Claudio Marzadori

Influence of urease and nitrification inhibitors on N losses from soils fertilized with urea

Abstract. The aim of this study was to evaluate how the N losses through volatilization and leaching from soils fertilized with urea can be affected by the application of a urease inhibitor or a urease plus a nitrification inhibitor. The experiment was carried out using lysimeters with 15N-labelled urea and N-(n-butyl) thiophosphoric triamide (NBPT) as urease inhibitor and dicyandiamide (DCD) as nitrification inhibitor, comparing three different treatments: urea alone (U), urea + NBPT (UN) and urea + NBPT + DCD (UND). Both volatilization and leaching were significantly different in the soils used, according to their physico-chemical characteristics. However, the pattern of the loss was similar: the volatilization was significantly reduced by NBPT (UN), but the presence of DCD (UND) significantly increased the loss, with respect to UN. Considering leaching, the highest amount of NO3– was lost with UND, the lowest with U. The greatest amount of N lost by leaching was soil-derived N produced by the N mineralization-immobilization turnover. We suggest that, by maintaining the NH4+ in the soils, the inhibitors, in particular DCD, caused a priming effect with a subsequent increase in the rate of soil organic matter mineralization and an extra release of soil organic N. The priming effect was real in the sandy loam (SL) soil where a net N release was observed, whereas in the clay loam (CL) soil the effect of the inhibitors was less pronounced and an apparent priming effect was observed; however, a real priming effect also cannot be excluded in this soil.

Urease from the soil bacterium Bacillus pasteurii: immobilization on Ca-polygalacturonate.

Abstract Urease purified from the soil bacterium Bacillus pasteurii was adsorbed and immobilized on a preformed network of Ca-polygalacturonate, a substrate which has a similar composition and morphology to the mucigel present at the root-soil interface. The adsorption proceeded with an essentially quantitative yield, and the immobilized enzyme showed no decrease of specific activity with respect to the free enzyme. The dependence of urease adsorption on NaCl concentration suggested that the enzyme is bound to the carrier gel through electrostatic interactions. The immobilized enzyme showed increased stability, with respect to the free enzyme, with increasing time or temperature, and in the presence of proteolytic enzymes. The pH activity profile revealed that the adsorbed enzyme showed no change in the optimum pH (8.0), but it was more active than the free form in the pH range 5–8. The Michaelis-Menten kinetic pararneters V max and K m were measured for the free ( V max = 1960 ± 250 units ml −1 ; K m = 235 ± 20 mM) and immobilized ( V max = 1740 ± 185 units ml −1 ; K m = 315 ± 25 mM) urease. The substantial similarity of V max in the two cases suggests that there were no conformational changes involving the active site upon enzyme immobilization, while substrate partitioning effects between the bulk solution and the micro-environment surrounding the immobilized enzyme must be operating so as to partly increase its K m . These results suggest that bacterial urease present in plant root mucigel plays a large role in the mobilization of urea N. Its activity is in fact significantly mantained and protected by immobilization on hydrophilic gels such as those produced by root exudates.

Effects of lead pollution on different soil enzyme activities

We studied the effects of Pb pollution on soil dehydrogenase and phosphatase activity. Samples of four soils (Saxe, Podestà, Porto Teulada, and Sa Xia Manna) were collected from various locations in southwestern Sardinia, Italy. The soils, which differ mainly in heavy metal contents of pedologic origin (Cu, Zn, Cd, and Pb), were treated with Pb (0, 100, 500, 1000, and 5000 μg Pb g-1 soil) and incubated in the laboratory. Samples of the incubated soils were collected periodically (0, 1, 2, 4, 8, and 16 weeks) and the enzymes were measured. Soil dehydrogenase activity was influenced by both the Pb additions and variations in soil moisture content. Only the addition of 5000 μg Pb g-1 soil led to a significant decrease in dehydrogenase activity compared to the controls, while the other doses of Pb did not always result in a clear reduction in enzyme activity. Drying the soil led to a considerable reduction in dehydrogenase activity, sometimes so far as to render the differences found between the various treatments not statistically significant. Soil phosphate activity was also influenced by the Pb additions, but the effect of the variation in soil moisture content was less than that found for the dehydrogenase. After the 2nd week of incubation, the phosphate activity in the Podestà and Saxe soils had decreased proportionally to the increase in Pb content. At the end of the incubation period, in the Porto Teulada and Sa Xia Manna soils, a net reduction in phosphatase activity versus controls was found only at the highest Pb concentration. Although both enzyme activities were influenced by the Pb additions, the phosphate activity was less sensitive to variations in the soil moisture content and may thus be a more suitable indicator for soil pollution by Pb.

Effects of the urease inhibitor N-(n-butyl)phosphorothioic triamide in low concentrations on ammonia volatilization and evolution of mineral nitrogen

Laboratory incubation experiments were conducted to study the influence of increasing concentrations of N-(n-butyl)phosphorothioic triamide (NBPT) on NH3 volatilization and rate of urea hydrolysis and evolution of mineral N in Ozzano, Rimini and Carpi soils with different physicochemical characteristics. Low concentrations of NBPT reduced NH3 losses due to volatilization after urea fertilization and the effectiveness of the inhibitor was related to the soil characteristics (e.g. high concentrations of organic C and sand). After 15 days of incubation, no significant reductions of losses were found for any of the NBPT concentrations employed in Rimini soil. The application of NBPT led to a considerable reduction of the formation of nitrite. This process was completely annulled with the highest dose of NBPT (0.5% w/wurea) in the Carpi soil after 15 days. In Rimini soil, however, the use of NBPT was less effective in influencing nitrite formation. The use of NBPT favoured accumulation of nitrate proportional to the NBPT concentration employed while it had no influence on the NHinf4sup+fixation by 2:1 layer silicates. The data obtained support previous evidence that NBPT is effective in reducing the problems encountered in using urea as fertilizer. However, environmental conditions and soil physicochemical characteristics may have an important influence on the effectiveness of NBPT.

Dynamics of Mineral Nitrogen in Soils Treated with Slow‐Release Fertilizers

Abstract In this study, five different slow‐release fertilizers were incubated in two soils, and their nitrogen (N) dynamics was followed for 4 months. Four of the fertilizers used were organic: two were hydrolyzed fur, one was made by hydrolyzed fur, farmyard manure, horn, and hoofs (Endurance), and one contained synthetic organic N [isobutylidendiurea (IBDU)]. The fifth, on the contrary, was a mineral fertilizer (Entec 26) containing both nitrate (NO3 −) and ammonium (NH4 +) and a nitrification inhibitor (3,4 DMPP). The two soils used were a sandy loam (SL) and a clay loam (CL). The fertilizer‐N release was conditioned by both the fertilizers and the soil characteristics. The highest accumulation of mineral N as NO3 − was observed in the SL soil for all the treatments. The lowest net N release was observed in both soils with End, which was found to be the most resistant to microbial attack and degradation. The two fur‐based fertilizers released similar amounts of N, which was higher in the SL soil than in the CL soil. The highest accumulation of fertilizer‐derived N in the CL soil resulted from IBDU; however, the net accumulation of N in the SL soil with this fertilizer was even higher than the amount of N initially added. This fact was attributed to an increase in mineralization of the soils native organic N. A real positive “priming effect” was also observed in the SL soil with E26. On the contrary, E26 caused a low level of N accumulation in the CL soil.

Kinetic properties and stability of potato acid phosphatase immobilized on Ca-polygalacturonate

Abstract Acid phosphatase from potato was adsorbed and immobilized on a pre-formed network of Ca-polygalacturonate, a substrate which has a composition and morphology similar to the mucigel present at the root-soil interface. The influence of different types of organic buffers on enzyme adsorption and activity was investigated. The highest enzyme activity, for free and adsorbed enzyme, was obtained with Na-maleate buffer at pH 6.0, which was used for all subsequent experiments. The Michaelis-Menten kinetic parameters, Vmax and Km, were determined for free and adsorbed phosphatase. Vmax showed a 60% decrease upon adsorption (2.09±0.30 U/mg, for the soluble form and 0.84±0.15 U/mg, for the adsorbed enzyme), whereas Km increased from 0.49±0.15 mM for the free enzyme to 0.99±0.20 mM for adsorbed phosphatase. Phosphatase adsorption decreased as the concentration of NaCl increased, indicating that the enzyme is bound to the carrier gel through coulombic interactions. Adsorption increased stability of the enzyme as compared with the free enzyme (t1/2 of the activity was 9.4 days and 5.8 days, respectively), but increased thermal and proteolytic inactivation. The pH/activity profile revealed no change in terms of shape or optimum pH (4.5) upon adsorption of the enzyme. These results indicate that adsorption of acid phosphatase on Ca-polygalacturonate induces changes in the kinetic properties and stability of the enzyme, and the same type of response can be extrapolated from these results for acid phosphatases of the rhizosphere.

Influence of the content of heavy metals and molecular weight of humic acids fractions on the activity and stability of urease

The aim of this work was to evaluate the effect of two different humic acid (HA) fractions, high molecular weight (HMW 100‐300 kDa) and low molecular weight (LMW 10‐20 kDa), extracted from peat on the activity and stability of Jack Bean urease. HMW HA significantly inhibited urease activity at pH 6.0 but did not influence the activity at pH 7.0 and 8.0. HMW HA stabilised urease activity over a period of 11 days to treatments with protease and with Cu 21 and Hg 21 (two powerful inhibitors of soluble urease activity). The LMW HA inhibited urease activity at pH 6.0, 7.0 and 8.0, and did not stabilise urease activity in the presence of protease. The residual activity of urease, at pH 7.0 and 8.0 in the presence of LMW HA and Cu 21 , was 90 and 69%; in the presence of LMW HA and Hg 21 it was 81 and 52%. The residual activity of urease, at pH 7.0 and 8.0, in the presence of HMW HA and Cu 21 was 99 and 88%; in the presence of HMW HA and Hg 21 it was 94 and 74%. These results showed that the two HA fractions influenced both the activity and stability of the urease differently. It is proposed that the inhibition of the urease by HMW and LMW HA is mainly due to the two heavy metals which, although immobilised on the HA, are still able to interact with the urease. q 2000 Elsevier Science Ltd. All rights reserved.

Role of Uronic Acid Polymers on the Availability of Iron to Plants

Abstract The interaction between polygalacturonic acid and Fe(III) was studied in the presence and in the absence of pyruvic, malic, and citric acids. Kinetical data and FT‐IR analyses show that the polysaccharidic matrix acts as an accumulator of Fe(III) and that the metal ion interacts electrostatically with both the carboxylic and other functional groups of the polysaccharidic matrix. Copper(II) ions, which have a high affinity towards the carboxylic groups of the polysaccharide, do not influence markedly the Fe(III) absorption indicating that the carboxylic groups are not determining in the Fe(III) accumulation process. Furthermore, the results suggest that iron inside the fibrils is under an hydrolyzed form or as Fe(III) hydroxy polymer. In the presence of malic and citric acids the amount of Fe(III) accumulated at pH 4.7 and 6.0 is markedly lower than that found in the presence of pyruvic acid what was attributed to the higher affinity of citric and malic acid towards the metal ion.

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

Abstract 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 (HANaPP and HANaOH) exhibited different elemental compositions, metal concentrations and structural modifications in the FT-IR spectra. Depending on the pH, HANaPP and HANaOH influenced both the urease activity and urease kinetic parameters (Vmax and Km) in the same way. They inhibited urease activity between pH 6 and 7 and reduced the Vmax of the reaction at pH 6 and 7. The presence of humic acids improved the affinity of the enzyme for the substrate (Km). The stability of the urease with time, and in the presence of pronase, was improved by HANaPP and HANaOH 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.

Activity and stability of urease-hydroxyapatite and urease-hydroxyapatite-humic acid complexes

In agricultural calcareous soils, hydroxyapatite (APA) may well represent an important support for urease immobilisation and could be present in both mineral and organo-mineral complexes. In this paper we studied the formation of APA-urease-humic acid (HA) complexes after the addition of urease either before or after HAs. We then proceeded to evaluate the role of HAs on the activity and stability of the complexes as compared to the APA-urease complexes and free urease. When increasing amounts of HAs were added after urease, they did not affect the activity of the enzymes that had already adsorbed onto the complexes. On the contrary, adding the same amount of HA before the enzyme caused a significant reduction in the amount of enzyme adsorbed. However, when urease adsorption onto the APA-HA complexes was carried out in the presence of NaCl, the enzyme activity of the complexes increased sharply to 86% of the initial activity. The immobilisation of the enzyme on the support increased urease stability against pronase treatment as well as directly in soil over time. The inhibition of urease activity by Cu2+ was reduced by urease immobilisation. However, the presence of HA did not alter the stabilisation capability of APA when alone.