Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Changes of Nitrifying Bacteria Depending on the Presence and Absence of Organic Pollutant in Nak-Dong River

낙동강에서의 유기성 오염 유무에 따른 질화세균의 변화

  • 진선영 (대구대학교 자연과학대학 생명과학과) ;
  • 이영옥 (대구대학교 자연과학대학 생명과학과)
  • Received : 2013.04.19
  • Accepted : 2013.05.14
  • Published : 2013.06.30
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Abstract

This study was performed at 2 sites of Nak-Dong River to investigate the changes of nitrifiers depending on the presence and absence of organic pollutants (due to the effluents of domestic wastewater treatment plant, WWTP). Conventional chemical parameters such as T-N, $NH_4$-N, $NO_2$-N, $NO_3$-N were measured and the quantitative nitrifiers at the 2 sites were analyzed comparatively by fluorescent in situ hybridization (FISH) with NSO190 and NIT3, after checking the presence of gene amoA of ammonia oxidizing bacteria (AOB) and 16S rDNA signature sequence for Nitrobacter sp. that belongs to nitrite oxidizing bacteria (NOB). Also ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria were detected using FISH to get a glimpse of the general bacterial community structure of the sites. Based on the distribution structure of the ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria and the measurement of nitrogen in different phases, it could be said that the site 2 was more polluted with organics than site 1. Corresponding to the above conclusion, the average numbers of AOB and NOB detected by NSO160 and NIT3, respectively, at site 2 [AOB, $9.3{\times}10^5$; NOB, $1.6{\times}10^6$ (cells/ml)] was more than those at site 1 [AOB, $7.8{\times}10^5$; NOB, $0.8{\times}10^6$ (cells/ml)] and also their ratios to total counts were higher at site 2 (AOB, 27%; NOB, 34%) than those at site 1 (AOB, 18%; NOB, 23%). Thus, it could be concluded that the nitrification at site 2 was more active due to continuous loading of organics from the effluents of domestic WWTP, compared to site 1 located closed to raw drinking water supply and subsequently less polluted with organics.

유기성 오염원(생활하수 처리시설 방류수) 유무에 따른 질화 세균의 변화를 알아보기 위해 낙동강의 2 조사수역에서 다양한 형태의 질소(T-N, $NH_4$-N, $NO_2$-N, $NO_3$-N) 농도를 측정하였고 조사 수역에 있는 질화세균 종류를 확인한 후, fluorescent in situ hybridization (FISH)법으로 질화세균 수를 정량 평가하였다. 즉 암모니아 산화세균의 종류는 amoA 유전자, 그리고 아질산 산화 세균인 Nitrobacter sp.는 SSU 16S rDNA 특정 기호서열을 목표로 한 PCR-DGGE를 수행한 후 염기서열 분석으로 그들이 각각 Nitrosomonas sp., Nitrobacter sp.임이 확인됨에 따라 그에 상응하는 gene probe, NSO190와 NIT3을 사용해 FISH법을 수행하여 각 수역의 질화세균수를 비교하였다. 아울러 전반적인 세균학적 수질을 모니터링하기 위해 수계에 많은 ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria도 FISH법으로 검출하였다. ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria 분포도와 모든 유형의 질소 측정 결과에 따르면 정점 1 보다 정점 2의 유기물 오염도가 높다고 할 수 있었다. 이에 상응해 NSO160과 NIT3로 검출한 평균 질화세균수도 정점 1 (암모니아산화세균, $7.8{\times}10^5$; 아질산산화세균, $0.8{\times}10^6$ cells/ml)보다 정점 2 (암모니아산화세균, $9.3{\times}10^5$; 아질산산화세균, $1.6{\times}10^6$ cells/ml)에 더 많았고 그들이 총세균수에서 차지하는 평균 비율(%) 역시 정점 1 (NSO190, 18%; NIT3, 23%)에 비해 정점 2 (NSO190, 27%; NIT3, 34%)에서 높았다. 따라서 유기오염도가 낮은 상수원수 취수장 인근 수역인 정점 1 보다 생활하수처리시설 방류수로 인해 유기성 오염원이 상존하는 정점 2에서의 질화작용이 더 활발하다고 결론지을 수 있었다.

Keywords

References

  1. Alfreider, A., Pernthaler, J., Amann, R., Sattler, B., Glöckner, F.O., Wille, A., and Psenner, R. 1996. Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization. Appl. Environ. Microbiol. 62, 2138-2144.
  2. Amann, R., Ludwig, W., and Schleifer, K.H. 1995. Phylogenetic and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143-169.
  3. Anderson, I.C., Poth, M., Homstead, J., and Burdige, D. 1993. A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis. Appl. Environ. Microbiol. 59, 3525-3533.
  4. Belser, I.W. 1979. Population ecology of nitrifying bacteria. Ann. Rev. Microbiol. 33, 309-333. https://doi.org/10.1146/annurev.mi.33.100179.001521
  5. Cebron, A. and Garnier, J. 2005. Nitrobacter and Nitrospira genera as representatives if nitrite-oxidizing bactria: Detection, quantification and growth along the lower Seine River (France). Wat. Res. 39, 4979-4992. https://doi.org/10.1016/j.watres.2005.10.006
  6. Degrange, V. and Bardin, R. 1995. Detection and counting of nitrobacter populations in soil by PCR. Appl. Environ. Microbiol. 61, 2093-2098.
  7. Glöckner, F.O., Fuchs, B.M., and Amann, R. 1999. Bacterioplankton composition of lake and oceans: A first comparison based on fluorescence in situ hybridization. Appl. Environ. Microbiol. 65, 3721-3726.
  8. Herrmann, M., Saunders, A.M., and Schramm, A. 2008. Archaea dominate the ammonia-oxidizing community in the rhizosphere of the freshwater macrophyte Littorella uniflora. Appl. Environ. Microbiol. 74, 3279-3283. https://doi.org/10.1128/AEM.02802-07
  9. Hicks, R., Amann, R., and Stahl, D.A. 1992. Dual staining of natural bacterioplankton with 4, 6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom level 16S rRNA sequences. Appl. Environ. Microbiol. 58, 2158-2163.
  10. Hornek, R., Pommerenign-Röser, A., Koops, H.P., Farnleitner, A., Kreuzinger, H., Kirschner, N., Mach, A., and Robert, L. 2006. Primers containing universal bases reduce multiple amoA gene specific DGGE base patterns when analyzing the diversity of beta-ammonia oxidizers in the environment. Microbiol. Methods 66, 147-155. https://doi.org/10.1016/j.mimet.2005.11.001
  11. Kim, D.J., Hong, S.H., and Ahn, T.S. 1999. Seasonal and vertical change of bacterial communities in Lake Soyang. Kor. J. Microbiol. 35, 242-247.
  12. Kowalchuk, G.A., Naoumenko, Z.S., Derikx, P.J.L., Felske, A., Stephen, J.R., and Arkhipchenko, I.A. 1999. Molecular analysis of ammonia-oxidizing bacteria of the $\beta$ subdivision of the class Proteobacteria in compost and composted materials. Appl. Microbiol. Biotechnol. 65, 396-403.
  13. Lee, Y.O. 2008. Changes of nitrifying bacteria in the different zone (upper․mid․lower part) of the Nak-Dong river. J. Kor. Society Wat. Environ. 24, 214-220.
  14. Lee, Y.O. and Lee, H.S. 2002. Seasonal variations of nitrifying bacteria in agricultural reservoir. Kor. J. Limnol. 35, 152-159.
  15. Lee, H.S., Park, C.W., Kim, M.K., and Lee, Y.O. 2002. Seasonal variation of eubacterial community structure and their structure affecting environmental parameters in reservoir. Kor. J. Microbiol. 38, 31-37.
  16. Liu, Y. and Capdeville, B. 1994. Kinetic behaviors of nitrifying biofilm growth in wastewater nitrification. Environ. Technol. 15, 1001-1013. https://doi.org/10.1080/09593339409385509
  17. Madigan, M.T., Martinko, J.M., Dunlap, P.W., and Clark, D.P. 2009. Anammox, pp. 603-604. In Biology of microorganisms. 12 ed. Pearson education.
  18. Manz, W., Amann, R., Ludwig, W., Wagner, M., and Schleifer, K.H. 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: Problems and solutions. Syst. Appl. Microbiol. 15, 593-600. https://doi.org/10.1016/S0723-2020(11)80121-9
  19. Manz, W., Szewzyk, U., Ericsson, P., Amann, R., Schleifer, K.H., and Stenström, T. 1993. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes. Appl. Environ. Microbiol. 59, 2293-2298.
  20. McCaig, A.E., Phillips, C.J., Stephen, J.R., Kowalchuk, G.A., Harvey, S.M., Herbert, R.A., Embley, T.M., and Prosser, J.I. 1999. Nitrogen cycling and community structure of proteobacterial beta-subgroup ammonia-oxidizing bacteria within polluted marine fish farm sediments. Appl. Environ. Microbiol. 65, 213-220.
  21. Minister of Environment. 2009. Manual for the Examination of Water quality.
  22. Park, H.D., Wells, G.F., Bae, H., Criddle, C.S., and Francis, C.A. 2006. Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors. Appl. Environ. Microbiol. 72, 5643-5647. https://doi.org/10.1128/AEM.00402-06
  23. Pernthaler, J., Glöckner, F.O., Unterholzner, S., Alfreider, A., Psenner, R., and Amann, R. 1998. Seasonal community and population dynamics of pelagic bacteria and archaea in a high mountain lake. Appl. Environ. Microbiol. 64, 4299-4306.
  24. Purkhold, U., Pommerening-Roser, A., Juretschko, S., Schmid, M.C., Koops, H.P., and Wagner, M. 2000. Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: Implications for molecular diversity surveys. Appl. Environ. Microbiol. 66, 5368-5382. https://doi.org/10.1128/AEM.66.12.5368-5382.2000
  25. Regan, J.M., Harrington, G.W., Baribeau, H., DeLeon, R., and Noguera, D.R. 2003. Diversity of nitrifying bacteria in full-scale chloraminated distribution systems. Wat. Res. 37, 197-205. https://doi.org/10.1016/S0043-1354(02)00237-3
  26. Rossello-Mora, R. and Amann, R. 2001. The species concept for prokaryotes. FEMS Microbiol. Rev. 25, 39-67. https://doi.org/10.1111/j.1574-6976.2001.tb00571.x
  27. Rotthauwe, J.H., Witzel, K.P., and Liesack, W. 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63, 4704-4712.
  28. Schmidt, I., Hermelink, C., van de Pas-Schoonen, K., Strous, M., den Camp, H.J., Kuenen, J.G., and Jetten, M. 2002. Anaerobic ammonia oxidation in the presence of nitrogen oxides (NOx) by two different lithotrophs. Appl. Environ. Microbiol. 68, 5351-5357. https://doi.org/10.1128/AEM.68.11.5351-5357.2002
  29. Schramm, A., de Beer, D., Wagner, M., and Amann, R. 1998. Identification and activities in situ of Nitrosospira and Nitirospira spp. as dominant populations in a nitrifying fluidized bed reactor. Appl. Environ. Microbiol. 64, 3480-3485.
  30. Schramm, A., Larsen, L.H., Revsbech, N.P., Ramsing, N.B., Amann, R., and Schleifer, K.H. 1996. Structure and function of a nitrifying biofilm as determined by in situ hybridi-zation and the use of microelectrodes. Appl. Environ. Microbiol. 62, 4641-4647.
  31. Stephen, J.R., Chang, Y.J., Macnaughton, S.J., Kowalchuk, G.A., Leung, K.T., Flemming, C.A., and White, D.C. 1999. Effect of toxic metals on indigenous soil beta-subgroup proteobacterium ammonia oxidizer community structure and protection against toxicity by inoculated metal-resistant bacteria. Appl. Environ. Microbiol. 65, 95 -101.
  32. Stephen, J.R., McCaig, A.E., Smith, Z., Prosser, J.I., and Embley, T.M. 1996. Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria. Appl. Environ. Microbiol. 62, 4147-4154.
  33. Su, J.J., Yeh, K.S., and Tseng, P.W. 2006. Piggery wastewater treatment systems with heterotrophic nitrification capability in Taiwan. Curr. Microbiol. 53, 77-81. https://doi.org/10.1007/s00284-006-0021-x
  34. Wagner, M., Rath, G., Koops, H.P., Floos, J., and Amann, R. 1996. In situ analysis of nitrifying bacteria in sewage treatment plants. Wat. Sci. Technol. 34, 237-244.
  35. Winkler, M.K., Bassin, J.P., Kleerebezem, R., Sorokin, D.Y., and van Loosdrecht, M.C. 2012. Unravel-ling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge. Appl. Microbiol. Biotechnol. 94, 1657-1666. https://doi.org/10.1007/s00253-012-4126-9
  36. You, J., Das, A., Dolan, E.M., and Hu, Z. 2009. Ammonia-oxidizing archaea involved in nitrogen removal. Wat. Res. 43, 1801-1809. https://doi.org/10.1016/j.watres.2009.01.016