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

摘要

Abstract: In the last few years several investigations, based on culture-dependent and -independenttechniques, have shown that salt-attacked stone surfaces present a habitat for extremely salt tolerantand 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 crystallizationin the porous space of the stone is causing decohesion of material and destruction of the originalmedieval paintings. The salt, together with the oligotrophic conditions, creates a very special andextreme habitat for halotolerant and halophilic microorganisms. In this study we investigate and monitor the cultivable and non-cultivable members of the microbialcommunity present on the stonework of the medieval Chapel of St. Virgil after several severedisturbances of the microbial environment caused by desalination and disinfection treatments. With thisfinality, a combination of culture-dependent and -independent techniques was selected. The geneticdiversity of a total of 104 bacterial strains isolated from the stone samples was analysed by denaturinggradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD) analysis and 16SrRNA 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 isolatedstrains 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 clonelibraries and sequencing analyses, were used to monitor and identify Archaea, the non-cultivablefraction of the microbial community. The detected archaeal sequences were closely related to differentuncultured archaeons as well as to the cultured genera Halococcus and Halalkalicoccus andHalobacterium. Cultivation and molecular analyses revealed the presence of highly specialized microorganisms thatwere able to thrive and survive after several desalination and disinfection treatments in the extremeenvironment presented by the salt-attacked Chapel of St. Virgil.