Applied Microbiology and Biotechnology

, Volume 101, Issue 12, pp 5149–5162 | Cite as

Changes in the microbial community of an anammox consortium during adaptation to marine conditions revealed by 454 pyrosequencing

  • Blanca M. Gonzalez-Silva
  • Are J. Rønning
  • Ingrid K. Andreassen
  • Ingrid Bakke
  • Francisco J. Cervantes
  • Kjetill Østgaard
  • Olav Vadstein
Environmental biotechnology


The anammox activity of a freshwater anammox consortium was strongly inhibited at low-salinity level. Stepwise adaptation from 0 to 3 g NaCl L−1 took 153 days. Further adaptation to high-salinity concentration (from 3 to 30 g L−1) took only 40 days, and no inhibition was observed. A comprehensive insight into the salinity-induced successions of the total and the anammox communities was obtained by 454 pyrosequencing of 16S rRNA gene amplicons and statistical analysis. A major succession in the anammox community was observed at 3 g L−1 where the dominating population shifted from Candidatus Brocadia fulgida to Ca. Kuenenia stuttgartiensis. The latter dominated at high salinity and seemed to be essential for the high (˃96%) ammonium and nitrite removal efficiencies achieved. SIMPER analysis indicated that these two dominating anammox species explained most to the differences in community structure among samples and helped in identifying other important members at different salinities.


Anammox Amplicon pyrosequencing Bacterial community Salinity 



This work is a part of a Ph.D. study funded by the Faculty of Natural Sciences and Technology and Strategiske Omstillingsmidler (SO) (grant number: 81733600), Norwegian University of Science and Technology (NTNU). The authors wish to thank Dr. Christian Vogelsang, from Norwegian Institute for Water Research (NIVA), for providing the anammox culture and all the scientific support given to this project in the early stage. Finally, we want to thank two anonymous reviewers for helpful suggestions to an earlier version of the paper.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2017_8160_MOESM1_ESM.pdf (254 kb)
ESM 1 (PDF 253 kb)


  1. Bakke I, Coward E, Andersen T, Vadstein O (2015) Selection in the host structures the microbiota associated with developing cod larvae (Gadus morhua). Environ Microbiol 17(10):3914–3924. doi: 10.1111/1462-2920.12888 CrossRefPubMedGoogle Scholar
  2. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Meth 7(5):335–336 CrossRefGoogle Scholar
  3. Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Aust J Ecol 18:117–117CrossRefGoogle Scholar
  4. Costa MC, Carvalho L, Leal CD, Dias MF, Martins KL, Garcia GB, Mancuelo ID, Hipolito T, Conell EF, Okada D, Etchebehere C, Chernicharo CA, Araujo JC (2014) Impact of inocula and operating conditions on the microbial community structure of two anammox reactors. Environ Technol 35(13–16):1811–1822. doi: 10.1080/09593330.2014.883432 CrossRefPubMedGoogle Scholar
  5. Dale OR, Tobias CR, Song B (2009) Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in cape fear river estuary. Environ Microbiol 11(5):1194–1207CrossRefPubMedGoogle Scholar
  6. Dapena-Mora A, Vázquez-Padín J, Campos J, Mosquera-Corral A, Jetten M, Méndez R (2010) Monitoring the stability of an anammox reactor under high salinity conditions. Biochem Eng J 51(3):167–171CrossRefGoogle Scholar
  7. Davis JC (1986) Statistics and data analysis in geology. Wiley, New York, pp 289–291Google Scholar
  8. Díaz V, Ibáñez R, Gómez P, Urtiaga AM, Ortiz I (2012) Kinetics of nitrogen compounds in a commercial marine recirculating aquaculture system. Aquac Eng 50:20–27. doi: 10.1016/j.aquaeng.2012.03.004 CrossRefGoogle Scholar
  9. Díez-Vives C, Gasol JM, Acinas SG (2012) Evaluation of marine Bacteroidetes-specific primers for microbial diversity and dynamics studies. Microb Ecol 64(4):1047–1055CrossRefPubMedGoogle Scholar
  10. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461. doi: 10.1093/bioinformatics/btq461 CrossRefPubMedGoogle Scholar
  11. Egli K, Bosshard F, Werlen C, Lais P, Siegrist H, Zehnder A, Van der Meer J (2003) Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon. Microb Ecol 45(4):419–432CrossRefPubMedGoogle Scholar
  12. Fu B, Liu J, Yang H, Hsu TC, He B, Dai M, Kao SJ, Zhao M, Zhang XH (2015) Shift of anammox bacterial community structure along the Pearl Estuary and the impact of environmental factors. J Geophys Res Oceans 120(4):2869–2883CrossRefGoogle Scholar
  13. Gao D-W, Tao Y (2011) Versatility and application of anaerobic ammonium-oxidizing bacteria. Appl Microbiol Biotechnol 91(4):887–894CrossRefPubMedGoogle Scholar
  14. Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaentol Electronica 4(1):9Google Scholar
  15. Heermann R, Jung K (2012) K+ supply, osmotic stress and the KdpD/KdpE two-component system two component systems in bacteria. Caister Academic Press, NorfolkGoogle Scholar
  16. Holtmann G, Bakker EP, Uozumi N, Bremer E (2003) KtrAB and KtrCD: two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. J Bacteriol 185(4):1289–1298CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hu Z, Speth DR, Francoijs KJ, Quan ZX, Jetten MS (2012) Metagenome analysis of a complex community reveals the metabolic blueprint of anammox bacterium “Candidatus Jettenia asiatica”. Front Microbiol 3:366. doi: 10.3389/fmicb.2012.00366 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Isanta E, Bezerra T, Fernández I, Suárez-Ojeda ME, Pérez J, Carrera J (2015) Microbial community shifts on an anammox reactor after a temperature shock using 454-pyrosequencing analysis. Bioresour Technol 181(0):207–213. doi: 10.1016/j.biortech.2015.01.064 CrossRefPubMedGoogle Scholar
  19. Jin R-C, Ma C, Mahmood Q, Yang G-F, Zheng P (2011) Anammox in a UASB reactor treating saline wastewater. Process Saf Environ Prot 89(5):342–348CrossRefGoogle Scholar
  20. Jin R-C, Yang G-F, Yu J-J, Zheng P (2012) The inhibition of the anammox process: a review. Chem Eng J 197:67–79. doi: 10.1016/j.cej.2012.05.014 CrossRefGoogle Scholar
  21. Kartal B, Koleva M, Arsov R, van der Star W, Jetten MS, Strous M (2006) Adaptation of a freshwater anammox population to high salinity wastewater. J Biotechnol 126(4):546–553CrossRefPubMedGoogle Scholar
  22. Kartal B, Kuenen J, Van Loosdrecht M (2010) Sewage treatment with anammox. Science 328(5979):702–703CrossRefPubMedGoogle Scholar
  23. Kartal B, van Niftrik L, Keltjens JT, Op den Camp HJ, Jetten MS (2012) Anammox—growth physiology, cell biology, and metabolism. Adv Microb Physiol 60:212Google Scholar
  24. Kindaichi T, Tsushima I, Ogasawara Y, Shimokawa M, Ozaki N, Satoh H, Okabe S (2007) In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms. Appl Environ Microbiol 73(15):4931–4939CrossRefPubMedPubMedCentralGoogle Scholar
  25. Laureni M, Weissbrodt DG, Szivák I, Robin O, Nielsen JL, Morgenroth E, Joss A (2015) Activity and growth of anammox biomass on aerobically pre-treated municipal wastewater. Water Res 80:325–336CrossRefPubMedPubMedCentralGoogle Scholar
  26. Liu C, Yamamoto T, Nishiyama T, Fujii T, Furukawa K (2009) Effect of salt concentration in anammox treatment using non woven biomass carrier. J Biosci Bioeng 107(5):519–523CrossRefPubMedGoogle Scholar
  27. Liu M, Peng Y, Wang S, Liu T, Xiao H (2014) Enhancement of anammox activity by addition of compatible solutes at high salinity conditions. Bioresour Technol 167:560–563. doi: 10.1016/j.biortech.2014.06.015 CrossRefPubMedGoogle Scholar
  28. Ma C, Jin RC, Yang GF, Yu JJ, Xing BS, Zhang QQ (2012) Impacts of transient salinity shock loads on anammox process performance. Bioresour Technol 112:124–130. doi: 10.1016/j.biortech.2012.02.122 CrossRefPubMedGoogle Scholar
  29. Malovanyy A, Plaza E, Trela J, Malovanyy M (2015) Ammonium removal by partial nitritation and anammox processes from wastewater with increased salinity. Environ Technol 36(5):595–604CrossRefPubMedGoogle Scholar
  30. McIntosh D, Fitzsimmons K (2003) Characterization of effluent from an inland, low-salinity shrimp farm: what contribution could this water make if used for irrigation. Aquac Eng 27(2):147–156CrossRefGoogle Scholar
  31. Nobu MK, Narihiro T, Rinke C, Kamagata Y, Tringe SG, Woyke T, Liu W-T (2015) Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor. ISME J 9(8):1710–1722. doi: 10.1038/ismej.2014.256 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Oshiki M, Satoh H, Okabe S (2016) Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environ Microbiol 18:2784–2796. doi: 10.1111/1462-2920.13134 CrossRefPubMedGoogle Scholar
  33. Pereira AD, Leal CD, Dias MF, Etchebehere C, Chernicharo CAL, de Araújo JC (2014) Effect of phenol on the nitrogen removal performance and microbial community structure and composition of an anammox reactor. Bioresour Technol 166(0):103–111. doi: 10.1016/j.biortech.2014.05.043 CrossRefPubMedGoogle Scholar
  34. Schmid MC, Maas B, Dapena A, van de Pas-Schoonen K, van de Vossenberg J, Kartal B, Van Niftrik L, Schmidt I, Cirpus I, Kuenen JG (2005) Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria. Appl Environ Microbiol 71(4):1677–1684CrossRefPubMedPubMedCentralGoogle Scholar
  35. Strous M, Heijnen JJ, Kuenen JG, Jetten MSM (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl Microbiol Biotechnol 50(5):589–596. doi: 10.1007/s002530051340 CrossRefGoogle Scholar
  36. Stumm W, Morgan JJ (1981) Aquatic chemistry: an introduction emphasizing chemical equilibria in natural waters. ​John Wiley & Sons, Inc., New YorkGoogle Scholar
  37. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599. doi: 10.1093/molbev/msm092 CrossRefPubMedGoogle Scholar
  38. van der Star WR, Miclea AI, van Dongen UG, Muyzer G, Picioreanu C, van Loosdrecht M (2008) The membrane bioreactor: a novel tool to grow anammox bacteria as free cells. Biotechnol Bioeng 101(2):286–294CrossRefPubMedGoogle Scholar
  39. Van Hulle SW, Vandeweyer HJ, Meesschaert BD, Vanrolleghem PA, Dejans P, Dumoulin A (2010) Engineering aspects and practical application of autotrophic nitrogen removal from nitrogen rich streams. Chem Eng J 162(1):1–20CrossRefGoogle Scholar
  40. van Niftrik L, Jetten MS (2012) Anaerobic ammonium-oxidizing bacteria: unique microorganisms with exceptional properties. Microbiol Mol Biol Rev 76(3):585–596CrossRefPubMedPubMedCentralGoogle Scholar
  41. Vik U, Logares R, Blaalid R, Halvorsen R, Carlsen T, Bakke I, Kolstø A-B, Økstad OA, Kauserud H (2013) Different bacterial communities in ectomycorrhizae and surrounding soil. Sci Rep 3:3471CrossRefPubMedPubMedCentralGoogle Scholar
  42. Wang J, Gu J-D (2013) Dominance of Candidatus Scalindua species in anammox community revealed in soils with different duration of rice paddy cultivation in Northeast China. Appl Microbiol and Biotechnol 97(4):1785–1798CrossRefGoogle Scholar
  43. Windey K, De Bo I, Verstraete W (2005) Oxygen-limited autotrophic nitrification–denitrification (OLAND) in a rotating biological contactor treating high-salinity wastewater. Water Res 39(18):4512–4520. doi: 10.1016/j.watres.2005.09.002 CrossRefPubMedGoogle Scholar
  44. Yang J, Zhang L, Hira D, Fukuzaki Y, Furukawa K (2011) Anammox treatment of high-salinity wastewater at ambient temperature. Bioresour Technol 102(3):2367–2372CrossRefPubMedGoogle Scholar
  45. Zhang Z, Chen S, Wu P, Lin L, Luo H (2010) Start-up of the Canon process from activated sludge under salt stress in a sequencing batch biofilm reactor (SBBR). Bioresour Technol 101(16):6309–6314. doi: 10.1016/j.biortech.2010.03.040 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Blanca M. Gonzalez-Silva
    • 1
  • Are J. Rønning
    • 1
  • Ingrid K. Andreassen
    • 1
  • Ingrid Bakke
    • 1
  • Francisco J. Cervantes
    • 2
  • Kjetill Østgaard
    • 1
  • Olav Vadstein
    • 1
  1. 1.Department of Biotechnology, Faculty of Natural Sciences and TechnologyNTNU Norwegian University of Science and TechnologyTrondheimNorway
  2. 2.División de Ciencias AmbientalesInstituto Potosino de Investigación Científica y Tecnológica (IPICyT)San Luis PotosiMexico

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