Chemoorganotrophic bacteria Isolated From Biodeteriorated Surfaces In Cave And Catacombs

International Journal of Speleology 41(2) 125-136 Tampa, FL (USA) July 2012 Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs Filomena De Leo1, Agnese Iero1, Gabrielle Zammit2, and Clara E. Urzì1 Abstract: De Leo F., Iero A., Zammit G. and Urzì C. 2012. Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs. International Journal of Speleology, 41(2), 125-136 Tampa, FL (USA). ISSN 0392-6672. http://dx.doi.org/10.5038/1827-806X.41.2.1 The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study. Keywords: biofilm; catacombs; caves; chemoorganotrophic bacteria; clustering; 16S rDNA sequencing Received 20 October 2011; Revised 2 January 2012; Accepted 10 January 2012 INTRODUCTION Any study of microbial communities colonizing biodeteriorated surfaces should involve a combination of several analytical techniques such as microscopy, culture techniques, biochemical tests and molecular tools, which are designed to give complementary results. Each individual technique while having its own limitations, contributes the necessary information to provide a better understanding of the microbial community as a whole and its role in the deterioration of inorganic and/or organic substrata (Urzì et al., 2003). Culture-based techniques are selective due to the limited choice of media used for the cultivation of microorganisms, and because the viable and culturable microflora (VCM) may be restricted to 1 to 5% of the whole population (Amann, 2000). However, the culture-based approach offers the possibility to isolate and thus analyze a great number of strains (Donachie et al.,2007). It is then possible to study the isolated strains, to cluster them into operational taxonomic units (OTUs), to compare different microbial communities on the basis of types of cultivable bacteria isolated in term of richness and the frequency of isolates, distribution or relative abundance of types. Furthermore, cultivation-based techniques in association with other complementary molecular techniques give a good idea of the “microorganisms in action” because, Department of Life Sciences, “M. Malpighi” University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy;����� Corresponding author: Clara Urzì () 1 Department of Physiology and Biochemistry, University of Malta, Biomedical Sciences Building, Msida, MSD2620, Malta. 2 in most cases, strains that grow in culture can prove to be metabolically active microorganisms, if the environment provides the required conditions. This is particularly true in the conservation of indoor Cultural Heritage monuments where most of the dangerous microorganisms for the artefact itself (biodeteriogens) are those that grow epilithically on the surfaces; they form a biofilm and cover areas that are the most valuable because are sculptured or painted. Very often, in fact, stone surfaces are hidden by unaesthetic colonization due to phototrophic and chemoorganotrophic biofilms (Roldán & HernándezMariné, 2009), and it is a common practice to treat those surfaces with biocides in order to eradicate the biodeteriogens present (Nugari et al., 2009). However, biocides commonly applied on valuable surfaces are not always completely successful to eradicate the complex community within the biofilm (Salvadori & Charola, 2011). Thus it is important to study the culturable fraction of microorganisms in order to test in the laboratory if the chemical compounds used are effective against the deteriogenic microflora. For this reason, when dealing with a large number of strains, it is imperative to use a reliable and easily implemented technique to group the strains into homogeneous clusters and reduce the amount of work to be carried out to characterize the isolates. In this research study a large number of cultivable bacteria isolated from three sites were analyzed through a multi-step approach that included the fre���� quency of types of colony, preliminary description of their micro-morphology,������������������������������� clusterization of all the isolates via ITS-PCR, and the identification of selected strains within each cluster. Chemoorganotrophic bacteria isolated from cave and catacombs 126 MATERIAL AND METHODS Areas of study Sampling campaigns were carried out in the Ocean Cubicle (CSC13), inside the Catacombs of St. Callistus (Rome, Italy), in the Cave of Bats (Z1 to Z8) close to the village of Zuheros (Córdoba, Southern Spain) in the frame of the research activity carried out during the European project CATS (EVK4-200000028), and in different areas of St. Agatha’s Crypt and Catacombs (SA) in Rabat, Malta, during the activity carried out in the framework of a COST Action G8 scientific mission (COST-STSM-G8-1435) (Zammit et al., 2009). In both catacombs the relative humidity (RH) was always above 90% while the Mediterranean climate of the areas did not influence the inner temperature of the catacombs due to their deep location (range 1520°C) (Albertano et al., 2003; Zammit et al., 2009). The Cave of Bats presented a RH variable from 95 (inner part of the cave) to 56% (near the exit) and an average temperature between 8 and 14ºC, depending on the area, at the time of sampling (Urzì et al., 2010). Samples were taken aseptically in correspondence to alterations on rock surface described as black spots, green patina, whitish/grey patinas with scalpel and/ or adhesive tape as shown in Table 1. Cultural analyses For the isolation of chemoorganotrophic microorganisms samples were processed as described by Urzì et al. (2010). The following agarized media were used: BRII medium (Bunt and Rovira, 1955 modified as reported in Urzì et al., 2001), SC (Starch Casein KNO 3 agar, Kuster & Williams, 1964) and R2A (Reasoner and Geldreich, 1985 Oxoid); in all media 0.05% cycloheximide was added to avoid/limit the growth of unwanted fungal contaminants. Incubation was carried out at 28° C up to one month to allow slow-growing strains. At the end of incubation time, enumeration of microorganisms as cfu/g of sample was carried out and randomly chosen bacterial strains (10/20 colonies per sample) were isolated on Trypticase Soy Agar (TSA, Ox (...truncated)