Molecular characterization of dissolved organic …organic matter which acts as carbon sink in the...

REVIEW

Molecular characterization of dissolved organicmatter (DOM): a critical review

Antonio Nebbioso & Alessandro Piccolo

Received: 30 June 2012 /Revised: 12 August 2012 /Accepted: 14 August 2012 /Published online: 11 September 2012# Springer-Verlag 2012

Abstract Advances in water chemistry in the last decadehave improved our knowledge about the genesis, com-position, and structure of dissolved organic matter, andits effect on the environment. Improvements in analyti-cal technology, for example Fourier-transform ion cyclo-tron (FT-ICR) mass spectrometry (MS), homo andhetero-correlated multidimensional nuclear magnetic res-onance (NMR) spectroscopy, and excitation emissionmatrix fluorimetry (EEMF) with parallel factor(PARAFAC) analysis for UV–fluorescence spectroscopyhave resulted in these advances. Improved purificationmethods, for example ultrafiltration and reverse osmosis,have enabled facile desalting and concentration of fresh-ly collected DOM samples, thereby complementing theanalytical process. Although its molecular weight (MW)remains undefined, DOM is described as a complexmixture of low-MW substances and larger-MW biomo-lecules, for example proteins, polysaccharides, and exo-cellular macromolecules. There is a general consensusthat marine DOM originates from terrestrial and marinesources. A combination of diagenetic and microbialprocesses contributes to its origin, resulting in refractoryorganic matter which acts as carbon sink in the ocean.Ocean DOM is derived partially from humified products

of plants decay dissolved in fresh water and transportedto the ocean, and partially from proteinaceous and poly-saccharide material from phytoplankton metabolism,which undergoes in-situ microbial processes, becomingrefractory. Some of the DOM interacts with radiationand is, therefore, defined as chromophoric DOM(CDOM). CDOM is classified as terrestrial, marine,anthropogenic, or mixed, depending on its origin. Ter-restrial CDOM reaches the oceans via estuaries, whereasautochthonous CDOM is formed in sea water by micro-bial activity; anthropogenic CDOM is a result of humanactivity. CDOM also affects the quality of water, byshielding it from solar radiation, and constitutes a car-bon sink pool. Evidence in support of the hypothesisthat part of marine DOM is of terrestrial origin, beingthe result of a long-term carbon sedimentation, has beenobtained from several studies discussed herein.

Keywords Dissolved organic matter . Carbon cycle .

Humic substances .Water . Dissolved organic carbon .

Natural products

Environmental relevance of DOM

Natural organic matter (NOM) is a product of plant andanimal tissue decay, and, together with the biota, is ofpivotal importance in the global carbon cycle. NOM isfound in soil, sediments, and natural water and itsbiological mineralization contributes, with anthropogenicemissions, to increasing the carbon dioxide content ofthe atmosphere. Any control of the environmental pro-cesses of NOM transformation would become possibleonly if additional knowledge about its molecular com-position were accumulated [1].

Molecular characterization of NOM has therefore be-come a primary research objective in environmental and

A. Nebbioso (*) :A. PiccoloCentro Interdipartimentale di Risonanza Magnetica Nucleare perl’Ambiente, l’Agroalimentare e i Nuovi Materiali (CERMANU),Università degli Studi di Napoli Federico II,via Università 100,80055 Portici, Italye-mail: [email protected]

A. PiccoloDipartimento di Scienze del Suolo, della Pianta, Dell’Ambiente edelle Produzioni Animali (DISSPAPA), Università degli Studi diNapoli Federico II,via Università 100,80055 Portici, Italy

Anal Bioanal Chem (2013) 405:109–124DOI 10.1007/s00216-012-6363-2

ecological chemistry [2]. The recent development of instru-mental techniques, for example NMR spectroscopy, hyphenat-ed mass spectrometry, and X-ray spectroscopic methods, hasgreatly enhanced our ability to detect and characterize singleorganic compounds and, occasionally, homogeneous mixtures.However, because of the great complexity and heterogeneouscomposition of NOM, its characterization remains a challenge.

Dissolved organic matter (DOM) cannot be correctlyregarded as a chemical solution; it is, rather, a very finecolloidal suspension. The distinction between particulate or-ganic matter (POM) and DOM is only operationally defined[3]: DOM is assumed to pass through a 0.45-μm filter porewhereas POM is blocked (Fig. 1). DOM and its compositionare important in natural-water ecosystems because of thenumber of processes in which it becomes involved. DOM actsas a strong chelating agent for metals, thus affecting theirsolubility, transport, and toxicity [4]. It is fundamentally in-volved in the transport of organic pollutants [5], formation ofcolloidal particles (and affects their surface area) [6], aqueousphotochemical reactions [7], nutrients cycling and availability[8], pH-buffering [9], and the distribution of ions betweenaqueous and solid phases [10]. DOM and POM are alsoimportant sources of energy in river-water ecosystems [11].

The most abundant pool of DOM is that in the oceans, alsoknown as marine DOM. This is generally believed to be thedecay products of phytoplankton and consists of 25–50 %proteins, 5–25 % lipids, and up to 40 % carbohydrates[12–14]. However, DOM from terrestrial sources, for examplebiomass, plant litter, and soil organic matter, also reachesocean waters by transport from rivers, lakes, glaciers, andother natural sources. Such transfer of terrestrial carbon is animportant link in the global carbon cycle. Estimates of totalglobal transport of organic carbon to the oceans range from0.4 to 0.9×1015g−1year−1 [15–17].

Genesis of DOM

Terrestrial DOM is the result of biological degradation andprogressive concentration of organic compounds particular-ly resistant to degradation. Degradation of vascular plantsfurnishes DOM containing approximately 10 % proteins,30–50 % carbohydrates (mainly cellulose), some lipids con-centrated in the roots and leaf cuticles [18], 15–25 % lignin[19], and other biomacromolecules. Moreover, it seemsthere is a correlation between environmental conditionsand type of terrestrial DOM derived from soil [20].

Lignin, an important tracer for terrestrial OM [21], con-sists of repeating phenylpropanoid units which are randomlylinked to each other by ether and carbon–carbon bonds. Thisbranched macromolecular network confers on lignin chem-ical stability that is assumed to resist extensive microbialdegradation [22, 23]. Proteins and carbohydrates are, incontrast, biolabile compounds, because of the susceptibilityof peptide and glycosidic bonds to hydrolysis by a variety ofenzymes [24–26]. However, even labile molecules are pre-served in marine sediments under specific conditions, e.g.,when they are protected in the frustules of marine organisms[27] and within recalcitrant complex structures formed withDOM [28–30]. Such highly complex OM is also referred toas molecularly uncharacterized (MU-OM), because its com-position can be hardly resolved at a molecular level byconventional analytical techniques [31, 32]. MU-OM mayaccount for up to 80 % of marine sediments [31, 33]. Highlyunsaturated compounds are also present in DOM of naturalwaters as strongly resistant organic components, and arealso referred to as black carbon (BC) or the product of eitherincomplete burning of biomass or recycled kerogen. Evenbacteria-derived OM, for example recalcitrant peptidogly-can from cell walls, can be found in DOM [34, 35].

Fig. 1 Size range of particulate(POM) and dissolved organicmatter (DOM) and organiccompounds in natural waters.AA, amino acids; CHO,carbohydrates; CPOM, coarseparticulate organic matter; FA,fatty acids, FPOM, fineparticulate organic matter; HA,hydrophilic acids; HC,hydrocarbon; VPOM, very fineparticulate organic matter.Adapted from Ref. [3]

110 A. Nebbioso, A. Piccolo

As a result of this heterogeneous and complex chemicalnature, research on molecular characterization of DOM isconducted widely by use of several different approaches andstrategies. The purpose of this review is, thus, to evaluatethe most recent advances in analytical methods for charac-terization of DOM in natural water bodies.

Characterization of DOM

State of the art of DOM isolation and purification

DOM in the environment is found, with rare exceptions (e.g.Suwannee River DOM), at extremely low concentrations(0.5–1.0 mgL−1 in the oceans); inorganic salts exceed thisvalue by several orders of magnitude. Therefore, specifical-ly designed techniques are generally used to increase theconcentration of DOM and to remove salts. Retention-basedmethods involving XAD resins [36] or C18 stationary phases[37] have been extensively investigated, only to reveal that avariable but substantial part of DOM is lost because ofincomplete retention [38].

Ultrafiltration (UF) [39–42] has been used to removelarge volumes of water through membrane pores which arerestrictive for DOM but not for water molecules and smallions. It is a formidable desalting method, that is also used topurify water from excessive DOM content [43]. UF is alsoaffected by incomplete recovery of organic carbon, but to alesser extent.

Reverse osmosis (RO) is an improvement of UF. ROoperates similarly to UF by allowing water through mem-branes with a restrictive cut-off for DOM. However, in ROthe solution is forced by a pressure gradient to flow againstosmotic flow (hence reverse osmosis). Different and morerestrictive membranes are used for RO than for UF, resultingin greater retention of ions in DOM samples. Such retentionconsists mainly of ions derived from H4SiO4 and H2SO4.Development of methods such as RO coupled with electro-dialysis [44] and pulsed electrodialysis [45] was, in fact,intended to minimize these inorganic impurities. These pro-cesses are now rapidly becoming conventional for DOMpurification [46] and are in constant development, optimi-zation, and standardization [47].

Treatment of sediments to enable solid-state cross-polarization magic-angle spinning (CPMAS) NMR spec-troscopy of their labile organic matter is routinely basedon acid washing and chemical digestion with HCl and/orHF [48]. However, adoption of sensitive instrumental meth-ods for DOM analysis, for example ultrahigh-resolutionmass spectrometry, may prevent such sample pretreatment[49], thus limiting the formation of artefacts resulting fromthe processes of DOM concentration and pH modification[50]. A promising application for DOM separation seems to

be the development of carbon nanotubes as solid-phaseextraction (SPE) stationary phases which exploit the affinityof nano-structures for organic compounds in solution [51].Such stationary phases have, however, so far resulted inlimited recovery that ranges between 30 and 80 %, depend-ing on DOM type, and specific selectivity for low-molecular-weight DOM fractions.

There is increasing interest in DOM from other sources,for example atmospheric aerosols [52]. Discovery, charac-terization and quantitative assessment of such alternativesources is critical for understanding the relevance of DOMin global carbon dynamics. This, however, is a challengingtask, because sampling and analysis of fog-water-derivedDOM is more difficult than for surface or ground water.Interest in this subject is increasing, and, recently, a thor-ough review of analytical methods for airborne DOM aero-sols was published by Duarte and Duarte [53]. Theseauthors emphasized the environmental significance of thisunderestimated source of organic carbon and discussed theinherent use of NMR, IR, and MS methods for its analysis.

Main analytical methods for DOM

Several problems may affect the analysis of NOM—diffi-culty of complete dissolution [54], lack of proper molecularseparation [54], extreme heterogeneity of samples, mutualinterference from different classes of compound, tendencyof association in complex superstructures [55].

DOM is no exception. However, substantial analyticalimprovement has recently resulted from the introduction ofFourier-transform ion cyclotron (FT-ICR) mass spectrome-try (MS). This is the most advanced instrumentation avail-able for detection of ionized organic compounds, because ofits ultrahigh resolution and because it is usually coupledwith non-destructive ion sources, for example electrosprayionization (ESI). The impact of FT-ICR MS on NOM anal-ysis has been outstanding [56], and it has rapidly becomeone of the first choices in DOM studies [57]. A consequenceof this breakthrough, the number of masses characterized inDOM analysis has increased to such an extent that resultscan be efficiently reported only in simplified diagrams, asplots sorting m/z ratios by homologous series (Kendrick)and by O/C and H/C ratios (Van Krevelen) [58]. An aroma-ticity (AI) index has also been proposed [59], to highlightaromatic (>0.5) or condensed aromatic (>0.67) empiricalformulae.

FT-ICR MS has substantially improved the capacity toidentify DOM molecules. Two specific applications of FT-ICR MS are worth mentioning—detection of eitherhydrogen-deficient aromatic compounds or nitrogenous or-ganic molecules. In fact, FT-ICR MS high resolution scan-ning is effective for detection of:

Molecular characterization of dissolved organic matter 111

1. ions with H/C ratios <0.9 and <0.25, which imply alarge number of double bond equivalents (DBE) andthus restricts possible structures to condensed aromaticrings; and

2. ions with heteroatoms such as nitrogen.

Analysis of condensed aromatic rings in sea water nearthe Antarctica continent [60] and the Southern Ocean be-tween South Africa and Antarctica [61], revealed 224 dif-ferent empirical formulae with large DBE. By use of basicorganic chemistry, the minimum and maximum number ofrings for the MS empirical formulae observed can be calcu-lated; it was found that only structures with five to eightcondensed rings were plausible [60]. Moreover, analysis ofvery hydrophilic (HPI) fractions from sea water of theAtlantic Ocean revealed the presence of nitrogen-containing compounds. The empirical formulae showed thisocean DOM contained compounds with up to three nitrogenatoms [62]. Analysis of sediments from Mangrove Lake,Bermuda, and Mud Lake, Florida, has confirmed the pres-ence of homologous series of CHNO compounds which arelikely to contain alkyl amide structures [63].

Unfortunately FT-ICR MS is not easily hyphenated,and coupling with liquid chromatography (LC) is onlypossible in the off-line mode [64]. Off-line FT-ICR MScoupled with HPLC is widely applied and yields out-standing results [65]. Hyphenated methods based ononline chromatographic configurations are only possiblewith other types of MS. High-performance direct andreversed-phase adsorption [66] and size-exclusion liquidchromatography [67] interface well with negative-modeESI-MS, which remains the most frequent option forMS analysis of dissolved humic substances. The ESIsource may be coupled with ion trap [67], quadrupoleTOF [66], or triple-quadrupole [68] MS, depending onthe type of information required. In fact, there is ageneral consensus that ion-trap mass spectrometers havesuperior sensitivity whereas quadrupole instruments, es-pecially those with the triple configuration, have better massaccuracy. Triple quadrupoles are also generally capable ofhigh-resolution analysis. Other high-resolution MS, for exam-ple isotope ratio MS, is often used to measure stable isotopes,e.g. to investigate the 13C signature of DOM, by breakingdown analytes and measuring atomic masses [69].

High-performance size-exclusion chromatography(HPLC) coupled with either the traditional UV detection[70] or combined multi-detectors [71] is also used for quali-tative and quantitative evaluation of DOM. HPLC has alsobeen coupled with NMR spectrometers in on-line mode, thusenabling investigation of the structure of separated DOMmolecules [72]. Finally, a very specific HPLC techniquenamed HPAEC-PAD (high-performance anion-exchangechromatography with pulsed amperometric detection),

applied within a rigorous analytical procedure, has enabledinvestigation the complexity of DOM polysaccharides [73].

These MS methods have become conventional for DOManalysis because dissolved organic molecules are readily ion-ized, especially in negative-ion mode. However, many prob-lems remain unsolved. First, non-ionizable compounds cannotbe characterized by MS. Second, ionization of terrestrial HSor DOM is a complex phenomenon prone to irreproducibleresults, because of molecular interferences as a result of com-plex inhomogeneous, supramolecular associations [68, 74,75]. These limitations thus prevent reliance on MS methodsalone to achieve structural identification of DOM molecules.

NMR spectroscopy has become fundamental in comple-menting DOM characterization, because ionization is notrequired for the NMR excitation and detection of 1H and13C nuclides. Both solution and solid-state NMR spectros-copy are well established tools in the environmental scien-ces [48, 76–78]. Molecular structural information has beenobtained from conventional mono, bi, and tri-dimensionalNMR spectra [79–81], and information about moleculardiffusion properties and stacking arrangements of organicmatter is obtained by use of DOSY [82] and relaxation time(T1H and T1ρH) techniques [83, 84]. Moreover, the high-resolution magic-angle spinning (HR-MAS) technique,which is applied to semi-solid samples, is increasingly beingused to characterize colloidal humic matter and DOM [85].

Ultraviolet and fluorescence spectroscopy of DOM

Because of several limitations of even the most advancedNMR and MS techniques in DOM analysis, there is anincreasing interest in advanced applications of UV and fluo-rescence spectroscopy, because of their qualitative andquantitative reliability. WETStar instrumentation is an ex-ample of modern fluorimetry applied to DOM[86]. Themain advantage of this approach over use of traditionalfluorimeters is the better accuracy achieved for unfilteredsamples. This advantage enables in-situ analysis of watersamples without preliminary purification steps [86]. In-situDOM measurements with WETStar fluorimeters are com-parable with values obtained on filtered samples by use oftraditional fluorimeters. Another innovative application ofspectrophotometry for DOM analysis is combination of 3Dspectrofluorimetry with HPLC and capillary electrophoresis[87]. This combined technique is capable of differentiatingmarine from fresh-water DOM, thereby extending its poten-tial to profiling of water samples.

DOM quantification methods have been also developedfor large-scale monitoring by remote sensing. This approachis simpler than methods requiring direct sampling; it is,therefore, attracting much interest. A remarkable applicationof this approach [88] uses a combination of:


1. below-sea-surface spectral downward scalar irradianceto calculate a radiative transfer model (STAR) correctedfor clouds by use of TOMS UV reflectivity;

2. surface-ocean spectral diffuse attenuation coefficientsand absorption coefficients for chromophoric dissolvedorganic matter retrieved from SeaWiFS ocean colour byuse of the SeaUV/SeaUVc algorithms; and

3. spectral apparent quantum yield for the inherent photo-chemical reactions.

This approach enabled resolution of the contribution ofsurface chromophoric DOM (CDOM) from total DOM.Unfortunately, one disadvantage is a lack of detailed struc-tural characterization of the DOM, so applicability is con-fined to large-scale environmental studies, for example CO2

management.Conversely, excitation emission matrix fluorimetry

(EEMF), although unsuitable for remote sensing, is a usefultool for DOM molecular characterization, because of thespecific spectrum obtained from each known fluorophore.Nevertheless, EEMF may be compatible with large-scaleanalysis when combined with a network of sampling sta-tions, for example that described by Singh et al. [89]. In fact,by coupling EEMF with parallel factor (PARAFAC) analy-sis, scientists have also been able to discriminate qualitativedifferences in CDOM throughout the Barataria Basin [89].The combination of EEMF with PARAFAC analysis hasbeen attracting attention as a reliable method, and literaturedescriptions of its application are increasing [90, 91].

Marine DOM

Size distribution and molecular structure

There is no model for characterization of marine DOM.DOM arising from natural bodies and stored in the oceanis a major carbon sink, and redistributes DOM in regionswhere it is less available, for example the oceans depths,glaciers, pores, or polar caps. Marine DOM is also formedas a product of the biochemistry of life forms, for examplediatoms, bacteria, algae, or microfauna. These may eitheractively release organic matter or undergo natural decayafter cell death; their metabolic diversity is one of the factorsresulting in the heterogeneity of DOM.

Unlike soil organic matter (SOM), which is deposited assolid, marine DOM is subjected to much faster dynamicsbecause it is dissolved in a water mass. DOM is thereforetransferred or transformed in larger amounts than SOM, andits availability for enzyme activity, oxidation, or metal com-plexation is significantly larger than that of SOM. More-over, DOM is usually found at lower concentrations andgreater ionic strength (salinity) than SOM, and both

conditions affect the mutual association capacity of DOM.These factors contribute to differentiation of natural organicmatter in its marine and terrestrial form. Unfortunately, bothSOM and marine DOM suffer from the same analyticallimitations with regard to determination of molecular weightand structure.

The molecular weight (MW) of marine DOM cannot bedefined simply. The current general consensus is that low-molecular-weight substances coexist with increasingly larg-er biomolecules, for example proteins, polysaccharides, andexocellular macromolecules [92], and with inorganic ions,which stabilize intermolecular association by formation ofcomplexes. Such a heterogeneous mixture of substances isinvolved in several mutual interactions and associations,which result in a wide range of apparent molecular weights.DOM scientists operationally distinguish low molecularweight (LMW <1 kDa) from high molecular weight(HMW >1 kDa) fractions when measuring the MW ofDOM associations.

The current view of DOM structure required an interdis-ciplinary approach, varying from marine geochemistry tomicrobiology and polymer physics. This was because of thethree-dimensional DOM network that arises from the wellknown interactions between small organic compounds, bio-macromolecules, and microgels [93]. Microgels are a prod-uct of the transformation of bacterial or diatomexopolymeric substances [94]. They acquire progressivelydifferent physicochemical properties and their particle sizechanges from the original size [93]. It is known that part ofDOM originates from humic matter, and thus shares itsproperties, including self-association in supramolecularstructures [55, 68]. Thus, one can assume that the self-associated molecules in DOM may also interact with micro-gels, and this may explain the observed changes in thechemical properties of the latter. The occurrence of interac-tions between DOM small molecules and polymers wasrevealed experimentally by in-vitro experiments with am-phiphilic exopolymers of Sagittula stellata [30]. Cationsseem to be of pivotal importance in the formation of micro-gels and to their stability, probably because of developmentof ion bridges across branches of polymers [30, 95]. Amphi-philicity has been observed in supramolecular associationsof small DOM molecules of terrestrial origin, suggestingthat interactions between suprastructures and microgels areplausible [29]. Moreover, coastal mixing zones have beenindicated as the most likely environment for these aggrega-tion phenomena. Transition to saline water has been sug-gested as a possible cause of the contraction of suchassociations, which may facilitate the intertwining withmicrogels in solution.

The hypothesis of coexistence of autochthonous andterrestrial substances in marine DOM was confirmed bystudies on the HMW fraction of marine DOM collected


from the mid-Atlantic Bight coastal region, in which anacylpolysaccharide (APS) and other marine and terrestrialhumic-like compounds were identified [40]. On the basis ofanalysis of its spatial distribution, the authors remarked thatAPS is most abundant in surface waters, being synthesizedthere, but rapidly decreases in deep waters. This suggeststhat the marine DOM transported as a microgel may be asignificant component of the oceanic carbon pool.

A more recent experiment compared 1H NMR spectraand analytical chemical profiles of DOM extracted from adiatom after microbial degradation with those of an APS-containing DOM obtained from sea water. It was found thatboth DOM had relatively high stability against microbialdegradation of hydrolysable sugars during incubation for40 days [96]. More evidence of bacterial polymers in marineDOM arises from analysis of samples from the Pacific,Atlantic, and northern oceans, which revealed the presenceof beta hydroxylated, branched, and other aliphatic acids oftypical bacterial origin [97]. This further suggests that di-verse procaryotic and eucaryotic species contribute withtheir biomacromolecules to marine DOM. In this case, it isinteresting that both free and ester-bound hydroxy acidswere found, implying that the extent of degradation ofbacterial membranes by hydrolysis is variable, and resultsin products with a range of molecular weights.

However, DOM size fractions are sometimes reported asstrongly related to their sugar chemical composition [92],with amino, deoxy, and methylated sugars being more abun-dant in HMW fractions and hexose in LMW [98]. This mayindicate that modified sugars are predominantly present asbuilding blocks of large polymeric structures, whereas hexosesugars are differently associated. In fact, periodate oxidationof HMWDOMyielded 6-deoxy andmethylmonosaccharides,and, because the consumption of oxidant was greater than fora linear polysaccharide, a branched structure was suggestedfor the HMW DOM [99]. It is plausible that additional infor-mation about diagenetic processes may contribute to elucida-tion of chemical implications of marine DOM. In fact, on thebasis of results from analysis of neutral and amino sugars, it isknown that diagenetic transformation affects HMWmore thanLMW DOM [100], thus implying that microgels probablyundergo the same fate. Further evidence comes from analysisof DOM harvested in the Hawaii sea water [77], whichrevealed a decrease in the relative contribution of carbohy-drates and a concomitant increase of lipids with increasingdepth.

Other biomacromolecules also found in DOM are pro-teins, which are of different significance to marine biochem-istry [92, 101, 102]. Degradation of proteins occursnaturally in the environment and produces progressivelysmaller fragments down to single amino acids. The survivalof proteins as refractory organic matter is a highly contro-versial subject and the mechanism underlying the partial

protection of proteinaceous materials is not yet clear. It ispossible that peptides have a specific function in the forma-tion of DOM associations, for example being part of asupramolecular structure or present simply hosted in un-bound forms. An experimental model with bovine serumalbumin incubated in sea water suggested that the fragmentsformed contained fewer than 40 amino acids, thus maintain-ing an intermediate MW between that of polymers discussedabove and that of single molecules [25].

The idea that proteins in marine DOM originate mainlyfrom bacterial membranes [101] was recently challenged[102], because of evidence based on sequences peculiar totypical cytosolic enzymes. After cell death, it is plausiblethat most of the protein pool is metabolized by microbes,and that only a fraction is protected as recalcitrant peptidesby either physical protection or selective preservation [28].However, studies on the interaction of proteinaceous mate-rials with microgel polymers has produced controversialresults. Microscopy of simulated marine aerosols has shownseparated clusters of stained proteinaceous material andexopolymers (Fig. 2), suggesting no reciprocal interactionamong these classes of compounds [103]. In contrast, char-acterization of microgels by fluorescence and proteomicmethods achieved detection of peptidases, proteases, andATPases [104]. Moreover, evidence for the presence ofwhole bacterial enzymes indicates other possible means ofstabilization of proteins and peptidoglycans, for example theformation of Pseudomonas-type vesicles [35]. Characteriza-tion of proteins in marine DOM has also been attempted bymarine proteomics [105]. In these methods peptides aresequenced by tandem mass spectrometry. This “catalogu-ing” of proteins has improved our understanding of:

1. evolution and cycling of carbon pools within the ocean;and

2. how different proteomes adapt to different conditions,

thereby providing insight into which proteins are preservedin the environment.

Macromolecules and molecules, though self-associatingin large suprastructures, are mostly constituents of HMWDOM fractions, whereas other low-MW species are pre-dominantly found in the LMW fraction of DOM. The clas-ses of compounds which have been found in DOM includelipids [97, 106], BC [60, 61], and carboxyl-rich alicyclicmolecules (CRAM) [107, 108], all supposedly of terrestrialthermogenic origin. There may also be smaller unboundcompounds in DOM, for example those found in soil organ-ic matter. However, whereas SOM requires extensive frac-tionation to yield mass spectra from which meaningfulstructures can be suggested [2], this requirement may beless strict for DOM.

At this stage, available data describe the structure ofmarine DOM as a system containing constituents with


polydispersed MW. The HMW DOM fraction contains exo-polymeric materials of autochthonous origin which formstable microgels with polysaccharides and lipids, and coex-ist, or even interact with, proteinaceous matter and withterrestrial-derived humic-like supramolecular structures.The LMW DOM fraction is composed of terrestrial materialoriginating from the decay of biomacromolecules, the de-gree of mutual binding of which is not yet clarified.

Chromophoric DOM (CDOM) in marine environments

CDOM is the fraction of organic matter capable of interact-ing with light and adsorbing its energy. The importance ofthis fraction is connected with the marine biota that needlight for photosynthesis and it is established as a primaryfactor that affects biological cycles and phytoplankton life[109]. For DOM to have chromophoric properties it mustcontain unsaturated and conjugated groups, for examplearomatic or quinoid structures. The conventional techniquefor investigating CDOM quality and dynamics is excitationemission matrix fluorescence (EEMF) spectroscopy com-bined with parallel factor (PARAFAC) analysis.

CDOM is classified as terrestrial, marine, anthropogenic,or mixed, depending on its origin. Terrestrial DOM is trans-ferred to sea water via estuaries, as suggested by the corre-la t ions found between river output and CDOMconcentration [110]. Conversely, autochthonous CDOM isdirectly formed in sea water, as indicated by its more ho-mogeneous marine distribution [111]. Recently evidencethat submarine hydrothermal vents release CDOM has beenobtained, revealing a new source for autochthonous CDOM

in sea water [112]. Terrestrial CDOM discharged to seawater also includes anthropogenic CDOM; unlike naturalCDOM, the anthropogenic material is likely to cause envi-ronmental stress because it contains PCBs [113].

It is generally agreed that normal CDOM fluorescencespectra contain peaks from the main chemical constituents,for example humic-like and protein-like materials. Modernfluorescence spectroscopy combined with PARAFAC easilydifferentiates these two types of CDOM in ocean waters[114], and reveals the dilution which estuarine CDOMundergoes when mixing with oceans. Numerous studieshave traced CDOM flows spatially and temporally, to char-acterize DOM dynamics along mixing zones and acrosswater columns. The approach has been used in a variety ofgeographical areas and this type of study is growing innumber. The results tend to agree, irrespective of geograph-ical area [110, 115–122]. These results further confirm thatCDOM is of pivotal importance in global carbon cycles andmore efforts should be made to characterize its dynamics.Moreover, CDOM in ocean ecosystems also acts as acarbon sink, because its interaction with light causespartial photochemical mineralization, leading to dis-solved inorganic carbon (DIC). This carbon, which ismostly produced off-shore, is not released into the at-mosphere and hence does not contribute to the green-house effect [123]. Photochemical oxidation products ofCDOM are not limited to DIC. In fact, carbon monox-ide (CO), which is of fundamental and often underesti-mated importance in carbon cycling, is also formed atdifferent depths and may be measured by means of thespectroscopic methods discussed herein [88].

Fig. 2 A, B, Proteinaceousparticles and C, D, transparentexopolymeric particles (TEPs)that contain polysaccharides insimulated aerosol (spray).Adapted from Ref. [103]


Molecular characterization of marine DOM

Molecular characterization of DOM is important to betterunderstand how DOM molecules are organized and mutuallyrelated. Moreover, detailed elucidation of the structure of thecomponents of DOMwould enable recognition of biomarkerspresent in specific geographical areas [124]. Study of bio-markers and their association with well characterized DOMhas great potential for understanding the environmental dy-namics of major carbon reservoirs by measuring.

Advanced analytical instrumentation nowadays enablescharacterization of single compounds from highly complexmolecular mixtures. Such a molecular identification is pos-sible for purified, desalted samples even without pre-treatment, thereby preserving the molecular structures asthey are found in the environment. Identification of poly-saccharides and proteins still requires hydrolytic pretreat-ment before instrumental determination of the structure ofsingle units. Pretreatment is followed by MS and NMRmolecular characterization to define the structures of thesugars and lipids composing marine DOM [40, 125, 126].However, a recent study on mucilage polysaccharide used adirect analytical approach without preliminary hydrolysisand achieved molecular characterization of the sequence ofmonosaccharides by use of a tandem MS technique [127].

Fourier transform ion cyclotron resonance (FT-ICR) massspectrometry is also becoming the instrument of choice formolecular characterization of marine DOM, because of theoutstanding quality of results obtained by use of this massspectrometric technique. Some studies using FT-ICR MShave focused on one particular class of molecule whereasothers have preferred to attempt comprehensive samplecharacterization. An example of the first approach is theidentification of hydroxy fatty acids, as lipid biomarkers,in ultrafiltered organic matter from ocean waters to evaluatepotential inputs from bacterial membrane lipids [97]. Thesecond approach consists in comprehensive characterizationof all the empirical formulae found by FT-ICR MS accuratemass measurements [60, 61, 128, 129]. Structures charac-terized with this method are reported in Fig. 3; substanceswith multiple condensed rings were attributed to BC, as aproduct of the partial oxidation of DOM [60].

FT-ICR MS may also be used as a complementary andconfirmative technique for characterization of natural organ-ic matter. This approach has been used in studies on partialproducts of DOM chemical oxidation, in which there is stillsignificant interest. Solid-state 13C NMR spectroscopy hasprovided experimental evidence of the existence of structur-al products of chemical oxidation in DOM [32]. Later, it wasshown [130] that oxidized products similar to black carbon,

Fig. 3 Molecular structuresand alternative isomersproposed for each (CH2)nhomologous series ofcondensed polyaromaticcompounds. It is assumed that amaximum number of oxygenatoms is present in carboxylfunctional groups. From Ref.[60]


including perylene and benzopyrene derivatives with a varietyof substituents, were present in DOM. Their detection wasalso accomplished by FT-ICRMS in DOM samples from verydifferent geographical areas [60, 61, 128, 129], suggestingthat the genesis and molecular structure of black carbon prod-ucts in DOM is consistent throughout the oceans. Hence, FT-ICR MS has been proved to suitable for molecular character-ization of DOM constituents, although its use in combinationwith other techniques, for example NMR spectroscopy,should be still recommended.

High-resolution mass spectrometry of complex samplescan provide a large number of empirical formulae fromDOM samples, including those of lignins, tannins, amines,amides, carboxyl-rich alicyclic molecules (CRAMs), lipids,amino sugars, and carbohydrates (Fig. 4). The inherent com-plexity of the analytical response obtained must be simplifiedby use of Kendrick and Van Krevelen (KvK) plots [65, 107].However, with only empirical formulae, although systema-tized in KvK plots, information about the chemical structureof DOM components remains rather poor. There is, thus,growing pressure to develop innovative scanning methodsinvolving tandem MS techniques. In fact, ion fragmentationand daughter ion scans achieved by secondary tandem massspectrometry may enable the required structural elucidation ofthe entire molecular complexity of DOM.

Application to DOM of the most recent advances in high-resolution tandem mass spectrometry have improved ourunderstanding of environmental carbon cycling. In fact,despite variability in oceanic environments, because of dif-ferent depth, geographic position, proximity of estuaries,and anthropogenic modifications, results obtained forDOM analysis have been quite consistent [110, 115–122].As a result of such advanced studies, there is a generalconsensus that marine DOM:

1. is a combination of components of terrestrial and marineorigin, which can be easily differentiated according totheir genesis;

2. is formed by multiple metabolic pathways which origi-nate in terrestrial and marine environments via a com-bination of natural processes (e.g. forest fires, plantdecay, etc.), microbial activity, and environmental phe-nomena (e.g. solar irradiation, oxidation); and

3. may have a long residence time, because of the refractorynature of its components, and is, thus, an ocean carbonsink.

Understanding fresh-water DOM

Size distribution and molecular architecture

Although fresh-water and oceanic DOM share somecharacteristics, they also differ substantially, because ofthe different environments in which they are formed,transformed, accumulated, or transported. A specificdifference is that fresh-water DOM normally flows intothe oceanic pool whereas the opposite flux is very rareand may only occur during natural disasters. Thus,marine DOM is found to have characteristics similar tothose of fresh-water DOM, because of to their commonterrestrial origin. Numerous studies have been conductedon estuarine mixing areas, and have shown that bothtypes of DOM are spatially distributed by streams andtides [131]. Because discrimination of these DOM typescan be challenging, here we emphasize the differencesbetween terrestrial DOM molecules before and aftercontact with the marine environment.

Fresh-water DOM is derived from terrestrial soil organicmatter (SOM) that underwent specific transformations toincrease its affinity for an aqueous environment. SOM istraditionally and operationally divided in three pools: fulvicacids, humic acids, and humin, according to their solubilityin acids and alkali. Contemporary understanding regardsSOM as an aggregate of numerous heterogeneous moleculesof relatively small molecular mass held together by weaknon-covalent bonds [2, 55, 132, 133]. There is experimentalevidence to show that DOM is also arranged in similarsupramolecular associations [68].

Because of the similarity with terrestrial SOM, genesis offresh-water DOM is therefore strongly related to terrestrialvegetation. This is suggested by measurements of δ13C ofriver sediments and autochthonous plants, which werefound to be strongly correlated [134]. In this work, analytesof plankton origin were found in both river and estuaryDOM, although in smaller amounts than in marine DOMsamples. Transportation of riverine DOM to the marineenvironment was also related to complex transformations.Combination of mass spectrometric characterization withfluorescence analysis of CDOM distribution indicated thatDOM apparent mass decreased as salinity increased [135].

Fig. 4 Molecular assignment of DOM components from ChesapeakeBay, by use of Van Krevelen diagrams based on FT-ICR MS experi-mental data. From Ref. [107]


The composition of fresh-water DOM is believed todepend on the transformation of plant compounds intohumic-like substances. Investigation of river and lakeDOM composition with different techniques supports thehypothesis of plant genesis. In particular, NMR spectrosco-py using two dimensional long-range correlation techniqueshas enabled characterization of compounds directly relatedto decay of terpenes [72], for example CRAM and materialderived from linear terpenoids (MDLT), in agreement withprevious DOM literature [136]. The same NMR techniquesalso enabled detection of hexopolysaccharides and aromaticstructures, possibly of lignin origin.

ESI FT-ICR MS investigation of river DOM revealedevidence of hydrogen-deficient compounds assigned toblack carbon (BC), thereby confirming the hypothesis oftransport of terrestrial material in marine DOM [128]. Onceformed, these black carbon substances have a dynamicdistribution in natural bodies. Recent FT-ICR MS studieson lacustrine DOM from Lake Superior emphasized differ-entiation among fresh-water areas such as swamp, creek,and river, depending on spatial and temporal distribution[137]. Furthermore, larger amounts of lignin-like com-pounds were reported in this lake than in its tributaries.Lignins are regarded as refractory substances and, in fact,their contribution to total DOM accumulated along theriverine paths, owing to slower mineralization, and wasfound to reach the greatest concentration in the oceans[137]. In this scenario, the chemical properties of com-pounds are bound to affect their distribution in water.Although the contribution of lipids is expected to be limitedbecause of limited aqueous solubility, they are, nevertheless,found in lacustrine DOM [138]. This finding inevitableleads to the inference that the supramolecular structure ofDOM enhances the solubility of specific hydrophobic mol-ecules by forming complex associations with them. Thedynamics of hydrophobic compounds in natural bodies iscomplex, because they are also important components ofparticulate organic matter (POM). However, although it iswell established that hydrophobic OM is an abundant com-ponent of POM and sedimentary matter [139], it is not yetclear whether hydrophobic DOM and POM are related toeach other.

Because of the further complexity introduced by thetendency of DOM molecules in solution to associate, as-sessment of the size of DOM particles is not straightforward.Interestingly, whereas mass spectrometry of DOM indicatesmolecular masses lower than 1000 Da for most compounds[68, 128, 137], size-exclusion chromatography (SEC) pro-files of the same sample suggests a much larger hydrody-namic volume. This discrepancy confirms that singlemolecules are prone to spontaneous association [55, 132],but more evidence should be gathered on how this structureis organized.

The findings reported here have led to a plausible struc-ture for fresh-water DOM, which is currently described asan aggregation in spontaneous self-associated superstruc-tures formed by plant-derived products of natural decay,for example lipids, amino sugars, sugars, CRAM and otherterpene derivatives, aromatic condensed structures (BC),and lignin-derived compounds.

Chromophoric DOM in the fresh-water environment

Interaction with solar radiation is a fundamental property offresh-water DOM and is very relevant in fresh-water environ-mental interactions. The formation of chromophoric DOM(CDOM) is still debated, but experimental evidence over thelast decade suggests that organic matter derived from phyto-plankton, initially colourless, is processed by microbial florainto fluorescent DOM. In fact, CDOM isolated after incuba-tion of algae was found to grow concomitantly with microbialmass [140]. Further evidence of the involvement of phyto-plankton in the formation of lacustrine DOM came fromquantitative assessment of average and daily rates of in-situproduction [141]. It has also recently been reported that fluo-rescence absorption peaks for humic and fulvic acids in-creased proportionally with the amount of DOM. Theseacids are probably formed under terrestrial conditions andthen transported in natural water bodies, thereby affectingfluorescence response [142]. The different chemical compo-sition of autochthonous and humic DOM in fresh waternecessitated more systematic description. Hence, the humifi-cation index (HIX) and the index of recent autochthonouscontribution (BIX) were usefully introduced [143].

The chemical origin of the colloidal properties of CDOMhave also been investigated by flow-field flow fractionation[144], assuming differentiation between humic and/orfulvic-like and protein-like compounds. Whereas the originof the latter was attributed to fresh-water autochthonous life,the sources of the former materials are believed to be terres-trial. Furthermore, it seems there is a strict correlation be-tween the size fraction and the composition of the colloidalphase, with protein-like materials occurring primarily in thesmaller size fraction and humic-type materials in the larger[145]. All these findings suggest that the genesis of fresh-water DOM is similar to that of marine DOM, because bothcontain compounds with well differentiated optical proper-ties. In fact, differentiation of riverine from marine DOM isalso possible in water sampled in estuarine mixing zones[131]. In this work, two rivers flowing into the same gulfsupplied DOM with different optical behaviour, therebyaffecting the spatial distribution of the resulting marineDOM. In agreement with this finding, an interesting exper-iment has further clarified the effect of salinity and dilutionon DOM fluorescence spectra, by revealing that changes insalinity has no significant effect on fluorescence lifetime


[146]. However, the transition in other optical characteristicsfrom riverine to marine DOM has been related to increasedsalinity [147], thereby indicating that ionic strength is ofpivotal importance in the association of DOM molecules. Itcan be concluded that although salt concentration seems toaffect DOM and its optical properties, fluorescence lifetimeis not affected. As a result of developments in fluorescencemethods, recent research has successfully differentiatedfresh-water DOM from several geographical sites [89, 90,148–150].

It is currently believed that CDOM degradation simulta-neously involves microbial metabolism, which seems tohave a preference for allochthonous CDOM [151], photo-degradation [152–154], and, to a lesser extent, adsorption onsuspended particles [155]. Recent evidence suggests thatprotein-like and humic CDOM are predominantly degradedby microbial and UV phenomena, respectively [156]. Par-ticularly enlightening is a report that CDOM shields aquaticlife from potentially harmful radiation [157]. Not surpris-ingly, techniques for large-scale monitoring of CDOM are inconstant development [158, 159].

Molecular characterization of fresh-water DOM

The molecular composition of fresh- DOM has been studiedless than that of marine DOM, probably owing to the greatereffect of oceanic DOM on the geochemical carbon balance.Nevertheless, several studies have tried to remedy this andcharacterize fresh- substances in detail. A noteworthy NMRspectroscopy experiment performed on lacustrine DOM[72] has shown the potential of this technique in recognizingand quantifying functional groups even in such a complexDOM system. In this study, by simple 1H monodimensionalspectroscopy, aliphatic, carbohydrate, aromatic, and CRAMmolecules were differentiated by chemical shift analysis.Furthermore, 13C investigation by multidimensional hetero-nuclear techniques, for example HMQC (heteronuclear mul-tiple quantum coherence) and HMBC (heteronuclearmultiple bonding coherence), enabled characterization ofspecific regions assignable to well known organic species,for example anomeric carbons from carbohydrates, conju-gated double bonds from aliphatic acids, CRAM, N-acetyls,and others. Interestingly, differentiation between terpene-derived CRAM and MDLT has been achieved by HMBC(Fig. 5), thereby providing a potential tool for investigation,in further detail, of terpene metabolism in DOM.

�Fig. 5 2D 1H–13C HMBC spectra of LO-DOM. (A) Expansion in-cluding the full CRAM (I) and MDLT (II) regions. (B) Expansion of(A) showing the 5–115 ppm (carbon) region. (C) Expansion of (A)showing the carboxyl region. The notation La, Lb, Lc, and Ld is used toidentify crosspeaks in the HMBC spectrum. From Ref. [72]


A different approach based on the use of pyrolysis cou-pled to MS has been proposed by Guo et al. [160]. Thiswork showed that pyrolysed products of the high molecular-weight fraction of DOM contained compounds, for examplefurfural, methylfurfural, dimethylbenzene, phenol, andmethylcyclopentenone, which may be assigned to sugarand aromatic structures. Another study on lacustrine DOMused Fourier-transform infra-red (FT-IR) spectroscopy incombination with direct temperature-resolved MS (DT-MS), and achieved excellent characterization of chemicalgroups by processing the spectroscopic data by use of math-ematical methods [161]. The last two are examples of inex-pensive but effective approaches which may act as asubstitute for more advanced instrumentation. However,the conventional technique for molecular investigation ofDOM is, again, FT-ICR MS, because of its resolving power,which is capable of revealing hundreds of empirical formu-lae and furnishing plausible molecular structures for eachunknown compound. Characterization of condensed aro-matic compounds (BC) [128] (Fig. 6) and amides [63] hasbeen achieved by use of FT-ICR MS.

Transport of terrestrial DOM to oceans

The long-debated hypothesis that part of marine DOM is ofterrestrial origin seems to have been confirmed by severalstudies. In particular, analysis of spatial distribution in

estuarine systems showed that riverine DOM is highly pre-served in the ocean, despite structural rearrangements,which occur mostly because of to changes in salinity[135]. In a review of recent breakthroughs in arctic biogeo-chemistry, Dittmar and Kattner [162] observed that terres-trial DOM was persistently refractory in DOM pools of thearctic ocean, being the result of a long-term carbon sedi-mentation and the end-product of a natural carbon sink.

A molecular investigation of transported DOM by HPLCand MS revealed it is composed of both hydrophilic andhydrophobic fractions [65]. Low-MW compounds were pre-dominantly found in the former whereas larger-MW constit-uents were identified in the latter. In the same work,comparison of the molecular composition of a wood extractand DOM revealed several characteristics in common, there-by indicating that lignin-derived compounds are a majorcomponent of refractory DOM. Further insight into lignintransformation [163] suggested the involvement of photo-chemical processes, implying that chemical transformationof DOM is not only a result of microbial activity. Consistentwith such findings, an increase of oxidation state was mea-sured in DOM from Chesapeake Bay flowing in-shore tooff-shore [107], confirming that environmental conditions inthe mixing zone affected the transformation of transportedDOM. The same work also suggested the presence of ter-restrial black-carbon structures in transported DOM, as apyrogenic contribution from carbonized OM leachingthrough soil and reaching fresh water. The complexity ofthe process of formation and transport of refractory DOM tooceanic carbon pools would greatly benefit from a dedicatedstudy which included samples from different estuaries.

From the published literature it can be inferred that oceanDOM is derived partially from:

1. the humified products of plant decay, which subsequent-ly dissolve in fresh water and are transported to theocean; and

2. phytoplankton proteins and polysaccharide productswhich are altered and transformed in situ by bacteriato form another pool of refractory organic matter.

During transport, chemical transformations may also oc-cur, thereby increasing its refractory properties. This organicmatter is subsequently included in the oceanic carbon poolfor a long-term storage.

DOM in pore water

Pore water is water in physical spaces isolated by sedimentsin which geochemical transformations differ from thoseoccurring in outside spaces. MALDI-TOF-MS evidenceindicates that protein breakdown in pores is not as extensiveas in open marine waters [25]. The accumulation and dy-namics of DOM are dramatically dependent on whether the

Fig. 6 Possible chemical structure of one peak (C27H18O8) in the massspectrum of DOM from McDonalds Branch Basin located in the NewJersey Pine Barrens (USA). From Ref. [128]


pore water conditions are anoxic, suboxic, or mixed-redox(suboxic, or oscillating between oxidizing and reducing),which result in very different reactive products and in var-iable rates of degradation [164]. Moreover, DOM accumu-lation has been found to be generally limited in the mixed-redox zone relative to the anoxic zone, and humic-likefluorescence intensity also differed between mixed-redoxand anoxic zones of the sediment, such that anoxic porewaters were relatively enriched in fluorescent, humic-likecompounds [164]. Pore waters are also found within peatsoils, in which they pose a problem to water quality. In fact,because of microbiological degradation of pore DOM,“tea”-coloured water is released, and requires purifyingtreatment before use [165].

Pore waters contain CDOM material, and, as for othertypes of DOM, humic-like and protein-like fluorophores[166]. Humic-like DOM apparently increased with depthwhereas no particular trends were observed for protein-likeDOM. Moreover, humic-like CDOM may be a low-molecular-weight fraction of refractory pore DOM. Asexpected, experimental evidence showed that humic-likeCDOM was better preserved under anoxic conditions [166].

Molecular characterization of pore-water DOM fromsediments of the Iberian peninsula revealed relatively highabundance of highly oxygenated aromatic compounds ornitrogen-bearing compounds with low H/C ratios [167].These structures were likely to originate from lignin phenolsof terrestrial origin. In fact, their amounts decreased as thematerial was transported along the shelf.

Pore DOM is, therefore, a material produced by geomor-phological phenomena similar to those which produce fresh-water and marine DOM, and its composition is similar inseveral ways to those of open-water types of DOM. How-ever, the compartment in which it is confined affects itschemical and chromophoric composition, which ultimatelydepend on conditions such as oxygen availability, depth,and microbial population [167].

Conclusions

Characterization of DOM has advanced substantially in thelast decade, mainly because of the development of advancedinstrumental analytical methods, for example FT-ICR MS,FEEM-PARAFAC, multidimensional NMR, tangential flowultrafiltration, and reverse osmosis, but also because of theintroduction of innovative ideas regarding DOM genesis andtransformation. The recognition of components from terrestri-al sources, the discovery of microgels, advances in under-standing the effect of microorganisms in reworking DOMand the supramolecular nature of humic substances, and thediscovery of new mechanisms for carbon storage in the envi-ronment, are the current milestones in the science of natural

OM. The knowledge acquired encompasses not only DOMitself and its fate, but also that of terrestrial and marine biota,and of larger environmental domains, for example continentalshelves, sediments, and natural streams. The content andstructures of proteins, lipids, polysaccharides, lipopolysac-charides, lignin, terpene derivatives, and black carbon compo-nents must be characterized to enable understanding of thegenesis, structure, and chemical reactivity of DOM. Determi-nation of chromophores in DOM has been useful in enablingextensive monitoring of fluxes and streams, and in revealinganthropogenic interference.

Future challenges in environmental science are:

1. standardization of procedures for assigning structureson the basis of MS, NMR, and fluorescence experimen-tal data;

2. clarification of the involvement of microorganisms intransforming and degrading DOM, i.e. the extent towhich they produce OM and the extent to which theytransform existing OM; and

3. understanding the correlation between DOM composi-tion and its properties, for example recalcitrance, actionas a radiation shield, and interaction with organic andmetal pollutants.

It is expected that research based both on the use ofadvanced instrumentation and on the methodological expe-rience acquired so far will enable successful tackling ofthese challenges in the future.

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Molecular characterization of dissolved organic …organic matter which acts as carbon sink in the...

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