Environmental Science and Pollution Research

, Volume 26, Issue 19, pp 19012–19024 | Cite as

Effects of short- and long-term exposures of humic acid on the Anammox activity and microbial community

  • Khadija Kraiem
  • Mohamed Ali WahabEmail author
  • Hamadi Kallali
  • Andrea Fra-vazquez
  • Alba Pedrouso
  • Anuska Mosquera-Corral
  • Naceur Jedidi
Advanced Oxidation Process for Sustainable Water Management


Humic acid has a controversial effect on the biological treatment processes. Here, we have investigated humic acid effects on the Anammox activity by studying the nitrogen removal efficiencies in batch and continuous conditions and analyzing the microbial community using Fluorescence in situ hybridization (FISH) technique. The results showed that the Anammox activity was affected by the presence of humic acid at a concentration higher than 70 mg/L. In fact, in the presence of humic acid concentration of 200 mg/L, the Anammox activity decreased to 57% in batch and under continuous condition, the ammonium removal efficiencies of the reactor decreased from 78 to 41%. This reduction of Anammox activity after humic acid addition was highlighted by FISH analysis which revealed a considerable reduction of the abundance of Anammox bacteria and the bacteria living in symbiosis with them. Furthermore, a total inhibition of Candidatus Brocadia fulgida was observed. However, humic acid has promoted heterotrophic denitrifying bacteria which became dominant in the reactor. In fact, the evolution of the organic matter in the reactor showed that the added humic acid was used as carbon source by heterotrophic bacteria which explained the shift of metabolism to the favor of heterotrophic denitrifying bacteria. Accordingly, humic acid should be controlled in the influent to avoid Anammox activity inhibition.


Anammox bacteria Heterotrophic bacteria Humic acid Inhibition Nitrogen 


Funding information

This research work is financially supported by the Tunisian Ministry of Higher Education, Scientific Research and Technology.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1995) Combination of 16S rRNA targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925. Google Scholar
  2. APHA (1998) Standard methods for the examination of water and wastewater, 20th edn. United Book Press, USAGoogle Scholar
  3. Buchanan RE, Gibbons CNE (1974) Bergey’s manual of determinative bacteriology, 8th edn. The William & Wilkins, Baltimor, p 1268Google Scholar
  4. Campos JL, Figueroa M, Mosquera-Corral A, Méndez R (2009) Aerobic sludge granulation: state-of-the-art. Int J Environ Eng 1:136–151. CrossRefGoogle Scholar
  5. Cao S, Du R, Li B, Ren N, Peng Y (2016) High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater. Appl Microbiol Biotechnol 100:6457–6467. CrossRefGoogle Scholar
  6. Chamchoi N, Suwanchai N (2007) Anammox enrichment from different conventional sludges. Chemosphere 66(11):2225–2232. CrossRefGoogle Scholar
  7. Cho S, Takahashi Y, Fujii N, Yamada Y, Satoh H, Okabe S (2010) Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor. Chemosphere 78:1129–1135.
  8. Costa MC, Carvalho L, Leal CD, Dias MF, Martins KL, Garcia GB, Mancuelo ID, Hipólito 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 35:1811–1822.
  9. Dapena-Mora A, Campos JL, Mosquera-Corral A, Jetten MSM, Méndez R (2004) Stability of the ANAMMOX process in a gas-lift reactor and a SBR. J Biotechnol 110:159–170. CrossRefGoogle Scholar
  10. Dapena-Mora A, Fernández I, Campos JL, Mosquera-Corral A, Méndez R, Jetten MSM (2007) Evaluation of activity and inhibition effects on Anammox process by batch tests based on gas production. Enzym Microb Technol 40:859–865. CrossRefGoogle Scholar
  11. Daverey A, Chen YC, Sung S, Lin JG (2014) Effect of zinc on anammox activity and performance of simultaneous partial nitrification, anammox and denitrification (SNAD) process. Bioresour Technol 165:105–110.
  12. Dong S, Li M, Chen Y (2017) Inherent humic substance promotes microbial denitrification of landfill leachate via shifting bacterial community, improving enzyme activity and up-regulating gene. Sci Rep 7:12215.
  13. Egli K, Bosshard F, Werlen C, Lais P, Siegrist H, Zehnder AJB, van der Meer JR (2003) Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon. Microb Ecolo 45:419–432.
  14. Fernandes T, van Lier JB, Zeeman G (2014) Humic acid-like and fulvic acid-like inhibition on the hydrolysis of cellulose and tributyrin. Bioenerg Res 8:821–831. CrossRefGoogle Scholar
  15. Fernández I, Dosta J, Fajardo C, Campos JL, Mosquera-Corral A, Méndez R (2012) Short- and long-term effects of ammonium and nitrite on the Anammox process. J Environ Manag 95:S170–S174. CrossRefGoogle Scholar
  16. Gonzalez-Gil G, Sougrat R, Behzad AR, Lens PNL, Saikaly PE (2014) Microbial community composition and ultrastructure of granules from a full-scale Anammox reactor. Microb Ecol 70:118–131. CrossRefGoogle Scholar
  17. Helmer C, Tromm C, Hippen A, Rosenwinkel KH, Seyfried CF, Kunst S (2001) Single stage biological nitrogen removal by nitration and anaerobic ammonium oxidation in biofilm systems. Water Sci Technol 43:311–320CrossRefGoogle Scholar
  18. Hou L, Zheng Y, Liu M, Gong J, Zhang X, Yin G, You L (2013) Anaerobic ammonium oxidation (anammox) bacterial diversity, abundance, and activity in marsh sediments of the Yangtze Estuary. J Geophys Res Biogeosci 118:1237–1246. CrossRefGoogle Scholar
  19. Hu BL, Zheng P, Tang CJ, Chen JW, van der Biezen E, Zhang L, Ni BJ, Jetten MSM, Yan J, Hu HQ, Kartal B (2010) Identification and quantification of anammox bacteria in eight nitrogen removal reactors. Water Res 44:5014–5020. CrossRefGoogle Scholar
  20. Jetten MSM, Horn SJ, vanLoosdrecht MCM (1997) Towards a more sustainable municipal wastewater treatment system. Water Sci Technol 35:171–180. CrossRefGoogle Scholar
  21. Jin RC, Yang GF, Yu JJ, Zheng P (2012) The inhibition of the Anammox process: a review. Chem Eng J 197:67–79. CrossRefGoogle Scholar
  22. Khadem AF, Azman S, Plugge CM, Zeeman G, van Lier JB, Stams AGM (2017) Effect of humic acids on the activity of pure and mixed methanogenic cultures. Biomass Bioenergy 99:21–30. CrossRefGoogle Scholar
  23. Kindaichi T, Yuri S, Ozaki N, Ohashi A (2012) Ecophysiological role and function of uncultured Chloroflexi in an Anammox reactor. Water Sci Technol 66:2556–2561. CrossRefGoogle Scholar
  24. Lackner S, Gilbert EM, Vlaeminck SE, Joss A, Horn H, van Loosdrecht MC (2014) Full-scale partial nitritation/Anammox experiences—an application survey. Water Res 55:292–303. CrossRefGoogle Scholar
  25. Lan CJ, Kumar M, Wang CC, Lin JG (2011) Development of simultaneous partial nitrification, Anammox and denitrification (SNAD) process in a sequential batch reactor. Bioresour Technol 102:5514–5519. CrossRefGoogle Scholar
  26. Li S, Hu J (2016) Photolytic and photocatalytic degradation of tetracycline: effect of humic acid on degradation kinetics and mechanisms. J Hazard Mater 318:134–144. CrossRefGoogle Scholar
  27. Meyer RL, Risgaard-Petersen N, Allen DE (2005) Correlation between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment. Appl Environ Microbiol 71:6142 6149. Google Scholar
  28. Nielsen PH, Daims H, Lemmer H (2009) FISH handbook for biological wastewater treatmentGoogle Scholar
  29. Oshiki M, Shimokawa M, Fujii N, Satoh H, Okabe S (2011) Physiological characteristics of the anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sinica. Microbiology 157:1706–1713. CrossRefGoogle Scholar
  30. Pereira AD, Leal CD, Dias MF, Etchebehere C, Chernicharo CAL, Araújo JC (2014) Effect of phenol on the nitrogen removal performance and microbial community structure and composition of an Anammox reactor. Bioresour Technol 166:103–111. CrossRefGoogle Scholar
  31. Quan ZX, Rhee SK, Zuo JE, Yang Y, Bae JW, Park JR, Lee ST, Park YH (2008) Diversity of ammonium oxidizing bacteria in a granular sludge anaerobic ammonium oxidizing (anammox) reactor. Environ Microbiol 10:3130–3139. CrossRefGoogle Scholar
  32. Sonthiphand P, Hall MW, Neufeld JD (2014) Biogeography of anaerobic ammonia-oxidizing (Anammox) bacteria. Front Microbiol 5:1–14. CrossRefGoogle Scholar
  33. Steinberg CW, Meinelt T, Timofeyev M, Bittner M, Menzel R (2008) Humic substances. Environ Sci Pollut Res 15:128–135. CrossRefGoogle Scholar
  34. 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 50:589–596. CrossRefGoogle Scholar
  35. Strous M, Kuenen JG, Jetten MSM (1999) Key physiology of anaerobic ammonium oxidation. Appl Environ Microbiol 65:3248–3250Google Scholar
  36. Sueoka K, Satoh H, Onuki M, Mino T (2009) Microorganisms involved in anaerobic phenol degradation in the treatment of synthetic coke-oven wastewater detected by RNA stable-isotope probe. FEMS Microbiol Lett 291:169–174. CrossRefGoogle Scholar
  37. Tang CJ, Zheng P, Wang CH, Mahmood Q, Zhang JQ, Chen XG, Zhang L, Chen JW (2010) Performance of high-loaded ANAMMOX UASB reactors containing granular sludge. Water Res 45:135–144.
  38. Toh SK, Ashbolt NJ (2002) Adaptation of anaerobic ammonium-oxidising consortium to synthetic coke-ovens wastewater. Appl Microbiol Biotechnol 59:344–352. CrossRefGoogle Scholar
  39. Ullhyan A, Ghosh UK (2012) Biodegradation of phenol with immobilized Pseudomonas putida activated carbon packed bio-filter power. Afr J Biotechnol 11:15160–15167. Google Scholar
  40. Van der Star WR, Miclea AI, van Dongen UG, Muyzer G, Picioreanu C, van Loosdrecht MC (2008) The membrane bioreactor: a novel tool to grow Anammox bacteria as free cells. Biotechnol Bioeng 101(2):286–294. CrossRefGoogle Scholar
  41. Wahab MA, Jellali S, Jedidi N (2010a) Effect of temperature and pH on the biosorption of ammonium onto Posidonia oceanica fibers: equilibrium and kinetic modeling studies. Bioresour Technol 101:8606–8615. CrossRefGoogle Scholar
  42. Wahab MA, Jellali S, Jedidi N (2010b) Ammonium biosorption onto sawdust: FTIR analysis, kinetics and adsorption isotherms modeling. Bioresour Technol 101:5070–5075. CrossRefGoogle Scholar
  43. Wahab MA, Hatem B, Jellali S, Jedidi N (2012) Characterization of ammonium retention processes onto cactus leaves fibers using FTIR, EDX and SEM analysis. J Hazard Mater 241:101–109. CrossRefGoogle Scholar
  44. Wahab MA, Habouzit F, Bernet N, Steyer JP, Jedidi N, Escudié R (2014) Sequential operation of a hybrid anaerobic reactor using a lignocellulosic biomass as biofilm support. Bioresour Technol 172:150–155. CrossRefGoogle Scholar
  45. Wang CC, Lee PH, Kumar M, Huang YT, Sung S, Lin JG (2010) Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill-leachate treatment plant. J Hazard Mater 175:622–628. CrossRefGoogle Scholar
  46. Xiong L, Wang YY, Tang CJ, Chai LY, Xu KQ, Song YX, Ali M, Zheng P (2013) Start-up characteristics of a granule-based Anammox UASB reactor seeded with anaerobic granular sludge. Biomed Res Int 2013:9. Google Scholar
  47. Zekker I, Rikmann E, Tenno T, Seiman A, Loorits L, Kroon K, Tenno T (2014) Nitritating-Anammox biomass tolerant to high dissolved oxygen concentration and C/N ratio in treatment of yeast factory wastewater. Environ Technol 35:1565–1576. CrossRefGoogle Scholar
  48. Zhang J, Zhang Y, Li Y, Zhang L, Qiao S, Yang F, Quan X (2012) Enhancement of nitrogen removal in a novel Anammox reactor packed with Fe electrode. Bioresour Technol 114:102–108. CrossRefGoogle Scholar
  49. Zhou S, Yang J, Xu G, Zhuang L (2014) Methanogenesis affected by the cooccurrence of iron (III) oxides and humic substances. FEMS Microbiol Ecol 88:107–120. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of Wastewater Treatment and Valorization, Water Research and Technologies CenterCERTESolimanTunisia
  2. 2.Faculty of Sciences of TunisUniversity of Tunis El ManarTunisTunisia
  3. 3.Department of Chemical Engineering, Institute of Technological Research, School of EngineeringUniversidade de Santiago de CompostelaSantiago de CompostelaSpain

Personalised recommendations