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

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Doctoral school in biology biodiversity and analysis of ecosystem

DOCTORAL SCHOOL IN BIOLOGY

“BIODIVERSITY AND ANALYSIS OF ECOSYSTEM”

Academicyear2012/2013

Cycle XXVI

“Impact of endolithic organisms on stone monuments”

Ph.DStudent:

Casanova MunicchiaAnnalauraTutor:

Prof.ssa Caneva


Doctoral school in biology biodiversity and analysis of ecosystem

Biodeterioration/ endolithic microorganisms

Introduction

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


Doctoral school in biology biodiversity and analysis of ecosystem

Biodeterioration/ endolithic microorganisms

Introduction

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.

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


Doctoral school in biology biodiversity and analysis of ecosystem

Aims

GeneralAim:

A major contribution about the knowledge of biodeterioration process due to endolithson the stone monuments by discovering new approaches and new techniques

In Detail :

Detection of endolithic traces on stone monuments in Temperate and Mediterranean bioclimates

Microorganismsadoptorganic or inorganicsurvivalstrategies under high stress conditions, leavingbiological or geological traces on rock

Study of the spatiality of endolithicmicroorganisms in relation with the stone


Doctoral school in biology biodiversity and analysis of ecosystem

Samples

Materials and Methods

Natural outcrops

Stone monuments

(Case studies)

7 samples – Marble rock from Carrara area.

1 sample- Black schistHebrew’s cemetery tombstone in Venice.

3 samples- Dolomitic limestone cliff of the Amalfi Coast.

11 samples- Carbonate limestone Church in Martvilli (Georgia)


Doctoral school in biology biodiversity and analysis of ecosystem

Optical Microscope/ SEM-EDS

Materials and methods

Experimental protocol

Observation of polished cross-section

Spread and the depth of the colonization

Observation of polished sections after staining with Periodic Acid Schiff (PAS)


Doctoral school in biology biodiversity and analysis of ecosystem

Optical Microscope/ SEM-EDS

Materials and methods

Experimental protocol

Observation in light trasmitted

Observation of thin-section

Morphology of microorganisms and bioalteration induce on stone

SEM observation of cross-section after acid attack and fractured sample


Doctoral school in biology biodiversity and analysis of ecosystem

RamanSpectroscopy

Materials and methods

Raman spectroscopy is an analytical technique that provides molecular structural information, based on inelastic scattering of monochromatic light.

to identify traces of the organic and inorganic compounds left by endolithic microorganisms


Doctoral school in biology biodiversity and analysis of ecosystem

Confocal Laser Scanning Microscope

Materials and methods

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

to determine the occurrence, the spatial organization and the volume of endolithic microorganisms


Doctoral school in biology biodiversity and analysis of ecosystem

ImageAnalysis

Materials and methods

Display, analyze, process the image obtained by CLSM.

ImageJ® Java-based image processing program

Quantify the bio-volume occupied inside the stone of each stack , develop profiles and isosurfaces in 3-Dimension.

ImageJ Plugin

3D viewer and voxels counter

Imaris software ,Trial version, (Bitplane).


Doctoral school in biology biodiversity and analysis of ecosystem

Papers

  • V. Lombardozzi, T. Castrignanò, M. D’Antonio, A. Casanova Municchia, G. Caneva, An interactive database foranecologicalanalysisofstonebioppitting, 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

  • .


Doctoral school in biology biodiversity and analysis of ecosystem

An interactive database for an ecological analysis of stone biopitting

Papers1/4

Interactive online database developed in order to :

-Synthesize the available information on the stone-pitting phenomena

-Identify the trends of stone-pitting phenomena, the most favorable environmental conditions, the most affected kind of stone, the most common biodeteriogens.

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


Doctoral school in biology biodiversity and analysis of ecosystem

Results/Conclusions

Papers1/4

Marble is often described as the most affected material

On carbonate and marble rocks is found the most evidence of appearance of cyanobacteria

Cyanobacteria are the dominant group associated with pitting

Biopitting is mainly described in vertical and subvertical surfaces, showing a preference for southern exposures


Doctoral school in biology biodiversity and analysis of ecosystem

Unusual differential erosion related to the presence of endolithic microorganisms (Martvili, Georgia)

Papers2/4

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)

Detail of the stone surface affected by biodeterioration phenomena on South-facing side of the Church.

AIMS

- Provides an interpretation of the differential erosion phenomenon

- Identify the ecological conditions which favor the phenomenon

-Impact on the stone conservation


Doctoral school in biology biodiversity and analysis of ecosystem

Results

Papers2/4

The stone is a fine-grained limestone.

Traces of fossils with relatively high values of porosity (23.28%).

The southern facade is the most intensely affected by the differential erosion phenomenon

Cyanobacteria are the most common microorganisms occuring

Through the analysis of images it was possible to estimate a considerable colonized area

The microorganisms appears to from an average depth of 200 μm

Black meristematic fungi are on and below the surface of the sample


Doctoral school in biology biodiversity and analysis of ecosystem

Conclusion

Papers2/4

This unusual differential erosion phenomenon is related to :

-The physical petrographic features of the rock, (heterogeneity and discontinuity) giving rise the deterioration phenomenon in specific preferential areas.

- Intense xeric conditions that permit the establishment of endolithic microorganisms (Southern facade )

-Biodeterioration due to the cyanobacteria and meristematic fungi endolithic activity


Doctoral school in biology biodiversity and analysis of ecosystem

Identification of endolithic traces on stone monuments

Papers3/4

Raman spectroscopic analysis applied to four endolithic samples from Temperate and Mediterranean bioclimate regions

Oral Presentation: 7th International Conference on the application of Raman spectroscopy in Art and Archaeology,Ljubljana 2-6 September 2013

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


Doctoral school in biology biodiversity and analysis of ecosystem

Materials and Methods

Papers3/4

Four samples colonized by endoliths

-Marble rock showing colonization of cyanobacteria and endolithic fungi from quarries in Carrara Area

-Black schist from Hebrew’s cemetery

tombstone in Venice. Endolithic lichens with perithecia completely sunken in the rock;

- Dolomitic limestone showing colonization of endolithic cyanobacteria from cliff of the Amalfi Coast

-Fine-grained limestone from the Church of the Virgin in Martvili in Western Georgia. The back side shows orange biological traces


Doctoral school in biology biodiversity and analysis of ecosystem

Materials and Methods

Papers3/4

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;

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


Doctoral school in biology biodiversity and analysis of ecosystem

Results/ Sample 1

Papers3/4

Calcite

Scytonemin

Marble rock

Inner side

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

Anthraquinonecoumpounds

against intense solar radiation

Chlorophyll

Surface


Doctoral school in biology biodiversity and analysis of ecosystem

Results/ Sample 2

Papers3/4

Fe

Black schist from Hebrew’s cemetery

  • Goethite

  • α-FeO(OH)

-Lepidocrocite

δ-FeO(OH)

Carbon-based substance

Dolomite

Calcite


Doctoral school in biology biodiversity and analysis of ecosystem

Results

Papers3/4

Black schist from Hebrew’s cemetery

Anthraquinone coumpounds

against intense radiation


Doctoral school in biology biodiversity and analysis of ecosystem

Results/Sample 3 and Sample 4

Papers3/4

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.

Dolomitic limestone

Carotenoid is an accessory pigment, usually produced under stress conditions within antioxidant strategy

limestone from the Church of the Virgin in Martvili


Doctoral school in biology biodiversity and analysis of ecosystem

Conclusions

All samples show traces of compounds known to be effective against UV-radiation damage

-Sample1-Marble from Carrara area.

Scytoneminappears in all spectra and at different depths of the sample.

Anthraquinonecoumpounds

-Sample2-Hebrew’s cemetery tombstone in Venice.

Iron oxide hydroxides due to a bioalteration by endolithic.

Anthraquinonecoumpounds

- Sample3-4 Dolomitic limestone and sample from Church in Martvili

Carotenoid compounds (antioxidant) useful against the high UV-radiation


Doctoral school in biology biodiversity and analysis of ecosystem

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

Papers4/4

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)


Doctoral school in biology biodiversity and analysis of ecosystem

Materials and Methods

Papers4/4

Rock flakes from the Marble rock samples stained with :

The nucleic acid stain, propidium iodide ( after permeabilization of the cell membranes)

DNA structures

excitation 543 nm emission 633 nm ( RED CHANNEL )

The glycoconjugates stain lectinConcanavalin-A Alexa Fluor 488

Extrapolymeric substances (EPS)

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)


Doctoral school in biology biodiversity and analysis of ecosystem

Results

Papers4/4

Isosurface presentations show the 3-D arrangement

760 μm

Microorganisms stained with red fluorescent (PI)

Extracellular matrix (EPS) stained with green fluorescence

(ConA- Alexa Fluor 488)

Overview in y-x directions shoving the penetrations

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

Overview in x-z direction of the cyanobacteria distribution


Doctoral school in biology biodiversity and analysis of ecosystem

Results

Papers4/4

Volume distribution; ConA- Alexa Fluor 488 stain

Volume distribution; propidium iodide stain

Total Volume

2 %

Total Volume

2.5 %

Volumes were calculated from each of six image stacks corresponding to the various depths


Doctoral school in biology biodiversity and analysis of ecosystem

Results/ Comparison PAS and SEM

Papers4/4

Entire cross-section partially decalcified by hydrochloric acid solution

3 mm

Cyanobacteria immersed in the substratum

Total Area

6 %

Calcite grains perforated by hyphae

Fungal hyphae embedded in extracellular matrix (white arrow)

Cross-section after PAS staining

After application of a threshold classification, the substratum (black area) and endolithic colonization (white areas).


Doctoral school in biology biodiversity and analysis of ecosystem

Conclusion

Papers4/4

The marble rock sample is colonized by cyanobacteria completely immersed in the stone.

Below the cyanobacteria a dense network of fungal hyphae is present

CLSM with the double staining distinguish and quantify the contribution of the extracellular matrixfrom that of DNA structure

The volume occupies by endolithiccyanobacteria and fungi is about 2.5% and the EPS Volume is about 2 %

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.

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


Doctoral school in biology biodiversity and analysis of ecosystem

GeneralConclusion

A increase of the information about the real effect on the stone monuments andof the potential damage by endolithic attack

New approaches and new techniques have been applied

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

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


Doctoral school in biology biodiversity and analysis of ecosystem

I wishtothanks: Di Giulio A. ; Ricci M.A.; Salvadori O; Sodo A.; PercarioZ.; and the LIME staff for the technicalsupport.

I thankall 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 wantover-please the laboratoryfor the support and the best moments (Alma, Roberto; Francoise, Flavia, Valentina; Giulia, Simona, Wawan).

Thanksforyourattention…


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