Identificazione di taxa batterici in reperti lignei di interesse archeologico
10.6092/issn.1973-9494/4748
application/pdf
1973-9494
4748
en
Enza Di Carlo
Conservation Science in Cultural Heritage
molecular techniques
Identification of bacterial taxa in archaeological waterlogged wood
Text.Serial.Journal
https://conservation-science.unibo.it/
14
Articles
2014-12-20
2015-02-25
2014-12-20
2015-11-02
247-254
CSCH
Enza Di Carlo
2014/12/20
10.6092/issn.1973-9494/4748
247
1973-9494
molecular techniques
en
254
http://www.eurofins.com
Identification of bacterial taxa in archaeological waterlogged wood
https://conservation-science.unibo.it/article/download/4748/4232
14
IDENTIFICATION OF BACTERIAL TAXA IN ARCHAEOLOGI-
 CAL WATERLOGGED WOOD

    Franco Palla•, Giovanna Barresi, Enza Di Carlo
    Laboratorio di Biologia e Biotecnologie per i Beni Culturali
    Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF)
    Università degli Studi di Palermo, Italy



    Keywords: submerged archeological wood, biodeterioration, SEM, molecular
    techniques



    1. Introduction

    The conservation of archaeological waterlogged artifacts presents manifold prob-
lems due to the vulnerability of the materials which are affected, often synergistically,
by physical, chemical and biological factors, particularly as a result of fungal and bac-
terial colonization [1 - 2].
    In characterizing the state of conservation of archaeological waterlogged wood,
studies mainly focalize on micro-morphological analysis, using Optical and Scanning
Electron Microscopy [3 - 4].
    In addition, information about the environment where the artifacts are found (pH,
temperature, water salinity, sediment characteristics) is required, as it, too, has con-




                                                                                                    CONSERVATION SCIENCE IN CULTURAL HERITAGE
tributed to their abiotic and biotic deterioration [2].
    It is of fundamental importance to detect and identify any morphological alterations
caused by biological colonization, so as to distinguish degradation processes that have
occurred during the time spent in the underwater site from those that have occurred
during maintenance and storage in museum deposits or exhibition spaces.
    The action of some microorganisms leads to important changes in the anatomical
structure, physical properties and chemical composition of wood, with implications for
the stability of the archaeological finds. As regards bacterial colonization, waterlogged
wooden finds provide an ideal habitat for many communities, but the identification of
the species is mainly carried out through in vitro culture or by evaluating the micro-
morphological alteration of the wooden structure [5 - 8].
    The main colonizing bacteria in waterlogged wood are, erosion bacteria (EB), which
are tolerant of low oxygen concentrations, and tunneling bacteria (TB). SEM analyses
revealed their erosion effects manifest as deep channels, parallel to micro-fibrils in the
cell wall [4] and loss of lingo-cellulosic structure. Tunneling bacteria are able to alter
middle lamella elements, rich in lignin, deteriorating tracheids, fibers and vessels, be-
fore reaching the lumen of the cell wall [2, 8 – 9].
    When a find is recovered from the seabed and brought into an aerobic environment,
with fluctuating humidity, iron sulfides start to oxidize, producing iron sulfate minerals,

• Corresponding author: franco.palla@unipa.it

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                                                                                                           such as melanterite, rozenite, jarosite. Moreover, the presence of sulfur oxidation can
                                                                                                           induce formation of yellow, orange and white mineral aggregates or pyrite [10 – 12].
                                                                                                               In anaerobic conditions, organic and cellulose residuals produced by erosion bacteria
                                                                                                           (EB) activity induce development of sulfur-reducing bacteria (SRB), able to produce hy-
                                                                                                           drogen sulfide (H2S). The HS- ions react with lignin-rich parts of the wood to form organic
                                                                                                           sulfur compounds (mainly thiols) or produce, together with iron ions that come from the
                                                                                                           corrosion of metal substrates, particles of iron sulfides deposited in wood cavities [10].
                                                                                                               Thus, microbial degradation of wood resting within marine sediments in low oxygen
                                                                                                           conditions is usually accompanied by accumulation of sulfur and iron compounds [12 – 15].
                                                                                                               In the last few decades, the use of molecular techniques has allowed microbial species
                                                                                                           to be detected and identified in archaeological finds too, based on the analysis of specific
                                                                                                           sequences of microbial genomic DNA, extracted directly from wood samples, correspond-
                                                                                                           ing to the regions 16S and ITS - Internal Transcribed Sequence rRNA [13 – 15].
F. Palla, G. Barresi, E. Di Carlo - Identification of bacterial taxa in archaeological waterlogged wood




                                                                                                               In this study, SEM analysis and molecular investigations have revealed specific
                                                                                                           structural alterations and cells in wood attributable to bacterial colonization, as well as
                                                                                                           the presence of framboidal pyrite (FeS2), also found in clusters.
                                                                                                              Molecular analysis based on in vitro amplification (Polymerase Chain Reaction) of
                                                                                                           16S and ITS rRNA sequences [16 -18], allowed the identification of microbial consor-
                                                                                                           tia with ligninolytic and cellulosolytic activity (Cellulomonas, Bacillus, Pseudomonas,
                                                                                                           Sphingomonas and Xanthomonas genera), in addition to iron-oxydizing and sulfate-
                                                                                                           reducing bacteria (Marinobacter spp., Desulforudis audaxviator), otherwise undetect-
                                                                                                           able with traditional in vitro culture methods.



                                                                                                            2. Material and methods

                                                                                                              2.1. Sampling

                                                                                                              Wood samples were collected by sterile scalpel and loops, from blackish–brown
                                                                                                           areas (Figure 1). The samples (w1, w2, w3, w4, w5) were utilized for SEM analysis and
                                                                                                           for microbial genomic DNA extraction, obtained directly from the wooden fragments or
                                                                                                           the single colonies isolated on Nutrient agar (Difco).




                                                                                                                                     Figure 1. Wooden finds sampled (w1-w5)



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 2.2. Microscopic analysis

  Morphology of wooden sections was investigated by Scanning Electron Microscope
(Leica, Cambridge Leo-400), coating the samples with gold micro-particles (13 nm), by
Agar-Auto-Sputter-Coater (B7341).

  2.3. DNA extraction and bacterial identification

    Bacterial genomic DNA was obtained from single colonies isolated on Nutrient Agar,
by means of rapid lysis in 1X TE (10mM Tris-HCl, pH 7.5/ 1mM EDTA) at 94°C for 3
min. using either the Stool mini Kit (Quiagen) or genomic DNA Purification kit (Fer-
mentas), modified as follows: incubation at 65°C for 4 hours in presence of 5 mg/ml
Proteinase K (Invitrogen).
    Genomic DNA molecules were utilized as a template in the PCRs (Polymerase
Chain Reaction) using 16S-rDNA primers specific for Pseudomonas, Cellulomonas
and Bacillus genera, or universal primers for the ITS (16S-23S, Internal Transcribed
Spacer) rDNA region [19]. The PCR reaction mixture consisted of: genomic bacterial
DNA (lysis solution 5 µl or DNA 40 ng); 1X Reaction Buffer; 10 µM forward primer; 10
µM reverse primer; 2 µM dNTP mix; 2 mM MgCl2; 5 U/µl Taq DNA polymerase (Invitro-
gen). The PCR reactions are performed by the following amplification profiles: 1 initial
cycle of denaturation at 95°C for 5 min; 30 cycles as follows: denaturation at 94°C for
1 min, annealing at 50°C (16S)–58°C (ITS) for 1 min, extension at 72°C for 2 min. A
final extension step (72°C for 7 min) was added to ensure that all PCR products are
full-length. DNA fragments were analyzed on 2% agarose gel in 1X TAE (50X TAE =
40 mM Trizma base, 20 mM acetic acid, 1 mM EDTA, pH 8.4), in 1X SYBR-safe DNA
gel stain (Invitrogen).
    The nucleotide composition of the PCR products was determined by Eurofins MWG
Operon Sequencing Service [20] and the homology research was undertaken by




                                                                                                 CONSERVATION SCIENCE IN CULTURAL HERITAGE
BLAST analyzer [21].


  3. Results

  3.1. Microscopy analyses


  SEM analyses of radial and transversal sections indicated middle lamella and cell
wall alteration (Figure 2 B). Structural deterioration of the wood is shown in Figure 3
A-B, where the cell walls appear more distorted and fractured, due also to the mechan-
ical action of crystalline aggregates inside the structure (Figure 3 C). The presence
of pyrite framboids, single or clustered, is evident in Figure 4 A-B-C. Erosion effects,
attributable to bacteria activity, are shown in Figure 5 A-B.




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F. Palla, G. Barresi, E. Di Carlo - Identification of bacterial taxa in archaeological waterlogged wood




                                                                                                           Figure 2. SEM micrographies: A) Transverse view; B) xilema degradation, red arrows indicate
                                                                                                           detachment of secondary cell walls S2 from middle lamella




                                                                                                           Figure 3. SEM micrographies: A) transverse section: the structure appear distorted for loss
                                                                                                           of mechanical resistance; B) radial section: cracks and fractures (red arrow) in cell walls; C)
                                                                                                           transverse view: the presence of crystalline aggregates induce breaking in cell wall




                                                                                                               Figure 4. SEM micrographies: single (A) or clustered (B) Pyrite framboids; diatoms (C)




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Figure 5. SEM micrographies: A- B) Cell walls erosion decay, probably induced by bacteria (red
arrows)




   3.2. DNA extraction and bacterial identification

    PCR reaction products were analyzed by electrophoresis on 2% agarose gel, the
sequencing and sequences analysis using the BLAST platform allowed cellulosolytic
bacteria belonging to Cellulomonas, Bacillus, Pseudomonas, to be identified in almost
all the samples analyzed.
    Specifically for sample w3, in Figure 6A fragments of 220 bp, 750 bp, 680 bp (PCR re-
actions using ITS rDNA primers) correspond to Desulforudis audaxviator; Marinobacter
spp. (sulfate - reducing and iron - oxidizing) and Xanthomonas spp. (ligninolytic) respec-




                                                                                                          CONSERVATION SCIENCE IN CULTURAL HERITAGE
tively. The presence of Cellulomonas, Bacillus and Pseudomonas genera was detected
using 16S rDNA primers, whose amplification products were 201 bp, 181 bp and 338 bp
in length respectively (Figure 6B). In addition, mono and polysaccharides hydrolyzing
bacteria, belonging to Sphingomonas genera, were revealed in sample w2.




Figure 6. Gel electrophoresis on 2% agarose gel of w3 sample PCR reactions: A) lanes 1-3), in
vitro amplification by universal ITS 16-23S rDNA primers; B) lanes 4-6, in vitro amplification by
16S-rDNA specific primers. M= DNA marker Sharpmass (Euroclone)


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                                                                                                           Table 1. Bacterial identification in five samples (w) through 16S and ITS amplification. Bacteria
                                                                                                           revealed in w3 are marked in red



                                                                                                               DNA (BACTERIAL COLONIES)                                  DNA (WOODEN FRAGMENTS)

                                                                                                                                         ITS – PCR                  ITS - PCR                       16S - PCR

                                                                                                                               w1   w2    w3   w4    w5   w1   w2   w3          w4   w5   w1   w2    w3     w4   w5

                                                                                                           Pseudomonas spp.    +    +     +    +                                          +    +     +      +

                                                                                                           Cellulomonas spp.   +    +                +                                    +    +     +

                                                                                                           Bacillus spp.       +    +          +                                                     +      +    +

                                                                                                           Xanthomonas spp.               +

                                                                                                           Desulforudis                                   +         +
F. Palla, G. Barresi, E. Di Carlo - Identification of bacterial taxa in archaeological waterlogged wood




                                                                                                           audaxviator

                                                                                                           Sphingomonas spp.                                   +

                                                                                                           Marinobacter spp.                                        +




                                                                                                               4. Conclusions

                                                                                                             The diagnostic tests performed on objects of historical and artistic interest provide in-
                                                                                                           formation essential for assessing the state of preservation and in particular for identify-
                                                                                                           ing microbial colonization related to processes of biodeterioration. Although not always
                                                                                                           easy to implement, the identification of the components of a microbial consortium is of
                                                                                                           considerable importance in the implementation of operations compatible with both the
                                                                                                           artifact / specimen, and with the operator, as well as with the environment; this allows
                                                                                                           microbial growth to be eliminated or counteracted in a targeted manner. In the present
                                                                                                           study protocols were used developed specifically for the characterization of bacterial
                                                                                                           taxa that colonize waterlogged archaeological wood. In particular, the study involved
                                                                                                           the use of a scanning electron microscope (SEM) to observe the structural changes
                                                                                                           in the wood, and the set up of molecular protocols, based on the analysis of microbial
                                                                                                           genomic DNA, for the detection and identification of bacterial colonization.

                                                                                                             In particular, bacteria were identified with ligninolytic or cellulolytic activity, belong-
                                                                                                           ing to the genera Cellulomonas, Bacillus, Pseudomonas, and Xanthomonas (besides
                                                                                                           Sphingomonas in sample w2) and iron-oxidising bacteria or iron sulfate - reducers,
                                                                                                           respectively Marinobacter sp. and Desulforudis audaxviator, otherwise undetectable
                                                                                                           with conventional in vitro culture methods.

                                                                                                             Molecular studies have allowed us to complete identification of the bacteria coloniz-
                                                                                                           ing the wooden finds through the analysis of microbial genomic DNA obtained from
                                                                                                           the bacteria colonies isolated on agar medium or directly extracted from the wood
                                                                                                           samples. Combining the results of the SEM observations and molecular studies, it
                                                                                                           is possible to correlate the microbial populations with the structural alterations with
                                                                                                           a view to compiling appropriate conservative restoration protocols which respect the
                                                                                                           integrity of the archaeological find.

                                                                                                             This microscopy and molecular biology integrated approach provides an overview on
                                                                                                           microbial colonization and related alterations, in order to define appropriate restoration
                                                                                                           protocols.


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      Acknowledgements

      Authors thank C. Di Liberto for SEM analysis.

      References

[1]     GELBRICH J., MAI C., MILITZ H., 2008, Chemical changes in wood degraded by bac-
        teria, International Biodeterioration & Biodegradation, 61, pp. 24-32.
[2]     BLANCHETTE R.A., 2000, A review of microbial deterioration found in archaeological
        wood from different environments, International Biodeterioration & Biodegradation, 46,
        pp. 189-204.
[3]     MACCHIONI N., CAPRETTI C., SOZZI L., PIZZO B., 2013, Grading the decay of water-
        logged archaeological wood according to anatomical characterization. The case of the
        Fiavé site (N-E Italy), International Biodeterioration & Biodegradation, 84, pp. 54-64.
[4]     SAFA A. M. HAMED, MONA F. ALI, NESRIN M. N. El HADIDI, 2012, Using SEM in
        monitoring changes in archaeological wood: A review, in A. Méndez-Vilas (ed.), Current
        Microscopy Contributions to Advances in Science and Technology.
[5]     NILSSON T., BJÖRDAL C.G., 2008, Culturing wood-degrading erosion bacteria, Inter-
        national Biodeterioration & Biodegradation, 61(1), pp. 3-10.
[6]     NILSSON T, BJÖRDAL C.G, 2008, The use of kapok fibers for enrichment cultures
        of lignocelluloses-degrading bacteria, International Biodeterioration & Biodegradation,
        61, pp. 11-16.
[7]     BJÖRDAL C.G., NILSSON T., 2008, Reburial of shipwrecks in marine sediments: a
        long -term study on wood degradation, Journal of Archaelogical Science, 35, pp. 862-
        872.
[8]     KIM Y.S., SINGH A.P., 2000, Micromorphological characteristics of wood biodegrada-
        tion in wet environments: a review, IAWA Journal, 21 (2), pp. 135-155.
[9]     BJÖRDAL C.G., NILSSON T., DANIEL G., 1999, Microbial decay of waterlogged ar-
        chaeological wood found in Sweden. Applicable to archaeology and conservation, In-
        ternational Biodeterioration & Biodegradation, 43, pp. 63-73.




                                                                                                         CONSERVATION SCIENCE IN CULTURAL HERITAGE
[10]    FORS Y., JALILEHVAND F., DAMIAN RISBERG E., BJÖRDAL C., PHILLIPS E., SAND-
        STRÖM M., 2012, Sulfur and iron analyses of marine archaeological wood in ship-
        wrecks from the Baltic Sea and Scandinavian water, Journal of Archaeological Science,
        39, pp. 2521-2532.
[11]    FORS Y., NILSSON T., RISBERG E.D., SANDSTRÖM M., TORSSANDER P., 2008,
        Sulfur accumulation in pinewood (Pinus sylvestis) induced by bacteria in a simulated
        seabed environment: Implications for marine archaeological wood and fossil fuels, In-
        ternational Biodeterioration & Biodegradation, 62, pp. 336- 347.
[12]    RÉMAZEILLES C., TRAN K., GUILMINOT E., CONFORTO E., REFAIT P., 2013, Study
        of Fe(II) sulphides in waterlogged archaeological wood, Studies in Conservation, 58
        (4), pp. 297-307.
[13]    HELMS A.C., MARTINY A.C., HOFMAN-BANG J., AHRING B.K., KILSTRUP M., 2004,
        Identification of bacterial cultures from archaeological wood using molecular biological
        techniques, International Biodeterioration & Biodegradation, 53, pp. 79-88.
[14]    LANDY E., BJÖRDAL C.G., NILSSON T., MITCHELL J. I., EATON R. A., 2004, Estima-
        tion of the diversity of bacterial communities in wooden foundation poles across Europe
        using molecular biology techniques, in Proc. from Biomolecular Archaeology Sympo-
        sium, Amsterdam.
[15]    PALLA F., MANCUSO F.P., BILLECI N., 2013, Multiple approaches to identify bacteria
        in archaeological waterlogged wood. Journal of Cultural Heritage 14s, pp. e41- e46.
[16]    PALLA F., 2012, Analytical techniques, in Science and Conservation for Museum Col-
        lections, Chap 14, pp. 459-470, Nardini, Firenze, Italy.
[17]    LANDY E., MITCHELL J, HOTCHKISS S., EATON R., 2008, Bacterial diversity as-


                                                                                                   253
                                                                                                                  sociated with archaeological waterlogged wood: ribosomal RNA clone libraries and
                                                                                                                  denaturing gradient gel electrophoresis (DGGE), International Biodeterioration & Bio-
                                                                                                                  degradation, 61 (1), pp.106-116.
                                                                                                           [18]   PALLA F., BILLECI N, MANCUSO F.P., PELLEGRINO L., LORUSSO L.C., 2010, Mi-
                                                                                                                  croscopy and Molecular biology techniques to study biocenosis diversity in semi-con-
                                                                                                                  fined environments, in Conservation Science in Cultural Heritage, vol.10, pp. 185-194.
                                                                                                           [19]   PALLA F., ANELLO L., MARINEO S., LOMBARDO G., 2006, Characterization of bacte-
                                                                                                                  rial community in indoor environment, in Taylor & Francis (ed.), Heritage, Weathering
                                                                                                                  and Conservation, vol.1 pp. 361-365. London.
                                                                                                           [20]   http://blast.ncbi.nlm.gov
                                                                                                           [21]   http://www.eurofins.com



                                                                                                              Biographical notes
F. Palla, G. Barresi, E. Di Carlo - Identification of bacterial taxa in archaeological waterlogged wood




                                                                                                              Franco Palla, is Associate Professor of “Environmental and Applied Botanical
                                                                                                           Sciences” at the University of Palermo, Italy. He is involved in the Academic Five-Year
                                                                                                           Degree Course in Conservation and Restoration of Cultural Heritage, both as teacher and
                                                                                                           Vice-Coordinator. He is a Member of the National Order of Biologists of the Commission
                                                                                                           for the Protection of Cultural Heritage; Scientific coordinator of UNIPA Research Unit -
                                                                                                           Project PON01_00625, IT@CHA (Italian Technology for Advanced Applications in Cultural
                                                                                                           Assets). He is one of the members of the working groups of experts in the Italy-Cambodia
                                                                                                           Cooperation Project “Training of Cultural Heritage Experts”, University of Palermo - Royal
                                                                                                           University of Fine Art and Ministry for Culture and Fine Arts, Angkor, Cambodia. He is a
                                                                                                           Scientific Consultant in the field of Biological deterioration, in restoration projects regarding
                                                                                                           archaeological sites, works of art and historical- artistic artifacts. Professor Palla is also
                                                                                                           a member of the Advisory Committee of the Journal “Conservation Science in Cultural
                                                                                                           Heritage”. Currently Professor Palla is the scientific coordinator of the Laboratory of
                                                                                                           Biology and Biotechnology for Cultural Heritage at the Department of Biological, Chemical,
                                                                                                           Pharmaceutical Sciences and Technology (STEBICEF) at the University of Palermo.

                                                                                                              Giovanna Barresi graduated in “Science for Conservation and Restoration” at the
                                                                                                           University of Florence. She works in the diagnostic field - analytical, and applied re-
                                                                                                           search for the enzymatic cleaning of the surface of artifacts of historical and artistic
                                                                                                           interest, at the Laboratory of Biology and Biotechnology for the Cultural Heritage of
                                                                                                           the Department of Biological, Chemical, Pharmaceutical Sciences and Technology
                                                                                                           (STEBICEF) at the University of Palermo.

                                                                                                             Enza Di Carlo has a degree in “Biological Sciences” at the University of Palermo
                                                                                                           and he specialized in “Microbiology and Virology” at the School of Medicine at the
                                                                                                           same university. She is Research Fellow at the Department of Biological, Chemical,
                                                                                                           Pharmaceutical Sciences and Technology (STEBICEF) at the University of Palermo,
                                                                                                           where she carried out a microbiological monitoring of environments of preservation
                                                                                                           and use of cultural heritage.




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