BIOFILM FORMING CYANOBACTERIA , ALGAE AND FUNGI ON TWO HISTORIC MONUMENTS IN BELGRADE , SERBIA

Biofilm on the sandstone substrata of the bridge “Brankov most” and on the granite substrata of the “Monument of the Unknown Hero” contains a complex consortia of cyanobacteria, algae, and fungi. Coccoid and filamentous cyanobacteria, green algae and diatoms make up the photosynthetic part of the biofilm while hyphal fragments, chlamydospores, fruiting bodies and spores take part as fungal components. These structures make a dense layer by intertwining and overlapping the stone surface. Five cyanobacterial, 11 algal and 23 fungal taxa were found. The interaction of the biofilm’s constituents results in the bioweathering of the stone substrata through mechanical penetration, acid corrosion and the production of secondary mycogenic biominerals.


INTRODUCTION
During the recent decades there has been a general concern about the deterioration of historic buildings.Along with chemical and physical weathering factors, microbial growth plays an important role in this process (Suihko et al., 2007).Microorganisms from all five kingdoms of the living world can colonize stone surfaces depending on stonebioreceptivity and form a microbial sub-aerial community biofilm.Bioreceptivity is a term which indicates the potential of a material such as stone for colonization by one or more groups of living organisms without necessarily undergoing any biodeterioration (Prieto & Silva, 2005).The microbial colonization of stones depends on environmental factors such as water availability, pH, climatic exposure, nutrient sources, and on petrologic parameters, such as mineral composition porosity and permeability of the rock material (Warscheid & Braams, 2000).The stone ecosystem is subjected to harsh environmental changes, especially temperature and moisture, which exerts extreme selective pressure on any developing microbial community (May, 2003).The bioreceptivity of stone depends on its structure and chemical composition, while the intensity of the microbial contamination is caused by the climatic conditions and the anthropogenic eutrophication of the atmosphere (Prieto & Silva, 2005).Biofilm formation on clean surfaces usually starts with phototrophic organisms (algae, cyanobacteria) which use CO 2 from the atmosphere and sunlight as their carbon and energy source.Heterotrophic organisms (most bacteria and all fungi) need some organic source for their growth, and this is provided by the metabolites of phototrophic organisms or by air-borne deposition.It has been shown that the very low nutrient requirements of some rock inhabiting heterotrophic microorganisms may be fulfilled by remains of polluted air and rain or animal remains and secretion (Suihko et al., 2007).
All biofilm-forming microorganisms may cause biodeterioration and degrade stone mechanically, chemically and aesthetically through the metabolic activities and biomineralization process in these biofilms (Suihko et al., 2007).Phototrophic microorganisms can grow on the stone surface (called epilithic phototrophs) or may penetrate some millimeters into the pore system (called endolithic phototrophs).Phototrophic microorganisms have a direct effect on the deterioration of stones due to their pigments which cause an aesthetically detrimental effect, and under certain climatic conditions they provide a protective film on the stone surface regulating humidity and temperature.The establishment of heterotrophic microbiota on rocks is possible even without the pioneering participation of phototrophic organisms.Chemoorganotrophic fungi are especially concentrated in stone crusts.They are able to penetrate into the rock material by hyphal growth and by biocorrosive activity, due to the excretion of organic acids or by the oxidation of mineralforming cations, preferably iron and manganese.Their deterioration activities also include the discoloration of stone surfaces, due to the excretion of melanin by dematiaceous fungi (Warscheid & Braams, 2000).

Sampling
The samples were collected from stone material with visible alteration and degradation on two localities in Belgrade: "The Monument of the Unknown Hero" on Mt.Avala and the "Brankov most" bridge (Fig. 1).

Mycological analyses
All samples from the granite and sandstone were taken for mycological analyses by swabbing the surfaces with sterile cotton swabs.The swab samples were diluted in 10 ml sterile distilled water and shaken for 10 min.A malt-streptomycin-agar medium (MA) prepared according to Boot (1971), with 500 mg streptomycin per liter, was inoculated with 1 ml of the resulting suspensions.Every sample was done in triplicate.The plates were incubated on 24°C in a thermostat.Isolation of the colonies that formed was done successively, using a standard mycological medium (Malt extract agar, Potato-dextrose agar and Czapek's solution agar).All the cultures were grown for 7 days in a thermostat at 24 °C and the macroscopic and microscopic characteristics of the obtained isolates were examined.Identification of fungi was based on the macroscopic features of the colonies growing on the agar plates and the micromorphology of the reproductive structures using identification keys (Raper & Fennel, 1965;Ainsworth et al., 1973;Arx, 1974;Pitt, 1979;Ellis & Ellis, 1997).

RESULTS
The organisms forming the biofilm were identified to the species or genus level, based on their macroand micromorphological characteristics.

The detected photosynthetic organisms
The photosynthetic organisms of the biofilm were comprised of 5 taxa of cyanobacteria, 5 taxa of green algae and 6 diatom taxa (Table 1).The most abundant biofilm-forming cyanobacteria on the sandstone substrata was coccoid Gloeocapsa novacekii while the only cyanobacteria detected on the granite substrata was a filamentous species belong to the Leptolyngbia genus which included subaerophytic species, inhabiting different types of wet stone surfaces.On both investigated sites, the dominant green alga was coccoid Desmococcus olivaceus while this species formed an association with filamentous Microspora sp. on the granite substrata.Coccoid and filamentous cyanobacteria and green algae formed a dense mucoid biofilm (Fig. 2).

DISCUSSION
Algological and mycological analyses showed that the structure of the microbiota was specific to different rock substrata, but that the total taxa for the granite and sandstone were very similar.The genera of phototrophic organisms identified are mainly of algae involved in gelatinous products, the presence of which is related to mineral fixation (Peraza Zurita et al., 2005).It was significant that Haematococcus pluvialis was found in a red resting stage coloring the incavation caused by bombs in Second World War.The red-stained granite surface had a dense layer consisting of cysts with asta-xanthin deposits.Although according to Tomaselli et al. (2000) the taxonomic diversity of stonedwelling photosynthetic micro-organisms appears to be rather wide, especially considering that the habitats they colonize can be classified as extreme habitats, our results emphasize the low photosynthetic biodiversity in these biofilms, with only 16 taxa found.Crispim et al. (2004) reported the presence of 11 cyanobacterial taxa on church walls in Porto Alegre (Brazil).The most dominant fungi among Deuteromycotina were dematiaceous hyphomycetes with melanized hyphae and reproductive structures (genus Alternaria, Aureobasidium, Cladosporium, Drechslera).The production of dark conidia and pigments was recorded in culture media during the cultivation of melanized fungi.The microfungi identified, especially melanized hyphomycetes from the genus Alternaria, Cladosporium, Drechslera, Epicoccum, cause discoloration, as well as the mechanical exfoliation of building stone material analyzed through mechanical hyphae penetration and the production of different pigments and organic acids (Milanesi et al., 2005).Aureobasidium pullulans var.melanigerum is a stain fungus that usually causes staining of different stone surfaces which decreases the aesthetic qualities of historic monuments.The microscopic analyses of the biofilm samples showed the presence of an initial association of potential photobionts and mycobionts able to form lichens.The ability of microcolonial fungi to form associations with potential photobionts present on  rock surfaces was demonstrated for pure cultures in vitro by Gorbuschina et al. (2005).Recently it was revealed that fungi comprise a significant component of microbiota in a wide range of rocks including sandstone, granite, limestone, marble and gypsum (Burford et al., 2003(Burford et al., ), Šimonovičova et al. (2004) ) reported the presence of 36 different microfungi on stone in a hypogean cemetery in Bratislava.Microbial biofilms modify the capillary water uptake of the porous stone material investigated, causing alterations in the water-vapor diffusion.Surface active compounds in the biofilm provoke a decrease in the pore water tension, changing the specific moisture relationship of the stone and protecting microorganisms against water loss and desiccation and favoring subsequent microbial contamination and their bio-corrosive activity (Warscheid, 1996).Besides balancing humidity changes, the biofilm protects the stone microbiota from extreme temperatures as well as the toxic impact by salt and heavy metal accumulation.This may explain the resistance of some microorganisms to biocidal treatments (Gaylarde & Morton, 1999).Prieto, B.and B. Silva (2005).Estimation of potential bioreceptivity of granitic rocks from their instrinsic properties.Int.Biodeter. Biodegr.56, 206-215. Raper, B.K., and D.I Fennel (1965).The Genus Aspergillus, The Williams and Wilkins Company, Baltimore., K. (1972).Chlorophyta III Flora Slodkowodna Polski, Tom 10, Panstwowe Wydawnictwo Naukowe, Warszawa.

Fig. 1 .
Fig. 1.Some sampling sites with visible stone alterations: a. Haematococcus pluvialis in red resting stage, covers the incavation of granite monument "The Monument of the Unknown Hero" on Avala; b.Exfoliation of sandstone substrata on the Brankov most bridge.

Table 1 .
Algae and cyanobacteria found on different stone material.

Table 2 .
Micromycetes found on different stone material.