Major Structural Components in Freshwater Dissolved Organic Matter
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Major Structural Components in Freshwater Dissolved Organic Matter

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Environ Sci Technol. 2007 Dec 15;41(24):8240-7. ...

Environ Sci Technol. 2007 Dec 15;41(24):8240-7.
Major structural components in freshwater dissolved organic matter.

Lam B, Baer A, Alaee M, Lefebvre B, Moser A, Williams A, Simpson AJ.

Department of Chemistry, University of Toronto Scarborough, Toronto, Ontario, Canada M1C 1A4.

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.

PMID: 18200846 [PubMed - indexed for MEDLINE]

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  • 1. Major Structural Components in Freshwater Dissolved Organic Matter Buuan Lam1, Mehran Alaee2, Brent Lefebvre3, Arvin Moser3, Antony Williams3 and André J. Simpson1 1 Department of Chemistry, University of Toronto Scarborough, Toronto, Ontario, Canada, M1C 1A4 2 National Water Research Institute, Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada, L7R 4A6 3 Advanced Chemistry Development Inc., 110 Yonge Street, 14th floor, Toronto, Ontario, Canada, M5C 1T4. Abstract 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 (NMR) spectroscopy to demonstrate the major structural components in Lake Ontario DOM. These include carboxyl-rich alicyclic molecules (CRAMs), heteropolysaccharides and aromatic compounds, which are consistent with components recently identified in marine dissolved organic matter (1). In addition, long-range proton- carbon correlations are obtained for DOM, which support the existence of carotenoid- derived aliphatic molecules (CDAMs). It is tentatively suggested that the bulk of freshwater dissolved organic matter is aliphatic in nature, with CRAMs derived from cyclic terpenoids, and CDAMs derived from carotenoids, which are a sub-category of linear terpenoids. This is in agreement with previous reports which identify terpenoids as major precursors of DOM (2). 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. Corresponding author. Tel: 1-416-287-7547; Fax: 1-416-287-7279; E-mail address:
  • 2. Introduction Dissolved organic matter (DOM) is a complex, heterogeneous mixture found ubiquitously in nature. It comprises a major mobile fraction of organic carbon on Earth and is an intimate link between the terrestrial and aquatic environment (3-5). Terrestrial and freshwater DOM experiences an annual flux of approximately 0.4 x 1015 gC/year via riverine discharge (4) to the marine environment. It is believed that DOM plays a significant role in the enhanced solubility (6) and binding (7) of chemical contaminants and may potentially be a shuttle for the long range transport of chemicals globally. Thus, the cycling of DOM from freshwater to marine sources is not only important in the global carbon cycle, but is a significant mediator in the fate and transport of pollutants in the environment. Despite this importance, there is still much to be revealed regarding the structural components that make up this complex environmental mixture and how these compounds vary between freshwater and marine environments. The difficulty in isolating sufficient quantities to conduct meaningful analytical studies, compounded by the limitation of analytical techniques to adequately provide detailed molecular information on DOM, have been key factors in hindering a more comprehensive understanding. The isolation and concentration of DOM on resins has greatly improved the access to larger sample quantities (8-10); however, improved analytical techniques still need to be actively developed to provide molecular information. Multidimensional solution-state nuclear magnetic resonance (NMR) spectroscopy is becoming a widely employed and very powerful technique to study structures and interactions in environmental chemistry (11-16). Here, dissolved organic matter from Lake Ontario, Canada is studied in detail. Lake Ontario covers just over 19,000 km2, contains over 1,600 cubic kilometers of freshwater (17) and is part of the Great Lakes, which represent the world‘s largest freshwater lakes system. Recently a pivotal paper by Hertkorn et al. (1) utilized a range of modern 1-D and 2-D NMR approaches to identify carboxyl-rich alicyclic molecules (CRAMs) in oceanic DOM. This pioneering paper has been essential in providing key assignments making further NMR based studies possible. Here, we build upon the work of Hertkorn et al. (1) who have reported on major structural and refractory components of marine DOM, extending these initial findings to show that marine and freshwater DOM share many structural similarities. Long-range correlations are collected for freshwater DOM which is extremely challenging given the relatively low sensitivity of the experiments and the fast relaxation of DOM. Combining recent improved long-range NMR experiments with relaxation optimized delays (18) permits weak long-range correlations to be recorded for DOM for the first time. The long-range proton-carbon correlations help confirm previous assignments of CRAMs and support the presence of an aliphatic material derived from carotenoids, a sub-category of linear terpenoids in freshwater DOM.
  • 3. Materials and Methods Sample Preparation. A freshwater DOM sample taken from Lake Ontario (Darlington Provincial Park, Ontario, Canada) was used in this study. Lake Ontario DOM (LDOM) was isolated as described by Simpson et al (19). Briefly, water from Lake Ontario was prefiltered through 0.22 μm PVDF filters. DOM was then isolated on site from this filtered water using diethylaminoethyl (DEAE)-cellulose resin. DOM was recovered from the resin using 0.1M NaOH, ion-exchanged using Amberjet 1200H Plus resin (note: pH was adjusted to ~6 after ion-exchanging), and freeze-dried. Excess salts were removed from the sample by excessive dialysis against double-distilled water using 100 molecular weight cut-off cellulose ester tubing. The sample was once again freeze- dried to obtain a dry powder. NMR Analysis. The sample (100 mg) was re-suspended in 1 mL of deuterium oxide (D2O) and NaOD (5 μL, 30% by weight) was added to ensure complete solubility for NMR analysis. Samples were analyzed using nuclear magnetic resonance (NMR) spectroscopy on a Bruker Avance 500 MHz spectrometer equipped with a 1H-BB-13C 5 mm, triple resonance broadbanded inverse (TBI) probe. 1-D solution state 1H NMR experiments were performed with 512 scans, a recycle delay of 3 s, and 32 K time domain points. Solvent suppression was achieved by Presaturation Utilizing Relaxation Gradients and Echoes (PURGE) (20). Spectra were apodized through multiplication with an exponential decay corresponding to 1 Hz line broadening, and a zero filling factor of 2. Diffusion-edited experiments were performed with a bipolar pulse longitudinal encode-decode sequence (21). Scans (1024) were collected using a 1.25 ms, 53.5 gauss/cm, sine-shaped gradient pulse, a diffusion time of 50 ms, 8192 time domain points and a sample temperature of 298 K. Spectra were apodized through multiplication with an exponential decay corresponding to 10 Hz line broadening using a zero filling factor of 2. Heteronuclear Multiple Quantum Coherence (HMQC) spectra were collected in phase-sensitive mode using Echo/Anti-echo gradient selection. 1024 scans were collected for each of the 256 increments in the F1 dimension. 1 K data points were collected in F2, a 1J 1H-13C value of 145 Hz and a relaxation delay of 1 s was employed. The F2 dimension was multiplied by an exponential function corresponding to a 15 Hz line broadening, while the F1 dimension was processed using a sine-squared function with a /2 phase shift and a zero-filling factor of 2. Heteronuclear Multiple Bond Correlation (HMBC) were carried out in phase- sensitive mode using Echo/Anti-echo gradient selection (18) and a relaxation optimized delay of 25 ms for the evolution of long-range couplings. 2048 scans were collected for each of the 128 increments in the F1 dimension. 2 K data points were collected in F2 and a relaxation delay of 1 s was employed. The F2 dimension was multiplied by an exponential function corresponding to a 15 Hz line broadening, while the F1 dimension was processed using a sine-squared function with a /2 phase shift and a zero-filling factor of 2. Spectral predictions were carried out using Advanced Chemistry Development’s ACD/SpecManager and ACD/2D NMR Predictor using the Neural Network Prediction algorithms (version 10.02). Parameters used for prediction including line shape, spectral
  • 4. resolution, sweep width, and base frequency were chosen to match those of the real datasets as closely as possible. Results and Discussion Characterization of LDOM from 1D and 2D NMR data Figure 1 shows the 1H NMR data of freshwater DOM from Lake Ontario (LDOM). The top spectrum (Fig. 1A) gives a general profile of the components present in the sample, including major resonances from aliphatics (Fig. 1A – I, note some CRAMs resonances may also resonate in this region), carboxyl-rich alicyclic molecules (CRAMs) (Fig. 1A – II), carbohydrates (Fig. 1A – III), and aromatics (Fig. 1A – IV). Further discussion is provided later in this paper. Signals from larger macromolecular and/or aggregated species can be further emphasized by the use of diffusion editing. Diffusion editing ―spatially encodes‖ molecules at the start of the experiment and then ―refocuses‖ these at the end of the experiment. Species that diffuse or exhibit a high degree of motion during the experiment are not refocused and are essentially ―gated‖ from the final spectrum (21). In essence the spectrum produced will contain only signals from species that undergo little or no self diffusion; hence structures identified will be in macromolecules and/or stable aggregates. The diffusion edited spectrum (Fig. 1B) compared to that of the conventional 1H NMR spectrum (Fig. 1A) shows a generally similar profile, indicating that most of the DOM components are present as either stable aggregates and/or macromolecular species. At this time it is not possible to distinguish whether the species are macromolecular in nature or simply aggregated/associated due to the high concentration of the sample. Future studies based on Diffusion Ordered Spectroscopy are planned to address this aspect of the DOM (13). Due to the large degree of overlap, as evident from the spectrum in Figure 1A, extracting detailed structural information from the 1D NMR alone is difficult. 2D NMR experiments provide increased spectral dispersion as well as additional connectivity information, which permits further characterization of the chemical functionalities present in DOM. Figure 2A and 2B shows Heteronuclear Multiple Quantum Coherence (HMQC) NMR for the LDOM sample. The HMQC experiment detects one bond 1H-13C couplings in an organic structure. A cross peak in an HMQC spectrum represents the chemical shift of both carbon and proton atoms in a C-H unit (12). The HMQC identifies a range of chemical constituents present including anomeric units in carbohydrates (1), conjugated unsaturated moieties (2), aromatics (3), N/O-acetylated components (likely algal or bacterial derived (4) (22, 23)), aliphatics (5), carboxyl-rich alicyclic molecules (6) (CRAMs) (1) (see later for discussion), methyl esters (7), methylene (CH2) from carbohydrates (8), and methine (CH) from carbohydrates (9). Note, assignments offered here are also consistent with TOCSY, NOESY, and edited heteronuclear correlations (data not shown), as well as literature assignments. It is interesting to note that the methoxy group from lignins, often the most intense signal in soil organic matter (12), is not present in LDOM, indicating that terrestrial inputs are quickly transformed in Lake Ontario. The methyl ester region (region 7, Figure 2B) should not be confused with the methoxy from lignin which is not present in LDOM sample. Heteronuclear Multiple Bond Correlation Spectroscopy (HMBC) provides long-range 1 13 H- C couplings (generally up to 3 bonds) and provides critical information as to how H-
  • 5. C units are structurally organized. Information gained from the HMBC (Figure 2C), is discussed below in relation to specific structural components. Identification of major components of LDOM Several structural components have been isolated and shown to comprise marine DOM, including polymeric carbohydrate moieties (1, 24-27), long chain aliphatic compounds (1, 27, 28), acetyl (1, 27, 28), aromatics (1, 27), and recently, carboxyl-rich alicyclic molecules (CRAMs) (1). In comparison, the 1H NMR obtained for LDOM in this study has a general overall profile that is similar to many of the DOM spectra presented in the literature from both marine and freshwater sources (1, 29). The LDOM sample appears remarkably similar to a DOM sample from the Pacific Ocean described by Hertkorn et al. (1). In this sample, Hertkorn et al. (1) described three major resonances attributed to carbohydrates, methyl/methylene resonances from purely aliphatic carbon, and CRAMs. Using the same model (see reference (1)), quantifications for LDOM (Figure 1A) were produced, yielding values of ~17% for the carbohydrate region, ~12% for the aliphatic region, and ~62% for the CRAMs region. These three regions combined comprised the majority (~91%) of the proton signals from LDOM, similar to the quantities reported by Hertkorn et al. (1) for marine DOM. Carbohydrates. Carbohydrate components have been shown to represent up to 50% of high-molecular weight (HMW) surface marine DOM but comprise a much smaller proportion of deeper ocean waters (30). Of these however, only a small fraction are simple carbohydrates structures, which contribute only a small percentage to the total composition of marine DOM (27). The majority of carbohydrates identified in the literature appear to comprise complex polymeric structures referred to as heteropolysaccharides (HPS) (25, 30) or acyl polysaccharides (APS) which contain carbohydrates, lipids and acetate to varying degrees (24). These polymeric carbohydrates have been shown to be major constituents of ultra-filtered DOM and a rapidly cycling component of marine surface waters (24). Similarly, the freshwater LDOM contains a considerable contribution from carbohydrates which are not removed during diffusion editing (see Figure 1B) and may potentially be associated with acetyl groups (see Figure 2B – 4). This suggests that the freshwater sample may indeed contain a similar proportion of large complex carbohydrate moieties compared with marine DOM. This is supported, in part, by comparison of the contour shapes for the carbohydrate region which are roughly similar in the Pacific Ocean DOM (1) and the LDOM considered here. Unfortunately, in the case of carbohydrates, the 2D HMBC data does not provide additional information as to the structures present in freshwater DOM, mainly due to the majority of carbohydrate signals being below the detection limit of the HMBC. Further work is needed to confirm the similarity of the carbohydrates in freshwater and marine DOM, both in terms of origin and structure. Carboxyl-rich alicyclic molecules (CRAMs). Characteristics of the region in the LDOM sample spanning approximately 1.7—3.3 ppm (Figure 1) has been shown to be prevalent in a vast majority of 1H NMR spectra in the literature for both marine and freshwater DOM where NMR profiles have been presented (1, 19, 24, 28-31). However, the components that comprise this region were not adequately defined until a recent study by Hertkorn et al. (1), who showed this region to largely contain carboxyl-rich alicyclic
  • 6. molecules (CRAMs). Herkorn et al. (1) describe CRAMs as a major refractory component of marine DOM which is likely derived from sterols and hopanoids (both categories of terpenoids) and is consistent with carboxylated alicyclic structures with carboxyl to aliphatic carbon ratios of approximately 1:2 to 1:7. Although not conclusively shown to be present in freshwater DOM, as Hertkorn et al. (1) points out, the presence of CRAMs in freshwater is likely due to the global distribution of biomolecules and the similarity in biogeochemical processes that occur within the environment. It is not surprising therefore, that evidence for CRAM-like structures in the LDOM sample are seen in both the 1D and 2D NMR spectra. Long-range correlations from the HMBC data (Fig. 2C – I) not only substantiates the presence of CRAMs in LDOM, but also provides strong evidence to corroborate the structure of CRAMs proposed by Hertkorn et al. (1). As previously noted, HMBC identifies long-range 1H-13C correlations. HMBC correlations (Fig. 2C – I) show carboxylic components (Fig. 2C, bottom cross-peak) directly coupled to alicyclic rings (Fig. 2C, top cross-peak) in the LDOM sample. This is consistent with CRAMs structures found by Hertkorn et al.(1) in marine DOM. In fact, it would appear that the CRAMs present in LDOM contain structures that are similar to large, fused, non-aromatic rings, with a high ratio of substituted carboxyl groups most consistent with Isomer I proposed by Hertkorn et al. (1). The HMBC data also shows that the CRAMs in LDOM contain few substitutions from other functionalized moieties (i.e. methyl, hydroxyl), with the majority of substitutions being those from carboxyl groups (hence the lack of other correlations in this region). This is further supported by predicted HMBC spectra using software available from Advanced Chemistry Development (data not shown). These simulations were conducted on a multitude of structures; however, only those simulations from alicyclic, non-aromatic rings with a high ratio of substituted carboxyl groups produced a similar HMBC profile. Figure 3A shows an example of a CRAM-type structure. Note that Hertkorn et al. (1) found over 600 ions with CRAM molecular compositions in their marine DOM sample; therefore it is impossible to provide an exact ―structure‖ of CRAMs, as numerous, structurally-different components are likely contributing to the signals in this region. The structure in 3A is shown as an example only, and contains two key features that are likely characteristic of all CRAMs structures; a cyclic terpenoid backbone and a high degree of carboxylation. Further details, for example, additional substitutions, cross-linkages, molecular size etc., cannot be determined from the NMR data at hand. Carotenoid-derived aliphatic molecules (CDAMs). Carotenoids are known to be prevalent in the aquatic environment (32-36). Over 650 individual species have currently been identified in aquatic organisms, with a net annual production estimated at over 100 million tons from photosynthetic organisms alone (35). Most of these species contain conjugated double bond systems with substituted methyl groups on the double bonds. The fate of these abundant materials is not well understood (32), especially in freshwater environments, in which they are known to be preferentially preserved (36). Interestingly, characteristic resonances from conjugated double bonds are visible in the HMQC spectrum of LDOM (see Figure 2A – 2). This resonance is consistent with a significant contribution of carotenoids in the LDOM sample (note the particular example shown in Figure 3C does not have a high degree of conjugation, but many carotenoid structures exhibit extensive conjugation). The HMBC data (see Figure 2C – II) contains a region consistent with functionalized aliphatic molecules that is likely derived from carotenoid precursors. Figure 3B shows a
  • 7. representation of a methyl substituted double bond (characteristic of those found in isoprene units, there are 8 isoprene units in most carotenoids). The carbon with the methyl group directly attached is tertiary (three carbon bonds) and thus more prone than the secondary carbon (two carbon bonds) of the double bond to undergo substitution reactions. Crosspeak ‗d‘ in the HMBC spectrum (Figure 2C – D) clearly shows that the methyl (and adjacent CH2 units in the chain) correlate strongly with a carboxylic acid group, indicating carboxylation at this carbon. Figure 3B (middle structure) shows the structural unit that likely forms regions a, b, and d, which correlate with the same regions denoted in the HMBC data (see Figure 2C – II). However, an additional region ‗c‘ is also present in the experimental data. We tentatively suggest that over time the methyl group may in some cases, become oxidized to an OH group (see Figure 3B, structure on right) thus giving rise to an additional carbon proton correlation at ~80 ppm (see Figure 2C – c). The chemical shift range observed in the HMBC for ‗c‘ (~80 ppm carbon) is rather unusual, and can only occur when two electronegative groups are directly attached to the carbon. NMR predictions indicate that this chemical shift most likely occurs when both a carboxyl and hydroxyl group are substituted on the same carbon. To confirm the assignments offered above, extensive simulations were carried out for carboxylated structures that would form from common carotenoids found in the aquatic environment (35). Figure 3C shows an example of a carotenoid precursor and a likely formation product. Phytoene (Fig. 3C – top) is synthesized by algae and microorganisms and is a precursor from which other carotenoids are derivatized (35). This precursor may, through biological or chemical processes, form the functionalized carotenoid-derived aliphatic material (CDAM) shown in Figure 3C – bottom. Figure 4C shows the simulation of this model CDAM (Fig. 3C – bottom) which matches very well to the experimental HMBC data (Figure 2C – II). Note for the purposes of simulation the double bond which is unlikely to remain intact (see later) is replaced by a single bond (other possibilities for example bond cleavage etc. are possible, also see later). To further confirm that other common groups in DOM cannot produce the experimental HMBC data, full simulations were carried out for the Open Chain Aliphatic Polycarboxylic acid Model (OCAPM) used by Hertkorn et al. (1). They created this OCAPM as a random open chain model that incorporated all common functionalities found in DOM. Ultimately, they concluded based on 1D and HSQC NMR data (1 bond 1H-13C correlations, similar to the HMQC employed here) that such material could not be present in high abundance in marine DOM. Indeed this conclusion is also supported by the suite of NMR data presented here for freshwater DOM. In particular, the match between the simulated HMBC data of OCAPM (Figure 4B) with the experimental HMBC data (Figure 2C) is particularly poor. This strongly suggests that randomly linked functionalities cannot account for the experimental HMBC data collected for LDOM, which ultimately matches very well with a mixture of carboxylated carotenoid-derived aliphatic molecules (CDAMs). While it is clear that CDAMs are strongly supported by the NMR data, it is not easy to decipher the fate of the remaining double bonds (i.e. double bonds in a straight aliphatic chain that have no methyl substitution) in carotenoids. Many carotenoid structures contain extensive conjugated networks of double bonds, and indeed these conjugated double bonds are clear in the HMQC data (see Figure 2A, region 2). However, if methyl-substituted double bonds are preferentially derivatized, while non- substituted straight chain double bonds (NSDB see Figure 3C) are left intact, a strong contribution in the HMQC region labeled NCDB (non-conjugated double bonds) in
  • 8. Figure 2A would be expected. This however, is not observed and no evidence for ―isolated‖ double bonds is observed in the HMBC data (region not shown). This seems to suggest that these double bonds undergo some sort of biological and/or chemical alteration. While it is known that such bonds in carotenoids can be easily cleaved in aquatic media (37), other potential reactions could also include methylation, hydrogenation, oxidation, and/or crosslinking with other DOM species. Unfortunately, from the NMR data at hand, it is impossible to determine the exact fate of these CDAM species. As such, the unit in the precursor species in Figure 3C (top) denoted as NSDB is annotated with a potential cleavage and R groups in the transformation product, essentially indicating the reaction at the NSDB is still to be determined. Further Considerations Here, it is demonstrated that carboxyl-rich alicyclic molecules (CRAMs) are potentially the largest contributor to freshwater DOM in the Great Lakes system. This is consistent with findings reported for Pacific Ocean DOM (1). While CRAMs appear to be derived from cyclic terpenoids, another fraction is identified in freshwater DOM that appears to be derived from linear carotenoids (32-38) (a sub-category of linear terpenoids). In addition, smaller contributions from heteropolysaccharides (22, 39) and aromatics are also present in LDOM. At present, little detail can be obtained from the aromatic compounds in LDOM, mainly due to their relatively low abundance in the sample. In addition to the species observed above, it is very likely that a considerable proportion of intact precursor molecules, mainly cyclic and linear terpenoids, are also present in LDOM. In the case of carotenoids, these contributions are evident from the conjugated double bond systems that are well resolved in the HMQC data (Fig. 2A – 2). The fact that aquatic DOM from Lake Ontario is mainly derived from terpenoids is consistent with earlier reports that suggests the majority of dissolved organic matter from landfill sites, surface water and groundwater is also of terpenoid origin (2). Terpenoids comprise the most abundant family of natural compounds in nature (40), with over 22,000 structures already defined (41, 42), many of which are derived from membrane constituents and secondary metabolites from various prokaryotic and eukaryotic organisms (43), as well as plant-derived species from both the terrestrial (44) and aquatic environments (45). Given the presence of structurally similar precursor components in both freshwater and marine environments, it is difficult to ascertain whether the constituents of DOM in freshwater are of terrestrial or aquatic origin. However, in light of this, it is known that certain terpenoid structures are specific to certain species (33, 35). Thus it will be interesting to observe whether the signatures of specific ―tracer‖ terpenoids are preserved in DOM over time, potentially providing a rich source of information as to the sources and dynamics of dissolved carbon on a global scale. Finally, it is important to point out that the multidimensional NMR approaches employed here and in other works (1, 12, 23, 46-48), are not just helping to unravel the key structural components present in a major global carbon pool, but these approaches are also permitting more detailed assignments of complex NMR datasets. This is critical, as once assignments can be made, the full arsenal of modern nuclear magnetic resonance techniques can be better employed to understand key processes, such as aggregation, flocculation and contaminant interactions – processes that have historically been hampered by a lack of understanding of the principal structural components present in major carbon pools such as dissolved organic matter.
  • 9. Acknowledgements We thank the National Science and Engineering Research Council of Canada (NSERC) (discovery grant to A.J.S.), the Canadian Foundation for Climate and Atmospheric Sciences and the International Polar Year (IPY) for providing funding.
  • 10. Figure 1. 1H NMR spectra showing A) freshwater DOM taken from Lake Ontario (LDOM), and B) the diffusion edited spectrum for LDOM. Resonances from I – aliphatics, II – CRAMs, III – carbohydrates, and IV – aromatics. Figure 2. 2D NMR spectra of A) Heteronuclear Multiple Quantum Coherence (HMQC) spectrum for the LDOM, B) zoom region of the HMQC spectrum from A, with contours reduced by a factor of 5 for clarity, C) Heteronuclear Multiple Bond Correlation (HMBC) of LDOM giving structural information for CRAMs (region I) and carotenoid-derived aliphatic molecules (CDAMs) (region II). Specific assignments are as follows : 1=anomeric carbon from carbohydrates, 2=conjugated unsaturated aliphatics, 3=aromatics, 4=N/O acetate, 5=aliphatics, 6=CRAMs, 7=methyl esters, 8=methylene from carbohydrates, and 9=methine from carbohydrates. NCDB = non-conjugated double bonds, see text. Notations a,b,c,d are used for identification of crosspeaks in the HMBC, see text for discussion. Figure 3. A = CRAM structure characterized by cyclic terpenoid rings highly substituted with carboxylic acids. Note this is one of possibly 1000‘s of CRAM structures and isomers that may be present in DOM and is shown as an example only. B = Schematic outlining the specific functionalities that produce the types of cross peaks observed in the HMBC data. Shaded regions (a-d) represent those substituents which give rise to the corresponding aliphatic cross peaks in HMBC (Fig. 2C – II a-d), see text for discussion. C = An example carotenoid structure which, through chemical or biological processes, may give rise to carotenoid-derived aliphatic molecules (CDAMs). The structure shown is an example only and demonstrates the types of units that are in high abundance in CDAMs. NSDB indicates a mid-chain double bond that has no substitution (non- substituted double bond). The fate of these units cannot be determined from the data at hand and is discussed further in the text. Figure 4: A) Expanded region of the 1H NMR of LDOM from Fig. 1A. B) Simulated HMBC with 1H NMR projection of Open Chain Aliphatic Polycarboxylic acid Model used by Hertkorn et al. to negate the presence of random linear substitutions in marine DOM (1). C) Simulated HMBC with 1D 1H NMR projection for the example CDAM structure shown in Fig. 3C.
  • 11. I II III A IV B 9 8 7 6 5 4 3 2 ppm
  • 12. ppm A 20 40 60 ppm 80 100 3 1 120 2 NCDB 140 8 7 6 5 4 3 2 1 ppm ppm ppm 4 B 7 20 5 40 ppm 9 6 60 8 80 Boxed region from above (A), Contours reduced by factor of 5 5 4 3 2 1 ppm ppm ppm C a 50 b 100 ppm I II c 150 d 200 4 3 2 1 ppm ppm
  • 13. B Tertiary HO d O HO d O Carbon a b a a c a A O OH OH OH a O C CH 3 CH 3 CH 3 CH 3 NSDB CH 3 OH H 3C CH 3 CH 3 CH 3 CH 3 O O HO O OH O OH R CH 3 CH 3 OH CH 3 HO O HO O HO O HO O CH 3 ≈ H 3C O OH O OH O OH O OH CH 3 CH 3 OH CH 3 R
  • 14. A 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 0 B 20 40 60 80 ppm 100 120 140 160 180 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm 0 C 20 40 60 80 ppm 100 120 140 160 180 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm
  • 15. References (1) Hertkorn, N.; Benner, R.; Frommberger, M.; Schmitt-Kopplin, P.; Witt, M.; Kaiser, K.; Kettrup, A.; Hedges, J. I., Characterization of a major refractory component of marine dissolved organic matter. Geochimica Et Cosmochimica Acta 2006, 70, (12), 2990-3010. (2) Leenheer, J. A.; Nanny, M. A.; McIntyre, C., Terpenoids as major precursors of dissolved organic matter in landfill leachates, surface water, and groundwater. Environmental Science & Technology 2003, 37, (11), 2323-2331. (3) Hedges, J. I.; Keil, R. G.; Benner, R., What happens to terrestrial organic matter in the ocean? Organic Geochemistry 1997, 27, (5-6), 195-212. (4) Hedges, J. I., Global Biogeochemical Cycles - Progress and Problems. Marine Chemistry 1992, 39, (1-3), 67-93. (5) Benner, R.; Benitez-Nelson, B.; Kaiser, K.; Amon, R. M. W., Export of young terrigenous dissolved organic carbon from rivers to the Arctic Ocean. Geophysical Research Letters 2004, 31, (5). (6) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E., Water Solubility Enhancement of Some Organic Pollutants and Pesticides by Dissolved Humic and Fulvic-Acids. Environmental Science & Technology 1986, 20, (5), 502-508. (7) Akkanen, J.; Kukkonen, J. V. K., Measuring the bioavailability of two hydrophobic organic compounds in the presence of dissolved organic matter. Environmental Toxicology and Chemistry 2003, 22, (3), 518-524. (8) Lam, B.; Simpson, A. J., Passive sampler for dissolved organic matter in freshwater environments. Analytical Chemistry 2006, 78, (24), 8194-8199. (9) Thurman, E. M.; Malcolm, R. L., Preparative Isolation of Aquatic Humic Substances. Environmental Science & Technology 1981, 15, (4), 463-466. (10) Aiken, G. R.; Thurman, E. M.; Malcolm, R. L.; Walton, H. F., Comparison of Xad Macroporous Resins for the Concentration of Fulvic-Acid from Aqueous-Solution. Analytical Chemistry 1979, 51, (11), 1799-1803. (11) Cardoza, L. A.; Korir, A. K.; Otto, W. H.; Wurrey, C. J.; Larive, C. K., Applications of NMR spectroscopy in environmental science. Progress in Nuclear Magnetic Resonance Spectroscopy 2004, 45, (3-4), 209-238. (12) Simpson, A., Multidimensional solution state NMR of humic substances: A practical guide and review. Soil Science 2001, 166, (11), 795-809. (13) Simpson, A. J., Determining the molecular weight, aggregation, structures and interactions of natural organic matter using diffusion ordered spectroscopy. Magnetic Resonance in Chemistry 2002, 40, S72-S82. (14) Simpson, A. J.; Kingery, W. L.; Spraul, M.; Humpfer, E.; Dvortsak, P.; Kerssebaum, R., Separation of structural components in soil organic matter by diffusion ordered spectroscopy. Environmental Science & Technology 2001, 35, (22), 4421-4425. (15) Simpson, A. J.; Lam, B.; Diamond, M. L.; Donaldson, D. J.; Lefebvre, B. A.; Moser, A. Q.; Williams, A. J.; Larin, N. I.; Kvasha, M. P., Assessing the organic composition of urban surface films using nuclear magnetic resonance spectroscopy. Chemosphere 2006, 63, (1), 142-152. (16) Simpson, M. J., Nuclear magnetic resonance based investigations of contaminant interactions with soil organic matter. Soil Science Society of America Journal 2006, 70, 995-1004.
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