, Volume 139, Issue 2, pp 179–195 | Cite as

Co-occurrence of in-stream nitrogen fixation and denitrification across a nitrogen gradient in a western U.S. watershed

  • Erin K. Eberhard
  • Amy M. Marcarelli
  • Colden V. Baxter


It is frequently assumed that nitrogen (N2) fixation and denitrification do not co-occur in streams because each process should be favored under different concentrations of dissolved inorganic nitrogen (DIN), and therefore these processes are rarely quantified together. We asked if these processes could co-exist by conducting a spatial survey of N2 fixation using acetylene reduction and denitrification using acetylene block [with and without amendments of carbon (C) as glucose and nitrogen (N) as nitrate]. Rates were measured on rocks and sediment in 8 southeastern Idaho streams encompassing a DIN gradient of 26–615 µg L−1. Sampling at each site was repeated in summer 2015 and 2016. We found that both denitrification and N2 fixation occurred across the gradient of DIN concentrations, with N2 fixation occurring primarily on rocks and denitrification occurring in sediment. N2 fixation rates on rocks significantly decreased 100× across the DIN gradient in 1 year of the study, and amended (with N and C) denitrification rates increased 10× across the DIN gradient in both years. Multiple linear regression and partial least squares models with environmental characteristics measured at the scale of entire stream reaches showed that C and phosphorus were positive predictors of amended and unamended denitrification rates, but no significant model could explain N2 fixation rates across all streams and years. This, coupled with the observation that detectable rates of N2 fixation occurred primarily on rocks and denitrification occurred primarily on sediment, suggests that microhabitat scale factors may better predict the co-occurrence of these processes within stream reaches. Overlooking the potential co-occurrence of N2 fixation and denitrification in stream ecosystems will impede understanding by oversimplifying the contribution of each process to the N cycle.


Nitrogen fixation Denitrification Dissolved inorganic nitrogen Streams 



We thank C. Allison, A. Copley, J. Cornell, E. Coscarelli, A. Eckersell, H. Harris, M. Kelly, D. Larson, K. Nevorski, J. Ortiz, J. Paris, and R. Van Goethem for field and laboratory assistance. We also thank J. Bump, S. Techtmann, and three anonymous reviewers for providing comments that helped to improve this manuscript. This research was funded by the National Science Foundation award DEB 14-51919 to A.M.M. and NSF-EPSCoR (IIA-1301792) support to C.V.B. This is contribution no. 48 of the Great Lakes Research Center at Michigan Tech.

Supplementary material

10533_2018_461_MOESM1_ESM.docx (22 kb)
Supplementary material 1 (DOCX 22 kb)


  1. American Public Health Administration (2005) Standard Methods for examination of water and wastewater. APHA, Washington DCGoogle Scholar
  2. An S, Gardner WS, Kana T (2001) Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with inlet mass spectrometry analysis. Appl Environ Microbiol 67:1171–1178CrossRefGoogle Scholar
  3. Arango CP, Tank JL, Schaller JL et al (2007) Benthic organic carbon influences denitrification in streams with high nitrate concentration. Freshw Biol 52:1210–1222CrossRefGoogle Scholar
  4. Barton GJ (2004) Surface and ground-water relations on the Portneuf River, and temporal changes in ground-water levels in the Portneuf Valley, Caribou and Bannock Counties, Idaho, 2001–2002. USGS Scientific Investigations Report 2004-5170Google Scholar
  5. Bechtold HA, Marcarelli AM, Baxter CV et al (2012) Effects of N, P, and organic carbon on stream biofilm nutrient limitation and uptake in a semi-arid watershed. Limnol Oceanogr 57:1544–1554CrossRefGoogle Scholar
  6. Benjamin JR, Lepori F, Baxter CV et al (2013) Can replacement of native by non-native trout alter stream-riparian food webs? Freshw Biol 58:1694–1709CrossRefGoogle Scholar
  7. Bergey EA, Getty GM (2006) A review of methods for measuring the surface area of stream substrates. Hydrobiologia 556:7–16CrossRefGoogle Scholar
  8. Brandes J, Devol A, Yoshinari T et al (1998) Isotopic composition of nitrate in the central Arabian Sea and eastern tropical North Pacific: a tracer for mixing and nitrogen cycles. Limnol Oceanogr 44:106–115Google Scholar
  9. Burgin AJ, Hamilton SK (2007) Have we overemphasized the role of denitrification in aquatic ecosystems? A review of nitrate removal pathways. Front Ecol Environ 5:89–96CrossRefGoogle Scholar
  10. Burnham KP, Anderson DR (2002) Model selection and multimodel inference: a practical information-theoretic approach, 2nd edn. Springer, New YorkGoogle Scholar
  11. Capone DG (1993) Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. In: Kemp PF, Sherr BF, Sherr EB, Cole JJ (eds) Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca RatonGoogle Scholar
  12. Capone DG (2001) Marine nitrogen fixation: what’s the fuss? Curr Opin Microbiol 4:341–348CrossRefGoogle Scholar
  13. Carrascal LM, Galvan I, Gordo O (2009) Partial least squares regression as an alternative to current regression methods uses in ecology. Oikos 118:681–690CrossRefGoogle Scholar
  14. Dent CL, Grimm NB (1999) Spatial heterogeneity of stream water nutrient concentrations over successional time. Ecology 80:2283–2298CrossRefGoogle Scholar
  15. Deutsch C, Sarmiento JL, Sigman DM et al (2007) Spatial coupling of nitrogen inputs and losses in the ocean. Nature 445:163–167CrossRefGoogle Scholar
  16. Dodds WK, Castenholz RW (1988) The nitrogen budget of an oligotrophic cold water pond. Arch Hydrobiol 79(Suppl):343–362Google Scholar
  17. Dodds WK, Smth VH, Lohman K (2002) Nitrogen and phosphorus relationship to benthic algal biomass in temperate streams. Can J Fish Aquat Sci 59:865–874CrossRefGoogle Scholar
  18. Dodds WK, Burgin AJ, Marcarelli AM et al (2017) Nitrogen transformations. In: Lamberti GA, Hauer FR (eds) Methods in stream ecology, vol 2. ecosystem function. Elsevier/Academic Press, Amsterdam/New York, pp 143–196Google Scholar
  19. Elwood JW, Newbold JD, Trimble AF et al (1981) The limiting role of phosphorus in a woodland stream ecosystem: effects of P enrichment on leaf decomposition and primary producers. Ecology 62:146–158CrossRefGoogle Scholar
  20. Fernandez C, Farias L, Ulloa O (2011) Nitrogen fixation in denitrified marine waters. PLoS ONE 6:e20539CrossRefGoogle Scholar
  21. Finlay JC, Hood JM, Limm MP et al (2011) Light-mediated thresholds in stream water nutrient composition in a river network. Ecology 92:140–150CrossRefGoogle Scholar
  22. Finlay JC, Small GE, Sterner RW (2013) Human influences on nitrogen removal in lakes. Science 342:247–250CrossRefGoogle Scholar
  23. Flores E, Herrero A (2010) Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat Rev Microbiol 8:39–50CrossRefGoogle Scholar
  24. Frissell CA, Liss WJ, Warren CE et al (1986) A hierarchical framework for stream habitat classification: viewing streams in a watershed context. Environ Manag 10:199–214CrossRefGoogle Scholar
  25. Fulweiler RW, Heiss EM (2014) (Nearly) a decade of directly measured sediment N2 fluxes: what can Nargansett Bay tell us about the global ocean nitrogen budget? Oceanography 27:184–195CrossRefGoogle Scholar
  26. Gettel GM, Giblin AE, Howarth RW (2007) The effects of grazing by the snail, Lymnaea elodes, on benthic N2 fixation and primary production in oligotrophic arctic lakes. Limnol Oceanogr 52:2398–2409CrossRefGoogle Scholar
  27. Grace JB, Anderson TM, Olff H et al (2010) On the specification of structural equation models for ecological systems. Ecol Monogr 56:1001–1014Google Scholar
  28. Grimm NB, Petrone K (1997) Nitrogen fixation in a desert stream ecosystem. Biogeochemistry 37:33–61CrossRefGoogle Scholar
  29. Groffman PM, Dorsey AM, Mayer PM (2005) N processing within geomorphic structures in urban streams. J N Am Benthol Soc 24:613–625CrossRefGoogle Scholar
  30. Groffman PM, Altabet MA, Bohlke JK et al (2006) Methods for measuring denitrification: diverse approaches to a difficult problem. Ecol Appl 16:2091–2122CrossRefGoogle Scholar
  31. Groffman PM, Butterbach-Bahl K, Fulweiler RW et al (2009) Challenges to incorporating spatially and temporally explicit phenomena (hot spots and hot moments) in denitrification models. Biogeochemistry 93:49–77CrossRefGoogle Scholar
  32. Henry JC, Fisher SG (2003) Spatial segregation of periphyton communities in a desert stream: causes and consequences for N cycling. J N Am Benthol Soc 22:511–527CrossRefGoogle Scholar
  33. Hiatt DL, Robbins CJ, Back JA et al (2017) Catchment-scale alder cover controls nitrogen fixation in boreal headwater streams. Freshw Sci. Google Scholar
  34. Holmes RM, Jones JB, Fisher SG et al (1996) Denitrification in a nitrogen-limited stream ecosystem. Biogeochemistry 33:125–146CrossRefGoogle Scholar
  35. Holmes RM, Aminot A, Kerouel R et al (1999) A simple and precise method for measuring ammonium in marine and freshwater ecosystems. Can J Fish Aquat Sci 56:1801–1808CrossRefGoogle Scholar
  36. Hopkins JM, Marcarelli AM, Bechtold HA (2011) Ecosystem structure and function are complementary measures of water quality in a polluted, spring-influenced river. Water Air Soil Pollut 214:409–421CrossRefGoogle Scholar
  37. Kemp MJ, Dodds WK (2001) Centimeter-scale patterns in dissolved oxygen and nitrification rates in a prairie stream. J N Am Benthol Soc 20:347–357CrossRefGoogle Scholar
  38. Kemp MJ, Dodds WK (2002) The influence of ammonium, nitrate, and dissolved oxygen concentrations on uptake, nitrification, and denitrification rates associated with prairie stream substrata. Limnol Oceanogr 47:1380–1393CrossRefGoogle Scholar
  39. Knowles R (1982) Denitrification. Microbiological Rev 46:43Google Scholar
  40. Kothawala DN, Stedmon CA, Muller RA et al (2014) Controls of dissolved organic matter quality: evidence from a large-scale boreal lake survey. Glob Change Biol 20:1101–1114CrossRefGoogle Scholar
  41. Kunza LA, Hall RO Jr (2013) Demographic and mutualistic responses of stream nitrogen fixers to nutrients. Freshw Sci 32:991–1004CrossRefGoogle Scholar
  42. Kunza LA, Hall RO Jr (2014) Nitrogen fixation can exceed inorganic nitrogen uptake fluxes in oligotrophic streams. Biogeochemistry 121:537–549CrossRefGoogle Scholar
  43. Lemmon PE (1956) A spherical densitometer for estimating forest overstory density. For Sci 2:314–320Google Scholar
  44. Lewis WM, Wurtsbaugh WA (2008) Control of lacutrine phytoplankton by nutrients: erosion of the phosphorus paradigm. Internat Rev Hydrobiol 93:446–465CrossRefGoogle Scholar
  45. Macko SA, Enzeroth L, Parker PL (1984) Regional differences in nitrogen and carbon isotopes on the continental shelf of the Gulf of Mexico. Naturwissenschaften 71:374–375CrossRefGoogle Scholar
  46. Marcarelli AM, Wurtsbaugh WA (2007) Effects of upstream lakes and nutrient limitation on periphytic biomass and nitrogen fixation in oligotrophic, subalpine streams. Freshw Biol 52:2211–2225CrossRefGoogle Scholar
  47. Marcarelli AM, Wurtsbaugh WA (2009) Nitrogen fixation varies spatially and seasonally in linked stream-lake ecosystems. Biogeochemistry 94:95–110CrossRefGoogle Scholar
  48. Marcarelli AM, Baker MA, Wurtsbaugh WA (2008) Is in-stream N2 fixation an important N source for benthic communities and stream ecosystems? J N Am Benthol Soc 27:186–211CrossRefGoogle Scholar
  49. Marcarelli AM, Van Kirk RW, Baxter CV (2010) Predicting effects of hydrologic alteration and climate change on ecosystem metabolism in a western U.S. river. Ecol Appl 20:2081–2088CrossRefGoogle Scholar
  50. McClain ME, Boyer EW, Dent L et al (2003) Biogeochemical hot spots and hot moments at the interface of terrestrial and aquatic ecosystems. Ecosystems 6:301–312CrossRefGoogle Scholar
  51. McGuire KJ, Torgersen CE, Likens GE et al (2014) Network analysis reveals multiscale controls on streamwater chemistry. Proc Natl Acad Sci USA 111(19):7030–7035CrossRefGoogle Scholar
  52. Minshall GW, Andrews DA (1973) An ecological investigation of the Portneuf River, Idaho: a semiarid-land stream subjected to pollution. Freshw Biol 3:1–30CrossRefGoogle Scholar
  53. Newell SE, McCarthy MJ, Gardner WS et al (2016) Sediment nitrogen fixation: a call for re-evaluating coastal N budgets. Estuar Coast. Google Scholar
  54. Paerl HW, Scott JT (2010) Throwing fuel on the fire: synergistic effects of excessive nitrogen inputs and global warming on harmful algal blooms. Environ Sci Technol 44:7756–7758CrossRefGoogle Scholar
  55. Paerl HW, Scott JT, McCarthy MJ et al (2016) It takes two to tango: when and where dual nutrient (N & P) reductions are needed to protect lakes and downstream ecosystems. Envrion Sci Technol 50(20):10805–10813CrossRefGoogle Scholar
  56. Pringle CM, Naiman RJ, Bretschko G et al (1988) Patch dynamics in lotic systems: the stream as a mosaic. J N Am Benthol Soc 7:503–524CrossRefGoogle Scholar
  57. Schindler DW (1977) Evolution of phosphorus limitation in lakes. Science 195:260–262CrossRefGoogle Scholar
  58. Schindler DW, Hecky RE, Findlay DL et al (2008) Eutrophication of lakes cannot be controlled be reducing nitrogen input: results of a 37-year whole-ecosystem experiment. Proc Natl Acad Sci USA 105:11254–11258CrossRefGoogle Scholar
  59. Scott JT, Grantz EM (2013) N2 fixation exceeds internal nitrogen loading as a phytoplankton nutrient source in perpetually nitrogen-limited reservoirs. Freshwater Sci 32:849–861CrossRefGoogle Scholar
  60. Scott JT, Marcarelli AM (2012) Cyanobacteria in freshwater benthic environments. In: Whitton BA (ed) Ecology of Cyanobacteria II: their diversity in space and time. Springer, DordrechtGoogle Scholar
  61. Scott JT, McCarthy MJ (2010) Nitrogen fixation may not balance the nitrogen pool in lakes over timescales relevant to eutrophication management. Limnol Oceanogr 55:1265–1270CrossRefGoogle Scholar
  62. Seitzinger SP (1988) Denitrification in freshwater and coastal marine ecosystems: ecological and geochemical significance. Limnol Oceanogr 33:702–724Google Scholar
  63. Seitzinger SP, Harrison JA, Bohlke JK et al (2006) Denitrification across landscapes and waterscapes: a synthesis. Ecol Appl 16:2064–2090CrossRefGoogle Scholar
  64. Smith VH (1983) Low nitrogen and phosphorus ratios favor dominance by blue-green algae in lake phytoplankton. Science 221:669–671CrossRefGoogle Scholar
  65. Taylor BW, Keep CF, Hall RO Jr (2007) Improving the fluorometric ammonium method: matrix effects, background fluorescence, and standard additions. J N Am Benthol Soc 26:167–177CrossRefGoogle Scholar
  66. Welter JR, Benstead JP, Cross WF et al (2015) Does N2 fixation amplify the temperature dependence of ecosystem metabolism? Ecology 96:603–610CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological SciencesMichigan Technological UniversityHoughtonUSA
  2. 2.Department of Biological Sciences, Stream Ecology CenterIdaho State UniversityPocatelloUSA

Personalised recommendations