AMMONIFICATION AND NITRIFICATION IN ZINC-CONTAMINATED SOIL

Magdalena Zaborowska, Jadwiga Wyszkowska, Jan Kucharski
Department of Microbiology, University of Warmia and Mazury in Olsztyn, Poland

ABSTRACT

Two laboratory experiments were conducted on samples of leached brown soil developed from light loam (pH_KCl 6.8) in order to determine the effect of zinc on the process of ammonification and nitrification. Ammonification was studied in experiment 1, where variable factors were zinc dose (mg Zn·kg^-1 soil): 0, 5, 500, 1000, 1500 and 2000, source of organic nitrogen: urea, L-aspartic acid, L-arginine and L-alanine, and time of soil incubation (h): 24, 48, 72, 96 and 120. Nitrification was studied in experiment 2, where variable factors were zinc dose (mg Zn ·kg^-1 soil): 0, 5, 500, 1000, 1500 and 2000, source of nitrogen: urea and ammonium sulfate, and time of soil incubation (days): 15, 30, 60, 90 and 120. The course of ammonification and nitrification was determined based on the concentrations of mineral nitrogen (N-NH₄ and N-NO₃). Soil pH was measured at the same time. Zinc decreased ammonification of urea, L-aspartic acid, L-arginine and L-alanine as well as of nitrification of ammonia nitrogen. The rate of mineralization was faster in the case of urea, compared with amino acids, while nitrification of the N-NH₄⁺ ion formed during urea hydrolysis was more efficient, compared to the same ion formed as a result of ammonium sulfate dissociation. Soil acidity had a negative effect on nitrification, particularly in soil fertilized with ammonium sulfate and urea.

Key words: zinc, soil contamination, ammonification, nitrification.

INTRODUCTION

Nitrogen compounds found in soil in the form of mineral and organic bonds are available to microbes and plants as NO₃^- and NH₄⁺, produced in consequence of ammonification, nitrification and N₂ fixation [13,20]. Since nitrogen is a component of proteins and nucleic acids, its presence in the cells of living organisms is required for their growth and development. Biological transformation of nitrogen may take place with the participation of both bacteria and fungi synthesizing enzymes that degrade organic compounds containing this element, such as proteases, lysozyme, urease, nuclease [15]. These enzymes may be adsorbed on soil colloids or found in bacterial cells following autolysis [3]. Except for urea hydrolysis, which occurs with the participation of the extracellular enzyme urease, organic nitrogen ammonification is an element of metabolism in live microbial cells, and may take place by way of hydrolytic, oxidative, reductive or desaturation desamination [1].The rate of ammonification and nitrification may be modified by a too low count of microbes responsible for these processes [21], which in turn is indirectly affected for instance by soil reaction, rapid uptake of nitrate ions by bacterial cells [3,12,21], temperature and soil moisture content [3,7,8,12]. Ammonium ion oxidation is also dependent on texture of soil [10], organic carbon content [8,14,16] and aerobic conditions [21]. Some results also show that the differences between the coefficients of nitrogen mineralization and immobilization depend on the rate of microbial respiration and ATP concentration rather than on the C : N ratio [6]. The rate of ammonification and nitrification is modified not only by the above factors, but also by soil contamination with heavy metals. Heavy metals have an inhibitory effect on the counts of nitrifying and ammonifying bacteria [4,11,22]. However, ammonification is carried out by a larger and more diverse group of microbes, and thus is less susceptible to the toxic effect of heavy metals than nitrification [9,13,17]. Nitrifying bacteria are most sensitive to soil contamination with xenobiotics of all microbial groups [2].

Due to the fact that the other metals (chromium, nickel, cadmium, copper) changed concentration of mineral nitrogen (N-NH₄⁺ and N-NO₃^-) in the soil [5,22,23], the objective of the present study was to determine the effect of soil contamination with zinc on the ammonification and nitrification process.

MATERIAL AND METHODS

Two four-factorial experiments were performed under laboratory conditions. Samples of leached brown soil developed from light loam (1.0-0.1 mm – 63%, 0.1-0.02 mm – 12%, < 0.02 mm – 25%; pH_KCl 6.8) were taken in humus horizon. The soil contained 7.9 g C_org·kg^-1, its hydrolytic acidity and cation exchange capacity were 13.2 mmol·kg^-1 and 109.9 mmol·kg^-1, respectively. Ammonification was studied in experiment 1, where variable factors were:

– zinc dose (mg Zn·kg^-1 soil): 0, 5, 500, 1000, 1500 and 2000,
– source of organic nitrogen: urea, L-aspartic acid, L-arginine and L-alanine,
– time of soil incubation (h): 24, 48, 72, 96 and 120.

Nitrification was studied in experiment 2, where variable factors were:

– zinc dose (mg Zn·kg^-1 soil): 0, 5, 500, 1000, 1500 and 2000,
– source of nitrogen: urea and ammonium sulfate,
– time of soil incubation (days): 15, 30, 60, 90 and 120.

Both experiments were conducted in six replications. 50 g samples of air-dried soil sifted through a 1 mm sieve were placed in 100 cm³ beakers. The samples were contaminated with zinc in the form of ZnSO₄ · 7H₂O (factor 1), and nitrogen compounds were introduced (factor 2) in the amount of 0 and 300 mg N·kg^-1 d.m. of soil. Following thorough mixing soil moisture content was brought to capillary water capacity of 60%. Over the entire experimental period the samples were stored in a thermostat at 25^oC. At a specified time (factor 3) the concentrations of N-NH₄⁺ and N-NO₃^- were determined in soil, and pH was measured in soil extract (1% aqueous solution of K₂SO₄). Each time determinations were made using a separate lot of beakers filled with soil.

In laboratory experiments 1 and 2 N-NH₄ and N-NO₃ were extracted using a 1% aqueous solution of K₂SO₄. 50 g soil samples were transferred quantitatively to plastic bottles using 250 cm³ of a 1% aqueous solution of K₂SO₄ (soil to extractor ratio of 1 : 5), and shaken for 30 min. N-NH₄ extracted from soil was determined with Nessler’s solution. Absorbance of amidomercury iodide oxide was measured with a spectrophotometer at a wavelength of 410 nm. The N-NO₃ content was also determined with a spectrophotometer (410 nm), measuring absorbance of nitrophenoldisulfonic acid obtained by reaction of N-NO₃ with phenoldisulfonic acid. A detailed description of soil analysis is given in a paper by Wyszkowska et al. [23]. Experimental results provided the basis for describing the course of ammonification and nitrification. The coefficients of ammonification and nitrification as well as the percentages of ammonified and nitrified nitrogen were calculated taking into account mineral nitrogen concentrations.

Coefficients of ammonification were calculated based on mineral nitrogen concentrations [22]:

N_am = [N₁ – N₀] · D^-1

where:

N₁ – N-NO₃^- + N-NH₄⁺ content in soil fertilized with organic nitrogen compounds, mg,
N₀ – N-NO₃^- + N-NH₄⁺ content in soil not fertilized with organic nitrogen compounds, mg,
D – dose of nitrogen (300 mg N·kg^-1),

and coefficients of nitrification [22]:

N_nit = [N₁ – N₀] · D^-1

where:

N₁ – N-NO₃^- content in soil fertilized with urea or ammonium sulfate, mg,
N₀ – N-NO₃^- content in soil not fertilized with urea or ammonium sulfate, mg,
D – dose of nitrogen (300 mg N kg^-1).

Results were verified statistically by Duncan’s test, using Statistica software [19].

RESULTS AND DISCUSSION

In the present study zinc negatively affected ammonification of urea, L-aspartic acid, L-arginine and L-alanine (Table 1, Figs. 1 and 2). This negative impact was related primarily to the degree of soil contamination with this metal and to the type of ammonified organic compound. Zinc inhibited both soil nitrogen transformation and decomposition of urea, L-aspartic acid, L-arginine and L-alanine. The negative effect of zinc on the ammonification process was confirmed by significant negative coefficients of correlation between the degree of soil contamination with Zn and the amount of ammonified nitrogen.

A particularly low rate of ammonification of urea and amino acids was observed during the first 24 hours of soil incubation in treatments contaminated with zinc in the amount of 500 to 2000 mg Zn·kg^-1 d.m. of soil (Fig. 2). In these samples 2000 mg Zn·kg^-1 soil inhibited ammonification of soil nitrogen, L-alanine, L-arginine, urea and L-aspartic acid by 15, 80, 73, 76 and 96%, respectively. However, after another 24 hours (in hour 48 of the experiment), the quantity of ammonified nitrogen increased significantly in all soil samples, regardless of nitrogen source. The highest increase (threefold in soil enriched with urea, fivefold in soil enriched with L-arginine, 14-fold in soil enriched with L-aspartic acid and 23-fold in soil enriched in L-alanine) was recorded in treatments containing the highest dose of zinc (2000 mg·kg^-1). Between 72 hours and 120 hours the amount of ammonified nitrogen did not change so rapidly. The negative impact of zinc on the ammonification process diminished as well. In consequence, in hour 120 of incubation, in soil samples contaminated with the highest dose of zinc (2000 mg Zn·kg^-1 soil) urea ammonification was not inhibited at all, whereas ammonification of L-aspartic acid, L-alanine and L-arginin was inhibited in 18, 33 and 37%, respectively. Soil nitrogen ammonification was inhibited in as much as 52%. It should be noted that over the entire experimental period (24 h – 120 h) the ammonification process was also hindered, though to a slight degree, by the lowest dose of zinc (5 mg Zn·kg^-1 soil).

Apart from soil contamination with zinc, the course of organic nitrogen mineralization was also considerably affected by the duration of the experiment (Fig. 2). Irrespective of zinc dose, in hour 24 of the study ammonification of urea, L-aspartic acid, L-arginine and L-alanine was at an average level of 34, 28, 36 and 17%, respectively. In hour 120 these values were much higher, i.e. 62, 55, 73 and 70% for urea, L-aspartic acid, L-arginine and L-alanine, respectively.

The low amount of ammonified nitrogen, especially during the first 24 hours of the experiment, could result from the acidifying effect of urea and amino acids on soil (Table 2), which most probably negatively affected the count and activity of ammonifying bacteria. The acidifying effect of ZnSO₄ · 7H₂O was observed in soil samples over the entire incubation period. This was confirmed by significant negative coefficients of correlation (from -0.94^** to -0.99^**) between the degree of soil contamination with zinc and soil pH.

The relationships between the degree of soil contamination with zinc, mineral nitrogen concentrations and soil pH were also reflected by Pearson’s simple correlation coefficients (Table 3). There was a negative correlation between zinc dose and the pH of soil enriched with organic nitrogen compounds. Mineralization of amino acids had a positive effect on soil acidity, which gradually decreased. This trend was confirmed by significant positive coefficients of correlation between soil pH and the amount of N-NH₄⁺ produced (r = 0.75^**, r = 0.46^** and r = 0.61^** for L-aspartic acid, L-arginine and L-alanine, respectively).

Similarly as in the case of ammonification, the inhibitory effect of zinc on the rate of nitrification was related primarily to the degree of soil contamination with this metal and the type of nitrified nitrogen compound (ammonium sulfate and urea). The negative impact of zinc on this process was confirmed by the quantities of nitrified nitrogen found in soil (Table 4, Figs. 3 and 4). Ammonia nitrogen nitrification was particularly negatively impacted by soil contamination with zinc in the amount of 1000 to 2000 mg Zn·kg^-1 soil, which significantly hindered the process for 120 days. The dose of 500 mg Zn·kg^-1 soil also restricted the rate of nitrification, but to a much lesser extent. The coefficients of correlation between the quantity of N-NO₃^- produced as a result of microbial oxidation of N-NH₄⁺ and the zinc content of soil were found to be highly significant, i.e. -0.63^** in soil enriched with urea and -0.65^** in treatments without nitrogen and with ammonium sulfate (Table 5). The inhibitory effect of zinc on the nitrification process diminished along with the time of experiment. On day 15 the dose of 2000 mg Zn·kg^-1 soil inhibited nitrification of soil nitrogen, ammonium sulfate and urea in 84, 95 and 97%, respectively, while on day 120 these values were much lower, i.e. 25, 44 and 19%, respectively.

It should be stressed that under experimental conditions, regardless of the degree of soil contamination with zinc, nitrification of the N-NH₄⁺ ion formed during urea hydrolysis was more efficient, compared to the same ion formed as a result of ammonium sulfate dissociation. This could be caused by the fact that the sulfate (VI) anion contributes to increased soil acidity, thus slowing down the rate of ammonium ion oxidation, whereas carbon dioxide produced during urea hydrolysis contributes to the formation of carbonates increasing soil pH. On average, irrespective of the dose of zinc sulfate, the amount of nitrified nitrogen in urea-enriched soil was higher, compared with samples containing ammonium sulfate, by 28, 26, 37, 17 and 19% on day 15, 30, 60, 90 and 120, respectively. The rate of nitrification was also dependent on soil acidity, positively correlated with the quantity of successively produced nitrates (Table 6). This concerns mainly soil fertilized with ammonium sulfate and urea. The coefficients of correlation between the amount of N-NO₃^- produced and soil pH were highly significant, i.e. r = 0.35^**, r = 0.40^** and r = 0.36^** in soil without nitrogen, with ammonium sulfate and with urea, respectively (Table 5). The strong negative effect of zinc on the nitrification process, observed in this study, may be attributed to the chemolitotrophic of nitrifying bacteria, characterized by high environmental requirements [15,22]. Benbi et al. [5] and Dhul et al. [10] also reported a slower rate of mineralization of organic compounds containing nitrogen in zinc-contaminated soil. However, this process is inhibited by heavy metals to a lower extent than nitrification [13]. Singha et al. [17] studied the effect of zinc on nitrogen transformation and demonstrated that this metal has a strongly toxic effect on nitrification. They found that zinc and cadmium reduced the activity of nitrification and ammonification to 86.1 and 44.2%, respectively, and that cadmium was a stronger inhibitor of both processes than zinc. The inhibitory effect of zinc and other heavy metals (Cu and Cd) on nitrification was also observed by Benbi et al. [5]. These authors proved that soil contamination with copper (50-200 mg·kg^-1), zinc (200-400 mg·kg^-1) and cadmium (0.5-5 mg·kg^-1) hindered nitrification for several weeks, contributing to N-NH₄⁺ accumulation. Like in the study conducted by Gupta and Chaudhry [12], in the present experiment a zinc dose of 400 mg·kg^-1 soil was sufficient to inhibit nitrification. Dhul et al. [10] demonstrated that a zinc dose of 1000 mg Zn·kg^-1 soil was required to stop microbial oxidation of N-NH₄⁺. Smolders et al. [18] performed a laboratory experiment and found that nitrogen nitrification was inhibited in as much as 95% in soil samples contaminated with zinc in the amount exceeding 500 mg Zn·kg^-1, while the same quantity of this metal accumulated in leached soil under natural conditions did not hinder nitrification to a comparable extent.

CONCLUSIONS

1. Zinc present in excessive amounts in soil (500-2000 mg Zn·kg^-1) had a negative effect on ammonification of L-aspartic acid, L-arginine, L-alanine and urea as well on ammonia nitrogen nitrification. Zinc was found to be a stronger inhibitor of nitrification than of ammonification.

2. Regardless of the degree of soil contamination with zinc, nitrification of the N-NH₄⁺ ion formed during urea hydrolysis was more efficient, compared to the same ion formed as a result of ammonium sulfate dissociation.

3. The lowest rate of ammonification of urea and amino acids was observed during the first 24 hours of soil incubation.

4. There was a significant negative correlation between soil acidity and the amount of nitrified nitrogen, especially in soil fertilized with ammonium sulfate and urea.

5. The reaction of soil enriched with L-aspartic acid, L-arginine, L-alanine and urea decreased along with an increase in the degree of soil contamination with zinc.

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Accepted for print: 17.10.2007

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