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Denitrification in sediment determined from nitrogen isotope pairing
FEMS MicrobiologyEcology86 (1992)357-362 © 1992Federationof European MicrobiologicalSocieties0168-6496/92/$05.00 Publishedby Elsevier
357
FEMSEC ~ 3 ~
Denitrification in sediment determined from nitrogen
isotope pairing Lars P e t e r Nielsen Department of Ecology and Genetics, Universityof Aarhus, Aarhus, Denmark
Received 17 October 1991 Revisionreceivedand accepted 19 December 1991 Key words: Denitrification; Nitrification; Sediment; Isotopes; ~5N; ~sNO~
1. SUMMARY
2. INTRODUCTION
A new method for accurate and easy measurement of denitrification in sediments is presented. The water overlying intact sediment cores was enriched with ~5NO3 which mixed with the ~4NO3 of the natural sources of NO3. The formation by denitrification of single-labeled (14N 15N) and double.-labeled (IZN mSN)dinitrogen pairs was measured by mass spectrometry after a few hours incubation. Total denitrification including the formation of unlabeled p4NI4N) dinitroden could be calculated assuming random isotope pairing by denitrification of the uniformly mixed N O j species. In contrast to previous approaches, by this method it is possible to measure denitrification of both N O j diffusing lrom the overlying water and NO~ from nitrification within the sediment.
In aquatic environments, combined nitrogen is recycled to the atmosphere by denitrification. Rates and regulations of this process are of increasing interest due to severe eutrophication problems caused by anthropogenic inputs of combined nitrogen [1]. Anoxic layers of sediments are the major sites of denitrification, which may reduce both NO3 diffusing from the overlying water column and NO3 produced by nitrification in the oxic layers. The supply of NO~- from the two sources is regulated quite differently; the former being greater at higher concentrations of nitrate in the water and lower penetration of oxygen in the sediment [2,3], the latter dependent on sediment N-mineralization and generally enhanced by deeper oxygen penetration [4]. Coupled nitrification-denitrification is thought to be quantitatively more important than influx of NO~ in most aquatic sediments [1], but the evidence is conflicting [2,5]. The lack of suitable methods has been a major obstacle in studies of denitrification. Attempts to
Correspondence to: L.P. Nielsen,Departmentof Ecologyand
Genetics, University of Aarhus, Ny Munkegade, DK-8000 Aarhus C, Denmark.
358 measure denitrification in sediments using the acetylene-block technique [6] or 15NO3 [7] have only quantified denitrifieation of NO~" taken up from the overlying water (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). The few attempts to specifically measure coupled nitrification-denitrification have been based on the production of 15N-labeled N2 after the addition of JSNH~" to the overlying water [7-9]. The results obtained by these attempts are, however, rather inaccurate due to very high NH~ enrichments or questionable assumptions about N-isotope ratios in the NH~ and NO 3 pools of the active layers (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). The best current methods for determining total denitrification rates are based on the measurement of N 2 fluxes from sediments [10,11] (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). These methods, however, depend on either week-long pre-incubation in the laboratory [10] or substantial depletions of NO~ and 02 in the overlying water during incubation [ll], thus altering in situ conditions. Using the new isotope pairing method presented here, both coupled nitrification-denitrification and denitrification of NO3 from the overlying water can be quantified in an easy and rapid batch mode assay with minimal deviation from in situ conditions.
3. MATERIALS AND METHODS Intact cores were sampled in April 1991 from the sediment of Salten A, a stream draining the productive lake Salten Langs¢, Denmark. The sediment consisted mainly of sand with a 0.5-cm organic-rich top layer. The average porosity of the top 3 cm was 43% (vol/vol). Sediment cores were collected in Plexiglas tubes (10 cm 2, 3-cm sediment, 7-cm water column) and stored overnight in a container with NO~-free freshwater. During storage and subsequent incubation, the in situ temperature of 13°C was maintained and the water column in each core was gently stirred by a small rotating magnet.
Ini6ally each core was closed with a rubber stopper and 2 mi of water was replaced with nitrogen-free air (21% O2 in He) using syringes. The elimination of N2 in the gas phase lowered the background N2 level thereby improving the final detection of 15N-enriched N 2. Incubations were started by injecting 15NO3 from a 10 mM stock solution of 99.6% ~5NO3 into the initially NO~'-free water column. Water free of 14NO3 was used in this specific study in order to make nitrification in the se,"-nent the only possible source of 14NO~. Four parallel time series with 6 cores each were incubated for 0-3.5 h. One series was incubated with 56/~M 15NO~ corresponding to the in situ concentration of NO~, and a second series was incubated with 19/~M 15NO~. To verify the measurements of coupled nitrification-denitrification, nitrification was blocked in a third series ~y adding 120/zM thiourea [12] to the water column 0.5 h prior to incubation with 19/~M ~sNO~. The fourth series was used only for the analysis of changes in 0 2 and NO~ concentrations during the incubation ( < 15 and 8%, respectively, after 3.5 h). The incubation of a core in each series was stopped at 40 min intervals and dissolved N 2 in the water phase and pore water were equilibrated with the gas phase by vigorous shaking for 4 rain. Controls showed that 95% equilibrium was obtained by this procedure. Denitrification was assumed to be inhibited instantly as 0 2 was mixed into the sediment, and shaking and gas sampling were done before the slurry went anoxic. The whole gas phase was sampled by syringe and stored in a pre-evacuated 3.5-ml blood collection tube (S. Rysgaard, N. Risgaard-Petersen and N.P. Revsbech, in preparation). The gas samples were analyzed on an isotope ratio mass spectrometer with three collectors (Sira Series II, VG Isotech, Middlewich, Chesire, U.K.). Subsamples of gas (200/~l) were injected into a carrler gas flow of He (30 ml rain- I) and possible interfering gasses were separated from dinitrogen in a freeze trap (copper-tube loop in liquid nitrogen) and by retention in a molecular sieve tube (2.4 m, 5 ,~, 60-80 mesh). The gas finally passed a splitter leading part of the flow (approx. 2%)
359 into the mass spectrometer. The retention time for N 2 was 150 s and the mass peaks were integrated over 110 s. All three N 2 species (t4Ni4N, t4NtSN and ~SN~SN, atom masses 28, 29 and 30 respectively) were measured and excess ;4N'SN and ~SN~SN was determined using air samples with the same total N 2 amount. In air t4NtSN and tSNtSN constitutes 0.7299 and 0.001% of total N2, respectively, and the maximal excesses recorded in the samples were 0.088 and 0.175% of total N 2. The accuracy of 14NISN and ISNtSN measurements were 0.0001 and 0.001% of total N2, respectively. In RESUt:rs AND DISCUSSION t4NlSN and iSNISN refer to the excess values. The production of 14NtSN and 15NtSN in each core expressed in nmol N was calculated knowing the distribution coefficient of N 2 in water and gas (0.018 at 13°C, ref. 13) and the volumes of gas and water in the cores.
120
.
.
,
m
..
.
~ . . . . - i
~
11f..I
oI
14N!,~
o
o
% TmneIh~
12~
I
8(
Z
c3
4. RESULTS AND DISCUSSION
t:z
14N1~ ......
4.1. Denitrification rates
The amount of ISN-labeled N 2 increased in all time series; the highest rate was in the series with 56 p,M tSNO3 in the overlying water (Fig. 1). Single labeled dinitrogen (14N 15N), in addition to double labeled (15NISN), was formed in the two first series without nitrification inhibitor, thus showing a contribution of 14NO3 from nitrification in the sediment. The variability between the cores was larger than seen in other sediments using the same method (L.P. Nielsen, in preparation), and the differences most likely reflected real variations in biological activity. The ratios of I4NISN/15NIsN did not change significantly during each of the 3 time series indicating a rapid and uniform equilibrium between 'SNO~ and 14NO3 in the denitrification zone of the sediment (Fig. 2). This can be explained by the microzonation of denitrification in the sediment: recent N20 microsensor studies in similar stream sediments with 100/zM N O i in the overlying water showed that denitrification was restricted to a narrow 0.5-mm thick zone immediately below a l-ram thick oxic surface layer [14]. The turnover time in the sediment of
Tee (h) 120
8c
4°
~
°
~4N~SN .....
o 1 2 3 Time(h) Fig. 1. Excessof tSNISN(solid lines)and 14NISN(dashed lines) in sedimentcores incubatedfor different times with 99.6 atom% tSNO~" in the overlyingwater. A: With 56/tM 15NO~"in the overlyingwater;,B: with 19/~MtsNO~";C: with 19 pM tsNO~" plus nitrificationinhibiior(n.i.). Each pair of data points represents one sediment cure. Lines represent linearregressions.
36O 3
rearranged to
f i4/f ts (14NISN)/2(15NIsN) ffi
(3)
Eqns. 1-3 were finally combined to express Di4 by the measured data:
Ig~SO~
Dr4 = ((14NISN)/2(15NISN)) o
o
• 19pMNO~+n.j.
_-
•
• "-
o
;
2 Twne {h)
Fig. 2. Ratio of 14NISNto ISNISNin the N 2 evolvedduring the 3 experimentsillustratedin Fig. 1.
NO 3 diffusing from the water could be calculated to 8 min, indicating a rapid development of steady-state profiles of m5NO~" in the present study. All NO;" diffusing from the overlying water to the anoxic denitrification zone has to pass through the oxic surface layer add to mix with NO3 produced in this layer. Rates of denitrification of tsNO3" (Dins) and t4NO~" (Dr4) were calculated from the measured productions of 14NlSN and ~SN~SN. D~5 was calculated simply as the sum of ~SN in the produced labeled dinitrogen: D15 = (14NtSN) + 2(15NtSN)
fl4/fl5
The calculated rates of denitrification at the three treatments are presented in Fig. 3. In the present study all 14NO3 in the overlying water was replaced with tsNO 3, and D~s therefore represented denitrification of NO 3 from the overlying water, while D14 represented denitrification coupled to nitrification in the sediment (Fig. 3). In y;ene~a', however, the differentiation between the two NO~ sources of denitrification may also be obtained in studies where ~5NO3 is added to a water column with ambient concentrations of 14NO3 (L.P. Nielsen, in preparation): if the ~4N and 15N frequencies of the nitrate in the
56"iNO; 90, 19pMNO~
-4--
(1)
19IJMNO3+n.J. 3(
DI4 was calculated from Dts: DI4 = DI5 *
* (('4NrsN) + 2('5N'SN))
(2)
where ft4 and fls represented the frequencies of 14N and 15N in the NO3 reduced by the denitrifying bacteria. The ratio ft4/fls could be calculated from the ratio of the produced labeled dinitrogen species (14NISN)/(ISNISN) as the denitrified NO 3 species were uniformly mixed, and t4NISN and tSN~SN therefore were formed with the probabilities 2ft4fts and ftsfls respectively [i5]. The ratio (14NISN)/(tSNlSN) could then be expressed as (14NISN)/(lSNtSN) ffi (2f14fls)/(ftsfts)
_ms
Fig. 3. Rates of denitrificationin stream sedimentas calculated from linearregressionsof the data presented in Fig. 1. Shaded bars indicatecouplednitrification-denitrification(denitrificationof 14NO~) and whitebars indicatedenitrification of nitratediffusingfromthe overlyingwater (denitrificationof IsNO~"). The 56/~M NO~" correspondsto in situ conditions in the stream. Note that coupled nitrification-denitrification was independentof overlyingNO~ concentrationand efficientlyblockedby the nitrificationinhibitor,whereasdenitrification of NO~" from the overlyingwater was proportionalto the NO~" concentrationand not affected by the nitrification inhibitor.Bars indicatestandarderrors derivedfromstandard errors of the regressioncoefficients.
361 overlying water become f ~ and f~5, then the ambient denitrification of t4NO3 from the water column (D~'4) is D~4 = Dr5 * f~4/f~s and coupled nitfification-denitrification (D~4) is calculated by difference: D~4 -- Dr4 - D~'4 The frequencies f ~ and f ~ may be obtained by measuring the NO~" concentration before and after the addition of lsNO3. If the frequencies may change significantly during incubation they can be measureo directly by mass spectrometry after chemical [16] or biological [17] (N. Risgaard-Petersen, S. Rysgaard and N.P. Revsbech, in preparation} conversion of nitrate in water samples to gaseous N. 4.2. Application of the isotope pairing technique This isotope pairing technique has been applied successfully to a number of different marine and freshwater sediments with denitrffication rates varying from 0.1 to 300 izmol N m -2 h -I, using variations of the presented batch mode assay (L.P. Nielsen, in preparation) and traditional flow-through systems (S. Rysgaard, N. Risgaard-Petersen, L.P. Nielsen and N.P. Revsbech, in preparation). A somewhat similar analysis of the mixing of lsNO~" and '4NO~ was introduced in studies of soil denitrification more than 33 years ago [15]. The application has been limited, however, primarily due to the problem of achieving an even mixing of the nitrate species in the soil matrix
umn to any nitrification sites in the animal burrows. Heterogeneity of the sediment or bioturbation could lead to different balances between nitrification and influx of NO~: at different spots of the sediment. This would result in higher production of the homogenous isotope pairs, S4Nt4N and ~5N~SN, relative to the mLxed pair ~4NtSN, compared to model predictions assuming uniform balance. The 14NI4N production and hence the total denitrification activity would therefore be underestimated by the method. This underestimation depends on the tsNO~ level applied, however, and may therefore be tested by using different concentrations of tsNO~. At big,her concentrations more of the 14NO~ will be trapped and measured directly as 14NtSN, and the possible miscalculation of 14Ni4N production will be of less significance compared to situations with lower concentrations of tsNO~. If the calculated rate of coupled nitrification-denitrification is independent of the applied tsNO~" concentrations, there would consequently be strong evidence for the correctness of the assumed uniform mixing. This is also indicated by the data presented here (Fig. 3). In general it is recommended that incubations for both different times and for different concentrations of tSNO3 be employed. In conclusion, the first results presented in this paper indicate that the nitrogen isotope pairing technique will become a powerful tool in studies of denitrification in almost any aquatic environment. ACKNOWLEDGEMENTS
[181. The fundamental limitation of the isotope pairing method is the demand for a uniform mixing of the added ~sNO 3 with the endogenous sources of 14NO3 . Coupled nitrification-denitrification around roots of aquatic macrophytes which secrete 02 in sediments cannot be measured, as the coupled processes will occur in microsites isolated from the. a,10ed 15NO3 in the water column and sediment, surface. Bioturbation by macrofauna, howe~er, does not prevent mixing of the NO~" species since ~sNO 3 will always be coupled to transport of 0 2 from the water col-
I thank Preben G. SOrensen and Sten P. Andersen for skillful technical assistance. T. Henry Blackburn, Niels P. Revsbech, Peter B. Christensen and Bo B. Jergensen are gratefully acknowledged for help with the manuscript. This work was supported by the EEC MAST program contract 0020, Danish Natural Science Research Council, Danish Technical Research Council (J. No. 11-8630 and 16-4806), and National Agency of Environmental Protection in Denmark in relation to HAV90-Marine Research Program in Denmark (contract No. 1.23).
362 REFERENCES [1] Seitzinger, S.P. (1990) Denitrifieation in aquatic sediments. In: Denitrification in Soil and Sediment (Revsbech, N.P. and Serensen, J., Eds.) pp. 301-322. FEMS Symposium No. 56, Plenum Press, New York. [2] Christensen, P.B., Nielsen, L.P., Sor~enson,J. and Revsbech, N.P. (1990) Denitrification in nltrate-rich streams: Diel and seasonal variation related to benthic oxygen metabolism. Limnol. Oceanogr. 35, 640-651. [3] Nielsen, L.P., Christensen, P.B., Revsbech, N.P. and S~rensen, J. (1990) Denitrification and photosynthesis in stream sediment studied with microsensor and whole-core techniques. Limnol. Oceanogr. 35, 1135-1144. [4] Henriksen, K. and Kemp, W.M. (1988) Nitrification in estuarine and coastal marine sediments. In: Nitrogen Cycling in Coastal Marine Environments (Blackburn, T.H. and S~rensen, J., Eds.) pp. 207-249, SCOPE 33, John Wiley, Chichester, U.K. [5] Blackburn, T.H. (1990) Denitrification model for marine sediments. In: Denitrification in Soil and Sediment (Revsbech, N.P. and S¢~rensen, J., Eds.) pp. 323-338. FEMS Symposium No. 56, Plenum Press, New York. [6] Revsbech, N.P. and S¢rensen, J. (1990) Combined use of the acetylene inhibition technique and microsensors for quantification of denitrifcation in sediments and biofilms. In: Denitrification in Soil and Sediment (Revsbech, N.P. and Serensen, J., Eds.) pp. 259-276. FEMS Symposium No. 56, Plenum Press, New York. [7] Koike, I. (1990) Measurement of sediment denitrifcation using ISN tracer method. In: Denitrification in Soil and Sediment (Revsbech, N.P. and S~rensen, J. Eds.), pp. 291-300. FEMS Symposium No. 56, Plenum Press, New York. [8] Nishio, T., Koike, I. and Hattori, A. (1983) Estimates of denitriflcation and nitrification in coastal and estuarine sediments. Appl. Environ. Microbiol. 45, 444-450.
[9] Jenkins, M.C. and Kemp, W.M. (1984) The coupling of nitrification and denitriflcation in two estuarine sediments. Limnol. Oceanogr. 29, 609-619. [10] Seitzinger, S.P., Nixon, S.W. and Pilson, M.E.Q. (1984) Denitrification and nitrous oxide production in a coastal marine ecosystem. Limnol. Oceanogr. 29, 73-83. [11] Devol, A.H. (1991) Direct measurement of nitrogen gas fluxes from continental shelf sediments. Nature 349, 319-321. [12] B~dard, C. and Knowles, R. (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH~ and CO oxidation by methanotrophs and nitriflers. Microbiol. Rev. 53, 68-84. [13] Broecker, W.S. anti Peng, T.-H. (1974) Gas exchange rates between air and sea. Tellus 26, 21-35. [14] Christensen, P.B., Nielsen, L.P., Revsbech, N.P. and Serensen, J. (1989) Microzonation of denitrification activity in stream sediments as studied with a combined oxygen and nitrous oxide microsensor. Appl. Environ. Microbiol. 55, 1234-1241. [15] Hauck, R.D., Melsted, S.W. and Yankwich, P.E. (1958) Use of N-isotope distribution in nitrogen gas in the study of denitrification. Soil. Sci. 86, 287-291. [16] Fiedler, R. and Proksch, G. (1975) The determination of nitrogen-15 by emission and mass spectrometry in biochemical analysis: a review. Anal. Chim. Acta, 78, 1-62. [17] Christensen, S. and Tiedje, J.M. (1988) Sub-parts-per-billion'nitrate method: use of an N20-producing denitrifier to convert NO~ or 15NO~ to N20. Appl. Environ. Microbiol. 54,1409-1413. [18] Myrold, D.D. (1990) Measuring denitrification in soils using 15N techniques. In: Denitrification in Soil and Sediment (Revsbech, N.P. and Serensen, J., Eds.) pp. 181-198. FEMS Symposium No. 56, Plenum Press, New York.
357
FEMSEC ~ 3 ~
Denitrification in sediment determined from nitrogen
isotope pairing Lars P e t e r Nielsen Department of Ecology and Genetics, Universityof Aarhus, Aarhus, Denmark
Received 17 October 1991 Revisionreceivedand accepted 19 December 1991 Key words: Denitrification; Nitrification; Sediment; Isotopes; ~5N; ~sNO~
1. SUMMARY
2. INTRODUCTION
A new method for accurate and easy measurement of denitrification in sediments is presented. The water overlying intact sediment cores was enriched with ~5NO3 which mixed with the ~4NO3 of the natural sources of NO3. The formation by denitrification of single-labeled (14N 15N) and double.-labeled (IZN mSN)dinitrogen pairs was measured by mass spectrometry after a few hours incubation. Total denitrification including the formation of unlabeled p4NI4N) dinitroden could be calculated assuming random isotope pairing by denitrification of the uniformly mixed N O j species. In contrast to previous approaches, by this method it is possible to measure denitrification of both N O j diffusing lrom the overlying water and NO~ from nitrification within the sediment.
In aquatic environments, combined nitrogen is recycled to the atmosphere by denitrification. Rates and regulations of this process are of increasing interest due to severe eutrophication problems caused by anthropogenic inputs of combined nitrogen [1]. Anoxic layers of sediments are the major sites of denitrification, which may reduce both NO3 diffusing from the overlying water column and NO3 produced by nitrification in the oxic layers. The supply of NO~- from the two sources is regulated quite differently; the former being greater at higher concentrations of nitrate in the water and lower penetration of oxygen in the sediment [2,3], the latter dependent on sediment N-mineralization and generally enhanced by deeper oxygen penetration [4]. Coupled nitrification-denitrification is thought to be quantitatively more important than influx of NO~ in most aquatic sediments [1], but the evidence is conflicting [2,5]. The lack of suitable methods has been a major obstacle in studies of denitrification. Attempts to
Correspondence to: L.P. Nielsen,Departmentof Ecologyand
Genetics, University of Aarhus, Ny Munkegade, DK-8000 Aarhus C, Denmark.
358 measure denitrification in sediments using the acetylene-block technique [6] or 15NO3 [7] have only quantified denitrifieation of NO~" taken up from the overlying water (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). The few attempts to specifically measure coupled nitrification-denitrification have been based on the production of 15N-labeled N2 after the addition of JSNH~" to the overlying water [7-9]. The results obtained by these attempts are, however, rather inaccurate due to very high NH~ enrichments or questionable assumptions about N-isotope ratios in the NH~ and NO 3 pools of the active layers (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). The best current methods for determining total denitrification rates are based on the measurement of N 2 fluxes from sediments [10,11] (S. Seitzinger, L.P. Nielsen, J. Caffrey and P.B. Christensen, submitted). These methods, however, depend on either week-long pre-incubation in the laboratory [10] or substantial depletions of NO~ and 02 in the overlying water during incubation [ll], thus altering in situ conditions. Using the new isotope pairing method presented here, both coupled nitrification-denitrification and denitrification of NO3 from the overlying water can be quantified in an easy and rapid batch mode assay with minimal deviation from in situ conditions.
3. MATERIALS AND METHODS Intact cores were sampled in April 1991 from the sediment of Salten A, a stream draining the productive lake Salten Langs¢, Denmark. The sediment consisted mainly of sand with a 0.5-cm organic-rich top layer. The average porosity of the top 3 cm was 43% (vol/vol). Sediment cores were collected in Plexiglas tubes (10 cm 2, 3-cm sediment, 7-cm water column) and stored overnight in a container with NO~-free freshwater. During storage and subsequent incubation, the in situ temperature of 13°C was maintained and the water column in each core was gently stirred by a small rotating magnet.
Ini6ally each core was closed with a rubber stopper and 2 mi of water was replaced with nitrogen-free air (21% O2 in He) using syringes. The elimination of N2 in the gas phase lowered the background N2 level thereby improving the final detection of 15N-enriched N 2. Incubations were started by injecting 15NO3 from a 10 mM stock solution of 99.6% ~5NO3 into the initially NO~'-free water column. Water free of 14NO3 was used in this specific study in order to make nitrification in the se,"-nent the only possible source of 14NO~. Four parallel time series with 6 cores each were incubated for 0-3.5 h. One series was incubated with 56/~M 15NO~ corresponding to the in situ concentration of NO~, and a second series was incubated with 19/~M 15NO~. To verify the measurements of coupled nitrification-denitrification, nitrification was blocked in a third series ~y adding 120/zM thiourea [12] to the water column 0.5 h prior to incubation with 19/~M ~sNO~. The fourth series was used only for the analysis of changes in 0 2 and NO~ concentrations during the incubation ( < 15 and 8%, respectively, after 3.5 h). The incubation of a core in each series was stopped at 40 min intervals and dissolved N 2 in the water phase and pore water were equilibrated with the gas phase by vigorous shaking for 4 rain. Controls showed that 95% equilibrium was obtained by this procedure. Denitrification was assumed to be inhibited instantly as 0 2 was mixed into the sediment, and shaking and gas sampling were done before the slurry went anoxic. The whole gas phase was sampled by syringe and stored in a pre-evacuated 3.5-ml blood collection tube (S. Rysgaard, N. Risgaard-Petersen and N.P. Revsbech, in preparation). The gas samples were analyzed on an isotope ratio mass spectrometer with three collectors (Sira Series II, VG Isotech, Middlewich, Chesire, U.K.). Subsamples of gas (200/~l) were injected into a carrler gas flow of He (30 ml rain- I) and possible interfering gasses were separated from dinitrogen in a freeze trap (copper-tube loop in liquid nitrogen) and by retention in a molecular sieve tube (2.4 m, 5 ,~, 60-80 mesh). The gas finally passed a splitter leading part of the flow (approx. 2%)
359 into the mass spectrometer. The retention time for N 2 was 150 s and the mass peaks were integrated over 110 s. All three N 2 species (t4Ni4N, t4NtSN and ~SN~SN, atom masses 28, 29 and 30 respectively) were measured and excess ;4N'SN and ~SN~SN was determined using air samples with the same total N 2 amount. In air t4NtSN and tSNtSN constitutes 0.7299 and 0.001% of total N2, respectively, and the maximal excesses recorded in the samples were 0.088 and 0.175% of total N 2. The accuracy of 14NISN and ISNtSN measurements were 0.0001 and 0.001% of total N2, respectively. In RESUt:rs AND DISCUSSION t4NlSN and iSNISN refer to the excess values. The production of 14NtSN and 15NtSN in each core expressed in nmol N was calculated knowing the distribution coefficient of N 2 in water and gas (0.018 at 13°C, ref. 13) and the volumes of gas and water in the cores.
120
.
.
,
m
..
.
~ . . . . - i
~
11f..I
oI
14N!,~
o
o
% TmneIh~
12~
I
8(
Z
c3
4. RESULTS AND DISCUSSION
t:z
14N1~ ......
4.1. Denitrification rates
The amount of ISN-labeled N 2 increased in all time series; the highest rate was in the series with 56 p,M tSNO3 in the overlying water (Fig. 1). Single labeled dinitrogen (14N 15N), in addition to double labeled (15NISN), was formed in the two first series without nitrification inhibitor, thus showing a contribution of 14NO3 from nitrification in the sediment. The variability between the cores was larger than seen in other sediments using the same method (L.P. Nielsen, in preparation), and the differences most likely reflected real variations in biological activity. The ratios of I4NISN/15NIsN did not change significantly during each of the 3 time series indicating a rapid and uniform equilibrium between 'SNO~ and 14NO3 in the denitrification zone of the sediment (Fig. 2). This can be explained by the microzonation of denitrification in the sediment: recent N20 microsensor studies in similar stream sediments with 100/zM N O i in the overlying water showed that denitrification was restricted to a narrow 0.5-mm thick zone immediately below a l-ram thick oxic surface layer [14]. The turnover time in the sediment of
Tee (h) 120
8c
4°
~
°
~4N~SN .....
o 1 2 3 Time(h) Fig. 1. Excessof tSNISN(solid lines)and 14NISN(dashed lines) in sedimentcores incubatedfor different times with 99.6 atom% tSNO~" in the overlyingwater. A: With 56/tM 15NO~"in the overlyingwater;,B: with 19/~MtsNO~";C: with 19 pM tsNO~" plus nitrificationinhibiior(n.i.). Each pair of data points represents one sediment cure. Lines represent linearregressions.
36O 3
rearranged to
f i4/f ts (14NISN)/2(15NIsN) ffi
(3)
Eqns. 1-3 were finally combined to express Di4 by the measured data:
Ig~SO~
Dr4 = ((14NISN)/2(15NISN)) o
o
• 19pMNO~+n.j.
_-
•
• "-
o
;
2 Twne {h)
Fig. 2. Ratio of 14NISNto ISNISNin the N 2 evolvedduring the 3 experimentsillustratedin Fig. 1.
NO 3 diffusing from the water could be calculated to 8 min, indicating a rapid development of steady-state profiles of m5NO~" in the present study. All NO;" diffusing from the overlying water to the anoxic denitrification zone has to pass through the oxic surface layer add to mix with NO3 produced in this layer. Rates of denitrification of tsNO3" (Dins) and t4NO~" (Dr4) were calculated from the measured productions of 14NlSN and ~SN~SN. D~5 was calculated simply as the sum of ~SN in the produced labeled dinitrogen: D15 = (14NtSN) + 2(15NtSN)
fl4/fl5
The calculated rates of denitrification at the three treatments are presented in Fig. 3. In the present study all 14NO3 in the overlying water was replaced with tsNO 3, and D~s therefore represented denitrification of NO 3 from the overlying water, while D14 represented denitrification coupled to nitrification in the sediment (Fig. 3). In y;ene~a', however, the differentiation between the two NO~ sources of denitrification may also be obtained in studies where ~5NO3 is added to a water column with ambient concentrations of 14NO3 (L.P. Nielsen, in preparation): if the ~4N and 15N frequencies of the nitrate in the
56"iNO; 90, 19pMNO~
-4--
(1)
19IJMNO3+n.J. 3(
DI4 was calculated from Dts: DI4 = DI5 *
* (('4NrsN) + 2('5N'SN))
(2)
where ft4 and fls represented the frequencies of 14N and 15N in the NO3 reduced by the denitrifying bacteria. The ratio ft4/fls could be calculated from the ratio of the produced labeled dinitrogen species (14NISN)/(ISNISN) as the denitrified NO 3 species were uniformly mixed, and t4NISN and tSN~SN therefore were formed with the probabilities 2ft4fts and ftsfls respectively [i5]. The ratio (14NISN)/(tSNlSN) could then be expressed as (14NISN)/(lSNtSN) ffi (2f14fls)/(ftsfts)
_ms
Fig. 3. Rates of denitrificationin stream sedimentas calculated from linearregressionsof the data presented in Fig. 1. Shaded bars indicatecouplednitrification-denitrification(denitrificationof 14NO~) and whitebars indicatedenitrification of nitratediffusingfromthe overlyingwater (denitrificationof IsNO~"). The 56/~M NO~" correspondsto in situ conditions in the stream. Note that coupled nitrification-denitrification was independentof overlyingNO~ concentrationand efficientlyblockedby the nitrificationinhibitor,whereasdenitrification of NO~" from the overlyingwater was proportionalto the NO~" concentrationand not affected by the nitrification inhibitor.Bars indicatestandarderrors derivedfromstandard errors of the regressioncoefficients.
361 overlying water become f ~ and f~5, then the ambient denitrification of t4NO3 from the water column (D~'4) is D~4 = Dr5 * f~4/f~s and coupled nitfification-denitrification (D~4) is calculated by difference: D~4 -- Dr4 - D~'4 The frequencies f ~ and f ~ may be obtained by measuring the NO~" concentration before and after the addition of lsNO3. If the frequencies may change significantly during incubation they can be measureo directly by mass spectrometry after chemical [16] or biological [17] (N. Risgaard-Petersen, S. Rysgaard and N.P. Revsbech, in preparation} conversion of nitrate in water samples to gaseous N. 4.2. Application of the isotope pairing technique This isotope pairing technique has been applied successfully to a number of different marine and freshwater sediments with denitrffication rates varying from 0.1 to 300 izmol N m -2 h -I, using variations of the presented batch mode assay (L.P. Nielsen, in preparation) and traditional flow-through systems (S. Rysgaard, N. Risgaard-Petersen, L.P. Nielsen and N.P. Revsbech, in preparation). A somewhat similar analysis of the mixing of lsNO~" and '4NO~ was introduced in studies of soil denitrification more than 33 years ago [15]. The application has been limited, however, primarily due to the problem of achieving an even mixing of the nitrate species in the soil matrix
umn to any nitrification sites in the animal burrows. Heterogeneity of the sediment or bioturbation could lead to different balances between nitrification and influx of NO~: at different spots of the sediment. This would result in higher production of the homogenous isotope pairs, S4Nt4N and ~5N~SN, relative to the mLxed pair ~4NtSN, compared to model predictions assuming uniform balance. The 14NI4N production and hence the total denitrification activity would therefore be underestimated by the method. This underestimation depends on the tsNO~ level applied, however, and may therefore be tested by using different concentrations of tsNO~. At big,her concentrations more of the 14NO~ will be trapped and measured directly as 14NtSN, and the possible miscalculation of 14Ni4N production will be of less significance compared to situations with lower concentrations of tsNO~. If the calculated rate of coupled nitrification-denitrification is independent of the applied tsNO~" concentrations, there would consequently be strong evidence for the correctness of the assumed uniform mixing. This is also indicated by the data presented here (Fig. 3). In general it is recommended that incubations for both different times and for different concentrations of tSNO3 be employed. In conclusion, the first results presented in this paper indicate that the nitrogen isotope pairing technique will become a powerful tool in studies of denitrification in almost any aquatic environment. ACKNOWLEDGEMENTS
[181. The fundamental limitation of the isotope pairing method is the demand for a uniform mixing of the added ~sNO 3 with the endogenous sources of 14NO3 . Coupled nitrification-denitrification around roots of aquatic macrophytes which secrete 02 in sediments cannot be measured, as the coupled processes will occur in microsites isolated from the. a,10ed 15NO3 in the water column and sediment, surface. Bioturbation by macrofauna, howe~er, does not prevent mixing of the NO~" species since ~sNO 3 will always be coupled to transport of 0 2 from the water col-
I thank Preben G. SOrensen and Sten P. Andersen for skillful technical assistance. T. Henry Blackburn, Niels P. Revsbech, Peter B. Christensen and Bo B. Jergensen are gratefully acknowledged for help with the manuscript. This work was supported by the EEC MAST program contract 0020, Danish Natural Science Research Council, Danish Technical Research Council (J. No. 11-8630 and 16-4806), and National Agency of Environmental Protection in Denmark in relation to HAV90-Marine Research Program in Denmark (contract No. 1.23).
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