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Effect of Urease Inhibitors on Volatile N Loss From Soil and Other N Transformations
M. S. Coyne
Department of Plant and Soil Sciences
One of the dominant concerns in environmental science is the impact of non-point source pollution from agricultural fertilization on water quality. Anything that preserves N, prevents its transformation to NO3-, and allows reduction in its use can have a positive impact on environmental quality. Urea is the dominant type of solid N- fertilizer sold globally. It is also a key element in commercial turfgrass production and lawn maintenance. Urea synthesis requires energy, and as the cost of energy rises, so does the cost of fertilizer N derived from urea.
There is much economic incentive to investigate ways of minimizing urea hydrolysis and the potential loss of volatile NH3-N. Rapid urea hydrolysis also releases NH3 and causes odor near confined animal operations. Rapid urea hydrolysis also releases N beyond the needs of crop uptake. Subsequent N-transformation processes can convert this N to NO3-, which is a soluble anion and has been noted to contribute to eutrophication in the Gulf of Mexico.
From an economic and environmental perspective the mechanisms by which urea hydrolysis is controlled in soil systems are important. Commercial urease inhibitors such as NBPT (N-(n-butyl) thiophosphoric triamide, trade name AGROTAINr) and N-Guardr have been used to retard urea hydrolysis. The subsequent influence of these compounds on other N-transformation processes has yet to be thoroughly investigated. Inhibition of urea hydrolysis can have an indirect effect on nitrification rates in soil environments because it decreases substrate availability. There could also be direct effects. New urease inhibitors reaching the market are based on the cation exchange capacity of polymers that are assumed to inhibit urease by adsorbing the nickel (Ni) in the active site. This has yet to be adequately demonstrated and shown to occur beyond the level of the urease itself, that is, can these polymers also affect the urease-producing organisms themselves or other significant N-transformation processes.
There are other enzymes in N-transformation processes that contain metal co-factors in active sites. Three such enzymes are ammonia monooxygenase (Cu), nitrate reductase (Mo), and nitrous oxide reductase (Cu). If the polymers adsorbing Ni from extracellular urease have a similar effect on these other enzymes, it could significantly alter N-transformation processes in soil. Retarding further conversion of N2O to N2 during denitrification could lead to increased trace gas evolution from urease-treated soil.
The research proposed in this study will make contributions to basic science by examining the potential for metal co-factors in N-transformations to be manipulated by chelating compounds. And on an applied level, it will investigate the efficacy and potential drawbacks of existing mechanisms for inhibiting urease in soil environments, thereby contributing to better N-use efficiency and environmental quality.
2009 Project Description
The following experiments were conducted in support of the proposed research activities:
1. ACTIVATION STUDIES. Purified jack bean urease was exposed to NBPT incubated with various compounds to determine the effect on suppressing urease activity in vitro: A) Activation by hydrogen peroxide. For activation by hydrogen peroxide NBPT solutions ranging from 0 to 0.03% (w/v) were incubated with concentrations of a commercial hydrogen peroxide mix ranging from 0 to 0.8% and incubated overnight. B) Activation by ferric chloride: For activation by ferric chloride, NBPT solutions ranging from 0 to 0.125% (w/v) were incubated with concentrations of ferric chloride ranging from 0 to 0.1% and incubated overnight.
2. CELL ASSAYS. A factorial experiment was performed to investigate whether NBPT could affect intracellular urease. A culture of LB3, a urealytic bacterium, was grown overnight in a factorial combination of two treatments: presence or absence of urea, presence or absence of 1% NBPT. Overnight turbid cell cultures were harvested by centrifugation: A) The cell-free broth from each incubation was used to evaluate the urease excreted into the cell culture during growth. B) One set of harvested cells was washed three times in buffer to remove extraneous urea, urease, and NBPT and then incubated in the presence of urea and the presence and absence of hydrogen peroxide-activated NBPT. C) A second set of harvested cells was washed three times in buffer, sonicated to release intracellular urease, and then incubated with urea in the presence and absence of hydrogen peroxide-activated NBPT.
3. SOIL ADSORPTION/INTERACTION STUDIES. Four soils were used in this study representing a range of SOM content and pH: Declo silt loam (ID), Flanagan silt loam (IL), KS, Richfield silt loam (KS), Amarillo fine sandy loam (TX). The soils were maintained, air-dried, at room temperature during use: A) Adsorption Study. The adsorption study was performed twice, each time yielding approximately the same result. B) Soil Activation Study. The activation study was performed by incubating inactivated or activated NPBT for 72 hr with increasing concentrations of soil. For the purposes of demonstrating urease activity in soil a demonstration was prepared and present at a bi-annual research day held at the University of Kentucky Spindletop farm.
The interaction of the urease inhibitor NBPT with soil, cells, and cellular enzyme is not completely understood. Better information about the interaction of NBPT with the soil environment could enhance its effectiveness. In this study a series of enzyme assays, cell studies, and adsorption assays were conducted to determine the behavior of NBPT with soil constituents. The project activities in this preliminary phase were primarily designed to create the background knowledge necessary to make suitable recommendations to manufacturers and consumers with respect to urea and urease inhibitor use in plant and soil systems.
NBPT could be rapidly transformed to its active form, NBO, by adding small amounts of hydrogen peroxide. Other mineral oxidizing agents in soil, such as ferric iron did not appear to have a similar activating effect. Cells grown in the presence of NBPT and urea appear to take up the compound and demonstrate reduced urease activity when exposed to urea. However, NBPT added without urea may actually induce urease synthesis with the result that total urease activity in the system increases.
Washed cell assays clearly indicate that NBPT is able to be taken up by cells and inhibit intracellular urease. Sonication of NBPT-incubated cells illustrates this point, because sonicated cells previously grown with NBPT have lower urease activity than cells grown without NBPT. The active form of NBPT can be deactivated or sorbed by soil constituents. This appears to be related to SOM content. On long term exposure to soil, however, unactivated NBPT can be activated in soil while activated NBPT becomes increasingly sorbed or deactivated. Therefore, part of the stability of NBPT in the soil environment depends not only on SOM content but the extent to which active and inactive forms are produced and sorbed. An activated NBPT added to a low SOM soil would be more effective than the parent compound. Conversely, in a high SOM soil, parent compound that requires activation by SOM appears to be more effective. For any given soil, therefore, there may be an optimum exposure/SOM relationship during which NBPT is most effective. The studies performed here suggest that it is between 24 and 72 hours.
Overall results to date inevitably raise additional questions about the interaction of NBPT with soil and cells. The apparent capacity of NBPT to inhibit intracellular urease has applications to the foliar application of NBPT in turf. Likewise, the complex adsorption/activation relationships of NBPT to soil constituents suggest the potential for fine tuning the application rates of NBPT based on SOM quantity and quality characteristics.
The results are promising in that future studies and increasing scale of research will make it possible to make specific suggestions with respect to change in action. Because of the high cost of fertilizer N, and the potential to reduce fertilizer urea losses through volatilization has important economic value.