Linking microbial immobilization of fertilizer nitrogen to in situ turnover of soil microbial residues in an agro-ecosystem

Agriculture, Ecosystems and Environment 229 (2016) 40–47

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Linking microbial immobilization of fertilizer nitrogen to in situ turnover of soil microbial residues in an agro-ecosystem Xiao Liua,b , Guoqing Hua,b , Hongbo Hea,c,* , Chao Lianga,d, Wei Zhanga , Zhen Baia , Yeye Wua , Guifeng Lina , Xudong Zhanga,* a

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China University of Chinese Academy of Science, Beijing 100049, China National Field Observation and Research Station of Shenyang Agro-ecosystems, Shenyang 110016, China d Great Lakes Bioenergy Research Centre, University of Wisconsin, Madison, WI 53706, USA b c

A R T I C L E I N F O

Article history: Received 26 September 2015 Received in revised form 13 May 2016 Accepted 17 May 2016 Available online xxx Keywords: N-labeled fertilizer Maize residue Soil amino sugar Microbial immobilization Agro-ecosystem 15

A B S T R A C T

Understanding long-term microbial immobilization of nitrogen (N) fertilizer is essential for N management in agricultural soils. Evaluating the transformation and accumulation of N fertilizer into microbial residues is critical for developing such an understanding due to the requirement of timeintegrated biomarkers and a 15N-labeling technique. By tracing the dynamics of amino sugars derived from annually applied fertilizer over 8 years, we investigated the influence of continuous maize residue mulching on the temporal immobilization of fertilizer N in an agricultural soil and quantified the turnover of microbial residues in situ. We found that the amino sugar transformation rate from fertilizer N was constant over time in both fertilization-only and maize residue mulching managements, but it was significantly higher in the upper cultivation layer (0–10 cm) after maize residue mulching. Mulching with maize residue facilitated initial fertilizer N transformation, while the subsequent 7-year application maintained the increased transformation rate. Consequently, the accumulation of fertilizer-derived amino sugars increased linearly in both managements within the 8 years of our field experiment. The mean residence time (MRT) of soil amino sugar-N was estimated by using extrapolation and first-order kinetics approaches, respectively. The calculated MRT of amino sugar-N using first-order kinetics (78 and 154 years at 0–10 and 10–20 cm, respectively) was slightly shorter than that estimated by the extrapolation (89 and 165 years at 0–10 and 10–20 cm, respectively) in the fertilization-only management. Mulching with maize residue did not change the MRT of amino sugar-N because maize residue addition enhanced the immobilization of maize residue-derived N or the transformation of indigenous soil N in addition to those of fertilizer N, leading to the same proportion of new N assimilated in microbial residues. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction The maintenance of crop production in intensively cultivated agricultural ecosystems depends largely on the input of nitrogen (N) fertilizer. However, approximately 25% of the applied fertilizer is emitted to the atmosphere and 20% is lost to aquatic systems through nitrate leaching (Cassman et al., 2002; Galloway et al., 2004), leading to a variety of adverse environmental effects, such as eutrophication and greenhouse gas emissions (Tilman et al.,

* Corresponding author at: Institute of Applied Ecology, Chinese Academy of Sciences, No. 72 Wenhua Road, Shenyang 110016, China. E-mail addresses: [email protected] (H. He), [email protected] (X. Zhang). http://dx.doi.org/10.1016/j.agee.2016.05.019 0167-8809/ã 2016 Elsevier B.V. All rights reserved.

2002). The assimilation of inorganic N by soil microorganisms is the most crucial biochemical process for N retention in soil, and this process could competitively reduce the loss of fertilizer N to the environment (Vinten et al., 2002; Tahovská et al., 2013), particularly in heavily degraded arable land. Thus, agricultural management practices that could enhance microbial immobilization of fertilizer N have attracted much attention. Crop residues (e.g., maize residue) are important resources involved in nutrient cycling in agro-ecosystems. Through the release of available carbon (C) during microbial decomposition (Szili-Kovács et al., 2007; Said-Pullicino et al., 2014), crop residues can stimulate the proliferation of starving microorganisms and enhance the biological immobilization of fertilizer N. However,

X. Liu et al. / Agriculture, Ecosystems and Environment 229 (2016) 40–47

despite the well-recognized activation of microorganisms stimulated by a single addition of maize residue (De Nobili et al., 2001; Blagodatskaya and Kuzyakov, 2008), the influences of continuous mulching with maize residue on the temporal microbial N immobilization over long-term is unknown. Because the initial addition of a substrate resulted in rapid responses of microorganisms while the subsequent additions led to gradual microbial adaptations to long-term substrate supply (Schimel et al., 2007; Blagodatskaya and Kuzyakov, 2013), we hypothesized that the initial and subsequent maize residue applications played different roles in promoting microbial immobilization of fertilizer N. Microorganisms are not only the mediators of microbial N immobilization but also the drivers of N turnover through their metabolic processes (Zeglin et al., 2013). The formation of microbial residues from fertilizer N is accompanied by the partial decomposition of the indigenous portion of microbial residues in soil, which results in the turnover of microbial residues (He et al., 2011). Understanding the turnover of microbial residues induced by fertilizer N is essential for evaluating in situ microbial immobilization of fertilizer N and microbial contributions to the turnover of soil N in agro-ecosystems (Perelo et al., 2006). In addition to the indigenous portion of soil N, the immobilization of maize residue-derived N also contributed to microbial residue accumulation (Bird et al., 2001; Ding et al., 2011). However, it remains unclear how the addition of maize residue influenced the turnover of N-containing microbial residues induced by fertilizer application. Amino sugars are important cell wall constituents of microorganisms (Guggenberger et al., 1999; Amelung et al., 2001). Compared with the cytoplasmic components, amino sugars can persist in soils for relatively longer time after cell death and thus could serve as time-integrated biomarkers for long-term microbial N immobilization and microbial residues accumulation (van Groenigen et al., 2010; Miltner et al., 2012). As the size of the total microbial residues carbon (C) pool could account for 80% of the organic C in soil estimated by the Absorbing Markov Chain model (Liang et al., 2011), the role of microbial residues in N immobilization is pivotal in soil N turnover. Although individual amino sugars allowed a rough comparison of fungal and bacterial contributions to N turnover, the information regarding the integrated function of microorganisms related to fertilizer N assimilation could only be indicated by evaluating total amino sugars. Studies on the turnover of amino sugars (based on C cycling) have been executed by applying artificial 13C-labeling or C3/C4 vegetation change (Glaser et al., 2006; Amelung et al., 2008; Derrien and Amelung, 2011; Schmidt et al., 2011), whereas under certain management practices in agro-ecosystems, such as maize residue return, the amino sugar turnover in situ induced by annual N fertilizer application was unknown. Therefore, by tracing fertilizer-derived amino sugars based on 15N-labeling and differentiating techniques under 8-year fertilization-only and maize residue managements, the aims of our study were 1) to estimate the temporal contribution of microbial processes related to the fertilizer N immobilization on a field scale, and 2) to explore the influence of maize residue management on fertilizer N immobilization, as well as fertilizer N induced microbial residues turnover in situ. 2. Materials and methods 2.1. Site description An 8-year field experiment, started in 2007, was established at the National Field Observation and Research Station of Shenyang Agro-ecosystems (N41
310, E123
240 ) in northeast China. The weather at the site is typical of a temperate, humid, continental

41

monsoon climate. The mean annual temperature is 7–8
C, and the mean annual precipitation is approximately 700 mm. The soil of the experimental field is classified as Alfisols (Soil Taxonomy) or Luvisols (World Reference Base). Maize (Zea mays L.) is planted annually at a mean density of 57,700 plants ha1 and harvested in late September. 2.2. Experimental design The experiment was conducted in a randomized design with three replicates. The micro-plots (1.6 m  1.3 m) were randomly arranged in the field at distances of approximately 2.5 m apart. The micro-plots were surrounded by polyvinyl-chloride (PVC) boards that had an aboveground height of 15 cm and were pressed into the soil to a depth of 35 cm. For each plot, maize was sown at a density of 12 plants. Two treatments were included in this study. Treatment 1 (T1): 200 kg N ha1 yr1 of total 15N-labeled (NH4)2SO4 (50 atom% 15N) was applied annually in three splits. The first 50 kg N ha1 yr1 was applied as the basal fertilizer before seeding stage, the second 100 kg N ha1 yr1 as the first topdressing at jointing stage and the last 50 kg N ha1 yr1 as the second topdressing at silking stage. Treatment 2 (T2): In addition to the same application rate and frequency of 15N-labeled (NH4)2SO4 (50 atom% 15N), unlabeled maize residue (with natural abundance of 15N) was cut into 10 cm long pieces and annually applied on field surfaces after seeding. The application rate of the unlabeled maize residue was 5.8 Mg ha1, equivalent to about 50% of annual average yield. Annual C and N input from the unlabeled maize residue was approximately 2507 and 48.3 kg ha1, respectively (Hu et al., 2015). In addition, two sets of treatment controls were conducted for differentiating fertilizerderived amino sugars. In these unlabeled micro-plots (three for each treatment), either (NH4)2SO4 was applied or (NH4)2SO4 was applied plus maize residue mulching. The unlabeled samples were collected at the same time as T1 and T2. In this experiment, phosphorus (P) and potassium (K) fertilizers, in the form of KH2PO4 and K2SO4 pellets, were applied at rates of 30 kg P and 58 kg K ha1 yr1 as basal fertilizers before seeding (Lü et al., 2013). Seedbeds in each plot were manually prepared with a reduced disturbance of the soil by hoes, and the base fertilizers were applied at a depth of 5–10 cm before maize seeding. After maize seeding, maize residue was applied on the soil surface at the corresponding plots. 2.3. Soil sampling After maize harvesting during the period 2007–2014, three individual soil samples were collected in each plot at depths of 0– 10 and 10–20 cm, respectively. Samples at each soil layer were taken with a 3-cm-diameter soil auger and then thoroughly mixed by hand to obtain a composite sample from each plot. Before trial, composite soil samples in each plot were collected in autumn of 2006 to obtain the initial contents of amino sugars in the corresponding micro-plots (n = 3). After visible plant and organic debris was removed, the air-dried soil samples were ground and then sieved to 0.25 mm for amino sugar analysis. 2.4. Analysis of amino sugars and determination of 15N isotope incorporation by gas chromatography/mass spectrometry Amino sugars were quantified according to the method of Zhang and Amelung (1996). Briefly, after hydrolysis with 6 M HCl for 8 h at 105
C, the solution was filtered and dried with a rotary evaporator under vacuum. By redissolving the residue with water, the solution was adjusted to neutral pH and then centrifuged. After freeze-drying the supernatant, methanol was added to remove

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amino sugars from the residues. The purified amino sugars were transformed into aldononitrile derivatives and extracted with dichloromethane from the aqueous solution. After evaporating the dichloromethane, the amino sugar derivatives were redissolved in a hexane and ethyl acetate solvent (v:v = 1:1) for quantification by gas chromatography (GC). Myo-inositol (100 mg) was added as an internal standard before hydrolysis, and N-methylglucamine (100 mg) was added before derivatization as the recovery standard. The 15N enrichment in each amino sugar was quantified using a gas chromatography/mass spectrometry (GC/MS) system (Finnigan Trace, Thermo Electron Co., Ltd., USA) equipped with a quadrupole MS supplied with chemical ionization (CI). The temperature and electron energy of the CI source were set at 180
C and 70 eV, respectively. The interface temperature was 250
C, and helium was used as a carrier gas with a flow rate set at 0.8 mL min1. The GC temperature program was followed as described by He et al. (2006), and the split ratio was 30:1. The reaction gas was methane, and its flow was 1.5 mL min1. In the mass spectrum, three parent fragments (m/z 148, 206 and 223) were obtained for GluN and GalN, while two parent fragments (m/z 264 and 324) were obtained for MurN. Any of the fragments can be equally used to calculate the 15N enrichment in a compound (He et al., 2006), but base fragments were preferred to obtain the highest intensity of the isotope fragment. Therefore, the 15N enrichment of individual amino sugars was quantified in the selected ion monitoring (SIM) mode, and the intensity of target fragments (F, i.e., 14N), as well as the corresponding F plus 1 (F + 1, i.e., 15N), were measured because only one N atom was observed in the amino sugar molecules. 15N enrichment in glucosamine (GluN) and galactosamine (GalN) was determined according to the intensity of m/z 206 and 207, whereas 15N enrichment in muramic acid (MurN) was estimated by monitoring the intensity of m/z 264 and 265.

where k is the decay constant and t is the years elapsed since start of experiment.

2.5. Calculations

2.6. Statistical analysis

The fertilizer-derived amino sugars (ASt-FD, mg kg1 soil) were calculated according to the following equations:

Statistical analysis was performed using the software package SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Repeatedmeasure analysis of variance (ANOVA) was used to examine the differences in the contents of total amino sugars (ASt), fertilizerderived amino sugars (ASt-FD), non-fertilizer-derived amino sugars (ASt-NFD), percentages of ASt-FD in ASt and amino sugar transformation rates (ATRt) with time between treatments. These data between treatments in each soil layer were then analyzed by independent-sample t-test, whereas the sampling year was analyzed by one-way ANOVA. Multiple comparisons were performed based on the least significance difference (LSD) test at a confidence level of 95%. The curve fits in calculating MRT were performed with Origin 8.6 software (OriginLab, Northampton, MA).

AStFD ¼ ð

3 X ASi  APEi Þ=ATf

ð1Þ

i¼1

APEi ¼ ðRsi  Rci Þ=½1 þ ðRsi  Rci Þ

ASt ¼

3 X ASi

ð2Þ

ð3Þ

i¼1

where ASi (mg kg1 soil) is the content of each amino sugar (GluN, GalN and MurN), APEi is the 15N atom% excess of each amino sugar, Rsi is the isotope ratio of each amino sugar in labeled soil samples and Rsi ¼ ½AðFþ1Þ =AðFÞ  (A is the area of the selected ion). Rci represents the ratio of each amino sugar in the corresponding treatment control analyzed on the same GC/MS assay (He et al., 2006), ATf (%) is the 15N abundance of the added fertilizer (50%) and ASt (mg kg1 soil) is the total amino sugar content as sum of GluN, GalN and MurN. As a result, the non-fertilizer-derived amino sugar concentrations (ASt-NFD, mg kg1 soil) were calculated by subtracting the ASt-FD from the ASt. The amino sugar transformation rates (ATRt, %) were calculated by: ATRt ¼ ½AStFD  N=½M  ðt  2006Þ  100%

ð4Þ

AStFD  N ¼ ð

3 X ASi  Ni  APEi =ATf Þ  a  h  d

ð5Þ

i¼1

where ASt-FD-N (g) is the N stock of ASt-FD in one layer of each plot, Ni (%) is the N content of each amino sugar (GluN and GalN: 7.82%; MurN: 5.57%), a (m2) is the area of each plot (2.08 m2), h (cm) is the height of each layer (10 cm) and d (g cm3) is the bulk density in each layer (1.17 g cm3 at 0–10 cm and 1.44 g cm3 at 10–20 cm soil depth). M is the annual applied rate of fertilizer N in each plot (41.6 g yr1) and t is the sampling year. Mean residence time (MRT) of amino sugars induced by fertilizer N was calculated by using two approaches. In the first one, the ASt were fitted to mono-exponential curves according to Roth et al. (2011) and ASt-FD were fitted in linear relationships, thus the MRT of soil amino sugar-N was calculated by extrapolating the intersections of the ASt-FD and ASt functions (Glaser et al., 2006). This approach was established on the assumption that all inherent amino sugars would be completely replaced by the fertilizerderived amino sugars. Secondly, the MRT of amino sugar-N was calculated by first-order kinetics (Bock et al., 2007; Amelung et al., 2008). This approach was established on the assumption that soil total amino sugars cannot be completely replaced by the fertilizerderived amino sugars and the non-fertilizer-derived amino sugars degrade exponentially according to first-order kinetics. k ¼ lnð1  AStFD =ASt Þ=t

ð6Þ

MRT ¼ 1=k

ð7Þ

3. Results 3.1. Amino sugars in the fertilization-only treatment The initial contents of amino sugars in the fertilization-only plots were 824  18 mg kg1 soil at 0–10 cm depth and 750  17 mg kg1 soil at 10–20 cm depth, contributing to 6.2% of the total N in the soil (Supplementary data, Fig. A1c and d). In T1, the contents of total amino sugars (ASt) increased exponentially during the 8 years, estimated according to the assumption of Roth et al. (2011). The increase was 155 mg kg1 soil in 0–10 cm layer and 95 mg kg1 soil in 10–20 cm layer, respectively (Fig. 1a and b). Fertilizerderived amino sugars (ASt-FD) increased linearly at an average rate of 11.9 and 5.5 mg kg1 yr1 in 0–10 cm and 10–20 cm soil layers, respectively. As a result, the concentrations of ASt-FD after 8 years

X. Liu et al. / Agriculture, Ecosystems and Environment 229 (2016) 40–47

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Fig. 1. Concentrations of total amino sugars (ASt), fertilizer-derived amino sugars (ASt-FD) and non-fertilizer-derived amino sugars (ASt-NFD) from 2006 to 2014 at the depths 0–10 cm and 10–20 cm with T1 and T2. T1: annual application of 15N-labeled (NH4)2SO4; and T2: annual application of 15N-labeled (NH4)2SO4 and maize residues. Bars represent the standard error (n = 3).

reached 96.5 mg kg1 soil at 0–10 cm depth and 41.2 mg kg1 soil at 10–20 cm depth (Fig. 1c and d), and these values were mainly attributed to the newly synthesized GluN (accounting for approximately 68–80% of the ASt-FD). Over the 8 years, the contents of ASt-NFD increased gradually by approximately 58.5 and 53.8 mg kg1 soil at the two depths, respectively (Fig. 1e and f). The percentage of ASt-FD in ASt increased annually to approximately 9.9% (0–10 cm layer) and 4.8% (10–20 cm layer), respectively, after 8 years (Fig. 2). In addition, the amino sugar transformation rate (ATRt) was calculated at 0.51  0.03% in 0– 10 cm layer and 0.30  0.04% in 10–20 cm layer and did not change over time at either depth (Fig. 3). 3.2. Amino sugars after fertilization plus continuous maize residue mulching The initial values of amino sugars in the plots for maize residue mulching were 815  16 mg kg1 soil and 753  16 mg kg1 soil at soil depths of 0–10 and 10–20 cm, respectively, showing no significant variations among the plots before fertilization trials. Compared with the fertilization-only treatment, the contents of ASt after continuous maize residue mulching increased exponentially by a larger percentage of 20.2% (p < 0.001; Fig. 1a and b). The ASt-FD content in 0–10 cm layer increased linearly at a rate of approximately 14.2 mg kg1 yr1, which was 18.5% higher than in fertilization-only treatment (p < 0.001; Fig. 1c and d). The concentrations of ASt-NFD (including both maize residue-derived

and the indigenous soil fractions) increased exponentially by 174 mg kg1 soil, which was significantly greater than the fertilization-only treatment in 0–10 cm soil layer in the 8th year (p < 0.001; Fig. 1e and f). The addition of maize residue did not have a significant influence on the dynamics of ASt, ASt-FD or ASt-NFD in 10–20 cm layer (p > 0.05; Fig. 1). Compared with the percentages of ASt-FD in the ASt in fertilization-only treatment, those percentages after maize residue mulching did not show significant changes at both depths (p > 0.05; Fig. 2). Therefore, the ATRt in the mulching treatment did not exhibit temporal variations at either soil depth except the first year in 0–10 cm layer. The value of ATRt in mulching maize residue was 0.62  0.04% at 0–10 cm depth, significantly higher than that in fertilization-only treatment (p < 0.001). However, there was no significant difference at depth of 10–20 cm between the two treatments (p > 0.05; Fig. 3). 3.3. Estimation of the turnover of soil amino sugars induced by fertilizer N addition By extrapolating the curves of ASt-FD and ASt (Fig. 1a–d) in this continuous 15N-isotope tracing field experiment, the MRT values of amino sugar-N in the fertilization-only treatment were estimated at approximately 89 and 165 years at 0–10 and 10–20 cm depths, respectively. The MRT of amino sugar-N after maize residue mulching treatment did not exhibit a significant difference to the fertilization-only treatment, being 90 and 171 years at the two soil

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Fig. 2. Percentages of fertilizer-derived amino sugars (ASt-FD) in the total amino sugars (ASt) from 2006 to 2014 at depths of 0–10 and 10–20 cm. T1: annual application of 15Nlabeled (NH4)2SO4; and T2: annual application of 15N-labeled (NH4)2SO4 and maize residues. Symbols and bars represent the mean and standard error (n = 3).

depths, respectively (Fig. 4, Table 1). The MRT of amino sugar-N yielded similar values when calculated by first-order kinetics. In the fertilization-only treatment, they were 78 and 154 years at 0– 10 and 10–20 cm depths, respectively, while they were 75 and 164 years at the two soil depths in maize residue addition treatment, respectively (Fig. 5, Table 1). 4. Discussion 4.1. Temporal pattern of in situ microbial immobilization of fertilizer N As time-integrated biomarkers, the increasing amount of the newly synthesized amino sugars in both treatments clearly indicated the accumulation of microbially immobilized fertilizer N (Fig. 1c and d) (He et al., 2011). The fertilizer-derived amino sugars increased linearly in both the treatments, and thus the ATR was unchanged during the 8-year experiment time (Fig. 3). This finding suggested that the efficiency of N transformation from

fertilizer to microbial residues remained constant over time although there was a saturation tendency of microbial residue accumulation in soil (Figs. 1a and b and 3). Compared to N fertilization application, the additional maize residue mulching can activate dormant microorganisms to a greater extent and enhance the microbial utilization of N (De Nobili et al., 2001; Fontaine et al., 2003). However, the significant increase in microbial immobilization rate of fertilizer N was only observed at the initial stage (first year) in the surface soil, although the immobilization of fertilizer N was always and across all years accelerated by continuous maize residue mulching relative to fertilization-only treatment (Fig. 3). Therefore, the retention rate of microbial residues was time-independent and treatment-specific during the 8 treatment years because of the gradual microbial adaption to long-term substrate supply (Schimel et al., 2007). Because the N fertilizer was supplied to the 5–10 cm layer in powder form and NH4+ does not readily move downward (Azam et al., 2001), the significantly higher production of amino sugars at

Fig. 3. Changes in amino sugar transformation rate (ATRt) at each depth from 2007 to 2014. T1: annual application of 15N-labeled (NH4)2SO4; and T2: annual application of 15 N-labeled (NH4)2SO4 and maize residues. Symbols and bars represent the mean and standard error (n = 3).

X. Liu et al. / Agriculture, Ecosystems and Environment 229 (2016) 40–47

45

Fig. 4. Mean residence time (MRT) of amino sugars estimated by extrapolating the curves of the fertilizer-derived amino sugars (ASt-FD) and total amino sugars (ASt). T1: annual application of 15N-labeled (NH4)2SO4; and T2: annual application of 15N-labeled (NH4)2SO4 and maize residues.

the surface than in the deeper soil was attributed to the reduced accessibility of fertilizer to microorganisms in deeper soils (Fig. 1c and d). In the surface soil, continuous mulching with maize residue produced “hotspots” during the maize residue decomposition, providing a readily available source of C, nutrient and energy for microorganisms to enhance fertilizer N immobilization (Bastian et al., 2009; Njeru et al., 2014), which contributed to the significantly higher transformation rates of fertilizer N into amino sugars in surface soil than those in the deeper soil layer (Fig. 3). Although maize residue mulching did not influence microbial immobilization of fertilizer N significantly in the deeper soil, the increased retention of fertilizer N in microbial residues in the entire cultivation soil layer (0–20 cm) indicated enhanced fertilizer N immobilization (Fig. 1c, 1d). This process favoured the formation of organic constituents while retarded the movement of inorganic N downward, and thus could significantly reduce the risk of leaching loss of fertilizer N (Vanlauwe et al., 2001; Gentile et al., 2009). 4.2. Turnover of microbial residues induced by fertilizer N under different managements Despite the increased amino sugar production accompanied with the immobilization of fertilizer N, the dynamics of total amino sugars conformed to exponential curve (Fig. 1a and b). This suggested that the accumulation of microbial residues was

essentially a balance between production of the newly synthesized portion (gain) and decomposition of the indigenous components (loss), which is termed as the turnover of microbial residues induced by fertilizer N application. Based on the assumption that soil total amino sugars would be completely replaced by the fertilizer-derived amino sugars, the estimated MRT of amino sugarN using the extrapolation approach was rather a minimum 15N renewal time of soil amino sugars theoretically. Essentially, amino sugars may form bound residues by interactions with mineral soil particles or by entrapment into more recalcitrant organic material (Kindler et al., 2006; von Lützow et al., 2008; Miltner et al., 2012), and thus they will never be completely replaced. As the approach of first-order kinetics has taken N saturation into consideration, the calculated MRT of amino sugar-N using first-order kinetics was generally shorter than that estimated by the extrapolation. However, the MRT values calculated by these two approaches were in the same range. As a whole, the fertilizer induced MRT of amino sugar-N was shorter than two-century scale, i.e., ranged 75– 90 years in 0–10 cm and 154–171 years in 10–20 cm soil depth in our temperate agro-ecosystem (Figs. 4 and 5 and Table 1). In our current research, we performed a fertilization experiment, in which a continuous 15N-labeling technique was applied to evaluate N turnover in microbial residues. The only comparable work was a 7-year 13C-depleted CO2 labeling experiment in a temperate grassland ecosystem. Based on the MRT of GluN and GalN, the MRT of total amino sugar-C in the surface soil of this

Table 1 Mean residence time (years) using two calculation approaches (extrapolation or first-order kinetics) for total amino sugars in two treatments. soil depth (cm)

0–10 10–20

MRT using first-order kinetics (year)

MRT using extrapolation (year) Annual application of 15Nlabeled (NH4)2SO4 (T1)

Annual application of 15N-labeled (NH4)2SO4 and maize residues (T2)

Annual application of 15Nlabeled (NH4)2SO4 (T1)

Annual application of 15N-labeled (NH4)2SO4 and maize residues (T2)

89 165

90 171

78 154

75 164

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Fig. 5. Mean residence time (MRT) of amino sugars calculated by first-order kinetics T1: annual application of labeled (NH4)2SO4 and maize residues.

grassland was at the decadal (ca. 6 years) scale because of the dominance of GluN and GalN in total amino sugars (Glaser et al., 2006; Derrien and Amelung, 2011). Therefore, the differences in the estimated MRT of amino sugar in different ecosystems were possibly caused by the specific isotope labeling experiment and variations in C and N transformation in microbial residues. As amino sugars are both stable microbial components and precursor for the formation of soil organic matter, their dynamics were closely related to the stoichiometric ratio between C and N in the soil, thus exhibiting different turnover patterns in different ecosystems (Amelung et al., 2001; He et al., 2011). However, the investigated grassland soil received N fertilizer at an annual rate of 140 kg N ha1 (except for 100 kg N ha1 in the first year) and had a C/N ratio in the range of 12–13 (Glaser et al., 2006), which was not significantly different to those in our field experiment. Thus, compared with the influence of different ecosystems on microbial residues turnover, the origins of C and N incorporated into microbial biomass, as well as the reutilization of microbial residues, might play more important roles in controlling the formation and decomposition dynamics of microbial residues. Photosynthetic C (13C-labeled) was allocated belowground and could act as a C skeleton for amino sugar synthesis via the tricarboxylic acid cycle (Paul, 2007), whereas NH2 in the amino sugar molecule was acquired from either fertilizer N (15N-labeled) or any other available N sources. The independent incorporation of C and N possibly resulted in the apparently non-synchronous turnover of amino sugar-C and amino sugar-N. Moreover, amino sugars could serve as both C and N sources in the soil matrix after depolymerization of microbial cell wall (Roberts et al., 2007). For the mineralization of N-containing available substrates, C is generally prone to loss from soil through microbial respiration, whereas N is more likely to be retained and reutilized in soil (Pansu et al., 2014). Hence, a recycling of 15N will retain the 15N signal more than the 13C signal. As a result, long-term retention dynamics of amino sugar-N may differ from that of amino sugar-C, which may be indicated by the longer MRT of amino sugar-N compared with amino sugar-C (Fig. 4).

15

N-labeled (NH4)2SO4; and T2: annual application of

15

N-

The available C released from the continuous addition of maize residue substantially enhanced microbial immobilization and accumulation of fertilizer N in microbial residues (Fig. 1c). On the other hand, the annual input of N derived from maize residue, which was equal to 48.3 kg N ha1, was also involved in microbial utilization and the production of microbial residues, thus facilitating the overall N retention and stabilization in soil (Fig. 1a). However, maize residue mulching did not apparently change the MRT of amino sugar-N at both soil layers (Figs. 4 and 5 and Table 1), implying that the production of fertilizer-derived microbial residues was proportionally diluted by the immobilization of maize residue-derived N or the transformation of indigenous soil N (Fig. 2). Unfortunately, although we speculated that the turnover of amino sugars after mulching with maize residue would be driven by both the application of fertilizer and maize residue, a method of evaluating the in situ turnover of soil amino sugars induced by these two N sources is not available despite the cross 15N-labeling of fertilizer or maize residue. 5. Conclusions The in situ microbial immobilization of fertilizer N over time was well represented by the dynamics of fertilizer N (15N) enrichment in soil amino sugars. The immobilization rate of fertilizer N was constant during the 8-year experiment with two fertilizer regimes, but it was significantly higher with maize residue mulching. Importantly, the application of maize residue facilitated the initial fertilizer N immobilization, and subsequent applications maintained the increased transformation rate. Mulching with maize residue enhanced the retention capacity of fertilizer N in microbial residues in the upper cultivation layer (0–10 cm) rather than in the deeper cultivation layer (10–20 cm). This process retarded the movement of inorganic N downward, and consequently could significantly reduce the risk of leaching loss of fertilizer N. The MRT of amino sugars induced by fertilizer N was between 70 and 150 years in this temperate agro-ecosystem, but it was still significantly longer than that reported in the grassland soil

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