aroF and cm2: potential molecular markers for the detection of stone-inhabiting Actinobacteria on cultural heritage sites.

Tangible archeological sites and stone monuments are naturally decayed and deteriorated over time, providing substances that can sustain life, although they provide a complicated ecosystem characterized by low nutrition and desiccation. Stone-inhabiting bacteria (SIB) and especially members of the phylum Actinobacteria dominate such environments, particularly the members of the family Geodermatophilaceae. We used the published data of two confirmed SIB species to mine their genomes for specific molecular markers to rapidly survey the presence of SIB in cultural heritage material prior to further analysis. The search focused on the mycosporine-like amino acids (MAAs) synthesis pathway. MAAs are intracellular compounds biosynthesized by the shikimic acid pathway to synthesize aromatic amino acids and were found related to abiotic resistance features in microorganisms. Based on genome mining, the DAHP II (aroF) and a homolog of the Chorismate mutase gene (cm2) were found mostly in Actinobacteria and few other species. After calibration on five stone-inhabiting Actinobacteria (SIAb) species using conventional PCR, newly designed primers were successfully applied to environmental DNA extracted from two Egyptian pyramidal sites using a qPCR approach. This is the first report of aroF and cm2 as qPCR markers to detect SIAb from cultural heritage material prior to proceeding with further analysis (e.g., metagenomics and meta-barcoding analyses).

Microbiome and nitrate removal processes by microorganisms on the ancient Preah Vihear temple of Cambodia revealed by metagenomics and N-15 isotope analyses.

Preah Vihear temple is one of the most significant representatives of the ancient Angkorian temples listed as United Nations Educational, Scientific and Cultural Organization (UNESCO) World Heritage Sites. The surfaces of this Angkor sandstone monument are covered with deteriorated materials, broadly called "sediments" here, resulting from a long time of weathering of the sandstone. The sediments might adversely affect the ancient sandstone substratum of this cultural heritage, and the potential risk from them is essential information for current strategies and on-going protection and management. The extracted DNA from the sediment samples of this temple was used for Illumina high-throughput sequencing analysis, resulting in approximately 12 Gb of metagenomic dataset. The results of this shotgun metagenomic analysis provided a thorough information of the phylogenetic groups presented in the microbiome of the sediment samples, indicating that potential metabolic activities, involving different geomicrobiological cycles, may occur in this microbiome. The phylogenetic result revealed that the majority of metagenomic reads were affiliated with Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes at the phylum level. The metabolic reconstruction results indicated that the important geomicrobiological cycling processes include carbon sequestration, and nitrogen and sulfur transformation as the potentially active ones in the sediments of the sampling sites. Specifically, the dissimilatory nitrate reduction to ammonium (DNRA) and the newly discovered complete ammonia oxidation (comammox) were retrieved from this metagenomic dataset. Furthermore, the genetic information on the presence of acid-producing processes by sulfur- and ammonia-oxidizing bacteria and archaea in this metagenomic dataset suggested that the microbial flora in these samples has the potential to participate in the degradation of sandstone cultural heritage by producing acids. N-15 isotope amendment and incubation analysis results confirmed the presence of active denitrification, but not anammox activity at this temple. These results are important for our knowledge on the microbial community composition and microbial biodeterioration processes affecting this sandstone cultural heritage and will aid in the protection and management of the ancient Preah Vihear temple.Key Points• Microbiota on Preah Viher temple was analyzed using NGS.• Nitrate-N transformation by DNRA, comammox, and denitrifcation was detected.• N-15 isotope analysis confirmed the active denitrifcation, but not Anammox.• Accumulation of nitrate is a result of less active removal by denitrification.

Correction to: Microbiome and nitrate removal processes by microorganisms on the ancient Preah Vihear temple of Cambodia revealed by metagenomics and N-15 isotope analyses.

The published online version contains mistake for the author the authors correction to change the name from Clara Uriz to Clara Urzì was missed.

Modestobacter italicus sp. nov., isolated from Carrara marble quarry and emended descriptions of the genus Modestobacter and the species Modestobacter marinus, Modestobacter multiseptatus, Modestobacter roseus and Modestobacter versicolor.

A Gram-reaction-positive, aerobic bacterial strain showing coccoid cells and designated as BC 501T was isolated from a black patina of the surface of a Carrara marble blockin the Gioia quarry in Tuscany, Italy. A polyphasic study was carried out to clarify the taxonomic status of BC 501T within the evolutionary radiation of the genus Modestobacter. Phenotypic and genotypic characteristics as well as phylogenetic distinctiveness confirmed that it represents a novel species of the genus Modestobacter, for which the name Modestobacteritalicus sp. nov. is proposed. The type strain is BC 501T (=DSM 44449T=CECT 9708T). Emended descriptions of the genus Modestobacter and the species Modestobacter marinus, Modestobacter multiseptatus, Modestobacter roseus and Modestobacter versicolor are also proposed.

Occurrence of the black yeast Hortaea werneckii in the Mediterranean Sea.

The occurrence of cultivable fungi was investigated along the water column (25-2500 m depth) of four off-shore stations in the Mediterranean basin. An unexpected high abundance of fungi, accompanied by a scarce biodiversity, was observed up to 2500 m depth. The black yeast Hortaea werneckii, known to be one of the most salt tolerant eukaryotic organisms, was isolated for the first time from the Mediterranean Sea, and it was the dominant fungus present in seawater in almost all stations and depths, suggesting its ubiquitous distribution. Isolation of cultivable strains allowed their phylogenetic and taxonomic characterization, and demonstrated that almost all the retrieved fungal species should be considered of terrestrial origin, but well adapted to survive and reproduce at temperature and salinity conditions of the Mediterranean seawater.

Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies.

Roman Catacombs are affected by different kinds of biofilms that were extensively investigated in the last 14 years. In particular, the areas far from the lamps are often covered by white biofilms of different extension, consistency and nature. The aim of this paper is to describe the profile of the microbial community present in two areas of the Ocean's Cubiculum (CSC13), characterized by similar alterations described as white biofilms, by using a multistep approach that included direct microscopy observations, culture-dependent and culture-independent methodologies through the extraction of DNA and RNA directly from the sampled areas. In addition to this, we extracted the DNA directly from the Petri dishes containing R2A and B4 media after incubation and growth of bacteria. Our results evidenced that a complex bacterial community (mainly constituted by filamentous Actinobacteria, as well as Firmicutes and Proteobacteria) colonizes the two different white biofilms, and its detection, quantitative and qualitative, could be revealed only by different approaches, each method giving different information that only partially overlap.

Description of Kribbella italica sp. nov., isolated from a Roman catacomb.

A novel actinobacterium, strain BC637(T), was isolated from a biodeteriogenic biofilm sample collected in 2009 in the Saint Callixstus Roman catacomb. The strain was found to belong to the genus Kribbella by analysis of the 16S rRNA gene. Phylogenetic analysis using the 16S rRNA gene and the gyrB, rpoB, relA, recA and atpD concatenated gene sequences showed that strain BC637(T) was most closely related to the type strains of Kribbella lupini and Kribbella endophytica. DNA-DNA hybridization experiments confirmed that strain BC637(T) is a genomic species that is distinct from its closest phylogenetic relatives, K. endophytica DSM 23718(T) (63 % DNA relatedness) and K. lupini LU14(T) (63 % DNA relatedness). Physiological comparisons showed that strain BC637(T) is phenotypically distinct from the type strains of K. endophytica and K. lupini. Thus, strain BC637(T) represents the type strain of a novel species, for which the name Kribella italica sp. nov. is proposed ( = DSM 28967(T) = NRRL B-59155(T)).

Kribbella albertanoniae sp. nov., isolated from a Roman catacomb, and emended description of the genus Kribbella.

A novel actinobacterium, strain BC640(T), was isolated from a biofilm sample collected in 2009 in the Saint Callistus Roman catacombs. Analysis of the 16S rRNA gene sequence showed that the strain belonged to the genus Kribbella. Phylogenetic analysis using the 16S rRNA gene and concatenated gyrB, rpoB, relA, recA and atpD gene sequences showed that strain BC640(T) was most closely related to the type strains of Kribbella yunnanensis and Kribbella sandramycini. Based on gyrB genetic distance analysis, strain BC640(T) was shown to be distinct from all Kribbella type strains. DNA-DNA hybridization experiments confirmed that strain BC640(T) represents a genomic species distinct from its closest phylogenetic relatives, K. yunnanensis DSM 15499(T) (53.5±7.8 % DNA relatedness) and K. sandramycini DSM 15626(T) (33.5±5.0 %). Physiological comparisons further showed that strain BC640(T) is phenotypically distinct from the type strains of K. yunnanensis and K. sandramycini. Strain BC640(T) ( = DSM 26744(T) = NRRL B-24917(T)) is thus presented as the type strain of a novel species, for which the name Kribbella albertanoniae sp. nov. is proposed.

Culturable and unculturable potential heterotrophic microbiological threats to the oldest pyramids of the Memphis necropolis, Egypt.

A large percentage of the world's tangible cultural heritage is made from stone; thus, it deteriorates due to physical, chemical, and/or biological factors. The current study explored the microbial community inhabiting two prehistoric sites with high cultural value in the Memphis necropolis of Egypt (Djoser and Lahun Pyramids) using amplicon-based metabarcoding and culture-dependent isolation methods. Samples were examined by epifluorescent microscopy for biological signs before environmental DNA extraction and in vitro cultivation. The metabarcoding analysis identified 644 bacterial species (452 genera) using the 16S rRNA and 204 fungal species (146 genera) using ITS. In comparison with the isolation approach, an additional 28 bacterial species (13 genera) and 34 fungal species (20 genera) were identified. A total of 19 bacterial and 16 fungal species were exclusively culture-dependent, while 92 bacterial and 122 fungal species were culture-independent. The most abundant stone-inhabiting bacteria in the current study were Blastococcus aggregatus, Blastococcus saxobsidens, and Blastococcus sp., among others. The most abundant rock-inhabiting fungi were Knufia karalitana and Pseudotaeniolina globosa, besides abundant unknown Sporormiaceae species. Based on previous reports, microorganisms associated with biodeterioration were detected on color-altered sites at both pyramids. These microorganisms are potentially dangerous as physical and chemical deterioration factors and require proper conservation plans from a microbiological perspective.

A Deep Insight into the Diversity of Microfungal Communities in Arctic and Antarctic Lakes.

We assessed fungal diversity in water and sediment samples obtained from five Arctic lakes in Ny-Ålesund (Svalbard Islands, High Arctic) and five Antarctic lakes on Livingston and Deception Islands (South Shetland Islands), using DNA metabarcoding. A total of 1,639,074 fungal DNA reads were detected and assigned to 5980 ASVs amplicon sequence variants (ASVs), with only 102 (1.7%) that were shared between the two Polar regions. For Arctic lakes, unknown fungal taxa dominated the sequence assemblages, suggesting the dominance of possibly undescribed fungi. The phylum Chytridiomycota was the most represented in the majority of Arctic and Antarctic samples, followed by Rozellomycota, Ascomycota, Basidiomycota, and the less frequent Monoblepharomycota, Aphelidiomycota, Mortierellomycota, Mucoromycota, and Neocallimastigomycota. At the genus level, the most abundant genera included psychrotolerant and cosmopolitan cold-adapted fungi including Alternaria, Cladosporium, Cadophora, Ulvella (Ascomycota), Leucosporidium, Vishniacozyma (Basidiomycota), and Betamyces (Chytridiomycota). The assemblages displayed high diversity and richness. The assigned diversity was composed mainly of taxa recognized as saprophytic fungi, followed by pathogenic and symbiotic fungi.

Analysis and comparison of the microflora isolated from fresco surface and from surrounding air environment through molecular and biodegradative assays.

The aim of this study was to find a correlation among the environmental isolated microflora and the fresco colonizators through the investigation of their biodegradative abilities and DNA characteristics. A molecular technique named RAMP (Random Amplified Microsatellite Polymorphisms) was utilized in order to analyze the DNA diversity of bacterial and fungal species isolated from fresco as well as from air samples. The RAMP-PCR results were combined with the screening of some biodegradative properties obtained through the use of specific agar plate assays detecting the proteolytic, solubilization and biomineralization abilities of the isolated microflora. This comparative analysis showed that only in few cases a direct link among the fresco and airborne isolates of specific microbial group existed. The investigation clearly evidenced that colonization of surface of Ladislav's fresco occurred in different time and by different strains than those observed at the moment of sampling campaign. Furthermore, the microflora investigation permitted the identification of taxonomically interesting bacteria with particular biodegradative properties, which had been less studied until now.

Fungal outbreak in the Catacombs of SS. Marcellino and Pietro Rome (Italy): From diagnosis to an emergency treatment.

The present study reports a sudden fungal outbreak that occurred in the corridor near the entrance of the Catacombs of SS. Marcellino and Pietro in Rome (Italy) observed after 1 year of a restoration treatment that interested the walls of the entrance of the Catacombs and some artifacts placed in situ. The colonization was observed on the vault at the entrance and in correspondence with the restored marble pieces displayed on the left side of the corridor. No growth was observed on the right side where similarly treated marble slabs were placed. Samples taken in correspondence with fungal biofilm were analyzed through the combined use of microscopical, cultural, and molecular tools and showed that the vault and the left side of the corridor entrance were colonized by a complex fungal biofilm consisting mainly of Coniophora sp. and other genera, such as Hypomyces, Purpureocillium, Acremonium, Penicillium, and Alternaria, many of which are well known as responsible of biodeterioration of stone surfaces. Regarding the brown-rot basidiomycete Coniophora, it was able to form very large colonies on the substrata with a diameter of up to 57 cm. Although the direct observation under a light microscope evidenced the presence of abundant brown fungal conidia, several attempts to cultivate the microorganism failed, therefore only through DNA sequencing analyses, it was possible to identify and characterize this fungus. There is very little literature on the genus Coniophora which is reported as one of the causes of wet-rot decay of wood in buildings. A connection with calcium-containing materials such as bricks and mortars was demonstrated, but no data were available about the possible role of this species in the biodeterioration of stones. This study features the first finding of a strain related to the basidiomycetous genus of Coniophora in the order Boletales in association with evident phenomena of biodeterioration.

Development of Eco-Friendly Hydrophobic and Fouling-Release Coatings for Blue-Growth Environmental Applications: Synthesis, Mechanical Characterization and Biological Activity.

The need to ensure adequate antifouling protection of the hull in the naval sector led to the development of real painting cycles, which involve the spreading of three layers of polymeric material on the hull surface exposed to the marine environment, specifically defined as primer, tie coat and final topcoat. It is already well known that coatings based on suitable silanes provide an efficient and non-toxic approach for the hydrophobic and antifouling/fouling release treatment of surfaces. In the present work, functional hydrophobic hybrid silica-based coatings (topcoats) were developed by using sol-gel technology and deposited on surfaces with the "doctor blade" method. In particular, those organic silanes, featuring opportune functional groups such as long (either fluorinated) alkyl chains, have a notable influence on surface wettability as showed in this study. Furthermore, the hydrophobic behavior of this functionalized coating was improved by introducing an intermediate commercial tie-coat layer between the primer and the topcoat, in order to decrease the wettability (i.e., decreasing the surface energy with a matching increase in the contact angle, CA) and to therefore make such coatings ideal for the design and development of fouling release paints. The hereby synthesized coatings were characterized by optical microscopy, contact angle analysis and a mechanical pull-off test to measure the adhesive power of the coating against a metal substrate typically used in the nautical sector. Analysis to evaluate the bacterial adhesion and the formation of microbial biofilm were related in laboratory and simulation (microcosm) scales, and assessed by SEM analysis.

Design and Development of Fluorinated and Biocide-Free Sol-Gel Based Hybrid Functional Coatings for Anti-Biofouling/Foul-Release Activity.

Biofouling has destructive effects on shipping and leisure vessels, thus producing severe problems for marine and naval sectors due to corrosion with consequent elevated fuel consumption and higher maintenance costs. The development of anti-fouling or fouling release coatings creates deterrent surfaces that prevent the initial settlement of microorganisms. In this regard, new silica-based materials were prepared using two alkoxysilane cross-linkers containing epoxy and amine groups (i.e., 3-Glycidyloxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane, respectively), in combination with two functional fluoro-silane (i.e., 3,3,3-trifluoropropyl-trimethoxysilane and glycidyl-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorononylether) featuring well-known hydro repellent and anti-corrosion properties. As a matter of fact, the co-condensation of alkoxysilane featuring epoxide and amine ends, also mixed with two opportune long chain and short chain perfluorosilane precursors, allows getting stable amphiphilic, non-toxic, fouling release coatings. The sol-gel mixtures on coated glass slides were fully characterized by FT-IR spectroscopy, while the morphology was studied by scanning electron microscopy (SEM), and atomic force microscopy (AFM). The fouling release properties were evaluated through tests on treated glass slides in different microbial suspensions in seawater-based mediums and in seawater natural microcosms. The developed fluorinated coatings show suitable antimicrobial activities and low adhesive properties; no biocidal effects were observed for the microorganisms (bacteria).

A New Extremotolerant Ecotype of the Fungus Pseudotaeniolina globosa Isolated from Djoser Pyramid, Memphis Necropolis, Egypt.

Most of the rock-inhabiting fungi are meristematic and melanized microorganisms often associated with monument biodeterioration. In previous microbial profiling of the Egyptian Djoser pyramid, a Pseudotaeniolina globosa isolate was found. The current study aimed to characterize the P. globosa isolated from the Djoser pyramid compared with an Italian isolate at morphological, physiological, and molecular levels. Experiments were carried out to test temperature, salinity, and pH preferences, as well as stress tolerance to UV radiation and high temperature, in addition to a multi-locus genotyping using ITS, nrSSU or 18S, nrLSU or 28S, BT2, and RPB2 markers. Morphological and molecular data confirmed the con-specificity of the two isolates. However, the Egyptian isolate showed a wider range of growth at different environmental conditions being much more tolerant to a wider range of temperature (4-37 °C) and pH values (3.0-9.0 pH) than the Italian (10-30 °C, 4.0-6.0 pH), and more tolerant to extreme salinity levels (5 M NaCl), compared to the lowest in the Italian isolate (0.2 M NaCl). Besides, the Egyptian isolate was more tolerant to high temperature than the Italian isolate since it was able to survive after exposure to up to 85 °C for 5 min, and was not affected for up to 9 h of UV exposure, while the Italian one could not regrow after the same treatments. The Pseudotaeniolina globosa species was attributed to the family Teratosphaeriaceae of the order Capnodiales, class Dothideomycetes. Our results demonstrated that the Egyptian isolate could be considered an ecotype well adapted to harsh and extreme environments. Its potential bio-deteriorating effect on such an important cultural heritage requires special attention to design and conservation plans and solutions to limit its presence and extension in the studied pyramid and surrounding archaeological sites.

Microbial diversity in paleolithic caves: a study case on the phototrophic biofilms of the Cave of Bats (Zuheros, Spain).

The biological colonization of rocks in the Cave of Bats (Cueva de Los Murciélagos, Zuheros, Spain) was studied in order to reveal the diversity of microorganisms involved in the biofilm formation. The culturable, metabolically active fraction of biodeteriogens present on surfaces was investigated focusing on morphological, ultrastructural, and genetic features, and their presence related to the peculiar environmental conditions of the underground site. PCR-ITS analysis and 16S rDNA sequences were used to clusterize and characterize the isolated strains. The presence of bacterial taxa associated to the photosynthetic microflora and fungi within the biofilm contributed to clarify the relationships inside the microbial community and to explain the alteration observed at the different sites. These results will contribute to the application of more successful strategies for the preventive conservation of subterranean archaeological sites.

Whole Genome Sequencing and Comparative Genome Analysis of the Halotolerant Deep Sea Black Yeast Hortaea werneckii.

Hortaea werneckii, an extreme halotolerant black yeast in the order of Capnodiales, was recently isolated from different stations and depths in the Mediterranean Sea, where it was shown to be the dominant fungal species. In order to explore the genome characteristics of these Mediterranean isolates, we carried out a de-novo sequencing of the genome of one strain isolated at a depth of 3400 m (MC873) and a re-sequencing of one strain taken from a depth of 2500 m (MC848), whose genome was previously sequenced but was highly fragmented. A comparative phylogenomic analysis with other published H. werneckii genomes was also carried out to investigate the evolution of the strains from the deep sea in this environment. A high level of genome completeness was obtained for both genomes, for which genome duplication and an extensive level of heterozygosity (~4.6%) were observed, supporting the recent hypothesis that a genome duplication caused by intraspecific hybridization occurred in most H. werneckii strains. Phylogenetic analyses showed environmental and/or geographical specificity, suggesting a possible evolutionary adaptation of marine H. werneckii strains to the deep sea environment. We release high-quality genome assemblies from marine H. werneckii strains, which provides additional data for further genomics analysis, including niche adaptation, fitness and evolution studies.

Preparation and characterization of photocatalytic Gd-doped TiO2 nanoparticles for water treatment.

In recent years, the photocatalytic process by using TiO2 nanoparticles (NPs) has produced a great interest in wastewater treatment due to its interesting features such as low-cost, environmental compatibility, and especially capacity to eliminate persistent organic compounds as well as microorganisms in water. In the present work, the photocatalytic activity of Gd-doped TiO2 nanopowders, with different doping amount 0.1, 1, and 5 mol% synthesized by the sol-gel method, was studied under UV/Visible irradiation for water treatment application. The Gd-doped TiO2 nanoparticles were investigated for their photocatalytic degradation of methylene blue (MB) dye and antibacterial activities against two bacterial strains namely Stenotrophomonas maltophilia (S. maltophilia) and Micrococcus luteus (M. luteus). MB dye was used as a pollutant model to estimate reactive oxygen species (ROS) generation and to correlate killing action of nanoparticles with the generation of ROS. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Raman spectroscopy were used to characterize the as-synthesized nanomaterials. Photocatalytic, as well as antibacterial tests, showed that doping with an appropriate amount of Gd could reduce the radiative recombination process of photogenerated electron-hole pairs in TiO2 and induce a significant enhancement in photocatalytic and consequently antibacterial activity. The experimental sequence of bactericidal activity and photocatalytic degradation efficiency exhibited by the different gadolinium-doped nanoparticles was the following: 0.1 mol% Gd-doped TiO2 > 1 mol% Gd-doped TiO2 > 5 mol% Gd-doped TiO2 > pure titania.

Global Molecular Diversity of the Halotolerant Fungus Hortaea werneckii.

A global set of clinical and environmental strains of the halotolerant black yeast-like fungus Hortaea werneckii are analyzed by multilocus sequencing and AFLP, and physiological parameters are determined. Partial translation elongation factor 1-α proves to be suitable for typing because of the presence/absence of introns and also the presence of several SNPs. Local clonal expansion could be established by a combination of molecular methods, while the population from the Mediterranean Sea water also responds differently to combined temperature and salt stress. The species comprises molecular populations, which in part also differ physiologically allowing further diversification, but clinical strains did not deviate significantly from their environmental counterparts.