Review and Future Development of New Culture Methods for Unculturable Soil Bacteria

난배양성 토양세균을 위한 신배양기술의 고찰과 향후 발전 방향

  • Kim, Jai-Soo (Department of Life Science, Kyonggi University)
  • 김재수 (경기대학교 자연과학대학 생명과학과)
  • Received : 2011.08.17
  • Accepted : 2011.12.14
  • Published : 2011.09.30


This review describes the characteristics of various unculturable soil bacteria, successfully-cultivating examples of those bacteria, and the diverse factors to be considered for successful cultivation. Most importantly, the selection of proper media is very important because unculturable bacteria demand different types of nutrients at various concentrations of substrates, nitrogens and phosphorus. To develop a new medium to successfully culture unculturable bacteria from soil, molecular ecological studies should be combined together. The inoculum size on a plate is also important: less than 50 bacterial cells are recommended to be plated on a single culture plate. The environmental factors such as pH and salt concentration of the medium need to be adjusted as similar as possible to mimic the original soil environments, and the trial of the various temperatures and extended period of cultivation are better. Since one cannot simply tell about which one was unculturable among a great number of colonies grown on a newly developed medium, some suitable detection methods and fast identification methods are required. Many soil bacteria live with cooperation one another in their communities, so that enrichment such as coculture of using other bacterial metabolites and subsequent pure cultures can also guarantee successful cultivation of the previously uncultured bacteria in soil. Here, this review will discuss for the future perspectives to culture the unculturable soil bacteria.


coculture;metagenomics;new culture method;soil bacteria;unculturable


Supported by : 경기대학교


  1. Alain, K. and J. Querellou. 2009. Cutivating the uncultured: limits, advances and future challenges. Extremophiles 13, 583-594.
  2. Allsopp, D., R.R. Colwell, and D.L. Hawksworth. 1995. Microbial diversity and ecosystem function. CAB International, Wallingford, UK.
  3. Andrews, J.H. and R.F. Harris. 1986. r- and K-selection and microbial ecology. Adv. Microb. Ecol. 9, 99-147.
  4. Balestra, G.M. and I.J. Misaghi. 1997. Increasing the efficiency of the plate count method for estimating bacterial diversity. J. Microbiol. Methods 30, 111-137.
  5. Bartscht, K., H. Cypionka, and J. Overmann. 1999. Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community. FEMS Microbiol. Ecol. 28, 249-259.
  6. Batchelor, S.E., M. Cooper, S.R. Chhabra, L.A. Glover, G.S. Stewart, P. Williams, and J.I. Prosser. 1997. Cell densityregulated recovery of starved biofilm populations of ammoniaoxidizing bacteria. Appl. Environ. Microbiol. 63, 2281-2286.
  7. Bremner, J.M. and L.A. Douglas. 1971. Use of plastic films for aeration on soil incubation experiments. Soil Biol. Biochem. 3, 289-296.
  8. Bruns, A., H. Cypionka, and J. Overmann. 2002. Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea. Appl. Environ. Microbiol. 68, 3978-3987.
  9. Bruns, A., U. Nubel, H. Cypionka, and J. Overmann. 2003. Effect of signal compounds and incubation conditions on the culturability of fresh-water bacterioplankton. Appl. Environ. Microbiol. 69, 1980-1989.
  10. Bussmann, I., B. Philipp, and B. Schink. 2001. Factors influencing the cultivability of lake water bacteria. J. Microbiol. Methods 47, 41-50.
  11. Calcott, P.H. and J.R. Postgate. 1972. On substrate-accelerated death in Klebsiella aerogenes. J. Gen. Microbiol. 70, 115-122.
  12. Camilli, A. and B.L. Bassler. 2006. Bacterial small-molecule signaling pathways. Science 311, 1113-1116.
  13. Casida, L.E. 1968. Methods for the isolation and estimation of activity of soil bacteria, pp. 97-122. In T.R.G. Gray and D. Parkinson (eds.), The Ecology of Soil Bacteria. Liverpool University Press, Liverpool, UK.
  14. Cayley, S., M.T. Record, and B.A. Lewis. 1989. Accumulation of 3 -(N-morpholino)-propanesulfonate by osmotically stressed Escherichia coli K-12. J. Bacteriol. 171, 3597-3602.
  15. Chin, K.J., D. Hahn, U. Hengstmann, W. Liesack, and P.H. Janssen. 1999. Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms. Appl. Environ. Microbiol. 65, 5042-5049.
  16. Christner, B.C., E. Mosley-Thompson, L.G. Thompson, V. Zagorodnov, K. Sandman, and J.N. Reeve. 2000. Recovery and identification of viable bacteria immured in glacial ice. Icarus 144, 479-485.
  17. Crocetti, G.R., J.F. Banfield, J. Keller, P.L. Bond, and L.L. Blackall. 2002. Glycogen-accumulating organisms in laboratoryscale and full-scale wastewater treatment processes. Microbiology 148, 3353-3364.
  18. Davis, K.E.R., S.J. Joseph, and P.H. Janssen. 2005. Effects of growth medium, inoculum size, and incubation time on the culturability and isolation of soil bacteria. Appl. Environ. Microbiol. 71, 826-834.
  19. Davis, K.E.R., P. Sangwan, and P.H. Janssen. 2011. Acidobacteria, Rubrobacteridae and Chloroflexi are abundant among very slow-growing and mini-colony forming soil bacteria. Environ. Microbiol. 13, 798-805.
  20. De Spiegeleer, P., J. Sermon, A. Lietaert, A. Aertsen, and C.W. Michiels. 2004. Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli. J. Appl. Microbiol. 97, 124-133.
  21. Eichorst, S.A., J.A. Breznak, and T.M. Schmidt. 2007. Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria. Appl. Environ. Microbiol. 73, 2708-2717.
  22. Eilers, H., J. Pernthaler, J. Peplies, F.O. Glöckner, G. Gerdts, and R. Amann. 2001. Isolation of novel pelagic bacteria from the German Bight and their seasonal contributions to surface picoplankton. Appl. Environ. Microbiol. 67, 5134-5142.
  23. Ensign, S.A., F.J. Small, J.R. Allen, and M.K Sluis. 1998. New roles for $CO_2$ in the microbial metabolism of a liphatic epoxides and ketones. Arch. Microbiol. 169, 179-187.
  24. Ferrari, B.C., S.J. Binnerup, and M. Gillings. 2005. Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl. Environ. Microbiol. 71, 8714-8720.
  25. Fischer, H. 1909. Bakteriologisch-chemishe Untersuchungen. Bakteriologischer Teil. Landw. Jahrb. 38, 355-364.
  26. Freeman, R., J. Dunn, J. Magee, and A. Barrett. 2002. The enhancement of isolation of mycobacteria from a rapid liquid culture system by broth culture supernate of Micrococcus luteus. J. Med. Microbiol. 51, 92-93.
  27. Frohlich, J. and H. König. 2000. New techniques for the isolation of single prokaryotic cells. FEMS Microbiol. Rev. 24, 567-572.
  28. Furlong, M.A., D.R. Singleton, D.C. Coleman, and W.B. Whitman. 2002. Molecular and culture-based and analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus. Appl. Environ. Microbiol. 68, 1265-1279.
  29. Gray, T.R.G., P. Baxby, I.R. Hill, and M. Goodfellow. 1968. Direct observation of bacteria in soil, pp. 171-197. In T.R.G. Gray and D. Parkinson (eds.), The Ecology of Soil Bacteria. Liverpool University Press, Liverpool, UK.
  30. Guan, L.L. and K. Kamino. 2001. Bacterial response to siderophore and quorum-sensing chemical signals in the seawater microbial community. BMC Microbiol. 1, 27.
  31. Harris, D. and E.A. Paul. 1994. Measurements of bacterial growth rates in soil. Appl. Soil Ecol. 1, 277-290.
  32. Hattori, T. 1976. Plate count of bacteria in soil on a diluted nutrient broth as a culture medium. Rep. Inst. Agric. Res. Tohoku Univ. 27, 23-30.
  33. Hattori, T. 1980. A note on the effect of different types of agar on plate count of oligotrophic bacteria in soil. J. Gen. Appl. Microbiol. 26, 373-374.
  34. Hattori, T. and R. Hattori. 1976. The physical environment in soil microbiology: an attempt to extend principles of microbiology to soil microorganisms. CRC Crit. Rev. Microbiol. 4, 423-461.
  35. Hattori, R. and T. Hattori. 1980. Sensitivity to salts and organic compounds of soil bacteria isolated on diluted media. J. Gen. Appl. Microbiol. 26, 1-14.
  36. Hattori, S., A.S. Galushko, Y. Kamagata, and B. Schink. 2005. Operation of the CO dehydrogenase/acetyl-CoA pathway both in acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum. J. Bacteriol. 187, 3471-3476.
  37. Hattori, S., Y. Kamagata, S. Hanada, and H. Shoun. 2000. Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium. Int. J. Syst. Evol. Microbiol. 50, 1601-1609.
  38. Huber, H., M.J. Hohn, R. Rachel, T. Fuchs, V.C. Wimmer, and K.O. Stetter. 2002. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63-67.
  39. Hugenholtz, P., B.M. Goegel, and N.R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765-4774.
  40. Ishikuri, S. and T. Hattori. 1985. Formation of bacterial colonies in successive time intervals. Appl. Environ. Microbiol. 49, 870-873.
  41. James, N. and M. Sutherland. 1940. Effect of numbers of colonies per plate on the estimate of the bacterial population in soil. Can. J. Res. Section C. 18, 347-356.
  42. Janssen, P.H., P.S. Yates, B.E. Grinton, P.M. Taylor, and M. Sait. 2002. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol. 68, 2391-2396.
  43. Jensen, V. 1962. Studies on the microflora of Danish beech forest soils. I. The dilution plate count technique for enumeration of bacteria and fungi in soil. Zentralbl. Bakteriol. Parasitenkd. Abt. 2. 116, 13-32.
  44. Jensen, V. 1968. The plate count technique, pp. 158-170. In T.R.G. Gray and D. Parkinson (eds.), The Ecology of Soil Bacteria. Liverpool University Press, Liverpool, UK.
  45. Joseph, S.J., P. Hugenholtz, P. Sangwan, C.A. Osborne, and P.H. Janssen. 2003. Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol. 69, 7210-7215.
  46. Kaeberlein, T., K. Lewis, and S.S. Epstein. 2002. Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Science 296, 1127-1129.
  47. Kell, D.B., A.S. Kaprellyants, and A. Grafen. 1995. On pheromones, social behaviour and the functions of secondary metabolism in bacteria. Trends Ecol. Evolution 10, 126-129.
  48. Kolter, R., D.A. Siegele, and A. Tormo. 1993. The stationary phase of the bacterial life cycle. Annu. Rev. Microbiol. 47, 855-874.
  49. Kushmaro, A. and S. Geresh. 2004. Method for isolating and culturing unculturable microorganisms. International Applicatiojn Published under the Patent Cooperation Treaty (PCT), International Publication Number: WO 2004/022698 A2.
  50. Leadbetter, J.R. 2003. Cultivation of recalcitrant microbes: cells are alive, well and revealing their secrets in the 21st century laboratory. Curr. Opin. Microbiol. 6, 274-281.
  51. Lilburn T.G., K.S. Kim, N.E. Ostrom, K.R. Byzek, J.R. Leadbetter, and J.A. Breznak. 2001. Nitrogen fixation by symbiotic and free-living spirochetes. Science 292, 2495-2498.
  52. Madigan, M.T., J.M. Martinko, P.V. Dunlap, and D.P. Clark. 2009. Brook Biology of Microorganisms, 12th ed., Pearson Benjamin Cummings, San Francisco, CA, USA.
  53. Mason, T.G. and G. Blunden. 1989. Quaternary ammonium and tertiary sulfonium compounds of algal origin as alleviators of osmotic stress. Bot. Mar. 32, 313-316.
  54. McCaig, A.E., S.J. Grayston, J.I. Prosser, and L.A. Glover. 2001. Impact of cultivation on characterization of species composition of soil bacterial communities. FEMS Microbiol. Ecol. 35, 37-48.
  55. Miller, M.B. and B.L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165-199.
  56. Mochizuki, M. and T. Hattori. 1986. Kinetics of microcolony formation of a soil olitrophic bacterium, Agromonas sp. FEMS Microbiol. Ecol. 38, 51-55.
  57. Monciardini, P., L. Cavaletti, P. Schumann, M. Rohde, and S. Donadio. 2003. Conexibacterwoesii gen. nov., sp. nov., a novel representative of a deep evolutionary line of descent within the class Actinobacteria. Int. J. Syst. Evol. Microbiol. 53, 569-576.
  58. Mukamolova, G.V., A.S. Kaprelyants, D.I. Young, M. Young, and D.B. Kell. 1998. A bacterial cytokine. Proc. Natl. Acad. Sci. USA 95, 8916-8921.
  59. Mukamolova, G.V., N.D. Yanopolskaya, D.B. Kell, and A.S. Kaprelyants. 1998. On resuscitation from the dormant state of Micrococcus luteus. Antonie van Leeuwenhoek 73, 237-243.
  60. Nadell, C.D., J.B. Xavier, and K.R. Foster. 2009. The sociobiology of biofilms. FEMS Microbiol. Rev. 33, 206-224.
  61. Novitsky, J.A. 1987. Microbial growth rates and biomass production in a marine sediment: evidence for a very active but mostly nongrowing community. Appl. Environ. Microbiol. 53, 2368-2372.
  62. Ohno, M., I. Okano, T. Watsuji, T. Kakinuma, K. Ueda, and T. Beppu. 1999. Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain. Biosci. Biotechnol. Biochem. 63, 1083-1090.
  63. Ohno, M., H. Shiratori, M.J. Park, Y. Saitoh, Y. Kumon, N. Yamashita, A. Hirata, H. Nishida, K. Ueda, and T. Beppu. 2000. Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth. Int. J. Syst. Evol. Microbiol. 50, 1829-1832.
  64. Olsen, R.A. and L.R. Bakken. 1987. Viability of soil bacteria: optimization of plate-counting techniques and comparisons between total counts and plate counts within different size groups. Microb. Ecol. 13, 59-74.
  65. Overmann, J. 2006. Principles of enrichment, isolation, cultivation and preservation of prokaryotes, pp. 80-136. In M. Doworkin, S. Falkow, E. Rosenberg, K.H. Schleifer, and E. Stackebrandt (eds.), The Prokaryotes, 3rd ed. Vol. 1: Symbiotic Associations, Biotechnology, Applied Microbiology. Springer, New York, NY, USA.
  66. Palumbo, A.V., C. Zhang, S. Liu, S.P. Scarborough, S.M. Pfiffner, and T.J. Phelps. 1996. Influence of media on measurement of bacterial populations in the subsurface. Appl. Biochem. Biotech. 57/58, 905-914.
  67. Plugge, C.M. and A.J.M. Stams. 2002. Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor. Microbiol. Ecol. 43, 379-387.
  68. Postgate, J.R. and J.R. Hunter. 1964. Accelerated death of Aerobacter aerogenes starved in the presence of growth limiting substrates. J. Gen. Microbiol. 34, 459-473.
  69. Reichenbach, H. and M. Dworkin. 1981. Introduction to the glidinh bacteria, pp. 315-327. In M.P. Starr, H. Stolp, H.G. Truper, A. Balows, and H.G. Schlegel (eds.), The Prokaryotes. A handbook on habitats, isolation, and identification of bacteria, vol. 1. Springer-Verlag, Heidelberg, Germany.
  70. Rosch, C., A. Mergel, and H. Bothe. 2002. Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Appl. Environ. Microbiol. 68, 3818-3829.
  71. Sait, M., K.E.R. Davis, and P.H. Janssen. 2006. Effect of pH on the isolation and distribution of members of subdivision 1 of the phylum Acidobacteria occurring in soil. Appl. Environ. Microbiol. 72, 1852-1857.
  72. Sait, M., P. Hugenholtz, and P.H. Janssen. 2002. Cultivation of globally-distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environ. Microbiol. 4, 654-666.
  73. Sangwan, P., X. Chen, P. Hugenholtz, and P.H. Hanssen. 2004. Chthoniobacter flavus gen. nov., sp. nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia. Appl. Environ. Microbiol. 70, 5875-5881.
  74. Sangwan, P., S. Kovac, K.E.R. Davis, M. Sait, and P.H. Janssen. 2005. Detection and cultivation of soil Verrucomicrobia. Appl. Environ. Microbiol. 71, 8402-8410.
  75. Schoenborn, L., P.S. Yates, B.E. Grinton, P. Hugenholtz, and P.H. Janssen. 2004. Liquid serial dilution is inferior to solid media for isolation of cultures representing the phylum level diversity of soil bacteria. Appl. Environ. Microbiol. 70, 4363-4366.
  76. Sexstone, A.J., N.P. Revsbech, T.P. Parkin, and J.M. Tiedje. 1985. Direct measurement oxygen profiles and denitrification rates in soil aggregates. Soil Sci. Soc. Amer. J. 49, 645-651.
  77. Shleeva, M.O., K. Bagramyan, M.V. Telkov, G.V. Mukamolova, M. Young, D.B. Kell, and A.S. Kaprelyants. 2002. Formation and resuscitation of "non-culturable" cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary. Microbiology 148, 1581-1591.
  78. Simon, H.M., C.E. Jahn, L.T. Bergerud, M.K. Sliwinski, P.J. Weimer, D.K. Willis, and R.M. Goodman. 2005. Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots. Appl. Environ. Microbiol. 71, 4751-4760.
  79. Simu, K. and A. Hagstrom. 2004. Oligotrophic bacterioplankton with a novel single-cell life strategy. Appl. Environ. Microbiol. 70, 2445-2451.
  80. Sorheim, R., V.L. Torsvik, and J. Goksøyr. 1989. Phenotypic divergences between populations of soil bacteria isolated on different media. Microb. Ecol. 17, 181-192.
  81. Stevenson, B.S., S.A. Eichorst, J.T. Wertz, T.M. Schmidt, and J.A. Breznak. 2004. New strategies for cultivation and detection of previously uncultured microbes. Appl. Environ. Microbiol. 70, 4748-4755.
  82. Streit, W.R. and R.A. Schmitz. 2004. Metagenomics-the key to the uncultured microbes. Curr. Opin. Microbiol. 7, 492-498.
  83. Sun, Z. and Y. Zhang. 1999. Spent culture supernant of Mycobacterium tuberculosis H37Ra improved viability of aged cultures of this strain and allows small inocula to initiate growth. J. Bacteriol. 181, 7626-7628.
  84. Suzuki, S., S. Horinouchi, and T. Beppu. 1988. Growth of a tryptophanas-producing thermophile, Symbiobacterium thermophilum gen. nov., sp. nov., is dependent on coculture with a Bacillus sp. J. Gen. Microbiol. 134, 2353-2362.
  85. Tanaka, Y., S. Hanada, A. Manome, T. Tsuchida, R. Kurane, K. Nakamura, and Y. Kamagata. 2004. Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth. Int. J. Syst. Evol. Microbiol. 54, 955-959.
  86. Thornton, H.G. 1992. On the development of a standardized agar medium for counting soil bacteria, with especial regard to the repression of spreading colonies. Ann. Appl. Biol. 9, 241-274.
  87. Tiedje, J.M. 1994. Microbioal diversity: of value to whom? ASM News 60, 524-525.
  88. Torsvik, V., R. Sorheim, and J. Goksoyr. 1996. Total bacterial diversity in soil and sediment connunities - a review. J. Ind. Microbiol. 17, 170-178.
  89. Ueda, K., H. Saka, Y. Ishikawa, T. Kato, Y. Takeshita, H. Shiratori, M. Ohno, K. Hosono, M. Wada, and T. Beppu. 2002. Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium, Symbiobacterium thermophilum. Appl. Microbiol. Biotechnol. 60, 300-305.
  90. Wang, J., C. Jenkins, R.I. Webb, and J.A. Fuerst. 2002. Isolation of Gemmata-like and Isophaera-like planctomycete bacteria from soil and freshwater. Appl. Environ. Microbiol. 68, 417-422.
  91. Waterbury, J.B. 1991. The cyanobacteria-isolation, purification, and identification, pp. 149-196. In M.P. Starr, H. Stolp, H.G. Truper, A. Balows, and H.G. Schlegel (eds.), The prokaryotes. A handbook on habitats, isolation, and identification of bacteria, vol. 1. Springer-Verlag, Heidelberg, Germany.
  92. West, S.A., S.P. Diggle, A. Buckling, A. Gardner, and A.S. Griffin. 2007. The social lives of microbes. Annu. Rev. Ecol. Evol. Syst. 38, 53-77.
  93. Widdel, F. 1987. New types of acetate-oxidazing, sulfatereducing Dessulfobacter species, D. hydrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov. Arch. Microbiol. 148, 286-291.
  94. Widdel, F. and F. Bak. 1992. Gram-negative mesophilic sulfate-reducing, pp. 3352-3378. In A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.H Schleifer (eds.), The Prokaryotes. A handbook on the biology of bacteria: Ecophysiology, isolation, and identification, application, 2nd ed., vol. 4. Springer-Verlag, New York, NY, USA.
  95. Winding, A., S.J. Binnerup, and J. Sørensen. 1994. Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl. Environ. Microbiol. 60, 2869-2875.
  96. Winogradsky, S. 1949. Microbiologie du Sol. Problemes et Methodes. Masson, Paris, France.
  97. Zengler, K., H.H. Richnow, R. Rossello-Mora, W. Michaelis, and F. Widdel. 1999. Methane formation from long-chain alkanes by anaerobic microorganisms. Nature 401, 266-269.
  98. Zengler, K., G. Toledo, M. Rappé, J. Elkins, E.J. Mathur, J.M. Short, and M. Keller. 2002. Cultivating the uncultured. Proc. Natl. Acad. Sci. USA 99, 15681-15686.