A comparative study on the chemical composition of humic acids from forest soil,agricultural soil and lignite deposit: Bound lipid,carbohydrate and amino acid distributions

Base- and acid-hydrolysable fractions of humic acids (HAs) isolated from a forest soil, an agricultural soil and a lignite deposit were analysed, and comparisons were made between the base hydrolysable lipid (bound lipid), carbohydrate and amino acid signatures of the different humic acids.Bound lipids differ depending on the humic acid origin. Their composition were rather similar for the two soil humic acids, with three main lipid classes identified: (i) aliphatic components, (ii) aromatic components and (iii) sterols and triterpenols. The aliphatic subfraction was the most abundant and consisted predominantly of cutin- and suberin-derived moieties some of which could be clearly related to the vegetation. A minor bacterial input was indicated by the presence of short chain α- and β-hydroxyalkanoic acids. Aromatic subfraction contributed to a low amount to the total base hydrolysates and consisted mainly of lignin-derived methoxyphenols. Present in trace amounts, sterols and triterpenols are mainly of higher plant origin. The base hydrolysate from lignite humic acid markedly differs. Bound lipids released from lignite HA comprised almost exclusively aliphatic components, largely dominated by long chain alkanoic acids. Lignin-derived moieties, hardly detected, consisted solely of vanillic and 4-hydroxybenzoic acids indicating a much higher degree of lignin alteration in lignite humic acid. Sterols and triterpenols were absent.Although the composition of monosaccharides released upon acid hydrolysis was rather uniform irrespective of the humic acid origin, the distribution changed with the degree of humification of the HAs. Ratios of (Galactose+Mannose) to (Xylose+Arabinose) increased from soil to lignite humic acids. The high values of the ratios indicate that carbohydrates are primarily of microbial origin.In all humic acids neutral and acidic protein amino acids dominated. Non protein amino acids were only minor components consisting mainly of hydroxy proline and ornithine. The amino acid distributions of both soil HAs were similar. The amino acid distribution of lignite HA resembled that of soil HAs except for the following differences: (1) the absence of hydroxy proline and the greater abundance of ornithine suggesting a higher microbial contribution to the amino acids as the degree of humification increases, (2) the higher contribution of polar amino acids suggesting a preferential preservation of these amino acids possibly by interaction with the humic acid surface through hydrogen bonds.     

溶液培养条件下腐殖酸对铁异化还原的影响

腐殖酸对土壤和水体环境中铁(Fe)的还原过程有重要影响。本文采用从山西大同风化煤、河南巩县褐煤、云南昆明滇池底泥中提取制备的腐殖酸,通过布置培育试验并接种土壤悬液,研究了不同来源的腐殖酸对无定形氧化铁异化还原的影响。结果表明:单独添加腐殖酸对氧化铁的还原几乎没有影响;而当同时添加腐殖酸与葡萄糖时,培养基质中氧化铁的还原过程显著加强;腐殖酸浓度越高对氧化铁还原的促进作用越明显。不同来源的腐殖酸因其复杂程度和结构不同,对氧化铁还原的促进作用有明显差异,其中山西大同风化煤提取的腐殖酸促进作用最大,云南昆明滇池底泥和河南巩县褐煤提取的腐殖酸之间则无显著差异。     

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.     

Humic acid reactions with amino acids

Reactions between five humic acids extracted from soils with widely differing pedological histories and 17 amino acids commonly occurring in proteins were investigated in aqueous solutions at pH 3.0 and 6.5. The reactions were affected by: (a) nature of the humic acid: (b) pH of the system: and (c) length of contact. The principal reaction appeared to be the microbiological oxidative degradation of the amino acids leading to the formation of substantial amounts of ammonium. While there was no evidence that the humic acids per se interacted with the amino acids, they did not appear to interfere with the microbiological degradation of the amino acids.     

有机酸对五种人工合成磷酸盐活化作用及活化途径的研究

采用实验室合成和化学浸提方法,研究了高分子量腐殖酸和低分子量柠檬酸在不同浓度条件下对五种人工合成磷酸盐的释磷效应及活化途径。结果表明:两种有机酸均能促进五种人工合成磷酸盐中磷素的释放,且释磷量随有机酸浓度的增加而增大;其中柠檬酸的活化能力明显强于腐殖酸;腐殖酸对三种钙磷的活化效果较好,而Fe-P和Al-P基本不受其影响或影响很小。柠檬酸通过质子和有机阴离子的共同作用来活化人工合成Ca2-P、Ca8-P和Al-P中的磷,Ca10-P中磷的活化几乎完全依靠质子作用,而低浓度有机酸作用下Fe-P的活化则来源于有机阴离子的作用。     

Persulfate oxidation of humic acids extracted from three different soils

Three soil humic acids were degraded by a new mild chemical oxidation method using potassium persulfate at pH 2.0. The method degraded 30–40% of the starting material. The type and relative quantities of the products or fragments released as determined by GC/MS varied with the source of the polymers. Humic acid extracted from an Inceptisol yielded fatty, benzenecarboxylic and phenolic acids; humic acid from a Typic Chromoxerert produced largely dialkyl phthalates, and humic acid from an Humic Haplorthod gave only branched and straight-chain fatty acids. It is suggested that the method may be used to advantage as a first oxidant in a sequential degradation.     

Chemical studies on soil humic acids

Seventeen samples of soil humic acids, two fractions of soil fulvic acid sample, and several related compounds such as lignin, tannin, flavonoid and artificial humic substances were decomposed in conc. KOH solution at 180°C. Succinic acid, glutaric acid, phloroglucin, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, 3,4-dihydroxy-5-methoxybenzoic acid, and gallic acid were detected in the degradation products of humic acids. The amounts of these degradation products were discussed in relation to the degree of humification or the sources of the humic acid samples. Succinic acid also resulted from glucose, polymaleic acid, and the humic acid and humin prepared from glucose, but glutaric acid resulted only from glucose humic acid and glucose humin but not from glucose and polymaleic acid. Succinic acid and glutaric acid were supposed to result from the same structural portions in humic acids because of the very significant positive linear correlation between their amounts. p-Hydroxybenzoic, vanillic, protocatechuic, and 3,4-dihydroxy-S-methoxybenzoic acids were presumed to result mainly from lignin structure in humic acids. Soil humic acids yielded small amounts of gallic acid although the yields by hydrolysable tannins were in large amounts. The yields of above-mentioned degradation products from humic acids decreased with increasing degree of humification. Phloroglucin resulting from ftavonoids including condensed tannins were also found in the degradation products of humic substances. Its yield showed no linear correlation with RF value of humic acid, and is presumed to be rather related to the vegetation at the sites of soil sampling.     

有机酸培养时间和种类对煤矸石碎屑组成及速效养分的影响

为研究有机酸添加后煤矸石碎屑组成及其速效养分变化,以淮南潘集矿区为研究区域,选取矿区内多处煤矸石山上大块煤矸石人工破碎后作为供试材料,向其中添加10 mmol/L低分子量有机酸和腐殖酸水溶液,恒温培养120 d,在不同培养阶段取出部分样品测定煤矸石碎屑颗粒组成、EC、pH和速效养分等指标。结果表明,有机酸的添加总体上促进了煤矸石碎屑颗粒的细化。其中,黏粒平均增幅达81.90%,极细砂增幅在42%以上,粉粒和极粗砂粒含量平均下降幅度分别为21.05%、62.34%。有机酸添加有助于煤矸石碎屑颗粒基质结构向好的方向发展,并且以柠檬酸和腐殖酸效果最好。培养后的各处理pH值、电导率、速效养分对有机酸种类和培养时间的响应存在显著差异。有机酸添加后,酒石酸、草酸和腐殖酸处理pH均上升较大,平均pH值分别为7.69、7.75和7.71,显著大于对照处理(P<0.05),达到微碱性水平。除苹果酸处理外,其他处理的pH随培养时间的变化表现“N”型特征。电导率的变化与pH值变化正好相反,以苹果酸处理上升最大,比培养前增幅达146.78%;有机酸添加极大促进了煤矸石碎屑碱解氮(AN)和速效磷(AP)的释放,前者以苹果酸效果较好(释放量最大为CK的1.56倍和培养前的13.03倍),后者以柠檬酸效果较好(释放量最大为培养前的2.56倍和CK的3.39倍)。随着培养时间延长,碱解氮和速效磷释放量均逐渐增加;水分浸润和有机酸溶液添加均在前期表现一定抑制煤矸石碎屑颗粒速效钾(AK)释放的作用,后期逐渐表现一定促进释放的作用,但各有机酸处理之间差异不显著。有机酸对煤矸石山生态修复肯有一定积极作用。     

Quinoid groups in humic acids

Quinoid groups of humic acids from various sources (soil, peat, brown and oxidized coals) can undergo reversible redox transformations, this being a fundamental property of this class of natural compounds.Kinetic oxidation curves for reduced humic acids indicate the presence of various types of quinoid groups.The specificity of a previously suggested technique for determining quinones in humic acids has been corroborated. Exhaustive carbonyl reduction by sodium borohydride combined with reductometric methods for determining quinones in alkaline media make possible a better estimation of the ketone content in humic acids.When humic acids are reduced in an acid solution of tin chloride, rigorous conditions (4 h, 120°C, strong acid media) lead to an irreversible conversion of part of the quinone carbonyls and, as a result, the reaction proceeds nonstoichiometrically.     

The contribution of lignin to the composition of humic acids from a wheat-straw amended soil during 3 years of incubation in pots

An Andisol from the Vesuvian volcanic area, low in organic matter, was treated with wheat straw (20/1 w/w) and urea (500/1 w/w) in pots, and maintained at 60–90% of field capacity in an open greenhouse in order to study the yields and properties of humic acids after 30, 122 and 224 d, and 1, 2 and 3 years of incubation. During the first year the yields increased, but subsequently they showed a decrease. However, the chemical and spectroscopic properties of humic acids remained substantially constant. The chemical characteristics of modified lignin from added wheat straw were recognized in the properties of humic acids by UV-visible, FT-IR and ¹³C CP-MAS NMR spectroscopic analysis. Evidence was obtained that these modifications included the microbial removal of carbohydrates, the oxidative destruction or transformation of the side chains of lignin units, the demethylation of mainly guaiacyl, and subsequent oxidation of catechol-like structures to o-quinone. The latter can be postulated to promote the nucleophilic addition of amino compounds with formation of aromatic amines in humic acids.     

In-source pyrolysis-field ionization mass spectrometry and Curie-point pyrolysis-gas chromatography/mass spectrometry of amino acids in humic substances and soils

With the aid of in-source pyrolysis-field ionization mass spectrometry (Py-FIMS) and Curie-point pyrolysis-gas chromatography/mass spectrometry (cPy-GC/MS) in the conventional electron impact mode, characteristic signals of 23 amino acid standards were described. Thermal and mass spectrometric fragmentation pathways of these amino acids differed with each method and complemented each other. Pyrolysis products assigned by Py-FIMS extended the range of signals for N-containing compounds in humic substances and soil organic matter and gave marker signals for free amino acids and their subunits in proteinaceous materials. These characteristic signals were correlated with the amino acid content in N-rich humic fractions consisting of seven fulvic acids and eight humic acids. The selected marker signals reflected 25–84% of the variances of the molar distribution of acidic, neutral, neutral aromatic, and basic amino acids in the humic fractions. In addition, a well described agricultural soil (0.08% amino acid N) was spiked with a standard amino acid mixture (0.08 mg amino acid N 100 mg^-1 dry soil) and produced enhancements of the relative abundances of the corresponding amino acid signals. Moreover, for 27 samples of whole agricultural soils of widely different origins, soil types, and vegetations, 15 selected amino acid indicators were correlated significantly with -amino N (r=0.76^***) and total N (r=0.65^***).     

Permanganate oxidation of methylated fulvic and humic acids in chloroform

Methylated fulvic and humic acids were oxidized by permanganate in chloroform in the presence of a cyclic polyether, 18-crown-6. The total amount of oxidation products identified was 6.5% for fulvic acid and 6.7% for humic acid. About 46% of the oxidation products found from both fractions were dicarboxylic acids (15 compounds), the rest were benzenecarboxylic acids and their methoxyl derivatives (15 compounds). Alkanes and fatty acids were also found.Nonandioic acid and 3,4-dimethoxy-benzenecarboxylic acid are found in almost equal amounts. Each accounts for 1.3% of the starting material, and together they represent 40% of the oxidation products. 3,4-Dimethoxy-benzoic acid and 4-methoxy-benzoic acid are believed to result from the oxidation of terminal groups in the humic polymer.Proton resonance spectra show that about 25% of the aliphatic protons are part of methylene chains. The most prominent chain lengths consist of 6–8 methylene groups as shown by the dicarboxylic acids formed by oxidation.     

Inhibition by soil humic acids of native and acetylated proteolytic enzymes

The activities of acetyltrypsin and acetylcarboxypeptidase were unaffected by neutralized solutions of soil humic acids in concentrations which markedly inhibited the non-acetylated enzymes. Further, acetyltrypsin, unlike trypsin, did not coprecipitate with humic acids. The results support the conclusion that humic acids bind the proteases by a cationexchange mechanism whereby protease amino groups are linked to humic acid carboxyl groups.     

Pyrolysis of a soil humic acid and of their hydrolyzed and methylated products

Pyrolysis of soil humic acid yields a variety of products arising from heterogeneous materials associated to the humic structure and from this self. After acid hydrolysis most of the compounds related to proteins, polysaccharides and lignin dissappear. Adsorbed compounds such as fatty and dicarboxylic acids are released after methylation and subsequent pyrolysis. In addition pyrolysis produces aromatic and substituted aromatic hydrocarbons. All the identified pyrolysis products have also been isolated by different complex and time-consuming chemical degradations and solvent extraction procedures. Pyrolysis provide some evidence of the humic acid structure.     

Relative importance of protons and solution calcium concentration in phosphate rock dissolution by organic acids

A series of experiments was conducted to quantify the relative contribution of protons and other mechanisms to the dissolution of phosphate rocks (PRs) from six countries in solutions of low-molecular-weight-aliphatic organic acids. The amounts of P and Ca released after 3 d of incubation at 28°C were determined in all the experiments. In the first experiment the solubility of the PRs < 500 μm particle size) in 100 M (25 mL g^-1 PR) oxalic, tartaric, and citric acids was compared with that in three mineral acids and four chelating compounds. There were no differences in the amount of P released by the mineral acids, but the organic acids released more P than could be accounted for by protonation. The chelating compounds were the least effective. In the second experiment, 1 g each of Sri Lanka and Togo PRs was incubated with 2.5 mmol of the organic acids using three acid concentration (mM) : acid volume ratios (250:10, 100:25, and 25:100). The amount of P dissolved from the PRs and the amount of acid remaining in the filtrate were about the same for all three treatments. A mixture of the organic acids and the respective Na-salts was prepared at six different acid: salt ratios in the third experiment. Total acid concentration in all the solutions was 100 mM. Higher proportion of the Na-salt in the mixture decreased PR dissolution, while the amount of the acid remaining in the filtrate increased. The Ca concentration in the filtrate showed an opposite trend. The results indicated that PR dissolution by the organic acids was initially dependent on protonation, which accounted for only 13–38% of the amount of P dissolved. Most of the amount of P dissolved could therefore be attributed to other mechanisms. Removal of dissolved Ca from the solution appears to be the major factor controlling the subsequent dissolution of the PRs. The ability of tartaric and oxalic acids to dissolve effectively the PRs was attributed to the formation of an insoluble calcium compound which was precipitated from the solution. For the use of organic acids as a potential amendment for improving the P availability of PRs, the Ca binding power of the organic acids should also be considered.     

Paramagnetic properties of humus acids of podzolic and bog-podzolic soils

The contents of free radicals in preparations of humic and fulvic acids extracted from virgin and plowed podzolic, surface gleyic podzolic, and peaty podzolic-gleyic soils were determined. The concentration of paramagnetic centers in the humic acids was 1.5–2 times higher than that in the fulvic acids. The agricultural use decreases the paramagnetic activity of the humus compounds and promotes the accumulation of biothermodynamically stable organic compounds in the plow horizons.     

The persulfate oxidation of fulvic acids

Persulfate oxidation of two fulvic acids from different soils was accomplished. This is a new mild chemical degradative method which differentiates between fulvic acids isolated from soils developed under widely differing environments. A fulvic acid isolated from an Humic Haplorthod gives as major oxidation products phenolic acids, whereas a fulvic acid from an Andic Hapludult yields benzenecarboxylic acids as most abundant compounds. The possibility to differentiate between fulvic acids from different soils, as chemical degradation with persulfate does, offers interesting prospects to the characterization of humic materials.     

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.     

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.     

Nitrogenous constituents in nest soils of harvester ants (messov ebeninus) and their influence on plant growth

Abstract

As a corollary of a detailed study on the ecology of the Harvester ants in soils of semi‐arid natural pastures, samples from a loessial Arid Brown soil were analyzed for the content of nitrogenous constituents (humic compounds, amino acids, nitrates, ammonia) as well as phenols and carbohydrates and the nutrient status of the soil.

While there was a significant increase in fulvic and humic acids in the ant nests, the simultaneous decrease in amino acids ‐ in relation to the surrounding soil ‐ indicates that the humic substances have been synthesized from amino acids and the abundantly present phenols and possibly carbohydrates.

The much higher concentration of nitrogen compounds in the ant mounds supports the idea that the soil mixing activity of the Harvester ants enhances microbiological processes and in turn the fertility of the mounds, as evident in the better growth of the pasture plants.