Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2379–2389 | Cite as

Single-stage autotrophic nitrogen removal process at high loading rate: granular reactor performance, kinetics, and microbial characterization

  • Feiyue Qian
  • Abebe Temesgen Gebreyesus
  • Jianfang Wang
  • Yaoliang Shen
  • Wenru Liu
  • Lulin Xie
Environmental biotechnology


For the possible highest performance of single-stage combined partial nitritation/anammox (PNA) process, a continuous complete-mix granular reactor was operated at progressively higher nitrogen loading rate. The variations in bacterial community structure of granules were also characterized using high-throughput pyrosequencing, to give a detail insight to the relationship between reactor performance and functional organism abundance within completely autotrophic nitrogen removal system. In 172 days of operation, a superior total nitrogen (TN) removal rate over 3.9 kg N/(m3/day) was stable implemented at a fixed dissolved oxygen concentration of 1.9 mg/L, corresponding to the maximum specific substrate utilization rate of 0.36/day for TN based on the related kinetics modeling. Pyrosequencing results revealed that the genus Nitrosomonas responsible for aerobic ammonium oxidation was dominated on the granule surface, which was essential to offer the required niche for the selective enrichment of anammox bacteria (genus Candidatus Kuenenia) in the inner layer. And the present of various heterotrophic organisms with general functions, known as fermentation and denitrification, could not be overlooked. In addition, it was believed that an adequate excess of ammonium in the bulk liquid played a key role in maintaining process stability, by suppressing the growth of nitrite-oxidizing bacteria through dual-substrate competitions.


Partial nitritation Anammox Granular sludge Microbial community structure Substrate competition 



This study was supported by the National Natural Science Foundation of China (51308367, 51578353) and the Natural Science Foundation of Jiangsu Province, China (BK20150284). The authors also acknowledge support from the Preponderant Discipline Construction Project in higher education of Jiangsu Province, China, and the Two-year International Master Program on Environmental Engineering authorized by the Chinese Ministry of Commerce (2015E0434).

Compliance with ethical standards

Ethical statement

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_2018_8768_MOESM1_ESM.pdf (506 kb)
ESM 1 (PDF 506 kb)


  1. Ali M, Okabe S (2015) Anammox-based technologies for nitrogen removal: advances in process start-up and remaining issues. Chemosphere 141:144–153. CrossRefPubMedGoogle Scholar
  2. APHA (1998) Standard methods for examination of water and wastewater, 20th edn. American Public Health Association, New YorkGoogle Scholar
  3. Chen FY, Liu YQ, Tay JH, Ning P (2015) Rapid formation of nitrifying granules treating high-strength ammonium wastewater in a sequencing batch reactor. Appl Microbiol Biotechnol 99(10):4445–4452. CrossRefPubMedGoogle Scholar
  4. Cho S, Fujii N, Lee T, Okabe S (2011) Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor. Bioresour Technol 102(2):652–659. CrossRefPubMedGoogle Scholar
  5. Chu ZR, Wang K, Li XK, Zhu MT, Yang L, Zhang J (2015) Microbial characterization of aggregates within a one-stage nitritation–anammox system using high-throughput amplicon sequencing. Chem Eng J 262:41–48. CrossRefGoogle Scholar
  6. Dosta J, Vila J, Sancho I, Basset N, Grifoll M, Mata-Álvarez J (2015) Two-step partial nitritation/Anammox process in granulation reactors: start-up operation and microbial characterization. J Environ Manag 164:196–205. CrossRefGoogle Scholar
  7. Gilbert EM, Agrawal S, Schwartz T, Horn H, Lackner S (2015) Comparing different reactor configurations for partial nitritation/anammox at low temperatures. Water Res 81:92–100CrossRefPubMedGoogle Scholar
  8. Gonzalez-Gil G, Sougrat R, Behzad AR, Lens PN, Saikaly PE (2015) Microbial community composition and ultrastructure of granules from a full-scale anammox reactor. Microb Ecol 70(1):118–131. CrossRefPubMedGoogle Scholar
  9. Gonzalez-Martinez A, Osorio F, Rodriguez-Sanchez A, Martinez-Toledo MV, Gonzalez-Lopez J, Lotti T, van Loosdrecht MCM (2015a) Bacterial community structure of a lab-scale anammox membrane bioreactor. Biotechnol Prog 31(1):186–193. CrossRefPubMedGoogle Scholar
  10. Gonzalez-Martinez A, Rodriguez-Sanchez A, Muñoz-Palazon B, Garcia-Ruiz MJ, Osorio F, van Loosdrecht MCM, Gonzalez-Lopez J (2015b) Microbial community analysis of a full-scale DEMON bioreactor. Bioprocess Biosyst Eng 38(3):499–508. CrossRefPubMedGoogle Scholar
  11. Grady CPL, Daigger GT, Love NG, Filipe CDM (2011) Biological wastewater treatment, 3rd edn. CRC Press, London, pp 99–102Google Scholar
  12. Guo JH, Peng YZ, Fan L, Zhang L, Ni BJ, Kartal B, Feng X, Jetten MSM, Yuan ZG (2016) Metagenomic analysis of anammox communities in three different microbial aggregates. Environ Microbiol 18(9):2979–2993. CrossRefPubMedGoogle Scholar
  13. Hubaux N, Wells G, Morgenroth E (2015) Impact of coexistence of flocs and biofilm on performance of combined nitritation-anammox granular sludge reactors. Water Res 68:127–139CrossRefPubMedGoogle Scholar
  14. Jemaat Z, Suárez-Ojeda ME, Pérez J, Carrera J (2014) Partial nitritation and o-cresol removal with aerobic granular biomass in a continuous airlift reactor. Water Res 48:354–362. CrossRefPubMedGoogle Scholar
  15. Ke Y, Azari M, Han P, Görtz I, Gu JD, Denecke M (2015) Microbial community of nitrogen-converting bacteria in anammox granular sludge. Int Biodeter Biodegr 103:105–115. CrossRefGoogle Scholar
  16. Lackner S, Gilbert EM, Vlaeminck SE, Joss A, Horn H, van Loosdrecht MCM (2014) Full-scale partial nitritation/anammox experiences—an application survey. Water Res 55:292–303. CrossRefPubMedGoogle Scholar
  17. Li JZ, Meng J, Li JL, Wang C, Deng KW, Sun K, Buelna G (2016a) The effect and biological mechanism of COD/TN ratio on nitrogen removal in a novel upflow microaerobic sludge reactor treating manure-free piggery wastewater. Bioresour Technol 209:360–368CrossRefPubMedGoogle Scholar
  18. Li W, Zheng P, Ji JY, Zhang M, Guo J, Zhang JQ, Abbas G (2014) Floatation of granular sludge and its mechanism: a key approach for high-rate denitrifying reactor. Bioresour Technol 152:414–419. CrossRefPubMedGoogle Scholar
  19. Li XJ, Sun S, Badgley BD, Sung S, Zhang H, He Z (2016b) Nitrogen removal by granular nitritation-anammox in an upflow membrane-aerated biofilm reactor. Water Res 94:23–31. CrossRefPubMedGoogle Scholar
  20. Luo JH, Chen H, Han XY, Sun YF, Yuan ZG, Guo JH (2017) Microbial community structure and biodiversity of size-fractionated granules in a partial nitritation-anammox process. FEMS Microbiol Ecol 93:1–10CrossRefGoogle Scholar
  21. Ma B, Bao P, Wei Y, Zhu GB, Yuan ZG, Peng YZ (2015) Suppressing nitrite-oxidizing bacteria growth to achieve nitrogen removal from domestic wastewater via anammox using intermittent aeration with low dissolved oxygen. Sci Rep 5(1):13048–13057. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Mines RO (2014) Environmental engineering principles and practice, 1st edn. John Wiley & Sons, Ltd, ChichesterGoogle Scholar
  23. Nowka B, Daims H, Spieck E (2015) Comparison of oxidation kinetics of nitrite-oxidizing bacteria: nitrite availability as a key factor in niche differentiation. Appl Environ Microbiol 81(2):745–753. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Oshiki M, Satoh H, Okabe S (2016) Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environ Microbiol 18:2784–2796CrossRefPubMedGoogle Scholar
  25. Park H, Sundar S, Ma Y, Chandran K (2015) Differentiation in the microbial ecology and activity of suspended and attached bacteria in a nitritation-anammox process. Biotechnol Bioeng 112(2):272–279. CrossRefPubMedGoogle Scholar
  26. Pérez J, Lotti T, Kleerebezem R, Picioreanu C, van Loosdrecht MCM (2014) Outcompeting nitrite-oxidizing bacteria in single-stage nitrogen removal in sewage treatment plants: a model-based study. Water Res 66:208–218CrossRefPubMedGoogle Scholar
  27. Poot V, Mithell H, Geleijnse MA, van Loosdrecht MCM, Pérez J (2016) Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge. Water Res 106:518–530. CrossRefPubMedGoogle Scholar
  28. Qian FY, Wang JF, Shen YL, Wang Y, Wang SY, Chen X (2017a) Achieving high performance completely autotrophic nitrogen removal in a continuous granular sludge reactor. Biochem Eng J 118:97–104. CrossRefGoogle Scholar
  29. Qian FY, Chen X, Wang JF, Shen YL, Gao JJ, Mei J (2017b) Differentiation in nitrogen-converting activity and microbial community structure between granular size fractions in a continuous autotrophic nitrogen removal reactor. J Microbiol Biotechnol 27(10):1798–1807. CrossRefPubMedGoogle Scholar
  30. Regmi P, Miller MW, Holgate B, Bunce R, Park H, Chandran K, Wett B, Murthy S, Bott CB (2014) Control of aeration, aerobic SRT and COD input for mainstream nitritation/denitritation. Water Res 57:162–171. CrossRefPubMedGoogle Scholar
  31. Shi XY, Sheng GP, Li XY, Yu HQ (2010) Operation of a sequencing batch reactor for cultivating autotrophic nitrifying granules. Bioresour Technol 101(9):2960–2964. CrossRefPubMedGoogle Scholar
  32. Speth DR, in't Zandt MH, Guerrero-Curz S, Dutilh BE, MSM J (2016) Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system. Nat Commun 7:11172–11182. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Third KA, Sliekers AO, Kuenen JG, Jetten MSM (2001) The CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation: interaction and competition between three groups of bacteria. Syst Appl Microbiol 24(4):588–596. CrossRefPubMedGoogle Scholar
  34. Van Hulle SWH, Vandeweyer HJP, 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):1–20. CrossRefGoogle Scholar
  35. Varas R, Guzman-Fierro V, Giustinianovich E, Behar J, Fernandez K, Roeckel M (2015) Startup and oxygen concentration effects in a continuous granular mixed flow autotrophic nitrogen removal reactor. Bioresour Technol 190:345–351. CrossRefPubMedGoogle Scholar
  36. Vázquez-Padín J, Mosquera-Corral A, Campos JL, Méndez R, Revsbech NP (2010) Microbial community distribution and activity dynamics of granular biomass in a CANON reactor. Water Res 44:4359–4370CrossRefPubMedGoogle Scholar
  37. Vlaeminck SE, De Clippeleir H, Verstraete W (2012) Microbial resource management of one-stage partial nitritation/anammox. Microb Biotechnol 5:433–448CrossRefPubMedPubMedCentralGoogle Scholar
  38. Vlaeminck SE, Terada A, Smets BF, De Clippeleir H, Schaubroeck T, Bolca S, Demeestere L, Mast J, Boon N, Carballa M, Verstraete W (2010) Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox. Appl Environ Microbiol 76:900–909CrossRefPubMedGoogle Scholar
  39. Wang JF, Qian FY, Liu XP, Liu WR, Wang SY, Shen YL (2016) Cultivation and characteristics of partial nitrification granular sludge in a sequencing batch reactor inoculated with heterotrophic granules. Appl Microbiol Biotechnol 100(21):9381–9391. CrossRefPubMedGoogle Scholar
  40. Wang L, Zheng P, Chen TT, Chen JW, Xing YJ, Ji QX, Zhang M, Zhang J (2012) Performance of autotrophic nitrogen removal in the granular sludge bed reactor. Bioresour Technol 123:78–85. CrossRefPubMedGoogle Scholar
  41. Wang L, Zheng P, Xing YJ, Li W, Yang J, Abbas G, Liu S, He ZF, Zhang JQ, Zhang HT, Lu HF (2014) Effect of particle size on the performance of autotrophic nitrogen removal in the granular sludge bed reactor and microbiological mechanisms. Bioresour Technol 157:240–246. CrossRefPubMedGoogle Scholar
  42. Wang S, Liu Y, Niu Q, Ji J, Hojo T, Li YY (2017) Nitrogen removal performance and loading capacity of a novel single-stage nitritation-anammox system with syntrophic micro-granules. Bioresour Technol 236:119–128. CrossRefPubMedGoogle Scholar
  43. Zhang BG, Liu Y, Tong S, Zheng MS, Zhao YX, Tian CX, Liu HY, Feng CP (2014a) Enhancement of bacterial denitrification for nitrate removal in groundwater with electrical stimulation from microbial fuel cells. J Power Sources 268:423–429. CrossRefGoogle Scholar
  44. Zhang JB, Zhou J, Han Y, Zhang XG (2014b) Start-up and bacterial communities of single-stage nitrogen removal using anammox and partial nitritation (SNAP) for treatment of high strength ammonia wastewater. Bioresour Technol 169:652–657. CrossRefPubMedGoogle Scholar
  45. Zhang XJ, Zhang HZ, Ye CM, Wei MB, Du JJ (2015) Effect of COD/N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors. Bioresour Technol 189:302–308. CrossRefPubMedGoogle Scholar
  46. Zheng BY, Zhang L, Guo JH, Zhang SJ, Yang AM, Peng YZ (2016) Suspended sludge and biofilm shaped different anammox communities in two pilot-scale one-stage anammox reactors. Bioresour Technol 211:273–279. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Feiyue Qian
    • 1
  • Abebe Temesgen Gebreyesus
    • 1
  • Jianfang Wang
    • 1
  • Yaoliang Shen
    • 1
  • Wenru Liu
    • 1
  • Lulin Xie
    • 1
  1. 1.School of Environmental Science and EngineeringSuzhou University of Science and TechnologySuzhouPeople’s Republic of China

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