Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

Hydrolases extracted from soil: Their properties and activities

The pH optima and stabilities of some hydrolases extracted by pyrophosphate from two different soils were determined for phosphatase, urease, casein and benzoylargininamide-hydrolysing proteases. Hydrolases activities of soil extracts were more resistant to thermal denaturation than their respective commercial preparations. Extracted phosphatases exhibited a marked stability to temperature and to proteolysis.By exhaustively ultrafiltering soil extracts against 0.1 M pyrophosphate at pH 7.1 fractions of mol. wt higher and lower than 100,000 were obtained. Ultrafiltration increased the total activities of extracted ureases and phosphatases while those of casein and benzoylargininamide-hydrolysing proteases were decreased. Both on an organic C and an N basis, specific activities of ureases and phosphatases increased after ultrafiltration.     

Transformation of organic matter from maize residues into labile and humic fractions of three European soils as revealed by 13C distribution and CPMAS-NMR spectra

The dynamics of incorporation of fresh organic residues into the various fractions of soil organic matter have yet to be clarified in terms of chemical structures and mechanisms involved. We studied by ¹³C‐dilution analysis and CPMAS‐¹³C‐NMR spectroscopy the distribution of organic carbon from mixed or mulched maize residues into specific defined fractions such as carbohydrates and humic fractions isolated by selective extractants in a year‐long incubation of three European soils. The contents of carbohydrates in soil particle size fractions and relative δ¹³C values showed no retention of carbohydrates from maize but rather decomposition of those from native organic matter in the soil. By contrast, CPMAS‐¹³C‐NMR spectra of humic (HA) and fulvic acids (FA) extracted by alkaline solution generally indicated the transfer of maize C (mostly carbohydrates and peptides) into humic materials, whereas spectra of organic matter extracted with an acetone solution (HE) indicated solubilization of an aliphatic‐rich, hydrophobic fraction that seemed not to contain any C from maize. The abundance of ¹³C showed that all humic fractions behaved as a sink for C from maize residues but the FA fraction was related to the turnover of fresh organic matter more than the HA. Removal of hydrophobic components from incubated soils by acetone solution allowed a subsequent extraction of HA and, especially, FA still containing much C from maize. The combination of isotopic measurements and NMR spectra indicated that while hydrophilic compounds from maize were retained in HA and FA, hydrophobic components in the HE fraction had chemical features similar to those of humin. Our results show that the organic compounds released in soils by mineralization of fresh plant residues are stored mainly in the hydrophilic fraction of humic substances which are, in turn, stabilized against microbial degradation by the most hydrophobic humic matter. Our findings suggest that native soil humic substances contribute to the accumulation of new organic matter in soils.     

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.     

Isoelectric focusing of β-glucosidase humic-bound activity in semi-arid Mediterranean soils under management practices

Changes in β-glucosidase enzyme–humic complexes and conventional parameters (pH, total organic C, total N, water-soluble C, and bulk density) were studied in an almond-cropped soil prone to erosion under a rehabilitation practice. The experimental plan included three soil slopes (0%, 2%, and 6%) and two type of fertilization (organic and mineral), with sampling of rhizosphere and inter-row soils. The enzyme humic complexes were extracted by pyrophosphate, purified by ultrafiltration of the organic extracts on molecular mass exclusion membranes (mol wt > 10⁴) and fractionated by isoelectric focusing technique (IEF). The IEF on polyacrylamide rod gels with a restricted pH gradient ranging between 6.0 and 4.0 gave five humic bands on the basis of the little differences of their electric charges (pI). Under both organic and mineral fertilization, β-glucosidase activity bound to the fractionated humic substances, especially in the pH range 4.5–4.2 of the rhizosphere soil, was higher than that of the inter-row soil. This also occurred in 6% slope where the enzyme activity was lower than in soil with lower slopes. The higher number of the β-glucosidase active humic bands in rhizosphere than inter-row soil, particularly for the 0% slope, may be due to the presence of humic molecules capable of preserving the enzyme molecules in the active form, other than to the higher microbial activity synthesizing and releasing the tested enzymes.     

Humic materials from an organic soil: A comparison of extractants and of properties of extracts

Five extraction procedures and thirteen extracting reagents, which included dipolar aprotic solvents, organic chelating agents, pyridine, ethylenediamine, sodium hydroxide, ion-exchange resins and two salts (sodium pyrophosphate and ethylenediamine hydrochloride), were used to extract humic materials from an organic soil. Extractabilities increased in the general order: salts < organic chelating agents < dipolar aprotic solvents < pyridine < ethylenediamine = sodium hydroxide, and the amounts of the soil organic matter extracted by the reagents in the series ranged from 13 to 63%. Gel chromatography techniques indicated that extracts in dipolar aprotic solvents were predominantly of intermediate and low molecular-weight values, and it is suggested that the more highly oxidised soil humic materials were extracted in these. The more efficient solvents extracted materials with a range (high—low) of molecular-weight values. Data from elemental analysis and from E.S.R. measurements indicated that ethylenediamine altered the chemical nature and the composition of extracts. Dipolar aprotics, by the same criteria, were found not to alter the humic extracts, and can be regarded as mild reagents for the extraction of a less representative (of the total) fraction of soil organic matter. Sodium hydroxide in solution, despite its oxidation effects, was the best of the reagents tested for isolating extracts which were representative of a wide range of soil humic substances.     

Microdetermination of protease activity in humic bands of different sizes after analytical isoelectric focusing

Summary Extracellular benzoyl-l,-argininamide (BAA)-hydrolysing protease was extracted with neutral pyrophosphate from an arable soil and fractionated by membrane ultrafiltration. There were three fractions: A₁ (molecular weight > 10⁵), A_II (molecular weight 10⁴–10⁵), and R (molecular weight < 10⁴). Analytical isoelectric focusing (IEF) of the fractions was carried out on polyacrylamide gels with a restricted pH gradient of 4.0 to 5.0. Two extracellular proteases characterized soil extract E, with one peak (Ip 4.44) bound to a large amount of humic matter and the other (Ip 4.06) bound to a small amount of humus. Following ultrafiltration, the humus-enzyme complex of extract E (Ip 4.44) split into the fractions A_I, A_II, and R, and was displaced at Ip values that depended on the electrophysical properties of bound organic matter, whereas that at Ip 4.06 was completely removed from the extract E and accumulated only into the low-molecular-weight fraction R. High recoveries of absolute activity were obtained after IEF of the whole extract E, and fractions A_II and R, but only about 50% was recovered from fraction A_I.It appears that humic substances have reversible inhibitory effects on extracellular proteases, since the maximum recoveries of activity were obtained from fractions where high amounts of protease non-active organic matter had been removed by IER IEF was able to fractionate humic molecules and purify humic-protease complexes on the basis of smaller differences in Ip, and even smaller differences of 0.05 pH units. The present results show that BAA hydrolysing proteases were preferentially linked with a specific class of humic molecules with an Ip of close to 4.44.Joint program CNR (Italy) — C.S.I.C. (Spain), no. 7, 1985–1986. This paper is part of the doctoral thesis of Prof. M. Bonmati     

Influence of land use on the characteristics of humic substances in some tropical soils of Nigeria

In highly weathered tropical conditions, soil organic matter is important for soil quality and productivity. We evaluated the effects of deforestation and subsequent arable cropping on the qualitative and quantitative transformation of the humic pool of the soil at three locations in Nigeria. Cultivation reduced the humic pool in the order: acetone‐soluble hydrophobic fraction (HE) > humic acid (HA) > humin (HU) > fulvic acid (FA), but not to the same degree at all three sites. The C and N contents, as well as the C/N ratios of humic extracts, were large and not substantially influenced by land use. The δ¹³C values of the humic extracts were invariably more negative in forested soils thereby showing a dilution of δ¹³C signature with cultivation from C3 to C4 plants. The δ¹³C values of apolar HE fractions were generally more negative, indicating a reduced sensitivity compared with other humic fractions to turnover of crop residues. The contents of hydrophobic constituents (alkyl and aromatic C), as revealed by cross‐polarization magic angle spinning (CPMAS) ¹³C‐NMR spectroscopy, in HA, FA and HU were generally < 50%, with the exception of larger hydrophobicity in HU in the forested soil at Nsukka and HA in that at Umudike. The HE fraction contained significantly more apolar constituents, and consequently had a larger intrinsic hydrophobicity than the other humic fractions. The larger reduction of apolar humic constituents than of the less hydrophobic humic fractions, when these soils were deforested for cultivation, indicates that at those sites the stability of accumulated organic matter is to be ascribed mainly to the selective preservation of hydrophobic compounds.     

珠江三角洲地区土壤与表层沉积物有机质的性质结构研究

通过分析海洋、河流、池塘和稻田四种热带 /亚热带土壤与表层沉积物及用化学方法分离的有机质 ,证明了土壤与沉积物有机质是高度不均匀的 ,除了包括腐殖酸和演化程度较深的干酪根外 ,强调了演化程度更深的碳黑的存在 ,并对不同有机质的来源、性质和结构进行了表征。腐殖酸和干酪根来源于生物体的演化 ,在结构上后者比前者致密 ;碳黑来源于化石燃料和生物体的不完全燃烧 ,具有更致密的结构 ,对有机质的非均质性影响很大。因此可认为 ,系统地研究土壤与表层沉积物有机质的非均质性及组分特征对了解地球表层的生物地球化学过程和疏水性毒害有机污染物在环境中的迁移具有重要的意义。     

从高山火山灰中提取铝, 铁和有机碳的可供选择的方法

Montane volcanic ash soils contain disproportionate amounts of soil organic carbon and thereby play an often underestimated role in the global carbon cycle.Given the central role of Al and Fe in stabilizing organic matter in volcanic ash soils,we assessed various extraction methods of Al,Fe,and C fractions from montane volcanic ash soils in northern Ecuador,aiming at elucidating the role of Al and Fe in stabilizing soil organic matter(SOM).We found extractions with cold sodium hydroxide,ammonium oxalate/oxalic acid,sodium pyrophosphate,and sodium tetraborate to be particularly useful.Combination of these methods yielded information about the role of the mineral phase in stabilizing organic matter and the differences in type and degree of complexation of organic matter with Al and Fe in the various horizons and soil profiles.Sodium tetraborate extraction proved the only soft extraction method that yielded simultaneous information about the Al,Fe,and C fractions extracted.It also appeared to differentiate between SOM fractions of different stability.The fractions of copper chloride-and potassium chloride-extractable Al were useful in assessing the total reactive and toxic Al fractions,respectively.The classical subdivision of organic matter into humic acids,fulvic acids,and humin added little useful information.The use of fulvic acids as a proxy for mobile organic matter as done in several model-based approaches seems invalid in the soils studied.     

Characterization by isoelectric focusing of the organic matter of a regenerated soil

Abstract

Regeneration of a soil with a high degree of desertification was conducted by the addition of different doses of municipal solid waste organic matter (MSW). Five years after this treatment, humic substances were extracted from these soils and characterized by spectroscopy and isoelectric focusing. No significant differences between E4/E6 ratio and ?log K (Log A400 nm‐log A600 nm) were observed for humic substances extracted from treated and untreated soils. However, the isoelectric focusing (IEF) technique established differences between the humic substances from control and treated soils. The focusing pattern of the former showed a well defined band at pH 9.1 which nearly disappeared in the soils with high doses of MSW (1–2 %). However the organic matter which focused at pH 5.8 was present in all soils.     

The distribution of nitrogen in some highly organic tropical volcanic soils

The qualitative and quantitative distribution of N-compounds in 10 tropical soils, and in a number of humic materials extracted from representative samples thereof, was determined after 6 N HCl hydrolysis.Eighty to 98% of the total N in the soils and humic materials was hydrolysable by 6n HCl. Slightly less than one half the hydrolysable N in the soils and humic fractions consisted of amino acids. Well-drained soils and fulvic acids extracted from them contained unusually high concentrations of the acidic amino acids, aspartic and glutamic acids. Between 80 and 95% of the amino acids in the soils was accounted for in the humic materials + NaOH-insoluble organic residues. NH⁺₄-N released by acid hydrolysis was generally higher for the soil samples than for the humic materials. Amino sugar-N constituted relatively small proportions of the total N in the soils and humic fractions.Our data suggest that large quantities of amorphous allophanic materials coupled with relatively high enzymic activity are responsible for the observed accumulation of acidic amino acids in the well-drained tropical volcanic soils.     

DISTRIBUTION OF PYROPHOSPHATE-EXTRACTABLE IRON AND ORGANIC CARBON IN SOILS OF VARIOUS GROUPS

Potassium pyrophosphate (0.1m ) removes very little Fe from crystalline Fe oxides at pH 10, but peptizes finely divided hydrous amorphous oxides and organic matter in soils. Fe and C contents of extracts from each horizon of twenty-six British soil profiles show distinctive patterns, independent of the residual dithionite-soluble Fe. Thus extracts of humus Fe podzols have maximum Fe and C in the B horizon, peaty gley podzol has maximum Fe in the B horizon but maximum C in the surface. These groups are differentiated from non-podzols which have maximum pyrophosphate extractable Fe and C in the surface horizon, decreasing with depth. Intermediate patterns help to quantify differences in soils of classes having properties of more than one soil group.     

Characterization of humic substances obtained from calcareous soils from Greece with various extractants. I. Chemical characterization

Humic substances of the Ah horizons of five differently calcareous soils from Greece under different vegetative covers were successively extracted with diethylether, acetone, ethanol and dioxane. Following these extractions, remaining humic substances were extracted with 0,1N NaOH. Humic substances were also directly extracted with 0,1N NaOH. The extracts of the organic solvents were separated by thin layer chromatography (T.L.C.) on silicagel plates, while the NaOH extracts were characterized by elemental and functional group analyses. The T.L.C. revealed that ?-acceptors and phenols were present in all extracts, while ?-donors and lignines were present only in the dioxane extracts. Lipides were present in the diethylether, dioxane and acetone extracts. Sugars were found only in the acetone extracts and reducing substances in the acetone and dioxane extracts. Some analytical characteristics of the humic substances were associated with the types of vegetative cover and/or soil properties. Humic acids (HA's) extracted with 0,1N NaOH from soil samples previously treated with organic solvents, generally varied in their carbon (C), carboxyl (COOH) and ash contents as compared with those extracted directly.     

Isolation by isoelectric focusing of humic-urease complexes from earthworm (Eisenia fetida)-processed sewage sludges

 Vermicomposting is an eco-biotechnological process that transforms energy-rich and complex organic substances into a stabilized humus-like product. In a laboratory experiment, Eisenia fetida (Sav.) earthworms were employed to process putrescible sewage sludges into a high-value biofertilizer, very rich in urease activity and humic-urease complexes (stabilized extracellular enzymes). Extracellular humic-urease complexes were extracted by a single 24-h extraction at 37  °C using neutral pyrophosphate (0.1 M); then, the extracts were dialysed and characterized by means of an analytical isoelectric focusing technique. This technique gave a multiplicity of humic bands enzymatically active, with isoelectric points ranging from 4.8 to 5.6. The results demonstrated that, after an 18-week incubation period, sewage sludge had undergone a biochemical evolution, which caused a doubling of absolute urease activity and a six-fold increase in specific activity (activity with reference to the humic C fraction). The biochemical evolution of the vermicompost was evaluated also from the sharp decrease in pyrophosphate-extractable C and water-soluble C. Stabilization of organic C during vermicomposting and the activity of humic-urease complexes expressed at low pH values are of extreme importance when organic wastes are used in acid soils for biochemical restoration purposes. Received: 10 June 1999     

Use of 0·1 m pyrophosphate to extract urease from a podzol

Sodium pyrophosphatc (0·1 m) at pH 7.1 and 37°C extracted a significant fraction of urease from a podzol. Maximum extraction values were obtained after 18 h. The yields of soil organic matter and urease activity during the extraction show a different pattern: the extraction of non-specific organic matter precedes and may facilitate the following extraction of an active urease organo-complex. The urease extracted by pyrophosphate is about 30 40 per cent of the total urease activity, as shown by plotting the urease activity against the population changes of ureolytic microorganisms, both in the original and extracted soil. The number of ureolytic microorganisms is unaffected by pyrophosphate, and the extracted urease is assumed to be extracellular.     

Retention of added copper by two soils as affected by organic matter,CaCO3, and exchangeable ions

The effects of addition and removal of organic matter and CaCO₃ and of saturating a loam and a loamy sand soil with hydrogen (HCl treated), with calcium and with sodium on the retention of added copper were studied. Removal of organic matter reduced the copper-retaining capacity in soils, while addition of 1 to 4% humic acid to H₂O₂-treated soils increased the retention. Soil organic matter had higher specific copper retaining as well as fixing capacity than the added humic acid. Humic acid fixed about 62 and 49% of the copper it retained, while soil organic matter fixed about 65 and 61% in the loam and loamy sand respectively, which could not be extracted with 0.1 N HCl. About 30% extra copper retained by the added humic acid was exchangeable. Saturating the soils with hydrogen (HCl treatment) decreased the retention of added copper considerably but addition of CaCO₃ up to 8% increased the retention by increasing the pH of the soil system. The CaCO₃ induced retention was lower than caused by humic acid additions. The specific copper retention by native CaCO₃ was slightly higher than that of added CaCO₃. However, other changes associated with the pre-treatment of the soil may have caused those differences in specif is copper retention. Although CaCO₃ had as high a copper fixing capacity as organic matter its contribution towards exchangeable copper was negligible.     

Degradation and humification of maize straw in soil microcosms inoculated with simple and complex microbial communities

Microbial communities are responsible for soil organic matter cycling and thus for maintaining soil fertility. A typical Orthic Luvisol was freed from organic carbon by thermal destruction at 600°C. Then the degradation and humification of ¹⁴C‐labelled maize straw by defined microbial communities was analysed. To study the role of microbial diversity on the humification of plant material, microcosms containing sterilized soil were inoculated with a natural microbial community or with microbial consortia consisting of bacterial and fungal soil isolates. Within 6 weeks, 41 ± 4% of applied ¹⁴C‐labelled maize straw was mineralized in the soil microcosms containing complex communities derived from a soil suspension, whilst the most efficient communities composed of soil isolates mineralized less than 35%. The humification products were analysed by solution state ¹³C‐NMR‐spectroscopy and gel permeation chromatography (GPC). The analyses of humic acids extracts by solution state ¹³C‐NMR‐spectroscopy revealed no difference in the development of typical chemical functional groups for humic substances during incubation. However, the increase in specific molecular size fractions of the extracted humic acids occurred only after inoculation with complex communities, but not with defined isolates. While it seems to be true that redundancy in soil microbial communities contributes to the resilience of soils, specific soil functions may no longer be performed if a microbial community is harshly affected in its diversity or growth conditions.     

Characteristics of the organic matter in a slightly and in a severely crusted soil

Chemical and spectroscopic characteristics were determined on the organic matter in two Luvisolic soils from northwestern Alberta, Canada. One of these (the Debolt soil) is known to crust only slightly while the other (the Demmitt soil) exhibits severe crusting after heavy rainfalls. The Debolt top soil is richer in total C, total N, carbohydrates and proteinaceous materials than the Demmitt top soil. Humic acid extracted from the Debolt soil is more aliphatic, and contains fewer CO₂H groups than does humic acid extracted from the Demmitt soil. The quality of the organic matter appears to play a role in soil crusting.     

APPLICATIONS OF NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY TO THE STUDY OF THE STRUCTURE OF SOIL ORGANIC MATTER

Recent developments in the application of nuclear magnetic resonance (n.m.r.) spectroscopy to soil science are reviewed. Progress in ¹H, ¹³C and CP-¹³C n.m.r. spectroscopy of humic substances is reported. Methods of determining the fraction of aromatic carbon in soil organic matter extracts are discussed, and methods by which structural group analysis can be carried out on in situ organic matter of whole soils are reviewed.