Research Accomplishment Reports 2011

Effect of Urease Inhibitors on Volatile N Loss From Soil and Other N Transformations

M. S. Coyne
Department of Plant and Soil Sciences

 

Non-Technical Summary

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.

2011 Project Description

The following four 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.

2) EFFECT OF POTENTIAL INHIBITORS ON SHORT-TERM NITRIFICATION. The purpose of these studies was to determine if various urease inhibitors reduced nitrification in short term potential assays.

3) LONG TERM NITRIFICATION TEST. The purpose of these tests was to determine if potential urease inhibitors affected long-term nitrification rates in amended soil.

4) INHIBITOR AGING TEST. The purpose of this study was to determine if putative urease inhibitors with multiple butyl group of cyclic structure decomposed with time to release active urease-inhibiting compounds. The initial premise of the study was that aging of the putative inhibitors would result in inactive compounds becoming active with time as constituents were activated or removed.

DISSEMINATION: An overview of urease inhibitors was presented at the 07/21/2011. Princeton KY Field Day. "Roadblocks in the Path of the Nitrogen Cycle: Inhibiting Nitrogen Change." PI Coyne also participated in a successful grant to the Kentucky Water Resources Research Inst., which is intended to evaluate urease inhibitors on the metagenome of urease producing organisms. Moe, L. and M.S. Coyne (Co-I) 2011. "Metagenomic Analysis of Microbial Urea Transformation in Soil." USGS 104b Student Research Enhancement Proposal.

2011 Impact

The research conducted this period evaluated whether variations of the parent compound with respect to the number of butyl groups had biological significance.

1) INHIBITORY PROPERTIES OF NBPT CO-PRODUCTS: In general, modification of the parent compound led to analogs that had less urease activity. Removal of the butyl compound entirely did not eliminate urease-inhibiting activity. DCD and DMPP, two commercial nitrification inhibitors, had no urease-inhibiting activity.

2) EFFECT OF POTENTIAL INHIBITORS ON SHORT-TERM NITRIFICATION: With the exception of the S. Concentrate, neither the known nitrification inhibitors nor the sample urease inhibitors - including NBPT and Agrotain - appreciably reduced short term nitrification rates. The short term nitrification enzyme assays suggested that the test compounds were not specific nitrification inhibitors.

3) LONG TERM NITRIFICATION TEST: Where a putative inhibitor was present, the net ammonification was lower than the control. By the end of incubation all but three of the putative urease inhibitors had lower ammonium concentrations than the control. DMPP, a known nitrification inhibitor, was the only sample that had a higher ammonium concentration than the control. Agrotain and S. Concentrate, the commercial urease inhibitors had the lowest ammonium concentrations. Net nitrification was delayed for approximately 1 week in all samples. Thereafter, the concentration of nitrate increased uniformly in all samples with time. DMPP had the lowest nitrate concentration, which is consistent with a known nitrification inhibitor. Net total mineralization is more revealing about the actual effect of the sample compounds on N transformations in the system.

The consistent trend was for the compounds to inhibit total N mineralization in the system. This was most pronounced with the Agrotain and the S. Concentrate. DMPP had the lowest fraction of mineralization attributable to nitrification, which is consistent with a nitrification inhibitor. Agrotain and S. Concentrate had the highest fraction of mineralization attributable to nitrification, which is not consistent with a nitrification inhibitor.

The long term nitrification assay do not support the idea that they are inhibitors. Instead, the compounds appear to exert their effect because they reduce the total amount of ammonium available to nitrification.

4) INHIBITOR AGING TEST: In most cases, compounds thought to be urease inhibitors proved to be urease inhibitors for at least one week after incubation, but with the exception of commercial Agrotain, urease activity rebounded to the level of the control after two weeks. Only three compounds appeared to support aging of urease inhibitors into an active form in situ.

IMPACTS: 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. For example, DCD and DMPP, two commercial nitrification inhibitors, had no urease-inhibiting activity. Because of the high cost of fertilizer N, and the potential to reduce fertilizer urea losses through volatilization this research has important economic value.