Microb Ecol (2010) 60:81–95 DOI 10.1007/s00248-010-9672-z ENVIRONMENTAL MICROBIOLOGY Biodiversity of Phototrophic Biofilms Dwelling on Monumental Fountains Oana A. Cuzman & Stefano Ventura & Claudio Sili & Cristina Mascalchi & Tulio Turchetti & Luigi P. D’Acqui & Piero Tiano Received: 24 September 2009 / Accepted: 3 April 2010 / Published online: 6 May 2010 # Springer Science+Business Media, LLC 2010 Abstract Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical–chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens Electronic supplementary material The online version of this article (doi:10.1007/s00248-010-9672-z) contains supplementary material, which is available to authorized users. O. A. Cuzman (*) : P. Tiano (*) CNR-ICVBC Istituto per la Conservazione e la Valorizzazione dei Beni Culturali, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy e-mail: cuzman@icvbc.cnr.it e-mail: p.tiano@icvbc.cnr.it S. Ventura (*) : C. Sili : C. Mascalchi : L. P. D’Acqui CNR-ISE Istituto per lo Studio degli Ecosistemi, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy e-mail: s.ventura@ise.cnr.it T. Turchetti CNR-IPP Istituto per la Protezione delle Piante, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of microenvironmental conditions for each fountain. Introduction Monumental fountains are particularly affected by microbial colonization due to constant presence of water, which permits a fast and intense growth in the presence of light. The developed photosynthetic patina gives rise to biodegradation processes combined with visual aspect alterations. Different micro-environmental conditions with areas constantly in contact with water or only sporadically wet, and others perpetually dry, offer an ideal growth substrate for a complex biocoenosis. These various habitats determine a different distribution of organisms on the fountain according to their specific requirements [28]. Special attention to the maintenance of monumental fountains is given in Italy and Spain but mainly when nonbiological alterations appear and their state of conservation must be evaluated [8, 24, 27, 39]. Usually, detailed data related with the microbial composition are not considered necessary for restoration processes, biological patina/biofilms being usually eliminated by mechanical removal, or by nonspecific biocide treatments. Taking into account that the colonization process is a natural event that restarts after each removal, a microbial ecology study can contribute to understand which are the most adapted genera/species dwelling on these artifacts, which ones are the most susceptible as biodeteriogens, and, therefore, what new methods can be developed to control unwanted biological colonization in its initial steps. Our aim was to identify the principal microbial components (algae, cyanobacteria, and fungi) of the phototrophic 82 biofilms developed on monumental fountains located in two different countries and to compare the biodiversity of different fountains biotopes in relationships to water availability, light exposure, and lithotypes. Materials and Methods Investigated Fountains A total of 31 samples were collected in sterile vials by scraping from different locations of four artistic fountains (Fig. 1) located in Italy and Spain, using a sterile scalpel. Table 1 contains the description of the collected samples. The samples coming from fountains of Florence were immediately transported to laboratory for microbiological analysis. The samples coming from Spain were either kept refrigerated or preserved in 2% formalin and immediately sent to our laboratories in Florence by air express courier where they were subsequently subjected to the microbiological procedures. Figure 1 Monumental fountains investigated in this study: a Tacca’s Fountain, Florence (Italy); b Second Fountain from Villa la Pietra, Florence (Italy); c Fountain from Patio de la Sultana, Generalife, Granada (Spain); d Fountain from Patio de la Lindaraja, Alhambra, Granada (Spain) O. A. Cuzman et al. Isolation, Identification of Microorganisms, and Biofilm Observation Aliquots of the collected samples suspended in standard saline solution were sown in Petri dishes containing the respective solid medium for the isolation of (1) cyanobacteria (BG-11), (2) nitrogen-fixing cyanobacteria (BG-110), (3) algae (modified BG-11 (5 ml/L NaNO3) diluted 1:1 with sterile water), and (4) fungi (PDA Difco). BG-11 type media were described in [32]. Petri dishes containing cultural media for phototrophs were incubated under continuous low white fluorescent light at a photosynthetic photon flux density of 10 μmol photon m−2 s−1 at 27°C, while those containing PDA for fungi were incubated in darkness, at 26°C. Developed cyanobacterial, algal, and diatom colonies were transferred into tubes containing their specified liquid cultural media, while fungal strains were isolated on Petri dishes with PDA medium. All isolated strains were maintained on slants with their specific cultural medium in appropriate light and temperature conditions. Biodiversity of Phototrophic Biofilms on Monumental Fountains 83 Table 1 The characteristics of collected samples from five investigated monumental fountains No. Fountain 1 Fountain characteristics Encodes, typology, and sampling position Tacca’s Fountains, Florence, Italy Made of marble (pedestal and border of 1T—light green biofilm continuous wet because of the flowing water, basins) and pietra Serena sandstone (basins) cylindrical marble pedestal, east orientation 2T—dark green biofilm sporadically wet due to the splashing of water, right corner of the parallelepiped pedestal, east orientation Running water from public water system O1—brown biofilm below the water level, internal part of the basin, south orientation O2—green-brown biofilm below the water level, internal part of the basin, south orientation N1—brown biofilm above the water level, sporadically wet due to the water splashing, internal part of the basin, north orientation N2—green-brown biofilm above the water level, sporadically wet due to the water splashing, internal part of the basin, north orientation 2 Second Fountain of Villa la Pietra, Made of concrete (all the fountain) Florence, Italy VP1—gray powder above the water level, dry internal part of the basin, northeast orientation VP2—black crust, dry internal part of the basin, northeast orientation Out of use, with stagnant water VP3—brown-green biofilm below the water level, internal part of the basin, north-east orientation VP4—brown-green biofilm, interface water/air, intermittent wetted area, internal part of the basin, northeast orientation VP5 and VP6—light green and turbid deposition, bottom of basin 3 Fountain from Patio de la Sultana, Sierra Elvira stone (all the fountain, Generalife, Granada, Spain surtidores) S1a—green biofilm under the water level, internal part of the central fountain, east orientation S1b—green biofilm, low part of the pedestal trunk of the central fountain with constant high wetness, south orientation Running water from Darro river S1c—green biofilm, the same level with the water one, corner of the pedestal basement, north orientation S1d—green biofilm, low part of the pedestal trunk of the central fountain with constant high wetness, east orientation S1e—green biofilm, low part of the pedestal trunk of the central fountain with constant high wetness, north orientation S1f—green biofilm, low part of the pedestal trunk of the central fountain with constant high wetness, west orientation S2—green spots on gray patina, sporadically wet by splashed water, continuous shaded “surtidor,” west orientation S5N—gray patina, rarely wet, continuous unshaded “surtidor,” north orientation S5W—brownish patina, rarely wet, continuous unshaded “surtidor,” west orientation S6N—dark-green patina, rarely wet, continuous unshaded “surtidor,” north orientation S6S—gray patina, rarely wet, continuous unshaded “surtidor,” south orientation S9W—red brownish patina, continuously wet, continuous shaded “surtidor,” west orientation S9N—dark-green patina, continuously wet, continuous shaded “surtidor,” north orientation S9S—green patina, continuously wet, continuous shaded “surtidor,” south orientation S9E—green patina, continuously wet, continuous shaded “surtidor,” east orientation 4 Fountain from Patio de la Lindaraja Alhambra, Granada, Spain Made of marble (pedestal) and Sierra Elvira stone (basin) 2L—green patina, sporadically wet, internal part of zigzag ornaments of superior marble basin, south-east orientation Running water from Darro river 3L—green patina, sporadically wet, internal part of superior marble basin, south orientation 5L—green biofilm, sporadically wet, internal horizontal part of pilaster ornaments, west orientation 6L—green patina, continuously wet, internal part of foundation, west orientation 84 The morphological characterization and morphotype identification were carried out by optical microscopy (Nikon Eclipse E600) according to [2, 6, 14, 17–19]. Representative isolated cyanobacterial strains were also characterized by phylogenetic analysis of the 16S rRNA gene. Cross-section of small solid parts of the samples collected from the Sultana Fountain were observed by epifluorescence microscopy (Nikon Eclipse E600) using a UV-2A filter cube (Ex 330–380 nm, DM 400 nm, BA 420 nm). A polystyrene resin (Mecaprex MA2, Presi, France) was used to embed the samples. Images were recorded using a Nikon DXM1200F digital CCD camera. Fourier transform-infrared (FT-IR) spectroscopy was used for the characterization of cross-section layers. The FT-IR spectra have been recorded using a Perkin Elmer System 2000 FT-IR spectrophotometer equipped with diamond cell. DNA Extraction Total DNA was extracted both from isolated cyanobacterial strains and from raw samples collected from the Second Fountain of Villa la Pietra, Florence, Italy and from the Sultana Fountain of Generalife, Granada, Spain. The extracted DNA from isolated strains was used for genesequencing purposes, while the DNA extracted from the raw samples was subjected to the automated ribosomal RNA intergene spacer analysis (ARISA). Thirty milliliters of liquid cultures (BG-11 or BG-110 media) of the isolated cyanobacterial strains were incubated with agitation in a Gallenkamp Orbital Incubator (atmosphere enriched with CO2 4%, 27°C, light/dark cycles 14/ 10 h). The biomass was harvested by centrifugation, and the pellet fraction was washed with standard saline solution (0.1% w/v) twice or more in case of mucilaginous cultures. Then, the pellet was divided into aliquots (≈ 30 mg) and frozen at −20°C. At the time of DNA extraction, pellets were thawed and treated with PowerPlant™ DNA Isolation Kit, according to the manufacturer’s protocol (Mo Bio Laboratories Inc.). Total community DNA of samples collected from the fountains was extracted using PowerSoil™ DNA Isolation Kit, according to the manufacturer’s protocol (Mo Bio Laboratories Inc.). All the DNA extracts were loaded onto 0.5% agarose gel previously stained with ethidium bromide (10 mg/ml). The quality of DNA extracts was examined under UV light after 15 min DNA migration at 150 V. PCR Amplification and Sequencing of the 16S rRNA Gene To amplify the 16S rRNA gene plus the adjacent ITS region, universal primer 16S27F (5′-AGAGTTTGATCCTGGCT CAG-3′) and cyanobacterial specific primer 23S30R O. A. Cuzman et al. (5′-CTTCGCCTCTGTGTGCCTAGGT-3′) were used [12, 36]. The genomic DNA of isolated cyanobacterial strains (1 µl) was amplified using Hot Start Master (Larova GmbH, Germany). PCR was carried out with an activation and initial denaturation step of 2 min at 94°C, followed by 30 cycles of denaturation of 45 s at 94°C, annealing of 1 min at 55°C, and elongation of 2 min at 72°C, followed by a final extension step of 7 min at 72°C. All amplification reactions were performed in a TGradient Thermal Cycler (Biometra, Goettingen, Germany). The amplicons were visually quantified by comparison with DNA Molecular Weight Marker VI (0.15–2.1 kbp; Roche, Mannheim, Germany) on agarose gel electrophoresis. The amplified DNA fragments were purified using ExoSAP-IT Clean-Up kit following the manufacturer’s instructions (USB Corporation, Affymetrix, Inc., Santa Clara, CA, USA) and then sent to BMR Genomics, Padua, Italy (www.bmr-genomics.it) for sequencing. The gene sequences of the 16S rRNA gene of the 30 strains isolated from three monumental fountains (Tacca’s Fountain, Second Fountain, and Fountain from Patio de la Sultana) were obtained using the three sequencing primers 16S979F, 16S544R, and 16S1092R [16]. Single reads were aligned in a unique 16S rRNA gene sequence using the software suite PHRED, PHRAP CONSED developed by the University of Washington [15] and consensus sequences imported in ARB [22], which was used for the subsequent analytical steps. Most similar sequences included in the analysis were retrieved from the latest SILVA database [29]. The SINA aligner of the SILVA website (http://www.arb-silva. de) was also used to produce the sequence alignment for the ARB software. The alignment was later visually checked and corrected under ARB. Phylogenetic relationships of the sequences were calculated with ARB using neighborjoining (NJ) [33], maximum likelihood (ML), and parsimony algorithms using sequence stretches over 1,200 bp long. A matrix of sequence distances produced by ARB was used to infer percent sequence similarities. PCR Amplification and ARISA PCR amplification of the rRNA 16S gene for ARISA was performed using two specific primer sets, for the eubacterial and cyanobacterial domains, respectively (see Table 2). The reaction mixture (50 µl each) for the PCR amplification contained: 5 µl ×10 DyNAzyme EXT buffer (with 15 mM MgCl2), 5 µl dNTPmix (2 mM), 1 µl of each primer (10 pmol/µl), 2.5 µl BSA (Bovine Serum Albumin, BioLabs, 10 mg/ml), 33.5 µl distilled water. Aliquots of the mix (48 µl) were placed in PCR tubes and sterilized under UV light (312 nm) for 8 min. Then, 1 µl of Taq DNA polymerase (Amersham Pharmacia) and 1 µl of genomic DNA were added. Reactions (50 µl final volume) were initially denaturated for 5 min at 94°C, followed by Biodiversity of Phototrophic Biofilms on Monumental Fountains Table 2 Primer sequences and target groups a R (reverse) and F (forward) designations refer to the primer orientation in relation to the rRNA 85 Primera Sequence (5′→3′) Target group Reference 16S1515F 23S30R-6FAM ITSF ITSReub-HEX AGT CGT AAC AAG GTA GCC GTA CC CTT CGC CTC TGT GTG CCT AGG T GTC GTA ACA AGG TAG CCG TA GCC AAG GCA TCC ACC Cyanobacteria Cyanobacteria Eubacteria Eubacteria [9] (modified) [21] [9] [9] 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, and then a final extension step at 72°C for 10 min was performed. The PCR products (5 µl each) mixed with bromophenol blue were visualized after 45 min of electrophoresis on 1.2% agarose gel stained with ethidium bromide (10 mg/ml). A standard DNA marker (Molecular Weight Marker VI, 0.15–2.1 kbp (Roche, Mannheim, Germany)) was loaded in the same gel (3 and 6 µl) in order to approximate the quantization of amplified DNA. The amplified DNA (2 ng/µl each) was then sent to BMR Genomics for fragment separation in a capillary chromatographer. A fluorescent dye-labeled size standard GeneScan™ ROX500 (35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, 500 bp fragments) was added, and the analysis was carried out with a GeneScan 3100 ABI Automated Capillary DNA Sequencer (Applied Biosystems). The computational analyses were performed with Peak Scanner ™ 1.0 (Applied Biosystems, 2006) and BioNumerics 2.5 (Applied Maths, Belgium) software. Only peaks higher than 50 relative fluorescence units and fragments between 100 and 650 bp long were included in the calculation. Principal component analysis (PCA) was performed using ARISA profiles obtained with BioNumerics software in order to evaluate the similarities of microbial communities of fountains samples. The intensity of peaks for each band was normalized in accordance with the total fluorescence of each sample ARISA profile. PCA was carried out using Unscrambler® Version 9.7 (Camo Software AS, Oslo, Norway) with the default parameters. Results Biodiversity of Photosynthetic Microorganisms on Monumental Fountains Microscopical observations of raw samples revealed that the main groups of phototrophic microorganisms occurring in the biofilm growing on the surface of the investigated monumental fountains belonged to Cyanobacteria, Chlorophyta, and Bacillariophyta. Table 3 contains the list of the organisms that could be microscopically determined in the samples retrieved from the fountains investigated in this work. The morphology of some phototrophic components of the biofilms present in the raw samples and some isolated strains is shown in Figs. 2 and 3, respectively. Cyanobacteria constituted an important group and were represented by coccoid (Chroococcus sp., Gloeocapsa spp.), filamentous (Leptolyngbya sp., Phormidium sp., Pseudophormidium sp.), and heterocytous forms (Calothrix spp., Nostoc spp.). Diatoms were identified in all investigated fountains (Navicula sp.), with a dominance in the Fountain of Patio de la Lindaraja. This could be due to the fact that this fountain is well kept and periodically cleaned; therefore, the stone surface is almost continuously sound and exposed to primary biological colonization. Some authors consider diatoms as primary colonizers of stone surfaces due to their ability to produce copious amounts of adhesive mucilage during the buildup of primary biofilms [38, 42]. Green algae were very well represented in all fountains, especially by Chlorella spp. and Cosmarium spp. In both fountains from Spain, Apatococcus lobatus is the most common species among the green algae, not observed in the Italian fountains. Some fungal hyphae were observed in the wet raw samples. Their isolation on specific cultural medium revealed the presence of Acremonium spp. and Phoma spp. as common genera in three different fountains. The black fungi such as Alternaria spp. and Torula spp. and some common fungal genera such as Aspergillus spp., Penicillium spp., and Fusarium spp. were also identified. Green algae such as Apatococcus sp., Chlorella spp., Cosmarium spp., Palmella sp., and diatoms as well, such as Achnanthes spp., Aulacoseira sp., Navicula spp., Nitzschia sp., have been preferentially observed in samples collected from submerged parts or from continuously and abundantly wet areas. In these habitats, Cyanobacteria were also represented, by genera such as Aphanothece spp., Borzia sp., Pseudanabaena sp. Some microorganisms such as Gloeocapsa novacekii, Gloeocapsa sanguinea, Calothrix parietina, and Leptolyngbya foveolarum have been observed on samples collected from dry habitats. The phylogenetic analysis of 30 cyanobacterial strains isolated from the studied fountains was performed on the basis of a nearly complete 16S rRNA gene sequence fragment of 1,230 bp. Forty-six cyanobacterial sequences from the public domain were added to the analysis for reference purposes; the 16S rRNA gene sequence of Chloroflexus aurantiacus was used to root the phylogenetic 86 O. A. Cuzman et al. Table 3 Occurrence of Cyanobacteria, Chlorophyta, Bacillariophyta, and Fungi in the four investigated monumental fountains Genus Table 3 (continued) Genus Fountain Fountain 1 1 CYANOBACTERIA Aphanocapsa sp. A. endophytica A. grevillei A. muscicola Aphanothece sp. A. stagnina Borzia trilocularis Calothrix spp. C. parietina Chroococcidiopsis sp. Chroococcus sp. C. minor C. turgidus Gloeobacter violaceus Gloeocapsa sp. G. novacekii G. sanguinea Leptolyngbya spp. L. foveolarum L. margaretheana Lyngbya martensiana Nostoc spp. Phormidium spp. P. aerugineo-caeruleum P. breve Pleurocapsa concharum Pseudanabaena sp. P. galeata Pseudophormidium tenue Rivularia sp. Schizothrix lacustris Staniera sp. CHLOROPHYTA Apatococcus sp. Apatococcus lobatus Chlorella sp. C. ellipsoidea Chlorella saccharophila Cosmarium sp. Dilabifilum prinzii Monoraphidium contortum Scenedesmus obtusus S. quadricauda Palmella sp. BACILLARIOPHYTA Achnanthes sp. Achnanthes affinis 2 + 3 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 3 4 Aulacoseira varians Cymbella leptoceros Diatoma sp. Epithemia zebrina Navicula spp. Nitzschia sp. N. communis Pinnularia sp. Synedra sp. FUNGI Acremonium spp. Alternaria spp. Aspergillus spp. Fusarium spp. Penicillium spp. Phoma spp. Torula spp. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1 Tacca’s Fountains; 2 Second Fountain from Villa la Pietra; 3 Sultana Fountain; 4 Lindaraja Fountain + + + 2 4 + trees. The three different approaches used, NJ, ML, and parsimony, resulted in nearly identical strain clustering. Bootstrap applied to NJ and Parsimony gave largely congruent values that demonstrated the stability of the phylogenetic inferences; thus, only the NJ tree, with the indication of bootstrap values, is given in Fig. 4, while parsimony and ML analyses can be found in Supplementary Figures 1 and 2, respectively. As shown on the right side of Fig. 4, 17 clusters that included sequences of strains isolated from the studied fountains, strongly supported by high bootstrap values, have been identified and in several instances assigned to known taxa as summarized in Supplementary Table 1. Six out of 17 clusters were also joined in couples in super-clusters with nearly complete bootstrap support. Genera Nostoc, Nodularia, and Anabaena, which belongs to Nostocaceae, also formed a supported cluster at 71/60% bootstrap, while members of the Nostocales included in the study clustered together at a 77/88% level. Taxonomic assignment and respective sequence similarities and bootstrap values of the clusters were given in Supplementary Table 1. Evidence of strain grouping related to the isolation place was not supported by the phylogenetic analysis. Microbial Community Study + Cross-sections of samples S1c and S2 collected from Sultana Fountain (Generalife Gardens, Alhambra, Granada) Biodiversity of Phototrophic Biofilms on Monumental Fountains Figure 2 Morphology under the optical microscope of different species of cyanobacteria (a– c, e, g–j), algae (k), and diatoms (d–f) observed in raw samples collected from Second Fountain from Villa la Pietra (a, b, g–j), Tacca’s Fountain (e), Fountain from Patio de la Sultana (c, f, k), and Fountain from Patio de la Lindaraja (d). a Schizothrix lacustris; b Calothrix parietina; c Lyngbya martensiana.; d Aulacoseira varians and Navicula spp.; e Achnantes affinis and Chroococcus turgidus; f Cymbella leptoceros.; g Aphanocapsa grevillei; h Gloeocapsa novacekii; i Chroococcus turgidus; j Borzia trilocularis; k Apatococcus lobatus. Scale bar 5 µm (a), 10 µm (b–k) 87 b a c d e f g i j revealed the presence of calcium carbonate layers alternating with the microorganisms layers. A stratification in the sample S2 which can be due to the alternation of dry/wet conditions which favored the precipitation was clearly h k visible in Fig. 5. The presence of the calcium carbonate was confirmed by FT-IR analysis of the white layers of these cross-sections. Recorded FT-IR spectrum with the specific peaks can be seen in Supplementary Figure 3. This 88 Figure 3 Isolated strains from the monumental fountains: a Chlorella ellipsoidea; b Scenedesmus obtusus; c Scenedsmus quadricauda; d Aphanocapsa muscicola; e Pseudanabaena galeata; f Aphanothece stagnina; g Leptolyngbya margaretheana; h L. foveolarum; i Chroococcus minor; j; Chroococcus sp.; k Pleurocapsa concharum; l Calothrix parietina; m Nostoc sp.; n Nitzschia communis. Scale bar 10 µm O. A. Cuzman et al. a d b c e f g h k i j l m process could be also enhanced by the presence of the microorganisms, especially cyanobacteria. In fact, the ARISA electropherograms of this sample (Fig. 6) revealed a certain diversity of cyanobacteria. The morphological identification of the microorganisms present in this sample revealed as dominant the presence of various types of Phormidium sp., an oscillatorialean cyanobacterium, reported for its ability of favoring calcium carbonate precipitation in layers [7]. n Figure 4 Neighbor-joining phylogenetic tree of cyanobacterial strains„ isolated from monumental fountains, based on 16S rRNA gene sequences. Percentages at nodes indicate bootstrap values obtained from 1,000 replicas. In bold: sequences from 30 cyanobacterial strains isolated from monumental fountains. Scale bar represents 0.01 substitution per nucleotide position. On the right, stable clusters 1 to 17 identified at the genus or species level. Nmusc: reference cluster for the species N. muscorum. Sequence accession numbers are given in parenthesis Biodiversity of Phototrophic Biofilms on Monumental Fountains Nostoc sp. 5N–02c Nostoc sp. 6S–05a 96 Nostoc sp. VP2–08 Nostoc sp. 2–07 73 Nostoc sp. 1b–05 Nostoc cf. verrucosum (AB245144) 72 Nostoc sp. 9E–03 51 Nostoc sp. CENA107 (EF088341) Nostoc sp. Al1 (AM711522) Nostoc calcicola III (AJ630447) Nostoc sp. 9S–06 Nostoc edaphicum X (AJ630449) Nostoc sp. Ev1 (AM711535) 94 Nostoc commune M–13 (AB088405) Nostoc commune O Brien 02011101 (DQ185223) 78 Nostoc muscorum I (AJ630451) Nostoc sp. Cc2 (AM711532) 56 Nostoc ellipsosporum V (AJ630450) 55 Nostoc sp. PCC 7120 (BA000019) 71 Nodularia sphaerocarpa UTEX B 2092 (AJ781151) 77 Anabaena planctonica 1tu28s8 (AJ630430) Calothrix parietina 2T10 Calothrix sp. 2T08 99 Calothrix sp. UAM 315 MU27 (EU009152) 91 Calothrix desertica PCC 7102 (AF132779) 100 Calothrix sp. PCC 7714 (AJ133164) 100 Rivularia sp. VP4–08 58 Rivularia sp. UAM 305 MU24 (EU009149) Chroococcidiopsis sp. 9E–07 100 Chroococcidiopsis cubana SAG 39.79 PCC 7326 (AJ344558) 48 Chroococcidiopsis thermalis PCC 7203 (AB039005) Chroococcidiopsis sp. BB79.2 SAG 2023 (AJ344552) 75 Chroogloeocystis siderophila 5.2 s.c.1 (AY380791) Pleurocapsa cf. concharum 1d–08 Microcoleus glaciei UTCC 475 (AF218374) 100 Phormidium murrayi ANT.ACEV5.2 (AY493627) 68 Phormidium aerugineo–caeruleum 9N–01 Microcoleus sp. HTT–U–KK5 (EF654070) Microcoleus chthonoplastes SAG 38.98 (EF654089) 100 Chroococcus sp. VP2–07 Chroococcus sp. VP2–04c Chroococcus sp. 9E–05 Chroococcus sp. VP2–02 Chroococcus sp. 9E–04 99 Chroococcus sp. 2T05h 71 Chroococcus sp. JJCM (AM710384) 98 Microcystis aeruginosa 0BB35S02 (AJ635430) Pleurocapsa concharum VP3–02 56 Pleurocapsa sp. PCC 7327 (AB039007) Pleurocapsa concharum VP3–02b 83 Pleurocapsa concharum VP4–07 Cyanothece (Aphanothece) sp. PCC 7822 (ABVE01000002) Leptolyngbya sp. VP3–07 Leptolyngbya sp. CENA112 (EF088337) 99 Leptolyngbya frigida ANT.LH52.2 (AY493575) 96 Leptolyngbya frigida ANT.MANNING.1 (AY493573) 61 Leptolyngbya sp. 1T12c Leptolyngbya boryana UTEX B 485 (AF132793) Leptolyngbya foveolarum VP1–08 100 52 Leptolyngbya foveolarum Komarek 1964/112 (X84808) Aphanocapsa muscicola VP3–03 97 Aphanocapsa muscicola 5N–04 Acaryochloris sp. JJ8A6 (AM710387) 76 Acaryochloris sp. JJ7–5 (AM710386) Acaryochloris marina MBIC 11017 (AY163573) Leptolyngbya margaretheana 1T12 Leptolyngbya sp. 0BB19S12 (AJ639895) 98 Leptolyngbya nodulosa UTEX 2910 (EF122600) 100 Leptolyngbya sp. PCC 7104 (AB039012) 96 Leptolyngbya antarctica ANT.ACE.1 (AY493588) Pseudanabaena sp. PCC 6802 (AB039016) Pseudanabaena sp. 1a–03 86 Pseudanabaena sp. 0tu30s18 (AM259268) Pseudanabaena sp. 63–1 (EF110976) 100 Gloeobacter violaceus VP3–01 Gloeobacter violaceus PCC 7421 (BA000045) Chloroflexus aurantiacus J–10–fl (CP000909) 0.01 89 1 2 3 Nmusc 4 5 6 7 8 9 10 11 12 13 14 15 16 17 90 O. A. Cuzman et al. The microbial communities of the pedestal collected from N, S, E, W orientations (S-1e, S-1b, S-1e, S-1f) formed a separate group which was also associated with the community of sample 9N, possibly because of the humidity retention, being located in shadow. This group was distinguished by the presence of a eubacterial operational taxonomic unit with an ARISA fragment 535 bp long (Supplementary Figure 4). Discussion Figure 5 Biofilm cross-section of the sample S2 collected from Sultana Fountain (Granada, Spain) observed in UV light. The layers closer to the stone are in the bottom, while the thick microorganisms layer from the upper part of the photo is the most recently developed. Arrows indicate the calcium carbonate layers. Scale bar 500 µm The complex ARISA profiles of cyanobacteria and eubacteria of the microbial communities from the monumental fountains were compared using PCA. The PCA score-1 versus score-2 plot has been shown in Fig. 7. Four PCA factors accounted for around 81% of the total variance, with the first PC mainly characterized by a eubacterial fragment (E 535) as indicated by the line plot of accumulated bars shown in Supplementary Figure 4, accounting for 47% of the variance. The second PC (characterized by cyanobacterial fragments C 440 and C 321) accounted for 15%, the third PC (E 342 and C362) and fourth PC (E 471) accounted for 11% and 8% of the total variance, respectively. Unsupervised classification using the K-means clustering method analysis (based on the Euclidean distance; an Unscrambler® utility) identified four clusters of scores: one for the Second Fountain (Villa la Pietra, Florence, Italy) and three for the Sultana Fountain (Generalife Gardens, Alhambra, Granada, Spain). These clusters were reported in Fig. 7. Two clusters of the Spanish fountain seems to be identified by the light regime of their members: microbial communities of the samples located in the illuminated areas (S-1a, S–6S, S–6N, S–5W) were grouped together, while most of the samples located in shadow formed a separate group (S-2, S–9S, S–9W). The water presence influenced also the similarity of the microbial communities of the samples. Figure 6 ARISA electropherogram of the cyanobacterial community of sample S2 Monumental fountains offer a favorable biotope for the development of a complex microbial biodiversity due to relative ease of colonization. A literature survey of the types of photosynthetic microorganisms occurring on this kind of habitats was performed in order to identify the most common taxa and to compare them with those retrieved from other kinds of stones monuments. Studies on fountains by several authors have been listed in Table 4, while phototrophic microorganisms reported for monumental fountains have been listed in Table 5. Many papers mainly reported the occurrence of cyanobacteria and green algae as colonizers of stone cultural assets [38]. Ecologically, cyanobacteria, green algae, and diatoms inhabiting stone cultural assets may belong to two main groups: one is composed of species that can withstand extreme conditions such as highlight intensity and drought periods while the second group contained species living under dim light and constant humidity conditions [26]. Both conditions were found in artistic fountains; therefore, many microorganisms that were reported for stone artifacts, like Aphanocapsa sp., Calothrix sp., Chroococcus sp., Gloeocapsa sp., Nostoc sp., Oscillatoria sp., Phormidium sp., Synechosystis sp., Synechococcus sp., Chlorella sp., Scenedesmus sp., Achnanthes sp., Navicula sp., Nitzschia sp., and Pinnularia sp. [10, 11, 13, 20, 26, 37, 38], can as well dwell on monumental fountains as ubiquitous members. Many of the colonizers (Table 5) reported for the fountains mentioned in Table 4 belonged to Cyanobacteria (Chlorogloea sp., Chroococcus sp, Pleurocapsa sp., Phormidium sp.). The green algae were well represented by Chlorella sp. and Chlorococcum sp., while among the diatoms, Navicula sp. was the most common genus. Biodiversity of Phototrophic Biofilms on Monumental Fountains 91 Figure 7 The PCA score-1 versus score-2 plot generated from ARISA profiles for monumental fountains microbial communities. Four clusters of scores (based on Euclidean distance) were identified Microorganisms such as Calothrix sp., Leptolyngbya sp., Nostoc sp., Scenedesmus sp., Achnanthes sp., and Nitzchia sp. that were observed in the fountains investigated in this research (Table 3) had been also reported for other monumental fountains (Table 5). The fungal genera which have been identified in the investigated artistic fountains (Table 3) are very common for airborne microflora (Alternaria spp., Aspergillus spp., Fusarium spp.). Their presence can be due to biofilm mucilaginous consistency, which is able to entrap the airborne spores. Cyanobacteria, bacteria, algae, and fungi embedded in a gel-like polymeric matrix (extracellular polymeric substances (EPS)) and organized in a phototrophic biofilm can develop on all kind of stone surfaces, giving rise to colored patinas, incrustations, and causing biodeteriogenic processes. Same cyanobacteria are capable of direct precipitation of calcium carbonate onto/into their sheaths, but many species, even without this capability, induce precipitation of calcium carbonate by providing nucleation sites for its crystallization [26]. This behavior is especially attributed to: (1) various metabolic processes such as photosynthetic uptake of CO2 and/or HCO3−, ammonification, denitrification, sulfate reduction; (2) the presence of EPS; and (3) biofilms trapping airborne particles [31]. Biomineralization processes (concre- tion, accretion) were reported for cultural assets both in marine and freshwater environments [4, 23]. In the present work, two approaches have been implemented to reveal the diversity of cyanobacteria as the main phototrophic microorganisms in the biofilms colonizing the surfaces of the studied fountains and to compare the cyanobacterial and eubacterial community composition to the local microhabitat conditions. In the first approach, strains of cyanobacteria have been isolated and carefully identified with an integrated approach putting together morphological characters and molecular phylogeny based on the analysis of their 16S rRNA gene sequences. The molecular phylogeny (Fig. 4) very precisely reflected the botanical identification of the cyanobacterial isolates (Supplementary Table 1). In a certain way, this contrasted with the repeatedly claimed inadequacy of the classical taxonomy of cyanobacteria to represent their evolutionary relationships, while it clearly showed that complex morphological characters, when accurately determined, can supply valuable taxonomic indications. To support this conclusion, a wide range of 46 reference sequences from the public domain were included in the analysis; they have been selected considering both their relatedness to the fountain isolates and also the quality of their sequences, Table 4 The investigated fountains reported by various authors Fountain References Trevi Fountain, Rome, Italy Pretoria Fountain, Palermo, Italy Fountain of Tritone , Rome, Italy Four Rivers Fountain, Rome, Italy Fountain of Bibatauin, Granada, Spain Lions Fountain, Alhambra Palace, Granada, Spain Fountain from Patio de la Lindaraja, Alhambra Palace, Granada Spain El Bano de Comares and La sala de los Abencerrajes, Alhambra Palace, Granada, Spain Patio del Cuarto Dorado and Patio de los Arrayanes, Alhambra Palace, Granada, Spain A [25] B [24] C [1] D [30] E [40] F [35] G [4] H [5] I [34] 92 O. A. Cuzman et al. Table 5 Most common taxa of Cyanobacteria, Chlorophyta and Bacillariophyta occurring in the fountains reported in Table 4 Genus Frequency (%) Species (no) Species Reference CYANOBACTERIA Aphanocapsa sp. Aphanothece sp. Calothrix sp. Chamaesiphon sp. Chlorogloea sp. Chroococcidiopsis sp. Chroococcus sp. Gloeocapsa sp. Hyella sp. Lyngbya sp. Microcystis sp. Myxosarcina sp. Nostoc sp. Oscillatoria sp. Phormidium sp. 11 22 22 33 44 33 44 33 11 33 11 33 11 33 78 1 2 3 4 3 1 4 2 1 3 1 2 1 3 9 D F,I B E,G,I E,F,G,I E,G,H A,B,D,E B,E,I G A,B,I A B,G,I I A,B,D A,D,E,F,H,I 11 56 22 33 1 3 3 2 33 44 55 44 22 11 22 22 22 22 22 22 33 2 1 1 2 4 3 1 1 1 4 1 1 1 44 44 22 22 11 11 22 11 67 2 4 5 1 1 1 2 1 7 22 11 11 22 3 1 5 2 Plectonema sp. Pleurocapsa sp. Schizothrix sp. Symploca sp. CHLOROPHYTA Apatococcus sp. Chlorella sp. Chlorococcum sp. Chlorosarcinopsis sp. Cosmarium sp. Oocystis sp. Palmella sp. Pleurastrum sp. Poloidion sp. Scenedesmus sp. Spirogyra sp. Stichococcus sp. Ulotrix sp. BACILLARIOPHYTA Achnanthes sp. Amphora sp. Cymbella sp. Diadesmis sp. Diatoma sp. Epithemia sp. Gomphonema sp. Melosira sp. Navicula sp. Nitzschia sp. Pinnularia sp. Staurastrum sp. Synedra sp. A. grevilllei A. saxicola C. elenkinii, C. fusca C. incrustans, C. polonicum, C. polymorphus C. microcystoides, C. purpurea C. minutus, C. turgidus, C. varius G. compacta H. fontana L. martensiana, L. tenue M. chroococcoides O. agardhii, O. lacustris P. ambiguum, P. autumnale, P. calidum, P. favosum, P. foveolarum, P. retzii, P. subfuscum, P. uncinatum P. battersii P. minor, P. fluviatilis S. gomontii, S. tenuis S. elegans, S. muralis A. lobatus C. minor C. depressum, C. granatum, C. reniforme O. crassa, O. lacustris, O. solitaria P. miniata P. didymos S. ecornis, S. obliquus, S. smithii A. lanceolata A. ovalis, A. perpusilla, A. veneta C. affinis, C. cystula, C. helvetica, C. hustedtii, C. ventricosa D. vulgare E. sorex G. intricatum, G. olivaceum M. varians N. cari, N. criptocephala, N. gracilis, N. menisculus, N. rhyncocephala, N. seminulum N. linearis, N. palea A B,E,F,G,I G,H E,H,I G,H,I A,B,C,F,G B,E,F,H,I E,F,I D,E A G,I E,I H A,I B,I A,E B,E,I D,E,I A,D,F,I D,I H,I I D D,I I D,E,F,G,H,I A,D A S. boreale, S. lunatum, S. lunatum var. planctonicum, S. manfeldtii, S. pingue A S. ulna A,D Biodiversity of Phototrophic Biofilms on Monumental Fountains determined through the tools of the SILVA rRNA database project [29]. The results showed an excellent bootstrap support for the obtained clusters and a clear indication that classical cyanobacterial genera often consisted in phylogenetically coherent units. Also, sequence similarity values fairly matched to the indication given for the species level in the bacterial taxonomy (97.5%), while similarity values for the genus level were generally around 5% lower than the suggested reference value of 95%. Should this 90–95% similarity inside well delimited genera be confirmed, a more flexible adaptive potential given by a larger genetic diversity inside cyanobacterial genera could be hypothesized. As expected, the distribution of strains belonging to the genera Nostoc and Leptolyngbya did not correspond to a unique cluster for each genus. Even if a unique, large branch of the tree, indeed with a very low bootstrap support (56%), included the strains identified as belonging to the genus Nostoc, it is known from a comprehensive study of a variety of Nostoc strains that they did not form a monophyletic group (Hrouzek and Ventura, personal communication). Moreover, in the present work, fountain strains identified as belonging to Nostoc were grouped into three highly supported separate clusters. Out of them, cluster 3 could be identified as Nostoc commune on the basis of included reference sequences, while in Fig. 4, it was also shown that not any fountain isolate from this study belonged to Nostoc muscorum. Three separate clusters (12, 13, 15 in Fig. 4 and Supplementary Table 1) housed strains identified as Leptolyngbya sp. It is well known that these very simple, thin filamentous morphotypes, with limited morphological differentiation, cannot be described in a unique valid genus. At present, efforts from several laboratories have substantially failed in the precise identification of reliable phenetic characters reflecting phylogenetically coherent clusters. Nevertheless, a few valuable information can be obtained from the present study. Cluster 13 of Fig. 4 included L. foveolarum strain VP1-08, along with reference sequences of Leptolyngbya boryana and L. foveolarum; also Leptolyngbya tenerrima and Leptolyngbya angustata belonged to it (data not shown). Since L. boryana is the type species of the genus Leptolyngbya, cluster 13 deserved the generic name Leptolyngbya, and the nearly complete similarity of its members, reported in Supplementary Table 1, supported their collective identification with the species L. boryana. This in turn would raise the question whether L. foveolarum, L. tenerrima, and L. angustata are indeed anything more than synonyms of L. boryana. Two more Leptolyngbya clusters, numbers 12 and 15, were identified in Fig. 4; cluster 12 was stably related to L. boryana cluster 13, while cluster 15 was phylogenetically unrelated to the other two. Given the attribution of cluster 13 to the type species of the genus 93 Leptolyngbya, the supported super-cluster 12+13 could coherently correspond to the true genus Leptolyngbya, but from the present work, no other morphological evidences could be found strengthening molecular phylogeny data. The phylogenetic analysis also confirmed the clear separation of Calothrix from Rivularia, as described by Berrendero and colleagues [3]. Among the unicellular cyanobacteria, one should notice cluster 6, housing sequences belonging to Chroococcidiopsis, a typical colonizer of rather dry stone surfaces, and cluster 11 with representatives of Pleurocapsa concharum, adapted to more wet conditions. These ecological characters of the two taxa were clearly reflected in the origins of the samples from which the strains included in clusters 6 and 11 were isolated. Typical Aphanocapsa sp. isolates VP3-03 and 5N-04 clustered along with freshwater cyanobacterial strains identified as Acharyochloris on the basis of their sequence relatedness to Acharyochloris marina. Representative sequences of A. marina and of freshwater Acharyochloris spp. have been included in the tree. The taxonomic assignment of these latter freshwater cyanobacteria would need a more detailed study, considering the peculiarities of the genus Acharyochloris, and the new findings here reported that suggested a strong correlation with Aphanocapsa. The isolation of a cyanobacterial strain identified as Gloeobacter violaceus and the strong relatedness of its sequence with other sequences in the public domain described as belonging to the same species, definitely established the real existence of this long disputed cyanobacterial species. Considering that the cyanobacterial strains isolated during the present work came from three fountains located in two different countries, built with different lithotypes and subjected to different water regimes and cleaning practices, their phylogenetic clustering did not show any trace of geographical distribution, but in several clusters (as an example, clusters 1, 10, 15), very similar strains from separate locations were found. A study of the relationships between the diversity of the phototrophic biofilms and the characters of their sampling sites has been, therefore, performed using ARISA profiles of the communities. The PCA analysis stressed that micro-environmental conditions, such as light and water availability, and the type of stone used to build the fountains had an influence on microbial diversity gathering. The microbial community of the Second Fountain from Italy, made of concrete, formed a separated group with respect to the Sultana Fountain from Spain, which is made of Sierra Elvira stone. The values obtained with the PCA analysis indicated the importance of some cyanobacterial and eubacterial fragments in the microbial population studied. For example, the fragment E535 is common in the 56% of the samples studied. 94 The microbiological studies of the investigated artistic fountains showed the presence of a superficial microbial colonization with a significant biodiversity, which was influenced by different microenvironmental conditions. The biodeteriogenic activity of microorganisms on the stone cultural assets is well known [41] and monumental fountains are exposed to a strong risk caused by the water availability which favors the development of a thick patina. Biodegradation forms like encrustations and esthetical damage and other risks like water system clogging and proliferation of some pathogenic or toxic microorganisms affect artistic fountains. It must be also taken into account that all stone treatments have an influence on the bioreceptivity of the stone itself, possibly giving an advantage to specific microorganisms. The understanding of fountains microbial ecology is the starting point for a scientific approach to develop suitable, effective, and longlasting control methods. Acknowledgments The authors thank F. Bolivar (Faculty of Fine Arts, Univ. of Granada, Spain), The Council of Alhambra and Generalife (Granada, Spain), Florence Municipality, and the management of the Villa la Pietra (Florence, Italy) for their assistance in collecting samples. This work has been made in the frame of the EPISCON Project (MEST-CT-2005-020559—European PhD in Science for Conservation). References 1. Barcellona Vero L, Tabasso Laurenzi M (1982) La Fontana del Tritone di L. Bernini, a Roma: un esempio di alterazione legato a fattori chimici, biologici e ambientali. In: Proceedings of 3rd International Congress on the Deterioration and Preservation of Stones, Venezia, 24-27 October 1979, Italy, Istituto di chimica industriale Press, Padova, pp 511–516 2. Barnett HL (1998) Taxonomy and identification. In: Hunter BB (ed) Illustrated genera of imperfect fungi. APS, Minnessota, pp 6–198 3. Berrendero E, Perona E, Mateo P (2008) Genetic and morphological characterization of Rivularia and Calothrix (Nostocales, Cyanobacteria) from running water. Int J Syst Evol Microbiol 58:447–460 4. Bolívar Galiano FC, Sánchez Castillo PM (1997) Biomineralization processes in the fountains of Alhambra, Granada, Spain. Int Biodet Biodegr 40:205–215 5. Bolívar Galiano FC, Sánchez Castillo PM (1998) Biodeterioro en el Baño de Comares y la Sala de los Abencerrajes en la Alhambra: estudio comparativo. In: Valenciana G (ed) Proceedings of Congreso de conservatión y restauración de bienes culturales. Martin Impresores Press, Alicante, pp 549–553 6. Bourrelly P (1966) Les algues d’eau douce. N. Boubée&Cie, Paris, pp 1–475 7. Brehm U, Krumbein WE, Palinska KA (2004) Biomicrospheres generate ooids in the laboratory. Geomicrob J 23:545–550 8. Cardilli Alloisi L, Di Bella I (1979) La Fontana del Tritone di G.L. Bernini – problemi di restauro e manutenzione. In: Proceedings of 3rd International Congress on the Deterioration and Preservation of Stones, Venezia, 24-27 October 1979, Italy, Istituto di chimica industriale Press, Padova, pp 517–529 O. A. Cuzman et al. 9. Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlinni C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol 70:6147–6156 10. Crispim CA, Gaylarde PM, Gaylarde CC (2003) Algal and cyanobacterial biofilms on calcareous historic buildings. Curr Microbiol 46:79–82 11. Crispim CA, Gaylarde CC, Gaylarde PM (2004) Biofilms on Church walls in Porto Alegre, RS, Brazil, with special attention to cyanobacteria. Int Biodet Biodegr 54:121–124 12. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16 S ribosomal RNA. Nuc Acids Res 17:7843–7853 13. Flores M, Lorenzo J, Gómez-Alarcón G (1997) Algae and bacteria on historic monuments at Alcala de Henares, Spain. Int Biodet Biodegr 40:241–246 14. Geitler L (1932) Cyanophyceae. In: Akad. Verslagsges (ed) Rabenhorst’s Kryptogamenflora von Deutschland, Österreich und der Schweiz, Johnson Press, Leipzig, 1–1196 15. Gordon D (2004) Viewing and editing assembled sequences using consed. In: Baxevanis AD, Davison DB (eds) Current protocols in bioinformatics. Wiley, New York, pp 1–43 16. Hrouzek P, Komarek J, Lukesova A, Mugnai MA, Turicchia S, Ventura S (2005) Diversity of soil Nostoc strains: phylogenetic and morphological variability. Arch Hydrobiol Suppl Algol Stud 117:251–264 17. Komarek J, Anagnostidis K (1988) Modern approach to the classification system of cyanophytes. Arch Hydrobiol Suppl Algol Stud 53:327–472 18. Komarek J, Anagnostidis K (1998) Cyanoprokaryota 1. Teil Chroococcales. In: Ettl H, Gärtner G, Heynig H; Mollenhauer D (ed) Süβwasser-flora von Mitteleuropa, Fisher Press, StuttgartJena 19/1:1–548 19. Komarek J, Anagnostidis K (2005) Cyanoprokaryota 1. Teil Oscillatoriales. In: Büdel B, Gärtner G, Krienitz L, Schageri M (eds) Süβwasser-flora von Mitteleuropa. Elsevier, Munich, pp 1–759 20. Lamenti G, Tiano P, Tomaselli L (1997) Microbial communities dwelling on marble statues. In: Monte M (ed) Proceedings of the 8th Workshop Eurocare Euromarble EU 496. CNR, Rome, pp 83–87 21. Lèpere C, Wilmotte A, Meyer B (2000) Molecular diversity of Microcystis strains (Cyanophyceae, Chroococcales) based on 16 S rDNA sequences. Syst Geogr Plants 70:275–283 22. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nuc Acids Res 32:1363–1371 23. McNamara CJ, Lee KB, Russel MA, Murphy LE, Mitchell R (2009) Analysis of bacterial community composition in concretions formed on the USS Arizona, Pearl Harbor, HI. J Cult Herit 10:232–236 24. Not R, Miceli G, Terranova F, Catalisano A, Lo Campo P (1995) Indagini sulla natura biotica delle alterazioni. In: Catalisano A, Di Natale R, Gallo E, Vergara F (eds) Fontana Pretoria, studi per un progetto di restauro. Arti Grafiche Siciliane, Palermo, pp 51–63 25. Nugari MP, Pietrini AM (1997) Trevi Fountain: an evaluation of inhibition effect of water-repellents on cyanobacteria and algae. Int Biodet Biodegr 40:247–253 26. Ortega-Calvo JJ, Hernandez-Marine M, Saiz-Jimenez C (1993) Cyanobacteria and algae on historic buildings and monuments. In: Garg KL, Garg N, Mukerji KG (eds) Recent advances in biodeterioration and biodegradation. Naya Prokash, Calcutta, pp 174–203 Biodiversity of Phototrophic Biofilms on Monumental Fountains 27. Pietrini AM (1991) Un’indagine conoscitiva sulla microflora. In: Segrete C (ed) Fontana di Trevi, la storia, il restauro. Assessorato alla Cultura, Rome, pp 187–189 28. Pietrini AM, Ricci S (2005) Fontane e Nimfee. In: Nardini (ed) La biologia vegetale per i beni culturali. Nardini, Florence, pp 189–201 29. Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J, Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nuc Acids Res 35:7188–7196 30. Ricci S, Pietrini AM (1994) Characterization of the microfloral algae present on the Fountain of Four Rivers, Rome. In: Fassina V, Ott H, Zezza F (ed) Proceedings of III International Symposium on the Conservation of Monuments in the Mediterranean Basin, Venice, Italy, Soprintendenza ai Beni Artistici e Storici di Venezia Press, pp 353–357 31. Riding R (2000) Microbial carbonates:the geological record of calcified bacterial-algal mats and biofilms. Sedimentology 47:179–214 32. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier R (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61 33. Saitou N, Nei M (1987) The neighbor-jointing method: a new method for reconstruction phylogenetic trees. Mol Biol Evol 4:406–425 34. Sánchez Castillo PM, Bolívar Galiano FC (1997) Caracterización de comunidades epilíticas en fuentes monumentales y su aplicación a la diagnosis del biodeterioro. Limnetica 13:31–46 95 35. Sarró MI, García A, Rivalta VM, Moreno DA, Arroyo I (2006) Biodeterioration of the Lions Fountain at the Alhambra Palace, Granada (Spain). Build Environ 41:1811–1820 36. Taton A, Grubisic S, Brambilla E, De Vit R, Wilmotte A (2003) Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurado Dry Valleys, Antarctica): a morphological and molecular approach. Appl Environ Microb 69:5157–5169 37. Tomaselli L, Lamenti G, Bosco M, Tiano P (2000) Biodiversity of photosynthethic micro-organisms dwelling on stone monuments. Int Biodet Biodegr 46:251–258 38. Uher B, Aboal M, Kovacik L (2005) Epilithic and chasmoendolithic phycoflora of monuments and buildings in South-Eastern Spain. Cryptogam Algol 26:275–358 39. Zurita Peraza Y, Bolívar Galiano FC (2002) Le fontane monumentali dell’Alhambra a Granada. In: Nardini (ed) Kermes, Nardini Press, Florence, pp 63–68 40. Zurita Peraza Y, Cultrone G, Sánchez Castillo PM, Sebastián E, Bolívar Galiano F (2005) Microalgae associated with deteriorated stonework of the Fountain of Bibatauín in Granada, Spain. Int Biodet Biodegr 55:55–61 41. Warscheid T, Braams J (2000) Biodeterioration of stone: a review. Int Biodeter Biodegr 46:343–368 42. Wetherbee R, Lind JL, Burke J (1998) The first-kiss: establishment and control of initial adhesion by raphid diatoms. J Phycol 34:9–15