Measurement of electrostatic and site-specific associations of alkali metal cations with humic acid

A discontinuous acidimetric titration method incorporating ultrafiltration was developed to measure the association of a soil humic acid with Li⁺ t, Na⁺ and K⁺ (pH 3 to 8). In addition, possible site-specific binding of these alkali metal cations was investigated using desorption experiments at pH 1. Li, Na and K cations behaved equivalently in the titrations and the amounts of these cations associated with the humic acid was measurable at all pH values between 3 and 8. Up to 90% of the total alkali metal cation was humate-associated at pH 8. The absolute amount of humic-associated cation did not depend on the alkali metal cation concentration, but rather on the solution alkalinity. In addition, the net charge of the humate polyanion made a negligible contribution to the electroneutrality of the bulk solution under all conditions. These results are consistent with a diffuse layer model of hydrated humic acid in which the alkali metal cations neutralize the humic charge. The association of Na+ andK+ with humic acid at pH 1 was successfully described by a Langmuir adsorption model. The number of sites per g of humic acid was very small, and greater for K+ than for Nat. Lithium cations exhibited no detectable humic association at pH 1. These differences suggest that humic acids may have a small number of specific binding sites for which the size of the hydrated cation is important.     

Study of the acid‐base properties of a peat soil and its humin and humic acid fractions

For accurate interpretation of cation binding to natural organic matter, the proton binding behaviour of both solid and dissolved natural organic matter must first be established. In the present study, potentiometric titrations of samples of humin and humic acid extracted from a peat soil were performed at different ionic strengths. Humic acid (HA) samples in solution (dissolved humic acid, DHA) and in suspension (aggregated humic acid, AHA) were titrated. The corresponding charge curves were analysed with the NICA‐Donnan model and the results were compared with those previously obtained for the peat soil. Good reproduction of the DHA charge curves was obtained, and in the case of the AHA, the exact forms of the charge curves were not reproduced at pH < 6 because of the conformational changes and dissolution of the sample that took place throughout the titration. The peat and humin displayed similar proton binding behaviour, which was different to that of the humic acid. With the Donnan approach, the value of parameter b, that relates the Donnan volume to the ionic strength, was the same for peat and humin and less than that of the humic acid, for both the dissolved samples and those in suspension, indicating that the effect of ionic strength on the charge is greater in the peat and humin than in the HA. The ratio between the contents of phenolic groups and of carboxylic groups was greater in peat and humin than in humic acid. The model used revealed that the acid‐base behaviour of the peat is closer to that of the humin than to that of the humic acid.     

Cation binding by acid-washed peat, interpreted with Humic Ion-Binding Model VI-FD

A sample of ombrotrophic peat from Moor House in northern England was extensively extracted with dilute nitric acid (pH 1) to free it of bound cations. Suspensions of the acid‐washed peat (5–30 g l^?1), prepared with different concentrations of background electrolyte (NaCl and KCl), were used to conduct batch acid–base titrations. A strong dependence of proton release on ionic strength (I) was observed, the apparent acid dissociation constant (pK_app) being found to decrease by approximately 1.0 for each tenfold increase in I. This behaviour could not be explained satisfactorily with Humic Ion‐Binding Model VI, a discrete‐site/electrostatic model of cation binding by humic substances, parameterized with data from laboratory studies on isolated samples. More success was obtained by abandoning the impermeable‐sphere electrostatic submodel used in Model VI, and instead assuming the peat to consist of aggregates with fixed internal volume, and with counterion accumulation described by the Donnan model, as proposed by Marinsky and colleagues. The fixed‐volume Donnan model (Model VI‐FD) could also approximately explain other reported results from acid–base titrations of peat, including the effects on the titrations of complexing cations (Al, Ca, Cu). Copper titrations of the Moor House sample were performed using an ion‐selective electrode, with peat suspensions in the acid pH range, at two ionic strengths, and in the presence of Al and Ca. The measured concentrations of Cu²⁺ were in the range 10^?13?10^?5 m . Model VI‐FD provided reasonable fits of the experimental data, after optimization of the intrinsic binding constant for Cu, the optimized value being close to the default value derived previously from data referring to isolated humic substances. The optimized constants for Al and Ca, derived from their competition effects, were also close to their default values. Additional experiments were performed in which the centrifugation‐depletion method was used to measure the binding of a cocktail of metals (Al, Ni, Cu, Zn, Cd, Eu, Pb) at a single pH. The model correctly predicted strong binding of Al, Cu, Eu and Pb, and weaker binding of Ni, Zn and Cd. For the strongly binding metals, the dissolved forms were calculated to be mainly due to complexes with dissolved humic matter, whereas the free ions (Ni²⁺, Zn²⁺, Cd²⁺) dominated for the weakly binding metals. Acid‐washed soil appears to provide a valuable intermediate between isolated humic substances and untreated soil for the investigation of cation binding by natural organic matter in the natural environment.     

An Experimental and Modelling Study of Cu2+ Binding on Humic Acids at Various Solution Conditions. Application of the NICA-Donnan Model

Humic substances are characterized by a strong binding capacity for both metals and organic pollutants, affecting their mobility and bioavailability. The understanding of the mechanisms of proton and metal binding to humic substances is of fundamental importance in geochemical modelling and prediction of cation speciation in the environment. This work reports results on copper binding on humic acids obtained through a thorough experimental and modelling approach. Two humic acids, a reference purified peat humic acid isolated by the International Humic Substances Society (IHSS) and a humic acid from a Greek soil, were experimentally studied at various pH values (4, 6 and 8), humic acid concentrations (ranging from 20 to 200 mg?L^?1) and ionic strength (0.1 and 0.01 M NaNO₃). The binding of copper to humic acids was determined over wide ranges of copper ion concentrations using a copper ion selective electrode. The copper binding isotherms obtained at different conditions have shown that copper binding is dependent on the pH and ionic strength of the solution and on the concentration of both humic acids. Copper binding experimental data were fitted to non-ideal competitive adsorption NICA-Donnan model and the model parameter values were calculated. Both Cu²⁺ and CuOH⁺ species binding to humic acid with different binding affinities were considered. Two sets of the NICA-Donnan parameters have been calculated: one for humic acid concentrations of ??100 mg?L^?1and one for humic acid concentration of 20 mg?L^?1. The meaning of the parameters values for each concentration level is also discussed.     

Sorption of cadmium by complexes of kaolinite with humic acid

Abstract

Levels of cadmium (Cd) in New Zealand pastoral soils have increased due to Cd impurities in applied fertilisers. As there is little information on the interaction of Cd with soil mineral‐organic matter complexes, the sorption of Cd by complexes of kaolinite with humic acid has been investigated. Sorption was measured at pH and ionic strength values typically found for solutions of pastoral soils in New Zealand. Sorption increased with the content of humic acid in the complex, and as the pH of the medium was raised from 4.2 to 6.3. Sorption was also influenced by the ionic strength of the ambient solution, notably by the nature of the cation in the added electrolyte. The experimental data were interpreted in terms of the effect of solution pH and ionic composition on the charge characteristics of kaolinite and humic acid. These factors, in turn, influence clay particle association as well as the clay‐humic and metal‐humic interaction.     

Analysis of the variable charge of two organic soils by means of the NICA-Donnan model

We have tested to see if the generic set of NICA‐Donnan model parameters, used to describe isolated humic substances, can also describe soil humic substances in situ. A potentiometric back‐titration technique was used to determine the variable surface charge of two organic peat soils at three different ionic strengths. The non‐ideal, competitive‐adsorption NICA‐Donnan model was used to simulate the surface charge, by assuming a bimodal distribution of H⁺ affinity on the soil solid phase. The model provided an excellent fit to the experimental data. The Donnan volume, V_D, varied slightly with ionic strength, although the variation was less than for humic substances in solution. The values obtained for the parameters that define the affinity distributions, the intrinsic proton binding constant (log K_i^int) and the heterogeneity of the site (m_i), were similar to those observed for isolated soil humic acids. The abundance of carboxylic groups in the whole soil represented 30% of the typical value for isolated soil humic acids. The composition of the organic matter of the whole soils, obtained by ¹³C CPMAS NMR, was comparable to the characteristic composition of soil humic acids.     

Proton interactions with soil organic matter: the importance of aggregation and the weak acids of humin

Samples of three organic‐rich soils (ombrotrophic peat, podzol H‐horizon, humic ranker) were extensively washed with dilute nitric acid, dialysed against deionised water, and then subjected to acid‐base titrations over the pH range 3–10, in 0.3–300 mm NaNO₃, and with soil concentrations in the range 2–150 g l^?1. The results for the three soils were quantitatively similar. Comparison of the titration data with previously published results for humic acids isolated from the same soils showed the soil organic matter to have a greater ionic strength dependency of proton binding and to possess relatively greater buffering capacity at high pH, attributable to weak acid groups (c. 2–5 mmol g^–1) in the humin fraction of the soils. To describe the soil titration data quantitatively, we modified Humic Ion‐Binding Model VI‐FD, which utilizes a fixed Donnan volume to describe counterion accumulation, by increasing the content of weak acid groups. When artefacts in pH measurement caused by the suspension effect were taken into account, the resulting Model VI‐FD2 provided good or fair simulations of all the titration data. The results suggest that soil structure, specifically aggregation, plays a significant role in cation binding by organic soils in situ. The lack of dependence of the titration results on soil suspension concentration suggests that the findings can be applied to soils in situ.     

ANION ADSORPTION BY GOETHITE AND GIBBSITE

The relationship between adsorption and pH, termed the adsorption envelope, varies with the pKa, of the conjugate acid of the anion, for several adsorbents in presence of an excess of specifically adsorbed anions. At pH values where the acid is fully dissociated, specific adsorption occurs only to the extent of the positive charge of the surface and little specific adsorption is found at pH values more alkaline than the zero point of charge (zpc). With incompletely dissociated acids, anion adsorption can also take place at pH values more alkaline than the zpc providing the pH is somewhere near a pKa value of the acid, where the energy required to abstract a proton from the acid is at a minimum. The proton is required for the removal of a surface OH which provides a site for the anion. Specific anion adsorption (i) from a fully dissociated acid can reduce the positive charge of the surface to zero and (ii) from a weak acid can make even a negative surface more negative. Thus specific adsorption shifts the zpc to more acid values. The relationship between anion adsorption and OH release is not necessarily simple. The ideas proposed for anion adsorption have implications for cation adsorption and incorporation in an oxide surface.     

Soil humic acid aggregation by dynamic light scattering and laser Doppler electrophoresis

Humic acids (HAs), similar to other fractions of humic substances (HSs), have a large number of reactive functional groups enabling them to aggregate in solutions. Regardless of the origin of humic acid (aqueous or soil), this aggregation process is dependent on environmental conditions and strongly influences the mobility of soluble ionic and molecular pollutants. The aim of this work was to monitor the aggregation process of two humic acids isolated from different mineral soils (IHSS Elliot soil HA standard and Rendzic Leptosol HA) in the 2–11 pH range. Changes in aggregate size in HA sols were followed up using dynamic light scattering (DLS), while zeta potential (ZP) measurements in the same pH range were performed applying laser Doppler electrophoresis (LDE) technique. The effect of HA sol concentration and soil source on aggregation was examined as well. Besides, HA samples were characterized using Fourier transform infrared (FT‐IR) spectroscopy. By inspecting HA‐particle‐size dependence on pH, it can be concluded that both HAs in corresponding sols behave as molecular aggregates or supramolecular structures, formed from small individual moieties (sizes < 10 nm) at higher pH values. The ZP vs. pH curve for both HAs revealed the ZP minimum in the 5–7 pH range, caused most likely by dissociation of acidic functional groups prevailing at lower pH values and deaggregation predominating over dissociation at higher pH values.     

Factors controlling the partitioning of pyrene to dissolved organic matter extracted from different soils

The mobility of hydrophobic organic compounds (HOCs) in soils can be influenced by the presence of dissolved organic matter (DOM). While numerous studies have determined interactions of HOCs with humic and fulvic acids, only few data exist on the partitioning of HOCs to natural, non‐fractionated DOM as it occurs in soil solutions. In this study, DOM was extracted from 17 soil samples with a broad range of chemical and physical properties, originating from different land uses. The partition coefficients of pyrene to DOM were determined in all soil extracts and for two commercial humic acids using the fluorescence quenching method. For the soil extracts, log K_DOC values ranged from 3.2 to 4.5 litres kg^?1. For the Aldrich and Fluka humic acids, log K_DOC was 4.98 and 4.96 litres kg^?1, respectively, thus indicating that they are not representative for soil DOM. After excluding these two values, the statistical analysis of the data showed a significant negative correlation between log K_DOC and pH. This was also shown for one sample where the pH was adjusted to values ranging from 3 to 9. A multiple regression analysis suggested that ultraviolet absorbance at 280 nm (an indicator for aromaticity) and the E4:E6 ratio (an indicator for molecular weight) had additional effects on log K_DOC. The results indicate that the partitioning of pyrene to DOM is reduced at alkaline pH, probably due to the increased polarity of the organic macromolecules resulting from the deprotonation of functional groups. Only within a narrow pH range was the K_DOC of pyrene mainly related to the aromaticity of DOM.     

Ionic-strength and pH effects on the sorption of cadmium and the surface charge of soils

Two Oxisols (Mena and Malanda), a Xeralf and a Xerert from Australia and an Andept (Patua) and a Fragiaqualf (Tokomaru) from New Zealand were used to examine the effect of pH and ionic strength on the surface charge of soil and sorption of cadmium. Adsorption of Cd was measured using water, 0.01 mol dmp^?3 Ca(NO₃)₂, and various concentrations of NaNO₃ (0.01–1.5 mol dm^?3) as background solutions at a range of pH values (3–8). In all soils, the net surface charge decreased with an increase in pH. The pH at which the net surface charge was zero (point of net zero charge, PZC) differed between the soils. The PZC was higher for soils dominated by variable-charge components (Oxisols and Andept) than soils dominated by permanent charge (Xeralf, Xerert and Fragiaqualf). For all soils, the adsorption of Cd increased with an increase in pH and most of the variation in adsorption with pH was explained by the variation in negative surface charge. The effect of ionic strength on Cd adsorption varied between the soils and with the pH. In Oxisols, which are dominated by variable-charge components, there was a characteristic pH below which increasing ionic strength of NaNO₃ increased Cd adsorption and above which the reverse occurred. In all the soils in the normal pH range (i.e. pH>PZC), the adsorption of Cd always decreased with an increase in ionic strength irrespective of pH. If increasing ionic strength decreases cation adsorption, then the potential in the plane of adsorption is negative. Also, if increasing ionic strength increases adsorption below the PZC, then the potential in the plane of adsorption must be positive. These observations suggest that, depending upon the pH and PZC, Cd is adsorbed when potential in the plane of adsorption is either positive or negative providing evidence for both specific and non-specific adsorption of Cd. Adsorption of Cd was approximately doubled when Na rather than Ca was used as the index cation.     

Effects of changes in pH, ionic strength, and sulphate concentration on the CEC of temperate acid forest soils

As the acidity of rain diminishes, changes in the pH, ionic strength, and ion activities of the soil solution will influence the charge characteristics of soil. We have investigated the response of cation exchange capacity (CEC) of three acid forest soils of variable charge to small changes in pH, ionic strength, and SO^2?₄ concentration. The variable charge for these temperate soils has the same significance as for tropical soils and those from volcanic ash. Maximum absolute increase in CEC on increasing pH by 0·2–0·5 units reached 5 cmol_c kg^-1 in O horizons. The increase in CEC on doubling ionic strength in EA and Bsh horizons of a Cambic Podzol was about half that amount, but relative gains compared to effective CEC were 65 and 46%, respectively. For other soil horizons, absolute changes were smaller, and relative changes were between 10 and 30%. Halving the SO^2?₄ concentration significantly influenced CEC only in some samples. Both pH and ionic strength must be adjusted with care when determining CEC_c of acid forest soils. Decreasing acid deposition will not inevitably increase CEC_c because in some soils pH effects may be balanced by simultaneous decrease in ionic strength.     

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.     

Effects of pH,solid/solution ratio,ionic strength,and organic acids on Pb and Cd sorption on kaolinite

Potentiometric and ion-selective electrode titrations together with batch sorption/desorption experiments, were performed to explain the aqueous and surface complexation reactions between kaolinite, Pb, Cd and three organic acids. Variables included pH, ionic strength, metal concentration, kaolinite concentration and time. The organic acids used were p-hydroxybenzoic acid, o-toluic acid, and 2,4-dinitrophenol. Titrations were used to derive previously unavailable aqueous conditional stability constants for the organometallic complexes. Batch results showed that aqueous lead-organic complexation reduced sorption of Pb by kaolinite. Cadmium behavior was similar, except for 2,4-dinitrophenol, where Cd sorption was increased. Metal sorption increased with increasing pH and decreasing ionic strength. Distribution ratios (K _d 's) decreased with increasing solid/solution ratio. The subsurface transport of lead and cadmium may be enhanced via complex interactions with organic wastes or their degradation products and sorbent mineral surfaces.

     

RATIONALIZATION OF IONIC STRENGTH AND CATION EFFECTS ON PHOSPHATE SORPTION BY SOILS

The amounts of inorganic phosphate (P) sorbed by four contrasting unfertilized soils during 40 h were influenced by the ionic strength and cation species of the contacting solution (support medium) used, as indicated by isotherms over the final P concentration range of 0 to 1 μg P/ml and 0 to 10 μg P/ml. An increase in ionic strength enhanced P sorption during 40 h but the species of cation also influenced the amount of P sorbed, as shown by the isotherms obtained in 10^?2M Ca and 3 × 10^?2M Na systems. Although pH affected the amounts of P sorbed, pH effects alone could not adequately explain the differences in P sorption. Kinetic studies indicated that within the range of P addition used for each soil, the equilibrium P concentration, at infinite time, was independent of ionic strength and cation species. Consequently, the composition of the solution affected only the rate at which equilibrium was attained. The results are attributed to the effects of ionic strength on the surface charge of retaining components and the thickness of the diffuse double layer, and the effects of specilic sorption of a divalent cation on surface charge, as they relate to the rate of P sorption.     

Effect of soil depth on acid properties of humic substances extracted from an ombrotrophic peat bog in northwest Spain

The most southerly ombrotrophic peat bogs in Europe are in Galicia (northwest Spain). The humic matter in them originates from chemical processes in anaerobic conditions. We studied the acid properties of fulvic acids and humic acids isolated from two peat horizons of an ombrotrophic peat bog by potentiometric titration. Solutions containing 25, 50 and 100 mg l⁻¹ of each humic substance were titrated at ionic strengths 0.005 m , 0.01 m and 0.1 m (with KNO₃ as the inert electrolyte). Charge curves were analysed with a Donnan model to determine the intrinsic proton binding parameters. The concentration of the humic substance affected the charge curves more significantly at pH exceeding 6, and tended to disappear at greater concentrations. The proton binding conditional constants decreased with increasing ionic strength, this effect being more significant in the carboxylic groups with less affinity for protons. The proton binding constant of the carboxyl groups in a fulvic acid was one order of magnitude less than the value for the corresponding humic acid, whereas for the phenolic groups the values for both fractions were similar. The total content of acid groups was approximately 2 mol kg⁻¹ greater in the fulvic fraction than in the humic fraction. Both humic fractions from the lower horizon contained more acid groups than those from the upper horizon, mainly because the content of carboxyl groups increases with soil depth. Therefore, the humic substances in the lower horizon of the peat will be more negatively charged, which will affect their solubility and the binding of metal ions.     

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.     

THE HUMIC ACIDS EXTRACTED BY VARIOUS REAGENTS FROM A SOIL

Solutions of o.5N NaOH, o.1M pyrophosphate (pH 7), and o.5N Na(CO^2?₃/HCO^?₃) [2:1] extract humic acid and organic matter from a soil with decreasing effectiveness. Pre-treating the soil with o.1N HC1 increased the yield of humic acid obtained with the alkaline extractants. An additional pre-treatment with a mixture, which was normal with respect to HC1 and HF, gave a slight reduction in yield. Increasing the temperature of extraction increased the yield of humic acid. The total C extracted was usually in excess of the humic acid recovered. The difference was obtained as ‘humins’. The sum of the Fe₂O₃, SiO₂, and A1₂O₃ contents of the humic acids was always less than 2 per cent. Where the extraction was carried out at room temperatures the SiO₂/Al₂O₃ ratio suggested that they might be present as clay mineral. When extraction was carried out at an elevated temperature this ratio was altered. Humic acids of low ash content (0.1–0.5 per cent) could be obtained by the use of hot reagents. Of all the extractants used at room temperature, pyrophosphate produced the humic acid of lowest ash content (～ 0.2 per cent). The Fe₂O₃ content of the humic acids was not correlated with their SiO₂ or A1₂O₃ content. The N-content of the humic acids was substantially independent of the method of extraction. The cation-exchange capacities (C.E.C.), average pK values and range of pK values, have been determined from the titration curves of the humic acids. These quantities vary with the method of extraction. There are good correlations between cation exchange capacity and both average pK values and the range of pK values. The within-molecule variation of pK values appears to be greater than the between-molecule variation. No correlation exists between C.E.C. and Fe, Al, Si, and N content of the humic acids.     

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