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The nitrification inhibitor DMPP had no effect on denitrifying enzyme activity
Soil Biology & Biochemistry 34 (2002) 1825–1827 www.elsevier.com/locate/soilbio
Short Communication
The nitrification inhibitor DMPP had no effect on denitrifying enzyme activity C. Mu¨llera,*, R.J. Stevensb, R.J. Laughlinb, F. Azama, J.C.G. Ottowa a Department of Applied Microbiology, University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany Agriculture and Environmental Science Division, The Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK
b
Received 4 April 2002; received in revised form 16 July 2002; accepted 19 August 2002
Abstract Denitrifying enzyme activity (DEA) and flux rates of nitrous oxide (N2O) and dinitrogen (N2) were studied in DEA assays on soils treated with 3,4-dimethylpyrazole phosphate (DMPP). Nitrous oxide and N2 fluxes were quantified by 15N gas-flux method with no additional enzymatic inhibitors, thus, overcoming problems associated with the use of chloramphenicol and acetylene. The nitrification inhibitor DMPP did not affect DEA or the measured gas emissions even when applied in concentrations 14 times higher than the recommended concentration. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: 15N; Nitrous oxide; Dinitrogen; Denitrifying enzyme activity; 3,4-Dimethylpyrazole phosphate
1. Introduction Field studies with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP, marketed by COMPO GmbH & Co. KG under the name ENTEC) showed that it decreased nitrous oxide (N2O) and carbon dioxide (CO2) emissions significantly and increased methane (CH4) uptake (Weiske et al., 2001). The aim of the study presented here was to investigate if the decrease of N2O could be related to an inhibitory effect on the enzymes of the non-target process, denitrification, by determining the denitrifying enzyme activity (DEA) in soils previously treated with DMPP. To measure whether DMPP affected nitrate reductase or N2O reductase, emissions of N2O and dinitrogen (N2) had to be made separately so the commonly used DEA technique had to be modified. DEA is usually determined under anaerobic conditions with a supply of carbon and nitrate to ensure that all existing denitrification enymes operate under optimum conditions (Smith and Tiedje, 1979). Acetylene is used to block N2O reductase so that the total N gas emitted (N2O þ N2) can be easily * Corresponding author. Address: Department of Plant Ecology, University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. Tel.: þ 49-641-9935315; fax: þ49-641-9935309. E-mail address: [email protected] (C. Mu¨ller).
measured as N2O (Yoshinari and Knowles, 1976). Smith and Tiedje (1979) observed two phases of denitrification rates after the start of the enzyme assay. Phase I was characterized by a linear N2O increase which lasted for approximately 1.5 h. They attributed this increase to activity of pre-existing enzymes. To extend this denitrification phase I, they proposed the application of chloramphenicol which inhibits the formation of new enzymes. However, work by Pell et al. (1996) provided evidence that chloramphenicol does not only inhibit the synthesis of new enzymes but affects also the activity of enzymes already present in soil. To overcome problems associated with chloramphenicol, we performed measurements within 1 h after the start of the assay which still lies in the linear range of denitrification phase I (we confirmed the existence of denitrification phase I in preliminary studies not reported here). Recent studies showed that acetylene in concentrations high enough to block the last enzymatic step in the denitrification sequence causes scavenging of the intermediate nitric oxide (NO). This occurs mainly under aerobic conditions thereby underestimating the overall denitrification N-gas loss (Bollman and Conrad, 1997). Under anaerobic conditions, however, acetylene causes artifacts, particularly at high levels of C substrate (McKenney et al., 1996). Therefore, to overcome problems associated with acetylene and separately
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C. Mu¨ller et al. / Soil Biology & Biochemistry 34 (2002) 1825–1827
determine the rates of N2O and N2 in DEA assays we used the 15N gas flux method (Stevens et al., 1993) in the linear range of denitrification phase I without any enzymatic inhibitors. In addition, this method allowed the determination of CO2 and CH4 emissions under anaerobic conditions which otherwise could have been affected by the inhibitors.
2. Materials and methods The soil (silty clay; organic C, 1.35%; total N, 0.15%; pH (CaCl2), 6.8) was the same as described by Azam et al. (2001). Prior to the DEA analysis, twenty-five grams of soil at a moisture content of 40% (vol/vol) 21 were pre-incubated for 10 days with 50 mg NO2 3 -N g 21 soil and 10 mg glucose-C g soil to develop denitrifying enzymes. Two hours before the DEA assay the soils were treated with DMPP at concentrations of 0, 3.5 (concentration used in practice), 10, 20 and 50 mg g21 soil and with three replicates per treatment. DEA assays were carried out in 250 ml flasks (Brand) with a septum fitted in the lid for gas sampling. At the start of the assay 75 ml of a nitrate – glucose solution were applied to each 21 flask resulting in concentrations of 50 mg KNO2 3 -N g 21 soil and 300 mg glucose-C g soil. The applied nitrate was enriched with 15N at 99 at.%. Flasks were immediately closed and the headspace flushed with He (Grade 5.0, Messer Griesheim) for 1 min with a double needle. The samples were placed at 20 8C on a rotary shaker at 120 rpm. Gas samples were taken at 30 and 60 min after flushing with disposable 60 ml syringes and analysed for O2, CH4, CO2 and N2O on a gas chromatograph (GC) equipped with an FID and ECD detector (Mosier and Mack, 1980) within 4 h after sampling. Gas fluxes were calculated between the two samplings. The extracted gas after the first sample was replaced by the same amount of He. The concentrations of the second sampling were adjusted for the dilution and also for dissolved gas in the soil solution using the Bunsen coefficient (Moraghan and Buresh, 1977). The concentration of N2O was calculated by the 15N gas – flux method as described by Stevens et al. (1993). The concentration of N2 in the He headspace was calculated from the sum of the ion currents (I) at m/z 28, 29 and 30 with reference to an air standard. The molecular ratios 29R (29I/28I) and 30R (30I/28I) were then determined and differences between the molecular ratios in enriched and normal atmospheres were calculated as D29R and D30R to calculate the enrichment of the denitrifying pool (15XN) and the N2 concentration according to Mulvaney and Boast (1986). The ratio between N2O and N2 was determined from samples taken after 60 min by the 15N gas flux method (Stevens et al., 1993) and the N2 flux was calculated by multiplying this ratio with the measured N2O flux.
3. Results and discussion No oxygen could be detected in any of the gas samples which showed that the He flushing method was successful in creating anaerobic conditions. Additional evidence for anaerobic conditions was provided by the positive methane flux (data not presented) showing that methanogenic rather than methanotrophic activity was prevalent. Results of N2O concentrations determined by gas chromatograph and mass spectrometer (MS) were in close agreement (Fig. 1, MSN2O ¼ 2 0.38 þ 0.99GCN2O; r 2 ¼ 0.99). The average enrichment of the N2O was 64.6 at.% 15N and was the same whether calculated from 45 R or 46R. The distribution of the 15N atoms in the N2O molecules was therefore random indicating that it was all derived from a single labeled nitrate pool. More than half of the nitrate added in the pre-incubation was still present at the start of the DEA assay and diluted the applied nitrate. The fluxes of N2O and N2 were statistically the same (P . 0.05) for all treatments (Fig. 2), i.e. DMPP application did not affect either nitrate reductase or N2O reductase capacity in soil even at concentrations more than 14 times of the recommended practice concentration. The average DEA
Fig. 1. Comparison and linear regression between N2O concentrations determined on GC and on MS.
Fig. 2. Nitrous oxide and dinitrogen fluxes (mean ^ SD) in soils treated with various concentrations of the nitrification inhibitor DMPP.
C. Mu¨ller et al. / Soil Biology & Biochemistry 34 (2002) 1825–1827
rate (N2O þ N2 flux) was 0.867 ^ 0.118 g N g21 h21 and the average ratio of N2 O=N2 O þ N2 was 0.165, i.e. 16.5% of the overall measured gaseous N flux was N2O the rest N2. The CH4 and CO2 emissions during anaerobic conditions and optimal supply of substrates were not affected by DMPP (data not presented). Therefore, the observed decrease of N2O and CO2 in field studies after DMPP application (Weiske et al., 2001) could not be explained by impacts on denitrification in soil. We conclude that the modified DEA method was successful in determining the N2O and N2 rates under anaerobic conditions and optimal substrate supply. The advantage is that N2O and N2 as well as CO2 and CH4 emissions can be determined separately on sub-samples of the same headspace.
Acknowledgements We would like to thank the German science foundation (Deutsche Forschungsgemeinschaft) and the Alexander von Humboldt Foundation for providing scholarships to C. Mu¨ller and F. Azam, respectively.
References Azam, F., Benckiser, G., Mu¨ller, C., Ottow, J.C.G., 2001. Release, movement and recovery of 3,4-dimethylpyrazole phosphate (DMPP), ammonium, and nitrate from stabilized nitrogen fertilizer granules in
1827
a silty clay soil under laboratory conditions. Biology and Fertility of Soils 34, 118– 125. Bollman, A., Conrad, R., 1997. Acetylene blockage technique leads to underestimation of denitrification rates in oxic soils due to scavenging of intermediate nitric oxide. Soil Biology & Biochemistry 29, 1067–1077. McKenney, D.J., Druruy, C.F., Wang, S.W., 1996. Effect of acetylene on nitric oxide production in soil under denitrifying conditions. Soil Science Society of America Journal 60, 811 –820. Moraghan, J.T., Buresh, R.J., 1977. Correction for dissolved nitrous oxide in nitrogen studies. Soil Science Society of America Journal 41, 1201–1202. Mosier, A.R., Mack, L., 1980. Gas chromatographic system for precise, rapid analysis of nitrous oxide. Soil Science Society of America Journal 44, 1121–1123. Mulvaney, R.L., Boast, C.W., 1986. Equations for determination of nitrogen-15 labeled dinitrogen and nitrous oxide by mass spectrometery. Soil Science Society of America Journal 50, 360– 363. Pell, M., Stenberg, B., Stenstro¨m, J., Torstensson, L., 1996. Potential denitrification activity assay in soil—with and without chloramphenicol? Soil Biology & Biochemistry 28, 393–398. Smith, M.S., Tiedje, J.M., 1979. Phases of denitrification following oxygen depletion in soil. Soil Biology & Biochemistry 11, 261–267. Stevens, R.J., Laughlin, R.J., Atkins, G.J., Prosser, S.J., 1993. Automated determination of 15N-labelled dinitrogen and nitrous oxide by mass spectrometry. Soil Science Society of America Journal 57, 981–988. Weiske, A., Benckiser, G., Herbert, T., Ottow, J.C.G., 2001. Influence of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) in comparison to dicyandiamide (DCD) on nitrous oxide emissions, carbon dioxide fluxes and methane oxidation during 3 years of repeated application in field experiments. Biology and Fertility of Soils 34, 109– 117. Yoshinari, T., Knowles, R., 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochemical and Biophysical Research Communications 69, 705– 710.
Short Communication
The nitrification inhibitor DMPP had no effect on denitrifying enzyme activity C. Mu¨llera,*, R.J. Stevensb, R.J. Laughlinb, F. Azama, J.C.G. Ottowa a Department of Applied Microbiology, University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany Agriculture and Environmental Science Division, The Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK
b
Received 4 April 2002; received in revised form 16 July 2002; accepted 19 August 2002
Abstract Denitrifying enzyme activity (DEA) and flux rates of nitrous oxide (N2O) and dinitrogen (N2) were studied in DEA assays on soils treated with 3,4-dimethylpyrazole phosphate (DMPP). Nitrous oxide and N2 fluxes were quantified by 15N gas-flux method with no additional enzymatic inhibitors, thus, overcoming problems associated with the use of chloramphenicol and acetylene. The nitrification inhibitor DMPP did not affect DEA or the measured gas emissions even when applied in concentrations 14 times higher than the recommended concentration. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: 15N; Nitrous oxide; Dinitrogen; Denitrifying enzyme activity; 3,4-Dimethylpyrazole phosphate
1. Introduction Field studies with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP, marketed by COMPO GmbH & Co. KG under the name ENTEC) showed that it decreased nitrous oxide (N2O) and carbon dioxide (CO2) emissions significantly and increased methane (CH4) uptake (Weiske et al., 2001). The aim of the study presented here was to investigate if the decrease of N2O could be related to an inhibitory effect on the enzymes of the non-target process, denitrification, by determining the denitrifying enzyme activity (DEA) in soils previously treated with DMPP. To measure whether DMPP affected nitrate reductase or N2O reductase, emissions of N2O and dinitrogen (N2) had to be made separately so the commonly used DEA technique had to be modified. DEA is usually determined under anaerobic conditions with a supply of carbon and nitrate to ensure that all existing denitrification enymes operate under optimum conditions (Smith and Tiedje, 1979). Acetylene is used to block N2O reductase so that the total N gas emitted (N2O þ N2) can be easily * Corresponding author. Address: Department of Plant Ecology, University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. Tel.: þ 49-641-9935315; fax: þ49-641-9935309. E-mail address: [email protected] (C. Mu¨ller).
measured as N2O (Yoshinari and Knowles, 1976). Smith and Tiedje (1979) observed two phases of denitrification rates after the start of the enzyme assay. Phase I was characterized by a linear N2O increase which lasted for approximately 1.5 h. They attributed this increase to activity of pre-existing enzymes. To extend this denitrification phase I, they proposed the application of chloramphenicol which inhibits the formation of new enzymes. However, work by Pell et al. (1996) provided evidence that chloramphenicol does not only inhibit the synthesis of new enzymes but affects also the activity of enzymes already present in soil. To overcome problems associated with chloramphenicol, we performed measurements within 1 h after the start of the assay which still lies in the linear range of denitrification phase I (we confirmed the existence of denitrification phase I in preliminary studies not reported here). Recent studies showed that acetylene in concentrations high enough to block the last enzymatic step in the denitrification sequence causes scavenging of the intermediate nitric oxide (NO). This occurs mainly under aerobic conditions thereby underestimating the overall denitrification N-gas loss (Bollman and Conrad, 1997). Under anaerobic conditions, however, acetylene causes artifacts, particularly at high levels of C substrate (McKenney et al., 1996). Therefore, to overcome problems associated with acetylene and separately
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 1 6 5 - 7
1826
C. Mu¨ller et al. / Soil Biology & Biochemistry 34 (2002) 1825–1827
determine the rates of N2O and N2 in DEA assays we used the 15N gas flux method (Stevens et al., 1993) in the linear range of denitrification phase I without any enzymatic inhibitors. In addition, this method allowed the determination of CO2 and CH4 emissions under anaerobic conditions which otherwise could have been affected by the inhibitors.
2. Materials and methods The soil (silty clay; organic C, 1.35%; total N, 0.15%; pH (CaCl2), 6.8) was the same as described by Azam et al. (2001). Prior to the DEA analysis, twenty-five grams of soil at a moisture content of 40% (vol/vol) 21 were pre-incubated for 10 days with 50 mg NO2 3 -N g 21 soil and 10 mg glucose-C g soil to develop denitrifying enzymes. Two hours before the DEA assay the soils were treated with DMPP at concentrations of 0, 3.5 (concentration used in practice), 10, 20 and 50 mg g21 soil and with three replicates per treatment. DEA assays were carried out in 250 ml flasks (Brand) with a septum fitted in the lid for gas sampling. At the start of the assay 75 ml of a nitrate – glucose solution were applied to each 21 flask resulting in concentrations of 50 mg KNO2 3 -N g 21 soil and 300 mg glucose-C g soil. The applied nitrate was enriched with 15N at 99 at.%. Flasks were immediately closed and the headspace flushed with He (Grade 5.0, Messer Griesheim) for 1 min with a double needle. The samples were placed at 20 8C on a rotary shaker at 120 rpm. Gas samples were taken at 30 and 60 min after flushing with disposable 60 ml syringes and analysed for O2, CH4, CO2 and N2O on a gas chromatograph (GC) equipped with an FID and ECD detector (Mosier and Mack, 1980) within 4 h after sampling. Gas fluxes were calculated between the two samplings. The extracted gas after the first sample was replaced by the same amount of He. The concentrations of the second sampling were adjusted for the dilution and also for dissolved gas in the soil solution using the Bunsen coefficient (Moraghan and Buresh, 1977). The concentration of N2O was calculated by the 15N gas – flux method as described by Stevens et al. (1993). The concentration of N2 in the He headspace was calculated from the sum of the ion currents (I) at m/z 28, 29 and 30 with reference to an air standard. The molecular ratios 29R (29I/28I) and 30R (30I/28I) were then determined and differences between the molecular ratios in enriched and normal atmospheres were calculated as D29R and D30R to calculate the enrichment of the denitrifying pool (15XN) and the N2 concentration according to Mulvaney and Boast (1986). The ratio between N2O and N2 was determined from samples taken after 60 min by the 15N gas flux method (Stevens et al., 1993) and the N2 flux was calculated by multiplying this ratio with the measured N2O flux.
3. Results and discussion No oxygen could be detected in any of the gas samples which showed that the He flushing method was successful in creating anaerobic conditions. Additional evidence for anaerobic conditions was provided by the positive methane flux (data not presented) showing that methanogenic rather than methanotrophic activity was prevalent. Results of N2O concentrations determined by gas chromatograph and mass spectrometer (MS) were in close agreement (Fig. 1, MSN2O ¼ 2 0.38 þ 0.99GCN2O; r 2 ¼ 0.99). The average enrichment of the N2O was 64.6 at.% 15N and was the same whether calculated from 45 R or 46R. The distribution of the 15N atoms in the N2O molecules was therefore random indicating that it was all derived from a single labeled nitrate pool. More than half of the nitrate added in the pre-incubation was still present at the start of the DEA assay and diluted the applied nitrate. The fluxes of N2O and N2 were statistically the same (P . 0.05) for all treatments (Fig. 2), i.e. DMPP application did not affect either nitrate reductase or N2O reductase capacity in soil even at concentrations more than 14 times of the recommended practice concentration. The average DEA
Fig. 1. Comparison and linear regression between N2O concentrations determined on GC and on MS.
Fig. 2. Nitrous oxide and dinitrogen fluxes (mean ^ SD) in soils treated with various concentrations of the nitrification inhibitor DMPP.
C. Mu¨ller et al. / Soil Biology & Biochemistry 34 (2002) 1825–1827
rate (N2O þ N2 flux) was 0.867 ^ 0.118 g N g21 h21 and the average ratio of N2 O=N2 O þ N2 was 0.165, i.e. 16.5% of the overall measured gaseous N flux was N2O the rest N2. The CH4 and CO2 emissions during anaerobic conditions and optimal supply of substrates were not affected by DMPP (data not presented). Therefore, the observed decrease of N2O and CO2 in field studies after DMPP application (Weiske et al., 2001) could not be explained by impacts on denitrification in soil. We conclude that the modified DEA method was successful in determining the N2O and N2 rates under anaerobic conditions and optimal substrate supply. The advantage is that N2O and N2 as well as CO2 and CH4 emissions can be determined separately on sub-samples of the same headspace.
Acknowledgements We would like to thank the German science foundation (Deutsche Forschungsgemeinschaft) and the Alexander von Humboldt Foundation for providing scholarships to C. Mu¨ller and F. Azam, respectively.
References Azam, F., Benckiser, G., Mu¨ller, C., Ottow, J.C.G., 2001. Release, movement and recovery of 3,4-dimethylpyrazole phosphate (DMPP), ammonium, and nitrate from stabilized nitrogen fertilizer granules in
1827
a silty clay soil under laboratory conditions. Biology and Fertility of Soils 34, 118– 125. Bollman, A., Conrad, R., 1997. Acetylene blockage technique leads to underestimation of denitrification rates in oxic soils due to scavenging of intermediate nitric oxide. Soil Biology & Biochemistry 29, 1067–1077. McKenney, D.J., Druruy, C.F., Wang, S.W., 1996. Effect of acetylene on nitric oxide production in soil under denitrifying conditions. Soil Science Society of America Journal 60, 811 –820. Moraghan, J.T., Buresh, R.J., 1977. Correction for dissolved nitrous oxide in nitrogen studies. Soil Science Society of America Journal 41, 1201–1202. Mosier, A.R., Mack, L., 1980. Gas chromatographic system for precise, rapid analysis of nitrous oxide. Soil Science Society of America Journal 44, 1121–1123. Mulvaney, R.L., Boast, C.W., 1986. Equations for determination of nitrogen-15 labeled dinitrogen and nitrous oxide by mass spectrometery. Soil Science Society of America Journal 50, 360– 363. Pell, M., Stenberg, B., Stenstro¨m, J., Torstensson, L., 1996. Potential denitrification activity assay in soil—with and without chloramphenicol? Soil Biology & Biochemistry 28, 393–398. Smith, M.S., Tiedje, J.M., 1979. Phases of denitrification following oxygen depletion in soil. Soil Biology & Biochemistry 11, 261–267. Stevens, R.J., Laughlin, R.J., Atkins, G.J., Prosser, S.J., 1993. Automated determination of 15N-labelled dinitrogen and nitrous oxide by mass spectrometry. Soil Science Society of America Journal 57, 981–988. Weiske, A., Benckiser, G., Herbert, T., Ottow, J.C.G., 2001. Influence of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) in comparison to dicyandiamide (DCD) on nitrous oxide emissions, carbon dioxide fluxes and methane oxidation during 3 years of repeated application in field experiments. Biology and Fertility of Soils 34, 109– 117. Yoshinari, T., Knowles, R., 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochemical and Biophysical Research Communications 69, 705– 710.