Organic carbon sequestration in soil humic substances as affected by

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Agricultural and Environmental Chemistry

Organic carbon sequestration in soil humic substances as affected by application of different nitrogen fertilizers in a vegetable-rotation cropping system Meng Li, Hualing Hu, Xiaosong He, Jinhu Jia, Marios Drosos, Guoxi Wang, Fulai Liu, Zhengyi Hu, and Beidou Xi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07114 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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Journal of Agricultural and Food Chemistry

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Organic carbon sequestration in soil humic substances as affected by

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application of different nitrogen fertilizers in a vegetable-rotation

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cropping system

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Meng Lia,b,d, Hualing Huc, Xiaosong Hea, Jinhu Jiae, Marios Drososf, Guoxi Wangb,

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Fulai Liud, Zhengyi Hub, Beidou Xia,*

6 a

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State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China

b

College of Resources and Environment, Sino-Danish College, University of Chinese

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Academy of Sciences, Beijing 100049, China c

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College of Environmental Sciences and Engineering, Tianjin University, Tianjin

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300035, China d

Department of Plant and Environmental Sciences, Crop Science Section, University

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of Copenhagen, Højbakkegaard Allé 13, DK-2630 Taastrup, Denmark e

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f

China Environmental Science Press, Beijing 100062, China

Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per

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l'Ambiente, l'Agroalimentare ed i Nuovi Materiali (CERMANU), Università di Napoli

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“Federico II”, via Università 100, 80055 Portici, Italy

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Corresponding author:

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ACS Paragon Plus Environment


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Prof. Dr. Beidou Xi, at State Key Laboratory of Environmental Criteria and Risk

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Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012,

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China

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E-mail addresses:

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[email protected] (M. Li), [email protected] (B. Xi)

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2


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Abstract

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Little is known on the effect of application of different nitrogen (N) fertilizers on soil

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organic carbon (SOC) sequestration in soil humic substances (HS). We investigated HS

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molecular characteristics in an Orthic Acrisol, southwestern China, under 2-year field

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fertilization of a urea (U), a polymer-coated urea (PCU) and a biochar-coated urea

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(BCU) using

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promoted SOC sequestration into HS and favoured alky-C and aromatic-C rather than

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O-alkyl-C and carbonyl-C for humic acids and humin in soil. Application of PCU and

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BCU may better enhance vegetable yield, SOC sequestration and HS stability than the

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U application, which may benefit from longer time of N existence and higher total N in

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soil. Among the N treatments, BCU application mostly affected the compositions and

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stability of SOC in the HS for the OC input and prime effect of biochar for SOC

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transformation.

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Keywords: Slow-release nitrogen fertilizers (SRNF), Biochar, Nitrogen addition, Soil

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organic matter, 13C-CPMAS-NMR spectroscopy.

13C-CPMAS-NMR

spectroscopy. Results showed that N fertilization

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3



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1. Introduction

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Organic carbon (OC) sequestration in arable soil is achieved by mainly receiving

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atmospheric CO2, C deposition, C fertilizers and plant residual C.1-2 It is significant to

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maintain soil fertility and potentially contribute to climate change mitigation.3-4 As a

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major of soil organic matter (SOM), humic substances (HS) can be operationally

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classified into three fractions: humic acids (HA), fulvic acids (FA) and humin (HU).

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Their chemical natures and molecular structures vary largely and affect the functioning

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of soil OC (SOC).5 Recent studies showed that the OC sequestration in soil HS was not

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always in an inert and stable form.1,6 Labile OC in the HS subfractions, such as alkyl-

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C, carbohydrate-C and so on, were easily mineralized into inorganic C and lost from

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soil.7 Meanwhile, some hydrophobic components in HS or soil may prohibit the OC

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sequestration in paddy soils of South China.8 It has been thus proposed that

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identification of OC sequestration in HS is essential to understand the process of SOC

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stabilization and global carbon cycle.9-10

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As an important agricultural practice to improve soil fertility, nitrogen (N)

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fertilization has been suggested to affect the chemical structures of SOM11 and then

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alter soil OC dynamics.12 Preston and Newman13 observed changes of O-alkyl-C and

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di-O-alkyl-C in HU with long-term of N fertilization in a coastal soil of British

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Columbia. Sjöberg et al.14 also found an accumulation of aromatic-C and phenolic C in

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HS when N fertilization was applied. Increasing C input of crop residues15 and slowing

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the SOC mineralization through stabilizing SOM against microbial decomposition16 in

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soil may be the potential reasons for the OC sequestration under N addition. However, 4


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the differences of OC sequestration in HS under different N fertilizers is still not clear.

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N in traditional N fertilizers, such as urea (one of the largest N fertilizers applied),

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tended to be lost with ammonia volatilization and transportation by runoff and leaching

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and only functioned with a low nutrient use efficiency in a limited time.17 Slow-release

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N fertilizer (SRNF) was therefore developed by coating urea with polymer or biomass

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materials to improve N supply management.18-19 Compared to traditional N fertilizer,

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slow-release N may participate in soil C and N cycles in a much longer time with higher

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N use efficiency, much lower N losses and stronger unbalanced C/N ratio.20 Even, a

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newly-developed biochar coated N fertilizer was applied in China to boost soil fertility

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and regulate C pools.21-23 In fact, there are increasing evidences that biochar amendment

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may lead either to a long-term C sequestration due to the sorption of SOM liable

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fraction promoting C balance in soil24 or to a rapid mineralization due to the utilization

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of liable fraction of SOM and biochar.25 We thus conceived that OC sequestration in

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HS may be greatly affected by application of different N fertilizers. Moreover, the usage

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of SRNF has increased remarkably in the last decade. It is necessary to investigate the

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effect of the application of SRNF on the SOC sequestration for a sustainable

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agricultural production.

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The objectives of this study were thus to apply different N fertilizers in the soil

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under a vegetable-rotation cropping system during two-year field experiment and to

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mainly investigate the OC molecular structure of HS in N fertilized soils using carbon-

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13 cross polarization magic angle spinning nuclear magnetic resonance (13C-CPMAS-

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NMR) spectroscopy. 5



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2. Materials and methods

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2.1. Site description

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The field experiment was performed in a cropland in Jinning town (24o36′N, 102o41′E)

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of Yunnan province, southwestern China. The farming in the cropland was under

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vegetable rotation system with triple cropping per year. The climate belongs to a low-

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latitude plateau subtropical monsoon with an average annual temperature of 14.6 oC

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and an average annual rainfall of 925.4 mm in conventional years. Initially, the soil in

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the cropland was classified into red soil (Orthic Acrisol, according to FAO soil catalog)

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and had 6.34 of pH, 21.1 mg g-1 of SOM, 0.53 mg g-1 of total N (TN), 1.78 mg g-1 of

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total phosphorus (TP), 0.54 mg g-1 of Olsen-P, and a soil texture of silty clay loam (clay

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62.3%, silt 27.4%, sand 10.3%).

99

2.2. N fertilizers

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Three N fertilizers were applied in the field experiment, including an ordinary urea (U,

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containing 45.8% N), a polymer coated urea (PCU, containing 43.1% N) and a biochar

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coated urea (BCU, containing 30.2% N). Among them, the U and PCU were purchased

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from Agrium Advanced Technologies Inc., Calgary, Canada, and local market,

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respectively. The BCU was prepared by mixing 65% urea, 20% biochar and oxidized

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corn starch with a granulation process to reach final C content of 33.2%. The biochar

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used in the BCU was derived from coconut shell by pyrolysis in a muffle furnace system

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at 600 oC under N2 atmosphere for 2 h. The obtained biochar was ground to pass through

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0.25-mm sieve for the preparation of the BCU and the properties of the biochar were: 6


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pH 6.89, total C 634.9 mg g-1, total hydrogen 21.2 mg g-1, total oxygen 143.4 mg g-1,

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TN 11.3 mg g-1, Olsen-P 13.8 mg kg-1, ash content 28.8%, and surface area 22.4 m2 g-

111

1.

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the U released nearly 100% N in three days, while the PCU and BCU released

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approximately 80% N in 40 days.

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2.3. Field experiment and sampling

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A two-year field experiment (from September 2014 to August 2016) was conducted in

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the cropland which was subdivided into 12 experimental plots (3.0 m × 6.5 m). The

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field experiment consisted of the following four treatments: 1) CK, no N fertilization;

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2) U, application of urea; 3) PCU, application of PCU; 4) BCU, application of BCU.

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All treatments were performed in triplicate.

In addition, the PCU and BCU were SRNF. When soaked in deionized water at 23oC,

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Except for the CK treatment, a rate of 300 kg N ha-1 year -1 was applied in the

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experimental plots. For the BCU treatment, approximately 200 kg ha-1 year -1 of biochar

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was added. A basal nutrients addition provided P as triple-superphosphate at the rate of

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250 kg P2O5 ha-1 year -1 and potassium (K) at the rate of 230 kg K2O ha-1 year-1. Snow

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pea (Pisum sativum L.), zucchini (Cucurbita pepo L.) and maize (Zea mays L.) were

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cultivated in rotation during the whole field experiment. The experimental plots were

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conducted with the same nutrient (except N fertilization) and agricultural management.

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After two-year of vegetable cultivation, all the vegetable plants (including root) were

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also collected, cleaned by de-ionized water, oven-dried at 60oC to constant weight, and

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then weighted to estimate vegetable yields. Three surface soils (0‒20 cm) from one plot

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were sampled and mixed into one soil sample. A total of 12 soil samples from the 12 7



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experimental plots were finally collected. The air-dried soil samples were ground to

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pass through 2-mm and 0.15-mm sieves prior to analysis. The oven-dried plant samples

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were smashed into < 1 mm with a grinder and then part of the smashed plant sample (5

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g) was ground to pass through 0.15-mm sieve by ball milling.

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2.4. Analysis of soil properties

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Soil pH was determined using a pH meter (S20K, Mettler Toledo, Switzerland) at a

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soil/water ratio of 1:2.5. Cation-exchange capacity (CEC) was measured by the method

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described by Rhoades.26 The contents of total C (TC) and N (TN) in the soil, plant and

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HS samples were measured on an element analyzer (Elementar, Germany). SOC

140

content was determined using a dichromate-oxidation method described by Nelson and

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Sommer.27

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2.5. HS extraction

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The isolation and purification of HA, FA and HU from soil samples were performed

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using procedures similar to those described elsewhere.28 Briefly, about 40 mL of

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deionized water was added into 20 g of air-dry soil, shaken at 25oC for 30 min, and

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centrifuged at 10,000 g for 15 min to remove poorly decomposed light fraction.13 The

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remaining was extracted with 0.5 M NaOH and 0.1 M Na4P2O7 at 25oC under N2 for 18

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h with a soil/solution ratio of 1:5 and centrifuged at 10,000 g for 30 min. The clear

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supernatant was transferred into a 250-mL centrifuge bottle and acidified with

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concentrated HCl to reach pH = 1.0±0.1. After being centrifuged at 25oC and 10,000 g

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for 15 min, the residue was washed for three times with deionized water to pH = 7 and

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freeze dried to obtain HA. The clear supernatant containing FA was eluted through a 8


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XAD-8 resign column (0.30-0.45 mm of particle size, Amberlite, USA) at a flow

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velocity of 1 mL min-1 for three times to remove non-humic impurities.29 The XAD-8

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resin column was pre-cleaned with deionized water, 0.1 M NaOH, and HCl (pH=1) for

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three times.30 The adsorbed FA fraction was then back eluted with 0.1 M NaOH. The

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eluted solution was acidified to pH = 1.0±0.1 again and purified by passing through a

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H+ saturated cation-exchange resin (AG50W-X8, Bio-Rad, USA) for three times, freeze

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dried, and then stored at -20oC prior to NMR analysis. The residue obtained from

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alkaline extraction was washed with deionized water for several times to pH = 7 and

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then mixed with 2 M HF at a soil/solution ratio of 1:10 at 25oC for 24 h. After another

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centrifuge at 25oC and 10,000 g for 15 min, the residue was washed with deionized

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water for several times to pH = 7 and extracted with 0.5 M NaOH and 0.1 M Na4P2O7

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at 25oC under N2 for 18 h and centrifuged at 10, 000 g for 30 min in a 1:5 of soil/solution

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ratio. The extractant was purified to obtain HU by the procedure as descripted above

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for HA.

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2.6. FT-IR and 13C-CPMAS-NMR spectroscopy

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Pellet of the biochar (used in BCU) mixed with potassium bromide in a 1:100 ratio was

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prepared to obtain transmission Fourier transfer-infrared spectra which was measured

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on a Bruker Tensor II spectrometer (Germany). The analysis was carried out in the mid-

171

infrared region from 4000 cm-1 to 400 cm-1 with 32 scans and a resolution of 4 cm-1.

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The data processing was performed using the OPUS 6.5 software (Bruker, Germany).

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13C-CPMAS-NMR

spectra for the HS samples and biochar were obtained using a

500 MHz Bruker AVANCE III HD spectrometer (Bruker, Switzerland) equipped with 9



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a 4-mm MAS BB probe, operating at 13C resonating frequency of 100.64 MHz, 10,000

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scans, a recycle time of 1 s, a rotating speed of 5.0 kHz, a contact time of 1 ms, and an

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acquisition time of 20 ms. All the NMR spectra were baseline corrected and processed

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by MestReNova v9.0.1 software (Mestrelab Research S. L., Spain). The 13C-CPMAS-

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NMR spectra was generally divided into five groups: Alkyl-C (0‒45 ppm), O-alkyl-C

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(45‒105 ppm), aromatic-C (105‒160 ppm), and carbonyl-C (160‒200 ppm).7,31 The

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integrated area of the functional C signals was taken to evaluate the relative C content.31

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The ratios of hydrophobic-C (alkyl-C plus aromatic-C, HB) to hydrophilic-C (O-alkyl-

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C plus carbonyl-C, HI) (HB/HI), alkyl-C to O-alkyl-C (A/O), and alkyl-C to aromatic-

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C (A/A) were calculated to assess the degree of hydrophobicity, humification and

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aromaticity of HS.28,32

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2.7. Data statistics

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One-way analysis of variance (ANOVA) following honest significance difference was

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performed to detect the significant difference (P < 0.05) of soil characteristics and C

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groups of HS between N treatments by using SPSS v22 (IBM, USA).

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3. Results

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3.1. FT-IR and 13C-CPMAS-NMR spectra of biochar used in the BCU fertilizer

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As shown in Figure 1a, the FT-IR spectra of the biochar used in the BCU was mainly

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consisted of a broad band at 3000‒3700 cm-1 representing stretching of O-H and N-H,

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two sharp peaks at 2922 cm-1 and 2852 cm-1 (aliphatic C-H stretching, mainly CH3- and

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-CH2-), a shoulder peak at 1715 cm-1 (stretching C=O), a sharp and strong peak at 1631 10


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cm-1 (aromatic C=C), and another broad peak around 850‒1315 cm-1 (stretching C-O

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of polysaccharides, C-N stretching, and aromatic ring bending).33-34 The 13C-CPMAS-

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NMR spectra of the biochar showed that alkyl-C (9.5%) and aromatic-C (87.7%) were

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the main OC fractions (Figure 1b). Two small peaks at 24 ppm and 29 ppm were

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assigned to be -CH3 and -CH2-, respectively. A very strong signal at 129 ppm and a

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weak shoulder peak at 152 ppm occurred in the aromatic-C region were related to aryl-C

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and phenolic-C, respectively. Both the FT-IR and 13C-CPMAS-NMR spectroscopy

204

found strong signals of alkyl-C (-CH3 and -CH2-) and aromatic-C (Figure 1).

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3.2. Basic soil properties and plant uptake of N and C

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All soil samples collected from the field experiment were weakly acidic ranging from

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6.42 to 6.75 (Table 1). The CK treated soils (6.50±0.07) had lower pH than the U treated

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soils (6.66±0.10). The U, PCU and BCU treated soils had higher (P < 0.05) CEC than

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the CK treated soils with the highest CEC (29.6±2.4 cmol kg-1) found in the BCU

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treated soils. Higher total N and C were found in the PCU and BCU treated soils than

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in the CK and U treated soils (Table 1). C/N ratio in the soils ranged from 7.92 to 9.13.

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The BCU treated soils had the highest total C (25.46±2.27 mg g-1) and C/N ratio

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(8.96±0.16). During the two-year of vegetable cultivation, a total of 51.33‒98.57 t ha-1

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(dry weight) of vegetable were harvested, while the plant uptake of N was

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382.69‒882.94 kg N ha-1 (Table 2). Compared with the CK treatment, higher vegetable

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yield and higher plant uptake of N were found in the other treatments.

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3.3. Contents and elemental composition of HS

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Ranges of 6.42‒9.63 mg g-1 of HA, 4.66‒7.65 mg g-1 of FA and 0.59‒0.87 mg g-1 of

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HU were extracted from soil samples (Table 3). The content of HA in the CK treated

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soils (6.69±0.42 mg g-1) were the lowest among the treatments. Meanwhile, BCU

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(8.98±0.88 mg g-1) and PCU (8.23±0.53 mg g-1) treated soils had higher HA content

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than the U treated soils (7.74±0.51 mg g-1). Remarkably, the BCU treated soils had the

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greatest content of FA (6.93±0.87 mg g-1) and HU (0.79±0.09 mg g-1) than the other

224

treatments.

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The contents of C and N in the HS samples were detected to be 150.2‒356.1 mg

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g-1 and 19.8‒38.2 mg g-1, respectively (Table 4). HA and HU contained higher C and N

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than FA. HS of the BCU treated soils had the highest C (348.7±23.3 mg g-1 in HA,

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196.5±19.9 mg g-1 in FA and 293.3±22.7 mg g-1 in HU), while the lowest C was found

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in HS samples of the CK treated soils. Higher N content was detected in the HS of the

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PCU and BCU treated soils than in those of the U and CK treated soils (P < 0.05, Table

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4). The C/N ratios of extracted HS from soil samples ranged from 6.4 to 10.3. No

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significant differences of HS C/N between treatments were detected.

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3.4. 13C-CPMAS-NMR spectra

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The

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with different N fertilizers are shown in Figure 2. Two shoulder peaks at 24 and 38

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ppm, a sharp and strong peak at 29 ppm in the alkyl-C region of all the HS samples

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were assigned to be -CH3, -(CH2)n-, and -CH2-, respectively.31 The two peaks appeared

238

at 55 and 71 ppm in the region of O-alkyl-C attributed to the signals of -CH2OH and -

239

CHOH-. A weak shoulder peak at 103 ppm attributed to be -OCHO- and can be

13C-CPMAS-NMR

spectra of HA, FA, and HU extracted from the soils treated

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classified into the di-O-alkyl-C group.35 In the region of aromatic-C, the signals of aryl-

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C and phenolic-C occurred at 129 and 152 ppm. A single and strong signal at 170 ppm

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was related to carbonyl-C, including carboxylic acids, amides and esters.36

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Aromatic-C (33.3%‒38.2%) was the prevailing C functional group in the HA and

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HU, followed by O-alkyl-C (23.2%‒30.7%) and alkyl-C (19.5%‒25.8%), and the

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smallest C group was carbonyl-C (13.8%‒17.3%) (Table 4). By contrast, higher

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proportions of alkyl-C (25.6%‒32.3%) and carbonyl-C (18.4%‒22.3%), lower

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proportion of aromatic-C (20.4%‒24.5%) were detected in the FA than in the HA and

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HU. As a result, FA had higher HB (44.2%‒54.6%) and lower HI (45.7%‒56.8%) than

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HA and HU (P < 0.05). For all the HA and HU samples, the PCU and BCU treated soils

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had significant lower O-alkyl-C and higher alkyl-C and aromatic-C than U and CK

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treated soils (P < 0.05, Table 4). In comparison with the PCU treated soils, HA and HU

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in the BCU treated soils had even lower O-alkyl-C and higher alkyl-C and aromatic-C.

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Moreover, in most cases, there were no significant differences of observed C groups

254

between in the U and the CK treated soils. In the HA and HU samples, the BCU treated

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soils had highest ratios of A/A, A/O and HB/HI (Figure 3). FA (0.79‒1.26 for A/O,

256

1.04‒1.55 for A/A, and 0.77‒1.19 for HB/HI) possessed the greatest variability of those

257

ratios among HA, FA and HU samples. Compared to the CK treated soils, HB increased

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while HI decreased in HA and HU of the N fertilized soils and reached a maximum

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increase of HB by 6.2% and a maximum decrease of HI by 7.8% (Table 4).

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4. Discussions 13



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4.1. Soil properties affected by different N fertilizers

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After N fertilizers were applied into soil, a urea hydrolysis process may result in a NH4+

264

release to the soil and thereafter a slight increase of soil pH (Table 1). The CEC was

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significantly enhanced by application of N fertilizers. Especially, the highest CEC was

266

found in the BCU treated soils, which may be attributed to the fact that biochar in the

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BCU can increase soil CEC.23 N fertilization also improved soil TN, which was in line

268

with most fertilization experiments.13,37-38 SRNF amendment can better improve TN

269

when compared with the U treatment. No significant difference of TN between in the

270

PCU and the BCU treated soils were found. Higher TC detected in the N fertilized soils

271

showed that N fertilization promoted soil C (Table 1). Similar results were also reported

272

by Song et al.39 and Wang et al.21 This may because that N fertilization may promote C

273

input into soil through increasing plant biomass and its residues, including roots and

274

fallen leaves.15 Moreover, TC was mostly enhanced by the BCU application among the

275

N treatments, which may be attributed to the C input along with the addition of biochar.

276

When applying PCU, high content of TN and lack of exogenous C source induced the

277

lowest C/N ratio (8.04±0.15) among the different N treatments (Table 1). In contrast,

278

BCU amendment brought about highest C/N ratios (8.96±0.16) with a high N

279

accumulation and C supply for soils. Application of N fertilizers enhanced vegetable

280

field and promoted N uptake by plant significantly (P < 0.05). In particular, the

281

amendment of PCU and BCU mostly promoted the yield of vegetable and plant N

282

uptake (Table 2).

283

4.2. Effect of N fertilization on OC sequestration in HS 14


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N fertilized soils had higher contents of HA (7.38‒9.77 mg g-1) and FA (4.93‒7.81 mg

285

g-1) than the CK treated soils (6.25‒7.10 mg g-1 of HA and 4.50‒5.02 mg g-1 of FA,

286

Table 3). Combined with the enrichment of SOC observed in the U, PCU and BCU

287

treated soils (Table 1), this may indicate that N fertilization may benefit for OC

288

sequestration potential, which corresponded with the result reported by Guo et al.38 in

289

a field experiment in the Loess Plateau. Meanwhile, no significant increase of HU

290

content was observed in soils when applied N fertilizers except BCU. Moreover, TC

291

and TN in the N fertilized soils trended to raise and showed changes of elemental

292

composition in the soil HS (Table 4). The result may confirm that N addition promote

293

the sequestration of C and N into HS.40

294

As revealed by 13C-CPMAS-NMR spectroscopy, the effects of N fertilization on

295

four functional C groups in the HS varied. O-alkyl-C tended to decrease accompanied

296

by an increase trend of alkyl-C and aromatic-C with the amendment of N fertilizers

297

(Table 5), indicating that N fertilization favoured OC sequestration in alky-C and

298

aromatic-C rather than O-alkyl-C and carbonyl-C. This was in line with the results

299

conducted by Zhang et al.31 in a Typic Hapludoll of northwest China and Song et al.4

300

in Eutric Cambisols of North China. The increase of alkyl-C may because that alkyl-C

301

derived from plant polyesters degradation and from soil micro-organisms metabolism

302

and N fertilization promoted input of plant residue and microbial activity in soil.36

303

Conversely, alkyl-C and carbonyl-C were suppressed in FA by N fertilization (Table

304

5). The observation may suggest that the OC sequestration may be apt to varied

305

functional C groups for different HS fractions when N fertilization occurred. Moreover, 15



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306

the decrease of carbonyl-C may be attributed to carbonyl-C was mainly labile SOC and

307

N fertilization may decrease the easily-oxidizable soil organic C (SOC).37,41

308

In all the HS of N fertilized soils, the relative percent of aromatic-C tended to

309

increase (Table 5), showing N fertilization promoted OC sequestration in aromatic-C.

310

Combined with higher A/A ratio found in HA and HU (Figure 3), N fertilization also

311

enhanced aromaticity of HA and HU and therefore the stability of SOC.42 As a result

312

of N fertilization, the ratios of A/O and HB/HI in HA and HU were improved, showing

313

higher humification and hydrophobicity degrees. It has been proposed that the larger

314

hydrophobicity of soil HS may indicate a lower mineralization of labile SOC, and

315

consequently, higher SOC stability.28 Also, higher humification and higher A/O showed

316

increase of resistant decomposable OC. Those results may imply that N fertilization

317

promoted SOC stability of HA and HU and may be due to greater microbial activity

318

and suitable C/N ratio with addition of N.31

319

4.3. Effect of application of different N fertilizer on OC sequestration in HS

320

When applying different N fertilizers, SRNF (PCU and BCU) fertilization better

321

promoted the contents of HA and FA in soils than U fertilization (Table 3). This may

322

be due to higher TN and TC and longer effective time induced by greater use efficiency

323

of N in SRNF (Table 1). No significant increase of HU content in the U and PCU

324

fertilized soils was detected. By contrast, highest contents of HA, FA and HU were

325

found in the BCU treated soils, suggesting that BCU fertilization mostly benefited the

326

OC sequestration in HS, which was attributed to the addition of biochar during BCU

327

application. 16


Page 17 of 40


328

The chemical compositions of HS varied with amendment of different N fertilizers

329

(Table 5). When compared with the CK treatment, relative amounts of alkyl-C and

330

aromatic-C increased in HA and HU with the U treatment (Table 5). By contrast, the

331

application of SRNF (PCU and BCU) may further promote alkyl-C in HA and HU and

332

decreased alkyl-C in FA. In particular, application of PCU and BCU further decreased

333

the A/A, A/O, and HB/HI ratios in FA and tended to increase these ratios in HU and

334

HA when comparing with the U treatment (Figure 3). The result revealed that the

335

amendment of PCU and BCU may enhance the stability of HU and HA, and

336

consequently, promoted the formation of a more stable SOC pool.43 A probable reason

337

may be that the SRNF application allowed a much longer time of N existence than the

338

U application in soil and reduced N volatilization, which may contribute to greater

339

utilization of N by plant, soil enzyme activity, and soil microbes.44 In addition, SOC

340

fractions have been proposed to respond differently to various TN levels in soils.39 The

341

higher TN in the SRNF fertilized soils may greater increase the belowground biomass,

342

such as root, and therefore improved the SOC stocks in soil.38 It is noted that the BCU

343

treated soils had higher content of aromatic-C than the other treatments (Table 5), which

344

may be attributed to high input of aromatic-C during biochar addition (Figure1).

345

Besides, the BCU treatment mostly promoted increase of alkyl-C and decrease of O-

346

alkyl-C in HA and HU. As a result, highest ratios of A/A, A/O and HB/HI were found

347

in the BCU treated soils, indicating a higher hydrophobicity, humification and

348

aromaticity for SOM (Table 5). Like PCU, the application of BCU also realized a longer

349

time of N existence in soil and an extent OC (mainly aromatic-C) input and contributed 17



Page 18 of 40

350

to higher TN content and OC sequestration.19 A biochar-soil interaction and the prime

351

effect of biochar also played an important role in the SOC transformation.45 A greatest

352

decrease of carbonyl-C was observed in the BCU treated soils. This may be due to the

353

mineralization of labile OC in soil primed by biochar, which was also found in previous

354

studies.46‒47 These results could have important implications for the SOC management

355

with the objectives to develop a sustainable agricultural production and mitigate global

356

climate change.

357 358

Acknowledgments

359

This work was jointly supported by National Natural Science Fund Projects of China

360

(41701561)

361

(2016M601095).

and

China

Postdoctoral

Science

Foundation

funded

Project

362 363

Conflict of Interest

364

The authors declare no competing interest.

365 366

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25



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511

Tables

512

Table 1. Basic properties of soils treated with different nitrogen fertilizers CEC †

Treatment

Total N

Total C

pH s

C/N / cmol

CK ‡

6.50±0.07b

kg-1

22.0±1.9d

mg

g-1

2.32±0.21c 19.61±1.56d 8.45±0.13b 2.61±0.23b 21.85±2.15c

U

6.66±0.10a

24.2±2.2c

8.37±0.14b

PCU

6.58±0.08ab

27.0±2.0b

2.89±0.20a 23.00±1.87b 8.04±0.15c

BCU

6.56±0.09ab

29.6±2.4a

2.84±0.24a 25.46±2.27a 8.96±0.16a

513

† CEC,

cation exchange capacity, ‡ CK, no N fertilization, U, application of urea, PCU,

514

application of polymer coated urea, BCU, application of biochar coated urea. The value

515

was given by mean ± standard deviation. Different letters (a, b, c, and d) alongside the

516

given values represent significant differences between different N treatments at P

Organic carbon sequestration in soil humic substances as affected by

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