Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Monitoring of Microorganisms Added into Oil-Contaminated Microenvironments by Terminal-Restriction Fragment Length Polymorphism Analysis

  • JUNG SEONG-YOUNG (Microbiology Laboratory, Korea Ocean Research & Development Institute, Department of Biochemistry, Hanyang University) ;
  • LEE JUNG-HYUN (Microbiology Laboratory, Korea Ocean Research & Development Institute) ;
  • CHAI YOUNG-GYU (Department of Biochemistry, Hanyang University) ;
  • KIM SANG-JIN (Microbiology Laboratory, Korea Ocean Research & Development Institute)
  • Published : 2005.12.01
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Abstract

Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor inoculated oil-degrading microorganisms during bioremedial treatability tests. A pair of universal primers, fluorescently labeled 521F and 1392R, was employed to amplify small subunit rDNA in order to simultaneously detect two bacterial strains, Corynebacterium sp. IC10 and Sphingomonas sp. KH3-2, and a yeast strain, Yarrowia lipolytica 180. Digestion of the 5'-end fluorescence/labeled PCR products with HhaI produced specific terminal-restriction fragments (T-RFs) of 185 and 442 bases, corresponding to Corynebacterium sp. IC10 and Y. lipolytica 180, respectively. The enzyme NruI produced a specific T-RF of 338 bases for Sphingomonas sp. KH3-2. The detection limit for oildegrading microorganisms that were inoculated into natural environments was determined to be $0.01\%$ of the total microbial count, regardless of the background environment. When three oil-degrading microorganisms were released into oil-contaminated sand microenvironments, strains IC10 and 180 survived for 35 days after inoculation, whereas strain KH3-2 was detected at 8 days, but not at 35 days. This result implies that T-RFLP could be a useful tool for monitoring the survival and relative abundance of specific microbial strains inoculated into contaminated environments.

Keywords

References

  1. Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 9: 143-169
  2. Avaniss-Aghajani, E., K. Jones, D. Chapman, and C. Brunk. 1994. A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences. BioTechniques 17: 144-149
  3. Bartha, R. and D. Pramer. 1965. Features of a flask and method for measuring the persistence and biological effects of pesticides in soil. Soil Sci. 100: 68-70 https://doi.org/10.1097/00010694-196507000-00011
  4. Bertani, G. 1951. Studies of lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62: 293-300
  5. Bruce, K. D. 1997. Analysis of mer gene subclasses within bacterial communities in soils and sediments resolved by fluorescent-PCR-restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 63: 4914-4919
  6. Chin, K.-J., T. Lukow, and R. Conrad. 1999. Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil. Appl. Environ. Microbiol. 65: 2341-2349
  7. Choi, S.-C., K. K. Kwon, J. H. Sohn, and S.-J. Kim. 2002. Evaluation of fertilizer additions to stimulate oil biodegradation in sand seashore mesocosms. J. Microbiol. Biotechnol. 12: 431-436
  8. Clement, B. G., L. E. Kehl, K. L. Debord, and C. L. Kitts. 1998. Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J. Microbiol. Meth. 31: 135-142 https://doi.org/10.1016/S0167-7012(97)00105-X
  9. Dunbar, J., L. O. Ticknor, and C. R. Kuske. 2000. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66: 2943-2950 https://doi.org/10.1128/AEM.66.7.2943-2950.2000
  10. Dunbar, J., L. O. Ticknor, and C. R. Kuske. 2001. Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Appl. Environ. Microbiol. 67: 190-197 https://doi.org/10.1128/AEM.67.1.190-197.2001
  11. Farrelly, V., F. A. Rainey, and E. Stackebrandt. 1995. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl. Environ. Microbiol. 61: 2798-2801
  12. Fogel, G. B., C. R. Collins, J. Li, and C. F. Brunk. 1999. Prokaryotic genome size and SSU rDNA copy number: Estimation of microbial relative abundance from a mixed population. Microbiol. Ecol. 38: 93-113 https://doi.org/10.1007/s002489900162
  13. Jackman, S. C., H. Lee, and J. T. Trevors. 1992. Survival, detection and containment of bacteria. Microbial Releases 1: 125-154
  14. Kim, J.-S., S.-W. Kwon, F. Jordan, and J.-C. Ryu. 2003. Analysis of bacterial community structure in bulk soil, rhizosphere soil, and root samples of hot pepper plants using FAME and 16S rDNA clone libraries. J. Microbiol. Biotechnol. 13: 236-242
  15. Kim, T.-H., J.-H. Lee, Y.-S. Oh, K.-S. Bae, and S.-J. Kim. 1999. Identification and characterization of an oil-degrading yeast, Yarrowia lipolytica 180. J. Microbiol. 37: 128-135
  16. Kim, T.-H., Y.-S. Oh, and S.-J. Kim. 2000. The possible involvement of the cell surface in aliphatic hydrocarbon utilization by an oil-degrading yeast, Yarrowia lipolytica 180. J. Microbiol. Biotechnol. 10: 333-337
  17. Kuske, C. R., K. L. Banton, D. L. Adorada, P. C. Stark, K. K. Hill, and P. J. Jackson. 1998. Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl. Environ. Microbiol. 64: 2463- 2472
  18. Lee, J.-H., H.-H. Shin, H.-K. Lee, K.-K. Kwon, and S.-J. Kim. 1998. Efficient removal of humic substances in preparing DNA extract from marine sediments. Kor. J. Microbiol. 34: 132-136
  19. Lee, J.-H., S.-Y. Jung, and S.-J. Kim. 2001. Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences. Biotechnol. Lett. 23: 1741-1748 https://doi.org/10.1023/A:1012440232320
  20. Leser, T. D., M. Boye, and N. B. Hendriksen. 1995. Survival and activity of Pseudomonas sp. strain B13 (FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton. Appl. Environ. Microbiol. 61: 1201-1207
  21. Liu, W.-T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63: 4516-4522
  22. Liu, W.-T., T. L. Marsh, and L. J. Forney. 1998. Determination of the microbial diversity of anaerobic-aerobic activated sludge by a novel molecular biological technique. Wat. Sci. Tech. 37: 417-422 https://doi.org/10.1016/S0273-1223(98)00141-3
  23. Ludemann, H., I. Arth, and W. Liesack. 2000. Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Appl. Environ. Microbiol. 66: 754-762 https://doi.org/10.1128/AEM.66.2.754-762.2000
  24. Maidak, B. L., J. R. Cole, C. T. Parker, Jr. G. M. Garrity, N. Larsen, B. Le, T. G. Lilburn, M. J. McCaughey, G. J. Olsen, R. Overbeek, S. Pramanik, T. M. Schmidt, J. M. Tiedje, and C. R. Woese. 1999. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res. 27: 171-173 https://doi.org/10.1093/nar/27.1.171
  25. Marsh, T. L. 1999. Terminal restriction fragment length polymorphism (T-RFLP): An emerging method for characterizing diversity among homologous populations of amplification products. Curr. Opin. Microbiol. 2: 323-327 https://doi.org/10.1016/S1369-5274(99)80056-3
  26. Marsh, T. L., W.-T. Liu, L. J. Forney, and H. Cheng. 1998. Beginning a molecular analysis of the eukaryal community in activated sludge. Wat. Sci. Tech. 37: 455-460 https://doi.org/10.1016/S0273-1223(98)00145-0
  27. Marsh, T. L., P. Saxman, J. Cole, and J. Tiedje. 2000. Terminal restriction fragment length polymorphism analysis program, a Web-based research tool for microbial community analysis. Appl. Environ. Microbiol. 66: 3616-3620 https://doi.org/10.1128/AEM.66.8.3616-3620.2000
  28. Matsuki, T., K. Watanabe, R. Tanaka, M. Fukuda, and H. Oyaizu. 1999. Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-genetargeted species-specific primers. Appl. Environ. Microbiol. 65: 4506-4512
  29. Meckenstock, R., P. Steinle, J. R. Van de Meer, and M. Snozzi. 1998. Quantification of bacterial mRNA involved in degradation of 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 from liquid culture and from river sediment by reverse transcriptase PCR (RT/PCR). FEMS Microbiol. Lett. 167: 123-129 https://doi.org/10.1111/j.1574-6968.1998.tb13217.x
  30. Moeseneder, M. M., J. M. Arrieta, G. Muyzer, C. Winter, and G. J. Herndl. 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 65: 3518-3525
  31. Moeseneder, M. M., C. Winter, J. M. Arrieta, and G. J. Herndl. 2001. Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes. J. Microbiol. Meth. 44: 159-172 https://doi.org/10.1016/S0167-7012(00)00247-5
  32. Osborn, A. M., E. R. B. Moore, and K. N. Timmis. 2000. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2: 39-50 https://doi.org/10.1046/j.1462-2920.2000.00081.x
  33. Nam, K., H. S. Moon, J. Y. Kim, and J. J. Kukor. 2002. Linkage between biodegradation of polycyclic aromatic hydrocarbons and phospholipid profiles in soil isolates. J. Microbiol. Biotechnol. 12: 77-83
  34. Oh, Y.-S., D.-S. Sim, and S.-J. Kim. 2003. Effectiveness of bioremediation on oil-contaminated sand in intertidal zone. J. Microbiol. Biotechnol. 13: 437-443
  35. Rainey, F. A., N. Ward, L. I. Sly, and E. Stackebrandt. 1994. Dependence of the taxon composition of clone libraries for PCR amplified, naturally occurring 16S rDNA, on the primer pair and the cloning system used. Experientia 50: 796-797
  36. Reysenbach, A.-L., L. J. Giver, G. S. Wickham, and N. R. Pace. 1992. Differential amplification of rRNA genes by polymerase chain reaction. Appl. Environ. Microbiol. 58: 3417-3418
  37. Shin, S. K., Y.-S. Oh, and S.-J. Kim. 1999. Biodegradation of phenanthrene by Sphingomonas sp. strain KH3-2. J. Microbiol. 37: 185-192
  38. Suzuki, M. T. and S. J. Giovannoni. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62: 625-630
  39. Tsai, Y.-L. and B. H. Olson. 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Appl. Environ. Microbiol. 58: 754-757
  40. van der Maarel, M. J. E. C., R. R. E. Artz, R. Haanstra, and L. J. Forney. 1998. Association of marine archaea with the digestive tracts of two marine fish species. Appl. Environ. Microbiol. 64: 2894-2898
  41. Vionis, A. P., E. A. Katsifas, and A. D. Karagouni. 1998. Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosm. Antonie Van Leeuwenhoek 73: 103-115 https://doi.org/10.1023/A:1000354323881
  42. Wagner, M., R. Amann, H. Lemme, and K. Schleife. 1993. Probing activated sludge with oligonucleotides specific for proteobacteria: Inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59: 1520-1525
  43. Zhou, J., A. V. Palumbo, and J. M. Tiedje. 1997. Sensitive detection of a novel class of toluene degrading denitrifiers, Azoarcus tolulyticus, with small-subunit rRNA primers and probes. Appl. Environ. Microbiol. 63: 2384-2390