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Revista de la ciencia del suelo y nutrición vegetal

On-line version ISSN 0718-2791

R.C. Suelo Nutr. Veg. vol.9 no.2 Temuco  2009 

Rev. Cienc. Suelo Nutr. /J. Soil. Sci. Plant Nutr. 9(2): 125-133 (2009)



Y.H. Juan1,2, L.J. Chen1*, Z.J. Wu1 and R Wang2

1institute of Applied Ecology, Chinese Academy of Sciences, P. O. Box 417, Shenyang 110016, People's Republic of China. 2Liaoning Academy of Agricultural Sciences, Shenyang 110161, People's Republic of China. *Corresponding author:



With black soil (Pachic Udic Argiboroll) of Northeastern China as the test object, an incubation test was conducted to investigate the effects of urease inhibitors, hydroquinone (HQ), phenyl phosphorodiamidate (PPD) and N-(n-Butyl) thiophosphoric triamide (NBPT), on the kinetic characteristics of soil urease under normal moisture and waterlogged conditions, aimed to study the changes of catalytic potential of soil urease and the inhibition mechanisms. The results showed that test urease exhibited typical Michaelis-Menten kinetic behaviors, and all test inhibitors increased the Km and decreased the Vmax of soil urease, behaving as mixed inhibitors to soil urease. Under both normal and waterlogged conditions, compared with HQ, PPD and NBPT made Km increase and Vmax and Vmaxand Vmax/Km decrease more greatly, and the duration of these effects was longer (ca. 30 days vs. 10 days). Under water-logging, PPD made more increment of Km and more decrement of Vmax and Vmax/Km than NBPT, compared with that under normal soil moisture condition, suggesting that NBPT was more available under normal soil moisture condition, while PPD was promising under water-logging condition. To apply urease inhibitors and to control soil moisture condition could be a feasible way in increasing fertilizer N use efficiency affected by soil urease.

Keywords: kinetic parameters, mixed inhibition, moisture regime, soil urease



Urea is the most frequently applied N-fertilizer in agriculture (Bremner, 1995), which accounts for 46% of the total world N-fertilizer consumption (Watson, 2000). However, due to the rapid hydrolysis of its amide N by reaction with the enzyme urease, a quantitatively impressive loss of urea N as NH3 volatilization and N03- leaching could occur, and thus, a definite decrease of urea N use efficiency is observed (Cai et al, 2002; Sharpe and Harper, 2002; Sommer et al, 2003; Blennerhassett et al., 2006; Parfitt et al., 2006).

To improve fertilizer N use efficiency, various attempts have been made to reduce the urea N loss (Vlek and Craswell, 1981; Byrnes and Freney, 1995; Blaise and Prasad, 1995), among which the use of urease and nitrification inhibitors in conjunction with urea fertilizer is an available option.

Currently, a number of compounds, e.g., hydroquinone            (HQ),            phenyl posphorodiamidate (PPD), and N-(n-butyl) phosporothioic triamide (NBPT), etc., have been tested for their availability in inhibiting soil urease activity and in retarding urea hydrolysis (Wang et al, 1991a; Luo et al., 1994; Keerthisinghe and Freney, 1994). The effectiveness of these compounds applied to soils was affected by environmental factors, such as pH (Hendrickson and Douglass, 1993), temperature (Hendrickson and O'Connor, 1987), and moisture content (Sigunga et al, 2002; Clough et al, 2004). However, little information is available about the inhibitor effects on soil urease kinetics, which is of significance in understanding the types of inhibition mechanisms and the effectiveness of urease inhibitors.



Soil and inhibitors

Surface black soil samples (0-20 cm) (Pachic Udic Argiboroll, US Soil Taxonomy) were collected from the Hailun Experimental Station of Ecology (47°25'N, 126°46'E), Chinese Academy of Sciences, in Heilongjiang Province of Northeastern China. After removing plant roots and debris, soil samples were sieved (< 2 mm), air-dried in shade, and stored for analysis.

The physicochemical properties determined as described by Lu (2000) were pH 6.45 (soil: water ratio, 1:2.5), organic C 29.20 g kg-1, total N 2.43 g kg-1, total P 0.82 g kg-1, total S 0.63 g kg-1, alkali-hydrolyzed N 130.15 mg kg-1, available P 105.24 mg kg-1, available S 22.9 mg kg-1, sand 13.85%, clay 34.61%, and silt 51.45%.

Urease inhibitors HQ (99%), PPD (97%), and NBPT (99.5%) were purchased from Sigma (USA), ACROS (Belgium), and Toronto Research Chemicals Inc. (Canada), respectively.

Incubation test

The air-dried soil samples were re-moistened at 15% soil moisture content (SMC), and pre-incubated at 25°C for 21 d to restore microbial activities (Bandick and Dick, 1999; Zornoza et al, 2006). After pre-incubation, the samples were amended with HQ, PPD, and NBPT at rate of 50 mg kg-1 dry soil (about 1 % on a urea weight basis), respectively. The samples under normal moisture condition were in plastic bags with the same amount (about 500 g inhibitor-added soil of 20% SMC), while those under water-logging were in 150 mL stoppered Erlenmeyer flasks (about 5 g inhibitor-added soil of 15% SMC for each flask). Then, soil samples were incubated at 25°C under both normal moisture (20% moisture content) and water-logging (with a 3-5 cm water layer) conditions.

During incubation, water loss (assessed by weight) was compensated daily by adding distilled water. Controls (without any urease inhibitor application) were incubated at the moisture conditions previously described. Three replicates were installed for each moisture regime.

Urease (EC activity assay

At 1, 10, and 30 d during incubation, 5 g incubated soil was thoroughly mixed with 5 ml urea solution with a series of concentrations (5, 10, 15, 25, 35, and 45 mmol L-1), and then incubated at 37±1°C for 5 h. After incubation, the residual urea was extracted with 50 mL 2 mol L-1 KC1 acetic phenyl mercury solution for 1 h on a rotary shaker, followed by filtration with quantitative filter paper (cpl5 cm) (Tabatabai, 1994), and determined by Continuum Flow Auto Analyzer 3 BRAN+LUEBBE, which involves the reaction of urea with diacetylmonoxime (DAM) in the presence of thiosemicarbazide (TSC), H3P04, and H2S04 under heating. The intensity of red color formed was measured at 527 run wavelength by spectrophotometer. Soil urease activity was expressed as mg of hydrolyzed urea-N kg-1 dry soil 5h-1

Michaelis   kinetic   parameters measurement

The kinetic parameters Km and Vmax were calculated by Lineweaver-Burk equation, the linear transformation of Michaelis-Menten equation (Segel, 1975):

Statistical analysis

All data were calculated on the basis of oven-dried soil, and represented as means ± standard deviation of 3x3 data. The effects of moisture regime, urease inhibitor, incubation time, and their interactions on the kinetic parameters of soil urease were analyzed by a two-way analysis of variance (ANOVA) with the General Linear Models (GLM) procedure of SPSS 11.5 for windows, and the differences among treatment means were performed by Duncan's multiple test at p < 0.05 (SPSS 2000).



The two-way ANOVA results of kinetic parameters showed that moisture regime, urease inhibitor, incubation time, and their combinations mostly had significant effects on the Km, Vmax, and VmJKm of soil urease (Table 1).

Compared with control, the amendment of test inhibitors made Km increase (Figure 1), possibly because of the formation of inhibitor-urease complex decreasing the affinity of urease for the substrate, or the partitioning effects between the bulk solution and the microsite of enzyme attachment (Goldstein, 1976). The increase of Km provided evidence that applying urease inhibitor in conjunction with urea could retard urea hydrolysis and increase the urea-N use efficiency (Wang et al, 1991b; Zhao and Zhou, 1991; Watson et al., 1994; Varel, 1997).

The effects of test inhibitors on Km differed with moisture regime, incubation time, and the kinds of inhibitors (Fig.1). Under water-logging, the effectiveness of test inhibitors in increasing Km followed the order of NBPT, PPD > HQ on the 1st day of incubation, and PPD > NBPT » HQ on the 10th day and by the end of the incubation. A similar trend was observed under normal moisture condition, except for the order of NBPT > PPD » HQ on the 10th day and by the end of the incubation. The differences in the effectiveness of test inhibitors were likely ascribed to their structural and functional properties. Hydroquinone (HQ), as a derívate of phenol, was easily soluble and oxidable, resulting in its shorter lasting time and weaker effectiveness on soil urease, while to the contrary, PPD and NBPT, as the derivates of phosphorylamide (Schlegel et al., 1986; Byrnes and Freney, 1995), have similar structure to urea (Van Cleemput and Wang, 1991), making them have higher potential to compete with the specific substrate for urease active sites (McCarty et al, 1990; Chaiwanakupt et al, 1996).

Compared with PPD, the better effectiveness of NBPT was due to its more functional groups (e.g., amino group, butyl, and thio group) (McCarty and Bremner, 1989) and a released sulfur group during its transformation (Blakeley and Zerner, 1984; Beninie et al., 1996).

With incubation time, the Km decreased, but the duration restored the control level differed with the kinds of inhibitors. In water-logged treatment, the duration was about lOd under HQ and about 30d under PPD and NBPT; while in normal moisture treatment, it was longer under NBPT than under HQ and PPD (30 d vs. 10 d). The longer effectiveness of PPD and NBPT in water-logged and normal moisture conditions was probably due to the formation of more effective products,           hydrolyzed          product diamidophosphate (DAP) and oxon analog N-(n-butyl) phosphoric triamide (BNPO), respectively (McCarty et al. 1989; Creason et al, 1990; Manunza et al, 1999; Krajewska and Zaborska, 2007). Therefore, the duration and effectiveness of urease inhibitor on soil urease may be related to the rate and time of effective product formation, and the synergetic effects of inhibitor itself and the product (Douglass and Hendrickson, 1989).


Figure 2 showed the variation of Fmm of urease. In principle, test inhibitors decreased the Vmax of soil urease, due to the formation of inhibitor-enzyme complex decreasing the formation and dissociation of enzyme-substrate complex (Lai and Tabatabai, 1992). Under waterlogging, the effectiveness of test inhibitors in decreasing Vmax was in the order of PPD > NBPT » HQ, while under normal moisture condition, the order was NBPT > PPD» HQ, due to their structural and functional characteristics mentioned above.

With incubation time, Vmax significantly increased (statistical results were not shown).

However, the time that Vmax recovered to the control level differed. In general, the duration under PPD and NBPT was longer than that under HQ in both moisture conditions (ca. 30 d vs. 10 d), which confirmed the previous studies that PPD and NBPT are more effective than HQ (Martens and Bremner, 1984; Luo et al., 1994).

VmJKm has been considered as an index of the catalytic capacity of enzyme through enzymatic reactions. Compared with control, VmJKm of soil urease significantly decreased with application of test inhibitors (data were not shown), indicating the decease of catalytic ability of test enzyme. Under soil moisture and incubation time conditions, the findings observed were similar to that of Fmax, suggesting that PPD and NBPT could greatly decrease the catalytic capability of urease under waterlogged and normal moisture conditions, respectively.






In general, the above results demonstrated that all test inhibitors were of mixed inhibition on soil urease, which was not in accordance with the previous study of PPD (Krajewska and Zaborska, 2007). Compared with HQ, PPD and NBPT made the kinetic parameters change more greatly, showing higher inhibitory effectiveness on soil urease. With incubation time, Km decreased, while Vmax and VmJKm increased; however, the duration under PPD and NBPT was greatly longer than that under HQ (ca. 30 d vs. 10 d). Compared with normal moisture, water-logging increased Km, but decreased Fmax and VmJKm under PPD, while under NBPT, the changes of kinetic parameters were in adverse, indicating that PPD and NBPT are the most promising in water-logging and normal moisture, respectively.

Despite the fact that only a soil was studied, the results obtained are of significance and weight to make reliable conclusions of general interest that are presumably extendable to other soils and agricultural management practices. It is seen that to apply urease inhibitors and to control soil moisture could be a feasible way in increasing fertilizer N use efficiency, and a new standard for choosing a promising urease inhibitor may be potential by its effects on urease kinetics.



Financial supports from National Basic Research Program of China (973 Program) (2007CB109307) and Chinese Government Science and Technology Supporting Program (2006BAD10B01) are gratefully acknowledged. We thank Professor L. K. Zhou for his critical review of our manuscript, and the staffs of Department Soil and Plant Nutrition, Institute of Applied Ecology under Chinese Academy of Sciences for their academic and technical assistance.



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