Amino sugars in fungal melanins and soil humic acids

Humic acids from six Brazilian topsoils (three Latosols and three Podzols) and five soil fungal melanins were hydrolysed and the released amino sugars qualitatively and quantitatively determined by capillary gas-liquid chromatography. Melanins were obtained from liquid culture media, synthesized with nitrate or asparagine as a source of N, of Stachybotrys atra, Aspergillus glaucus, Eurotium echinulatum and Hendersonula toruloidea. Glucosamine (48–60%), galactosamine (39–52%), and traces of mannosamine were detected in all humic acids. The total amino sugar content ranged from 1351 to 2287 mg kg^?1. In the fungal melanins analysed, mostly glucosamine (80–99%) and trace to small amounts of galactosamine and mannosamine (<10%) were found in amounts ranging from 192 to 635 mg kg^?1. The Occurrence of mannosamine in fungal melanins and fungal polysaccharides had not previously been found. The present study gives additional data to the theory that fungal melanins may play a role, as precursors, in the formation of soil organic matter.     

Effect of fungal humic-like polymers and their phenolic units on auxin destruction

Humic-like substances synthesized by Aspergillus sydowi, Epicoccum nigrum, Hendersonula toruloidea and Stachyhotrys atra inhibited indoleacetic acid (IAA) destruction by lentil roots extracts. The fungus polymers were similar to soil humic acids with respect to the effect on IAA-oxidase activity. Model polymers prepared using pure phenols reduced the IAA oxidation too. The relationships between the effect of fungal substances on IAA-oxidase activity and their phenolic units content are briefly discussed.     

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.     

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.     

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.     

腐殖酸-锌络合物稳定性的研究

腐殖酸-金属络合物在土壤形成和植物营养等方面具有重要的作用。六十年代以来,这方面的研究日益增多。锌是植物所需的微量元素之一,在土壤中常以络合态存在。因此,以络合态存在的锌的数量及其稳定性对土壤的供锌能力将有较大的影响。不少研究者虽曾研究了腐殖酸一锌的稳定性^[4,6,7,10],但所测得的稳定常数值差异甚大,有的可差几个甚至高达十个数量级。导致这种差异的原因很多,其中之一是各研究者所用的腐殖酸各人不同。一些研究者认为络合物的log K值因供试腐殖酸的腐殖化程度而异,另一些研究者认为log K值因腐殖酸分子大小不同而不同。本工作测定了不同来源的腐殖酸的某些性质及其与锌形成的络合物的稳定性,试图对它们之间的关系作一初步探讨。     

Elemental,functional group and infrared spectroscopic analysis of actinomycete melanins from brazilian soils

Eleven actinomycete melanins were characterized by elemental and functional group and infrared analysis. A soil humic acid from a Brazilian topsoil, a darkred latosol under savanna grassland, analysed previously, was used for comparative purposes. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for soil humic acids and fungal melanins. Compared to the soil humic acid, the actinomycete melanins showed greater detail, indicative of higher aliphaticity. Most of these were, in varying degrees, similar to the type III IR spectra of humic acids, which are characteristically high in proteinaceous material, and with variable amounts of polysaccharides. The exceptions were two melanin spectra that showed more resemblance to the humic acid from the dark-red latosol, which belongs to the type I spectra of soil humic acids, a category that includes most soil humic acids. The probable participation of melanic actinomycetes in the formation of humic polymers in discussed.     

Characterization of lake sediment humic acids from the Ćelije Lake system near Kruševac (Central Serbia) by13C and 1H solution NMR and 13C cpmas NMR

Humic acids were extracted from the bottom sediment of the three lakes (Zlatari, Vasici and Vodozahvat) that together comprise Lake ?elije a regional drinking water reservoir. The humic acids were examined by high resolution ¹³C and ¹H solution NMR and ¹³C CPMAS. The aromaticity expressed as the number of benzene rings per 1000 g of humic acid is 1.8 for Zlatari, 2.3 for Vasici and 1.9 for Vodozahvat. Oxygen-substituted aromatic carbon structures in humic acids from the first two lakes (Zlatari and Vasici) are similar and resemble p-coumaryl units in lignin while those in Lake Vodozahvat more closely resemble coniferyl or sinapyl units in lignin. The aromatic rings of all three humic acids are highly substituted. Humic acids from the first two lakes have very similar carbohydrate carbon contents (19.9 and 19.5% respectively) compared to 23.3% for Vodozahvat humic acid. Zlatari and Vodozahvat humic acids have a higher alkyl chain content which indicates a greater potential for forming hydrophobic cavities. The carboxyl and aromatic OH group content of each humic acid influences their capacity for forming complexes with metal ions.     

我国几种土壤中腐殖质性质的研究

研究了几种土壤腐殖酸的基本性质,其中包括土壤的腐殖质组成,胡敏酸以及富里酸的元素组成,含氧官能团含量,光密度值与芳化度等,计算了E₄与E₄/E₆比值与上述性质的相关系数。结果表明土壤腐殖质组成和性质与其形成条件有着密切的联系,并且有一定的地带规律性。水稻土中有机质含量增加,HA/FA比值变高,其活性HA显著降低,说明水稻土有利于有机质的积累并改变了腐殖质的组成。E₄、E₄/E₆比值与元素组成,C/H比值,含氧官能团及芳化度之间的相关系数表明E₄除了与C%,O%,酚羟基-O%,醌基-O%呈极显著或显著相关外,还与C/H比值及芳化度呈极显著相关,因此E₄值可作为腐殖物质芳化度的一个指标。     

黄土区土壤腐殖物质的化学性质及其与成土条件的关系

土壤腐殖质是土壤发生、发育的结果,是土壤区别于成土母质的主要标志。特定的生物气候条件(以及其它成土条件)形成特定的土壤类型,也形成具有特定性质的土壤腐殖质。因此,研究土壤中腐殖物质的组成、特性可以在一定程度上估计土壤生物活动的强弱与成土过程的趋向,从而为土壤分类提供重要的依据。     

Humus quantity and quality of an entic Haplustoll under different soil‐crop management systems

Abstract

The effects of different management systems on the level and composition of humified organic matter in an entic Haplustoll from the semiarid Pampean region were studied. The systems were: TPc, wheat‐mixed pasture; TV, wheat (Triticum aestivum), oat (Avena sativa), corn (Zea mays) and triticale grasses; TP, wheat‐cattle grazing; and V, virgin, non cultivated. Humic acids were extracted, fractionated, and analyzed for their organic carbon (OC) content, elemental composition, and E4:E6 spectral ratios. The infrared (IR), electron spin resonance (ESR). and ¹³C‐NMR spectra were registered on these humic acids. The TP rotation showed the lowest humic acid‐carbon to fulvic acid‐carbon (HA‐C:FA‐C) ratio. The lower O:C ratio of humic acids from the cropped soils indicates a higher level of oxidation than that of the virgin one. The comparison of the different methodologies allowed us to conclude that crop rotations and conservation tillage were adequate to mantain the level and composition of the soil organic matter and humus which affected the soil fertility and level of productivity     

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.     

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.     

Humic acid applications to lettuce do not improve yield but do improve phosphorus availability

The effects of humic acid (HA) and phosphorus (P) applications on plant growth and nutrient content of lettuce, together with available soil phosphorus remaining after harvest were examined. The amounts of phosphorus used were 0, 120, and 240 kg ha^?1 and those of humic acid were 0, 100, 200, and 300 kg ha^?1. Phosphorus increased the nitrogen content of lettuce significantly (P<0.01) while the application of humic acid did not have significant effect. Humic acid, phosphorus, and HA×P interaction increased the nitrogen content of lettuce significantly (P<0.05). All effects were not significant for plant K, Ca, Mg, Fe, Cu, and Mn contents. Application of phosphorus decreased Zn content significantly (P<0.05). Increased amount of phosphorus caused significant increases in residual soil phosphorus (P<0.01). It was concluded that the application of 120 kg ha^?1 of phosphorus together with 300 kg ha^?1 of humic acid was convenient for the head weight of lettuce.     

Soil organic matter beyond molecular structure Part II: Amorphous nature and physical aging

Glassy, rubbery, and crystalline phases are representatives of supramolecular structures which strongly differ in order, density, and other characteristics. In this contribution, the amorphous nature of soil organic matter (SOM) is reviewed with respect to the glassy/rubbery model, glass transition mechanisms, interactions of SOM with water, and physical aging. Glass‐transition behavior and physical aging are inherent properties of amorphous solids, and numerous spectroscopic investigations give insights into different domain mobilities of humic substances (HS). The correlation between sorption nonlinearity and glassiness of polymers and HS supports a relation between sorption and amorphicity in Aldrich humic acid. Further evidence is still required for the transfer to soil HS and SOM. Sorption and differential scanning calorimetry (DSC) data suggest a correlation between aromaticity and glassiness in HS, and the available data do currently not allow to decide unambiguously between specific sorption and hole filling as explanation. This needs to be verified in future research. Although parts of the investigations have up to now only been conducted with humic substances, the collectivity of available data give strong support for the glassy/rubbery conception of SOM. They clearly indicate that amorphous characteristics cannot be excluded in SOM. This is further supported by the observation of different types of glass‐transition behavior in samples of whole humous soil. In addition to classical glass transitions in water‐free soil samples, water surprisingly acts in an antagonistic way as short‐term plasticizer and long‐term antiplasticizer in a second, nonclassical transition type. Latter is closely connected with physico‐chemical interactions with water and suggests water bridges between structural elements of SOM (HBCL‐model). The gradual increase of T_g* in SOM indicates physico‐chemical aging processes, which are not restricted to polymers. They may be responsible for contaminant aging, changes in surface properties and increased soil compaction in agricultural soils.     

Effect of cultivated plants on physico‐chemical characteristics of humic substances of soils amended by sewage sludge

Abstract

Humic substances were extracted from raw sewage sludge samples as well as an alluvial slightly alkaline soil [Typic Xerofluvent (So)], a clay loam soil [Calcixerollic Xerochrept (M)] and the corresponding field plots amended with different rates of sewage sludge and cultivated with corn and cotton respectively, in a two‐year field experiment. These substances have been characterized by chemical and spectroscopic methods. The chemical analyses showed that humic acids (HAs) and fulvic acids (FAs), were effected by the cultivated plant. Humic substances extracted from field plots with cotton showed higher carbon, but less nitrogen (N), total acidity and carboxylic groups content, as compared with those under corn. Generally HAs and FAs extracted from the fields plots amended with sewage sludge had higher N content, lower values of total acidity, carboxylic groups and carbon contents than those from the unamended soils. The FT‐IR spectra of humic (HAs) and fulvic (FAs) acids extracted from sewage sludge indicated the presence of high percentage of aliphatic carbon, polysaccharides and proteinaceous materials. The spectra of the humic matter in the field plots showed less aliphatic but increased amide stretch in comparison with the sludge. The absorption due to C=O in carboxylic groups of the FAs was not pronounced, though the presence of polysaccharides and protein decomposition products was clearly indicated.     

Temporal changes in humic acids in cultivated soils with continuous manure application

Abstract

Although the application of manure to upland fields is believed to induce changes in the quality of humic substances in soil as well as the quantity, the direction and extent of these changes have not been elucidated. To understand temporal variations in humic acids, periodically collected soil samples from two fields, a Typic Hapludult (Togo) and a Pachic Melanudand (Kuriyagawa), with cattle manure and chemical fertilizer (CF) were examined. The content and degree of humification (darkening) of the humic acids were distinctly greater in Kuriyagawa than in Togo soil. Corresponding to the difference in the degree of humification, molecular size distribution, elemental composition, infrared (IR) spectra, and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra of humic acids differed between the two soils. Manure application at 40 Mg ha^?1 year^?1 for 16 years (Togo) and at 80 or 160 Mg ha^?1 year^?1 for 19 years (Kuriyagawa) resulted in greater humic acid content compared with plots with CF only because of its increase in the manured plots and/or decrease in the CF plots. Manure application at an extremely high rate (160 Mg ha^?1 year^?1) resulted in higher H content and greater signal intensities of alkyl C, O-alkyl C and amide C=O in the ¹³C CPMAS NMR and/or IR spectra. Although humic acids with larger molecule sizes increased in all the manured plots, differences between the humic acids from the plots with and without manure applied at practical levels in the elemental and spectroscopic analyses were small or scarce. These results were considered to be because of the similarity between the indigenous soil humic acids and the manure-derived ones in Togo soil (a low degree of humification) and because of the abundance of highly-humified humic acids in Kuriyagawa soil.     

Humic and fulvic acids as indicators of soil and water pollution

Humic substances are the major organic components of soil and sediments, but little is known on how they are affected by environmental and industrial pollution. To find out whether such effects could be recognized, a number of analytical characteristics were compared of humic and fulvic acids extracted from unpolluted and polluted soils and sediments. The main differences were that, per unit weight, polluted humic and fulvic acids contained more N, S and H but fewer CO₂H groups, and were more aliphatic than unpolluted samples. Unusually high N and S contents of humic materials appear to be the most valid indicators of pollution. Humic acids are preferred to fulvic acids as indicators of pollution because the former are more readily separated and purified.     

Influence of clay minerals on the formation of humic substances by Epicoccum nigrum and Stachybotrys chartarum

The influence of montmorillonite, kaolinite and finely ground quartz on the formation of humic acid-type polymers by Epicoccum nigrum and Stachybotrys chartarum was studied. The fungi were grown in shake and in deep (4 cm) or shallow (1.5 cm) stationary cultures. In general, clay shortened the time required for the formation of dark colored substances and increased the amounts of humic acid-type polymers in the culture solutions or extracted from the cells with NaOH. In some tests, the time of maximum humic polymer accumulation was much earlier in the presence of clay but total amounts formed in check cultures eventually equalled that of the cultures with clay additions. The ratios of the total humic acid to the total cell substance or to the glucose consumed were also generally increased by montmorillonite. Kaolinite and quartz exerted a similar but less pronounced effect. In deep stationary cultures of S. chartarum, total growth and humic acid formation was sometimes depressed by the higher concentrations of montmorillonite but in shallow cultures biomass and humic acid formation were increased. In cultures with an initial pH of 6.0, humic acid polymers were formed in the cells before they appeared in the solutions. During autolysis, some of the cellular polymers were either released into the medium or became more readily extractable with NaOH. The clays did not appreciably alter the chemical properties of the humic polymers, namely, C and N contents, exchange capacity, COOH groups, total acidity, or phenols released upon sodium amalgam reduction. In the presence of clays, phenols were formed more quickly in the culture solutions, but the kinds and relative amounts did not appear to be altered. Clays did not significantly affect oxygen consumption during autoxidation of phenol mixtures. The observations indicate that, by affecting growth and metabolism, the clays indirectly influence phenolic polymer formation.     

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

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