Estuaries and Coasts

, Volume 42, Issue 5, pp 1265–1280 | Cite as

Quantifying Effluent Dissolved Organic Nitrogen (EDON) Uptake by Microbial Communities Along a Salinity Gradient in the York River

  • Xiaolong Yao
  • Rachel E. Sipler
  • Brianna C. Stanley
  • Quinn N. Roberts
  • Marta P. Sanderson
  • Charles B. Bott
  • Deborah A. BronkEmail author


Effluent discharged from water reclamation facilities (WRFs) contains dissolved organic nitrogen, termed effluent dissolved organic nitrogen (EDON), that subsequently enters coastal waterways. It is still unclear at what rate EDON can be taken up by microbial communities relative to other nitrogen (N) substrates. Bench-scale sequencing batch reactors (SBRs), used to mimic WRFs, were supplied with 15N-labeled ammonium (15NH4+) to produce 15N-labeled EDON (EDO15N) that was subsequently used to measure uptake rates along a salinity gradient of the York River, Virginia, USA, in the spring and summer. Although NH4+ dominated influent N pools, only a small fraction (4.1%) of EDON was produced from NH4+ microbial assimilation in biological treatment processes. When added as a short-term (4-h) tracer, the EDO15N was taken up by estuarine microbes at rates 0.01–0.434 μmol N L−1 h−1, which are similar to rates of NH4+ and nitrate uptake. When added to 48-h bioassays, EDON stimulated phytoplankton growth more at the lower salinity (0–8‰) sites (8.5–13.8 μg Chl a L−1) than at the higher salinity (20‰) site (up to 0.4 μmol Chl a L−1). The microbes in the 0.7–5 μm size fraction had significantly higher EDO15N uptake rates than the larger size fraction (e.g., > 5 μm, p < 0.05). Taken together with urea and amino acids, DON plays a more important role in N nutrition for microbes during the summer months. This study provides the first EDO15N uptake rates using EDO15N produced from 15NH4+ in SBRs, and the results provide conclusive evidence that organic N in effluent is biologically available to estuarine microbes.


Effluent Dissolved organic nitrogen (DON) Uptake Microbial communities York River 



We are grateful to anonymous reviewers for their insightful suggestions. This paper is Contribution No. 3824 of the Virginia Institute of Marine Science, College of William & Mary.

Funding Information

This study was supported by The Water Environment Research Foundation grant no. U1R11 to DAB and RES. XY was supported by the China Scholarship Council (CSC) (No. 201704910658).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12237_2019_563_MOESM1_ESM.docx (2.7 mb)
ESM 1 Supporting Information Tables showing specific and absolute uptake rates of N substrates measured on the 0.3–0.7, 0.7–5, and > 5 μm size fractions at each site during the summer and difference values between 4 and 48 h of the spring and summer 48-h bioassays for the concentrations of nutrients, DOC, Chl a, and phaeopigments. Figures showing NH4+ regeneration rates at each site during the tracer studies, specific and absolute uptake rates of the 0.3–0.7, 0.7–5, and > 5 μm size fractions during the summer, and changes in the concentrations of nutrients, DOC, Chl a, and phaeopigments under each treatment during the 48-h bioassays (DOCX 2773 kb)


  1. Aquino, S.F., and D.C. Stuckey. 2003. Production of soluble microbial products (SMP) in anaerobic chemostats under nutrient deficiency. Journal of Environmental Engineering 129 (11): 1007–1014. Scholar
  2. Berman, T., and S. Chava. 1999. Algal growth on organic compounds as nitrogen sources. Journal of Plankton Research 21 (8): 1423–1437. Scholar
  3. Bronk, D.A. 2002. Dynamics of DON, p. 153–247. In Biogeochemistry of marine dissolved organic matter, ed. D.A. Hansell and C.A. Carlson. Cambridge: Academic Press.Google Scholar
  4. Bronk, D.A., and P.M. Glibert. 1993. Application of a 15N tracer method to the study of dissolved organic nitrogen uptake during spring and summer in Chesapeake Bay. Marine Biology 115 (3): 501–508. Scholar
  5. Bronk, D.A., P.M. Glibert, and B.B. Ward. 1994. Nitrogen uptake, dissolved organic nitrogen release, and new production. Science 265 (5180): 1843–1846. Scholar
  6. Bronk, D.A., P.M. Glibert, T.C. Malone, S. Banahan, and E. Sahlsten. 1998. Inorganic and organic nitrogen cycling in Chesapeake Bay: Autotrophic versus heterotrophic processes and relationships to carbon flux. Aquatic Microbial Ecology 15: 177–189. Scholar
  7. Bronk, D.A., J.H. See, P. Bradley, and L. Killberg. 2007. DON as a source of bioavailable nitrogen for phytoplankton. Biogeosciences 4 (3): 283–296.CrossRefGoogle Scholar
  8. Bronk, D.A., Q.N. Roberts, M.P. Sanderson, E.A. Canuel, P.G. Hatcher, R. Mesfioui, K.C. Filippino, M.R. Mulholland, and N.G. Love. 2010. Effluent organic nitrogen (EON): Bioavailability and photochemical and salinity-mediated release. Environmental Science & Technology 44 (15): 5830–5835. Scholar
  9. Bronk, D.A., L. Killberg-Thoreson, R.E. Sipler, M.R. Mulholland, Q.N. Roberts, P.W. Bernhardt, M. Garrett, J.M. O’Neil, and C.A. Heil. 2014. Nitrogen uptake and regeneration (ammonium regeneration, nitrification and photoproduction) in waters of the West Florida Shelf prone to blooms of Karenia brevis. Harmful Algae 38: 50–62. Scholar
  10. Conley, D.J., H.W. Paerl, R.W. Howarth, D.F. Boesch, S.P. Seitzinger, K.E. Havens, C. Lancelot, and G.E. Likens. 2009. Controlling eutrophication: Nitrogen and phosphorus. Science 323 (5917): 1014–1015. Scholar
  11. Dittmar, T., B. Koch, N. Hertkorn, and G. Kattner. 2008. A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater. Limnology and Oceanography: Methods 6 (6): 230–235. Scholar
  12. Dudek, N., M.A. Brzezinski, and P.A. Wheeler. 1986. Recovery of ammonium nitrogen by solvent extraction for the determination of relative 15N abundance in regeneration experiments. Marine Chemistry 18 (1): 59–69. Scholar
  13. Eom, H., D. Borgatti, H.W. Paerl, and C. Park. 2017. Formation of low-molecular-weight dissolved organic nitrogen in predenitrification biological nutrient removal systems and its impact on eutrophication in coastal waters. Environmental Science & Technology 51 (7): 3776–3783. Scholar
  14. Fan, C., P.M. Glibert, and J.M. Burkholder. 2003. Characterization of the affinity for nitrogen, uptake kinetics, and environmental relationships for Prorocentrum minimum in natural blooms and laboratory cultures. Harmful Algae 2 (4): 283–299. Scholar
  15. Fan, L., M.T. Brett, W. Jiang, and B. Li. 2017. Dissolved organic nitrogen recalcitrance and bioavailable nitrogen quantification for effluents from advanced nitrogen removal wastewater treatment facilities. Environmental Pollution 229: 255–263. Scholar
  16. Filippino, K.C., M.R. Mulholland, P.W. Bernhardt, G.E. Boneillo, R.E. Morse, M. Semcheski, H. Marshall, N.G. Love, Q. Roberts, and D.A. Bronk. 2011. The bioavailability of effluent-derived organic nitrogen along an estuarine salinity gradient. Estuaries and Coasts 34 (2): 269–280. Scholar
  17. Funkey, C.P., R.J. Latour, and D.A. Bronk. 2015. Abiotic effects on effluent dissolved organic nitrogen along an estuarine transect. Water Environment Research 87 (3): 258–265. Scholar
  18. Gagnon, R., M. Levasseur, A.M. Weise, J. Fauchot, P.G.C. Campbell, B.J. Weissenboeck, A. Merzouk, M. Gosselin, and B. Vigneault. 2005. Growth stimulation of Alexandrium Tamarense (dinophyceae) by humic substances from the Manicouagan river (eastern Canada). Journal of Phycology 41 (3): 489–497. Scholar
  19. Galloway, J.N., A.R. Townsend, J.W. Erisman, M. Bekunda, Z. Cai, J.R. Freney, L.A. Martinelli, S.P. Seitzinger, and M.A. Sutton. 2008. Transformation of the nitrogen cycle: Recent trends, questions, and potential solutions. Science 320 (5878): 889–892. Scholar
  20. Gilbert, P.M., F. Lipschultz, J.J. McCarthy, and M.A. Altabet. 1982. Isotope dilution models of uptake and remineralization of ammonium by marine plankton1. Limnology and Oceanography 27 (4): 639–650. Scholar
  21. Glibert, P.M., F.P. Wilkerson, R.C. Dugdale, J.A. Raven, C.L. Dupont, P.R. Leavitt, A.E. Parker, J.M. Burkholder, and T.M. Kana. 2016. Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions. Limnology and Oceanography 61 (1): 165–197. Scholar
  22. Grady, C.P.L., G.T. Daigger, N.G. Love, and C.D. Filipe. 2011. Biological wastewater treatment. Boca Raton: CRC press.Google Scholar
  23. Hansell, D.A., P.M. Williams, and B.B. Ward. 1993. Measurements of DOC and DON in the Southern California Bight using oxidation by high temperature combustion. Deep Sea Research Part I: Oceanographic Research Papers 40 (2): 219–234. Scholar
  24. Heil, C.A. 2005. Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) schiller. Harmful Algae 4 (3): 603–618. Scholar
  25. Heisler, J., P.M. Glibert, J.M. Burkholder, D.M. Anderson, W. Cochlan, W.C. Dennison, Q. Dortch, C.J. Gobler, C.A. Heil, E. Humphries, A. Lewitus, R. Magnien, H.G. Marshall, K. Sellner, D.A. Stockwell, D.K. Stoecker, and M. Suddleson. 2008. Eutrophication and harmful algal blooms: A scientific consensus. Harmful Algae 8 (1): 3–13. Scholar
  26. Hounshell, A.G., B.L. Peierls, C.L. Osburn, and H.W. Paerl. 2017. Stimulation of phytoplankton production by anthropogenic dissolved organic nitrogen in a coastal plain estuary. Environmental Science & Technology 51 (22): 13104–13112. Scholar
  27. Hu, H., H. Ma, L. Ding, J. Geng, K. Xu, H. Huang, Y. Zhang, and H. Ren. 2016. Concentration, composition, bioavailability, and N-nitrosodimethylamine formation potential of particulate and dissolved organic nitrogen in wastewater effluents: A comparative study. Science of the Total Environment 569-570: 1359–1368. Scholar
  28. Huisman, J., G.A. Codd, H.W. Paerl, B.W. Ibelings, J.M.H. Verspagen, and P.M. Visser. 2018. Cyanobacterial blooms. Nature Reviews Microbiology 16 (8): 471–483. Scholar
  29. Khan, E., M. Awobamise, K. Jones, and S. Murthy. 2009. Method development for measuring biodegradable dissolved organic nitrogen in treated wastewater. Water Environment Research 81 (8): 779–787. Scholar
  30. Killberg-Thoreson, L.M. 2011. A tale of two blooms: Dynamics of nitrogen uptake by harmful algae in the Eastern Gulf of Mexico and York River, Virginia, USA. Doctor of Philosophy, The College of William and Mary.Google Scholar
  31. Kirchman, D.L., R.G. Keil, and P.A. Wheeler. 1989. The effect of amino acids on ammonium utilization and regeneration by heterotrophic bacteria in the subarctic Pacific. Deep Sea Research Part A. Oceanographic Research Papers 36 (11): 1763–1776. Scholar
  32. Koroleff, F. 1983. Determination of ammonium. In Methods of Seawater Analysis, ed. K. Grasshoff, M. Ehrhardt, and K. Kremling, 2nd ed., 150–157. Weinheim: Verlag Chemie.Google Scholar
  33. Liu, H., J. Jeong, H. Gray, S. Smith, and D.L. Sedlak. 2012. Algal uptake of hydrophobic and hydrophilic dissolved organic nitrogen in effluent from biological nutrient removal municipal wastewater treatment systems. Environmental Science & Technology 46 (2): 713–721. Scholar
  34. Lomas, M.W., T. Mark Trice, P.M. Glibert, D.A. Bronk, and J.J. McCarthy. 2002. Temporal and spatial dynamics of urea uptake and regeneration rates and concentrations in Chesapeake Bay. Estuaries 25 (3): 469–482. Scholar
  35. Marshall, H.G., L. Burchardt, and R. Lacouture. 2005. A review of phytoplankton composition within Chesapeake Bay and its tidal estuaries. Journal of Plankton Research 27 (11): 1083–1102. Scholar
  36. Marshall, H.G., R.V. Lacouture, C. Buchanan, and J.M. Johnson. 2006. Phytoplankton assemblages associated with water quality and salinity regions in Chesapeake Bay, USA. Estuarine, Coastal and Shelf Science 69 (1-2): 10–18. Scholar
  37. Mesfioui, R., N.G. Love, D.A. Bronk, M.R. Mulholland, and P.G. Hatcher. 2012. Reactivity and chemical characterization of effluent organic nitrogen from wastewater treatment plants determined by Fourier transform ion cyclotron resonance mass spectrometry. Water Research 46 (3): 622–634. Scholar
  38. Mesfioui, R., H.A.N. Abdulla, and P.G. Hatcher. 2015. Photochemical alterations of natural and anthropogenic dissolved organic nitrogen in the York River. Environmental Science & Technology 49 (1): 159–167. Scholar
  39. Nam, S.-N., and G. Amy. 2008. Differentiation of wastewater effluent organic matter (EfOM) from natural organic matter (NOM) using multiple analytical techniques. Water Science and Technology 57 (7): 1009–1015. Scholar
  40. Paerl, H.W., T.G. Otten, and R. Kudela. 2018. Mitigating the expansion of harmful algal blooms across the freshwater-to-marine continuum. Environmental Science & Technology 52 (10): 5519–5529. Scholar
  41. Pagilla, K.R., M. Urgun-Demirtas, K. Czerwionka, and J. Makinia. 2008. Nitrogen speciation in wastewater treatment plant influents and effluents—The US and polish case studies. Water Science and Technology 57 (10): 1511–1517. Scholar
  42. Parsons, T.R., Y. Maita, and C.M. Lalli. 1984. A manual of biological and chemical methods for seawater analysis. Oxford: Pergamon Press.Google Scholar
  43. Pehlivanoglu-Mantas, E., and D.L. Sedlak. 2008. Measurement of dissolved organic nitrogen forms in wastewater effluents: Concentrations, size distribution and NDMA formation potential. Water Research 42 (14): 3890–3898. Scholar
  44. Perminova, I.V., I.V. Dubinenkov, A.S. Kononikhin, A.I. Konstantinov, A.Y. Zherebker, M.A. Andzhushev, V.A. Lebedev, E. Bulygina, R.M. Holmes, Y.I. Kostyukevich, I.A. Popov, and E.N. Nikolaev. 2014. Molecular mapping of sorbent selectivities with respect to isolation of Arctic dissolved organic matter as measured by Fourier transform mass spectrometry. Environmental Science & Technology 48 (13): 7461–7468. Scholar
  45. Price, N.M., and P.J. Harrison. 1987. Comparison of methods for the analysis of dissolved urea in seawater. Marine Biology 94 (2): 307–317. Scholar
  46. Qin, C., H. Liu, L. Liu, S. Smith, D.L. Sedlak, and A.Z. Gu. 2015. Bioavailability and characterization of dissolved organic nitrogen and dissolved organic phosphorus in wastewater effluents. Science of the Total Environment 511: 47–53. Scholar
  47. Raphael, M.K., and P.C. William. 2000. Nitrogen and carbon uptake kinetics and the influence of irradiance for a red tide bloom off southern California. Aquatic Microbial Ecology 21: 31–47.CrossRefGoogle Scholar
  48. Sattayatewa, C., K. Pagilla, P. Pitt, K. Selock, and T. Bruton. 2009. Organic nitrogen transformations in a 4-stage Bardenpho nitrogen removal plant and bioavailability/biodegradability of effluent DON. Water Research 43 (18): 4507–4516. Scholar
  49. See, J.H., D.A. Bronk, and A.J. Lewitus. 2006. Uptake of Spartina-derived humic nitrogen by estuarine phytoplankton in nonaxenic and axenic culture. Limnology and Oceanography 51: 2290–2299. Scholar
  50. Seitzinger, S.P., R.W. Sanders, and R. Styles. 2002. Bioavailability of DON from natural and anthropogenic sources to estuarine plankton. Limnology and Oceanography 47 (2): 353–366. Scholar
  51. Seitzinger, S.P., J.A. Harrison, E. Dumont, A.H.W. Beusen, and A.F. Bouwman. 2005. Sources and delivery of carbon, nitrogen, and phosphorus to the coastal zone: An overview of Global Nutrient Export from Watersheds (NEWS) models and their application. Global Biogeochemical Cycles 19 (4): n/a–n/a. Scholar
  52. Sharp, J.H., R. Benner, L. Bennett, C.A. Carlson, R. Dow, and S.E. Fitzwater. 1993. Re-evaluation of high temperature combustion and chemical oxidation measurements of dissolved organic carbon in seawater. Limnology and Oceanography 38 (8): 1774–1782. Scholar
  53. Sharp, J.H., A.Y. Beauregard, D. Burdige, G. Cauwet, S.E. Curless, R. Lauck, K. Nagel, H. Ogawa, A.E. Parker, O. Primm, M. Pujo-Pay, W.B. Savidge, S. Seitzinger, G. Spyres, and R. Styles. 2004. A direct instrument comparison for measurement of total dissolved nitrogen in seawater. Marine Chemistry 84 (3-4): 181–193. Scholar
  54. Sin, Y., R.L. Wetzel, and I.C. Anderson. 2000. Seasonal variations of size-fractionated phytoplankton along the salinity gradient in the York River estuary, Virginia (USA). Journal of Plankton Research 22 (10): 1945–1960. Scholar
  55. Sipler, R.E., and D.A. Bronk. 2015. Chapter 4 - dynamics of dissolved organic nitrogen A2 - Hansell, Dennis a. In Biogeochemistry of marine dissolved organic matter (second edition), ed. C.A. Carlson, 127–232. Boston: Academic Press.CrossRefGoogle Scholar
  56. Stücheli, P.E., J. Niggemann, and C.J. Schubert. 2018. Comparison of different solid phase extraction sorbents for the qualitative assessment of dissolved organic nitrogen in freshwater samples using FT-ICR-MS. Journal of Limnology 77: 400–411. Scholar
  57. Suter, E.A., K.M.M. Lwiza, J.M. Rose, C. Gobler, and G.T. Taylor. 2014. Phytoplankton assemblage changes during decadal decreases in nitrogen loadings to the urbanized Long Island Sound estuary, USA. Marine Ecology Progress Series 497: 51–67. Scholar
  58. The Chesapeake Bay Resource Library. 2009. Special Report URL (last accessed 23 October 2011):
  59. Vaquer-Sunyer, R., D.J. Conley, S. Muthusamy, M.V. Lindh, J. Pinhassi, and E.S. Kritzberg. 2015. Dissolved organic nitrogen inputs from wastewater treatment plant effluents increase responses of planktonic metabolic rates to warming. Environmental Science & Technology 49 (19): 11411–11420. Scholar
  60. Varela, M.M., A. Bode, E. Fernández, N. Gónzalez, V. Kitidis, M. Varela, and E.M.S. Woodward. 2005. Nitrogen uptake and dissolved organic nitrogen release in planktonic communities characterised by phytoplankton size–structure in the Central Atlantic Ocean. Deep Sea Research Part I: Oceanographic Research Papers 52 (9): 1637–1661. Scholar
  61. Veuger, B., J.J. Middelburg, H.T.S. Boschker, J. Nieuwenhuize, P. van Rijswijk, E.J. Rochelle-Newall, and N. Navarro. 2004. Microbial uptake of dissolved organic and inorganic nitrogen in Randers Fjord. Estuarine, Coastal and Shelf Science 61 (3): 507–515. Scholar
  62. Wiegner, T.N., S.P. Seitzinger, P.M. Glibert, and D.A. Bronk. 2006. Bioavailability of dissolved organic nitrogen and carbon from nine rivers in the eastern United States. Aquatic Microbial Ecology 43: 277–287. Scholar

Copyright information

© Coastal and Estuarine Research Federation 2019

Authors and Affiliations

  1. 1.Virginia Institute of Marine ScienceCollege of William & MaryGloucester PointUSA
  2. 2.State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and LimnologyChinese Academy of SciencesNanjingChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.Department of Ocean SciencesMemorial University of NewfoundlandSt. John’sCanada
  5. 5.Hampton Roads Sanitation DistrictVirginia BeachUSA
  6. 6.Bigelow Laboratory for Ocean SciencesBoothbayUSA

Personalised recommendations