Chemical Force Spectroscopy Evidence Supporting the Layer-by

Article pubs.acs.org/est

Chemical Force Spectroscopy Evidence Supporting the Layer-byLayer Model of Organic Matter Binding to Iron (oxy)Hydroxide Mineral Surfaces Alexander W. Chassé,† Tsutomu Ohno,*,† Steven R. Higgins,∥ Aria Amirbahman,‡ Nadir Yildirim,§ and Thomas B. Parr¶ †

School of Food and Agriculture, ‡Department of Civil and Environmental Engineering, and §School of Forest Resources, Advanced Structures and Composite Center, University of Maine, Orono, Maine 04469-5722, United States ∥ Department of Chemistry, Wright State University, Dayton, Ohio 45435, United States ¶ Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *

ABSTRACT: The adsorption of dissolved organic matter (DOM) to metal (oxy)hydroxide mineral surfaces is a critical step for C sequestration in soils. Although equilibrium studies have described some of the factors controlling this process, the molecular-scale description of the adsorption process has been more limited. Chemical force spectroscopy revealed differing adhesion strengths of DOM extracted from three soils and a reference peat soil material to an iron (oxy)hydroxide mineral surface. The DOM was characterized using ultrahighresolution negative ion mode electrospray ionization Fourier Transform ion cyclotron resonance mass spectrometry. The results indicate that carboxyl-rich aromatic and N-containing aliphatic molecules of DOM are correlated with high adhesion forces. Increasing molecular mass was shown to decrease the adhesion force between the mineral surface and the DOM. Kendrick mass defect analysis suggests that mechanisms involving two carboxyl groups result in the most stable bond to the mineral surface. We conceptualize these results using a layer-by-layer “onion” model of organic matter stabilization on soil mineral surfaces.

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INTRODUCTION Soils provide an important ecosystem service by storing an estimated 1461 Pg of C globally as organic matter.1 This quantity of C is greater than the combined atmospheric (760 Pg) and vegetative (560 Pg) contributions to the C storage pool.2 Organic matter is a chemically complex heterogeneous mixture of microbially processed molecules, and this complexity has led to the use of two different approaches for isolating organic matter and studying its reactions. First, studies have used low molecular weight synthetic aliphatic and aromatic acids as models of natural organic matter.3,4 Second, studies have used operationally extracted (on a pH solubility basis) humic substances to represent natural organic matter in chemical studies.5,6 These humic and fulvic acids are thought to be primarily high molecular weight aromatic polymers formed through enzyme-mediated microbial processes.7 However, recent studies have questioned the similarity of humic substances to natural soil organic matter.8 A new paradigm views natural organic matter as associations of low molecular weight components held together by hydrogen bonding and hydrophobic interactions.9 Sorption of the dissolved organic matter (DOM) fraction to mineral surfaces is the initial reaction in soil C sequestration.10 © XXXX American Chemical Society

Short-ranged ordered (SRO) minerals have been shown to adsorb DOM in soils through interactions with the SRO−OH functional groups.11 One conceptual model of organic matter stabilization is the layer-by-layer “onion” model which is based on the observation of decreasing C/N ratio of soil organicmineral particles with increasing particle density.12 The first layer directly adjacent to the mineral surface in this model is composed of N-containing amphiphilic components rich in functional groups containing lone electron pairs such as amines, amides, and pyrroles.12 Amines have been shown to react with Fe (oxy)hydroxide surfaces through a nucleophilic substitution of the surface Fe−OH by R-NH2 to form a covalently bound Fe-NHR and H2O as the leaving group.13 In addition, the first layer also has amphiphilic components rich in carboxylic functional groups that adsorb through ligand-exchange mechanisms. This initial layer provides a surface on which more hydrophobic and less polar organic matter molecules can then be adsorbed to form subsequent layers of organic matter. Received: April 15, 2015 Revised: July 23, 2015 Accepted: July 27, 2015

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DOI: 10.1021/acs.est.5b01877 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

the DOM solution was coated for 24 h. The coated glass disk was rinsed with DI-H2O and used immediately for the force measurements. Interaction Force Determination. The CFS measurements were made on an Asylum Research MFP-3D AFM instrument with a closed fluid cell enclosed in an environmental chamber kept at 23 ± 1 °C. Probes were calibrated using the GetReal algorithm which combines the Sader and the thermal noise method to obtain the spring constant prior to each F-D measurement. The F-D curves were obtained in 0.01 M sodium acetate buffer at pH 4.65, a representative pH value of acid soils. The probes were equilibrated in the buffer solution for 30 min prior to data collection. The tip was engaged onto the surface and 100 F-D curves were obtained on 10 × 10 grid within a 3 μm × 3 μm area for each DOM material. The maximum individual adhesion force values were composed into a histogram, and the distribution was fit to a Gaussian function to obtain the mean and standard deviation of distribution using MATLAB. Mass Spectra Data Post-Processing. The DOM extracts were processed through Agilent PPL solid-phase extraction cartridges to desalt the extract for subsequent electrospray ionization FT-ICR-MS.24 Further details about the FT-ICR-MS analysis are provided in the Supporting Information. For molecular formula calculation, m/z values with a signal-to-noise ratio above 5 were used and assigned using the formula extension approach.25 The assigned formulas were constrained to combinations of C8−52, H6−100, O1−30, N0−5, S0−3, and P0−2. A MATLAB script was used to parse the assigned formulas into the appropriate van Krevelen space which consisted of six discrete regions:23 (1) condensed aromatic molecules (AImod > 0.66); (2) aromatic molecules (0.66 ≥ AImod > 0.50); (3) lignin/phenolics (AImod ≤ 0.50 and H/C < 1.5); (4) aliphatic molecules without N (2.0 > H/C ≥ 1.5 and N = 0); (5) saturated molecules (H/C ≥ 2.0 or O/C ≥ 0.9); and (6) aliphatic molecules containing N (2.0 > H/C ≥ 1.5 and N > 0). The modified aromaticity index (AImod) is calculated as (1 + C − 1/2O − S − 1/2H)/(C − 1/2O − S − N − P) and the double bond equivalent (DBE) is calculated as 1 + 1/2(2C − H + N + P).26 The AImod assumes that 1/2 of the oxygen is bound through π-bonds which corresponds to NMR speciation oxygen content of organic matter.26,27 Further postprocessing details can be found elsewhere.28 The Kendrick mass defect (KMD) analysis is a chemically meaningful method to determine molecules of a homologous series that differ only by the numbers of a given functional group (i.e., a COO− group).20 The Kendrick mass and KMD of the formula assigned molecules were calculated as

Chemical force spectroscopy (CFS) with functionalized atomic force microscope (AFM) tips can directly measure the interaction of organic matter with mineral surfaces14,15 and viruses16 by determining the force between the tip and the sample surface as a function of separation distance (i.e., forcedisplacement (F-D) curve). The observed F-D curve reflects the strength of the binding interaction between the functionalized surface of the probe and the mineral surface substrate. Although the bulk chemical properties such as UV absorptivity, molecular weight, and functional group content can be used to characterize DOM, they cannot provide molecular-scale information about the individual components of complex mixtures such as DOM.17 Ultrahigh-resolution and accuracy capabilities of electrospray ionization Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) typically allow unique formula assignments for thousands of individual DOM components.18 Plots of the H:C versus O:C ratios of the formulas (van Krevelen diagram) have been used to classify each component into biomolecular chemical classes.19 In addition, Kendrick mass defect (KMD) analysis can be used to assess the effect of functional group homologue series’ on reactivity, providing further information on structure−reactivity relationships.20 In this study, we used CFS to investigate the interaction of DOM extracted from three soil organic horizons and a reference International Humic Substances Society (IHSS) Pahokee peat soil with iron (oxy)hydroxide surfaces. We used a correlation-based approach21−23 to elucidate relationships between the FT-ICR-MS-determined DOM components with the CFS-derived adhesion strengths. The molecular-scale information provided by the combination of CFS and FTICR-MS methods promises to increase our understanding of the processes driving carbon sequestration in soils which is an important component of the global carbon cycle.

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EXPERIMENTAL SECTION Details about the DOM source materials and extraction procedures are provided in the Supporting Information. Preparation of Fe (oxy)Hydroxide AFM Tips and DOM Substrate. Olympus TR800PSA silicon nitride cantilevers with a spring constant of 0.57 (range: 0.27−1.19) N m−1 and a foursided tip with a radius of 20 ± 5 nm were used in this study. The probes were functionalized with iron (oxy)hydroxide using the method of Aubry et al.14 Briefly, 30 mL of a stirred 0.01 M FeCl3 solution was slowly raised to pH ∼ 5.5 using dropwise addition of 0.1 N NaOH to the initial formation of Fe (oxy)hydroxide flocs, and 300 μL of the suspension was transferred to a 35-mm glass-well Petri dish. Using clean tweezers, two AFM probes were placed in the well to be coated for ∼20 h. The probes were removed from the coating solution and rinsed by dipping the probes into deionized distilled water (DI-H2O) three times. The tips were then allowed to dry prior to use. The coating of the DOM on the glass substrate was conducted using poly-L-lysine hydrobromide (PLL) as a binder.16 A solution of 0.1 g L−1 PLL was prepared with DIH2O. The DOC concentrations of the soil extracts ranged from 237 to 393 mg C L−1. The solutions were diluted to a fixed DOC concentration of 100 mg C L−1 to equalize the quantity of C coated. First, 400 μL of the PLL solution was pipetted onto a clean glass disk which served as bottom plate for the closed AFM fluid cell. After a PLL coating period of 24 h, the glass disk was carefully rinsed with DI-H2O, and then 400 μL of

Kendrick mass = observed mass × (nominal mass of COO− /exact mass of COO−) KMD = nominal mass − Kendrick mass −

(1) (2) −

where nominal mass of COO = 44, exact mass of COO = 43.989828, and nominal mass is the integer value of the observed mass. Members of a homologous series that differ only in the number of a carboxyl groups in the formula have the same KMD. CFS and Mass Spectra Correlation Analysis. The absolute intensities of the mass spectra peaks were used as an estimate of their concentration in the extracts because in all four samples a fixed amount of DOC (25 mg) was processed B


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Figure 1. Histogram of force distribution with the Gaussian fits of its mean and standard deviation for the (A) deciduous Oa DOM, (B) coniferous Oa DOM, (C) barren soil DOM, and (D) IHSS Pahokee peat DOM with inserted figure showing force distribution with an uncoated, bare Si3N4 tip. The numbers in parentheses are the limits of the 95% confidence interval.

through the solid-phase extraction cartridges and eluted with 1 mL of methanol. A script written in R identified 1424 formulas present in all samples. The use of correlation analysis to explore relationships between ultrahigh-resolution mass spectrometry data and other external data is limited to the components which are present in all the samples.29 For each of the 1424 components, a Pearson’s correlation coefficient was calculated between the mass spectra intensities and the CFS-derived adhesion force values for the four samples studied. For the n = 4 of this experimental design, a r value of 0.80 is the threshold for significance at the p = 0.1 level. Two examples of the correlation analysis are shown in Supporting Information Table S1. Surface and DOM Charge Modeling. The four DOM source materials were extracted at a 1:10 (g/mL) sample/ deionized water ratio. The filtered extract was passed through a H+-saturated cation exchange resin to remove free metals present in the solution. The total DOC concentrations of the extracts were determined using a Shimadzu 5000 analyzer. The acid functional group content of each material was determined by potentiometric titration in a glass reaction beaker maintained at 25.0 ± 0.1 °C. Titration solutions were the undiluted extracts which ranged in concentration from 237 to 393 mg DOC L−1. The solutions were adjusted to 20 mM ionic strength with NaCl, and N2 was bubbled through the solutions for 15 min prior to and throughout titration to minimize CO2

contamination. The solutions were titrated with standardized 0.10 mol L−1 NaOH to pH 10.5 using a Metrohm 907 Titrando autotitrator. We used a fixed discrete acidity constant (Ka) spectrum model to fit the experimental acid−base titration data of the DOM samples.30 The DOM was represented as HLi ⇔ Li− + H+

i = 4; log Ka , i fixed at 4, 6, 8, and 10 (1)

The experimental data were fit with computer program FITEQL with total concentrations of reactive sites, ∑ (HLi + Li−), as the fitting parameters.31 The input consisted of equilibrium constants, total or free concentrations of chemical components and species, and the stepwise added strong base concentration and the corresponding H+ concentration as the experimental data. The model minimized the difference between the total calculated (THcalcd) and total experimental (THexp) proton balance equations, TH calcd = [H+] − [OH−] −

TH exp = TH 0 − C b

∑ Li −

(2) (3)

where TH0 is the excess of strong acid anions to strong base cations initially, which is equal to the measured H + concentration before the start of titrations, and Cb is the concentration of strong base added at each titration step.30 C


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samples which may suggest that the IHSS peat is composed of more diverse DOM components involved in bonding to the iron (oxy)hydroxide-coated tip. The inset in Figure 1D shows the adhesion value of 0.23 nN for the uncoated Si3N4 tip interaction with IHSS peat DOM which was an order of magnitude lower than the 2.72 nN for the coated tip, indicating that our coating process has functionalized our tips to allow determination of the interaction between the DOM surface and the iron (oxy)hydroxide surface. The AFM probes, prior to coating with iron (oxy)hydroxide films, were most likely coated in a thin layer of silicon oxide (SiOx) based on previous studies of Si3N4 surfaces exposed to air and/or water. The X-ray photoelectron spectroscopy characterization of freshly fractured (air-exposed) and chromic-acid tribochemically polished Si3N4 surfaces have shown little difference in the thicknesses of the oxide layers found to develop on the polished and fractured surfaces.35 The authors estimated the Si3N4 surfaces were coated with 1−1.5 nm of a mixed oxide−nitride (SiOxNy) and an outer layer of SiO2 with an estimated thickness of 0.2−0.5 nm. The outermost layer consisted of an adventitious carbon coating. In a study conducted to investigate the surface properties of AFM tips, adhesion forces between Si3N4 AFM tips and various substrates in water were determined.36 Strong adhesion forces observed were ascribed to the presence of SiO2 on the Si3N4 surface, which was presumably formed from reaction with the water. Assuming that the Si3N4 AFM probes used in the present work were coated with a thin oxide film, we next consider the iron (oxy)hydroxide coating of these tips. Silica microspheres were coated with iron oxide films by Aubry et al.14 using a procedure similar to what we have employed in this study. Electrokinetic data from the iron oxide-coated microspheres indicated behavior consistent with a surface terminated by an iron oxide. In addition, attempts to measure the thickness of the iron oxide film, using scanning electron microscopy of focused ion beam-milled tips, were unsuccessful due to the film’s very small dimension.14 Whereas the tips used in the present investigations were coated with an iron oxide film, an assertion supported by the evidence in Figure 1 comparing pulloff force histograms of iron oxide-coated tips with an uncoated tip (inset, Figure 1D), we assume that the iron oxide film has chemical properties (e.g., point of zero charge, surface acidity constants) similar to those of bulk iron oxide and that the underlying silica-coated Si3N4 does not affect these surface properties. CFS and Mass Spectrometry Data Relationships. We used a correlation-based approach21−23,29 to relate the normalized intensity of FT-ICR-MS formulas present in all four samples to the CFM-derived adhesion strengths. The DOM coating on the iron (oxy)hydroxide-coated AFM tips were conducted with a fixed amount of DOC by using the same volume of coating solution and the same DOC concentration for all four DOM samples. Similarly, the DOM solution processing prior to the mass spectrometry analysis was also conducted on an equivalent DOC amount basis by applying 25 mL of 100 ppm DOC solution for each of the DOM solutions to the PPL solid-phase extraction cartridges to desalt the DOM solution. The DOM for all of the samples was eluted with 1 mL of methanol, resulting in a constant amount of DOM across all four samples in a fashion identical to the coating for the CFS study. Therefore, the FT-ICR-MS spectra are effectively on a mass C basis, reporting the units on that basis would be a

Surface complexation modeling was also used to determine the pH dependence of surface charge distribution of Fe(III) (oxy)hydroxide.32 To this end, we used the existing data from the literature based on the acid−base titration for goethite (αFeOOH).33,34 The model considered a specific surface area of 45 m2 g−1, a functional group site density of 1.68 nm−2, and the following surface acid−base reactions with intrinsic equilibrium surface binding constants (Kint) and the diffuse double layer Coulombic term: >SOH + H+ ⇔ >SOH 2+ >SOH ⇔ > SO− + H+

log K1int = 6.19 ± 0.06

(4)

log K 2 int = −10.14 ± 0.12 (5)

where >SOH denotes a reactive surface functional group that undergoes proton complexation and dissociation. The model generated concentrations of >SOH, >SOH2+, and >SO− surface groups at different pH values.

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RESULTS AND DISCUSSION DOM Chemical Characteristics. The distribution of the van Krevelen diagram-derived classification groupings of the DOM molecules found in the four samples are shown in the Supporting Information Figure S1. Across all samples the lignin/phenolic group was dominant with 52−64% of the assigned formulas, while the saturated molecule group consisted of less than 1% of formulas in all the samples. The condensed aromatic molecules in the three soil organic horizon DOMs averaged 4.1%, considerably less than 11.1% for the IHSS peat DOM. The contrast was smaller for the aromatic molecule content with the three soil DOM samples averaging 12.5% of formulas which was slightly below the 16.6% aromatic content for the IHSS peat DOM. The total aliphatic group (both non N- and N-containing) contents were similar across the four soils, ranging between 17.1 and 24.4%. However, differences in the distribution of aliphatic formulas containing N and those without N were found in the four samples (Figure S1). The three field organic soil horizon DOM samples contained more non-N containing aliphatic molecules, while the IHSS peat soil DOM contained more of the N-containing aliphatic molecules. The biggest difference can be seen with the IHHS peat DOM which had 65% of the aliphatic formulas containing 1 or more N atoms, while only 7% of the barren soil DOM formulas contained N atoms. The Pahokee peat soils were formed from freshwater marsh organic deposits and represent an end-member of the soil C stabilization process. The high N-containing aliphatic content of the DOM of the IHHS peat DOM supports the conjecture that N-containing functional group form strong bonds with mineral surfaces leading to their more refractory nature. Chemical Force Measurements. In the CFS measurement, iron (oxy)hydroxide coated Si3N4 AFM tips were brought into contact with the layered DOM−PLL−glass disk in pH 4.65, 0.01 M sodium acetate buffer solution, then retracted. A representative F-D curve for the IHSS Pahokee peat soil DOM is shown in Figure S2. The histograms of maximum adhesion force and the Gaussian fit of the four DOM samples are shown in Figure 1A−D. The three soil organic horizon DOM samples fit the Gaussian function well with the rsquare values >0.95 with the mean adhesion force range of 0.64−1.06 nN. The IHSS Pahokee peat soil DOM had a higher mean adhesion value of 2.72 nN, and a greater spread in distribution around the mean value than the other three soil D


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Figure 2. (A) Average m/z values, (B) modified aromaticity index values, and (C and D)chemical composition of the top and bottom quintile of the correlation coefficients with respect to CFS adhesion strength representing the significant positively and negatively correlated DOM components, respectively. For the box plots in A and B, the bottom boundary of the box indicates the 25th percentile, the line within the box marks the median, and the top boundary of the box indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles, respectively. The points above and below the whiskers are the outlying points. In the pie charts C and D, the number in each pie wedge is the number of molecules found in the top and bottom quintile which were significantly correlated to adhesion strength.

uniform linear transformation across all spectra that would affect the intensity of peaks, but have no effect on the variance and no effect on the statistical correlations. The molecular weight and modified aromaticity index values of the components which were significantly (r > 0.8, p = 0.1) positively and negatively correlated to CFS adhesion strength are shown in Figure 2A and B, respectively. The DOM molecules with high positive correlations to adhesion strength have lower molecular mass and higher modified aromaticity index values than those that were negatively correlated. The chemical composition differed as well, with condensed aromatic and aromatic components together accounting for 60% of the positively correlated components, compared to 9% for the negatively correlated components (Figure 2C and D). These data support laboratory-scale studies which have shown that aromatic DOM molecules are preferentially adsorbed to SRO mineral surfaces.11,37 In addition, N-containing aliphatic molecules accounted for 9% of the most positively correlated components, but were absent in the most negatively correlated components supporting the important role of N-containing functional groups of DOM adsorption to mineral surfaces. The van Krevelen distribution of the DOM components not significantly correlated (|r| < 0.8) to adhesion force are shown in Figure S3 and had chemical composition which was intermediate of those positively and negatively correlated to adhesion force (Figure S4, 2C and D). These results also provide strong support for the layer-by-layer “onion” model12

of organic matter structuring onto mineral surfaces where Ncontaining aliphatic components and aromatic structures are involved in the initial adsorption of DOM to SRO mineral surfaces. These data demonstrate that both molecular mass and chemical composition of the DOM are important factors in determining its adhesion strength to iron (oxy)hydroxide surfaces. The average correlation coefficient between the adhesion strength as a function of molecular mass (binned in 25 Da windows) for the five van Krevelen diagram-derived classification groups are shown in Figure 3. The dashed line at r = 0.8 indicates the threshold for significance at the p = 0.1 level. The condensed aromatic and aromatic molecules remained significantly correlated to adhesion strength to 397 and 370 Da, respectively. The N-containing aliphatic molecules which were also shown to be strongly related to the adhesion strength retained its significant effect to 328 Da. The lignin/phenolic and non-N-containing aliphatic molecules which were not strongly related to adhesion strength had the lowest molecular mass thresholds at 293 and 290 Da, respectively. The results suggesting that the first layer may be primarily composed of low molecular weight DOM molecules (Figure 2A) is opposite to numerous equilibrium adsorption isotherm studies which have shown that higher molecular mass components are preferentially adsorbed by SRO surfaces.37,38 This discrepancy is likely due to the different time scales of the CFS and adsorption isotherm studies. The results from this E


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Figure 3. Relationship of the correlation coefficient between adhesion force and molecular mass of the condensed aromatic, aromatic, lignin/ phenolic, non-N-containing aliphatic, and N-containing aliphatic molecules in the DOM extracts. The average correlations were calculated in bin sizes of 25 Da units. The dashed line indicates the threshold correlation for significance (r > 0.8, p = 0.1), and the molecular mass of the crossing point for each of the molecular classes is noted.

study likely interrogate the very initial stages of DOM adsorption with the subsecond time scale for the CFS determinations onto freshly coated iron (oxy)hydroxide AFM tips. In contrast, the equilibrium isotherms typically are on the ∼24−48 h time scale to show responses that approach quasiequilibrium. The reaction of DOM with SRO surfaces is likely to be composed of multiple processes over different temporal scales, each with differing selectivity. Different experimental methodologies are required to probe the diverse range of time scales involved in environmental processes. The results here show how the chemical composition of DOM affects its initial interaction with Fe (oxy)hydroxide mineral surfaces. Surface and DOM Acid−Base Modeling. Although the correlational analysis above suggests the existence of relationships between adhesion force and various DOM chemical properties, it provides no insight into the mechanisms involved in the relationship. To gain a more mechanistic understanding of the observed DOM interactions with the Fe (oxy)hydroxidecoated AFM tips, we used surface complexation modeling to determine the pH dependence of surface charge distribution of iron (oxy)hydroxide coating with respect to the concentrations of >SOH, >SOH2+, and >SO− surface groups at different pH values (Figure 4A).32 At the experimental pH of 4.65 used in the CFS studies, 28.5% of the surface−OH sites are modeled to be in the positively charged protonated form (e.g., >SOH2+). The selected experimental pH of 4.65 was chosen because it is the average pH of the organic soil horizon at the sites from which the deciduous and coniferous Oa horizon samples and the native barren soils were collected. The adsorption of DOM does alter surface properties, however the changes are not accessible with titration methods because the adsorbed DOM will desorb as pH increases which precludes the direct measurement of the DOM adsorbed surface.

Figure 4. (A) Modeled surface speciation of the coated iron (oxy)hydroxide AFM tip and (B) total negatively charged acid functional group content as a function of pH for the four DOM extracts investigated.

Additionally, we characterized the acid−base properties of each DOM sample using potentiometric titrations. The fixed discrete acidity constant (Ka) spectrum model using four proton affinity classes with log Ka values of 4, 6, 8, and 10 was used to fit the titration data of each DOM extract. The model fit the titration curves well as shown by the close agreement between the experimental and model TH values across the entire titration pH range (Figure S5). The average carboxyl content (sum of log Ka 4 and 6) of the three organic soil DOM was 3.67 ± 0.27 mmol g−1 C, as compared to 6.95 mmol g−1 C for the IHSS peat DOM (Table S2). The total quantity of negatively charged DOM functional group content (sum of L1−, L2−, L3−, and L4−) as a function of pH is shown in Figure 4B. At pH 4.65, the three soil DOM samples have negatively charged functional group content ranging from 0.98 to 1.95 mmol g−1 C, while the IHSS peat DOM has higher negatively charged content of 3.69 mmol g−1 C due to its relatively high carboxyl group content (Figure 4B). The functional group information we used constitutes a maximum value, since some of the groups are used to bind to the substrate. The positively charged surface functional group and negatively charged DOM molecules F


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Environmental Science & Technology would favor the formation of electrostatic associations in the pH range typical of acidic forest soils. DOM Adsorption Mechanisms. Aromatic carboxylic acids serve as a common model for DOM, as such structures are a common component of natural organic matter.3,4 Infrared spectroscopy studies have shown evidence suggesting that inner-sphere ligand exchange complexes exist below pH 6, while outer-sphere complexes occur in the pH 3−9 range.39 The mechanism of the DOM ligand exchange reaction with metal (oxy)hydroxide mineral surfaces involves three sequential steps.40 (A) >SOH + H+ ⇔ > SOH 2+

(Surface protonation)

(B) > SOH 2+ + R − COO− ⇔ >SOH 2+ −− OOC − R (Outer‐sphere electrostatic bond) (C) >SOH 2+ −− OOC − R ⇔ SOOC − R + H 2O (Inner‐sphere ligand exchange)

One method to differentiate between the outer- and innersphere mechanisms is to conduct adsorption experiments with different background ionic strength solutions.41 Outer-sphere complexes are sensitive to background electrolyte concentrations because both the adsorbing ion and electrolyte ions are in the diffuse layer whereupon they both contribute to changes in the activity coefficient. In contrast, the inner-sphere complexes are located in the surface-adjacent Stern layer and have no interaction with the background electrolytes in the diffuse layer. The F-D measurements of the IHSS Pahokee DOM were conducted at 1, 10, and 100 mM NaCl to determine the effects of ionic strength on the adhesion force (Figure S6). Field measurements suggest that 1−100 mM ionic strength is a typical range with respect to ionic strength found in soil solution.42 The regression fit was not significant (p = 0.56) indicating the absence of ionic strength effects on the adhesion strength which further supports the argument the adhesion force is due primarily to the formation of inner-sphere complexes through ligand exchange reactions. The CFS results of this study support the empirical findings based on the presence of differing background salts to compete with DOM for bonding sites on goethite.43 It was reported that 92% of organic matter molecules extracted from a forest soil organic horizon interact through inner-sphere ligand exchange reactions involving the carboxyl and phenolic OH groups of DOM.43 Kendrick Mass Defect Analysis. Kendrick mass defect analysis with the carboxyl group as the unit block was used to investigate the role of the DOM carboxyl group in the adsorption process in greater detail.20 The results for the homologous series with greater than four members for the condensed aromatic, aromatic, and lignin/phenolic classes of molecules are shown in Figure 5A. The trend of decreasing correlation with adhesion force with increasing nominal mass for the carboxyl KMD series mirrors the average results shown for all members of the classification group (Figure 3) where correlation value decreases with increasing molecular mass. To investigate how multiple carboxyl groups affect the interaction with the Fe (oxy)hydroxide-coated tips, the average change in the coefficient value as additional carboxyl groups are added for the condensed aromatic, aromatic, and lignin/phenolic classes of DOM molecules are shown in Figure 5B. Each carboxyl group addition adds 44 Da to the molecular mass. The initial

Figure 5. (A) Kendrick mass defect analysis (carboxyl group basis) for the condensed aromatic, aromatic, and lignin/phenolic DOM molecules showing homologous series with greater than four members and (B) the effects of increasing carboxyl groups on the correlation of the carboxyl-containing molecules with mean adhesion force strength.

addition of a carboxyl group decreases the average correlation coefficient by less than 0.07 for the two aromatic structures and the lignin/phenolic compounds, suggesting that the additional charge introduced into the DOM molecules compensates for the increased molecular mass. This result extends the observation made with the model benzenecarboxylates that two carboxylates are involved in the formation of inner-sphere complexes to natural DOM molecules.37 With the addition of two carboxyl groups, only the condensed aromatic structure shows a minimal decrease in the correlation coefficient value, indicating that steric effects from the larger molecular mass is greater than the potential gains from ligand exchange between the additional carboxyl group and surface functional groups. The understanding of the mechanisms of organic matter interaction with mineral surfaces is crucial to the development of strategies that enhance carbon sequestration in soils. Using CFS and ultrahigh-resolution FT-ICR-MS we show how individual molecules of DOM are correlated to its adhesion G



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strength to iron (oxy)hydroxide mineral surfaces. We show that the abundance of condensed aromatic, aromatic, and Ncontaining aliphatic molecules of DOM are correlated with the adhesion force between the DOM and iron (oxy)hydroxide functionalized AFM tips. This work presents data that support the layer-by-layer “onion” model of organic matter stabilization where the initial layer is composed of carboxyl-groupcontaining aromatic compounds and N-containing aliphatic compounds. The results show that the force of DOM binding to the mineral surfaces, at least at the initial stages of adsorption, decreases with increasing molecular mass. KMD analysis also extends the previously reported results based on substituted benzoic acids that two carboxyl groups are involved in the inner-sphere bond formation to natural organic matter. This work suggests that changes in DOM composition due to the environmental processes generating DOM (e.g., forest cover change or land use change) may affect stabilization of DOM at a molecular level with landscape-scale consequences for carbon sequestration and loss.

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REFERENCES

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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/acs.est.5b01877. Extended details about the DOM source material and FT-ICR-MS analysis. Percentage distribution of the van Krevelen diagram-derived classification groups for the four DOM sources studied (Figure S1). Representative F-D curve for IHSS Pahokee DOM (Figure S2). Distribution of chemical classifications of the DOM components not significantly correlated to adhesion force (Figure S3). van Krevelen diagram of DOM chemical composition of the DOM components not significantly correlated to adhesion force (Figure S4). Experimental and model total proton balance condition (TH) values for the four DOM extracts (Figure S5). Effect of ionic strength on the measured adhesion force between the iron (oxy)hydroxide coated AFM tip and the IHSS Pahokee peat soil DOM (Figure S6). Chemical force spectroscopy and mass spectra correlation analysis (Table S1). Potentiometric titration-derived acidic functional group contents for the four DOM extracts (Table S2). (PDF) Excel spreadsheet of experimental data (XLSX)

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone 207-581-2975; fax 207-5812999. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.W.C. and T.O. extend our appreciation to Dr. Leonardo Gutierrez at the University of Illinois at Urbana−Champaign and Dr. Jean Philippe Croue at the King Abdullah University of Science and Technology for discussion and assistance with the preparation of the iron (oxy)hydroxide coated AFM probes. This project was supported by USDA-NIFA-AFRI 2013-6701921368 and the Maine Agricultural and Forest Experiment Station Hatch Project ME0-H-1-00472-11. This is Maine Agricultural and Forest Experiment Station Journal no. 3432. H


Article

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