Community structures and comparison of nosZ and 16S rRNA genes from culturable denitrifying bacteria

  • Cumhur AvşarEmail author
  • E. Sümer Aras
Original Article


The objectives of this study were (i) to isolate and characterize of cultivable denitrifying bacteria using classic microbiological and molecular methods, (ii) to compare of 16S rRNA and nosZ genes as molecular markers, (iii) to determine bacterial community structure and diversity in soil samples using single-strand conformation polymorphism (SSCP) analysis. In this study, 49 bacterial isolates were cultivated and phylogenetic analyses grouped them into two phyla: Proteobacteria (37 species) and Firmicutes (12 species). Our study showed that the nosZ functional gen could be used to identify denitrifying bacteria abundance in environment but could not be used to identify pure bacterial cultures. In addition, the bacterial community structure showed significant differences among the various soil types. Phylogenetic analysis of community structure indicated that 51 clones could be divided into 2 phylotypes. Uncultured bacteria (80.4%) and Gammaproteobacteria (19.6%) were the dominant components of the soil bacterial community. For 16S rRNA, PCR products of 49 bacteria were obtained with 27F-1492R primer pairs. For nosZ, PCR products were obtained with primers 1F-1R (259 bp), 2F-2R (267 bp), and F-1622R (453 bp) of 39 bacteria that the single nosZ band provided on the agarose gel. The bacterial 16S rRNA gene clone library was dominated by Gammaproteobacteria and Bacilli. The nosZ clone sequences did not represent the bacteria from which they were obtained but were found to be closer to the environmental clones. Our study showed that the nosZ functional gene could be used to identify denitrification abundance in environment but could not be used to identify pure bacterial cultures. It was also found that the nosZ sequences showed uncultured denitrifier species.


Authors’ contributions

CA and ESA made a contribution to designing the study. CA was responsible for completing the experiments and data analysis. CA and ESA made a contribution to writing the manuscript. All authors read and approved the final manuscript.

Funding information

This research has been supported by Ankara University Scientific Research Project Coordination Unit. Project Number: 17L0430004, 2017-2018.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12223_2019_754_MOESM1_ESM.docx (55 kb)
ESM 1 (DOCX 55 kb)


  1. Avşar C (2018) Quantification of denitrifier population size in soil, bacteria community structures and comparison of nosZ and 16S rRNA genes from culturable denitrifying. PhD Thesis, Ankara University, Ankara, Turkey, 188p.Google Scholar
  2. Awong-Taylor J, Craven KS, Griffiths L, Bass C, Muscarella M (2008) Comparison of biochemical and molecular methods for the identification of bacterial isolates associated with failed loggerhead sea turtle eggs. J Appl Microbiol 104(5):1244–1251. CrossRefPubMedGoogle Scholar
  3. Backman JS, Hermansson A, Tebbe CC, Lindgren PE (2003) Liming induces growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest soil as determined by SSCP and DGGE. Soil Bio Biochem 35(10):1337–1347. CrossRefGoogle Scholar
  4. Belak Á, Kovács M, Hermann Z, Holczman Á, Márta D, Stojakovič S, Maraz A (2011) Molecular analysis of poultry meat spoiling microbiota and heterogeneity of their proteolytic and lipolytic enzyme activities. Acta Aliment 40(1):3–22. CrossRefGoogle Scholar
  5. Benga L, Benten WPM, Engelhardt E, Köhrer K, Gougoula C, Sager M (2014) 16S ribosomal DNA sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics. Lab Anim 48(4):305–312. CrossRefPubMedGoogle Scholar
  6. Bosshard PP, Abels S, Altwegg M, Böttger EC, Zbinden R (2004) Comparison of conventional and molecular methods for identification of aerobic catalase-negative Gram-positive cocci in the clinical laboratory. J Clin Microbiol 42(5):2065–2073. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bowles MW, Nigro LM, Teske AP, Joye SB (2012) Denitrification and environmental factors influencing nitrate removal in Guaymas Basin hydrothermally altered sediments. Front Microbiol 3:1–11. CrossRefGoogle Scholar
  8. Bunge M, Lechner U (2004) Studying the microbial dynamics of a trichlorobenzene-dechlorinating community by single-strand conformation polymorphism. Kapitel 6:127–140. CrossRefGoogle Scholar
  9. Byun SO, Fang Q, Zhou H, Hickford JGH (2009) An effective method for silver-staining DNA in large numbers of polyacrylamide gels. Analytic Biochem 385(1):174–175. CrossRefGoogle Scholar
  10. Carlson JM, Leonard AB, Hyde ER, Petrosino JF, Primm TP (2017) Microbiome disruption and recovery in the fish Gambusia affinis following exposure to broad-spectrum antibiotic. Infect Drug Res 10:143. CrossRefGoogle Scholar
  11. Čuhel J, Šimek M, Laughlin RJ, Bru D, Chèneby D, Watson CJ, Philippot L (2010) Insights into the effect of soil pH on N2O and N2 emissions and denitrifier community size and activity. Appl Environ Microbiol 76(6):1870–1878. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cui P, Fan F, Yin C, Song A, Huang P, Tang Y, Liang Y (2016) Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes. Soil Bio Biochem 93:131–141. CrossRefGoogle Scholar
  13. Dandie CE, Burton DL, Zebarth BJ, Trevors JT, Goyer C (2007) Analysis of denitrification genes and comparison of nosZ, cnorB, and 16S rDNA from culturable denitrifying bacteria in potato cropping systems. Syst Appl Microbiol 30:128–138. CrossRefPubMedGoogle Scholar
  14. Delorme S, Philippot L, Edel-Hermann V, Deulvot C, Mougel C, Lemanceau P (2003) Comparative genetic diversity of the narG, nosZ, and 16S rRNA genes in fluorescent pseudomonads. Appl Environ Microbiol 69(2):1004–1012. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gaimster H, Alston M, Richardson D, Gates A, Rowley G (2017) Transcriptional and environmental control of bacterial denitrification and N2O emissions. FEMS Microbiol Let fnx277.
  16. Graf DRH, Jones CM, Hallin S (2014) Intergenomic comparisons highlight modularity of the denitrification pathway and underpin the importance of community structure for N2O emissions. PLoS ONE 9:e114118. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hayatsu M, Tago K, Saito M (2008) Various players in the nitrogen cycle: diversity and functions of the microorganisms involved in nitrification and denitrification. Soil Sci Plant Nut 54:33–45. CrossRefGoogle Scholar
  18. Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soil. Appl Environ Microbiol 72:5181–5189. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N (2006) The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012–2021. CrossRefPubMedGoogle Scholar
  20. Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y (2006) Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments. J Microbiol Meth 66:165–169. CrossRefGoogle Scholar
  21. Horn MA, Drake HL, Schramm A (2006) Nitrous oxide reductase genes (nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar. Appl Environ Microbiol 72(2):1019–1026. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Ishii S, Ashida N, Otsuka S, Senoo K (2011) Isolation of oligotrophic denitrifiers carrying previously uncharacterized functional gene sequences. Appl Environ Microbiol 77(1):338–342. CrossRefPubMedGoogle Scholar
  23. Jones CM, Graf DRH, Bru D, Philippot L, Hallin S (2013) The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. The ISME J 7:417–426. CrossRefPubMedGoogle Scholar
  24. Mills HJ, Hunter E, Humphrys M, Kerkhof L, McGuinness L, Huettel M, Kostka JE (2008) Characterization of nitrifying, denitrifying, and overall bacterial communities in permeable marine sediments of the northeastern Gulf of Mexico. Appl Environ Microbiol 74(14):4440–4453. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Morkved PT, Dörsch P, Bakken LR (2007) The N2O product ratio of nitrification and its dependence on long-term changes in soil pH. Soil Bio Biochemist 39(8):2048–2057. CrossRefGoogle Scholar
  26. Morley N, Baggs EM, Dörsch P, Bakken L (2008) Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2 and O2 concentrations. FEMS Microbe Eco 65(1):102–112. CrossRefGoogle Scholar
  27. Philippot L, Hallin S, Schloter M (2007) Ecology of denitrifying prokaryotes in agricultural soil. Adv Agro 96:249–305. CrossRefGoogle Scholar
  28. Philippot L, Spor A, Henault C, Bru D, Bizouard F, Jones CM, Sarr A, Maron P (2013) Loss in microbial diversity affects nitrogen cycling in soil. The ISME J 7:1609–1619. CrossRefPubMedGoogle Scholar
  29. Pihlatie M, Syväsalo E, Simojoki A, Esala M, Regina K (2004) Contribution of nitrification and denitrification to N2O production in peat, clay and loamy sand soils under different soil moisture conditions. Nut Cycle Agro 70(2):135–141. CrossRefGoogle Scholar
  30. Richardson D, Felgate H, Watmough N, Thomson A, Baggs E (2009) Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle could enzymic regulation hold the key. Trends Biotech 27:388–397. CrossRefGoogle Scholar
  31. Rossmann B, Müller H, Smalla K, Mpiira S, Tumuhairwe JB, Staver C, Berg G (2012) Banana-associated microbial communities in Uganda are highly diverse but dominated by Enterobacteriaceae. Appl Environ Microbiol 78(14):4933–4941. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Saggar S, Jha N, Deslippe J, Bolan NS, Luo J, Giltrap DL, Tillman RW (2013) Denitrification and N2O:N2 production in temperate grasslands: processes, measurements, modelling and mitigating negative impacts. Sci Total Environ 465:173–195. CrossRefPubMedGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Clonning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York, U.S.A., p 1102.Google Scholar
  34. Sanford RA, Wagner DD, Wu Q, Chee-Sanford JC, Thomas SH, Cruz-García C, Nissen S (2012) Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils. PNAS 109(48):19709–19714. CrossRefPubMedGoogle Scholar
  35. Scala DJ, Kerkhof LJ (1999) Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments. Appl Environ Microbiol 65(4):1681–1687. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Schmalenberger A, Schwieger F, Tebbe CC (2001) Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling. Appl Environ Microbiol 67(8):3557–3563. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Schwieger F, Tebbe CC (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64:4870–4876. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Schwieger F, Tebbe CC (2000) Effect of Field Inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria. Appl Environ Microbiol 66:3556–3565. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Shcherbak I, Millar N, Robertson GP (2014) Global metaanalysis of the nonlinear response of soil nitrous oxide (N2O) emissions to fertilizer nitrogen. PNAS 111(25):9199–9204. CrossRefPubMedGoogle Scholar
  40. Skiba UM, Sheppard LJ, MacDonald J, Fowler D (1998) Some key environmental variables controlling nitrous oxide emissions from agricultural and seminatural soils in Scotland. Atmospheric Environ 32(19):3311-3320.
  41. Smalla K, Oros-Sichler M, Milling A, Heuer H, Baumgarte S, Becker R, Neuber G, Kropf S, Ulrich A, Tebbe CC (2007) Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: do the different methods provide similar results. J Microbiol Met 69:470–479. CrossRefGoogle Scholar
  42. Song B, Leff LG (2005) Identification and characterization of bacterial isolates from the Mir space station. Microbiol Res 160(2):111–117. CrossRefPubMedGoogle Scholar
  43. Stach JE, Bathe S, Clapp JP, Burns RG (2001) PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. FEMS Microbiol Eco 36(2-3):139–151. CrossRefGoogle Scholar
  44. 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 Eco 49(3):401–417. CrossRefGoogle Scholar
  45. Tsai S, Selvam A, Chang Y, Yang S (2009) Soil bacterial community composition across different topographic sites characterized by 16S rRNA gene clones in the Fushan Forest of Taiwan. Bot Stud 50(1):57–68Google Scholar
  46. Wallenstein MD, Myrold DD, Firestone M, Voytek M (2006) Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods. Ecological Appl 16(6):2143-2152.
  47. White JD, Scott NA (2006) Specific leaf area and nitrogen distribution in New Zealand forests: species independently respond to intercepted light. Forest Ecol Manage 226:319–329. CrossRefGoogle Scholar
  48. Wittorf L, Bonilla-Rosso G, Jones CM, Bäckman O, Hulth S, Hallin S (2016) Habitat partitioning of marine benthic denitrifier communities in response to oxygen availability. Environ Microbiol Rep 8(4):486–492. CrossRefPubMedGoogle Scholar
  49. Wolińska A, Górniak D, Zielenkiewicz U, Goryluk-Salmonowicz A, Kuźniar A, Stępniewska Z, Błaszczyk M (2017) Microbial biodiversity in arable soils is affected by agricultural practices. Int Agrophys 31(2):259–271. CrossRefGoogle Scholar
  50. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Molec Bio Rev 61(4):533–536Google Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  1. 1.Department of Biology, Faculty of Arts and SciencesSinop UniversitySinopTurkey
  2. 2.Department of Biology, Faculty of ScienceAnkara UniversityAnkaraTurkey

Personalised recommendations