Major Structural Components in Freshwater Dissolved Organic Matter

Buuan Lam1, Mehran Alaee

2, Brent Lefebvre

3, Arvin Moser

3, Antony Williams

3 and

André J. Simpson1

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

2National Water Research Institute, Environment Canada, 867 Lakeshore Road, P.O. Box 5050,

Burlington, Ontario, Canada, L7R 4A6

3Advanced 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:

andre.simpson@utoronto.ca

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.

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

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 1H-

13C couplings (generally up to 3 bonds) and provides critical information as to how H-

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

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

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

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.

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.

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.

B

A

23456789 ppm

I

II

III

IV

B

A

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Boxed region from above (A),

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CH3 CH3

CH3 CH3

CH3 CH3

CH3

CH3

CH3

CH3

CH3 CH3

OH CH3

OH CH3

CH3

CH3

CH3

CH3

O OHO OH O OH O OH

OOH OOH OOH OOHR

R

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OH O

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CH3 CH3

CH3 CH3

CH3 CH3

CH3

CH3

CH3

CH3

CH3 CH3

OH CH3

OH CH3

CH3

CH3

CH3

CH3

O OHO OH O OH O OH

OOH OOH OOH OOHR

R

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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.

(17) Great Lakes Environmental Research Laboratory: National Oceanic and

Atmospheric Association. http://www.glerl.noaa.gov/pr/ourlakes/lakes.html (May 2007),

(18) Cicero, D. O.; Barbato, G.; Bazzo, R., Sensitivity enhancement of a two-

dimensional experiment for the measurement of heteronuclear long-range coupling

constants, by a new scheme of coherence selection by gradients. Journal of Magnetic

Resonance 2001, 148, (1), 209-213.

(19) Simpson, A. J.; Tseng, L. H.; Simpson, M. J.; Spraul, M.; Braumann, U.; Kingery,

W. L.; Kelleher, B. P.; Hayes, M. H. B., The application of LC-NMR and LC-SPE-NMR

to compositional studies of natural organic matter. Analyst 2004, 129, (12), 1216-1222.

(20) Simpson, A. J.; Brown, S. A., Purge NMR: Effective and easy solvent

suppression. Journal of Magnetic Resonance 2005, 175, (2), 340-346.

(21) Wu, D.; Chen, A.; Johnson, C. S., Jr., An improved diffusion-ordered

spectroscopy experiment incorporating bipolar-gradient pulses. J. Magn. Reson., Ser. A

1995, 115, (2), 260-4.

(22) Aluwihare, L. I.; Repeta, D. J., A comparison of the chemical characteristics of

oceanic DOM and extra-cellular DOM produced by marine algae. Marine Ecology-

Progress Series 1999, 186, 105-117.

(23) Simpson, A. J.; Song, G. X.; Smith, E.; Lam, B.; Novotny, E. H.; Hayes, M. H.

B., Unraveling the structural components of soil humin by use of solution-state nuclear

magnetic resonance spectroscopy. Environmental Science & Technology 2007, 41, (3),

876-883.

(24) Aluwihare, L. I.; Repeta, D. J.; Chen, R. F., Chemical composition and cycling of

dissolved organic matter in the Mid-Atlantic Bight. Deep-Sea Research Part Ii-Topical

Studies in Oceanography 2002, 49, (20), 4421-4437.

(25) Aluwihare, L. I.; Repeta, D. J.; Chen, R. F., A major biopolymeric component to

dissolved organic carbon in surface sea water. Nature 1997, 387, (6629), 166-169.

(26) Pakulski, J. D.; Benner, R., Abundance and Distribution of Carbohydrates in the

Ocean. Limnology and Oceanography 1994, 39, (4), 930-940.

(27) Benner, R., Chemical Composition and Reactivity. In Biogeochemistry of Marine

Dissolved Organic Matter, Hansell, D. A.; Carlson, C. A., Eds. Academic Press: New

York, 2002; pp 59-85.

(28) Hedges, J. I.; Eglinton, G.; Hatcher, P. G.; Kirchman, D. L.; Arnosti, C.; Derenne,

S.; Evershed, R. P.; Kogel-Knabner, I.; de Leeuw, J. W.; Littke, R.; Michaelis, W.;

Rullkotter, J., The molecularly-uncharacterized component of nonliving organic matter in

natural environments. Organic Geochemistry 2000, 31, (10), 945-958.

(29) Morris, K. F.; Cutak, B. J.; Dixon, A. M.; Larive, C. K., Analysis of diffusion

coefficient distributions in humic and fulvic acids by means of diffusion ordered NMR

spectroscopy. Analytical Chemistry 1999, 71, (23), 5315-5321.

(30) Benner, R.; Pakulski, J. D.; McCarthy, M.; Hedges, J. I.; Hatcher, P. G., Bulk

Chemical Characteristics of Dissolved Organic-Matter in the Ocean. Science 1992, 255,

(5051), 1561-1564.

(31) Sannigrahi, P.; Ingall, E. D.; Benner, R., Cycling of dissolved and particulate

organic matter at station Aloha: Insights from C-13 NMR spectroscopy coupled with

elemental, isotopic and molecular analyses. Deep-Sea Research Part I-Oceanographic

Research Papers 2005, 52, (8), 1429-1444.

(32) Louda, J. W.; Liu, L.; Baker, E. W., Senescence- and death-related alteration of

chlorophylls and carotenoids in marine phytoplankton. Organic Geochemistry 2002, 33,

(12), 1635-1653.

(33) Liaaenjensen, S., Marine Carotenoids - Recent Progress. Pure and Applied

Chemistry 1991, 63, (1), 1-12.

(34) Liaaenjensen, S., Marine Carotenoids - Selected Topics. New Journal of

Chemistry 1990, 14, (10), 747-759.

(35) Matsuno, T., New Structures of Carotenoids in Marine Animals. Pure and

Applied Chemistry 1985, 57, (5), 659-666.

(36) Moss, B., Studies on Degradation of Chloro-Phyll Alpha and Carotenoids in

Freshwaters. New Phytologist 1968, 67, (1), 49-&.

(37) Kanasawud, P.; Crouzet, J. C., Mechanism of Formation of Volatile Compounds

by Thermal-Degradation of Carotenoids in Aqueous-Medium .2. Lycopene Degradation.

Journal of Agricultural and Food Chemistry 1990, 38, (5), 1238-1242.

(38) Fox, D. L., Some Biochemical Aspects of Marine Carotenoids. Fortschritte Der

Chemie Organischer Naturstoffe 1948, 5, 20-39.

(39) Repeta, D. J.; Quan, T. M.; Aluwihare, L. I.; Accardi, A. M., Chemical

characterization of high molecular weight dissolved organic matter in fresh and marine

waters. Geochimica Et Cosmochimica Acta 2002, 66, (6), 955-962.

(40) Devon, T. K.; Scott, A. I., Handbook of Naturally Occurring Compounds.

Academic Press: New York, 1972; Vol. II: Terpenes.

(41) McGarvey, D. J.; Croteau, R., Terpenoid Metabolism. Plant Cell 1995, 7, (7),

1015-1026.

(42) Connolly, J. D.; Hill, R. A., Dictionary of Terpenoids. Chapman and Hall:

London, 1991.

(43) Ourisson, G.; Rohmer, M.; Poralla, K., Prokaryotic Hopanoids and Other

Polyterpenoid Sterol Surrogates. Annual Review of Microbiology 1987, 41, 301-333.

(44) Harborne, J. B.; Tomas-Barberan, F. A., Ecological Chemistry and Biochemistry

of Plant Terpenoids. Oxford University Press: New York, 1991.

(45) Faulkner, D. J. In Some Terpenoids from Marine Organisms, Proceedings of the

symposium on sources, effects, and sinks of hydrocarbons in the aquatic environment,

The American Univeristy, Washington DC, August, 1976; The American Univeristy,

Washington DC, 1976; pp 200-212.

(46) Hertkorn, N.; Claus, H.; Schmitt-Kopplin, P. H.; Perdue, E. M.; Filip, Z.,

Utilization and transformation of aquatic humic substances by autochthonous

microorganisms. Environmental Science & Technology 2002, 36, (20), 4334-4345.

(47) Kaiser, E.; Simpson, A. J.; Dria, K. J.; Sulzberger, B.; Hatcher, P. G., Solid-state

and multidimensional solution-state NMR of solid phase extracted and ultrafiltered

riverine dissolved organic matter. Environmental Science & Technology 2003, 37, (13),

2929-2935.

(48) Kelleher, B. P.; Simpson, A. J., Humic substances in soils: Are they really

chemically distinct? Environmental Science & Technology 2006, 40, (15), 4605-4611.