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갯지렁이(Perinereis aibuhitensis)에서 분리한 Bacillus spp.의 생리생화학적 특성 분석

Physiological and Biochemical Characterization of Bacillus spp. from Polychaete, Perinereis aibuhitensis

  • 신세연 (순천대학교 환경교육과) ;
  • ;
  • 이상석 (순천대학교 동물자원학과) ;
  • 강경호 (전남대학교 수산양식학부) ;
  • 강형일 (순천대학교 환경교육과)
  • Shin, Seyeon (Department of Environmental Education, Sunchon National University) ;
  • Yundendorj, Khorloo (Department of Environmental Education, Sunchon National University) ;
  • Lee, Sang-Suk (Department of Animal Science & Technology, Sunchon National University) ;
  • Kang, Kyoung-Ho (Department of Aqualife Science, Chonnam National University) ;
  • Kahng, Hyung-Yeel (Department of Environmental Education, Sunchon National University)
  • 투고 : 2012.12.26
  • 심사 : 2013.02.25
  • 발행 : 2013.03.30

초록

본 연구에서는 연안 갯벌에서 유기물 분해능이 매우 우수한 것으로 알려진 갯지렁이(Perinereis aibuhitensis)에 내생하고 있는 균주 중 호기성과 혐기성 조건에서 생장 가능한 5 종류의 Bacillus 균주 CBW3, CBW4, CBW9, CBW14 그리고 EBW10를 선별하여 그 특성을 비교 분석하였다. 16S rRNA 염기서열에 기초하여 동정한 결과, CBW3과 CBW14는 B. nanhaiensis, B. arsenicus 그리고 B. barbaricus와 99.8% 이상의 높은 상동성을, CBW4, CBW9 그리고 EBW10 균주는 B. anthracis, B. algicoa 그리고 B. thuringiensis와 각각 92.7%, 99.8% 그리고 99.8%의 상동성을 보였다. 이들 대부분 균주들의 생장온도 범위는 $4-45^{\circ}C$, 염도는 0-17%, pH는 5-9 범위로 매우 다양하게 나타났다. 모든 균주들이 casein, starch 분해 효소를 가지고 있었으며 특히 균주 EBW10은 시험한 모든 고분자 물질을 분해할 수 있는 protease, amylase, cellulase. lipase 등의 효소 활성을 가지고 있을 가능성이 높음을 제시해주었다. 대상 균주 5 종 모두 alkaline phosphatase 활성을 가지며, CBW3, CBW4 그리고 EBW10은 acid phospatase 활성을 동시에 가지고 있었으며, CBW3, CBW14, EBW10은 esterase (C4) 활성을, CBW3, CBW9, EBW10은 CBW4는 esterase lipase (C8) 활성을 나타냈다. 지방산 분석 결과 CBW3, CBW9, CBW14, EBW10 균주에서는 anteiso $C_{15:0}$이 균주에 따라 차이가 있었지만 약 42.8%에서 55.7%까지 가장 많은 비율을 차지하는 지방산으로 분석되었고, 오직 CBW4에서만 iso $C_{15:0}$이 전체 지방산의 46.9%로 가장 많은 비율로 나타났다.

This study compared the characteristics of five Bacillus strains capable of aerobic and anaerobic growth, CBW3, CBW4, CBW9, CBW14 and EBW10. They were isolated and selected from a polychaete, Perinereis aibuhitensis, which is known as a good degrader of organic compounds in marine wetland. Based on a 16S rRNA sequence, CBW3 and CBW14 were found to share more than 99.8% similarity with B. nanhaiensis, B. arsenicus and B. barbaricus. CBW4, CBW9 and EBW10 shared 92.7%, 99.8%, and 99.8% similarity with B. anthracis, B. algicoa and B. thuringiensis, respectively. The temperature, salinity, and pH ranges of the cell growth of the Bacillus strains were $4-45^{\circ}C$, 0-17%, and pH 5-pH 9, respectively. All Bacillus strains were found to exhibit enzyme activities for the degradation of casein and starch. Notably, strain EBW10 exhibited the enzyme activities for all the tested macromolecules, DNA, casein, starch, cellulose, and four kinds of Tweens, which suggests the possibility that it had protease, amylase, cellulose, and lipase. All five Bacillus strains had alkaline phosphatase activities, and the strains CBW3, CBW4, and EBW10 also had acid phospatase. Strains CBW3 and EBW10 exhibited the enzyme activities both for esterase (C4) and esterase lipase (C8). The analysis of fatty acids revealed that in all strains, major fatty acids were anteiso $C_{15:0}$ and iso $C_{15:0}$.

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참고문헌

  1. Arena, A., Maugeri, T. L., Pavone, B., Iannello, D., Gugliandolo, C. and Bisignano, V. 2006. Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis. Int Immunopharmacol 6, 8-13. https://doi.org/10.1016/j.intimp.2005.07.004
  2. Bagheri, T., Hedayati, S. A., Yavari, V., Alizade, M. and Farzanfar, A. 2008. Growth, survival and gut microbial load of rainbow trout (Onchorhynchus mykiss) fry given diet supplemented with probiotic during the two months of first feeding. Turk J Fish Aquat Sci 8, 43-48.
  3. Banerjee, S., Devaraja, T. N., Shariff, M. and Yusoff, F. M. 2007. Comparison of four antibiotics with indigenous marine Bacillus spp. in controlling pathogenic bacteria from shrimp and artemia. J Fish Dis 30, 383-389. https://doi.org/10.1111/j.1365-2761.2007.00819.x
  4. Bishop, P. J. and Neuman, G. 1970. The history of the Ziehl-Neelsen stain. Tubercle 51, 196. https://doi.org/10.1016/0041-3879(70)90073-5
  5. Boulygina, E. S., Ignatov, A. N., Tsygankova, S. V., Korotkov, E. V. and Kuznetsov, B. B. 2009. Interspecies relations between Bacillus thuringiensis strains studied by AP-PCR and sequence analysis of ribosomal operon regions. Microbiol 78, 703-710. https://doi.org/10.1134/S002626170906006X
  6. Bryan, G. W. and Gibbs, P. E. 1987. Polychaete common ragworms as indicators of heavy-metal availability in marine deposits. In : Biological Processes and Wastes in the Oceans (Capuzzo J. M., Kester D. R., eds.). Malabar, Florida : Robert E Krieger Publishing Co, 37-39.
  7. Campos, L. C., Su, M. F. J., Graham, N. J. D. and Smith, S. R. 2002. Biomass development in slow sand filters. Water Res 36, 4543-4551. https://doi.org/10.1016/S0043-1354(02)00167-7
  8. Chun, J., Lee, J. H., Jung, Y., Kim, M., Kim, S., Kim, B. K. and Lim, Y. W. 2007. EzTaxon : a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57, 2259-2261. https://doi.org/10.1099/ijs.0.64915-0
  9. Davidson, J., Helwig, N. and Summerfelt, S. T. 2008. Fluidized sand biofilters used to remove ammonia, biochemical oxygen demand, total coliform bacteria, and suspended solids from an intensive aquaculture effluent. Aquacult Engineer 39, 6-15. https://doi.org/10.1016/j.aquaeng.2008.04.002
  10. Edwards, P. R. and Bruner, D. W. 1942. Serological identification of Salmonella cultures. Circ Ky Agric Exp Sta 54, 35.
  11. Felsenstein, J. 1985. Confidence limits on phylogenies : an approach using the bootstrap. Evol 39, 783-791. https://doi.org/10.2307/2408678
  12. Fourcy, D., Jumel, A., Heydorff, M. and Lagadic, L. 2002. Esterases as biomarkers in Nereis (Hediste) diversicolor exposed to temephos and Bacillus thuringiensis var. israelensis used for mosquito control in coastal wetlands of Morbihan (Brittany, France). Mar Environ Res 54, 755-759. https://doi.org/10.1016/S0141-1136(02)00153-8
  13. Fujioka, Y., Shimoda, T. and Srithong, C. 2007. Diversity and community structure of macrobenthic fauna in shrimp aquaculture ponds of the Gulf of Thailand. Jap Agric Res Quart 41, 163-172. https://doi.org/10.6090/jarq.41.163
  14. Gatesoupe, F. J. 1999. The use of probiotics in aquaculture. Aquacult 180, 147-165. https://doi.org/10.1016/S0044-8486(99)00187-8
  15. Ghosh, K., Sen, S. K. and Ray, A. K. 2002. Characterization of bacilli isolated from the gut of rohu, Labeo rohita fingerlings and its significance in digestion. J Appl Aquacult 12,33-42. https://doi.org/10.1300/J028v12n03_04
  16. Gili, J. M. and Coma, R. 1998. Benthic suspension feeders: their paramount role in littoral marine food webs. Trends Ecol Evol 13, 316-321. https://doi.org/10.1016/S0169-5347(98)01365-2
  17. Gomez-Gil, B., Roque, A. and Turnbull, J. F. 2000. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquacult 191, 259-270. https://doi.org/10.1016/S0044-8486(00)00431-2
  18. Guncheva, M. and Zhiryakova, D. 2011. Catalytic properties and potential applications of Bacillus lipases. J Mol Cat B: Enzym 68, 1-21. https://doi.org/10.1016/j.molcatb.2010.09.002
  19. Guo, H., Luo, S., Chen, L., Xiao, X., Xi, Q., Wei, W., Zeng, G., Liu, C., Wan, Y. and Chen, J. 2010. Bioremediation of heavy metals by growing hyperaccumulaor endophytic bacterium Bacillus sp. L14. Bioresour Technol 101, 8599-8605. https://doi.org/10.1016/j.biortech.2010.06.085
  20. Hall, T. 2007. BioEdit. Biological sequence alignment editor for Win95/98/ NT/2K/XP. Carlsbad, CA : Ibis Biosciences.
  21. Hovda, M. B., Sivertsvik, M., Lunestad, B. T., Lorentzen, G. and Rosnes, J. T. 2007. Characterisation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE. Food Microbiol 24, 362-371. https://doi.org/10.1016/j.fm.2006.07.018
  22. Hucker, G. J. and Cohn, H. J. 1923. Methods of gram staining. N.Y. State Agr Exp Sta Tech Bull 93, 123.
  23. Hugh, R. and Leifson, E. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various Gram negative bacteria. J Bacteriol 66, 24-26.
  24. Ivanova, E. P., Alexeeva1, Y. A., Zhukova, N. V., Gorshkova, N. M., Buljan, V., Nicolau, D. V., Mikhailov, V. V. and Christen, R. 2004. Bacillus algicola sp. nov., a novel filamentous organism isolated from brown alga Fucus evanescens. Syst Appl Microbiol 27, 301-307. https://doi.org/10.1078/0723-2020-00269
  25. Jeffries, C. D., Holtman, D. F. and Guse, D. G. 1957. Rapid method for determining the activity of microorganisms on nucleic acids. J Bacteriol 75, 590-591.
  26. Joo, H.-S., Kumar, C. G., Park, G.-C., Paik, S. R. and Chang, C.-S. 2004. Bleach-resistant alkaline protease produced by a Bacillus sp. isolated from the Korean polychaete, Periserrula leucophryna. Pro Biochem 39, 1441-1447. https://doi.org/10.1016/S0032-9592(03)00260-7
  27. Kennedy, S. B., Tucker, J. W., Neidig, C. L., Vermeer, G. K., Cooper, V. R., Jarrell, J. L. and Sennett, D. G. 1998. Bacterial management strategies for stock enhancement of warm water marine fish : a case study with common snook (Centropomus undecimalis). Bull Mar Sci 62, 573-588.
  28. Kovacs, N. 1928. Eine vereinfachte methode zum Nachweis der indolbildung durch bakterien. Z Immum Forsch Exp Ther 55, 311.
  29. Kumar, S., Nei, M., Dudley, J. and Tamura, K. 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9, 299-306. https://doi.org/10.1093/bib/bbn017
  30. Lane, D. J. 1991. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115-175. In Stackebrandt, E. and Goodfellow, M. (eds.), New York : Wiley.
  31. Mikesell, M. D., Kukor, J. J. and Olsen, R. H. 1993. Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer. Biodegradation 4, 249-259.
  32. Moeller, M. 1891. Ueber eine neue Methode der Sporenfarbung. Zbl Bakt, I Abt Orig 10, 273-277.
  33. Moriarty, D. J. W. 1998. Control of luminous Vibrio species in penaeid aquaculture ponds. Aquacult 164, 351-358. https://doi.org/10.1016/S0044-8486(98)00199-9
  34. Orejas, C., Gili, J. M., Arntz, W. E., Ros, J. D., Lo"Lpez, P. J., Teixido"L, N., Filipe, P. 2000. Benthic suspension feeders, key players in Antarctic marine ecosystems. Contribut Sci 1, 299-311.
  35. Palacios, G. L. and Timmons, M. B. 2001. Determining design parameters for recovery of aquaculture wastewater using sand beds. Aquacult Engineer 24, 289-299. https://doi.org/10.1016/S0144-8609(01)00070-X
  36. Perez, E., Blasco, J. and Sole, M. 2004. Biomarker responses to pollution in two invertebrate species : Scrobicularia plana and Nereis diversicolor from the Cadiz bay (SW Spain). Mar Environ Res 58, 275-279. https://doi.org/10.1016/j.marenvres.2004.03.071
  37. Prieur, D., Mevel, G., Nicolas, J. L., Plusquellec, A. and Vigneulle, M. 1990. Interactions between bivalve molluscs and bacteria in the marine environment. Oceanography Mar Biol : Annual Rev 28, 277-352.
  38. Raida, M. K., Larsen, J. L., Nielsen, M. E. and Buchmann, K. 2003. Enhanced resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), against Yersinia ruckeri challenge following oral administration of Bacillus subtilis and B. licheniformis (BioPlus2B). J Fish Dis 26, 495-498. https://doi.org/10.1046/j.1365-2761.2003.00480.x
  39. Ryu, C., Lee, K., Hawng, H.-J., Yoo, C.-K., Seong, W.-K. and Oh, H.-B. 2005. Molecular characterization of Korean Bacillus anthracis isolates by amplified fragment length polymorphism analysis and multilocus variable-number tandem repeat analysis. Appl Environ Microbiol 71, 4664-4671. https://doi.org/10.1128/AEM.71.8.4664-4671.2005
  40. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
  41. Sambrook, J. and Russel, D. W. 2001. Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  42. Shivaji, S., Suresh, K., Chaturvedi, P., Dube, S. and Sengupta, S. 2005. Bacillus arsenicus sp. nov., an arsenic-resistant bacterium isolated from a siderite concretion in West Bengal, India. Int J Syst Evol Microbiol 55, 1123-1127. https://doi.org/10.1099/ijs.0.63476-0
  43. Sierra, G. 1957. A simple method for the detection of lipolytic activity of microorganisms and some observations on the influence of the contact between cells and fatty substrates. Ant van Leeuwen 23, 15. https://doi.org/10.1007/BF02545855
  44. Stabili, L., Licciano, M., Giangrande, A., Fanelli, G. and Cavallo, R. S. 2006. Sabella spallanzanii filter-feeding on bacterial community: ecological implications and applications. Mar Environ Res 61, 74-92. https://doi.org/10.1016/j.marenvres.2005.06.001
  45. Sugita, H., Hirose, Y., Matsuo, N. and Deguchi, Y. 1998. Production of the antibacterial substance by Bacillus species strain NM12, an intestinal bacterium of Japanese coastal fish. Aquacult 165, 269-280. https://doi.org/10.1016/S0044-8486(98)00267-1
  46. Taubel. M., Kampfer, P., Buczolits, S., Lubitz, L. and Busse, H.-J. 2003. Bacillus barbaricus sp. nov., isolated from an experimental wall painting. Int J Syst Evol Microbiol 53, 725-730. https://doi.org/10.1099/ijs.0.02304-0
  47. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. 1997. The clustal_x windows interface : flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  48. Vaseeharan, B. and Ramasamy, P. 2003. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett Appl Microbiol 36, 83-87. https://doi.org/10.1046/j.1472-765X.2003.01255.x
  49. Verschuere, L., Rombaut, G., Sorgeloos, P. and Verstraete, W. 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Res 64, 655-671. https://doi.org/10.1128/MMBR.64.4.655-671.2000
  50. Vigneswaran, S., Ngo, H. H. and Wee, K. L. 1999. Effluent recycle and waste minimisation in prawn farm effluent. J Cleaner Prod 7, 121-126. https://doi.org/10.1016/S0959-6526(98)00048-1
  51. Yoon, J. H., Kim, I.-G., Kang, K. H., Oh, T.-K. and Park, Y.-H. 2004. Bacillus hwajinponensis sp. nov. and an unnamed Bacillus genomospecies, novel members of Bacillus rRNA group 6 isolated from sea water of the East Sea and the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 803-808. https://doi.org/10.1099/ijs.0.02678-0
  52. Ziaei-Nejad, S., Rezaeib, M. H., Takamic, G. A., Lovettd, D. L., Mirvaghefia, A. and Shakourie, M. 2006. The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquacult 252, 516-524. https://doi.org/10.1016/j.aquaculture.2005.07.021