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N2O production from hydroxylamine oxidation and corresponding hydroxylamine oxidoreductase involved in a heterotrophic nitrifier A. faecalis strain NR


N2O production from NH2OH oxidation involved in a heterotrophic nitrifier Alcaligenes faecalis strain NR was studied. 15N-labeling experiments showed that biological NH2OH consumption by strain NR played a dominant role in N2O production, although chemical reaction between NH2OH and O2 indeed existed. Hydroxylamine oxidoreductase (HAO) from strain NR was partially purified by (NH4)2SO4 fractionation and DEAE Cartridge chromatography. The maximum activity of HAO was 9.60 mU with a specific activity of 92.04 mU/(mg protein) when K3Fe(CN)6 was used as an electron acceptor. The addition of Ca2+ promoted the HAO activity, while the presence of Mn2+ inhibited the enzyme activity. The optimal temperature and pH for HAO activity were 30 °C and 8. Analysis of enzyme-catalyzed products demonstrated that NH2OH oxidation catalyzed by HAO from strain NR played significant role in the production of N2O.

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  1. 1.

    Stein LY (2011) Surveying N2O-producing pathways in bacteria. Methods Enzymol 486:131–152

  2. 2.

    Ravishankara AR, Daniel JS, Portmann RW (2009) Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326:123–125

  3. 3.

    Wunderlin P, Mohn J, Joss A, Emmenegger L, Siegrist H (2012) Mechanisms of N2O production in biological wastewater treatment under nitrifying and denitrifying conditions. Water Res 46:1027–1037

  4. 4.

    Soler-Jofra A, Picioreanu C, Yu R, Chandran K, van Loosdrecht MCM, Pérez J (2018) Importance of hydroxylamine in abiotic N2O production during transient anoxia in planktonic axenic Nitrosomonas cultures. Chem Eng J 335:756–762

  5. 5.

    Zhang QL, Liu Y, Ai GM, Miao LL, Zheng HY, Liu ZP (2012) The characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium, Bacillus methylotrophicus strain L7. Bioresour Technol 108:35–44

  6. 6.

    Zhao B, Tian M, An Q, Ye J, Guo JS (2017) Characteristics of a heterotrophic nitrogen removal bacterium and its potential application on treatment of ammonium-rich wastewater. Bioresour Technol 226:46–54

  7. 7.

    Feng L, Jia R, Zeng Z, Yang G, Xu X (2018) Simultaneous nitrification-denitrification and microbial community profile in an oxygen-limiting intermittent aeration SBBR with biodegradable carriers. Biodegradation 29:473–486

  8. 8.

    Wang T, Dang Q, Liu C, Yan J, Fan B, Cha D, Yin Y, Zhang Y (2016) Heterotrophic nitrogen removal by a newly-isolated alkalitolerant microorganism, Serratia marcescens W5. Bioresour Technol 211:618–627

  9. 9.

    Wehrfritz J, Reilly A, Spiro S, Richardson DJ (1993) Purification of hydroxylamine oxidase from Thiosphaera pantotropha Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification. FEBS Lett 335:246–250

  10. 10.

    Moir JW, Wehrfritz J, Spiro S, Richardson DJ (1996) The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17. Biochem J 319:823–827

  11. 11.

    Carantoa JD, Lancastera KM (2017) Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase. Proc Natl Acad Sci USA 114:8217–8222

  12. 12.

    Zhang S, Sha C, Jiang W, Li W, Zhang D, Li J, Meng L, Piao Y (2015) Ammonium removal at low temperature by a newly isolated heterotrophic nitrifying and aerobic denitrifying bacterium Pseudomonas fluorescens wsw-1001. Environ Technol 36:2488–2494

  13. 13.

    Lei Y, Wang Y, Liu H, Xi C, Song L (2016) A novel heterotrophic nitrifying and aerobic denitrifying bacterium, Zobellella taiwanensis DN-7, can remove high-strength ammonium. Appl Microbiol Biotechnol 100:4219–4229

  14. 14.

    Zheng M, He D, Ma T, Chen Q, Liu S, Ahmad M, Gui M, Ni J (2014) Reducing NO and N2O emission during aerobic denitrification by newly isolated Pseudomonas stutzeri PCN-1. Bioresour Technol 162:80–88

  15. 15.

    Zhao B, Cheng DY, Tan P, An Q, Guo JS (2018) Characterization of an aerobic denitrifier Pseudomonas stutzeri strain XL-2 to achieve efficient nitrate removal. Bioresour Technol 250:564–573

  16. 16.

    Stein LY (2019) Insights into the physiology of ammonia-oxidizing microorganisms. Curr Opin Chem Biol 49:9–15

  17. 17.

    Ni BJ, Yuan Z (2015) Recent advances in mathematical modeling of nitrous oxides emissions from wastewater treatment processes. Water Res 87:336–346

  18. 18.

    Papen H, Von BR, Hinkel I, Thoene B, Rennenberg H (1989) Heterotrophic nitrification by Alcaligenes faecalis: NO2 , NO3 , N2O, and NO production in exponentially growing cultures. Appl Environ Microbiol 55:2068–2072

  19. 19.

    Otte S, Schalk J, Kuenen JG, Jetten MSM (1999) Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis. Appl Microbiol Biotechnol 51:255–261

  20. 20.

    Joo HS, Hirai M, Shoda M (2005) Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4. J Biosci Bioeng 100:184–191

  21. 21.

    Pilegaard K (2013) Processes regulating nitric oxide emissions from soils. Philos Trans R Soc Lond B Biol Sci 368:1–8

  22. 22.

    Zhao B, He YL, Hughes J, Zhang XF (2010) Heterotrophic nitrogen removal by a newly isolated Acinetobacter calcoaceticus HNR. Bioresour Technol 101:5194–5200

  23. 23.

    Richardson DJ, Wehrfritz J, Keech A, Crossman LC, Roldan MD, Sears HJ, Butler CS, Reilly A, Moir JWB, Berks BC, Ferguson SJ, Thornson AJ, Spiro S (1998) The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification. Biochem Soc Trans 26:401–408

  24. 24.

    Zhao B, An Q, He YL, Guo JS (2012) N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR. Bioresour Technol 116:379–385

  25. 25.

    Hooper AB, Maxwell PC, Terry KR (1978) Hydroxylamine oxidoreductase from Nitrosomonas: absorption spectra and content of heme and metal. Biochemistry 17:2984–2989

  26. 26.

    Cabail MZ, Pacheco AA (2003) Selective one-electron reduction of Nitrosomonas europaea hydroxylamine oxidoreductase with nitric oxide. Inorg Chem 42:270–272

  27. 27.

    Jetten MSM, de Bruijn P, Kuenen JG (1997) Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase. Anton Leeuw 71:69–74

  28. 28.

    Zhang SM, Li WG, Zhang DY, Huang XF, Qin W, Sha CQ (2014) Purification and characterization of a low-temperature hydroxylamine oxidase from heterotrophic nitrifier Acinetobacter sp. Y16. Biomed Environ Sci 27:515–522

  29. 29.

    APHA (1998) Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, Washington

  30. 30.

    Frear DS, Burrell RC (1955) Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal Chem 27:1664–1665

  31. 31.

    Ono Y, Makino N, Hoshino Y, Shoji K, Yamanaka T (1996) An iron dioxygenase from Alcaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite. FEMS Microbiol Lett 139:103–108

  32. 32.

    Arciero DM, Hooper AB (1993) Hydroxylamine oxidoreductase from Nitrosomonas europaea is a multimer of an octa-heme subunit. J Biol Chem 268:14645–14654

  33. 33.

    Wehrfritz J, Carter JP, Spiro S, Richardson DJ (1997) Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of a hydroxylamine-cytochrome c oxidoreductase from a Pseudomonas species. Arch Microbiol 166:421–424

  34. 34.

    Stüven R, Vollmer M, Bock E (1992) The impact of organic matter on nitric oxide formation by Nitrosomonas europaea. Arch Microbiol 158:439–443

  35. 35.

    Su JJ, Liu BY, Liu CY (2001) Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol 90:457–462

  36. 36.

    Wang X, An Q, Zhao B, Guo JS, Huang YS, Tian M (2018) Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL. Appl Microbiol Biotechnol 102:2019–2030

  37. 37.

    Kim JK, Park KJ, Cho KS, Nam SW, Park TJ, Bajpai R (2005) Aerobic nitrification-denitrification by heterotrophic Bacillus strains. Bioresour Technol 96:1897–1906

  38. 38.

    Goldstein S, Czapski G (1995) The reaction of NO. with O2 and HO2 : a pulse radiolysis study. Free Radical Bio Med 19:505–510

  39. 39.

    Hughes MN (2008) Chemistry of nitric oxide and related species. Methods Enzymol 436:3–19

  40. 40.

    Shafirovich V, Lymar SV (2002) Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide. Proc Natl Acad Sci USA 99:7340–7345

  41. 41.

    Hughes MN (1999) Relationships between nitric oxide, nitroxyl ion, nitrosonium cation and peroxynitrite. Biochim Biophys Acta 1411:263–272

  42. 42.

    Kuenen J, Robertson L (1994) Combined nitrification-denitrification processes. FEMS Microbiol Rev 15:109–117

  43. 43.

    Huang X, Li W, Zhang D, Qin W (2013) Ammonium removal by a novel oligotrophic Acinetobacter sp. Y16 capable of heterotrophic nitrification-aerobic denitrification at low temperature. Bioresour Technol 146:44–50

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This research was supported by the National Natural Science Foundation of China (Grant No. 51208534), Technical Innovation and Application Demonstration Project of CQ CSTC (Grant No. cstc2018jscx-msybX0308), and Fundamental Research Funds for the Central Universities (Grant No. 106112016CDJXY210007).

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Correspondence to Qiang An.

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Zhao, B., Ran, X.C., An, Q. et al. N2O production from hydroxylamine oxidation and corresponding hydroxylamine oxidoreductase involved in a heterotrophic nitrifier A. faecalis strain NR. Bioprocess Biosyst Eng 42, 1983–1992 (2019). https://doi.org/10.1007/s00449-019-02191-w

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  • N2O production
  • NH2OH oxidation
  • Heterotrophic nitrifier
  • Hydroxylamine oxidoreductase
  • Nitrogen removal