Molecular Characterization of the Bacterial Community in Activated Sludges by PCR­RFLP

PCR-RFLP 방법을 이용한 활성 슬러지의 세균군집 분석

  • Lee Hyun-Kyung (Department of Microbiology and Basic Science Research Institute, Chungbuk National University) ;
  • Kim Jun-Ho (Department of Microbiology and Basic Science Research Institute, Chungbuk National University) ;
  • Kim Chi-Kyung (Department of Microbiology and Basic Science Research Institute, Chungbuk National University) ;
  • Lee Dong-Hun (Department of Microbiology and Basic Science Research Institute, Chungbuk National University)
  • 이현경 (충북대학교 미생물학과 및 기초과학연구소) ;
  • 김준호 (충북대학교 미생물학과 및 기초과학연구소) ;
  • 김치경 (충북대학교 미생물학과 및 기초과학연구소) ;
  • 이동훈 (충북대학교 미생물학과 및 기초과학연구소)
  • Published : 2004.12.01

Abstract

Diversity of the bacterial communities and the relation between community structure and components of waste­water were analyzed by 16S rRNA-based molecular techniques. Clone libraries of the 16S rDNAs from the sludges were constructed by PCR and cloning. The 1,151 clones from a sludge sample of sewage treatment plant were clustered into 699 RFLP phylotypes and the 1,228 clones from the wastewater disposal plant of chemical industry were clustered into 300 RFLP phylotypes. Shannon-Weiner diversity indices of two sampling sites were 8.7 and 6.1, indicating that the bacterial community structure of sewage treatment plant was more diverse than that of wastewater disposal plant of chemical industry. Forty clones belonging to predominant RFLP types were selected and sequenced. Seventy percent (28 clones) of the sequenced clones were related to the uncultured bacteria in public databases. The ${\beta}-Proteobacteria$ dominated in the bacterial communities of investigated two sludge samples. 16S rDNA sequences of the sewage treatment plant were similar to those of other activated sludges, while the bacterial community in wastewater disposal plant of chemical industry rep­resented the strains identified from high-temperature, anaerobic, hydrocarbon-rich, and sulfur-rich environ­ments. This result suggested that bacterial communities depended upon the components of wastewater.

폐수처리에 중요한 역할을 담당하고 있는 세균 군집의 다양성과 폐수종류에 따른 군집차이를 알아보기 위해 분자생물학적 분석방법을 사용하였다. 국내 폐수처리장 슬러지 시료로부터 16S rDNA 클론 라이브러리를 구축하였고, HaeIII RFLP pattern과 염기서열을 분석하였다. 하수처리장 시료에서는 총 1,151개의 클론에서 699개의 서로 다른 RFLP pattern이, 화학산업 폐수처리장 시료에서는 총 1,228개의 클론에서 300개의 서로 다른 RFLP pattern이 관찰되었다. Shannon-Weiner diversity index의 계산결과 하수처리장 슬러지 시료는 8.7,화학산업 폐수처리장 슬러지 시료는 6.1로 하수처리장시료가 더 다양한 군집을 구성하고 있었다. 두 시료에서 우점하는 RFLP pattern에 해당되는 40개의 클론을 선정하여 염기서열 분석과 상동성 검색을 수행하였다. 분석된 서열의 $70\%$인 28개의 클론은 배양이 보고되지 않은 균주의 16S rRNA와 유사도가 높았고, 두 시료 모두 ${\beta}-Proteobacteria$가 우점하였다. 그러나, 하수처리장의 경우 활성슬러지에서 분리된 균주들과 유사한 군집이 많았던 반면, 화학산업 페수처리장의 경우 고온이며, 혐기성이고,탄화수소나 황이 많이 존재하는 환경에서 분리된 균주들과 유사한 군집이 많았다. 이러한 결과는 유입수의 조성에 따른 차이로 생각된다.

Keywords

References

  1. Amann, R., H. Lemmer, and M. Wagner. 1998. Monitoring the community structure of wastewater treatment plants: a comparison of old and new techniques. FEMS Microbiol. Ecol. 25, 205- 215
  2. Coates, J.D., U. Michaelidou, R.A. Bruce, S. M. O’connor, J. N. Crespi, and L. A. Achenbach. 1999. Ubiquity and diversity of dissimilatory (per) chlorate- reducing bacteria. Appl. Environ. Microbiol. 65, 5234-5241
  3. Curds, C.R. and H. A. Hawkes. 1975. Ecological aspects of usedwater treatment. Vol. 1. The organisms and their ecology. Academic Press, London
  4. Dabert, P., B. Sialve, J.P. Delgenès, R. Moletta, and J.J. Godon. 2001. Characterization of the microbial 16S rDNA diversity of an aerobic phosphorous-removal ecosystem and monitoring of its transition to nitrate respiration. Appl. Microbiol. Biotechnol. 55, 500-509
  5. Dojka, M.A., P. Hugenholtz, S.K. Hack, and N.R. Pace. 1998. Microbial diversity in a hydrocarbon- and chlorinated-solventcontaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64, 3869-3877
  6. Dunbar, J., L.O. Ticknor, and C.R. Kuske. 2000. Assement of microbial diversity in four southwestern united states soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66, 2943-2950
  7. Elshahed, M., J.M. Senko, F.Z. Najar, S.M. Kenton, B.A. Roe, T. A. Dewers, J.R. Spear, and L.R. Krumholz. 2003. Bacterial diversity and sulfur cycling in mesophilic sulfide-rich spring. Appl. Environ. Microbiol. 69, 5609-5621
  8. Etchebehere, C., M.I. Errazquin, P. Dabert, and L. Muxi. 2002. Community analysis of a denitrifying reactor treating landfill leachate. FEMS Microbiol. Ecol. 40, 97-106
  9. Felsenstein, J. 1985. Confidence limits on phylogenics, an approach using the bootstrap. Evolution 39, 783-791
  10. Felsenstein, J. 2004. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle
  11. Hugenholtz, P., C. Pitulle, K.L. Hershberger, and N. R. Pace. 1998. Novel division level bacteria diversity in a Yellowstone hot spring. J. Bacteriol. 180, 366-376
  12. Jeon, C.O., D.S. Lee, and J.M. Park. 2003. Microbial communities in activated sludge performing enhanced biological phosphorous removal in a sequencing batch reactor. Water Res. 37, 2195-2205
  13. Jukes, T.H. and C.R. Cantor. 1969. Evolution of protein molecules, p. 21-132. In: H. N. Munro (ed.), Mammalian Protein Metabolism. Academic Press, New York
  14. Kanagawa, T., Y. Kamagata, S. Aruga, T. Kohno, M. Horn, and M. Wagner. 2000. Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge. Appl. Environ. Microbiol. 66, 5043-5052
  15. Krebs, C.J. 1989. Ecological Methodology. Harper & Row, New York
  16. LaPara, T.M., C.H. Nakatsu, L. Pantea, and J.E. Alleman. 2000. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreators treating pharmaceutical wastewater. Appl Environ. Microbiol. 66, 3951-3959
  17. Lee, H.W., S.Y. Lee, J.W. Lee, J.B. Park, E.S. Choi, and Y.K. Park. 2002. Molecular characterization of microbial community in nitrate-removing activated sludge. FEMS Microbiol. Ecol. 41, 85- 94 https://doi.org/10.1111/j.1574-6941.2002.tb00969.x
  18. Manz, W., M. Wagner, R. Amann, and K.H. Schleifer. 1994. In situ characterization of the microbial consortia active in two wastewater treatment plants. Water Res. 28, 1715-1723
  19. Miller, D.N., J.E. Bryant, E.L. Madsen, and W.C. Ghiorse. 1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 65, 4715-4724
  20. Orphan, V.J., L.T. Taylor, D. Hafenbradl, and E.F. Delong. 2000. Culture-dependent and culture-independent characterization of microbial assemblage associated with high-temperature petroleum reservoirs. Appl. Environ. Microbiol. 66, 700-711
  21. Pillips, C.A. 2001. Arcobacter spp in food: isolation, identification and control. Trens in Food Sci & Technol. 12, 263-275
  22. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstruction phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  23. Snaidr, J., R. Amann, I. Huber, W. Ludwig, and K.H. Schleifer. 1997. Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Environ. Microbiol. 63, 2884-2896
  24. Soddell, J.A. and R.J. Seviour. 1990. Microbiology of foaming in activated sludge plants. J. Appl. Bacteriol. 69, 145-176
  25. Teske, A., K.U. Hinrichs, V. Edgcomb, A. de V. Gomez, D. Kysela, S. P. Sylva, M. L. Sogin, and H. W. Jannasch. 2002. Microbial diversity of hydrothermal sediments in the Guaymas Basin: Evidence for anaerobic methanotrophic communities. Appl. Environ. Microbiol. 68, 1994-2007
  26. Urakawa, H., K. Kita-Tsukamoto, and K. Ohwada. 1999. Microbial diversity in marine sediments from Sagami Bay and Tokyo Bay, Japan, as determined by 16S rRNA gene analysis. Microbiology 145, 3305-3315
  27. Ventura, M., M. Elli, R. Reniero, and R. Zink. 2001. Molecular microbial analysis of Bifidobacterium isolate from different environments by the species-specific amplified ribosomal DNA restriction analysis. FEMS Microbiol. Ecol. 36, 113-121
  28. Wagner, M. and A. Loy. 2002. Bacterial community composition and function in sewage treatment systems. Curr. Opin. Biotechnol. 13, 218-227
  29. Wintzingerode, F., B. Selent, W. Hegemann, and U.B. Göbel. 1999. Phylogenetic analysis of an anaerobic, trichlorobenzenetransformation microbial consortium. Appl. Environ. Microbiol. 65, 283-286
  30. Zilles, J.L., J. Peccia, M.W. Kim, C.H. Hung, and D.R. Noguera. 2002. Involvement of Rhodocyclus-related organisms in full-scale wastewater treatment plants. Appl. Environ. Microbiol. 68, 2763- 2769