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Article Cite This: ACS Earth Space Chem. XXXX, XXX, XXX−XXX

Relationship between Molecular Components and Reducing Capacities of Humic Substances Jitao Lv,† Ruixia Han,†,‡ Zaoquan Huang,†,‡ Lei Luo,† Dong Cao,† and Shuzhen Zhang*,†,‡ †

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ‡ University of Chinese Academy of Sciences, Beijing, 100049, China S Supporting Information *

ABSTRACT: Humic substances (HSs) are collections of diverse organic compounds with broad redox capacities, which directly or indirectly affect the biogeochemical behaviors and fates of almost all the pollutants in the environment. The present study investigates the relationships between the molecular characteristics of HSs and their reducing capacities or electron-donating capacities (EDCs) by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), total phenolic assay, and mediated electrochemical oxidation analysis. For decreasing the molecular heterogeneity of bulk HSs, HSs were first separated into three fractions according to their polarities. The results demonstrated that compounds in HS fractions with moderate polarity possessed a high content of total phenols and consistently had high EDCs. A strong linear correlation (R2 = 0.97) existed between EDCs and the total phenolic content, which confirmed that phenols contributed to the EDCs of HSs. Further analysis of molecular components confirmed that polyphenol-like compounds with medium oxygen content were the major moieties acting as electron donors in HSs. This study provides a linkage between the molecular components of HSs and their EDCs, which will help us to understand the molecular-dependent reducing properties of HSs or other dissolved organic matters under oxic conditions. KEYWORDS: humic, electron donating capacity, reducing capacity, phenol, FT-ICR-MS

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INTRODUCTION Humic substances (HSs) are the most widely distributed organic materials in the environment, including waters, soils, and sediments.1−3 Because of their chemical diversity, HSs have reversible redox capacities within a wide range of redox potentials and therefore are able to act as redox buffers and play very important roles in numerous biogeochemical processes.4,5 Depending on how many redox-active functional groups are reduced or oxidized, HSs possess oxidation capacities to take up electrons by the oxidized moieties (electron-accepting capacities, EACs) or reducing capacities to release electrons from reduced moieties to oxidants with a more positive redox potential (electron-donating capacities, EDCs).6−10 These electron-transfer capacities of HSs further affect the biogeochemical redox reactions and the fate of almost all the redox-sensitive pollutants in the environment directly or indirectly.11−13 For example, it has been reported that HSs can serve as electron mediators to promote microbial reductive dehalogenation of pentachlorophenol (PCP) and tetrabromobisphenol-A (TBBPA) in anaerobic environments.14,15 HSs can also act as electron donors and complexation agents toward © XXXX American Chemical Society

Hg(II), playing a dual role in abiotic dark reduction of Hg(II) to Hg(0) in waters, soils, and sediments.16 In addition, HSs can mediate the redox transformation of iron oxides,17,18 thus likely influencing the transformation of environmental pollutants. Therefore, clarifying the electron-transfer capacities of HSs is critically important to understand their effects on the behaviors and fates of pollutants in the environment. The redox capacities of HSs have been studied for decades.19−24 As early as 1996, Lovley et al. found that HSs could act as terminal electron acceptors in anaerobic microbial respiration6 and that the reduced HSs could also serve as electron donors for the reduction of electron acceptors with a more positive redox potential, such as O2, Fe(III), Mn(IV), selenite, arsenate, and Cr(VI), etc.;21,25,26 the role of HSs in this process was very similar to that of the anthraquinone-2,6disulfonate (AQDS)/AHQDS redox couple.26,27 Furthermore, Received: Revised: Accepted: Published: A

December 27, 2017 February 6, 2018 February 13, 2018 February 13, 2018 DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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ACS Earth and Space Chemistry

Bond Elute PPL, 10 g/20 mL) were rinsed with one cartridge volume each of methanol (MeOH, MS grade, Fisher) and 0.01 M HCl (GR grade, Sinopharm Chemical Reagent Co., Ltd.) immediately before use. Fifty milliliters of each HS sample (100 mg of HS L−1) was acidified with pure HCl to pH 2 and passed through the SPE cartridges by a peristaltic pump at ∼1 mL/ min. Then, the cartridges were rinsed with two cartridge volumes of 0.01 M HCl for the complete removal of salts and then dried under a stream of N2. HS entrapped by the cartridges was then collected by stepwise elution, which was performed by eluting with 50 mL of 20% MeOH (MS grade), 50 mL of 50% MeOH, and 50 mL of pure MeOH at a flow rate of 5 mL/min sequentially. The eluted HS fractions were then lyophilized and stored in the dark at −20 °C. These dried samples were collected and redissolved at a designed concentration in 50:50 methanol/water (v/v) for electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) analysis or redissolved in ultrapure water for electrochemical and total phenolic analysis. Solutions of HS fractions at the concentration of 50 mg of HS L−1 were prepared by dissolving the dried samples in ultrapure water. The total organic carbon (TOC) content of the prepared solutions was determined with a TOC analyzer (vario TOC, Elementar, Hanau, Germany). The specific UV absorbance at 254 nm (SUVA254) of prepared solutions was measured with a Shimadzu UV-2600 UV−vis spectrometer and calculated by the ratio of UVA254 to the TOC content and was reported in units of liter per milligram carbon per meter (L mgC−1 m−1). Negative Ionization ESI-FT-ICR-MS Analysis. Ultra-highresolution mass spectra were acquired using a Bruker SolariX FT-ICR-MS equipped with a 15.0 T superconducting magnet and an ESI ion source as described in our previous study.39 Samples for ESI-FT-ICR-MS analysis were continuously infused into the ESI unit by syringe infusion at a flow rate of 120 μL h−1. The ESI needle voltage was set to −3.8 kV. All of the samples were analyzed in negative ionization mode with broadband detection. Ions were accumulated in a hexapole ion trap for 0.2 s before being introduced into the ICR cell. Four M words of data were recorded per broadband mass scan. The lower mass limit was set to m/z = 200 Da and the upper mass limit to m/z = 1000 Da. One hundred mass spectra were averaged per sample. The spectra were externally calibrated with 10 mM sodium formate (GR grade, Merck) solution in 50% isopropyl alcohol (HPLC grade, Fisher) using a linear calibration and then internally recalibrated using an in-house reference mass list. Peaks with a signal-to-noise ratio (S/N) ≥ 6 were used to identify C5−50H10−100O0−40N0−3 molecular formulas using Bruker Data Analysis software based on the requirement that the mass error for a given chemical formula between the measured mass and calculated mass was ≤0.5 ppm and that the formulas must be a part of a homologous series. Elements P and S were excluded because of their low levels in all the humic samples. The elemental ratios of H/C < 2.2 and O/C < 1.2 were used as further restrictions for formula calculation. The following parameters for data analysis were calculated according to the literature, and the detailed equations are available in the previous studies:39,40 H/C ratio and O/C ratio, parameters for constructing a van Krevelen diagram;35 doublebond equivalence (DBE), a measure of the number of double bonds and rings in a molecule (eq 1);41 the nominal oxidation state of carbon (NOSC), a parameter that can be used to

Scott et al. found a positive correlation between the EACs of various HSs and the concentrations of quinone-like organic radicals as determined by electron spin resonance.28 Compared to EACs, only scant knowledge has been obtained about the electron-donating properties of HSs due to the limitations of methods to quantify the EDCs of HSs. Recently, Aeschbacher et al. developed a highly sensitive mediated electrochemical oxidation (MEO) method to directly quantify the EACs and EDCs of HSs.21 Using this method, phenolic moieties were confirmed as the major electron-donating moieties in HSs, which was supported by the positive linear correlation of the EDCs with the titrated phenol contents of HSs.20,29 Despite this, the inherent mechanism by which the molecular compositions of HSs determine their electron transfer properties is still poorly understood,22,30 largely because of the compositional heterogeneity and chemical diversity of HSs as well as the lack of methods to reveal heterogeneous molecular properties of organic materials such as HSs. According to the new view, HSs are collections of diverse organic molecules with relatively low molecular mass, forming dynamic associations stabilized by hydrophobic interactions and hydrogen bonds.31−33 At present, only Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) can offer the possibility to detect the thousands of individual molecular formulas in HSs, which provides the opportunity to interpret the redox properties of HSs at the molecular level.34,35 On the other hand, given the high molecular diversity of HSs, it is hard to use bulk samples to determine the specific contributions of different kinds of compounds in HSs to their redox capacities.33 A common strategy to resolve this problem is to fractionate bulk HSs into a series of subfractions.36−38 Through fractionation, relatively homogeneous molecules are collected in one subfraction, and the hierarchical differences between subfractions are manifested. Therefore, the present study selected four kinds of HSs and separated each bulk HS into three fractions, respectively, according to their polarity by a facile solid-phase extraction-stepwise elution (SPE-SE) method.39 The molecular components, total phenolic contents, and EDCs of all the HS fractions were analyzed, and a direct linkage was established between the molecular characteristics and EDCs of HSs, which will help to clarify how molecular components of HSs determine their reducing capacities. The results of this study will enhance our understanding of the molecular-dependent electron donating capacity of HSs under oxidizing conditions.

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MATERIALS AND METHODS Materials. Suwannee River Fulvic Acid Standard II (SRFA, 2S101F), Suwannee River Humic Acid Standard II (SRHA, 2S101H), Nordic Aquatic Fulvic Acid Reference (NAFA, 1R105F), and Pahokee Peat Fulvic Acid Standard II (PFA, 2S103F) were acquired from the International Humic Substances Society (IHSS). Vitamin C and 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) diammonium salt (>99%) (ABTS) were obtained from J&K Scientific, China. All solutions were prepared with ultrapure water (>18.5 MΩ cm−1, Millipore). Fractionation of Humic Substances. The bulk HSs were subjected to component fractionation into three fractions, F1, F2, and F3, by solid phase extraction-stepwise elution (SPE-SE) processes reported previously.39 All the eluting solutions and ultrapure water were predeoxygenated by purging with high purity N2 for 10 min prior to use. The SPE cartridges (Varian B

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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ACS Earth and Space Chemistry Table 1. Molecular Characteristics of HS Fractions Identified by Negative ESI-FT-ICR-MS SRFAF1 SRFAF2 SRFAF3 SRHAF1 SRHAF2 SRHAF3 NAFAF1 NAFAF2 NAFAF3 PFAF1 PFAF2 PFAF3

Cwa

Hwa

Owa

Nwa

H/Cwa

O/Cwa

DBEwa

NOSCwa

24.91 27.62 24.32 24.01 27.21 22.46 25.05 28.70 25.45 24.15 27.28 26.83

17.74 21.27 23.78 18.05 21.98 24.23 18.84 22.97 25.17 20.41 25.76 30.80

16.17 15.75 11.16 15.48 14.87 9.58 15.95 15.80 11.27 15.83 15.21 11.63

0.28 0.25 0.19 0.20 0.12 0.06 0.19 0.12 0.06 0.27 0.16 0.19

0.72 0.78 1.01 0.76 0.83 1.09 0.76 0.81 1.02 0.85 0.95 1.16

0.65 0.57 0.46 0.65 0.55 0.43 0.64 0.55 0.44 0.66 0.56 0.44

17.18 18.11 13.52 16.08 17.28 11.38 16.72 18.28 13.90 14.96 15.41 12.45

0.62 0.39 −0.07 0.57 0.29 −0.22 0.54 0.31 −0.13 0.47 0.17 −0.28

a

Refers to magnitude-weighted average number of C, H, O, N atoms and the magnitude-weighted average values of H/C, O/C, DBE, and NOSC in identified molecular formulas (S/N ≥ 6).

estimate the polarity of a molecule (eq 2);42 and a modified aromaticity index (AImod), which was calculated from eq 3 proposed by Koch and Dittmar to estimate the fraction of aromatic and condensed aromatic structures.43 DBE = 1 + (2c − h + n)/2

(1)

NOSC = 4 − [(4c + h − 3n − 2o)/c]

(2)

AI mod = (1 + c − o/2 − h/2)/(c − o/2 − n)

(3)

assessed according to the Folin−Ciocalteu (F−C) method44 with a minor modification. Briefly, 1 mL of HS fractions (5−50 mgHS L−1) was mixed with 50 μL of F−C reagent, and 150 μL of 20% (w/v) sodium carbonate solution. After 60 min at 25 °C, the absorbance was measured at 750 nm. Quantification was carried out using a standard curve for gallic acid (GA). The content of polyphenols was calculated from the slope of the plot of moles of gallic acid versus mass of HS fraction added and expressed as millimoles of gallic acid per gram of HS fraction (mmolGA (gHS)−1). All samples were analyzed in triplicate. Mediated Electrochemical Oxidation (MEO). Electrochemical experiments were controlled with a CHI630D electrochemical workstation (Chenhua Co. Ltd., Shanghai, China). Potentials were measured versus a Ag/AgCl reference electrode but were reported versus the standard hydrogen electrode (SHE). The EDCs of HSs were evaluated using the MEO method developed by Aeschbacher et al.21 and conducted in a glovebox at ambient temperature. The experiments were carried out with a conventional threeelectrode configuration with a cylindrical vitreous carbon (46 mm (d) × 50 mm (h)) working electrode (WE), a Pt wire counter electrode separated from the WE compartment by a glass frit, and a Ag/AgCl reference electrode. ABTS was used as the electron transfer mediator between HSs and the WE. For the EDCs to be determined, the electrochemical cell was first filled with 80 mL of pH 7 buffer (0.1 M KCl, 0.1 M phosphate) and polarized to an oxidizing potential of +0.725 V (vs SHE). Then, 520 μL of ABTS (20 mM) was added to the electrolyte solution, resulting in an oxidative peak current due to oxidation of ABTS to ABTS radical cation (ABTS•+). After the redox equilibrium was attained (∼60 min), 300 μL of HSs solutions were successively spiked to the cell. Oxidation of electrondonating moieties in the added HSs by ABTS•+ resulted in the formation of reduced ABTS, which was subsequently reoxidized at the WE to ABTS•+ to re-establish redox equilibrium. The resulting oxidative current peak was integrated to yield the EDC values of the added HSs

where c, h, o, and n refer to the stoichiometric numbers of carbon, hydrogen, oxygen, and nitrogen atoms per formula, respectively. The magnitude-averaged c, h, o, n, and O/C, H/C, DBE, and NOSC values for each sample can be determined by eq 440 ⎛ ⎞ (M )w = ⎜⎜∑ Ii × (M )i ⎟⎟ /∑ Ii ⎝ i ⎠ i

(4)

where M represents parameters c, h, o, n, and O/C, H/C, DBE, and NOSC, respectively, w signifies a magnitude-weighted calculation, and Ii and (M)i are the relative abundance and M value of peak i, respectively. Molecular groups were further delineated by the AImod and H/C cutoffs according to Rossel et al. with a minor modification:40 (1) formulas of saturated compounds including fatty acids and fatty amines (H/C > 2), (2) formulas of peptides (2 ≥ H/C > 1.5, O/C < 0.9, and N > 0), (3) formulas of unsaturated aliphatics (2 ≥ H/C > 1.5, O/C < 0.9, and N = 0), (4) formulas of carbohydrates and aminosaccharides (H/C ≤ 2, O/C > 0.9), (5) formulas of highly unsaturated compounds (AImod ≤ 0.5 and H/C ≤ 1.5), (6) formulas of polyphenols (0.5 < AImod < 0.67), and (7) formulas of condensed aromatics (AImod ≥ 0.67). Formulas in groups 5−7 were further divided into three subgroups with poor-oxygen (O/C < 0.4), midoxygen (0.4 ≤ O/C ≤ 0.67), and rich-oxygen (O/C > 0.67) because 0.4 was approximately the starting point of the O/C rising edge of the formulas in F1, which contained the formulas with highest O/C, and 0.67 was the ending point of the O/C dropping edge of formulas in F3, which contained the formulas with lowest O/C. The proportion of molecules in each group was calculated based on the intensity-weighted abundance of the formulas in the group to the total. Determination of Polyphenols via the Folin−Ciocalteu Assay. Polyphenolic substances in HS fractions were

EDC =

∫ FI dt /mHS

where I [A] is the baseline-corrected current and F (= 96485 s A/mole−) is the Faraday constant, and mHS [mgHS] is the mass of HS fractions analyzed. The reactions of each HS fraction were analyzed in triplicate with t = 60 min between replicate C

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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to PFA, which might be due to the fact that PFA was derived from a terrigenous source, whereas the others were from aquatic sources. The results clearly demonstrate that fractionation increased the chemical differences between different fractions but decreased the molecular heterogeneity within a fraction, although overlap of molecular formulas between fractions is inevitable due to the high molecular diversity of HSs (Figure S3). Such fractionation is beneficial to investigation of the relationship between molecular components and specific chemical properties, such as the reducing capacities of HSs. The relative distributions of O/C, H/C, DBE, and NOSC of the identified molecular formulas in the HS fractions are displayed in Figure S4, and their magnitude-weighted average values are listed in Table 1. DBE is a measure of the number of double bonds and rings in a molecule;41 NOSC is defined as the charge a carbon atom would take if it were to lose all electrons in bonds with more electronegative atoms but gain all electrons in bonds with less electronegative atoms45 and can be used as a parameter to estimate the polarity of compounds in DOM.42,45 It clearly seemed that the three separated fractions had very different molecular components, especially regarding O/C and NOSC. Fraction F1 was dominated by compounds with positive NOSC and high O/C (above 0.67), whereas fraction F3 was dominated by compounds with negative NOSC and lower O/C (below 0.4), and compounds in fraction F2 were dominated by intermediate NOSC and O/C. The polarity of the primary compounds in the three fractions identified by their NOSC followed the order F1 > F2 > F3, whereas the H/C ratios of compounds in the three fractions followed the order F3 > F2 > F1, which implied that the proportion of alkyl C in compounds increased from F1 to F3. The DBEw was much lower in fraction F3 than in fractions F1 and F2, and the DBEw of F2 was higher than that of F1 by ∼1 unit. In HSs, the contribution of DBE is mainly derived from aromatic rings or carboxyl and carbonyl groups. The lower DBE and higher H/C in the F3 fraction indicated that compounds in this fraction contained more alkyl C but less aromatic C than those in the F1 and F2 fractions. Compounds in the F1 fraction contained more O-containing groups but low DBE, suggesting that compounds contained less aromatic C in F1 than F2. These conclusions were confirmed by the specific UV absorbance at 254 nm (SUVA254), which followed the order F2 > F1 > F3 (Figure S5). The van Krevelen diagrams of all of the identified CcHhOoN0−3 formulas in the HS fractions are displayed in Figure 2. Molecular formulas such as aliphatics, aromatics, polyphenols, and so forth are distinctly located in different regions in the van Krevelen diagram. Therefore, formulas were further categorized into seven groups based on their AImod and H/C cutoffs (shown in dotted lines in Figure 2). As shown in Figure 3, the relative percentages of formulas in groups 1−4 (H/C above 1.5 and O/C above 0.9) were below 10% of the ones in all HS fractions, and these molecular groups were therefore not included in further discussion. The majority of formulas (above 90%) belonged to groups 5−7, which represent formulas of highly unsaturated compounds, polyphenols, and condensed aromatics, respectively. Considering the fact that many chemical properties, such as the reducing capacities of compounds, are dependent on their oxidation degree, the formulas in groups 5−7 were further divided into poor-, mid- and rich-oxygen subgroups according to their O/C values. It was found that rich-oxygen formulas were mainly presented in the groups 5 and 6 in the F1 fraction, and pooroxygen formulas were mainly presented in groups 5 and 6 in

analyses to ensure baseline separation of individual current peaks. The experimental setup was first tested using standard vitamin C (VC), which has an expected EDC of 2. The measured EDC for VC was 1.88 ± 0.06 mole− (molVC)−1 using the present system, and the differences between the EDCs of SRFA, SRHA, NAFA, and PFA determined in this study and their values reported in literature were less than 15%,20 confirming the applicability and reliability of this system for MEO (as shown in Figure S1). The electrolyte solution and ABTS were renewed after six successive analyses to avoid a decrease in mediation over time.20 Statistical Analysis. Hierarchal cluster analysis (HCA) was used to indicate the similarity of HS fractions to each other based on the magnitude of the weighted parameters in Table 1. Squared Euclidean distance was calculated as the measure of the distance with the cluster method of Ward’s linkage between groups using SPSS software (IBM company, USA). Person’s correlation analysis was conducted between the molecular characteristics of HS fractions and their chemical features, including total phenol, SUVA254, and EDC using the corrplot package (version 0.77) in R version 3.4.0. Linear regressions between selected parameters were further performed using Origin 8.0.

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RESULTS AND DISCUSSION Component Fractionation and Molecular Formulas of HS Fractions Identified by ESI-FT-ICR-MS. For relatively homogeneous collections of molecules in HSs to be obtained, four bulk HSs (SRFA, SRHA, NAFA, and PFA) were first subjected to component fractionation into three fractions, F1, F2, and F3, by the SPE-SE method using MeOH as eluent with different concentration gradients (20, 50, and 100%), respectively. Twelve HS fractions were therefore obtained, and the mass percentage of HSs in each fraction followed the order F3 > F2 > F1 for all of the HSs (as shown in Figure 1).

Figure 1. Mass percentage of the HS fractions (F1, F2, F3) isolated from SRFA, SRHA, NAFA, and PFA.

The molecular compositions of these HS fractions were analyzed using ESI-FT-ICR-MS, and molecular formulas with CcHhOoN0−3 were identified. Molecular characteristics of the identified molecular formulas in each fraction are presented in Table 1. Using hierarchical cluster analysis based on these molecular characteristics, the 12 HS fractions were grouped into three clusters: F1 fractions (SRFAF1, SRHAF1, NAFAF1, and PFAF1), F2 fractions (SRFAF2, SRHAF2, NAFAF2, and PFAF2), and F3 fractions (SRFAF3, SRHAF3, NAFAF3, and PFAF3) (Figure S2). The clustering distances of the fractions of SRFA, SRHA, and NAFA were always closer to each other than D

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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Figure 2. van Krevelen diagrams for CcHhOoN0−3 formulas obtained in HS fractions (F1, F2, F3) isolated from SRFA (A−C), SRHA (D−F), NAFA (G−I), and PFA (J−L) by SPE-SE. Color bars represent the S/N of peaks, and bubbles represent the DBE values.

formulas in group 6 observed in the ESI-FT-ICR-MS analysis. However, inconsistent results were obtained between these measurements in F1 and F2. Analysis further showed that the total phenolic concentrations were significantly correlated to the proportion of medium-oxygen-containing molecules (g6 Om) (R2 = 0.83) but not correlated to the proportions of pooror rich-oxygen-containing molecules (g6 Op and g6 Or) in group 6 (Figure S6). This implied that formulas with low or high O/C in group 6 made little contribution to the total contents of phenols in HSs. As a result, including all the formulas in group 6 as polyphenols may overestimate their actual contribution. For example, many compounds, such as aromatic poly(carboxylic acid)s with methoxy or ethoxy group mainly concentrated in F1 according to their polarity and molecular characteristics, have formulas containing low H/C and high O/C and could be mis-grouped into polyphenol-like compounds (Group 6). Therefore, subgrouping the formulas according to their O/C was necessary when delineating the group of formulas of polyphenols.

the F3 fraction. The majority of the formulas presented in F2 fractions contained midoxygen. Relationship between the Total Phenolic Contents in HS Fractions and Their Molecular Components. Quantification of the total phenolic contents in HS fractions was conducted via the modified Folin−Ciocalteu method.44 This method is nonspecific and measures all the forms of phenolics, including monophenols, polyphenols, and lignin phenols; therefore, the results represent the total phenolic contents in HS fractions.46 Selecting a standard phenol compound as a representative of the total phenolic composition in HSs is impossible, so the concentration in equivalents of gallic acid (GA) is commonly used (mmol GA(gHS)−1).47 The total phenolic concentrations determined are shown in Figure 4A. In comparison, the total phenolic concentration in fractions followed the order of NAFA > SRFA ≈ SRHA > PFA among the HSs, and the order among fractions was F2 > F1 > F3. It is necessary to note that, in F3 of all the HSs, a low phenolic concentration detected by the Folin−Ciocalteu method was consistent with a low proportion of polyphenol E

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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Figure 3. Proportions of molecular groups in HS fractions (F1, F2, F3) of SRFA (A−C), SRHA (D−F), NAFA (G−I), and PFA (J−L) obtained from van Krevelen diagram chemical classes. Blank bar represents the total molecular groups without distinguishing oxygen content.

Relationships between Reducing Capacities of HS Fractions and Their Molecular Components. The EDCs of HS fractions measured using the MEO method are presented in Figure 4A. The EDCs of the three fractions in each HS followed the order F2 > F1 > F3, indicating that compounds with high EDCs were enriched in the medium polarity fractions of HSs, whereas the F3 fractions, which were composed of compounds with higher proportions of alkyl moieties and lower DBE, had much lower EDCs than the other two fractions. F1 had a higher number of oxygenic moieties and a higher degree

of unsaturation but lower EDCs than F2 fractions. This was due to the fact that plenty of oxygenic moieties were presented as carboxyl groups, which had no reducing activity. It was worth noting that a strong positive correlation existed between the EDCs of the fractions in each HS and the SUVA254, a simple indicator of the DOC aromaticity determined by UVA254 per unit carbon (R2 = 0.86−1.0, as shown in Figure 5), which implied that molecular collections separated from a single HS with high aromaticity made a major contribution to EDCs, but this relationship cannot be extended to fractions among F

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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Figure 4. Electron-donating capacities (EDCs) of HS fractions (F1, F2, F3) of SRFA, SRHA, NAFA, and PFA at an applied potential of Eh = 0.725 V and pH 7.0 (A) and plot of their EDCs versus total phenols measured by the Folin−Ciocalteu method (B); red band is the 95% confidence interval for linear fitting (red solid line). Data are presented as mean ± standard deviations (n = 3).

phenolic contents showed the potential for use of the Folin− Ciocalteau method to evaluate the EDCs of HSs. Compared to MEO or other conventional methods using chemical oxidants,7,10,21,26 the Folin−Ciocalteau assay is a method saving both time and materials; therefore, it is expected to be useful as a high-throughput assay for EDC analysis. However, further confirmation of its reliability is necessary with more HSs from various sources. Figure 6 displays the Pearson correlation matrix of the EDCs with the molecular characteristics of the fractions of SRFA,

Figure 5. Plots of EDCs versus SUV254 (A) of HS fractions (F1, F2, F3) isolated from SRFA, SRHA, NAFA, and PFA by SPE-SE. Data are presented as mean ± standard deviation (n = 3).

different HSs, especially for HSs from different sources and different humic forms (FA/HA). With comparable aromaticity, the EDCs of different kinds of HS fractions followed the order of aquatic FA (SRFA and NAFA) > aquatic HA (SRHA) > terrestrial FA (PFA). A similar result was found by Aeschbacher et al. using 15 natural HSs.20 However, they observed that the EDCs of terrestrial HSs had a weak correlation with their aromaticity determined by 13C NMR analysis (R2 = 0.32). Instead of aromaticity, a strong linear correlation was found between the total phenolic concentrations and the EDCs of all the HS fractions (R2 = 0.97, Figure 4B) in this study, which indicated that phenolic moieties acted as the major electrondonating moieties in HS fractions. This result was consistent with a previous study,20 which observed a strong linear correlation between the EDCs and titrated phenolic contents of 10 natural HSs. Therefore, it is very likely that phenolic moieties rather than aromatic moieties determine the EDCs of HSs. Compounds with high aromaticity in aquatic FA, such as SRFA and NAFA, may contain more phenolic moieties than in HA or terrestrial FA because compounds containing polyphenol moieties were found to be preferentially sequestered by Fe and Al oxyhydroxides in terrestrial environments,40,48,49 which reduced the extractable proportion of polyphenolcontaining compounds in HSs. Furthermore, the hydroxylation extent of the aromatic nucleus of compounds is higher in aquatic HSs than in terrestrial HSs because aquatic HSs exist in a more oxidized microenvironment.50,51 In addition, the strong linear correlation (R2 = 0.97) between EDCs and the total

Figure 6. Pearson correlation matrix of the electron-donating capacities (EDCs) with the molecular characteristics of HS fractions (F1, F2, F3) of SRFA, SRHA, NAFA, and PFA with p < 0.05.

SRHA, NAFA, and PFA with p < 0.05. This revealed that the proportion of compounds in group 6 with medium oxygen content (g6 Om), the proportion of compounds in group 7 with medium oxygen content (g7 Om), DBE, and NOSC were positively correlated with EDCs, whereas H/C, the proportions of compounds in groups 5 and 6 with poor oxygen (g5 Op and g6 Op) and compounds in other groups were negatively correlated with EDCs. Accordingly, the relative contributions of formulas distributed in the different molecular collections to their EDCs could be estimated intuitively from the van Krevelen diagrams (Figure S7). In more detail, among the characteristics, only DBE and the proportion of g6 Om were linearly correlated with the EDCs (R2 = 0.74 and 0.83, respectively). The plot of EDCs versus DBE and the G

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

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Figure 7. Plot of electron-donating capacities (EDCs) versus double-bond equivalence (DBE) (A) and EDCs versus the proportions of polyphenollike molecules with midoxygen (Om: 0.4 ≤ O/C ≤ 0.67) in HS fractions (F1, F2, F3) of SRFA, SRHA, NAFA, and PFA; red band is the 95% confidence interval for linear fitting (red solid line). Data are presented as mean ± standard deviation (n = 3).

distribution of the photolabile molecular formulas in the van Krevelen diagram, and the highly EDC contributive area overlapped with each other, suggesting that photodegradation may induce changes in the reducing capacity of DOM.52

proportions of g6 Om are further displayed in Figure 7. As mentioned above, compounds in group 6 with medium oxygen content (g6 Om) made major contributions to the total contents of phenols in HSs (Figure S6B), and a strongly linear correlation between the proportion of molecules in g6 Om and EDCs further indicated that midoxygen-containing polyphenollike compounds made more contribution to EDCs than other phenols in HSs. The linear correlation between DBE and EDCs may be due to the strong correlation between DBE and the proportion of g6 Om (Figure S8). Compounds with highly phenolic moieties were those with a high degree of unsaturation due to their rich contents of aromatic rings. Thus, the DBE value alone was inappropriate for use in evaluating the EDCs of HS fractions. Only the proportion of g6 Om, among all the molecular characteristics, can be used as a suitable indicator to evaluate the EDCs of HSs. It is necessary to note that this study revealed the different contributions of molecular components to the reducing capacities of HSs based on their polarity fractionation. Further investigation is necessary to explain the variation in EDCs of HS or DOM according to their bulk molecular components. However, above all, comprehensively revealing the molecules present in DOM is the precondition when linking their molecular components to the reducing capacities, and particular attention should be paid to the fact that the proportion of molecules with low polarity tends to be underestimated, whereas molecules with high polarity tend to be overestimated by FT-ICR-MS analysis of bulk DOM.39 Nevertheless, the obtained relationship between molecular components of HSs and their electron-donating capacity in the present study is very helpful for gaining an understanding of the variation of the reducing capacity of a DOM in environmental processes, such as interfacial adsorptive fractionation, photocatalytic transformation, biodegradation and chlorination disinfection, and so forth. For example, previous studies have reported that the adsorption on Fe/Al-containing minerals can induce molecular fractionation of DOM. Combining the results of the present study, we can speculate that most of the molecules that would preferably be sequestered by Fe/Al minerals are those having high EDCs, and the remaining molecules in water are those having low or no EDCs.40,42 Therefore, it is expected that interfacial adsorptive fractionation is an important process that will influence the redox properties of organic matter in pore water and mineral-organic associations in soils or sediments. Stubbins et al. have used FT-ICR-MS to characterize the photolabile, photoproduced, and photoresistant molecular formulas in a river DOM, the

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CONCLUSIONS The reducing capacity is a fundamental property of HSs pertinent to their mediation of reductive transformations, which is defined as the capacity for donating electrons to an added oxidant. It is highly dependent on the chemical composition and characteristics of HSs. The present study, for the first time, investigated relationships between the molecular components of HSs and their reducing capacities. The results indicated that HS fractions with moderate polarity possessed high contents of total phenols and had the highest EDCs. A strong linear correlation was found among EDCs, Folin−Ciocalteu-detected total phenols, and the proportion of polyphenol-like formulas containing medium oxygen content (0.4 ≤ O/C ≤ 0.67) identified by ESI-FT-ICR-MS, which further confirmed the major contribution to electron donation by phenols, especially polyphenolic compounds with medium oxygen content. In addition, the relationship between formulas in HSs identified by ESI-FT-ICR-MS and their electron-donating capacity will help us to understand changes in the reducing capacities of HSs or other DOM in dynamic environmental processes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsearthspacechem.7b00155. Oxidative current responses, hierarchical cluster analysis, formula compositions, relative frequencies, SUVA254 of the HS fractions, plots of the total phenol concentration versus the proportion of molecules, correlation coefficients, and plots of DBEw versus the proportion of molecules in group 6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ruixia Han: 0000-0003-2357-8019 Dong Cao: 0000-0001-8793-4401 Shuzhen Zhang: 0000-0002-3494-9674 H

DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX

Article

ACS Earth and Space Chemistry Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the 973 Program of China (Grant 2014CB441102), the National Natural Science Foundation of China (Projects 21537005, 41773119, and 21621064), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDB14020202).

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DOI: 10.1021/acsearthspacechem.7b00155 ACS Earth Space Chem. XXXX, XXX, XXX−XXX