A. Z. Miller

Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: an overview

The presence and deteriorating action of micro-organisms on monuments and stone works of art have received considerable attention in the last few years. Knowledge of the microbial populations living on stone materials is the starting point for successful conservation treatment and control. This paper reviews the literature on cyanobacteria and chlorophyta that cause deterioration of stone cultural heritage (outdoor monuments and stone works of art) in European countries of the Mediterranean Basin. Some 45 case studies from 32 scientific papers published between 1976 and 2009 were analysed. Six lithotypes were considered: marble, limestone, travertine, dolomite, sandstone and granite. A wide range of stone monuments in the Mediterranean Basin support considerable colonization of cyanobacteria and chlorophyta, showing notable biodiversity. About 172 taxa have been described by different authors, including 37 genera of cyanobacteria and 48 genera of chlorophyta. The most widespread and commonly reported taxa on the stone cultural heritage in the Mediterranean Basin are, among cyanobacteria, Gloeocapsa, Phormidium and Chroococcus and, among chlorophyta, Chlorella, Stichococcus and Chlorococcum. The results suggest that cyanobacteria and chlorophyta colonize a wide variety of substrata and that this is related primarily to the physical characteristics of the stone surface, microclimate and environmental conditions and secondarily to the lithotype.

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.

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.

Relating physical and chemical properties of four different biochars and their application rate to biomass production of Lolium perenne on a Calcic Cambisol during a pot experiment of 79 days.

Three pyrolysis biochars (B1: wood, B2: paper-sludge, B3: sewage-sludge) and one kiln-biochar (B4: grapevine wood) were characterized by determining different chemical and physical properties which were related to the germination rates and to the plant biomass production during a pot experiment of 79 days in which a Calcic Cambisol from SW Spain was amended with 10, 20 and 40 t ha(-1) of the four biochars. Biochar 1, B2 and B4 revealed comparable elemental composition, pH, water holding capacity and ash content. The H/C and O/C atomic ratios suggested high aromaticity of all biochars, which was confirmed by (13)C solid-state NMR spectroscopy. The FT-IR spectra confirmed the aromaticity of all the biochars as well as several specific differences in their composition. The FESEM-EDS distinguished compositional and structural differences of the studied biochars such as macropores on the surface of B1, collapsed structures in B2, high amount of mineral deposits (rich in Al, Si, Ca and Fe) and organic phases in B3 and vessel structures for B4. Biochar amendment improved germination rates and soil fertility (excepting for B4), and had no negative pH impact on the already alkaline soil. Application of B3, the richest in minerals and nitrogen, resulted in the highest soil fertility. In this case, increase of the dose went along with an enhancement of plant production. Considering costs due to production and transport of biochar, for all used chars with the exception of B3, the application of 10 t ha(-1) turned out as the most efficient for the crop and soil used in the present incubation experiment.

Reproducing stone monument photosynthetic-based colonization under laboratory conditions

In 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.

Rubrobacter bracarensis sp. nov., a novel member of the genus Rubrobacter isolated from a biodeteriorated monument.

Three actinobacteria strains isolated from a green biofilm covering the biodeteriorated interior walls of Vilar de Frades Church (Portugal) were studied using a polyphasic approach. The three strains were aerobic, non-spore forming and Gram-positive. Phylogenetically, the most closely related described species was Rubrobacter radiotolerans (94.2-94.3% and 81.9-82.5% similarities for the 16S rRNA and rpoB gene sequences, respectively). The fatty acid profile was dominated by anteiso-C(17:1) ω9c, and MK-8 was the only menaquinone present. These data clearly showed that the three strains could represent a new species, for which we propose the name Rubrobacter bracarensis sp. nov., with strain VF70612_S1(T) (=CECT 7924=DSMZ 24908) as the type strain.

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

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.

Uncovering the origin of the black stains in Lascaux cave in France

Lascaux Cave in France was discovered in 1940. Since being opened to visitors the cave has suffered three major microbial outbreaks. The current problem is the fast dissemination of black stains which are threatening the Palaeolithic paintings. Previous data pointed to the involvement of new fungal species in the formation of black stains on the rock walls and ceiling. However, it appears that there could be other reasons for the formation of different and extensive black stains coating the surface of the clayey sediments. Our analyses reveal that black stains on clayey sediments are mainly produced by Acremonium nepalense, a manganese oxide-depositing fungus, widely distributed in the cave. Thus, in Lascaux Cave, the black stains have a dual origin: on limestone rocks they are mainly produced by the accumulation of fungal melanins, and on clayey sediments by the biogenic deposition of black manganese oxides.

Halophilic microorganisms are responsible for the rosy discolouration of saline environments in three historical buildings with mural paintings

A number of mural paintings and building materials from monuments located in central and south Europe are characterized by the presence of an intriguing rosy discolouration phenomenon. Although some similarities were observed among the bacterial and archaeal microbiota detected in these monuments, their origin and nature is still unknown. In order to get a complete overview of this biodeterioration process, we investigated the microbial communities in saline environments causing the rosy discolouration of mural paintings in three Austrian historical buildings using a combination of culture-dependent and -independent techniques as well as microscopic techniques. The bacterial communities were dominated by halophilic members of Actinobacteria, mainly of the genus Rubrobacter. Representatives of the Archaea were also detected with the predominating genera Halobacterium, Halococcus and Halalkalicoccus. Furthermore, halophilic bacterial strains, mainly of the phylum Firmicutes, could be retrieved from two monuments using special culture media. Inoculation of building materials (limestone and gypsum plaster) with selected isolates reproduced the unaesthetic rosy effect and biodeterioration in the laboratory.

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.