Article Cite This: J. Agric. Food Chem. 2019, 67, 8107−8118
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Demonstration of Chemical Distinction among Soil Humic Fractions Using Quantitative Solid-State 13C NMR Jisheng Xu,†,‡ Bingzi Zhao,*,† Zengqiang Li,† Wenying Chu,§ Jingdong Mao,§ Dan C. Olk,∥ Jiabao Zhang,† Xiuli Xin,† and Wenxue Wei⊥
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†
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China ‡ University of Chinese Academy of Science, Beijing 100049, P. R. China § Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, United States ∥ USDA-ARS, National Laboratory for Agriculture and the Environment, Ames, Iowa 50011, United States ⊥ Institute of Subtropical Agriculture, Chinese Academy of Sciences, No. 1071 Yuandaer Road, Changsha 410125, P. R. China S Supporting Information *
ABSTRACT: Humic substances (HS) are vital to soil fertility and carbon sequestration. Using multiple cross-polarization/ magic-angle spinning (multiCP/MAS) NMR combined with dipolar dephasing, we quantitatively characterized humic fractions, i.e., fulvic acid (FA), humic acid (HA), and humin (HM), isolated from two representative soils (upland and paddy soils) in China under six long-term (>20 years) fertilizer treatments. Results indicate that each humic fraction showed chemical distinction between the upland and paddy soils, especially with much greater aromaticity of upland HMs than of paddy HMs. Fertilizer treatment exerted greater influence on chemical natures of upland HS than of paddy HS, although the effect was less than that of soil type. Organic manure application especially decreased the percentages of aromatic C in the upland HAs and HMs compared with the control. We concluded that humic fractions responded in chemical nature to environmental conditions, i.e., soil type/cropping system/soil aeration and fertilizer treatments. KEYWORDS: multiCP/MAS NMR, humic substances, humin, oxidation degree, nonprotonated C, fertilization
1. INTRODUCTION Humic substances (HS) are the largest pool of soil organic matter (SOM), making up 60−80% of SOM.1 Traditionally, HS can be classified into fulvic acids (FAs), humic acids (HAs), and humin (HM) based on their solubilities in acid and base,2 although the exact boundaries between these fractions have not been clarified in structural terms.3 In addition to the differences of solubilities among humic fractions, FAs, HAs, and HMs also generally differ from each other in chemical natures.2,4 Humic acids usually contain more aromatics than do the FAs,5,6 although in some cases HAs were not mainly composed of aromatic structures.7,8 For example, the percentages of aromatic C were 37−44% in HAs but only 12−14% in FAs from an agricultural experimental station in Japan.6 Fulvic acid generally features a peak of COO/N−C O at ∼173 ppm in its 13C NMR spectrum whether it was extracted from agricultural soils6,9 or forest soils.5 Recently, by using the dipolar dephasing (DD) technique, Barros Soares et al.5 reported that the FAs in eucalypt plantation soils contained considerable nonprotonated aromatic C, which may be from highly modified lignin molecules or condensed aromatic structures. However, there is scarce information whether agricultural soils have these nonprotonated aromatics in their FAs. Humin has prominently displayed an aliphatic nature with abundant methylene or O-alkyl C,10,11 and these compounds are less abundant in FAs and HAs.11,12 In previous studies the chemical compositions of HS were also found to respond to field treatments. For the FAs, the © 2019 American Chemical Society
aromatic C contents tended to be higher and those of O-alkyl C were lower in the NPK treatment than in an unfertilized Fulvisol soil in a field subjected to double cropping (rice and barley).6 Jindo et al.7 reported that organic amendments to a semiarid soil enriched aromatic C in the HAs compared with those from the native soil after 360-day incubation. The aromatic content, however, decreased after 5-year13 or longterm (25 years)6 application of organic manures to agricultural soils. Humin, as the major fraction of HS,2 is the least understood fraction due to difficulties in its isolation and purification.4,10,14 Hence, there is scarce information on the impacts of long-term fertilization on the chemical compositions of HM.15 In one study, the alkyl-C/O-alkyl-C ratio of HM was found to be slightly decreased after long-term N fertilization in a forest soil.16 In a broader review of nearly 30 published studies, Plaza and Senesi17 concluded that changes in the chemical nature of humic fractions following organic amendments accurately depict partial incorporation of the organic amendment into native soil organic matter. Hence, the impact of an organic amendment on the chemical nature of HS will depend on the extent that the organic amendment differs chemically from native soil organic matter. Received: Revised: Accepted: Published: 8107
April 11, 2019 June 23, 2019 July 1, 2019 July 1, 2019 DOI: 10.1021/acs.jafc.9b02269 J. Agric. Food Chem. 2019, 67, 8107−8118
Article
Journal of Agricultural and Food Chemistry
Table 1. Annual Application Rates for the Fertilizer Treatments, Cropping Systems, and Climates for the Upland and Paddy Soils mineral fertilizer −1
N (kg ha ) upland soil OFa NPKOF NPK NP NK control paddy soil OF NPKOF NPK NP NK control
−1
−1
P (kg ha ) K (kg ha )
0, 0b 75, 75 150, 150 150, 150 150, 150 0, 0
10.4, 21.5, 32.7, 32.7, 0, 0 0, 0
0, 0c 54, 72 54, 72 54, 72 54, 72 0, 0
0, 0 20, 0 20, 0 20, 0 0, 0 0, 0
3.9 15 26.2 26.2
organic fertilizer (Mg ha−1)
cropping system
climate
70.7, 70.7 97.6, 97.6 124.5, 124.5 0, 0 124.5, 124.5 0, 0
2.758, 2.758 1.379, 1.379 0, 0 0, 0 0, 0 0, 0
winter wheat (grown from October to May)− annual average precipitation, 597 mm; summer maize (from June to September) annual average temperature, 13.9 °C
0, 0 30, 50 30, 50 0, 0 30, 50 0, 0
8.5, 8.5 8.5, 8.5 0, 0 0, 0 0, 0 0, 0
early rice (from April to July)−late rice (from annual average precipitation, 1448 mm; July to November) annual average temperature, 16.5 °C
a
OF, organic fertilizer; NPKOF, combined NPK mineral fertilizer with organic fertilizer; NPK, balanced N, P, and K mineral fertilizer; NP, N and P mineral fertilizer; NK, N and K mineral fertilizer; control, without fertilizer. bThe first value for each upland soil is the fertilizer rate for maize, and the second value is the rate for wheat. cThe first value for each paddy soil is the fertilizer rate for early rice, and the second value is the rate for late rice.
Table 2. Soil Chemical Properties under Long-Term Application of Mineral and Organic Fertilizers on Upland and Paddy Soils pH (H2O) upland soil OFa NPKOF NPK NP NK control paddy soil OF NPKOF NPK NP NK control
SOCb (g kg−1)
TN (g kg−1)
TP (g kg−1)
TK (g kg−1)
AN (mg kg−1)
AP (mg kg−1)
AK (mg kg−1)
8.40 8.40 8.44 8.28 8.51 8.73
b b b b b a
10.64 8.05 5.76 5.45 3.91 3.98
a b c c d d
1.13 0.88 0.66 0.63 0.48 0.44
a b c c d d
0.68 0.69 0.64 0.65 0.46 0.47
a a a a b b
19.10 18.86 18.94 18.89 19.68 18.76
b b b b a b
16.75 13.57 9.81 13.15 23.85 7.46
b bc cd bc a d
21.43 17.17 12.27 12.20 1.30 1.03
a b c c d d
244.90 201.43 210.65 48.62 385.86 69.69
b c c e a d
4.73 4.57 4.59 4.65 4.66 4.65
a a a a a a
22.47 27.20 21.40 21.80 18.10 17.50
b a bc bc cd d
2.37 2.82 2.37 2.39 2.18 1.93
b a b b bc c
0.49 0.74 0.60 0.63 0.43 0.43
c a b b c c
14.54 16.47 15.63 12.50 15.17 12.25
c a b d bc d
124.05 128.09 127.42 114.04 157.79 122.40
bc b b c a bc
12.97 29.65 21.23 28.57 6.19 10.68
c a b a d c
111.01 168.35 120.22 57.84 181.81 53.89
b a b c a c
a
OF, organic fertilizer; NPKOF, combined NPK mineral fertilizer with organic fertilizer; NPK, balanced N, P, and K mineral fertilizer; NP, N and P mineral fertilizer; NK, N and K mineral fertilizer; control, without fertilizer. bSOC, soil organic carbon; TN, total N; TP, total P; TK, total K; AN, available N; AP, available P; AK, available K. Values within the same column for both soils followed by the same letter do not differ at P < 0.05.
Although the 13C CP/MAS NMR technique has been frequently used on SOM or HS research,21−23 its limitations in quantitative studies have hindered better assessment of roles of humic structure in soil humification. The 13C multiCP/MAS NMR technique developed by Johnson and Schmidt-Rohr24 provides an efficient approach to obtain quantitative spectra with good signal-to-noise ratios. Dipolar dephasing is very useful to differentiate nonprotonated and protonated C and mobile groups such as rotating CH3 and long-chained (CH2)n.13 Nonprotonated C can serve as an index of humification, a process that leads to greater substitution, branching, and cross-linking.25 Other humification indices have also been used to compare humic fractions and to establish their responsiveness to location or field treatments.26 The objectives of this study were to quantitatively distinguish the chemical compositions of HS, including FAs, HAs, and HMs,
However, other studies detected only small differences in the chemical compositions of HS.18 For example, using 13C crosspolarization/magic-angle spinning (CP/MAS) NMR, Nogueirol et al.19 found that the HAs in tropical soils were structurally similar, irrespective of tillage systems or amendments (limestone, gypsum, compost, and sludge). Different humic fractions, such as HM and HA extracted from the surface horizon of the Bainsville soil, were also reported to have similar chemical compositions, as determined by CP/MAS NMR.20 Hayes et al.10 reviewed the concept that HM was similar in structural features and chemical composition to soluble humic fractions, differing only in molecular weight and contents of functional groups. Therefore, additional studies are needed to clarify the differences among humic fractions in their chemical compositions and as affected by variable land uses. 8108
DOI: 10.1021/acs.jafc.9b02269 J. Agric. Food Chem. 2019, 67, 8107−8118
Article
Journal of Agricultural and Food Chemistry
Figure 1. Flowchart for extraction and purification of humic substances. samples were ground to pass through a 2 mm sieve and stored at 4 °C until further analysis. Soil chemical properties at the time of sampling are presented in Table 2. Soil pH was measured in deionized water (1:2.5, w/v), soil organic C was measured by dichromate oxidation, total N was measured by Kjeldahl digestion,28 and total P and total K were digested by HF−HClO4.29 Available N was measured by the method of 2 M KCl extraction, available P was measured by sodium bicarbonate extraction, and available K was measured by CH3COONH4 extraction.28 2.2. Extraction of Humic Substances. The FAs, HAs, and HMs were isolated and purified by the protocols described by the International Humic Substances Society (IHSS) and Preston et al.30 with minor modifications. The three fractions were separated through the classic acid−base fractionation,2 as summarized in Figure 1. More details are presented by Xu et al.14 The masses and C contents of the HS samples were determined to calculate the corresponding humic C contents on a soil basis. Then, the percentage of alkaline-extractable HS recovered as HA, or precipitation quotient (PQ), is considered as a humification index and was calculated as follows: PQ = CHA/(CFA + CHA) × 100.14 For each fertilizer treatment, the HS were extracted from each plot separately, but the HS replicates were combined into one composite sample for NMR measurements. 2.3. 13C Nuclear Magnetic Resonance Spectroscopy. All humic fractions were characterized by 13C multiCP/MAS NMR using a Bruker Avance 400 spectrometer. The technique provides quantitative spectra with good signal-to-noise ratios, very small (
