Riccardo Spaccini

Increased soil organic carbon sequestration through hydrophobic protection by humic substances

Abstract We studied the effect of humified organic matter (OM) on the mineralization of a representative labile organic compound in soil. In an incubation experiment, a 13C-labeled 2-decanol was added to soil either alone (2-dec∗) or in mixture with two humic acids from compost (HAC∗) and lignite (HAL∗) which had different hydrophobic properties. Isotopic dilution (δ13C) showed that after 3 months of incubation about 58, 40 and 28% of the added 13C was retained in the whole soil treated with HAL∗, HAC∗ and 2-dec∗, respectively. The higher the hydrophobicity of the employed humic material, the larger was the sequestration of organic carbon in soil. Fractionation of incubated samples revealed that the labeled carbon progressively accumulated in the finest particle-size fractions. However, the high hydrophobicity of the lignite HA favored accumulation of 13C also in the sand-sized fraction. The NMR spectra of humic extracts showed that the 13C-methyl group in the original 2-decanol had been oxidized to a 13C-carboxyl group during incubation for all treatments. This indicated that despite its hydrophilicity, the resulting carboxyl carbon was sequestered into the hydrophobic domains of the humic pool in soil. In fact, the residual 13C was larger in humic than in fulvic extracts for the control sample (2-dec∗) and even more so in extracts from soil treated with both exogenous humic acids. Our results suggest that labile organic compounds may be effectively protected in soil by humified OM and their microbial mineralization substantially reduced. Innovative soil management practices employing hydrophobic humic substances may increase the biological stability of soil OM and thus contribute to significantly mitigate CO2 emissions from agricultural soils.

Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid.

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients (D), while carbon distribution was evaluated by solid state (CP/MAS) (13)C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions.

Bioactivity of chemically transformed humic matter from vermicompost on plant root growth.

Chemical reactions (hydrolysis, oxidation, reduction, methylation, alkyl compounds detachment) were applied to modify the structure of humic substances (HS) isolated from vermicompost. Structural and conformational changes of these humic derivatives were assessed by elemental analyses, size exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance ((13)C CPMAS-NMR), and diffusion ordered spectroscopy (DOSY-NMR), whereas their bioactivity was evaluated by changes in root architecture and proton pump activation of tomato and maize. All humic derivatives exhibited a large bioactivity compared to original HS, both KMnO(4)-oxidized and methylated materials being the most effective. Whereas no general relationship was found between bioactivity and humic molecular sizes, the hydrophobicity index was significantly related with proton pump stimulation. It is suggested that the hydrophobic domain can preserve bioactive molecules such as auxins in the humic matter. In contact with root-exuded organic acids the hydrophobic weak forces could be disrupted, releasing bioactive compounds from humic aggregates. These findings were further supported by the fact that HS and all derivatives used in this study activated the auxin synthetic reporter DR5::GUS.

Carbohydrates and aggregation in lowland soils of Nigeria as influenced by organic inputs

We evaluated the influence of several organic matter management practices on the characteristics of carbohydrates in water-stable aggregates and soil aggregate stability at three Nigerian locations (Abakiliki, Nsukka and Umudike) where forests had been converted to arable farming. The effect of management practices to enhance aggregate stability was site-specific. The highest aggregate stability was obtained with Gliricidia sepium at Abakiliki, with Cajanus cajan followed by rice mill wastes (RW) at Nsukka and with the forested soil at Umudike. While none of the treatments at all sites was able to enhance the C and N contents of the soils to the levels obtained in the forested sites, a net improvement in carbohydrate and organic carbon (OC) content was found for some management practices. The carbohydrate status increased with G. sepium at Abakiliki, and with Dactylodenae bacteriialone or in combination with Pentaclethra species at Umudike, while at Nsukka all organic inputs increased carbohydrate content over the control and forested soils. However, neither total OC nor the carbohydrate content were significantly correlated to the variability in aggregate stability of these soils. The δ 13 C values found for acidic hydrolysates were constant within the soil aggregate sizes and generally distributed around −29 to −30‰, suggesting that the OC from these sites originated from C3 plants. Our results indicate that in these tropical Nigerian soils, aggregate stability and OC content are generally preserved by alley-cropping in well structured soil, whereas treatments with organic wastes are sustainable management practices in more fragile soils.

Polymerization of humic substances by an enzyme-catalyzed oxidative coupling

Abstract A novel understanding of the structural features of humic substances supports the self-assembly supramolecular association of relatively small molecules rather than their polymeric nature. An increase in the conformational stability of humus may thus be achieved through promotion of intermolecular covalent bondings between heterogeneous humic molecules by an enzyme-catalyzed oxidative reaction. We present evidence from high performance size exclusion chromatography (HPSEC) and diffuse reflectance infrared spectrometry (DRIFT) that oxidation of a humic material catalyzed by horseradish peroxidase stabilizes the humic structure by the formation of aryl and alkyl ethers and permanently enhances its molecular size.

Effects of some dicarboxylic acids on the association of dissolved humic substances

Changes in molecular size distributions of four different humic materials were evaluated through high performance size-exclusion chromatography (HPSEC), before and after treating humic solutions with naturally occurring dicarboxylic (oxalic, malonic, succinic and glutaric) acids. Chromatograms of dissolved humic substances showed a decrease in peak absorbance as well as an increase in peak elution volume when the solution pH was lowered from 7 to 3.5 by addition of dicarboxylic acids before HPSEC analysis. The resulting reduction in molecular size is explained by the disruption of unstable humic superstructures into smaller-sized associations stabilized by the formation of strong intra- and inter-molecular hydrogen bonds among humic molecules. The extent of humic size variation was in the order: oxalic<malonic<succinic<glutaric acid. Dicarboxylic acids with an increasing number of C atoms and pKas, had progressively larger degrees of protonation which increasingly reduced the electrostatic repulsion from humic molecules. The less dissociated dicarboxylic acids enabled a closer contact with the hydrophobic domains of weakly bound humic superstructures and produced more extensive conformational rearrangements. Recognition of a process controlling the conformational structure of humic molecules by organic acids exuded in the soil solution by either plant roots or soil microbes may be important in understanding the role of humus in soil-plant interactions.

Decomposition of maize straw in three European soils as revealed by DRIFT spectra of soil particle fractions

Diffuse Reflectance Infrared Fourier Transform DRIFT technique was used to investigate decomposition of maize straw residues in particle-size fractions of three European soils in a 1-year incubation experiment that simulated both incorporation and mulching practices. The aliphatic bands in the 2920-2860 cm y1 regions were used to derive decomposition curves and mineraliza- tion rates of maize residues. Evaluation of soil organic matter changes by DRIFT in particle-size fractions depended on soil texture and soil aggregate stability. Decomposition could be followed over the full incubation period only in three size-fractions of the silty German soil because of its intermediate aggregate stability. DRIFT spectra were not useful in the least stable sandy Danish soil and in the most stable clayey Italian soil. Decomposition rates derived from DRIFT results were similar to those obtained by isotopic techniques. DRIFT spectroscopy may represent a rapid and accurate method to follow fresh organic matter degradation directly in soil matrix or particle-size fractions in some soils provided that adequate soil dispersion is obtained. q 2001 Elsevier Science B.V. All rights reserved.

Separation of molecular constituents from a humic acid by solid-phase extraction following a transesterification reaction

A selective removal of humic constituents involved in ester groups was conducted by a boron trifluoride-methanol transesterification reaction after removal of structurally unbound lipids. An analytical separation of subfractions containing specific classes of compound liberated from the humic matrix simplifies their identification by gas chromatography-mass spectrometry. We compared the traditional liquid-liquid separation into phenolic and aliphatic fractions with the modern and versatile fractionation technique using solid-phase extraction (SPE) on aminopropyl-bonded phases. Our results showed that both methods ensured separation of the same compounds, such as di- and tri-hydroxyalkanoic acids, alpha-, beta- and omega-hydroxy fatty acids, alkanoic acids, alpha,omega-alkanedioic acids, n-alkanols, phenolic acids and sterols. Moreover, the SPE method not only provided a larger recovery of compounds, but involved smaller sample and solvent requirements, and larger ease and rapidity of sample handling than the traditional liquid-liquid separation. The SPE method should be thus recommended in structural studies of natural organic matter.

Interactions of three s-triazines with humic acids of different structure.

The adsorption of three s-triazines (atrazine, terbutylazine, and simazine) on three different humic acids (HAs), before and after acidic hydrolysis, was studied at four diverse pH values from 3 to 4.5. The Freundlich sorption affinity (K(f)) and intensity (1/n) of s-triazines were related to the chemical and structural composition of HA and used in a multivariate statistical analysis. At low pH, the sorption values for s-triazines were not directly justified by the content of carboxyl groups in original HA, while only an increase of phenolic carbons in hydrolyzed HA supported the increased s-triazine adsorption. The structural composition of both s-triazines and humic samples explained 86% of the significance in multivariate analyses, whereas the role of pH remained hidden in only 14% of the statistical significance. Adsorption of s-triazines was mainly related to carbon content, hydrophobicity, and aromaticity of HA, thereby implying a predominant binding role of weak dispersive forces. Steric properties explained the larger adsorption affinity of atrazine and terbutylazine with respect to simazine. The occurrence of charge-transfer interactions between atrazine and mainly hydrolyzed HA was further suggested by the increased number of free radicals detected in atrazine-HA complexes at different pH values. Charge-transfer interactions were postulated to occur when HA conformations became progressively controlled by aromatic components. This work indicates that humic matter rich in hydrophobic and aromatic constituents is more likely to adsorb s-triazines and reduce their environmental mobility.

The molecular characteristics of compost affect plant growth, arbuscular mycorrhizal fungi, and soil microbial community composition

Compost amendment to agricultural soils influences plant growth and soil quality by affecting activity of arbuscular mycorrhizal fungi (AMF) and composition of microbial community. We related the molecular composition of compost of different maturity added to soils to their effects on maize growth, N and P uptake, AMF root colonization and growth, and composition of soil microbial community. The characteristics of compost after different days of maturation (C60, C90, C120) were provided by 13C-solid state NMR spectroscopy, while neutral (NLFA) and phospholipid (PLFA) fatty acid analyses were used to evaluate the effects of compost on the composition of soil microbial communities. Multivariate elaboration was used to determine the relationships between microbial groups, as identified by PLFA analysis, and molecular properties of composts. Although compost amendments increased soil total C and N, and available P, soil addition of both C60 and C120 compost samples was detrimental to plant and AMF growth. Compost amendments modified the composition of soil microbial communities. The high content of biolabile compounds in C60 and C120 compost samples decreased the C16:1ω5 NLFA that was related to AMF and Gram(+)/Gram(−) and AMF/saprotrophic fungi ratios. A linear correlation was found between the molecular indexes in compost and the microbial groups in soil, thereby suggesting that the molecular composition of compost strictly controls the development and abundance of soil microbial communities. These findings highlight the importance of controlling the molecular quality of recycled biomass added to soil, in order to predict the effect on crop yields and biotic composition of soil.