Multivariate analysis of CPMAS 13C-NMR spectra of soils and humic matter as a tool to evaluate organic carbon quality in natural systems

A series of humic and fulvic acids isolated from different sources, size‐fractions separated from a humic acid, and three soils of different origin were subjected to CPMAS ¹³C‐NMR spectroscopy to obtain the distribution of their carbon contents. The relative areas of chemical shift regions in NMR spectra were used to apply a principal component analysis (PCA) to the three sets of samples. The multivariate analysis was successful in efficiently differentiating samples on the basis of the quality of their organic carbon content. The PC biplots based on two principal components distinguished objectively among samples as accurately as it was possible to do by subjective qualitative evaluation of the original spectra. In the case of the soils, a discriminant analysis (DA) was applied to build a classification model that allowed the validation of the three soils according to their origin. Percentage of validation in the classification model is expected to increase when a large number of NMR spectra are accumulated and/or the concentration of organic carbon in samples is enhanced. The multivariate analyses described are likely to become a useful tool to increase the importance of CPMAS ¹³C‐NMR spectra in the appraisal of natural organic matter variations in heterogeneous natural systems.     

A 13C-NMR study of the interactions of soil organic matter with aluminium and allophane in podzols

The stabilization of organic matter in soil by interaction with aluminium (Al) or allophane is important in maintaining soil quality, and in retarding the decomposition of soil organic matter. Complexation of Al by soil organic matter may also ameliorate Al toxicity. Here we use ¹³C-NMR spectroscopy to assess the interaction of soil organic matter with both Al and allophane in two poorly drained podzols containing only trace amounts of iron. The ¹³C-NMR spectrum of the subsoil of the allophane-rich One Tree Point podzol shows an intense peak at 179 p.p.m., assigned to carbon in carboxylic acids. This peak shifts to 177 p.p.m. after removal of allophane (11% of the soil mass) by treatment with HF. We infer that the carboxyl groups in the organic matter are bonded to structural Al on the surface of allophane spherules. In the non-allophanic Te Kopuru podzol, on the other hand, the organic matter apparently interacts with Al ions in the soil solution. This soil also has more aromatic carbon and fewer carbons in carboxyl and carbohydrate structures than the allophanic sample. There is an indication that allophane stabilizes carbohydrate groups as well as carboxyl groups.     

Dissolved organic matter sorption on sub soils and minerals studied by 13C-NMR and DRIFT spectroscopy

Sorption on the mineral matrix is an important process restricting the movement of dissolved organic matter (DOM) in soils. In this study, we aimed to identify the chemical structures responsible for the retention of DOM by sorption experiments with total DOM and acidic humic substances (AHS), containing humic and fulvic acids, on soil samples and minerals (goethite, ferrihydrite, and amorphous Al(OH)₃). The AHS remaining in solution after sorption were studied by ¹³C nuclear magnetic resonance (NMR) analysis, and total DOM and AHS for bed on the surfaces of minerals by diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy. The soil samples were taken from strongly sorbing Bw horizons of two Inceptisols rich in pedogenetic Fe (29 and 35 g kg ^?1) and containing little C (7 and 22 g kg^?1). The ¹³C-NMR spectra showed that sorption causes a preferential removal of aromatic and carboxyl C from the solution, whereas alkyl-C accumulates in the solution. No change was observed for O-alkyl C. The DRIFT spectra of sorbed total DOM and AHS showed a relative increase of the band intensity of carboxyl groups compared to DOM in the initial solution, confirming the importance of those groups for the sorption to mineral surfaces. The spectra also indicated reactions of carboxyl groups with metals at the mineral surfaces. The extent to which the carboxyl groups are bound depended on the surface coverage with DOM and the type of mineral.     

Solid-state 13C-NMR dipolar dephasing experiments for quantifying protonated and non-protonated carbon in soil organic matter and model systems

Dipolar dephasing was investigated as a means for quantitatively differentiating protonated and non‐protonated and molecularly mobile and rigid components in soil organic matter. We experimented on cellulose, hemicelluloses, lignin, a protein, chitin, charcoal, palmitic acid and two waxes which served as model systems for components of soil organic matter. Dipolar dephasing decay curves showed significant deviation from the expected exponential decay (for non‐protonated and methyl carbons) and Gaussian decay (for non‐methyl protonated carbons), partly due to rotational and dipolar modulation. No signal is observed for most protonated carbon (excluding methyl carbon) for a dipolar dephasing delay of 45 μs, and so we generated three subspectra, representing non‐protonated, non‐methyl protonated and methyl carbon classes, requiring the acquisition of just the 0‐ and 45‐μs dipolar dephased spectra. This methodology was applied to eight samples of soil organic matter, allowing the determination of relative contributions of overlapping resonances such as C‐substituted and H‐substituted aromatics (110–145 p.p.m.), carbohydrate anomeric and tannin (90–110 p.p.m.), and amino acid and methoxyl (45–65 p.p.m.). The waxes behaved aberrantly, probably because some of their components are highly mobile. We determined accurately the exponential dipolar dephasing decay rates of non‐protonated carbon resonances, free from the interference of rotational and dipolar modulations.     

Effect of barley plants on the decomposition of 14C-labelled soil organic matter

The effect of barley plants on the rate of decomposition of soil organic matter over a 6-week period was studied using soil that had been previously labelled by incubation with ¹⁴C-labelled ryegrass for 1 year. The plants reduced the loss of ¹⁴CO₂, from soil by 70 per cent over 42 days. About half of the reduction was accounted for by the uptake of labelled C by the plant roots, very little ¹⁴C label being associated with the shoot. Chemical fractionation of the root showed that the ¹⁴C was chemically incorporated into cell wall materials such as cellulose and holocellulose. The reduction in organic matter decomposition in the presence of plants has been explained by earlier workers in terms ofa reduction in microbial activity as a result of a soil moisture deficit caused by plant transpiration. This explanation does not account for all the reduction in decomposition noted in the present experiments. Control soil (without a plant, but amended with glucose or yeast extract to simulate the effect of root exudates) showed a small positive priming effect, the release of ¹⁴CO₂, being increased. Thus the mechanism by which plants conserve organic matter is complex and cannot be explained merely by analogy to an increased level of nutrients available for microbial metabolism.     

Land-use related organic matter dynamics in North Cameroon soils assessed by 13C analysis of soil organic matter fractions

Topsoil samples from cultivated and adjacent non‐cultivated fields on three major agricultural soils in North Cameroon were fractionated into particle‐size fractions that were analysed subsequently for their C and ¹³C contents. The aim was to obtain further insight into the dynamics of soil organic matter (SOM) in relation to land use in Cameroon. Since organic carbon contents of the fractions were often very small, samples and analyses were extensively replicated to obtain robust statistical estimates of observed differences. For each soil type, differences in δ¹³C values between fields could be related to changes in the input and decomposition of organic matter arising from soil type, land management and, for example, the nature and abundance of weeds. Turnover of organic matter appeared to be fastest in the sand fraction, which is in line with results from earlier studies. In the finer fractions, clear differences in reaction to changes in input and decomposition were observed, that seem to be linked to differences in clay mineralogy. The results illustrate that SOM in the various fractions is much less stable and more strongly affected by changes in land use than might be assumed on the basis of changes in total SOM contents alone. At the same time, they demonstrate the relevance of ¹³C isotope analyses of SOM for studies on the impact of land use on these savannah soils with little SOM that are highly susceptible to degradation.     

The dynamics of organic matter in rock fragments in soil investigated by 14C dating and measurements of 13C

Rock fragments in soil can contain significant amounts of organic carbon. We investigated the nature and dynamics of organic matter in rock fragments in the upper horizons of a forest soil derived from sandstone and compared them with the fine earth fraction (<2 mm). The organic C content and its distribution among humic, humin and non‐humic fractions, as well as the isotopic signatures (Δ¹⁴C and δ¹³C) of organic carbon and of CO₂ produced during incubation of samples, all show that altered rock fragments contain a dynamic component of the carbon cycle. Rock fragments, especially the highly altered ones, contributed 4.5% to the total organic C content in the soil. The bulk organic matter in both fine earth and highly altered rock fragments in the A1 horizon contained significant amounts of recent C (bomb ¹⁴C), indicating that most of this C is cycled quickly in both fractions. In the A horizons, the mean residence times of humic substances from highly altered rock fragments were shorter than those of the humic substances isolated in the fine earth. Values of Δ¹⁴C of the CO₂ produced during basal respiration confirmed the heterogeneity, complexity and dynamic nature of the organic matter of these rock fragments. The weak ¹⁴C signatures of humic substances from the slightly altered rock fragments confirmed the importance of weathering in establishing and improving the interactions between rock fragments and surrounding soil. The progressive enrichment in ¹³C from components with high‐¹⁴C (more recent) to low‐¹⁴C (older) indicated that biological activity occurred in both the fine and the coarse fractions. Hence the microflora utilizes energy sources contained in all the soil compartments, and rock fragments are chemically and biologically active in soil, where they form a continuum with the fine earth.     

Effects of mineral surface iron on the CPMAS 13C-NMR spectroscopic detection of organic matter from soil fractions in an agricultural topsoil with different amendments

The decrease of NMR visibility of the C signal in soil samples due to the association between organic carbon (OC) and the topsoil mineral surface was investigated. CPMAS ¹³C‐NMR spectra were obtained for soil particle‐size fractions (< 2 μm, 2–20 μm, > 20 μm) and bulk soils from an agricultural topsoil (Chernozem) that had received three different amendments (no fertilization, mineral fertilization (NPK), mineral (NPK) and organic (cattle manure) fertilizations) at Bad Lauchstädt, Germany. The soil organic carbon content of the three soils depended on the degree of soil fertilization. There was no constant relationship between the total NMR signal intensity and the total amount of organic carbon (TOC) for all size fractions. Indeed, a key role played in the C signal intensity by the paramagnetic ferric ion from the clay content in soil fractions and bulk soils was confirmed. Thus, we describe the variations of C signal intensity by taking into account the distribution of clay‐associated OC and non‐associated OC pools. Depending on the amendment, the C signal visibility was weakened by a factor of 2–4 for the clay‐associated OC. This estimation was rendered possible by combining mineral specific surface area (SSA) measurements with the N₂ gas adsorption method (BET method) and determination of TOC and iron concentrations. This approach contributes to the quantitative evaluation of the CPMAS ¹³C‐NMR detection.     

Dynamics of soil organic matter turnover and soil respired CO2 in a temperate grassland labelled with 13C

The fate of carbon (C) in grassland soils is of particular interest since the vast majority in grassland ecosystems is stored below ground and respiratory C‐release from soils is a major component of the global C balance. The use of ¹³C‐depleted CO₂ in a 10‐year free‐air carbon dioxide enrichment (FACE) experiment, gave a unique opportunity to study the turnover of the C sequestered during this experiment. Soil organic matter (SOM), soil air and plant material were analysed for δ¹³C and C contents in the last year of the FACE experiment (2002) and in the two following growing seasons. After 10 years of exposure to CO₂ enrichment at 600 ppmv, no significant differences in SOM C content could be detected between fumigated and non‐fumigated plots. A ¹³C depletion of 3.4‰ was found in SOM (0–12 cm) of the fumigated soils in comparison with the control soils and a rapid decrease of this difference was observed after the end of fumigation. Within 2 years, 49% of the C in this SOM (0–12 cm) was exchanged with fresh C, with the limitation that this exchange cannot be further dissected into respiratory decay of old C and freshly sequestered new C. By analysing the mechanistic effects of a drought on the plant‐soil system it was shown that rhizosphere respiration is the dominant factor in soil respiration. Consideration of ecophysiological factors that drive plant activity is therefore important when soil respiration is to be investigated or modelled.     

13C NMR characterization of humic acids from soils of a development sequence

Humic acids were isolated from nine topsoils in New Zealand tussock grasslands. Cross-polarization ¹³C NMR spectra of solid samples were used to estimate fractions of carbon contained in different types of chemical functional groups. The degree of oxygen substitution of aromatic rings showed a strong negative correlation with soil development. Aromaticities greater than 0.25 were found in humic acids from only the two least-developed soils.     

The effect of water content on solid-state 13C NMR quantitation and relaxation rates of soil organic matter

Two hydrofluoric acid‐treated soils were prepared with water contents ranging up to 22% by exposing them to a range of atmospheric humidities. There was no effect of water content on the chemical shift distribution of nuclear magnetic resonance (NMR) signal in ¹³C cross‐polarization (CP) NMR spectra. The sensitivity of the ¹³C CP NMR spectra decreased slightly with increasing water content. Much of this decrease could be attributed to decreases in T_1ρH relaxation rates, caused by enhanced molecular mobility of the organic matter in the presence of absorbed water. Rates of T_1H relaxation were very sensitive to water content, and average T_1H relaxation rates decreased four‐ to five‐fold from the smallest to the largest water content. Rates of T_1H relaxation were non‐uniform, and were better modelled by two‐T_1H component fits than one‐T_1H component fits. The ratio of rapidly to slowly relaxing components increased with increasing water content. Proton spin relaxation editing (PSRE) subspectra revealed substantial changes in the nature of these two components with increasing water content. These results indicate the presence of an organic matter component that is very sensitive to water content, transforming from slowly relaxing at a small water content to rapidly relaxing at a greater water content. This component was shown to be rich in O–alkyl and carbonyl C, and may be hemicellulosic root exudates and microbial mucilages. The slowly relaxing PSRE component was a mixture of ligno‐cellulose and alkyl biopolymers, whereas the rapidly relaxing component was primarily charcoal for one of the soils, and was reminiscent of dissolved organic carbon for the other soil. These findings show that care must be taken in controlling water contents when using PSRE to study organic matter.     

Transformation of organic matter from maize residues into labile and humic fractions of three European soils as revealed by 13C distribution and CPMAS-NMR spectra

The dynamics of incorporation of fresh organic residues into the various fractions of soil organic matter have yet to be clarified in terms of chemical structures and mechanisms involved. We studied by ¹³C‐dilution analysis and CPMAS‐¹³C‐NMR spectroscopy the distribution of organic carbon from mixed or mulched maize residues into specific defined fractions such as carbohydrates and humic fractions isolated by selective extractants in a year‐long incubation of three European soils. The contents of carbohydrates in soil particle size fractions and relative δ¹³C values showed no retention of carbohydrates from maize but rather decomposition of those from native organic matter in the soil. By contrast, CPMAS‐¹³C‐NMR spectra of humic (HA) and fulvic acids (FA) extracted by alkaline solution generally indicated the transfer of maize C (mostly carbohydrates and peptides) into humic materials, whereas spectra of organic matter extracted with an acetone solution (HE) indicated solubilization of an aliphatic‐rich, hydrophobic fraction that seemed not to contain any C from maize. The abundance of ¹³C showed that all humic fractions behaved as a sink for C from maize residues but the FA fraction was related to the turnover of fresh organic matter more than the HA. Removal of hydrophobic components from incubated soils by acetone solution allowed a subsequent extraction of HA and, especially, FA still containing much C from maize. The combination of isotopic measurements and NMR spectra indicated that while hydrophilic compounds from maize were retained in HA and FA, hydrophobic components in the HE fraction had chemical features similar to those of humin. Our results show that the organic compounds released in soils by mineralization of fresh plant residues are stored mainly in the hydrophilic fraction of humic substances which are, in turn, stabilized against microbial degradation by the most hydrophobic humic matter. Our findings suggest that native soil humic substances contribute to the accumulation of new organic matter in soils.     

Organic matter composition in sewage farm soils: Investigations by 13C-NMR and pyrolysis-field ionization mass spectrometry

The composition of soil organic matter (SOM) on sewage farms south of Berlin was investigated by solid-state CP/MAS ¹³C-NMR and pyrolysis-field ionization mass spectrometry (Py-FIMS) of freeze-dried sewage solids and soil samples of differing contamination. These were an untreated soil (USOIL), a former sewage farm used as arable land since 1990 (SF90A), and a recent sewage farm (SF1994). The CP/MAS ¹³C-NMR spectra showed enrichments of the sewage-treated soils with aliphatic C and C in OCH₃-groups and amino acids. In the Py-FI mass spectra the major markers of sewage and SOM in sewage farm soils were (i) N-containing compounds, in particular peptides, (ii) dimethylphthalate (m/z 194), (iii) sterols, and (iv) signals in the mass range m/z 502 to 554 of mono- and diaryl esters which were substituted by long aliphatic chains. The latter signals were intense in the sewage solids, increased in intensity from sample SF90A to SF1994; but they were not present in the USOIL, thus clearly indicating anthropogenic origin. Temperature-resolved Py-FIMS showed that the SOM compounds in the sewage farm soils were generally incorporated into bonds with widely different stabilities which could be relevant for SOM turnover and environmental effects. This is demonstrated for the trapping of dimethylphthalate in a modelled humic substance.     

Effect of tillage on soil organic carbon mineralization estimated from 13C abundance in maize fields

Three methods of cultivation, conventional tillage (CT), superficial tillage (ST) and no-tillage (NT), were applied for 17 years to continuous maize. Their effect on soil organic carbon content was investigated through measurements of carbon and ¹³C/¹²C ratios, using the natural difference in ¹³C content between C₃ plants and maize, which is a C₄ plant. Because the soil had carried C₃ plants before the experiment started, the organic carbon remaining from that time (C₃,-carbon), was distinguished from the carbon derived from maize. Comparison between continuous wheat and maize plots showed that organic matter from both maize and wheat decomposed without significant ¹³C enrichment, whereas older C₃-carbon was enriched by 1.5% compared to that of fresh wheat material. From the initial 3.6 kg C m^?2 in the topsoil (0–30 cm), 0.95 were mineralized in the CT treatment, but only 0.45 in NT. The mineralization was the same in the tilled layer of ST as in CT. The CT treatment accumulated 1.1 kg C m^?2 of maize-derived carbon and the NT treatment 0.8. The mineralization of initial C₃-carbon was the same at all depths between 0 and 30 cm in the NT treatment; 75% of the carbon derived from maize was found in the 0–5 cm layer.     

The influence of humus fractionation on the chemical composition of soil organic matter studied by solid-state 13C NMR

Four samples of soil organic matter and their humic acids, fulvic acids and humin were studied with solid-state ¹³CP MAS NMR. The whole soil samples were fractionated using NaOH and HCl in order to extract humic acids, fulvic acids and humin. This investigation indicates that conventional humus fractionation does not significantly change the content of different functional groups in soil.     

1H-nuclear magnetic resonance study of a soil humic acid

The structure of a humic acid from a New Zealand pasture topsoil has been investigated by ¹H-n.m.r. spectroscopy. The work complements a recent ¹³C-n.m.r. study on the same humic acid. Irradiating the water peak in the spectrum greatly improves spectral quality. The results show that the proton aromaticity of the humic acid (excluding exchangeable protons) is 17%. Considerable amounts of protons in the humic acid are associated with carbohydrates (35%). Useful structural parameters describing the average molecular structure of the humic acid were derived by combining both ¹H and ¹³C data. Further work is needed to determine both the percentage exchange of protons in alkali and the percentage of inorganic hydrogen in humic acids, before the condensation and the degree of substitution of aromatic rings can be quantitatively estimated.     

Quantitative solid-state 13C NMR spectroscopy of organic matter fractions in lowland rice soils

Spin counting on solid‐state ¹³C cross‐polarization (CP) nuclear magnetic resonance (NMR) spectra of two humic fractions isolated from tropical lowland soils showed that only 32–81% of potential ¹³C NMR signal was detected. The observability of ¹³C NMR signal (C_obs) was higher in the mobile humic acid (MHA) than in the calcium humate (CaHA) fraction, and increased with increasing intensity of irrigated rice cropping. NMR observability appeared to be related to the nature of the organic carbon, with phenol‐ and methoxyl‐rich samples having the higher values of C_obs. The Bloch decay (BD) technique provided more quantitatively reliable ¹³C NMR spectra, as evidenced by values of C_obs in the range 91–100% for seven of the eight humic fractions studied. The BD spectra contained considerably more aryl and carbonyl signal, and less O–alkyl and alkyl signal, with the greatest differences between CP and BD spectra observed for the samples with low C_obs(CP). The causes of low CP observability were investigated using the spectral editing technique RESTORE ( RE storation of S pectra via T _CH and T O ne R ho (T_1ρH) E diting). Rapid T_1ρH relaxation was found to be primarily responsible for the under‐representation of carbonyl carbon, whereas inefficient cross‐polarization was primarily responsible for the under‐representation of aryl carbon in CP spectra. Proton NMR relaxation rates T_1H and T_1ρH were found to correlate with other NMR properties and also with cropping management. Non‐uniform rates of T_1H relaxation in two of the CaHA fractions enabled the generation of proton spin relaxation editing subspectra.     

Sludge-derived organic carbon in an agricultural soil estimated by 13C abundance measurements

The objective of this study is to develop a method to follow the dynamics of sludge‐derived organic carbon, which will allow us to understand the behaviour of trace metals in the sludge‐treated soils. We studied, in a sandy agricultural soil of southwest France, cultivated with maize and amended with sewage‐sludge over 20 years, the dynamics of different sources of organic matter and compared this with a control, which had never received any treatment. For the first time, a method is proposed that will distinguish and quantify sludge‐derived organic carbon, maize‐derived organic carbon, and native organic carbon. This method is based on the mean differences in δ¹³C abundances between native (−26.5‰), maize (−12.5‰) and sludge (−25.4‰) organic carbon. Three hypotheses on the dynamics of soil organic matter sources are proposed: (i) isotopic differences observed between control and sludge‐treated soils are due only to the incorporation of sludge C, whereas in the others, the control was used to model the incorporation of (ii) maize C or (iii) native C in the sludge‐treated soils. The comparison of the stocks of each source (native C, maize C and sludge C) found in the bulk soil with the sum of corresponding stocks found in particle‐size fractions allowed us to reject the two first hypotheses and to validate the last one. Repeated applications of sewage‐sludge induced accumulation of sludge‐derived organic carbon in the topsoil, and simultaneously contributed to the preservation of maize‐derived organic carbon. When sludge applications ceased, the rapid decrease in soil organic matter stocks was mostly caused by the degradation of the sludge‐derived organic carbon sources. At the same time, the maize‐derived organic carbon shifted from the coarsest fraction (200–2000 μm) to the finest fraction (0–50 μm). Therefore, this study has shown that repeated applications of sewage‐sludge induced changes in soil organic matter dynamics over time.     

13C NMR of humic substances: pH and solvent effects

Since the concentration of free radicals in humic subtances increases at high pH the use of basic solutions for ¹³C NMR spectroscopy may cause broadening and loss of aromatic signals, with distortion of intensity distributions. No such effects were found in ¹³C spectra of soil humic and fulvic acid, an aquatic fulvic acid, and two phenolic polymers run in aqueous solutions at different pH values, and in dimethylsulphoxide. With increasing pH, the peak in the carboxyl region shifted in a manner consistent with greater dissociation of carboxyl and phenolic groups, and also certain features in the aliphatic and carboxyl regions were enhanced under some solution conditions. Elevated solution temperature (70°C) caused only slight improvement in the resolution of some lines. Chemical shifts were determined for some known phenolic and benzenecarboxylic acid compounds in DMSO and NaOD. The range for phenolic carbons extended to 173 ppm in NaOD, while some aromatic carbons occurred around 105 ppm, in the same region as anomeric carbons. Thus, even under quantitative acquisition conditions, relative areas may be used only to estimate proportions of different types of carbons and functional groups.