Fluorescence excitation-emission matrix regional integration to quantify spectra for dissolved organic matter

Excitation-emission matrix (EEM) fluorescence spectroscopy has been widely used to characterize dissolved organic matter (DOM) in water and soil. However, interpreting the > 10,000 wavelength-dependent fluorescence intensity data points represented in EEMs has posed a significant challenge. Fluorescence regional integration, a quantitative technique that integrates the volume beneath an EEM, was developed to analyze EEMs. EEMs were delineated into five excitation-emission regions based on fluorescence of model compounds, DOM fractions, and marine waters or freshwaters. Volumetric integration under the EEM within each region, normalized to the projected excitation-emission area within that region and dissolved organic carbon concentration, resulted in a normalized region-specific EEM volume (phi(i,n)). Solid-state carbon nuclear magnetic resonance (13C NMR), Fourier transform infrared (FTIR) analysis, ultraviolet-visible absorption spectra, and EEMs were obtained for standard Suwannee River fulvic acid and 15 hydrophobic or hydrophilic acid, neutral, and base DOM fractions plus nonfractionated DOM from wastewater effluents and rivers in the southwestern United States. DOM fractions fluoresced in one or more EEM regions. The highest cumulative EEM volume (phi(T,n) = sigma phi(i,n)) was observed for hydrophobic neutral DOM fractions, followed by lower phi(T,n) values for hydrophobic acid, base, and hydrophilic acid DOM fractions, respectively. An extracted wastewater biomass DOM sample contained aromatic protein- and humic-like material and was characteristic of bacterial-soluble microbial products. Aromatic carbon and the presence of specific aromatic compounds (as indicated by solid-state 13C NMR and FTIR data) resulted in EEMs that aided in differentiating wastewater effluent DOM from drinking water DOM.     

Characteristics of dissolved organic matter in Baltic coastal sea ice: allochthonous or autochthonous origins?

The origin of dissolved organic matter (DOM) within sea ice in coastal waters of the Baltic Sea was investigated using parallel factor (PARAFAC) analysis of DOM fluorescence. Sea ice DOM had distinctly different fluorescence characteristics than that of the underlying humic-rich waters and was dominated by protein-like fluorescence signals. PARAFAC analysis identified five fluorescent components, all of which were present in both sea ice and water. Three humic components were negatively correlated to salinity and concluded to be terrestrially derived material. Baltic Sea ice DOM was found to be a mixture of humic material from the underlying water column incorporated during ice formation and autochthonous material produced by organisms within the ice. Dissolved organic carbon (DOC) and nitrogen (DON) concentrations were correlated to the humic fluorescence, indicating that the majority of the organic carbon and nitrogen in Baltic Sea ice is bound in terrestrial humic material trapped within the ice. This has implications for our understanding of sea ice carbon cycling in regions influenced by riverine input (e.g., Baltic and Arctic coastal waters), as the susceptibility of DOM to degradation and remineralization is largely determined by its source.     

Characterization and copper binding of humic and nonhumic organic matter isolated from the South Platte River: evidence for the presence of nitrogenous binding site

Humic substances typically constitute 40-60% of the dissolved organic matter (DOM) in surface waters. However, little information is available regarding the metal binding properties of the nonhumic hydrophilic portion of the DOM. In this study, humic and nonhumic DOM samples were isolated from the South Platte River (Colorado, DOC = 2.6 mg x L(-1), SUVA254 = 2.4 L/mg x m) using a two-column array of XAD-8 and XAD-4 resins. The three major isolated fractions of DOM, which accounted for 57% of the bulk DOM,were characterized using a variety of analytical tools. Proton and copper binding properties were studied for each fraction. The main objective of this work was to compare the structural and chemical characteristics of the isolated fractions and test models describing DOM reactivity toward metal ions. The characterization work showed significant structural differences between the three isolated fractions of DOM. The hydrophobic acid fraction (i.e., humic substances isolated from the XAD-8 resin) gave the largest C/H, C/O, and C/N ratios and aromatic carbon content among the three isolated fractions. The transphilic acid (TPHA) fraction ("transphilic" meaning fraction of intermediate polarity isolated from the XAD-4 resin) was found to incorporate the highest proportion of polysaccharides, whereas the transphilic neutral (TPHN) fraction was almost entirely proteinaceous. The gradual increase of the charge with pH for the three DOM fractions is most likely caused by a large distribution of proton affinity constants for the carboxylic groups, as well as a second type of group more generally considered to be phenolic. In the case of the DOM fraction enriched in proteinaceous material (i.e., TPHN fraction), the results showed that the amino groups are responsible for the charge reversal. For low copper concentrations, nitrogen-containing functional groups similar to those of amino acids are likely to be involved in complexation, in agreement with previously published data.     

Ultrafiltration of nonionic surfactants and dissolved organic matter

A brownwater sample with a high content of humic substances (HS) was fractionated by multistage ultrafiltration (mst-UF) into five fractions with nominal molecular weights ranging from >30 to <1 kDa. Fractions were characterized with respect to molecular size distribution and structure. Size exclusion chromatography with online DOC detection revealed that mst-UF yielded fractions with decreasing Mp (molecular weight at peak maximum) and polydispersities from nominally large to small mst-UF fractions. 13C MAS NMR analysis showed that the content of carbohydrate structures decreased from the original sample toward smaller molecular weight (MW) fractions, which in turn contained more carboxylic groups and branched aliphatic structures. Specific UV absorbances (SUVA254) were highest in the >30 kDa fraction and decreased with decreasing MW. To evaluate whether separation mechanisms other than size exclusion were of importance during the fractionation, the behavior of low molecular weight model compounds (MC) with a range of polarities was studied. Recoveries decreased with increasing hydrophobicity of the MC. For selected nonylphenol ethoxylates and 4-nonylphenol the recovery correlated well with the hydrophile-lipophile balance value. The presence of dissolved organic matter (DOM) caused an additional loss of hydrophobic MC, possibly because of sorption of the compounds onto DOM fouling layers. The hydrophilic MC caffeine was recovered almost completely (85-86%) regardless of the DOM content of the model solution. It was concluded that size exclusion was the dominant fractionation mechanism for caffeine, whereas hydrophobic interactions played a major role during the mst-UF fractionation of nonpolar contaminants. For a better understanding of the behavior of polyfunctional molecules such as HS, the effect of other physicochemical properties needs to be investigated in further studies.     

Molecular fractionation of dissolved organic matter with metal salts

Coagulation of dissolved organic matter (DOM) by hydrolyzing metals is an important environmental process with particular relevance, e.g., for the cycling of organic matter in metal-rich aquatic systems or the flocculation of organic matter in wastewater treatment plants. Often, a nonremovable fraction of DOM remains in solution even at low DOM/metal ratios. Because coagulation by metals results from interactions with functional groups, we hypothesize that noncoagulating fractions have a distinct molecular composition. To test the hypothesis, we analyzed peat-derived dissolved organic matter remaining in solution after mixing with salts of Ca, Al, and Fe using 15 T Electrospray Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FT-ICR-MS). Addition of metals resulted in a net removal of DOM. Also a reduction of molecular diversity was observed, as the number of peaks from the ESI-FT-ICR-MS spectra decreased. At DOM/metal ratios of ～9 Ca did not show any preference for distinct molecular fractions, while Fe and Al removed preferentially the most oxidized compounds (O/C ratio >0.4) of the peat leachate. Lowering DOM/metal ratios to ～1 resulted in further removal of less oxidized as well as more aromatic compounds ("black carbon"). Molecular composition in the residual solution after coagulation was more saturated, less polar, and less oxidized compared to the original peat leachate and exhibited a surprising similarity with DOM of marine origin. By identifying more than 9200 molecular formulas we can show that structural properties (saturation and aromaticity) and oxygen content of individual DOM molecules play an important role in coagulation with metals. We conclude that polyvalent cations not only alter the net mobility but also the very molecular composition of DOM in aquatic environments.     

Solid-state and multidimensional solution-state NMR of solid phase extracted and ultrafiltered riverine dissolved organic matter

In this study we used multidimensional solution-state NMR to elucidate the differences in the chemical composition of solid phase extracted and ultrafiltered DOM isolates. DOM was isolated from water sampled from an oligotrophic river, the River Tagliamento (Italy). The recovery of total DOM was up to 42% with both isolation techniques. In addition to 1- and 2-D solution-state NMR, we also applied 1-D solid-state 13C NMR spectroscopy for DOM characterization. 13C NMR spectroscopy only produced broad overlapping resonances, thus allowing a bulk characterization of DOM composition. However, it demonstrated that the bulk chemical composition of the two DOM fractions exhibited minor spatial-temporal changes. The 2-D experiments (TOCSY, HMQC) showed that the solid phase extracted hydrophobic DOM contained predominantly aliphatic esters, ethers, and hydroxyl groups, whereas the ultrafiltered DOM was comprised partially of peptides/protein, with further evidence for a small amount of aliphatic/fatty acid material. Sugars were present in both DOM fractions. The results show the two isolation techniques selected for different suites of compounds within the bulk DOM pool.     

Molecular characterization of dissolved organic matter in glacial ice: coupling natural abundance 1H NMR and fluorescence spectroscopy

Glaciers and ice sheets are the second largest freshwater reservoir in the global hydrologic cycle, and the onset of global climate warming has necessitated an assessment of their contributions to sea-level rise and the potential release of nutrients to nearby aquatic environments. In particular, the release of dissolved organic matter (DOM) from glacier melt could stimulate microbial activity in both glacial ecosystems and adjacent watersheds, but this would largely depend on the composition of the material released. Using fluorescence and (1)H NMR spectroscopy, we characterize DOM at its natural abundance in unaltered samples from a number of glaciers that differ in geographic location, thermal regime, and sample depth. Parallel factor analysis (PARAFAC) modeling of DOM fluorophores identifies components in the ice that are predominantly proteinaceous in character, while (1)H NMR spectroscopy reveals a mixture of small molecules that likely originate from native microbes. Spectrofluorescence also reveals a terrestrial contribution that was below the detection limits of NMR; however, (1)H nuclei from levoglucosan was identified in Arctic glacier ice samples. This study suggests that the bulk of the DOM from these glaciers is a mixture of biologically labile molecules derived from microbes.     

Preliminary study of lake dissolved organic matter in light of nanoscale supramolecular assembly

Large-scale preparative high-performance size-exclusion chromatography (HPSEC) was performed to separate different molecular size fractions in milligram quantities from strongly colored dissolved organic matter (DOM) of a freshwater using a very mild conjugate acid-base pair (10 mM acetic acid-sodium acetate solution at pH 7.0 with an ionic strength of 6 x 10(-5)) as the mobile phase. The homogeneity-uniformity of different molecular size fractions in relation to their combined original mixture was verified by an analytical HPSEC system. In addition to molecular size distribution and basic spectroscopic characteristics, Fourier transform infrared spectroscopy was applied to specify structural features for different size fractions. The results demonstrate clearly that only a very small amount of conjugate organic acid-base pair is required to generate a powerful resolution for a DOM mixture, and very strong treatments with organic acids are not necessarily needed to reach a better SEC resolution. Most essential is the combined outcome of different HPSEC experiments and determined structural functionalities which indicate that almost all original DOM solutes are aggregated mixtures consisting of different associations possessing various molecular size ranges, which can be separated from their integrated whole as nearly homogeneous and uniform species. In summary, the present study strongly speaks for the need to direct the research of natural dissolved and colloidal organic carbon more strongly toward a nanoscale study of supramolecular assemblies. More precise knowledge about the primary, secondary, and tertiary structure of dissolved DOM constituents has its essential function, e.g., for environmental protection and utilization of surface waters.     

Terpenoids as major precursors of dissolved organic matter in landfill leachates,surface water,and groundwater

13C NMR analyses of hydrophobic dissolved organic matter (DOM) fractions isolated from a landfill leachate contaminated groundwater near Norman, OK; the Colorado River aqueduct near Los Angeles, CA; Anaheim Lake, an infiltration basin for the Santa Ana River in Orange County, CA; and groundwater from the Tomago Sand Beds, near Sydney, Australia, found branched methyl groups and quaternary aliphatic carbon structures that are indicative of terpenoid hydrocarbon precursors. Significant amounts of lignin precursors, commonly postulated to be the major source of DOM, were found only in trace quantities by thermochemolysis/gas chromatography/mass spectrometry of the Norman Landfill and Tomago Sand Bed hydrophobic DOM fractions. Electrospray/tandem mass spectrometry of the Tomago Sand Bed hydrophobic acid DOM found an ion series differing by 14 daltons, which is indicative of aliphatic and aryl-aliphatic polycarboxylic acids. The product obtained from ozonation of the resin acid, abietic acid, gave a similar ion series. Terpenoid precursors of DOM are postulated to be derived from resin acid paper sizing agents in the Norman Landfill, algal and bacterial terpenoids in the Colorado River and Anaheim Lake, and terrestrial plant terpenoids in the Tomago Sand Beds.     

Major structural components in freshwater dissolved organic matter

Dissolved organic matter (DOM) contains a complex array of chemical components that are intimately linked to many environmental processes, including the global carbon cycle, and the fate and transport of chemical pollutants. Despite its importance, fundamental aspects, such as the structural components in DOM remain elusive, due in part to the molecular complexity of the material. Here, we utilize multidimensional nuclear magnetic resonance spectroscopy to demonstrate the major structural components in Lake Ontario DOM. These include carboxyl-rich alicyclic molecules (CRAM), heteropolysaccharides, and aromatic compounds, which are consistent with components recently identified in marine dissolved organic matter. In addition, long-range proton-carbon correlations are obtained for DOM, which support the existence of material derived from linear terpenoids (MDLT). It is tentatively suggested that the bulk of freshwater dissolved organic matter is aliphatic in nature, with CRAM derived from cyclic terpenoids, and MDLT derived from linear terpenoids. This is in agreement with previous reports which indicate terpenoids as major precursors of DOM. At this time it is not clear in Lake Ontario whether these precursors are of terrestrial or aquatic origin or whether transformations proceed via biological and/ or photochemical processes.