Soil autotrophic and heterotrophic respiration in response to different N fertilization and environmental conditions from a cropland in Northeast China

Soil Biology & Biochemistry 110 (2017) 103e115

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Soil autotrophic and heterotrophic respiration in response to different N fertilization and environmental conditions from a cropland in Northeast China Zengming Chen a, b, Yehong Xu a, b, Jianling Fan a, Hongyan Yu a, Weixin Ding a, * a b

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 December 2016 Received in revised form 17 March 2017 Accepted 18 March 2017

Partitioning soil respiration (Rs) into its heterotrophic (Rh) and autotrophic (Ra) components is crucial to evaluate the effects of inorganic and organic nitrogen (N) fertilization on carbon (C) cycling in agricultural ecosystems. We carried out a field experiment in a maize cropland in Northeast China using the root exclusion method to separate Rh and Ra, and investigate their responses to different fertilization regimes. These included no N fertilization (CK), inorganic N fertilizer (NPK), 75% urea N plus 25% pig (PM1) or chicken (CM1) manure N, and 50% urea N plus 50% pig (PM2) or chicken (CM2) manure N. Annual Rs was significantly increased from 314 g C m
2 in CK to 389, 366, and 371 g C m
2 in NPK, CM1, and PM2, respectively, and further to 420 g C m
2 in PM1, whereas a similar value to CK was observed in CM2 (327 g C m
2). N-induced increases in Rs were largely attributable to the response of Ra (except CM2), which increased by 18e54% due to higher nitrate supply. Rh increased from 183 to 192e209 g C m
2 in plots receiving N fertilizer, with significant increases observed in PM1 and PM2, likely due to the high ammonium and labile organic C concentrations in these treatments. Manure type and application rate had significant effects on Rs and Ra, but not Rh. Compared with CM, PM was more effective in stimulating Ra due to its greater decomposability. Rs and Ra decreased in the order of PM1 > PM2 and CM1  CM2, presumably because of the lower inorganic N supply with increasing manure application rate. The estimated C sequestration rate shifted from negative in CK and NPK to positive in the manure treatments, especially in PM2 and CM2 that gained 0.44 and 0.49 Mg C ha
1 yr
1, respectively. These results suggested that combined application of half inorganic N plus half organic N might have potential to enhance soil C sequestration in cropland of Northeast China. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Autotrophic respiration Heterotrophic respiration Maize Nitrogen fertilization Organic fertilizer Soil respiration

1. Introduction Soil respiration (Rs), i.e., the carbon dioxide (CO2) efflux from soil, is estimated at 50e98 Pg C yr
1 and is the second largest carbon (C) flux after photosynthesis between terrestrial ecosystems and the atmosphere (Bond-Lamberty and Thomson, 2010; Xu and Shang, 2016). Therefore, even small changes in Rs are expected to have a large impact on the global C cycle and its feedbacks to climate change (Schlesinger and Andrews, 2000). The large uncertainty in Rs estimation is partly because Rs is regulated by multiple abiotic and biotic factors, such as soil temperature,

* Corresponding author. E-mail addresses: [email protected] (Z. Chen), [email protected] (Y. Xu), jlfan@ issas.ac.cn (J. Fan), [email protected] (H. Yu), [email protected] (W. Ding). http://dx.doi.org/10.1016/j.soilbio.2017.03.011 0038-0717/© 2017 Elsevier Ltd. All rights reserved.

moisture, nutrient availability, and plant productivity (Hanson et al., 2000; Davidson et al., 2006; Xu and Shang, 2016). Furthermore, Rs consists of two main components, heterotrophic (Rh) and autotrophic (Ra) respiration, which respond differently to changes in influencing factors (Bond-Lamberty et al., 2004; Savage et al., 2013; Chen et al., 2014a). Rh is derived from the decomposition of soil organic matter (SOM) and plant residues, which depends on the activity of soil microbial communities and labile substrate availability (Whitaker et al., 2014; Ding et al., 2016). Ra includes respiration by live roots and heterotrophic respiration from rhizosphere microorganisms, and is primarily regulated by the root activity and plant photosynthate supply (Tang et al., 2005; Vargas et al., 2011). Among the various environmental factors, soil temperature and moisture are generally acknowledged as the dominant drivers of Rs

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due to their direct effects that alter the activities of soil microbes and plant roots, and indirect effects through changing substrate supply and plant growth (Reichstein et al., 2003; Davidson et al., 2006; Wan et al., 2007). However, many studies have shown that soil temperature (e.g., Li et al., 2013a; Matteucci et al., 2015) or moisture (e.g., Zimmermann et al., 2010; Pang et al., 2013) have minor effects on Rs. These inconsistent results are likely related to the various responses of Rh and Ra. For example, Ra has been found to be more sensitive to variations in soil temperature and moisture €kiranta et al., 2008; Zhang et al., 2013). Balogh et al. than Rh (Ma (2016) reported that Ra was repressed by drought more than Rh in a grassland in central Europe. In contrast, other studies demonstrated that Rh was more strongly controlled by soil temperature and moisture than Ra, which was more tightly coupled with plant physiological activity (Hartley et al., 2007; Li et al., 2010). Baldocchi et al. (2006) found that the summer rain events could cause a pulse in the flux of Rh but not Ra. Thus, partitioning Rs into its components and assessing their responses to soil temperature and moisture are essential to improve our mechanistic knowledge and model prediction of Rs under various environmental conditions and management practices (Subke et al., 2006; Hartley et al., 2007; Hopkins et al., 2013). Nitrogen (N) enrichment can have profound effects on C cycling in terrestrial ecosystems (Janssens and Luyssaert, 2009). Although numerous experiments have been conducted, the impact of N addition on Rs is still debated in the literature, mainly due to the different responses of each of the Rs components (Janssens et al., 2010; Zhou et al., 2014). N fertilization can stimulate Ra due to increased plant productivity (Cleveland and Townsend, 2006; Tu et al., 2013), and can enhance Rh as a result of higher soil microbial biomass or activity (Gong et al., 2012; Xu et al., 2016). In contrast, Phillips and Fahey (2007) found that Ra was lower after fertilization because of a reduction in root biomass and mycorrhizal colonization in a hardwood forest soil. It was suggested that belowground C allocation might be reduced after N addition as a result of decreased C and energy costs of foraging for nutrients (Olsson et al., 2005). Likewise, N addition may reduce Rh by suppressing soil microbial biomass and oxidative enzyme activities, promoting recalcitrant compound formation or inducing soil acidification (Treseder, 2008; Janssens et al., 2010). Therefore, it is essential to investigate the different responses of Rh and Ra to N addition to fully evaluate and understand soil C dynamics with increasing N inputs. Croplands store more than 10% of global soil organic C (SOC) and have a large potential for C sequestration and climate change gy and Jackson, 2000; Amundson et al., 2015). mitigation (Jobba Cultivated Mollisols of Northeast China are highly fertile and cover ~300 000 km2 of farmland that is largely devoted to grain production, but these soils have experienced significant SOC loss in the past decades (Sun et al., 2010). Application of organic fertilizer along with inorganic N fertilizer has been recommended to increase SOC content and simultaneously ensure N supply to crop growth (Ding et al., 2012). Organic amendments have been widely demonstrated to increase Rs (e.g., Brye et al., 2006; Ding et al., 2007b; McMullen et al., 2015). However, Meijide et al. (2010) showed that organic fertilizers could increase, decrease, or have no effect on Rs depending on fertilization types. Brye et al. (2006) revealed that Rs was unaffected by the form of poultry litter, but generally affected by its application rate. Conversely, Li et al. (2013b) pointed out that the impact of organic input on Rs was primarily related to the type of organic amendments but not the application rate. It is very likely that the composition and application rate of organic materials can exert an impact on C decomposition and N mineralization, and thus microbial substrate  et al., 2012; Weber et al., availability and plant N supply (Romanya

2014). As a consequence, the heterotrophic and autotrophic component of Rs will be altered, which may complicate the responses of total Rs. However, evidence of the effects of organic amendments on Rh and Ra is scarce. The root exclusion method has been widely used to separate Rh and Ra because it is easier and cheaper to implement compared with other methods, such as the isotopic (13C or 14C) technique (Hanson et al., 2000; Hopkins et al., 2013). Nevertheless, this method may underestimate Rh because organic C inputs to soil from roots are excluded and the rhizosphere priming effect on SOM decomposition is absent. In addition, soil temperature and moisture may be altered due to the lack of roots. Any method to partition Rs has unavoidable biases (Subke et al., 2006) but uncertainty associated with Rs components could be lower when roots are excluded without disturbance of the soil environment, such as by leaving unplanted areas in cropland (Suleau et al., 2011). In the present study, we conducted a field experiment to measure the annual fluxes of Rs, Rh, and Ra with the root exclusion method, from a cropland under different fertilization regimes. The main aims were to investigate how Rs and its components respond to the variation of environmental factors, specific to soil temperature and moisture, and combined application of inorganic N fertilizer and manure with different types and application rates. 2. Material and methods 2.1. Study site This study was conducted in a rainfed maize-cultivated cropland at the Hailun National Agro-ecological Experimental Station, Heilongjiang Province, China (47 260 N, 126 380 E). The climate of this region is temperate sub-humid and is influenced by the continental monsoon, characterized by a short hot summer and long cold winter. From 1953 to 2013, the mean annual air temperature was 1.9  C, and the monthly mean air temperature was lowest in January (
21.6  C) and highest in July (21.6  C). The long-term mean annual precipitation is 556 mm, of which 87% occurs during the crop growing season from May to October. Prior to the establishment of our experiment, the field was continuously cultivated under a maize-soybean rotation. The soil is derived from loamy loess and classified as Typic Hapludoll in the USDA soil taxonomy. The soil texture is clay loam with 8% sand, 72% silt, and 20% clay. The bulk density is 1.0 g cm
3, and other soil properties are listed in Table 1. 2.2. Experimental design In May 2011, eighteen plots (12 m  4.2 m) were established in three blocks (¼ replicates). Six treatments were assigned randomly per block: no N fertilizer as control (CK), 100% urea N (NPK), 75% urea N plus 25% pig (PM1) or chicken (CM1) manure N, and 50% urea N plus 50% pig (PM2) or chicken (CM2) manure N (Table S1). The N fertilizer application rate was 150 kg N ha
1 in each fertilized treatment. As a common practice, N fertilizers were split into preplanting and post-emergence fertilization with a ratio of 1:1 for N application rate. In pre-planting fertilization, 75, 37.5, and 37.5 kg N ha
1 urea was applied for NPK, PM1, and CM1, respectively; manure was applied at 37.5 kg N ha
1 for PM1 and CM1, and 75 kg N ha
1 for PM2 and CM2. For the post-emergence fertilization, 75 kg N ha
1 urea was side-dressed in all fertilized treatments. Organic fertilizers were obtained from commercial companies and their main characteristics are given in Table 1. Ammonium (NHþ 4 ) and nitrate (NO
3 ) content in the manure were measured using the MgO-Devarda alloy distillation method (Lu, 2000). The hemicellulose (HEM), cellulose (CEL), and lignin (LIG) fractions were

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Table 1 Selected properties of the soil (0e20 cm), and pig (PM) and chicken (CM) manure. pH

OC

TN

C/N

(g kg
1) Soil PM CM

5.86 7.68b 7.97a

27.5 87.9b 119.3a

NHþ 4

NO
3

(mg N kg
1) 2.2 10.2b 14.8a

12.5 8.6a 8.1a

6.7 85.2b 397.6a

30.7 5.0b 163.5a

LOC

HEM

(g kg
1)

(g kg
1)

0.67 2.71a 1.99b

e 98a 55b

CEL

LIG

e 101a 94a

e 31b 55a

Different letters within the same column indicate significant differences between the two types of manure at P < 0.05.
OC, organic carbon; TN, total nitrogen; C/N, the ratio of OC to TN; NHþ 4 , ammonium; NO3 , nitrate; LOC, labile organic carbon; HEM, hemicellulose; CEL, cellulose; LIG, lignin.

determined using the acid detergent fiber method (van Soest et al., 1991). Other manure properties were measured in the same methods used for soil samples. In the study region, the field is split into ridges and furrows at a distance of 70 cm after harvest in autumn each year. The preplanting fertilizers were banded in the ridges on 14 May 2011, and maize seeds were then sown at a plant spacing of 25 cm. An area of 1.4 m  2.0 m (six plant places and three ridges) in each plot was left unplanted. Pre-emergence herbicide was applied for weed control and visible weeds were removed by hand throughout the experimental period. Post-emergence N fertilizer application occurred at the maize V6eV8 growth stage on 15 June 2011. Mature maize was harvested on 27 September 2011. All crop residues were removed from the plots followed by manual tillage. Samples of maize grain and straw were oven-dried at 60  C until a constant weight to calculate the grain yield and aboveground biomass. 2.3. Soil respiration and environmental measurements Soil CO2 fluxes were measured over the experimental period of a whole year by the static closed chamber method. In each plot, a cylindrical polyvinyl chloride (PVC) tube (10 cm height and 10 cm inner diameter) was inserted to 5 cm depth of soil at the center of one ridge including one maize plant. A PVC base frame (30 cm length, 70 cm width, and 20 cm height) was embedded into the soil at a depth of 10 cm, making the PVC tube at its center. The PVC tube and base frame were fixed before maize sowing and kept in place throughout the experimental period. Fertilizers were weighed and applied separately for the PVC base area to ensure accurate application rates. When taking gas samples, a PVC pipe (30 cm height and 10 cm outer diameter) was inserted to the existing PVC tube. A custom-made stainless steel chamber (30 cm length, 70 cm width, and 25 cm height) insulated by white foam was fitted to the PVC base frame. The chamber consisted of two separate parts that were linked by two hinges and sealed with rubber strips. An opening (10 cm diameter) was made in the middle of the chamber top to fit the PVC pipe with sealing strips to form an airtight seal. For the unplanted area (1.4 m  2.0 m), the PVC base frame was inserted 20 cm into the soil at the center of this area and a PVC chamber was used for gas sampling. Liedgens and Richner (2001) reported that maize roots are most abundant up to 40 cm from the stem and Fan et al. (2016) recovered most maize roots from the top 20 cm of the soil profile. Thus, we assumed that there was no root respiration measured from the base frame in the unplanted area. See Ding et al. (2007b) for more detailed information on the devices. Headspace gas samples were taken twice a week during the growing season, weekly after harvest before the soil was frozen and during spring thaw, and biweekly during the frozen period. In total, gas sampling was performed 69 times. The sampling order of blocks was chosen at random, but treatments were always sampled in the same order within a block. All headspace gas samples were collected between 09:00 a.m. and 12:00 p.m. Every time, four gas samples were collected from the chamber headspace using a plastic syringe equipped with a three-way stopcock at 0, 10, 20, and 30 min after the chamber was installed and immediately transferred to

pre-evacuated 20-mL glass vials. Meanwhile, the chamber air temperature was recorded with thermometers. Gas samples were analyzed for CO2 concentration using a gas chromatograph equipped with a flame ionization detector (Agilent 7890, Santa Clara, USA). The CO2 fluxes were calculated from the slope of the linear increase in the CO2 concentration over the sampling time. Data were accepted based on a coefficient of determination (R2 value) above 0.90. Cumulative CO2 emissions were calculated by linear interpolation between measurement dates. Ra was estimated by subtracting Rh from Rs that were measured from the unplanted and planted area, respectively. Daily precipitation, air (AT) and soil (ST) temperature were recorded at a meteorological station. Soil particle size distribution was determined by a laser particle size analyzer (LS13320, Beckman Coulter, Brea, USA). The pH was measured in a 1:2.5 soil-water ratio. Bulk density was determined by the core method. The organic C (OC) and total N (TN) contents in soil were analyzed using the wet oxidation-redox titration and micro-Kjeldahl method, respectively (Lu, 2000). For every gas sampling, soil temperature at 5 (ST5) and 10 (ST10) cm were measured with a digital thermometer, or geothermometer (during the frozen period), in each plot. Simultaneously, volumetric soil water content (SWC) at a depth of 0e10 cm was determined using a time domain reflectometry probe during the non-frozen period. Soil samples (0e20 cm) were taken
weekly after fertilization until soil freeze-up. Soil NHþ 4 and NO3 were extracted with 2 M potassium chloride solution and analyzed on a continuous-flow autoanalyzer (Sanþþ, Breda, the Netherlands). Soil labile organic carbon (LOC) concentrations were determined by a potassium permanganate oxidation method (Mirsky et al., 2008). The inorganic N and LOC were measured weekly and biweekly, respectively, resulting in a total of 26 and 14 repeated measures during the study. 2.4. Data analyses and statistics Crop residues were removed from the field as a common practice in the study region, and were not included in the estimation of plant C input. According to the literature review by Bolinder et al. (2007), if all recoverable aboveground materials of grain maize were exported from the field, the relative proportion of net primary productivity returned to soil was 27%, mainly including litter fall during the growing season, harvest losses, roots, and root-derived organic materials from root exudates and root turnover. For estimating plant C input, it is assumed that the C concentration in maize plant was 0.45 g g
1 and the shoot-to-root ratio of maize was 5.6 (Bolinder et al., 2007). Then the plant C input to soil (Cin-plant) was calculated as follows:

Cin
plant ¼ Aboveground biomass  ð1 þ 1=5:6Þ  0:45  0:27 (1) The total C input was calculated as the sum of plant C input and applied organic manure C. The C output was defined as C loss in the form of Rh (Li et al., 2010). The net SOC sequestration was calculated by subtracting the C output from C input.

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The intensity of soil NHþ 4 (NH4I), NO3 (NO3I), inorganic N
(NHþ þ NO ) (IONI), and LOC (LOCI) were calculated by a linear 4 3 interpolation of the concentrations between measurement days (Maharjan and Venterea, 2013). The intensity index combines the
magnitude of NHþ 4 , NO3 , or LOC accumulation over time and thus predicts the cumulative exposure of soil microbial communities to available N or C sources (Burton et al., 2008). The normal distribution of the data was examined using the Kolmogorov-Smirnov test before analysis, and ln-transformation was used if necessary. A constant of 2 was added to Rs and Rh fluxes before transformation. The response ratio of a variable to N fertilization (RR) was calculated as the ln-ratio of the value in the fertilized treatment to that in the control (CK). Significant differences among treatments were tested using the one-way analysis of variance (ANOVA) followed by the least significant difference test. Two-way ANOVA analysis was performed to evaluate the effects of manure type and application rate, and their interaction on annual Rs, Rh, Ra, Rh/Rs, and their RR. Pearson correlation and regression analyses were conducted to explore the relationships between CO2 flux and other factors. Binary regression models were employed to examine the combined effects of ST10 and SWC on Rs, Rh, and Ra. The standardized regression coefficient (beta coefficient) was used to express the relative importance of ST10 and SWC in influencing CO2 flux (Niknahad Gharmakher et al., 2009). Cubic regression models (y ¼ b0 þ b1  x þ b2  x2 þ b3  x3) were developed to describe the temporal pattern of the RR of Rh fluxes. The temperature sensitivity of Rs, Rh, and Ra were assessed by the Q10 value calculated by the following functions (Zhou et al., 2007):

R ¼ R0 eaT

(2)

where R is the rate (mg C m
2 h
1) of Rs, Rh or Ra of each treatment, T is soil temperature ( C) at 10 cm depth, R0 is the basal respiration rate at 0  C, and a represents the temperature sensitivity. The Q10 value was then calculated by:

Q10 ¼ e10a

(3)

Statistical significance was set at P < 0.05 unless otherwise indicated. All statistical analyses were conducted with SPSS 18.0 (SPSS Inc. Chicago, USA) or Origin Pro 8.5 (OriginLab, Northampton, USA). 3. Results 3.1. Environmental conditions Total precipitation was 544 mm during the one-year experimental period, which was equivalent to 98% of the long-term mean value (Fig. 1a). Summer (JuneeAugust) precipitation was 418 mm, accounting for 76.7% of the annual amount. Daily AT was highest on 19 June 2011 (28.3  C), lowest on 24 December 2011 (
30.1  C), and averaged at 2.0  C. ST followed the trend of AT, except during the soil frozen period when ST was obviously higher than AT. Throughout the duration of the experiment, there were no significant differences in SWC among treatments. The mean SWC of all plots ranged from 7.7% to 41.7%, with an average of 24.4% (Fig. 1a). Soil inorganic N (ION) was mainly in the form of NO
3 (Fig. S1), which varied from 4.4 to 41.2 mg N kg
1 during the measurement period. The NO
3 intensity (NO3I) increased significantly (P < 0.01) from 2.26 to 3.45e3.94 g N d kg
1 by N fertilization (Fig. S2). Soil
1 NHþ and was less 4 concentration ranged from 1.0 to 8.6 mg N kg variable than the NO
concentration. Fertilization did not affect soil 3 NHþ 4 concentration at each measurement time, but significantly

increased NH4I from 0.42 to 0.46e0.50 g N d kg
1. Similarly, soil LOC concentration was not significantly different among treatments, but LOCI was significantly higher in the fertilized treatments (104.2e104.9 g C d kg
1) than CK (103.3 g C d kg
1). Manure treatments had higher LOCI than NPK; however, only the difference between CM1 and NPK was significant. 3.2. Soil respiration under various environmental factors and N fertilizations The fluxes of Rs and Rh exhibited the same clear seasonal variation in all treatments (Fig. 1b and c). Rs and Rh were low at the beginning of the experiment, then steadily increased, and reached their maximum in JulyeAugust when the temperature was high and rainfall was sufficient. The highest fluxes of Rs (263.6 mg C m
2 h
1) and Rh (118.3 mg C m
2 h
1) were measured in the CM1 and PM1 treatment, respectively. From mid-August, Rs and Rh gradually declined and became less than 28 mg C m
2 h
1 after maize harvest. The temporal trend of Ra was similar to that of Rs and Rh during the growing season (Fig. S3). Ra ranged from 2.0 to 180.0 mg C m
2 h
1, and averaged at 48.7 mg C m
2 h
1 for all treatments. No clear variation patterns were observed for the RR of Ra flux in all fertilized treatments (Fig. S4). However, the RR of Rh flux showed a consistent pattern in each fertilized treatment, i.e., it gradually decreased to a minimum (below zero) on approximately 60th measured day, then increased above zero after maize harvest, and peaked during the spring thaw period in 2012 (Fig. 2). The temporal pattern of the RR of Rh flux could be depicted by a cubic model in NPK (P ¼ 0.0007), PM1 (P ¼ 0.0005), and CM1 (P ¼ 0.002), but not in PM2 or CM2. Precipitation (Pr) on the day prior to CO2 measurement was significantly correlated with Rs in NPK, PM1, PM2, and CM2 (Table S2), and Rh in each treatment (Table S3), but not with Ra (Table S4). Rs, Rh, and Ra were all highly correlated with ST5 and, in particular, ST10 in all treatments at P < 0.01. Likewise, there were significant correlations between SWC and Rs, Rh, or Ra (all P < 0.01, except Ra). However, the correlations between soil NHþ 4 and LOC with Rs and its components were much weaker in each individual treatment. When all the data were combined, the correlations between Rs and LOC, and between Rh and NHþ 4 or LOC were significant (P < 0.01). Soil NO
3 and ION were negatively correlated with Rs, Rh, and Ra, especially for Rs and Rh in the fertilized treatments. With the regression analysis, we found that all the fluxes of Rs, Rh, and Ra increased exponentially with increasing ST (P < 0.001; Fig. 3). The Q10 value was 2.59e2.98, 2.08e2.43, and 2.42e2.99 for Rs, Rh, and Ra, respectively. Rs and Ra had higher Q10 values than Rh in each treatment, and no significant differences were observed among treatments. There was a linear relationship between soil moisture, Rs and its components (Fig. 4). Interestingly, larger R2 values were found for the linear models that fitted SWC with Rh than Rs or Ra in all treatments, with the exception of CM1. The regression models including both ST10 and SWC explained 94e98% of the seasonal variations of Rs, Rh, and Ra (Table 2). The beta coefficient of SWC was higher than ST10 in the regression functions for Rs or Rh, while ST10 had a larger beta coefficient than SWC for Ra. Annual Rs was 314 g C m
2 from soils without N fertilization, and significantly increased to 389 g C m
2 in NPK (Fig. 5a). The highest Rs was measured in PM1 with a value of 420 g C m
2, which was significantly higher than that of CK and other manure treatments, but similar to NPK. Rs under PM application was significantly higher than CM at the identical application rate (i.e., PM1 > CM1 and PM2 > CM2), but decreased with increasing manure application rate (i.e., PM2 < PM1 and CM2 < CM1). There were no significant differences in annual Rh among the CK, NPK,

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Fig. 1. Precipitation, air temperature (AT), soil temperature at 5 (ST5) and 10 cm (ST10), volumetric soil water content (SWC) (a), total soil respiration (Rs) (b), and heterotrophic respiration (Rh) (c) from soils under different fertilization treatments. Vertical bars are the standard errors of the means (n ¼ 3).

CM1, and CM2 treatments. Rh was 209 g C m
2 for both PM1 and PM2, which was significantly higher than CK, and slightly higher than CM1 and CM2. N fertilization enhanced annual Ra from 137 g C m
2 for CK to 161 and 168 g C m
2 for PM2 and CM1, respectively, and significantly further to 190 and 211 g C m
2 for NPK and PM1, respectively. However, CM2 exhibited almost the same Ra as CK. Similar to Rs, Ra was higher in the PM than CM treatments at the same application rate, and reduced with an increasing manure application rate. Annual Rs and Ra (both P < 0.0001), but not Rh, increased exponentially with increasing NO3I, while there were linear relationships between NH4I and LOCI with annual Rh (P ¼ 0.01 and 0.004, respectively), but not with Rs and Ra (Fig. 6). The ratio of Rh/Rs was largest in CK (58.2%) and CM2 (60.5%), and

significantly decreased to 49.5e56.4% in other treatments (Fig. 5a). The RR of annual Rs was highest and lowest in PM1 and CM2, respectively, which was also the case for the RR of Ra (Fig. 5b). For the manure treatments, the RR of both Rs and Ra decreased in the following order: PM1 > CM1  PM2 > CM2. The RR of Rh was higher in the manure treatments than NPK, with significant differences observed between PM and NPK. The RR of Rh/Rs was below zero in all fertilized treatments (except CM2), and significantly lower in NPK and PM1 than in the other treatments (P < 0.01). Two-way ANOVA analysis showed that the type and application rate of organic fertilizer had significant effects of on annual Rs, Ra, Rh/Rs, and their RR, but not on Rh (Table 3).

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Fig. 2. Seasonal variation of the response ratio (RR) of the flux of heterotrophic respiration (Rh) to nitrogen fertilization. The relationships between the RR and experimental day could be fitted with a cubic model for NPK (a), PM1 (b), and CM1 (c), but not the PM2 and CM2 (d) treatments. Shaded sections indicate the 95% confidence intervals of the regression models.

3.3. Plant biomass and carbon balance The maize grain yield and aboveground biomass increased with N fertilization, but not significantly (P > 0.05) (Fig. S5). The estimated C input was not significantly different between CK and NPK (1.79 vs. 1.87 Mg C ha
1), but significantly increased to 2.24e2.53 Mg C ha
1 by manure application. The net SOC sequestration rate was below zero in CK and NPK, and increased to 0.149 and 0.268 Mg C ha
1 for PM1 and CM1, respectively, with further significant (P < 0.05) gains of 0.444 and 0.486 Mg C ha
1 for PM2 and CM2, respectively (Fig. 7).

In the present study, the root exclusion method was used to partition the heterotrophic and autotrophic components of Rs. As presented above, the Rh/Rs results observed in this study and those by Ding et al. (2007a) and Ni et al. (2012) using the root exclusion method were very close to those measured by Rochette and Flanagan (1997) and Gong et al. (2012) with the 13C tracing technique. Gavrichkova and Kuzyakov (2008) reported that the results of maize root respiration obtained by 14C labeling and in unplanted soils were similar. Therefore, although some uncertainties may exist, it was feasible to partition Rs into Rh and Ra using the root exclusion method, and then assess their responses to various environmental factors and N fertilization.

4. Discussion 4.2. Temporal variation of soil respiration and its components 4.1. Soil respiration and its partitioning Annual Rs measured in our maize-planted field was 314e420 g C m
2, which was at the lower end of the global range of 120e1500 g C m
2 from cropland reported by Chen et al. (2014a). However, it was close to the values measured in maize croplands in Midwest USA (350e430 g C m
2; Al-Kaisi et al., 2008) and Eastern Canada (374e492 g C m
2; Gagnon et al., 2016). The contribution of annual Rh to Rs (49.5e60.5%) fell well within the range of 27e86% in agriculture ecosystems based on Subke et al. (2006), and was in line with other results obtained from soils under maize cultivation during a whole year or growing season, e.g., 54.6% (Rochette and Flanagan, 1997), 54.5% (Ding et al., 2007a), 55.2e57.3% (Gong et al., 2012) and, 45.8e56.1% (Ni et al., 2012).

Soil temperature and moisture are the two dominant environmental factors controlling Rs (Savage and Davidson, 2001; Reichstein et al., 2003). In the present study, the fluxes of Rs, Rh, and Ra all exhibited a bell-shaped curve, and followed the variations of ST and SWC (Fig. 1 and Fig. S3). Over the experiment duration, Rs and its components displayed close relationships with ST (Tables S2e4). The Q10 values of Rs, Rh, and Ra ranged from 2.08 to 2.99 (Fig. 3). These values were within the range of 2e5 that has been normally reported in previous studies (Bahn et al., 2010). In general, Rs was stimulated by rainfall events, especially during summer, which could be supported by the linear increases in Rs, Rh, and Ra with increasing SWC (Fig. 4). It has been shown that when soil moisture is greater than 60e70% water-filled pore space

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Fig. 3. Exponential relationships between total soil respiration (Rs), heterotrophic (Rh) and autotrophic (Ra) respiration and soil temperature at 10 cm in the treatment of CK (a), NPK (b), PM1 (c), CM1 (d), PM2 (e), and CM2 (f).

(WFPS), it can suppress Rs due to restricted aeration (Kiese and Butterbach-Bahl, 2002; Ding et al., 2007b). Soil moisture was always less than 69% WFPS in our study, and probably did not reach a level that would inhibit aeration. Regression models including both ST and SWC explained more than 94% of the seasonal variations of Rs, Rh, or Ra (Table 2), suggesting that a better prediction was achieved by including both temperature and moisture than from a temperature response function. Interestingly, the beta coefficient of SWC was higher than ST10 in the regression functions of Rh, whereas the opposite was true for Ra. Consistently, for each individual treatment, Ra had a higher Q10 value than Rh (Fig. 3), while Rh was more closely related with precipitation (Tables S3 and S4) or SWC (Fig. 4) than Ra. These results suggested that Ra was more temperature-sensitive than Rh and that Rh was more moisture-sensitive than Ra. Parton et al. (2012) reported that Rh could be greatly enhanced by rainfall with daily amount greater than 5 mm; however, at least 10 mm

rainfall was needed to significantly stimulate Ra (Chen et al., 2009). Rh has been suggested to be more responsive to changes in moisture than Ra (Zimmermann et al., 2010). This is because Rh represents microbial respiration throughout the soil profile and microorganism populations are most abundant in the top few centimeters of the soil, whereas Ra includes root respiration and those roots tend to be located deeper in the profile. Increased soil moisture could stimulate the mineralization of labile organic compounds and enhance the diffusion of substrates, enzymes, and microbial cells (Casals et al., 2011; Manzoni et al., 2012). Moreover, the microbial biomass and activity, and thus the heterotrophic respiration could be increased with increasing soil moisture within a certain range (Moyano et al., 2013; Matteucci et al., 2015). Higher temperature sensitivity of Ra than Rh has also been observed in a tallgrass prairie (Zhou et al., 2007), a well-drained €kiranta et al., 2008), and a wheat-maize afforested peatland (Ma rotation cropland (Zhang et al., 2013). Ra principally depends on

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Fig. 4. Linear relationships between total soil respiration (Rs), heterotrophic (Rh) and autotrophic (Ra) respiration and volumetric soil water content (SWC) in the treatment of CK (a), NPK (b), PM1 (c), CM1 (d), PM2 (e), and CM2 (f).

the supply of recent photosynthates, and the plant photosynthetic rate is highly linked to temperature variation (Tang et al., 2005; Baldocchi et al., 2006; Hopkins et al., 2013). Thus, the effects of temperature change on Ra may be enlarged as a result of the responses of various factors, including substrate supply, live roots activity and growth, and rhizospheric microorganisms (Schindlbacher et al., 2009). Therefore, the Q10 values used here, and in most studies, express the “apparent” rather than the “real” temperature sensitivity, therefore the Q10 values cannot be extrapolated directly to predict Rs under climate warming (Davidson et al., 2006; Subke and Bahn, 2010). 4.3. Effects of fertilization on soil respiration and its components Annual Rs increased from 314 g C m
2 in CK to 327e420 g C m
2 by N fertilizer application, with significant effects observed in all treatments with the exception of CM2 (Fig. 5a). This finding is

consistent with previous studies that observed higher Rs after N fertilization from the cropland ecosystems (Meijide et al., 2010; Morell et al., 2010; Li et al., 2013b). Zhou et al. (2014) conducted a meta-analysis and found that N addition positively affected Rs in cropland; however, great variation existed in the responses of Rh or Ra, largely because of the limited number of studies separating the Rs components. Our results showed that the stimulation effect of fertilization on Rs was mainly the result of an increase in Ra and its contribution to Rs, and the RR of annual Ra was higher than that of Rh in each fertilized treatment, except CM2 (Fig. 5). It has been acknowledged that Ra is driven by belowground C allocation and generally increases with the increase of plant productivity (Bond-Lamberty et al., 2004; Savage et al., 2013). Aboveground biomass was higher, albeit not significantly, in the fertilized treatment compared with CK (Fig. S5). Increased root biomass and N content after N fertilization can also contribute to the enhanced Ra (Tu et al., 2013; Zhang et al., 2014). In a previous study

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111

Table 2 Regression models of soil respiration (Rs), and heterotrophic (Rh) and autotrophic (Ra) respiration against soil temperature at 10 cm (ST10) and volumetric soil water content (SWC). The flux data of Rs, Rh, and Ra were ln-transformed, and a constant of 2 was added to Rs and Rh before transformation. All models are significant at P < 0.0001. Treatment

Rs

Rh

Ra

CK NPK PM1 CM1 PM2 CM2 CK NPK PM1 CM1 PM2 CM2 CK NPK PM1 CM1 PM2 CM2

Equation

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0.107 ST10 þ 0.0834 SWC 0.112 ST10 þ 0.0883 SWC 0.101 ST10 þ 0.0984 SWC 0.0986 ST10 þ 0.0952 SWC 0.0985 ST10 þ 0.0959 SWC 0.0980 ST10 þ 0.0902 SWC 0.0815 ST10 þ 0.0825 SWC 0.0852 ST10 þ 0.0821 SWC 0.0795 ST10 þ 0.0893 SWC 0.0859 ST10 þ 0.0838 SWC 0.0744 ST10 þ 0.0932 SWC 0.0841 ST10 þ 0.0823 SWC 0.113 ST10 þ 0.0473 SWC 0.122 ST10 þ 0.0526 SWC 0.115 ST10 þ 0.0595 SWC 0.110 ST10 þ 0.0534 SWC 0.0941 ST10 þ 0.0586 SWC 0.0935 ST10 þ 0.0582 SWC

performed in the same region, Ni et al. (2012) found that both the maize root biomass and N content were increased by N addition. It was interesting to find that annual Ra increased exponentially with

R2

0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.97 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.97 0.94 0.97

F

1428 1489 1737 1655 1753 1542 1378 1018 1478 1247 1300 1326 1067 1019 881 567 284 558

Beta coefficients ST10

SWC

0.461 0.465 0.418 0.415 0.416 0.417 0.395 0.413 0.382 0.411 0.355 0.401 0.620 0.623 0.582 0.589 0.524 0.519

0.548 0.546 0.594 0.596 0.596 0.594 0.612 0.592 0.628 0.597 0.653 0.608 0.385 0.382 0.422 0.409 0.459 0.481

increasing NO3I, but not NH4I (Fig. 6). An increased NO
3 supply could promote plant growth and thus Ra, as discussed above. On the other hand, as the predominant form of inorganic N in test soil,

Fig. 5. Annual total soil respiration (Rs), heterotrophic (Rh) and autotrophic (Ra) respiration, and the ratio of annual Rh to Rs (Rh/Rs) (a); and the response ratio of annual Rs, Rh, Ra and Rh/Rs to nitrogen fertilization (b). Vertical bars are the standard errors of the means (n ¼ 3). Significant differences between treatments at P < 0.05 are indicated by different letters.

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Fig. 6. Relationships between annual total soil respiration (Rs; a, b, and c), heterotrophic (Rh; d, e, and f) and autotrophic (Ra; g, h, and i) respiration and soil intensities of ammonium (NH4I), nitrate (NO3I), or labile organic carbon (LOCI) for all treatments.

þ NO
3 rather than NH4 was suggested be the preferred N source for crop plant uptake (Inselsbacher et al., 2013). It has been proved that þ NO
3 should first be reduced to NH4 before plant assimilation, which needs energy supply from the decomposition of carbohydrates and has large effects on respiratory costs (Tischner, 2000). For maize, almost half of the NO
3 is reduced in the roots, which in turn strongly increases the rate of root-derived CO2 fluxes (Gavrichkova and Kuzyakov, 2008).

Table 3 Results (P values) of two-way ANOVA analysis on the effects of the type and application rate of organic fertilizer and their interaction on annual soil respiration (Rs), heterotrophic (Rh) and autotrophic (Ra) respiration, annual Rh/Rs, and their response ratios (RR) to nitrogen fertilization. Annual amount

Type Rate Type  rate

Response ratio

Rs

Rh

Ra

Rh/Rs

Rs

Rh

Ra

Rh/Rs

0.007 0.012 0.733

0.288 0.736 0.762

0.047 0.024 0.570

0.002 0.001 0.498

< 0.001 0.001 0.769

0.060 0.627 0.519

0.008 0.003 0.649

< 0.001 < 0.001 0.259

Bold value indicates significant effect at P < 0.05.

Compared with CM, PM contains more hemicellulose and cellulose, and less lignin (Table 1), indicating that PM was more decomposable than CM in our study (Morvan and Nicolardot, 2009). The LOC concentration was also significantly higher in PM than CM. More available substrates contents in PM than CM likely contributed to higher Rh from the plots applied with PM than CM under the identical application rate (Fig. 5). Therefore, it could be deduced that more organic fertilizer was decomposed and released more N in PM than CM at the same application rate (Chen et al., 2014b). This could partly explain higher Ra in PM1 than CM1 and in PM2 than CM2. The decrease in Ra with increasing manure application rate was likely caused by the lower inorganic N supply due to the simultaneously reduced urea N application rate (Fig. S2). There was no significant difference of annual Rh between the CK and NPK treatment (Fig. 5a). Similarly, Sun et al. (2014) reported that urea addition did not affect soil microbial biomass and thus Rh in a larch plantation. In the manure treatments, Rh was enhanced by 8.1e14.1% in comparison to CK. Higher Rh is generally observed as a result of increased organic substrates supply (Iqbal et al., 2009; McMullen et al., 2015). In this study, Rh was found to increase with increasing LOCI (Fig. 6f). Different from plants, microorganisms

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113

Fig. 7. Annual soil organic carbon (SOC) sequestration under different fertilization treatments. Vertical bars are the standard errors of the means (n ¼ 3). Significant differences between treatments at P < 0.05 are indicated by different letters.


þ preferentially utilize NHþ 4 over NO3 , and NH4 can inhibit microbial assimilation of NO
3 even at a low concentration (Booth et al., 2005). We found that there was a linear relationship between Rh and NH4I, but not NO3I (Fig. 6). Therefore, higher LOCI and NH4I may contribute to greater stimulation effect on Rh in the treatments receiving manure than NPK as indicated by higher response ratio of Rh (Fig. 5). However, significant increment of Rh only exhibited in the PM, but not CM treatments, probably due to higher labile organic C content in PM than CM (Table 1). Although more organic C was applied in PM2 and CM2 than PM1 and CM1, Rh was almost the same between PM2 and PM1, and between CM2 and CM1. Niklasch and Joergensen (2001) also found that the decomposition of applied compost C reduced with an increasing addition rate in a cropland soil. Decreased manure decomposition at higher application rate in our study was also probably due to a reduced stimulation effect of urea N at lower application rates (Zhang et al., 2014). Therefore, more studies, especially in situ experiments, are needed to further investigate the effects of application rate of inorganic or organic N fertilizers on the decomposition of organic C. A clear temporal variation of the RR of Rh flux was found in each fertilized treatment. This was more significant in NPK, PM1, and CM1 with a higher urea application rate, which could be described by cubic functions (Fig. 2). The RR was initially above zero and then dropped below zero on approximately 60th measured day, indicating that the fertilization effect on Rh shifted from positive to negative. Similarly, Xu et al. (2016) found that N addition enhanced pine litter decomposition during the first 56 days, but suppressed it from day 57 afterwards. The initial stimulation effect was mainly due to the increased decomposition of labile organic compounds in response to N addition (Xu et al., 2017). As the labile substrates were gradually consumed, the positive RR declined and became negative when the heterotrophic CO2 was more likely sourced from the fungal decomposition of recalcitrant organic C, which might be inhibited by N addition (Neff et al., 2002; Janssens et al., 2010). During the frozen period, the RR of Rh shifted to positive again. It was suggested that under the frozen conditions, the availability of substrate and nutrient, and the microbial utilization capacity became rather low (Schimel and Mikan, 2005), which could be alleviated by fertilization. In a previous study, we found that both

the concentration and decomposability of soil dissolved organic C increased after freeze-thaw cycles (Chen et al., 2016). Hence, the stimulation effect of N fertilization on Rh was greatly increased during the spring thaw.

4.4. Implications for carbon sequestration Crop residues are traditionally removed from the field in the study region, which was a main reason for SOC loss of Mollisol in Northeast China over the past several decades (Sun et al., 2010). We found that the C input was lower than the output in CK and NPK (Fig. 7), suggesting that the study soils without organic amendments would be a net C source. Annual C lost contributed 0.11% of SOC in the 0e20 cm layer for NPK. This value was lower than the annual SOC decrease for Mollisol (0.74%) in Northeast China over the past thirty years, estimated by Yan et al. (2011). However, it was comparable with the result (0.16%) measured in a cropland receiving inorganic fertilizer during 2001e2011 at the same site as ours (Ding et al., 2012). The SOC loss rate has been reported to decline with increasing cultivation year in Northeast China (Zhang et al., 2007). The C input was higher than the output in the manure treatments, and the SOC sequestration rate was estimated at 0.149e0.486 Mg C ha
1 yr
1, with a significant increment observed in PM2 and CM2. Similarly, Jiang et al. (2014) predicted that the average SOC sequestration rate was 0.287 Mg C ha
1 yr
1 under combined application of inorganic fertilizer with manure for the uplands of Northern China, based on the results from long-term field trials. Accordingly, combined application of inorganic and organic N with a 1:1 ratio would have potential to enhance the SOC stock of the study region. It should be noted that high uncertainty may exist in our calculation of soil C balance. The proportion of net primary productivity returned to soil, plant C concentration, and shoot-to-root ratio were assumed to be the averages of grain maize according to Bolinder et al. (2007). This can introduce biases in plant C input estimation because high variations exist in these variables across different sites (Bolinder et al., 2007). Long-term experiment is recommended to make a direct measurement of SOC storage changes under different N fertilization regimes.

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5. Conclusions Our results emphasized the importance of partitioning the Rs components because Rh and Ra had different responses to changes in environmental factors and N fertilization. Ra was found to be more temperature-sensitive than Rh, while Rh was more moisturesensitive than Ra. The type and application rate of manure had significant effects on Rs and Ra, but not Rh. Further studies with more types and application rate gradients of organic N fertilizers should be carried out to better understand their effects on soil CO2 fluxes. Studies using C isotope tracing technique are recommended to provide mechanistic insight into the dynamics of applied organic C in soil. A shift of the net C sequestration rate from negative to positive after replacing urea partly with manure, especially at the higher rate, suggested that combined application of inorganic and organic fertilizers might have potential to increase the SOC stock in upland Mollisols. However, extrapolating this result to other regions in Northeast China should be done with caution, and more multi-year experiments are necessary to confirm our findings at a larger spatial scale. Acknowledgements The authors would like to thank the colleagues at Hailun National Agro-ecological Experimental Station, Chinese Academy of Sciences for their helpful assistance in the field experiment. Many thanks also to Chief Editor Prof. Joann Whalen and three anonymous reviewers for their insightful and constructive comments and suggestions that really helped us to improve the manuscript quality greatly. This work was financially supported by the Chinese Academy of Sciences (XDB15020100) and the National Natural Science Foundation of China (31561143011). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2017.03.011. References Al-Kaisi, M.M., Kruse, M.L., Sawyer, J.E., 2008. Effect of nitrogen fertilizer application on growing season soil carbon dioxide emission in a corn-soybean rotation. Journal of Environmental Quality 37, 325e332. Amundson, R., Berhe, A.A., Hopmans, J.W., Olson, C., Sztein, A.E., Sparks, D.L., 2015. Soil and human security in the 21st century. Science 348, 1261071. Bahn, M., Reichstein, M., Davidson, E.A., Grünzweig, J., Jung, M., Carbone, M.S., Epron, D., Misson, L., Nouvellon, Y., Roupsard, O., Savage, K., Trumbore, S.E., Gimeno, C., Curiel Yuste, J., Tang, J., Vargas, R., Janssens, I.A., 2010. Soil respiration at mean annual temperature predicts annual total across vegetation types and biomes. Biogeosciences 7, 2147e2157. Baldocchi, D., Tang, J.W., Xu, L.K., 2006. How switches and lags in biophysical regulators affect spatial-temporal variation of soil respiration in an oak-grass savanna. Journal of Geophysical Research: Biogeosciences 111, G02008. r, K., Fo ti, S., Posta, K., Eugster, W., Nagy, Z., 2016. AutoBalogh, J., Papp, M., Pinte trophic component of soil respiration is repressed by drought more than the heterotrophic one in dry grasslands. Biogeosciences 13, 5171e5182. Bolinder, M.A., Janzen, H.H., Gregorich, E.G., Angers, D.A., VandenBygaart, A.J., 2007. An approach for estimating net primary productivity and annual carbon inputs to soil for common agricultural crops in Canada. Agriculture, Ecosystems and Environment 118, 29e42. Bond-Lamberty, B., Thomson, A., 2010. Temperature-associated increases in the global soil respiration record. Nature 464, 579e582. Bond-Lamberty, B., Wang, C.K., Gower, S.T., 2004. A global relationship between the heterotrophic and autotrophic components of soil respiration? Global Change Biology 10, 1756e1766. Booth, M.S., Stark, J.M., Rastetter, E., 2005. Controls on nitrogen cycling in terrestrial ecosystems: a synthetic analysis of literature data. Ecological Monographs 75, 139e157. Brye, K.R., Golden, B., Slaton, N.A., 2006. Poultry litter decomposition as affected by litter form and rate before flooding for rice production. Soil Science Society of America Journal 70, 1155e1167. Burton, D.L., Li, X.H., Grant, C.A., 2008. Influence of fertilizer nitrogen source and

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