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Cultivation and molecular monitoring of halophilic microorganisms inhabiting an extreme environment presented by a salt-attacked monument

Published online by Cambridge University Press:  15 December 2009

Jörg Ettenauer ,
Katja Sterflinger  and
Guadalupe Piñar
Jörg Ettenauer
Affiliation:
Institute of Applied Microbiology, Department of Biotechnology, Vienna Institute of Bio Technology (VIBT). University of Natural Resources and Applied Life Sciences, Muthgasse 11, A-1190Vienna, Austria
Katja Sterflinger
Affiliation:
Institute of Applied Microbiology, Department of Biotechnology, Vienna Institute of Bio Technology (VIBT). University of Natural Resources and Applied Life Sciences, Muthgasse 11, A-1190Vienna, Austria
Guadalupe Piñar*
Affiliation:
Institute of Applied Microbiology, Department of Biotechnology, Vienna Institute of Bio Technology (VIBT). University of Natural Resources and Applied Life Sciences, Muthgasse 11, A-1190Vienna, Austria
*
e-mail: [email protected] / [email protected]
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Abstract

In the last few years several investigations, based on culture-dependent and -independent techniques, have shown that salt-attacked stone surfaces present a habitat for extremely salt tolerant and moderate halophilic microorganisms. The inner walls of the Chapel of St. Virgil in Vienna (Austria) are an example of this phenomenon. Salt crusts cover most of the wall surfaces and salt crystallization in the porous space of the stone is causing decohesion of material and destruction of the original medieval paintings. The salt, together with the oligotrophic conditions, creates a very special and extreme habitat for halotolerant and halophilic microorganisms.

In this study we investigate and monitor the cultivable and non-cultivable members of the microbial community present on the stonework of the medieval Chapel of St. Virgil after several severe disturbances of the microbial environment caused by desalination and disinfection treatments. With this finality, a combination of culture-dependent and -independent techniques was selected. The genetic diversity of a total of 104 bacterial strains isolated from the stone samples was analysed by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD) analysis and 16S rRNA gene sequencing. Strains were distributed over 29 groups on the basis of their RAPD patterns. Only 19 groups were differentiated by DGGE. Comparative sequence analyses showed that the isolated strains belong to related species of the genera Halobacillus (47.1%), Bacillus (35.6%), Acinetobacter (4.8%), Halomonas (3.9%), Nesterenkonia (2.9%), Paucisalibacillus (2.9%), Paenibacillus (1%), Staphylococcus (1%) and Exiguobacterium (1%).

In addition, polymerase chain reaction DGGE fingerprints, in combination with the creation of clone libraries and sequencing analyses, were used to monitor and identify Archaea, the non-cultivable fraction of the microbial community. The detected archaeal sequences were closely related to different uncultured archaeons as well as to the cultured genera Halococcus and Halalkalicoccus and Halobacterium.

Cultivation and molecular analyses revealed the presence of highly specialized microorganisms that were able to thrive and survive after several desalination and disinfection treatments in the extreme environment presented by the salt-attacked Chapel of St. Virgil.

Keywords

ArchaeaDNA-fingerprintsHalobacillushalophilic microorganismsmolecular monitoringphylogenetic identificationsalt efflorescences
Type
Research Article
Information
International Journal of Astrobiology , Volume 9 , Issue 1 , January 2010 , pp. 59 - 72
Copyright
Copyright © Cambridge University Press 2009

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References

Amann, R., Ludwig, W. & Schleifer, K.H. (1995). Microbiol. Rev. 59, 143–169.CrossRefGoogle Scholar
Amoroso, G.G. & Fassina, V. (1983). Stone Decay and Conservation. Elsevier, Amsterdam.Google Scholar
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, J.D. (1997). Nucleic Acids Res. 25, 3389–3402.CrossRefGoogle Scholar
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G, Smith, J.A. and Struhl, K. (1991). Current Protocols in Molecular Biology. John Wiley and Sons, New York.Google Scholar
Buchholz-Cleven, B.E.E., Rattunde, B. & Straub, K.L. (1997). Syst. Appl. Microbiol. 20, 301–309.CrossRefGoogle Scholar
Busse, J.-H., Denner, E.B.M. & Lubitz, W. (1996). J. Biotechnol. 47, 3–38.CrossRefGoogle Scholar
El-Rahman, H.A.A., Fritze, D., Spröer, C. and Claus, D. (2002). Int. J. Syst. Evol. Microbiol. 52, 2127–2133.Google Scholar
Fajardo-Cavazos, P. & Nicholson, W. (2006). Appl. Environ. Microbiol. 72, 2856–2863.CrossRefGoogle Scholar
Fendrihan, S., Berces, A., Lammer, H., Musso, M., Ronto, G., Polacsek, T.K., Holzinger, A., Kolb, C. & Stan-Lotter, H. (2009). Astrobiology 9, 104–112.CrossRefGoogle Scholar
Giovannoni, S.J., Britschgi, T.B., Moyer, C.L. & Field, K.G. (1990). Nature 345, 60–63.CrossRefGoogle Scholar
Gonzalez, J.M. & Saiz-Jimenez, C. (2005). Int. Microbiol. 8, 189–194.Google Scholar
Hansen, A.A. (2007). ROME Response of Organisms to the Martian Environment, pp. 1–18. European Space Agency.Google Scholar
Head, I.M., Saunders, J.R. & Pickup, R.W. (1998). Microbiol. Ecol. 35, 1–21.CrossRefGoogle Scholar
Hein, I., Mach, R.L., Farnleitner, AH. & Wagner, M. (2003). J. Microbiol. Meth. 52, 305–313.CrossRefGoogle Scholar
Heyrman, J., Logan, N.A., Rodriguez-Diaz, M., Scheldeman, P., Lebbe, L., Swings, J., Heyndrickx, M. & De vos, P. (2005). Int. J. Syst. Evol. Microbiol. 55, 119–131.CrossRefGoogle Scholar
Heyrman, J. & Swings, J. (2001). Syst. Appl. Microbiol. 24, 417–422.CrossRefGoogle Scholar
Hugenholtz, P., Goebel, B.M. & Pace, N.R. (1998). J. Bacteriol. 180, 4765–4774.CrossRefGoogle Scholar
Incerti, C., Blanco-Varela, M.T., Puertas, F. and Saiz-Jimenez, C. (1997). Protection and conservation of the European cultural heritage. Research Report n° 4, pp. 225–232.Google Scholar
Klose, V. (2001). Diploma Thesis, University of Vienna, 2001.Google Scholar
Lane, D.J. (1991). Nucleics Acid Techniques in Bacterial Systematics, ed. Stackebrandt, E. & Goodfellow, M., pp. 115–175. John Wiley & Sons, Chichester, UK.Google Scholar
Laiz, L., Piñar, G., Lubitz, W. & Saiz-Jimenez, C. (2003). Environ. Microbiol. 5, 72–74.CrossRefGoogle Scholar
Lazar, I. (1971). Rev. Roum. Biol. Scr. Bot. 16, 437–444.Google Scholar
Li, W.J., Zhang, Y.Q., Schumann, P., Liu, H.Y., Yu, L.Y., Zhang, Y.Q., Stackebrandt, E., Xu, L.H. & Jiang, C.L. (2008). Int. J. Syst. Evol. Microbiol. 58, 1359–1363.CrossRefGoogle Scholar
Muyzer, G., De Waal, E.C. & Uitterlinden, A.G. (1993). Appl. Environ. Microbiol. 59, 695–700.CrossRefGoogle Scholar
Muyzer, G. & Smalla, K. (1998). Anton. Leeuw. 73, 127–141.CrossRefGoogle Scholar
Nübel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R.I., Ludwig, W. & Backhaus, H. (1996). J. Bacteriol. 178, 5636–5643.CrossRefGoogle Scholar
Nicholson, W.L. (2002). Cell. Mol. Life. Sci. 59, 410–416.CrossRefGoogle Scholar
Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. & Setlow, P. (2000). Microbiol. Mol. Biol. Rev. 64, 548–572.CrossRefGoogle Scholar
Nunes, I., Tiago, I., Pires, A.L., da Costa, M.S. & Verissimo, A. (2006). Int. J. Syst. Evol. Microbiol. 56, 1841–1845.CrossRefGoogle Scholar
Piñar, G., Gurtner, C., Lubitz, W. & Rölleke, S. (2001a). Meth. Enzymol. 336, 356–366.CrossRefGoogle Scholar
Piñar, G., Ramos, C., Rölleke, S., Schabereiter-Gurtner, C., Vybiral, D., Lubitz, W. & Denner, E.B.M. (2001b). Appl. Environ. Microbiol. 67, 4891–4895.CrossRefGoogle Scholar
Piñar, G., Ripka, K., Weber, J. & Sterflinger, K. (2009). Int. Biodeter. Biodeg. 63, 851–859.CrossRefGoogle Scholar
Piñar, G., Saiz-Jimenez, C., Schabereiter-Gurtner, C., Blanco-Valera, M.T., Lubitz, W. & Rölleke, S. (2001c). FEMS Microbiol. Ecol. 37, 45–54.CrossRefGoogle Scholar
Power, E.G.M. (1996). J. Hosp. Infect. 34, 247–265.CrossRefGoogle Scholar
Rappé, M.S. & Giovannoni, S.J. (2003). Ann. Rev. Microbiol. 57, 369–394.CrossRefGoogle Scholar
Raskin, L., Stromley, J.M., Rittmann, B.E. & Stahl, D.A. (1994). Appl. Environ. Microbiol. 60, 1232–1240.CrossRefGoogle Scholar
Ripka, K. (2005). Diploma Thesis, University of Vienna.Google Scholar
Ripka, K., Denner, E.B.M., Michaelsen, A., Lubitz, W. & Piñar, G. (2006). Int. Biodeter. Biodegr. 58, 124–132.CrossRefGoogle Scholar
Rölleke, S., Muyzer, G., Wawer, C., Wanner, G. & Lubitz, W. (1996). Appl. Environ. Microbiol. 62, 2059–2065.CrossRefGoogle Scholar
Rölleke, S., Witte, A., Wanner, G. & Lubitz, W. (1998). Int. Biodeter. Biodegr. 41, 85–92.CrossRefGoogle Scholar
Romano, I., Nicolaus, B., Lama, L., Manca, M.C. & Gambacorta, A. (1996). Syst. Appl. Microbiol. 19, 326–333.CrossRefGoogle Scholar
Saiz-Jimenez, C. & Laiz, L. (2000). Int. Biodeter. Biodegr. 46, 319–326.CrossRefGoogle Scholar
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor Laboratory Press, New York.Google Scholar
Schabereiter-Gurtner, C., Piñar, G., Lubitz, W. & Rölleke, S. (2001). J. Microbiol. Meth. 45, 77–87.CrossRefGoogle Scholar
Spring, S., Ludwig, W., Marquez, M.C., Ventosa, A. & Schleifer, K.-H. (1996). Int. J. Syst. Bacteriol. 46, 492–496.CrossRefGoogle Scholar
Stan-Lotter, H., Radax, C., McGenity, T.J., Legat, A., Pfaffenhuemer, M., Wieland, H., Gruber, C. & Denner, E.B.M. (2004). Halophilic Microorganisms, pp. 89–102. Springer Verlag, Berlin.CrossRefGoogle Scholar
Täubel, M. (2001). Diploma Thesis, University of Vienna.Google Scholar
Teske, A., Sigalevich, P., Cohen, Y. & Muyzer, G. (1996). Appl. Environ. Microbiol. 62, 4210–4215.CrossRefGoogle Scholar
Tomlinson, G.A. & Hochstein, L.I. (1976). Can. J. Microbiol. 22, 587–591.CrossRefGoogle Scholar
Ventosa, A., Marquez, M.C., Garabito, M.J. & Arahal, D.R. (1998). Extremophiles 2, 297–304.CrossRefGoogle Scholar
Ward, D.M., Weller, R. & Bateson, M.M. (1990). Nature 345, 63.CrossRefGoogle Scholar
Welsh, J. & McClelland, M. (1990). Nucleic Acids Res. 18, 7213–7218.CrossRefGoogle Scholar
Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. & Tingey, S.V. (1990). Nucleic Acids Res. 18, 6531–6535.CrossRefGoogle Scholar
Yoon, J.H., Kim, I.G., Kang, K.H., Oh, T.K. & Park, Y.H. (2003). Int. J. Syst. Evol. Microbiol. 53, 1297–1303.CrossRefGoogle Scholar