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

Introduction 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

Morphology of pitting from the Trajan Column. Caneva et al., 1994

Introduction 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.

Aims

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 :

SamplesMaterials and Methods

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

Materials and methods 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

Materials and methods 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

Materials and methods 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.

Materials and methods 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

Materials and methods 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 Plugin3D viewer and voxels counter

Papers

 

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

 

. 

Papers 1/4 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

Papers 1/4 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

Papers 2/4 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

Papers 2/4 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

Papers 2/4 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

Papers 3/4 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-protectionCalcium oxalates Aridity tolerance

Papers 3/4 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

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

Papers 3/4 Results/ Sample 1Inner side

Scytonemin

Marble rock

Calcite

Scytonemin is synthesized by cyanobacteria as extracellular sheath pigment, against UV radiation

Anthraquinone coumpoundsagainst intense solar radiation

Surface

Chlorophyll

Dolomite Calcite Carbon-based substance

Papers 3/4

Black schist from Hebrew’s cemetery

- Goethite α-FeO(OH)

-Lepidocrocite δ-FeO(OH)

Fe

Results/ Sample 2

Papers 3/4 Results

Black schist from Hebrew’s cemetery

Anthraquinone coumpoundsagainst intense radiation

Papers 3/4 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

-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

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

Papers 4/4 Detection of endolithic spatial distribution in marble stone using Confocal laser scanning microscopy

AimsDetection 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)

Papers 4/4 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)

Papers 4/4 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 penetrationsOverview in x-z direction of the cyanobacteria distribution

Overview in x-z directions shoving the penetrations in the sample thickness

760

μm

Papers 4/4 Results

Total Volume2.5 %

Total Volume2 %

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

3 m

mPapers 4/4 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)

Papers 4/4 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 %

General Conclusion

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

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).