The biodiversity of subaerophytic phototrophic biofilms from Maltese hypogea

The present study focuses on a description of the biodiversity of subaerial phototrophic biofilms occurring on archaeological surfaces in Maltese hypogean environments, namely St Paul's, St Agatha's and Abbatija tad-Dejr Catacombs, all situated in Rabat and the ancient Hal Saflieni Hypogeum at Paola, Malta. Direct observation of the biofilms, carried out using light (LM), epifluorescent and confocal laser scanning microscopy (CLSM), allowed the description of the major cyanobacterial and microalgal taxa, and also the associated heterotrophic microorganisms, mainly actinobacteria. Some biofilm microorganisms were able to grow in culture and this allowed the isolation of cyanobacterial, microalgal and chemoorganotrophic bacterial strains. Thylakoid arrangement and cell division were examined using transmission electron microscopy (TEM). The cytomorphology of isolated microorganisms was described. The undisputed phototrophic protagonists in these subaerial biofilms of Maltese hypogean environments were the non heterocytous (Oscillatorialean) cyanobacteria. In order to increase the limited data available for Leptolyngbya spp. from aerophytic epilithic biofilms in catacombs, the 16S rRNA genes of isolated Leptolyngbya strains were sequenced and compared with those obtained for related strains. Phylogenetic trees of cyanobacterial 16S rRNA sequences were constructed using parsimony and Bayesian analyses. Microorganisms forming biofilms in Maltese hypogea were found to be similar, both cytomorphologically and genetically, to those colonising lithic surfaces of caves and catacombs in other Mediterranean countries.