N-15 NMR spectra of naturally abundant nitrogen in soil and aquatic natural organic matter samples of the International Humic Substances Society

The naturally abundant nitrogen in soil and aquatic NOM samples from the International Humic Substances Society has been characterized by solid state CP/MAS ¹⁵N NMR. Soil samples include humic and fulvic acids from the Elliot soil, Minnesota Waskish peat and Florida Pahokee peat, as well as the Summit Hill soil humic acid and the Leonardite humic acid. Aquatic samples include Suwannee River humic, fulvic and reverse osmosis isolates, Nordic humic and fulvic acids and Pony Lake fulvic acid. Additionally, Nordic and Suwannee River XAD-4 acids and Suwannee River hydrophobic neutral fractions were analyzed. Similar to literature reports, amide/aminoquinone nitrogens comprised the major peaks in the solid state spectra of the soil humic and fulvic acids, along with heterocyclic and amino sugar/terminal amino acid nitrogens. Spectra of aquatic samples, including the XAD-4 acids, contain resolved heterocyclic nitrogen peaks in addition to the amide nitrogens. The spectrum of the nitrogen enriched, microbially derived Pony Lake, Antarctica fulvic acid, appeared to contain resonances in the region of pyrazine, imine and/or pyridine nitrogens, which have not been observed previously in soil or aquatic humic substances by ¹⁵N NMR. Liquid state ¹⁵N NMR experiments were also recorded on the Elliot soil humic acid and Pony Lake fulvic acid, both to examine the feasibility of the techniques, and to determine whether improvements in resolution over the solid state could be realized. For both samples, polarization transfer (DEPT) and indirect detection (¹H–¹⁵N gHSQC) spectra revealed greater resolution among nitrogens directly bonded to protons. The amide/aminoquinone nitrogens could also be observed by direct detection experiments.     

Comparative characterization of humic substances from the open ocean, estuarine water and fresh water

Humic substances were isolated from ocean, estuarine water and fresh water using a two column array of XAD-8 and XAD-4 resins in series. The extracted fulvic acids and XAD-4 fraction from different origins were characterized using UV–vis., molecular fluorescence, Fourier transform infrared (FTIR) spectroscopy and cross polarization magic angle spinning (CPMAS)-¹³C nuclear magnetic resonance (NMR) spectroscopy. The isolation procedure allowed us to obtain the necessary amount of sample for characterization, even in the case of open ocean water, which has a very low amount of dissolved organic carbon (DOC). Humic substances from the open ocean showed the lowest chromophore and fluorophore contents and showed relatively greater fluorescence at lower wavelengths than those from fresh water. FTIR and ¹³C NMR spectra highlighted the idea that humic substances from a marine environment have a more branched aliphatic structure and less aromatic structure than those highly influenced by terrestrial sources. The spectra also suggest that the open ocean humic substances have a higher content of olefinic carbons than aromatic- or alkyl-substituted carbons.     

CPMAS 13C NMR spectra of estuarine sedimentary humic acids

CPMAS ¹³C NMR spectra of two estuarine sedimentary humic acids were recorded on a Bruker WP-SY 200 spectrometer. Both samples were found to contain similar aromatic and aliphatic carbon fractions. The sedimentary humic acids have unusually high methoxyl contents and more than one type of methoxyl is indicated. The high methoxyl content may be related to relatively low values for estuarine sedimentary humic acid-metal complexes. Carbohydrate contents of the humic acid samples were also found to be low.     

Preparation and characterization of humic acid cross-linked with organic bridging groups

Cross-linking of humic substances with organic bridging groups is thought to contribute to the humification of soil organic matter. Model cross-linked humic substances were prepared by cross-linking Amherst soil humic acid by a diepoxide and a polycarboxylic acid, applying procedures established for cross-linking of polymers and textile fabrics. Products of the cross-linking reactions were analyzed by FTIR and ¹³C CPMAS NMR. Physicochemical properties of the products were determined by solubility experiments and thermal analysis. The incorporation of the cross-linker into the matrix of the humic acid by covalent linkages was confirmed by both the disappearance of bands of the reactive functional groups of the cross-linker in the FTIR spectrum and the increase of signals related to the incorporation of the cross-linker into the matrix of the humic acid in the FTIR and ¹³C CPMAS NMR spectra. The formation of covalent ester and ether linkages by the cross-linking reaction was indicated. Water solubilities at pH 6.2 of the cross-linked samples as determined by UV/Vis spectrometry were reduced compared to controls. Fewer water molecule bridges were formed in the cross-linked samples, which was attributed to a lower number of available functional groups and increased distances between humic acid strands caused by the cross-linking molecules. Reduced reactivities of humic acid strands in the cross-linked samples further indicated successful cross-linking. The reactions investigated in this study can be regarded as models for reactions occurring in natural soils to test the significance of cross-linking reactions in the humification process of soil organic matter and the physico-chemical properties and ecological function of organic matter in geosolids.     

[1H] and [13C]NMR studies on the importance of aromatic structures in fulvic and humic acids

To obtain information on the contribution of aromatic fragments to the chemical structure of humic substances, we carried out a study on the [¹H]NMR and [¹³C]NMR spectra of humic and fulvic acids and their oxidative degradation products extracted from an Andosol soil.[¹H]NMR spectra of all organic fractions present considerable adsorption between 7.4 and 8.8 ppm, due to the presence of aromatic protons.The percentages of aromatic protons in respect to the total amount of protons are as follows: FA 20%, HA 19%, degraded FA < 12%, degraded HA 14%. The values indicate that the contribution of aromatic structures to the humic substances is significant, also considering that they are highly substituted.The degraded fractions contain smaller amounts of aromatic protons, because degradation causes the opening of the aromatic rings. Thus results obtained from the degradation do not seem to be reliable for defining the importance of aromatic structures in humic substances.Also the [¹³C]NMR spectra show signals in the aromatic region which derive from unsubstituted carbon atoms, while signals originating from tertiary carbon atoms merge with the noise. We believe that, at present, [¹H]NMR spectroscopy is more suited for studying the role played by aromatic compounds in organic soil fractions.     

Adsorption of dissolved organic matter on clay minerals as assessed by infra-red, CPMAS C NMR spectroscopy and low field T1 NMR relaxometry

Dissolved organic matter (DOM) is a very important environmental constituent due to its role in controlling factors for soil formation, mineral weathering and pollutant transport in the environment. Prediction of DOM physical-chemical properties is achieved by studying its chemical structure and spatial conformation. In the present study, dissolved organic matter extracted from compost obtained from the organic fraction of urban wastes (DOM-P) has been analysed by FT-IR, CPMAS ¹³C NMR spectroscopy and ¹H T₁ NMR relaxometry with fast field cycling (FFC) setup. While the first two spectroscopic techniques revealed the chemical changes of dissolved organic matter after adsorption either on kaolinite (DOM-K) or montmorillonite (DOM-S), the latter permitted the evaluation of the conformational variations as assessed by longitudinal relaxation time (T₁) distribution at the fixed magnetic field of 500 mT. Alterations of T₁ distributions from DOM-P to DOM-K and DOM-S were attributed to a decreasing molecular complexity following DOM-P adsorption on the clay minerals. This study applied for the first time solid state ¹H T₁ NMR relaxometry to dissolved organic matter from compost obtained from the organic fraction of urban wastes and revealed that this technique is very promising for studying environmentally relevant natural organic systems.     

The usefulness of UV–visible and fluorescence spectroscopies to study the chemical nature of humic substances from soils and composts

The aim of this work is to study the suitability of the use of fluorescence and UV–visible spectroscopies to evaluate the humification degree of different organic alkaline extracts obtained from diverse materials (humic substances, composted and non-composted materials). Our results indicate that the complementary utilization of the fluorescence index proposed by Milori et al. [Milori, D., Martin-Neto, L., Bayer, C., Mielniczuk, J., Vagnato, V., 2002. Humification degree of soil humic acids determined by fluorescence spectroscopy. Soil Science, 167, 739–749] (the total area of the emission spectra collected over a range of 460–650 nm using an excitation wavelength of 465 nm), the ratio between the electron-transfer band and benzenoid band of benzene UV light absorption, and the molar absorption coefficients at 600 nm and 280 nm proved capable of discriminating between extracts obtained from non-humified, partially humified and very humified materials.Likewise, our results suggest that humification is related to increments in the aromatic character of the molecules, which may be reflected in the presence of more aromatic rings and/or more condensed polyaromatic structures, and the degree of substitution in aromatic rings with polar functional groups, as well as to the possible increase in the conjugation degree in unsaturated aliphatic chains.     

1H and 13C NMR of marine humic acids

¹³C and ¹H NMR spectra were obtained for humic acids isolated from marine sediments. NMR shows great promise in identifying structural components of humic acids as some new and interesting structural features are identified. Aliphatic structures were found to constitute a much larger fraction of humic acids than previously thought, and they appeared to be highly branched. Although the aromatic content of terrestrial humic acid was found to be lower than expected, the aromaticity appears to be a specific discriminator of terrestrial/aquatic source types. A humic acid isolated from an anoxic algal sapropel was found to be composed predominantly of polyuronic acids and different than other aquatic sedimentary humic substances.     

Comment on the paper: “[1H] and [13C]NMR studies on the importance of aromatic structures in fulvic and humic acids” (Ruggieroet al., 1979)

Some statements referring to our work on ¹³C NMR of humic and fulvic acids have been recently reported in this journal by Ruggiero and co-workers. They are discussed in order to avoid possible misinterpretations.     

Methylation patterns of aquatic humic substances determined by13C NMR spectroscopy

¹³C NMR spectroscopy is used to examine the hydroxyl group functionality of a series of humic and fulvic acids from different aquatic environments. Samples first are methylated with¹³C-labeled diazomethane. The NMR spectra of the diazomethylated samples allow one to distinguish between methyl esters of car☐ylic acids, methyl ethers of phenolic hydroxyls, and methyl ethers of phenolic hydroxyls adjacent to two substituents. Samples are then permethylated with¹³C-labeled methyl iodide/NaH.¹³C NMR spectra of permethylated samples show that a significant fraction of the hydroxyl groups is not methylated with diazomethane alone. In these spectra methyl ethers of carbohydrate and aliphatic hydroxyls overlap with methyl ethers of phenolic hydroxyls. Side reactions of the methyltion procedure including carbon methylation in the CH₃I/NaH procedure, are also examined. Humic and fulvic acids from bog, swamp, groundwater, and lake waters showssome differences in their distribution of hydroxyl groups, mainly in the concentrations of phenolic hydroxyls, which may be attributed to their different biogeochemical origins.     

1H NMR spectra of humic and fulvic acids and their peracetic oxidation products

¹H NMR spectra of humic (HA) and fulvic (FA) acids and their oxidative degradation products are reported. The HA shows the presence of -(CH₂)_n - CH₃ (n > 6) chemical fragments belonging to n-alkanes and/or n-fatty acids physically adsorbed onto the macromolecule structure. These fragments are absent in the FA fraction. Both humic fractions reveal the presence of similar amounts of aromatic protons which partly undergo exchange phenomena. The importance of this experimental observation is discussed. Oxidative degradation seems to cause partial cleavage of aromatic rings, more pronounced in the FA than in the HA. The degraded FA shows a higher total acidity and a higher phenolic OH content than the degraded HA. Both degraded fractions display some sharp singlet signals at 1.9 and 3.9 ppm arising from protons belonging to repetitive chemical fragments probably formed during the oxidation reaction. Tentative assignments of these signals are given. A general analysis of the HA and FA degraded spectra seems to indicate that the chemical fragments which undergo peracetic oxidation are substantially similar. The extent of oxidation of the two humic fractions is different. The HA degradation products reveal the presence of oligomeric structures, whereas the degraded FA appears less resistant to the oxidizing agent.     

Characterization of sinking particles from the northwest Mediterranean Sea using advanced solid-state NMR

Sinking particles are the major transporter of organic carbon from surface to the deep ocean, and their chemical composition changes dramatically with depth. However, the exact mechanism controlling the chemical transformation as particles sink is not well understood, and little detail is known about the structural changes. This is mainly due to the paucity of techniques available to analyze the major macromolecular components of sinking particles. Here we applied advanced solid-state NMR techniques, including cross polarization/magic angle spinning (CPMAS), direct polarization/magic angle spinning (DPMAS), two-dimensional ¹H-¹³C heteronuclear correlation (2D HETCOR) and ¹H T₁ inversion recovery, on sinking particles collected in the northwest Mediterranean Sea. The CPMAS ¹³C NMR spectrum of the 200-m particles is significantly different from that of the DPMAS ¹³C NMR spectrum: CPMAS overestimates the NCH and CHO groups, but underestimates the aliphatic components, which is attributed to the high mobility of polymethylene units. Thus DPMAS is more suitable for quantifying organic composition of sinking particles. Using 2D HETCOR with ¹H spin diffusion, we estimated that the size of domains (similar structural entities either physically or chemically grouped together) in the 200-m sinking particles can be as large as tens of nanometers. The results of CPMAS ¹³C NMR and ¹H inversion recovery on sinking particles from 200, 520 and 920 m indicate that the macromolecular heterogeneity observed in surface particles virtually disappears as particles sink into the deep ocean. This suggests that the macromolecular components at depth are different in structural composition than those in surface waters, and may be compositionally homogenized as particles sink.     

Ultrafiltration of fulvic and humic acids,a comparison of stirred cell and hollow fiber techniques

The ultrafiltration of solutions of well characterized samples of fulvic and humic acids or their salts, using two different techniques, show that these materials are retained by commercial ultrafiltration membranes with molecular weight cut-off values far higher than the reported molecular weights of fulvic or humic acid. The lower molecular weight fulvic acid or its Na⁺ -salt is retained even better than the higher molecular weight Na⁺-salt of humic acid. Both stirred cell and hollow fiber techniques can be employed to concentrate or desalt solutions of these humic materials, with relatively small solute losses. Very high simple electrolyte backgrounds (above 1 M) lower the performance of a given membrane considerably, moderate concentrations of simple electrolyte do not influence the ultrafiltration efficiency.     

Detailed investigation of organic matter components in extracts and drainage waters from a soil under long term cultivation

Aqueous solutions of increasing pH (7.0, 10.6 and 12.6) were used to extract exhaustively the organic matter (OM) from a pelo-stagnogley (heavy clay) soil in long term cultivation. OM yield was 1.7 times greater when the extracts were processed using an XAD-8 and XAD-4 resin-in-tandem procedure than that from the procedure of the International Humic Substances Society (IHSS). The substantial difference can be attributed to the amount retained by the XAD-4 resin, which is lost in the IHSS process. Elemental, δ¹³C, δ¹⁵N, cation exchange capacity, neutral sugars, amino acids and solid state CPMAS ¹³C NMR analyses indicated significant, but rational similarities and differences between the various fractions isolated. There was strong NMR evidence for material derived from lignin in all the humic and fulvic acid isolates. The signals were attenuated in the more transformed/oxidized fractions isolated at lower pH. Novel humic acid fractions enriched in carbohydrate/peptide functionalities were isolated from the more hydrophobic extracts at pH 10.6 and 12.6. Isolates from XAD-4, of microbial origin, were enriched in neutral sugars but not in amino acids, and had minimal aromaticity. Components isolated from the cultivated soil were broadly similar to those from a comparable soil in long term grassland. The compositions of fractions isolated from the drainage water were similar to those extracted from the soil but had higher carboxyl content. The amount and composition of the various organic fractions in grassland and the continuously cropped soil are discussed in terms of their potential to contribute to carbon sequestration by soil under similar management regimes.     

Investigation of marine and terrestrial humic substances by H and C, nuclear magnetic resonance and infrared spectroscopy

Humic acids were isolated from 5 sediments in which the origin nature of the organic matter are both typical and different. The humic acids were characterized on the basis of elemental compositions, infrared spectra and ¹H and ¹³C NMR. This last technique, especially ¹³C NMR, provides qualitative and semi-quantitative information regarding aromatic structure. Combined data from the three techniques permits differentiation of marine and terrestrial organic matter as well as identification of mixtures of humic acids from the two sources.     

Sources of sedimentary humic substances: vascular plant debris

A modern Washington continental shelf sediment was fractionated densimetrically using either an organic solvent, CBrCl₃, or aqueous ZnCl₂. The resulting low density materials (<2.06 g/ml) account for only 1% of the sediment mass but contain 25% of the sedimentary organic carbon and 53% of the lignin. The C/N ratios (30–40) and lignin phenol yields ($\text{Λ = 8}$) and compositions indicate that the low density materials are essentially pure vascular plant debris which is slightly enriched in woody (versus nonwoody) tissues compared to the bulk sediment. The low density materials yield approximately one-third of their organic carbon as humic substances and contribute 23% and 14% of the total sedimentary humic and fulvic acids, respectively. Assuming that the lignin remaining in the sedimentary fraction is also contained in plant fragments that yield similar levels of humic substances, then 50% and 30% of the total humic and fulvic acids, respectively, arise directly from plant debris.Base-extraction of fresh and naturally degraded vascular plant materials reveals that significant levels of humic and fulvic acids are obtained using classical extraction techniques. Approximately 1–2% of the carbon from fresh woods and 10–25% from leaves and bark were isolated as humic acids and 2–4 times those levels as fulvic acids. A highly degraded hardwood yielded up to 44% of its carbon as humic and fulvic acids. The humic acids from fresh plants are generally enriched in lignin components relative to carbohydrates and recognizable biochemicals account for up to 50% of the total carbon. Humic and fulvic acids extracted directly from sedimentary plant debris could be responsible for a major fraction of the biochemical component of humic substances.     

New evidence for covalent coupling of peptides to humic acids based on 2D NMR spectroscopy: A means for preservation

Nitrogen immobilization in soils and sediments involving the preservation of peptides is an important yet not well-understood process. Several hypotheses have been proposed for the preservation of peptides in these systems; however, to date, there is no direct molecular-level evidence for any one of several hypotheses. In this study, a synthesized peptide with ¹⁵N-labeled amino acid residues was utilized to examine the mechanism by which humic substances from a peat might chemically interact with peptides to induce preservation. Two-dimensional ¹⁵N-¹H HSQC NMR experiments were performed to evaluate the bonding and nonbonding interactions between ¹⁵N-labeled peptide and an Everglades peat humic acid. The observed changes in cross peaks provide the first direct spectroscopic evidence for what we believe to be covalent binding between these substances. Noncovalent interactions between the ¹⁵N-labeled peptides and humic acid molecules are also observed in the spectra. Quinone structures in humic acids are suggested to be the important reactive groups, based on reaction of the labeled peptide with a model quinone which shows similar nitrogen-proton correlated peak patterns as is observed in the HSQC NMR spectra of humic acids reacted with the peptide. The Michael reaction with quinone structures is proposed, and this type of reaction provides a mechanism that is consistent with previous observation for the chemistry of sedimentary nitrogen species.     

Use of solid-state 13C NMR in structural studies of humic acids and humin from Holocene sediments

¹³C NMR spectra of solid humic substances in Holocene sediments have been obtained using cross polarization with magic-angle sample spinning techniques. The results demonstrate that this technique holds great promise for structural characterizations of complex macromolecular substances such as humin and humic acids. Quantifiable distinctions can be made between structural features of aquatic and terrestrial humic substances. The aliphatic carbons of the humic substances are dominant components suggestive of input from lipid-like materials. An interesting resemblance is also noted between terrestrial humic acid and humin spectra.     

Structural analysis of sea loch sedimentary humic substances by 13C1H dipolar dephasing NMR

Nuclear magnetic resonance (NMR) techniques have been applied to the structural characterization of humic substances isolated from an organic-rich sediment in Loch Thurnaig, northwest Scotland. Both the sedimentary humic acid (SHA) and sedimentary fulvic acid (SFA) fractions from Loch Thurnaig contained substantial quantities of carboxylic and alkyl carbon with a small contribution (26 and 22% respectively) of aromatic/olefinic carbon atoms. The latter structures were shown by dipolar dephasing ¹³C NMR to be largely non-protonated. Differences in the alkyl structures of the two fractions were noted, with the SHA containing a larger amount of branched chain aliphatic material. As branched chain compounds are more resistant to microbial degradation than straight chain compounds, this may indicate that SHA is the older, more refractory fraction of the sedimentary organic matter, although it is possible that the results reflect different algal precursor material. The fraction of aromatic carbon was higher in the SHA than in the SFA as is generally found for humic and fulvic acids isolated from the same source. The low aromaticities and highly branched aliphatic structures show that the sedimentary humic substances from the loch resemble dissolved marine humic substances rather than their terrestrial counterparts.     

Metal distribution and nature of some Cu,Mn and V complexes in humic and fulvic acid fractions of soil organic matter

Organic matter from an arable soil derived from base rich parent material was extracted by alkali and fractionated on the basis of solubility in 0.1 N HCl, hot water and hot 6 N HCl and by selective adsorption on charcoal. The distribution of associated metals was determined and Cu had the largest proportion, 15%, associated with the organic matter. Moderate proportions of the total Al, Co, Ni, and V (3–8%) but only small amounts (?1%) of the Mn, Fe, Ti, Cr, Ba and Sr were extracted from the soil by alkali. The Fe and Ti were concentrated mainly in the humic fraction whereas Mn and V were both found largely in the fulvic acid.Electron paramagnetic resonance spectra of the various fractions were examined and attempts made to relate the spectra to the forms of some of the metals present. In the humic acid fraction Cu was present partly as a copper porphyrin-type complex but in the fulvic acid it was in some other complexed form. VO²⁺ occurred in complexed forms in the fulvic acid which were more covalent than VO²⁺ humic acid complexes, whereas the Mn²⁺ components of the humic and fulvic acids all had a high degree of ionicity.