Isolation and Characterization of Novel Denitrifying Bacterium Geobacillus sp. SG-01 Strain from Wood Chips Composted with Swine Manure

  • Yang, Seung-Hak ;
  • Cho, Jin-Kook ;
  • Lee, Soon-Youl ;
  • Abanto, Oliver D. ;
  • Kim, Soo-Ki ;
  • Ghosh, Chiranjit ;
  • Lim, Joung-Soo ;
  • Hwang, Seong-Gu
  • Received : 2013.04.03
  • Accepted : 2013.06.04
  • Published : 2013.11.01


Nitrate contamination in ground and surface water is an increasingly serious environmental problem and only a few bacterial strains have been identified that have the ability to remove nitrogen pollutants from wastewater under thermophilic conditions. We therefore isolated thermophilic facultative bacterial strains from wood chips that had been composted with swine manure under aerated high temperature conditions so as to identify strains with denitrifying ability. Nine different colonies were screened and 3 long rod-shaped bacterial strains designated as SG-01, SG-02, and SG-03 were selected. The strain SG-01 could be differentiated from SG-02 and SG-03 on the basis of the method that it used for sugar utilization. The 16S rRNA genes of this strain also had high sequence similarity with Geobacillus thermodenitrificans $465^T$ (99.6%). The optimal growth temperatures ($55^{\circ}C$), pH values (pH 7.0), and NaCl concentrations (1%) required for the growth of strain SG-01 were established. This strain reduced 1.18 mM nitrate and 1.45 mM nitrite in LB broth after 48 h of incubation. These results suggest that the G. thermodenitrificans SG-01 strain may be useful in the removal of nitrates and nitrites from wastewater generated as a result of livestock farming.


Compost;Denitrification;Geobacillus thermodenitrificans;Nitrates;Nitrites;Swine Manure;Wood Chips


  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389-3402.
  2. APHA (American Public Health Association). 1989. Standard methods for the examination of water and wastewater, 17th ed. American Public Health Association, Washington, DC, pp. 4.123, 4.129.
  3. Anthonisen, A. C., R. C. Loehr, T. B. Prakasam and E. G. Srinath. 1976. Inhibition of nitrification by ammonia and nitrous acid. J. Water Pollut. Control Fed. 48:835-852.
  4. Beffa, T., M. Blanc, P. F. Lyon, G. Vogt, M. Marchiani, J. L. Fischer, and M. Aragno. 1996. Isolation of Thermus strains from hot composts (60 to 80 degrees C). Appl. Environ. Microbiol. 62:1723-1727.
  5. Cheneby, D., S. Perez, C. Devroe, S. Hallet, Y. Couton, F. Bizouard, G. Iuretig, J. C. Germon, and L. Philippot. 2004. Denitrifying bacteria in bulk and maize-rhizospheric soil: diversity and $N_{2}0$-reducing ability. Can. J. Microbiol. 50:469-474.
  6. Choi, E., Y. Yu, M. Cui, Z. Yun, and K. Min. 2008. Effect of biologically mediated pH change on phosphorus removal in BNR system for piggery waste treatment. J. Environ. Sci. Health. Part A: Tox Hazard. Subst. Environ. Eng. 43:154-160.
  7. Clause, D. and R. C. W. Berkeley. 1986. The genus Bacillus. In: Bergey's Manual of Systematic Bacteriology (Ed. P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt). Baltimore, USA: Williams & Wilkins Co. pp. 1105-1139.
  8. Dees, P. M. and W. C. Ghiorse. 2001. Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA. FEMS Microbiol. Ecol. 35:207-216.
  9. Deflaun, M. F., J. K. Fredrickson, H. Dong, S. M. Pfiffner, T. C. Onstott, D. L. Balkwill, S. H. Streger, E. Stackebrandt, S. Knoessen, and E. Van, Heerden. 2007. Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine. Syst. Appl. Microbiol. 30:152-164.
  10. De, Guardia, A., C. Petiot, D. Rogeau, and C. Druilhe. 2008. Influence of aeration rate on nitrogen dynamics during composting. Waste Manag. 28:575-587.
  11. Ezeji, T. C., A. Wolf, and H. Bahl. 2005. Isolation, characterization, and identification of Geobacillus thermodenitrificans HRO10, an $\alpha$-amylase and $\alpha$-glucosidase producing thermophile. Can. J. Microbiol. 51:685-693.
  12. Felsenstein, J. 1993. PHYLIP (phylogeny interference package), version 3.5c. Distributed by the author. Department of Genome Science, University of Washington, Seattle, USA.
  13. Feng, L., W. Wang, J. Cheng, Y. Ren, G. Zhao, C. Gao, Y. Tang, X. Liu, W. Han, X. Peng, R. Liu, and L. Wang. 2007. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc. Natl. Acad. Sci. USA 104:5602-5607.
  14. Galkiewicz, J. P. and C. A. Kellogg. 2008. Cross-kingdom amplification using bacteria-specific primers: complications for studies of coral microbial ecology. Appl. Environ. Microbiol. 74:7828-31.
  15. Gilbert, Y., Y. Le Bihan, G. Aubry, M. Veillette, C. Duchaine, and P. Lessard. 2008. Microbiological and molecular characterization of denitrification in biofilters treating pig manure. Bioresour. Technol. 99:4495-4502.
  16. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [$^{15}N$]nitrate in biological fluids. Anal. Biochem. 126:131-138.
  17. Hatsu, M., J. Ohta, and K. Takamizawa. 2002. Monitoring of Bacillus thermodenitrificans OHT-1 in compost by whole cell hybridization. Can. J. Microbiol. 48:848-852.
  18. Henry, S., E. Baudoin, J. C. Lopez-Gutierrez, F. Martin-Laurent, A. Brauman, and L. Philippot. 2004. Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J. Microbiol. Methods 59:327-335.
  19. Horiuchi, J. I., K. Ebie, K. Tada, M. Kobayashi, and T. Kanno. 2003. Simplified method for estimation of microbial activity in compost by ATP analysis. Bioresour. Technol. 86:95-98. DOI:
  20. Kariminiaae-Hamedaani, H. R., K. Kanda, and F. Kato. 2004. Denitrification activity of the bacterium Pseudomonas sp. ASM-2-3 isolated from the Ariake Sea tideland. J. Biosci. Bioeng. 97:39-44.
  21. Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge, UK.
  22. McGowin, A. E., K. K. Adom, and A. K. Obubuafo. 2001. Screening of compost for PAHs and pesticides using static subcritical water extraction. Chemosphere 45:857-864. DOI:
  23. Manachini, P. L., D. Mora, G. Nicastro, C. Parini, E. Stackebrandt, R. Pukall, and M. G. Fortina. 2000. Bacillus thermodenitrificans sp. nov., nom. rev. Int. J. Syst. Evol. Microbiol. 50:1331-1337.
  24. Nazina, T. N., D. S. Sokolova, A. A. Grigoryan, N. M. Shestakova, E. M. Mikhailova, A. B. Poltarius, T. P. Tourova, A. M. Lysenko, G. A. Osipov, and S. S. Belyaev. 2005. Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high temperature petroleum reservoir, and the validation of the Geobacillus species. Syst. Appl. Micribiol. 28:43-53.
  25. Peters, S., S. Koschinsky, F. Schwieger, and C. C. Tebbe. 2000. Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66:930-936.
  26. Philippot, L. 2005a. Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol. 13:191-192.
  27. Philippot, L. 2005b. Tracking nitrate reducers and denitrifiers in the environment. Biochem. Soc .Trans. 33:200-204.
  28. Rinaldo, S., A. Arcovito, M. Brunori, and F. Cutruzzola. 2007. Fast dissociation of nitric oxide from ferrous Pseudomonas aeruginosa $cd_{1}$ nitrite reductase: A novel outlook on the catalytic mechanism. J. Biol. Chem. 282:14761-14767.
  29. Saitou, N. and M. Nei. 1987. The neighboring-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.
  30. Sogin, M. L. 1990. Amplification of ribosomal RNA genes for molecular evolution studies. In: PCR Protocols: A Guide to Methods and Applications (Ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White). London, United Kingdom: Academic Press, Inc. pp. 307-314.
  31. Strom, P. F. 1985. dentification of thermophilic bacteria in solid-waste composting. Appl. Environ. Microbiol. 50:906-913.
  32. Suler, D. J. and M. S. Finstein. 1977. Effect of temperature, aeration, and moisture on $CO_{2}$ formation in bench-scale, continuously thermophilic composting of solid waste. Appl. Environ. Microbiol. 33:345-350.
  33. Suthersan, S. and J. J. Ganczarczyk. 1986. Inhibition of nitrite oxidation during nitrification: Some observations. Water. Pollut. Res. J. Can. 21:257-226.
  34. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22:4673-4680.
  35. Tiquia, S. M., N. F. Y. Tam, and I. J. Hodgkiss. 1997. Composting of spent pig litter at different seasonal temperatures in subtropical climate. Environ. Pollut. 98:97-104.
  36. Tiquia, S. M. and N. F. Y. Tam. 2000. Fate of nitrogen during composting of chicken litter. Environ. Pollut. 110:535-541. DOI:
  37. Turk, O. and D. S. Mavinic. 1986. Preliminary assessment of a shortcut in nitrogen removal from wastewater. Can. J. Civ. Eng. 13:600-605.
  38. Van, De, Pas-Schoonen, K. T., S. Schalk-Otte, S. Haaijer, M. Schmid, H. Op Den, Camp, M. Strous, J. Gijs Kuenen, and M. S. M. Jetten. 2005. Complete conversion of nitrate into dinitrogen gas in co-cultures of denitrifying bacteria. Biochem. Soc. Trans. 33:205-209.
  39. Wang, C. M., C. L. Shyu, S. P. HO, and S. H. Chiou. 2007. Species diversity and substrate utilization patterns of thermophilic bacterial communities in hot aerobic poultry and cattle manure composts. Microb. Ecol. 54:1-9.
  40. Ward, B. B. 1995. Diversity of culturable denitrifying bacteria. Limits of rDNA RFLP analysis and probes for the functional gene, nitrite reductase. Arch. Microbiol. 163:167-175.
  41. Zhou, Q., S. Takenada, S. Murakami, P. Seesuriyachan, A. Kuntiya, and K. Aoki. 2007. Screening and characterization of bacteria that can utilize ammonium and nitrate ions simultaneously under controlled cultural conditions. J. Biosci. Bioeng. 103: 185-191.
  42. Zoes, V., H. Dinel, T. Pare, and A. Jaouich. 2001. Growth substrates made from duck excreta enriched wood shavings and source-separated municipal solid waste compost and separates: physical and chemical characteristics. Bioresour. Technol. 78:21-30.

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Grant : Cooperative Research Program for Agriculture Science & Technology Development

Supported by : Rural Development Administration