Antonio Nebbioso

Molecular characterization of dissolved organic matter (DOM): a critical review

Advances in water chemistry in the last decade have improved our knowledge about the genesis, composition, and structure of dissolved organic matter, and its effect on the environment. Improvements in analytical technology, for example Fourier-transform ion cyclotron (FT-ICR) mass spectrometry (MS), homo and hetero-correlated multidimensional nuclear magnetic resonance (NMR) spectroscopy, and excitation emission matrix fluorimetry (EEMF) with parallel factor (PARAFAC) analysis for UV–fluorescence spectroscopy have resulted in these advances. Improved purification methods, for example ultrafiltration and reverse osmosis, have enabled facile desalting and concentration of freshly collected DOM samples, thereby complementing the analytical process. Although its molecular weight (MW) remains undefined, DOM is described as a complex mixture of low-MW substances and larger-MW biomolecules, for example proteins, polysaccharides, and exocellular macromolecules. There is a general consensus that marine DOM originates from terrestrial and marine sources. A combination of diagenetic and microbial processes contributes to its origin, resulting in refractory organic 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 transported to the ocean, and partially from proteinaceous and polysaccharide material from phytoplankton metabolism, which undergoes in-situ microbial processes, becoming refractory. Some of the DOM interacts with radiation and is, therefore, defined as chromophoric DOM (CDOM). CDOM is classified as terrestrial, marine, anthropogenic, or mixed, depending on its origin. Terrestrial CDOM reaches the oceans via estuaries, whereas autochthonous CDOM is formed in sea water by microbial activity; anthropogenic CDOM is a result of human activity. CDOM also affects the quality of water, by shielding it from solar radiation, and constitutes a carbon sink pool. Evidence in support of the hypothesis that part of marine DOM is of terrestrial origin, being the result of a long-term carbon sedimentation, has been obtained from several studies discussed herein.

Advances in humeomics: Enhanced structural identification of humic molecules after size fractionation of a soil humic acid

We size fractionated a soil humic acid (HA) by preparative high performance size exclusion chromatography (HPSEC) and evaluated the analytical capacity of humeomics to isolate and identify humic molecular components in the separated size-fractions. HA and its three size-fractions were chemically fractionated to extract non-covalently bound organosoluble compounds (ORG1), weakly ester-bound organosoluble (ORG2) and hydrosoluble constituents (AQU2), strongly ester-bound organosoluble components (ORG3), and final unextractable residues (RES4). According to their solubility, the extracts were characterized by either GC-MS or on-line thermochemolysis/GC-MS techniques. The humeomic sequence showed that the analytical yields of identified compounds in either ORG or AQU extracts of size-fractions were invariably larger than for the unfractionated HA. This was attributed to a weaker conformational stability of humic suprastructures obtained by HPSEC fractionation, thereby enabling an improved separation and identification of single humic molecules. In line with the supramolecular understanding of humic substances, we found that hydrophobic compounds were mainly distributed in the largest size-fraction, while hydrophilic components were eluted in the smallest size-fraction. Furthermore, compounds with linear chains or stackable aromatic rings associated in regular structures were more abundant in the former fraction, whereas irregularly shaped compounds, that hindered association in larger size, were mostly found in the latter fraction. Thus the structural characteristics of single humic molecules determined their mutual association in humic suprastructures, as well as their conformational strength and shape. The lack of de novo synthesized macropolymers in the unfractionated soil humic matter was confirmed by the absence of RES4 fractions in the separated size-fractions. Our results indicate that humeomics capacity to reveal the complex molecular composition of humic suprastructures was significantly improved by subjecting humic matter to a preliminary HPSEC fractionation.

Limitations of electrospray ionization in the analysis of a heterogeneous mixture of naturally occurring hydrophilic and hydrophobic compounds

A model heterogeneous mixture of a hydrophilic tripeptide (phenylalanine-glycine-glycine, PGG) and hydrophobic organic acids ((12)C- and (13)C-octanoic acid and pentadecanoic acid) was subjected to electrospray ionization mass spectrometry (ESI-MS). The objective was to verify the previously noted inconsistencies in ESI-MS of complex environmental samples such as humic materials from either aquatic or terrestrial origins. The hydrophobic organic acids, either alone or together, reduced significantly the ESI-MS detection of the tripeptide molecular and self-associated ions at a concentration that was an order of magnitude lower than that of PGG. The most intense peaks were invariably those of the octanoic acid as either deprotonated, self-associated, or acetate-clustered molecules. The presence of equimolar amounts of PGG and organic acids yielded similar results, but with a significant increased detection of PDA and a smaller depression of the PGG signals. This behaviour is attributed to a different electrospray ionization of the mixture compounds depending on their most probable positioning at the surface of the evaporating droplet. The most favoured positioning of hydrophobic molecules at the aqueous-gas interphase allows preferential evaporation of hydrophobic ions whereas the hydrophilic molecules are retained in the droplet interior, and, their ESI-MS detection depressed. These findings suggest that the electrospray ionization of different molecules present in complex heterogeneous mixtures of environmental significance such as humic substances is limited by their concentration and reciprocal attracting forces.

Unveiling the molecular composition of the unextractable soil organic fraction (humin) by humeomics

We applied humeomics to a soil unextractable humin fraction (HUM1) and its derived humin (HUM2) after removal of minerals by an HF/HCl treatment. Humeomics implies progressive separation and structural characterization of molecules released from complex humin matrices as (i) unbound, (ii) weakly ester-bound, (iii) strongly ester-bound, and (iv) ether-bound. Molecular characterization of fractions was achieved by GC-MS, thermochemolysis-GC-MS, and 13C-CPMAS-NMR. Both weight and chromatographic yields were higher for the clay-depleted HUM2 than those for HUM1, and this increased molecular detection in HUM2. Saturated and unsaturated alkanoic, α,ω-alkanedioic, hydroxyalkanoic acids, alkanols, and hydrocarbons were found in both HUM1 and HUM2. Abundant odd-C numbered n-alkanoic acids in unbound fractions indicated accumulation of free microbial metabolites, whereas plant-derived acids remained in fractions more tightly bound to the humin matrix. Unsaturated, n-alkanedioic, and hydroxyalkanoic acids were detected after hydrolysis of complex esters. The aromatic character in humin residues progressively increased with humeomics sequential steps, while alkyl and hydroxy-alkyl compounds were reduced. Humins contained similar components as a humic acid extracted from the same soil, implying that traditional humic pools differed in supramolecular arrangement rather than in molecular composition. The humeomic approach enables the determination of the molecular composition of humic matter and may improve knowledge of the structure-activity relations of organic matter in soil.

Reduced complexity of multidimensional and diffusion NMR spectra of soil humic fractions as simplified by Humeomics

BackgroundHumeomics is a sequential step-wise chemical fractionation that simplifies the complex matrix of a humic acid (HA) and weakens its supramolecular interactions, thereby allowing a detailed characterization of the involved molecules. A recalcitrant residual end product of Humeomics, namely RES4, was successfully solubilized here in alkaline conditions and subjected to a semi-preparative high-performance size exclusion chromatography (HPSEC).ResultsThe resulting six size fractions separated by HPSEC were analyzed by different NMR techniques. 1D 1H-NMR spectra did not reveal significant molecular differences among size fractions, although all of them differed from the spectrum of the bulk RES4 especially in signal intensity for aliphatic materials, which were assigned by 2D NMR to lipidic structures. Diffusion-ordered spectroscopy (DOSY)-NMR spectra showed that the homogeneity of RES4 was significantly changed by the HPSEC separation. In fact, nominally large size fractions, rich in lipidic signals, had significantly lower and almost constant diffusivity, due to stable supramolecular associations promoted by hydrophobic interactions among alkyl chains. Conversely, diffusivity is gradually increased with the content of aromatic and hydroxyaliphatic signals, which accompanied the reduction of fractions sizes and was related to smaller superstructures.ConclusionsThis study not only confirmed the occurrence of supramolecular structures in the recalcitrant humic residue of Humeomics, but also highlighted that more homogeneous size fractions were more easily characterized by NMR spectroscopy.

A molecular zoom into soil Humeome by a direct sequential chemical fractionation of soil

A Humeomics sequential chemical fractionation coupled to advanced analytical identification was applied directly to soil for the first time. Humeomics extracted ~235% more soil organic carbon (SOC) than by the total alkaline extraction traditionally employed to solubilise soil humic molecules (soil Humeome). Seven fractions of either hydro- or organo-soluble components and a final unextractable humic residue were separated from soil. These materials enabled an unprecedented structural identification of solubilised heterogeneous humic molecules by combining NMR, GC-MS, and ESI-Orbitrap-MS. Identified molecules and their relative abundance were used to build up structure-based van Krevelen plots to show the specific contribution of each fraction to SOC. The stepwise isolation of mostly hydrophobic and unsaturated molecules of progressive structural complexity suggests that humic suprastructures in soil are arranged in multi-molecular layers. These comprised molecules either hydrophobically adsorbed on soil aluminosilicate surfaces in less stable fractions, or covalently bound in amorphous organo-iron complexes in more recalcitrant fractions. Moreover, most lipid molecules of the soil Humeome appeared to derive from plant polyesters rather than bacterial metabolism. An advanced understanding of soil humic molecular composition by Humeomics may enable control of the bio-organic dynamics and reactivity in soil.

Water-soluble lignins from different bioenergy crops stimulate the early development of maize (Zea mays, L.).

The molecular composition of water-soluble lignins isolated from four non-food bioenergy crops (cardoon CAR, eucalyptus EUC, and two black poplars RIP and LIM) was characterized in detail, and their potential bioactivity towards maize germination and early growth evaluated. Lignins were found to not affect seed germination rates, but stimulated the maize seedling development, though to a different extent. RIP promoted root elongation, while CAR only stimulated the length of lateral seminal roots and coleoptile, and LIM improved only the coleoptile development. The most significant bioactivity of CAR was related to its large content of aliphatic OH groups, C-O carbons and lowest hydrophobicity, as assessed by 31P-NMR and 13C-CPMAS-NMR spectroscopies. Less bioactive RIP and LIM lignins were similar in composition, but their stimulation of maize seedling was different. This was accounted to their diverse content of aliphatic OH groups and S- and G-type molecules. The poorest bioactivity of the EUC lignin was attributed to its smallest content of aliphatic OH groups and largest hydrophobicity. Both these features may be conducive of a EUC conformational structure tight enough to prevent its alteration by organic acids exuded from vegetal tissues. Conversely the more labile conformational arrangements of the other more hydrophilic lignin extracts promoted their bioactivity by releasing biologically active molecules upon the action of exuded organic acids. Our findings indicate that water-soluble lignins from non-food crops may be effectively used as plant biostimulants, thus contributing to increase the economic and ecological liability of bio-based industries.

Molecular Properties and Functions of Humic Substances and Humic-Like Substances (HULIS) from Biomass and Their Transformation Products

Agricultural and biorefinery byproducts should be regarded as important sources of chemicals and materials, instead of being disposed or burnt. Humic substances (HS) and humic-like substances (HULIS) isolated by such materials may be employed as plant biostimulants, due to their surprising bioactivity on plant development, either after their direct extraction from such byproducts or after composting them. In order to shed light on both the biological activity of HS and HULIS on plant physiology and on soil carbon dynamics, a number of analytical chemical techniques have been employed, thus, providing a detailed insight on their molecular nature. This chapter is intended to provide a comprehensive overview of the more advanced chemical techniques applied in the chemical characterization of HS and HULIS structure, such as GC-MS, NMR, HPSEC, EPR and thermal analyses. Each of these tools provides different but incomplete information on HS and HULIS molecular composition, due to both the intrinsic limitation of each technique and the large molecular heterogeneity and structural complexity of HS and HULIS. Thus, in order to elucidate the chemical nature of such substrates, the various analytical tools should be always exploited concomitantly and critically discussed, thus, offering a comprehensive understanding of HS and HULIS at a molecular level. Achieving this purpose will also allow to efficaciously exploit HS and HULIS as plant biostimulants in sustainable agriculture and/or biomass-based material chemistry.

Molecular Understanding of a Humic Acid by “Humeomic” Fractionation and Benefits from Preliminary HPSEC Separation

A mild stepwise fractionation of molecular components of a humic acid (HA) is here aimed to address the need of a systematic and reproducible method of separation and characterisation of humic substances. This approach may be defined as “humeomics” in analogy with genomics and is aimed at (1) characterising natural humic molecules and (2) clarify their relations with ecosystem functions. Moreover, the same HA was further processed with high-performance size exclusion chromatography (HPSEC) in three size fractions. Both HA and its three size fractions underwent the “humeomic” chemical fractionation to extract non-covalently bound organosoluble compounds (ORG1), weakly ester-bound organosoluble (ORG2) and hydrosoluble constituents (AQU2), strongly ester-bound organosoluble components (ORG3), and final unextractable residues (RES4). Structural identification of initial and final material, separated organosoluble and hydrosoluble fractions, and subfractions was conducted by GC-MS (gas chromatography–mass spectrometry), HPSEC-ESI-MS (high-performance size exclusion chromatography electrospray ionization mass spectrometry) (high resolution, Orbitrap), and solid- and liquid-state NMR. GC-MS revealed in organosoluble unbound fractions the presence of both saturated and unsaturated, linear and branched, alkanoic, hydroxyalkanoic and alkandioic acids, n-alkanes, and n-alkanols. Quantisation of analytes showed that (I) the sum of compound classes in separated fractions was greater than that for the initial HA and (II) that analytical yields of identified compounds in size fractions were invariably larger than for the unfractionated HA, thereby showing that both size exclusion and stepwise chemical fractionation increased significantly the analytical identification of humic molecules. Our results suggest this “humeomic” approach as a valid path for mapping humic molecular composition and assess humus origin and formation.

Rate of a Click Chemistry reaction under catalysis by trace‐amounts of copper as evaluated by NMR spectroscopy

Since its introduction, click chemistry revolutionized many fields of synthetic chemistry such as material science and molecular biology. It is considered the ‘cream of the crop’ of synthesis because of its speed and success rate. This notwithstanding, many kinetic and thermodynamic aspects of the typical Cu-catalysed [3 + 2] azide–alkyne cycloaddition (CuAAC) remain vaguely characterized at the present time. There are a number of studies focusing also on the kinetics of typical CuAAC reactions, showing how these follow first-order or, in some chain reaction mechanisms, secondorder kinetics, and no regioselectivity is observed. In the absence of any Cu catalyst, it was established that a cycloaddition reaction between azide and alkyne groups may only occur by using high temperatures (>70 °C) which are capable of overcoming the relatively high-energy activation barrier by increasing the collision energy between these groups. In such instances, second-order kinetics are observed. Conversely, owing to the introduction of a Cu catalyst, the cycloaddition step follows a different mechanism which requires a lower activation energy. Specifically, the Cu catalyst binds with the alkyne group in the initial step and facilitates conjugation of the azido group to the Cu–alkyne complex in the subsequent step. This alternative mechanism operates at milder temperatures (~40 °C) and follows first-order kinetics. Furthermore, the reaction implies a preferential orientation of the ligands of the azide and the alkyne to minimize steric hindrance, thereby yielding a regioselective product. One of the most critical disadvantages deriving from a nocatalyst approach is represented by the costs of heating required to improve the rate of cycloaddition and the loss of regioselectivity. Conversely, the catalytic approach brings the advantage of milder reaction temperatures, albeit with the requirement of a costly and time consuming Cu removal step, which is necessary in most cases. This conundrum, however, may be overcome by a method using very low amounts of catalyst, only for those applications where traces of Cu in the final product are acceptable. Therefore, we hereby report the reaction rate, monitored through NMR spectroscopy, of a [3+ 2] cycloaddition reaction between a watersoluble alkynol and a poly(ethylene glycol) oligomer, capped with an azido and an amine group at each end of the chain, and catalysed by trace amounts of copper (Scheme 1). Here, we aimed to demonstrate that despite the relevantly low amount of catalyst, the cycloaddition reaction may be carried out maintaining regioselectivity and at acceptable rates under the same mild temperature conditions as ordinarily used for click chemistry.