Characterization of humic substances obtained from calcareous soils from Greece with various extractants. I. Chemical characterization

Humic substances of the Ah horizons of five differently calcareous soils from Greece under different vegetative covers were successively extracted with diethylether, acetone, ethanol and dioxane. Following these extractions, remaining humic substances were extracted with 0,1N NaOH. Humic substances were also directly extracted with 0,1N NaOH. The extracts of the organic solvents were separated by thin layer chromatography (T.L.C.) on silicagel plates, while the NaOH extracts were characterized by elemental and functional group analyses. The T.L.C. revealed that ?-acceptors and phenols were present in all extracts, while ?-donors and lignines were present only in the dioxane extracts. Lipides were present in the diethylether, dioxane and acetone extracts. Sugars were found only in the acetone extracts and reducing substances in the acetone and dioxane extracts. Some analytical characteristics of the humic substances were associated with the types of vegetative cover and/or soil properties. Humic acids (HA's) extracted with 0,1N NaOH from soil samples previously treated with organic solvents, generally varied in their carbon (C), carboxyl (COOH) and ash contents as compared with those extracted directly.     

Chemical and physical characteristics of humic substances extracted from greek soils varying in classification order and natural drainage

Abstract

Humic and fulvic acids were extracted from the surface horizons of Alfisols, Vertisols, and Entisols with good and impeded natural drainage. These profiles are located in the temperate subhumid zone. The extracted humic substances were characterized by elementary and functional group analyses, IR‐spectroscopy, differential thermal analysis (DTA) and electron spin resonance spectroscopy (ESR). Some differences in the humic substances seem to be associated with differences in the natural drainage of the soils. When data from samples of the same soil order are compared, indicate that humic (HAs) and fulvic acids (FAs) extracted from soils with impeded drainage, have higher contents of carbon and spin concentration, but less total acidity and fewer carboxylic groups (COOH), than the respective humic substances extracted from well drained soils. The IR‐spectra of the HAs extracted from soils with impeded drainage showed more aliphatic groups than those of the well drained soils. No essential differences, which could be related to the drainage conditions of the studied soils, were observed in the IR‐spectra of FAs. DTA‐diagrams shows that the thermal stability of the HAs and the FAs are related to their carbon and ash contents. Iron was the dominant element in the ash of the humic acids, aluminum and silicon were the most abundant elements in the ash of the fulvic acids.     

Protease extraction from soil by sodium pyrophosphate and chemical characterization of the extracts

Two arable soils and one pasture soil had previously been air-dried for 6 d and stored at room temperature. The enzyme activities remaining after this treatment were constant. The soils were then extracted with 140 mM sodium pyrophosphate at pH 7.1. Amino acid N and total organic C content of soils and soil extracts, together with humic and fulvic acids content of soil extracts were determined. Total organic C was determined in soil residues obtained after extraction. Chemical characterization of the organic matter of soils, soil extracts and soil residues was carried out by pyrolysis–gas chromatography (Py–GC). Protease activity was determined in soil extracts and soil residues by using three different substrates: N-benzoyl- -argininamide (BAA), specific for trypsin; N-benzyloxycarbonyl- -phenylalanyl -leucine (ZPL), specific for carboxypeptidases, and casein, essentially non-specific. Comparative studies between specific activities referred to organic C in soils, soil extracts and soil residues and their corresponding pyrogram composition, and also between total extracted or residual activity and the humine or unhumified organic matter content of the corresponding soil, allowed us to establish hypotheses about the type of organic matter the enzymes are associated with. From 12% to 21% of the soil organic C (33% to 39% of which were humic acids) and from 3% and 18% of amino acid N were extracted from soil using pyrophosphate. Py–GC analyses showed that pyrophosphate was effective in extracting condensed humic substances and glycoproteins and that the organic matter present in soil extracts was especially rich in intact or partially-decomposed fresh residues of carbohydrate origin and also in certain humus-associated proteins. Extracted BAA-hydrolysing activity accounted for 11% to 36% of the soil activity, depending on soil type. Extracted ZPL- and casein-hydrolysing activities were, with one exception, remarkably high, accounting for about 100% or even more of the soil activity, depending on soil type. According to the results BAA-hydrolysing proteases are probably mostly associated with highly condensed humus, ZPL-hydrolysing proteases with less condensed humic substances and casein-hydrolysing proteases with fresh organic matter.     

Humic materials from an organic soil: A comparison of extractants and of properties of extracts

Five extraction procedures and thirteen extracting reagents, which included dipolar aprotic solvents, organic chelating agents, pyridine, ethylenediamine, sodium hydroxide, ion-exchange resins and two salts (sodium pyrophosphate and ethylenediamine hydrochloride), were used to extract humic materials from an organic soil. Extractabilities increased in the general order: salts < organic chelating agents < dipolar aprotic solvents < pyridine < ethylenediamine = sodium hydroxide, and the amounts of the soil organic matter extracted by the reagents in the series ranged from 13 to 63%. Gel chromatography techniques indicated that extracts in dipolar aprotic solvents were predominantly of intermediate and low molecular-weight values, and it is suggested that the more highly oxidised soil humic materials were extracted in these. The more efficient solvents extracted materials with a range (high—low) of molecular-weight values. Data from elemental analysis and from E.S.R. measurements indicated that ethylenediamine altered the chemical nature and the composition of extracts. Dipolar aprotics, by the same criteria, were found not to alter the humic extracts, and can be regarded as mild reagents for the extraction of a less representative (of the total) fraction of soil organic matter. Sodium hydroxide in solution, despite its oxidation effects, was the best of the reagents tested for isolating extracts which were representative of a wide range of soil humic substances.     

Characterization by isoelectric focusing of the organic matter of a regenerated soil

Abstract

Regeneration of a soil with a high degree of desertification was conducted by the addition of different doses of municipal solid waste organic matter (MSW). Five years after this treatment, humic substances were extracted from these soils and characterized by spectroscopy and isoelectric focusing. No significant differences between E4/E6 ratio and ?log K (Log A400 nm‐log A600 nm) were observed for humic substances extracted from treated and untreated soils. However, the isoelectric focusing (IEF) technique established differences between the humic substances from control and treated soils. The focusing pattern of the former showed a well defined band at pH 9.1 which nearly disappeared in the soils with high doses of MSW (1–2 %). However the organic matter which focused at pH 5.8 was present in all soils.     

Aerobic short-term microbial utilization and degradation of humic acids extracted from soils of long-term field experiments

A humic acid (HA) fraction of the soil organic matter (SOM) was extracted with alkali from soil samples originated in non-fertilized and fertilized (NPK + organic manure) plots of long-term (45 years) field experiments. The HA preparations served as supplemental sources of nutrients or as sole source of either C or N for soil micro-organisms indigenous to the same soils. Under aerobic conditions (shake cultures) between 15% and 45% of HA were degraded in 21 days. The degradation was minimum if HAs were added supplementary, although the biomass formation was strongly enhanced. Preparations of HA from long-term fertilized soils appeared somewhat less susceptible to microbial degradation but they were capable of supporting microbial growth. Under copious nutritional conditions some novel HA-like substances were formed. The HA preparations re-isolated from individual cultures exhibited differences in elemental and structural characteristics. The FTIR spectra indicated an increasing proportion of aromatic structures that appeared as associated with mineral moieties. Conclusively, HAs from long-term fertilized and manured soils could be considered as more resistant to microbial activities than those from control soil, but under limited nutrient conditions their aliphatic constituents appear utilizable by micro-organisms.     

Further investigations on the chemistry of fungal “humic acids”

In view of the considerable interest in laboratory-prepared fungal “humic acids” as possible precursors or incorporated structural components of soil humic substances, we degraded four fungal “humic acids” by the relatively mild alkaline cupric oxide oxidation. The oxidation products were extracted into organic solvents, methylated, separated by thin-layer chromatography and identified on a gas chromatographic-mass spectrometric-computer system.Average yields of major degradation products were: (a) aliphatic compounds, 38 per cent; (b) benzene-carboxylic compounds, 25 per cent; and (c) phenolic compounds, 21 per cent. The remaining 16 per cent consisted of a number of dialkyl phthalates. Our data agree with those that we reported earlier when we degraded a number of fungal “humic acids” by the more drastic alkaline permanganate oxidation and show that fungal “humic acids” are enormously complex organic materials containing aliphatic and aromatic structures, (some of which contain N), but only a relatively small proportion of which is phenolic. Most of the aliphatics isolated consisted of alkanes and fatty acids, which are known to persist in soils over long periods of time and are frequently firmly retained by soil humic substances.     

Distinguishing black carbon from biogenic humic substances in soil clay fractions

Most models of soil humic substances include a substantial component of aromatic C either as the backbone of humic heteropolymers or as a significant component of supramolecular aggregates of degraded biopolymers. We physically separated coarse (0.2–2.0 μm e.s.d.), medium (0.02–0.2 μm e.s.d.), and fine (> 0.02 μm e.s.d.) clay subfractions from three Midwestern soils and characterized the organic material associated with these subfractions using ¹³C-CPMAS-NMR, DTG, SEM-EDX, incubations, and radiocarbon age. Most of the C in the coarse clay subfraction was present as discrete particles (0.2–5 μm as seen in SEM images) of black carbon (BC) and consisted of approximately 60% aromatic C, with the remainder being a mixture of aliphatic, anomeric and carboxylic C. We hypothesize that BC particles were originally charcoal formed during prairie fires. As the BC particles aged in soil their surfaces were oxidized to form carboxylic groups and anomeric and aliphatic C accumulated in the BC particles either by adsorption of dissolved biogenic compounds from the soil solution or by direct deposition of biogenic materials from microbes living within the BC particles. The biogenic soil organic matter was physically separated with the medium and fine clay subfractions and was dominated by aliphatic, anomeric, and carboxylic C. The results indicate that the biogenic humic materials in our soils have little aromatic C, which is inconsistent with the traditional heteropolymer model of humic substances.     

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.     

Chemical and physical characteristics of humic and fulvic acids extracted from soils of the Mediterranean region

Humic and fulvic acids were extracted from two soils from Israel and from four soils from southern Italy. Following purification, the humic substances were characterized by elementary and functional group analyses, spectrophotometric and spectroscopic methods, thermal analysis and by scanning electron microscopy.The analytical characteristics of the humic and fulvic acids extracted from the Israeli and Italian soils were generally similar to those of humic materials originating from soils formed under widely differing geographic and pedologic conditions. There were some differences in the content and composition of inorganics in the fulvic acids which were reflected in functional group analyses, IR and ESR spectra, DTA curves and SEM micrographs.     

固态13C和15N核磁共振法研究15N标记土壤的腐殖质组分

Five humic fractions were obtained from a uniformly ^15N-labelled soil by extraction with 0.1 mol L^-1 Na4P2O7,0.1mol L^-1 NaOH ,and HF/HCl-0.1 mol L^-1 NaOH,consecutively,and analyzed by ^13C and ^15N CPMAS NMR (cross polarization and magic angle spinning nuclear magnetic resonace).Compared with those of native soils humic fractions studied as a whole contained more alkyls ,methoxyls and O-alkyls,being 27%-36%,17%-21%and 36%-40%,respectively,but fewer aromatics and carboxyls(bein 14%-20% and 13%-90%,respectively),Among those humic fractions ,the humic acid(HA)and fulvic acid(FA) extracted by 0.1 mol L^-1 Na4P2O7 contained slightly more carboxyls than corresponding humic fractions extracted by 0.1 mol L^-1 NaOH ,and the HA extacted by 0.1 mol L^-1 NaOH after treatment with HF/HCl contained the least aromatics and carboxyls.The distribution of nitrogen functional groups of soil humic fractions studied was quite similar to each other and also quite similar to that of humic fraction from native soils.More than 75% of total N in each fraction was in amide from,with 9%-13% present as aromatic and /or aliphatic amines and the remainder as heerocyclic N.     

Absorption spectra of humic acids

For the purpose of explaining the forming process of soil humic acids, the author determined the absorption spectra of various humic acids. From soils and peats which were pretreated with 5% HCl at 70°C for 30 minutes or from those which were not, humic acids were extracted by treating with 0.5% NaOH at boiling temperature for 30 minutes. In these humic acids, the one which is extracted after acid pretreatment is provisionally designated as SrL humic acid and the other as L humic acid. The supernatant alkaline solutions obtained by centrifuging the above mentioned extracts were acidified with hydrochloric acid, and precipitated humic acids were filtered and washed with water until Cl' free. Humic acids were dissolved in 0.1% NaOH and ultrafiltered using collodion membrane. The filtrates were acidified with hydrochloric acid and humic acids were collected by centrifuging, transferred on the filter paper, washed with dilute hydrochloric acid and water successively, then air-dried and pulverized.     

Analysis of total inositol phosphates in municipal solid waste compost-treated soils by two extraction methods

 Humic substances are the most dynamic component of agricultural soils. In this study, humic substances were extracted from soils based on standard acid/base solubility and by gel filtration using Sephadex G25. Organic P is a component of humic substances, and inositol phosphates are considered to have high prominence in organic P. The objectives of the study were to determine the effects of municipal solid waste (MSW) compost on the amount of inositol phosphates (IP) present in soils, and compare methods of extracting humic substances from soils. Total IP (IP_T) in soil extracts was determined following separation using a AG-1× 8 anion exchange resin, acid digestion and ICAP analysis for P. The percentage of IP (%IP) of the total P in the soil was also determined by colorimetric analysis. The soils were part of an experiment to study the effects of three rates of MSW compost and fertilizers on three crops grown in a Pugwash sandy loam (Humo-Ferric Podzol). The data were compared using ANOVA atP≤0.05; extraction methods, fractions [humic/fulvic, high/low molecular (MW)], and five treatments (three different rates of compost, fertilizer, and untreated soil) were compared. The gel filtration method extracted significantly more IP_T and %IP than the solubility method. In the solubility method, humic and fulvic acids contained the same amounts of IP_T and %IP, while the high MW fraction always contained more IP_T and %IP than the low MW fraction in the gel filtration method. Fulvic acids and the low MW fraction contained similar amounts of IP_T and %IP. There were differences in %IP between compost-treated soils and the non-compost-treated soils, although there were no differences in IP_T due to rate of compost addition. Received: 20 October 1999     

Humic and fulvic acids as indicators of soil and water pollution

Humic substances are the major organic components of soil and sediments, but little is known on how they are affected by environmental and industrial pollution. To find out whether such effects could be recognized, a number of analytical characteristics were compared of humic and fulvic acids extracted from unpolluted and polluted soils and sediments. The main differences were that, per unit weight, polluted humic and fulvic acids contained more N, S and H but fewer CO₂H groups, and were more aliphatic than unpolluted samples. Unusually high N and S contents of humic materials appear to be the most valid indicators of pollution. Humic acids are preferred to fulvic acids as indicators of pollution because the former are more readily separated and purified.     

Ein neues Verfahren zur Extraktion von organischen Stoffen aus Böden mit überkritischen Gasen

A new procedure for the extraction of organic matter from soils by supercritical gases Organic solvents beyond critical temperature and pressure have excellent properties for solving polymere organic substances. For soil samples yields of organic material with low ash content are much higher than those obtained by the common NaOH/HCl-extraction. Fractionation of the samples is easily achieved by a simple change of solvent bottles. In comparison to the original material oxygen contents are lower, due to a thermal or solvolytical split off of methanol or water. Extraction with supercritical gases – in addition to oxidative degradation –may be regarded as a useful method to enlighten the structure of humic acids. The effective extraction of firmly bound pesticides in soil is another point of interest.     

不同年代的埃特纳火山土壤上用甘油萃取有机质的特性

We characterized humic acids (HAs) and glycerol-extractable organic fractions (GEOFs) extracted from four Andisols, taken from comparable soil-climate conditions on the east side of Mount Etna. The soils were formed on old lava (about 9 000 years ago), old tephra (about 8 700 years ago), recent lava (about 2 600 years ago) and recent tephra (about 3 600 years ago). A part of the organic matter of the soils, deprived of HAs and fulvic acids (FAs), was isolated by glycerol extraction. The GEOF can not be extracted with alkaline solutions, probably because it is closely bound to the mineral component of the soil. The characterization of the extracted organic fraction was carried out using elementary and functional group analysis and Fourier transform infrared (FT-IR) spectroscopy. About 20 extractions were necessary to extract the HA and FA from the older soils and about 10 extractions to extract them from the younger soils. Data showed that the GEOFs had a greater ash content and a smaller N content, as well as a greater presence of aliphatic compounds and carboxylic groups as compared to the HA extracted from the same soil. The GEOFs extracted from younger soils also had a lower yield, ash and COOH-group content, and were more aliphatic than the GEOF extracted from older soils. Finally, the GEOFs were more closely bound to the amorphous component of the soil (‘short-range’ minerals) and consequently less subject to biodegradation.     

Unterscheidbarkeit von Humusfraktionen in Böden bei unterschiedlicher Bewirtschaftung

Differences in the properties of soil organic matter as a result of differing soil managements The organic matter of 62 soil samples from 20 farms was analyzed by the following methods: total organic C, “light” C (density < 2 g/ml) and C content and Q 4/6 of extracts obtained with NaF and NaOH solutions under different conditions. The soil samples were obtained from 10 pairs of adjacent plots; in each pair one of the plots was under “conventional”, the other under “bio-dynamic” management. The results obtained by the different laboratory methods evidenced more or less clearly the different managements. C extracted by shaking the soil with 0,5 % NaOH was most significant in that respect. The results as a whole are coherent in showing that in the soils under bio-dynamic treatment of higher content of the more easily decomposable fractions of the organic matter as compared with the soils under conventional treatment can be detected by rather simple means.     

A simple wet chemical extraction procedure to characterize soil organic matter (SOM). I. application and recovery rate

Abstract

The knowledge of soil organic matter (SOM) composition is important for research in soil science. This is why two classical wet chemical extraction procedures were tested and combined to characterize SOM. Twenty‐five samples from typical forest and arable soils in Schleswig‐Holstein, Northwest Germany, were investigated in the laboratory. Lipids were extracted using a pre‐step method. Several polysaccharide fractions were extracted sucessively with inorganic acids in a litter compound analysis (LCA). Proteins and lignins were determined in the bulk soil sample. In a humic compound analysis (HCA), fulvic and humic acids were extracted in the classical way with NaOH, and the non‐humic substances were removed with the aid of the “Sulfacetolysis” from the residues (= usually “humins")‐ The combination of these two wet chemical extraction proce dures (LCA and HCA) permitted quantitative estimations of the SOM composition in several soil horizons. The LCA method produced a better recovery rate (104%±4%) than the HCA methode (95%±15%). The litter compound/humic compound ratio of both analyses, and the combination of both correlated with visible humification grades in a significant way (r = ‐0.733 to ‐0.742***). LCA may be sufficient for solving special pedogenetic problems, because of its high recovery rate and the strong correlation between LCA and HCA.     

Comparative evaluation of methods employing alkali and sodium pyrophosphate to extract humic substances from peat

Abstract

Humic substances from two different types of peat ‐ Sphagnum and Carex ‐ were obtained by using either alkali extractants, O.IM NaOH, O.IM KOH, and 0.25M KOH, or solvents containing sodium pyrophosphate (O.IM Na₄P₂O₇, or O.IM NaOH/Na₄P₂O₇). The alkalis released considerably greater amounts of humic substances from the Sphagnum peat than the extractants containing pyrophosphate, whereas the opposite occurred in the Carex peat, the two greatest yields being obtained with 0.25M KOH in Sphagnum peat and with O.IM NaOH/Na₄P₂O₇ in Carex. As shown by the spectroscopic and chemical analyses, humic acids released from both peats by extractants containing Na₄P₂O₇ are generally more oxidized than those obtained by any of the alkali solutions. Also, humic acids extracted from the Sphagnum peat by any of the solvents generally exhibit lower molecular sizes, more pronounced aliphatic characteristics, and a higher degree of oxidation than those extracted from the Carex peat.

Results of gel permeation chromatography indicate that humic acids obtained from Sphagnum peat by either O.IM NaOH or O.IM KOH extraction show a similar distribution of molecular sizes with a predominance of compounds with a molecular weight greater than 50,000, whereas the use of 0.25M KOH enhances the release of partially degraded humic substances. Also, humic acids extracted from the same peat with 0.1M Na4P2O7 show a considerable amount of molecules of low molecular weight, but much fewer of these compounds are obtained when 0.1M NaOH/Na₄P₂O₇ extraction is employed. In Car ex peat, molecular size distribution does not seem to depend on the type of extractant used for solubilization of their humic acids.     

Pyrolysis-(gas chromatography/) mass spectrometry of agricultural soils and their humic fractions

Curie-point pyrolysis-gas chromatography/mass spectrometry and temperature-programmed in-source pyrolysis-field ionization mass spectrometry have been used for the chemical characterization of the organic material in agricultural soils with organic carbon contents between 1 and 1.5%. The results obtained from whole-soil samples, extractable humic substances and nonextractable humin by both techniques indicate for the Ap of a Gleysol thermal degradation products of carbohydrates, intact and/or biodegraded lignin, flavonoids, tannins and/or polyphenols, lipids, aliphatic polymers, and nitrogen-containing compounds. On a qualitative basis similar results have been obtained for the Ap of a Luvisol. The alkylbenzenes, alkylnaphthalenes, (di)alkylphenols, and alkyltoluenes identified are of special interest because these aryl-alkyl compounds demonstrate covalent linkages between aliphatic chains and aromatic subunits in soil organic matter. This type of linkage between individual subunits of the complex and polydisperse organic material enables the flexibility of rigid aromatic systems.