10.6092/issn.1973-9494/12805
Le chiese in legno e pietra dal XVII al XIX secolo sono un simbolo iconico del patrimonio nazionale rumeno. Il presente studio indaga con metodi qualitativi e quantitativi le comunità microbiche provenienti dalle superfici biodeteriorate di chiese monumentali in legno e pietra incluse nell'elenco dei beni culturali di importanza locale o nazionale. Da un totale di dodici monumenti, sono stati prelevati campioni con tamponi di cotone sterili, inoculati su colture specifiche e identificati con metodi classici, automatizzati e molecolari. Un totale di 133 ceppi appartenenti ad Ascomycota phylum sono stati identificati e confermati a livello di specie dalle chiese di legno, tra cui Penicillium spp. I ceppi (principalmente P. corylophylum, P. chrysogenum) sono i più frequenti, seguiti da Alternaria alternata e dalle specie dei generi Trichoderma, Aspergillus, Rhizopus, Mucor e Fusarium. Dalle chiese in pietra sono stati isolati in totale 100 ceppi appartenenti ai generi Aspergillus, Alternaria, Mucor, Penicillium, Aspergillus, Trichoderma, Fusarium e Rhizopus.
Un totale di 55 ceppi batterici è stato isolato e identificato come specie Bacillus, Artrobacter e Pseudomonas. La carica microbica dei campioni varia tra 2,18x107 e 3x105 CFU / mL. Un numero molto piccolo di ceppi fungini (6/77) isolati da chiese in legno (principalmente A. alternata, seguito da P. corylophilum e un ceppo Cladosporium spp.) e da chiese in pietra (5/55) (principalmente A. alternata, seguito di A. versicolor, ceppo di A. nidulans) sono stati coinvolti nella formazione del biofilm.
I risultati di questo studio possono aiutare a migliorare la comprensione del deterioramento microbico delle chiese del patrimonio rumeno e consentire la definizione di strumenti di decontaminazione, conservazione e conservazione più affidabili.
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Conservation Science in Cultural Heritage
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Multi-Level Characterization of Microbial Consortia Involved in the Biodeterioration of Wooden and Stone Romanian Heritage Churches 
Text.Serial.Journal
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Articles
2021-05-13
2021-04-27
2021-05-13
2021-05-13
289-308
CSCH
Irina Balotescu
2020
10.6092/issn.1973-9494/12805
289
UB-ICUB, IEF-Romanian Academy, Bucharest, Romania
1973-9494
dependent /independent cultivation methods
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Multi-Level Characterization of Microbial Consortia Involved in the Biodeterioration of Wooden and Stone Romanian Heritage Churches 
https://conservation-science.unibo.it/article/download/12805/12522
20
M     ULTI-LEVEL CHARACTERIZATION OF MICROBIAL
      CONSORTIA INVOLVED IN THE BIODETERIORATION
      OF WOODEN AND STONE ROMANIAN HERITAGE
      CHURCHES


   Irina Gheorghe, Ionela Sârbu*
   Botany-Microbiology and Genetics Departments, Faculty of Biology UB-ICUB
   University of Bucharest, Bucharest, Romania

   Ionut Pecete
   Central Reference Synevo-Medicover Laboratory
   Bucharest, Romania

   Ion Blăjan
   Romanian Peasant Museum
   Bucharest, Romania

   Irina Balotescu
   UB-ICUB, IEF-Romanian Academy
   Bucharest, Romania


   Keywords: Romanian heritage churches, biodegradation, fungi, bacteria, dependent
   /independent cultivation methods




   1. Introduction

    According to several studies biodeterioration represents the process of modifica-
tion of material properties due to the activity of organisms from different systematic
groups: bacteria, archaea, actinomycetes, algae, fungi, lichens, magnoliophytes, and
animals [1,2]. The present study aims to investigate, by qualitative and quantitative
methods, the microbial communities from biodeteriorated surfaces of wooden and stone
monument churches, most of them included in the list of Romanian cultural heritage of
local (Class B) and national (Class A) importance.
    The Romanian wooden churches are a category of monuments which belong to the
great family of European wood architecture [3].
    Placed since 1991 under the protection of the National Museum of the Romanian
Peasant (MNȚR) of Bucharest, Arad and Hunedoara, the wooden churches represent
monuments of national importance (Class A). Unfortunately, they are today in various
stages of degradation, with minimum interventions being made as regards their state
of conservation. In most cases, restoration has been carried out as an emergency in-


   * Corresponding author: ionela24avram@yahoo.com
                                                                                                                                                   tervention, as no national programs are dedicated to the restoration of monuments in
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   critical situations requiring immediate restoration.
                                                                                                                                                        The cultural and historical importance for the village communities in the areas
                                                                                                                                                   where these churches are located is a major one, given the multiethnic component
                                                                                                                                                   specific to the region, which include small Orthodox communities. For the Roma-
                                                                                                                                                   nians from the villages of Mureş, Arieş and Târnave (Transylvania region), the wood-
                                                                                                                                                   en churches bear witness to the serious consequences of denationalization, often
                                                                                                                                                   accompanied by religious persecution. These abusive interventions, on both the lo-
                                                                                                                                                   cal population and the churches, have strongly affected community life and the con-
                                                                                                                                                   dition of church edifices up to the present. Some members of the religious communi-
                                                                                                                                                   ties even left the country or converted to other confessions, therefore the duty of
                                                                                                                                                   preserving the monuments remained the concern of the parish or the state (Class A
                                                                                                                                                   Monuments).
                                                                                                                                                        Thus, from a historical perspective, wooden churches have been a way for the
                                                                                                                                                   Orthodox communities in the area to assert their ethnic and religious identity, whereas
                                                                                                                                                   from a cultural perspective, the aesthetic, architectural and artistic value of the build-
                                                                                                                                                   ings demonstrates an obvious concern for the construction of sustainable religious
                                                                                                                                                   establishments, marking the spiritual life of the practicing communities.
                                                                                                                                                        At present, these places of worship have a strictly heritage and historical value, and
                                                                                                                                                   religious rites specific to the Orthodox cult are no longer practised. In all the villages
                                                                                                                                                   where the churches, under the protection of MNȚR are located, new stone churches
                                                                                                                                                   have been built to better withstand the climatic and geographical conditions specific to
                                                                                                                                                   the area (i.e. low temperatures in cold seasons and high humidity, creating favorable
                                                                                                                                                   conditions for severe wood damage and the development of various species of fungi,
                                                                                                                                                   both outdoors and indoors).
                                                                                                                                                        Being an integral part of the peasant communities, these churches have a series of
                                                                                                                                                   features in common with traditional houses: similar dimensions, similar raw materials
                                                                                                                                                   (wood logs) and techniques (such as “blokbau” or interlocking) were used for their
                                                                                                                                                   construction. The design of the wooden churches included: the narthex, the nave and
                                                                                                                                                   the altar, arranged on the longitudinal axis of the construction to which, in some cases,
                                                                                                                                                   the porch is added (as is found especially in Transylvania and sometimes in Oltenia,
                                                                                                                                                   Muntenia or Moldova). These monuments have an obvious spatial architectural unity,
                                                                                                                                                   as they are often small and measured from the entrance to the narthex to the altar, so
                                                                                                                                                   the church is generally the size of a house.
                                                                                                                                                        The architecture, building techniques and decorations of the wooden churches are
                                                                                                                                                   similar to those of the peasant houses. The main feature is represented by the austere
                                                                                                                                                   style of the exterior, as the buildings were conceived to adapt to the landform and cli-
                                                                                                                                                   mate of the area. Massive wooden pillars whose tops are often shaped like a stylized
                                                                                                                                                   horse’s head and the heads of the rafters that support the eaves of the roof, are also
                                                                                                                                                   specific to these monuments.
                                                                                                                                                        Some aspects related to the execution technique or iconographic development,
                                                                                                                                                   are imposed by the special form of the wooden architecture and the way this mate-
                                                                                                                                                   rial is prepared. The painting registers (the series of iconografic images and their
                                                                                                                                                   order in the church) generally include the temple, icons, altar, vaults and paintings
                                                                                                                                                   of the founders. The latter are present in some of the churches, as the Byzantine
                                                                                                                                                   painting hermēneia (manual) considers them optional. Also, it is important to men-
                                                                                                                                                   tion that they cannot be attested in the case of monuments where the painting has
                                                                                                                                                   been completely removed or destroyed, especially where there is no photographic
                                                                                                                                                   evidence of the original painting. As an execution technique, tempera, frequently ap-


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plied directly on wood, canvas, plastered wood, or even fresco on wood covered with
plaster, are common.
    The pictorial ensembles specific to the wooden churches from Arad (Groşii Noi,
Juliţa and Troaş) and Hunedoara (Lunca Moţilor and Bejan) under the protection of
MNȚR (The Romanian Peasant Museum – http://www.muzeultaranuluiroman.ro/
museum.html), date from the 18th and the beginning of the 19th century and are the
work of the peasant painters.
    Of all the mentioned churches, the one in the MNȚR museum courtyard is in the
most advanced stage of deterioration. Both the outside and inside bear evident signs
caused by unfavorable weather conditions and the lack of emergency conservation
and restoration interventions. Currently, all churches require restoration work and are
inaccessible to the general public.
    A number of studies around the world have documented the degradation of wood-
en cultural heritage objects [4-10]. However, there is still insufficient information
regarding identification at species level and the enzymatic activity of the filamen-
tous fungi species involved in the biodeterioration of cultural heritage. Molecular
methods available to identify fungi, by sequencing conserved regions of rDNA,
could facilitate identification and provide information on the diversity of fungi caus-
ing the degradation. Fungal agents are also involved in the deterioration of stone
monuments [11-13], causing discoloration and degradation due to fungal growth on
their stone surfaces, physical intrusion or penetration of fungal hyphae and produc-
tion of pigments and organic acids (e.g. oxalate) [14-16], while bacterial coloniza-
tion could lead to crust formation, exfoliation and color alteration [17,18]. It must also
be taken into account that several environmental parameters, such as tempera-
ture, relative humidity or light intensity, have a great impact on fungal development
[19-21].
    Therefore, geographically located studies are required in order to elucidate the
diversity of the bio-deteriorating species and to further develop efficient tools for re-




                                                                                               Conservation science in Cultural Heritage
ducing their negative impact. Moreover, the presence of filamentous fungi inside the
heritage churches could be a major cause of respiratory diseases and allergies in
humans [3,22].


   2. Materials and methods

   2.1. Phenotypic (macroscopic and microscopic) identification of microbial
   isolates

     Based on the observed changes, samples were taken with cotton sterile swabs
from a total number of thirteen churches of wooden (n=5) and stone (n=8) from
Arad (AR), Hunedoara (HD) and Bucharest, between July and August 2018. Most
of the investigated churches (Table 1) are included in the national cultural heritage
of Romania.
     The samples were taken from areas of about 10 cm2 of the walls of the narthex,
nave and altar, to which aero-microbiota samples were added. A microbial attack was
suspected as the majority of the isolated points in the investigated churches presented
visible alterations, colored spots, discolored areas, deposits or patina on the surfaces;
for this reason, several samples were taken. Areas without visible changes were consid-
ered as negative controls.


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                                                                                                                                                   Table 1. Name, code, location and geographical coordinates of the investigated churches.
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration




                                                                                                                                                                                                                         On the list of national
                                                                                                                                                                             Village,
                                                                                                                                                         Church                             Geographical coordinates      cultural heritage of
                                                                                                                                                                             county
                                                                                                                                                                                                                               Romania
                                                                                                                                                                                           Wooden churches
                                                                                                                                                       Saint Nicolae
                                                                                                                                                                           Bucharest          44.4364°N;26.08783°E                 No
                                                                                                                                                         Hierarch
                                                                                                                                                      The wooden
                                                                                                                                                                             Troaș,
                                                                                                                                                      Church of the
                                                                                                                                                                          Săvârșin, AR       46.09050°N;22.29510°E        Yes, AR-II-m-A-00655
                                                                                                                                                       Three Holy
                                                                                                                                                                            county
                                                                                                                                                        Hierarchs
                                                                                                                                                       The wooden          Groșii Noi,
                                                                                                                                                       Church of the      Bârzava, AR       46.106532°N;21.993263°E       Yes, AR-II-m-A-00607
                                                                                                                                                      Lord’s greeting       county
                                                                                                                                                      The wooden       Julița, Vărădia
                                                                                                                                                    Church entrance of de Mureș, AR         46.039275°N;22.134529°E         AR-II-m-A-00615
                                                                                                                                                     the Virgin Mary        county
                                                                                                                                                      The wooden
                                                                                                                                                      Church of the       Lunca Moților,
                                                                                                                                                                                             46.170306°N;22.68047°E         HD-II-m-A-03360
                                                                                                                                                    Assumption of the      HD county
                                                                                                                                                       Virgin Mary

                                                                                                                                                                                           Stone churches

                                                                                                                                                    The stone Church
                                                                                                                                                                            Strei, HD
                                                                                                                                                    of the Assumption                       45.716650°N;22.988115°E      Yes, HD-II-m-A-03452
                                                                                                                                                                             county
                                                                                                                                                    of the Virgin Mary
                                                                                                                                                    The stone Church
                                                                                                                                                                           Ostrov, HD
                                                                                                                                                    Descent of the Holy                     45.527907°N; 22.846613°E      Yes, HD-II-a-A-03400
                                                                                                                                                                            county
                                                                                                                                                          Spirit
                                                                                                                                                    The stone Church       Orlea, HD
                                                                                                                                                                                            45.590715°N; 22.969964°E     Yes, HD-II-m-A-03445.
                                                                                                                                                      of Sântămărie         county
                                                                                                                                                                            Near to
                                                                                                                                                                           Silvașu de
                                                                                                                                                    Prislop monastery                      45.6317548°N;22.8503078°E     Yes, HD-II-m-A-03447
                                                                                                                                                                            Sus, HD
                                                                                                                                                                             county
                                                                                                                                                    The stone Church
                                                                                                                                                                           Paroș, HD
                                                                                                                                                    of the Descent of                       45.495998°N; 22.969807°E     Yes, HD-II-m-A-03401
                                                                                                                                                                            county
                                                                                                                                                      the Holy Spirit
                                                                                                                                                    The St. Nicholas      Densuș, HD
                                                                                                                                                                                            45.582631°N; 22.805392°E        HD-II-m-A-03454
                                                                                                                                                    Church of Densuș        county
                                                                                                                                                    The Church of the
                                                                                                                                                                          Nucșoara, HD
                                                                                                                                                     Ascension of the                       45.480841°N; 22.916338°E        HD-II-m-B-03370
                                                                                                                                                                             county
                                                                                                                                                          Lord
                                                                                                                                                      The Church of       Sînpetru, HD
                                                                                                                                                                                            45.551702°N; 22.912181°E        HD-II-m-A-03307
                                                                                                                                                      Saint George           county



                                                                                                   292
    After sampling, cotton swabs were submerged in 1 mL of sterile distilled water
solution for spore suspension, dilutions were performed and then plated onto two dif-
ferent media: PDA (Potato Dextrose Agar) and Rose Bengal (Liofilchem) incubated at
22 ± 1◦C for 5-7 days, allowing fungal growth and PCA (Plate Count Agar) for bacterial
growth. After this period, colonies were then purified (using the same culture medium
from where they were first isolated) and subcultures were prepared for morphological
and automated identification. Fungal isolates were identified to genus or species level
using biometric parameters (colony diameter) and microscopic features. Microscopic
examination was performed using blue cotton lactophenol staining. Bacterial strains
were preliminarily identified by Gram staining, catalase and oxidase tests.


   2.2. Quantitative assessment of microbial contamination by cultivation
   dependent methods

   In the next step, the number of colony-forming units (CFU/mL) of bacteria, yeasts,
and filamentous fungi was determined, according to the following equation: CFU/m2 =
(number of the developed colonies x 100)/dilution.


   2.3. Automated identification using MALDI-TOF system

    The identification of microbial consortia (filamentous fungi and bacteria) by the
MALDI-TOF system was based on a database that requires a permanent update for
the most accurate identification.


   2.4. Quantitative evaluation of the fungal adherence to inert substrata




                                                                                              Conservation science in Cultural Heritage
    Evaluation of biofilm development on the inert substrate was assessed by the mi-
crotiter broth method. Overnight cultures were grown in 96 multi-well plates containing
Potato Dextrose Broth (PDB) medium for fungi and Muller Hinton Broth (MHB) supple-
mented with 2% glucose for bacteria at 22°C for 48-72 h, then the content was re-
moved and washed three times with phosphate-buffered saline (PBS). The adherent
cells were then fixed with cold methanol, stained with an alkaline 1 % violet crystal
solution for 15 min., washed with water and resuspended in a 33 % acetic acid solution.
The intensity of the suspension was spectrophotometrically assessed, the amount of
adhered biomass being proportional to the absorbance value read at 492 nm [23].


   2.2. Evaluation of capsule and spore forming bacteria

    Staining of the spores with Malachite green according to the Benito Trujillo protocol
was performed on all Gram-positive strains. The microbial strains were grown in Luria
Broth (LB) medium supplemented with 0.5 M CaCl2 and incubated at 300 C for up to 72
hours. After incubation, microbial cultures were stained according to protocol and
spores were examined under the optical microscope. Depending on the stage of devel-
opment, the endospores appeared colored in different shades of green and the vegeta-
tive cells in red.


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                                                                                                                                                       2.3. Nucleic acid-based methods for fungal identification and diversity
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration




                                                                                                                                                        The fungal strains not identified by MALDI analysis were taxonomically identified by
                                                                                                                                                   sequencing the intergenic region of ribosomal genes. The genetic diversity of the fun-
                                                                                                                                                   gal species was studied by the PCR method of the Internal Transcribed Spacer (ITS)
                                                                                                                                                   region, a highly conserved rDNA region. The DNA was extracted by the classical phe-
                                                                                                                                                   nol/chloroform method and subsequently amplified using the following primers: ITS-1
                                                                                                                                                   (5’- TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’).
                                                                                                                                                   20ng of DNA and 0.5µM of each primer were introduced into the amplification reaction.
                                                                                                                                                   The amplification program included: initial denaturation of 95°C, 5min and 35 cycles
                                                                                                                                                   which included (95°C, 1 min; 55°C, 60 sec; 72°C, 8 min). The obtained amplicons
                                                                                                                                                   were verified in 1.5% agarose gel and subsequently sequenced using the ABI 3730xl
                                                                                                                                                   DNA sequencer – Applied Biosystems. The obtained sequences were analyzed
                                                                                                                                                   using ChromasLite 2.1.1 (http://www.technelysium.com.au) and compared with the
                                                                                                                                                   sequences from the National Center for Biotechnology Information (NCBI) data-
                                                                                                                                                   base using the Basic Local Alignment Search Tool (BLAST) program for species
                                                                                                                                                   identification.


                                                                                                                                                       3. Results and discussion

                                                                                                                                                       3.1. Microbial load of the collected samples

                                                                                                                                                       The comparative study of the microbial load of the samples taken from six wooden
                                                                                                                                                   (n=4) and stone (n=2) churches located in the counties of AR and HD (Bârzava, Lunca
                                                                                                                                                   Moților, Troaș, Densuș, Julița and Strei churches) between July and August 2018 from
                                                                                                                                                   different sampling points, revealed a two-log variation of the total microbial load, among
                                                                                                                                                   different churches and sampling points inside each church, ranging from 2.18x107 to
                                                                                                                                                   3x105 CFU/mL (Table 2).

                                                                                                                                                   Table 2. Comparative representation of total microbial loads (log CFU/mL) in different churches and
                                                                                                                                                   sampling points.

                                                                                                                                                                                      Church 1 Bârzava (AR county)
                                                                                                                                                        Pronaos              Naos               Altar             Objects           Entrance door
                                                                                                                                                         1x 10 5
                                                                                                                                                                            1x 10 5
                                                                                                                                                                                               1x 10 5
                                                                                                                                                                                                                     -                    -
                                                                                                                                                         3x 105             3x 105             2x 105
                                                                                                                                                        201x105            200x105             3x 106
                                                                                                                                                                                Church 2 Lunca Moților (HD county)
                                                                                                                                                        Pronaos              Naos               Altar             Objects           Entrance door
                                                                                                                                                         1x 105                                1x 105                -                    -
                                                                                                                                                         3x 105                                2x 105
                                                                                                                                                                                Church 3 Troaș-Săvârșin (AR county)
                                                                                                                                                        Pronaos              Naos               Altar             Objects           Entrance door
                                                                                                                                                         8x 105             7x 105              2x 105            1x 105                  -
                                                                                                                                                                                                8x 105
                                                                                                                                                                                               12x 105



                                                                                               294
                                Church 4 Densuș (HD county)
     Pronaos             Naos             Altar           Objects         Entrance door
      1x 105                                                                  3x 105
                                 Church 5 Julița (AR county)
     Pronaos             Naos             Altar           Objects         Entrance door
     1x 105            3x 105             3x 105         141x 105             1x 105
     76x 105          218x 105           12x 105
                                 Church 6 Strei (HD county)
     Pronaos             Naos             Altar           Objects         Entrance door
        -                 -                 -                  -              1x 105



   3.2. Phenotypic characterization of filamentous fungi

    One of the main goals of our study was to characterize the fungal strains isolated
from the wooden and stone monument churches showing various degrees of biodete-
rioration and to investigate some features associated with their survival and resistance
in different conditions, taking into account the results of many studies, showing that
both microclimate and materials affect microbial density [24] (Fig. 1 A and B; 2 A and B;
3 A and B).




                                                                                                Conservation science in Cultural Heritage


                     A                                              B
Figure 1. Wooden churches: iconostasis of the Saint Hierarch Nicolae Church (A); and the
Iconostasis of the wooden The Lord’s greeting, Barzava (B).

    It is well known that microorganisms, particularly fungi can inhabit, alter and degrade
different types of materials [25]. Cotton can be degraded when it is exposed to ultra-
violet light and chemical and biological attacks, as well as to contamination and me-
chanical stress [26]. These are all materials that support the growth of fungi throughout
the years. Based on the literature data, the majority of fungi species involved in the
deterioration of the cultural heritage belonged to Ascomycota phylum, and the occur-
rence of basidiomycetes restricted to wood degradation in churches or other protected
historical monuments. Zygomycetes are frequently isolated from pieces of art, but in
most cases their presence is only transitory [25].


                                                                                              295
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration




                                                                                                                                                                                A                                                B

                                                                                                                                                   Figure 2. The naves. A: the wooden Church of the Assumption of the Virgin Mary in Lunca Motilor;
                                                                                                                                                   and B, the stone Church of the Ascension of the Lord in Nucșoara.




                                                                                                                                                                         A                                                B

                                                                                                                                                   Figure 3. The stone Church of Sântămărie (A); an orthodox book from the stone Church of Saint
                                                                                                                                                   George in Sînpetru (B).


                                                                                                                                                       Preliminary phenotypic identification of the filamentous fungi was performed based
                                                                                                                                                   on the examination of macroscopic characters observable after 5-7 days of incubation
                                                                                                                                                   at 22°C, analyzing the obverse of colonies and their reverse and taking into account
                                                                                                                                                   the time of culture onset, the degree of development (biometric parameter), consistency
                                                                                                                                                   of the culture, the pigmentation, the shape of the colonies, their profiles, etc. Micro-
                                                                                                                                                   scopic examination allowed us to evidence the presence of vegetative and reproduc-
                                                                                                                                                   tive structures ensuring the correct interpretation of results, as there are many species
                                                                                                                                                   which, though present on the surface of the heritage object or in the environment,
                                                                                                                                                   might be dormant and thus not directly responsible for the decay of heritage objects
                                                                                                                                                   (Fig. 4. A1-A8).


                                                                                                296
Figure 4. The macro- and microscopic aspects of some representative filamentous fungi ge-
nus isolated from the investigated churches. A1: A. alternata – obverse aspect of the colonies;
A2: microscopic aspect of A. alternata – blue cotton staining; A3: Fusarium spp. – macroscopic
aspects of the colonies on PDA culture media; A4: Fusarium spp. – clamidospores – micro-
scopic aspect; A5: M. circinelloides – macroscopic aspects of the colonies on PDA culture
media; A6: M. circinelloides – microscopic aspects of the sporangiospores; A7: P. corylophy-
lum – macroscopic view; A8: P. corylophylum – microscopic aspects of the conidiophores and
conidia.



   3.3. Taxonomic assignment of the filamentous fungi isolated from wooden




                                                                                                    Conservation science in Cultural Heritage
        and stone churches

    The sampling from the wooden church belonging to the Romanian Peasant Mu-
seum in Bucharest allowed the identification of filamentous fungi distributed on the
following sources of isolation/species: ascomycetes belonging to the Aspergillus ter-
reus and Rhizopus oryzae species, as well as two basidiomycetes belonging to the
Schizophyllum commune species in the narthex area; Penicillium digitatum and Alter-
naria alternata predominated in the nave area, while the church altar was colonized
with isolates belonging to R. oryzae and Cladosporium sp. In the aero-microbiota sam-
ples, the R. oryzae and Trichoderma orientale strains were dominant. In the wooden
church, The Lord’s greeting in Bârzava, Groșii Noi village, the nave was colonized by
Penicillium spp., the narthex by P. chrysogenum and A. alternata and the altar by R.
stolonifer and Cladosporium spp.
    In the wooden church in Săvârșin village, the predominating species in the nave and
altar was P. corylophilum.
    The Julița village church was contaminated in the nave with P. corylophilum and
P. chrysogenum, in the narthex and attic with P. corylophilum and Mucor circinelloides,
while from the altar P. corylophilum and P. expansum were isolated. As regards the
wooden church, The Assumption of the Virgin Mary Church in Lunca Moților, Hunedoara
County, transferred to the Bucharest Romanian Peasant Museum, P. Corylophilum
prevailed in all sampling points (Table 3).


                                                                                                  297
                                                                                                                                                   Table 3. Comparative representation of the fungal species isolated from wooden churches and
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   sampling points.

                                                                                                                                                                  Church 1- Bucharest: Frequency of species with percentages
                                                                                                                                                          Pronaos                Naos                 Altar             Object            Others:
                                                                                                                                                                                                                                       aeromicrobiota
                                                                                                                                                   A. terreus, R. oryzae,     P. digitatum,        R. oryzae,                            R. oryzae, T.
                                                                                                                                                   S. commune (16.16%         A. alternata      Cladosporium sp.                      orientale (22.22%
                                                                                                                                                           each)              (25% each)         (5.71% each)                               each)
                                                                                                                                                                 Church 2 – Bârzava (AR): Frequency of species with percentages
                                                                                                                                                          Pronaos                Naos                 Altar              Attic             Others
                                                                                                                                                     P. chrysogenum, A.      Penicillium sp.    R. stolonifer and
                                                                                                                                                     alternata (33.33%          (50%)           Cladosporium sp.
                                                                                                                                                            each)                                (12.5% each)
                                                                                                                                                            Church 3 – Troaș – Săvârșin (AR): Frequency of species with percentages
                                                                                                                                                          Pronaos                Naos                 Altar              Attic             Others
                                                                                                                                                    P. corylophilum (20%)   P. corylophilum     P. corylophilum
                                                                                                                                                                                 (20%),               (20%),
                                                                                                                                                                                A. flavus       P. chrysogenum,
                                                                                                                                                                              (10% each)          R. stolonifer
                                                                                                                                                                                                   (10% each)
                                                                                                                                                                    Church 4 – Julița (AR): Frequency of species with percentages
                                                                                                                                                          Pronaos                Naos                 Altar              Attic          Others: roof
                                                                                                                                                      P. corylophilum M.    P. corylophilum     P. corylophilum,    P. corylophilum   A. flavus (2.77%)
                                                                                                                                                    circinelloides (5.55%      (13.88%),         P. expansum            (5.55%)
                                                                                                                                                             each)                               (5.55% each)
                                                                                                                                                              Church 5 – Lunca Moților (HD): Frequency of species with percentages
                                                                                                                                                          Pronaos                Naos                 Altar              Attic        Others: outer wall
                                                                                                                                                       P. corylophilum       P. corylophilum    P. corylophilum                        P. corylophilum
                                                                                                                                                          (13.88%)                (25%),           (16.66%)                                (5.55%)
                                                                                                                                                                            A. niger (13.88%)

                                                                                                                                                      As regards the stone churches from the Hunedoara county, the following genera
                                                                                                                                                   and species were isolated:
                                                                                                                                                           - Nucșoara church - Aspergillus versicolor strains were dominant followed by
                                                                                                                                                      A. alternata, M. circinelloides, P. chrysogenum and Aspergillus sydowii;
                                                                                                                                                           - the church of “Sfântul Mare Mucenic Gheorghe” in Sînpetru village - Aspergil-
                                                                                                                                                      lus niger, Penicillium spp., A. nidulans, and Purpureocillium lilacinum;
                                                                                                                                                           - In the church in Ostrov village - A. alternata was the dominant species, fol-
                                                                                                                                                      lowed in decreasing order by Penicillium roqueforti, P. digitatum, and P. expansum;
                                                                                                                                                           - in Prislop Monastery - the dominant species was A. niger, followed by A. al-
                                                                                                                                                      ternata, Trichoderma spp., Penicillium commune, and Mucor circinelloides;
                                                                                                                                                           - in the Church of Strei – the majority of the isolates belonged to A. alternata,
                                                                                                                                                      followed by A. niger and A. arundinis;


                                                                                                298
      - in the Sânămarie Church, in Orlea village - A. alternata and Fusarium cerealis
   culmorum group strains were dominant, followed by R. oryzae, Penicillium citri-
   num, Aspergillus flavus oryzae group, and P. corylophylum;
      - in the Church of Paroș village - A. versicolor and other species of the genus
   Aspergillus were dominant, followed by Cladosporium sp. and A. alternata strains
   (Table 4).

Table 4. Comparative representation of the fungal species isolated from stone churches.

                                         Identified species                  Frequency (%)
                                             A. versicolor                       32.55%
  Church 6 – Nucșoara
                                             A. alternata                         9.30%
         (HD)
                            M. circinelloides, P. chrysogenum. A. sydowii      6.97%each
                                          Aspergillus niger                        19%

  Church 7– Sînpetru          A. nidulans, P. lilacinum, Penicillium spp.       18% each
        (HD)                                Rhizopus spp.                          9%
                             Arthrinium phaeospermum, Aspergillus spp.           9% each

    Church 8 Ostrov                          A. alternata                          40%
         (HD)                  P. roqueforti, P. digitatum, P. expansum         20% each
                                          Aspergillus niger                        34%
   Church 9 – Prislop
                                   Trichoderma spp., A. alternata               22% each
         (HD)
                               Penicillium commune, M. circinelloides           11% each

   Church 10 – Strei                         A. alternata                          72%




                                                                                               Conservation science in Cultural Heritage
        (HD)                        A. niger, Arthrinium arundinis              14% each
                           A. alternata, Fusarium cerealis culmorum group          25%
      Church 11 –
      Sântămărie                     P. corylophylum, R. oryzae                 13% each
        (HD)
                                 A. flavus oryzae group, P. citrinum            12% each
                                     A. versicolor, Aspergillus sp.             17% each
                              Cladosporium sp., A. alternata, A. sydowii        12% each
      Church 12 –
        Paroș                             Aspergillus niger                      6% each
        (HD)                           Stachybotrys chartarum
                                        Penicillium expansum
                                           Penicillium spp.

    In Romania, only several studies have investigated the above-mentioned topic.
In 2013, Dăneasă et al. investigated one wooden church from Hunedoara County
(Boz village) included in the list of monuments of local importance (Class B). The study
demonstrated that several parts of the investigated church were in an advanced state
of degradation because of water infiltration through the highly damaged roof and re-
quired emergency interventions for its conservation, preservation and restoration [27].
In 2018, another group investigated the diversity of filamentous fungi and bacteria
isolated from one of the wooden churches (Bihor County) included in Class B of the


                                                                                             299
                                                                                                                                                   national list of historical monuments and demonstrated – similarly to our study – the
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   presence of Aspergillus, Rhizopus, Penicillium and Mucor genera; these fungi/bacteria
                                                                                                                                                   are also associated with a major risk to human health and can cause infections, aller-
                                                                                                                                                   gies, and toxicity [28]. The same group performed a similar study on the paintings
                                                                                                                                                   found on the interior walls of the Orthodox wooden Church of Saint Martyrs Constantin
                                                                                                                                                   Brȃncoveanu and His Sons, on the University of Oradea campus, also a Class B his-
                                                                                                                                                   torical monument; in this case, they identified Penicillium, Aspergillus, Alternaria or
                                                                                                                                                   Cladosporium strains [29].
                                                                                                                                                       Using culture-based methods to determine the filamentous fungi involved in the
                                                                                                                                                   biodeterioration of the mural paintings in the old orthodox church named “Annuncia-
                                                                                                                                                   tion”, located in the Sibiu area, two species of the Penicillium genus (P. rugulosum and
                                                                                                                                                   P. chrysogenum) were identified [30].
                                                                                                                                                       Several genera of filamentous fungi (Alternaria, Penicillium, Aspergillus, and Clad-
                                                                                                                                                   osporium), diverse Bacillus species, Planomicrobium and Variovorax genera, not pre-
                                                                                                                                                   viously reported in paintings or on wood, were identified in samples recovered from the
                                                                                                                                                   wall and icon surfaces of a Class A monument, the seventeenth-century wooden church
                                                                                                                                                   of Nicula, located in Cluj County, Romania [31].
                                                                                                                                                       The filamentous strains not identified by phenotypic approach to the species level
                                                                                                                                                   were submitted to molecular analysis. The ITS (internal transcribed spacer) region was
                                                                                                                                                   used as a target region in the phylogenetic analysis as it generally exhibits sequence
                                                                                                                                                   variations between species, but only minor variations within strains of the same spe-
                                                                                                                                                   cies. Molecular identification of the fungi up to species level was largely based on an
                                                                                                                                                   analysis of the ITS regions including ITS1, 5.8S rDNA and ITS4. The DNA sequences
                                                                                                                                                   in the ITS region are also highly variable and could serve as a marker of the phyloge-
                                                                                                                                                   netically distant taxonomic groups. The molecular investigation of the Penicillium spp.,
                                                                                                                                                   Trichoderma spp. Cladosporium spp. and Alternaria spp. strains allowed the identifica-
                                                                                                                                                   tion of P. chrysogenum, P. expansum, P. crustosum, P. colylophylum, T. longibrachyatum,
                                                                                                                                                   C. cladosporoides, and A. alternata species, with 100% identity (molecular identifica-
                                                                                                                                                   tion presented 100% identity with similar species from NCBI database – those with
                                                                                                                                                   99% were excluded from the study).


                                                                                                                                                      3.4. Bacterial yeast associations present in the analyzed samples

                                                                                                                                                       There are few studies regarding bacterial diversity associated with the biodegrada-
                                                                                                                                                   tion of wooden and stone objects. Initial tests such as Gram staining revealed that
                                                                                                                                                   Gram-positive bacilli are the predominant group, the results being confirmed by MALDI
                                                                                                                                                   analysis. According to the MALDI results, most of the bacterial strains isolated from the
                                                                                                                                                   wood churches belonged to the Bacillus genus.
                                                                                                                                                        A total number of 27 bacterial strains and 3 yeast strains belonging to the Crypto-
                                                                                                                                                   coccus genus were isolated from the wood churches included in the Bucharest Roma-
                                                                                                                                                   nian Peasant Museum, the predominant species being Bacillus pumilus, followed by
                                                                                                                                                   B. megaterium species and B. subtilis. From the less frequently isolated Gram-negative
                                                                                                                                                   strains, the predominant genus was Arthrobacter. More than 50% of the bacterial
                                                                                                                                                   strains not identified by using the MALDI-TOF technique, revealed the great diversity
                                                                                                                                                   of the bacterial strains found in these samples, as compared, for example, with clinical
                                                                                                                                                   samples. In addition, 28 bacterial strains and 5 yeast strains belonging to the Candida
                                                                                                                                                   genus (3) and Cryptococcus (2) genera were isolated from different stone churches
                                                                                                                                                   located in the Hațeg region. The predominant species was B. megaterium followed by
                                                                                                                                                   B. pumillus and B. thuringiensis (Table 5).


                                                                                              300
Table 5. Comparative representation of the bacterial species isolated from wooden and stone
churches.

                                         Identified species             Frequency (%)
                                          Bacillus pumillus                  11%
                                        Bacillus megaterium                  7%
                                           Bacillus subtilis                 3%
                                      Arthrobacter globiformis               4%
  Wood churches – AR county           Arthrobacter aurescens                 4%
                                        Arthrobacter bergeri                 4%
                                     Rhodococcus erythropolis                4%
                                         Ps. enthomophila                    4%
                                           Lb. alimetarius                   4%
                                           S. epidermidis                    4%
                                         Identified species             Frequency (%)
                                        Bacillus megaterium                  36%
                                          Bacillus pumillus                  14%
  Stone churches – HD county            Bacillus thuringiensis               4%
                                            P. koreensis                     4%
                                           Penibacillus sp.                  7%
                                             Candida sp.                     11%




                                                                                                Conservation science in Cultural Heritage
    Some strains belonging to Staphylococcus genera were considered anthropic con-
taminants of objects without direct impact on their degradation.
    In the wooden churches the bacterial diversity was higher compared to the stone
churches, probably due to the higher humidity and the presence of cellulose, which is
an excellent nutritional support for many microorganisms. However, similar to stone
churches, Gram-positive bacteria were predominant, as expected, given the physio-
logical conditions such as low humidity, pH variation, and the presence of mostly inor-
ganic nutrients.
    It was also noted that the bacterial community increased with the advancement of
the deterioration process.


   3.5. Feature related to microbial survival and resistance strategies

   3.5.1. Biofilm development

    The quantitative evaluation of the degree of biofilm development on the inert sub-
strate was performed for 77 fungal strains isolated from the wooden churches located
in Bucharest, Arad and Hunedoara, and 55 strains isolated from the stone churches
located in Hunedoara County. Only 6 fungal strains out of 77 fungal strains from wood-
en churches in Bucharest, Arad and Hunedoara showed the capability to form biofilms.


                                                                                              301
                                                                                                                                                   The main species recurrently found to harbor this feature was A. alternata, followed by
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   P. corylophilum and Cladosporium spp (Fig.5). The results of our study are in agree-
                                                                                                                                                   ment with those reported in the scientific literature regarding the heterotrophic compo-
                                                                                                                                                   nents of biofilms formed on wooden artifacts [32]. The capacity of the fungal strains
                                                                                                                                                   isolated from wood and stone churches to form biofilms seems to be correlated with
                                                                                                                                                   the high microbial load and the advanced degree of deterioration, an aspect highlight-
                                                                                                                                                   ing the need for intervention measures based on anti-biofilm compounds for prevent-
                                                                                                                                                   ing the biodegradation of heritage objects.




                                                                                                                                                   Figure 5. Graphic representation of the biofilm forming ability of the strains isolated from wooden
                                                                                                                                                   churches.


                                                                                                                                                      In Serbia, a research group investigated the biofilm-forming community isolated
                                                                                                                                                   from the stone Church of the Holy Ascension and revealed that the dominant genera
                                                                                                                                                   were Alternaria, Arthrinium and Cladosporium, also isolated in our study both from
                                                                                                                                                   wooden and stone churches. It must be mentioned that the Cladosporium species rep-
                                                                                                                                                   resents one of the main biological agents involved in the biodeterioration of the mural
                                                                                                                                                   paintings [33].




                                                                                                                                                   Figure 6. Graphic representation of the biofilm forming ability of the bacterial strains isolated from
                                                                                                                                                   wooden churches.


                                                                                                    302
    A similar study performed in Italy, on deteriorated wall paintings from seven medi-
eval churches revealed the presence of different species belonging to Penicillium, As-
pergillus, Fusarium, and Alternaria genera [34].
    Species such as Aspergillus versicolor, Cladosporium, or Penicillium spp. revealed by
other research groups, such as Trovão et al., in 2019, demonstrated the presence of
these species together with molds in samples taken from an old cathedral in Portugal [35].
    As for wooden churches, the highest biofilm producers were B. megaterium and B. pu-
millus strains, followed by Arthrobacter globiformis, Ps. entomophila and B. subtilis (Fig. 6).
    The bacterial strains isolated from stone churches exhibited a higher biofilm forma-
tion ability compared with strains isolated from wooden churches, half of them being
intensive biofilm producers. The most prolific in biofilm formation were Paenibacillus
peoriae, B. megaterium and Ps. koreensis strains.




Figure 7. Graphic representation of the biofilm forming ability of the bacterial strains isolated from
stone churches.




                                                                                                           Conservation science in Cultural Heritage
    3.5.2. Capsule and spore formation

   Spores, along with capsule, are considered additional mechanisms of resistance/
persistence of bacterial strains to extreme environmental conditions.




Figure 8. Microscopic aspect endospores – B. megaterium – Green Malachite staining; 1000X.


                                                                                                         303
                                                                                                                                                        As stated above, the dominant bacteria isolated from wooden and stone churches
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   were B. subtilis, B. megaterium, and B. mojavensis. All these species presented extremely
                                                                                                                                                   resistant inert endospores in response to nutrient deficiency and other environmental stress
                                                                                                                                                   conditions. All B. megaterium strains produced spores after 28 hours of incubation in
                                                                                                                                                   medium supplemented with CaCl2, while the other bacterial strains belonging to the Ba-
                                                                                                                                                   cillus genus started the sporulation process later, i.e., after 48-72h of incubation (Fig. 8).
                                                                                                                                                        As expected, the presence of the capsule was correlated with the ability of the
                                                                                                                                                   bacterial strains to form biofilm, the capsular polysaccharides facilitating the adhesion
                                                                                                                                                   process.
                                                                                                                                                        Besides, the presence of different features acting as protecting factors against harm-
                                                                                                                                                   ful external factors (capsule, spore, biofilm), half of the bacterial strains produce cellu-
                                                                                                                                                   lases, that could be involved in the biodegradation process of wood objects.


                                                                                                                                                         4. Conclusion

                                                                                                                                                       This multi-level characterization of microbial consortia from the wooden and stone
                                                                                                                                                   Romanian heritage churches was carried out by culture dependent (qualitative and
                                                                                                                                                   quantitative assessment of the microbial contamination, direct microscopic examination,
                                                                                                                                                   MALDI-TOF identification, quantitative evaluation of the fungal and bacterial adherence
                                                                                                                                                   to inert substrata, evaluation of capsule and spore forming bacteria) and independent
                                                                                                                                                   (PCR and sequencing of ITS intergenic regions in fungal strains) methods. The results
                                                                                                                                                   of this study could improve the understanding of the microbial biodeterioration process
                                                                                                                                                   of the heritage churches from Romania and allow reliable decontamination methods to
                                                                                                                                                   be defined.


                                                                                                                                                         Acknowledgments

                                                                                                                                                       Special thanks to PhD Prof. Mariana Carmen Chifiriuc who designed the study and
                                                                                                                                                   corrected the manuscript, and to master students Octavia-Emilia Zetu and Andreea-
                                                                                                                                                   Ștefania Dumbravă from the University of Bucharest, Faculty of Biology, who performed
                                                                                                                                                   some of the laboratory work. The authors also thank Daniela Zaharia from the Faculty of
                                                                                                                                                   History, Venus Mateescu (Romanian Peasant Museum) who facilitated the collection
                                                                                                                                                   of the samples from the cultural heritage churches belonging to the Romanian Peasant
                                                                                                                                                   Museum and to Violeta Corina Cristea from the Central Reference Synevo-Medicover
                                                                                                                                                   Laboratory for the identification of the strains.
                                                                                                                                                       We acknowledge the financial support of the Research Projects no. 52 PCCDI/2018”
                                                                                                                                                   Multidisciplinary and complex platform for the integrative and systematic research of the
                                                                                                                                                   identity of the tangible and non-tangible cultural heritage of Romania. Subproject 3 –
                                                                                                                                                   New technologies for preservation, conservation, recovering and restauration of the cul-
                                                                                                                                                   tural heritage” and 15PFE/2018 “Interdisciplinary institutional platform of excellence in
                                                                                                                                                   research, development, innovation and professional training in the field of archaeological
                                                                                                                                                   sciences” – “ArchaeoScience”≠RO. The funding had no role in the study design, data
                                                                                                                                                   collection and analysis, the decision to publish or in the preparation of the manuscript.


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   Biographical notes

   Irina Gheorghe is a PhD Lecturer of Applied Micology and General Micology at the
University of Bucharest, Faculty of Biology.

   Sârbu Ionela is a PhD Lecturer at the University of Bucharest, Faculty of Biology.

   Irina Balotescu is a PhD researcher at the Research Institute of the University of
Bucharest and “Constantin Brailoiu” Institute of Ethnigraphy and Folklore of the Roma-
nian Academy, Bucharest.

    Ion Blăjan is a PhD and Head of the Heritage and Scientific Evidence Section at




                                                                                                 Conservation science in Cultural Heritage
the Romanian Peasant Museum, Bucharest.

   Ionuț Pecete is a PhD student and biologist at the Central Reference Synevo-Medi-
cover Laboratory, Bucharest.


   Summary

    The 17th to 19th century wooden and stone churches are an iconic symbol of Ro-
manian national heritage. The present study investigates by qualitative and quantita-
tive methods the microbial communities from the biodeteriorated surfaces of wooden
and stone monument churches included in the cultural heritage list of local or national
importance. From a total of twelve monuments, samples were taken with cotton sterile
swabs, inoculated on specific culture and identified by classical, automated and mo-
lecular methods. A total of 133 strains belonging to Ascomycota phylum were identified
and confirmed at species level from the wooden churches, amongst which, Penicillium
spp. strains (mostly P. corylophylum, P. chrysogenum) were the most frequent, followed
by Alternaria alternata and species of Trichoderma, Aspergillus, Rhizopus, Mucor, and
Fusarium genera. From the stone churches a total of 100 strains belonging to Aspergil-
lus, Alternaria, Mucor, Penicillium, Aspergillus, Trichoderma, Fusarium and Rhizopus
genera were isolated.


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                                                                                                                                                       A total of 55 bacterial strains were isolated and identified as Bacillus, Artrobacter
I. Gheorghe, I. Sârbu, I. Pecete, I. Blăjan, I. Balotescu - Multi-level characterization of microbial consortia involved in the biodeterioration



                                                                                                                                                   and Pseudomonas species. The microbial load of the samples ranged between 2.18x107
                                                                                                                                                   and 3x105 CFU/mL. A very small number of fungal strains (6/77) isolated from wooden
                                                                                                                                                   churches (mostly A. alternata, followed by P. corylophilum and one Cladosporium spp.
                                                                                                                                                   strain) and from stone churches (5/55) (mostly A. alternata, followed by A. versicolor,
                                                                                                                                                   A. nidulans strain) were involved in biofilm formation.
                                                                                                                                                       The results of this study can help to improve understanding of the microbial dete-
                                                                                                                                                   rioration of Romanian heritage churches and allow more reliable decontamination, con-
                                                                                                                                                   servation and preservation tools to be defined.


                                                                                                                                                      Riassunto

                                                                                                                                                        Le chiese in legno e pietra dal XVII al XIX secolo sono un simbolo iconico del pa-
                                                                                                                                                   trimonio nazionale rumeno. Il presente studio indaga con metodi qualitativi e quantita-
                                                                                                                                                   tivi le comunità microbiche provenienti dalle superfici biodeteriorate di chiese monu-
                                                                                                                                                   mentali in legno e pietra incluse nell’elenco dei beni culturali di importanza locale o
                                                                                                                                                   nazionale. Da un totale di dodici monumenti, sono stati prelevati campioni con tampo-
                                                                                                                                                   ni di cotone sterili, inoculati su colture specifiche e identificati con metodi classici, au-
                                                                                                                                                   tomatizzati e molecolari. Un totale di 133 ceppi appartenenti ad Ascomycota phylum
                                                                                                                                                   sono stati identificati e confermati a livello di specie dalle chiese di legno, tra cui Peni-
                                                                                                                                                   cillium spp. I ceppi (principalmente P. corylophylum, P. chrysogenum) sono i più fre-
                                                                                                                                                   quenti, seguiti da Alternaria alternata e dalle specie dei generi Trichoderma, Aspergil-
                                                                                                                                                   lus, Rhizopus, Mucor e Fusarium. Dalle chiese in pietra sono stati isolati in totale 100
                                                                                                                                                   ceppi appartenenti ai generi Aspergillus, Alternaria, Mucor, Penicillium, Aspergillus,
                                                                                                                                                   Trichoderma, Fusarium e Rhizopus.
                                                                                                                                                        Un totale di 55 ceppi batterici è stato isolato e identificato come specie Bacillus,
                                                                                                                                                   Artrobacter e Pseudomonas. La carica microbica dei campioni varia tra 2,18x107 e
                                                                                                                                                   3x105 CFU / mL. Un numero molto piccolo di ceppi fungini (6/77) isolati da chiese in
                                                                                                                                                   legno (principalmente A. alternata, seguito da P. corylophilum e un ceppo Cladosporium
                                                                                                                                                   spp.) e da chiese in pietra (5/55) (principalmente A. alternata, seguito di A. versicolor,
                                                                                                                                                   ceppo di A. nidulans) sono stati coinvolti nella formazione del biofilm.
                                                                                                                                                        I risultati di questo studio possono aiutare a migliorare la comprensione del dete-
                                                                                                                                                   rioramento microbico delle chiese del patrimonio rumeno e consentire la definizione di
                                                                                                                                                   strumenti di decontaminazione, conservazione e conservazione più affidabili.




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