS NS NS NS * NS NS NS NS

NS NS NS NS NS NS NS NS NS * NS NS

WFPS: water-filled pore space; BD: bulk density; LOI: loss on ignition; TC: total carbon; TN: total nitrogen; TP: total phosphorus; ext-OC: extractable organic carbon; MBC: micro+ bial biomass carbon; NO− 3 : 2 M KCl extractable nitrate; and NH4 : 2 M KCl extractable ammonium.

was higher than Upland soils. Microbial biomass C was significantly higher (P b 0.05) for Edge than Upland soils. Soil NO− 3 content of Edge soils was significantly higher (P b 0.05) than Upland soils. 3.2. Initial N2O production and consumption rates

Table 3 Summary of effects of hydrological zones, grazing, treatment and their interaction on initial rates of N2O production, consumption, and net production. * — P b 0.05, ** — P b 0.01, and *** — P b 0.001. Source

DF

F-value Production

Consumption

Net production

Hydrological zone (H) Grazing (G) Treatment (T) H×G H×T G×T H×G×T

2 1 3 2 6 3 6

0.02 0.49 99.5*** 0.60 25.3*** 0.09 0.46

1.15 0.12 16.8*** 1.68 4.5** 1.67 0.78

1.75 0.16 121.5*** 2.78 33.6*** 0.47 2.09

Stepwise multiple regression analysis identified NO− 3 as the only main predictor, which explained 92% and 91% of the variation in ambient N2O production and consumption rates, respectively. Ambient N2O production and consumption rates, as well as NO− 3 , followed Gaussian functions of WFPS (Fig. 3). The peaks of Gaussian curves for ambient N2O production, consumption and NO− 3 all fell in the range of 70–75% WFPS. Principal component analysis (PCA) on soil biogeochemical parameters and the ambient rates of N2O production, consumption and net production indicated that 54% of the data variability was explained by principal component 1, while principal component 2 explained 23% (Fig. 4). Component 1 was significantly correlated (P b 0.01) with

Initial rates of N2O production and consumption were significantly influenced by treatment (P b 0.001) and its interaction with hydrological zones (P b 0.01). But neither grazing nor its interaction with other variables showed significant impacts (Table 3). Therefore, soils from both grazed and ungrazed areas in the same hydrological zone were considered as replicates.

1.5

3.2.1. Ambient N2O production and consumption rates Although the rates of N 2O production and consumption under ambient conditions (AMB) were not significantly different within three hydrological zones, Edge soils appeared to have relatively higher N2O production and consumption rates than Center and Upland soils (Fig. 2). Net production rate of N2O was significantly correlated (r = 0.97, P b 0.001) with production rate (Table 4). The highest ambient N2O net production rate was also observed for Edge soils (0.09 ± 0.03 mg N2O–N kg−1 dry soil h− 1), followed by Center (0.04 ± 0.03 mg N2O–N kg− 1 dry soil h− 1) and Upland soils (not detectable). The ambient N2O production and consumption rates were positively correlated (P b 0.05) with NO− 3 , MBC, LOI, TN, TC and TP (Table 4).

0.3

standard error, n = 6). Different letters denote significantly different means (Tukey's test, α = 0.05). Upland Moisture (%) WFPS (%) pH BD (g cm−3) LOI (%) TC (g C kg−1) TN (g N kg−1) TP (mg P kg−1) ext-OC (mg C kg−1) MBC (g C kg−1) −1 NO− ) 3 (mg N kg −1 NH+ ) 4 (mg N kg

Edge b

31.7 ± 1.9 41 ± 2 c 4.72 ± 0.12 b 0.87 ± 0.03 a 9.3 ± 0.9 b 43 ± 4 b 3.2 ± 0.3 b 182 ± 24 b 711 ± 90 a 1.1 ± 0.2 b 0.03 ± 0.01 b 5.4 ± 1.2 b

54.6 ± 7.0 65 ± 6 b 4.80 ± 0.05 b 0.84 ± 0.04 a 12.6 ± 1.4 ab 57 ± 8 ab 4.7 ± 0.7 ab 256 ± 32 b 1057 ± 125 a 1.9 ± 0.2 a 3.67 ± 1.31 a 5.7 ± 0.6 b

Production Consumption

0.9 0.6

a

71.5 ± 2.7 94 ± 2 a 5.29 ± 0.08 a 0.88 ± 0.02 a 15.1 ± 1.1 a 74 ± 5 a 5.9 ± 0.3 a 347 ± 27 a 858 ± 77 a 1.7 ± 0.2 ab 1.69 ± 0.73 ab 24.6 ± 6.4 a

WFPS: water-filled pore space; BD: bulk density; LOI: loss on ignition; TC: total carbon; TN: total nitrogen; TP: total phosphorus; ext-OC: extractable organic carbon; MBC: micro+ bial biomass carbon; NO− 3 : 2 M KCl extractable nitrate; NH4 : and 2 M KCl extractable ammonium.

A

B

a

C b

a

C b

0.0 1.5

Edge

1.2 0.9 A

0.6 0.3

AB B

B a

a

a

a

0.0 1.5

A

Center

1.2 B

0.9

Center a

Upland

1.2

N2O rate (mg N2O-N kg-1 dry soil h-1)

Table 2 Selected biogeochemical parameters for soils from Center, Edge and Upland (mean ±

705

0.6 0.3

C

ab b

a C

b

0.0 AMB

NIT

GLU GAN

Treatment Fig. 2. N2O production (gray) and consumption (white) rates under different treatments (AMB: ambient treatment; NIT: treatment with NO− 3 ; GLU: treatment with glucose-C; GAN: treatment with glucose-C and NO− 3 ). Bars represent mean (n = 6); error bars represent standard error of the mean. Different uppercase and lowercase letters represent significantly different means (Tukey's test, α = 0.05) of production and consumption rates between treatments within each hydrological zone.

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J. Hu et al. / Science of the Total Environment 532 (2015) 702–710

6

Table 4 Pearson's product correlation coefficients (r) for correlation of ambient rates of N2O production, consumption, and net production with selected biogeochemical parameters (n

Edge - Ungrazed

4

Moisture WFPS pH BD LOI TP TN TC ext-OC MBC NO− 3 NH+ 4 Production

Consumption

0.29 0.23 −0.08 −0.22 0.61 0.49 0.59 0.57 0.40 0.72 0.96 −0.25 –

NS NS NS NS ** * ** * NS *** *** NS

0.31 0.19 −0.10 −0.40 0.61 0.40 0.62 0.58 0.42 0.76 0.96 −0.18 0.93

Net production NS NS NS NS ** NS ** * NS *** *** NS ***

0.26 0.24 −0.06 −0.10 0.57 0.51 0.54 0.53 0.36 0.65 0.90 −0.27 0.97

NS NS NS NS * * * * NS ** *** NS ***

WFPS: water-filled pore space; BD: bulk density; LOI: loss on ignition; TC: total carbon; TN: total nitrogen; TP: total phosphorus; ext-OC: extractable organic carbon; MBC: micro+ bial biomass carbon; NO− 3 : 2 M KCl extractable nitrate; NH4 : and 2 M KCl extractable ammonium.

parameters including moisture, WFPS, LOI, TC, TN, TP, ext-OC, MBC, NO− 3 , ambient rates of N2O production, consumption, and net production, in which LOI, TN and TC contributed the most to Component 1. The parameters significantly correlated (P b 0.05) with Component 2 + were moisture, WFPS, pH, NO− 3 , NH4 , and ambient rates of N2O

0.4

-1 -1

N2O-N mg kg dry soil h

2

R =0.445 P = 0.012

0.2 0.1 0.0

0.4 N O consumption rate 2 Upland Edge Center

0.3 0.2 2

R =0.614 P < 0.001

0.1 0.0 -1 -

NO3 -N mg kg

Center - Ungrazed Upland - Grazed

2

Edge - Grazed

Center

Center - Grazed

0 -2

Upland

Edge

-4 -6 -6

-4

2 0 -2 Component 1 (54%)

4

6

Fig. 4. Score plot of principal components analysis (PCA) on soil biogeochemical parameters, and ambient rates of N2O production, consumption and net production. Solid and open markers represent soils collected from ungrazed and grazed areas, respectively; Circles, squares and triangles represent Upland, Edge, and Center soils, respectively.

production and consumption, but pH, NH+ 4 and WFPS had the highest loading on Component 2. The score plot indicated that Center soils were distinct from Upland soils on the ordination axis Component 1. Edge soils could be hardly separated from other soils on neither ordination axis.

N2O production rate

0.3

10

Component 2 (23%)

= 18). * — P b 0.05, ** — P b 0.01, *** — P b 0.001, and NS — no significant difference. Production

Upland - Ungrazed

NO3

-

8 2

R =0.615 P < 0.001

6 4 2 0 20

40

60

80

100

Water-filled pore space (%) Fig. 3. Three-parameter Gaussian model fit for ambient N2O production rate (top), N2O consumption rate (middle), and soil NO− 3 content (bottom). Black diamonds, open squares, and gray triangles represent Upland, Edge and Center soils, respectively.

3.2.2. N2O production and consumption rates under amended conditions When NO− 3 was amended (NIT), N2O production rates were significantly increased (P b 0.001) for Center and Upland soils and a slight increase (P = 0.081) was observed for Edge soils as compared to the measurements under ambient conditions (AMB) (Fig. 2). Unlike that under ambient conditions, N2O production rates significantly varied among hydrological zones (P b 0.001), with the highest rates observed for Center soils, followed by Edge and Upland soils (Fig. 2). In contrast to N2O production rates, differences in N2O consumption rates were not significant within hydrological zones under treatment NIT, with slightly higher (P = 0.081) values for Center soils than Upland soils (Fig. 2). Compared to ambient conditions, NO− 3 addition significantly increased N2O consumption rates for Upland soils (P b 0.001), moderately increased the rates for Center soils (P = 0.062), but not affected Edge soils. In contrast to the addition of NO− 3 , amendment of glucose-C only (GLU) did not vary the rates of N2O production and consumption from ambient conditions (Fig. 2). When soils were amended with both NO− 3 and glucose-C (GAN), N2O production rates were significantly increased for Center (P b 0.001), Edge (P b 0.01) and Upland (P b 0.001) soils as compared to the ambient conditions (AMB). Moreover, compared to the treatment with only addition of NO− 3 (NIT), N2O production rates under treatment GAN were significantly higher (P b 0.001) for Center and Upland soils but not for Edge soils (Fig. 2). Significant differences (P b 0.001) in N2O production rate were observed among hydrological zones even both NO− 3 and glucose-C were not limited under treatment GAN, with Center soils having N2O production rates 2 and 6 times higher than Edge and Upland soils, respectively (Fig. 2). On the other hand, N2O consumption rates were significantly increased (P b 0.001) by addition of both NO− 3 and glucose-C for Center and Upland soils, but not for Edge soils, compared to the ambient conditions (AMB). In addition, N2O consumption rates under treatment GAN were not significantly different from treatment NIT, with only a slight increase (P = 0.087) for Center soils (Fig. 2). Nitrous oxide consumption rates were not identical between hydrological zones under treatment GAN, with significantly higher (P b 0.05) rates for Center soils than Edge and Upland soils.

J. Hu et al. / Science of the Total Environment 532 (2015) 702–710

significantly (P b 0.05) increased from less than 0.4 during the early stage of incubation to higher than 0.7 at 68 h.

3.3. Cumulative N2O production and consumption

-1

Cumlative N2O production/consumption (mg N2O-N kg dry soil)

Cumulative N2O production and consumption over 5 days of the incubation period were significantly (P b 0.05) influenced by hydrological zones, treatment, and their interaction, but were not affected by grazing or its interaction with other factors. Therefore, samples from grazed and ungrazed area in the same hydrological zone were also treated as replicates, with the focus on the effects of hydrological zones (Fig. 5). When NO− 3 was amended (NIT), cumulative N2O production and consumption followed similar increasing patterns throughout the experimental period except for cumulative consumption for Center soils. For the Center soils, cumulative consumption of N2O had a faster increase after 6 h, while cumulative net production of N2O reached a plateau at 20 h, and appeared to maintain constantly in the range of 8.4–11.9 mg N2O–N kg−1 dry soil (Fig. 5). This pattern resulted in a significant increase (P b 0.05) in ratio of consumption to production for Center soils, whereas no significant difference in consumption to production ratio was observed for Upland and Edge soils throughout the incubation. At the end of days of the incubation, Center soils had significantly higher (P b 0.05) cumulative N2O production and consumption than Edge and Upland soils, but Edge soils had the highest cumulative N2O net production, which was 2.7 and 2.2 times of that for Center and Upland soils, respectively. The ratio of cumulative consumption to production was 0.87 ± 0.05, 0.43 ± 0.06, and 0.50 ± 0.05 for Center, Edge, and Upland soils, respectively. Cumulative N2O production and consumption under treatment GAN significantly differed from treatment NIT (P b 0.05) (Fig. 5). Increased N2O production was observed at the beginning of the incubation by further addition of glucose-C when NO− 3 was not limited; but N2O consumption did not respond as fast as N2O production. Nitrous oxide consumption increased rapidly after 20 h for all soils, and net consumption, i.e. a decrease of cumulative net production with time, even occurred later. The consumption to production ratio reached 0.91 ± 0.09 for Center soils at 44 h; while the ratio for Edge and Center soils

100

Upland - GAN

Upland - NIT

75

Production Consumption Net Production

50

707

25

4. Discussion 4.1. Effects of grazing on N2O production and consumption Effects of grazing on denitrification appear to be inconclusive in previous studies. Stimulating effects of grazing on denitrification have been observed in several grassland ecosystems (Le Roux et al., 2003; Luo et al., 1999a,b), which are predominantly brought about by high quantities of N in urine and dung exceeding the plant requirement, and compaction (high BD) of soil caused by treading (Oenema et al., 1997). Other studies have reported that long-term grazing reduced NO− 3 , and organic N and C content in soils, and resulted in decreased denitrification (Xu et al., 2008). No effect of grazing on denitrification has also been reported (Groffman et al., 1993). In our study, we observed no significant effect of grazing on most of the soil biogeochemical parameters based on a random soil sampling from both grazed and ungrazed areas (Table 1, Fig. 4). Grazing impacts were not found on N2O production and consumption either (Table 3). A low stocking density in our study could be a primary reason why grazing did not influence denitrification and associated soil biogeochemical properties. Grasslands that have experienced intensive grazing generally have higher denitrification enzyme activity as compared to those subjected to light grazing (Le Roux et al., 2003). The stocking rate in our study site was approximately 1 head ha− 1, which is relatively lower than that reported in studies where significant effects of grazing on denitrification were observed, e.g. 8 head ha−1 (Le Roux et al., 2003). Furthermore the short duration of ungrazed treatment might be another important reason for no effect of grazing. The exclosure fence was installed in February 2011 and our soil samples were collected in January 2012. Prior to the installation of exclosure fences, the whole area was under grazing with a low cattle density. Approximately one year duration might not be long enough for the natural recovering process in ungrazed areas from the impacts of grazing. Significant effects of grazing on denitrification have been commonly observed in ecosystems that have experienced long-term grazing, for example, 13 years (Le Roux, et al., 2003), 17 years (Xu et al., 2008), and more than 33 years (Frank and Groffman, 1998; Frank et al., 2000). 4.2. Effects of hydrology on N2O production and consumption

0 100

Edge - NIT

Edge - GAN

Center - NIT

Center - GAN

75 50 25 0 100 75 50 25 0 0

20 40 60 80 100 120 Time (h)

0

20 40 60 80 100 120 Time (h)

Fig. 5. Cumulative N2O production (black circles), consumption (open squares), and net production (gray triangles) under two treatments (NIT: treatment with NO− 3 ; GAN: treatment with glucose-C and NO− 3 ). Points represent means (n = 6), and error bars represent standard error of the mean.

4.2.1. Ambient N2O production and consumption rates The differences in initial rates of N2O production and consumption among hydrological zones under ambient conditions were probably due to greater NO− 3 for Edge soils than Center and Upland soils (Table 2). Both N2O production and consumption rates were strongly correlated with NO− 3 content (P b 0.001). Nitrate, as an electron acceptor for microorganisms involved in denitrification, has been reported to be directly proportional to N2O production (Senbayram et al., 2012; Weier − et al., 1993). The presence of N2O or NO− 3 (possible N2O from NO3 reduction) is required to induce the increase in N2O consumption during the anaerobic incubation (Firestone and Tiedje, 1979). Higher NO− 3 , therefore, also led to a higher N2O consumption for Edge soils. Ambient rates of N2O production and consumption, and NO− 3 showed similar Gaussian functions of WFPS (Fig. 3). Most likely, the high WFPS (94.4 ± 2.3%) for Center soils created anaerobic conditions in soils (Reddy and DeLaune, 2008), inhibiting the nitrification process (Ullah and Moore, 2009). Nitrification has been reported to decrease when WFPS is higher than 60% (Linn and Doran, 1984). Low capability of nitrification for Center soils resulted in low NO− 3 , subsequently low N2O production and consumption rates, and accumulation of NH+ 4 (Table 2). On the contrary, low WFPS (40.8 ± 2.3%) for Upland soils ceased the aeration limitation, but the low water content in soils could also lead to slower microbial activities and low inorganic N

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− (NH+ 4 + NO3 ) content (Table 2). Optimal rates of microbial nitrification and respiration (microbial activity) were reported at WFPS of 60% (Linn and Doran, 1984). Therefore, Edge soils had WFPS (65 ± 6%) for producing higher NO− 3 , leading to higher N2O production and consumption rates. Our study also showed regulation of organic C on denitrification, as evidenced by a significant and positive correlation (P b 0.01, Table 4) between organic matter content (LOI) and ambient N2O production and consumption rates. However, ext-OC, which is presumed to be an index of available organic C to denitrifiers, was not significantly correlated with N2O production or consumption rates. Hill and Cardaci (2004) and Jahangir et al. (2012) also found poor relationships between water-soluble organic C and N2O production, whereas N2O production has been reported to be significantly correlated with soil total organic C content (Well et al., 2001; Jahangir et al., 2012). This suggests that some of the dissolved organic C may not be available for denitrifiers (Dodla et al., 2008). The quality of organic C, such as chemical structure, rather than the solubility determines the availability of organic C to denitrifiers (Dodla et al., 2008). The organic C quality is likely to be distinct because of distinct vegetation compositions (organic C input), and varying decomposition rates (organic C output) for various hydrological zones. Community composition of microorganisms involved in denitrification could also contribute to the different ambient N2O production and consumption rates in various hydrological zones (Cavigelli and Robertson, 2000), as evidenced by the significantly different production and consumption rates when availability of NO− 3 and organic C were not limited (under treatment GAN). The effects of microbial community composition on N2O production and consumption rates are discussed below.

4.2.2. Limitation on N2O production and consumption rates Results from amendment studies further revealed the effects of three proximal factors, including NO− 3 , available organic C, and activity of denitrifiers, on N2O production and consumption along the hydrological gradient. With the addition of NO− 3 , soils exhibited significant increases in N2O production and consumption rates but not with addition of glucose-C, suggesting the available NO− 3 limited rates of N2O production and consumption in all hydrological zones, which is in agreement with previous studies (Hernandez and Mitsch, 2007; Song et al., 2014). The availability of NO− 3 has been found as a principal factor limiting N2O production rates in pastures (Luo et al., 1999b). As expected, soils with lower indigenous NO− 3 content, Center and Upland soils, responded to a greater extent to NO− 3 amendment than Edge soils in N2O production rates (Fig. 2). Stronger limiting effects of NO− 3 on N2O production for Upland and Center soils were likely to result in more constrained N2O reductive enzyme, i.e., Nos, and subsequently trigger more limited N2O consumption (Firestone and Tiedje, 1979), as evidenced by greater increases of N2O consumption rates with NO− 3 addition for Upland and Center soils than Edge soils. However, the addition of NO− 3 decreased the ratio of consumption to production rates, which is consistent with previous studies (Cai et al., 2002; Zaman et al., 2008). High NO− 3 content inhibited the portion of N2O reduced to N2, and resulted in lower ratio of N2O consumption to production rates, since NO− 3 is a preferential electron acceptor over N2O to denitrifiers (Firestone et al., 1979; Weier et al., 1993). Addition of glucose-C when NO− 3 was not limited (coupling addition of glucose-C) increased N2O production rates by 73%, 44%, and 60% for Upland, Edge and Center soils, respectively. Increasing labile organic C could increase N2O production rates when sufficient NO− 3 is available (Jahangir et al., 2012; Miller et al., 2009). It is speculated that the existence of high NO− 3 impeded the consumption of N2O (Firestone et al., 1979); therefore, the coupling addition of glucose-C did not significantly affect N2O consumption rates (Fig. 2). Nitrous oxide production and consumption rates were not identical between hydrological zones when NO− 3 and glucose-C have been

provided to soils (GAN in Fig. 2). The significant differences in N2O production and consumption rates were likely due to the differences in composition of the denitrifying communities in soils from various hydrological zones (Cavigelli and Robertson, 2000, 2001; Kjellin et al., 2007). Denitrifier population size is a possible reason of decreasing N2O production and consumption rates along the hydrological gradient when substrates are not limited, since larger microbial population size has been observed in the location with longer hydroperiod (Balasooriya et al., 2008). The contribution of denitrifying community structure, especially fungi to bacteria ratio, to differences in N2O rates also appears plausible. Fungi have been found capable of denitrification, but many fungi are lack of N2O reductase (Chapuis-Lardy et al., 2007). Compared to bacteria, fungi are less tolerant to wet conditions. Abundance of fungi is negatively correlated with soil water content (Reichardt et al., 2001), and fungal activity is also depressed by flooding (Bossio and Scow, 1995). Therefore, it is likely that the higher portion of denitrification was contributed by fungi for Upland and Edge soils compared to Center soils. The lack of capacity in consuming N2O could result in significantly lower N2O consumption rates for Upland and Edge soils than Center soils (Fig. 2). Further studies on composition of microbial communities are needed to validate this hypothesis. 4.2.3. Cumulative N2O production and consumption Cumulative N2O production and consumption could provide insights into the potential of soils in removing NO− 3 by denitrification and its end products after high NO− 3 has been applied. Our results showed substantial differences in cumulative N2O production and consumption among the soils from three hydrological zones. Distinct denitrifying community compositions, as previously discussed, could be a possible reason leading to the different N2O kinetics in various hydrological zones. The pattern of N2O cumulative curves (Fig. 5) is similar to those observed by Cavigelli and Robertson (2000), and Muñoz-Leoz et al. (2011). When NO− 3 was applied, N2O consumption to production ratio for Center soils increased during the incubation period (Fig. 5, NIT). It is likely that more N2O consumption enzyme (Nos) was induced or became more active for Center soils under the anaerobic conditions (Cavigelli and Robertson, 2000). Further addition of labile organic C probably increased NO− 3 depletion via denitrification (Jahangir et al., 2012), resulting in the occurrence of net consumption which was first observed for Center soils (Fig. 5, GAN). The results of cumulative N2O production and consumption indicate that Center soils are more capable of removing NO− 3 by denitrification with a low N2O emission due to the concurrently high potentials for N2O production and consumption. The Edge zone is a highly possible “hot spot” of N2O emission. Wetlands act as both water and nutrient storage systems at the landscape scale (Moreno et al., 2007). The NO− 3 from uplands would be brought into wetland through lateral movement, and be denitrified in wetlands with gaseous end products of high N2:N2O ratio. Otherwise, without wetlands, the NO− 3 would leave the system through leachate before being denitrified since upland soils have much lower capacity of NO− 3 removal compared to wetland soils. Wetlands, therefore, have the ecological functions of reducing NO− 3 leachate and N2O emissions. 5. Conclusions This study found that grazing did not appear to affect N2O production and consumption in this site possibly due to a low stock density, and short duration of ungrazed treatment. Hydrological conditions influenced N2O production and consumption mainly through regulating NO− 3 content by WFPS in soils. Ambient N2O production and consumption rates followed Gaussian functions on WFPS. Therefore, WFPS could be potentially used as an indicator for N2O production and consumption by denitrification. Soils in the transient zone (Edge) had the highest ambient N2O production and consumption rates, which is likely due to the − relatively high NO− 3 content. The proximal factors, NO3 , availability of

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labile organic C, and active denitrifying microorganisms appeared to interactively regulate the N2O production and consumption. But NO− 3 was the key factor limiting N2O production and consumption rates in all hydrological zones while the transient zone was less limited by NO− 3 as compared to the wetland (Center) and upland. The transient zone might be the “hot spot” of N2O emissions. Wetland within agricultural landscapes could be an ecological solution for NO− 3 leaching and N2O emissions from agroecosystems because of its concurrently high N2O production and consumption potentials. Acknowledgments This research was supported in part by the International Atomic Energy Agency (Technical Contract No. 15624), Vienna, Austria. The authors would like to thank Mr. Patrick Moran, Ms. Yu Wang, and Mr. Gavin Wilson for laboratory and field assistances during this project. References Balasooriya, W.K., Denef, K., Peters, J., Verhoest, N.E.C., Boeckx, P., 2008. 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