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

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 RAPID 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.