Skip to main content
SpringerLink
Log in

Nitrogen removal from wastewater by anaerobic methane-driven denitrification in a lab-scale reactor: heterotrophic denitrifiers associated with denitrifying methanotrophs

  • Environmental biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Nitrite-dependent anaerobic methane oxidation (n-damo) is a newly discovered bioprocess that reduces nitrite to dinitrogen with methane as electron donor, which has promising potential to remove nitrogen from wastewater. In this work, a lab-scale sequencing batch reactor (SBR) was operated for 609 days with methane as the sole external electron donor. In the SBR, nitrite in synthetic wastewater was removed continuously; the final volumetric nitrogen removal rate was 12.22±0.02 mg N L−1 day−1 and the percentage of nitrogen removal was 98.5 ± 0.2 %. Microbial community analysis indicated that denitrifying methanotrophs dominated (60–70 %) the population of the final sludge. Notably, activity testing and microbial analysis both suggested that heterotrophic denitrifiers existed in the reactor throughout the operation period. After 609 days, the activity testing indicated the nitrogen removal percentage of heterotrophic denitrification was 17 ± 2 % and that of n-damo was 83 ± 2 %. A possible mutualism may be developed between the dominated denitrifying methanotrophs and the associated heterotrophs through cross-feed. Heterotrophs may live on the microbial products excreted by denitrifying methanotrophs and provide growth factors that are required by denitrifying methanotrophs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • APHA, AWWA, WEF (2005) Standard methods for the examination of water and wastewater, 21 ed. American Public Health Association, Washington

  • Chen J, Zhou ZC, Gu JD (2014) Occurrence and diversity of nitrite-dependent anaerobic methane oxidation bacteria in the sediments of the South China Sea revealed by amplification of both 16S rRNA and pmoA genes. Appl Microbiol Biotechnol 98(12):5685–5696

  • Chen J, Zhou Z, Gu JD (2015) Complex community of nitrite-dependent anaerobic methane oxidation bacteria in coastal sediments of the Mai Po wetland by PCR amplification of both 16S rRNA and pmoA genes. Appl Microbiol Biotechnol 99(3):1463–1473

  • de Klerk A (2015) Engineering evaluation of direct methane to methanol conversion. Energ Sci Eng 3(1):60–70

    Article  Google Scholar 

  • Deutzmann JS, Stief P, Brandes J, Schink B (2014) Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake. Proc Natl Acad Sci U S A 111(51):18273–18278

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MS, Strous M (2009) Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Appl Environ Microbiol 75(11):3656–3662

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ettwig KF, Butler MK, Le Paslier D, Pelletier E, Mangenot S, Kuypers MM, Schreiber F, Dutilh BE, Zedelius J, de Beer D, Gloerich J, Wessels HJ, van Alen T, Luesken F, Wu ML, van de Pas-Schoonen KT, Op den Camp HJ, Janssen-Megens EM, Francoijs KJ, Stunnenberg H, Weissenbach J, Jetten MS, Strous M (2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464(7288):543–548

    Article  CAS  PubMed  Google Scholar 

  • Foley J, de Haas D, Yuan Z, Lant P (2010) Nitrous oxide generation in full-scale biological nutrient removal wastewater treatment plants. Water Res 44(3):831–844

    Article  CAS  PubMed  Google Scholar 

  • Gomez-Silvan C, Vilchez-Vargas R, Arevalo J, Gomez MA, Gonzalez-Lopez J, Pieper DH, Rodelas B (2014) Quantitative response of nitrifying and denitrifying communities to environmental variables in a full-scale membrane bioreactor. Bioresour Technol 169:126–133

    Article  CAS  PubMed  Google Scholar 

  • Haroon MF, Hu S, Shi Y, Imelfort M, Keller J, Hugenholtz P, Yuan Z, Tyson GW (2013) Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 500(7464):567–570

    Article  CAS  PubMed  Google Scholar 

  • He Z, Cai C, Geng S, Lou L, Xu X, Zheng P, Hu B (2013) Modeling a nitrite-dependent anaerobic methane oxidation process: parameters identification and model evaluation. Bioresour Technol 147:315–320

    Article  CAS  PubMed  Google Scholar 

  • He Z, Cai C, Shen L, Lou L, Zheng P, Xu X, Hu B (2015a) Effect of inoculum sources on the enrichment of nitrite-dependent anaerobic methane-oxidizing bacteria. Appl Microbiol Biotechnol 99(2):939–946

    Article  CAS  PubMed  Google Scholar 

  • He Z, Geng S, Cai C, Liu S, Liu Y, Pan Y, Lou L, Zheng P, Xu X, Hu B (2015b) Anaerobic oxidation of methane coupled to nitrite reduction by halophilic marine NC10 bacteria. Appl Environ Microbiol 81(16):5538–5545

    Article  CAS  PubMed  Google Scholar 

  • He Z, Geng S, Shen L, Lou L, Zheng P, Xu X, Hu B (2015c) The short- and long-term effects of environmental conditions on anaerobic methane oxidation coupled to nitrite reduction. Water Res 68:554–562

    Article  CAS  PubMed  Google Scholar 

  • Hommes RWJ, Postma, P. W., Neijssel, O. M., Tempest, D. W., Dokter, P., & Duine, J. A. (1984) Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli. FEMS Microbiol Lett 24(2–3):329–333

    Article  CAS  Google Scholar 

  • Hu B, He Z, Geng S, Cai C, Lou L, Zheng P, Xu X (2014a) Cultivation of nitrite-dependent anaerobic methane-oxidizing bacteria: impact of reactor configuration. Appl Microbiol Biotechnol 98(18):7983–7991

    Article  CAS  PubMed  Google Scholar 

  • Hu BL, Shen LD, Lian X, Zhu Q, Liu S, Huang Q, He ZF, Geng S, Cheng DQ, Lou LP, Xu XY, Zheng P, He YF (2014b) Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands. Proc Natl Acad Sci U S A 111(12):4495–4500

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 68:127–139

    Article  CAS  PubMed  Google Scholar 

  • Kampman C, Hendrickx TL, Luesken FA, van Alen TA, Op den Camp HJ, Jetten MS, Zeeman G, Buisman CJ, Temmink H (2012) Enrichment of denitrifying methanotrophic bacteria for application after direct low-temperature anaerobic sewage treatment. J Hazard Mater 227-228:164–171

    Article  CAS  PubMed  Google Scholar 

  • Kloos K, Mergel, A., Rösch, C., & Bothe, H. (2001) Denitrification within the genus Azospirillum and other associative bacteria. Aust J Plant Physiol 28(9):991–998

    Google Scholar 

  • Lu H, Chandran K, Stensel D (2014) Microbial ecology of denitrification in biological wastewater treatment. Water Res 64:237–254

    Article  CAS  PubMed  Google Scholar 

  • Luesken FA, Sanchez J, van Alen TA, Sanabria J, Op den Camp HJ, Jetten MS, Kartal B (2011) Simultaneous nitrite-dependent anaerobic methane and ammonium oxidation processes. Appl Environ Microbiol 77(19):6802–6807

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Modin O, Fukushi K, Yamamoto K (2007) Denitrification with methane as external carbon source. Water Res 41(12):2726–2738

    Article  CAS  PubMed  Google Scholar 

  • Ni BJ, Ruscalleda M, Smets BF (2012) Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in Anammox biofilm. Water Res 46(15):4645–4652

    Article  CAS  PubMed  Google Scholar 

  • Okabe S, Kindaichi T, Ito T (2005) Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms. Appl Environ Microbiol 71(7):3987–3994

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Raghoebarsing AA, Pol A, van de Pas-Schoonen KT, Smolders AJ, Ettwig KF, Rijpstra WI, Schouten S, Damste JS, Op den Camp HJ, Jetten MS, Strous M (2006) A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440(7086):918–921

    Article  CAS  PubMed  Google Scholar 

  • Shen LD, He ZF, Zhu Q, Chen DQ, Lou LP, Xu XY, Zheng P, Hu BL (2012) Microbiology, ecology, and application of the nitrite-dependent anaerobic methane oxidation process. Front Microbiol 3:269

    PubMed Central  PubMed  Google Scholar 

  • Shen LD, Huang Q, He ZF, Lian X, Liu S, He YF, Lou LP, Xu XY, Zheng P, Hu BL (2015) Vertical distribution of nitrite-dependent anaerobic methane-oxidising bacteria in natural freshwater wetland soils. Appl Microbiol Biotechnol 99(1):349–357

    Article  CAS  PubMed  Google Scholar 

  • Shi Y, Hu S, Lou J, Lu P, Keller J, Yuan Z (2013) Nitrogen removal from wastewater by coupling anammox and methane-dependent denitrification in a membrane biofilm reactor. Environ Sci Technol 47(20):11577–11583

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30(12):2725–2729

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Throbäck IN, Enwall, K., Jarvis, Å., & Hallin, S. (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49(3):401–417

    Article  PubMed  Google Scholar 

  • Wu ML, Ettwig KF, Jetten MS, Strous M, Keltjens JT, van Niftrik L (2011) A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus ‘Methylomirabilis oxyfera’. Biochem Soc Trans 39(1):243–248

    Article  CAS  PubMed  Google Scholar 

  • Wu ML, Wessels HJ, Pol A, Op den Camp HJ, Jetten MS, van Niftrik L, Keltjens JT (2015) An XoxF-type methanol dehydrogenase from the anaerobic methanotroph ‘Candidatus Methylomirabilis oxyfera’. Appl Environ Microbiol 81(4):1442–1451

    Article  PubMed Central  PubMed  Google Scholar 

  • Zhu B, van Dijk G, Fritz C, Smolders AJ, Pol A, Jetten MS, Ettwig KF (2012) Anaerobic oxidization of methane in a minerotrophic peatland: enrichment of nitrite-dependent methane-oxidizing bacteria. Appl Environ Microbiol 78(24):8657–8665

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhu G, Zhou L, Wang Y, Wang S, Guo J, Long XE, Sun X, Jiang B, Hou Q, Jetten MS, Yin C (2015) Biogeographical distribution of denitrifying anaerobic methane oxidizing bacteria in Chinese wetland ecosystems. Environ Microbiol Rep 7(1):128–138

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was funded by the National Natural Science Foundation of China (No. 51478415 and No. 41276109), the Fundamental Research Funds for the Central Universities (No. 2015QNA6012), and the Key Science and Technology Innovation Team Project of Zhejiang Province (2013TD12).

Conflict of interest

The authors declare that they have no competing interests.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Author information

Authors and Affiliations

  1. Department of Environmental Engineering, Zhejiang University, Hangzhou, 310058, China

    Zhanfei He, Jiaqi Wang, Xu Zhang, Chaoyang Cai, Sha Geng, Ping Zheng, Xinhua Xu & Baolan Hu

Authors
  1. Zhanfei He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiaqi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chaoyang Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sha Geng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ping Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinhua Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Baolan Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Baolan Hu.

Electronic supplementary materials

ESM 1

(PDF 200 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

He, Z., Wang, J., Zhang, X. et al. Nitrogen removal from wastewater by anaerobic methane-driven denitrification in a lab-scale reactor: heterotrophic denitrifiers associated with denitrifying methanotrophs. Appl Microbiol Biotechnol 99, 10853–10860 (2015). https://doi.org/10.1007/s00253-015-6939-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6939-9

Keywords

Search

Navigation