organic amendments and groundwater pollution implications

Accepted Manuscript Soluble organic nitrogen cycling in soils after application of chemical/organic amendments and groundwater pollution implications

Leyun Wang, Xilai Zheng, Feifei Tian, Jia Xin, Hui Nai PII: DOI: Reference:

S0169-7722(18)30085-8 doi:10.1016/j.jconhyd.2018.08.003 CONHYD 3413

To appear in:

Journal of Contaminant Hydrology

Received date: Revised date: Accepted date:

21 March 2018 27 June 2018 10 August 2018

Please cite this article as: Leyun Wang, Xilai Zheng, Feifei Tian, Jia Xin, Hui Nai , Soluble organic nitrogen cycling in soils after application of chemical/organic amendments and groundwater pollution implications. Conhyd (2018), doi:10.1016/j.jconhyd.2018.08.003

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ACCEPTED MANUSCRIPT Soluble organic nitrogen cycling in soils after application of chemical/organic amendments and groundwater pollution implications Leyun Wanga, Xilai Zheng*a,b, Feifei Tiana, Jia Xin*a,b, Hui Naia a

Key Laboratory of Marine Environment Science and Ecology, Ministry of Education

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and College of Environmental Science and Engineering, Ocean University of China,

Shandong Provincial Key Laboratory of Marine Environment and Geological

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b

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Qingdao 266100, China;

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Engineering, Ocean University of China, Qingdao 266100, China;

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*Corresponding author:

Dr. Xilai Zheng; phone: +86-532-6678-1759; email: [email protected] Dr. Jia Xin; phone: +86-532-6678-6568; email: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Nitrogen (N) fertilizers have been extensively used to maintain soil fertility in intensively agricultural soils, creating serious environmental pollution. In this study, a 70-day incubation experiment was conducted to investigate the effects of different N

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fertilizers (urea, manure, straw) on N mineralization, soluble organic nitrogen (SON)

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dynamics and its leaching potential in typical agricultural soils of the Shandong

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Peninsula. The results showed that the addition of N fertilizers affected the SON pools and soil N mineralization in different ways owing to their various properties and

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interaction with soils. When comparing treatments, urea application was found to

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decrease SON content, whereas manure and straw addition increased the SON content after long-term incubation. Considering that SON content depended on a complicated

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formation process and consumption process, no direct link between SON content and

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N mineralization capacity was observed in different treatments. Additionally, we analyzed free amino acids (FAAs) in SON and found that FAA content was negatively correlated with N mineralization, except for the straw treatment. This suggested that

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FAAs were important substrates of N mineralization in soils. In addition, the composition of SON was determined by 3-dimensional excitation-emission matrix and ultraviolet- visible absorbance spectrophotometer after long-term incubation. The PIII+V/PI+II+IV ratio, SUVA254 , and A253 /A203 ratio decreased after fertilizer application. This indicated that fertilizer addition decreased the SON humification degree and increased SON leaching. Therefore, SON should be taken into account when optimizing fertilization management and evaluating the risk of N leaching in

ACCEPTED MANUSCRIPT groundwater systems. Keywords Nitrogen fertilizers; Soluble organic nitrogen; Free amino acids; Microbial biomass

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nitrogen; Soluble organic nitrogen leaching; Groundwater system

ACCEPTED MANUSCRIPT 1 Introduction In agricultural ecosystems, fertilization is widely considered as a way to improve soil quality and productivity (Sun et al., 2015). Chemical fertilizers are commonly used in intensive agricultural soils because of their high nutrient content and ease of

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availability. However, overuse of chemical fertilizers has caused a diversity of

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environmental problems in unsaturated and saturated zones, such as low fertilizer use

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efficiency, soil acidification, and groundwater contamination (Ju et al., 2006; Watts et al., 2010; Zhao et al., 2014). To avoid environmental risks and enhance resource

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utilization, the application of exogenous organic amendments (EOAs) (e.g., animal

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manures, straw) is encouraged (Mohanty et al., 2011; Masunga et al., 2016). Although organic amendments can modify soil physical conditions (e.g., bulk density, soil

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structure), the nutrient and its release are too low to satisfy crop growth. In order to

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support sustainable nutrients, the combination of organic amendments and chemical fertilizers has become the predominant approach in intensive agriculture (Sun et al., 2015). Notably, adding different types of fertilizers to soils may influence nitrogen

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(N) mineralization, and sequentially alter the N supply in soils (Azeez and Van Averbeke, 2010). An inappropriate N supply limits vegetation growth and leads to N losses (Amanullah, 2007). Therefore, a complete understanding of the effects of different fertilizers on N mineralization in agricultural soils will help us to optimize fertilization management and associated strategies. Soil N mineralization is the biological process of organic N (ON) to inorganic N (IN), which influenced by the physical-chemical properties of exogenous materials

ACCEPTED MANUSCRIPT (e.g., chemical fertilizer, animal manures, and crop residues) (Masunga et al., 2016). Some studies showed that the effects of exogenous materials on soil N mineralization were not consistent (Watts et al., 2010; Mohanty et al., 2011). The exogenous material with low carbon (C)/N ratio mineralizes surplus N. Conversely, available N is

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immobilized by microorganisms with high C/N ratio (Chaves et al. 2008; Miller et al.

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2008).

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Previous soil N mineralization-related research has mainly focused on the dynamics of total (TN) and IN pools after fertilization in agricultural systems (Fan et

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al., 2018), but there is little information regarding the evolution of soluble organic N

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(SON) pools. Soluble organic N is the organic N which can be extracted by water, salt solutions, or electro- ultra- filtration (EUF) (Matsumoto and Ae, 2004; Bregliani et al.,

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2010). As the available pools of total organic nitrogen (TON), SON has the potential

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for rapid turnover and plays a key role in regulating N mineralization (Murphy, 2000; Christou et al., 2006). Although recent studies have focused on the relationship between SON and N mineralization, the conclusions remain controversial (Christou et

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al., 2005; Christou et al., 2006; Chen and Xu, 2008). Murphy (1998) found a significant negative correlation between SON and N mineralization in arable soils. Mengel (1999) confirmed the same results in forest and grassland soils. Conversely, numerous studies have concluded that there was no direct link between SON turnover and N mineralization (Mariano et al., 2016). These opposing results regarding the relationships between SON and N mineralization resulted from the complex components and intricate SON sources and sinks. Soluble organic N is extremely

ACCEPTED MANUSCRIPT complex and contains numerous nitrogenous organic constituents, including free amino acids (FAAs), peptides, and proteins (Jones and Kielland, 2012; Ge et al., 2012). The chemical composition and structure of SON determine its bioavailability in N cycling (Xu et al., 2003). Some studies have shown that FAA is the key

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component in N mineralization (Jones and Kielland, 2002; Brant et al., 2006;

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Engelking et al., 2007; Roberts et al., 2007). Smolander (1995) indicated that only a

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fraction of SON would be mineralized, because a proportion of organic compounds was soluble yet recalcitrant to microbial decomposition. Furthermore, SON is not only

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the product derived from the conversion of TON but also the substrate for N

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mineralization (Perakis and Hedin, 2002; Kessel et al., 2009; Liang et al., 2015), and these processes determine the role of SON in N cycling. Thus, investigating the role

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of SON in N mineralization should not only involve its quantities and qualities but

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also consider its cycling in soils. To our knowledge, limited information is available on SON pools, especially as affected by different fertilizers. Besides nutrient accumulation in soils, N loss into groundwater is another

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concern that drives us to explore the N mineralization process. It is conventionally postulated that N losses in agricultural systems are dominated by inorganic N. However, an increasing number of studies have claimed that leaching leads to losses of dissolved organic nitrogen (DON) from agricultural systems and is a crucial pathway of N loss (Perakis and Hedin, 2002; Vinther et al., 2006; Kessel et al., 2009; Liang et al., 2015). Siemens and Kaupenjohann (2002) reported that seepage losses of DON accounted for 6–21% of total N fluxes from agricultural lands. Lapworth (2008)

ACCEPTED MANUSCRIPT pointed out that DON was abundant in groundwater, accounting for 47–80% of the total dissolved N. Large amounts of DON loss leach into streams and underground water and lead to eutrophication and acidification (Vandenbruwane et al., 2007; Xu et al., 2011; Watanabe et al., 2014). In particular, DON in drinking water can react with

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chlorine during disinfection and release toxic byproducts (Lee et al., 2007; Gu et al.,

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2011). Thus, the contents and extent of DON leaching from surface soils to

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groundwater and streams is of concern (Liu et al., 2012; Qin et al., 2015). DON is defined as the fraction of SON which is collected in situ with no extractant used

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(Kessel et al., 2009). We postulated that constituents of SON in surface soils may

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determine the extent of DON loss. However, to date, little has been discovered about the mechanistic processes of SON leaching.

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The Shandong Peninsula is an intensive farmland area, and the vegetation yield

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accounts for 50% of the total yield in China (Chai et al., 2017). Excessive N application resulted in nitrate accumulation in soils and groundwater (Liang et al., 2015). Therefore, the effects of fertilization on soil N mineralization have become a

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vital research requirement in this region. In our study, a typical chemical fertilizer (urea) and two organic amendments (manure and straw) were adopted to explore the effects of different fertilizers on SON pools in agricultural soils in Shandong Peninsula. Specifically, the goals of the study were to (1) determine the effects of different fertilizers on soil N mineralization, (2) investigate the contents and components of SON involved in N cycling when different fertilizers are added to soils, and (3) discuss the potential of SON leaching to groundwater by component

ACCEPTED MANUSCRIPT analysis in agricultural systems. 2 Materials and methods 2.1 Materials 2.1.1 Soil

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Soil samples were collected in August 2016 from a 0–20 cm depth from a field

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that was an intensive agricultural area in Laixi City, Shandong Province (36°43′N,

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120°20′E). The mean annual temperature is 11.7 °C and mean annual precipitation is 690 mm. The field has been under the summer maize and winter wheat rotation.

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The annual N fertilizers application rate is 340 kg/hm2 (approximately equivalent to

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120 mg N/kg). Large plant material was manually removed, and the samples were sieved through 2 mm mesh. The soil was stored in glass jars at 4 °C for further

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analysis.

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The particle size distribution of the soil was determined by densitometer method. The soil pH and electrical conductivity (EC) were measured in deionized water (1:2.5 w/v) by using a multi-parameter water quality meter (HQ40d, HACH, America). The

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content of soluble organic carbon (SOC) was determined in 0.50 M K 2 SO 4 (1:10, w/v) using an organic carbon autoanalyzer (TOC-Vario, Elementar, Germany). Total (TN) and organic N (TON) was measured by Kjeldahl method (K9840, Hanon, China). The percentage of sand, silt, and clay was 58.7, 17.0, and 24.0%, respectively. The soil from the field was classified as sandy clay loam according to FAO (Food and Agriculture Organization). Soil physical-chemical characteristics are: pH 5.94, EC 345 μS /cm, SOC 88.39 mg C/kg, TON 565.59 mg N/kg, TN 680.00 mg N/kg.

ACCEPTED MANUSCRIPT 2.1.2 Fertilizers The chemical fertilizer (urea) is the commonly used fertilizer in this site, and manure and wheat straw are spread over the field. Thus, we used three exogenous fertilizers in an incubation experiment. The EOA (manure and straw) was chopped

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into small pieces using a grinder and passed a 2 mm sieve. Total C (TC), total N (TN)

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and organic N (TON) were measured by an elemental analyzer (FLASH-2000,

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Thermo, America). Moreover, these fertilizers were extracted with 0.50 M K 2 SO4 (1:10, w/v) in polypropylene tubes on a reciprocating shaker (IS-RDD3, Crystal,

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America) at 220 rev/min for 1 h. After shaking, samples were centrifuged (15 min;

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4000 rpm) and the supernatant recovered. Mineral N (NO 3 --N and NH4 +-N) was determined using the ultraviolet spectrophotometric method and Nessler’s reagent

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colorimetric method, respectively (Jones et al., 2004). Total soluble nitrogen (TSN) was measured using the persulfate oxidation method. Soluble organic N (SON) was

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calculated by the difference between the TSN and the mineral N (sum of NO 3 --N and NH4 +-N). Table 1 shows the physical-chemical properties of the exogenous fertilizers

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used in this experiment.

2.2 Experimental methods 2.2.1 Mineralization of soil organic N Before the incubation experiment, the soil was adjusted to 75% of the maximum water holding capacity by gravimetric method and pre- incubated at constant temperature (25 °C) to recover microbial activity. The incubation experiment was conducted in plastic containers (upper caliber × lower caliber × height = 74 mm × 57

ACCEPTED MANUSCRIPT mm × 47 mm) filled with pre- incubated soil (equivalently 60 g of air-dried soil). There were three types of N supplies: one chemical fertilizer (urea) and two EOAs, manure and straw. The treatments were: (1) control (CK), no application; (2) application of chemical fertilizer (U); (3) application of manure (M); (4) application

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of straw (S). For each sample, exogenous fertilizers mixed with 60 g soil to give a

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field application rate in topsoil equivalent to approximately 340 kg/hm2 . In order to

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maintain the soil N supply at the same level, total N added by each exogenous N material was consistent.

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The soils were incubated in the dark at 25 (±1) °C. Soil moisture content was

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maintained at 75% of the maximum water holding capacity by gravimetric method during the entire incubation period. Three replicates of each treatment were prepared

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for each sampling date (1, 4, 7, 12, 22, 36, and 70 days after incorporation) to allow

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for destructive sampling. Briefly, 5 g of soil were extracted with 50 mL 0.50 M K 2 SO4 in polypropylene tubes by shaking at 220 rev/min for 1 h. After shaking, samples were centrifuged (15 min; 4000 rpm) and the supernatant recovered for further analysis.

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2.2.2 Measurements of mineral N, FAA, SON, and microbial organic nitrogen (MBN) The contents of the different N forms in the soils were measured in this study. Mineral N

(NO 3 --N

and

NH4 +-N) was determined

using

the

ultraviolet

spectrophotometric method and Nessler’s reagent colorimetric method, respectively. Total soluble nitrogen (TSN) was measured using the persulfate oxidation method. Soluble organic N (SON) was calculated by the difference between the TSN and the mineral N (sum of NO 3 --N and NH4 +-N). FAA was determined by the

ACCEPTED MANUSCRIPT spectrophotometric ninhydrin method (Moore, 1968). Microbial organic nitrogen (MBN) was measured using the fumigation extraction method (Ross, 1990). To determine the composition of SON, a fluorescence spectrometer (F-4600, Hitachi, Japan) was used to characterize the 3-dimensional excitation emission matrix

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(3D-EEM). The spectroscopy conditions were as follows: excitation (Ex) and

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emission (Em) wavelengths, 200–400 and 200–500 nm, respectively, at 5 nm

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intervals; scan rate, 2400 nm/min.

Ultraviolet–visible (UV-vis) absorbance spectrophotometer (2800 UV/VIS,

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ONICO, Australia) was selected to determine SON fractions using a 1 cm quartz

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cuvette at wavelengths ranging from 200 to 700 nm. 2.3 Calculations and statistical analyses

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The mineral N or SON accumulation was best described by a zero-order kinetic

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model: N(t) = N0 +k0t, where t is the time (in days), N(t) is the amount of mineral N or SON at time t, N0 is the initial amount of mineral N or SON (mg N/kg), and k 0 is the zero-order mineral N or SON accumulation rate (mg N/(kg day)).

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All results were presented as the mean values and tested by analysis of variance (ANOVA). The significant differences between pairs were determined using Duncan’s multiple range test (P = 0.05), and the Statistical Product and Service Solutions Software (SPSS, version 20.0) was used. 3 Results 3.1 The effects of exogenous fertilizers on soil physical-chemical properties Table 2 shows soil characteristics with exogenous fertilizer addition at the

ACCEPTED MANUSCRIPT beginning of experiment. Exogenous fertilizer application caused statistically significant shifts in soil characteristics (P < 0.05). Soil pH significantly increased with exogenous fertilizers addition. The pH in the control treatment was 5.94, and the values in exogenous fertilizer amended soils ranged from 6.45 to 6.69. The electrical

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conductivity (EC) also increased along with the exogenous fertilizer addition,

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especially in the straw-amended soil. The EC in straw-amended soil was 1.59- fold

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greater than in the control treatment. The content of soluble organic carbon (SOC) and TON in soils were significantly higher in treatments with exogenous fertilizers than in

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the control. The highest SOC content was 207.94 mg C/kg in the straw-amended soil.

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TON contents were higher in exogenous fertilizer amended soils (ranging from 653.35 to 690.32 mg N/kg) than in the control (565.59 mg N/kg). However, TON

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content did not differ significantly between any two exogenous organic amendments.

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3.2 Soil mineral N dynamics

The mineral N dynamics varied in different treatments (Fig. 1). In the control, the mineral N content increased slightly during the first 12 days, but remained

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relatively constant after 22 days. In the urea-amended soil, during the entire incubation, soil mineral N concentrations rapidly increased and peaked at day 12, and then remained stable. Generally, the mineral N content in manure-amended soil initially decreased during the first 7 days, and increased from 7 to 50 days. Finally, the mineral N content remained constant after 50 days. However, in the straw-amended soil, the mineral N content decreased rapidly during the first 7 days, and then remained constant after 7 days. The mineral N content was influenced by the N

ACCEPTED MANUSCRIPT fertilizer type (P < 0.05). During the entire incubation period, the ranges of mineral N were 114.41–124.73, 136.65–232.59, 104.78–120.62, and 0.83–99.68 mg N/kg in the control, urea-amended, manure-amended, and straw-amended soils, respectively. During the entire incubation period, the mineral N content in the urea-amended soil

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was markedly higher than that of other treatments. The mineral N content was always

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lower in the straw-amended soil than in the control.

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Cumulative N mineralization resulted in the increase in soil mineral N content beyond the content already present before incubation. Cumulative N mineralization

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showed similar trends in all treatments. After a 70-day incubation period, the amount

urea-amended,

and

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of cumulative N mineralization was 9.78, 89.09, and 12.14 mg N/kg in the control, manure-amended

soils,

respectively.

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to

the

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non-amended soil, adding urea increased the amount of cumulative N mineralization,

mineralization.

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whereas manure addition had little effect on the amount of cumulative N

3.3 Soil SON dynamics

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The SON contents were significantly (P < 0.05) influenced by exogenous fertilizers (Fig. 2). The SON content in control soil significantly increased from 104.42 to 128.83 mg N/kg during the first 12 days, but slightly decreased from 12 to 22 days. Then, the SON content remained relatively constant after 22 days (Fig. 2). The final SON was 115.66 mg N/kg after the 70-day incubation period. In the urea-amended soil, the SON content notably decreased from 206.70 to 107.55 mg N/kg during the entire incubation. In the EOA-amended soils, the SON contents

ACCEPTED MANUSCRIPT significantly increased during the first 7 days, then remarkably decreased thereafter. The SON content increased by 7.51 and 12.01 mg N/kg at 7 days, whereas the SON content decreased by 5.49 and 19.08 mg N/kg from 7 to 70 days in manure-amended and straw-amended soils, respectively. Overall, the SON content was lower in

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urea-amended than non-amended soils after 7 days. Conversely, manure and straw

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application increased SON contents by 5.85 and 2.40 mg N/kg compared with the

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control after 70 days, following the order of manure-amended > straw-amended soils. 3.4 Soil FAA dynamics

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The FAA content initially increased for a short time, and then decreased with

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increasing incubation days, except in the urea-amended soil (Fig. 3). The maximum FAA content was 31.57, 59.01, 37.94, and 85.60 mg N/kg in the control,

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urea-amended, manure-amended, and straw-amended soils, respectively. After the

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70-day incubation period, the FAA content decreased to 7.96, 20.83, 23.52, and 37.66 mg N/kg in the above treatments, respectively. At the end of incubation, compared with that of the control, the FAA content increased in all fertilizer-amended

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treatments. On average, the proportion of FAA to SON ranged from 17.39 to 44.26% in all treatments during the entire incubation period. The proportion of FAA to SON was higher after fertilizer addition than that of the control. At the end of incubation period, the proportion of FAA to SON decreased to 6.88%, 19.37%, 19.35%, and 31.87% in the control, urea-amended, manure-amended, and straw-amended soils, respectively. 3.5 Soil MBN dynamics

ACCEPTED MANUSCRIPT During all incubation treatments, we found that the MBN content increased during first 7 days, and decreased thereafter (Fig. 4). The maximum values of MBN in the control, urea-amended, manure-amended, and straw-amended treatments were 125.10, 167.34, 218.67, 176.73 mg N/kg, respectively. At the end of incubation

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period, the MBN content in the above treatments decreased to 18.79, 19.11, 19.05,

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and 51.43 mg N/kg, respectively. Pairwise comparisons among treatment revealed a

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significant difference for MBN content between all the treatments during the entire incubation. The MBN content in the soils with exogenous fertilizer addition was

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significantly higher than that the control treatment. In particular, in straw-amended

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soils, the MBN content was always higher than the non-amended soil. 3.6 Composition of SON investigated by 3D-EEM and UV-vis absorbance

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spectroscopy

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The 3D-EEM spectrum of SON in soils is shown in Fig. 5. Based on fluorescence peak characteristics, the 3D- EEM spectra were divided into five regions. Generally, peaks of regions I and II occurred at shorter emission wavelengths (< 380

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nm) and shorter excitation wavelengths (< 250 nm). Peaks of region III appeared at shorter emission wavelengths (> 380 nm) and shorter excitation wavelengths (< 250 nm). Peaks of region IV exhibited shorter emission wavelengths (< 380 nm) and longer excitation wavelengths (> 250 nm). In addition, peaks of region V emerged at longer emission wavelengths (> 380 nm) and longer excitation wavelengths (> 250 nm). The 3D-EEM spectrum of SON in soils exhibited fluorescence peak characteristics, namely protein- like compounds (regions I and II), fulvic acid- like

ACCEPTED MANUSCRIPT substances (region III), soluble microbial byproduct materials (region IV), and humic- like substances (region V) (Wang et al., 2015). Our results showed that peaks of 3D-EEM spectrum were located at region II in all treatments after long-term incubation. Table 3 shows the percent distribution of volumetric fluorescence among

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the five regions (i.e., Pi) for SON at the end of incubation. P I+II accounted for 47.65–

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58.18% of the total in all treatments. Compared with different treatments,

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PIII+V/PI+II+IV ratios were lower in fertilizer-amended soils than in the control. The lowest ratio was in urea-amended soil.

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In this study, we also monitored the SUVA254 values and A253 /A203 ratios (Table

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3). The SUVA254 values and A253 /A203 ratios ranged from 0.10 to 0.45 and 0.03 to 0.22, respectively. The values and ratios displayed a declining trend in

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fertilizer-amended soils. Comparisons between different fertilizers revealed that the

4 Discussion

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values and ratios were lower for urea amendment than organic amendment.

4.1 The effects of adding different fertilizers on soil N mineralization

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In this study, soil N mineralization differed among all treatments, with the highest amount of N mineralization in the urea-amended soil and the lowest amount of N mineralization in the straw-amended soil. The extent of soil N mineralization depended on the physical-chemical properties of exogenous fertilizers (e.g., N content, content of labile C, C/N ratio) (Kieloaho et al., 2016). In particular, the effect of the C/N ratio of exogenous fertilizers on soil N mineralization is complicated (Chaves et al. 2008; Miller et al. 2008). The C/N ratio of exogenous fertilizers will

ACCEPTED MANUSCRIPT influence soil microbial growth when added to soils. Adding organic amendments with a higher C/N ratio (C/N ratio > 30) leads to the shortage of N in soils, and available N is immobilized by microorganisms. In contrast, adding organic amendments with a lower C/N ratio (C/N < 20) causes soil N surpluses, and sufficient

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N mineralizes (Chaves et al. 2008; Miller et al. 2008). In the present study, the

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extremely low C/N ratio (C/N < 20) in urea-amended soil facilitated soil N

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mineralization, whereas the higher C/N ratio (C/N ratio > 30) after straw application accounted for greater N immobilization than N mineralization.

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In addition, we analyzed FAA and SON to determine their roles in N

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mineralization. Table 4 shows the relationships between both FAA and SON with cumulative N mineralization. In our findings, there were significant differences

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between the relationships between FAA (or SON) and cumulative N mineralization in

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different treatments. Our results illustrated that FAA was negatively related to cumulative N mineralization (r = -0.77, P < 0.01; r = -0.63, P > 0.05; r = -0.42, P > 0.05) in the control, urea-amended, and manure-amended treatments, respectively,

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suggesting that amino acid N was the important substrate for N mineralization. To our knowledge, amino acid N compounds are the important substrates of N mineralization in soils, because they can be utilized by microorganisms (Jones and Kielland, 2002; Mariano et al., 2016). However, FAA had no significant relationship with cumulative N mineralization in the straw-amended soil. This might be because numerous FAAs were not available for microbial decomposition in the straw-amended soil (Smolander et al., 1995). In addition, the relationship of SON and cumulative N mineralization

ACCEPTED MANUSCRIPT varied because of the complicated components of SON. A strongly negative linear relationship between SON and cumulative N mineralization was observed (r = -0.98, P < 0.01) in the urea-amended soil. This may be because the rapid decomposition of urea when it was incorporated into soils. However, in other treatments, SON exhibited

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both positive and negative relationships with cumulative N mineralization. No

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significant relationship between SON and N mineralization was detected, except in

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urea-amended soil. This might have been caused by the complicated components and conversion of SON.

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To unveil the governing mechanism for distinct soil N mineralization behaviors

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with the addition of different fertilizers, we investigated soil microorganisms, which play the dominant role in soil N mineralization. During the entire incubation

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experiment, we found that the growth of microbes increased during the first 7 days,

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and decreased thereafter in all treatments (Fig. 4). Comparing between different treatments, we found that addition of exogenous fertilizers markedly stimulated the growth of microorganisms, especially in straw-amended soil. This could be explained

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by the improvement in soil conditions and the input of labile C in soils (Cookson et al., 2005; Hoyle, 2008). Generally, soil microorganisms were stimulated by improvement of their environment (e.g., pH, nutrient content) with the addition of exogenous fertilizers. In particular, SOC in exogenous fertilizers can be utilized as a C source by soil microorganisms, resulting in enhancement of microbial growth (Shand et al., 2002). Our findings indicated that exogenous fertilizer application caused a statistically significant increase in SOC content (P < 0.05) with the highest level in

ACCEPTED MANUSCRIPT straw-amended soil (Table 2). These results implied that soil N mineralization was stimulated by a rapid increase in microbes with fertilizer addition. Our results were consistent with previous reports (Michel et al., 2006; Kemmitt et al., 2006). 4.2 SON dynamics in soils

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In our research, no direct link between SON turnover and N mineralization was

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established, except in urea-amended soils. It resulted from the complicated

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components of SON, including amino acid N, amino sugar, nucleotides etc. (Greenfield 2001; Friedel and Scheller 2002). Only a fraction of SON can mineralize.

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Moreover, as median products, SON is consumed as substrates for N mineralization,

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although it originates from the transformation of complex organic N. Thus, to completely understand the role of SON pools involved in N cycling, a hypothetical

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model for the SON cycling in agricultural ecosystems was proposed (Fig. 6). SON

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measured in our study can include two parts: (1) dissolved organic N (DON) in the soil solution and (2) soluble organic N that was adsorbed (AON) in soils, but could be acquired by specific solutions (e.g., KCl, CaCl2 , K 2 SO4 ). Compared to AON, DON is

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more easily to be mineralized. In our study, the potential sources of SON as shown in Fig. 6, include (1) depolymerization and decomposition of complex organic N compounds (e.g., high molecular weight, HMW) and (2) microbial debris and metabolites. The fates of SON include (1) decomposition into inorganic N, (2) humification into recalcitrant organic N compounds, and (3) microbial cell uptake (if the inorganic N in soils is insufficient) (Jones et al., 2004). Because NO 3 - is rich in soils, there may be little reason for microorganisms to take up low molecular weight

ACCEPTED MANUSCRIPT (LMW) DON (Jones et al., 2004). In this study, NO 3 - was rich in soils. Thus, the microbial cell uptake of SON was eliminated. In our study, we analyzed the sources and fates of SON to assess its role in N cycling in agricultural ecosystems. 4.2.1 SON dynamics in a typical agricultural soil

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In the control treatment, we observed that SON increased with inorganic N

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accumulation during the first 12 days. The rate of SON increase was 2.22 mg N/kg/d,

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and the rate of mineral N accumulation was 0.94 mg N/kg/d during the first 12 days. The rate of SON increase was 2.36-fold greater than the rate of mineral N

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accumulation. It confirmed that the process of depolymerization and decomposition of

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complex organic N compounds was enhanced because of rapid microbia l growth during the first 12 days of incubation. After 12 days, the rates of SON increase and

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mineral N accumulation decreased to very low levels.

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4.2.2 SON dynamics after adding the chemical fertilizer Addition of exogenous fertilizers to soils stimulated soil microbial activity by improving soil conditions (e.g., soil pH, nutrient retention), and affected the rates of

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SON production and mineral N accumulation. In the urea-amended treatment, cumulative N mineralization was significantly negatively correlated with SON content (r = -0.98, P < 0.01) (Table 4). Urea was considered LMW-DON to be amended to soils (Berman and Bronk, 2003; Wang et al., 2008). Because of its chemical structure, an amide can be transformed into mineral N, which can be utilized directly by microorganisms. Therefore, SON containing a large number of amides was depleted, and it became the major substrate for N

ACCEPTED MANUSCRIPT mineralization. The results showed that the contents of SON in urea-amended soils was lower than in the control soils after 7 days, implying that urea application stimulated rapid microbial growth and further accelerated native SON mineralization in soils. After 70 days of incubation, the rates of SON increase and mineral N

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accumulation decreased to very low levels. This can be explained by the minimization

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of microbial biomass and activity with decomposition and depletion of the labile C.

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4.2.3 SON dynamics with EOA

In the present study, manure and straw addition increased the content of SON in

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soils during the entire incubation. This finding was confirmed by previous studies

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(Embacher et al., 2008; Ros et al., 2009; Liang et al., 2015). In our study, the content of SON in manure and straw were 9591.85 and 1526.31 mg/kg, respectively. If all the

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SON in EOA was released into the soil, the theoretical maximum content of SON in

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the soil was equal to 108.74 and 105.80 mg/kg at the total N application rate (120 mg/kg) for manure and straw, respectively. The experimental maximum content of SON in the soils with manure and straw amendment was 129.39 and 137.24 mg/kg,

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respectively. The experimental values were significantly higher than the theoretical. This indicated that SON increased when EOA were added to soils. The sources of increased SON may include (1) the increased decomposition of complex organic N compounds by high microbial biomass and activity and (2) microbial debris and metabolites. Our data provided supporting evidence for these two sources. Firstly, our data showed that the contents of MBN with EOA addition were higher than with chemical fertilizer addition during the entire incubation (Fig.

ACCEPTED MANUSCRIPT 4). In particular, the maximum MBN content was 1.31 and 1.06- fold higher in manure-amended and straw-amended soils, respectively, than in urea-amended soil at day 7. Our results demonstrated that manure and straw addition accelerated the process of complex organic N to SON by rapid growth of microorganisms. This

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process has been considered the dominant pathway for N supply in agricultural soils

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(Hadas et al., 1992; Schimel and Bennett, 2004). Secondly, our results showed that

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mineral N decreased during the entire incubation period in straw-amend soils and at an earlier stage in manure-amended soils, indicating that mineral N immobilization in

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soils with a high C/N ratio occurred in EOA-amended soils (Fig. 1). Meanwhile, it

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was also found that the content of MBN and SON in EOA-amended soils increased with decreasing mineral N and were higher than in other treatments after long

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incubation periods, which suggested that the application of EOA could promote the

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immobilization of excessive nitrates by stimulating microbial growth (Qiu et al., 2013). The immobilized N in live or dead microbial cells entered SON pools, and thus became another key source for the increased SON pools (Qiu et al., 2016).

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Thus, if excessive EOA are added to soils, it will increase the SON pools and may promote its leaching potential (Murphy et al., 2000; Carswell et al., 2016). To further analyze the distinct effects of d ifferent EOA amendments, the results in both treatments (manure and straw) were compared. Unlike in the straw-amended treatment, the C/N ratio decreased with the depletion of labile C at a later period, and mineral N accumulated

in manure-amended soil. This suggested that in

manure-amended soil, nitrification was the dominant process at the end of incubation.

ACCEPTED MANUSCRIPT After 70-day incubation period, the content of SON in manure-amended soil was higher than that of straw-amended soil. In this work, manure contained much more soluble forms of N than straw. Furthermore, soil N immobilization was stronger than N mineralization over time in straw-amended soil. Little of the immobilized N would

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later be mineralized (Xu et al., 2003). Thus, decomposition of complex organic N

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compounds was easier in manure-amended soil than in straw-amended soil in the later

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period, contributing to the higher SON content in manure-amended soil. 4.3 The potential of SON leaching to groundwater from soils

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SON is a dynamic participant in agriculture ecosystems, and its compositional

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characteristics can influence its bioavailability and mobility (Pehlivanoglu and Sedlak, 2006). In this study, the 3D-EEM spatial characteristics of SON and its

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fractions displayed some differences because of the complexity of their structural

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composition in different treatments. SON was mainly comprised of protein- like substances and fulvic- like materials by the end of incubation (Fig. 5). The protein- like substances are usually eliminated by biodegradation, although fulvic-like and

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humic- like materials are not easily utilized by microbes. The P III+V/PI+II+IV ratio was regarded as the humification index. A higher humification index suggests that SON contains more complex and stable organic N forms. By the end of incubation, PIII+V/PI+II+IV ranged from 0.41 to 0.62 (Table 3). Compared to the control, exogenous fertilizer addition resulted in lower ratios. This indicated a lower proportion of humic material with exogenous fertilizer application. SUVA254 values have been widely used as an index to estimate the aromatic

ACCEPTED MANUSCRIPT content of dissolved organic matter (DOM) (Li et al., 2014). Values less than 3 indicate that organic matter contains a specific hydrophilic substance. A253 /A203 ratios are usually used to reflect the concentration of substitution groups (Li et al., 2014). The higher ratios indicate that substitution groups in aromatic rings contain more

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carbonyl, carboxyl, hydroxyl, and ester groups. Conversely, lower A253 /A203 values

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indicate that substitution groups contain more aliphatic chains (Wang et al., 2015).

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Our study showed that SUVA254 values and A253 /A203 ratios ranged from 0.10 to 0.45 and 0.03 to 0.22, respectively (Table 3). This indicated that SON was mainly

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comprised of hydrophilic substances and substitution groups of aromatic rings with

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more aliphatic chains. The hydrophilic SON could be easily leached because of its low molecular weight (Kušlienė et al., 2015).

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To evaluate the potential of SON leaching, both its properties and total content

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should be taken into consideration. On the one hand, compared with organic amendments, the extent of humification with chemical fertilizer addition was lower (Table 3), indicating that substantial SON leaching would be triggered if extensive

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chemical fertilizers were added to soils under specific hydrological conditions (e.g., heavy rainfall, excessive irrigation) (Barton et al., 2006). On the other hand, in terms of SON contents, chemical fertilizer addition decreased SON content, whereas EOA addition increased SON content. The application of EOA resulted in much more SON accumulated in soils. Hence, EOA addition also contributed to a high potential of SON leaching. Finally, our study indicated that SON leaching in intensive agricultural soils should not be ignored when evaluating the risk of N leaching.

ACCEPTED MANUSCRIPT 4.4 The implications of fertilization management in agricultural soils In our research, two fertilization strategies (chemical fertilizer alone and organic amendment alone) were investigated in terms of the nutrient efficiency and leaching risks. For chemical fertilizer, it was found that the addition of chemical fertilizer alone

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increased the contents of mineral N in soils after 70-day incubation. The increased

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mineral N in soils could be utilized by plants. However, when the increased mineral N

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following excessive chemical fertilizer addition, exceeded plants requirements, the extra mineral N would accumulate in soils and leach into deeper profiles under

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irrigation. For organic amendment, the application of organic amendments alone

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resulted to mineral N immobilization by microorganisms due to a high C/N ratio in soils. Thus, the organic amendments, as slow releasing mineral N source, fail to

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guarantee enough mineral N for plants growth, particularly for some key growth

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stages. Moreover, the contents of SON increased after long-time incubation, and the increased SON was mainly comprised of hydrophilic substances and substitution groups of aromatic rings with more aliphatic chains. Accordingly, SON leaching

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potential was enhanced. Considering the advantages and disadvantages of above two fertilization strategies, we suggested the combination of chemical and organic fertilizers should be considered in agricultural practice and SON should not be ignored when evaluating the risk of N leaching. In future studies, the mechanisms of how plants compete for SON, and how their presences affect the N cycling or release of N in the field need to be further addressed. 5 Conclusion

ACCEPTED MANUSCRIPT Various properties of fertilizers and their distinct interactions with soil made them influence soil N in different ways. Chemical fertilizer addition increased cumulative N mineralization, whereas organic amendments with higher C/N ratio stimulated soil N immobilization with enhanced MBN in the soil. No direct link

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between SON contents and N mineralization capacity was observed in different

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treatments because of the intricate components and conversion processes of SON. The

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chemical fertilizer addition decreased the content of SON, and organic amendment application increased the content of SON. In addition, the increased SON in

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to a high potential for SON leaching.

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EOA-amended soils, containing large amounts of hydrophilic substances, contributed

ACCEPTED MANUSCRIPT Acknowledgements This study was supported by the National Program on the Key Project of Natural Science Foundation of China, NSFC (41731280) and the National Key Research

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Project (2016YFC0402810).

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Em (nm)

0

400

450

500

ED

Em (nm) Fig. 5 3D-EEM of the SON collected from the incubated soils after 70 days. (A) CK, (B) U, (C) M,

EP T

(D) S.

AC C

Ex (nm)

400

100

C

200

20

Ⅲ

Em (nm)

400

Ⅱ

30

0

200 200

Em (nm)

Ⅰ

Ⅱ

40

Ⅴ

0

PT

Ex (nm)

100

B

90

SC

RI

PT

ACCEPTED MANUSCRIPT

AC C

EP T

ED

MA

NU

Fig. 6 Schematic of soil SON cycling

ACCEPTED MANUSCRIPT Highlights 1. Soil N mineralization differed with different fertilizers addition. 2. The chemical fertilizer decreased the SON content, and EOA addition increased it. 3. Fertilizers addition altered the sources and sink of SON involved in N cycling.

AC C

EP T

ED

MA

NU

SC

RI

PT

4. EOA addition increased the potential of SON leaching.

ACCEPTED MANUSCRIPT Tables Table 1 Characteristics of exogenous fertilizers

(%)

(%)

ratio

urea

20.00

46.67

0.43

manure

20.93

1.55

straw

40.51

0.84

pH

SON

TON

mineral N

(mg N/kg)

(g N/kg)

(mg N/kg)

7.07

4.67×105

467.00

-

13.50

9.64

9591.85

48.23

8.15

MA ED EP T AC C

PT

C/N

RI

N

SC

C

1526.31

NU

Fertilizers

15.19

310.00

8.26

89.18

ACCEPTED MANUSCRIPT Table 2 Effects of exogenous fertilizer addition on soil chemical properties

CK

pH

5.94 ±

EC

SOC

TON

TN

(μS/cm)

(mg C/kg)

(mg N/kg)

(mg N/kg)

345.00 ± 1.00d

88.39 ± 0.01d

565.59 ±

680.00 ±

1.13c

1.96b

6.48 ±

362.00 ± 1.00c

93.71 ± 0.01c

SC

U

RI

0.01d

0.01b 6.45 ±

373.00 ± 1.00b

S

6.69 ±

MA

0.01c

102.13 ± 0.01b

NU

M

547.00 ± 1.00a 207.94 ± 0.01a

653.35 ±

790.00 ±

4.08b

4.94a

694.66 ±

800.00 ±

2.45a

2.62a

690.32 ±

790.00 ±

1.96a

3.20a

ED

0.01a

PT

Treatments

EP T

Soil chemical properties were determined after incorporation of different fertilizers into soils. Different letters among different treatments for the same parameter indicate s ignificant differences,

AC C

as determined by Duncan’s multiple range test (P < 0.05) using SPSS 20.0.

ACCEPTED MANUSCRIPT Table 3 UV-vis spectra parameters and percent distribution of SON at the end of incubation Treatments

SUVA254

A253 /A203

Percentage distribution (%) PI

PII

PIII

PIV

PV

PIII+V/PI+II+IV

0.45

0.03

2.29

47.37

26.11

12.06

12.18

0.62

U

0.10

0.03

1.64

56.55

18.06

12.82

10.94

0.41

M

0.15

0.06

4.10

50.30

21.60

S

0.44

0.22

3.90

43.76

12.12

RI

0.51

17.40

15.89

0.54

11.89

SC 19.06

NU MA ED EP T AC C

PT

CK

ACCEPTED MANUSCRIPT Table 4 Pearson’s correlation relationships between both FAA and SON with cumulative N mineralization (r) Index

CK

U

M

S

-0.63

SON

0.65

-0.98**

-0.42

0.24

RI

-0.77**

0.15

-0.67

SC

FAA

PT

cumulative N mineralization

AC C

EP T

ED

MA

NU

* Correlation is significant at the 0.05 level, ** Correlation is significant at the 0.01 level. (n=7)

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6