Chemical heterogeneity of humic substances: characterization of size fractions obtained by hollow-fibre ultrafiltration

To investigate the chemical heterogeneity of humic substances in relation to molecular size, fulvic and humic acids were extracted and purified from the surface horizon of a Humic Gleysol in northern Switzerland. A fractionation scheme using hollow‐fibre ultrafiltration cartridges was developed and used to obtain four size fractions of the humic acid with nominal molecular weight ranges > 300 kDa, 100–300 kDa, 30–100 kDa, and 10–30 kDa. The fulvic acid and all humic acid fractions were characterized by size exclusion chromatography, elemental analysis (C, H, N, S), as well as spectroscopic techniques including UV‐VIS, CP‐MAS ¹³C‐NMR, FT‐IR, and fluorescence spectroscopy. Clear chemical differences between the humic acid size fractions were observed. Smaller size fractions of the soil humic acid contained more chargeable functional groups and a larger percentage of aromatic carbon than the larger size fractions. Conversely, the percentage of aliphatic carbon increased with increasing apparent molecular weight. The chemical composition of the smallest humic acid fraction differed clearly from the fulvic acid fraction, despite similar apparent molecular size and carboxyl carbon content. Small humic acids contained much more aromatic carbon and less aliphatic carbon than the fulvic acid fraction. Apparently, humic size fractions differ in their chemical composition, which can have important implications for their environmental behaviour.     

土壤中钙结合和铁铝结合腐殖质的组成及结构特征

Calcium-bound and iron- and aluminium-bound humus extracted from different soils collected from north to south of China were characterized by chemical and spectroscopic methods. Meaningful differences in the composition and structure between them were revealed by ¹³C NMR, visible spectroscopy and elemental analysis. Results showed that the contents of carbon, hydrogen and nitrogen were higher in iron- and aluminium-bound humus than in calcium-bound humus while oxygen content in calcium-bound humus was shown to be higher. The calcium-bound humus had higher C/N and O/C ratios than iron- and aluminium-bound humus. The calcium-bound humic acid (HA1) showed higher E₄/E₆ ratios than iron- and aluminum-bound humic acid (HA2) while iron- and aluminum-bound fulvic acid (FA2) showed higher E₄/E₆ ratios than calcium-bound fulvic acid (FA1). An inverse relationship between E₄/E₆ ratios and aromaticity as determined by ¹³C NMR spectra was observed for HA and FA from black soil. The ¹³C NMR spectroscopy revealed that HA2 was more aromatic than HA1. On the other hand, FA1 exhibited a higher aromaticity than FA2.     

Comparisons of soil organic matter and its fractions by pyrolysis mass-spectrometry

Pyrolysis mass-spectra from a sample of the A₁-horizon of a soil from southern Spain showed predominant peaks related to furan derivatives similar to those observed from complex polysaccharides in which not only hexoses but also pentoses and deoxyhexoses were constituent units. Smaller peaks, typical for protein materials and phenolic units, were also observed. On the other hand, typical peaks for the methoxyphenols of lignins were very small and indicated only limited amounts of undecomposed lignin residues in this soil sample. Peaks related to benzene or toluene were also very small.Humic acid samples from this soil showed much more prominent signals related to protein materials, benzene and phenolic derivatives and weaker polysaccharide-related signals than did the entire sample. Typical lignin related peaks were small or insignificant. Spectra from the grey or brown humidic acid fractions were much like those of the parent humic acid. Brown humic acid, however, showed stronger signals for nitrogen and sulphur compounds, indicating a higher content of protein-like materials in this fraction. Preparations of humic acid hydrolyzed by 6 N HCl showed in their pyrolysis products a marked increase in phenols and methoxyphenols.In its pyrogram, humin resembled humic acid, but signals for complex polysaccharides were more evident. Lignin-like materials seem not to be higher in this fraction. Hymatomelanic acid showed prominent signals related to polysaccharides and lignin. Pyrograms from the soil polysaccharides showed the characteristic pattern of a complex polysaccharide with the presence of fragments from polymers of amino acids or amino sugars. Fulvic acid spectra showed obvious dissimilarities to those from humic acid in that signals for protein, as well as those related to phenols, were low. Depending upon the isolation method, the fulvic acid preparations showed differing signals related to polysaccharide or phenolic materials.     

MOLECULAR WEIGHT DISTRIBUTION OF SOIL ORGANIC MATTER AS AFFECTED BY ACID PRE-TREATMENT AND FRACTIONATION INTO HUMIC AND FULVIC ACIDS

Effects of acid pre-treatment and fractionation on the molecular weight distribution of OIM Na₄P₂O₇ organic matter extracts were investigated in a chronosequence of weakly weathered soils developed on aeolian sand in New Zealand. Acid pre-treatment of soils with OIM HCl followed by OIM HCI:03M HF was found to enhance the polydispersion in the nominal molecular weights of the extracts. The same treatment resulted in significant increases in yield and reduction in ash content. However, prolonged standing of extracts in the acids led to acid-induced polymerization, resulting in a predominance of organic matter in the higher nominal molecular weight ranges. Fractionation of organic matter extracts by acid precipitation into humic and fulvic acids did not separate them according to molecular weight as commonly believed. Instead, fulvic acids from most soils were found to have similar nominal molecular weight distributions to those of their humic acid counterparts. A large proportion of soil fulvic acid compounds was in the > 100 000 nominal molecular weight range.     

THE ACCUMULATION OF SOIL ORGANIC MATTER AND ITS CARBON ISOTOPE CONTENT IN A CHRONOSEQUENCE OF SOILS DEVELOPED ON AEOLIAN SAND IN NEW ZEALAND

Soil organic matter was extracted by a mixture of O.IM Na₄P₂O: O.IM NaOH from a chronosequence of weakly weathered soils developed on aeolian sand, and fractionated into humin (non-extractable), humic acid, and fulvic acid. The mass of total organic carbon in the profiles, the ¹⁴C content and the ¹³C/12C ratios were also determined. The weight of total carbon increased rapidly at first and then gradually without attaining a steady state. This trend was also shown by the humin and fulvic acid fractions, but the humic acid fraction appeared to have reached a maximum after about 3000 years. The order of total weights of the organic fractions was humin > fulvic acid > humic acid. The evidence suggests that the proportions of the humic fractions formed by decomposition are related to soil differences but not to vegetation. The greater part of the plant material found in the soils appears in the humin and fulvic acid fractions.     

FTIR‐spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH,Na4P2O7, and Na2CO3 extraction procedures

Aim of our study was the development of the methodological basis for the characterization of humic fractions of a long‐term field experiment. Humic acids (HAs) were extracted from three layers of a nontilled soil using three different extractants (1 M NaOH, 0.1 M Na₄P₂O₇, 1 M Na₂CO₃), and the humin fraction was enriched. NaOH as extractant for FTIR analysis of humic substances yields higher resolved IR spectra, especially in the important regions of stretching vibrations including aromatic and aliphatic groups and in the fingerprint area including amides, aliphats, and aromats than the other extractants. The NaOH extraction has lower extraction yields as compared to Na₄P₂O₇ and Na₂CO₃ and represents a different part of the soil organic matter (SOM). This is reflected by lower C : N ratios and higher E₄ : E₆ and fulvic acid–to–humic acid ratios as compared to the other extractants. The FTIR band areas of HA fraction obtained by NaOH showed an increase of the aromatic and carbonyl groups and a decrease of amide groups with increasing soil depth. Aliphatic groups showed contradicting results: The bands of the stretching vibrations increased, and the band of the bending vibrations decreased. We assume that band interactions in the bending vibrations were responsible for that phenomenon under the assumption of an increase of aliphatic groups with increasing soil depth. The IR bands of the enriched humin fraction showed a decreasing trend in case of both aliphatic bands deriving from stretching vibrations and an increase of aromatic characteristics with depth. Our study led to the conclusion that HA fractions obtained by 1 M NaOH represent a small and dynamic fraction indicated by the measured yields in combination with values of N_t, C : N, E₄ : E₆ ratios, and ratios of fulvic acids (FA) to HA. The humin fraction has a high contribution to the total organic C and represents a more stabilized fraction of SOM which still shows changes in its aromatic and aliphatic characteristics with soil depth.     

Chemical characteristics and microbial degradation of humate

Abstract

Humates are often used in agriculture as a source of organic matter. This study was conducted to characterize a commercial humate and to evaluate its chemical and decomposition characteristics. Characterization methods included fractionization of humic and fulvic acids of the humate, based on their alkali/acid insolubility; elemental analysis; acidic functional group analysis; and E₄/E₆ ratio determinations. The humate consisted of the following: 58% organic matter, 32% ash, and 10% moisture. Humic fraction was mostly humic acid (76%), with some fulvic acid (18%). Organic elemental composition [59% carbon (C), 5% hydrogen (H), and 36% oxygen (O)] also suggests a humic‐acid nature. Inorganic elemental content of this humate, which was primarily aluminum (Al) [4.9%] and iron (Fe) [0.46%], reflects its spodic origin. Much of the Al present, however, results from the flocculant (alum) used at the mining site to precipitate the humate. The relatively low total acidity of this humate (250 cmol/kg) suggests the blockage of some of its functional groups by Al, Fe, and associated clay minerals. After purification, total acidity increased to 510 cmol/kg and acidity associated with carboxyl groups increased to 280 cmol/kg. Results of E₄/E₆ determinations for the humate (2.5) and the humic‐acid fraction (4.8) also suggested that the organic fraction was predominately humic acid. Decomposition of the humate was estimated by measuring the quantity of carbon dioxide (CO₂) evolved during a four‐week incubation. Results suggested a relative resistance to microbial degradation. However, results also suggested the presence of some readily decomposable C compounds associated with humate. Agricultural use of this humate reguires some modifications to produce a more reactive material. This may include a change of flocculant and a lowering of its ash content.     

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.     

Effect of soil depth on acid properties of humic substances extracted from an ombrotrophic peat bog in northwest Spain

The most southerly ombrotrophic peat bogs in Europe are in Galicia (northwest Spain). The humic matter in them originates from chemical processes in anaerobic conditions. We studied the acid properties of fulvic acids and humic acids isolated from two peat horizons of an ombrotrophic peat bog by potentiometric titration. Solutions containing 25, 50 and 100 mg l⁻¹ of each humic substance were titrated at ionic strengths 0.005 m , 0.01 m and 0.1 m (with KNO₃ as the inert electrolyte). Charge curves were analysed with a Donnan model to determine the intrinsic proton binding parameters. The concentration of the humic substance affected the charge curves more significantly at pH exceeding 6, and tended to disappear at greater concentrations. The proton binding conditional constants decreased with increasing ionic strength, this effect being more significant in the carboxylic groups with less affinity for protons. The proton binding constant of the carboxyl groups in a fulvic acid was one order of magnitude less than the value for the corresponding humic acid, whereas for the phenolic groups the values for both fractions were similar. The total content of acid groups was approximately 2 mol kg⁻¹ greater in the fulvic fraction than in the humic fraction. Both humic fractions from the lower horizon contained more acid groups than those from the upper horizon, mainly because the content of carboxyl groups increases with soil depth. Therefore, the humic substances in the lower horizon of the peat will be more negatively charged, which will affect their solubility and the binding of metal ions.     

Macromolecule‐P associations and inositol phosphates in some chilean volcanic soils of temperate regions

Abstract

Total organic P, humic and fulvic acid‐P associations and inositol phosphates in nine volcanic soils of southern Chile were determined. The concentration of organic P (Po) ranged from 654 to 1942 ppm accounting for 49% to 64% of total soil P. Phosphorus associated to humic (HA‐P) and fulvic acids (FA‐P) accounted for 51–68% and 32–49% of Po, respectively. Inositol penta‐ and hexaphosphates represented 42% to 67% of Po suggesting that significant amounts are associated with both humic and fulvic acids. Po content was significantly correlated to organic C, total soil P and HA‐P. HA‐P and FA‐P fractions obtained from the most representative soil were examined by dyalisis and gel filtration. While approximately 96% of HA‐P presented a molecular weight higher than 100,000 daltons, 53% of FA‐P had a molecular weight under 12,000 daltons. It is suggested that these more labile organic P forms would be more easily mineralized, thus increasing the available P pool.     

Dynamics of Organic C Mineralization and the Mobile Fraction of Heavy Metals in a Calcareous Soil Incubated with Organic Wastes

Organic wastes such as sewage sludge and compost increase the input of carbon and nutrients to the soil. However, sewage sludge-applied heavy metals, and organic pollutants adversely affect soil biochemical properties. Therefore, an incubation experiment lasting 90 days was carried out to evaluate the effect of the addition of two sources of organic C: sewage sludge or composted turf and plant residues to a calcareous soil at three rates (15, 45, and 90 t of dry matter ha^–1) on pH, EC, dissolved organic C, humic substances C, organic matter mineralization, microbial biomass C, and metabolic quotient. The mobile fraction of heavy metals (Zn, Cd, Cu, Ni, and Pb) extracted by NH₄NO₃ was also investigated.The addition of sewage sludge decreased soil pH and increased soil salinity to a greater extent than the addition of compost. Both sewage sludge and compost increased significantly the values of the cumulative C mineralized, dissolved organic C, humic and fulvic acid C, microbial biomass C, and metabolic quotient (qCO₂), especially with increasing application rate. Compared to compost, the addition of sewage sludge caused higher increases in the values of these parameters. The values of dissolved organic C, fulvic acid C, microbial biomass C, metabolic quotient, and C/N ratio tended to decrease with time. The soil treated with sewage sludge showed a significant increase in the mobile fractions of Zn, Cd, Cu, and Ni and a significant decrease in the mobile fraction of Pb compared to control. The high application rate of compost resulted in the lowest mobility of Cu, Ni, and Pb. The results suggest that biochemical properties of calcareous soil can be enhanced by both organic wastes. But, the high salinity and extractability of heavy metals, due to the addition of sewage sludge, may limit the application of sewage sludge.     

Copper(II) complexes with refractory anionic surfactants found in sewage sludge

Electron spin resonance (ESR) spectrometry was used to study Cu(II) complexes with anionic surfactant compounds of the types that have been detected in sewage sludge or in the fulvic acid fraction of anaerobically-digested sewage sludge and found to resist biodegradation in the soil environment. The ESR spectra of frozen (77 °K), aqueous solutions of linear alkyl benzene sulfonates and fatty alcohol sulfate esters at Cu-ligand molar ratios ranging from 0.1 to 1 exhibited anisotropic patterns indicative of a d _x 2?y ² groundstate of Cu(II) bound into innersphere complexes with the ligands arranged in square planar coordination. Sulfonate-type surfactants, both in the acid and salt forms, appeared to complex Cu(II) more efficiently than ester sulfate-type surfactants. Conventional physical parameters calculated from the ESR spectra were consistent with fully oxygenated, 4 O-ligand binding sites for the Cu(II) ions and indicated the formation of similar unidentate or bidentate complexes between Cu(II) and the surfactants at any Cu/ligand molar ratio investigated. The spectral lineshapes and related parameters of the Cu(II)-anionic surfactant complexes, however, were not very similar to those observed previously for Cu(II)-sewage sludge FA complexes. It was concluded that anionic surfactants involved in Cu(II) complexation by sewage sludge or sewage sludge fulvic acid do not behave as isolated, independent ligands, but instead may participate as co-ligands with other O-containing functional groups and/or as moities incorporated into the fulvic acid structure.     

Effects of humic and fulvic acids on release of fixed potassium

Release of potassium fixed by expanding silicate clays is considered of practical importance in soil fertility. Humic and fulvic acids are expected to play a definite role in liberating this fixed K, because of their chelating power, but not much is known in this respect. The following investigation was conducted to study release of fixed K by montmorillonite and illite, using humic and fulvic acids isolated from the surface horizon of a Cecil soil (Typic Hapludult, Red Yellow Podzolic soil) as extractants. For comparison, extraction was also done with 1 N neutral NH₄ -acetate, a mixture of 0.05 N HCl and 0.025 N H₂SO₄ or H₂ O. Supporting analyses of clays were carried out with X-ray diffraction to establish changes, if any, in crystal structure due to fixation, release of K, or adsorption of humic compounds. The results indicated that humic and fulvic acids released some of the K fixed by montmorillonite or illite. In terms of percentage of the total K fixed, 9 to 28% were released by the various extractants. The percentages K released by humic and fulvic acids were similar from both montmorillonite and illite, but based on absolute values, humic and fulvic acids extracted less K (mg/100 g) from illite than montmorillonite. Although statistically significant at the 5% level of probability, the capacity of humic compounds to liberate fixed K was not different markedly from those of NH₄ -acetate and the double acid mixture. Differences in pH of humic solutions had no influence on extraction of fixed K. X-ray diffraction analysis yielded curves showing an increase in spacing from 10.4 Å for K-montmorillonite to 13.2 Å as a result of extraction with the double acid mixture.     

The distribution of the stable carbon isotope (13C/12C) in fractions of soil organic matter

The carbon-isotopic composition of fulvic and humic acid from the A horizons of eight soil types, developed under a wide variety of climatological conditions, was measured. The fulvic acid is always enriched in ¹³C as compared with the humic acid from the same soil by a rather constant factor of 0.9?. The fulvic acids are isotopically closer to the plant source of the organic matter and thus represent an intermediate stage in the formation of humic substances. Depth sections of peat soil showed that carbon isotopes can be used to evaluate the dynamic nature of the fulvic-acid fraction. With depth, a transfer of carbon groups from polysaccharides to fulvic acid is seen. Based on isotopic evidence it is shown that in addition to formation of β-humus, part of the fulvic acid is condensed with depth to a stable humic fraction — humin.     

不同施肥条件土壤胡敏酸级分变异及性质的研究

以１７年长期定位试验土壤为材料,在研究土壤有机质氧化稳定性基础上,采用酒精沉淀分级方法,对不同施肥处理土壤胡敏酸的级分及光学性质进行了研究,并对胡敏酸及其级分的类型进行了划分。结果表明长期施用有机（堆肥及秸秆）土壤有机质含量明显增加,有机质的氧化稳定系数降低,土壤胡敏酸组成分布中心向高浓度酒精析出的级分偏移,颗粒小的级分比例增大,土壤胡敏酸的Ｅ４,Ｅ６值减小Ｅ４／Ｅ６增大,而单施化肥则有相反趋势。     

Soil organic carbon storage and quality are impacted by corn cob biochar application on a tropical sandy loam

Purpose

Humic substances, which are integral components of total organic carbon (TOC), influence soil quality. The study aimed to investigate whether humic and non-humic fractions exhibit early, consistent, and measurable changes and affect TOC sensitivity and storage in a tropical sandy loam soils amended with corn cob biochar.

Materials and methods

There were four treatments with four replicates established in a randomized complete block design. Composite soil samples were taken from plots without biochar (CT), from plots incorporated with 15 t biochar ha^?1 (BC-15), and 30 t biochar ha^?1 without or with phosphate fertilizer (BC-30 and BC-30+P). The TOC, and humin, humic acid (HA), and fulvic acid (HA) fractions of soil organic carbon were determined for each treatment. The optical densities (400–700 nm) were measured on the soil-free extracts by spectrophotometry; the densities measured at 465 and 665 nm were used to calculate the E₄₆₅/E₆₆₅ ratios.

Results and discussion

The BC-30 and BC-30+P plots recorded the highest TOC, humin, humic acid (HA), and fulvic acid (FA) contents with respect to the lowest in the CT. The total exchangeable carbon stratification was significantly higher in all the biochar-treated plots relative to the CT. Spectral analysis showed higher values of E₄₆₅/E₆₆₅ (5.02 and 5.15) in the CT and BC-15-treated soils, respectively, compared with the BC-30 and BC-30+P-amended soils with E₄₆₅/E₆₆₅ ratios of 2.76 and 2.98, respectively.

Conclusions

Corn cob biochar applied to a tropical sandy loam:

? increased the concentrations of HA and FA and led to increased stratification of TOC, with a stronger effect on HA compared with FA;

? significantly lowered E₄₆₅/E₆₆₅ at the high biochar application rate of 30 t ha^?1, implying the dominance of high molecular weight humic acid-like substances, and increased degree of aromaticity of the TOC.

     

Decomposition and distribution of residual activity of some 13C-microbial polysaccharides and cells,glucose, cellulose and wheat straw in soil

Studies were made to determine the rate of decomposition of some ¹⁴C-labeled microbial polysaccharides, microbial cells, glucose, cellulose and wheat straw in soil, the distribution of the residual ¹⁴C in various humic fractions and the influence of the microbial products on the decomposition of plant residues in soil. During 16 weeks from 32 to 86 per cent of the C of added bacterial polysaccharides had evolved as ¹⁴CO₂. Chromobacterium violaceum polysaccharide was most resistant and Leuconostoc dextranicus polysaccharide least resistant. In general the polysaccharides, microbial cells, and glucose exerted little effect on the decomposition of the plant products. Upon incubation the ¹⁴C-activity was quickly distributed in the humic. fulvic and extracted soil fractions. The pattern of distribution depended upon the amendment and the degree of decomposition. The distribution was most uniform in the highly decomposed amendments. After 16 weeks the bulk of the residual activity from Azotobacter indicus polysaccharide remained in the NaOH extracted soil. From C. violaceum polysaccharide both the extracted soil and the humic acid fraction contained high activity. About 50–80 per cent of the residual activity from the ¹⁴C-glucose, cellulose and wheat straw amended soils could be removed by hydrolysis with 6 n HCl. The greater part of this activity in the humic acid fraction was associated with the amino acids and that from the fulvic acids and residual soils after NaOH extraction with the carbohydrates. About 8 16 per cent of the activity of the humic acid fraction was present in substances (probably aromatic) extracted by ether after reductive or oxidative degradation.     

The distribution of nitrogen in some highly organic tropical volcanic soils

The qualitative and quantitative distribution of N-compounds in 10 tropical soils, and in a number of humic materials extracted from representative samples thereof, was determined after 6 N HCl hydrolysis.Eighty to 98% of the total N in the soils and humic materials was hydrolysable by 6n HCl. Slightly less than one half the hydrolysable N in the soils and humic fractions consisted of amino acids. Well-drained soils and fulvic acids extracted from them contained unusually high concentrations of the acidic amino acids, aspartic and glutamic acids. Between 80 and 95% of the amino acids in the soils was accounted for in the humic materials + NaOH-insoluble organic residues. NH⁺₄-N released by acid hydrolysis was generally higher for the soil samples than for the humic materials. Amino sugar-N constituted relatively small proportions of the total N in the soils and humic fractions.Our data suggest that large quantities of amorphous allophanic materials coupled with relatively high enzymic activity are responsible for the observed accumulation of acidic amino acids in the well-drained tropical volcanic soils.     

The chemistry of humic and fulvic acids extracted from argentine soils—I. Analytical characteristics

Five humic and three fulvic acids were extracted from Argentine Chestnut, Brunizem, Solod and Solonetz soils and analysed by electrometric, spectroscopic and spectrometric methods. There were great similarities in elementary analysis, functional group content, absorption characteristics in the visible region and in the IR, and in the ESR measurements between the humic acids and, similarly, between the fulvic acids. There were distinct differences between the humic and fulvic acids in C, H, N and O content, distribution of oxygen in functional groups, E₄/E₆ ratios and free radical content.