Natural Organic Matter and the Event Horizon of Mass Spectrometry
Soils, sediments, freshwaters and marine waters contain natural organic matter (NOM) - an exceedingly complex mixture of organic compounds that collectively exhibit a nearly continuous range of properties (size- reactivity continuum). NOM is composed mainly of carbon, hydrogen and oxygen, with minor contributions from heteroatoms such as sulphur and phosphorus. Suwannee River fulvic acid (SuwFA) is a fraction of NOM that is relatively depleted in heteroatoms. Ultrahigh resolution Fourier transform ion cyclotron (FTICR) mass spectra of SuwFA reveal several thousand molecular formulae, corresponding in turn to several hundred thousand distinct chemical environments of carbon even without accountancy of isomers. The mass difference ƒ´m among adjoining C,H,O-molecules between and within clusters of nominal mass is inversely related to molecular dissimilarity: any decrease of ƒ´m imposes an ever growing mandatory difference in molecular composition. Molecular formulae that are expected for likely biochemical precursor molecules are notably absent from these spectra, indicating that SuwFA is the product of diagenetic reactions that have altered the major components of biomass beyond the point of recognition. The degree of complexity of SuwFA can be brought into sharp focus through comparison with the theoretical limits of chemical complexity, as constrained and quantized by the fundamentals of chemical binding. The theoretical C,H,O-compositional space denotes the isomer-filtered complement of the entire, very vast space of molecular structures composed solely of carbon, hydrogen and oxygen. The molecular formulae within SuwFA occupy a sizable proportion of the theoretical C,H,O-compositional space. A one-hundred percent coverage of the theoretically feasible C,H,O-compositional space by SuwFA molecules is attained throughout a sizable range of mass, H/C and O/C elemental ratios. The substantial differences between (and complementarity of) the SuwFA molecular formulae that are observed using six different modes of ionization (APCI, APPI, ESI in positive and negative modus) imply considerable selectivity of the ionization process and suggest that the observed mass spectra represent simplified projections of still more complex mixtures. N. Hertkorn, M. Frommberger, M. Witt, B. Koch, Ph. Schmitt-Kopplin, E. M. Perdue, Natural Organic Matter and the Event Horizon of Mass Spectrometry, Anal. Chem., 80 (2008) 8908-8919.
Pushing the Limits of NMR Spectroscopy: In Situ analysis of Organic Matter in Natural Waters
Dissolved Organic Matter (DOM) is ubiquitous in all natural waters and is known to play important roles in the carbon and nitrogen cycles, the transport and transformation of contaminants and nutrients, and health and biodiversity of aquatic species. Thus there is a great scientific need to further understand the composition, variability and reactivity of dissolved organic matter in the environment. Of all the analytical approaches employed to study DOM, NMR spectroscopy has provided the greatest insights into its general composition. However, conventional NMR studies often require a considerable amount of isolated DOM (mg quantities) and are adversely influenced by high salt and/or metal content which can result from sample concentration. Also there is concern that DOM can be altered during chemical isolation to varying extents and may not be completely representative of the material in its natural state. Here we demonstrate, that while very difficult, it is possible to obtain NMR spectra of Organic Matter in situ for practically all major bodies of water including groundwater, rainwater, seawater, and water from lakes and rivers. In sea water DOM is present at ~1ppm, and thus with a standard 5mm NMR probe (assuming ~300µL volume inside the coil), only ~300ng of DOM is present. Furthermore, considering that the intensity of the water signal is many orders of magnitude greater than the weak signals from the DOM (itself a heterogeneous mixture) it is clear that such applications challenge the limits of modern NMR spectroscopy.
Investigations of Online HPSEC-NMR for the Separation and Elucidation of Dissolved Organic Matter (DOM)
Dissolved organic matter (DOM) is the largest reservoir of actively cycling organic carbon on earth, plays a role in the fate of contaminants, releases CO2 as the primary degradation product, and yet the structural analysis of this globally-significant material is hindered by its molecular complexity. The heterogeneity of DOM makes it difficult to study using chromatography with conventional detectors as little structural information can be obtained in the presence of extensively co-eluting components. This presentation describes the direct hyphenation of high performance size exclusion chromatography (HPSEC) with nuclear magnetic resonance spectroscopy (NMR) to determine whether size-distinguished fractions differ in composition. The results support the applicability of using high performance liquid chromatography (HPLC) to generate more homogenous fractions of DOM prior to NMR analysis. The findings further suggest that the various size- fractions of DOM do exhibit structural variability. The largest fractions are enriched in carbohydrate- and aromatic-type structures, while the smallest material has a strong aliphatic signature. The mid-sized material is characterized by carboxylated alicyclic material (CRAM) - likely derived from secondary metabolites of terpenoids. CRAM is known to play an important role in the aggregation of DOM constituents. Accordingly, the bulk of the DOM eluted from the HPSEC system was found to elute within the range of the mid-sized material and was found to contain a strong signature from the carboxylated alicyclic material. Further development will likely include exploring various HPLC stationary phases for which to separate DOM according to other physical and chemical properties. These separations will focus on the dissaggregation of constituents into even more homogenous fractions for further structural elucidation.
Revealing Aquatic Dissolved Organic Matter Composition using Direct 1H NMR Spectroscopy
Dissolved organic matter (DOM) is ubiquitous in all aquatic ecosystems, and comprises a variety of chemically heterogeneous molecular structures and functional groups. DOM is often considered to be a major ligand for metals in most natural waters. However DOM reactivity is thought to be strongly dependent on its chemical structure. The purpose of this study is to evaluate the variability in molecular composition of aquatic DOM from different sources. Quantitative proton NMR spectra were obtained without any preconcentration using water suppression techniques. The reproducibility on the determination of aromatic and aliphatic proton was better than 3%. The structural information of DOM from northern rivers was compared to IHSS humic substances.
Biomarkers of Canadian High Arctic Litoral Sediments for Assessment of Organic Matter Sources and Degradation
Carbon stocks in the High Arctic are particularly sensitive to global climate change, and investigation of variations in organic matter (OM) composition is beneficial for the understanding of the alteration of organic carbon under anticipated future elevated temperatures. Molecular-level characterization of solvent extractable compounds and CuO oxidation products of litoral sedimentary OM at the Cape Bounty Arctic Watershed Observatory in the Canadian Arctic Archipelago was conducted to determine the OM sources and decomposition patterns. The solvent extracts contained a series of aliphatic lipids, steroids and one triterpenoid primarily of higher plant origin and new biomarkers, iso- and anteiso-alkanes originating from cerastium arcticum (Arctic mouse-ear chickweed, a native angiosperm) were discovered. Carbon preference index (CPI) values for the n-alkanes, n-alkanols and n-alkanoic acids suggests that the OM biomarkers result from fresh material input in early stage of degradation. The CuO oxidation products were comprised of benzyls, lignin phenols and short-chain diacids and hydroxyacids. High abundance of terrestrial OM biomarkers observed at sites close to the river inlet suggests fluvial inputs as an important pathway to deliver OM into the lake. The lignin phenol vegetation index (LPVI) also suggests that the OM origin is mostly from non-woody angiosperms. A relatively high degree of lignin alteration in the litoral sediments is evident from the abundant ratio of acids and aldehydes of the vanillyl and syringyl monomers. This suggests that the lignin contents have been diagenetically altered as the result of a long residence time in this ecosystem. The molecular-level characterization of litoral sedimentary OM in Canadian High Arctic region provides insight into current OM composition,potential responses to future disturbances and the biogeochemical cycling of carbon in the Arctic.
Molecular Characterization of Cryoconite Organic Matter from the Athabasca Glacier, Canadian Rocky Mountains
Cryoconite is a dark-colored, dust-like material found on the surfaces of glaciers. Cryoconite holes, which are produced by accelerated ice melt due to more solar radiation absorption by cryoconite than bare ice, act as habitats for microbial life and biologically mediated chemical reactions on otherwise relatively inert glacier surfaces. Cryoconite holes may behave as bacterial shelters during "Snowball Earth" events postulated for the Neoproterozoic Earth. In this study organic matter (OM) biomarkers and a host of one- and two-dimensional NMR techniques were used to characterize cryoconite organic matter (COM) collected from the Athabasca Glacier in the Canadian Rocky Mountains. Solvent extracts contain large quantities of fatty acids, n-alkanols, n- alkanes, wax esters and sterols. A large contribution of C23 and C25 relative to C29 and C31 n-alkanes ([C23/(C23+C29)] = 0.51) suggests that allochthonous COM is derived mainly from lower order plants such as mosses and lichens. This is confirmed by the absence of lignin-derived phenols, a biomarker of terrestrial vascular plants, after copper (II) oxidation in extracts and NMR analyses of COM. Solution-state 1H NMR reveals prominent peptide/protein structures which are characteristic of microbial inputs, while solid-state 13C CP/MAS NMR analysis shows a very high alkyl/O-alkyl ratio (2.16), suggesting that COM is unique compared to organic matter found in nearby soils which have alkyl/O-alkyl ratio of ~0.39. Our NMR results suggest that COM is dominated by microbial-derived compounds, which is also confirmed by phospholipid fatty acid results (6,950µg/gOC) which show significant microbial contributions to COM primarily from bacteria and minor microeukaryotes. Both biomarker and NMR data suggest that COM likely supports active microbial communities on the Athabasca Glacier. Given that such material is incorporated within the glacier in the accumulation zone or flushed by meltwaters into subglacial environments, reworked COM may provide nutrient sources for active microbial communities found within and under glaciers.
Molecular-Level Analysis of Organic Matter Structure and Composition from Different Soil Mineral Fractions
The formation and turnover of soil organic matter (SOM) depends on the inherent chemical characteristics of biomolecular inputs (lignin, proteins, carbohydrates, macromolecular lipids, etc.) as well as the interactions between biomolecules and soil mineral fractions. The objective of this study is to characterize organic matter associated with the light, sand, silt and clay fractions of a Canadian agricultural soil. And, because lignin is believed to be a major contributor in SOM formation and preservation, the oxidation state of lignin in the different mineral fractions was measured using mild alkaline copper oxidation and gas chromatography - mass spectrometery which releases lignin phenols that are indicative of lignin sources and stage of degradation. For example, an increase in the acid/aldehyde (Ad/Al) ratio of lignin phenols has been observed with increased lignin degradation (and oxidation). In this study, lignin phenols from organic matter associated with the clay fraction had higher Ad/Al ratios for both syringyl and vanillyl lignin monomers when compared to that associated with silt, sand and the whole soil. These results suggest that either lignin degradation is enhanced by SOM association with clay surfaces or that oxidized lignin is preserved on clay mineral surfaces via sorption after partial degradation has occurred. The structural characteristics of organic matter from the soil fractions will also be examined by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Organic matter associated with each mineral fraction will be extracted with NaOH for high resolution solution-state NMR spectroscopy. Results from NMR analysis will determine the relative abundance of functional groups (alkane, aromatic, carbonyl, alkoxy) in each of the soil fractions. Relative intensities of the functional groups are indicative of relative contributions of biomolecular classes such as lipids, lignin, fatty acids, and sugars to the organic matter associated with each fraction. The study comprises our initial steps in characterizing protection mechanisms responsible for the long-term retention and stability of biomolecules and their degradation intermediates in soil.
Organic Matter Stabilization in Surface and Subsurface Soil Horizons: C Mineralization and Early Formation of Water-Stable Macroaggregates
Fine-textured Gleysolic soils in Eastern Canada are widespread and productive. Their high silt and clay content (>80%) suggests that they may have a high C storage capacity. Recent research has shown that while the absence of tillage increases the level of macroaggregation and the amount of C stored in surface soil, incorporation of crop residues by full inversion tillage commonly practiced in Eastern Canada can increase the level of organo-mineral interaction and the amount of C retained in deeper soil layers. The objective of this experiment was to evaluate the intrinsic potential of surface and subsurface horizons of fine-textured Gleysolic soils to retain additional C input. Topsoil (0-20 cm) and subsoil (30-70 cm) samples were taken from a cultivated clayey soil profile of the Kamouraska serie located near Québec and incubated with 0, 2.5, 5.0, 10.0, 20.0, and 40.0 g C kg-1 soil of 13C-15N-labelled corn residues. Large amounts of residues were added to the soils in order to saturate SOC pools. Soils and residues were mixed and incubated under optimal temperature (25°C), water potential (-38 kPa), and nutrient (C:N = 10) conditions for 52 d. More C was lost as CO2 in the topsoil than in the subsoil for all residue-C treatment, except of the highest level (40 g C kg-1 soil). At each level of C input, the ratio of C lost to C added was lower in the subsoil than in the topsoil indicating higher C protection in the subsoil than in the topsoil, especially with lower plant C input rates. The effect of C input on macroaggregate formation and stabilization was greater in subsoil than topsoil, but both soils reach a plateau of 90% macroaggregates in the 20 g C kg-1 soil treatment. The formation of new water-stable macroaggregates was greater in the subsoil than topsoil, and was related to lower C lost through respiration. These preliminary results clearly show that the subsoil is responding differently to C additions than does the topsoil. Upcoming results on isotopic signature will give us more information on the incorporation of added-C within specific soil fractions. Moreover, the use of molecular analytical techniques to trace biomarkers within specific soil fractions could help us elucidate preferential stabilization mechanisms operating under C saturated and unsaturated conditions, and during the early C stabilization period.
Ecological and Molecular Study of Soil Chemoautotrophic Microorganisms via the Sequestration of Atmospheric 13CO2
Soil Organic Matter (SOM) is the most complicated biomaterial on Earth and stores significantly more carbon than is currently present in the atmosphere . It has been recently reported that humic material in SOM is a highly complex mixture of microbial and plant biopolymers and not a distinct chemical fraction as previously thought . Furthermore, it has been reported that the microbial biomass contribution to SOM is not comprised of mainly humic materials and that in fact the contribution to SOM by soil microorganisms has been seriously underestimated . Therefore, the question arises if we underestimate microbial biomass in soil do we also underestimate carbon sequestration by soil microbes? Soil microorganisms consist of a large range of diverse species with soil bacteria contributing a large proportion of the biomass content. Autotrophs are organisms that can produce organic compounds from CO2 as the sole carbon source, using either light (photoautotroph) or inorganic reactions (chemoautotroph) as the energy source. The aim of this project is to enrich chemoautotrophic soil microbes with carbon-13 (13C) sequestered as 13CO2. Once labeled, these target microbes can be differentiated from other microbes using techniques such as Stable Isotope Probing (SIP) and carbon NMR. This enrichment is facilitated via incubation in a custom built environmental chamber and the controlled introduction of 13CO2. Before introduction of 13CO2 the chambers capabilities had to be fully characterized to ensure that it was fit for purpose. Mixed cultures of soil chemoautotrophic microorganisms were propagated from different soils and data collected using the environmental chamber demonstrated that CO2 fluctuations mimicked the natural activity of actively growing chemoautotrophic cultures. Therefore using this soil slurry approach, a mixed culture of soil autotrophs will be exposed to 13CO2 prior to the harvesting of the microbial biomass. Ion chromatographic analysis of the soil slurry during the inactive and active growth phases has shown that the inorganic energy source provided to the mixed culture had been utilized in a complimentary manner to the chamber atmospheric CO2 data. Future work will involve the labeling of autotrophic soil microorganisms using the CO2 incubation chamber, extract the total DNA, carry out stable isotope probing (SIP) and identify species responsible for the 13C uptake. This data shall then be compared to carbon NMR spectra generated from the same sample set using solid state Magic Angle Spinning (MAS) NMR to determine microbial metabolic products and exudates. References:  Young, I. M. and J. W. Crawford, Interactions and self-organization in the soil-microbe complex. Science. 2004. 304 (5677), 1634-1637.  Kelleher, B. P. and Simpson, A. J., Humic Substances in Soils: Are they really chemically distinct? Environmental Science and Technology, 40(15), 4605-4611, 2006.  Simpson, A. J., Simpson M. J., Smith E. and Kelleher B. P., Microbially-derived inputs to soil organic matter: Are current estimates too low? Environmental Science Technology 2007, 41, 8070-8076
Assessment of Various Organic Matter Properties by Infrared Reflectance Spectroscopy of Sediments and Filters
The goal of this work was to evaluate the capability of infrared reflectance spectroscopy for a fast quantification of the elemental and molecular compositions of sedimentary and particulate organic matter (OM). A partial least-squares (PLS) regression model was used for analysis and values were compared to those obtained by traditional methods (i.e., elemental, humic and HPLC analyses). PLS tools are readily accessible from software such as GRAMS (Thermo-Fisher) used in spectroscopy. This spectroscopic-chemometric approach has several advantages including its rapidity and use of whole unaltered samples. To predict properties, a set of infrared spectra from representative samples must first be fitted to form a PLS calibration model. In this study, a large set (180) of sediments and particles on GFF filters from the St. Lawrence estuarine system were used. These samples are very heterogenous (e.g., various tributaries, terrigenous vs. marine, events such as landslides and floods) and thus represent a challenging test for PLS prediction. For sediments, the infrared spectra were obtained with a diffuse reflectance, or DRIFT, accessory. Sedimentary carbon, nitrogen, humic substance contents as well as humic substance proportions in OM and N:C ratios were predicted by PLS. The relative root mean square error of prediction (%RMSEP) for these properties were between 5.7% (humin content) and 14.1% (total humic substance yield) using the cross-validation, or leave-one out, approach. The %RMSEP calculated by PLS for carbon content was lower with the PLS model (7.6%) than with an external calibration method (11.7%) (Tremblay and Gagné, 2002, Anal. Chem., 74, 2985). Moreover, the PLS approach does not require the extraction of POM needed in external calibration. Results highlighted the importance of using a PLS calibration set representative of the unknown samples (e.g., same area). For filtered particles, the infrared spectra were obtained using a novel approach based on attenuated total reflectance, or ATR, allowing the direct analysis of the filters. In addition to carbon and nitrogen contents, amino acid and muramic acid (a bacterial biomarker) yields were predicted using PLS. Calculated %RMSEP varied from 6.4% (total amino acid content) to 18.6% (muramic acid content) with cross-validation. PLS regression modeling does not require a priori knowledge of the spectral bands associated with the properties to be predicted. In turn, the spectral regions that give good PLS predictions provided valuable information on band assignment and geochemical processes. For instance, nitrogen and humin contents were greatly determined by an absorption band caused by aluminosilicate OH group. This supports the idea that OM-clay interactions, important in humin formation and OM preservation, are mediated by nitrogen-containing groups.
Analysis of organic matter at the soil-water interface by NMR spectroscopy: Implications for contaminant sorption processes
Contaminant sorption to soil organic matter (OM) is the main fate of nonionic, hydrophobic organic contaminants in terrestrial environments and a number of studies have suggested that both soil OM structure and physical conformation (as regulated by the clay mineral phase) govern contaminant sorption processes. To investigate this further, a number of soil samples were characterized by both solid-state 13 C Cross Polarization Magic Angle Spinning (CPMAS) NMR and 1H High Resolution Magic Angle Spinning (HR- MAS) NMR. HR-MAS NMR is an innovative NMR method that allows one to examine samples that are semi- solid using liquid state NMR methods (ie: observe 1H which is more sensitive than 13C). With HR-MAS NMR, only those structures that are in contact with the solvent are NMR visible thus one can probe different components within a mixture using different solvents. The 1H HR-MAS NMR spectrum of a grassland soil swollen in water (D2O) is dominated by signals from alkyl and O-alkyl structures but signals from aromatic protons are negligible (the peak at ~8.2ppm is attributed to formic acid). When the soil is swollen in DMSO-d6, a solvent which is more penetrating and capable of breaking hydrogen bonds, aromatic signals are visible suggesting that the aromatic structures are buried within the soil matrix and do not exist at the soil-water interface. The 13C solid-state NMR data confirms that aromatic carbon is present in substantial amounts (estimated at ~40% of the total 13C signal) therefore, the lack of 1H aromatic signals in the HR-MAS NMR spectrum indicates that aromatic structures are buried and that the soil-water interface is dominated by aliphatic chains, carbohydrates, and peptides. The NMR data indicates that the mineral component of soils governs the physical conformation of OM at the soil-water interface.
Molecular-level interactions in soils and sediments: the role of aromatic pi- systems
This review intends to reduce uncertainties regarding the mechanisms by which molecules with aromatic moieties attach to organic and mineral components of terrestrial environments. We present published evidence for the existence of specific, sorptive interactions of aromatic moieties with environmental sorbents. We find that aromatic pi-systems within organic compounds have the capacity to adsorb to minerals and organic soil and sediment components such as natural organic matter (NOM) and fire-derived black carbon (BC) through strong sorptive forces other than hydrophobic interactions. Specific, polar interactions of aromatic pi-donor and -acceptor compounds show adsorption energies between 4 and 167 kJ mol-1. Bonding strengths of cation-pi interactions and pi-pi electron donor-acceptor (EDA) interactions appear to be larger than H bonding strengths and comparable to inner- and outer-sphere complex formation. We conclude that - in analogy to polar and ionizable functional groups - components with aromatic pi-donor and -acceptor systems equip organic molecules with a substantial sorptive potential. This observation has important implications for the fate and transport of aromatic contaminants. The resulting sorptive interactions might also play a yet- overlooked functional role in the complex chain of processes which preserve NOM against decomposition.
1H NMR Metabolomics: A New Molecular Level Tool for Assessment of Organic Contaminant Bioavailability to Earthworms in Soil
At contaminated field sites, the complete removal of polycyclic aromatic hydrocarbons (PAHs) is rarely achieved since a portion of these compounds remain tightly bound to the soil matrix. The concentration of PAHs in soil typically decreases until a plateau is reached, at which point the remaining contaminant is considered non- bioavailable. Numerous soil extraction techniques, including cyclodextrin extraction, have been developed to estimate contaminant bioavailability. However, these are indirect methods that do not directly measure the response of organisms to chemical exposure in soil. Earthworm metabolomics offers a promising new way to directly evaluate the bioavailability and toxicity of contaminants in soil. Metabolomics involves the measurement of changes in small-molecule metabolites, including sugars and amino acids, in living organisms due to an external stress, such as contaminant exposure. The objective of this study was to compare cyclodextrin extraction of soil (a bioavailability proxy) and 1H NMR metabolomic analysis of aqueous earthworm tissue extracts as indicators of contaminant bioavailability. A 30 day laboratory experiment was conducted using phenanthrene-spiked sphagnum peat soil and the OECD recommended earthworm species for toxicity testing, Eisenia fetida. The initial phenanthrene concentration in the soil was 320 mg/kg. Rapid biodegradation of phenanthrene occurred and concentrations decreased to 16 mg/kg within 15 days. After 15 days, phenanthrene biodegradation slowed and cyclodextrin extraction of the soil suggested that phenanthrene was no longer bioavailable. Multivariate statistical analysis of the 1H NMR spectra for E. fetida tissue extracts indicated that the metabolic profile of phenanthrene exposed earthworms differed from control earthworms throughout the 30 day experiment. This suggests that the residual phenanthrene remaining in the soil after 15 days continued to elicit a metabolic response, even though it was not extractable using cyclodextrin. Hence, while cyclodextrin extraction may serve as a good proxy for microbial bioavailability, our results suggest that it may not serve as a good proxy for earthworm bioavailability. 1H NMR metabolomics therefore offers considerable promise as a novel, molecular-level method to directly monitor earthworm bioavailability of potentially toxic and persistent compounds in the environment.