Divergent Accumulation of Microbial Residues and Amino Sugars in Loess Soil after Six Years of Different Inorganic Nitrogen Enrichment Scenarios

Amino sugars are key microbial biomarkers for determining the contribution of microbial residues in soil organic matter (SOM). However, it remains largely unclear as to what extent inorganic nitrogen (N) fertilization can lead to the significant degradation of SOM in alkaline agricultural soils. A six-year field experiment was conducted from 2013 to 2018 to evaluate the effects of chronic N enrichment on microbial residues, amino sugars, and soil biochemical properties under four nitrogen (urea, 46% N) fertilization scenarios: 0 (no-N, control), 75 (low-N), 225 (medium-N), and 375 (high-N) kg N ha−1. The results showed that chronic N enrichment stimulated microbial residues and amino sugar accumulation over time. The medium-N treatment increased the concentration of muramic acid (15.77%), glucosamine (13.55%), galactosamine (18.84%), bacterial residues (16.88%), fungal residues (11.31%), and total microbial residues (12.57%) compared to the control in 2018; however, these concentrations were comparable to the high-N treatment concentrations. The ratio of glucosamine to galactosamine and of glucosamine to muramic acid decreased over time due to a larger increase in bacterial residues as compared to fungal residues. Microbial biomass, soil organic carbon, and aboveground plant biomass positively correlated with microbial residues and amino sugar components. Chronic N enrichment improved the soil biochemical properties and aboveground plant biomass, which stimulated microbial residues and amino sugar accumulation over time.


Introduction
Amino sugars (AS) are important constituents of microbial cell walls in soil, and their accretion influences the contribution of microbial residues to soil organic matter (SOM) stability [1]. More than 90% of AS are found in dead cells [2], with the characteristic function of quantifying microbial residues, which is not a surrogate for animate microbial activity and biomass [3]. Thus, the amount of AS in the soil correlates with the amount of microbial residues accumulated in the SOM. Soil microbes use amino sugars as an alternative nitrogen (N) source in the absence of N due to their nutrient-dependent bioavailability. In addition, AS serves as a stable nitrogen pool in soils with high N content or as a labile nitrogen pool in N-limited soils [4], and they contribute significantly to bioavailable organic nitrogen in the soil [5]. Amino sugars account for about 5.0% to 12.0% of soil organic nitrogen [6,7], and 6.2% to 8.3% of soil organic carbon (SOC) [8] in the surface layer of most soils. In general, twenty-six (26) AS have been discovered in microorganisms and their synthesized products, of which only four are abundant in soils, namely D-glucosamine (GluN), D-muramic acid (MurN), D-galactosamine (GalN), and D-mannosamine (ManN) [9]. On average, GluN accounts for about 60% of the total AS in soils, followed by GalN (30%), MurN acid (7%), and ManN (4%) [10]. Muramic acid and glucosamine are primarily derived from bacteria and fungi, respectively, and their accumulation correlates with metabolic activities and microbial growth [10,11]. Galactosamine is derived from bacteria [12], but other authors assume that it is derived from fungi [13]. Ratios of AS can be used to quantify the accumulation of bacterial and fungal residues in soils [2]. The ratio of glucosamine to galactosamine is used to determine the contribution of fungi to SOM stability, while the ratio of glucosamine to muramic acid is used to determine the contribution of bacteria to AS accumulation [3,14].
Inorganic N enrichment influences the retention of microbial residues and regulates key processes in SOC turnover [15]. Microbial residues are the main carbon resource for persistent soil carbon pool, accounting for about 50% of SOC [16]. The balance between the synthesis and decomposition of microbial metabolites is directly related to the accumulation of microbial residues in the soil [10]. The amount of microbial residue C in SOC is used to quantify the contribution of microbe-derived carbon to SOC [16]. Microbial residues serve as an important source of stable carbon; thus, its contribution to SOC pool variations will influence predictions of global change on SOC pools. Therefore, studying the accumulation of microbial residues in soils is important in understanding the influence of microbes on SOC stabilization and the global C cycle [17]. Moreover, N enrichment leads to a shift from fungi to bacteria-dominated microbial communities [18]; this affects the ratio of fungal to bacterial residues, which in turn affects AS accumulation in soils. Nitrogen enrichment has also been reported to reduce microbial residues and amino sugar accumulation in N-limited acidic soils as a result of soil acidification or N saturation [19,20]. Soils with low pH (acidic soils) suppress microbial activities and growth [21][22][23], which negatively affects microbial residues and amino sugar accumulation. Soil pH is an important factor regulating the degradation of microbial cell walls, with a low pH favoring the degradation of chintin, while a high pH stimulates the degradation of peptidoglycan [5]. The response of microbial residues and amino sugar accumulation to inorganic N enrichment in acidic soils has been extensively studied [24,25]. Nevertheless, the effects of chronic N enrichment on microbial residues and amino sugar found in alkaline soils are poorly understood. We hypothesized that chronic N enrichment would promote microbial growth and activity as well as improve the biochemical properties of the alkaline soil, which in turn would stimulate microbial residues and AS accumulation over time. Adequate N enrichment enhances the ability of soil microbes to sequester N or C into their residues, which consequently increases net microbial AS accumulation over time [26]. Hence, it is important to investigate how different chronic N enrichment rates affect carbon and nitrogen sequestration in agricultural alkaline soils in order to understand the contribution of microbial residues and amino sugars to SOM stability. The study was therefore conducted to determine the effects of chronic N enrichment on microbial residues, amino sugar concentrations, and biochemical properties of an alkaline loess soil.

Site and Treatment Description
The study was conducted from May 2013 to October 2018 at the Xiangquan experimental site in Dingxi, Gansu Province, China. The site is located at latitude 35 • 27 N and longitude 104 • 30 E. It has a mean annual precipitation of 400 mm, a mean temperature of 6.9 • C, and a frost-free period of 140 days. The soil biochemical properties at the site are presented in Table 1. In this study, four nitrogen (urea, 46% N) application rates were investigated, namely: no-N application (0 kg N ha −1 , control), low-N application (75 kg N ha −1 ), medium-N application (225 kg N ha −1 ), and high-N application (375 kg N ha −1 ).

Soil Sampling and Analysis
Post-harvest soil samples were collected from five sampling points at a depth of 0-20 cm in each plot and then bulked to form a composite sample. Stones, animal and plant debris, and visually detectable fauna were removed, and each sample was divided into two parts. One part was kept in the refrigerator (4 • C) for microbial biomass and available nitrogen (nitrate and ammonium) analyses, while the other part was air-dried for 14 days and ground through 1 mm and 0.15 mm meshes before soil chemical analysis.
Soil pH (1:2.5 soil:water suspension) was determined using a portable pH meter (Shanghai Precision and Scientific Instrument Company Limited, China). Total nitrogen content was measured using a modified Kjeldahl Method, as described by Bremner [27]. Soil organic carbon content was determined using the potassium dichromate digestion method [28]. Ammonium (NH 4 + -N) and nitrate (NO 3 − -N) concentrations in the soil were extracted with 2M KCl at a ratio of 1:10. The concentrations of NH 4 + -N and NO 3 − -N in the filtrate were measured using the Continuous-Flow Analysis-AA3 analyzer, Seal, Germany. Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were determined using the chloroform fumigation extraction method [29], with slight modifications. Briefly, 10 g (oven-dry basis) of fresh soil was weighed in duplicates; one part was fumigated with ethanol-free chloroform (CHCI 3 ), while the other part was not fumigated. A total of 40 mL of 0.5 mol L −1 K 2 SO 4 was added to the fumigated and non-fumigated soil samples, and then shaken at 300 r min −1 for 60 min. The solutions were then filtered and the carbon and nitrogen content in the filtrates were determined using a TOC analyzer (German Jena, Multi N/C 3000). The MBC and MBN concentrations were calculated using the following formulas: Microbial biomass C = Fumigated C content -Non fumigated C content/kEC (1) Microbial biomass N = Fumigated N content -Non fumigated N content/kEN (2) where kEC = 0.45 [30] and kEN = 0.54 [31].
The carbon and nitrogen contents in the non-fumigated samples were used to calculate dissolved organic carbon (DOC) and dissolved organic nitrogen (DON), respectively, as described by Boyer and Groffman [32].

Microbial Residues and Amino Sugars Analysis
The concentration of amino sugars (GluN, GalN, and MurN) in the soil was determined according to the method described by Zhang and Amelung [33]. Briefly, soil samples were hydrolyzed at 105 • C with 6 M HCl for 8 h. The solutions were filtered and centrifuged after adjusting their pH to a level between 6.6 and 6.8. The supernatant solutions were then freeze-dried, and methanol was used to wash out the AS from the residues. The recovered AS were converted to aldononitrile derivatives and the excess derivatization reagents were removed by a two-step reaction. In the first step, a derivatization reagent containing 40 mg mL −1 4-(dimethylamino) pyridine in pyridine methanol (4:1 v/v) and 32 mg mL −1 hydroxylamine hydrochloride was added to the dry sample in the airlock, while in the second step, acetic anhydride was added. Dichloromethane was used to extract the derivatives, and 1M HCl and distilled water were used to remove excess anhydride. The final AS derivatives were then dissolved in 300 µL ethyl acetate-hexane (v:v = 1:1) for analysis. Internal standard (myo-inositol) was added before hydrolysis and the derivatives of AS were determined using an Agilent 6890A GC (Agilent Tech. Co., Wilmington, DE, USA) equipped with a flame ionization detector and an HP-5 (25 m × 0.32 mm × 0.25 µm) fused silica column. Fungal residues in SOC were calculated as the difference between total GluN and bacterial GluN [13,34], while the bacterial residues in SOC were determined by multiplying the MurN content by 45 [34]. The total microbial residues in SOC were calculated as the sum of bacterial and fungal residues.

Aboveground Plant Biomass Determination
Aboveground plant biomass was determined after potato plants were harvested in the 2018 season. In each experimental unit, six plants were cut from the soil surface, excluding the border plants. The shoots were then oven-dried at a constant temperature of 80 • C until a constant weight was reached and recorded as aboveground biomass (g m −2 ).

Data Analysis
A one-way analysis of variance was conducted using SPSS software version 21 for Windows (IBM Corp., Chicago, IL, USA). Treatment means were compared by the least significant difference test at a 0.05 probability level. Regression analysis was conducted using SPSS to determine the functional relationships among N rate, aboveground plant biomass, SOC, and total N. The correlation matrix analysis was performed using SPSS to determine the relationship between microbial amino sugar components and soil biochemical properties. Principal component analysis (PCA) was conducted using Canoco 5 (Microcomputer Power, Ithaca, NY, USA) to explore the effects of nitrogen enrichment on microbial residues, amino sugars, and soil biochemical properties. Figures and tables were drawn using GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA, USA) and Microsoft Word version 365 (Microsoft Corporation, Redmond, Washington, USA), respectively.

Effects of Chronic Nitrogen Enrichment on Amino Sugar and Microbial Residue Components from 2014 to 2018
Chronic N enrichment significantly affected the concentration of amino sugar components in the soil ( Figure 1). Glucosamine content followed a linear trend from 2014 to 2018 and significantly varied by N enrichment (Figure 1a). The GluN content ranged from 148.73 to 18.90 mg kg −1 and increased with increasing N rate; however, the GluN content under the high-N and medium-N treatments was at par throughout the study period. The medium-N treatment stimulated GluN content by 8.33%, 11.18%, and 13.55%  Figure 1d). The total AS content increased with increasing N rate, and it ranged from 223.54 to 281.04 mg kg −1 . There was no significant difference between the high-N and medium-N treatments in terms of total AS content. The total AS content in the soil was higher under the medium-N treatment than the control by 9.82%, 13.71%, and 15.31% in 2016, 2017, and 2018, respectively. The ratio of glucosamine to galactosamine (GluN/GalN) varied significantly by the nitrogen enrichment only in 2017 and 2018, and it decreased over time ( Table 2). The GluN/GalN ratio ranged from 1.99 to 2.25 and decreased with increasing N application rate. Compared to the control, the high-N treatment decreased the GluN/GalN ratio by 8.29% and 7.41% in 2017 and 2018, respectively. Similar to the GluN/GalN ratio, the ratio of glucosamine to muramic acid (GluN/MurN) was significantly affected by the nitrogen enrichment only in 2017 and 2018, and it decreased over time ( Table 2). The Glu/MurN ratio ranged from 18.38 to 20.79 and decreased with increasing N application rate. The GluN/MurN ratio was 5.64% and 6.18% higher under the control than under the high-N treatment in 2017 and 2018, respectively.  Nitrogen enrichment significantly affected the bacterial residues in SOC from 2015 to 2018 (Figure 2a). The bacterial residues in SOC ranged from 3.78 to 4.83 mg kg −1 , and it increased with increasing N rate; however, the high-N and medium-N treatments had statistically similar bacterial residues in SOC throughout the study period. Compared to the control, the medium-N treatment increased bacterial residues in SOC by 4.73% in 2015, 11.49% in 2016, 16.02% in 2017, and 16.88% in 2018. The fungal residues in SOC were also significantly varied by the N enrichment from 2015 to 2018 (Figure 2b). Fungal residues in SOC ranged from 14.15 to 16.40 mg kg −1 and it increased with an increase in N rate; but the fungal residues recorded by the high-N and medium-N treatments were statistically similar throughout the study period. The medium-N treatment stimulated fungal residues in SOC by 3.01%, 6.16%, 10.75%, and 11.31% in 2015, 2016, 2017, and 2018, respectively, compared to the control. Moreover, N enrichment significantly affected total microbial residues in SOC from 2015 to 2018 (Figure 2c). The total microbial residues in SOC ranged from 17.93 to 21.23 mg kg −1 and it increased with increasing N rate. The high-N and medium-N rates had statistically similar total microbial residues throughout the experiment; however, the medium-N treatment significantly stimulated total microbial residues in SOC by 3.38%, 7.35%, 11.94%, and 12.57% compared to the control in 2015, 2016, 2017, and 2018, respectively. The ratio of fungal residues to bacterial residues in SOC significantly varied by nitrogen enrichment from 2016 to 2018 and decreased with an increase in N rate (Figure 2d). The ratio of fungal residues to bacterial residues ranged from 3.40 to 3.74 and decreased over time. The ratio decreased by 6.25% in 2016, 6.28% in 2017, and 6.85% in 2018 under the high-N treatment compared to the control.

Effects of Chronic Nitrogen Enrichment on Microbial Biomass, and Dissolved Organic Carbon and Nitrogen Concentrations in the Soil from 2014 to 2018
Nitrogen enrichment significantly affected microbial biomass (MBN and MBC) from 2015 to 2018 (Figure 3a,b). The MBN concentration in the soil ranged from 5.66 to 13.1 mg kg −1 , while the MBC concentration ranged from 79.36 to 117.11 mg kg −1 . Microbial biomass followed a linear trend and increased with increasing N rate; however, the high-N and medium-N treatments had statistically similar microbial biomass throughout the study period. Compared to the control, the medium-N treatment stimulated MBN by 28 (Figure 3c). The MBC/SOC ratio ranged from 9.18 to 12.19 and ranked as: high-N > medium-N > low-N > no-N, throughout the experiment. The effect of N enrichment on the MBC/MBN ratio was also significant from 2014 to 2018 and decreased over time (Figure 3d). The ratio of MBC to MBN ranged from 8.59 to 14.47, and it decreased with increasing N rate and followed this trend: no-N > low-N > medium-N > high-N, respectively, throughout the study. Dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) were significantly stimulated by N enrichment and increased from 2015 to 2018 (Figure 4a,b). The DOC and DON contents followed a linear trend and ranged from 179.3 to 266.0 mg kg −1 and 129.5 to 223.8 mg kg −1 , respectively. Similar to MBC and MBN, DOC and DON increased with an increasing N application rate. The DOC and DON contents were ranked as: high-N > medium-N > low-N > no-N, throughout the study period. Similar to the MBC/SOC ratio, the ratio of dissolved organic carbon to soil organic carbon (DOC/SOC) varied significantly from 2016 to 2018 by nitrogen enrichment (Figure 4c). All treatments increased the DOC/SOC ratio over time; however, it increased with an increase in N rate. The DOC/SOC ratio ranged from 20.78 to 27.68. The high-N and medium-N treatments had statistically similar ratio throughout the experimental period; however, the DOC/SOC ratio was stimulated by 6.60% in 2016, 9.12% in 2017, and 11.07% in 2018 under the medium-N treatment compared to the control. The ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON) was significantly affected by nitrogen enrichment and decreased with increasing N rate (Figure 4d). The DOC/DON ratio ranged from 1.09 to 1.45 and decreased over time. The DOC/DON ratio was ranked as: no-N > low-N > medium-N > high-N, respectively.
Soil organic carbon was significantly affected by nitrogen enrichment from 2016 to 2018 ( Table 2). The SOC content ranged from 8.63 to 9.51 g kg −1 and it increased with increasing N application rate; however, the low-N, high-N, and medium-N treatments had statistically similar SOC throughout the study period. Compared to the control, the medium-N treatment significantly increased the SOC content by 6.71% in 2018. Soil pH was also significantly varied by N enrichment from 2016 to 2018, and it decreased over time ( Table 2). Soil pH ranged from 8.01 to 8.31 and it decreased with an increase in the N application rate. Compared to the control, soil pH significantly decreased by 1.71% and 2.32% under the medium-N and high-N treatments, respectively, in 2018. Moreover, total soil nitrogen significantly varied by N enrichment from 2016 to 2018 (Table 3). The total N content in the soil ranged from 0.95 to 1.28 g kg −1 . All the treatments increased total N content over time, but it increased significantly with an increase in N application rate. However, the medium-N and the high-N treatments had statistically similar total N content throughout the experiment. The medium-N treatment significantly increased total N content by 11.11%, 14.04%, and 17.89% in the 2016, 2017, and 2018 seasons, respectively, compared to the control. Soil available nitrogen (NH 4 + -N and NO 3 − -N) concentration was significantly increased by N enrichment from 2014 to 2018 (Table 3). The NH 4 + -N concentration ranged from 0.76 to 1.25 mg kg −1 , whereas NO 3 − -N concentration ranged from 4.68 to 19.18 mg kg −1 . The concentrations of NH 4 + -N and NO 3 − -N in the soil were ranked as high-N > medium-N > low-N > no-N throughout the experiment. In 2018, the high-N treatment increased NH 4 + -N and NO 3 − -N concentration by 38.40% and 75.50%, respectively, compared to the control.   The functional relationships between the N rate and aboveground plant biomass (APB), SOC and APB, and total N and APB are showed in Figure 5. There was a significant relationship between the N rate and APB (R 2 = 0.99). Similarly, APB significantly related to SOC (R 2 = 0.97) and total N (R 2 = 0.96).

Effects of Chronic Nitrogen Enrichment on Microbial Residues and Amino Sugar Accumulation
Amino sugars are usually derived from the turnover of microbial residues in soils, as plants cannot produce a large amount of AS [34,35]. Previous studies reported that amino sugars decreased significantly with N enrichment due to calcium or magnesium deficiency resulting from soil acidification, N saturation, or altered carbon availability [19,20]. Thus, N enrichment decreases microbial AS accumulation in acidic soils. Griepentrog et al. [24] and Zhang et al. [25] reported that nitrogen enrichment had an insignificant effect on AS concentrations in soil. However, the results of the present study showed that chronic N enrichment increased microbial residues and amino sugar concentrations in the soil (Figures 1 and 2), which is consistent with previous studies [17,26,36]. The discrepancy between the previous studies [24,25] and our study may be attributed to differences in soil pH. The pH of the soil used in the present study is above 8, while that of the previous studies was less than 5. Zhang et al. [25] documented that N enrichment lowered the pH of the acidic soil, which hindered microbial activities and production. Soils with low pH suppress the growth and activity of microbes, especially bacteria [22,37], thereby reducing amino sugar accumulation in N-fertilized soils [25,38]. In the present study, N enrichment reduced soil pH close to the neutral range (Table 2), which might have facilitated microbial growth and activity; consequently, this stimulated microbial residues and amino sugar accumulation. Soils with a neutral pH or a near-neutral range promote microbial growth and activity [22,23,39]. In addition, N enrichment stimulated microbial residues and AS probably due to its high microbial biomass, dissolved organic C, and SOC concentrations in the soil. Microbial residues and AS showed a positive correlation with microbial biomass, dissolved organic carbon, and SOC ( Table 4). The accumulation of microbial residues and AS was higher under the high-N and medium-N treatments, where the N requirement was sufficient for microbial growth. The lower microbial residues and AS accumulation in the low-N and the control treatments probably occurred due to low N availability (Table 3), thereby reducing microbial growth and their residual metabolic production [39]. Ding et al. [26] found that adequate N enrichment facilitated soil microbes to sequester N or C into their residues, and consequently increased their net microbial AS accumulation over time. Moreover, aboveground plant biomass, which was returned to the soil annually after harvest, increased with an increasing N rate ( Figure 5a); this in turn might have caused an increase in the microbial residues and AS accumulation under the high and medium N rates. The aboveground plant biomass also showed a positive and significant correlation with microbial AS components (Table 4). This finding concurs with Arnebrant et al. [40] and Corre et al. [41].
The concentration of the individual amino sugars (GluN, GalN, and MurN) increased with time and paralleled to the gradual accumulation of total N and SOC, which concurs with Pronk et al. [42]. The concentrations of the individual amino sugars were ranked as GluN > GalN > MurN and a similar trend was reported by previous studies [2,[24][25][26]. The ratios of GluN to MurN and GluN to GalN decreased with time, since the increase in GalN and MurN over time was greater than the increase in GluN due to bacteria dominance in the alkaline soil. In a high pH environment, bacteria dominate over fungi because bacteria are preferred at high pH levels [23,39,43]. This could explain why the ratio of fungal to bacterial residues decreased over time (Figure 2d). In general, bacteria require more nutrients than fungi [44], and therefore dominate in soils where N is abundant. This could also explain why the medium-N and the high-N treatments had the lowest ratio of GluN to MurN and GluN to GalN. The lower ratios of GluN to MurN and GluN to GalN indicate that bacteria contribute significantly to soil organic matter stability and amino sugar accumulation in the alkaline soil. Our findings are in agreement with Zhang et al. [45], who postulated that nitrogen enrichment lowers the ratio of fungi to bacteria due to different reactions between bacterial biomass and fungal biomass.

Effects of Chronic Nitrogen Enrichment on Soil Biochemical Properties
Nitrogen enrichment improved the soil biochemical properties, which is in line with previous studies [46,47]. Soil microbial biomass (MBC and MBN) increased with an increasing N rate, which concurs with Raiesi [48]. The high-N and the medium-N treatments had the highest microbial biomass, probably due to their high available N concentration (Table 3), which enhanced the degradation of SOM, thereby stimulating microbial biomass concentration in the soil. However, our result contradicts previous studies that reported that N enrichment reduces microbial biomass in soils [45,[49][50][51][52]. The discrepancy between our study and previous studies may be attributed to variations in soil pH [53], experimental duration and biome type [49], and nutrient availability [54]. The previous studies were conducted in forest and grassland ecosystems, while the present study was conducted in a cropland ecosystem; nitrogen enrichment decreases microbial biomass in forest and grassland ecosystems, while it increases microbial biomass in cropland and desert ecosystems [49]. Cropland and desert ecosystems are generally limited by nitrogen; therefore, nitrogen enrichment increases soil N availability and promotes microbial growth [49]. Soil DOC plays a key role in the formation of SOM and the movement of nutrients within ecosystems [55]. Nitrogen enrichment decreases soil DOC concentration due to the chemical stabilization of organic carbon [56] and the stimulation of the microbial mineralization rate, which increases DOC consumption [57]. Results of the current study, however, revealed that nitrogen enrichment stimulated soil DOC concentration, which is consistent with Shi et al. [58] and Wei et al. [52]; this could be attributed to the nutrient status of the soil [59,60]. Rosa et al. [60] pointed out that soil DOC significantly correlated with SOC and total N. Soil DOC increased with an increasing N rate (Figure 4b), probably due to aboveground plant biomass, which was returned to the soil annually. Increasing N rate significantly increased aboveground plant biomass (Figure 5a), which in turn stimulated DOC concentration in the soil. This finding corresponds with previous studies that reported that soil DOC increases as N rate increase due to litter production [61,62]. Nitrogen enrichment increased soil DON, which agrees with Currie et al. [63]. The soil DON content increased with an increasing N rate, probably due to the effect of nitrogen enrichment on soil pH. Felip and Rekasi [59] documented that soil pH negatively correlated with soil DON concentration. The MBC/MBN and DOC/DON ratios decreased with an increasing N rate (Figures 3d and 4d), since the increase in DON and MBN along the N gradient was higher than that of DOC and MBC contents, respectively. The nitrate concentration in the soil was higher than that of ammonium throughout the experimental period, as nitrate is the main form of available N in soils [46,64]. High-N treatment had the highest nitrate (NO 3 − -N) concentration, partly due to its high rate exceeding the N requirement of the plants, which is consistent with previous studies [46,65,66]. However, the linear accumulation of residual soil NO 3 − -N content by the high-N treatment could pollute underground water, and thereby affect human health [66][67][68].
Nitrogen enrichment decreased soil pH over time and could be attributed to the process of nitrification, which resulted in the production and release of hydrogen ions into the soil solution [53]. Lu et al. [69] pointed out that N enrichment increases the leaching of basic cations such as Ca 2+ and Mg 2+ in the charge balance of the soil solution, thereby decreasing soil pH. An increase in N rate decreased soil pH, which agrees with previous studies [47,49,52,66,70]. The slight decrease in soil pH in the control occurred probably as a result of the depletion of basic cations by plants and the decomposition of organic matter [70]. The soil organic carbon content increased over time, including in the control, and this may be partly due to carbon sequestered from plant biomass returned to the soil, which concurs with previous studies [71][72][73]. The SOC content increased with an increasing N rate, which can partly be attributed to aboveground plant biomass. The increase in N rate increased aboveground plant biomass, which was returned to the soil after harvest (Figure 5b) and therefore might have supplied additional carbon to the soil after decomposition [74,75]. Li et al. [74] found a positive and significant correlation between SOC and aboveground plant biomass. Moreover, Fontaine et al. [76] observed that high N enrichment stores more carbon in the soil due to the prime effect. Our results are in agreement with previous studies where increasing N enrichment significantly increased SOC [37,70,74]. Total N content in the soil increased with an increasing N rate and could probably be attributed to N accumulation from the chronic N enrichment, as crop N depletion was less than N inputs over time. This finding is consistent with Sainju et al. [66], Aula et al. [70], and Anning et al. [77], who reported that total soil N content increased with increasing N fertilization.

Conclusions
The results of the study support our hypothesis that chronic nitrogen enrichment could stimulate microbial residue and amino sugar accumulation in alkaline soil. Microbial residue and amino sugar accumulation increased with an increasing N rate; however, the high-N and medium-N treatments had statistically similar accumulation. Total amino sugar was significantly higher under high-N and medium-N treatments, where N requirement for microbial growth was sufficient. The ratios of glucosamine to muramic acid and glucosamine to galactosamine decreased over time as a result of the more substantial increase in bacterial residues than that in fungal residues. Thus, bacteria contribute significantly to soil organic matter stability, microbial necromass, and AS accumulation in alkaline soil. Nitrogen enrichment improved the soil biochemical properties by increasing soil organic carbon, total nitrogen, available nitrogen, microbial biomass, and dissolved organic carbon concentrations, while lowering soil pH. Microbial residues and AS components showed a positive and significant correlation with microbial biomass, dissolved organic carbon, soil organic carbon, and aboveground plant biomass. However, soil pH showed a negative and significant correlation with glucosamine, muramic acid, total amino sugar, bacterial residues, and total microbial residues in soil organic carbon. Chronic nitrogen enrichment improved the soil biochemical properties and aboveground plant biomass, which stimulated microbial residue and amino sugar accumulation over time. Soil enzymes and microbial turnover have been reported to influence microbial residues and AS accumulation; however, these parameters were not measured in the present study. Therefore, we recommend future studies to focus on how chronic N enrichment affects soil enzyme activity and microbial turnover, and their contribution to microbial residues and AS accumulation in alkaline agricultural soils.