Scientific registration n : 214 Symposium n : 26 Presentation : poster. AULAKH Milkha (1), KHERA Tejinder (2), DORAN John (3)

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1 Scientific registration n : 214 Symposium n : 26 Presentation : poster Effect of nitrate and ammoniacal N on denitrification in upland, nearly-saturated and flooded subtropical soils Effet de l'azote nitrique et ammoniacal sur la dénitrification dans les hautes terres, les terrains humides et les zones inondées des régions subtropicales AULAKH Milkha (1), KHERA Tejinder (2), DORAN John (3) (1) Soil & Water Sci. Div., Internat. Rice Res. Institute, P.O. Box 933, Manila 1099, Philippines (2) Department of Soils, Punjab Agricultural University, Ludhiana , Punjab, India (3) USDA-ARS, East Campus, University of Nebraska, Lincoln 68583, Nebraska, USA Summary The key role played by fertilizer N in augmenting world foodgrain production is well recognized. However, less attention has been focused on the environmental consequences of fertilizer use, especially in developing countries. With the increasing use of fertilizers in tropical and subtropical regions, it becomes increasingly more important to seriously consider the fertilizer-induced environmental impacts such as nitrate contamination of groundwater and pollution of the atmosphere by N 2 O emission from fertilizer use and denitrification. Fertilizer N is supplied to crops through nitrate and ammoniacal sources. As denitrifying organisms use nitrate as an alternate oxidant when oxygen is lacking, ammoniacal fertilizers must first be nitrified before they are subject to loss via denitrification. In soils remaining submerged, nitrification may proceed slowly, so that nitrate availability limits denitrification. Under the climatic conditions prevailing in subtropical regions, temperature is often favorable for denitrifying organisms. In addition to traditional upland soils used for arable agriculture and submerged soils used for rice farming, coarse-textured porous soils are also now used for raising both arable as well as irrigated rice crops. Frequent irrigations applied to rice often keep such soils in a "nearly-saturated" condition. In the present study, we have investigated the influence of fertilizer N applied through nitrate and ammonium sources on the availability of nitrates and gaseous N losses via denitrification in upland, nearly-saturated, and flooded soils. The rate of denitrification was very low in the upland soil conditions [60% water-filled pore space (WFPS)], irrespective of fertilizer N treatments. Increasing water content to nearly-saturated (90% WFPS) and flooded conditions (120% WFPS) resulted in four to six fold higher rates of denitrification within 2 days suggesting that the denitrifying activity commences within a few hours and 1

2 would be expected to continue until nitrate is exhausted or organic carbon supply becomes limited. The nearly-saturated soil behaved more or less like flooded soil. Rather than being partially aerobic, this soil system supported greater nitrification of applied ammoniacal fertilizer N than in flooded soil which resulted in higher relative rates of denitrification. In conclusion, the results of this study reveal that (i) semi-arid subtropical soils low in organic carbon (38 mg kg -1 in the soil used) under restricted aeration can support high rates of denitrification for short periods when sufficient nitrate is present; (ii) application of fertilizer N as nitrate enhances N losses via denitrification, however, the supply of available C determines the intensity and duration of denitrification; and (iii) when fertilizer N is applied as an ammoniacal form, nitrification proceeds slowly and nitrate availability limits denitrification in flooded soil, but, in nearly-saturated soils (or with alternative flooded and dry spells), nitrification supplies adequate NO 3 -N for high rates of denitrification. Introduction Over the past five decades, the key role of fertilizer nitrogen (N) has in augmenting foodgrain production was widely recognized in both the developed and developing world. However, little attention has been focused on environmental consequences of fertilizer use especially in developing countries. Increased fertilizer use in tropical and subtropical regions to meet the ever increasing needs for food and fiber for growing populations, mandates evaluation of fertilizer-induced environmental impacts such as groundwater contamination due to nitrates leaching and destruction of stratospheric ozone layer due to N 2 O emission, in part from denitrification. Soil denitrification is determined predominantly by temperature, soil aeration status, concentration of nitrate, organic carbon supply, soil ph, and texture (Aulakh et al., 1992). Under the climatic conditions prevailing in subtropical regions, temperature is often favorable for denitrifying organisms. In upland (arable) farming, most soils remain aerobic and therefore, diffusion of gases into and out of soil is not restricted. In the flooded soils commonly used for rice farming, submergence of soils results in the depletion of oxygen creating conditions congenial for denitrification. Soils that remain submerged for prolonged periods of time are common in traditional rice cultivation i.e. soils having low water percolation rates. A third category of soil for rice cultivation is the coarse-textured porous soils which are now used for raising both arable and irrigated rice (Aulakh and Bijay-Singh, 1997). Frequent irrigations applied to the rice crop often keep such soils in a "nearlysaturated" condition. When properly managed, porous soils are some of the most productive soils in the world (Aulakh et al., 1997a). Recently, we monitored nitrate and ammonium dynamics in such soils under a four-year rice-wheat cropping system (Aulakh et al., 1997b) and observed substantial movement of nitrate to lower soil depths beyond the rooting zone and a potential danger for NO 3 contamination of groundwater. However, direct measurements of gaseous N losses via denitrification in semi-arid subtropical soils under such unique cropping systems has seldom been undertaken. 2

3 Fertilizer N is supplied to crops through nitrate and ammoniacal sources such as potassium nitrate, calcium ammonium nitrate, ammonium sulfate, diammonium phosphate, urea, anhydrous ammonia etc. Denitrifying organisms use nitrate as an oxidant when oxygen is limited. Ammoniacal fertilizers must first be nitrified before they can be lost as a gas by denitrification. In soils remaining submerged, nitrification may proceed slowly, so that nitrate availability limits denitrification. The present study was undertaken to investigate the influence of fertilizer N applied through nitrate and ammoniacal sources on availability of nitrates and gaseous N losses via denitrification in upland, nearly-saturated, and flooded semiarid subtropical soils. Materials and Methods The soil used in this study was a Fatehpur sandy loam (Typic Ustochrepts) collected from the A horizon of a field under rice-wheat rotation at Punjab Agricultural University Research Farm, Ludhiana, India. The soil contained 780 g sand, 110 g silt and 110 g clay kg -1 soil on oven dry basis. It had an initial ph of 7.6 and contained 3.8 g organic C, 0.4 g total N, 25.7 mg NO 3 -N and 3.5 mg NH 4 -N kg -1. Field moist soil (0.12 g H 2 O g -1 soil) was passed through a 2 mm sieve and thoroughly mixed but was not allowed to dry. The soil was further preconditioned by wetting to 40% water-filled pore space (WFPS) and incubating for 2 weeks at 25 o C. Water-filled pore space, synonymous with relative saturation, was calculated as: WFPS = [(gravimetric water content x soil bulk density)/total soil porosity], where soil porosity = [1-(soil bulk density/2.65)] and 2.65 is the assumed particle density of the soil. Treatments consisted of 3 moisture regimes (60, 90 and 120 % WFPS which simulated upland, nearly-saturated and flooded soil conditions, respectively) and 2 N rates (check or without N and 100 mg N kg -1 through 2 nitrogen sources - potassium nitrate and ammonium sulfate). Each treatment was run in triplicate in seven batches to incubate for 0, 1, 2, 4, 8, 12 and 16 d periods in a randomized complete block design (RCBD). One hundred and eighty nine repacked soil cores (3 moisture regimes x 3 N treatments x 7 sampling times x 3 replications) were prepared by placing moist pre-conditioned soil (80 g on oven dry basis) in "see-through" plastic vials with a 40-mm inside diameter. The soil in each vial was then hand-compacted to bulk density of 1.55 g cm -3, the value commonly measured in the field. In fertilizer N treatment vials, 100 mg N kg -1 soil through either potassium nitrate or ammonium sulfate was added in solution. Distilled water was added dropwise using a fine jet pipette to obtain 60, 90 and 120% WFPS. Our previous studies (Aulakh et al. 1991a, 1991b) and preliminary tests in the present study revealed that surface applied fertilizer N solution was uniformly distributed in the soil core. Then each vial was covered with perforated polyethylene sheet so as to permit gaseous exchange but restrict evaporation of water. Thereafter, the vials were incubated at 35 o C which is the optimum temperature for 3

4 microbial activity, including nitrification, in semiarid subtropical soils (Bhupinderpal-Singh et al., 1993). Water loss through evaporation, if any, was replaced every 2 d. For each periodic gas sampling, individual vials of a batch were placed in a 0.87 L glass jar and sealed with screw-cap lid in which a serum stopper had been fitted. Then (10%,V/V) of jar head air was replaced with C 2 H 2. After 24 h, 2 cm 3 sample of the headspace atmosphere of each jar was removed in duplicate for determination of N 2 O and CO 2 by Gas Chromatography (Aulakh et al., 1991b). Data were corrected for the solubility of N 2 O and CO 2 in the soil water (Moraghan and Buresh, 1977). At the termination of each incubation period (0, 1, 2, 4, 8, 12 and 16 d), whole soil from each vial was extracted immediately with 2M KCl (1 h shaking) followed by filtration. These extracts were analyzed for (NO 3 +NO 2 )-N and NH 4 -N using a micro-kjeldahl procedure (Keeney and Nelson, 1982). Statistical analysis of experimental data was accomplished by standard Analysis of Variance in RCBD (Cochran and Cox, 1950) followed by mean separation within experimental soils using the least significant difference (LSD) test for significance at the 0.05 level of probability. Results and Discussion Denitrification The rate of denitrification was very low in the upland soil system (60% WFPS), irrespective of fertilizer N treatments and never exceeded 1 to 1.5 mg N 2 O-N kg -1 soil d -1. Increasing water content to near-saturation (90% WFPS) and flooded conditions (120% WFPS) apparently resulted in the depletion of oxygen creating anaerobic conditions leading to four to six fold higher rates of denitrification than the upland system. The increase in the rate of denitrification in most cases occurred within the first day. In check soil (without fertilizer N application), the highest peaks of 1.1 (upland), 6.3 (nearly-saturated), and 6.5 mg N 2 O-N kg - 1 d -1 (flooded) were recorded on the second day of incubation. Application of 100 mg NO 3 - N kg -1 soil increased the rate of denitrification almost two-fold, with maximum rates of 0.9, 10.5 and 10.7 mg N 2 O-N kg -1 d -1 occurring on the second day in upland, nearly-saturated and flooded soil, respectively. Similar trends were observed for the application of 100 mg NH 4 -N kg -1, except that rate of denirification was less for the nearly-saturated soil. After 4 d, the rate of denitrification decreased in both nearly-saturated and flooded soils. Of the total N lost in these two soil systems, 34-54% was lost during the initial 4 d followed by 20-26% in 5 to 8 d period, and 20-42% in the 9 to 16 d period (Table 1). Total N losses via denitrification without fertilizer N during the 16-d incubation were lowest (9.2 mg N kg -1 ) in upland and greatest (47.7 mg N kg -1 ) in flooded soil (Table 1).With 100 mg fertilizer N kg -1 soil added as NO 3 -N, the losses ranged from 8.8 mg N kg -1 in upland to 83.3 mg N kg -1 in flooded soil. With the addition of 100 mg NH 4 -N kg -1, total N losses from upland soil increased to 16.1 mg N kg -1 whereas the losses from flooded soil were 32% 4

5 lower than with 100 mg NO 3 -N kg -1. The N losses from nearly-saturated soil were 14% lower than from flooded soil both under check and 100 mg NO 3 -N kg -1 treatments but 18% higher than that from the flooded soil with 100 mg NH 4 -N kg -1 treatment. Availability of nitrate and organic C supply In the check soil, the amount of NO 3 -N at the beginning of the incubation was 38 mg kg -1. Continuous mineralization of soil organic N (and negligible denitrification loss) resulted in the accumulation of 54 mg NO 3 -N kg -1 in upland soil (Table 2). On the other hand, nitrate was exhausted quickly in nearly-saturated and flooded soils after 8 d, and the rate of denitrification became very low. In the 100 mg NO 3 -N kg -1 treatment, the amount of NO 3 -N in both nearly-saturated and flooded soils remained sufficiently high throughout the study period to support high rates of denitrification, but still the rate of denitrification fell after 8 d. Under these anaerobic or aeration-impeded soil systems, the pattern of CO 2 -C production was similar to that of N 2 O-N emission especially during the initial period of high activity. Like N 2 O-N emission, the rate of CO 2 -C production also peaked (8.2 to 11.1 mg CO 2 -C kg -1 d -1 ) by the second day. During the later part of study (8-16 d), C mineralization continued at relatively low rates ( mg CO 2 -C kg -1 d -1 ). Thus, the relatively low but almost constant rates of denitrification throughout the study period after 8 d may have been limited by continuously low supply of organic C to denitrifying organisms through the mineralization of native soil organic matter. Therefore, the supply of available C was a major factor influencing denitrification, especially when soil aeration was restricted and adequate amounts of NO 3 were present. These results illustrate that in such soils with restricted aeration, depending upon the amount of nitrate present and organic carbon supply, denitrifying activity could commence quickly and expected to continue until nitrate is exhausted or organic C supply becomes limited. Nitrification of the added 100 mg NH 4 -N kg -1 was very fast in upland soil and was essentially complete within 16 d as evidenced by the decline in soil NH 4 -N of 92 mg N kg -1 within 12 d (Table 3) and corresponding increase in soil NO 3 -N of 88 mg N kg -1 within the same period (Table 2). The NH 4 -N pool at 16 d in ammonium treated nearly-saturated and flooded soils was 44.7 and 55.0 mg N kg -1 greater, respectively, than NH 4 -N levels in the corresponding check soils (Table 3). As shown by a study with 15 N-labeled ammonium sulfate (Aulakh, 1989), a small fraction of the unaccounted for amount of applied 100 mg NH 4 -N kg -1 (65.3 and 45.0 mg NH 4 -N kg -1 in nearly-saturated and flooded soil, respectively) may have been converted to fixed NH 4 -N (non-exchangeable) and organic N pools but a major portion of it was presumably nitrified; NH 4 could oxidize to NO 3 in the thin O 2 - containing surface soil layer and in the overlying water phase of flooded soils (Engler and Patrick, 1974). That the nearly-saturated soil was partially aerobic is supported by a relatively higher nitrification of applied fertilizer ammoniacal N; this soil contained 20.1 mg NO 3 -N kg -1 as compared to only 1 mg NO 3 -N kg -1 in flooded soil on 16 d. Consequently the concurrent nitrification in nearly-saturated soil continued to supply NO 3 -N to denitrifiers and resulted in higher rates of denitrification especially during the later part of the incubation period (after 8 d), with 18% more cumulative N losses than flooded soil. Otherwise, the 5

6 nearly-saturated soil (that simulated percolating porous soil under irrigated rice cultivation) behaved more or less like flooded soil supporting 86% of the total denitrification losses estimated in flooded soil in no-n and 100 mg NO 3 -N kg -1 treatments. Acknowledgments We gratefully acknowledge the financial assistance received from the Office of International Cooperation and Development, USDA. References Aulakh, MS (1989) Transformations of ammonium nitrogen in upland and flooded soils amended with crop residues. J. Indian Soc. Soil Sci. 37: Aulakh, MS, and Bijay-Singh (1997) Nitrogen losses and fertilizer N use efficiency in irrigated porous soils. Nutrient Cycling Agroecosys. 43(3): Aulakh, MS; Doran, JW; and Mosier, AR (1992) Soil denitrification - significance, measurement and effects of management. In: Steward BA (ed.) Adv. Soil Sci., Springer-Verlag. New York. vol. 18, pp Aulakh, MS; Doran, JW; Walters, DT; Mosier, AR and Francis, DD (1991a) Crop residue type and placement effects on denitrification and mineralization. Soil Sci. Soc. Am. J. 55: Aulakh, MS; Doran, JW; Walters, DT; and Power, JF (1991b) Legume residue and soil water effects on denitrification in soils of different textures. Soil Biol. Biochem. 23: Aulakh, MS; Khera, TS; Kuldip-Singh; Bijay-Singh and Doran, JW (1997a) Effects of integrated management of legume green manuring and fertilizer N in a rice-wheat cropping system in an irrigated porous soil. 1. Crop yield, N uptake and N-use efficiency. Agron. J. (submitted). Aulakh, MS; Khera, TS; Kuldip-Singh; Gurjit-Singh and Doran, JW (1997b) Effects of integrated management of legume green manuring and fertilizer N in a rice-wheat cropping system in an irrigated porous soil. 2. Nitrate and Ammonium N Dynamics in the Soil During Four years and Their Leaching Beyond Rooting Zone. Agron. J. (submitted). Bhupinderpal-Singh; Bijay-Singh; Yadvinder-Singh (1993) Potential and kinetics of nitrification in soils from semi arid regions of north-western India. Arid Soil Res. Rehabil. 7: Cochran, WG and Cox, GM (1950). Experimental Designs, John Wiley and Sons, Inc., New York. Engler, RM; and Patrick, WH, Jr (1974) Nitrate removal from flooded water overlying flooded soils and sediments. J. Environ. Qual. 3: Keeney, DR and Nelson, DW (1982) Nitrogen - inorganic forms. In: A.L. Page et al. (eds), Methods of Soil Analysis. Volume 2. Chemical and Microbiological Properties, American Society of Agronomy, Madison, Wisconsin pp

7 Moraghan JT and Buresh R (1977) Correction for dissolved nitrous oxide in the nitrogen studies. Soil Sci. Soc. Am. J. 41: Keywords : N2O emissions, green-house effects, rice soils, fertilizer N loss Mots clés : émission de N20, effet de serre, sol de rizière, lessivage de l'azote Table 1. Effect of fertilizer N applied through nitrate and ammonium sources on cumulative denitification losses (mg N kg -1 soil) in upland, nearly-saturated and flooded soils. Soil Incubation period condition 0-4 d 5-8 d 9-12 d d Total 0-16 d Check (without fertilizer N) Upland Nearly-saturated Flooded LSD (0.05) o mg N kg -1 soil through KNO 3 Upland Nearly-saturated Flooded LSD (0.05) mg N kg -1 soil through (NH 4 ) 2 SO 4 Upland Nearly-saturated Flooded LSD (0.05)

8 Table 2. Effect of fertilizer N applied through nitrate and ammonium sources on NO 3 -N (mg N kg -1 ) in upland, nearly-saturated and flooded soils. Soil Incubation period (d) condition Check (without fertilizer N) Upland Nearly-saturated Flooded LSD (0.05) NS mg N kg -1 soil through KNO 3 Upland Nearly-saturated Flooded LSD (0.05) NS mg N kg -1 soil through (NH 4 ) 2 SO 4 Upland Nearly-saturated Flooded LSD (0.05) NS Table 3. Effect of fertilizer N applied through nitrate and ammonium sources on NH 4 -N (mg N kg -1 ) in upland, nearly-saturated and flooded soils. Soil Incubation period (d) condition Check (without fertilizer N) Upland Nearly-saturated Flooded LSD (0.05) NS mg N kg -1 soil through KNO 3 Upland Nearly-saturated Flooded LSD (0.05) NS mg N kg -1 soil through (NH 4 ) 2 SO 4 Upland Nearly-saturated Flooded LSD (0.05) NS NS