FTIR‐spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH,Na4P2O7, and Na2CO3 extraction procedures

Aim of our study was the development of the methodological basis for the characterization of humic fractions of a long‐term field experiment. Humic acids (HAs) were extracted from three layers of a nontilled soil using three different extractants (1 M NaOH, 0.1 M Na₄P₂O₇, 1 M Na₂CO₃), and the humin fraction was enriched. NaOH as extractant for FTIR analysis of humic substances yields higher resolved IR spectra, especially in the important regions of stretching vibrations including aromatic and aliphatic groups and in the fingerprint area including amides, aliphats, and aromats than the other extractants. The NaOH extraction has lower extraction yields as compared to Na₄P₂O₇ and Na₂CO₃ and represents a different part of the soil organic matter (SOM). This is reflected by lower C : N ratios and higher E₄ : E₆ and fulvic acid–to–humic acid ratios as compared to the other extractants. The FTIR band areas of HA fraction obtained by NaOH showed an increase of the aromatic and carbonyl groups and a decrease of amide groups with increasing soil depth. Aliphatic groups showed contradicting results: The bands of the stretching vibrations increased, and the band of the bending vibrations decreased. We assume that band interactions in the bending vibrations were responsible for that phenomenon under the assumption of an increase of aliphatic groups with increasing soil depth. The IR bands of the enriched humin fraction showed a decreasing trend in case of both aliphatic bands deriving from stretching vibrations and an increase of aromatic characteristics with depth. Our study led to the conclusion that HA fractions obtained by 1 M NaOH represent a small and dynamic fraction indicated by the measured yields in combination with values of N_t, C : N, E₄ : E₆ ratios, and ratios of fulvic acids (FA) to HA. The humin fraction has a high contribution to the total organic C and represents a more stabilized fraction of SOM which still shows changes in its aromatic and aliphatic characteristics with soil depth.     

Effects of humic and fulvic acids on release of fixed potassium

Release of potassium fixed by expanding silicate clays is considered of practical importance in soil fertility. Humic and fulvic acids are expected to play a definite role in liberating this fixed K, because of their chelating power, but not much is known in this respect. The following investigation was conducted to study release of fixed K by montmorillonite and illite, using humic and fulvic acids isolated from the surface horizon of a Cecil soil (Typic Hapludult, Red Yellow Podzolic soil) as extractants. For comparison, extraction was also done with 1 N neutral NH₄ -acetate, a mixture of 0.05 N HCl and 0.025 N H₂SO₄ or H₂ O. Supporting analyses of clays were carried out with X-ray diffraction to establish changes, if any, in crystal structure due to fixation, release of K, or adsorption of humic compounds. The results indicated that humic and fulvic acids released some of the K fixed by montmorillonite or illite. In terms of percentage of the total K fixed, 9 to 28% were released by the various extractants. The percentages K released by humic and fulvic acids were similar from both montmorillonite and illite, but based on absolute values, humic and fulvic acids extracted less K (mg/100 g) from illite than montmorillonite. Although statistically significant at the 5% level of probability, the capacity of humic compounds to liberate fixed K was not different markedly from those of NH₄ -acetate and the double acid mixture. Differences in pH of humic solutions had no influence on extraction of fixed K. X-ray diffraction analysis yielded curves showing an increase in spacing from 10.4 Å for K-montmorillonite to 13.2 Å as a result of extraction with the double acid mixture.     

Comparative evaluation of methods employing alkali and sodium pyrophosphate to extract humic substances from peat

Abstract

Humic substances from two different types of peat ‐ Sphagnum and Carex ‐ were obtained by using either alkali extractants, O.IM NaOH, O.IM KOH, and 0.25M KOH, or solvents containing sodium pyrophosphate (O.IM Na₄P₂O₇, or O.IM NaOH/Na₄P₂O₇). The alkalis released considerably greater amounts of humic substances from the Sphagnum peat than the extractants containing pyrophosphate, whereas the opposite occurred in the Carex peat, the two greatest yields being obtained with 0.25M KOH in Sphagnum peat and with O.IM NaOH/Na₄P₂O₇ in Carex. As shown by the spectroscopic and chemical analyses, humic acids released from both peats by extractants containing Na₄P₂O₇ are generally more oxidized than those obtained by any of the alkali solutions. Also, humic acids extracted from the Sphagnum peat by any of the solvents generally exhibit lower molecular sizes, more pronounced aliphatic characteristics, and a higher degree of oxidation than those extracted from the Carex peat.

Results of gel permeation chromatography indicate that humic acids obtained from Sphagnum peat by either O.IM NaOH or O.IM KOH extraction show a similar distribution of molecular sizes with a predominance of compounds with a molecular weight greater than 50,000, whereas the use of 0.25M KOH enhances the release of partially degraded humic substances. Also, humic acids extracted from the same peat with 0.1M Na4P2O7 show a considerable amount of molecules of low molecular weight, but much fewer of these compounds are obtained when 0.1M NaOH/Na₄P₂O₇ extraction is employed. In Car ex peat, molecular size distribution does not seem to depend on the type of extractant used for solubilization of their humic acids.     

Evaluation of the possibilities of using humic acids obtained from lignite in the production of commercial fertilizers

Purpose

Technological progress and high market demand contributed to a significant interest in the production of fertilizers based on humic acids. The aim of this study was to evaluate the possibilities of using humic acids obtained from lignite in the production of new commercial products. For this purpose, it is necessary to determine the quality standard requirements for such material. Properties of humic acids depend on source of origin as well as method of its extraction.

Materials and methods

The humic acids were extracted from polish deposit of lignite–Sieniawa Lubuska by alkaline extraction using for this purpose six kinds of extractants: 0.1 M NaOH and 0.25 M NaOH, 0.1 M KOH and 0.25 M KOH, and 0.1 M Na₄P₂O₇ and 0.25 M Na₄P₂O₇. The humic samples were used in solid powder form and characterized by UV-Vis spectroscopy, ¹³C NMR spectroscopy, fluorescence spectroscopy, and thermal analysis.

Results and discussion

The determining factor influencing a degree of humic acids extraction from lignite and their structure is type of extractant. The largest efficiency of extraction (about 50%) was obtained with the use of NaOH solutions. All examined humic acids were generally characterized by simple and heterogeneous molecularly structure with low molecular weight and low aromatic polycondensation. Therefore, it can be concluded that humic acids extracted with NaOH and KOH solutions are less condensed than those extracted with Na₄P₂O₇ solutions. It can suggest that humic acids obtained from lignite using solutions of Na₄P₂O₇ are characterized by a low transformation degree and greater amount of carboxyl groups.

Conclusions

Low rank coal can be successfully used in agriculture as a rich source of humic acids. Reagent used in their extraction, apart from high efficiency should have a neutral impact on their structure. Studies on the physicochemical properties of humic acids can be helpful in predicting behaviors of such fertilizer components in the environment and in inventing new products taking the principles of sustainable development into consideration.

     

AUTOXIDATION OF HUMIC ACID UNDER ALKALINE CONDITIONS

Humic acid was treated for 30 days with normal NaOH under either N₂ or O₂. In both cases some acid-soluble material resulted which was enriched with free and peptide α-amino N and also with total N. The molecular weight of the humic acid fell under alkaline conditions, while there was a decrease in total UV and visible absorption due to the production of less-coloured material which was largely present in the acid soluble fraction. Under O₂ these effects were considerably enhanced and simultaneously a significant arnount of O₂ was consumed. Other changes produced by O₂ were an increase in the cation-exchange capacity, particularly of the acid-soluble fraction, the production of carbonate, an increase in the Oxidation state of the acid, and a lessening of the resemblance of the acid to lignin as shown by the infra-red and UV spectra. In the main the changes produced by O₂ are similar to those observed for humic acids of decreasing molecular weight that can be extracted from a soil, either by differential extraction or by extraction followed by fractionation into different molecular-weight classes.     

RADIOCARBON DATING OF PALEOSOLS USING SOIL ORGANIC MATTER COMPONENTS

Soil organic matter components of classical humic acid and humic fractions were extracted from two paleosols using 0.1 M Na₄ P₃ O₂:0.1 M NaOH. These fractions were subjected to chemical treatment with 6 M HC1 or 70 per cent HNO, before radiocarbon assay, and their radiocarbon dates were compared with those of similar but untreated fractions, untreated buried soils, and previously published charcoal dates. Hydrolysis of humic acid and humin fractions with mineral acids, particularly 70 per cent HNO₃ led to older radiocarbon dates, comparable to, though slightly older than, those of charcoal derived from the same buried layers. In contrast, the dates of soils and their humic acid and humin fractions before acid hydrolysis were younger and therefore less reliable. The application of this procedure to studies of paleosols in New Zealand is discussed.     

Properties of soil humic substances in fractions obtained by sequential extraction with pyrophosphate solutions at different pHs

Abstract

Humic acids (HAs) obtained from four different soils by sequential extraction with pyrophosphate solutions at different pH values (i.e. at pH 3, 5, 7, 9, 11, and 13), were examined by ultraviolet and visible (UV-VIS) spectroscopy and elemental analysis. UV-VIS spectra from 230 to 700 nm were measured, and absorbance coefficients of 1% humic acid solutions at 600 nm (E ^1% ₆₀₀) and Δlog K were calculated. The shape of the spectra of humic acids depended on the soil types rather than on the pH values. The (E ^1% ₆₀₀) values were higher in the neutral region, and lower in the alkaline region, for each pH value they decreased in the following order: buried Andosol > Andosol > Histosol ≧ Cambisol. The Δlog K values for each pH value were higher in the following order: Histosol> Cambisol > buried Andosol≧Andosol. Although the linear correlation was found to be significant between the E ^1% ₆₀₀ and Δlog K values, the relation between these optical properties and pH values differed among the soil types. However, in the neutral region, the higher E ^1% ₆₀₀ values of humic acids from buried Andosol and Andosol and the lower Δlog K values of humic acids from Cambisol and Histosol suggest that the humification degree was higher in the neutral region. Data of elemental composition and atomic ratios were obtained for each humic acid. The H/C ratios in the four soils increased gradually with increasing pH values from 5 to 13, and those for each pH value were commonly higher in the following order: Cambisol> Histosol> Andosol> buried Andosol. The O/C and OIH ratios decreased with increasing pH values. The results of the H/C versus O/C diagram suggested that decarboxylated humic acids were extracted at the higher pH values. A highly significant correlation coefficient was obtained between the H/C and OIH ratios (r= ?0.891***). Results showing that the decrease of the pH values corresponded to the order of the humic acids in the correlation curve in the H/C versus O/H diagram suggest that the humic acids with a higher content of unsaturated bonds and a higher degree of oxidation were extracted at the lower pH values within the range from 5 to 13.     

Chemical studies on soil humic acids

Abstract

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

EXPERIMENTAL CONDITIONS CONCERNING POTENTIOMETRIC TITRATION OF HUMIC ACID

The humic acids of four soils from Denmark have been extracted by EDTA. The humic acids were separated from EDTA by precipitation with Ba²⁺. The ash content was lowered by treatment with HF and HC1. The ash content in the four humic acids was less than 0.6 per cent. Chemical analysis of the humic acids supply some evidence for the suggestion that EDTA extracted organic matter is a more definite fraction than humic acid extracted by NaOH. It was shown that an alkaline humate solution stored in sterile, sealed ampoules under N₂ does not change its initial pH, indicating that the protolytic equilibrium is established instantly. It is therefore proposed that the titration of humic acid should be carried out as quickly as a normal acid-base titration.     

Evaluation of several methods for determining the potassium content in diverse plant materials

Abstract

Different plant analysis methods including varied incubation times with 0.5N and IN hydrochloric acid (HCl), diacid [nitric (HNO₃) and perchloric (HClO₄) acids], triacid [HNO₃, sulfuric acid (H₂SO₄) and HClO₄], H₂SO₄+hydrogen peroxide (H₂O₂) (Wolf method), and 0.5N and lN ammonium acetate (NH₄OAc) were evaluated for measuring the potassium (K) concentration in straw and grain samples of cereal, legumes, oilseed crops, and fruit‐tree leaves. The average K concentration in nine plant materials indicated that K extracted by 0.5N and lN HCl for 5 minutes, 1 hour, and 17 hours contact periods did not differ significantly. But the amount of K extracted by these acidic solutions gradually decreased during incubation, possibly due to reabsorption of released K by the plant material. The amount of K released in to the 0.5N HCl and IN HCl extractants was in close agreement with that obtained with the standard triacid method. The IN NH₄OAc extraction method slightly overestimated the K concentration in the materials compared to the other methods. Differences were observed among the methods in extracting K from different plant materials. In case of grain samples, the triacid method gave slightly higher values than that obtained by IN NH₄OAc extraction. The results suggest that the 0.5N HCl and lN NH₄OAc extraction methods can be used for the determination of K in plant tissues as these methods gave lower standard deviation and coefficient of variation values compared to the triacid method.     

Decomposition and distribution of residual activity of some 13C-microbial polysaccharides and cells,glucose, cellulose and wheat straw in soil

Studies were made to determine the rate of decomposition of some ¹⁴C-labeled microbial polysaccharides, microbial cells, glucose, cellulose and wheat straw in soil, the distribution of the residual ¹⁴C in various humic fractions and the influence of the microbial products on the decomposition of plant residues in soil. During 16 weeks from 32 to 86 per cent of the C of added bacterial polysaccharides had evolved as ¹⁴CO₂. Chromobacterium violaceum polysaccharide was most resistant and Leuconostoc dextranicus polysaccharide least resistant. In general the polysaccharides, microbial cells, and glucose exerted little effect on the decomposition of the plant products. Upon incubation the ¹⁴C-activity was quickly distributed in the humic. fulvic and extracted soil fractions. The pattern of distribution depended upon the amendment and the degree of decomposition. The distribution was most uniform in the highly decomposed amendments. After 16 weeks the bulk of the residual activity from Azotobacter indicus polysaccharide remained in the NaOH extracted soil. From C. violaceum polysaccharide both the extracted soil and the humic acid fraction contained high activity. About 50–80 per cent of the residual activity from the ¹⁴C-glucose, cellulose and wheat straw amended soils could be removed by hydrolysis with 6 n HCl. The greater part of this activity in the humic acid fraction was associated with the amino acids and that from the fulvic acids and residual soils after NaOH extraction with the carbohydrates. About 8 16 per cent of the activity of the humic acid fraction was present in substances (probably aromatic) extracted by ether after reductive or oxidative degradation.     

STUDIES ON THE DECOMPOSITION OF PLANT MATERIAL IN SOIL

The organic matter in soils containing decomposing ¹⁴C-labelled ryegrass was fractionated chemically. Earlier work on these soils had shown that they contained a small fraction, heavily labelled relative to the rest of the soil organic matter, that was mineralized when the partially sterilized soils were incubated. Reagents effective in extracting heavily labelled-C included cold o.in HC1, boiling saturated CaSO₄ solution, and o.in Ba(OH)₂, but neither these nor any other reagent tested could extract material as heavily labelled as that mineralized when partially sterilized soil was incubated. Reagents that extract heavily labelled-C are poor extractants for humified material and are not strongly hydrolytic: the more vigorous the hydrolysis the smaller the proportion of labelled-C in the hydrolysate. The amounts of labelled-C dissolved by Ba(OH)₂ from soils sampled after different periods in the field were directly proportional to the amounts of labelled-C mineralized by those soils when partially sterilized (by exposure to CHC1₃ vapour), inoculated and incubated. Balance sheets are presented for the distribution of labelled and unlabelled-C in fractions separated by hydrolysis with 6N HC1, by NaOH extraction, by neutral pyrophosphate extraction, and by oxidation with H₂O₂. The fraction remaining after hydrolysis with 6N HC1 was the most lightly labelled and had the widest C/N ratio. The percentage of labelled-C in the material dissolved by alkali or by pyrophosphate was little more than in the material not dissolved, despite the presence in the soil of fractions differing at least twenty-fold in intensity of labelling.     

Activity and stability of jack bean urease in the presence of peat humic acids obtained using different extractants

 The effects of two humic acid extractants, 0.1 M Na₄P₂O₇ plus 0.1 M NaOH (NaPP) and 0.1 M NaOH (NaOH), on the activity and stability of a humic-urease complex were studied. The two humic acids isolated (HA_NaPP and HA_NaOH) exhibited different elemental compositions, metal concentrations and structural modifications in the FT-IR spectra. Depending on the pH, HA_NaPP and HA_NaOH influenced both the urease activity and urease kinetic parameters (V _max and K _m) in the same way. They inhibited urease activity between pH 6 and 7 and reduced the V _max of the reaction at pH 6 and 7. The presence of humic acids improved the affinity of the enzyme for the substrate (K _m). The stability of the urease with time, and in the presence of pronase, was improved by HA_NaPP and HA_NaOH with respect to free enzyme. These results confirm the importance of the interaction of urease with humic acids as a fundamental gateway for extracellular urease stabilisation. Since no difference in the extent of urease inhibition and urease stabilisation was observed for the two humic acids, it may be concluded that neither urease activity nor stability are influenced by the humic acid extractant used. Received: 5 November 1999     

Evaluation of Selected Extractants for Plant-Available Silicon in Rice Soils of Southern India

The extractable silicon (Si) using selected extractants irrespective of the soils used for the study was in the order of 0.005 M sulfuric acid (H₂SO₄) > 0.1 M citric acid > N sodium acetate (NaOAc) 2 > N NaOAc 1 > 0.5 M acetic acid 3 > 0.5 M acetic acid 2 > 0.5 M acetic acid 1> 0.01 M calcium chloride (CaCl₂) > 0.5 M ammonium acetate (NH4OAc) > distilled water 4 > distilled water 1. Silicon extraction with N NaOAc 1 appeared to be the most suitable for evaluating Si, followed by extraction with 0.5 M acetic acid 2 and N NaOAc 2. These extractants showed the greatest degree of significant correlation with the percentage of Si in straw and grain, as well as Si uptake by straw and grain. These methods also rapidly extract soil Si in comparison to the other methods and appear to be the most suitable for routine soil testing for plant-available Si in the rice soils of southern India.     

Adsorption and desorption of Cd on humic acid fraction of soils

The adsorption of ionic Cd has been investigated on three humic acids isolated from podzol, rendzina and brown Mediterranean soils of Tuscany. The adsorption isotherms have been determined at 5 and 25°C. Cadmium adsorption was described by the Langmuir adsorption equation. Langmuir parameters were related to the functional groups content of humic acids and decreased in the following order: rendzina>brown Mediterranean soil>podzol. Adsorption was independent on temperature and increased with pH. Desorption experiments with 0.1 N NH₄OAc and 0.25 M Cu (OAc)₂ proved that Cd is adsorbed on humic acid about 50% in an exchangeable form and 50% in coordination complexes.     

Determination of phosphorus in calcareous soils by mehlich 3, mehlich 2, cal,and egner extractants

Abstract

Acid extractants were found to be a problem when determining phosphorus in some soils especially in soils with a higher content of carbonates. Carbonate content, soil pH or extractable calcium cannot exactly predict the behavior of a particular soil. On the other hand, measurement of pH after extraction reflects the ability of soil to change the properties of an extractant. The objective of this study was to find the relationships between pH changes during the extraction and the extraction of phosphorus for Mehlich 3, Mehlich 2, CAL, and Egner extractants. Twelve soil samples from various regions of the Czech Republic were taken for investigation. The pH/KCl of the samples was in the range 4.9–7.3. Calcium carbonate was added into the extracting bottles to the soil samples before extraction. The CaCO₃/soil ratio was from 0 to 0.5. The content of phosphorus and pH were measured in filtrates after extraction. The ratio P₁ =P_meas /P_O was calculated for all measurements, all additions of calcium carbonate and all soils. P_meas is the measured value of phosphorus after extraction with the addition of calcium carbonate and P_o is the value of phosphorus determined after the extraction without addition of calcium carbonate. The ratio P_r reflects the changes in the extractability of phosphorus and it is independent of the absolute content of phosphorus in soil. The relationships between P_r and pH after extraction for CAL, Mehlich 2, and Mehlich 3 extractants were found. No such relationship was found for Egner extractant. Correction equations (x=pH after extraction, y=P_r):     

Effect of humic acid and fulvic acid based liquid and foliar fertilizers on the yield of wheat crop

Abstract

An organic-mineral liquid fertigation fertilizer containing humic acid, nitrogen (N)–phosphorus (P)–potassium (K), zinc (Zn), sulfur (S), manganese (Mn) and a liquid foliar fertilizer consisting of fulvic acid and gibberellic acids were formulated and applied to wheat. The purpose of this study was to develop an efficient and cost effective liquid and foliar fertilizer and compare their potential with commercially available urea and DAP. The fulvic acid and humic acids were obtained by alkaline extraction of lignite coal. All the treatments were significant over the control. The application of the liquid fertigation fertilizer and liquid foliar fertilizer along with 50?kg of urea per acre showed the best results biological yield (grain plus straw yield). It was found that the highest yield was obtained in T₆ treatment and the second best results were obtained with T₂ treatment, but has a high cost, while treatment T₆ was the most significant economical and yielded a high income when compared to other treatments.     

Humus degradation under the influence of simulated ‘acid rain’

The purpose of these investigations was to describe changes occurring in soil under the influence of simulated ‘acid rains’. It was achieved by treating three kinds of soil with water solutions of pH: 2.0, 3.0, 4.0, 5.0, 6.0, 7.8 for two vegetation periods. Differences were found in the pH of soils reaching 2.5 values in the sorption complex. The content of H⁺ increased and the sum of bases considerably decreased. Significant changes in number of bacteria, fungi, macromycetes were found. In most acid solutions they decreased by 100% as compared with the reference surface. The processes of nitrification, cellulose decomposition and soil respiration were limited, whereas ammonification was stimulated. Under the influence of ‘acid rains’ the content of C and N remained unchanged, while the content both of fulvic and grey and brown humic acids was reduced. With the increase in soil acidity the content of hymatomelanic acids increased. So significant changes in the content of bitumen, humins, humic C and the Q_4/6 color quotient were observed under the influence of simulated ‘acid rains’.     

Comparison of carbon skeletal structures in black humic acids from different soil origins

Andosols are characterized by an abundance of black humic acids (HAs) belonging to Type A with a high content of aromatic carbon (C) in particular condensed aromatic C. Black HAs are also observed in other soils, such as Chernozems and the subsoil of paddy field, and extracted after washing with an acid or using chelating agent such as sodium pyrophosphate (Na₄P₂O₇). However, contribution of condensed aromatic structures to those soil HAs are unknown. To obtain the information about C skeletal structures of black HAs in soils other than Andosols, HAs were obtained from 2 Chinese Chernozem samples, 2 subsoil samples from Japanese paddy fields (Fulvisols), and a Rendzina-like soil (Cambisols) as well as an Andosol sample (reference) by successive extraction with 0.1 M NaOH (HAs₁) and 0.1 M Na₄P₂O₇ (HAs₂), and ¹³C nuclear magnetic resonance and X-ray diffraction 11-band profile analyses were applied. In the black HAs₂ from the non-Andosol samples, the proportion of C present as aromatic C, size of C layer planes, and relative C layer plane content ranged from 52 to 59%, 0.48 to 1.92 nm (mean size, 0.76–0.91 nm), and 58 to 100 AU (arbitrary unit) mg^?1, respectively, with a positive correlation between total C layer plane content and the degree of humification. Those ranges were similar to the distribution ranges of Andosols HAs₁ reported by our previous study.     

Proprietes des diphenol oxydases extraites des sols

Properties of diphenol oxidases extracted from salts. Salmine and SP-Sephadex C-25 were used to separate the enzyme system associated with humic materials in the neutral extracts of fresh soils (NAFS Extract). Electrophoresis on polyacrylamide gel shows that this preparation is heterogeneous. The elementary analysis of the soil enzyme is C 43·13%; N 5·09%; H 7·21%; O 44·58%. Chromatographic analyses indicate that the soil enzyme contains 53 per cent amino acids, 36 per cent sugars and amino sugars and 10 per cent ammonium and inorganic materials. The soil enzyme has a maximum absorption at 270–280 nm. The soil enzyme degrades the following substrates at the relative rate mentioned in parentheses : d-catechin (298);p-cresol (251); catechol (156); dl-DOPA (100);p-phenylene diamine (59);p-quinol (20) in terms of rate of oxygen absorption. This enzymatic preparation has the properties of an o- and p-diphenol oxidase. The rate of decarboxylation was measured using a radiorespirometer. The following relative values are dl-DOPA-l-¹⁴C (100); dl-tyrosine-l-¹⁴C (35) ; dl-tyrptophan-1-¹⁴C (7); dl-phenylalanine-l-¹⁴C (2). The dl-DOPA-2-¹⁴C was partially degraded to ¹⁴CO₂. The O₂ absorbed and CO₂ (carboxyl) evolved in case of dl-DOPA was in the ratio of 1·8 at 37°C. The activation energy on dl-DOPA was 3·1 and 7·9 kcal/mole/°C for oxygen absorption and decarboxylation respectively. The enzymatic activity on dl-DOPA-l-¹⁴C was optimum in air and inhibited in a N₂ atmosphere. Decarboxylation on dl-DOPA-l-¹⁴C followed the Michaelis-Menten law, from which we found that K_m = 8·3 × 10^?4M for decarboxylation. The oxidative decarboxylation was inhibited by H₂O₂ (74%); KCN (75%); ascorbate (92%); BAL (97%);DIECA(90%).Melanogenesis of dl-DOPA followed first order kinetics. The maximum absorption at 305 nm during melanogenesis shows the formation of dopachrome.