Major Structural Components in Freshwater Dissolved Organic Matter

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:

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
1 13
H- C 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.

I

II

III

A IV

B

9 8 7 6 5 4 3 2 ppm

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

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

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

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.

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