A non-invasive approach to the polyphasic study of biodeteriogenic biofilms at St Agatha Crypt and Catacombs at Rabat, Malta

Abstract

The Maltese islands are rich in hypogean archaeological sites dating back to 3600 B.C., as well as late Roman and Byzantine catacombs dating back to the third century A.D. Even though the occurrence of subterranean sites is common on the Maltese islands and biofilms colonising stone widespread, limited studies have been conducted to date into the diversity of these epilithic microbial communities in such environments. Microorganisms are known to deteriorate cultural heritage, especially subterranean archaeological sites where the high relative humidity, limited air circulation and relatively constant temperature throughout the year support their development. These multispecies communities are characterised by complex interactions among organisms and with the mineral substrata. The main aim of this study was to define the microbial diversity at St. Agatha’s Crypt and Catacombs and to identify the alteration they cause on stone by using non-invasive sampling methods. A variety of microscopy techniques and culture methods were used to characterise the microflora. A total of 24 samples were collected at different locations by removing the superficial microbial film only and not the underlying substratum. Observation by epifluorescence, and light microscopy evidenced the abundant presence of the cyanobacteria Chroococcidiopsis, Leptolyngbya and Fischerella, some green algae, moss protonemata and associated populations of filamentous and rod-shaped bacteria. The high diversity of the chemoorganotrophs was revealed by culture techniques. Fungi were not observed in fresh samples, though mostly represented in culture by ubiquitous species of Penicillium, Aspergillus, Fusarium and Cladosporium. Only in the case of a resin statue was an extensive covering of black Cladosporium spores observed. Non-invasive sampling methods were useful in resolving the spatial and patchy distribution of biofilm on different substrata, in assessing the biofilm diversity and revealing the intimate relationships between biofilm-forming microorganisms in Maltese catacombs.