Organic matter fractions within macroaggregates in response to long-term fertilization in calcareous soil after reclamation

CAO Han-bing ,XlE Jun-yu ,HONG Jie,WANG Xiang,HU Wei,HONG Jian-ping

1 College of Resources and Environment,Shanxi Agricultural University,Taigu 030801,P.R.China

2 Shanxi Province Key Laboratory of Soil Environment and Nutrient Resources,Taiyuan 030031,P.R.China

3 College of Environment,Zhejiang University of Technology,Hangzhou 310014,P.R.China

4 College of Land Science and Technology,China Agricultural University,Beijing 100193,P.R.China

5 New Zealand Institute for Plant &Food Research Limited,Christchurch 8140,New Zealand

Abstract Soil organic carbon (SOC) plays a key role in improving soil quality and optimizing crop yield. Yet little is known about the fate of macroaggregates (＞0.25 mm) under long-term fertilization and their relative importance in SOC sequestration in reclaimed calcareous soil. Therefore,the effects of mineral fertilizers and organic manure on the mechanisms of organic carbon (OC) stabilization in macroaggregates were investigated in this study. Four treatments were used:unfertilized control(CK),mineral fertilizer (NPK),compost chicken manure alone (M),and mineral fertilizers plus manure (MNPK). Samples from the 0–20 cm layer of soil receiving 11-year-long fertilization were separated into four fractions based on the macroaggregates present (unprotected coarse and fine particulate organic matter,cPOM and fPOM;physically protected intra-microaggregate POM,iPOM;and biochemically protected mineral associated OM,MOM) by the physical fractionation method. Compared with the control,the long-term application of NPK had little effect on SOC content,total nitrogen (TN) content,and OC and TN contents of macroaggregate fractions. In contrast,incorporation of organic manure (MNPK) significantly increased SOC(45.7%) and TN (24.3%) contents. Application of MNPK increased OC contents within macroaggregate-extracted fractions of cPOM (292.2%),fPOM (136.0%) and iPOM (124.0%),and TN contents within cPOM (607.1%),fPOM (242.5%) and iPOM(127.6%),but not the mineral associated organic carbon (MOM-C) and nitrogen (MOM-N) contents. Unprotected C fractions were more strongly and positively correlated with SOC increase than protected C fractions,especially for cPOM-C,indicating that SOC sequestration mainly occurred via cPOM-C in the studied calcareous soil. In conclusion,MNPK increased the quantity and stability of SOC by increasing the contents of cPOM-C and cPOM-N,suggesting that this management practice(MNPK) is an effective strategy to develop sustainable agriculture.

Keywords:long-term fertilization,carbon sequestration,macroaggregate,physical fractionation,coarse particulate organic carbon,calcareous soil

1.lntroduction

Soil organic carbon (SOC) is a key indicator of soil quality and plays an important role in improving crop productivity by providing energy for microbial nutrient cycling (Gaiet al.2018). Fertilization is the most widespread agricultural management practice for improving soil quality,and is often recommended to increase SOC and soil fertility (Abraret al.2020). Furthermore,soils have great potential to mitigate increasing global atmospheric greenhouse gas (i.e.,CO2and N2O) emissions by sequestrating SOC (Macbean and Peylin 2014). Thus,there is a great need to understand the factors that contribute to high level SOC and to seek optimal fertilization practices to enhance SOC storage in soil.

The effects of long-term mineral fertilization or organic manure application on SOC and total nitrogen (TN) content have been widely investigated (Dattaet al.2018;Wenet al.2019). Studies have shown that SOC and TN concentrations tend to respond positively to manure amendments (Blanchetet al.2016;Ghoshet al.2018). However,long-term applications of mineral fertilizers not always had positive effects on SOC or TN concentrations (Shahidet al.2017;Wanget al.2018;Liuet al.2019),and the drivers behind the associated SOC sequestration mechanism are not clearly understood.

SOC is composed of several fractions based on their different intrinsic degradability features and factors controlling their decomposition rates (von Lützowet al.2007;Liet al.2017). Labile SOC fractions,such as microbial biomass C(Heet al.2015) and light fraction organic carbon (OC) (Jiaoet al.2018),are characterized by their rapid turnover rates and are considered to be parameters that are more sensitive to the effects of management practices than total SOC (von Lützowet al.2007). In contrast,stable SOC fractions,such as heavy fraction OC (Jiaoet al.2018) and recalcitrant OC(Jhaet al.2014),make greater contributions to sustained soil fertility than soil biological activities and nutrient supply.These fractions are generally and loosely linked with measurable quantities (Sixet al.2002). Sixet al.(1998,2000) proposed the use of aggregate physical fractionation and the associated conceptual SOC model to examine the effects of fertilizer management on SOC fractions to assess whether sequestered SOC can be stored in the long term.In this method,aggregates were divided into four conceptual fractions:coarse particulate organic matter (POM) (cPOM),fine POM (fPOM),intra-microaggregate POM (iPOM) and mineral-associated OM (MOM). The cPOM and fPOM were considered as unprotected soil organic matter (SOM),iPOM was physically protected SOM,and MOM was biochemically protected SOM. Previous studies have focused on the effects of land-use conversion (Sheehyet al.2015) and agricultural management practices,such as sludge or biochar addition (Nicolaset al.2014;Wang Det al.2017),on OC or TN within aggregate fractions with different protections. These studies have shown that iPOM was the primary fraction involved in SOC sequestration. Physical fractionation has also been extensively applied to examine the effects of long-term fertilization on the stability of SOC in red soil (Yang Fet al.2018),non-calcareous fluvo-aquic soil (Tianet al.2017),Luvic Phaeozems (Yang H Bet al.2018),and Anthrosols (Xieet al.2018). Yang Fet al.(2018)concluded that biochemically protected mineral-associated OC was the primary fraction after the addition of manure combined with mineral fertilizers (MNPK) in both upland and paddy soils that developed from the quaternary red clay parent material. However,Tianet al.(2017) and Yang H Bet al.(2018) observed that the unprotected cPOM fraction was the most important fraction for assessing the impact of long-term distinct fertilization management practices on SOC or TN availability in non-calcareous fluvo-aquic soil or Luvic Phaeozems after application of manure in combination with mineral fertilizer (MN or MNPK). In addition,Xieet al.(2018) found that SOC sequestration mainly occurred in the form of physically protected microaggregates (iPOM)in an Anthrosol of northwestern China after the application of MNP for 35 years. Yaoet al.(2019) reported that the protected OC fraction (both iPOM-C and MOM-C) was the important parameter to indicate the stability of SOC after growing leguminous green manure to replace summer fallow in Cumulic Haplustolls. SOC sequestration in different soils in response to fertilization regimes may be associated with different mechanisms of aggregate stabilization,such as physical protection,chemical protection,and biochemical stabilization (Sixet al.2002). Thus,further investigation is essential to improve our understanding of how aggregates store and protect OC under long-term fertilization regimes in a specific soil type.

Calcareous soils reclaimed after mining are typical in the eastern Loess Plateau of northern China,and they are widely distributed in the southeastern region of Shanxi Province. Two soil handling methods are used during the reclamation process,one is topsoil replacement (TSR,where topsoil is stripped and replaced immediately after mining onto a rehabilitation area),and the other one is soil horizon mixing (SHM,where the soil horizon is mixed during soil handling) (Liet al.2018). However,soil profile reconstruction has resulted in the degradation of soil physical properties (such as bulk density),nutrient contents and microbial diversity (Liuet al.2017;Yuanet al.2017).In 2017,the gob area caused by underground mining was nearly 500 000 ha (approximately 3% of the province’s land area),including 300 000 ha of subsidence land,108 000 ha of damaged cropland and 4 000 ha of forestry land,and the affected population was approximately 2.3 million(SSB 2017). Damaged mining land can be restored with appropriate reclamation techniques and post-reclamation measures (Liet al.2019),and the reclamation of coal mines plays an important role in restoring the ecological integrity of these degraded lands in the face of a growing population and urbanization. Reclaimed soils are similar to an“empty cup”with a large SOC sequestration potential(Yuanet al.2018). Studies of this large and increasing area of reclaimed land have focused on aspects of SOC,such as determination of the heavy and light fractions associated with SOC and TN contents (Jiaoet al.2018),calcium (Ca)-mediated stabilization of SOC (Huanget al.2019),and SOC pooling and CO2emissions (Liet al.2019). However,how SOC sequestration responds to different long-term fertilization regimes in calcareous soil after reclamation remains unknown,especially at the soil aggregate level.In this study,a long-term (11-year) experiment of reclaimed cropland in a mine reclamation region was used to estimate:(1) the impact of fertilization on crop yield,stocks of SOC,and TN;(2) variation in OC and TN accumulation in different macroaggregate-extracted fractions;and (3) relationships between the differences of the SOC and OC and TN contents within macroaggregate-extracted fractions.

2.Materials and methods

2.1.Study site and experimental design

A long-term experiment was established in March 2008 in Xiangyuan County (36°28´N,113°00´E,the elevation of 980 m a.s.l.),Shanxi Province,China. This site has a warm temperate continental monsoon climate with mean annual temperature of 9.5°C and mean annual precipitation of 550 mm (with more than 60% falling in the summer from July to September). The coal mine area began collapsing in the 1970s,but became stable near the year of 2000.Land rehabilitation was initiated in 2008 mainly using the TSR method. According to natural terrain conditions,topsoil was firstly removed,and then higher ground areas were excavated to fill the low-lying areas. Finally,stored soil was spread on top of the overburden to a depth of 30 cm. The whole reconstructed soil depth exceeded 100 cm. The area has been cultivated since the start of the long-term experiment with spring maize (ZeamaysL.)cropping system. The soil at the site is a silt clay loam(15% clay;55% silt;and 30% sand) and is classified as a Calcaric Cambisol (IUSS Working Group WRB 2015). The major physical-chemical properties of the 0–20 cm soil layer at the experimental site in 2008 were as follows:SOC concentration of 4.20 g kg–1,TN of 0.50 g kg–1,Olsen-P of 2.01 mg kg–1and exchangeable K+(extracted with 1.0 mol L–1ammonium acetate at pH 7) of 101.74 mg kg–1.

The experiment included four treatments arranged in a completely randomized block design in triplicate. Each replicate plot covered an area of (100 m2(10 m×10 m). The first treatment included no fertilizer or manure inputs (control,hereafter referred to as CK). The second treatment (NPK)consisted of annual mineral fertilizer inputs of 201.5 kg ha–1nitrogen (N),184.8 kg ha–1phosphorus (P2O5),and 98.4 kg ha–1potassium (K2O). The third treatment was the application of chicken manure compost alone (M),and the M plot received 12 000 kg ha–1of chicken manure compost annually. The compost was prepared by mixing chicken manure,tobacco straw,and rice chaff at a ratio of 80:15:5 and by composting the mixture for two months at Honghao Biotechnology Co.,Ltd.,Shanxi Province,China. The OC,TN,P2O5,and K2O concentrations of the manure compost were 25.8,1.7,1.5,and 0.8%,respectively. The fourth treatment was the application of chicken manure compost plus NPK (MNPK),where the MNPK plot received half-rates of N,P,and K from inorganic fertilizers and organic manure compost,as applied to the NPK treatment and M treatment,respectively. The mineral fertilizer sources were urea (46%N),superphosphate (12% P2O5),potassium chloride (60%K2O). The details of the quantities of each type of fertilizer used in the different treatments are listed in Table 1. Urea was applied as a basal fertilizer before sowing,and as a topdressing at the 12-leaf (V12) and silking (R1) stages,however,the other chemical and organic materials were applied as basal fertilizers before the soil was plowed.The spring maize was sown in early May and harvested approximately four months later at the end of September or early October. At maturity,crops were harvested manually with sickles,cutting close to the ground,in a 30-m2area to estimate the yields of each treatment. Then,maize straw from all treatments was returned to the field to mitigate soil erosion,which is a typical agricultural management practice for the Loess Plateau of China.

2.2.Soil sampling and analyses

Soil samples were collected at the end of September 2018,one day before the maize was harvested. In each plot,three undisturbed soil cores were taken using a cylinder of 10 cm in height and 10 cm in diameter to a depth of 0–20 cm. The core samples were carefully mixed and sealed immediately in a plastic bag. Moist soil samples collected from the field were gently broken apart along natural breakpoints and passed through an 8-mm sieve for macroaggregateanalysis. Additionally,three soil samples (0–20 cm) were taken from each plot using an auger (inner diameter of 2.5 cm) after the maize harvest for determination of the SOC and TN concentrations. The samples were bulked and plant residues and organic debris were carefully removed and then air-dried and stored at room temperature before analysis.

Table 1 Application rates of N,P,K and manure in different treatments per year (kg ha–1)

2.3.Aggregate isolation

The aggregates were separated by wet sieving following the method of Elliott (1986). The air-dried soil was passed through 2-,0.25-,and 0.053-mm sieves (Fig.1). Briefly,50 g of air-dried soil sample was submerged in deionized water on the top of a 2-mm sieve for 5 min before sieving. The sieving was performed by gently moving the sieve up and down by 3 cm for 50 times over 2 min. The fraction that remained on the 2-mm sieve was collected in a pre-weighed aluminum pan. Water plus soil＜2 mm was poured onto the 0.25-mm sieve,and the sieving procedure was repeated and repeated for the 0.053-mm sieve. All fractions were gently back-washed into a pre-weighed aluminum pan and dried overnight at 50°C. Thus,four size aggregates were obtained:i) ＞2 mm (large macroaggregates);ii) 0.25–2 mm (small macroaggregates);iii) 0.053–0.25 mm(microaggregates),and iv)＜0.053 mm (silt+clay fraction).

Fig.1 Physical fractionation scheme for the isolation of organic matter fractions within macroaggregates. cPOM,unprotected coarse particulate organic matter;fPOM,unprotected fine particulate organic matter;iPOM,physically protected intramicroaggregate particulate organic matter;MOM,biochemically protected mineral associated organic matter (composed of s+c_m and s+c_f fractions).

2.4.Physical fractionation of macroaggregates

To isolate microaggregates occluded in the macroaggregates,we used the method outlined by Sixet al.(2000) (Fig.1).Large macroaggregates and small macroaggregates were mixed proportionally in a 10 g subsample and were then soaked in 50 mL deionized water in a refrigerator overnight.The macroaggregates were immersed in water on a 0.25-mm mesh screen and gently shaken with 50 glass beads(4 mm in diameter). Continuous and steady water flow through the device ensured that microaggregates were immediately flushed onto a 0.053-mm sieve and were not exposed to any further disruption by the beads. When all the macroaggregates were broken up (achieved after approximately 5 min),the material collected on the 0.053-mm sieve was wet sieved following the procedure described above to ensure that the isolated microaggregates (0.053–0.25 mm)were water-stable. The coarse particulate organic matter(cPOM) collected on the 0.25-mm mesh sieve,microaggregates occluded in macroaggregates (0.053–0.25 mm),and the silt and clay within macroaggregates (s+c_f) were collected and then oven-dried at 50°C,weighed and stored for further analyses.

The intra-microaggregate particulate organic matter(iPOM) and the mineral-associated organic matter (MOM)fraction within the microaggregates were isolated by density flotation following Sixet al.(1998) (Fig.1). Firstly,a 5-g subsample of microaggregates occluded in the macroaggregates (0.053–0.25 mm) was oven-dried at 110°C before being suspended in a 100-mL centrifuge tube with 35 mL of 1.85 g cm-3sodium iodide (NaI) and slowly shaken by hand. Secondly,the samples were placed under a vacuum (138 kPa) for 10 min to evacuate air entrapped within the aggregates. After that,they were equilibrated for 20 min. Thirdly,the samples were centrifuged at 1 250×g for 60 min at 25°C . The floating material (fPOM) was aspirated and filtered using a 20-μm nylon filter. While the heavy fraction was rinsed twice with 50 mL deionized water to remove the remaining NaI and dispersed by shaking with 30 mL of 0.5% sodium hexametaphosphate for 18 h,the dispersed heavy fractions were passed through a 0.053-mm sieve to separate the iPOM and silt and clay fractions within the microaggregates (s+c_m). Finally,we mixed the s+c_f fraction and s+c_m fraction,and the resulting mixture was considered as the MOM fraction. All fractions were washed into preweighed aluminum containers and dried at 50°C,then weighed and stored for further analysis.

The OC and TN contents/concentrations in the subsamples of both bulk soil and extracted macroaggregate fractions (cPOM,fPOM,iPOM,and MOM fractions) were analyzed using a Vario MACRO cube elemental analyzer(Elementar Analysensysteme GmbH,Hanau,Germany).

2.5.Data calculation and statistical analysis

The SOC in the top 20 cm of the soil was converted to SOC stock (SOCstock,Mg ha–1) according to eq.(1):

where SOC is the content of SOC (g kg–1),BD is the soil bulk density (g cm–3),0.2 is the soil depth (m),and 10 is a unit conversion factor.

The amount of SOC gain (ΔSOCstock,Mg ha–1) due to fertilization was calculated according to eq.(2):

where SOCstock-treatmentand SOCstock-CKrepresent the SOC stocks in the treatments with manure or/and NPK and the control treatment,respectively.

The SOC sequestration rate (SOCSR,kg ha–1yr–1) was calculated according to eq.(3):

wherenis the duration of the reclaimed experiment (year).

The OC contents in the different sub-fractions within the macroaggregates were calculated according to eq.(4):

where OCcontent-subis the organic carbon content in the different sub-fractions within the macroaggregates (g C kg–1aggregate),OCcon-subis the organic carbon concentration of the sub-fraction,and Masssubis the mass proportion of the sub-fractions within the macroaggregates (% aggregate).

TN contents in the different sub-fractions within the macroaggregates were calculated in a similar way as in eq.(4).

Significant differences between the means were identified by performing one-way analysis of variance,and the least significant difference (LSD) was computed to compare the means of the above variables (P＜0.05) using SPSS Software (ver.18). Correlation analysis was performed using the corrplot package and lm function of R (ver.3.3.3)to test the correlations of SOC,TN,OC and TN contents among the fractions and between the differences in the contents of the unprotected/protected C and the SOC in the bulk soil. Pearson linear correlations between differences in OC contents within macroaggregate fractions and SOC were determined. Graphs were prepared using Origin Software (ver.8.1).

3.Results

3.1.Grain yield and biomass yield

The long-term fertilizer application significantly increased the annual mean maize grain yield compared with the control (Fig.2). The annual mean grain yield ranged from 3 746 to 9 124 kg ha–1,with the highest yield occurring under the MNPK treatment and the lowest occurring under the control. Compared with the control,chemical fertilizers(NPK) significantly increased the grain yield by 106.5%.Application of manure alone (M) and application of manure combined with chemical fertilizers (MNPK) significantly increased grain yields by 135.2 and 143.6%,respectively,over the control yield. In addition,the mean yield of the MNPK treatment was notably higher than that of the NPK treatment. However,no significant difference in maize yield was found between M and MNPK. The effect of different fertilizer treatments on biomass yield was generally similar to that of grain yield (Fig.2).

3.2.SOC stocks and sequestration rate

Compared with the control,the soil bulk density was significantly decreased by 6.0% under the MNPK treatment(Table 2),while the TN and SOC contents were significantly increased by 11.2–45.6% and 23.8–43.7% under all fertilizer treatments,respectively,leading to significant increases in SOC stocks. SOC stocks and sequestration rates showed the following decreasing trend:MNPK＞M＞NPK (all differences significant atP＜0.05) (Table 2).

3.3.Mass distribution of extracted fractions within macroaggregates

The mass distribution of macroaggregate-extracted fractions under different fertilization regimes is shown in Table 3.Generally,the MOM fraction was the dominant fraction,which ranged from 58.8 to 79.3%,followed by the iPOM fraction (9.1–16.6%) and the cPOM fraction (8.0–18.4%),while the fPOM fraction was the smallest,representing 3.6–6.2% for all treatments.

The NPK treatment did not affect the proportion of extracted macroaggregate fractions. Compared with the control,the proportions of cPOM,fPOM,and iPOM fractions were notably increased by 69.9–130.1%,46.3–72.2%,and 61.9–82.4% after long-term application of manure (M and MNPK),respectively (except for the proportion of iPOM fraction under the M alone treatment).However,the proportion of the MOM fraction was significantly decreased by 16.2–25.8% under the M andMNPK treatments (Table 3).

Fig.2 Mean annual summer maize grain yield (left-hand panel) and biomass yield (right-hand panel) during the 11-year application of mineral fertilizers (NPK),manure (M),mineral fertilizers plus manure (MNPK) and under the control treatment receiving no fertilizers (CK) (2009–2018). The solid and dotted lines,the lower and upper limits,and the lower and upper bars inside or outside the boxes represent median and mean values,25th and 75th,and 5th and 95th percentile yield values,respectively. The circle indicates the ＜5th and ＞95th percentiles of all data,respectively. Different lowercase letters indicate significant differences among different treatments (P＜0.05).

Table 2 Soil bulk density,TN content,SOC content,SOC stock and SOC sequestration rate in the 0–20 cm soil layer in 20181)

Table 3 Effects of long-term fertilization on the weight distribution of soil macroaggregate-extracted fractions (w/w,%)1)

3.4.Organic carbon contents of macroaggregateextracted fractions

For each treatment,the OC contents in the macroaggregateextracted fractions generally showed the following declining trend:cPOM or fPOM＞MOM＞iPOM (Fig.3). Compared with the control,the NPK treatment significantly decreased the OC content in the MOM fraction by 12.1%,but it had no significant effect on the OC contents in the other three macroaggregate-extracted fractions. Manure application alone (M) significantly increased the OC contents within fPOM and iPOM fractions by 193.0 and 73.0%,respectively.MNPK significantly increased the OC contents within the cPOM,fPOM and iPOM fractions by 292.2,136.0 and 124.0%,respectively. However,the OC contents in the MOM fraction were significantly decreased by 16.3–22.6%under the M and MNPK treatments (Fig.3).

3.5.Total nitrogen contents of macroaggregateextracted fractions

Generally,the TN contents in the macroaggregate-extracted fractions decreased in the order MOM＞cPOM＞fPOM≈iPOM for a given treatment (Fig.4). Fertilization did not affect the TN contents of the MOM fraction,and the NPK treatment did not affect the TN contents of any of the macroaggregateextracted fractions. The M alone treatment significantly increased the TN content within the fPOM fraction above the control,and the MNPK treatment significantly increased the TN contents in the cPOM,fPOM,and iPOM fractions by 607.1,242.5,and 127.6%,respectively (Fig.4). The average C:N ratios significantly decreased as follows:cPOM (25.1)＞fPOM (22.7)＞iPOM (10.2)＞MOM (2.4)(Figs.3 and 4).

Fig.3 Effects of long-term fertilization on organic carbon contents of extracted fractions within macroaggregates.cPOM,unprotected coarse particulate organic matter;fPOM,unprotected fine particulate organic matter;iPOM,physically protected intra-microaggregate particulate organic matter;MOM,biochemically protected mineral associated organic matter. CK,control;NPK,mineral fertilizers;M,compost chicken manure alone;MNPK,mineral fertilizers plus manure.Bars represent SD (n=3). Different lowercase letters indicate significant differences for a given fraction among different treatments (P＜0.05).

3.6.Relationship between different OC and TN fractions and SOC

Correlation matrix analysis suggested that the cPOM-C,iPOM-C,cPOM-N,fPOM-N,and iPOM-N contents in the macroaggregates were positively correlated with the SOC concentration (Fig.5-A). Significant positive linear correlations were also observed between the difference in the contents of the unprotected C and SOC in the bulk soil between the control and manure and/or NPK treatments,but there was no relationship with protected C (Fig.5-B).Moreover,the content difference in cPOM-C had a closer relationship with the difference in SOC concentration than the difference in fPOM-C (Fig.5-C). We also found several negative correlations mainly with the fractions from the macroaggregates (such as MOM-C) (Fig.5-D). The steepest slope of the linear equation for the cPOM-C content was 2.06,followed by the fPOM-C and iPOM-C contents,while the smallest value for the MOM-C content was–0.09.These results suggest that the greatest accumulation of organic carbon occurred in the cPOM fraction.

Fig.4 Effects of long-term fertilization on total nitrogen contents of extracted fractions within macroaggregates.cPOM,unprotected coarse particulate organic matter;fPOM,unprotected fine particulate organic matter;iPOM,physically protected intra-microaggregate particulate organic matter and MOM,biochemically protected mineral associated organic matter. CK,control;NPK,mineral fertilizers;M,compost chicken manure alone;MNPK,mineral fertilizers plus manure.Bars represent SD (n=3). Different lowercase letters indicate significant differences for a given fraction among different treatments (P＜0.05).

4.Discussion

4.1.Effects of fertilization on crop productivity and SOC sequestration

Fertilization has been an essential practice to maintain soil fertility and enhance crop productivity. In our study,the application of NPK and organic manure significantly increased maize yield compared with the control,especially the combined MNPK treatment with the highest yield (Fig.2).A similar result was reported by Ghoshet al.(2019),who found that long-term application of MNPK led to higher maize yield than the control and mineral fertilizer treatments in a hyperthermic Typic Udorthen soil. As the nutrient content and structure of the soil were extremely poor in the mining area,the favorable effect of MNPK on maize yield might be due to more absorption and utilization of macro-and micro-nutrients by maize,as well as improved soil physical structure (Huanget al.2019). Although the mineral NPK fertilizers also significantly increased maize yield compared to the control,there was probably no beneficial effect of these fertilizers on the soil physical structure (Huanget al.2019),thus NPK fertilization is not recommended over a long-term period.

It is important to stabilize SOC in the topsoil layer of reclaimed mining soils because SOC is a key factor affecting many other soil properties (Yuanet al.2017). In our study,the continuous application of NPK,M,and MNPK for 11 years significantly increased the SOC stock in the 0–20 cm soil layer by 10.4,18.1,and 36.9%,respectively,compared to the control. The SOC sequestration rate under the MNPK treatment (561.6 kg ha–1yr–1) was three times higher than the NPK treatment (158.3 kg ha–1yr–1) and two times higher than the M treatment (275.7 kg ha–1yr–1) (Table 2). The input of manure resulted in greater C storage than the addition of mineral fertilizers as found by Bharaliet al.(2017). Xieet al.(2017) estimated a SOC sequestration rate of 353–1 087 kg ha–1yr–1in soils subjected to NPK fertilizers,either alone or in combination with organic manure over a 21-year period under irrigated winter wheat–summer maize systems in an Anthrosol. In India,the application of NPK or NPK combined with organic manure resulted in SOC sequestration rates of 253 or 827 kg ha–1yr–1during 30 years under a rain-fed soybean–wheat rotation (Kunduet al.2007). However,the SOC sequestration rates in our study were lower than those in Xieet al.(2017) and Kunduet al.(2007),which may be largely explained by the relatively lower C input in the reclaimed mining soils (unpublished data). A higher OC input coupled with relatively favorable soil moisture conditions in irrigated plots may have stimulated soil microbial activity(Wanget al.2020),potentially increasing the annual SOC sequestration rate (Xieet al.2017). The rain-fed condition and the greater amount of crop residues returned to the soil may have contributed to the higher SOC sequestration rate in the study of Kunduet al.(2007) than in the present study.

4.2.Organic carbon and total nitrogen in macroaggregate-extracted fractions

Fig.5 Correlation matrix of content among different organic carbon (OC) and total nitrogen (TN) fractions (A),the correlations of the differences in the soil OC (SOC) content and unprotected/protected C contents (B),SOC content and cPOM-C and fPOM-C contents (C),and SOC content and iPOM-C and MOM-C contents (D) between different fertilization treatments and the control.SOC-LM and TN-LM,SOC and TN content in the ＞2 mm size macroaggregate,respectively;SOC-SM and TN-SM,SOC and TN content in the 0.25–2 mm size macroaggregate,respectively;SOC-m and TN-m,SOC and TN content in the 0.053–0.25 mm size microaggregate,respectively;SOC-s+c and TN-s+c,SOC and TN content in the silt and clay fraction (＜0.053 mm),respectively.cPOM,unprotected coarse particulate organic matter;fPOM,unprotected fine particulate organic matter;iPOM,physically protected intra-microaggregate particulate organic matter and MOM,biochemically protected mineral associated organic matter. For the correlation matrix,only the correlation coefficients with P＜0.05 are shown,and the size and color of the dots indicate the intensity of the correlation between different fractions. For the three inserts,＊ indicates P＜0.05.

The cPOM and fPOM fractions together constitute the conceptual ‘unprotected’ SOC pool,which consists of fresh plant residues and roots and is thought to be more sensitive to agricultural management practices than other fractions(Sixet al.2002). In the present study,the application of manure combined with synthetic fertilizers (MNPK)significantly increased the OC and TN contents in the cPOM fraction compared with the control. The OC and TN contents within the fPOM fraction were markedly higher in M and MNPK treatments. However,the treatment receiving synthetic fertilizers alone (NPK) showed no effects on the above two fractions (Figs.3 and 4). Our results regarding the OC and TN contents in the cPOM fraction were in agreement with those of Tianet al.(2017) and Heet al.(2015),who also found that there was a clearer increasing trend in OC and TN concentrations within the cPOM fraction where MNPK was applied than in their control treatment at the bulk soil scale. The increase found in the present study is attributed to direct inputs of C and indirect crop effects after the application of MNPK. The better crop growth may have increased OC or TN contents in the cPOM fraction under the MNPK treatment. Indeed,the average aboveground biomass from 2009 to 2018 was the highest under the MNPK treatment (20 736 kg ha–1) (Fig.2). Thus,the cPOM fraction was mostly enhanced by the regular supply of easily metabolized OC and TN under the MNPK treatment.

Similar to the results for the OC and TN contents of the fPOM fraction in our study,Jianget al.(2017) and Heet al.(2015) observed that continuous addition of organic manure integrated with synthetic NPK significantly increased the fPOM-C content within macroaggregates in gray desert soil and the fPOM-N concentration in Luvic Phaeozems,Calcaric Cambisols,and Ferralic Cambisols at the bulk soil scale. Sixet al.(2002) proposed that the cPOM and fPOM fractions,which are not occluded within microaggregates,are mainly derived from plant residues,fungal hyphae and spores,and in some cases,charcoal. As such,the current status of the OC and TN in the unprotected fractions here may be due to the decomposition of maize residues or increased activity of fungal hyphae and spores (Liet al.2016).

Among the different treatments,the unprotected C in the macroaggregates reveals their varied efficacies in stabilizing SOC and improving soil fertility over the long term (Yaoet al.2019). Our results indicated that the increase in unprotected C was strongly correlated with the increase in SOC (P＜0.05)(Fig.5-B),which implies that unprotected C was more crucial to SOC sequestration than protected C in the reclaimed calcareous soil of northern China. Although cPOM and fPOM were composed of fresh plant residue inputs (Sixet al.2002),our study indicated that they were not equal fractions because the MNPK treatment induced a more significant increase in the OC and TN contents within cPOM than that within fPOM (Figs.3 and 4) and the correlation analysis suggested that the difference in cPOM-C content between control and manure and/or NPK was positively and linearly correlated with the difference in SOC concentration(P＜0.05) (Fig.5-C). Moreover,the increase in the OC or TN contents of the cPOM fraction was the largest among the four fractions (Figs.3 and 4). Consequently,the increase in the cPOM-C and cPOM-N contents indicated that long-term application of MNPK increased SOC sequestration. The cPOM fraction might be the main form of SOC sequestration in the tested soil. Similarly,Xuet al.(2016) observed that SOC stabilization occurred primarily in the form of cPOM(＞0.25 mm),and it was also more significantly and linearly correlated with SOC than other fractions after long-term fertilization in a brown earth soil. We observed that there was no relationship between the difference in fPOM-C content and SOC concentration between the control and manure and/or NPK treatments (Fig.5-C). Sixet al.(2002)also suggested that the OC in the light fraction (referred to as fPOM in our study) did not increase with increasing C inputs. One possible explanation could be that the saturation behavior of the unprotected fraction depends on the balance between the C input and the specific decomposition rate of the fraction,while the different saturation behaviors of the unprotected fraction might rely on various factors,such as soil temperature,moisture and substrate biodegradability(Stewartet al.2008).

Examination of the iPOM is another applicable diagnostic tool to determine SOC storage in response to positive changes in agricultural management practices (Six and Paustian 2014). In our study,NPK application had no significant effects on the OC and TN contents occluded in the iPOM fraction,but these were 124.0 and 127.6% higher under the MNPK treatment than the control,respectively(Figs.3 and 4). This was consistent with the results obtained in previous studies (Heet al.2015;Tianet al.2017),where the OC and TN contents in the iPOM fraction were more strongly affected by the addition of manure combined with synthetic fertilizers (MNPK) than the application of synthetic fertilizers alone (NPK). As SOM is a major binding agent of soil aggregates (Sixet al.2004),long-term manure input may provide binding materials for improving microaggregation (Heet al.2015),which may decrease the organic matter turnover rate and increase the iPOM fraction associated with the OC and TN contents. Mineral fertilizer application increased root biomass and intensified mineralization of SOC that could not form sufficient complex organic compounds associated with minerals (Heet al.2015),which accounts for the lack of a response in the OC and TN of the iPOM fraction under mineral fertilization in the present study. Previous studies indicate that stabilization of the within-macroaggregate POM fraction (iPOM in this study) was one of the major protective mechanisms for SOC(Huanget al.2010;Six and Paustian 2014). The relationship of the differences in the iPOM-C and SOC between the control and treatments receiving manure or/and NPK fitted the linear model well (Fig.5-D),but our results revealed that the linear equation slope of iPOM-C (0.24) was less steep than for cPOM-C (2.06) (Figs.5-C and D). Therefore,the iPOM fraction may be the second most important fraction in SOC sequestration here.

The MOM fraction is acquired through condensation and complexation reactions or simply as a result of the inherent complex biochemical nature of the materials (Sixet al.2002).The present study showed that the MOM fraction constituted the largest proportion among the macroaggregate-extracted fractions under different fertilization treatments,but the OC contents in the MOM faction were significantly decreased after addition of NPK,M or MNPK (Fig.3),and the TN contents in the MOM fraction were not affected by various long-term fertilization regimes (Fig.4). A similar observation for the MOM-C content was reported by Wang Xet al.(2017),who also found that adding manure resulted in a marked decline in the OC content of the MOM fraction.However,other studies reported that long-term application of NPK showed no effects on MOM-C in red soil and noncalcareous fluvo-aquic soil,while application of MNPK significantly increased the OC concentration of the MOM fractions (Huanget al.2010;Tianet al.2017). This increase may be attributed to the increase in the abundance of soil fungi following the application of organic manure,promoting the conversion of the silt and clay fractions to aggregate fractions. Additionally,Wang Xet al.(2017) found that the content of minerals associated with the OC (MOM-C)increased by microbial metabolism and secretion was less than the content of MOM-C transferred to iPOM-C.Consequently,the biochemically protected OC content was reduced. Our results for MOM-C indicated improvement in SOC quality (Haynes 2000;Malhiet al.2005). The changed SOC quality was closely related to aggregate distribution,especially the macroaggregate content,as found by Heet al.(2018),which might imply that macroaggregates contained more labile C (cPOC) than other forms in our investigated soil (Fig.3). Our results further support the carbon saturation concept (Sixet al.2002) and hierarchical carbon saturation(Guldeet al.2008),indicating that the mineral fraction of macroaggregates became saturated,and consequently,additional C inputs accumulated in labile (unprotected) SOC pools (Guldeet al.2008).

In addition,the C:N ratio is an indicator of nutrient cycling and storage capacity (Simon 2008). In general,the soil C:N ratio decreases with intensified degradation and humification of organic matter. During the decomposition process,carbon is released through respiration,and part of the mineral N is lost through leaching or gas emissions,while part of the mineral N is recombined into the SOM pool (Chapinet al.2002). Our study revealed that the average C:N ratios significantly decreased as follows:cPOM (25.1)＞fPOM(22.7)＞iPOM (10.2)＞MOM (Figs. 3 and 4). The highest C:N ratios in the cPOM and fPOM fractions were close to the C:N ratios for plants (20:1 to 30:1),suggesting that they mainly consisted of plant residues (Diekowet al.2005). The iPOM fraction with intermediate C:N ratio was the core product of organic matter mineralization. The low C:N ratios of the MOM fraction might be due to the high degree of humification and stability (Tonget al.2014). The decrease in the C:N ratios in the four fractions indicated that the MOM fraction had more N than C stored in this fraction. The changes in C:N ratios among the four fractions also clearly illustrate the process of SOC accumulation and sequestration in soil,which is similar to the results of Tonget al.(2014) and Heet al.(2015).

5.Conclusion

In this study,continuous 11-year application of mineral fertilizers (NPK) had relatively few effects on the SOC and TN accumulation in bulk soil and each macroaggregateextracted fraction in reclaimed mine land soil. However,incorporation of NPK with organic manure (MNPK)significantly increased SOC and TN stocks and the OC and TN contents within the macroaggregate-extracted fractions of cPOM,fPOM,and iPOM,with most sequestration observed in the cPOM fraction. Positive linear correlations between the unprotected or protected C fractions and increased SOC,confirmed that the cPOM fraction was the main fraction involved in the sequestration of SOC in reclaimed calcareous soil under long-term fertilization.Therefore,manure combined with mineral fertilizer (MNPK)application was the best fertilization management strategy to drive sustainable crop production.

Acknowledgements

This research was supported financially by the National Natural Science Foundation of China (41807102 and U1710255-3),the Shanxi Province Key Laboratory Open Fund of Soil Environment and Nutrient Resources,China(2019003),the Science and Technology Innovation Fund of Shanxi Agricultural University,China (2019004) and the Incentive Funding Research Project for Excellent Doctors Settle Down to Work in Shanxi Province,China(SXYBKY201805).

Declaration of competing interest

The authors declare that they have no conflict of interest.