Effect of soil invertebrates on the formation of humic substances under laboratory conditions

The complete polymerization of phenols and proteins (one of the processes involved in the formation of humic substances) was explained. It was shown that fly (Bibio marci) larvae and earthworms (Aporrectodea caliginosa) participate in the complete polymerization of phenols and proteins. In a laboratory experiment, invertebrates participated in the degradation of organic matter and the synthesis of humic substances, which was proved in experiments with ¹⁴C-labeled phenols and proteins. The same organic substances (phenols and proteins) without the impact of invertebrates were used as the control substances. The distributions of the ¹⁴C isotope in alkaline extracts separated by solubility in acids (humic and fulvic acids) was compared to those of the control substances. The portion of the ¹⁴C isotope in the humic acids in the excrements of Bibio marci was higher than that in the control substances. The content of ¹⁴C-labeled humic substances in the excrements of the earthworm Aporrectodea caliginosa exceeded the control values only in the experiment with proteins. When clay material was added to the organic substances, the portion of the ¹⁴C isotope in the humic acids increased in both experiments with phenols and proteins. When these substrates passed through the digestive tracts of the invertebrates, the polymerization of organic substances and the inclusion of proteins and phenols into humic acids occurred.     

The origin of oxygen in soil humic substances

The ¹⁸O/¹⁶O ratios of a number of soil humic and fulvic acids were measured and compared with those of lignin and cellulose samples originating from the same area. The average enrichments above ground water were: cellulose 32%₀ humic and fulvic acid 29%₀ and lignin 14%₀, suggesting that the oxygen in humic and fulvic acid originates pricipally from cellulose or other plant carbohydrates and not lignin as has been suggested.     

Nature of soil carbohydrate and its association with soil humic substances

The fulvic acid and alkali-soluble polysaccharide fractions of a sandy loam arable soil of the Countesswells series have been subjected to acid hydrolysis or methylation and the products examined by infra-red and NMR spectroscopy, pyrolysis mass spectrometry and chemical analysis. Infra-red and NMR spectroscopy of the polysaccharide fraction indicated that the substance was predominantly carbohydrate, although sugars accounted for less than one-third of the weight by chemical analysis. Pyrolysis mass spectrometry con-firmed the presence of sugars by sugar anhydride formation, but also showed the presence of ‘secondary’ or ‘pseudo’ polysaccharide. The fulvic acid contained only 2–3% sugars by chemical analysis, whereas a much larger carbohydrate component was suggested by physicochemical analyses. Infra-red and pyrolysis mass spectrometry difference spectra for the residues after acid hydrolysis indicated the release of material with some of the characteristics of glycoprotein. Most of the carbohydrate present in the fulvic acid was of the secondary or pseudo polysaccharide type. The hypothesis that the secondary polysaccharide could be a degraded polysaccharide structure in which some sugar residues have been partly transformed to melanoidins by Maillard reaction is explored.     

The sodium-amalgam reduction of soil and fungal humic substances

Two soil humic acids and a “humic acid” synthesized in the laboratory by Stachybotrys chartarum were reduced with Na-amalgam. The reduction products were methylated, separated by preparative gas chromatography and identified by matching their mass and micro-infrared spectra with those of authentic specimsens.Yields of reduction products identified ranged from 2.7 to 4.2% of the initial weights of the humic materials. Major products identified were N-methyl-benzylsulfonamide, methylated phenolic acids, aromatic aldehydes and C₆C₂ - and C₆C₃ - compounds with 0 in the side chains. Since the Na-amalgam reduction of both soil and fungal humic materials produces the same or similar compounds, the method provides little information on the origin of these compounds, that is, whether they are lignin- or flavonoid-derived or synthesized by microorganisms. Compared with oxidative degradation methods, Na-amalgam reduction appears to be inefficient and tells little about the chemical structure of humic acid polymers.     

土壤腐殖质氮结构的X-光电能谱研究

X－ray photoelectron spectroscopy(XPS) was applied to examine the N structures of soil humic substances and some of their analogues.It was found that for soil humic substances XPS method gave similar results as those obtained by ^15N CPMAS NMR (cross-polarization magic-angle spinning nuclear magnetic resonance) method.70%-86% of total N in soil humic substances was in the form of amide,and 6%-13% was presented as ammes,with the remaining part as heterocyclic N.There was no difference in the distribution of the forms of N between the humic substances from soils formed over hundreds or thousands of years and the newly formed ones.For fulvic acid from weathered coal and benzoquinone-(NH4)2SO4 polymer the XPS results deviated significantly from the ^15N CPMAS NMR data.     

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.     

Isoelectric focusing of soil humic substances in the presence of 8M urea

Summary The isoelectric focusing (IEF) of different-molecular-size humic fractions, either separately or recombined, was carried out in the presence and absence of 8M urea. In the presence of urea, the fractions were recognizable from both their characteristic microheterogeneity and ranges of focalization. Urea caused a slight shift of the bands towards lower pH values. The refocusing of characteristic band of one fraction produced a slight discrepancy in the isoelectric points (0.2–0.3 pH units). However, the maintenance of the banding in 8M urea and the homogeneity of the band under refocusing demonstrated the reliability of the IEF in fractionating structurally different humic substances. Such experiments should provide useful information on the association/dissociation properties of soil organic matter under controlled conditions.Joint program of C.N.R. (Italy) and C.S.I.C. (Spain), no. 7     

Recent advances in the spectroscopic characterization of soil humic substances and their ecological relevance

Non destructive spectroscopic methods such as ¹³C- and ¹⁵N-NMR provide new insights into the structure of humic substances. Solid state NMR is capable of studying complete native soils by the CPMAS-technique. By means of nitrogen 15-enriched composts it is suggested that nitrogen in soil organic matter is mainly located in amide and peptide structures. Most probable assignments are given. The investigation of humification processes in forest soils is possible via an approach which uses several fractionation techniques in combination with degradative and non-degradative analytical techniques. Besides structure analysis, the ¹³C-NMR-spectroscopy provides important information about the functions of humic substances. By using ¹³C-enriched xenobiotics the binding mechanism to organic matter can be elucidated on a molecular basis. Molecular fluorescence spectrometry, a non-invasive method, is a powerful tool for the quantitative characterization of metal ion complexation by dissolved organic matter in aqueous leaf litter extracts in terms of conditional stability constants and metal binding capacities.     

Current ideas on the possible pathways for the formation of humic substances in soils

In the current approach to the study of humic substances (HSs), they are usually considered as complex chemical polymeric compounds with a specific combination of properties. This concept of HSs cannot explain their role in soils as a depot of nutrients for microorganisms and some experimental data. The main problem is related to the low energy efficiency of this depot of nutrients, because microorganisms have to consume energy for synthesizing enzymes that destroy HS polymers (macromolecules). At the same time, the recently proposed consideration of HSs as a system of supramolecular compounds completely eliminates this contradiction. In this work, an attempt has been made to consider HSs from other positions based on their possible functional role in soils and entire ecosystems. A scheme has been proposed for the transformation of the litterfall that is based on the supramolecular nature of the HSs and confirmed by reported experimental results.     

Quantitative differences in evaluating soil humic substances by liquid- and solid-state C-NMR spectroscopy

We compared the quantitative responses of liquid-state (LS) and solid-state (CPMAS) ¹³C-NMR spectroscopy of four different soil humic substances. The intensities of signals for the alkyl carbons (0–40 ppm) were significantly larger in CPMAS than in LS spectra. This difference is in agreement with the pseudo-micellar model of the conformational nature of humic substances. By this view, the hydrophobic interactions holding together the heterogeneous molecules of humic micelles inhibit the molecular motions of the alkyl carbons, thereby enhancing the spin-lattice relaxation times and consequently lowering the sensitivity of liquid-state NMR. Conversely, regardless of their position in the humic conformation, a better estimation of the number of alkyl carbons can be obtained by CPMAS-NMR because of the cross-polarization of hydrogen nuclei in CH₂ and CH₃ groups. The intensity of the 40–110 ppm region is also slightly lower in LS than in CPMAS-NMR spectra, despite the hydrophilicity of the oxidized and peptidic carbons resonating in this chemical shift interval. Their molecular motion may also be reduced by either the formation of intra- and inter-molecular hydrogen bondings due to poorly acidic hydroxyl groups of saccharides, or the degree of conformational rigidity that a pseudo-micellar arrangement confers even to hydrophilic domains. The higher content of aromatic carbons (110–160 ppm) found in the LS spectra was attributed partly to the high degree of substitution of the aromatic ring that slows down cross-polarization in CPMAS experiments and partly to the relative overestimation of this region by LS-NMR due to a lack of signal in the aliphatic interval. The slightly lower content of carboxyl carbons estimated in CPMAS spectra as compared to LS spectra was also attributed to slow cross-polarization. This work shows that the combined use of both NMR techniques is profitable in conformational analysis of humic substances and of dissolved organic matter in general.     

Role of inorganic anions in the structural transformation of molecules of humic substances in soil

It was shown that inorganic anions of exogenic compounds affected the structure of humic and fulvic acids in gray forest soil, as well as the yield of humic acids and their contents of carbon and metals. Humic substances in arable soil more readily interacted with the anions of extractants and fertilizers compared to virgin soil. Changes were revealed in the energy state of the electronic system of P- and N-modified fragments of humic acids from the arable soil. New ligand groups and chelate nodes were found in modified fragments using differential electronic spectra.     

Specificity of humic substances extracted from fissures and genetic horizons of peat-podzolic soil

A comparative characterization of humic acids (HAs) and fulvic acids (FAs) isolated from the material of fissures and genetic horizons of peat-podzolic soil is presented. It is shown that HAs from separate soil horizons differ sharply in their composition and properties, and the portion of hydrophilic fractions in their composition decreases with depth. This suggests the absence (or very slow) frontal migration of HAs in the soil profile. On the contrary, HAs isolated from the material filling the fissures are very homogeneous and resemble HAs isolated from the A1E horizon in their composition and properties. This confirms the probability of downward humus migration along the fissures from the upper soil horizons in the form of suspensions or colloidal solutions. An increase in the portion of hydrophilic HAs in the deep parts of fissure zones also supports this conclusion.     

长期施肥对黑土团聚体中腐殖物质组成的影响

腐殖物质(HS)是土壤有机质的主体,在土壤固碳方面具有重要作用[1],近些年来,在土壤有机质化学研究中越来越受到重视。团聚体是土壤有机质(SOM)分解转化和HS形成的最主要“场所”,土壤中的一切生物化学活动均在这一骨架内进行(包括土壤腐殖化作用)。在正常的或特定的条件下,不仅微生物主导的腐殖化作用需要合适的“场所”,而且所形成的HS也只有在合适的“场所”或者说与土壤矿质部分相结合才能长时间保存[2-3]。这种“场所”或者说有机无机结合状况,是制约整个土壤固碳反应的关键。但至今为止关于这种“场所”(团聚体)的固碳机制尚不清楚,特别是很少注重HS化学方面。团聚体和HS二者关系密切,不可分割。可以假     

Depth distribution of humic substances in Andosols in relation to land management and soil erosion

Although Andosols are relatively resistant to water erosion, they can be severely affected by changes in land use, resulting in accelerated erosion and loss of soil organic matter (SOM). We hypothesized that if the contents of specific components of SOM and organo–metallic complexes (humic acids –HAs–, fulvic acids –FAs–, sodium pyrophosphate extractable carbon –C_p–, aluminium –Al_p–, and iron –Fe_p–) consistently tend towards certain ratios in A and B horizons, they could be used to identify soils denuded by erosion. To test this hypothesis, we investigated the vertical distribution of humus components and certain ratios, namely C‐HA/C‐FA, C‐FA/total organic C (TOC), C_p/TOC and (Fe_p + Al_p)/C‐FA, in representative profiles of andic soils located in natural ecosystems with different degrees of human disturbance. Furthermore, we analysed these parameters in the topsoil of a natural protected area and in adjacent soils under different land use scenarios (natural reserve vs. traditional exploitation). We found that the ratios of C‐HA/C‐FA and, to a lesser extent, of C‐FA/TOC and C_p/TOC changed with depth in the selected soil profiles, but the values were characteristic of each type of soil horizon. The values of these ratios in the topsoils of the disturbed areas were closer to a B horizon than an A horizon. This pattern may be superimposed on pre‐existing gradients, such as those related to the type of natural vegetation. The use of these indices emerges as a possible land use and erosion indicator.     

Inhibitory action of dissolved humic substances on the growth of soil bacteria degrading DDT

Abstract

Dissolved humic acid (HA) and fulvic acid (FA) prepared from a Dando brown forest soil (Typic Dystrochrept) inhibited the growth of soil bacteria degrading DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) in the culture. The population of DDT-degrading Gram-variable rod Bll6 decreased by the application of both HA and FA, suggesting the presence of bactericidal effect. Such inhibitory effect was stronger for HA and resulted in a lower degrading activity of DDT in the culture of Bll6. No inhibitory effect was observed on the growth of DDT-degrading Bacillus sp. B75. The electron spin resonance spectra showed the presence of organic free radicals in both HA and FA. The relative concentration of the radicals was higher in HA. Storage of HA solution for 3 months at 4°C decreased the concentration of the radicals as well as the inhibitory action. The addition of catalase decreased the inhibitory effect of humic acid. It is suggested that a hydroxy radical, which is derived from free radicals of humic substances, is involved in the inhibition of bacterial growth and degradation of DDT.     

Gel filtration chromatography of humic substances in soil solutions using HPLC-determination of the molecular weight distribution

The molecular weight distribution of humic substances in soil solutions from a dystric cambisol and an orthic podzol were determined by high-performance gel filtration chromatography (Toyo Soda TSKgel G2000 SWXL column). The retention volumes of humic substances were compared to those of narrow molecular weight distributed poly-styrenesulphonates with known molecular weight. A change in mobile phase pH or ionic strength usually resulted in an almost identical change in retention volume for the polystyrenesulphonates and the bulk of humic substances. The possibility of using the former to calibrate the column is discussed. The molecular weight at peak maximum was 1120–1190 and 1800 for the cambisol and podzol samples, respectively, using polystyrenesulphonates as molecular weight standards. No pretreatment of samples was used, but ion exchange is recommended for samples with high Al concentrations.     

Growth and development of pepper are affected by humic substances derived from composted sludge

A greenhouse experiment was conducted to evaluate the effects of humic substances extracted from composted sewage sludge on growth, phenological development, and photosynthetic activity of pepper (Capsicum annuum L. cv. Piquillo) plants. Humic substances derived from composted sludge (HSS) were compared with those derived from leonardite (HSL). Two doses of both humic substances were assayed (200 and 500 mg C [L substrate]^–1) and compared with a control (C). HSS showed higher nitrogen content and a higher percentage of aliphatic carbon, as well as a lower content of aromatic and phenolic carbon than HSL. HSS significantly increased plant dry‐matter production (up to 560%), plant height (86%–151%), and leaf area (436%–1397%) during the early stages of pepper development. Net photosynthesis and stomatal conductance increased in the treatments with HSS (up to 48% and 63%, respectively) at the vegetative stage. In addition, HSS accelerated the phenological development of pepper plants, reducing significantly the number of days to flowering and ripening, which occurred 12 and 14 d earlier than in control plants, respectively. In general, the treatments with HSS and HSL did not markedly affect chlorophyll and nutrient concentrations in the leaves. At maturity, only small differences in total fruit yield, number of fruits per plant, and fruit size were observed between amended and control plants. The results suggest that the mechanisms through which HSS affected plant growth and development were not associated with an improved nutrient uptake. Although the identity of the growth‐promoting factors remains to be found, the results suggest that they may be linked to the chemical structure of the humic substances.     

A comparative survey of recent results on humic-like fractions in organic amendments and effects on native soil humic substances

The main scope of the wide use of organic amendments of various origin and nature in modern agriculture is to increase and/or restore the organic matter content in organically poor or depleted soils in order to maintain and/or increase crop production and reduce soil exposure to degradation, erosion, desertification and pollution. The agronomically efficient and environmentally safe use of any organic amendment requires, however, an adequate control not only of the chemical quality of the humic substance (HS)-like fractions contained, which is an important indicator of the maturity and stability achieved by organic matter in the amendment, but also of the effects that these HS may have on native soil HS. In this review, the most recent results obtained on this topic by the research groups operating in Bari, Italy, and in Madrid, Spain, are discussed comparatively with results recently published by other research groups. Overall, HS-like components of organic amendments are characterized by higher aliphatic character and molecular heterogeneity, lower contents of O, acidic functional groups and organic free radicals, and lower degrees of aromatic ring polycondensation, polymerization and humification than native soil HS. These differences are less evident for composted materials. The composition, structure and functionalities of HS in amended soils may be affected in different ways and at various extents on dependence of the nature, origin and rate of amendment. In general, these properties are intermediate between those of the unamended soil HS and the HS-like fractions in the amendment, but generally resemble more the former than the latter, especially with increasing time after amendment application.     

Fertigation of humic substances improves yield and quality of broccoli and nutrient retention in a sandy soil

There is lack of information available concerning the effect of humic substances (HS) applied via fertigation on plant growth in sandy soils. Therefore, a field experiment was carried out at El‐Saff district (20 km southwest of Cairo), Egypt, to investigate the role of HS fertigation on water retention of a sandy soil, yield and quality of broccoli (Broccoli oleracea L.) as well as on soil nutrient concentration retained after harvest. The experiment consisted of six fertigation treatments (50%, 75%, and 100% of the recommended NPK‐fertilizer rate for broccoli combined with and without HS application at 120 L ha^–1) in a complete randomized block design with three replicates. Humic substances affected spatial water distribution and improved water retention in the root zone. Furthermore, application of HS increased total marketable yield and head diameter of broccoli as well as quality parameters (i.e., total soluble solids, protein, and vitamin C). Higher nutrient concentrations were found in the broccoli heads and concentrations of plant‐available nutrients in soil after harvesting were also higher, indicating an improvement in soil fertility. In conclusion, HS fertigation can be judged as an interesting option to improve soil water and nutrient status leading to better plant growth.     

Structural changes in humic substances after long-term fertilization of a calcareous soil with pig slurries

Pig slurries are widely used on calcareous soils in European rainfed systems. Here we assess their impact on the amount of soil organic carbon (SOC) and on the composition of humic-type substances (HTS). Seven doses of slurry (five from fattening pigs and two from sows) ranging from 1.0 to 4.8 Mg ha⁻¹ yr⁻¹ of organic matter were evaluated after a period of 12 years and compared with mineral fertilizer treatment. At the end of the last annual cropping season (September), SOC was quantified, and HTS were isolated by alkaline extraction followed by acid precipitation, and studied by visible spectroscopy (800–400 nm) and Fourier-transformed infrared spectroscopy (4000–400 cm⁻¹). Following the trend in the slurry organic matter applied rates, SOC increased from 9.5 g C kg⁻¹ (mineral treatment) to 13.8 g C kg⁻¹. This SOC increase was equivalent to c. 25.4% of the slurry organic carbon applied. The incorporation of aliphatic structures, mainly polyalkyl, from slurries into the HTS tends to modify the composition of the soil organic matter (SOM), which is reflected in a decrease in the intensity of FT-IR peaks related to aromatic structures. Despite the trend of significant increase in SOC with fattening slurries, mainly from the organic matter rate of 1.6 Mg ha⁻¹ yr⁻¹ (c. 185 kg N ha⁻¹), the composition of the HTS showed an important aliphatic enhancement. The FTIR results showed that using exclusively the relative intensities of specific peaks (alkyl, carboxyl, aromatic and amide groups) as variables for the discriminant analysis, it is possible to identify HA between different groups of soils treated with progressive levels of slurry. Although the new aliphatic components could be considered important to improve soil physical quality, after the incorporation of additional SOM, the spectroscopic characteristics of HTS in soils treated with slurries suggested a weak effect in long-term C sequestration, as the newly incorporated OC forms are not qualitatively similar to the presumably stable native SOM. These potential changes in SOC and SOM composition at field level are constrained by the maximum allowed N rates from organic origin in some agricultural systems.