Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes

Soil Biology & Biochemistry 93 (2016) 131e141

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Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes Peiyuan Cui a, 1, Fenliang Fan a, 1, Chang Yin a, Alin Song a, Pingrong Huang b, Yongjun Tang b, Ping Zhu c, Chang Peng c, Tingqiang Li d, Steven A. Wakelin e, Yongchao Liang a, d, * a

Ministry of Agriculture Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China School of Chemistry and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China c Center of Agricultural Environment and Resource, Jilin Academy of Agricultural Sciences, Changchun 136000, China d Ministry of Education Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China e AgResearch, Private Bag 4749, Lincoln, Christchurch 8140, New Zealand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 July 2015 Received in revised form 5 November 2015 Accepted 7 November 2015 Available online 23 November 2015

Emissions of the greenhouse gas nitrous oxide (N2O) from soil are sensitive to many factors including temperature, nutrient status, pH and bulk-density. Interactions among these are complex, particularly in agricultural systems where fertilizer-addition has interactive influences across many soil properties. Under laboratory conditions, the temperature sensitivity of N2O emissions, as measured by Q10 values (fractional change in rate with a 10
C increase in temperature), was higher in soils receiving long-term fertilizer addition compared with control. Different fertilization regimes significantly influenced the emission pathways. Application of manure increased the proportion of potential N2O derived from denitrification although the incubation condition used in the present study might not be favorable for anaerobic denitrification. The abundance of archaeal amoA gene copies increased under all fertilizer treatments, but that of bacterial amoA only increased under mineral (NPK) fertilization. Meanwhile, abundance of nirS, nirK and nosZ only increased under OM and MNPK fertilization. T-RFLP analyses showed that both ammonia oxidizing and denitrifier community structures were altered by fertilization, but only the nirS community structure was sensitive to temperature change. Furthermore, a strong correlation was observed between nirS gene abundance and potential N2O emissions. Relationships between AOA, nirK gene abundances and potential N2O emission were significant but relatively weak. PLS path model revealed that besides direct effect, potential N2O emission was also indirectly influenced by temperature through mediation of NHþ 4 concentration and nirS-type denitrifier. Our work suggests that warming-induced elevation of potential N2O emission could be strengthened by long-term application of fertilizers, especially organic manure, via shifting community abundance and structure of nirStype denitrifier. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Nitrous oxide (N2O) emission Long-term fertilization Temperature sensitivity qPCR T-RFLP PLS path model

1. Introduction

* Corresponding author. Ministry of Education Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China. Tel.: þ86 571 88982073; fax: þ86 571 88982907. E-mail address: [email protected] (Y. Liang). 1 Peiyuan Cui and Fenliang Fan contributed equally to this work. http://dx.doi.org/10.1016/j.soilbio.2015.11.005 0038-0717/© 2015 Elsevier Ltd. All rights reserved.

Nitrous oxide (N2O) is a potent greenhouse gas (GHG), with a global warming potential approximately 320 fold that of CO2 (IPCC, 2007), and is also an important precursor to compounds contributing to depletion of stratospheric ozone (IPCC, 1995). Over half of the global emissions of N2O derive from agricultural soils (IPCC, 2007), and these are increasingly driven through elevated soil nitrogen (N) status, largely due to the increase of mineral fertilizer

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and animal manures (Bouwman et al., 2002). Potential N2O emissions are projected to grow exponentially as N inputs exceed the ability of crops to capture or use the N at any point in time, resulting in an accumulation of labile-N in agricultural soils (Shcherbak et al., 2014). N2O emissions from soils primarily result from two microbial processes central to soil N biogeochemical cycling: nitrification and denitrification (Conrad, 1996; Baggs and Philippot, 2010). In addition to the rates of these processes being temperaturesensitive (Avrahami et al., 2003; Abdalla et al., 2009; Braker et al., 2010; Szukics et al., 2010), the molar-quotient of N2O, compared with other nitrogenous products, also varies with temperature (Smith et al., 1998; Dobbie and Smith, 2003; Koponen and Martikainen, 2004; Schindlbacher et al., 2004; Dijkstra et al., 2012). Although efforts have been made to reveal the impact of temperature on different N2O emission pathways and their regulatory microorganisms (Avrahami and Bohannan, 2009; Billings and Tiemann, 2014), the underlying mechanisms mediating N2O emission sensitivity to temperature remain unclear (Dobbie and Smith, 2001). In addition to temperature, a range of other soil factors are known to moderate the rate and molar-quotient of N2O from microbial N-cycling processes. Primarily, these include levels of soil organic matter (SOM), labile soil carbon (C) and nitrogen (N) levels, soil pH, and factors regulating water filling pore space (bulk density, porosity, and hydrological balances) (Baggs and Bateman,
2005; Baggs et al., 2010; Cuhel et al., 2010; Harrison-Kirk et al., 2013). During agricultural management, many or all of these may be affected, resulting in complex interactions. Although processbased modeling has led to a better understanding of N2O-based emission factors in agricultural systems, the modeling is far from complete and is yet to account for biological variation among soils and the diverse ranges of farming systems (Grant and Pattey, 2008). Furthermore, there remains a significant knowledge gap in how temperature-based sensitivity interacts with edaphic and biological components of the soil ecosystem to regulate N2O emissions. To elucidate how temperature and soil fertility affect potential N2O emission rates via microbiological N-cycling process, we conducted temperature-controlled incubation experiments using field soils receiving long-term fertilizer inputs. We hypothesized that the temperature sensitivity of potential N2O would be interactive with fertilizer regimes. To link changes in process rates with microbial groups, functional genes are often used. In the context of this study, functional genes are those that encode proteins associated in a discrete nitrogen transformation phase. These include nirS and nirK (cytochrome cd1 and copper-containing nitrite reductase, respectively; Braker et al., 1998), nosZ (nitrous oxide reductase; Rich et al., 2003; Henry et al., 2006) and bacterial and archaeal forms of the amoA gene (ammonia monooxygenase; Leininger et al., 2006; He et al., 2007). The targeting of different genes spanning a range of discrete transformations provides for broader coverage of the processes that have evolved across phylogenetically diverse groups. For example, various taxa of nitrate-reducing denitrifiers exclusively possess either nirS or nirK gene types. By targeting both Nirvariants, the different ecologies of these groups have been shown to vary along with their respective contribution to denitrification (e.g. Braker et al., 2000; Kandeler et al., 2006; Yoshida et al., 2009). Furthermore, the ratio of nir and nosZ-genes has been linked to the final N2O/N2 molar ratios from the denitrification pathway (Regan et al., 2011). As such, the simultaneous assessment of multiple Ncycling genes provides a powerful method to understand the link between the variations of N2O emission rate and nitrifiers (via the rate limiting ammonia oxidation step) and denitrifiers (Morales et al., 2010).

Assessed as Q10 (fractional change in rate with a 10
C increase in temperature), the temperature sensitivity of N2O emission was previously reported to range from 2.3 to 50 for different soil types under different soil moisture conditions (Jones et al., 2008). Elevated temperature may result in enhanced denitrification activity (Braker et al., 2010). Both the denitrification gene (nirS) expression and N2O emission rate were higher at an increased temperature in a liquid culture experiment with Pseudomonas mandelii (Saleh-Lakha et al., 2009). Meanwhile, the nitrification rate and the activity of ammonia oxidizing bacteria (AOB) can be strongly affected by temperature (Avrahami and Bohannan, 2009). However, few studies have offered a rigorous assessment of the microbial community coupled with a rigorous assessment of N2O production rates, and different microbial sources of N2O emission (Butterbach-bahl et al., 2013). 2. Materials and methods 2.1. Long-term fertilization experiment and soil sampling Soil samples were taken from a long-term fertilization experiment with continuous maize cropping which was initiated in 1980 on a black soil (Mollisol) at a long-term experimental station at Gongzhuling (43
300 N, 124
480 E), Jilin province, China. With an altitude of 220 m, the site receives 450e600 mm of annual mean precipitation and has an annual mean temperature of 4e5
C. The initial properties of the surface soil (0e20 cm depth) were as follows: pH, 7.8; soil organic matter, 28.1 g kg1; total nitrogen (TN), 1.9 g kg1; total phosphorus (TP), 1.4 g kg1; available phosphorus (Olsen P), 27.0 mg kg1. Four fertilization treatments were used: no added fertilizer (CK), organic manure (OM), mineral fertilizer (combination of nitrogen, phosphorus and potassium; NPK), and organic manure plus mineral fertilizer (MNPK). The OM treatment received pig manure at 1300 kg ha2 year1. The NPK treatment received nitrogen, phosphorus and potassium fertilizers at 165, 37 and 67 kg ha2 year1 in the form of urea, double superphosphate, and potassium chloride, respectively. The MNPK treatment was NPK treatment plus pig manure applied at 1300 kg ha2 year1. All the treatments were replicated three times. Five soil samples from each replicate of each treatment were taken as a composite sample from the 0e20 cm depth. A total of twelve composite samples were sieved (2 mm) and immediately used for incubation. Part of the soil samples were air-dried in the laboratory for one week for determining selected physicochemical properties by standard procedures (Tan, 2005). 2.2. Incubation experiments Two incubation experiments were conducted to investigate the effects of temperature and fertilization regimes on potential N2O emission and soil microbial community. For the first incubation experiment, fresh soil samples were weighed (20 g on an oven dryweight basis) into individual glass bottles (125 ml). The moisture content of the soils was adjusted to 50% water holding capacity. The bottles were sealed with butyl stoppers and pre-incubated at room temperature (25
C) for one week, and then incubated at 5
C, 15
C, 25
C and 35
C for 30 days. There were four replicates for each fertilization and temperature treatment. Gas samples (10 ml) were taken from the headspace with a syringe at 3, 6, 9, 12, 16, 21 and 30 days after incubation (DAI). N2O concentrations were measured with a gas chromatograph (HP7890A, Agilent Technologies, CA, USA) according to Lam et al. (2011). All bottles were ventilated for 5 min after gas sampling, and then resealed. The bottles were destructively sampled at 30 DAI. Ammonium (NHþ 4 ) and nitrate (NO 3 ) concentrations were determined by a Lachat flow-injection

P. Cui et al. / Soil Biology & Biochemistry 93 (2016) 131e141

auto-analyzer (Lachat Instrument, Mequon, WI, USA) after extraction of soil samples with 0.01 M CaCl2. Iron and copper were extracted from soils according to the DTPA method (Lindsay and Norvell, 1978) and analyzed using ICP-OES (OPTIMA 5300DV, PerkinElmer, USA). The soil samples for molecular analysis were stored at 80
C until DNA extraction. For the second incubation experiment, C2H2 at 10 kPa was applied to inhibit nitrification and potential N2O reduction due to denitrification (Klemedtsson et al., 1988). Control treatments were included which contained no addition of C2H2. Meanwhile, the fertilization and temperature treatments were the same as above. Four replicates of 10 g dry-equivalent soil were weighed into individual glass bottles; other details were the same as described above. The incubation was undertaken under different temperature regimes (5
C, 15
C, 25
C and 35
C) for 3 days. Gas samples were taken and measured with the same method as given in the first experiment.

2.5. Quantitative real-time PCR (qPCR) The abundances of ammonia oxidizers and denitrifiers were quantified by qPCR. All qPCR assays were carried out in triplicate on an IQ5 instrument (BioRad, USA), using reaction mixtures previously described (Cui et al., 2013) with primer sets and thermocycling conditions listed in HYPERLINKTable S1. Standard curves were constructed with a 10-fold serial dilution of known copy numbers of plasmids harboring the gene of interest. 2.6. Statistical analysis 2.6.1. Q10 calculation To determine the soil temperature response of potential N2O emission, a common measure of temperature sensitivity, (Q10) was estimated based on the modified van't Hoff's equation, which is shown as follows: van't Hoff:

2.3. DNA extraction, PCR amplification and cloning DNA was extracted from 0.5 g of each soil sample using the FastDNA™ SPIN Kit for Soil and FastPrep-24 machine (Qbiogene, Canada) according to the manufacture's instructions. Successful DNA exaction was confirmed by agarose gel electrophoresis and extracts were stored at 20
C until use. Genes encoding ammonia monooxygenase (amoA) were used to study ammonia oxidizing bacteria (AOB) and archaea (AOA). For denitrifying bacteria, the nirS-, nirK- and the nosZ-type denitrifier genes, which encode cytochrome cd1 heme-nitrite-reductase (nirS), copper-nitrite-reductase (nirK), and nitrous oxide reductase (nosZ) were targeted. PCRs were performed on a DNA Engine Peltier Thermal Cycler (Bio-Rad) with the primer sets and thermocycling conditions listed in HYPERLINKTable S1. For denitrifier genes, each PCR reaction mixture consisted of 1 ml of 10-fold diluted soil DNA as template, 2.5 ml of 10  TaKaRa Ex Taq buffer, 0.625 U of TaKaRa Ex Taq polymerase, 200 mM of each dNTP, 0.4 mM of each primer and 1.5 mM of MgCl2 in a total volume of 25 ml. For archaea and bacteria amoA genes, PCR reaction mixtures were described previously (Cui et al., 2013). After agarose gel purification, PCR products were ligated into the pMT19-T vector according to the manufacture's instruction (TaKaRa, Dalian, China) for 5 h at 16
C. Transformations were performed using competent cells of Escherichia coli JM109; transformants were selected via blue-white screening. Sequencing of inserts was conducted on an ABI 3730 sequencer using BigDye terminator cycle sequencing chemistry (Applied Biosystems, CA, USA). 2.4. Terminal restriction fragment length polymorphism (T-RFLP) analysis The diversity of both ammonia oxidizers and denitrifiers was estimated by T-RFLP analysis. PCR amplifications of amoA, nirS, nirK and nosZ genes for T-RFLP analysis were the same as described above except that the forward primers were fluorescently labeled with 6-FAM (TaKaRa). After gel purification, PCR products were digested with restriction enzymes listed in HYPERLINKTable S1, which were selected by analyzing sequences in the clone library using program REPK (Collins and Rocap, 2007). The digested products were purified with a PCR product purification kit (Tiangen, Beijing, China) and analyzed with an ABI 3730 sequencer using an internal size standard MapMarker®1000 (Bioventures). Peak heights of terminal restriction fragments (T-RFs) were automatically quantified using Peak Scanner Software v1.0 (Applied Biosystems).

133

R ¼ a ebT



where Q10 ¼ eb10



(1)

modified van't Hoff: ððTTbasal Þ=10Þ

R ¼ Rbasal Q10



  ½10=ðT2 T1 Þ  where Q10 ¼ RT2 RT1 (2)

where R is N2O emission, T is incubation temperature, Tbasal is measured temperature, a and b are fitted parameters, and Q10 is the factor by which N2O emission is multiplied when temperature increases by 10
C. Based on equation (1), Q10-total was calculated to express the temperature sensitivity of potential N2O emission over whole incubation range (5e35
C). Also, based on equation (2), we calculated Q10-partial to express the temperature sensitivity for intervals of 5e15, 15e25 and 25e35
C. 2.6.2. T-RFLP data processing Data exported from Peak Scanner software were processed with T-REX, an online software for the analysis of T-RFLP data (http:// trex.biohpc.org) (Culman et al., 2009). After filtering noise and aligning T-RF, T-RFs were omitted if they occurred either in less than 2% of the total numbers of samples or with a relative abundance of less than 1% within a specific sample. Clean T-RFs were used to form data matrices, and then to run additive main effect and multiplicative interaction testing (AMMI; Culman et al., 2008). Interaction principal component (IPC) scores were obtained and used for analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) analysis exploring the effect of long-term fertilization and temperature on community structure. The data matrix was further used to calculate the relative abundance of the detected T-RFs of each gene. The TRFs differing by less than 1 bp were considered identical. Sizes of T-RFs were verified by T-RFLP analysis of representative clones. 2.6.3. Statistical tests ANOVA was conducted to test the significance of treatment effects on N2O emission rates, mineral nitrogen concentrations, amoA, nirS, nirK and nosZ gene copies numbers, and T-RFs' relative abundances. Correlations of N2O emission with gene abundances and T-RFs' relative abundances were assessed by Pearson's correlation procedure using SAS 8.02 (SAS Institute Inc., Cary, NC, USA). Partial least squares path modeling (PLS-PM) is a statistical method for studying cause and effect relationships among observed and latent variables, and has been previously applied to

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infer potential direct and indirect effects of soil biodiversity on ecosystem functions (Zhu et al., 2013; Barber an et al., 2014; Wagg et al., 2014). We used PLS-PM to explore relationships between incubation temperature, different soil conditions caused by longterm fertilization (pH, SOM and TN), microbial communities and potential N2O emissions, with the aim of evaluating the effect of temperature and long-term fertilization on potential N2O emission through nitrification and denitrification pathways. The model path used 1000 bootstraps to validate the estimates of path coefficients and the coefficients of determination (R2). Direct effects, represented by path coefficients, indicate the direction and strength of the linear relationships between variables. Indirect effects are the multiplied path coefficients between a predictor and a response variable, adding the product of all paths except the direct effect (Barber an et al., 2014). Because all measures of mineral nitrogen and nitrifier and denitrifier community were influenced by temperature and longterm fertilization, we assessed the variation in soil conditions and temperature as observed variables in the path model. The effect of  these latent variables on mineral nitrogen (NHþ 4 and NO3 concentration), gene abundance and community structure (IPC score of axis 1 and 2 of T-RFLP analysis, explained 60.1e94.3% of the total variation) may have consequently indirectly influenced potential N2O emission. Path model was evaluated using the ‘goodness of fit’ (GoF) statistic to measure its overall predictive power. The model was performed in R using the package “plspm” (Sanchez and Trinchera, 2012).

Fig. 1. Cumulative N2O emission during the 30-day incubation in soils of four fertilizer treatments (CK, OM, NPK and MNPK) under different incubation temperatures (5
C, 15
C, 25
C and 35
C). Data presented as mean values with standard errors (n ¼ 4).

11.2% with the NPK treatment (Table 1). Other soil properties such as total nitrogen, total and available phosphorus, were only increased by long-term application of OM and MNPK but not of NPK (Table 1).

2.7. Sequence analysis

3.2. Soil N2O emission and temperature sensitivity

Sequences were aligned with MEGA version 5.2 (Tamura et al., 2011) and the invalid fragments were erased. Similarity of sequences was calculated with MOTHUR (Schloss et al., 2009) and operational taxonomy units (OTUs) were defined as sharing 97% similarity. The translated amino acid sequences of representative sequences of OTUs and the related sequences obtained by BLAST were used to construct neighbor-joining trees with MEGA5 by performing 1000 bootstrapping replicates. The amoA, nirS, nirK and nosZ genes sequences were deposited to the Genebank database under accession number KM520761 to KM520915 and KM520977 to KM521149.

The cumulative N2O emissions varied significantly between fertilizations and incubation temperatures (Fig. 1). Overall, cumulative N2O emissions were influenced by the interaction between the three treatments (F ¼ 9.495, P < 0.001) (HYPERLINKTable S2), indicating that the effects of temperature, OM and NPK were significant but not additive. For all fertilizer treatments, cumulative N2O emissions increased with temperature (Fig. 1). At 5
C, cumulative N2O emissions of different fertilized soils were all very low (< 0.52 mM N2O). As incubation temperature increased to 35
C, the N2O emission increased by 11.5 times on average. For soils under diverse fertilization regimes, the dynamic patterns of N2O emission under different incubation temperature regimes differed greatly. The N2O emissions of soils treated with NPK fertilizer were lower than those of the control at 5
C and 15
C, but were not significantly different from those of the control at higher temperatures (Fig. 1). In contrast, N2O emissions were much higher in soils treated with OM and MNPK than in the control at 15e25
C. The N2O emission at 25
C and 35
C was higher in soils treated with OM than in soils treated with MNPK (Fig. 1). For each treatment, we calculated the temperature sensitivity (Q10) of N2O emission, which represents the fractional change in rate of N2O emission with a 10
C increase in temperature (Table 2). Over the whole incubation temperature interval (5e35
C), the Q10total was higher in the NPK, OM and MNPK treatments than in the

3. Results 3.1. Soil properties The chemical characteristics of soils subjected to 30-yearfertilization were altered greatly (Table 1). Chemical fertilizer and organic manure both significantly decreased soil pH but increased soil organic matter (SOM) contents. The soil treated with NPK had the highest pH (7.86), followed by the soil treated with OM (pH 7.70) and the soil treated with MNPK (pH 7.52). Soil organic matter content was increased by 56.4% and 66.7% with OM and MNPK treatment compared with the control, while it was increased by

Table 1 Soil pH, soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP), available phosphorus (Olsen P) of the four soils tested sampled from long-term fertilization experiment. Treatment

pH (H2O)

SOM (mg kg1)

TN (%)

TP (g kg1)

Olsen P (mg kg1)

CK OM NPK MNPK

7.94 7.70 7.86 7.52

23.18 36.26 25.78 38.63

0.14 0.21 0.16 0.24

0.48 1.05 0.46 1.69

6.82 188.66 11.01 208.82

d b c a

a c b d

Data followed by different letters indicates significant difference (P < 0.05).

a b a c

a b a c

a b a b

P. Cui et al. / Soil Biology & Biochemistry 93 (2016) 131e141

135

Table 2 Mean values of Q10 of N2O emission with different fertilizer treatments (CK, OM, NPK, MNPK) and C2H2 conditions (with or without 10 kPa C2H2 addition) (n ¼ 4). Q10-partial indicates the temperature sensitivity for intervals of 5e15
C, 15e25
C and 25e35
C and Q10-total indicates the temperature sensitivity for the whole incubation temperature interval (5e35
C). Treatment

CK OM NPK MNPK CK OM NPK MNPK

C2H2 addition (10 kPa)

e

þ

Q10-partial

Q10-total

5e15
C

15e25
C

25e35
C

2.10 4.02 5.19 2.29 1.51 2.15 3.07 2.04

1.64 2.76 2.14 2.30 2.18 1.82 2.00 2.94

1.57 1.98 1.64 2.16 2.35 2.93 1.83 2.15

a b c a a ab b ab

a c b b a a a b

a b a b ab b a a

1.77 2.80 2.74 2.29 1.97 2.23 2.23 2.42

a b b b a ab ab b

Data followed by different letters indicates significant difference (P < 0.05).

CK treatment (Table 2; P < 0.05). Meanwhile, in the MNPK treatments, Q10-partials showed no significant differences among the three temperature intervals (P > 0.05). In contrast, for the OM and NPK treatments, Q10-partials of 5e15
C were significantly higher than those of 15e25
C and those of 25e35
C (P < 0.01), probably due to the relatively low N2O emission at 5
C. To gain further insights into the relative contribution of nitrification and denitrification to total soil N2O emission, the second incubation experiment used 10% acetylene to inhibit the potential N2O emission from nitrification and potential N2O reduction from denitrification (Fig. 2). Overall, N2O emissions due to denitrification (DenN2O) showed a similar trend to the total N2O emission rate. DenN2O emission rates were influenced by temperature, OM, and their interaction, but not by NPK fertilization. Under different fertilizer treatments, the ratio of DenN2O emission rate to the whole N2O emission rate differed (Fig. 2), which was 45% and 40% in the CK and NPK treatments, respectively, compared to 81% and 74% in the OM and MNPK treatments. The temperature sensitivity (Q10) of DenN2O emission was calculated (Table 2). Unlike that of whole N2O emission, Q10-total was significantly higher in the MNPK treatment than in the CK treatment (P < 0.05). However, in the OM and NPK treatments, Q10total showed no significant variances compared with the control treatment.

Fig. 2. Histograms of the dynamics of N2O emission rate at different incubation temperatures (5
C, 15
C, 25
C and 35
C) and with (DenN2O) or without (N2O) 10 kPa C2H2 addition in soils of four fertilizer treatments: (a) CK, (b) OM, (c) NPK and (d) MNPK. Line charts represent the DenN2O/N2O ratio. Data presented as mean values with standard errors (n ¼ 4).

Long-term application of all the fertilizers significantly þ increased the soil NO 3 and NH4 concentrations. In all the fertilizer  treatments, NO3 concentrations were greater at 35
C than at 5e25
C (Fig. 3a). However, NO 3 concentrations were significantly lower in the OM and MNPK treatments than in the control at 25
C (P < 0.01). The variation in NHþ 4 concentration with fertilization and temperature was similar to that of the N2O emission rate (Fig. 3b). On average, NHþ 4 concentrations were significantly higher in the OM and MNPK treatments than in the CK and NPK treatments (P < 0.05). Additionally, in the OM and MNPK treatments, the NHþ 4 concentrations increased with incubation temperature. Thus, in the CK and NPK treatments, the NHþ 4 concentrations were 3.5 and 3.2 times greater at 35
C than at 5
C, but in the OM and MNPK treatments, NHþ 4 concentrations were 6.7 and 5.0 times greater at 35
C than at 5
C.

to 58.9  106 copies g1 soil, and from 5.1  108 to 25.3  108 copies g1 soil, separately. The amoA genes from both AOB and AOA were not strongly altered by incubation temperature, but differed among fertilizer treatments. The AOB abundance was higher in the NPK treatment than in other treatments, and AOA abundance was always higher in the NPK, OM and MNPK treatments than in the control (P < 0.01). The abundance of nirS genes ranged from 3.7  106 to 15.8  106 copies g1 soil (Fig. 4c). The long-term application of organic manure significantly increased nirS (P < 0.01), while NPK fertilizer had not effect (P > 0.05). In the OM and MNPK treatments, the nirS gene copies increased with increasing incubation temperature. The numbers of nirK and nosZ gene copies ranged from 7.3  106 to 36.3  106 copies g1 soil and 14.8  106 to 42.6  106 copies g1 soil, respectively (Fig. 4d and e). The gene abundances of nirK and nosZ of all the fertilization treatments were not strongly affected by incubation temperature, while long-term application of organic manure significantly increased the copies of these two genes.

3.4. Abundances of amoA, nirS, nirK and nosZ genes

3.5. Nitrifier and denitrifier community structure

The abundances of amoA genes for AOB and AOA were calculated based on the qPCR results (Fig. 4a and b), and ranged from 4.0  106

Long-term fertilization significantly influenced the relative abundances of the major nitrifier (ammonia oxidizing) and

 3.3. NHþ 4 and NO3 concentration

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þ

Fig. 3. Dynamics of (a) NO 3 and (b) NH4 concentrations in soils receiving four fertilizer treatments (CK, OM, NPK and MNPK) under different incubation temperatures (5 C, 15 C, 25
C and 35
C). Data presented as mean values with standard errors (n ¼ 4).

denitrifier genes as indicated by T-RFs (HYPERLINKFig. S1). Based on digestion with Hha I and Afa I enzymes, the PCR products of amoA gene in AOA and AOB were grouped across 13 and 14 T-RFs respectively (Fig. S1a and b). For archaea, 46.6% of the T-RFs (148, 222, 543, 556, 595 and 629 bp) increased in relative abundance following NPK or OM addition, and were related to Cluster I and IV in soil lineage (Fig. S2a). Meanwhile, 46.4% of the T-RFs (44, 66, 73, 84, 106, 166, and 191 bp) were significantly reduced in relative abundance with fertilization, and were related to taxa in Cluster II

Fig. 4. Dynamics of amoA gene copies of (a) ammonia oxidizing archaea (AOA) and (b) bacteria (AOB), (c) nirS, (d) nirK and (e) nosZ gene copies in soils receiving four fertilizer treatments (CK, OM, NPK and MNPK) at different incubation temperatures (5
C, 15
C, 25
C and 35
C). Data were presented as mean values with standard errors (n ¼ 4).

and III (Fig. S2a). For bacteria, 45.0% of the T-RFs (19, 250, 254, 270 bp) were increased in relative abundance due to application of NPK or OM, relating to Nitrosospira-like Cluster I, II and VI and Nitrosomonas-like Cluster I taxa (Fig. S2b). Approximately 50.4% of the T-RFs (44, 58, 112 and 211 bp) were decreased in relative abundance; these were affiliated with Nitrosospira-like Cluster III, IV and V taxa, and Nitrosomonas-like Cluster II taxa (Fig. S2b). PCR products of nirS, nirK and nosZ genes were grouped across 20, 5 and 16 T-RFs with the digestion of Afa I, Asp I and Msp I enzymes respectively (Fig. S1cee). With the nirS gene, 16 out of 20 T-RFs showed a significant variation due to long-term fertilization (Fig. S1c). About 22.4% T-RFs decreased in relative abundance in the fertilization treatments compared to the control, and 47.0% of T-RFs were increased. With the nirK gene, T-RF of 199 bp accounted for 71% of the total in relative abundance, and was significantly decreased due to fertilization (Fig. S1d). Phylogenetic analysis showed that, the 199 bp T-RF gene corresponded to the HT-nirK-7 clone, which was clustered in the vicinity of nirK from Mesorhizobium sp. (Fig. S3b). With the nosZ gene, all of the detected T-RFs were influenced by long-term fertilization, demonstrating that 60.4% of the T-RFs increased in relative abundance under fertilization (Fig. S1e). Based on the phylogenetic analysis, these T-RFs corresponded to the clones related to nosZ sequences from Rhizobiales of a-Proteobacteria such as Bosea sp., Chelatococcusdaeguensis, Mesorhizobium, Bradyrhizobium and Rhodospirillales of a-Proteobacteria such as Azospirillum brasilense (Fig. S3c). NPK fertilization had a significant effect (P < 0.05) on the community structure of nirK-type denitrifier, ammonia oxidizing bacteria and ammonia oxidizing archaea, and resulted in the detection of different community structures (Fig. S4a, b and d; Table 3). Application of organic manure also resulted in significant (P < 0.001) distinct communities of ammonia oxidizing bacteria and nirS-type denitrifier (Fig. S4b and c; Table 3). Incubation under different temperature regimes had a significant effect on the nirStype denitrifier (Fig. S4c; Table 3) (P < 0.05 [MANOVA] on IPC axis 1 and 2, which explained 90% of total variation). 3.6. PLS-PM analysis Although higher TN and SOM usually cause larger potential N2O emissions, soil condition appears to have a negative direct effect

P. Cui et al. / Soil Biology & Biochemistry 93 (2016) 131e141 Table 3 T-RFLP analyses of nitrifiers (archaea and bacteria amoA genes) and denitrifiers (nirS, nirK and nosZ genes) as affected by fertilization and temperature. The data are P values corresponding to the first two IPC scores of the AMMI analyses, and were obtained by ANOVA and MANOVA.

AOA

AOB

nirS

nirK

nosZ

Variation (%) NPK OM Temperature Variation (%) NPK OM Temperature Variation (%) NPK OM Temperature Variation (%) NPK OM Temperature Variation (%) NPK OM Temperature

IPCA1

IPCA2

37.8 0.479 0.01 0.771 77.5 0.665 <0.001 0.740 82.8 0.719 <0.001 0.885 51.0 0.044 0.024 0.310 42.7 0.313 <0.001 0.842

22.3 0.002 0.126 0.713 16.8 <0.001 0.745 0.936 7.7 0.262 0.440 0.414 35.6 0.039 0.975 0.146 26.2 0.331 0.049 0.401

Datas in bold indicate statistically significant values (P  0.05).

MANOVA 0.031 0.322 0.881 0.002 <0.001 0.997 0.773 <0.001 0.038 0.028 0.084 0.339 0.410 0.286 0.276

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(0.64) on potential N2O emission (Fig. 5). However, with larger  indirect effects (1.00) through NHþ 4 , NO3 and nirS community, the negative effect of soil condition on potential N2O emission can be  offset (Fig. 5). Meanwhile, temperature (0.20), NHþ 4 (0.47) and NO3 (0.33) concentrations have larger direct effects on potential N2O emission, indicating that concentration of substrates in the enzymatic reaction in nitrification and denitrification are of vital importance in the production of N2O (Fig. 5). Within the total effect of temperature on potential N2O emission (0.81), the indirect effects through NHþ 4 (0.35) and nirS-type denitrifier community (0.24) are dominant (Fig. 5).

4. Discussion In this work, we explored the impacts of organic and inorganic fertilization and global warming on potential N2O emission and its associated biological processes. To address this topic, we conducted a set of incubation experiments under controlled temperatures using long-term fertilized black soil. Two regular patterns of N2O emission were observed in the laboratory experiment: (1) elevated soil temperature resulted in a higher emission rate in the OMamended treatments than in the CK and NPK treatments; and (2) different fertilization patterns significantly influenced the emission

Fig. 5. Directed graph of the Partial Least Squares Path Model (PLS-PM). Each box represents an observed variables or latent variables. The loading for the microbial community and soil condition that create the latent variables are shown in the dashed rectangle. Larger path coefficients are reflected in the width of the arrow with blue indicating a positive effect and red a negative effect. Path coefficients are calculated after 1000 bootstraps. Coefficients differ significantly from 0 are indicated by *P  0.05, **P  0.01, ***P  0.001. The model is assessed using the Goodness of Fit statistic, a measure of the overall prediction performance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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pathways with application of OM increasing the proportion of potential N2O emission due to denitrification. Potential N2O emission was expected to be increased by N addition, which has been previously reported in both field and laboratory experiments (Bouwman et al., 2002; Barnard et al., 2005). In this study, SOM was significantly increased by the application of organic manure (Table 1). Due to accelerated mineralization under higher temperatures, NHþ 4 concentration increased dramatically under elevated temperature in the OM and MNPK treatments. As the primary substrate for ammonia oxidation,

the initial and rate-limiting step of nitrification (Martens-Habbena et al., 2009), the availability of NHþ 4 is of vital importance to the enzymatic processes and consequently affect the following oxidization and reduction in N cycle. Therefore, as by-products of nitrification and denitrification, N2O emission was strongly corre2 lated with NHþ 4 concentration (R ¼ 0.92, P < 0.001) (Fig. 6f). The proportion of DenN2O emission to total N2O emission was higher in the OM and MNPK treatments than in the CK and NPK treatments (Fig. 2), which is consistent to the previous finding that nitrification was more dominant in urea-treated soils, whereas

Fig. 6. Relationships of cumulative N2O emissions with abundances of (a) ammonia oxidizing archaea (AOA), (b) bacteria (AOB), (c) nirS, (d) nirK, (e) nosZ gene and NHþ 4 concentration (f).

P. Cui et al. / Soil Biology & Biochemistry 93 (2016) 131e141

denitrification was more dominant in organic-manure-amended soils than in control (Akiyama et al., 2004). Meanwhile, soil respiration was significantly increased by higher incubation temperature and application of organic manure (data not shown), which can induce soil anaerobiosis (Dobbie and Smith, 2001; Butterbachbahl et al., 2013) and create a more suitable condition for denitrification. However, the condition needed for active anaerobic denitrification might not be well maintained in this study because the bottles were regularly aerated during incubation. Moreover, the balance between nitrification and denitrification processes might also be affected by change in soil pH resulting from long-term application of organic manure (Table 1). The function of some denitrification enzymes is sensitive to the environmental pH because their active sites lie toward the outside (nitric oxide reductase [Nor]) or are actually outside (periplasmic nitrate reductase [Nap] and nitrite reductase [Nir]) of the cytoplasm membrane (Richardson et al., 2009). Long-term application of organic manure has a significant impact on nitrite reducers and nitrous oxide reducer, as the nirS, nirK and nosZ gene copy numbers were all increased in the OM and MPNK treatments (Fig. 4c, d and e). Consistent with our result, the work by Tatti et al. (2013) showed increased abundances of denitrifier function genes (nirS, nirK and nosZ) under long-term organic fertilization, supporting the contention that organic carbon is the most important factor explaining denitrifier abundance (Kandeler et al., 2006). Long-term fertilization of organic manure was reported to shift both nirS- and nirK-denitrifying bacterial communities in an aquic soil of Northeast China (Yin et al., 2014). In this study, significant variance due to long-term application of organic manure was only detected in nirS gene community structure (Fig. S4c, Table 3). Meanwhile, the application of NPK and OM showed significant effects on elevating the amoA gene abundance and shifting the nitrifier community structure, which is in

139

agreement with previous studies (Chu et al., 2007; MartensHabbena et al., 2009; Wang et al., 2009). The responses of potential N2O emission to temperature are microbiologically-dependent in different soils (Holtan-Hartwig et al., 2002; Stres et al., 2008; Szukics et al., 2010). Inconsistent responses of abundance and community structure to temperature have been reported in both AOA and AOB (Avrahami and Conrad, 2003; Tourna et al., 2008; Jiwon et al., 2013; Aiglsdorfer et al., 2014), but temperature-dependent change was not reflected in the qPCR data (Fig. 4a and b) or T-RFLP profiles (Fig. S1a and b, Table 3). The absence of temperature-dependent change in amoA gene may result from high abundance and redundancy of AOA. In our study, the AOA/AOB ratio was very high, ranging from 26 to 167. According to a previous report, AOA would dominate the ammonia utilization activity if this ratio was >10 (Prosser and Nicol, 2012). With high maximum specific growth rate when substrate is in excess (mmax) and low Ks (the substrate concentration at which m ¼ mmax/2), the growth rate of AOA is very sensitive to ammonium concentration (Prosser and Nicol, 2012). The exponential regression between AOA abundance and N2O emission (R2 ¼ 0.41, P < 0.05) (Fig. 6b) also supports that, as the initial substrate of nitrification, ammonium concentration has a strong indirect effect on potential N2O emission. The temperature-dependent change in microbial community abundance and structure of nirS-type denitrifiers was found to be the most significant. Two distinct patterns of nirS gene abundance were observed: (1) in the OM and MNPK treatments, elevated soil temperature resulted in an increase in gene abundance, and (2) in the CK and NPK treatments, gene abundance did not change with elevated temperature. Higher SOM concentration in the OM and MNPK treatments provided more complex C and N substrates than in CK and NPK treatments. Moreover, it is well known that iron and copper are necessary for

Fig. 7. Relative abundances of the T-RFs in the nirS gene T-RFLP analysis, which were significantly effected by soil incubation temperature in different fertilizer treatments. Asterisks designate T-RFs that are significantly correlated with N2O emissions in relative abundance (*Significant at the 0.05 probability level, **Significant at the 0.01 probability level, ***Significant at the 0.001 probability level).

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the activity of type S and type K nitrite reductases, and these two structurally different nitrite reductases are reported to be never  et al., 2002; Zumft, 1997). Soil copper in the same cell (Prieme and iron concentrations were strongly correlated (R2 ¼ 0.71, P < 0.001; R2 ¼ 0.47, P < 0.001) with nirS and nirK gene abundances respectively, indicating that the development and activity of denitrifiers also depend on trace metal availability (HYPERLINKFig. S5). Therefore, changes in temperature will have a different influence on the denitrifier substrate use efficiency and resource allocation under these conditions, and microbial resource requirements and the fate of those resources, including N2O, will likely change (Frey et al., 2013). The strong correlation between N2O emission and nirS gene abundance (R2 ¼ 0.63, P < 0.001) (Fig. 6) indicates that temperature had influenced the physiological process within nirS-type denitrifying microbes. The link between the N2O emission and the abundance of denitrifier communities under temperature variation has been reported before (Billings and Tiemann, 2014). Furthermore, according to TRFLP analysis of nirS gene, 10 out of 20 T-RFs were significantly correlated with N2O emission (Fig. 7). The 88 and 410 bp T-RFs, which affiliate with Azoarcus sp., Rubrivivax gelatinosus, Dechlorospirillum sp. and Dechloromonas sp. (Fig. S3a), were positively correlated with N2O emission, and were the most abundant fragments of all the treatments. This suggests that these nirStype denitrifiers are the main taxa regulating the N2O emission temperature sensitivity through the denitrification pathway. Soil NHþ 4 concentrations are a product of organic nitrogen mineralization, a temperature-sensitive process, or mineral fertilizer addition. The rate-limiting step in nitrification (formation of nitrate and coupling to other parts of the N cycle, such as denitrification) is regulated through the activity of ammonia oxidization bacteria and archaea. The formation of NHþ 4 and regulation of the activity of AOB and AOA are both moderated by temperature, which therefore affects potential nitrifier-N2O emissions under fertilization. In the denitrification pathway, our results show that temperature effects on potential N2O emissions are driven through regulation of the nirS-type denitrifier. Critically, among all the taxa targeted in this study, only nirS-type denitrifier responds physiologically to temperature and also has a larger effect (0.59) on N2O emission. The significant correlation of nirS gene abundance with potential N2O emission suggests that, in the black soil tested, nirS-type denitrifier rather than nirK-type denitrifier is more active in denitrification. Path analysis showed that potential N2O emission could be ‘directly’ influenced by nirS-type denitrifier community and ‘indirectly’ affected by temperature through it. This result indicates that a community shift can play an important role in linking agricultural practices to nitrogen loss. Furthermore, as temperature changes in response to global warming, the denitrifying microbial species and denitrifier community structure may be of great importance to the feedback of potential N2O emission.

Acknowledgments This work was jointly supported by the grants from The National Natural Science Foundation of China (Approved No. 41171208), National Key Basic Research Support Foundation of China (NKBRSF) (Approved No. 2015CB150502), the 12th Five-Year Key Programs for Science and Technology Development of China “Study of Key Technologies for Alleviating Obstacle Factors and Improving Productivity of Low-yield Cropland” (Approved No. 2012BAD05B06), National Nonprofit Institute Research Grant of CAAS (IARRP-201429) and Scientific Research Fund of Hunan Provincial Education Department (14B043).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2015.11.005.

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