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Stimulatory effect of nitrate on reduction of N2O to N2 by soil microorganisms
ski Bml. khchem. Vol. il. pp. 313 10 31s 8 Pergamon Press Ltd LY79. Prmted in Great fhtam
SHORT
COMMUNICATION
Stimulatory effect of nitrate on reduction of N20 to N, by soil microorganisms A. M. BLACKMER and J. M. Department
of Agronomy,
(Accepred
IO December
Recent research by atmospheric scientists has suggested that increased N fertilization of soils may increase emissions of N,O from soils to the atmosphere via denitrification of fertilizer N and thereby promote destruction of the stratospheric ozone layer protecting the earth from biologically harmful ultraviolet radiation from the sun (Johnston, 1972; McElroy et al., 1976; Crutzen and Ehhalt, 1977; Hahn and Junge, 1977; Liu VI ni., 1977). This has greatly stimulated research on denitri~~tion of nitrate in soils and has emphasized the need for a better understanding of factors affecting the relative amounts of NzO and Nz released from soils by this process. We recently demonstrated that NO; inhibits anaerobic reduction of N,O to N, by soil microorganisms and that, because of this inhibitory effect, the proportion of NO;-N emitted to the atmosphere as N,O when NO, undergoes denitrification in soil increases markedly with the concentration of NO; (Blackmer and Bremner, 1978). Our purpose here is to report work showing that. if the denitrifying microorganisms in soils have not had an opportunity to adapt for anaerobic reduction of NJ0 to N,, a small amount of nitrate has a stimulatory effect on their ability to perform this reduction. The soils used (Table I) were surface (O-15 cm) samples of Iowa soils selected to obtain a range in pH, texture and organic-matter content. Before use, each sample was air-dried, crushed and sieved (<2-mm). The analyses reported in Table I were performed as described by Blackmer and Bremner (1977a). To obtain samples of NO;-free soils, 100-g samples of each soil were treated with 30mf water and incubated under He at 30YY for 48 h. The samples were then dried rapidly by exposing them in a thin layer to a stream of air at room temperature. Analyses by the method of Bremncr and Keeney (1966) showed that the air-dried soil samples contained only trace amounts of NO; (~0.5 pg NO; .g-’ soil). To study the effect of nitrate on the ability of these soil samples to reduce N,O to Nz under anaerobic conditions. 30g subsamples were placed in 1.2-l flasks fitted with standard-taper (34145) ground-glass joints and
Table
FR.“. II,3
G
BREMNER
Iowa State University,
Ames, IA 50011, U.S.A.
1978)
treated with water or with water containing nitrate (as KNOJ). Each flask was then sealed with a glass stopper fitted with a standard-taper (34/45) ground-glass joint and glass stopcock (l-mm dia bore) and was connected to a manifold system for replacement of air by He or by He containing N,O (1 mg N,O-N.g-’ soil). The flasks were then placed in an incubator at 3o’C, and their atmospheres were sampled at intervals for determination of N, and N,O by gas chromatography (Blackmer and Bremner, 1977b). AH incubation experiments and analyses were performed in duplicate or triplicate. Table 2 shows that treatment of NOT-free soils with a small amount of NO; (5 pg NO;-N.g-’ soil) increased their ability to reduce NzO to N, under anaerobic conditions and that the stimuiatory effect of NO; on N,O reduction increased very markedly with decrease in soil pH. This stimulatory effect cannot be attributed to reduction of the added NO; to N, because Table 2 shows that addition of 5 pg NOT-N .g-’ soil increased production of N, by as much as 127 pg N, g -’ soil. Several investigations have indicated that cells of denitrifying microorganisms adapted for reduction of NO; are simultaneously adapted for reduction of N,O to N, (Allen and van Niel. 1952: Matsubara and Mori, 1968: Payne and Riley, 1969: Matsubara and Iwasaki, 1971). The work reported here indicates that NO; is more effective than N,O for stimulation of N,O-reductase activity in soil microorganisms. This would explain the apparent contradiction between the data in Table 2, indicating that NO; has a stimulatory effect on reduction of NzO to N, by soil m~~oorganisms, and the data reported in Table 3 of our previous paper (Blackmer and Bremner, l97S), indicating that NO; has an inhibitory effect on this process. The key difference between our experiments showing the stimulatory effect of NO; and those showing the inhibitory effect is that the soils showing the inhibitory effect contained NOj when they were preincubated under He before exposure to N,O, whereas those showing the stimulatory effect did not contain NO; before this preincubation. We
I. Analyses
of soils
Soil
PH
Organic C (“4)
Sand (%)
Clay (‘X)
Nitrate-N @g.g-’ soil)
Harps Webster Ames Marshall Monona Sparta Okoboji
7.8 6.8 6.5 5.8 5.1 5.4 4.8
4.3 3.8 2.9 2.4 2.0 0.7 6.7
21 35 33 2 4 75 7
36 25 26 32 26 9 40
20 7 9 29 11 5 0
313
314
Short Table 2. Amounts
communications
of N, produced on exposure of nitrate-free soils to N,O under anaerobic conditions*
and nitrate-treated
Amount of N, produced (pg N. g- ’ soil) Soil
PH
Harps
7.8
Webster
6.8
Marshall
5.8
Monona
5.7
Sparta
5.4
Okoboji
4.8
Nitrate-N (kg.g-’
added soil) 0 5 0 5 0 5 0 5 0 5 0 5
12h
24h
60 73 14 24 28 57 II 21 2 3 3 4
141 170 54 68 95 150 22 49 5 23 4 41
48 hf 322 331 145 166 229 307 86 145 24 73 26 153
(3) (14) (34) (69) (204) (488)
* Samples (30g) of nitrate-free soils were placed in 1.2-l flasks, treated with 10 ml water and preincubated under He for 12 h. They were then treated with 8 ml water containing 0 or 15O/dg N as KNOs and incubated at 30°C for various times under He containing N,O (1 mg N,O-N.g-’ soil). t Figures in parentheses indicate percentage stimulation of N,O reduction by nitrate. Table 3. Effects of different amounts of added on exposure of nitrate-free soil to N20 Amount
nitrate on amount of Nz produced under anaerobic conditions* of N, produced
(pg.g-’
soil)
Amount of nitrate-N added (~g.g~’ soil)
18h
24h
36h
0 5 10 20 50
4.2 12.1 7.6 3.2 3.0
6 38 32 19 5
28 126 124 104 22
* Samples (30 g) of nitrate-free Ames soil were placed in 1.2-I flasks. treated with 18 ml water containing @15OO~g N as KNO,, and incubated for various times under He containing N,O (I mg NLO-N.g-’ soil). conclude that, because NO; was absent, the denitrifying microorganisms in the soils showing the stimulatory effect did not adapt for reduction of N,O to N, during this preincubation. Tests with several soils showed that, when nitrate is added to a NO;-free soil containing microorganisms not adapted for reduction of N,O to N,, its effect on this reduction depends upon the amount added. This is illustrated by Table 3, which shows that both stimulatory and inhibitory effects of NO, on N,O reduction were observed when NO;-free Ames soil was treated with different amounts of NO; before exposure to NzO. The data reported are readily explained if NO; has two effects on N,O reduction by soil microorganisms, one being that it stimulates production of N,O reductase. the other being that it inhibits the activity of this enzyme. This would account for our finding that the stimutatory effect of NO; on N,O reduction decreased as the amount of NOT-N added was increased from 5 to 20 pg. g- ’ soil and that only the inhibitory effect of NO; on N,O reduction was evident when the amount of NO;-N added was increased to 5Ofig.g-’ soil.
Acknowledgemenrs-Journal Paper No. J-9209 of the Iowa Agriculture and Home Economics Experiment Station. Project 2096. This work was supported in part by the U.S. Department of Energy.
REFERENCES ALLEN M. B. and VAN NIEL C. B. (1952) Experiments on bacterial denitrification. J. Bact. 64, 397--412. BLACKMERA. M. and BREMNER J. M. (1977a) Nitrogen isotope discrimination in denitrification of nitrate in soils. Soil Bioi. Biochem. 9, 73377. BLACKMERA. M. and BREMNERJ. M. (1977b) Gas chromatographic analysis of soil atmospheres. Soil Sri. Sm. Am. J. 41, 908-912. BLACKMER A. M. and BREMNER J. M. (1978) Inhibitory effect of nitrate on reduction of N20 to NZ by soil microorganisms. Snif Bioi. Bjoc~~~. 10, 187~191. BREMWR J. M. and KEENEY D. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils. 3. Exchangeable ammonium, nitrate, and nitrite by extraction-distillation methods. Prcrc. Soil Sci. Sot. Am. 30, 577-582. CRUTZEN P. J. and EHHALT D. H. (1977) Effects of nitrogen fertilizers and combustion on the stratospheric ozone layer. An&o 6, 112-117. HAHN J. and JUNGE C. (1977) Atmospheric nitrous oxide: A critical review. Z. Naturf. 32a, 190-214. JOHNSTON H. (1972) Newly recognized vital nitrogen cycle. Proc. nutn. Acad. Sci.. U.S.A. 69, 2369-2372. Lru S. C., CICERONER. J., DONAHUE T. M. and CHAMEIUES W. L. (1977) Sources and sinks of atmospheric N20 and the possible ozone reduction due to industrial fixed nitrogen fertilizers. Teilzts 29, 251.--263.
Short MATXTBARAT. and IWASAKI H. (1971) Enzymatic
communications
steps of dissimilatory nitrite reduction in Alcdigenes faecalis. J. Biochem., Tokyo 69, 859-868. MA~SUBARA T. and MORI T. (1968) Studies on denitrification. IX. Nitrous oxide, its production and reduction to nitrogen. J. Biochem., Tokyo 64, 863-871.
MCELROY M. B., ELKINS J. Y. L. (1976) Sources and Reo. Geophys. Space Phys. PAYNE W. J. and RILEY P. S. of enzymatic reduction of Biol. Med. 132, 258-260.
315 W., WOFSY S. C. and
YUNG
sinks for atmospheric N,O. 14, 143-150. (1969) Suppression by nitrate nitric oxide. Proc. Sot. exp.
SHORT
COMMUNICATION
Stimulatory effect of nitrate on reduction of N20 to N, by soil microorganisms A. M. BLACKMER and J. M. Department
of Agronomy,
(Accepred
IO December
Recent research by atmospheric scientists has suggested that increased N fertilization of soils may increase emissions of N,O from soils to the atmosphere via denitrification of fertilizer N and thereby promote destruction of the stratospheric ozone layer protecting the earth from biologically harmful ultraviolet radiation from the sun (Johnston, 1972; McElroy et al., 1976; Crutzen and Ehhalt, 1977; Hahn and Junge, 1977; Liu VI ni., 1977). This has greatly stimulated research on denitri~~tion of nitrate in soils and has emphasized the need for a better understanding of factors affecting the relative amounts of NzO and Nz released from soils by this process. We recently demonstrated that NO; inhibits anaerobic reduction of N,O to N, by soil microorganisms and that, because of this inhibitory effect, the proportion of NO;-N emitted to the atmosphere as N,O when NO, undergoes denitrification in soil increases markedly with the concentration of NO; (Blackmer and Bremner, 1978). Our purpose here is to report work showing that. if the denitrifying microorganisms in soils have not had an opportunity to adapt for anaerobic reduction of NJ0 to N,, a small amount of nitrate has a stimulatory effect on their ability to perform this reduction. The soils used (Table I) were surface (O-15 cm) samples of Iowa soils selected to obtain a range in pH, texture and organic-matter content. Before use, each sample was air-dried, crushed and sieved (<2-mm). The analyses reported in Table I were performed as described by Blackmer and Bremner (1977a). To obtain samples of NO;-free soils, 100-g samples of each soil were treated with 30mf water and incubated under He at 30YY for 48 h. The samples were then dried rapidly by exposing them in a thin layer to a stream of air at room temperature. Analyses by the method of Bremncr and Keeney (1966) showed that the air-dried soil samples contained only trace amounts of NO; (~0.5 pg NO; .g-’ soil). To study the effect of nitrate on the ability of these soil samples to reduce N,O to Nz under anaerobic conditions. 30g subsamples were placed in 1.2-l flasks fitted with standard-taper (34145) ground-glass joints and
Table
FR.“. II,3
G
BREMNER
Iowa State University,
Ames, IA 50011, U.S.A.
1978)
treated with water or with water containing nitrate (as KNOJ). Each flask was then sealed with a glass stopper fitted with a standard-taper (34/45) ground-glass joint and glass stopcock (l-mm dia bore) and was connected to a manifold system for replacement of air by He or by He containing N,O (1 mg N,O-N.g-’ soil). The flasks were then placed in an incubator at 3o’C, and their atmospheres were sampled at intervals for determination of N, and N,O by gas chromatography (Blackmer and Bremner, 1977b). AH incubation experiments and analyses were performed in duplicate or triplicate. Table 2 shows that treatment of NOT-free soils with a small amount of NO; (5 pg NO;-N.g-’ soil) increased their ability to reduce NzO to N, under anaerobic conditions and that the stimuiatory effect of NO; on N,O reduction increased very markedly with decrease in soil pH. This stimulatory effect cannot be attributed to reduction of the added NO; to N, because Table 2 shows that addition of 5 pg NOT-N .g-’ soil increased production of N, by as much as 127 pg N, g -’ soil. Several investigations have indicated that cells of denitrifying microorganisms adapted for reduction of NO; are simultaneously adapted for reduction of N,O to N, (Allen and van Niel. 1952: Matsubara and Mori, 1968: Payne and Riley, 1969: Matsubara and Iwasaki, 1971). The work reported here indicates that NO; is more effective than N,O for stimulation of N,O-reductase activity in soil microorganisms. This would explain the apparent contradiction between the data in Table 2, indicating that NO; has a stimulatory effect on reduction of NzO to N, by soil m~~oorganisms, and the data reported in Table 3 of our previous paper (Blackmer and Bremner, l97S), indicating that NO; has an inhibitory effect on this process. The key difference between our experiments showing the stimulatory effect of NO; and those showing the inhibitory effect is that the soils showing the inhibitory effect contained NOj when they were preincubated under He before exposure to N,O, whereas those showing the stimulatory effect did not contain NO; before this preincubation. We
I. Analyses
of soils
Soil
PH
Organic C (“4)
Sand (%)
Clay (‘X)
Nitrate-N @g.g-’ soil)
Harps Webster Ames Marshall Monona Sparta Okoboji
7.8 6.8 6.5 5.8 5.1 5.4 4.8
4.3 3.8 2.9 2.4 2.0 0.7 6.7
21 35 33 2 4 75 7
36 25 26 32 26 9 40
20 7 9 29 11 5 0
313
314
Short Table 2. Amounts
communications
of N, produced on exposure of nitrate-free soils to N,O under anaerobic conditions*
and nitrate-treated
Amount of N, produced (pg N. g- ’ soil) Soil
PH
Harps
7.8
Webster
6.8
Marshall
5.8
Monona
5.7
Sparta
5.4
Okoboji
4.8
Nitrate-N (kg.g-’
added soil) 0 5 0 5 0 5 0 5 0 5 0 5
12h
24h
60 73 14 24 28 57 II 21 2 3 3 4
141 170 54 68 95 150 22 49 5 23 4 41
48 hf 322 331 145 166 229 307 86 145 24 73 26 153
(3) (14) (34) (69) (204) (488)
* Samples (30g) of nitrate-free soils were placed in 1.2-l flasks, treated with 10 ml water and preincubated under He for 12 h. They were then treated with 8 ml water containing 0 or 15O/dg N as KNOs and incubated at 30°C for various times under He containing N,O (1 mg N,O-N.g-’ soil). t Figures in parentheses indicate percentage stimulation of N,O reduction by nitrate. Table 3. Effects of different amounts of added on exposure of nitrate-free soil to N20 Amount
nitrate on amount of Nz produced under anaerobic conditions* of N, produced
(pg.g-’
soil)
Amount of nitrate-N added (~g.g~’ soil)
18h
24h
36h
0 5 10 20 50
4.2 12.1 7.6 3.2 3.0
6 38 32 19 5
28 126 124 104 22
* Samples (30 g) of nitrate-free Ames soil were placed in 1.2-I flasks. treated with 18 ml water containing @15OO~g N as KNO,, and incubated for various times under He containing N,O (I mg NLO-N.g-’ soil). conclude that, because NO; was absent, the denitrifying microorganisms in the soils showing the stimulatory effect did not adapt for reduction of N,O to N, during this preincubation. Tests with several soils showed that, when nitrate is added to a NO;-free soil containing microorganisms not adapted for reduction of N,O to N,, its effect on this reduction depends upon the amount added. This is illustrated by Table 3, which shows that both stimulatory and inhibitory effects of NO, on N,O reduction were observed when NO;-free Ames soil was treated with different amounts of NO; before exposure to NzO. The data reported are readily explained if NO; has two effects on N,O reduction by soil microorganisms, one being that it stimulates production of N,O reductase. the other being that it inhibits the activity of this enzyme. This would account for our finding that the stimutatory effect of NO; on N,O reduction decreased as the amount of NOT-N added was increased from 5 to 20 pg. g- ’ soil and that only the inhibitory effect of NO; on N,O reduction was evident when the amount of NO;-N added was increased to 5Ofig.g-’ soil.
Acknowledgemenrs-Journal Paper No. J-9209 of the Iowa Agriculture and Home Economics Experiment Station. Project 2096. This work was supported in part by the U.S. Department of Energy.
REFERENCES ALLEN M. B. and VAN NIEL C. B. (1952) Experiments on bacterial denitrification. J. Bact. 64, 397--412. BLACKMERA. M. and BREMNER J. M. (1977a) Nitrogen isotope discrimination in denitrification of nitrate in soils. Soil Bioi. Biochem. 9, 73377. BLACKMERA. M. and BREMNERJ. M. (1977b) Gas chromatographic analysis of soil atmospheres. Soil Sri. Sm. Am. J. 41, 908-912. BLACKMER A. M. and BREMNER J. M. (1978) Inhibitory effect of nitrate on reduction of N20 to NZ by soil microorganisms. Snif Bioi. Bjoc~~~. 10, 187~191. BREMWR J. M. and KEENEY D. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils. 3. Exchangeable ammonium, nitrate, and nitrite by extraction-distillation methods. Prcrc. Soil Sci. Sot. Am. 30, 577-582. CRUTZEN P. J. and EHHALT D. H. (1977) Effects of nitrogen fertilizers and combustion on the stratospheric ozone layer. An&o 6, 112-117. HAHN J. and JUNGE C. (1977) Atmospheric nitrous oxide: A critical review. Z. Naturf. 32a, 190-214. JOHNSTON H. (1972) Newly recognized vital nitrogen cycle. Proc. nutn. Acad. Sci.. U.S.A. 69, 2369-2372. Lru S. C., CICERONER. J., DONAHUE T. M. and CHAMEIUES W. L. (1977) Sources and sinks of atmospheric N20 and the possible ozone reduction due to industrial fixed nitrogen fertilizers. Teilzts 29, 251.--263.
Short MATXTBARAT. and IWASAKI H. (1971) Enzymatic
communications
steps of dissimilatory nitrite reduction in Alcdigenes faecalis. J. Biochem., Tokyo 69, 859-868. MA~SUBARA T. and MORI T. (1968) Studies on denitrification. IX. Nitrous oxide, its production and reduction to nitrogen. J. Biochem., Tokyo 64, 863-871.
MCELROY M. B., ELKINS J. Y. L. (1976) Sources and Reo. Geophys. Space Phys. PAYNE W. J. and RILEY P. S. of enzymatic reduction of Biol. Med. 132, 258-260.
315 W., WOFSY S. C. and
YUNG
sinks for atmospheric N,O. 14, 143-150. (1969) Suppression by nitrate nitric oxide. Proc. Sot. exp.