Infrared spectroscopic evidence supporting heterogeneous site binding models for humic substances

Infrared spectroscopy was used to corroborate predictions made by newly developed heterogeneous site binding models for humic substances. Experimental conditions to acquire the spectra of soil humic substances (humic and fulvic acid and a polysaccharide fraction) in an aqueous state using horizontal attenuated total reflectance Fourier transform infrared spectroscopy (HATR-FTIR) were established. Elimination of the water spectrum from that of the sample was achieved by spectral subtraction of the water peak at 2020 cm(-1). A KSCN internal standard with an absorption band at 2067 cm(-1) was used to verify the efficacy of the subtraction procedure. Spectral artifacts produced by the water spectrum subtraction and from contaminants within the humic materials have been identified. Three fulvic and one humic acid solution were examined in solutions of varying pH. Results show that the observed proportion of ionized carboxylate in relation to pH is consistent with models that assume electrostatic effects and a continuous distribution of proton association constants (log KH). The spectroscopic data were in accordance with calculations made using the generic humic and fulvic acid NICA-Donnan model parameters.     

Antimony(III) binding to humic substances: influence of pH and type of humic acid

Conditional distribution coefficients (Dom) for Sb(III) binding to three commercial humic acids (terrestrial, coal, and aquatic) were measured at environmentally relevant Sb(III)/DOC ratios and as a function of pH using an equilibrium dialysis method. Maximum binding of Sb(III) was observed around pH 6 for two of the humic acids. The third humic acid showed constant Dom values up to pH 6 and decreasing Dom values for pH > 6. Sb(III)/DOC ratio was found to be important for Dom (20 times higher Dom for 60 times lower Sb(III)/DOC ratio). Moreover, Dom depends on the individual humic acid, suggesting that different functional groups are involved and/or different degrees of stabilization by chelation or H-bridges. Chemical modeling of Sb(III)-humics binding at different pH values is consistent with two binding sites involving (i) a phenolic entity forming a neutral complex and (ii) a carboxylic entity forming a negatively charged complex. Under environmentally relevant conditions, over 30% of total Sb(III) may be bound to natural organic matter.     

Birnessite-induced binding of phenolic monomers to soil humic substances and nature of the bound residues

The nature of the abiotic birnessite (δ-MnO(2))-catalyzed transformation products of phenolic compounds in the presence of soil organic matter is crucial for understanding the fate and stability of ubiquitous phenolic carbon in the environment. (14)C-radioactive and (13)C-stable-isotope tracers were used to study the mineralization and transformation by δ-MnO(2) of two typical humus and lignin phenolic monomers-catechol and p-coumaric acid-in the presence and absence of agricultural and forest soil humic acids (HAs) at pH 5-8. Mineralization decreased with increasing solution pH, and catechol was markedly more mineralized than p-coumaric acid. In the presence of HAs, the mineralization was strongly reduced, and considerable amounts of phenolic residues were bound to the HAs, independent of the solution pH. The HA-bound residues were homogeneously distributed within the humic molecules, and most still contained the unchanged aromatic ring as revealed by (13)C NMR analysis, indicating that the residues were probably bound via ester or ether bonds. The study provides important information on δ-MnO(2) stimulation of phenolic carbon binding to humic substances and the molecular distribution and chemical structure of the bound residues, which is essential for understanding the environmental fates of both naturally occurring and anthropogenic phenolic compounds.     

Binding of mercury(II) to aquatic humic substances: influence of pH and source of humic substances

Conditional distribution coefficients (K(DOM')) for Hg(II) binding to seven dissolved organic matter (DOM) isolates were measured at environmentally relevant ratios of Hg(II) to DOM. The results show that K(DOM') values for different types of samples (humic acids, fulvic acids, hydrophobic acids) isolated from diverse aquatic environments were all within 1 order of magnitude (10(22.5 +/-1.0)-10(23.5 +/- 1.0)) L kg(-1)), suggesting similar Hg(ll) binding environments, presumably involving thiol groups, for the different isolates. K(DOM') values decreased at low pHs (4) compared to values at pH 7, indicating proton competition for the strong Hg(II) binding sites. Chemical modeling of Hg(II)-DOM binding at different pH values was consistent with bidentate binding of Hg(II) by one thiol group (pK(a) = 10.3) and one other group (pK(a) = 6.3) in the DOM, which is in agreement with recent results on the structure of Hg(II)-DOM bonds obtained by extended X-ray absorption fine structure spectroscopy (EXAFS).     

Influence of humic substance adsorptive fractionation on pyrene partitioning to dissolved and mineral-associated humic substances

Changes in pyrene binding by dissolved and mineral-associated humic substances (HS) due to HS adsorptive fractionation processes were examined in model environmental systems using purified Aldrich humic acid (PAHA) and Suwannee River fulvic acid (SRFA). For PAHA, carbon-normalized pyrene binding coefficients for nonadsorbed, residual fractions (Koc(res)) were different from the original dissolved PAHA Koc value (Koc(orig)) prior to contact with the mineral suspensions. A strong positive correlation between pyrene log Koc(res) and log weight-average molecular weight (MWw) for residual PAHA fractions was observed, which was relatively independent of the specific mineral adsorbent used and hypothesized fractionation processes. A strong positive correlation between log Koc(ads) and log MWw was also found for PAHA fractions adsorbed to kaolinite at low mass fraction organic carbon levels, although the relationship was statistically different from the one found with residual PAHA fractions. The same trends and correlations found for PAHA were not observed with SRFA, suggesting that the impacts of HS adsorptive fractionation on changes in hydrophobic organic contaminants binding are also influenced by the source and other biogeochemical characteristics of HS.     

Application of molecular modelling and spectroscopic approaches for investigating binding of vanillin to human serum albumin

In the present study, the interaction of vanillin and human serum albumin (HSA) has been characterised by molecular modelling, fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic methods. The results of molecular modelling suggested that vanillin was located within the binding pocket of subdomain IIA of HSA mainly by hydrophobic forces. The quenching of HSA fluorescence takes place with a binding constant (K) of 8.8, 7.7, 5.7, 4.2 × 10⁴ M⁻¹ at four different temperatures (288, 298, 308, 318 K), respectively. Meanwhile, the number of binding site (n ≈ 1) was also obtained from fluorescence titration data. The enthalpy change ΔH⁰ and the entropy change ΔS⁰ were calculated to be −20 kJ mol⁻¹ and 5.8 J mol⁻¹ K⁻¹ according to the Van’t Hoff equation. Furthermore, the alterations of protein secondary structure in the presence of vanillin were explored by FT-IR and CD spectra.     

Reversed-phase chromatography fractionation tailored to mass spectral characterization of humic substances

Large-scale structural characterization of humic substances via mass spectrometry requires reduction of complexity within nominal mass and separation of isomers, i.e., prefractionation. Humic substances (here loosely defined to encompass all humic, humic-like, and humic-containing material) are notoriously difficult to fractionate. Equally challenging is deriving information on whether and howfractionation has occurred. Here, reversed-phase high-performance liquid chromatography was used to induce tailored fractionation of Suwannee River fulvic acid (SRFA) within nominal mass. The fractionation was optimized on synthetic standards thatdiffered in polarity and had elemental formulas similar to SRFA. Fractions were analyzed via electrospray ionization ion-cyclotron resonance mass spectrometry. Kendrick and Van Krevelen comparisons showed that fractionation occurred as predicted based on known molecular formula patterns.     

Adsorption of humic substances on goethite: comparison between humic acids and fulvic acids

The adsorption of humic acids (HA) to goethite (at pH 3-11) and the proton co-adsorption (at pH 4.0, 5.5, and 7.0) were measured, and the results were compared to those of fulvic acids (FA). Compared to FA, the adsorption of HA is stronger and more ionic strength dependent. The adsorption of both HA and FA decreases with increasing pH. The relative change of the adsorption with pH is bigger for HA than for FA at relatively low pH. At relatively high pH, it is the opposite. Protons are released at pH 4.0 and co-adsorbed at pH 5.5 and 7.0 upon the adsorption of both HA and FA. The observed pH dependency of HA and FA adsorption is in agreement with the proton co-adsorption data. Model calculations show that the adsorbed FA particles are on average located in the Stern layer, whereas the adsorbed HA particles protrude beyond the Stern layer. The closer location to the surface of the adsorbed FA leads to stronger electrostatic interactions between the FA particles and the surface, which explains the larger amount of protons released at low pH and co-adsorbed at high pH with each mass unit of FA adsorbed than that with HA adsorbed. The model also revealsthatfor FA a mean-field (smeared-out) approximation is reasonable, but for HA a patchwise approach is more appropriate at relatively low loading.     

Utilization and transformation of aquatic humic substances by autochthonous microorganisms

Aquatic humic substances (HS) from a bog lake water, a riverwater, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3. The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater. The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 degrees C on a shaker. HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls. In full strength nutrient broth up to 27% of HS were utilized. Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures. Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars. The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures. After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/MS or capillary electrophoresis. 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available. The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS. The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS.     

Characterization and quantification of reversible redox sites in humic substances

Cyclic oxidation and reduction reactions using oxygen and palladium with H2, respectively, of dissolved humic and fulvic acids (HA and FA) and model quinone compounds were used to structurally characterize and quantify the electron-carrying capacity (ECC) of reversible redox sites present in humic substances. This technique was used to examine 8 quinone compounds and 14 HA and FA samples and identified 3 redox sites as a function of their stability against the Pd-catalyzed hydrogenolysis process. Six highly aliphatic HA and FA isolated from landfill leachate did not contain redox sites under any conditions; however, the other HA and FA demonstrated reversible redox properties characterized by a combination of three redox sites. On the basis of the model compound results, it is proposed that one site consists of a non-quinone structure (NQ) and the other two sites have quinone structures. The two quinone sites differ in that one group (Q1) has electron-withdrawing groups adjacent to the quinone functional group while the second group (Q2) contains either no substituents near the quinone or has nearby electron-donating groups with additional substitutents hindering hydrogenolysis through steric interactions. The reversible ECC of NQ sites ranged from 25 to 265 microequiv e- transferred/g HA or FA, representing 21-56% of the total ECC of the HA and FA when measured with the mildest reducing method (pH 8.0, pure Pd). Q1 redox sites resistant to hydrogenolysis at pH 8.0 using Pd/Al2O3 accounted for 13-58% of the total ECC and ranged from 40 to 120 microequiv e-/ g HA or FA. The most sensitive O2 reversible redox sites accounted for 8-50% of the total ECC (20-220 microequiv e-/ g HA or FA). These results directly demonstrate that HA and FA are capable of acting as reversible electron-transfer agents using different functional groups, some of which may not be quinones.     

Evidence for nonlinear binding of PAHs to dissolved humic acids

Binding of pyrene, fluoranthene, and phenanthrene to dissolved humic acids (HA) was determined by the fluorescence quenching (FQ) and complexation-flocculation (CF) methods. Determinations by the CF method, using varying contaminant concentrations and a constant HA concentration, yielded nonlinear Freundlich-type isotherms (n = 0.65-0.84). Experiments using both the CF and the FQ methods with varying HA concentrations and a constant contaminant concentration yielded curved "Stern-Volmer"-type plots that also indicate nonlinear binding. These findings suggest that linear partitioning or site complexation in the presence of excess available sites cannot fully describe the interactions of hydrophobic compounds with dissolved humic material. Site-specific hydrophobic interactions at limited interior or external molecular surfaces may be considered.