A multiproxy approach to evaluate biocidal treatments on biodeteriorated majolica glazed tiles.

The Fishing House located on the grounds of the Marquis of Pombal Palace, Oeiras, Portugal, was built in the 18th century. During this epoch, Portuguese gardens, such as the one surrounding the Fishing House, were commonly ornamented with glazed wall tile claddings. Currently, some of these outdoor tile panels are covered with dark colored biofilms, contributing to undesirable aesthetic changes and eventually inducing chemical and physical damage to the tile surfaces. Phylogenetic analyses revealed that the investigated biofilms are mainly composed of green algae, cyanobacteria and dematiaceous fungi. With the aim of mitigating biodeterioration, four different biocides (TiO2 nanoparticles, Biotin® T, Preventol® RI 80 and Albilex Biostat® ) were applied in situ to the glazed wall tiles. Their efficacy was monitored by visual examination, epifluorescence microscopy and DNA-based analysis. Significant changes in the microbial community composition were observed 4 months after treatment with Preventol® RI 80 and Biotin® T. Although the original community was inactivated after these treatments, an early stage of re-colonization was detected 6 months after the biocide application. TiO2 nanoparticles showed promising results due to their self-cleaning effect, causing the detachment of the biofilm from the tile surface, which remained clean 6 and even 24 months after biocide application. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Combining stable isotope (δ13C) of trace gases and aerobiological data to monitor the entry and dispersion of microorganisms in caves.

Altamira Cave (north of Spain) contains one of the world's most prominent Paleolithic rock art paintings, which are threatened by a massive microbial colonization of ceiling and walls. Previous studies revealed that exchange rates between the cave and the external atmosphere through the entrance door play a decisive role in the entry and transport of microorganisms (bacteria and fungi) and nutrients to the interior of the cave. A spatial-distributed sampling and measurement of carrier (CO2) and trace (CH4) gases and isotopic signal of CO2 (δ(13)C) inside the cave supports the existence of a second connection (active gas exchange processes) with the external atmosphere at or near the Well Hall, the innermost and deepest area of the cave. A parallel aerobiological study also showed that, in addition to the entrance door, there is another connection with the external atmosphere, which favors the transport and increases microorganism concentrations in the Well Hall. This double approach provides a more complete knowledge on cave ventilation and revealed the existence of unknown passageways in the cave, a fact that should be taken into account in future cave management.

First case of cerebral abscess due to a novel Nocardia species in an immunocompromised patient.

We report the first case of cerebral abscess due to a novel species of Nocardia in a heart transplant patient and describe the antimicrobial susceptibility of this isolate. As our patient was intolerant to trimethoprim-sulfamethoxazole, we also discuss alternative therapeutic options in brain abscess due to Nocardia sp.

Bioreceptivity of building stones: a review.

In 1995, Guillitte defined bioreceptivity, a new term in ecology, as the ability of a material to be colonized by living organisms. Information about the bioreceptivity of stone is of great importance since it will help us to understand the material properties which influence the development of biological colonization in the built environment, and will also provide useful information as regards selecting stones for the conservation of heritage monuments and construction of new buildings. Studies of the bioreceptivity of stone materials are reviewed here with the aim of providing a clear set of conclusions on the topic. Definitions of bioreceptivity are given, stone bioreceptivity experiments are described, and finally the stone properties related to bioreceptivity are discussed. We suggest that a standardized laboratory protocol for evaluating stone bioreceptivity and definition of a stone bioreceptivity index are required to enable creation of a database on the primary bioreceptivity of stone materials.

Fungal Planet description sheets: 128-153.

Novel species of microfungi described in the present study include the following from Australia: Catenulostroma corymbiae from Corymbia, Devriesia stirlingiae from Stirlingia, Penidiella carpentariae from Carpentaria, Phaeococcomyces eucalypti from Eucalyptus, Phialophora livistonae from Livistona, Phyllosticta aristolochiicola from Aristolochia, Clitopilus austroprunulus on sclerophyll forest litter of Eucalyptus regnans and Toxicocladosporium posoqueriae from Posoqueria. Several species are also described from South Africa, namely: Ceramothyrium podocarpi from Podocarpus, Cercospora chrysanthemoides from Chrysanthemoides, Devriesia shakazului from Aloe, Penidiella drakensbergensis from Protea, Strelitziana cliviae from Clivia and Zasmidium syzygii from Syzygium. Other species include Bipolaris microstegii from Microstegium and Synchaetomella acerina from Acer (USA), Brunneiapiospora austropalmicola from Rhopalostylis (New Zealand), Calonectria pentaseptata from Eucalyptus and Macadamia (Vietnam), Ceramothyrium melastoma from Melastoma (Indonesia), Collembolispora aristata from stream foam (Czech Republic), Devriesia imbrexigena from glazed decorative tiles (Portugal), Microcyclospora rhoicola from Rhus (Canada), Seiridium phylicae from Phylica (Tristan de Cunha, Inaccessible Island), Passalora lobeliae-fistulosis from Lobelia (Brazil) and Zymoseptoria verkleyi from Poa (The Netherlands). Valsalnicola represents a new ascomycete genus from Alnus (Austria) and Parapenidiella a new hyphomycete genus from Eucalyptus (Australia). Morphological and culture characteristics along with ITS DNA barcodes are also provided.

Enigmatic reticulated filaments in subsurface granite.

In the last few years, geomicrobiologists have focused their researches on the nature and origin of enigmatic reticulated filaments reported in modern and fossil samples from limestone caves and basalt lava tubes. Researchers have posed questions on these filaments concerning their nature, origin, chemistry, morphology, mode of formation and growth. A tentative microbial origin has been elusive since these filaments are found as hollow tubular sheaths and could not be affiliated to any known microorganism. We describe the presence of similar structures in a 16th century granite tunnel in Porto, Northwest Portugal. The reticulated filaments we identify exhibit fine geometry surface ornamentation formed by cross-linked Mn-rich nanofibres, surrounded by a large amount of extracellular polymeric substances. Within these Mn-rich filaments we report for the first time the occurrence of microbial cells.

Microbiological study of bulls of indulgence of the 15th-16th centuries.

During the restoration of the church of "San Esteban" in Cuéllar (Segovia, Spain) a few sepulchres were opened. Among them was that of Doña Isabel de Zuazo, from the 16th century. Together with the corpse was found a series of printed documents from the 15th-16th centuries, most of which were bulls of indulgence. A microbiological study of the documents was carried out using techniques of isolation and molecular microbiology, together with scanning electron microscopy. Most of the identified bacteria were highly suggestive of a human origin, particularly the predominance of Clostridium species consistent with the flora of the human intestinal tract. Our results demonstrate that appreciable post-mortem migration of bacteria has taken place from the corpse to the historic documents. This can be explained considering that the documents were found on pelvic region, and were contaminated by body fluids and putrefaction.

Fungal outbreak in a show cave.

Castañar de Ibor Cave (Spain) was discovered in 1967 and declared a Natural Monument in 1997. In 2003 the cave was opened to public visits. Despite of extensive control, on 26 August 2008 the cave walls and sediments appeared colonized by long, white fungal mycelia. This event was the result of an accidental input of detritus on the afternoon of 24 August 2008. We report here a fungal outbreak initiated by Mucor circinelloides and Fusarium solani and the methods used to control it.

Laboratory-induced endolithic growth in calcarenites: biodeteriorating potential assessment.

This study is aimed to assess the formation of photosynthetic biofilms on and within different natural stone materials, and to analyse their biogeophysical and biogeochemical deterioration potential. This was performed by means of artificial colonisation under laboratory conditions during 3 months. Monitoring of microbial development was performed by image analysis and biofilm biomass estimation by chlorophyll extraction technique. Microscopy investigations were carried out to study relationships between microorganisms and the mineral substrata. The model applied in this work corroborated a successful survival strategy inside endolithic microhabitat, using natural phototrophic biofilm cultivation, composed by cyanobacteria and algae, which increased intrinsic porosity by active mineral dissolution. We observed the presence of mineral-like iron derivatives (e.g. maghemite) around the cells and intracellularly and the precipitation of hausmannite, suggesting manganese transformations related to the biomineralisation.

The microbiology of Lascaux Cave.

Lascaux Cave (Montignac, France) contains paintings from the Upper Paleolithic period. Shortly after its discovery in 1940, the cave was seriously disturbed by major destructive interventions. In 1963, the cave was closed due to algal growth on the walls. In 2001, the ceiling, walls and sediments were colonized by the fungus Fusarium solani. Later, black stains, probably of fungal origin, appeared on the walls. Biocide treatments, including quaternary ammonium derivatives, were extensively applied for a few years, and have been in use again since January 2008. The microbial communities in Lascaux Cave were shown to be composed of human-pathogenic bacteria and entomopathogenic fungi, the former as a result of the biocide selection. The data show that fungi play an important role in the cave, and arthropods contribute to the dispersion of conidia. A careful study on the fungal ecology is needed in order to complete the cave food web and to control the black stains threatening the Paleolithic paintings.

The impact of arthropods on fungal community structure in Lascaux Cave.

AIMS: To determine the major components of the fungal population present in Lascaux Cave, France. The ceiling, walls, sediments and soil were colonized by Fusarium solani in 2001 and later, in 2006, black stains appeared. However, the origin of the successive fungal invasions is unknown as well as the ecology of the cave. METHODS AND RESULTS: The primers nu-SSU-0817F and nu-SSU-1536R were used for the direct amplification of fungal 18S-rDNA sequences from 11 samples. A total of 607 clones were retrieved. Eight out of the ten most abundant phylotypes corresponded to fungi associated with arthropods and represented about 50% of the clones. CONCLUSIONS: Entomophilous fungi play an important role in the cave and arthropods contribute to the dispersion of spores and fungal development. SIGNIFICANCE AND IMPACT OF THE STUDY: Choosing appropriate targets for control of fungal dispersal is dependent on knowing the causes of fungal colonization. A control of the arthropod populations seems to be a need in order to protect the rock art paintings in Lascaux Cave.

Isolation of five Rubrobacter strains from biodeteriorated monuments.

In the last few years, the microbial colonisation of mural paintings in ancient monuments has been attracting the attention of microbiologists and conservators. The genus Rubrobacter is commonly found in biodeteriorated monuments, where it has been reported to cause rosy discolouration. However, to date, only three species of this genus have been isolated, all from thermophilic environments. In this paper, we studied three monuments: the Servilia and Postumio tombs in the Roman Necropolis of Carmona (Spain), and Vilar de Frades church (Portugal), in search of Rubrobacter strains. In all cases, biodeterioration and the formation of efflorescences were observed, and five Rubrobacter strains were isolated. These isolates showed different physiology and migration in denaturing gradient gel electrophoresis, suggesting they might represent new species within this genus. The isolates reproduced some biodeterioration processes in the laboratory and revealed their biomediation in crystal formation.

The laser-induced discoloration of stonework; a comparative study on its origins and remedies.

For understanding the phenomena associated with the discoloration observed in some cases of infrared laser cleaned stonework surfaces, a comparative study of three different types and morphologies of pollution encrustation and stone substrates was undertaken. Fragments originating from monuments with historic and/or artistic value, bearing homogeneous thin soiling on Pentelic marble (Athens, Greece), thick encrustation on Hontoria limestone (Burgos, Spain) and compact thin crust on gypsum decorations (Athens, Greece), have been studied on the basis of their composition and origin, together with the conditions that may induce yellowing effects upon their laser cleaning with IR wavelengths. While irradiation in the UV (i.e. at 355 nm) could not effectively remove the encrustations studied, irradiation at 1,064 nm was found efficient to remove all the studied pollution accumulations. Discoloration towards yellow was evident in all cases and at different levels, including the samples with intentional patination layer. To the limit of Raman detection no chemical alterations were detected on the irradiated areas while the presence of yellow polar compounds in all the pollution crusts studied supports the argument that the discoloration of the stone surfaces upon their IR irradiation may be due to the uncovering of existing yellow layers as result of the migration of these compounds inwards to the original stone surface. To correct and/or prevent such undesired coloration the use of IR and UV radiation both in sequential and synchronous mode was considered, with positive results.

Role of specific microbial communities in the bioavailability of iron in Doñana National Park.

The Doñana National Park contains highly complex microbial communities that play critical roles in the bioavailability and biogeochemical cycling of elements. In this study, we analyzed the formation of Fe-rich films on the surface of shallow waters at Doñana National Park as a model for future applications in bioremediation. Phytoplankton composition and abundance, physicochemical measurements, culture enrichments, and molecular detection of microorganisms were determined during this study. Natural samples were incubated in the laboratory and enrichments producing similar phenomena were obtained. Bacteria belonging to the genera Enterobacter were the major participants in surface Fe-film formation in laboratory enrichments. Film formation was dependent on wind exposure and only those sites protected from the direct influence of wind developed these films. A higher concentration of euglenophytes was found in the proximity of film-covered waters. Available iron (Fe2+) and total Fe concentrations were high (18.8 and 46.6 mg/L, respectively) underneath the surface film, and higher than in film-free waters and pore-sediment water. The concentration of phosphate and its availability were higher in the iron-rich film sediment, as 61% of total P was bound to iron oxy-hydroxides. Our results provide evidence of the link between P and Fe biogeochemical cycling in aquatic systems on sandy soils at the Doñana National Park. In addition, they provide significant insights into the bioavailability of these elements with potential interest in the role of microorganisms for metal sequestration in natural environments and utilization in bioremediation processes.

Metabolically active microbial communities of yellow and grey colonizations on the walls of Altamira Cave, Spain.

AIMS: To determine the major components of total and metabolically active microbial communities of yellow and grey colonizations threatening the conservation of palaeolithic paintings in Altamira Cave (Spain). METHODS AND RESULTS: Micro-organisms present in yellow and grey colonizations were determined from DNA analysis with those showing metabolic activity determined from RNA analysis. Microbial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA libraries were constructed from PCR amplified products. Proteobacteria was the most frequent bacterial phylum. Other phyla detected from RNA-based microbial surveys were Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae and Gemmatimonadetes. The detected metabolically active micro-organisms represented only a fraction of the total bacterial community present in the studied colonizations as compared from DGGE analysis. CONCLUSIONS: The major bacterial participants in the development of yellow and grey colonizations in Altamira Cave were determined using RNA-based molecular techniques. Micro-organisms showing undetectable activity represent a potential risk for the conservation of these paintings if environmental conditions experience variations. SIGNIFICANCE AND IMPACT OF THE STUDY: Caves with palaeolithic paintings are affected by microbial deterioration. Assessing the composition of the microbial communities colonizing these caves represents a first stage to understand and control these colonizations.

Does green light influence the fluorescence properties and structure of phototrophic biofilms?

Artificial illumination can harm works of art by inducing the development of photosynthetic biofilms. With the aim of preventing biodeterioration or esthetic damage to such surfaces, we evaluated and compared the effects of illuminating biofilms formed by Gloeothece membranacea (cyanobacteria) and Chlorella sorokiniana (Chlorophyta) using exclusively white or green light.

Surface-enhanced Raman spectroscopy of chernozem humic acid and their fractions obtained by coupled size exclusion chromatography-polyacrylamide gel electrophoresis (SEC-PAGE).

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.

Performance and microbial communities of a continuous stirred tank anaerobic reactor treating two-phases olive mill solid wastes at low organic loading rates.

A study of the performance and microbial communities of a continuous stirred tank reactor (CSTR) treating two-phases olive mill solid wastes (OMSW) was carried out at laboratory-scale. The reactor operated at a mesophilic temperature (35 degrees C) and an influent substrate concentration of 162 g total chemical oxygen demand (COD)L(-1) and 126 g volatile solids (VS)L(-1). The data analyzed in this work corresponded to a range of organic loading rates (OLR) of between 0.75 and 3.00 g CODL(-1)d(-1), getting removal efficiencies in the range of 97.0-95.6%. Methane production rate increased from 0.164 to 0.659 L CH(4)L(reactor)(-1)d(-1) when the OLR increased within the tested range. Methane yield coefficients were 0.225 L CH(4)g(-1) COD removed and 0.290 L CH(4)g(-1) VS removed and were virtually independent of the OLR applied. A molecular characterization of the microbial communities involved in the process was also accomplished. Molecular identification of microbial species was performed by PCR amplification of 16S ribosomal RNA genes, denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Among the predominant microorganisms in the bioreactor, the Firmicutes (mainly represented by Clostridiales) were the most abundant group, followed by the Chloroflexi and the Gamma-Proteobacteria (Pseudomonas species as the major representative). Other bacterial groups detected in the bioreactor were the Actinobacteria, Bacteroidetes and Deferribacteres. Among the Archaea, the methanogen Methanosaeta concilii was the most representative species.

Deterioration of building materials in Roman catacombs: the influence of visitors.

In the last decades, damages on building materials and mural paintings were observed in Roman catacombs. The damages were due to extensive formation of biofilms induced by artificial illumination and humidity. Microenvironmental data (temperature, CO(2) concentration, humidity, and atmospheric pressure) clearly showed the negative influence of visitors. Increasing heat, light and water vapour condensation into corridors and cubicles favoured biofilm development. The composition of biofilms was different and depended mainly on distance to illumination sources and humidity, thus denoting the influence of light on the growth of phototrophic microorganisms in the catacombs. In addition, biofilm distribution was governed by the type of material to be colonised. This study shows that countermeasures are needed to prevent deterioration of hypogean environments.

Phyllobacterium catacumbae sp. nov., a member of the order 'Rhizobiales' isolated from Roman catacombs.

Two strains were isolated from tuff, a volcanic rock that forms the walls of the Roman Catacombs of Saint Callixtus in Rome, Italy. A polyphasic approach using nutritional and physiological tests, reactions to antibiotics, fatty acid profiles, DNA base ratios, DNA-DNA reassociation and 16S rRNA gene sequence comparisons showed that the two isolates belong to a novel species within the genus Phyllobacterium. The species Phyllobacterium catacumbae sp. nov. is proposed. The type strain is CSC19(T) (=CECT 5680(T)=LMG 22520(T)).