The effect of humic acids on the availability of phosphorus fertilizers in alkaline soils

Abstract. The effect of humic acids on transformation of phosphorus fertilizer was studied in an alkaline soil. Soil P was fractionated following 4 and 15 days incubation after humic acids were applied with phosphorus fertilizer to the soil. The availability of phosphate in the soil and total phosphorus in plants were determined at earing stage and at maturity in a pot experiment, and wheat yield was examined in a field trial. Addition of humic acids to soil with P fertilizer significantly increased the amount of water soluble phosphate, strongly retarded the formation of occluded phosphate and increased P uptake and yield by 25%.     

Effect of humic acids on the activity of two peroxidases

The activity of two peroxidases-from horseradish roots and Streptococcus faecalis-is inhibited by synthetic and natural humic acids. Kinetic analyses revealed that inhibition of the horseradish enzyme is due to binding of the donor substrate guaiacol to humic acids. Peroxidase from Streptococcus faecalis is inhibited by competitive interactions between the donor substrate NADH and humic acids for the enzyme. Binding of H₂O₂ to any of the enzymes was not influenced. The rate of inhibition by synthetic and natural humic acids was different; hydroquinone humic acid and an acetylated derivative were considerably more effective than humic acids from white peat.     

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。     

Regularities of extracting humic acids from soils using sodium pyrophosphate solutions

Regularities of extracting humic acids from different soil types (soddy-podzolic soil, gray forest soil, and all chernozem subtypes) with sodium pyrophosphate solutions at different pH values (from 5 to 13) have been studied. It is found that, regardless of soil type, the process occurs in two stages through the dissociation of carboxylic groups and phenolic hydroxyls, each of which can be described by a logistic function. Parameters of the logistic equations approximating the extraction of humic acids from soils at different pH values are independent of the content and composition of humus in soils. Changes in the optical density of humic acids extracted from soils using sodium pyrophosphate solutions with different pH values are described in the first approximation by the Gaussian function. The optically densest humic acids are extracted using sodium pyrophosphate solutions at pH 10. Therefore, it is proposed to use an extract with pH 10 for the characterization of organic matter with the maximum possible degree of humification in the given soil.     

Structure of humic acids isolated by sequential alkaline extraction from a typical chernozem

It was shown with the isolation of a humic acid (HA) preparation from a typical chernozem by sequential alkaline extraction as an example that the preparative yield of HAs decreased at each sequential extraction stage by 3–4 times. On the basis of studying the obtained preparations using elemental analysis, gel-penetration chromatography, and ¹³C NMR spectroscopy, the tendencies of the changes in the structural-group and molecular-weight compositions of the HAs from one extraction stage to the next one were revealed. The conclusion was drawn that a single extraction is sufficient for obtaining a representative HA sample.     

Studies on the colour of humic acids

Based on their solubility, humus has been often divided into three fractions, i.e. fulvic acids, humic acids and humins. On the other hand, it seems to be generally accepted that humus can be divided into two groups (1) (2), non-humic substances and humic substances. The term humic substances (3) is used as a generic expression to describe the dark coloured, amorphous material in humus, or any of its above-mentioned fractions.     

The alkaline hydrolysis of humic substances

A humic and a fulvic acid were subjected to four successive hydrolyses with 2NNaOH solution at 170°C for 3 h. Following each hydrolysis, the degradation products were extracted into ethyl acetate, methylated, separated by chromatographic techniques and identified by mass spectrometry and micro-IR spectrophotometry.One g of humic acid yielded 113.5 mg of aliphatic compounds (mainly n-C₁₆ and n-C₁₅) fatty acids), 72.1 mg of phenolics (principally guaiacyl and syringyl derivative) 17.1 mg of benzenepolycarboxylic acid esters and 30.4 mg of N-containing compounds (mainly secondary aromatic amides). Repeated attacks by the alkali on 1.0 g of fulvic acid released 103.8 mg of aliphatics, 133.8 mg of phenolics, 27.0 mg of benzenecarboxylic but 181.8 mg of N-containing compounds. Most of the latter appeared to have been formed during the second, third and fourth hydrolysis which produced reactive compounds that could abstract N from diazomethane which was used as methylating reagent. While 25.0% of the initial humic acid resisted repeated attacks by the alkali, practically all of the fulvic acid was converted into ethyl acetate-soluble products. The alkali-resistant humic acid residue produced high yields of benzenepolycarboxylic acids on alkaline permanganate oxidation.Alkaline hydrolysis, which is known to cleave CO bonds, was found to be relatively specific, although not very efficient, for the degradation of structural phenolic humic and fulvic acid components and for the concomitant liberation of adsorbed aliphatics and N-containing compounds, but was relatively ineffective for degrading aromatic structures linked by CC bonds.     

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.     

溶液体系中非生物因素对胡敏酸还原汞的影响

As a global pollutant process,the reduction of mercury(Hg)is especially important.One pathway is through an abiotic reduction with humic acids(HAs),which is controlled by different factors,including initial Hg and HA concentrations,pH,temperature and light.In this study,three humic acids were selected to illustrate the Hg~(2+)abiotic reduction mechanisms by HAs,and to identify the key limiting factors for reduction rates and amounts.In addition,the initial status of the HAs as a solid or in an aqueous solution were also compared,to help explain why HAs show different dominant characteristics(e.g.complexation or reduction)in the reaction process with Hg.Results indicated that HAs were able to reduce Hg abiotically.Higher initial Hg,higher HA concentrations and either high(8.1)or low(3.6)solution pH decreased the HA reduction capacity.In addition,Hg°production rates increased with increasing temperature,and the same trend was observed with light exposure.Humic acids added as an aqueous solution resulted in significantly greater Hg°production than addition as a bulk solid.Finally,the Hg reduction rate and capacity varied significantly(P0.05)with HAs from different sources.These findings helped to explain why HAs showed different dominant characteristics(e.g.complexation or reduction)in the reaction process with Hg,and evidentially demonstrated the existence of a possible pathway of Hg~(2+)reduction,which indicated that humic substances in natural environments,especially in water bodies,could act either as a sink or a source for Hg.     

Chemical studies on soil humic acids

The levels of alcoholic and phenolic hydroxyl, methoxyl, carboxyl, and carbonyl groups of 33 to 38 humic acids obtained from various types of soils were determined to analyze the relationships between the amounts of these functional groups and the degree of humification or the types of soils. The amounts of various oxygen-containing functional groups examined were all proven to be significantly different among the various types of humic acids by analyses of variance. During humification. generally, the carboxyl and carbonyl groups increased while alcoholic and phenolic hydroxyl and methoxyl groups decreased. Linear and logarithmic regression analyses of carboxyl group contents on RF values (optical density of the alkaline solution of humic acids at 600 nm) gave very significant positive correlations. The carboxyl group contents of Rp type humic acids and humic acids from calcareous soils were largely distributed in the upper side of the regression curve. The carbonyl group contents showed a very significant linear positive correlation with carboxyl group contents. and both of them showed high linear positive correlation with RF values. Phenolic hydroxyl group contents decreased with humification in Rp(l). B. and A type humic acids. Alcoholic hydroxyl group contents showed a significant negative linear association with _RF values. Methoxyl group contents decreased rapidly with increasing humification in low humified humic acids, and their negative correlation with _RF values were proven to be very significant by logarithmic regression analysis. The complicated relationship between oxygen content and RF value which was reported previously (7) has been accounted for by the results obtained in the experiments conducted here.     

Effect of soil pollution with polycyclic aromatic hydrocarbons on the properties of humic acids

Purpose

Interestingly, soil is the component of the natural environment in which most hydrophobic organic pollution including polycyclic aromatic hydrocarbons (PAHs) gets accumulated. The aim of the present paper was to determine the effect of soil pollution with PAHs on the elemental composition, spectral properties, and hydrophobic and hydrophilic properties of humic acids. The research was performed on different types of soil samples that were artificially polluted with selected PAHs (anthracene, pyrene, fluorene and chrysene).

Materials and methods

The soil samples were polluted with selected PAHs in an amount corresponding to 10 mg PAHs kg^?1. The PAHs-polluted soil samples were incubated for 180 and 360 days at a temperature of 20–25 °C and fixed moisture (50 % of field water capacity). Humic acids (HAs) were extracted from the soil samples prior to the incubation (additionally, soils not polluted with PAHs) and after 180 and 360 days of incubation. For isolated HAs, the following analyses were performed: elemental composition, UV–Vis and IR spectra, susceptibility to oxidation, and hydrophilic (HIL) and hydrophobic (HOB) properties were determined using high-performance liquid chromatography.

Results and discussion

The research demonstrated that introducing anthracene, fluorene, pyrene and chrysene to soil samples resulted in a change in some of the quality parameters of humic acids. However, the intensity and the direction of those changes were determined by soil properties. The changes of the parameters, once PAHs were introduced, that did not depend on the soil properties were ΔA _665u and ΔA _465u (susceptibility to oxidation at wavelengths of 465 and 665 nm) as well as HIL/ΣHOB. The same tendency in changes in the structure of humic acids, once PAHs were introduced, was also observed based on the Fourier transform infrared spectra pattern.

Conclusions

A single pollution of soils with PAHs that leads to changes in the quality parameters of humic acids shows that, as for the soils permanently exposed to pollution with those compounds, significant changes can occur in the properties of humic acids. As a result, it can lead to a change in the functions played by humic acids in the environment.     

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.     

Chemical studies on soil humic acids

Abstract

Relationships between elementary compositions and types of humic acids or the "degrees of humification were studied statistically using 39 humic acids prepared from various types of soils. Mean values of C%. H%. N%, 0%, H/C, N/C, and O/C of the different types of humic acids (A. B. Rp(l), Rp(2). and Po) were compared. The elementary compositions of various types of humic acids were proven by variance analyses to be significantly distinct. Linear regression analyses of C%, H%, N%. 0%, H/C, N/C, or O/C on RF or AlogK values were also carried out on 35 humic acids excluding the P_+~+++ type humic acids. The linear associations were found to be significant between C%-RF (5% level), H%-RF, H%-?logK, H/C-RF and HIC-?logK (0.1% level), N/C-RF(O.1% level). N%-RF(l% level), N%-?logK N/C-?logK(5% level), and ?logK (1% level), while no significant relationship was found with regard to C%;-?logK, 0%-?logK (both 5% level) and O/C-RF and O/C-?logK (l0% level). Carbon and oxygen contents of humic acids may be apt to reflect the different conditions of soils. The deeper the visible light absorption of humic acids and so the higher the degrees of humification, the lower the hydrogen contents of humic acids were. Though nitrogen content showed a trend similar to the trend of hydrogen content against RF values as a whole. the nitrogen content of less humified humic acids (Rp type) varied from very low to very high values. suggesting the enrichment of nitrogen into humic acid molecules in the early stage of humification. In the H/C versus O/C diagram, humic acids occupied an area reserved for the oxydation products of lignins. The area was fairly wide and “J”-shaped, that is, in the early to the middle stage of humification humic acids were arranged in the direction of dehydrogenation or demethanation, and in the later stage they were arranged in the direction of dehydration.     

Comparison of humic acids derived from city refuse with more developed humic acids

Abstract

The humic acids (HA) from composted and uncomposted city refuse (CR) were characterized by degradative (oxidation with persulphate and permanganate) and non-degradative techniques (FT-IR and ¹³C-NMR) in order to analyze the effect of the composting process on these HA. They were also compared with commercial HA extracted from leonardite. The carboxyl and carbonyl group content of the HA from CR increased slightly during composting. Since the HA from the composted CR showed a lower N and H content, the FT-IR spectra showed a lower intensity in the bands corresponding to peptides and carbohydrates. Differences were revealed when the HA from both CR were compared with those from leonardite which showed a much lower N and H content and a less aliphatic character. The percentage of degraded products by persulphate was higher for the HA from uncomposted CR. For the HA from both CR the major components among the oxidation products were dicarboxylic acids and normal fatty acids. In the leonardite HA, the major components consisted of benzene polycarboxylic acids. ¹³C-NMR revealed an attenuation of the aliphatic character of the HA from CR with composting.     

Effects of humic acids on the growth of bacteria

The influence of humic acids of different origins on the growth of bacterial cultures of different taxa isolated from the soil and the digestive tracts of earthworms (Aporrectodea caliginosa)—habitats with contrasting conditions—was studied. More than half of the soil and intestinal isolates from the 170 tested strains grew on the humic acid of brown coal as the only carbon source. The specific growth rate of the bacteria isolated from the intestines of the earthworms was higher than that of the soil bacteria. The use of humic acids by intestinal bacteria confirms the possibility of symbiotic digestion by earthworms with the participation of bacterial symbionts. Humic acids at a concentration of 0.1 g/l stimulated the growth of the soil and intestinal bacteria strains (66 strains out of 161) on Czapek’s medium with glucose (1 g/l), probably, acting as a regulator of the cell metabolism. On the medium with the humic acid, the intestinal bacteria grew faster than the soil isolates did. The most active growth of the intestinal isolates was observed by Paenibacillus sp., Pseudomonas putida, Delftia acidovorans, Microbacterium terregens, and Aeromonas sp.; among the soil ones were the representatives of the Pseudomonas genus. A response of the bacteria to the influence of humic acids was shown at the strain level using the example of Pseudomonas representatives. The Flexom humin preparation stimulated the growth of the hydrocarbon-oxidizing Acinetobacter sp. bacteria. This effect can be used for creating a new compound with the elevated activity of bacteria that are destroyers of oil and oil products.     

Fate of humic acids isolated from natural humic substances

Composition of humic acids (HA) is a function of plant-derived inputs, degradation processes regulated by microorganisms, organo-mineral interactions and age. Characterization of different origin humic substances is important for evaluation of their contribution to stabile and labile carbon pool in the environment. The relative abundance of chemical components in HA isolated from soils, compost, commercial lignohumates, alginite, acadiane and lignite was studied with aim to quantify content of important biomarkers such as amino acid, lipids and polyphenols. HA were considered as a heterogeneous complex and high concentration of peptides, polyphenols and lipids was determined in acadian-HA to compare with soil-HA. Compost-HA contained much more amino acids to compare with soil-HA samples. Alginite-HA and lignite-HA were similar in biomarkers content to soil-HA. Fourier transform infrared spectroscopy confirmed that chemical composition and functional groups content differs with the origin, humification degree and the age of studied samples. Soil-HA are typically composed of a variety of ?OH, COOH?, C–O, C–H₂, (aliphatic and aromatic) groups, quinines, lignin fragments, polysaccharide, monosaccharide and proteins fragments, which are linked together by ?O?, ?NH?, ?H=, >C=O, metal ions and –S? groups. ¹³C NMR spectroscopy showed that aromatic carbon content was the highest in lignite-HA and soil-HA.     

Molecular parameters of humic acids

Several molecular parameters of humic acids isolated from the soils of the most important genetic types are discussed.The material obtained makes it possible to establish limiting values of the molecular weights of humic acids and indicate the tendencies in their changes.The selected parameters characterise the submolecular, molecular and supramolecular levels of the organisation of humic acids. It has been shown that the most important indices include the molecular-weight distribution, molecular weight and the molecular form of humic acids.     

Several properties of humic acids

In the preceding paper¹), it was shown that the elementary composition of soil humic acid changed regularly with the progress of humification. Afterwards, the author conducted several experiments on the base exchange capacity, the content of hydrolysable nitrogen, the resistance to oxidizing reagent, the electrolyte coagulation and X-ray analysis of various humic acids. These experimental resu1ts will be given in this report.     

Elementary composition of humic acids

The author made a study of the elementary composition of various humic acids; L and SrL humic acids used in the previous report¹⁾, and two lowmoor humic acids.     

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.