Effects of copper on nitrous oxide (N2O) reduction in denitrifiers and N2O emissions from agricultural soils

  • Weishou Shen
  • Huaiwen Xue
  • Nan GaoEmail author
  • Yutaka Shiratori
  • Takehiro Kamiya
  • Toru Fujiwara
  • Kazuo Isobe
  • Keishi Senoo
Original Paper


Biochemical reduction of nitrous oxide (N2O) to dinitrogen (N2) by N2O reductase (N2OR) is the only known sink to consume N2O. Copper (Cu) and pH are two key factors determining the activity of the enzyme N2OR. We hypothesized that changes in the Cu level could enhance the reduction of N2O to N2 in denitrifier strains and decrease the N2O emissions from agricultural soils. To test this hypothesis, Cu-modified culture medium was applied to denitrifier strains, and Cu-modified organic fertilizer was applied to both soil microcosms and fields. Of 46 denitrifier strains, 25 showed higher denitrifying activities and 30N2/(46N2O + 30N2) after the addition of Cu under pure culture conditions. Among 10 genera, Azospirillum and Herbaspirillum were the most responsive to the Cu level changes. The N2O flux was significantly reduced 4 or 8 days onwards after the application of 130 mM CuSO4-modified organic fertilizer (vol:wt = 1:1) into Andosol or Fluvisol, respectively, under soil microcosm conditions. In addition, the cumulative N2O emissions were significantly reduced after the application of 130 mM CuSO4-modified organic fertilizer. They were moderately reduced after the application of 130 mM CuSO4-modified organic fertilizer (vol:wt = 1:1) into a Fluvisol field. They were significantly lower in Azospirillum sp. UNPF1-inoculated soils after the application of 130 mM CuSO4-modified organic fertilizer when compared with that in dual non-inoculated and unmodified soils. Soils inoculated with Herbaspirillum sp. UKPF54 showed results similar to non-inoculated Fluvisol fields. These results suggest that Cu may enhance N2O conversion to N2 in denitrifiers and that Cu-modified organic fertilizer may enhance N2O consumption or decrease N2O emissions in agricultural soils.


Copper Denitrifier Greenhouse gas Nitrous oxide (N2O) emission N2O reductase N2O reduction to dinitrogen 



We thank Shigeto Otsuka for his helpful discussion and Chie Hayakawa for her help with gas sampling in the fields. We also thank the technical staffs from the Niigata Agricultural Research Institute and Institute for Sustainable Agro-ecosystem Services, The University of Tokyo for their assistance with fieldwork.

Funding information

This study was supported by grants from the National Natural Science Foundation of China (41771291, 31972503), Japan Society for the Promotion of Science through a Postdoctoral Fellowship (14F04390), JSPS KAKENHI (JP15KT0024), and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (26037B and 27004C), Japan.

Supplementary material

374_2019_1399_MOESM1_ESM.docx (830 kb)
ESM 1 (DOCX 829 kb)


  1. Aguilera E, Lassaletta L, Sanz-Cobena A, Garnier J, Vallejo A (2013) The potential of organic fertilizers and water management to reduce N2O emissions in Mediterranean climate cropping systems. A review. Agric Ecosyst Environ 164:32–52CrossRefGoogle Scholar
  2. Ashida N, Ishii S, Hayano S, Tago K, Tsuji T, Yoshimura Y, Otsuka S, Senoo K (2010) Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria. Appl Microbiol Biotechnol 85:1211–1217CrossRefGoogle Scholar
  3. Black A, Hsu PL, Hamonts KE, Clough TJ, Condron LM (2016) Influence of copper on expression of nirS, norB and nosZ and the transcription and activity of NIR, NOR and N2OR in the denitrifying soil bacteria Pseudomonas stutzeri. Microb Biotechnol 9:381–388CrossRefGoogle Scholar
  4. Butterbach-Bahl K, Baggs EM, Dannenmann M, Kiese R, Zechmeister-Boltenstern S (2013) Nitrous oxide emissions from soils: how well do we understand the processes and their controls? Philos Trans R Soc Lond Ser B Biol Sci 368:20130122CrossRefGoogle Scholar
  5. Diacono M, Montemurro F (2010) Long-term effects of organic amendments on soil fertility. A review. Agron Sustain Dev 30:401–422CrossRefGoogle Scholar
  6. Felgate H, Giannopoulos G, Sullivan MJ, Gates AJ, Clarke TA, Baggs E, Rowley G, Richardson DJ (2012) The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways. Environ Microbiol 14:1788–1800CrossRefGoogle Scholar
  7. Gao N, Shen WS, Camargo E, Shiratori Y, Nishizawa T, Isobe K, He XH, Senoo K (2017) Nitrous oxide (N2O)-reducing denitrifier-inoculated granular organic fertilizer mitigates N2O emissions from agricultural soils. Biol Fertil Soils 53:885–898CrossRefGoogle Scholar
  8. Gao N, Shen WS, Kakuta H, Tanaka N, Fujiwara T, Nishizawa T, Takaya N, Nagamine T, Isobe K, Otsuka S, Senoo K (2016) Inoculation with nitrous oxide (N2O)-reducing denitrifier strains simultaneously mitigates N2O emission from pasture soil and promotes growth of pasture plants. Soil Biol Biochem 97:83–91CrossRefGoogle Scholar
  9. Gui MY, Chen Q, Ma T, Zheng MS, Ni JR (2017) Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1. Appl Microbiol Biotechnol 101:1717–1727CrossRefGoogle Scholar
  10. Hayakawa A, Akiyama H, Sudo S, Yagi K (2009) N2O and NO emissions from an Andisol field as influenced by pelleted poultry manure. Soil Biol Biochem 41:521–529CrossRefGoogle Scholar
  11. Hu HW, Chen DL, He JZ (2015) Microbial regulation of terrestrial nitrous oxide formation: understanding the biological pathways for prediction of emission rates. FEMS Microbiol Rev 39:729–749CrossRefGoogle Scholar
  12. IPCC (2013) Observations: atmosphere and surface. In: Stocker TF, Qin D, Plattner GK, Tignor M, Allen SK, Boschung J, Nauels A, Xia Y, Bex V, Midgley PM (eds) Climate change 2013: the physical science basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, pp 159–254Google Scholar
  13. Ishii S, Ashida N, Otsuka S, Senoo K (2011a) Isolation of oligotrophic denitrifiers carrying previously uncharacterized functional gene sequences. Appl Environ Microbiol 77:338–342CrossRefGoogle Scholar
  14. Ishii S, Ohno H, Tsuboi M, Otsuka S, Senoo K (2011b) Identification and isolation of active N2O reducers in rice paddy soil. ISME J 5:1936–1945CrossRefGoogle Scholar
  15. Itakura M, Uchida Y, Akiyama H, Hoshino YT, Shimomura Y, Morimoto S, Tago K, Wang Y, Hayakawa C, Uetake Y, Sánchez C, Eda S, Hayatsu M, Minamisawa K (2013) Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation. Nat Clim Chang 3:208–212CrossRefGoogle Scholar
  16. Klein CAM, Harvey MJ (2012) Nitrous oxide chamber methodology guidelines. Ministry of Primary Industries, WellingtonGoogle Scholar
  17. Lu RK (2000) Methods of soil agricultural chemical analysis. China Agriculture Science & Technology Press, BeijingGoogle Scholar
  18. Magalhães CM, Machado A, Matos P, Bordalo AA (2011) Impact of copper on the diversity, abundance and transcription of nitrite and nitrous oxide reductase genes in an urban European estuary. FEMS Microbiol Ecol 77:274–284CrossRefGoogle Scholar
  19. Nishimura S, Sawamoto T, Akiyama H, Sudo S, Cheng W, Yagi K (2005a) Continuous, automated nitrous oxide measurements from paddy soils converted to upland crops. Soil Sci Soc Am J 69:1977–1986CrossRefGoogle Scholar
  20. Nishimura S, Sudo S, Akiyama H, Yonemura S, Yagi K, Tsuruta H (2005b) Development of a system for simultaneous and continuous measurement of carbon dioxide, methane and nitrous oxide fluxes from croplands based on the automated closed chamber method. Soil Sci Plant Nutr 51:557–564CrossRefGoogle Scholar
  21. Nishizawa T, Quan AH, Kai A, Tago K, Ishii S, Shen WS, Isobe K, Otsuka S, Senoo K (2014) Inoculation with N2-generating denitrifier strains mitigates N2O emission from agricultural soil fertilized with poultry manure. Biol Fertil Soils 50:1001–1007CrossRefGoogle Scholar
  22. Nishizawa T, Tago K, Uei Y, Ishii S, Isobe K, Otsuka S, Senoo K (2012) Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil. AMB Express 2:50CrossRefGoogle Scholar
  23. Nishizawa T, Uei Y, Tago K, Isobe K, Otsuka S, Senoo K (2013) Taxonomic composition of denitrifying bacterial isolates is different among three rice paddy field soils in Japan. Soil Sci Plant Nutr 59:305–310CrossRefGoogle Scholar
  24. Pauleta SR, Dell’Acqua S, Moura I (2013) Nitrous oxide reductase. Coord Chem Rev 257:332–349CrossRefGoogle Scholar
  25. Qin HL, Tang YF, Shen JL, Wang C, Chen CL, Yang J, Liu Y, Chen XB, Li Y, Hou HJ (2018) Abundance of transcripts of functional gene reflects the inverse relationship between CH4 and N2O emissions during mid-season drainage in acidic paddy soil. Biol Fertil Soils 54:885–895CrossRefGoogle Scholar
  26. Qin SP, Ding KR, Clough TJ, Hu CS, Luo JF (2017) Temporal in situ dynamics of N2O reductase activity as affected by nitrogen fertilization and implications for the N2O/(N2O + N2) product ratio and N2O mitigation. Biol Fertil Soils 53:723–727CrossRefGoogle Scholar
  27. Qu Z, Wang JG, Almøy T, Bakken LR (2014) Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification, primarily due to acidification of the soils. Glob Chang Biol 20:1685–1698CrossRefGoogle Scholar
  28. Reay DS, Davidson EA, Smith KA, Smith P, Melillo JM, Dentener F, Crutzen PJ (2012) Global agriculture and nitrous oxide emissions. Nat Clim Chang 2:410–416CrossRefGoogle Scholar
  29. Saito T, Ishii S, Otsuka S, Nishiyama M, Senoo K (2008) Identification of novel betaproteobacteria in a succinate-assimilating population in denitrifying rice paddy soil by using stable isotope probing. Microbes Environ 23:192–200CrossRefGoogle Scholar
  30. Sullivan MJ, Gates AJ, Appia-Ayme C, Rowley G, Richardson DJ (2013) Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism. Proc Natl Acad Sci U S A 110:19926–19931CrossRefGoogle Scholar
  31. Tago K, Ishii S, Nishizawa T, Otsuka S, Senoo K (2011) Phylogenetic and functional diversity of denitrifying bacteria isolated from various rice paddy and rice-soybean rotation fields. Microbes Environ 26:30–35CrossRefGoogle Scholar
  32. Thomson AJ, Giannopoulos G, Pretty J, Baggs EM, Richardson DJ (2012) Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Philos Trans R Soc Lond Ser B Biol Sci 367:1157–1168CrossRefGoogle Scholar
  33. Uchida Y, Wang Y, Akiyama H, Nakajima Y, Hayatsu M (2014) Expression of denitrification genes in response to a waterlogging event in a Fluvisol and its relationship with large nitrous oxide pulses. FEMS Microbiol Ecol 88:407–423CrossRefGoogle Scholar
  34. Wei W, Isobe K, Shiratori Y, Nishizawa T, Ohte N, Otsuka S, Senoo K (2014) N2O emission from cropland field soil through fungal denitrification after surface applications of organic fertilizer. Soil Biol Biochem 69:157–167CrossRefGoogle Scholar
  35. Zhou BB, Wang YM, Feng YZ, Lin XG (2016) The application of rapidly composted manure decreases paddy CH4 emission by adversely influencing methanogenic archaeal community: a greenhouse study. J Soils Sediments 16:1889–1900CrossRefGoogle Scholar
  36. Zhu XY, Chen YG, Chen H, Li X, Peng YZ, Wang SY (2013) Minimizing nitrous oxide in biological nutrient removal from municipal wastewater by controlling copper ion concentrations. Appl Microbiol Biotechnol 97:1325–1334CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, and School of Environmental Science and EngineeringNanjing University of Information Science and TechnologyNanjingChina
  2. 2.National Engineering Research Center for Biotechnology and School of Biological and Pharmaceutical EngineeringNanjing Tech UniversityNanjingChina
  3. 3.Department of Applied Biological Chemistry, Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan
  4. 4.Niigata Agricultural Research InstituteNiigataJapan
  5. 5.Collaborative Research Institute for Innovative MicrobiologyThe University of TokyoTokyoJapan

Personalised recommendations