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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.
2010 Project Description
The following experiments were conducted in support of the proposed research activities:
1) INHIBITORY PROPERTIES OF NBPT CO-PRODUCTS. The purpose of these studies was to compare the inhibitory effect of various NBPT analogs and synthesis products on jack bean urease in laboratory enzyme assays to determine if the compounds had potential for further development or consideration as inhibitors. Thiophosphoric triamide, and mono-, di-, and tri- butyl thiophosphoric triamides were prepared at concentrations of approximately 0.09% v/v in the presence and absence of hydrogen peroxide. Solvents used to solubilize test compounds (N-methylpyrollidone) were also examined. Carbon black was investigated as a potential activating agent.
2) EFFECT OF LIQUID HUMUS ON N-CYCLING PROCESSES IN SOIL. A commercial liquid humus was added to two soils (Hanford and Panoche series) at rates ranging from recommended application to 16 times recommended application to observe the influence on nitrification, urease activity, denitrification, anaerobic mineralization, and long term aerobic mineralization. Nitrification and denitrification studies were 24-hour soil slurry incubations. Urease studies were conducted as a 24-hour soil slurry study and as an enzyme assay with jack bean urease. The anaerobic mineralization study was a 10-day incubation in saturating conditions and the long-term aerobic mineralization was a 4-week aerobic incubation.
The project activities in this year of research 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.
Under aerobic conditions NBPT [C4H9NHP(S)(NH2)2] is converted to an oxygen analog NBPTO [C4H9NHP(O)(NH2)2], which is a much stronger inhibitor. The NBTO fits the active site of urease and forms a stable complex. NBPT, while it may fit the active site, apparently does not form a stable complex, and so is less effective. The size and type of groups bound to the phosphoric triamides influences the inhibition. One butyl group may not influence the geometry of NBPT binding to the active site of urease. It is conceivable that two butyl groups would also not affect the tetrahedral geometry required for binding, so that dNBPT would also be effective. Three butyl groups, whether they interfere with tetrahedral geometry or (more likely) interfere with the placement of -NH2 at the active site, probably prevent tNBPT from associating with the active site. Consequently, there is no inhibition.
Several thiophosphoramide compounds were tested for their inhibitory effect on jack bean urease in short term assays. Commercial NBPT was superior to all other compounds tested. Each compound was a better inhibitor in the presence of hydrogen peroxide than in its absence, which indicates that control of the rate of oxidation of the inhibitors is potentially an important feature of regulating their activity and persistence in different soil environments. As the number of butyl groups increased on the thiophosphoric triamides, the activity decreased. The potential that increasing the number of butyl groups on the compounds will affect their persistence in soil was not addressed, but is a logical avenue of future research. Carbon black does not appear to actively oxidize thio groups in the inhibitors to the same extent as peroxide, but may have some influence. N-methylpyrollidone, a solvent used to solubilize the inhibitors, is inhibitory to urease at high concentrations, but in commercial formulations is unlikely to have a significant effect. Liquid humus at application rates 16 times that recommended had little effect on N transformations in soil.
The exception was urease activity, which significantly increased when humus rates increased. However, in a strongly buffered enzyme assay with jack bean urease no such effect was observed. The probable explanation was that the alkaline pH of the liquid humus improved pH conditions in the soil environment for urease activity. Because the liquid humus was added separately from the N added to each system, it is possible that there are some process rates that may be affected.
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.