Laboratory grown subaerial biofilms on granite: application to the study of bioreceptivity.

Simulated environmental colonisation of granite was induced under laboratory conditions in order to develop an experimental protocol for studying bioreceptivity. The experimental set-up proved suitable for producing subaerial biofilms by inoculating granite blocks with planktonic multi-species phototrophic cultures derived from natural biofilms. The ability of four different cultures to form biofilms was monitored over a three-month growth period via colour measurements, quantification of photosynthetic pigments and EPS, and CLSM observations. One of the cultures under study, which comprised several taxa including Bryophyta, Charophyta, Chlorophyta and Cyanobacteria, was particularly suitable as an inoculum, mainly because of its microbial richness, its rapid adaptability to the substratum and its high colonisation capacity. The use of this culture as an inoculum in the proposed experimental set-up to produce subaerial biofilms under laboratory conditions will contribute to standardising the protocols involved, thus enabling more objective assessment of the bioreceptivity of granite in further experiments.

Response surface optimization of a method for extracting extracellular polymeric substances (EPS) from subaerial biofilms on rocky substrata.

The aim of the present study was to optimize a protocol for extracting extracellular polymeric substances (EPS) from biofilms on rocky substrata, as the EPS matrix is considered key to understanding the biofilm mode of life. For this purpose, we tested the extraction efficacy of NaOH and H2SO4 at different concentrations, temperatures and times for obtaining EPS from multi-species subaerial biofilms grown on granite blocks under laboratory conditions. Two experimental designs (Box-Behnken design and full factorial design) were used in testing each extractant. The extraction efficiency was determined by analysing the carbohydrate, protein and DNA contents of the extracts obtained. H2SO4 proved unsuitable as an extractant as it caused excessive cell lysis. However, response surface optimization of NaOH-mediated extraction enabled cell lysis to be minimized. Confirmation experiments were performed under the optimal conditions established and a protocol for extracting EPS is proposed, yielding the first quantitative data on EPS extracted from subaerial biofilms developed on rocky substrata. Graphical abstract Development of a method for extracting EPS from subaerial biofilms on rocky substrata.

The susceptibility of weathered versus unweathered schist to biological colonization in the Côa Valley Archaeological Park (north-east Portugal).

This study addresses the primary and secondary bioreceptivity of schist used as a support for prehistoric rock art in the Côa Valley Archaeological Park (north-east Portugal) and provides some parameters that can be related to the risk of biologically induced schist weathering. Samples of freshly quarried and naturally weathered schist were characterized in terms of their intrinsic properties and maintained in controlled environmental conditions after inoculation with biofilm-forming cyanobacteria. The physical properties of the studied schist, as well as its abrasion pH, all varied according to the weathering degree of the samples and so did its susceptibility to colonization by biofilm-forming cyanobacteria. Complete separation between weathered and unweathered schist samples in terms of laboratory-induced photosynthetic biomass was obtained by measuring total colour change in the CIE (International Commission on Illumination) L*a*b* colour space. Weathered schist was more bioreceptive than unweathered schist, associated with increased open porosity, water saturation, capillary water and capillarity coefficient and decreased abrasion pH. In the future, it might be possible to determine the susceptibility of schist surfaces to biological colonization through evaluation of colour differences associated with the different weathering degrees presented by those surfaces prior to colonization.

Influence of the properties of granitic rocks on their bioreceptivity to subaerial phototrophic biofilms.

As any stone substrate is susceptible to biological colonisation, the choice of lithotype used for construction is a key strategy for preventing biodeterioration. For this purpose, a comprehensive evaluation of the primary bioreceptivity to phototrophic biofilms of eleven varieties of granitic rocks, commonly used as building material, was carried out. Blocks were inoculated with a multi-species phototrophic culture and subjected to standardised growth conditions for three months. Biofilm formation was assessed by chlorophyll (chl) fluorescence, colour measurements and extracellular polymeric substances (EPS) quantification. Relationships between the biofilm growth indicators and the properties of the different rocks studied were then analysed. Results showed that the bioreceptivity of the granites is more strongly affected by the physical characteristics of the stones than by their chemical and mineralogical properties, possibly because of the similar composition of the rocks studied. Growth of phototrophic biofilms was enhanced by high open porosity, capillary water content and surface roughness, and the bioreceptivity of weathered granites was higher than that of sound granites. The results obtained can therefore help in the selection of appropriate lithotypes for building purposes. The amounts of EPS produced by subaerial biofilms primarily depended on the requirements and/or characteristics of the biofilm-forming microorganisms, rather than on the bioreceptivity of the substratum, and microorganisms produce the amounts of EPS required at the initial stage of establishment on the stone surface, independently of the subsequent biomass development. These findings are especially important from the point of view of biodeterioration, in which the EPS matrix plays a central role.