View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Middlesex University Research Repository Available online at www.sciencedirect.com Journal of Cultural Heritage 10 (2009) 124e133 Case study Bacterial community analysis on the Mediaeval stained glass window ‘‘Nativita`’’ in the Florence Cathedral Massimiliano Marvasi a, Elisabetta Vedovato a, Carlotta Balsamo a, Azzurra Macherelli b, Luigi Dei b, Giorgio Mastromei a, Brunella Perito a,* a Dipartimento di Biologia Evoluzionistica ‘‘Leo Pardi’’, University of Florence, Via Romana 19, 50125 Florence, Italy b Dipartimento di Chimica, University of Florence, Via della Lastruccia 3, 50019 Sesto F.no, Florence, Italy Received 7 November 2007; accepted 5 August 2008 Abstract Microbial corrosion of glass causes problems on delicates antique glass samples. Until now, the effect of microbial activity on corrosion phenomena has not been well documented. Only a few studies have been published concerning the microflora growing on glass surfaces. The present study deals with the characterization of cultivable aerobic bacteria isolated from the historical glass window ‘‘Nativita`’’ in the Florence Cathedral, designed by Paolo Uccello and realized by Angelo Lippi between 1443 and 1444. Microbial strains were sampled from four of the 25 panels of the ‘‘Nativita`’’ in the occasion of a recent conservation treatment, due to the presence of various kinds of crusts. One hundred microorganisms were isolated, about 50% bacteria and 50% fungi. Bacteria were submitted to morphological characterization and classified in the Gram group. For twenty strains, from different glass panels, the 16S rDNA gene was amplified and sequenced. Sequence analysis showed genus Bacillus, Arthrobacter and Paenibacillus as the most representative. In particular Bacillus and Paenibacillus are crusts associated. Phylogenetic relationship among isolates was determined. Chemical analysis of the glass and crusts completed the study. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Glass; Bacteria; Microbial communities; 16S rDNA 1. Introduction Microorganisms, furthermore, are able to acquire the elements needed for growth from the glass itself [2]. Glass, like any other material, is damaged by aging. Drastic The microbial flora is usually the result of successive changes in the appearance, colour, and structure occur on colonisations by different microorganisms. A variety of stained glasses which have a large surface exposed to the microorganisms, including lichens (Diploica, Pertusaris, atmosphere. Physicochemical mechanisms of deterioration are Lepraria sp.), fungi (Aspergillus sp., Penicillium sp.) and known [1] and microorganisms could accelerate physico- bacteria (Flexibacter sp., Nitrosospira sp., Arthrobacter sp., chemical phenomena leading to decay processes. Recently, Streptomyces sp., Micrococcus sp., Frankia sp., Geo- various papers have addressed the role of microorganisms in dermatophilus sp.) were shown to grow on the glass surface glass biodeterioration. Microorganisms can enhance the dete- [4,5,7e10]. Moreover, it has been demonstrated that bacterial rioration process by excretion of chemically aggressive and fungal communities on biodeteriorated glass surfaces are substances and by physical attack on the glass [2e6]. much more complex than previously believed [10,11]. The study of microbial communities on mediaeval glass is interesting to understand the relationship between the micro- organisms and the glass surface. Studies about microbial * Corresponding author. communities are useful for monitoring microorganisms after E-mail address: brunella.perito@unifi.it (B. Perito). completion of the conservation procedure [12], they are also 1296-2074/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.culher.2008.08.010 M. Marvasi et al. / Journal of Cultural Heritage 10 (2009) 124e133 125 useful in identifying biocides able to eliminate sampled with ten plates: panels 6, 14 and 17 on the outside microorganisms. surface of the window and panel 7 on the inside surface of the In the last fifteen years molecular techniques based on window (Fig. 1). On each panel, different glass type and crusts rDNA sequences have been used to identify microorganisms were chosen for sampling. The plates were incubated at room on artistic glasses [10,11]. temperature for three days. Bacterial colonies were re-isolated In this paper we report the characterization of bacteria isolated several times, to obtain pure cultures, on Nutrient Agar from the historic window ‘‘Nativita`’’ in the Florence Cathedral. medium. Strains were named by a number, corresponding to The window ‘‘Nativita`’’ belongs to a wide iconographic the panel, followed by a letter, corresponding to the sampled plan to glorify the Virgin Mary. The window, of extreme area of the panel, and by a last number identifying the strain. artistic value, was designed by Paolo Uccello and created between 1443 and 1444 by Angelo Lippi. It is located in the 2.2. Phenotypical characterization north-west position, in the tambour under Brunelleschi’s dome in the Florence Cathedral. Colonies were examined under the stereomicroscope in The window has recently been dismantled and restored. order to characterize their shape. Cell morphology was Microorganisms were isolated from glasses before restoration observed in fresh samples with a phase contrast Nikon and characterized. Chemical analysis of glass and crusts have Alphaphot YS microscope at 400 and 1000 magnifications. completed this case study. Lysis test was performed with KOH 3% (water solution) to classify the isolates into the Gram group [13]. 2. Materials and methods 2.3. Test of copper resistance [14] 2.1. Sampling and growth condition Single colonies were inoculated in 10 ml Nutrient Broth Contact plates (BIOSTER) filled with Nutrient Agar and incubated at 28e37 C with shaking at 150 rpm. Cells (OXOID) with 1% glucose were used for microbiological were counted in a Petroff Counting Chamber (Hausser sampling. Four of the twenty-five panels of the windows were Scientific Company). An aliquot (10 ml) of a cell suspension Fig. 1. Window of ‘‘Nativita`’’ in the Florence Cathedral. It was made between 1443 and 1444 by Angelo Lippi. It represents the birth of Jesus. The numbers represent the sampled panels. 126 M. Marvasi et al. / Journal of Cultural Heritage 10 (2009) 124e133 containing 5 Â 108 cells/ml was spotted on Nutrient Agar divergences among strains were quantified by using the plates added with various concentration of CuSO4 (0; 0.2; 0.4; Kimura-2-parameter distance model [19]. For treatment of 0.8; 1.6 and 3.2 mM). Plates were incubated at two different gaps ‘‘Complete Deletion’’ option was chosen. Bootstrap temperatures of 28 and 37 C, depending on the growth analysis (1000 replicates) was used to test the topology of the conditions. Minimal inhibitory concentration (MIC) was neighbour-joining method data. expressed as the concentration that inhibited confluent growth of the culture after 24, 48 or 72 h (depending on the growth 2.6. Chemical analysis on green glass surfaces and on speed of strains). Each strain was tested at least twice. decay crusts 2.4. Molecular techniques The green glass was analyzed by Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-rays To extract DNA, bacteria were grown on Nutrient Agar as (EDX) analysis. The two most common types of grey and red confluent patina. DNA extraction was performed with crusts on panels, besides of SEM/EDX, were characterized FastDNA Kit (Q-BIOgene) according to the manufacturer’s also by Fourier Transform InfraRed spectroscopy (FTIR). Two specifications. different regions of the green glasses were analyzed. The first The 16S rDNA amplification was performed using primers named ‘‘external side’’, referring to the surface that in the case P0 and P6 which anneal to positions 8e27 and 1495e1515, of green glass was not affected by the crusts, and the second respectively, of the Escherichia coli 16S rDNA gene [15]. one named ‘‘tickness side’’, that was the part of the glass Primers have a tail (underlined) of Universal M13 Forward where the lead bars (removed for the analysis) supported the primer preceding P6 (50-GTAAAACGACGGCCAGTC- glass. TACGGCTACCTTGTTACGA-30) and of Universal M13 For infrared measurements a Bio-Rad FTS 40 spectrometer reverse primer preceding P0 (50-CAGGAAACAGCTATGACC was used; measurements were carried out in the frequency GAGAGTTTGATCCTGGCTCAG-30). region from 4000 to 400 cmÀ1 with 4 cmÀ1 resolution and 32 A ‘‘touch-down PCR’’ program was used with annealing scans. The samples (powder coming form external crusts temperature decreasing in progressive cycles. PCR conditions present onto the glass) for infrared analysis were prepared consisted of an initial denaturing step of 5 min at 95 C fol- according to the KBr pellet technique. Microscopic observa- lowed by 5 cycles of 95 C for 30 s; 60 C for 30 s, 72 C for tion was done by SEM Cambridge stereoscan 360 fitted with 2 min; 5 cycles of 95 C for 30 s; 55 C for 30 s, 72 C for an Oxford Instrument energy dispersive X-ray analyzer model 2 min and 25 cycles of 95 C for 30 s; 50 C for 30 s and INCAX-sight 7060 on the above mentioned glass regions after 72 C for 2 min. The reaction was completed with a final graphitisation. extension at 72 C for 10 min and then cooled and held at 8 C. PCR was carried out with 1 unit of EuroTaq polymerase 3. Results (Euroclone) on Primus Thermal Cycles machine (MWG). A small amount of all PCR products was analyzed by electro- The window of ‘‘Nativita`’’ measures about 25 m2 (Fig. 1), phoresis on 0.8% agarose gel in TBE buffer. All PCR products it is made with several stained pieces of glass and it is sup- were purified with High Product Purification Kit (ROCHE). ported by lead bars. The pictorial decoration and the grisailles Purified fragments were sequenced with Universal M13 are inside the Cathedral. The window is cemented to the Forward and Reverse primers by the analyzer ABIPrism310 tambour structure with lead bars.