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Biocleaning of cultural heritage items is mainly performed using living microorganisms. Approaches utilizing the enzymes of isolated microorganisms have not been frequently investigated. To find an enzymatic alternative for the removal of an oil-based overpainting, we focused on the characterization and use of a yeast Extracellular Enzymatic Mixture (EEM). A historical silk yeast was selected for its lipolytic properties and its EEM was extracted after cultivation on a medium supplemented with linseed oil. The EEM protein content was visualized by SDS-PAGE, its concentration assessed by fluorimeter and the enzymatic activity evaluated by p-NPP spectrophotometric lipase assay. The yeast growth was suppressed by adding diverse metal ions (Cd, Zn, Cr and Cu) in Reasoner's 2A (R2A) broth, while the quantity and activity of EEM were affected by adding Fe and Pb. Various delivery systems (agar-agar, tylose and klucel G) alone or in a combination with EEM were assayed on the historical painting surface. The colorimetric measurements and the ATR-FTIR analysis indicated that the combinations tylose-EEM and klucel G-EEM can be easily and effectively applied as biocleaning procedures to remove oil-based overpainting from fragile and valuable historical painting surfaces.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Wine reflects the specificity of a terroir, including the native microbiota. In contrast to the use of Saccharomyces cerevisiae commercial starters, a way to maintain wines’ microbial terroir identities, guaranteeing at the same time the predictability and reproducibility of the wines, is the selection of autochthonous Saccharomyces and non-Saccharomyces strains towards optimal enological characteristics for the chosen area of isolation. This field has been explored but there is a lack of a compendium covering the main methods to use. Autochthonous wine yeasts from different areas of Slovakia were identified and tested, in the form of colonies grown either on nutrient agar plates or in grape must micro-fermentations, for technological and qualitative enological characteristics. Based on the combined results, Saccharomyces cerevisiae PDA W 10, Lachancea thermotolerans 5-1-1 and Metschnikowia pulcherrima 125/14 were selected as potential wine starters. This paper, as a mixture of experimental and review contributions, provides a compendium of methods used to select autochthonous wine yeasts. Thanks to the presence of images, this compendium could guide other researchers in screening their own yeast strains for wine production.

Essential oils (EOs) of Thymus capitatus (Th) carvacrol chemotype and Origanum vulgare (Or) thymol and carvacrol chemotype were encapsulated in biocompatible poly(e-caprolactone) nanocapsules (NCs). These nanosystems exhibited antibacterial, antifungal, and antibiofilm activities against Staphylococcus aureus, Escherichia coli, and Candida albicans. Th-NCs and Or-NCs were more effective against all tested strains than pure EOs and at the same time were not cytotoxic on HaCaT (T0020001) human keratinocyte cell line. The genotoxic effects of EO-NCs and EOs on HaCaT were evaluated using an alkaline comet assay for the first time, revealing that Th-NCs and Or-NCs did not induce DNA damage compared with untreated control HaCaT cells in vitro after 24 h. The cells morphological changes were assessed by label-free live cell Raman imaging. This study demonstrate the ability of poly(e-caprolactone) nanocapsules loaded with thyme and oregano EOs to reduce microbial and biofilm growth and could be an ecological alternative in the development of new antimicrobial strategies.

The catabolism of milk protein in cheese is one way how the microorganisms influence the sensorial characteristics of the final product. In this investigation, we paid attention to four genes [prtP (cell-envelope proteinase gene), pepX (X-prolyl dipeptidyl aminopeptidase gene), pepN (aminopeptidase gene) and bcaT (branched chain aminotransferase gene)] responsible for the production of volatile aroma-active compounds from milk proteins by lactic acid bacteria (LAB). We studied the dynamics of these genes and their corresponding LAB host, during the maturation of a raw ewes' milk-based cheese, using metagenomics and metatranscriptomics approaches. The transcriptome-oriented experiments included the analysis of total RNA (at three stages of cheese maturation) and also the construction of specific cDNA sub-libraries of the abovementioned genes. The proteolytic transcriptome analysis was supported by following the transcription activity of 16S rRNA gene and by metagenomic investigation. The combination of the described methods permitted to screen the dynamics of targeted genes throughout the cheese production. Lactococci were the major players in the LAB group, but the analysis provided also information on the role and properties of members of the genus Lactobacillus, such as Lb. rhamnosus, Lb. helveticus, Lb. pentosus, Lb. curvatus, Lb. parabuchneri, Lb. plantarum, Lb. brevis, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. heilongjiangensis, proteolysis-related genes of which were active during cheese ripening.

Natural or anthropogenic disturbances in the forest ecosystem alter ecological conditions and lead to shifts in microbial diversity. We focused on the topsoil properties of specific sites in Tatra National Park (Slovakia) that were affected by windthrow, wildfire and wood extraction. We analyzed soil organic matter content (SOM), dry weight (DW), enzymatic activity including dehydrogenase activity (DHA), fluorescein diacetate hydrolysis (FDH) and phosphomonoesterase (PME). Bacterial community profiles were analyzed using the PCR-DGGE approach, and Ellenberg’s indicator values (EIV) were used as eco-indices of studied sites. Dystric Cambisol was sampled at sites distinguished by management approaches established after windthrow in 2004 and wildfire in 2005. We focused on the windthrow site where wooden debris was extracted (EXT); the site left for self-recovery with no intervention (NEX); the site affected by wildfire (FIR), a former reference site damaged by the windstorm in 2014 followed by the extraction of wooden debris (REX), and a non-affected reference spruce stand (REF). The windthrow sites with different management (EXT, NEX) showed relative similarities based on enzymatic activity, bacterial community profiling (85%) and EIV comparison. The FIR site exhibited no similarities (0%) with EXT and NEX in the bacterial community structure. The SOM content correlated only with PME (r2 = 0.42) and soil moisture (r2 = 0.64). Based on RDA analysis, certain EIVs seem to be suitable indicators of selected soil properties. The lack of differences between NEX and EXT could be caused by the diminishing of post-disturbance effects on microbial communities. The effect of wildfire on microbial activity and bacterial community structure seems to be longer than different management approach.

The potential of K2FeO4 as a pretreatment agent of a lignocellulosic material was examined on leaves of Acer platanodides as the sole substrate for biogas production by anaerobic digestion carried out through modelling laboratory-scaled semi-continuous reactors differing in loading rates and substrate (pretreated and untreated leaves). The quality of bioagas produced by K2FeO4-pretreated leaves was significantly better in terms of higher methane content and lower content of H2S. K2FeO4 had no crucial influence on growth inhibition of biogas-producing bacteria, which were analysed by comprehensive culture-independent methods utilising high-throughput sequencing of specific genes [bacterial and archaeal 16S rRNA, formyltetrahydrofolate synthetase gene (fhs), methyl-coenzyme M reductase α subunit gene (mcrA) and fungal internal transcribed spacers (ITS)]. The higher amount of CH4 in biogas utilising pretreated leaves as substrate could be caused by a shift to acetoclastic methanogenesis pathway, which was indicated by the higher amount of homoacetogenic bacteria and acetotrophic methanogens detected in those reactors.

Nanotechnology is a new frontier of this century that finds applications in various fields of science with important effects on our life and on the environment. Nanoencapsulation of bioactive compounds is a promising topic of nanotechnology. The excessive use of synthetic compounds with antifungal activity has led to the selection of resistant fungal species. In this context, the use of plant essential oils (EOs) with antifungal activity encapsulated in ecofriendly nanosystems could be a new and winning strategy to overcome the problem. We prepared nanoencapsules containing the essential oils of Origanum vulgare (OV) and Thymus capitatus (TC) by the nanoprecipitation method. The colloidal suspensions were characterized for size, polydispersity index (PDI), zeta potential, efficiency of encapsulation (EE) and loading capacity (LC). Finally, the essential oil nanosuspensions were assayed against a panel of fourteen fungal strains belonging to the Ascomycota and Basidiomycota phyla. Our results show that the nanosystems containing thyme and oregano essential oils were active against various fungal strains from natural environments and materials. In particular, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were two to four times lower than the pure essential oils. The aqueous, ecofriendly essential oil nanosuspensions with broad-spectrum antifungal activity could be a valid alternative to synthetic products, finding interesting applications in the agri-food and environmental fields.

A historical crypt offers us a particular view of the conditions of some buried materials (in this case textiles) and the various biogenic phenomena to which they were subjected over the centuries. In addition, significant knowledge can come by studying the DNA of buried objects which allows the recognition of materials, but also to reveal some practice of the funeral ceremony. In this study, the deteriorating microbial communities colonizing various funeral textile items were identified and characterized using microscopic observation, cultivation, polymerase chain reaction (PCR) and sequencing, hydrolytic tests, and culture-independent analysis (high-throughput sequencing, MinION platform). Different PCR assays and consequent sequencing of amplicons were employed to recognize the animal origin of bodice reinforcements and the type of plant used to embellish the young girl. The analysis of ancient DNA (aDNA from animal and plant) was also completed by the application of high-throughput sequencing through Illumina platform. The combination of all these techniques permitted the identification of a complex microbiota composed by dangerous degradative microorganisms able to hydrolyze various organic substrates such as fibroin, keratin, and cellulose. Bacteria responsible for metal corrosion and bio-mineralization, and entomopathogenic and phytopathogenic fungi. The analysis of aDNA identified the animal component used in bodice manufacturing, the plant utilized as ornament and probably the season of this fatal event.

Thymol is a substance with a great therapeutic potential possessing antibacterial and antifungal activity, with a characteristic odour that remains long after application but is not pleasant at higher concentrations. In this study, attention has been focused on describing the chemical and biological properties of the simply prepared trimethylsilyl ether of thymol (kubicin). Interestingly, kubicin has similar volatility as thymol, undergoes hydrolysis in the water (moisture; forming thymol and trimethylsilanol) and can be used at 6,000 times higher concentration than thymol without any negative and irritating odour. Kubicin showed diverse fungistatic and fungicidal activities when tested by direct contact assay, or in vapour phase. The volatile vapour of kubicin was effective on all tested fungal strains. These results suggest that vapours of kubicin might provide an alternative way to fight against fungal contamination.

Essential oils (EOs) are well-known for their beneficial properties against a broad range of microorganisms. For the better understanding of their mechanism of action in fungi, a microarray approach was used in order to evaluate the gene expression of Penicillium chrysogenum (recently renamed P. rubens) exposed to the indirect contact (vapors) of eight EOs. The selection of assayed EOs was based on their antifungal activity. The extraction of RNA and the microarray hybridization procedure were optimized for the analysis of P. rubens. Gene ontology annotation was performed to investigate the functional analysis of the genes. To uncover the metabolic pathway of these differentially expressed genes, they were mapped into the KEGG BRITE pathway database. The transcriptomic analysis showed that, from a total of 12,675 genes, only 551 genes are annotated, and the other 12,124 genes encoded hypothetical proteins. Further bioinformatic analysis demonstrated that 1350 genes were upregulated and 765 downregulated at least with half (four) of the utilizing EOs. A microarray investigation has confirmed the main impact of EOs to metabolic processes in P. rubens involved in vital functions. Presumably, this is the first time that a microarray hybridization analysis was performed in order to evaluate the gene expression of P. rubens exposed to various EOs.

Wine production is a complex procedure in which an important role is played by many microorganisms, particularly yeasts and bacteria. In modern wineries, alcoholic fermentation is usually carried out by adding microbial starter cultures of Saccharomyces cerevisiae strains for precisely controlled production. Nowadays, in the Slovak Republic, autochthonous vinification is getting more popular. The present article deals with the comparison of two vinification approaches, namely spontaneous fermentation and fermentation controlled by a standard commercial S. cerevisiae starter, from the point of view of microbiota dynamics and the chemical characteristics of the wines produced. The dynamics of microbial populations were determined during the fermentation process by a 16S and 28S rRNA next-generation sequencing approach. A profile of the volatile compounds during these fermentation processes was identified by solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In summary, the microbial diversity in the m1 phase (initial must) was higher, despite the presence of the starter culture. In the m3 phase (young wine), the microbiome profiles of both batches were very similar. It seems that the crucial phase in order to study the relationship of the microbiome and the resulting product should be based on the m2 phase (fermented must), where the differences between the autochthonous and inoculated batches were more evident.

In this study, we determined the antimicrobial activity of ten essential oils (EOs)&mdash, oregano, thyme, clove, arborvitae, cassia, lemongrass, melaleuca, eucalyptus, lavender, and clary sage&mdash, against drug-resistant microorganisms previously isolated from patients with skin infections. The essential oil compositions were determined using gas chromatography coupled to mass spectrometry (GC/MS). The assayed bacteria included Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter koseri, and Klebsiella pneumoniae. Two drug-resistant yeasts (Candida albicans and Candida parapsilosis) were also involved in our survey. Oregano, thyme, cassia, lemongrass and arborvitae showed very strong antibacterial and antifungal activity against all tested strains. These results show that these essential oils may be effective in preventing the growth of the drug-resistant microorganisms responsible for wound infections. In this study, the genotoxic effects of tested essential oils on healthy human keratinocytes HaCaT were evaluated using the comet assay for the first time. These results revealed that none of the essential oils induced significant DNA damage in vitro after 24 h. Moreover, the treatment of HaCaT cells with essential oils increased the total antioxidant status (TAS) level. The obtained results indicate that EOs could be used as a potential source of safe and potent natural antimicrobial and antioxidant agents in the pharmaceutical and food industries.

This microbiological survey was performed to determine the conservation state of a mummy in the Slovak castle of Krasna Horka and its surrounding environment. Culture-dependent identification was coupled with biodegradation assays on keratin, gelatin and cellulose. Next Generation Sequencing (NGS) using Illumina platform was used for a deeper microbial investigation. Three environmental samples were collected: from the glass of the sarcophagus, from the air inside it, and from the air of the chapel where the mummy is located. Seven different samples were taken from mummy's surface: from the left ear, left-hand palm, left-hand nail, left instep, right hand, abdomen and mineral crystals embedded within the skin. Three internal organ samples, from the lung, pleura and stomach, were also included in this study. Together, the culture-dependent and culture-independent analyses revealed that the bacterial communities present had fewer taxa than the fungal ones. The mycobiome showed the largest variability and included Epicoccum nigrum, Penicillium spp., Alternaria spp., Aspergillus spp., Cladosporium spp. and Aureobasidium pullulans; many other Ascomycota and Basidiomycota genera were detected by NGS. The most interesting results came from the skin mineral crystals and the internal organs. The hydrolytic assays revealed those microorganisms which might be considered dangerous 'mummy pathogens'. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Multi-drug resistant uropathogens are responsible for urinary tract infections. The antibacterial activity of seven essential oils, oregano, thyme, clove, arborvitae, cassia, lemongrass, tea tree) was investigated by agar diffusion method, followed by determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations against five multidrug resistant isolates namely Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloaceae, Morganella morganii, Proteus mirabilis. Oregano, thyme, cassia had antibacterial activity with inhibition zones ranging 25–39 mm; clove, arborvitae, tea tree and lemongrass 12–15 mm. The essential oils showed antibacterial activities with MICs ranged from 0.005% (w/v) to 0.5% (w/v). Thyme had the same MIC and MBC on all strains. The effects of the vapors of the essential oils were also tested by placing the oils on the underside of the Petri dish lid. Thyme, oregano and cassia essential oils strongly inhibited the growth of the clinical strains of bacteria tested in vapor phase. This study demonstrates the potential of investigated essential oils as natural alternatives for further application in hospital therapies in order to retard or inhibit the bacterial growth. For the first time antibacterial effects of essential oils (clove, arborvitae, tea tree, lemongrass, and cassia) were evaluated against Enterobacter cloaceae and Morganella morganii clinical isolates.

Different identification strategies employing new methodologies, namely, culture-dependent (using matrix-assisted laser desorption/ionization – time-of-flight mass spectrometry, MALDI-TOF MS) and culture-independent (using high-throughput sequencing on Illumina MiSeq platform) approaches, were applied for the first time to analysis of the deteriorating microflora of book samples. We compared two different identification techniques coupled to microbial cultivation: DNA sequencing and MALDI-TOF MS. The two identification systems produced almost the same results. DNA sequencing was able to better identify all the recovered isolates, while MALDI-TOF MS failed to properly recognize Streptomyces ambofaciens and Lysinibacillus fusiformis, the latter method permitting identification at least at genus level. In addition, the isolates Myxotrichum deflexum and Oidiodendron cerealis were not identified by the MALDI-TOF MS approach. This incongruence was caused by the absence of different kinds of microorganisms in the MALDI BioTyper reference database. The Illumina high-throughput sequencing coupled to non-invasive sampling allowed the identification of a complex bacterial and fungal community. The study presents the satisfactory performance of two novel alternative identification methods (MALDI-TOF MS and Illumina MiSeq) for the investigation of microflora colonizing archival items.

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.

Culture-dependent and culture-independent strategies were applied to investigate the microbiota of autumn undamaged and damaged berries, winter berries and ice wine must samples of Grüner Veltliner (Veltlínske zelené) from Small Carpathian wine-producing region. One hundred twenty-six yeasts and 242 bacterial strains isolated from several microbiological media (YPD, PDA, R2A, GYC, MRS and MRS-T) were clustered by ITS-PCR and subsequent Qiaxcel electrophoresis. Representatives of each cluster were identified by sequencing. The extracellular hydrolytic properties and intracellular activities of esterase and β-glucosidase of isolates were assayed. The culture-independent approach permitted the analysis of extracted DNA and RNA coupling DGGE fingerprinting with construction of clone libraries (bacterial and fungal; DGGE-cloning). The combination of the two approaches provided comprehensive data that evidenced the presence of a complex microbiota in each analyzed sample. RNA and DNA analyses facilitated differentiation of living microorganisms from the entire microbiota. Diverse microbial communities colonized the autumn and winter berries. Generally, the combination of results obtained by the methods suggested that the must samples contained mainly Saccharomyces cerevisiae, Metschnikowia spp., Hanseniaspora uvarum, Lactococcus lactis and Leuconostoc spp. The strains exhibited interesting esterase and β-glucosidase properties, which are important for aroma formation in wine. Fermentation strategies utilising these microorganisms, could be attempted in the future in order to modulate the ice wine characteristics.

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.

Wooden pipe organs are precious objects of cultural heritage the degradation of which has been very poorly studied. Fungal strains recovered from an organ dating back to the 19th century included Alternaria mali , Eurotium cristatum , Aspergillus amstelodami , Penicillium crustosum and Aspergillus sydowii . These isolates were used to artificially contaminate wooden pipes treated with linseed oil varnish and varnish-free. The pipes were incubated at different temperatures and relative humidity for one year. The pipes, incubated at a high relative humidity (70–75%), collapsed due to the biodegradation of glued joints. After one year, inoculated strains ( Alternaria mali , Penicillium crustosum and Aspergillus sydowii ) as well as some non-inoculated Talaromyces rugulosus, Paecilomyces formosus , Cladosporium cladosporioides , Aspergillus versicolor and Epicoccum nigrum were recovered from the pipes. Few strains exhibited lignolytic activity, while almost all were able to hydrolyze the rabbit glue used to join the wood elements of the pipes. A newly developed portable pipe-sound controller did not detect differences of sound spectra between the uncontaminated and the few surviving contaminated pipes. In this study we have demonstrated that glue degradation was critical for pipe preservation and it can be tremendously accelerated above a certain relative humidity threshold. Therefore, it is important to apply a monitoring strategy able to check the environmental conditions, microbial contamination and pipe sound alteration in order to preserve these valuable and rare cultural heritage objects.

In this study the biodegradation characteristics of aromatic aliphatic copolyester Ecoflex was assayed with five different bacterial strains isolated from different kinds of environments and living at diverse optimum of temperature. The biodegradation properties of selected strains (Brevibacterium luteolum, Bacillus thuringiensis, Aeromonas media, Geobacillus kaustophilus and Serratia sp.) were assessed through several plate assays oriented to their proteolytic, lipolytic, esterolytic and cellulolytic abilities; catalase activities were measured too. In addition the bacterial hydrolytic abilities for polyhydroxybutyrate, polylactic acid and polybutyrate adipate terephthalate were obtained using specific culture media including also a specific Ecoflex agar. The biodegradation dynamics of selected strains were evaluated mainly by the incubation of Ecoflex foils with a specific bacterium during a period from 3 days until 16 weeks and the change of the degradability of the Ecoflex surface in nitrogen has been assessed by nonisothermal chemiluminescence measurements from 40 to 250 °C. Scanning electron microscope observation of inoculated foils was applied too. The comparison of the results showed the complexity of biodegradation phenomena. The concentration of oxygenated products measured by reached the maximum after 2 weeks of cultivation for strains G. kaustophilus and B. thurigiensis, after 4 weeks of cultivation for strain B. luteolum, and 8 weeks of cultivation for strains Serratia sp. and A. media. Each strain evidenced its own hydrolytic optimum at different periods, but almost all tested strains demonstrated a decline of hydrolysis up to 16 weeks of incubation. This study demonstrated that nonisothermal chemiluminescence examining momentaneous oxidation state of the polymer surface is a valuable technique to follow the biodegradation dynamics on polymeric film, while the Ecoflex agar is able to select useful polymer-degrading bacteria.

The research aim was to determine the effectiveness of disinfection methods: thyme essential oil microatmosphere (TEO), silver nanoparticles misting (AgNPs), and low temperature plasma (LTP) by culture-dependent method and RNA analysis. In addition, we examined the influence of disinfection on mechanical and optical properties of paper from historical books with different levels of microbial contamination. All disinfection methods were generally bacteriostatic and fungistatic. The AgNPs misting method was more effective for bacterial inhibition ( R = 60–100%), than LTP ( R = 25–100%) and TEO ( R = 12–100%); all tested methods were less effective for fungi ( R = 0–99.8%). TEO exhibits a broader spectrum of fungicidal activity compared to AgNPs and LTP. The antimicrobial effectiveness depended on the area from where the microorganisms were isolated and their sensitivity to the active agent. A higher disinfection effectiveness was observed for books with higher levels of microbial contamination. RNA concentration was a good marker of antimicrobial activity of disinfection. RNA amount decreased by 95–100% after disinfection with LTP and TEO, and by 29–89% after AgNPs disinfection. Disinfection of paper from historical books with LTP, AgNPs and TEO did not significantly influence or have a positive effect on the mechanical and optical properties of paper from tested historical books. We show that LTP, TEO, and AgNPs can be used as microbiostatics, alternatively to the currently available methods.

Microbial diversity and volatile odour-active compounds were studied in barrelled ewes' cheese, which is a long-ripened intermediate product in the production of winter bryndza cheese, a type of PGI Slovakian bryndza cheese produced from unpasteurized ewes' milk. Microbial consortia were studied by both culture-based and culture-independent approaches. Microbiological analysis demonstrated that lactococci, lactobacilli and Geotrichum spp. were dominant, while levels of coliforms and coagulase-positive staphylococci were acceptably low. Culture-independent analysis facilitated identification of 26 prokaryotic taxa, with the majority of clones being Lactococcus lactis subsp. lactis and Streptococcus thermophilus, and 23 eukaryotic taxa, with the majority of clones being members of Galactomyces/Geotrichum group. Profiles of odour-active compounds were studied using gas chromatography – olfactometry supported by gas chromatography – mass spectrometry. Thirty-nine odour-active compounds, or mixtures in case of co-elution, were identified. Butanoic acid, ethyl butanoate, isovaleric acid, hexanoic acid, octanoic acid, decanoic acid, methyl octanoate, ethyl hexanoate, ethyl octanoate, p-cresol, and δ-decalactone were detected at high or moderate intensities in all samples. The data obtained demonstrate that barrelled ewes' cheese is a product with specific microflora and a very specific rich profile of odour-active volatile compounds.

The microbial communities responsible for the degradation of poly(lactic acid)/poly(3-hydroxybutyrate) (PLA/PHB) blend foils were investigated in 1 year long laboratory soil burial experiments. Different PLA/PHB foils were tested: (a) PLA/PHB original transparent foil, (b) PLA/PHB carbon black filled foil and (c) PLA/PHB black foil previously exposed for 90 days to sun light. The microbiome diversity of these three types of foil was compared with that identified from soil/perlite sample at the beginning of experiment and that developed on a cellulose mat. Culture-dependent and culture-independent (DGGE-cloning) approaches together with PLA, PHB and PLA/PHB degradation plate assays were employed. The cultivation strategy combined with degradation tests permitted the isolation and evaluation of several PLA/PHB blend degrading microorganisms such as members of the genera Bacillus, Paenibacillus, Streptomyces, Rhodococcus, Saccharothrix, Arthrobacter, Aureobasidium, Mortierella, Absidia, Actinomucor, Bjerkandera, Fusarium, Trichoderma and Penicillium. The DGGE-cloning investigation increased the information about the microbial communities occurring during bioplastic degradation detecting several bacterial and fungal taxa and some of them (members of the orders Anaerolineales, Selenomonadales, Thelephorales and of the genera Pseudogymnoascus and Pseudeurotium) were revealed here for the first time. This survey showed the microbiome colonizing PLA/PHB blend foils and permitted the isolation of several microorganisms able to degrade the tested polymeric blends.

This microbiological survey was performed to determine the conservation state of a mummy in the Slovak castle of Krásna Hôrka and its surrounding environment. Culture-dependent identification was coupled with biodegradation assays on keratin, gelatin and cellulose. Next Generation Sequencing (NGS) using Illumina platform was used for a deeper microbial investigation. Three environmental samples were collected: from the glass of the sarcophagus, from the air inside it, and from the air of the chapel where the mummy is located. Seven different samples were taken from mummy's surface: from the left ear, left-hand palm, left-hand nail, left instep, right hand, abdomen and mineral crystals embedded within the skin. Three internal organ samples, from the lung, pleura and stomach, were also included in this study. Together, the culture-dependent and culture-independent analyses revealed that the bacterial communities present had fewer taxa than the fungal ones. The mycobiome showed the largest variability and included Epicoccum nigrum, Penicillium spp., Alternaria spp., Aspergillus spp., Cladosporium spp. and Aureobasidium pullulans; many other Ascomycota and Basidiomycota genera were detected by NGS. The most interesting results came from the skin mineral crystals and the internal organs. The hydrolytic assays revealed those microorganisms which might be considered dangerous 'mummy pathogens'. © 2018 Society for Applied Microbiology and John WileySons Ltd.

The microbial spoilage and correlated surface changes of a cellulose nitrate negative film, a gelatine–silver positive print, a cardboard frame and a cellulosic envelope dated back to 1938–1940 was assessed by means of molecular methods and scanning electron microscopy (SEM). Materials characterisation was obtained with Raman, Infrared and Electronic dispersion spectroscopies. DNA was extracted from bacteria and fungi, amplified through PCR oriented to bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS), and clone libraries were constructed for each investigated material. The ITS fungal cloning was able to detect a bigger spectrum of species respect to bacterial one. Correspondence between molecular results and SEM observations was used to address the cause of biodeterioration to single species, and to map the presence of different organisms in separate niches. This investigation highlighted a co-occurrence of both bacteria and fungi on most of the substrata, and a spatial partitioning according to the different photographic materials. Moreover, for the first time, the effects of a biological attack on glassine paper were documented.

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.

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.

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.

The microbial diversity of different kinds of stains present on the surface of 14 historical documents (nine parchments and five paper letters) was evaluated through a combination of cultural and molecular methods. The samples were recovered using adhesive tape and swabs and were afterwards treated in two different ways: (1) direct inoculation on agar plates; or (2) suspension in physiological solution and plating in specific plates for the growth of bacteria and fungi. The isolated microorganisms, before identification, were selected by two different PCR-based methods – f-ITS and f-CBH, for bacteria and fungi, respectively. The f-ITS method is based on the amplification of the internal transcribed sequence between the bacterial 16S and 23S rDNA. The f-CBH method is a new molecular selection tool oriented to the fungal cellobiohydrolase gene. Both PCR selection methods produced typical profiles, which clustered the isolates in order to reduce them for subsequent sequencing identification through the amplification of the fungal 28S rRNA and the bacterial 16S rRNA genes. The cellulolytic and proteolytic abilities were screened through the use of three plate assays, the Ostazin Brilliant Red H-3B (OBR-HEC), milk agar, and gelatin agar. Massilia timonae , Lysobacter dokdonensis , and strains belonging to the genus Bacillus sp., Microbacterium sp., and Curtobacterium sp. with different fungal members such as Aspergillus fumigatus , Penicillium commune , Mucor spinosus , and Phoma herbarum (all recovered from paper) displayed both biodegradative activities. The parchment isolates with a marked proteolytic activity were Bacillus cereus , Staphylococcus epidermidis , Pseudomonas oryzihabitans , Virgibacillus sp., Micromonospora sp., and again members of the fungal genera Penicillium , Mucor , and Phoma .

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. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f-CBH-PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1-5.8S rDNA-ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f-CBH-PCR permitted the discrimination of fungal species, producing typical f-CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f-CBH-PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments.

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.

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.

This is the first study focused to bacterial diversity and dynamic during the vinification of two important Central Europe grape vines: Blaufrankisch and Gruner 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.

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.

Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a traditional Slovak cheese produced from unpasteurized ewes' milk. Quantitative culture-based data were obtained for lactobacilli, lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylococci, coagulase-positive staphylococci, yeasts, fungi and Geotrichum spp. in ewes' milk, curd produced from it and ripened for 0 - 10 days, and in bryndza cheese produced from the curd, in three consecutive batches. Diversity of prokaryotes and eukaryotes in selected stages of the production was studied by non-culture approach based on amplification of 16S rDNA and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and sequencing. The culture-based data demonstrated an overall trend of growth of the microbial population contributing to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing up to densities of 10(8) CFU/g, 10(9) CFU/g and 10(5) CFU/g, respectively, in all three consecutive batches of bryndza cheese. The diversity of bacteria encompassed Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter sp., Acinetobacter johnsonii, Citrobacter braakii, Clostridium bartlettii, Corynebacterium callunae, Corynebacterium maris, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter hormaechei, Enterococcus faecium, Enterococcus pallens, Escherichia coli, Haemophilus haemolyticus, Hafnia alvei, Kluyvera cryocrescens, Lactobacillus helveticus, Lactococcus garvieae, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, "Leuconostoc garlicum", Mannheimia glucosida, Mannheimia haemolytica, Pseudomonas sp., Ps. fluorescens, "Ps. reactans", Raoultella ornithinolytica, R. terrigena, "Rothia arfidiae", Staphylococcus aureus, Staph. epidermidis, Staph. felis, Staph. pasteuri, Staph. sciuri, Staph. xylosus, Streptococcus parauberis, Str. thermophilus and Variovorax paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, "Ascomycete sp.", Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica.

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 Pazmany, 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.