Miloš Stupar

Cyanobacteria, algae and microfungi present in biofilm from Božana Cave (Serbia)

Caves represent very specific worldwide environments (Lamprinou et al., 2012) and many of them are included in the UNESCO World Heritage List (Czerwik-Marcinkowska, 2013). Cave habitats are characterized by almost constant temperature (T) and relative humidity (RH) all year round. A typical cave can be divided into three primary zones: the entrance-, transition-, and a deep cave zone (Tobin et al., 2013). Light intensity is the major factor which determines whether the microbial communities of cave stone substratum will be autotrophic (cyanobacteria and algae) or heterotrophic (bacteria and fungi) (Albertano, 2012). The above mentioned factors (T, RH, LI) determine which microbial communities can grow in a given location, but many other characteristics of caves, such as location, entrance dimensions, morphology, orientation, intrinsic substratum properties, and other microclimate parameters (air circulation, hydrological conditions, etc.) are also important (Martinez & Citation:

A sub-aerial biofilms investigation and new approach in biocide application in cultural heritage conservation: Holy Virgin Church (Gradac Monastery, Serbia).

Sub-aerial biofilms (SAB) on frescoes and façade of the Holy Virgin Church (Gradac Monastery, Serbia) were investigated to study the effectiveness of benzalkonium chloride (BAC) as a biocide. Antifungal activity of Origanum vulgare essential oil as a natural product with confirmed high fungicidal potential was also tested against fungi causing alterations on the frescoes. Chemical and mineralogical composition of the church historical mortar showed the dominance of calcareous minerals. In large areas on the church façade, coloured biopatinas formed by different groups of coccoid and filamentous cyanobacteria, green algae and filamentous fungi were found. Epilithic lichens and bryophytes were also present on the church exterior suggesting long-term ecological succession. The presence on the frescoes, of human pathogenic fungus Aspergillus fumigatus were found in the internal environment. Aspergillus and Penicillium species isolated from painted walls were subject to an in vitro biocidal assay with the biocide BAC. An aqueous solution of 0.25% and 0.15% BAC was able to reduce the viability of all the tested fungal isolates after exposure to the solution for 5 min, while exposure to an aqueous solution of 0.05% for 30 min reduced conidial viability. Origanum vulgare essential oil also showed strong antifungal potential against selected fungal isolates.

Inhibitory effect of essential oil from Nepeta rtanjensis on fungal spore germination

The ability of Nepeta rtanjensis essential oil to inhibit conidia germination of fungi was evaluated in vitro. Tested fungi included in research were Cladosporium cladosporioides, Trichoderma viride and two Alternaria species originally isolated from N. rtanjensis. The conidia of Cladosporium cladosporioides were most susceptible to the N. rtanjensis essential oil treatment, and the oil concentration of 0.1 µg ml−1 caused the maximum of conidia germination inhibition. The highest concentration used in experiment that caused the maximum of conidia germination inhibition was 0.6 µg ml−1 for Alternaria isolated from leaf surface of N. rtanjensis. The germ tube elongation of Alternaria isolates significantly decreased in response of different concentrations of oil used in experiment.

Biodeteriogenic and toxigenic agents on 17th century mural paintings and façade of the old church of the Holy Ascension (Veliki Krčimir, Serbia):

Biofilm forming microbial community dwelling on the surface of façade and biodeteriorated 17th century wall paintings of the old Church of the Holy Ascension (Veliki Krčimir, Serbia) was evaluated through microscopic analyses, in order to detect toxigenic agents and assess potential damage caused by biodeteriogens. Initial optical examination of adhesive tape samples revealed complex microbial community flourishing on deteriorated mural surfaces highly enriched with inorganic impurities and organic contaminants of plant and animal origin. A variety of fungal structures, such as melanized hyphae, chlamydospores, fruiting bodies, as well as spores for sexual and asexual reproduction, were predominant in all samples analysed. In addition, among actively growing micromycetes, dematiaceous microcolonial fungi and toxigenic Chaetomium and Cladosporium species were most frequent. Scanning electron microscopy analysis revealed the presence of actinomycetes and highly developed mycelium network within the plaster and paint layers, as well as numerous Cladosporium-like conidial chains and Chaetomium lemon shaped ascospores. Such findings confirm our hypothesis that pulverization of painted layers was most likely due to hyphal penetration and formation of fruiting bodies and other fungal structures. Interestingly, although several green algae were documented on the surface of wall paintings and façade, no cyanobacteria were observed in any of the analysed samples.

Biogenesis of secondary mycogenic minerals related to wall paintings deterioration process

Present study addresses potential of fungal strains, isolated from deteriorated mural paintings and surrounding air environment of the Church of the Holy Ascension in Veliki Krčimir (Serbia), to precipitate mycogenic minerals, when cultivated on agarized B4 medium. Utilizing culture-based isolation methods, 38 filamentous fungi were obtained in total, 23 from mural paintings and 15 from air, respectively, mainly ascomycetes, while Bjerkandera adusta and Thanatephorus cucumeris were only basidiomycetes. A total of 31 of 38 fungal isolates, more than 80%, were able to form minerals of different morphologies and variable size, determined via SEM-EDS and XRPD, to be either calcite or calcite and weddellite association. Among screened fungi, all Penicillium, Chaetomium and Cladosporium isolates, as well as most of the Aspergillus isolates (8/11) precipitated minerals, whereas cultures of Bionectria, Bjerkandera, and Seimatosporium isolates lacked any observable crystal forms. With the exception of two Alternaria alternata strains, no apparent disparity in potential to precipitate minerals in general, or form particular crystal phase was documented among the air and mural paintings isolates. Possible mechanisms of fungal mineralization of calcite and weddellite are further proposed. In addition to providing experimental evidence for fungal induced precipitation of oxalate and carbonate minerals, presented data suggest that fungal activity could be an important factor in a weathering process affecting cultural heritage exhibited and stored in inadequate conditions. Implementation of B4 plate assay for screening of mineralization potential of the isolated fungi could be used to assess biodegradative risk mycobiota pose to the mural paintings, so appropriate conservation measures may be utilized.

Biodegradative potential of fungal isolates from sacral ambient: In vitro study as risk assessment implication for the conservation of wall paintings

The principal purpose of the study was to evaluate in vitro the potential ability of fungal isolates obtained from the painted layer of frescoes and surrounding air to induce symptoms of fresco deterioration, associated with their growth and metabolism, so that the risk of such deterioration can be precisely assessed and appropriate conservation treatments formulated. Biodegradative properties of the tested microfungi were qualitatively characterized through the use of a set of special agar plates: CaCO3 glucose agar (calcite dissolution), casein nutrient agar (casein hydrolysis), Czapek-Dox minimal medium (pigment secretion); and Czapek-Dox minimal broth (acid and alkali production). Most of the tested isolates (71.05%) demonstrated at least one of the degradative properties, with Penicillium bilaiae as the most potent, since it tested positive in all four. The remaining isolates (28.95%) showed no deterioration capabilities and were hence considered unlikely to partake in the complex process of fungal deterioration of murals via the tested mechanisms. The obtained results clearly indicate that utilization of fast and simple plate assays can provide insight into the biodegradative potential of deteriogenic fungi and allow for their separation from allochthonous transients, a prerequisite for precise assessment of the amount of risk posed by a thriving mycobiota to mural paintings.

Antifungal activity of the pygidial gland secretion of Laemostenus punctatus (Coleoptera: Carabidae) against cave-dwelling micromycetes

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.

Diversity of Terrestrial Cyanobacteria Colonizing Selected Stone Monuments in Serbia

ABSTRACT The presence of biofilms on stone monuments can lead to biodeterioration processes that significantly lower not only the aesthetic value of cultural heritage sites but also cause considerable physical and chemical damage to stone surfaces. In terrestrial environments, cyanobacteria have a significant role in biofilm formation on a variety of natural and artificial stone substrata, including granite, sandstone, limestone, lime mortar, etc. Studies on cyanobacterial diversity and estimation of biodeteriorative activity on stone cultural heritage objects can be very important for conservators and restorers, and cyanobacteria removal from stone monuments is currently a great challenge for conservation science. In that sense, the diversity of terrestrial cyanobacteria was investigated at six localities in Serbia. Samples for cyanobacterial analyses were taken from surface biofilms covering different substrata: granite and lime mortar (The Monument to the Unknown Hero), sandstone (Brankos Bridge, Eiffel’s Lock) and carbonate rock (Medieval tombstones from Mramorje and Rastište, Gradac Monastery). A total of 18 cyanobacterial taxa were identified. The highest diversity was found on carbonate rock, followed by sandstone. Cyanobacteria were also recorded on an artificial substratum – lime mortar, while cyanobacteria were not found on granite. All detected cyanobacteria seriously impaired the structural and aesthetic integrity of the studied monuments via mechanical and chemical actions, so their presence must not be overlooked.

Frankincense and myrrh essential oils and burn incense fume against micro-inhabitants of sacral ambients. Wisdom of the ancients?

ETHNOPHARMACOLOGICAL RELEVANCE Essential oils obtained from resins of Boswellia carteri Birdw. and Commiphora myrrha (Nees) Engl., commonly known as frankincense and true myrrh respectively, have been used extensively since 2800 BCE for the treatment of skin sores, wounds, teeth, inflammation, and urinary tract diseases in traditional medicine; for preparation of mummification balms and unguents; and also as incense and perfumes. Since ancient times, burning of frankincense and myrrh in places of worship for spiritual purposes and contemplation (a ubiquitous practice across various religions) had hygienic functions, to refine the smell and reduce contagion by purifying the indoor air. AIM OF THE STUDY The general purpose of the study was to assess the in vitro antimicrobial potential of the liquid and vapour phases of B. carteri and C. myrrha essential oils and burn incense, as well as to test the effectiveness of their in situ application to cleanse microbially-contaminated air within the ambient of an investigated 17th-century church. MATERIALS AND METHODS The chemical composition of B. carteri and C. myrrha essential oils, obtained by hydrodistillation of frankincense and true myrrh oleo gum resins was determined using GC/MS, and antimicrobial properties of their liquid and vapour phases were assessed by the broth microdilution and microatmosphere diffusion methods. Chemical analysis of burn incense fume obtained using bottle gas washing with dichloromethane as a solvent was performed by GC/MS, while its antimicrobial activity was evaluated using a modified microatmosphere diffusion method to evaluate germination inhibition for fungi and CFU count reduction for bacteria. The in situ antimicrobial activity of B. carteri burn incense and essential oil vapour phase was assessed in the sealed nave and diaconicon of the church, respectively. RESULTS The dominant compounds of B. carteri EO were α-pinene (38.41%) and myrcene (15.21%), while C. myrrha EO was characterized by high content of furanoeudesma-1,3-diene (17.65%), followed by curzerene (12.97%), β-elemene (12.70%), and germacrene B (12.15%). Burn incense fume and soot had α-pinene (68.6%) and incensole (28.6%) as the most dominant compounds, respectively. In vitro antimicrobial assays demonstrated high bacterial and fungal sensitivity to the liquid and vapour phases of EOs, and burn incense fume. In situ application of B. carteri EO vapour and incense fume resulted in reduction of air-borne viable microbial counts by up to 45.39 ± 2.83% for fungi and 67.56 ± 3.12% for bacteria (EO); and by up to 80.43 ± 2.07% for fungi and 91.43 ± 1.26% for bacteria (incense fume). CONCLUSIONS The antimicrobial properties of essential oil derived from frankincense, a compound with well-known traditional use, showed that it possesses a clear potential as a natural antimicrobial agent. Moreover, the results suggest possible application of B. carteri EO vapour and incense fume as occasional air purifiers in sacral ambients, apart from daily church rituals.

Cave biofilms: characterization of phototrophic cyanobacteria and algae and chemotrophic fungi from three caves in Serbia

Cyanobacteria, algae (Chlorophyta and Bacillariophyta), and fungi were identified from biofilm samples from three caves in western Serbia: Ribnička, Hadži Prodanova, and Rćanska. Temperature, light intensity, and relative humidity varied from 16.9 8C to 24.9 8C, 61% to 87%, and 215 Lux to 4400 Lux, respectively. In general, the highest number of documented taxa belonged to Cyanobacteria, with chroococcalean taxa prevailing and Gloeocapsa species as the most diverse. A large percentage of observed fungi were Ascomycetes or Zygomycetes, while the only representative of Basidiomycetes was Rhizoctonia s. lat. However, a redundancy analysis revealed that different taxonomic groups were dominant at different localities: cyanobacteria and fungi in Ribnička and Hadži Prodanova, and Chlorophyta and Bacillariophyta in Rćanska. The statistical analysis showed that relative humidity is an important physical parameter influencing the development of various microbial communities in different caves. Cyanobacteria were mostly found in places with lower relative humidity, while Chlorophyta and Bacillariophyta were found in places with higher humidity. The documented physical parameters did not have a significant impact on the distribution of fungi. Measured chlorophyll-a content was highest on horizontal surfaces, where the highest content of organic/inorganic matter were also recorded. The highest water content was observed in biofilm samples from which many cyanobacteria taxa were identified.