Pellegrino Conte

Potential availability of heavy metals to phytoextraction from contaminated soils induced by exogenous humic substances

Effective phytoremediation of soils contaminated by heavy metals depends on their availability to plant uptake that, in turn, may be influenced by either the existing soil humus or an exogenous humic matter. We amended an organic and a mineral soil with an exogenous humic acid (HA) in order to enhance the soil organic carbon (SOC) content by 1% and 2%. The treated soils were further enriched with heavy metals (Cu, Pb, Cd, Zn, Ni) to a concentration of 0, 10, 20, and 40 microg/g for each metal and allowed to age at room temperature for 1 and 2 months. After each period, they were extracted for readily soluble and exchangeable (2.5% acetic acid), plant-available (DTPA, Diethylentriaminepentaacetic acid), and occluded (1 N HNO(3)) metal species. Addition of HA generally reduced the extractability of the soluble and exchangeable forms of metals. This effect was directly related to the amount of added HA and increased with ageing time. Conversely, the potentially plant-available metals extracted with DTPA were generally larger with increasing additions of exogenous HA solutions. This was attributed to the formation of metal-humic complexes, which ensured a temporary bioavailability of metals and prevented their rapid transformation into insoluble species. Extractions with 1 N HNO(3) further indicated that the added metals were present in complexes with HA. The observed effects appeared to also depend on the amount of native SOC and its structural changes with ageing. The results suggest that soil amendments with exogenous humic matter may accelerate the phytoremediation of heavy metals from contaminated soil, while concomitantly prevent their environmental mobility.

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.

Increased retention of polycyclic aromatic hydrocarbons in soils induced by soil treatment with humic substances

The analytical recovery of a mixture of polycyclic aromatic hydrocarbons (PAHs) was determined from a soil before and after oxidation with hydrogen peroxide, and subsequently treated with increasing amounts of an exogenous humic acid and subjected to different incubation periods. The release of PAHs from soil depended on the specific structure and physico-chemical properties of each PAH, and increased with additions of exogenous humic materials for both the oxidized and non-oxidized soil as well as with time of PAH permanence in soil. PAH recoveries were lower in the non-oxidized soil, thereby revealing the importance of native organic matter in increasing PAH retention in soils. This study shows that mobility of PAHs in soils can be controlled by soil conditioning with humic substances.

Plant growth improvement mediated by nitrate capture in co-composted biochar

Soil amendment with pyrogenic carbon (biochar) is discussed as strategy to improve soil fertility to enable economic plus environmental benefits. In temperate soils, however, the use of pure biochar mostly has moderately-negative to -positive yield effects. Here we demonstrate that co-composting considerably promoted biochars’ positive effects, largely by nitrate (nutrient) capture and delivery. In a full-factorial growth study with Chenopodium quinoa, biomass yield increased up to 305% in a sandy-poor soil amended with 2% (w/w) co-composted biochar (BCcomp). Conversely, addition of 2% (w/w) untreated biochar (BCpure) decreased the biomass to 60% of the control. Growth-promoting (BCcomp) as well as growth-reducing (BCpure) effects were more pronounced at lower nutrient-supply levels. Electro-ultra filtration and sequential biochar-particle washing revealed that co-composted biochar was nutrient-enriched, particularly with the anions nitrate and phosphate. The captured nitrate in BCcomp was (1) only partly detectable with standard methods, (2) largely protected against leaching, (3) partly plant-available, and (4) did not stimulate N2O emissions. We hypothesize that surface ageing plus non-conventional ion-water bonding in micro- and nano-pores promoted nitrate capture in biochar particles. Amending (N-rich) bio-waste with biochar may enhance its agronomic value and reduce nutrient losses from bio-wastes and agricultural soils.

High pressure size exclusion chromatography (HPSEC) of humic substances : Molecular sizes, analytical parameters, and column performance

High Pressure Size Exclusion chromatography (HPSEC) is increasingly used to evaluate molecular sizes of humic substances from different sources. Asymmetry factors (As), number of theoretical plates (N), coefficient of distribution (k(d)), and column resolution (Rs) were determined for two different HPSEC columns (TSK G3000SW and Biosep S2000) and polysaccharides of known molecular weights were used as standards. Calibration curves were equivalent for both columns whereas analytical parameters revealed that the TSK column was only slightly more efficient in separating polysaccharide standards. Mw and Mn values for humic substances differed according to the molecular weight range of each column but relative standard deviation never exceeded 5% for both columns. Variations between columns were attributed to intrinsic humic properties such as the stability of conformational structures. These results suggested that humic substances in solutions are loosely-bound association of small molecules that may be consistently dispersed by diffusion through size-exclusion pores. HPSEC is confirmed to represent a highly precise method to evaluate the relative molecular-size distribution of dissolved humic substances.

Quantitative aspects of solid-state 13C-NMR spectra of humic substances from soils of volcanic systems

Copyright (c) 1997 Elsevier Science B.V. All rights reserved. Cross-Polarisation Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance spectroscopy (CPMAS 13 C-NMR) represents one of the most powerful tools to investigate soil organic matter (SOM) mainly because of its inherent capacity to provide a semi-quantitative evaluation of carbon distribution. A critical parameter during acquisition of CPMAS 13 C-NMR spectra is the contact time required to obtain the cross-polarisation between proton and carbon nuclei. The procedure to evaluate the best contact time for the acquisition of a quantitative CPMAS 13 C-NMR spectrum is to perform Variable Contact Time (VCT) experiments. In this work the structural features of a number of purified humic substances from Italian and Costarican volcanic soils were investigated by CPMAS 13 C-NMR spectroscopy after having performed preliminary VCT experiments. The VCT experiments showed that the average contact times vary according to the origin and chemical structure of the humic material. The optimal contact times (OCT) for nine humic samples were between 250 and 800 μs. These values were different from the time of 1000 μs that is commonly applied as the best average contact time for humic materials. Moreover, by comparing the NMR data to those obtained by elemental analysis (C/H ratio), it appeared that the efficiency of the cross-polarisation between protons and carbons, and hence the contact time, is affected not only by the number of protons, but also by their distribution over the molecules. The evaluation of errors in quantitative estimation of the different carbons revealed that the use of OCT generally reduced by half the loss of signals occurring when the average contact time of 1000 μs is used in CPMAS 13 C-NMR spectra of humic substances.

CHROMATOGRAPHIC AND SPECTROPHOTOMETRIC PROPERTIES OF DISSOLVED HUMIC SUBSTANCES COMPARED WITH MACROMOLECULAR POLYMERS

The commonly accepted polymeric nature of humic substances is still a matter of debate. In this work, both humic matter of different origin and undisputed neutral (polysaccharides) and negatively charged (polystyrenesulphonates) polymers of known molecular weight were studied by high performance size exclusion chromatography after dissolution in mobile phases of different composition but constant ionic strength. Modification of the control mobile phase with methanol, hydrochloric acid, and acetic acid showed a progressive alteration of the chromatographic behavior of humic materials, but it did not alter that of polymers. Absolute size reduction of bulk humic material revealed by a refractive index detector was accompanied by a substantial decrease in absorbance of peaks indicated by the UV detector. Reduction of molecular absorptivity of humic substances with changes in solution composition was confirmed by UV spectroscopy over a wide range of wavelengths. Differences in chromatographic behavior between undisputed polymers and humic samples suggest that humic matter reflects, rather than a polymeric nature, a supramolecular association of heterogeneous molecules held together mainly by weak hydrophobic forces. The content of hydrophilic and hydrophobic carbons in humic substances appears to control the intermolecular hydrogen and dispersive bondings that ultimately stabilize humic conformations in different mobile phases.

Conformational changes of humic substances induced by some hydroxy-, keto-, and sulfonic acids

We studied the effect of organic acids of plant, microbial, or anthropic origin on the molecular size distribution of dissolved humic acids (HAs). High Performance Size Exclusion Chromatography (HPSEC) was used to evaluate size changes in four different HAs upon addition of hydroxy- (glycolic and malic), keto- (glyoxylic), and sulfonic (benzenesulfonic and methanesulfonic) acids. All humic substances showed a decrease of peaks absorbance when the pH of HAs dissolved in HPSEC mobile phase was lowered from 7 to 3.5 by acid addition before analysis. This effect, combined with an increase of peaks elution volumes in most cases, was interpreted as a disruption of supramolecular humic associations into smaller-size but energy-richer conformations brought about by the formation of mixed intermolecular hydrogen bonding upon acid treatment. The extent of size variation was related to the pKa of acids and also to the chemical and stereochemical affinity of humic components with the chemical structure of the acids. Dicarboxylic malic acid was the most effective in modifying humic conformations in all HAs whereas the aromatic-rich superstructure of HA from an oxidized coal was effectively disrupted by the relatively small methanesulfonic acid and the chemically akin benzenesulfonic acid. These results suggest that the conformational association of humus dissolved in the soil solution may be systematically altered by organic acids present in the rhizosphere and might have effects on plant and microbial activities. q 2001 Elsevier Science Ltd. All rights reserved.

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.

Quantitative differences in evaluating soil humic substances by liquid- and solid-state 13C-NMR spectroscopy

Abstract We compared the quantitative responses of liquid-state (LS) and solid-state (CPMAS) 13 C-NMR spectroscopy of four different soil humic substances. The intensities of signals for the alkyl carbons (0–40 ppm) were significantly larger in CPMAS than in LS spectra. This difference is in agreement with the pseudo-micellar model of the conformational nature of humic substances. By this view, the hydrophobic interactions holding together the heterogeneous molecules of humic micelles inhibit the molecular motions of the alkyl carbons, thereby enhancing the spin-lattice relaxation times and consequently lowering the sensitivity of liquid-state NMR. Conversely, regardless of their position in the humic conformation, a better estimation of the number of alkyl carbons can be obtained by CPMAS-NMR because of the cross-polarization of hydrogen nuclei in CH 2 and CH 3 groups. The intensity of the 40–110 ppm region is also slightly lower in LS than in CPMAS-NMR spectra, despite the hydrophilicity of the oxidized and peptidic carbons resonating in this chemical shift interval. Their molecular motion may also be reduced by either the formation of intra- and inter-molecular hydrogen bondings due to poorly acidic hydroxyl groups of saccharides, or the degree of conformational rigidity that a pseudo-micellar arrangement confers even to hydrophilic domains. The higher content of aromatic carbons (110–160 ppm) found in the LS spectra was attributed partly to the high degree of substitution of the aromatic ring that slows down cross-polarization in CPMAS experiments and partly to the relative overestimation of this region by LS-NMR due to a lack of signal in the aliphatic interval. The slightly lower content of carboxyl carbons estimated in CPMAS spectra as compared to LS spectra was also attributed to slow cross-polarization. This work shows that the combined use of both NMR techniques is profitable in conformational analysis of humic substances and of dissolved organic matter in general.