1. DOCTORAL SCHOOL IN BIOLOGY “BIODIVERSITY AND ANALYSIS OF ECOSYSTEM” “Impact of endolithic organisms on stone monuments” Academic year 2012/2013 Cycle XXVI Ph.D Student: Casanova Municchia Annalaura Tutor: Prof.ssa Caneva

2. Biodeterioration/ endolithic microorganisms Endolithic growth form can be present in different microorganisms groups: cyanobacteria, green and red algae, fungi, and lichens. Their grow inside the stone in order to protect themselves from adverse conditions (high solar radiation, adverse temperature and desiccation conditions) Can penetrate some millimeters or centimeters inside the rock

3. Morphology of pitting from the Trajan Column. Caneva et al., 1994 Biodeterioration/ endolithic microorganisms The stone monuments, as well as rocks, can be colonized by endolithic microorganisms showing biodeterioration phenomenon. Often it is not recognized and is confused with abiotic cause Understimated question despite is the most dangerous biological deterioration processes affecting stone monuments.

4. Study of the spatiality of endolithic microorganisms in relation with the stone Detection of endolithic traces on stone monuments in Temperate and Mediterranean bioclimates Microorganisms adopt organic or inorganic survival strategies under high stress conditions, leaving biological or geological traces on rock A major contribution about the knowledge of biodeterioration process due to endoliths on the stone monuments by discovering new approaches and new techniques General Aim: In Detail :

5. Samples 11 samples- Carbonate limestone Church in Martvilli (Georgia) 1 sample- Black schist Hebrew’s cemetery tombstone in Venice. 3 samples- Dolomitic limestone cliff of the Amalfi Coast. 7 samples – Marble rock from Carrara area. Stone monuments (Case studies) Natural outcrops

6. Optical Microscope/ SEM-EDS Experimental protocol Observation of polished cross-section Observation of polished sections after staining with Periodic Acid Schiff (PAS) Spread and the depth of the colonization

7. Optical Microscope/ SEM-EDS SEM observation of cross-section after acid attack and fractured sample Experimental protocol Observation of thin-section Morphology of microorganisms and bioalteration induce on stone Observation in light trasmitted

8. Raman Spectroscopy to identify traces of the organic and inorganic compounds left by endolithic microorganisms Raman spectroscopy is an analytical technique that provides molecular structural information, based on inelastic scattering of monochromatic light.

9. Confocal Laser Scanning Microscope to determine the occurrence, the spatial organization and the volume of endolithic microorganisms Provide series of thin optical sections of the sample, at different intervals along the Z axis 3-D image of fluorescent organisms or stained with fluorescent labels

10. Image Analysis ImageJ® Java-based image processing program Display, analyze, process the image obtained by CLSM. Quantify the bio-volume occupied inside the stone of each stack, develop profiles and isosurfaces in 3-Dimension. Imaris software,Trial version, (Bitplane). ImageJ Plugin 3D viewer and voxels counter

11. 1.V. Lombardozzi, T. Castrignanò, M. D’Antonio, A. Casanova Municchia, G. Caneva, An interactive database for an ecological analysis of stone bioppitting, Int. Biodeterioration & Biodegradation, 2012, 73, 8. 2. G. Caneva, V. Lombardozzi, S. Ceschin, A. Casanova Municchia, O. Salvadori, Unusual differential erosion related to the presence of endolithic microorganisms ( Martvili, Georgia), Journal of Cultural Heritage, 2013, in Press 3. A. Casanova Municchia, G. Caneva, M. A. Ricci, A. Sodo, Identification of endolithic traces on stone monuments, in review to the Journal of Raman Spectroscopy 4. A. Casanova Municchia, Z. Percario, G. Caneva, Detection of endolithic spatial distribution in marble stone using Confocal laser scanning microscopy, submitted to the Journal of Microscopy.

12. An interactive database for an ecological analysis of stone biopitting Interactive online database developed in order to : - Identify the trends of stone-pitting phenomena, the most favorable environmental conditions, the most affected kind of stone, the most common biodeteriogens. - Synthesize the available information on the stone-pitting phenomena 24 are the papers used to building up the database; 83 the total number of sites ; 249 the total number samples. Most of the sampling sites are in the Mediterranean Basin

13. Results/Conclusions Marble is often described as the most affected material Cyanobacteria are the dominant group associated with pitting Biopitting is mainly described in vertical and subvertical surfaces, showing a preference for southern exposures On carbonate and marble rocks is found the most evidence of appearance of cyanobacteria

14. Unusual differential erosion related to the presence of endolithic microorganisms (Martvili, Georgia) - Identify the ecological conditions which favor the phenomenon AIMS Detail of the stone surface affected by biodeterioration phenomena on South- facing side of the Church. A differential erosion phenomenon was observed on the walls of the Church of the Virgin in Martvilli. Characterized by the circular imprints left in the stone (from 1 cm to 3 cm in diameter) - Provides an interpretation of the differential erosion phenomenon - Impact on the stone conservation

15. Results The southern facade is the most intensely affected by the differential erosion phenomenon Cyanobacteria are the most common microorganisms occuring Black meristematic fungi are on and below the surface of the sample The microorganisms appears to from an average depth of 200 μm The stone is a fine-grained limestone. Traces of fossils with relatively high values of porosity (23.28%). Through the analysis of images it was possible to estimate a considerable colonized area

16. Conclusion This unusual differential erosion phenomenon is related to : - Intense xeric conditions that permit the establishment of endolithic microorganisms (Southern facade ) -The physical petrographic features of the rock, (heterogeneity and discontinuity) giving rise the deterioration phenomenon in specific preferential areas. - Biodeterioration due to the cyanobacteria and meristematic fungi endolithic activity

17. Identification of endolithic traces on stone monuments Oral Presentation: 7th International Conference on the application of Raman spectroscopy in Art and Archaeology,Ljubljana 2-6 September 2013 Raman spectroscopic analysis applied to four endolithic samples from Temperate and Mediterranean bioclimate regions to identified the traces of organic and inorganic compounds present in the stone monuments High stress conditions inducing the microorganisms to adopt survival strategies protection from desiccation and high solar radiation Scytonemin UV-protection Calcium oxalates Aridity tolerance

18. Materials and Methods Four samples colonized by endoliths - Marble rock showing colonization of cyanobacteria and endolithic fungi from quarries in Carrara Area - Dolomitic limestone showing colonization of endolithic cyanobacteria from cliff of the Amalfi Coast - Black schist from Hebrew’s cemetery tombstone in Venice. Endolithic lichens with perithecia completely sunken in the rock; -Fine-grained limestone from the Church of the Virgin in Martvili in Western Georgia. The back side shows orange biological traces

19. Papers 3/4 All measurements have been performed with a Renishaw In ‑ Via Reflex Raman microscope 785 nm near-infrared and the green laser line at 514 nm; objectives 50X; Materials and Methods Raman spectra have been recorded on the surface and in the inner part of the samples, to identify a possible difference of the bio- and geotraces detected at different depth

20. Results/ Sample 1 Inner side Scytonemin Marble rock Calcite Scytonemin is synthesized by cyanobacteria as extracellular sheath pigment, against UV radiation Anthraquinone coumpounds against intense solar radiation Surface Chlorophyll

21. DolomiteCalcite Carbon-based substance Black schist from Hebrew’s cemetery -Goethite α-FeO(OH) -Lepidocrocite δ-FeO(OH) Results/ Sample 2

22. Results Black schist from Hebrew’s cemetery Anthraquinone coumpounds against intense radiation

23. Results/Sample 3 and Sample 4 Dolomitic limestone Raman spectra recorded using the 785 nm laser line, show only the substrate 514 nm laser excitation to identify the organic traces typical spectrum of a carotenoid with bands centered at 1522 and 1154 cm−1. Carotenoid is an accessory pigment, usually produced under stress conditions within antioxidant strategy limestone from the Church of the Virgin in Martvili

24. - Sample1-Marble from Carrara area. Scytonemin appears in all spectra and at different depths of the sample. -Sample2-Hebrew’s cemetery tombstone in Venice. Iron oxide hydroxides due to a bioalteration by endolithic. Conclusions Anthraquinone coumpounds - Sample3-4 Dolomitic limestone and sample from Church in Martvili All samples show traces of compounds known to be effective against UV-radiation damage Carotenoid compounds (antioxidant) useful against the high UV- radiation

25. Detection of endolithic spatial distribution in marble stone using Confocal laser scanning microscopy Aims Detection of the endolithic spatial distribution and quantify the bio- volume occupied using the confocal laser scanning microscopy (CLSM) with a double- staining. Understand the real impact of on the stone conservation Compare the results with those acquired from microscopy techniques (SEM and light microscope)

26. Materials and Methods Rock flakes from the Marble rock samples stained with : The nucleic acid stain, propidium iodide ( after permeabilization of the cell membranes) The glycoconjugates stain lectin Concanavalin-A Alexa Fluor 488 DNA structures Extrapolymeric substances (EPS) excitation 543 nm emission 633 nm ( RED CHANNEL ) excitation 543 nm emission 633 nm ( GREEN CHANNEL) The CSLM images were collected in a set of optical cross- sectional image in the x-y plane obtained at different intervals along the z-axis. ( Total Z path 160 μm)

27. Results Microorganisms stained with red fluorescent (PI) Extracellular matrix (EPS) stained with green fluorescence (ConA- Alexa Fluor 488) Isosurface presentations show the 3-D arrangement Overview in y-x directions shoving the penetrations Overview in x-z direction of the cyanobacteria distribution Overview in x-z directions shoving the penetrations in the sample thickness 760 μm

28. Results Total Volume 2.5 % Total Volume 2 % Volumes were calculated from each of six image stacks corresponding to the various depths Volume distribution; propidium iodide stain Volume distribution; ConA- Alexa Fluor 488 stain

29. 3 mm Results/ Comparison PAS and SEM Cross-section after PAS staining After application of a threshold classification, the substratum (black area) and endolithic colonization (white areas). Total Area 6 % Cyanobacteria immersed in the substratum Entire cross-section partially decalcified by hydrochloric acid solution Calcite grains perforated by hyphae Fungal hyphae embedded in extracellular matrix (white arrow)

30. Conclusion Microscope CSLM results provided a good information about the 3-D spatiality of the endolithic microorganisms, the real volume occupied, the distribution between the grains and the penetration into the calcite grains. CLSM with the double staining distinguish and quantify the contribution of the extracellular matrix from that of DNA structure This is the first study of the biodeterioration phenomenon due to endolithic microorganism aimed at stone monuments by the use of CLSM microscope with double staining The marble rock sample is colonized by cyanobacteria completely immersed in the stone. Below the cyanobacteria a dense network of fungal hyphae is present The volume occupies by endolithic cyanobacteria and fungi is about 2.5% and the EPS Volume is about 2 %

31. A prove of the potentiality of the Raman Spectroscopy, which is here applied for the first time on stone monuments, in the identification of traces of biological compounds from endolithic microorganisms New approaches and new techniques have been applied A useful contribution for a clear identification of the presence of endolithic microorganisms on stone monuments A new approach, with the employment of 3-D technologies, in the evaluation the real impact of endolithic microorganisms on the stone monuments A increase of the information about the real effect on the stone monuments andof the potential damage by endolithic attack

32. Thanks for your attention … I wish to thanks : Di Giulio A. ; Ricci M.A.; Salvadori O; Sodo A.; Percario Z.; and the LIME staff for the technical support. I thank all the doctoral school and all the Ph.D students A big thank to my tutor Prof.ssa G. Caneva for her help and for all the experiences of this three years and I want over- please the laboratory for the support and the best moments ( Alma, Roberto; Francoise, Flavia, Valentina; Giulia, Simona, Wawan ).