Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy

Summary Humic acids from two Brazilian topsoils under savanna grassland and five soil fungal melanins were characterized by elemental, functional group and infrared analysis. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for several other fungal melanins and soil humic acids. Compared with the soil humic acids, the infrared spectra of the fungal melanins showed greater detail, indicative of higher aliphaticity. They were similar to the type III infrared spectra of humic acids, which are characteristically high in proteinaceous material and polysaccharides. The infrared spectra of the humic acids from the two Brazilian soils studied were classified as type I, which includes most soil humic acids. Notwithstanding the greater detail, in some areas the fungal melanin spectra were similar to those reported for other fungal melanins and humic acids of different origins. The probable contribution of the melanic fungi to the formation of soil humic polymers is discussed.     

Neutral sugars in melanins synthesized by actinomycetes from Brazilian soils

 Nine actinomycete melanins synthesized under various culture conditions, eight of them by actinomycete samples isolated from Brazilian topsoils under savanna (cerrado) vegetation and one from an ATCC sample, were subjected to a two-step hydrolysis procedure and the sugars released qualitatively and quantitatively determined by capillary gas-liquid chromatography (GLC). Humic acids (HAs) extracted from these soils, analysed previously, were used for comparison. The neutral sugars glucose, galactose, mannose, xylose, arabinose, ribose, rhamnose and fucose and the alcohol sugar inositol were present in varying amounts in most of the melanins analysed. The same sugars were present in the HAs used for comparison, except for ribose. Some qualitative and quantitative differences observed in the two types of macromolecules would be expected, considering their origins. The results indicate that the actinomycete melanins have a qualitative sugar distribution pattern similar to that of the HAs from Brazilian tropical soils and of HAs reported for soils from other climatic regions. The possible participation of actinomycete melanins in the formation of soil humic substances is discussed. Received: 4 April 1997     

Comparative studies of pyrolysis-mass spectra of melanins,model phenolic polymers,and humic acids

Curie-point pyrolysis in direct combination with a fast-scanning, quadrupole mass-spectrometer was used for the characterization of humic acids from soils, peats, and composted straw, fungal humic acid-type polymers (melanins), model phenolic polymers and lignins. All the humic acids, most of the fungal melanins and the model polymer with linked peptone yielded complex but highly similar spectra. Prominent homologous ion series were: m/e 34, 48 and 62 (“sulfides”); m/e 67, 81 and 95 (“pyrroles”); m/e 78, 92 and 106 (“benzenes”); m/e 94, 108 and 122 (“phenols”); and m/e 117, 131 and 145 (“indoles”). “Humic acid” fractions from fresh straw and straw composted for various periods of time yielded relatively strong but gradually diminishing lignin series peaks at m/e 94, 108, 120 and 122 (phenols); m/e 124, 138, 150 and 164 (methoxyphenols); and at m/e 154 and 168 (dimethoxyphenols). Weak signals for the methoxy and dimethoxyphenol series were also present in the soil humic acid spectra. In addition to the series peaks for the other fungal melanins, the S. chartarum spectrum showed a pronounced ion series at m/e 68, 82, 86 and 96 which is indicative of polysaccharides and at m/e 136, 150 and 164 which suggests the presence of alkylphenols.     

Electron paramagnetic resonance and ultraviolet-visible spectroscopic evidence for copper porphyrin in actinomycete melanins

The coordination chemistry of iron (III) (Fe³⁺) and copper (II) (Cu²⁺) in melanins synthesized by seven actinomycetes isolated from Brazilian latosol soils under savanna (cerrado) vegetation was investigated using electron paramagnetic resonance (EPR) and ultraviolet-visible (UV-Vis) spectroscopy. The EPR spectra indicated the presence of Cu²⁺ ions bound to oxygenated and nitrogenous functional groups, and rhombic coordinated Fe³⁺ ions. In some of the actinomycete melanins the EPR hyperfine splitting of Cu⁺² ion complexes was well resolved, and indicated four magnetically equivalent nitrogen atoms in a plane. This result suggested the presence of Cu⁺² porphyrin complexes, which was confirmed by Soret bands in the 400-nm region of the UV-Vis spectra. The concentration of Mn in actinomycete melanins, determined by inductively coupled plasma atomic emission spectroscopy, was lower than those of Cu and Fe. Received: 9 April 1997     

Elemental,functional infrared and free radical characterization of humic acid-type fungal polymers (melanins)

Summary Humic acid-type polymers (melanins) synthesized in culture media by the fungi Aspergillus glaucus, Eurotium echinulatum, Hendersonula toruloidea, Stachybotrys atra and Aspergillus sydowi were analysed for elemental composition, functional group content, infrared (IR) and electron spin resonance (ESR) properties. Results were discussed in comparison with range values referred for soil humic acids. The fungal polymers showed significant differences in carboxyl and nitrogen content and C/H atomic ratios, reflecting a different degree of condensation (aromaticity) among the various samples. IR analysis gave evidence of: (a) the predominant aromatic character of melanins from A. glaucus, E. echinulatum and H. toruloidea; (b) the high content of aliphatic and olefinic components of S. atra melanin; (c) the typical presence of amide bonds in the nitrogen-richest melanins from A. sydowi and H. toruloidea; and (d) the generally low amount of free carboxyl groups, which often appeared involved in hydrogen bonds. ESR spectra showed that all the melanins studied contained appreciable concentrations of organic free radicals of prevailing semiquinonic nature and of the same order of magnitude commonly measured in humic acids from soil and other sources. The free electron concentration was shown to be directly related to the C/H atomic ratio and to the degree of aromaticity shown by IR analysis. This indicated that the highest free radical content in the melanins from E. echinulatum and A. glaucus was associated with the highest presence of condensed aromatic structures. Humic acid-type polymers synthesized by soil fungi may, therefore, contribute to the total free radical content of soil humic substances and play important roles in all reactions involving free radicals in soils and related environments.     

紫色水稻土胡敏酸的形成——稻草腐解试验

土壤腐植酸类物质的形成是土壤固碳的重要过程,但对腐植酸类物质形成过程的了解仍不甚清楚,为了丰富土壤腐植酸类物质形成理论,采用富立叶变换红外光谱和固态交叉极化-魔角旋转^13C-核磁共振光谱技术分析了紫色水稻土稻草腐解过程中胡敏酸的波谱学特征。结果表明,稻草腐解的前期,胡敏酸的红外光谱所有吸收峰（3364、2933、1653、1599、1508、1461、1421、1331、1225、1126、1033cm^-1）强度皆有明显减弱,核磁共振光谱的烷基、多羟基和芳基的共振峰明显减弱且甲氧基的共振峰显著增强,即表明提取的胡敏酸为类胡敏酸的木质素;随着腐解的进行,胡敏酸的红外光谱的吸收峰强度皆显著增强,核磁共振光谱的烷基、芳基和羰基的共振峰增强,即表明此时的胡敏酸已是以木质素残体为核心并结合烷基、酰胺以及糖类物质反应形成的高分子聚合体;稻草腐解的后期,胡敏酸的红外光谱的2933cm^-1处的吸收峰强度减弱,1651、1599、1508、1461、1422和1224cm^-1处的吸收峰小幅增强,核磁共振光谱的烷基共振峰减弱,甲氧基共振峰增强,表明此时的胡敏酸发生脱烷基（主要是甲基）过程。因此,红外光谱吸收峰强度与核磁共振光谱共振峰强度的规律性变化反映了稻草腐解过程紫色水稻土胡敏酸的形成过程具有阶段性,紫色水稻±胡敏酸的形成过程符合木质素学说。     

Effect of low-molecular-weight organic acids on Cl- adsorption by variable charge soils

Low-molecular-weight (LMW) organic acids exist widely in soils and have been implicated in many soil processes.The objective of the present paper was to evaluate effect of two LMW organic acids, citric acid and oxalic acid, on Cl^- adsorption by three variable charge soils, a latosol, a lateritic red soil and a red soil, using a batch method. The results showed that the presence of citric acid and oxalic acid led to a decrease in Cl- adsorption with larger decreases for citric acid. Among the different soils Cl- adsorption in the lateritic red soil and the red soil was more affected by both the LMW organic acids than that in the latosol.     

几种有机酸对可变电荷和恒电荷土壤吸附镉的影响

The objectives of this study were to illustrate the reaction processes, to identify and quantify the precipitates formed, and to estimate the porosity losses in order to eliminate drawbacks during remediating monochlorobenzene （MCB） and trichloroethylene （TCE）-contaminated aquifers using the ORC-GAC-Fe^0-CaCO3 system. The system consisted of four columns （112 cm long and 10 cm in diameter） with oxygen-releasing compound （ORC）, granular activated carbon （GAC）, zero-valent iron （Fe^0）, and calcite used sequentially as the reactive media. The concentrations of MCB in the GAC column effluent and TCE in the Fe^0 column effluent were below the detection limit. However, the concentrations of MCB and TCE in the final calcite column exceeded the maximum contaminant level （MCL） under the Safe Drinking Water Act of the US Environmental Protection Agency （US EPA） that protects human health and environment. These results suggested that partitioning of MCB and TCE into the gas phase could occur, and also that transportation of volatile organic pollutants in the gas phase was important. Three main precipitates formed in the ORC-GAC-Fe^0-CaCO3 system： CaCO3 in the ORC column along with Fe（OH）2 and FeCO3 in the Fe^0 column. The total porosity losses caused by mineral precipitation corresponded to about 0.24% porosity in the ORC column, and 1% in the Fe^0 column. The most important cause of porosity losses was anaerobic corrosion of iron. The porosity losses caused by gas because of the production and entrapment of oxygen in the ORC column and hydrogen in the Fe^0 column should not be ignored. Volatilization, precipitation and porosity losses were considered to be the main drawbacks of the ORC-GAC-Fe^0-CaCO3 system in remediating the MCB and TCE-contaminated aquifers. Thus, measurements such as using a suitable oxygen-releasing compound, weakening the increase in pH using a buffer material such as soil, stimulating biodegradation rates and minimizing the plugging caused by the relatively high dissolved oxygen levels should be taken to eliminate the drawbacks and to improve the efficiency of the ORC-GAC-Fe^0-CaCO3 system.     

七种钾释放动力学方程的比较和动力学参数的应用

Batch equilibrium experiments were conducted to investigate cadmium （Cd） sorption by two permanent-charge soils, a yellow-cinnamon soil and a yellow-brown soil, and two variable-charge soils, a red soil and a latosol, with addition of selected organic acids （acetate, tartrate, and citrate）. Results showed that with an increase in acetate concentrations from 0 to 3.0 mmol L^-1, Cd sorption percentage by the yellow-cinnamon soil, the yellow-brown soil, and the latosol decreased. The sorption percentage of Cd by the yellow-clnnamon soil and generally the yellow-brown soil （permanent-charge soils） decreased with an increase in tartrate concentration, but increased at low tartrate concentrations for the red soil and the latosol. Curves of percentage of Cd sorption for citrate were similar to those for tartrate. For the variable-charge soils with tartrate and citrate, there were obvious peaks in Cd sorption percentage. These peaks, where organic acids had maximum influence, changed with soil type, and were at a higher organic acid concentration for the variable-charge soils than for the permanent charge soils. Addition of cadmium after tartrate adsorption resulted in higher sorption increase for the varlable-charge soils than permanent-charge soils. When tartrate and Cd solution were added together, sorption of Cd decreased with tartrate concentration for the yellow-brown soil, but increased at low tartrate concentrations and then decreased with tartrate concentration for the red soil and the latosol.     

Chromatographic Separation of Green Humic Acid from Podzol Humus

One of the authors, Kumada ¹⁾ has found that absorption spectra of podzol humic acids, remarkably different from the other soil humic acids, have fairly distinct absorption bands near 620, 570 and 450 mµ, and he tentatively named them. P type humic acid. P type humic acid seems specific not only in its visible absorption spectra, but also in infra. red absorption spectra, in susceptible to electrolytes, etc. The authors are now engaged in the study of the nature and properties of P type humic acid, and recently they have found that it is composed of at least two main fractions, the one is reddish brown, and the other green, in alkaline solution. The experimental results are briefly presented.     

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 EFFECT OF LIGHT ON THE ESR SPECTRA OF HUMIC ACIDS

Photo-induced Electron Spin Resonance signals have been detected in basic aqueous solutions of humic acids from soil and from synthetic and commercial sources. Parameters of these signals under atmospheres of air, O₂ and N₂ were the same as or very similar to those of the already known ‘dark’ stable ESR-signals of humic acids and melanins. The maximum increase of signal amplitude under the influence of 395–780 nm wavelength irradiation was one order of magnitude. The influence of microwave power on ESR-signal amplitude was examined under different atmospheres. It has been found that oxygen strongly diminishes the saturability of signals. This indicates that oxygen has an influence upon relaxation properties of paramagnetic centres of humic acid. Photoinduced ESR signal is a newly discovered feature of humic acids, similar to that of melanin and may be expected to affect physico-chemical processes occurring in soils.     

Effects of mineral and monocarboxylic acids on the molecular association of dissolved humic substances

The conformational structure of dissolved humic substances is an important property that controls the reactivity of humus in the soil solution. High performance size-exclusion chromatography was used here to study the changes in molecular size of different humic substances brought about by addition of mineral (HCl) and monocarboxylic (formic, acetic, propionic, and butyric) acids. The CPMAS-NMR spectra showed that humic substances had varying chemical composition and that the ratio of hydrophilic to hydrophobic carbon (HI/HB) was greater for a humic acid from soil than for ones from oxidized coal and lignite. All humic substances showed a decrease in UV absorbance of chromatographic peaks when treated with either HCl or monocarboxylic acids. This was due to the hypochromic effect by which the absorptivity of associated molecules is decreased when they are separated. We attributed the molecular separation upon acid treatment to the formation of intermolecular hydrogen bonding that alters the original conformation stabilized mainly by weaker hydrophobic interactions. Addition of organic acids not only further decreased peak absorbances of humic acids but also caused their shift to larger elution volumes, indicating a larger conformational disruption than with HCl. The extent of the molecular size changes showed a relation to the number of carbons of monocarboxylic acids and to the HI/HB ratios of humic materials. The larger the carbon content of organic acids and the smaller the HI/HB ratio of humic materials, the larger was the decrease of the average molecular size of humic acids. These results suggest that dissolved humic substances associate predominantly by hydrophobic forces and that the apolar components of humic substances largely control their aggregation and reactivity in the environment.     

ELECTRON-SPIN RESONANCE OF HUMIC ACIDS FROM CULTIVATED SOILS

An examination has been made of the electron-spin resonance spectra of humic acids from (i) two groups of cultivated soils from the north-east of Scotland, and the related natural soils from which the cultivated soils are considered to be derived, and (ii) some Rothamsted soils. With the Scottish soils the humic acids from the more acid natural soils (pH <4.6) show e.s.r. spectra with a four-peak structure (class I), and those of the less acid soils (pH >4.6) almost structureless spectra (class II). The cultivated soils contain humic acids with the same class of spectrum as that of the soils from which they are derived, even though they are now much less acid (_PH 5.9 –6.5). Humic acids from Rothamsted soils, sampled recently, were compared with those from the same site sampled 80 years previously. Where the soil has been allowed to revert towards a natural state but still contains free calcium carbonate the pH remained high (7.8–7.9) and the spectrum of the humic acid remains class II. Where the soil contained no free calcium carbonate the pH has fallen from 7.1 to 4.5 on reversion and the spectrum class of the humic acid has changed from II to I. The results support the hypothesis that the class of spectrum of the humic acid depends on the pH at which it is formed.     

Effect of organic amendment on amount and chemical characteristics of humic acids in upland field soils

To assess the effect of continuous organic material (OM) application on soil humic acids, the amount and chemical characteristics of humic acids in various types of soils (n = 10) were compared between plots treated with farmyard manure (FYM) or rice straw compost (RSC) plus chemical fertilizer (CF) and plots treated with CF alone. The degree of humification (degree of darkening), molecular size distribution and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectra of humic acids from CF‐treated soils showed wide variation among the soils. Humic acid content was generally larger in OM + CF soils than in corresponding CF soils, and the stable C isotopic ratio suggested partial replacement of indigenous humic acids with OM‐derived ones even where no apparent increase in humic acid content was observed. The rate of OM application and the indigenous humic acid content were related positively and negatively, respectively, to the apparent accumulation rate of humic acids among soils. The degree of humification of humic acids was generally smaller in OM + CF soils than in CF soils. Humic acids extracted from FYM and RSC exhibited chemical characteristics typical of humic acids having a smaller degree of humification, which suggested the contribution of OM‐derived humic acids to the differences between OM + CF and CF soil humic acids, such as larger average molecular sizes and smaller and larger proportions of aromatic C and O‐alkyl C, respectively, relative to total C in the OM + CF soil humic acids. Little change was observed in the chemical characteristics of humic acids when the degree of humification of indigenous humic acids was small. The effect of OM application on the chemical characteristics of humic acids was most conspicuous in soils containing humic acids having an intermediate degree of humification, possibly resulting from the combination of accelerated degradation of indigenous humic acids and the accumulation of OM‐derived humic acids.     

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.     

Fe2+与胡敏酸的络合特征及其抗氧化性和生物有效性研究

在研究不同来源胡敏酸性质的基础上,研究了Fe2+与胡敏酸的络合特征以及络合态Fe2+的抗氧化性和生物有效性。结果表明,来源于土的胡敏酸属于A型,而来源于腐解秸秆、粪肥的胡敏酸属于RP型。A型胡敏酸与Fe2+络合能力大于RP型。HA-Fe2+络合物的络合稳定常数越大,络合物中Fe2+抗氧化性越强,玉米叶片中全铁含量也越高,但存在活性铁含量和全铁含量不一致现象。     

Multivariate statistical analysis of mass spectra as a tool for the classification of the main humic substances according to their structural and conformational features

The aim of this work is to explore the suitability of the complementary use of mass spectra and the corresponding statistical analysis (principal components-Pareto analysis (PCA) and discriminant analysis (DA)) of these spectra to differentiate diverse humic samples as a function of their structural and conformational features. To this end, the mass spectra of humic samples belonging to the main humic fraction types (gray humic acid, brown humic acid, and fulvic acid) were obtained by electrospray ionization mass spectrometry (ESI-MS). The results obtained showed that the application of PCA yielded a clear separation between blanks and humic samples. However, a clear differentiation among the humic fraction types was not achieved. The DA of PCA data, however, yielded a clear separation among the humic substances (HS) samples belonging to each HS fraction type considered: gray humic acids, brown humic acids, and fulvic acids. These results showed that the mass spectra of each humic sample include characteristic mass/charge (m/z) distribution values that can be considered as a "fingerprint" representative of its specific structural features. Our results also indicate that, although the m/z values principally corresponded to single-charged ions, we cannot identify these molecular weight distributions with those of humic samples, since sample molecular fragmentation, as well as partial molecular ionization, cannot be ruled out under our experimental and instrumental conditions.     

Comparisons of IR-spectra from melanins of microscopic soil fungi,humic acids and model phenol polymers

Microscopic soil fungi, such as Epicoccum nigrum, Stachybotrys chartarum, Hendersonula toruloidea and others when cultivated on glucose form high molecular weight melanins in the culture media and in the cells. The melanins resemble humic acids with respect to certain chemical properties and the IR-spectra also indicate similarities in functional groups. However, bands at 2900, 1460 and 1380 cm^?1 suggest a higher content of aliphatic CH-components, and a pronounced shoulder at 1540 cm^?1 suggests peptide-like material. A shoulder at 1720 cm^?1 and a broad band at 1630–1650 cm^?1 form evidence of fewer free carboxyl groups than in most soil humic-acid preparations. Upon hydrolysis with 6N HCl methylation or a short heat treatment, distinct adsorptions at 1720 and in the 1600 cm^?1 region appeared in the residues, indicating C=O vibrations of free carboxyl groups and aromatic C=C vibration. Comparisons with the variability of the carboxyl band in model polymers from phenols of various structures and prepared either by phenolase or autoxidative polymerization in the presence or absence of amino-acid compounds, suggested that the carboxyl group in fungal melanins may be bonded by H-bridges which shift the C=O absorption into lower frequences.Addition of clay minerals to the culture media of fungi accelerated or increased biomass and melanin formation. Comparison of band locations and shape of the spectra from the melanins with or without clays indicated that no basic alteration occurred in the composition of the melanins from clay culture. However, a higher ash content and the presence of specific bands from the added clays, together with sharpening and a shifting of the broad 1650 cm^?1 band from the controls into higher frequencies, suggest chemical bonding of the melanins with clays through participation of the carboxyl groups. The model polymers from specific mixtures of phenols together with peptones and prepared either by oxidation with phenolase or by autoxidation, closely resembled the fungal melanins. This represents strong evidence for a similarity in the structural units and their linkages in both the melanins and the model polymers. The similarity of fungal melanins to some humic acids from recent lake and marine sediments and soil humic-acid fractions is discussed.     

Über die Hemmung der Aminopeptidase K (aus Tritirachium album Limber) durch Huminstoffe

The Inhibition of Aminopeptidase K (from Tritirachium album Limber) by Humic Acids Interactions between Aminopeptidase K (from Tritirachium album Limber) and thirteen humic acids were examined. Ten of the humic acids were extracted from different types of soil; three were preparations based on hydroquinone, two of these were chemically modified by methylation and acetylation. All of the humic acids were found to cause complete inhibition of the enzyme, with no significant difference between the effects of natural and synthetic humic acids. The reaction constants showed the type of inhibition to be noncompetitive. The inhibited enzyme could be partially reactivated by the addition of zinc. It is assumed that zinc causes enlargement of the humic acid particles and thus a decrease in the number of inhibiting particles. An interesting relation between the average particle weight and the number of humic acid particles could be pointed out. With a molecular weight smaller than 10000, an increasing number of humic acid particles is necessary for affecting the activity of Aminopeptidase K.