Growth of phototrophic biofilms from limestone monuments under laboratory conditions

International Biodeterioration & Biodegradation,xxx (2009) 1–8In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano,Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm(Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the threemonth incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work,a multiple-species community assemblage was obtained for further stone colonisation experiments

The influence of inherent properties of building limestones on their bioreceptivity to phototrophic microorganisms

Annals of Microbiology, 59 (4) 705-713 (2009)The influence of open porosity, water absorption capillarity, water vapour permeability, surface roughness, stone pH and chemical composition on stone bioreceptivity to phototrophic microorganisms was assessed by means of a thorough stone characterisation with subsequent artificially inoculation of limestone samples with a multi-species phototrophic culture and placing them inside a growth chamber for 90 days. A principal component analysis and an analysis of variance(ANOVA) were carried out in order to evaluate the direct relationships between stone bioreceptivity and petrophysical properties. From the principal component analysis, two main components were obtained and assigned a petrophysical/photosynthetic biomass meaning. Stone bioreceptivity, quantified by the amount of chlorophyll a and intensity of chlorophyll a fluorescence present on the stone samples after 90 days-incubation, was included in both principal components. The first component was linked to the amount of chlorophyll a and was highly and linearly associated to capillarity and roughness,and less associated with open porosity and water vapour permeability. The second component, linked to the intensity of chlorophyll a fluorescence measured on the stone surfaces, was not linearly associated with the petrophysycal properties,showing the fallibility of this in vivo chlorophyll quantification technique on the estimation of photosynthetic biomass growing on stone materials, particularly when endolithic growth occurs

Microbiology of the stalactites from Grotta dei Cervi, Porto Badisco, Italy

The active stalactites from Grotta dei Cervi, Porto Badisco, southeastern Italy, were sampled to investigate the microbial communities present in these speleothems. Sampling was carried out in a transect about 150 m long in the central gallery, where numerous Gram-positive bacteria were isolated. Actinomycetes of the genus Streptomyces were the most abundant, followed by members of the genus Bacillus. Further isolates were assigned to the genera Amycolatopsis, Arthrobacter, Agromyces, Micrococcus, Nocardiopsis and Rhodococcus of the order Actinomycetales. The ability of actinomycetes to colonize subterranean environments is discussed

Reproducing stone monument photosynthetic-based colonization under laboratory conditions

Science of the total environment 405(2008) 278-285In order to understand the biodeterioration process occurring on stone monuments, we analyzed the microbial communities involved in these processes and studied their ability to colonize stones under controlled laboratory experiments. In this study, a natural green biofilm from a limestone monument was cultivated, inoculated on stone probes of the same lithotype and incubated in a laboratory chamber. This incubation system, which exposes stone samples to intermittently sprinkling water, allowed the development of photosynthetic biofilms similar to those occurring on stone monuments. Denaturing gradient gel electrophoresis (DGGE) analysis was used to evaluate the major microbial components of the laboratory biofilms. Cyanobacteria, green microalgae, bacteria and fungi were identified by DNA-based molecular analysis targeting the 16S and 18S ribosomal RNA genes. The natural green biofilm was mainly composed by the Chlorophyta Chlorella, Stichococcus, and Trebouxia, and by Cyanobacteria belonging to the genera Leptolyngbya and Pleurocapsa. A number of bacteria belonging to Alphaproteobacteria, Bacteroidetes and Verrucomicrobia were identified, as well as fungi from the Ascomycota. The laboratory colonization experiment on stone probes showed a colonization pattern similar to that occurring on stone monuments. The methodology described in this paper allowed to reproduce a colonization equivalent to the natural biodeteriorating process

Crossiella, a Rare Actinomycetota Genus, Abundant in the Environment

The genus Crossiella contains two species, C. equi, causing nocardioform placentitis in horses, and C. cryophila, an environmental bacterium. Apart from C. equi, which is not discussed here, environmental Crossiella is rarely reported in the literature; thus, it has not been included among “rare actinobacteria”, whose isolation frequency is very low. After C. cryophila, only five reports cover the isolation of Crossiella strains. However, the frequency of published papers on environmental Crossiella has increased significantly in recent years due to the extensive use of next-generation sequencing (NGS) and a huge cascade of data that has improved our understanding of how bacteria occur in the environment. In the last five years, Crossiella has been found in different environments (caves, soils, plant rhizospheres, building stones, etc.). The high abundance of Crossiella in cave moonmilk indicates that this genus may have an active role in moonmilk formation, as evidenced by the precipitation of calcite, witherite, and struvite in different culture media. This review provides an overview of environmental Crossiella, particularly in caves, and discusses its role in biomineralization processes and bioactive compound production.This research was funded by the projects PID2020-114978GB-I00 and PID2019-110603RB-I00. The Malaga City Council financed data from the archaeological basement of the Thyssen Museum of Malaga through a conservation contract for this Roman site

Analytical pyrolysis evidences the presence of granaticins in the violet stains of a Roman tomb

The walls of the Circular Mausoleum tomb (Roman Necropolis of Carmona, Spain) exhibit an important number of violet stains of unknown origin. Analytical pyrolysis detected in the tomb walls granaticin A, a violet pigment with an isobenzochromanequinone structure, as well as in the extracts of two bacterial strains isolated from the walls. The bacterium was tentatively identified as Streptomyces sp. High performance liquid chromatography confirmed that this Streptomyces synthesized as major pigments dihydrogranaticin A, granaticin A and granaticin B.JAE Research Fellowship from CSIC GCL2010-17183 and 201230E125. M.D.H. and A.Z.M.Marie Curie Intra-European Fellowship of the European Commission’s 7th Framework Programme PIEF-GA-2012-32868

Pathogenic and opportunistic microorganisms in caves

With today’s leisure tourism, the frequency of visits to many caves makes it necessary to know about possible potentially pathogenic microorganisms in caves, determine their reservoirs, and inform the public about the consequences of such visits. Our data reveal that caves could be a potential danger to visitors because of the presence of opportunistic microorganisms, whose existence and possible development in humans is currently unknown