Abstract
In batch experiments, we studied the isotope fractionation of nitrogen and oxygen during denitrification of two bacterial strains (Azoarcus sp. strain DSM 9056 and Pseudomonas pseudoalcaligenes strain F10). Denitrification experiments were conducted with succinate and toluene as electron donor in three waters with a distinct oxygen isotope composition. Nitrate consumption was observed in all batch experiments. Reaction rates for succinate experiments were more than six times higher than those for toluene experiments. Nitrogen and oxygen isotopes became progressively enriched in the remaining nitrate pool in the course of the experiments; the nitrogen and oxygen isotope fractionation varied between 8.6–16.2 and 4.0–7.3‰, respectively. Within this range, neither electron donors nor the oxygen isotope composition of the medium affected the isotope fractionation process. The experimental results provide evidence that the oxygen isotope fractionation during nitrate reduction is controlled by a kinetic isotope effect which can be quantified using the Rayleigh model. The isotopic examination of nitrite released upon denitrification revealed that nitrogen isotope fractionation largely follows the fractionation of the nitrate pool. However, the oxygen isotope values of nitrite are clearly influenced by a rapid isotope equilibration with the oxygen of the ambient water. Even though this equilibration may in part be due to storage, it shows that under certain natural conditions (re-oxidation of nitrite) the nitrate pool may also be indirectly affected by an isotope equilibration.
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References
Aravena R, Robertson WD (1998) Use of multiple isotope tracers to evaluate denitrification in ground water: study of nitrate from a large-flux septic system plume. Ground Water 36:975–982
Böhlke JK, Denver JM (1995) Combined use of groundwater dating, chemical, and isotopic analyses to resolve the history and fate of nitrate contamination in 2 agricultural watersheds, Atlantic Coastal-Plain, Maryland. Water Resour Res 31:2319–2339
Böhlke JK, Wanty R, Tuttle M, Delin G, Landon M (2002) Denitrification in the recharge area and discharge area of a transient agricultural nitrate plume in a glacial outwash sand aquifer, Minnesota. Water Resour Res 38:1105–1131
Böhlke JK, Mroczkowski SJ, Coplen TB (2003) Oxygen isotopes in nitrate: new reference materials for O-18: O-17: O-16 measurements and observations on nitrate-water equilibration. Rapid Commun Mass Spectrom 17:1835–1846
Böhlke JK, Smith RL, Hannon JE (2007) Isotopic analysis of N and O in nitrite and nitrate by sequential selective bacterial reduction to N2O. Anal Chem 79:5888–5895
Böttcher J, Strebel O, Voerkelius S, Schmidt HL (1990) Using isotope fractionation of nitrate nitrogen and nitrate oxygen for evaluation of microbial denitrification in a sandy aquifer. J Hydrol 114:413–424
Bozau E, Knöller K, Strauch G (2006) Nitrate degradation without N-15 enrichment: a hydrochemical and isotopic study of a fractured rock aquifer including embedded lakes. Isotopes Environ Health Stud 42:251–260
Brandes JA, Devol AH, Yoshinari T, Jayakumar DA, Naqvi SWA (1998) Isotopic composition of nitrate in the central Arabian Sea and eastern tropical North Pacific: a tracer for mixing and nitrogen cycles. Limnol Oceanogr 43:1680–1689
Brunner B, Bernasconi SM, Kleikemper J, Schroth MH (2005) A model for oxygen and sulfur isotope fractionation in sulfate during bacterial sulfate reduction processes. Geochim Cosmochim Acta 69:4773–4785
Casciotti KL, McIlvin MR (2007) Isotopic analyses of nitrate and nitrite from reference mixtures and application to Eastern Tropical North Pacific waters. Mar Chem 107:184–201
Casciotti KL, Sigman DM, Hastings MG, Böhlke JK, Hilkert A (2002) Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. Anal Chem 74:4905–4912
Casciotti KL, Böhlke JK, McIlvin MR, Mroczkowski SJ, Hannon JE (2007) Oxygen isotopes in nitrite: analysis, calibration, and equilibration. Anal Chem 79:2427–2436
Chen DJZ, MacQuarrie KTB (2005) Correlation of delta N-15 and delta O-18 in NO3 − during denitrification in groundwater. J Environ Eng Sci 4:221–226
Chiba H, Sakai H (1985) Oxygen isotope exchange-rate between dissolved sulfate and water at hydrothermal temperatures. Geochim Cosmochim Acta 49(4):993–1000
Dolfing J, Zeyer J, Binder-Eicher P, Schwarzenbach RP (1990) Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch Microbiol 154:336–341
Einsiedl F, Mayer B (2006) Hydrodynamic and microbial processes controlling nitrate in a fissured-porous karst aquifer of the Franconian Alb, Southern Germany. Environ Sci Technol 40:6697–6702
Einsiedl F, Maloszewski P, Stichler W (2005) Estimation of denitrification potential in a karst aquifer using the N-15 and O-18 isotopes of NO3. Biogeochemistry 72:67–86
Granger J, Sigman DM, Prokopenko MG, Lehmann MF, Tortell PD (2006) A method for nitrite removal in nitrate N and O isotope analyses. Limnol Oceanogr Methods 4:205–212
Granger J, Sigman DM, Lehmann MF, Tortell PD (2008) Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria. Limnol Oceanogr 53:2533–2545
Kendall C (1998) Tracing nitrogen sources and cycling in catchments. In: Kendall C, McDonnell JJ (eds) Isotope tracers in catchment hydrology. Elsevier, Amsterdam, pp 519–576
Knöller K, Strauch G (1999) Assessment of the flow dynamic of a mining lake by stable isotope investigations. Isotopes Environ Health Stud 35:75–83
Knöller K, Vogt C, Richnow HH, Weise SM (2006) Sulfur and oxygen isotope fractionation during benzene, toluene, ethyl benzene, and xylene degradation by sulfate-reducing bacteria. Environ Sci Technol 40:3879–3885
Lehmann MF, Reichert P, Bernasconi SM, Barbieri A, McKenzie JA (2003) Modelling nitrogen and oxygen isotope fractionation during denitrification in a lacustrine redox-transition zone. Geochim Cosmochim Acta 67:2529–2542
Lloyd RM (1968) Oxygen isotope behavior in the sulfate-water-system. J Geophys Res 73:6099–6110
Mariotti A, Germon JC, Hubert P, Kaiser P, Letolle R, Tardieux A, Tardieux P (1981) Experimental-determination of nitrogen kinetic isotope fractionation—some principles—illustration for the denitrification and nitrification processes. Plant Soil 62:413–430
Martienssen M, Schöps R (1997) Biological treatment of leachate from solid waste landfill sites—alterations in the bacterial community during the denitrification process. Water Res 31(5):1164–1170
Mengis M, Schiff SL, Harris M, English MC, Aravena R, Elgood RJ, MacLean A (1999) Multiple geochemical and isotopic approaches for assessing ground water NO3 − elimination in a riparian zone. Ground Water 37:448–457
Moreno-Vivián C, Cabello P, Martínez-Luque M, Blasco R, Castillo F (1999) Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol 181(21):6573–6584
Northrop DB (1981) The expression of isotope effects on enzyme catalyzed reactions. Annu Rev Biochem 50:103–131
Olleros T (1983) Kinetische Isotopeneffekte der Arginase- und Nitratreduktase-Reaktion: Ein Beitrag zur Aufklärung der entsprechenden Reaktionsmechanismen. Ph.D. dissertation, Technische Universität München-Weihenstephan
Osenbrück K, Fiedler S, Knöller K, Weise SM, Sültenfuss J, Oster H, Strauch G (2006) Timescales and development of groundwater pollution by nitrate in drinking water wells of the Jahna-Aue, Saxonia, Germany. Water Resour Res 42:W12416. doi:10.1029/2006WR004977
Pfennig N, Eimhjell Ke, Jensen SL (1965) A new isolate of Rhodospirillum fulvum group and its photosynthetic pigments. Archiv Für Mikrobiologie 51:258–268
Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27–36
Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ (2001) Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58:165–178
Sigman DM, Casciotti KL, Andreani M, Barford C, Galanter M, Böhlke JK (2001) A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Anal Chem 73:4145–4153
Song BK, Ward BB (2003) Nitrite reductase genes in halobenzoate degrading denitrifying bacteria. FEMS Microbiol Ecol 43:349–357
Sturchio NC, Böhlke JK, Beloso AD Jr, Streger SH, Heraty LJ, Hatzinger PB (2007) Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies. Environ Sci Technol 41:2796–2802
Voss M, Dippner JW, Montoya JP (2001) Nitrogen isotope patterns in the oxygen-deficient waters of the Eastern Tropical North Pacific Ocean. Deep Sea Res I 48:1905–1921
Widdel F, Rabus R (2001) Anaerobic biodegradation of saturated and aromatic hydrocarbons. Curr Opin Biotechnol 12:259–276
Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616
Acknowledgements
This work is integrated in the research and development program of the Helmholtz Centre for Environmental Research. We would like to thank Daniela Reichert and Wolfgang Städter of the Stable Isotope Laboratory Halle/Saale for conducting numerous nitrogen and oxygen isotope analyses. Special thanks are addressed to Ramona Hoffman who conducted the hydrochemical analyses of our experimental samples and to Stephanie Hinke for preparing the cultivation media. The study is part of the DFG research unit 580 “electron transfer processes in anoxic aquifers (etrap)” (FOR 580 grant Ri903/3-2) supporting Stefan Feisthauer.
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Knöller, K., Vogt, C., Haupt, M. et al. Experimental investigation of nitrogen and oxygen isotope fractionation in nitrate and nitrite during denitrification. Biogeochemistry 103, 371–384 (2011). https://doi.org/10.1007/s10533-010-9483-9
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DOI: https://doi.org/10.1007/s10533-010-9483-9