10.48255/1973-9494.JCSCH.21.2021.09
I cianobatteri o alghe azzurre sono ubiquitariamente presenti in ambienti sia di acqua dolce sia salmastra, sviluppandosi anche in condizioni di elevata umidità, colonizzando pietre o superfici di monumenti e fontane, e generando complessi biofilm, in grado di indurre il biodeterioramento dei materiali costitutivi del manufatto. Così come per altri organismi fotoautotrofi, i cianobatteri appartengono ai microrganismi identificati come colonizzatori primari, svolgendo un ruolo importante nella formazione di consorzi microbici in grado di indurre processi di deterioramento. In questo studio si è analizzato il biofilm presente sulla superficie di una fontana in materiale lapideo, rivelando, mediante microscopia ottica sia in campo chiaro sia in fluorescenza, la presenza di colonie di cianobatteri con peculiari morfologie e strutture. Inoltre, la tecnica qPCR (quantitative Polymerase Chain Reaction) ha permesso di confermare il gene della Tioredossina reduttasi (TrxR) come marcatore molecolare adatto per studi di filogenesi. I risultati evidenziano la presenza di cianobatteri come principali taxa, la cui crescita è indotta dal microclima e dalle condizioni ambientali, oltre che dal litotipo e dalle caratteristiche fisiche della superficie lapidea. La più completa identificazione delle popolazioni microbiche che colonizzano manufatti storico-artistici è peculiare per la definizione di un’adeguata strategia per il controllo delle attività microbiche, per la rimozione del biofilm, oltre al contrasto di eventuali ricolonizzazioni.
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1973-9494
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Franco Palla
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Conservation Science in Cultural Heritage
cultural heritage
Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface: a case study in Palermo (Sicily, Italy)
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2022-08-02
2022-07-19
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Franco Palla
2021
10.48255/1973-9494.JCSCH.21.2021.09
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Department of Biological Research Chemical and Pharmaceutical Sciences and Technologies (BCPhSET), University of Palermo, Italy
1973-9494
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Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface: a case study in Palermo (Sicily, Italy)
https://conservation-science.unibo.it/article/download/15251/14477
21
F   RESHWATER CYANOBACTERIA, IDENTIFIED BY
    MICROSCOPIC AND MOLECULAR INVESTIGATIONS ON
    A COLONIZED FOUNTAIN SURFACE: A CASE STUDY IN
    PALERMO (SICILY, ITALY)

   Roberta Russo, Marco Chiaramonte
   Institute for Biomedical Research and Innovation (IBRI),
   National Research Council, Palermo, Italy

   Franco Palla * 1




   Department of Biological Research Chemical and Pharmaceutical Sciences and
   Technologies (BCPhSET),
   University of Palermo, Italy

   Keywords: blue-green algae, biodeterioration, molecular biology, fluorescence
   microscopy, cultural heritage
   DOI: 10.48255/1973-9494.JCSCH.21.2021.09



   1. Introduction

     Cyanobacteria, more commonly known with the name of blue or blue-green algae
(Cyanophyceae), are a heterogeneous group of single-cell photoautotrophic organisms
[1]. They are the only group of prokaryotic algae, and it has been ascertained that they
were the first organisms to produce organic matter on earth and the first photosynthetic
organisms to release oxygen into the atmosphere [2]. They are widespread in several
habitats with various ecological conditions, being able to develop both in marine and ter-
restrial ecosystems, and also in extreme environments such as glaciers, deserts, marine
habitats and freshwater, and rocks or sediments [3]. Cyanobacterial growth and replica-
tion time depend on environmental factors such as temperature (optimal range being
35-40°C), light, water, carbon dioxide and inorganic substrates, except for some species
that are able to survive in complete darkness [4]. Most cyanobacteria are autotrophic
aerobic organisms, but some show a distinct ability for heterotrophic nutrition, and some
are anaerobic. These organisms are of different sizes, ranging from microscopic to mac-
roscopic and have a coccoid or filamentous shape. Among the filamentous species there
are some that develop particular cell types, called heterocysts, surrounded by a very
thick wall, which depend on other cells for nourishment and performing atmospheric ni-
trogen fixation. Many filamentous cyanobacteria also develop a second cell type called
akinetes, similar to the spores that germinate when there are favourable conditions for
growth [3]. Their photosynthetic activity, similar to that of higher plants, contain pigments
such as chlorophyll-a, phycoerythrin, carotenoid, xanthophylls and phycocyanin [3]. Phy-
cobiliproteins are responsible for the characteristic blue-green colouring of cyanobacte-
ria and are able to absorb visible light with a wavelength ranging from 500 to 650 nm, in
order to perform photochemical reactions [5].


   *Corresponding author: franco.palla@unipa.it
                                                                                                                                                             The classification of cyanobacteria has been, and still is, the subject of numerous re-
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                        views both from a bacteriological and botanical point of view [6]. The most common clas-
                                                                                                                                                        sification code is based on their morphological and cytological characteristics, by which
                                                                                                                                                        the Cyanophyceae class is divided into four orders: Chroococcales, Oscillatoriales, Nos-
                                                                                                                                                        tocales, Stigonematales [7-8].
                                                                                                                                                             Cyanobacteria have potential application in the field of human healthcare as they are
                                                                                                                                                        known to produce a variety of bioactive compounds. Therefore, it may be possible to hy-
                                                                                                                                                        pothesize their potential use in many applications in the fields of bioenergy, natural prod-
                                                                                                                                                        ucts, medicine, agriculture, and the environment [9-10]. On the contrary, the spread of
                                                                                                                                                        cyanobacteria in fresh water represents an increasing problem related to the quality of
                                                                                                                                                        surface water. In fact, the excess supply of nutrients (phosphorus and nitrogen) deter-
                                                                                                                                                        mines the phenomenon of water eutrophication. In particular, some species belonging to
                                                                                                                                                        the genera Oscillatoria, Microcystis, Anabaena, are able to form large blooms on the sur-
                                                                                                                                                        face of rivers, lakes and seas, known by the term water bloom [3]. Cyanobacteria are
                                                                                                                                                        able to produce toxic substances (cyanotoxins), representing a risk for human health [11]
                                                                                                                                                        and acting on many ecosystems. They are also responsible for the large number of
                                                                                                                                                        deaths of fish, crustaceans and other animals [12]. Frequently, cyanotoxins such as mi-
                                                                                                                                                        crocystins, nodularins, cylindrospermopsin, Lyngbya toxins, anatoxins can be present.
                                                                                                                                                        Toxicosis from marine phycotoxin producers in animals can occur from direct contact via
                                                                                                                                                        skin, inhalation or ingestion of toxigenic organisms, or by ingestion through the food
                                                                                                                                                        chain. The effects of different phycotoxins range from respiratory paralysis to acute liver
                                                                                                                                                        or nervous system damage, skin irritation and tumor promotion [13].
                                                                                                                                                             Moreover, cyanobacteria form an external mass of mucilaginous material, which
                                                                                                                                                        serves as protection for the structure (biofilm) that holds the microbial cell colonies to-
                                                                                                                                                        gether, allowing them to adhere to the substrate, inducing biodeterioration processes
                                                                                                                                                        [14]. Both macro-organisms (animals, plants) and microorganisms (bacteria, fungi, cy-
                                                                                                                                                        anobacteria, algae and lichens) can trigger the biodeterioration of cultural assets [15].
                                                                                                                                                        Identifying the biological systems on artwork surfaces is essential to accurately evalu-
                                                                                                                                                        ate biodeterioration processes, perform proper sampling and integrated investigations
                                                                                                                                                        based on microscopy, in vitro microbial culture and molecular biology analysis [16].
                                                                                                                                                             The aim of this study was to reveal and identify, through an integrated approach
                                                                                                                                                        (microscopy analysis and molecular biology investigation), cyanobacteria colonies in
                                                                                                                                                        the complex biofilm colonizing the surface of the marble fountain of the Two Dragons
                                                                                                                                                        (Fontana dei Due Draghi) in Palermo city centre (Sicily, Italy). In particular, combining
                                                                                                                                                        optical microscopy analysis, in bright and fluorescence fields, and molecular biology
                                                                                                                                                        investigation by quantitative Polymerase Chain Reaction (qPCR), information on the
                                                                                                                                                        presence and amount of cyanobacteria colonies was gathered. Specifically, Thiore-
                                                                                                                                                        doxin reductase protein was utilized to perform the corresponding phylogenetic tree of
                                                                                                                                                        plants, animals and cyanobacteria, showing that it can be a good molecular marker for
                                                                                                                                                        the identification of cyanobacteria colonies.


                                                                                                                                                           2. Materials and methods

                                                                                                                                                           2.1. Microscopy analysis

                                                                                                                                                            Biofilm samples were taken from selected areas of a historical 17th century marble
                                                                                                                                                        fountain and stored (in water at room temperature and exposed to the daily light cycle)
                                                                                                                                                        until their use for investigation.


                                                                                                 206
   Micro-fragments were directly observed with an Axioscop2 plus microscope (Zeiss,
Jena, Germany) and images acquired using a Zeiss Axiocam camera (Zeiss 412-312,
magnification of 20X, 40X and 60X), in bright and fluorescence fields. The excitation and
emission filter sets were:
   i) blue light excitation with emission in green (BP450-490/FT510/LP520; FITC,
       Fluorescein isothiocyanate), exciting chlorophyll-a (which appears red) and phy-
       coerythrin type I (which appears yellow-orange);
   ii) green light excitation with emission in yellow/orange (BP520-560/FT580/LP590;
       TRITC, Tetra-methyl-rhodamine), exciting both phycocyanin and phycoerythrin
       type II, emitting fluorescence in red and orange-red respectively.


   2.2. Extraction of genomic DNA

    Biofilm fragments were first dehydrated (keeping them between absorbent paper
sheets for two hours), then removed and pulverized by mortar and pestle in the pres-
ence of liquid nitrogen; 200 ng of this powder were used for extraction of the genomic
DNA (gDNA) by GeneElute Blood Genomic DNA Kit (Sigma Aldrich, St Louis, MO,
USA), following the instructions in the technical manual. The RNA-free DNA (RNase-A
incubation for 20 min at 37°C) was quantified using a Bio-photometer D30 (Eppendorf,
Hamburg, Germany) at 260 nm (1 O.D. equal to 50 ng/μL). Aliquots of gDNA (20 ng)
were the template molecules in Real-Time qPCR reactions.


   2.3. Real-Time quantitative PCR (qPCR)

     The quantitative analysis of the genomic DNA target sequences was carried out us-
ing the StepOnePlus Real-Time PCR System (Applied Biosystems, Life Technologies,
Carlsbad, CA, USA) according to the manufacturer’s manual. A comparative threshold




                                                                                              CONSERVATION SCIENCE IN CULTURAL HERITAGE
cycle method with SYBR Green master mix was performed [17]. To perform the qPCR,
the following parameters were used: a denaturation cycle at 95 ° C for 10 minutes to al-
low activation of the polymerase; 38x cycles: melting at 95 ° C for 15 seconds, anneal-
ing/extension at 60 ° C for 60 seconds. The 16S-rRNA was the housekeeping gene,
while the Thioredoxin reductase (TrxR) was the target gene. DNA amplification was per-
formed by specific primers for 16S-rRNA and TrxR coding genes. For the 16S-rRNA
gene, primers were: Forward = 5’-AGTAGCTGGTCTGAGAGGATGA-3’ and Reverse = 5’-
CGGTATTGCTCCGTCAGGCTTTC-3’; amplicon was 118 bp in length. The primers specif-
ic for the TrxR target sequence were: Forward = 5’-AAGGCTTGGAGTGGGATGGCGAA-3
‘and Reverse = 5’- TGCCACAGATACTTGATTCAAGCC -3 ‘; amplicon size was 116 bp
in length.
     Results are the average of two qPCR experiments.


   2.4. Phylogenetic analysis of Thioredoxin reductase proteins

    TrxR proteins sequences from different organisms, cyanobacteria, vertebrates and
invertebrates, were analysed using the BLAST program (Basic Local Alignment Search
Tool) (http://blast.ncbi.nlm.nih.gov/); their accession numbers are indicated in Table 1.
Scientific research produces a large volume of complex data, which is collected and
made available in databases that represent peculiar sources of information on different


                                                                                            207
                                                                                                                                                        biological molecules (DNA, RNA, Protein), very frequently used in science. In this case
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                        study, the Genbank accession numbers are related to Thioredoxin reductase protein
                                                                                                                                                        sequences, allowing the identification of macro or micro biological species. Specifical-
                                                                                                                                                        ly, multiple sequence alignments were performed by Clustal Omega (http://www.ebi.
                                                                                                                                                        ac.uk/Tools/msa/clustalo/) and the evolutionary analysis was conducted with MEGA-X
                                                                                                                                                        software [18] we report a transformation of MEGA to enable cross-platform use on Mi-
                                                                                                                                                        crosoft Windows and Linux operating systems. MEGA X does not require virtualization
                                                                                                                                                        or emulation software and provides a uniform user experience across platforms. MEGA
                                                                                                                                                        X has additionally been upgraded to use multiple computing cores for many molecular
                                                                                                                                                        evolutionary analyses. MEGA X is available in two interfaces (graphical and command
                                                                                                                                                        line, using the Neighbour-Joining (NJ) method [19].
                                                                                                                                                            The ExPASy proteomic server (http://www.expasy.org/) was used to perform L-Align,
                                                                                                                                                        in order to find the similarity percentage among TrxR proteins.

                                                                                                                                                        Table 1. Thioredoxin reductase proteins Genbank accession numbers and organisms used in the
                                                                                                                                                        phylogenetic analysis. The percentage of similarity refers to Cyanobacterium sp.

                                                                                                                                                                    Organisms               Genbank Accession                  Percentage
                                                                                                                                                                                                 number               of similarity (%) / number of
                                                                                                                                                                                                                               amino acids
                                                                                                                                                         Cyanobacterium sp.                     AFZ46444.1
                                                                                                                                                         Synechococcus sp.                    MBF2080429.1                      97/328
                                                                                                                                                         Oscillatoria sp.                       TAF50584.1                      96/328
                                                                                                                                                         Rivularia sp.                       WP_015121190.1                     96/329
                                                                                                                                                         Leptolyngbya sp.                    WP_075598049.1                     96/328
                                                                                                                                                         Synechocystis sp.                   WP_066342850.1                     97/328
                                                                                                                                                         Homo sapiens                           AAB35418.1                      58/112
                                                                                                                                                         Drosophila melanogaster               NP_524216.1                      58/190
                                                                                                                                                         Bacillus sp.                           KRF49594.1                      57/331
                                                                                                                                                         Arabidopsis thaliana                   CAA80656.1                      53/308
                                                                                                                                                         Chlorella vulgaris                     BAH29954.1                      55/332



                                                                                                                                                            2.5. Statistical analysis

                                                                                                                                                            The statistical analysis of the qPCR results was performed using the ANOVA one-
                                                                                                                                                        way analysis of variance test, with R statistical software (http: //www.r-project.org, Vien-
                                                                                                                                                        na, Austria) and significance at p < 0.05, followed by Tukey’s multiple comparison test.


                                                                                                                                                            3. Results

                                                                                                                                                            3.1. Microscopy analysis

                                                                                                                                                            In order to evaluate the presence of cyanobacteria, which are potentially toxic for
                                                                                                                                                        human and animal health and /or harmful for cultural assets, pigmented green biofilms
                                                                                                                                                        were collected from the marble fountain surface, as shown in Figure 1A (indicated by
                                                                                                                                                        the white square).


                                                                                                208
      Tiny fragments of biofilm were observed under optical microscope as described in
Materials and Methods by seeing them in bright and fluorescence fields (Figure 1 and 2),
taking advantage of the fact that cyanobacteria naturally emit fluorescence (in green
and red), as they have pigments such as chlorophyll, carotenoid and phycobilin-pro-
teins exhibiting different colours [20].
      In Figure 1B the filamentous Oscillatoria sp., Lyngbya sp. and the coccoid cyanobac-
teria Chroococcus sp., surrounded by other unidentified organisms, were observed.
      Figure 1C shows a wide field of Gloeocapsa sp., a unicellular colonial alga whose
colonies are small and irregularly aggregated and enclosed in a mucilaginous matrix;
it is sometimes found covering large areas of wet stony substrates. It counts many spe-
cies as reported by the Taxonomic outlines of Bergey’s Manual of Systematic Bacteri-
ology [21] and counts 130 species as reported by AlgaeBase (a global algal database,
available at: https://www.Algaebase.org).
      Figure 1D highlights the presence of cyanobacteria, such as Chroococcus sp. and
the tuft of a filamentous branching Dichothrix sp., in particular, which is clearly recog-
nizable in the red fluorescence field.




                                                                                                         CONSERVATION SCIENCE IN CULTURAL HERITAGE




Figure 1. Sampling of the Two Dragons biofilm. A) Due to their autofluorescence (red and green),
microscopy analysis in the bright and fluorescence fields highlights several cyanobacteria mor-
phologies, indicated by coloured arrows; B) black arrow: Lyngbya sp; white arrow: Chroococcus
sp.; light blue arrow: Oscillatoria sp.; yellow arrow: Lyngbya sp.; C) white square: Gloeocapsa sp.;
D) black and yellow arrows: Chroococcus sp.; white and light blue arrows: Dichothrix sp.


                                                                                                       209
                                                                                                                                                            In Figure 2A, the Oscillatoria sp. cyanobacteria is recognizable. Since hundreds
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                        of species are attributable to the Oscillatoria genus, as recorded in the AlgaeBase
                                                                                                                                                        database, correct identification of the species is quite difficult. The filamentous cya-
                                                                                                                                                        nobacterium Microcoleus sp. is shown in Figure 2B. The filament of this genus is
                                                                                                                                                        composed of a gelatinous colourless sheath, in which are packed parallel trichomes,
                                                                                                                                                        the number of which usually changes from 2 to more than 100 within one sheath. Fig-
                                                                                                                                                        ure 2C shows a colony of Chroococcus turgidus, located in the biofilm edge, in red
                                                                                                                                                        and green fields. They are spherical unicellular or colonial organisms, usually few-
                                                                                                                                                        celled, forming a gelatinous biofilm [22]. Figure 2D shows the sheath of the cyano-
                                                                                                                                                        bacteria Lyngbya sp.




                                                                                                                                                        Figure 2. Morphological profiles in bright and fluorescence fields (red and green) of cyano-
                                                                                                                                                        bacterial species revealed in the fountain biofilm. A) black and yellow arrows: Oscillatoria sp.;
                                                                                                                                                        B) black and yellow arrows: Microcoleus sp.; C) Chroococcus turgidus; D) yellow arrow:
                                                                                                                                                        Lyngbya sp.


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    3.2. Quantitative analysis of cyanobacteria by quantitative polymerase chain
         reaction (qPCR)

    A molecular biology investigation was performed using aliquots of genomic DNA
(gDNA), extracted as described in Materials and Methods and utilized as templates in the
qPCR, in order to evaluate the relative amount of cyanobacteria present in the biofilm
fragments. Using specific ad hoc primers designed for this study on the Cyanobacterium
stanieri gDNA sequence, the target (TrxR) or housekeeping (16S-rRNA) genes were
quantified by qPCR experiments. As a control sample (CTRL), gDNA was extracted from
the cyanobacteria Phormidium sp., collected from freshwater aquaria (Figure 3A and B),
and analysed in bright, red and green fluorescence fields. The histogram in Figure 3B
shows the result of the qPCR experiments, in which the amount of the TrxR DNA mole-
cules in the fountain biofilm (*) and control (CTRL) are defined. Results highlight that the
presence of TrxR DNA molecules found in the control (Phormidium sp.) are five times
more abundant than in the biofilm samples. This is related to the DNA target molecules
available; in fact, starting from an equal amount of gDNA (20 ng), in the control sample
only cyanobacteria target sequences are present, instead in the fountain biofilm sample
gDNA molecules are available not only from cyanobacteria but also from other organisms.




                                                                                                       CONSERVATION SCIENCE IN CULTURAL HERITAGE

                    C




Figure 3. Morphological profile of Phormidium sp. colonies, observed in bright and fluorescence
(red and green) fields; A) = 40X; and B) 60X magnification; C) the result of the qPCR experiments
is reported in the histogram: CRTL= amplification product using Phormidium sp. gDNA as tem-
plate; Fountain= amplification product using fountain biofilm gDNA as template. * indicates a sig-
nificant difference in amount of DNA target sequence, amount was set equal to 1 on an arbitrary
scale of values (y-axis shows Fold Change values).
                                                                                                     211
                                                                                                                                                            3.3. Phylogenetic analysis of TrxR proteins
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface




                                                                                                                                                            In order to select an efficient and specific cyanobacteria target gene to perform
                                                                                                                                                        qPCR reactions, a phylogenetic tree was defined (Figure 4). Specifically, the TrxR
                                                                                                                                                        protein sequence from six different cyanobacteria were aligned with related sequenc-
                                                                                                                                                        es of Homo sapiens (vertebrate), Arabidopsis thaliana (plant), Chlorella vulgaris
                                                                                                                                                        (green alga), Bacillus sp. (bacteria) and Drosophila melanogaster (invertebrate) by
                                                                                                                                                        Clustal-O alignment program (Figure 5). All the organisms are summarized in Table 1
                                                                                                                                                        with their accession numbers. The phylogenetic tree shows that all cyanobacteria
                                                                                                                                                        TrxR proteins are in the same clade (red square). Instead, H. sapiens and D. melan-
                                                                                                                                                        ogaster, A. thaliana, C. vulgaris and Bacillus sp. proteins are separated from cyano-
                                                                                                                                                        bacteria. This result indicates a great difference between cyanobacteria and animals,
                                                                                                                                                        plants or bacteria protein sequences, confirming the specific qPCR amplification of
                                                                                                                                                        cyanobacteria target sequences.




                                                                                                                                                        Figure 4. Phylogenetic analysis of TrxR proteins from different organisms. The percentage of rep-
                                                                                                                                                        licate trees (1000 replicates) are shown next to the branches. The red square indicates all cyano-
                                                                                                                                                        bacteria species.


                                                                                                                                                            Alignment of TrxR amino acid sequences (Figure 5) confirms the specificity of
                                                                                                                                                        amplification performed by primers designed in this study. Specifically, amino acid
                                                                                                                                                        sequences corresponding to Cyanobacterium sp. are highlighted in pink, while yel-
                                                                                                                                                        low indicates the amino acid sequences of other cyanobacteria (Rivularia, Oscillato-
                                                                                                                                                        ria, Geminocystis, Synechococcus, Leptolyngbya); clear differences are detectable
                                                                                                                                                        for the other organisms analysed (Homo, Bacillus, Chlorella, Arabidopsis). Moreover,
                                                                                                                                                        the percentage of similarity among the TrxR proteins is reported in Table 1, showing
                                                                                                                                                        a high percentage of similarity among cyanobacteria (96-97%) and a low percentage
                                                                                                                                                        of similarity in other organisms, bacteria, animals and plant sequences (ranging from
                                                                                                                                                        53 to 58%).


                                                                                                                  212
Figure 5. Clustal-O Multiple Alignment of TrxR protein portions from different organisms: pink= ami-
no acid sequence specific for Cyanobacterium sp., amplified by qPCR; yellow=amino acid sequenc-
es of other Cyanobacteria genera (percentage of similarity = 96-97%). The other organisms show
an evident difference in amino acid sequences of TrxR protein (percentage of similarity = 53-58%).


    4. Discussion

    The present study provides supplementary information to a previous investiga-
tion carried out by Di Carlo et al [23] on cyanobacteria-rich biofilms colonizing the
surfaces of an ancient marble fountain (dating back to 1630) located in the city cen-
tre of Palermo, Sicily, Italy. In Sicily, winters are mild and summers are very hot
which, together with the high levels of humidity on the fountain’s surfaces, strongly
influence microbial growth giving rise to remarkably complex biofilms. A biofilm or bi-
ological patina represents a widespread phenomena in nature and is a three-dimen-
sional structure in general established by a consortium of microorganisms in which
cells stick to each other [24]. In the biofilm, microorganisms are fully surrounded by




                                                                                                         CONSERVATION SCIENCE IN CULTURAL HERITAGE
an extracellular matrix composed of polysaccharides, proteins, lipids and DNA. This
mucilaginous material is a fundamental feature for the protection and survival of the
organisms, allowing them to adhere to the substrate [14]. Environmental conditions
determine the degree of colonization, the type of community, the biofilm composition
and structure.
    The marble of the fountain is a rock of calcium carbonate, with porosity, rough-
ness, and water absorption, which acts as an appropriate support for the growth of mi-
croorganisms due to the source of readily available water [25]. Environmental param-
eters, like humidity, pollution and temperature, also influence the colonization process
of the microorganisms [26]. Consequently, as an example, significant differences be-
tween the cyanobacterial populations in polluted cities compared to rural unpolluted
sites were observed. Different habitats or polluted environments can cause lower bio-
diversity, thus promoting the growth of only the more resistant taxa, as reported for the
genus Scytonemin [27].
    Cyanobacteria are present in a wide variety of terrestrial habitats (including rocks
and deserts) as well as in modern and ancient buildings [28]. It is well known that cya-
nobacteria induce deterioration of stone cultural heritage (monuments and stone art-
works) and, in European countries of the Mediterranean Basin, about 172 taxa have
been described, including 37 cyanobacteria (blue algae) and 48 chlorophyta (green al-
gae) genera. The most widespread and commonly reported were Gloeocapsa, Phor-
midium and Chroococcus as cyanobacteria taxa [29-30].


                                                                                                       213
                                                                                                                                                            The cyanobacteria found in our case study are the most widespread in the Med-
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                        iterranean basin, e.g. Gloeocapsa, Chroococcus, Oscillatoria, Lyngbya and Microco-
                                                                                                                                                        leus, which have a gelatinous sheath that is sometimes pigmented and acts as a res-
                                                                                                                                                        ervoir of water, and play an important role in adhesion to the substratum. The
                                                                                                                                                        deteriorating action of microorganisms on works of art have received substantial
                                                                                                                                                        consideration in the last few years. Knowledge of the microbial populations living on
                                                                                                                                                        works of art is essential in order to monitor and keep any deterioration process un-
                                                                                                                                                        der control, as well as programme appropriate consequential conservation treat-
                                                                                                                                                        ments [29]. Moreover, Chroococcales, Oscillatoriales and Nostocales, are also tar-
                                                                                                                                                        geted as toxic cyanobacteria, since they produce toxins that can be detrimental for
                                                                                                                                                        human health [31]. Therefore, early identification of cyanobacteria colonies allows an
                                                                                                                                                        adequate strategy to be set up, which can act on microbial vitality and provide for the
                                                                                                                                                        removal of undesired biofilm.
                                                                                                                                                            However, cyanobacteria are characterized by notable morphological, physio-
                                                                                                                                                        logical and genetic diversity, consequently methods for identifying cyanobacteria
                                                                                                                                                        need to be specifically calibrated. Morphological profiles on cyanobacteria per-
                                                                                                                                                        formed by Scanning Electron Microscopy (SEM) and Transmission Electron Mi-
                                                                                                                                                        croscopy (TEM) have been reported by Marinè 2004 [32] and Ranalli 2005 [33], but
                                                                                                                                                        in very few studies has fluorescence microscopy been applied [34]. In addition, dif-
                                                                                                                                                        ferent methods have been applied for microscopy observation, such as those meth-
                                                                                                                                                        ods that use the fixing of microbial cells with formalin [2] or staining them with Lu-
                                                                                                                                                        gol’s Iodine reactive [35].
                                                                                                                                                            Indeed, the direct analysis of secreted cyanotoxins by analytical methods, such as
                                                                                                                                                        liquid chromatography/mass spectrometry (LC/MS) and immunoassays such as ELISA
                                                                                                                                                        (Enzyme Linked Immunosorbent Assay) [36], has been implemented with respect to
                                                                                                                                                        monitoring strategies. These methods are sensitive enough to reveal cyanobacteria
                                                                                                                                                        toxins but can have limited application and represent only a first level for cyanobacte-
                                                                                                                                                        ria identification. However, by combining different investigation methods, exhaustive
                                                                                                                                                        results can be obtained [37].
                                                                                                                                                            Here, we exploited the known autofluorescence of cyanobacterial cells [34]. We
                                                                                                                                                        used an optical microscope with fluorescence to unequivocally detect the genus of cy-
                                                                                                                                                        anobacteria present in the biofilm taken from the marble fountain of the Two Dragons
                                                                                                                                                        in Palermo. Based on the presence of photosynthetic pigments (chlorophylls and phy-
                                                                                                                                                        cobiliproteins), cyanobacteria were revealed in the biofilms on the marble fountain, us-
                                                                                                                                                        ing specific fluorescence filters [9,34].
                                                                                                                                                            Furthermore, cyanobacteria are very heterogeneous also from a genetic point of
                                                                                                                                                        view. In spite of a general genetic divergence between genera, there is considerable
                                                                                                                                                        intragenus diversity and there is no unanimous consensus on which molecular mark-
                                                                                                                                                        ers are eligible to reach an unambiguous identification.
                                                                                                                                                            Therefore, another approach to detect the presence of cyanobacteria was to con-
                                                                                                                                                        duct a molecular investigation using DNA-related assays, mainly based on PCR reac-
                                                                                                                                                        tion tests followed by restriction enzyme analysis or DNA sequencing. The 16S-rRNA
                                                                                                                                                        (16S-ribosomal RNA) molecular locus is an effective marker for the molecular identifi-
                                                                                                                                                        cation of cyanobacteria at the genus level, as well as ITS (Internal Transcribed Se-
                                                                                                                                                        quence of rRNA) [38].
                                                                                                                                                            In the last decade, qPCR has been used to quantify cyanobacteria populations
                                                                                                                                                        present in biofilms, including toxic ones [39]. Indeed, the quantitative estimation of
                                                                                                                                                        bacterial cells within an environmental sample by real-time quantitative PCR (qPCR)
                                                                                                                                                        represents an excellent strategy to reveal the potentially toxic cyanobacteria popula-


                                                                                                              214
tion [40]. Al-Tebrineh and co-workers, applied the qPCR method for the detection and
quantification of hepato-toxigenic cyanobacteria in bloom samples, using mcyE and
ndaF gene primers, indicating that this technology does effectively offer advantages
over traditional protocols used to assess water quality [41]. Although many target/ref-
erence genes were selected to identify cyanobacteria species [42], in this study,
qPCR was performed using TrxR as the target gene and 16S-rRNA as the house-
keeping gene, in order to quantify cyanobacteria in toto. The TrxR is a homodimeric
70-kDa flavoprotein in bacteria, fungi and plants with redox active enzymes able to
reduce thioredoxin (Trx). Two classes of TrxR were previously identified, one in bac-
teria and one in eukaryotes. In the higher eukaryotes, TrxR are of about 120 kDa,
with a wide substrate specificity that also reduces non-disulphide substrates such as
vitamin C [43] thioredoxin reductase and NADPH, the thioredoxin system, is ubiqui-
tous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC. As a
control sample we used the cyanobacteria Phormidium sp. collected in a freshwater
aquarium. Compared to the control, a decrease in the quantity of the TrxR gene was
found in the fountain biofilm, indicating a reduced quantity of cyanobacteria (due to the
content of other organisms in addition to the cyanobacteria) with respect to the control
sample containing only cyanobacteria.
    Phylogenetic analysis demonstrated that the qPCR amplified product was specif-
ic for cyanobacteria and not for other contaminant species, as the TrxR proteins of
the cyanobacteria clustered separately from other species such as bacteria, inverte-
brates, vertebrate animals and plants. The use of the TrxR gene in qPCR experi-
ments does not discriminate the single species of cyanobacteria but determines the
specific quantization of cyanobacteria when compared with other taxa found in the
microbial consortium.



   5. Conclusion




                                                                                              CONSERVATION SCIENCE IN CULTURAL HERITAGE
     In this study we have attempted to define easy but efficacious methods to detect
cyanobacteria in biofilms collected from different sites, as well as stonework surfaces,
based on fluorescence microscopy and molecular biology (qPCR) investigation. More-
over, we propose the use of the TrxR gene as an efficient molecular marker for quan-
titative analysis.
     It is known that cyanobacteria are the most important colonizers of stonework sur-
faces in outdoor environments, as they are able to survive changes in climatic and en-
vironmental conditions. They are responsible for biodeterioration, but can create a bio-
film which encourages the growth of other deteriogens such as fungi or lichens [27], as
well as representing a potential danger to human health [39-40].
     Therefore, early diagnosis, also using biotechnological protocols, allows adequate
prevention strategies to be defined in safe conditions, in addition to taking action be-
fore irreversible surface changes occur.



   Acknowledgements

   The authors thank the Ufficio Città Storica Metropolitana di Palermo and the Fon-
dazione Salvare Palermo. The study is part of the PON 01_00625 research project.


                                                                                            215
                                                                                                                                                              References
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface




                                                                                                                                                        [1]     Warscheid, Th., Braams, J. (2000). Biodeterioration of stone: a review. Interna-
                                                                                                                                                                tional Biodeterioration & Biodegradation, 46, pp. 343-368.
                                                                                                                                                        [2]     Chorus, I., Bartram, J. eds. (1999). Toxic Cyanobacteria in Water: A guide to their
                                                                                                                                                                public health consequences, monitoring and management, London: Routledge.
                                                                                                                                                        [3]     Saad, A., Atia, A. (2014). Review on freshwater blue-green algae (cyanobacte-
                                                                                                                                                                ria): Occurrence, classification and toxicology. Biosci. Biotechnol. Res. Asia. 11,
                                                                                                                                                                pp. 1319-1325.
                                                                                                                                                        [4]     Haney, P.J., Badger, J.H., Buldak, G.L., Reich, C.I., Woese, C.R., Olsen G.J.
                                                                                                                                                                (1999). Thermal adaptation analyzed by comparison of protein sequences from
                                                                                                                                                                mesophilic and extremely thermophilic Methanococcus species. Proc. Natl.
                                                                                                                                                                Acad. Sci. USA. 96, pp. 3578-3583.
                                                                                                                                                        [5]     Samsonoff, W.A., Mac Coll, R. (2001). Biliproteins and phycobilisomes from cy-
                                                                                                                                                                anobacteria and red algae at the extremes of habitat, Arch. Microbiol. 176, pp.
                                                                                                                                                                400-405.
                                                                                                                                                        [6]     Marquardt. J., Palinska, K.A. (2007). Genotypic and phenotypic diversity of cya-
                                                                                                                                                                nobacteria assigned to the genus Phormidium (Oscillatoriales) from different
                                                                                                                                                                habitats and geographical sites, Arch. Microbiol. 187, pp. 397–413.
                                                                                                                                                        [7]     Komárek, J., Kaštovský, J., Mareš, J., Johansen, J.R. (2014). Taxonomic classi-
                                                                                                                                                                fication of cyanoprokaryotes (cyanobacterial genera), using a polyphasic ap-
                                                                                                                                                                proach, Preslia 86, pp. 295–335.
                                                                                                                                                        [8]     Kurmayer, R. (2012). The toxis cyanobacterium nostoc sp . strain 152 produces
                                                                                                                                                                highest amounts of microcystin and nostophycin under stress conditions, Eu-
                                                                                                                                                                rope PMC Funders Group 47, pp. 200–207.
                                                                                                                                                        [9]     Zhang, T., Shen, Z., Xu, P., Zhu, J., Lu, Q., Shen, Y., Wang, Y., Yao, C., Li, J.,
                                                                                                                                                                Wang, Y., Jiang, H. (2012). Analysis of photosynthetic pigments and chlorophyll
                                                                                                                                                                fluorescence characteristics of different strains of Porphyra yezoensis, J. Appl.
                                                                                                                                                                Phycol. 24, pp. 881–886.
                                                                                                                                                        [10]    Zahra, Z., Choo, D.H., Lee, H., Parveen, A. (2020). Cyanobacteria: Review of
                                                                                                                                                                Current Potentials and Applications, Environments. 7, p. 13.
                                                                                                                                                        [11]    Ressom, R., Soong, F., Fitzgerald, J., Turczyonowicz, L., El Saadi O., Roger, D.,
                                                                                                                                                                Maynard, T., Falconer, I. (1994). Health effects of toxic cyanobacteria (blue-
                                                                                                                                                                green algae). Camberra: National Health and medical Research Council.
                                                                                                                                                        [12]    Duy, T.N., Lam, P.K., Shaw, G.R., Connell, D.W. (2000). Toxicology and risk as-
                                                                                                                                                                sessment of freshwater cyanobacterial (blue-green algal) toxins in water. Rev.
                                                                                                                                                                Environ. Contam. Toxicol. 163 , pp. 113–185.
                                                                                                                                                        [13]    Russeaux, C.G., Gad, S.C. (2013) Statistical Assessment of Toxicologic Pathol-
                                                                                                                                                                ogy Studies. In Haschek.and Rousseaux’s Handbook of Toxicology Pathology,
                                                                                                                                                                3rd Edition, Academic Press Elsevier, pp. 893-988.
                                                                                                                                                        [14]    Gantt, E. (1994). Supramolecular Membrane Organization, in: Molecular Biolo-
                                                                                                                                                                gy Cyanobacteria. Cham: Springer International Publishing AG, pp. 119–138.
                                                                                                                                                        [15]    Bartoli, F., Municchia, A.C., Futagami, Y., Kashiwadani, H., Moon, K.H., Caneva
                                                                                                                                                                G. (2014). Biological colonization patterns on the ruins of Angkor temples (Cam-
                                                                                                                                                                bodia) in the biodeterioration vs bioprotection debate, International Biodeterior.
                                                                                                                                                                Biodegradation. 96, pp. 157-165.
                                                                                                                                                        [16]    Palla, F. and Barresi, G. eds. (2017). Biotechnology and conservation of cultur-
                                                                                                                                                                al heritage. Cham: Springer International Publishing AG.
                                                                                                                                                        [17]    Livak, K.J., Schmittgen, T.D. (2001). Analysis of relative gene expression data us-
                                                                                                                                                                ing real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, pp. 402-408.


                                                                                                              216
[18]    Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018). MEGA X: Molecular
        evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35,
        pp. 1547-1549.
[19]    Saitou, N., Nei, M.(1987). The neighbor-joining method: a new method for re-
        constructing phylogenetic trees. Mol. Biol. Evol. 4, pp. 406-425.
[20]    Saini, D.K., Pabbi, S., Shukla, P. (2018). Cyanobecterial pigments: Persepctive and
        biotechnological approaches. Food and Chemical Toxicology. 120, pp. 616-624.
[21]   Garrity, G.M., Bell, J.A., Lilburn, T.G., Lansing E. (2004). Taxonomic Outline of the
        Prokaryotes Bergey’s Manual ® of Systematic Bacteriology, 2nd edition, Cham:
        Springer International Publishing AG.
[22]    McGregor, G.B. (2013) Freshwater cyanobacteria of North-Eastern Australia: 2.
        chroococcales. Phytotaxa. 133, pp. 1-130.
[23]    Di Carlo, E., Lombardo, G., Barresi, G., Manachini, B., Palla, F., D’Ondes, G.C.,
        Lo Giudice, S.L. (2015) Biological macro and micro systems co-existing in the
        “fountain of the two dragons” in Palermo. Conserv. Science Cultural Heritage 15,
        95-107.
[24]    Hall-Stoodley, L., Costerton, J.W., Stoodley, P. (2004). Bacterial biofilms: From the
        natural environment to infectious diseases, Nat. Rev. Microbiol. 2, pp. 95-108.
[25]    Prieto, B., Silva, B. (2005). Estimation of the potential bioreceptivity of granitic
        rocks from their intrinsic properties, International Biodeterioration and Biodegra-
        dation. 56, pp. 206-215.
[26]    Ortega-Morales, O., Montero-Muñoz, J.L., Baptista Neto, J.A., Beech, I.B., Sun-
        ner, J., Gaylarde, C. (2019). Deterioration and microbial colonization of cultural
        heritage stone buildings in polluted and unpolluted tropical and subtropical cli-
        mates: A meta-analysis, International Biodeterior. Biodegrad. 143, pp. 104734.
[27]    Gaylarde, C.C. (2020). Influence of Environment on Microbial Colonization of
        Historic Stone Buildings with Emphasis on Cyanobacteria, Heritage, 3(4),
        pp. 1469-1482.




                                                                                                  CONSERVATION SCIENCE IN CULTURAL HERITAGE
[28]    Crispim, C.A., Gaylarde, C.C. (2005). Cyanobacteria and Biodeterioration of Cul-
        tural Heritage: A Review. Microbial Ecology, 49, pp. 1-9.
[28]    Macedo, M.F., Zelia-Mille, r A., Dionisio, A., Saiz-Jimenez, C. (2009). Biodiversi-
        ty of cyanobacteria and green algae on monuments in the Mediterranean Ba-
        sin : an overview. Microbiology, 155, pp. 3476-3490.
[30]    Ortega-Calvo, J.J., Ariño, X., Hernandez-Marine, M., Saiz-Jimenez, C. (1995).
        Factors affecting the weathering and colonization of monuments by phototrophic
        microorganisms. Sci. Total Environ. 167, pp. 329–341.
[31]    Martins, R., Pereira, P., Welker, M., Fastner, J., Vasconcelos, V.M. (2005). Toxici-
        ty of culturable cyanobacteria strains isolated from the Portuguese coast. Toxicon.
        46, pp. 454–464.
[32]    Mariné, M.H., Clavero, E., Roldán, M. (2004). Microscopy methods applied to
        research on cyanobacteria Cyanobacteria are abundant throughout the world
        and contribute significantly to global primary. Limnetica, 23, pp. 179-185.
[33]    Ranalli, G., Alfano, G., Belli, C., Lustrato, G., Colombini, M.P., Bonaduce, I., Za-
        nardini, E., Abbruscato, P., Cappitelli, F., Sorlini, C. (2005). Biotechnology ap-
        plied to cultural heritage : biorestoration of frescoes using viable bacterial cells
        and enzymes. J. Applied Microbiology 98, pp. 73–83.
[34]    Grigoryeva, N., Chistyakova. L. (2018). Fluorescence Microscopic Spectrosco-
        py for Investigation and Monitoring of Biological Diversity and Physiological State
        of Cyanobacterial Cultures. In Cyanobacteria. London: IntechOpen, pp. 11-44.


                                                                                                217
                                                                                                                                                        [35]   Hawkins, P.R, Holliday, J., Kathuria, A., Bowling, L. (2005). Change in cyano-
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                               bacterial biovolume due to preservation by Lugol’s Iodine. Harmful Alagae 4, pp.
                                                                                                                                                               1033-1043.
                                                                                                                                                        [36]   Yu, F.Y., Liu, B.H., Chou, H.N., Chu, F.S. (2002). Development of a sensitive
                                                                                                                                                               ELISA for the determination of microcystins in algae. J. Agric. Food Chem. 50,
                                                                                                                                                               pp. 4176-4182.
                                                                                                                                                        [37]   Triantis, T., Tsimeli, K., Kaloudis, T., Thanassoulias, N., Lytras, E., Hiskia, A.
                                                                                                                                                               (2010). Development of an integrated laboratory system for the monitoring of cy-
                                                                                                                                                               anotoxins in surface and drinking waters. Toxicon 55, pp. 979-989.
                                                                                                                                                        [38]   Webb ,V.L., Maas, E.W. (2002). Sequence analysis of 16S rRNA gene of cyano-
                                                                                                                                                               bacteria associated with the marine sponge Mycale (Carmia ) hentscheli. FEMS
                                                                                                                                                               Microbiology Letters 207, pp. 43-47.
                                                                                                                                                        [39]   Sabart, M., Pobel, D., Briand, E., Combourieu, B., Salençon, M.J., Humbert,
                                                                                                                                                               J.F., Latour, D. (2010). Spatiotemporal Variations in Microcystin Concentrations
                                                                                                                                                               and in the Proportions of Microcystin-Producing Cells in Several Microcystis
                                                                                                                                                               aeruginosa Populations. Appl. Environ. Microbiol. 76, pp. 4750-4759.
                                                                                                                                                        [40]   Pearson, L.A., Neilan, B.A. (2008). The molecular genetics of cyanobacterial
                                                                                                                                                               toxicity as a basis for monitoring water quality and public health risk., Curr. Opin.
                                                                                                                                                               Biotechnol. 19, pp. 281-288.
                                                                                                                                                        [41]   Al-Tebrineh, J., Gehringer. M.M., Akcaalan, R., Neilan, B.A. (2011). A new quan-
                                                                                                                                                               titative PCR assay for the detection of hepatotoxigenic cyanobacteria. Toxicon 57,
                                                                                                                                                               pp. 546-554.
                                                                                                                                                        [42]   Pinto, F., Pacheco, C.C., Ferreira, D., Moradas-Ferreira, P., Tamagnini, P. (2012).
                                                                                                                                                               Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria.
                                                                                                                                                               PLOS ONE 7, pp. 1-9.
                                                                                                                                                        [43]   Arnér, E.S.J., Holmgren, A. (2000). Physiological functions of thioredoxin and
                                                                                                                                                               thioredoxin reductase. Eur. J. Biochem. 267, pp. 6102-6109.


                                                                                                                                                         Biographical notes

                                                                                                                                                          Roberta Russo is a Researcher at the Institute for Biomedical Research and Inno-
                                                                                                                                                        vation (IRIB), National Research Council (CNR), Palermo, Italy. She graduated in Bio-
                                                                                                                                                        logical Sciences at the University of Palermo, with a thesis on Experimental proce-
                                                                                                                                                        dures on Molecular Biology; she has a PhD in Cellular and Developmental Biology;
                                                                                                                                                        she had a post-Doc fellowship in Palermo, at the CNR. Her research fields include the
                                                                                                                                                        study of cell signaling pathways and analysis of differential gene expression, in physi-
                                                                                                                                                        ological conditions and in response to stress and bioactive molecules, through sea ur-
                                                                                                                                                        chin embryos and vertebrate cells in vitro; pathway analysis of genes involved in os-
                                                                                                                                                        teoclastogenesis of murine cells; the study of evolutionary relationships of groups of
                                                                                                                                                        organisms by phylogeny of proteins isolated from bacteria, animals and plants. She is
                                                                                                                                                        a training tutor on Basic and Master degrees.

                                                                                                                                                          Marco Chiaramonte graduated in Biological Sciences at the University of Palermo
                                                                                                                                                        and has a PhD in Environmental Biology and Biodiversity from the same University. He
                                                                                                                                                        had a Fellowship at the National Council Research (CNR), Palermo, Italy, on the study
                                                                                                                                                        of skeletogenesis in sea urchin embryos; a Fellowship at the CNR of Palermo on the
                                                                                                                                                        study of immune effects of bioactive molecules; a post-Doc fellowship at the Universi-
                                                                                                                                                        ty of Palermo on the extraction and analysis of bioactive molecules from fish waste. He


                                                                                                              218
is a tutor for the Basic and Master degrees in Biodiversity and Evolution. His research
fields focus on phylogenetic studies on proteins/genes in various organisms; the study
of cell signaling pathways and analysis of differential gene expression, in physiological
conditions and in response to pollution and to bioactive molecules.

  Franco Palla is Associate Professor of Applied and Environmental Botany at the Uni-
versity of Palermo, Italy. He is Coordinator of the five-year Degree in Conservation and
Restoration of Cultural Property (LMR/02, certified professional restorer). He was the
scientific head of the UNIPA Research Unit, on the Research Project PON01_00625,
It@cha (Italian Technology for Advanced Applications in Cultural Assets) and was a
member of the working groups in the joint project between Italy-Cambodia for Training
Experts of Cultural Heritage, University of Palermo – Royal University of Fine Arts and
the Ministry of Culture and Fine Arts, Angkor, Cambodia. He is the Scientific Coordinator
of the Laboratory of Biology and Biotechnologies for Cultural Heritage at the Department
of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) at
the University of Palermo, Italy, performing applied research and scientific consulting in
the field of the conservation and restoration of natural and cultural heritage. He is corre-
sponding and author of 198 papers published in international and national scientific jour-
nals, books and proceedings, related to the field of Molecular Biology and Biotechnolo-
gy for studying and conservation of cultural and natural Heritage.


 Summary

  Cyanobacteria or blue algae are ubiquitously present in both fresh and brackish wa-
ter environments. They also grow in conditions of high humidity, colonizing stones or
monuments and fountain surfaces, and creating thick biofilms able to induce biodete-
rioration in the constituent materials of artefacts. As well as several photoautotrophic
organisms, cyanobacteria belong to the microorganisms identified as primary coloniz-




                                                                                                 CONSERVATION SCIENCE IN CULTURAL HERITAGE
ers, playing an important role in stone artwork deterioration.
  In this study, an analysis was made of the biofilm collected from the stone fountain of
the Two Dragons in Palermo (Italy), revealing the presence of cyanobacterial colonies by
optical microscopy, due to their peculiar auto-fluorescence. Furthermore, molecular in-
vestigations by qPCR (quantitative Polymerase Chain Reaction) were utilized to gather
quantitative information, and phylogenetics analysis was used to confirm the Thioredox-
in reductase (TrxR) gene as a suitable molecular marker. The results highlight the pres-
ence of cyanobacteria as the main taxa, whose growth is induced by microclimatic and
environmental conditions, and by the physical characteristics of the stone surface.
  Identification of microbial populations living on stone artworks is the starting point for
successful control and conservation strategies, which can help to define the correct
protocols to block cellular activity and to find appropriate methods for removing biofilm,
as well as counteracting possible recolonization.


 Riassunto

  I cianobatteri o alghe azzurre sono ubiquitariamente presenti in ambienti sia di acqua
dolce sia salmastra, sviluppandosi anche in condizioni di elevata umidità, colonizzan-
do pietre o superfici di monumenti e fontane, e generando complessi biofilm, in grado
di indurre il biodeterioramento dei materiali costitutivi del manufatto. Così come per al-


                                                                                               219
                                                                                                                                                        tri organismi fotoautotrofi, i cianobatteri appartengono ai microrganismi identificati come
R. Russo, M. Chiaramonte, F. Palla - Freshwater cyanobacteria, identified by microscopic and molecular investigations on a colonized fountain surface



                                                                                                                                                        colonizzatori primari, svolgendo un ruolo importante nella formazione di consorzi mi-
                                                                                                                                                        crobici in grado di indurre processi di deterioramento.
                                                                                                                                                           In questo studio si è analizzato il biofilm presente sulla superficie di una fontana in
                                                                                                                                                        materiale lapideo, rivelando, mediante microscopia ottica sia in campo chiaro sia in
                                                                                                                                                        fluorescenza, la presenza di colonie di cianobatteri con peculiari morfologie e struttu-
                                                                                                                                                        re. Inoltre, la tecnica qPCR (quantitative Polymerase Chain Reaction) ha permesso di
                                                                                                                                                        confermare il gene della Tioredossina reduttasi (TrxR) come marcatore molecolare
                                                                                                                                                        adatto per studi di filogenesi. I risultati evidenziano la presenza di cianobatteri come
                                                                                                                                                        principali taxa, la cui crescita è indotta dal microclima e dalle condizioni ambientali, oltre
                                                                                                                                                        che dal litotipo e dalle caratteristiche fisiche della superficie lapidea.
                                                                                                                                                           La più completa identificazione delle popolazioni microbiche che colonizzano manu-
                                                                                                                                                        fatti storico-artistici è peculiare per la definizione di un’adeguata strategia per il control-
                                                                                                                                                        lo delle attività microbiche, per la rimozione del biofilm, oltre al contrasto di eventuali ri-
                                                                                                                                                        colonizzazioni.




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