Formation of humic acids during plant residue humification and their nature

The process of humic acid formation during humification of plant residues (clover leaves and roots as well as timothy roots) was studied. According to some characteristics (elementary composition, IR-spectra, the content of COOH-groups) the newly-formed humic acids may be defined as “young forms”. The comparison of the new forms with more mature soil humic acids revealed the following specific features of the latter: (a) a higher degree of aromatic nuclei condensation; (b) a higher content of carboxyl groups; and (c) a more uniform structure.The authors discuss some defects of the methods used for extraction of humic acids from fresh and slightly humified plant residues by alkaline solutions. An artificial formation of humus-like substances and their extraction as admixtures together with true humic acids are inevitable with these methods. This should be taken into account in studying and systematizing such forms of humic acids.     

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     

ADVERSE EFFECTS OF HUMIC SUBSTANCES FROM DIFFERENT ORIGIN ON LUPIN AS RELATED TO IRON SOURCES

Humic substances improve the efficiency of different iron (Fe) sources overcoming Fe deficiency chlorosis of plants. However, applied at high rates, they can promote negative effects on plants. The main objective of this work was to study the potential adverse effect of three humic acids from different origin when they were applied with two effective Fe sources for plants: Fe- ethylenediaminedihydroxyphenylacetic acid (EDDHA) and Vivianite. To this end, an experiment with lupin (Lupinus albus L.) was performed involving two factors: (i) Fe source, and (ii) humic substances from three different origin (composted cork, leonardite, and compost obtained from a mixture of olive husk with cotton gin trash) applied at 0, 0.1, and 0.5 g organic carbon (C) kg^?1 of growing media. At the rates used, humic substances promoted adverse effects on plant development, chlorophyll meter readings, and Fe content in lupin grown in calcareous media. Overall, the effect on dry matter and Fe content in plants was more relevant when Fe was supplied with Vivianite, the effect on chlorophyll meter readings being more significant when Fe was applied as Fe-EDDHA. Differences were also observed depending on the source of humic substances, those from leonardite promoting the greatest decrease in dry matter in roots and shoots. These humic substances possessed the highest values of spectroscopy index for aromaticity (A₂₅₄ ). On the other hand, the application of humic substances from olive husk compost, which exhibited the lower aromaticity index, resulted in the smallest decrease in dry matter production and chlorophyll meter readings. Dry matter in roots decreased logarithmically with increased values of the estimates of the amounts of aromatic compounds accumulated in the growing media (R² = 0.92; P < 0.01) with Vivianite as Fe source. Thus, the effects decreasing dry matter production, particularly in roots, and chlorophyll meter readings can be ascribed at least partially to the presence of phytotoxic aromatic compounds in humic substances.     

接种菌剂对鸡粪堆肥腐殖酸的影响

以鸡粪和稻草为原料进行条垛式堆肥,接种芽孢杆菌菌剂,堆肥共持续42 d。堆肥过程中接种菌剂处理和对照的总腐殖酸含量呈现先下降然后上升趋势。堆肥前3 d接种菌剂处理和对照的总腐殖酸含量基本相同,第7d接种菌剂处理总腐殖酸含量开始大于对照,堆肥结束时接种菌剂堆肥的总腐殖酸含量为23.1%,而对照的为17.2%。游离腐殖酸、水溶性腐殖酸、胡敏酸含量与总腐殖酸含量的变化趋势基本一致,呈现先降后升的趋势。富里酸含量呈先升后降趋势。堆肥结束时接种菌剂处理的各种腐殖酸含量均显著高于对照,说明接种菌剂可以促进堆肥中腐殖酸的产生。     

Composted biosolids enhance fertility of a sandy loam soil under dairy pasture

Field and pot trials were established to assess potential benefits and adverse effects of amending a sandy loam soil, under grazed ryegrass-clover pasture, with compost manufactured from wastewater biosolids, wood waste and green waste. Compost was applied to the field trial site annually for 4 years and the pot trials used soil from the field trial site each year after compost application. The pot trials demonstrated that yield of silver beet (Beta vulgaris L.) increased with increasing compost application rate and that plant metal uptake was (except for Zn) unrelated or inversely related to soil metal concentrations. In samples from the field trial, soil total C, N, P and Olsen P increased markedly with increasing compost application rate. Cation exchange capacity, exchangeable cations and total-extractable and EDTA-extractable metals (Cd, Cr, Cu, Ni, Pb and Zn) were also elevated, total Cu to the limit allowable in biosolids-amended soil. Soil basal respiration, microbial biomass C and anaerobically mineralisable N were significantly increased in the amended plots. Anaerobically mineralisable N was highly correlated with respiration (r =0.98, n =24) and only weakly related to microbial biomass C, probably indicating that a high proportion of the N mineralised was from the compost organic matter. Sulphatase and phosphatase activities increased, but not significantly, and there were no measurable effects on rhizobial numbers or on sensitive microbial biosensors (Rhizotox C and lux-marked Escherichia coli). Biosolids compost application enhanced soil fertility, productivity and microbial biomass and activity, with no apparent adverse effects attributable to heavy metals.     

How relevant is recalcitrance for the stabilization of organic matter in soils?

Traditionally, the selective preservation of certain recalcitrant organic compounds and the formation of recalcitrant humic substances have been regarded as an important mechanism for soil organic matter (SOM) stabilization. Based on a critical overview of available methods and on results from a cooperative research program, this paper evaluates how relevant recalcitrance is for the long‐term stabilization of SOM or its fractions. Methodologically, recalcitrance is difficult to assess, since the persistence of certain SOM fractions or specific compounds may also be caused by other stabilization mechanisms, such as physical protection or chemical interactions with mineral surfaces. If only free particulate SOM obtained from density fractionation is considered, it rarely reaches ages exceeding 50 y. Older light particles have often been identified as charred plant residues or as fossil C. The degradability of the readily bioavailable dissolved or water‐extractable OM fraction is often negatively correlated with its content in aromatic compounds, which therefore has been associated with recalcitrance. But in subsoils, dissolved organic matter aromaticity and biodegradability both are very low, indicating that other factors or compounds limit its degradation. Among the investigated specific compounds, lignin, lipids, and their derivatives have mean turnover times faster or similar as that of bulk SOM. Only a small fraction of the lignin inputs seems to persist in soils and is mainly found in the fine textural size fraction (<20 µm), indicating physico‐chemical stabilization. Compound‐specific analysis of ¹³C : ¹²C ratios of SOM pyrolysis products in soils with C3‐C4 crop changes revealed no compounds with mean residence times of > 40–50 y, unless fossil C was present in substantial amounts, as at a site exposed to lignite inputs in the past. Here, turnover of pyrolysis products seemed to be much longer, even for those attributed to carbohydrates or proteins. Apparently, fossil C from lignite coal is also utilized by soil organisms, which is further evidenced by low ¹⁴C concentrations in microbial phospholipid fatty acids from this site. Also, black C from charred plant materials was susceptible to microbial degradation in a short‐term (60 d) and a long‐term (2 y) incubation experiment. This degradation was enhanced, when glucose was supplied as an easily available microbial substrate. Similarly, SOM mineralization in many soils generally increased after addition of carbohydrates, amino acids, or simple organic acids, thus indicating that stability may also be caused by substrate limitations. It is concluded that the presented results do not provide much evidence that the selective preservation of recalcitrant primary biogenic compounds is a major SOM‐stabilization mechanism. Old SOM fractions with slow turnover rates were generally only found in association with soil minerals. The only not mineral‐associated SOM components that may be persistent in soils appear to be black and fossil C.     

Abbau von Huminstoffen durch Bodenmikroorganismen — eine Übersicht

Degradation of humic substances by soil microorganisms — a review Humic substances which represent differently extractable fractions of the soil organic matter exert multifarious effects on soil as a site for plant growth and a part of terrestrial environments. Among them especially humic acids and fulvic acids are subject to degradation and/or transformation by soil microorganisms. Several authors demonstrated the participation of different species of fungi, actinomycetes and also of non-mycelial aerobic or anaerobic bacteria in those processes under laboratory conditions. Indications exist that humic substances irrespective of their structure undergo degradation on cell surfaces due to the activity of exoenzymes. In this respect microbial phenoloxidases play an extraordinarily important role. The degradation rate of humic substances can be followed by optical, gravimetric and chemoanalytical methods as well as using biochemical and microbiological procedures (CO₂ release, microbial growth, biomass formation). An objective evaluation, however, can be hindered by the adsorption of humic substances on microbial biomass and sometimes also by formation of novel humic-like microbial metabolites. Therefore it is necessary to apply a multifactorial approach in the study of the degradation of humic substances which includes both quantitative and qualitative parameters. To better elucidate how these processes may occur under natural conditions, mixed populations of soil microorganisms should be predominantly involved in future studies.     

On the Probable Nature of Biological Activity of Humic Substances

It is common knowledge that humic substances extracted from different sources are characterized by high biological activity (BA), though the nature of this phenomenon is not quite clear up to now. To expand our understanding of the BA of humic substances, we studied the effect of humates prepared from humic acids of different origin on the germination of seeds. The efficiencies of seed treatment by humate solutions obtained from preliminary purified humic acids extracted from peat, coal, and soil differed insignificantly. At the same time, the solutions of salts of humic substances obtained via alkaline extraction from peat without subsequent purification did not lead to statistically significant biological effects. The analysis of literature and our own data allowed us to conclude that the biological activity of humic acids could be related to their capacity to regulate the growth processes via binding growth inhibitors released into the solution upon seed swelling into the supramolecular complexes.     

Microbial release and degradation of nonextractable anilazine residues.

Humic substance fractions obtained from a degraded loess soil taken from a long-term lysimeter experiment with the fungicide anilazine were incubated in aerated liquid cultures together with native soil microorganisms. Biomineralization, remobilization of [U-phenyl-(14)C]anilazine, respectively, its metabolites, and changes of the humic matrix were observed under variable nutrient conditions. Stimulated microbial activity favored the degradation of nonextractable (14)C-anilazine residues. However, nitrogen deficiency enhanced structural changes in the humic substances, which seemed to be used then as a nitrogen source. Along with the microbial degradation of the humic substances, parts of the bound anilazine residues became remobilized. Furthermore with the use of AMD-TLC, dihydroxy anilazine was detected within the nonextractable residues. The portion of rather weak bondings between the soil organic acids and the anilazine residues turned out to be considerably lower in the humic acids fractions than in the fulvic acids fraction.     

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.     

On artificial humic acids

I. Absorption Spectra of Some Artificial Humic Acids

It is a well known fact that numerous dark brown or black and amorphous substances cap be produced from organic compounds by purely chemical processes. Some of them are called artificial humic acids, and have been used as a model of soil humic acids by many workers. But it is doubtful whether such artificial humic acids are considered to be a model of soil humic acids, even if there can be found any similarities in their properties. However it may be an useful method for the study of humus formation to compare artificial humic acids with soil humic acids, and to make efforts to find a good model or to prepare artificial humic acids comparable to soil humic acids under laboratory conditions. For this purpose, first of all, the author prepared artificial humic acids from glucose, hydroquinone and lignin, and their absorption spectra were determined.     

Molecular characteristics of humic acids extracted from compost at increasing maturity stages

The molecular composition of humic acids (HA) extracted from compost at increasing maturity stages was determined by off-line TMAH-thermochemolysis-GC-MS, in combination with solid-state nuclear magnetic resonance (NMR) and infrared (IR) spectroscopies. While spectroscopy measurements followed the bulk changes, thermochemolysis provided a detailed molecular variation of HA composition. Both thermochemolysis and spectroscopy indicated that polysaccharides, alkyl, cyclic, and aromatic compounds were the predominant components of HA, the stable fraction of compost. NMR dipolar dephasing (DD) experiments confirmed that HA extracts contained lignin in lower amount than its oxidized degradation products. The progressive compost maturity was reflected in HA extracts by a decrease of carbohydrate content and a selective preservation of hydrophobic alkyl molecules, such as medium- and long-chain fatty acids, aliphatic alcohols, linear hydrocarbons, and plant polyester derivatives, like long-chain alkyl dicarboxylic acids, and ω-hydroxyacids. Spectroscopy results showed a concomitant entrapment in HA of biolabile compounds, such as peptidic moieties. The wide range of identified lipid components and plant biomarkers may represent useful tools to trace origin, quality, and transformation of amended compost in soil ecosystems.     

Alleviation of noxious effects of cattle ranch composts on wheat seed germination by inoculation with Azospirillum spp.

Two commonly-used composts from dairy cow manure that are used to improve poor structure and fertility of desert soils have inhibitory effects on wheat seed germination, probably as a result of their high levels of humic acids. Inoculation of wheat seeds with two species of the plant growth-promoting bacteria Azospirillum brasilense Cd and A. lipoferum JA4 (separately) prior to sowing in these amended soils improved germination, similar to the natural level of germination of seeds in desert soil without compost amendment. Both compost amendments increased height of wheat seedlings in the range of 20–25%, increased shoot dry weight by 15–19%, but severely decreased (51–54% less) root dry weight. Inoculation of wheat seeds with A. brasilense Cd, but not with A. lipoferum JA4, significantly increased plant growth parameters (height, shoot and root dry weight) over control plants grown in soil-compost mixtures. This bacterial species could survive for a period of 20 days in compost humic acid solution, could increase its population when the humic acids served as the sole carbon source, and may change the composition of humic acids in which it grows. We suggest that inoculation with A. brasilense may alleviate noxious effects on germinating seeds caused by compost application by possibly transforming the composition of humic acids in the compost.     

Die Veränderung von Huminsäuren in alkalischer Lösung

The change of humic acids in alkaline solution Five natural humic acids were fragmented under the conditions of the conventional method of extraction of humic substances with sodium hydroxide. Fulvic acid, hymatomelanic acid and a water soluble fraction (at pH 2) could be isolated as fragmentation products. The three fractions were formed directly after the treatment with sodium hydroxide, their quantities depend on the concentration and time of treatment with sodium hydroxide and from the function of the system. The deviating, differentiating and reproducing formation of fragments is explained by partial reversible reactions. Therefore fulvic acid and hymatomelanic acid must be regarded as artefacts. existing during the procedure of isolation of humic substances at high pH-values. Therefore the conventional isolation of humic substances from soils should always be accompanied by experiments, which inform about the arteficial production of ‘fulvic acids’ and ‘hymatomelanic acids’ during the procedure of isolation.     

Humus composition of soils under forest, coffee and arable cultivation in hilly areas of south Sumatra, Indonesia

To understand the effect of land use changes on the composition of humus in tropical soils, samples from land under primary forest, secondary forest, coffee plantation, and arable crops were investigated at three sites in south Sumatra, Indonesia. Total carbon and total nitrogen contents were 1.7 to 4.3 times and 1.1 to 2.8 times greater in the topsoil under primary forest than under the other types of land use. Following change from primary forest to other uses, the proportion of humic acids in the organic matter of the topsoils decreased while that of the fulvic acid fraction increased. Within the range of land uses, differences in the yields of humic acids and fulvic acid fractions were, respectively, larger and smaller than those in total carbon content. The humic acids were classified into the low and middle classes in the degree of humification. Absorption due to the green fraction of humic acids, Pg, was detected in the UV‐visible spectra of almost all the humic acids. No relation was observed between the degree of humification of humic acids or the strength of Pg absorption in their spectra and land use change. The fulvic acid fractions were fractionated on insoluble polyvinylpyrrolidone (PVP) into the adsorbed fractions consisting of humic substances and the non‐adsorbed fractions consisting of non‐humic substances. A positive correlation between the amount of the fulvic acid fraction and the percentage of the PVP‐adsorbed fraction within it indicated that the variation in the amount of the fulvic acid fraction was attributable to acid‐soluble humic substances. The ionization difference spectra of solutions between pH 12 and pH 7 suggested that the chemical structures of the PVP‐adsorbed fulvic acids have been altered by land use change.     

Comparison of Halogenation Potential Among Various Fractions of TOC in Water from Jingmi Canal,Beijing

Naturally occurring organic compounds in water from Jingmi Canal were sequentially fractionated into five fractions using filtration and adsorption columns of XAD-8 and XAD-4 resins in tandem: the fractions being particulates, hydrophobic compounds, humic substances, XAD-4 acids and other hydrophilic neutral solutes. Then each fractions was chlorinated simultaneously with sodium hypochlorite, and the dominant halogenated product determined in this experiment was chloroform. Brominated THMs were detected as well. Both TOC abundance and ratio of CHCl₃ product to corresponding fraction of TOC showed that dissolved humic substances and particulate-adsorbed organics were the major precursors of chloroform. Low-molecular-weight hydrophilic XAD-4 acids also possessed noticeable halogenation activity. Other aquatic organic solutes, however, were relatively inert with respect to the three fractions mentioned above. These results suggested that in addition to humic substances, other potential precursors which have not been studied thoroughly before, such as XAD-4 acids, should be considered during water chloronation.     

3C-NMR structural features of soil humic acids and their methylated,hydrolyzed and extracted derivatives

¹³C-NMR spectra of aqueous solutions of three humic acids (1 Typic Chromoxerert (HA-V), 1 Humic Haplorthod (HA-P), and 1 Inceptisol (HA-D)) are presented. The influence of chemical modifications (hydrolysis with 6N HCl, diazomethane methylation, and organic solvent extraction) of the humic substances and their spectra is studied. The spectra of the various humic substances show significant differences.Methylation of the compounds introduces changes in the aromatic and aliphatic (sp³) region of the spectra. Acid hydrolysis removes residual carbohydrates and amino acid signals from the spectra, whereas solvent extraction has the most pronounced influence upon the aliphatic high field region of the spectra, and shows also, that a significant amount of the paraffinic carbons is bound covalently to the aromatic nuclei of the compounds.     

Chromatographische Unterscheidung von Huminstoffen und Nichthuminstoffen aus Schwarzerdehumus

Chromatographic distinction of humic and non humic substances in chernozem-humus Fulvic acids, brown humic acids and grey humic acids from a chernozem from the Austrian Marchfeld were analyzed chromatographically on controlled pore glass with pore diameters of 177 and 259 Å. Concentration of coloured material and concentration of carbon were recorded in the chromatogramms. Specific extinctions at 400 nm were calculated for unit of C and maximal values were taken as constants specific for “pure” humic substances. These were 445,6 ± 10,5,388,7 ± 19,3 and 128,9 ± 0,8 for “pure” grey humic acids, brown humic acids and fulvic acids, respectively. With the aid of these constants, “pure” humic substances could be distinguished from accompanying non humic substances. “Pure” humic substances amounted to 63%, non humic substances to 37% of extractable organic C. 53%, 22% and 25% of the carbon of “pure” humic substances were from grey humic acids, brown humic acids and fulvic acids, respectively.     

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.     

Ein Vergleich der Huminstoffsysteme einer Parabraunerde aus der Kölner Bucht und einer österreichischen Schwarzerde

A comparison of the humic systems from a Luvisol from the area of Köln and from an Austrian Chernozem Grey humic acids, brown humic acids and fulvic acids have been extracted from a Luvisol after removal of the “light fraction”. The humic fractions were investigated chromatographically using controlled pore glass with a pore diameter of 17,7 nm. Concentration of both coloured material and carbon were simultaneously recorded in the chromatograms. Specific extinctions at 400 nm were calculated and maximal values were taken as constants specific for “pure” humic substances. These were 349,6; 155,8 and 39,4 for “pure” grey humic acids, brown humic acids and fulvic acids, respectively, and were in part considerably below those determined earlier for an Austrian Chernozem. Using these constants “pure” humic substances could be distinguished numerically from accompanying non-humic substances. The ratio of “pure” humic to non-humic substances was approximately 2:1 for both soils. Within the “pure” humic substances Chernozem humus was dominated by grey humic acids amounting up to 52% whereas in the Luvisol brown humic acids had the same level.