Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome.

Different types of biofilms are widespread on lithic faces of the Catacombs of Domitilla (Rome, Italy) due to the favorable microclimatic conditions (temperature, high RH% and low irradiance). The biofilm, once established, becomes particularly dangerous due to the coverage of valuable surfaces causing spoilage, softening of materials and mineral precipitation. It is common practice to treat these surfaces with biocides in order to eradicate the microorganisms present. The aim of the present research was to compare the changes occurring to the microbial community present in the biofilm in one site of the Catacombs of Domitilla (CD15) before and after a biocide treatment (a mixture of quaternary ammonium compounds and octylisothiazolone, OIT), applied for a one month period. A multistep approach was followed, based on microscopy, cultural methods and molecular techniques (f-ITS and 16S rDNA sequencing), for the phenotypic and genetic analysis of the culturable microbial population. Our results highlighted that the biocide treatments had little effect against cyanobacteria, while the bacterial population increased in numbers but changed drastically in terms of diversity. In fact, some bacteria proliferate at the expense of the organic matter released by dead microorganisms as demonstrated by laboratory tests. Further, our data describe how the microbial interaction can have different responses depending on the favorable conditions for one kind of microorganism in respect to the others. This study exemplifies the real risks of applying biocide treatments on complex microbial communities and pinpoints the necessity of subjecting treatments to monitoring and reassessment. Moreover, the work showed the potential of bacteria isolated after the treatment for use, under controlled conditions, in combatting unwanted microbial growth in that they possess a positive tropism toward stressed microorganisms and high hydrolytic enzymatic activity against cell components (e.g. cellulose, chitin and pectin). A tentative protocol is proposed.