Andrea Puškárová

Disclosing a crypt: Microbial diversity and degradation activity of the microflora isolated from funeral clothes of Cardinal Peter Pázmány

A crypt can be considered as a particular environment where different microbial communities contribute to decomposition of organic materials present inside during a long interval of time. The textile remains of the funeral clothes (biretta and tunic) of Cardinal Pázmány, an important historic figure dead in Bratislava the 19th March 1637, conserved in this kind of environment were subjected to microbial investigation. The sampling comprised three different approaches and the use of various kinds of cultivation media. Two different PCR-based clustering methods, f-ITS and f-CBH, were employed in order to select the bacterial and fungal microfloras which were identified in a second step by the 16S rRNA and ITS sequencing respectively. The isolated microflora was tested for its proteolytic, keratinolytic and cellulolytic activities and for its ability to grow on Fibroin agar medium. The combination of cultural, molecular and biodegradative assays was able to isolate and characterize a bacterial community composed mainly by members of the phyla Firmicutes and Actinobacteria. The fungal community appeared more diversified, together with several Penicillium and Aspergillus strains, members belonging to the species Beauveria bassiana, Eurotium cristatum, Xenochalara juniperi, Phialosimplex caninus and Myriodontium keratinophilum were isolated. Bacteria, especially the Bacillus members, showed their strong ability to degrade keratin and gelatin and a large portion of them was able to growth on Fibroin agar. The fungal isolates displayed a widespread cellulolytic activity and fibroin utilization, although they possessed a weaker and slower proteolytic and keratinolytic properties respect to bacterial counterpart. The present study can be considered perhaps as the first or among the few microbial investigations which treated the textile biodegradation from such unusual environment.

Biodeterioration of epoxy resin: a microbial survey through culture-independent and culture-dependent approaches.

During the 20th century, synthetic polymers were greatly used in the field of art. In particular, the epoxy resins were used for both conservation and for creating sculptures. The biodeterioration of these polymers has not been adequately studied. The aim of this investigation was to examine the microflora responsible for the deterioration of an epoxy statue exposed to outdoor conditions. Fungal and bacterial microflora were isolated from the art object, clustered by fluorescence-ITS (internal transcribed spacer), identified by ITS and 16S rRNA sequencing and tested for their lipolytic abilities by three agar assays. Different algal, bacterial, cyanobacterial and fungal clone libraries were constructed. The surrounding airborne microflora was analyzed using culture-dependent and culture-independent approaches. The results indicated the presence, on the statue surface, of an interesting and differentiate microbial community composed of rock-inhabiting members, algal photobionts (Trebouxia spp., Chloroidium ellipsoideum and Chlorella angustoellipsoidea), Cyanobacteria (Leptolyngbya sp., Phormidium sp., Cylindrospermum stagnale, Hassallia byssoidea and Geitlerinema sp.), black yeasts related to the species Friedmanniomyces endolithicus, Pseudotaeniolina globosa, Phaeococcomyces catenatus and Catenulostroma germanicum and several plant-associated fungi. This investigation provides new information on the potential microfloral inhabitants of epoxy resin discovering a new ecological niche, occupied mainly by several members of rock-colonizing microbial species.

Bacterial strains isolated from PCB-contaminated sediments and their use for bioaugmentation strategy in microcosms

This study was focused on the characterization of 15 bacterial strains isolated from long‐term PCB‐contaminated sediment located at the Strážsky canal in eastern part of Slovakia, in the surroundings of a former PCB producer. PCB‐degrading strains were isolated and identified as Microbacterium oleivorans, Stenotrophomonas maltophilia, Brevibacterium sp., Ochrobactrum anthropi, Pseudomonas mandelii, Rhodococcus sp., Achromobacter xylosoxidans, Stenotrophomonas sp., Ochrobactrum sp., Pseudomonas aeruginosa, and Starkeya novella by the 16S rRNA gene sequence phylogenetic analysis. This study presents a newly isolated bacterial strain S. novella with PCB‐degrading ability in liquid medium as well as in sediment. For A. xylosoxidans, the bphA gene was identified. The best growth ability in the presence of all sole carbon sources (biphenyl and PCBs vapor) was obtained for Ochrobactrum sp. and Rhodococcus sp. Uncultured Achromobacter sp. showed the highest potential for bioaugmentation of PCB‐contaminated sediment.

A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.

Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.

Microbial communities affecting albumen photography heritage: a methodological survey

This study is one of the few investigations which analyze albumen prints, perhaps the most important photographic heritage of the late 19th and early 20th centuries. The chemical composition of photographic samples was assessed using Fourier-transform infrared spectroscopy and X-ray fluorescence. These two non-invasive techniques revealed the complex nature of albumen prints, which are composed of a mixture of proteins, cellulose and salts. Microbial sampling was performed using cellulose nitrate membranes which also permitted the trapped microflora to be observed with a scanning electron microscope. Microbial analysis was performed using the combination of culture-dependent (cultivation in different media, including one 3% NaCl) and culture-independent (bacterial and fungal cloning and sequencing) approaches. The isolated microorganisms were screened for their lipolytic, proteolytic, cellulolytic, catalase and peroxidase activities. The combination of the culture-dependent and -independent techniques together with enzymatic assays revealed a substantial microbial diversity with several deteriogen microorganisms from the genera Bacillus, Kocuria, Streptomyces and Geobacillus and the fungal strains Acrostalagmus luteoalbus, Bjerkandera adusta, Pleurotus pulmonarius and Trichothecium roseum.

A novel PCR‐based approach for the detection and classification of potential cellulolytic fungal strains isolated from museum items and surrounding indoor environment

Aims:  To develop a novel PCR‐based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh‐I) and its polymorphism.

Bacterial consortia at different wine fermentation phases of two typical Central European grape varieties: Blaufränkisch (Frankovka modrá) and Grüner Veltliner (Veltlínske zelené).

This is the first study focused to bacterial diversity and dynamic during the vinification of two important Central Europe grape vines: Blaufränkisch and Grüner Veltliner. The investigation strategy included culture-dependent and culture-independent approaches. Four different agar media were utilized for the isolation of various bacteria occurring in several fermentation stages. The isolates were clustered by fluorescent-ITS PCR and, one or more representatives of each cluster, were identified by 16 rRNA gene sequencing. The culture-independent approach, based on 16S rRNA gene amplification, combined the denaturing gradient gel electrophoresis (DGGE) method and the construction of bacterial clone library for each wine fermentation step. A complex bacterial community was identified, comprising different lactic acid bacteria and acetic acid bacteria, such as Leuconostoc spp., Lactobacillus spp. and Gluconobacter spp. Other OTUs and bacterial isolates embraced the Actinobacteria, Bacilli, Alpha-, Beta- and Gamma-proteobacteria classes. Different taxa already detected by recent studies, such as Sphingomonas, Variovorax, Pantoea, Enterobacter and Tatumella, were detected confirming the continuous occurrence of these kinds of bacteria in wine environment. Moreover, novel genera (Amycolatopsis, Hydrogenophilus, Snodgrassella, Telluria, Gilliamella, Lelliottia, and Lonsdale quercina) never detected before were recognized, too. The role of these, until now anonymous, bacteria during vinification deserves investigation, which could open a new research field in wine technology.

The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells

Six essential oils (from oregano, thyme, clove, lavender, clary sage, and arborvitae) exhibited different antibacterial and antifungal properties. Antimicrobial activity was shown against pathogenic (Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis) and environmental bacteria (Bacillus cereus, Arthrobacter protophormiae, Pseudomonas fragi) and fungi (Chaetomium globosum, Penicillium chrysogenum, Cladosporium cladosporoides, Alternaria alternata, and Aspergillus fumigatus). Oregano, thyme, clove and arborvitae showed very strong antibacterial activity against all tested strains at both full strength and reduced concentrations. These essential oils showed different fungistatic and fungicidal activities when tested by direct application and in the vapor phase. The genotoxic effects of these oils on HEL 12469 human embryo lung cells were evaluated using an alkaline comet assay for the first time, revealing that none of the oils induced significant DNA damage in vitro after 24 h. This study provides novel approaches for assessing the antimicrobial potential of essential oils in both direct contact and the vapor phase and also demonstrates the valuable properties of the phenol-free arborvitae oil. These results suggest that all the tested essential oils might be used as broad-spectrum anti-microbial agents for decontaminating an indoor environment.

Biochemical and proteomic characterization of the extracellular enzymatic preparate of Exiguobacterium undae, suitable for efficient animal glue removal

In this work, we describe the preparation and characterization of a biopreparate for efficient and rapid animal glue removal. The biopreparate is based on the extracellular proteolytic enzymes of an Exiguobacterium undae environmental isolate. Liquid chromatography-mass spectrometry analysis showed that the biopreparate is predominantly composed of hydrolytic enzymes—proteases and peptidases, nucleases, peptide ABC transporter substrate-binding proteins, and a phosphatase. The two main proteins present are bacillolysin and a peptide ABC transporter substrate-binding protein. Inhibition and proteomic analyses of the biopreparate revealed that bacillolysin, a neutral metalloendopeptidase, is mainly responsible for its proteolytic activity. This biopreparate was able to satisfactorily remove two types of animal glue from different kinds of material surfaces. These results suggest that this biopreparate could serve as a potential new tool for the restoration of historical objects rather than living microorganisms.