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Nitrogen removal by co-occurring methane oxidation, denitrification, aerobic ammonium oxidation, and anammox

Abstract

The pathway for removing NO 3 and NH 4 + from wastewater in the presence of both CH4 and O2 was clarified by studying microbial activity and community. Batch incubation tests were performed to characterize the microbial activity of the sludge, which was acclimatized in a bioreactor in which O2 and CH4 were supplied to treat wastewater containing NO 3 and NH 4 + . The tests showed that the sludge removed significant amounts of NO 3 and NH 4 + in the presence of CH4 and O2, and the presence of the activity of methane oxidation, denitrification, nitrification, and anammox in the sludge. It was estimated that the total inorganic nitrogen removal was attributed to denitrification associated with methane oxidation as 53.4%, microbial assimilation as 37.9%, and anammox as 8.7%. Nitrification also contributed to NH 4 + decrease as 34.5% and anammox as 6.4%. Anammox activity was unambiguously demonstrated by 29N2 production in anaerobic batch incubation with 15N-labeled inorganic nitrogen compounds. The presence of methane-oxidizing bacteria and candidate denitrifiers in the sludge was shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. Clone library analysis of the PCR-amplified 16S rRNA gene fragment using specific primers for aerobic ammonium oxidizer and anammox revealed the presence of these bacteria. The results reveal that complex nitrogen-removal processes occur in the presence of CH4 and O2 by methanotroph, denitrifier, aerobic ammonium oxidizer, and anammox.

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References

  1. Amano T, Yoshinaga I, Okada K, Yamagishi T, Ueda S, Obuchi A, Sako Y, Suwa Y (2007) Detection of anammox activity and diversity of anammox bacteria-related 16S rRNA genes in coastal marine sediment in Japan. Microb Environ 22:232–242

  2. Bédard C, Knowles R (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH 4 + , and CO oxidation by methanotrophs and nitrifiers. Microbiological Rev 53:68–84

  3. Costa C, Dijkema C, Friedrich M, Garcia-Encina P, Fernandez-Polanco F, Stams AJ (2000) Denitrification with methane as electron donor in oxygen-limited bioreactors. Appl Microbiol Biotechnol 53:754–762

  4. Eisentraeger A, Klag P, Vansbotter B, Heymann E, Dott W (2001) Denitrification of groundwater with methane as sole hydrogen donor. Water Res 35:2261–2267

  5. Ettwig KF, Shima S, van de Pas-Schoonen KT, Kahnt J, Medema MH, op den Camp HJM, Jetten MSM, Strous M (2008) Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environ Microbiol 10:3164–3173

  6. Garrido JM, van Benthum WAJ, van Loosdrecht MCM, Heijnen JJ (1997) Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor. Biotechnol Bioeng Vol 53:168–178

  7. Graham DW, Chaudhary JA, Hanson RS, Arnold RG (1993) Factors affecting competition between type I and type II methanotrophs in two-organism, continuous-flow reactors. Microbial Ecol 25:1–17

  8. Houbron E, Torrijos M, Capdeville B (1999) An alternative use of biogas applied at the water denitrification. Water Sci and Technol 40:115–122

  9. Islas-Lima S, Thalasso F, Gomez-Hernandez J (2004) Evidence of anoxic methane oxidation coupled to denitrification. Water Res 38:13–16

  10. Kalyuzhnyi S, Gladchenko M, Mulder A, Versprille B (2006) DEAMOX—new biological nitrogen removal process based on anaerobic ammonia oxidation coupled to sulphide-driven conversion of nitrate into nitrite. Water Res 40:3637–3645

  11. Kartal B, Kuypers MMM, Lavik G, Schalk J, Op den Champ HJM, Jetten MSM, Strous M (2007) Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ Microbiol 9:635–642

  12. Khin T, Annachhatre AP (2004) Nitrogen removal in a fluidized bed bioreactor by using mixed culture under oxygen-limited conditions. Water Sci and Technol 50:313–320

  13. Kohlerstaub D, Frank S, Leisinger T (1995) Dichloromethane as the sole carbon source for Hyphomicrobium sp strain DM2 under denitrification conditions. Biodegradation 6:229–235

  14. Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW (1997) Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63:1489–1497

  15. Lee HJ, Bae JH, Cho KM (2001) Simultaneous nitrification and denitrification in a mixed methanotrophic culture. Biotechnol Lett 23:935–941

  16. Lieu PK, Hatozaki R, Homan H, Furukawa K (2005) Single-stage nitrogen removal using anammox and partial nitrification (SNAP) for treatment of synthetic landfill leachate. Jpn J Water Treat Biol 41:103–112

  17. Mergaert J, Cnockaert MC, Swings J (2003) Thermomonas fusca sp nov and Thermomonas brevis sp nov., two mesophilic species isolated from a denitrification reactor with poly(epsilon-caprolactone) plastic granules as fixed bed, and emended description of the genus Thermomonas. Int J Syst Evol Microbiol 53:1961–1966

  18. Modin O, Fukushi K, Yamamot K (2007) Denitrification with methane as external carbon source. Water Res 41:2726–2738

  19. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reaction-amplified genes-coding for 16S rRNA. Appl Environ Microbiol 59:695–700

  20. Osaka T, Yoshie S, Tsuneda S, Hirata A, Iwami N, Inamori Y (2006) Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microb Ecol 52:253–266

  21. Osaka T, Ebie Y, Tsuneda S, Ianamori Y (2008) Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing. FEMS Microbiol Ecol 64:494–506

  22. Pynaert K, Smets BF, Wyffels S, Beheydt D, Siciliano SD, Verstraete W (2003) Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl Environ Microbiol 69:3626–3635

  23. Raghoebarsing AA, Pol A, van de Pas-Schoonen KT, Smolders AJP, Ettwig KF, Rijpstra WIC, Schouten S, Damste JSS, Op den Camp HJM, Jetten MSM, Strous M (2006) A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440:918–921

  24. Rajapakse JP, Scutt JE (1999) Denitrification with natural gas and various new growth media. Water Res 33:3723–3734

  25. Rhee GY, Fuhs GW (1978) Wastewater denitrification with one-carbon compounds as energy source. J Water Pollution Control Federation 9:2111–2119

  26. Schmid MC, Maas B, Dapena A, de Pas-Schoonen KV, de Vossenberg JV, Kartal B, van Niftrik L, Schmidt I, Cirpus I, Kuenen JG, Wagner M, Damste JSS, Kuypers M, Revsbech NP, Mendez R, Jetten MSM, Strous M (2005) Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria. Appl Environ Microbiol 71:1677–1684

  27. Schmidt I, Sliekers O, Schmid M, Bock E, Fuerst J, Kuenen JG, Jetten MSM, Strous M (2003) New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS Microbiol Rev 27:481–492

  28. 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

  29. Sumino T, Isaka K, Ikuta H, Saiki Y, Yokota T (2006) Nitrogen removal from wastewater using simultaneous nitrate reduction and anaerobic ammonium oxidation in single reactor. J Biosci Bioeng 102:346–351

  30. Suzuki K, Tanaka Y, Kuroda K, Hanajima D, Fukumoto Y, Yasuda T, Waki M (2007) Removal and recovery of phosphorous from swine wastewater by demonstration crystallization reactor and struvite accumulation device. Bioresour Technol 98:1573–1578

  31. Tanaka Y, Kitayama K, Fukunaga S, Haga K (1999) Performance of a process consisted of UASB reactor and trickling filter for swine wastewater treatment. Jpn J Water Treat Biol (in Japanese) 35:177–188

  32. Thalasso F, Vallecillo A, García-Encina P, Fdz-Polanco F (1997) The use of methane as a sole carbon source for wastewater denitrification. Water Res 31:55–60

  33. van de Graaf AA, de Bruijn P, Robertson LA, Jetten MSM, Kuenen JG (1996) Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiol 142:2187–2196

  34. Waki M, Tanaka Y, Osada T, Suzuki K (2002) Effects of nitrite and ammonium on methane-dependent denitrification. Appl Microbiol Biotechnol 59:338–343

  35. Waki M, Suzuki K, Osada T, Tanaka Y (2004a) Effect of CH4 and O2 concentrations on methane-dependent denitrification. Jpn J Water Treat Biol 40(2):53–61

  36. Waki M, Suzuki K, Osada T, Tanaka Y, Ike M, Fujita M (2004b) Microbiological activities contributing to nitrogen removal with methane: effects of methyl fluoride and tungstate. Bioresour Technol 94:339–343

  37. Waki M, Yokoyama H, Ogino A, Suzuki K, Tanaka Y (2008) Nitrogen removal from purified swine wastewater using bio-gas by semi-partitioned reactor. Bioresour Technol 99:5335–5340

  38. Werner M, Kayser R (1991) Denitrification with biogas as external carbon source. Water Sci and Technol 23:701–708

  39. Xiao Y, Zeng GM, Yang ZH, Liu YS, Ma YH, Yang L, Wang RJ, Xu ZY (2009) Coexistence of nitrifiers, denitrifiers and anammox bacteria in a sequencing batch biofilm reactor as revealed by PCR-DGGE. J Appl Microbiol 106:496–505

  40. Yokoyama H, Waki M, Moriya N, Yasuda T, Tanaka Y, Haga K (2007) Effect of fermentation temperature on hydrogen production from cow waste slurry by using anaerobic microflora within the slurry. Appl Microbiol Biotechnol 74:474–483

  41. You J, Das A, Dolan EM, Hu Z (2009) Ammonia-oxidizing archaea involved in nitrogen removal. Water Res 43:1801–1809

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Acknowledgments

We would like to thank Dr. E. Mikami (formerly of the National Institute of Advanced Industrial Science and Technology) for his valuable advice. We would also like to thank Ms. K. Sumiya (National Institute of Livestock and Grassland Science, Japan) for her skilful assistance with our laboratory work. This research was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan through a research project on the biorecycling of wastes from the agriculture, forestry, and fisheries sectors.

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Correspondence to Miyoko Waki.

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Waki, M., Yasuda, T., Yokoyama, H. et al. Nitrogen removal by co-occurring methane oxidation, denitrification, aerobic ammonium oxidation, and anammox. Appl Microbiol Biotechnol 84, 977–985 (2009). https://doi.org/10.1007/s00253-009-2112-7

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Keywords

  • Methanotroph
  • Denitrifier
  • Nitrifier
  • Anammox
  • Simultaneous removal of nitrate and ammonium