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COLONISATION PATTERNS OF STONE SURFACES ANALYSED BY FRACTAL GEOMEfRY

C. Urzl*, S. Trusso0, A. Kopecky#

(*) Istituto di Microbiologia, Facolta di Scienze, Universita di Messina (

0) Istituto di Tecniche Spettroscopiche, CNR, Messina

Salita Sperone, 31 1-981()6 Villaggio S.Agata Messina, Italy (#) lnstitut ftir Biochemische Technologie und Mikrobiologie, Technical Universitat, Wien,

Austria

ABSTRACT

Fractal geometry as first pointed out by Mandelbrot (1983) is able to describe various biological phenomena including processes related to the biological attack.

The ~stribution of microorganisms on stone surface can be random, like the distribution of ~ust particles on a plane surface, or dependent on a construction law, determined by the environment.

The aim of our investigations was to elucidate the mechanism of microbial colonisation spreading on stone surfaces of several monuments in Europe, by means of fractal geometry.

In this way we should get useful information about the growth pattern of microorganisms related to the composition of colonised surf ace, nutritional requirement and environmental factors.

For this purpose, photographic documentation of several monument surfaces located over all Europe, in which was evident a biological colonisation was chosen.

Digitised images of the colonisation patterns have been analysed by means of an image processing algorithm in order to obtain both surface and mass fractal dimension.

Our preliminary results seem to evidence that in most cases microbial colonisation of rock surface has both a surface as well as a mass fractal dimension.

INTRODUCTION

Many natural phenomena, in living and non-living systems seem to obey laws regulating their growth and their spatial and temporary extension.

Microbial growth in nature depends on environmental, climatic and nutritional factors and on the nature of substrate to which they are related and/or attached.

In unfavourable conditions they adapt their metabolism to a life different from their optimum and they form metabolically active cells and anabiotic structures as well as aggregates as already observed by Winogradsky.

It is well known that microbial reproduction rate is limited by the nutrient concentration and this latter is limited by the diffusion around and into the colony (Ben-Jacob et al., 1996).

In an ideal situation growth occurs in all the directions and without restrictions and organisms decide for the most favourable configuration. This fact, however, is very improbable in natural conditions for a biological system.

In environmental non controlled situations prevails the growth at non equilibrium conditions in which the microbial morphophites and the aggregation types are highly complex and controlled by a diffusion limited aggregation (DLA) process ~Krumbein et al., ~989).

Fujikawa ( 1994) showed that practically any m1croorgamsms grow accordmg to DLA process if they are under poor nutrient conditions. . . . .

Despite the fact that bacterial growth can be compared to the d~ffus1on hm1ted growth of the non-living systems, they present an additional level of C?~plex1ty due to the fact th~t. the single constituents (block) of the colony are themsel.v~s hvmg. systems. Thus .an .eff1c1ent adaptation of the whole colony to adverse growth cond1t1ons reqmres a self-orgamsatton to all levels (Ben-Jacobs, 1996).

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Fractal analysis, introduced in microbiology, has described morphological differences correlated with growth, metabolic activities, pigmentation (Obert et al., 1990). .

Concerning the microbial colonisation of rock surfac~s and stone monu~ents, ex~ens1on, thickness and biomineralisation processes can be also studied through a phys1co-chem1cal law and especially through diffusion limited aggregation law. . . . .

Considering the "habitat" monument surfaces as a natural environment, Its colomsation by organisms and the consequent biodeteriorative process is controlled by the same conditions above discussed.

In fact, oligotrophic nutritional conditions often occur on the rock surfaces as well as limiting climatic conditions and they affect the type of organisms (e. g well adapted to survive in low water availability, in high sun exposure) and the modality of growth and the spreading of the colonisation (Urzl et al. 1995).

If we consider the first moment in which microorganisms enter in contact with the rock surface (as primary bioreceptivity, sensu Guillitte, 1995), their distribution will be random and can be compared to the distribution of dust inorganic particles on a plane surface (Kaye, 1989).

After it, adhesion processes render more stable the connection between cells and substrate and if growth conditions occur the colonisation process starts.

Microbial colonisation of rock surfaces can occur both on the surface and in depth. Surf ace can be colonised by microorganisms because of their ability of movement and by

the production of tiny branched structures (hyphae or similar ones) described as explorative structures.

When microorganisms can find better conditions in the deeper layers, their mobilisation can also occur through solubilisation process and mechanical penetration.

Both surficial and deeper colonisation processes can be studied in the frame of fractal geometry.

In this contribution, the Authors tried, by means of fractal geometry, to elucidate the mechanism of microbial colonisation spreading on stone surfaces of several monuments in the Mediterranean Basin.

MATERIALS AND METHODS

Photographic documentation at macro- and stereomicroscopic level were chosen from: a) monuments and rock surfaces showing a clear biological colonisation, b) rock slabs infected in laboratory condition and, c) fungal colonies growing into different media

Photos were digitised in black and white by a UMAX(UC840) Scanner with 200 to 450 dpi (pixels per inch) resolution. The choice of the resolution was dependent on the size of the selected area

A threshold algorithm was used to transform images in black and white (colonies are white while mar~le surf ace is black). The threshol~ level was adjusted in order to separate exactly the colomes from the marble surface. The size of the images depends on the selected area but it was never less than 400x400 pixels.

The evaluation of the fractal dimension was performed by the box counting method (Obert et al., 1990). . Such a method is carried out as follows: the image of the colony is divided in boxes of

s1delength _I, the boxes that_ c?ver the colony are counted, and the relative value N(l) is stored. Then the size of the boxes is mcreased and the counting procedure repeated. A distinction can be made between boxes that cover the colony and that which cover the border of the colony so that at the end of the procedure we have two distinct values for mass Nmass(/) and surf~ce Nsurf (I), both behave as a power law:

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where Cm and Cs are proportionality constant, dm and ds are respectively the mass and surf ace dimension of the colony.

The counting box method, implemented both in basic language and with matlab image processing toolbox, was tested on images of Euclidean objects like circles, lines and squares in order to reproduce the correct values of dm and ds. The same test was performed on fractal objects which have analytically calculable fractal dimensions between one and two such as the Koch curve and a DLA aggregate (Witten and Sander, 1981). When the logarithm of Nmass(l) is plotted versus the logarithm of l, a straight line is obtained, the slope of which is dm.

RESULTS

In Table 1 are summarised the results obtained by analysing images of colonisation pattern in natural and in laboratory conditions as well as the growth of single fungal colony growing in a petri dish.

Table 1 - Summary of results carried out on the digitised images. Description of origin, characteristic of stone, colonisation pattern as well as dm and ds value and fractal dimension obtained are showed.

Reference Origin dm 4v Description of fractal dimension

Fracl Carrara marble slab infected with a 1.44 1.21 mass & surface fractal dematiaceous strain

Frac2 Carrara marble sarcophage (SI) in l.53 1.37 mass & surface fractal the Messina Museum garden

Frac3 Carrara marble statue in the Messina 1.58 1.57 mass fractal Museum garden, colonised by Sarcynomyces petricola strain (specie nova)

Frac4 Carrara marble from Munich 1.46 1.12 mass & surface fractal cemetery. Inner part with a fungal colonisation.

Frac5 Marble from LNEC garden, Llsbona, 1.57 1.31 mass & surf ace fractal with evident fungal clusters.

Frac6 Carrara marble sarcophage (S2) from 1.48 1.16 mass & surface fractal the garden of Messina Museum Noto's calcarenite from the quarry 1.98 1.01 Euclidean Frac7 with algal and fungal colonisation

Frac9 Nigrospora colony growing in 1.69 1.36 mass & surface fractal

Czapekagar Fracll Carrara marble sarcophage (S3) from 1.39 1.15 mass & surface fractal

the garden of Messina Museum Frac12 Pentelic marble from Parthenon, 1.66 1.33 mass & surface fractal

Athens with lichen colonisation and biopitting

The dm value is related with the density of colonisation pattern or colony growth; a higher value reflects a high density of colony pattern, thus the ability to occupy all the available space.

For example, in our results, the highest dm ~bserve~ for Frac 7 (dm = 1,98) assumed an Euclidean value: in fact, almost all the rock surface is colomsed.

Some examples are shown in Figures 1, 2, 3 and 4.

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Fig l. a) Stereomicroscopic evidentiation of an experimental fungal infection on Carrara marble slab (by Ute Wollenzien, Oldenburg) in laboratory controlled and limited nutrient conditions. b) Thresholded image of previous one, in which the algorithm was carried out. White pixels represent the fungal colonisation while the black ones the marble surface. Two different patterns can be recognised. The arrow indicates the presence of resistance structures (sclerotia), while the tiny ramified structures demonstrate the spreading ability of the fungal strain on the marble surface.

10' d,.=l.44

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Fig. 2. Plot of the experimental values (o) of Nmass(l) versus l determined by the box counting method (Obert et al., 1990). The regression line extends well over a decade, the slope furnishes a mass fractal dimension of 1.44.

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Fig. ~ · .a) Blow up of a. fungal colonisation on a marble statue located in the garden of the Messina Museum. Idenllf1cat10n of strams isolated m correspondence of black spots demonstrated the colonisation of a sole strain of a black yeast Sarcynomyces petricola, specie nova. b) its black and white image shows a fractal dimension (frac 3 drn = 1.58 and <is= 1.57). Being these two values quite similar, only a mass fractal dimension is recognised. This means that the fungal colonisation is spreading homo eneousl all over the marble surface.

Fig. 4. a) Nigrospora colony isolated from calcareous stone of St. Trophime Portal growing in Czapek Agar.; b) black and white image of the fungal colony from which the fractal dimension was taken (frac 9) d,n 1,69 ds l ,36. Both fractal mass and surface fractal dimension occurri . The colony patterns characterised by a higher density in the inner part, while in the periferical area the growth occurs as branched structures. The image obtained is a classical fractal one.

DISCUSSION

As already reported in a previous paper (Urzi et al. , 1995), rock substrate exhibits drastic local disparities of climatic, environmental and nutritional conditions. This enormously affects the colonisation of microorganisms and their biodiversity.

If we analyse more closely our results, different environmental situations are clearly evidenced.

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In the majority of cases examined, the colonisation pattern is characterised by the presence of clusters of higher density more or less diffused all over th~ rock surface. U:s~ally clusters represent survival structures that better react to adverse environmental conditions. Dispersive behaviour occurs during growth under high nutrient conditions; while under low nutrient or starvation conditions population expansion becomes of secondary importance and cells displays an aggregative behaviour (James et al., 1995). Under limiting conditi~ns, cell aggregates occupy microniches, that sometimes are below the surface. These depressions are caused by the microflora through solubilisation or mechanical processes causing the crystals detachment and the formation of biopitting. In this micro-habitat, colonisators first occupy all the space available (roundish structures, with internal high density) and only later they start a new colonisation of the remaining surface. The phenomenon can lead in the time to a homogenous colonisation of the rock surf ace.

Production of aerial mycelia on the one hand is an energy consuming process, whilst on the other, it is necessary for the population maintenance. Consequently, in more favourable conditions on the rock surface and near by the surface hyphae are produced and they colonise faster the rock surface. Usually the growth of such structures is controlled by a certain anisotropy given by the size of crystals.

In conclusion, our preliminary results, have shown the possibility to study the microbial colonisation of rock surfaces through fractal geometry. In fact, due to the self-similarity of fractal structures, the phenomena studied are independent of the magnification level and some hypotheses on colonisation behaviour of microorganisms can be carried out.

Other studies are currently being carried out in order to better understand the microbial colonisation and in general the microbial growth under non-controlled and controlled conditions. The help of image analysis, and computer assistance can increase enormously our knowledge on those processes.

ACKNOWLEDGEMENT

This research was supported by EEC Programme Environment through the grant n° EV5V-CT94-0569 "Atmospheric Eutrophication and Saecular Organic Pollution (Biological and Mineralogical Reactions of Mediterranean Monuments)" and in part by a C.N.R. (National Council of Research) contribution for the 1995 "Biologia applicata alla conservazione del patrimonio monumentale: indagini e interventi" (Urzl) and Progetto Strategico Beni culturali CNR (Trusso). We are grateful to William Fenton for his advice in the improvement of English form.

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