The primary purpose of the study, as part of the planned conservation work, was to uncover all aspects of autochthonous biofilm pertaining to the formation of numerous deterioration symptoms occurring on the limestone Rožanec Mithraeum monument in Slovenia. Using state-of-the-art sequencing technologies combining mycobiome data with observations made via numerous light and spectroscopic (FTIR and Raman) microscopy analyses pointed out to epilithic lichen Gyalecta jenensis and its photobiont, carotenoid-rich Trentepohlia aurea, as the origin of salmon-hued pigmented alterations of limestone surface. Furthermore, the development of the main deterioration symptom on the monument, i.e., biopitting, was instigated by the formation of typical endolithic thalli and ascomata of representative Verrucariaceae family (Verrucaria sp.) in conjunction with the oxalic acid-mediated dissolution of limestone. The domination of lichenized fungi, as the main deterioration agents, both on the relief and surrounding limestone, was additionally supported by the high relative abundance of lichenized and symbiotroph groups in FUNGuild analysis. Obtained results not only upgraded knowledge of this frequently occurring but often overlooked group of extremophilic stone heritage deteriogens but also provided a necessary groundwork for the development of efficient biocontrol formulation applicable in situ for the preservation of similarly affected limestone monuments.

Stone monuments can be difficult environments for life, particularly with respect to liquid water access. Nevertheless, microbial communities are found on them with apparent ubiquity. A variety of strategies for access to liquid water have been proposed. Regardless of their water-retention mechanisms details, though, we argue that water activity (a key indicator for cell viability) is constrained by environmental conditions, largely independently of community structure, and is predicted by the local temperature and relative humidity. However, direct measurement of water activity in SABs, particularly those growing on stone surfaces, is difficult. A method for estimating water activity within SABs is presented that uses a minimally invasive combination of conservative sampling, weather data, confocal imaging, and mathematical modeling. Applying the methodology to measurements from the marble roofs of the Federal Hall National Memorial and of the Thomas Jefferson Memorial, estimations are made for water activity in their subaerial stone communities over the course of an approximately one year period.

The safeguarding of Australian outdoor stone heritage is currently limited by a lack of information concerning mechanisms responsible for the degradation of the built heritage. In this study, the bacterial community colonizing the stone surface of an outdoor sculpture located at the Church of St. John the Evangelist in Melbourne was analysed, providing an overview of the patterns of microbial composition associated with stone in an anthropogenic context. Illumina MiSeq 16S rRNA gene sequencing together with confocal laser microscope investigations highlighted the bacterial community was composed of both phototrophic and chemotrophic microorganisms characteristic of stone and soil, and typical of arid, salty and urban environments. Cardinal exposure, position and surface geometry were the most important factors in determining the structure of the microbial community. The North-West exposed areas on the top of the sculpture with high light exposure gave back the highest number of sequences and were dominated by Cyanobacteria. The South and West facing in middle and lower parts of the sculpture received significantly lower levels of radiation and were dominated by Actinobacteria. Proteobacteria were observed as widespread on the sculpture. This pioneer research provided an in-depth investigation of the microbial community structure on a deteriorated artistic stone in the Australian continent and provides information for the identification of deterioration-associated microorganisms and/or bacteria beneficial for stone preservation.

Pigment-based color is one of the most important phenotypic traits of biofilms at the mineral-air interface (subaerial biofilms, SABs), because it reflects the physiology of the microbial community. Because color is the hallmark of all SABs, we argue that pigment-based color could convey the mechanisms that drive microbial adaptation and coexistence across different terrestrial environments and link phenotypic traits to community fitness and ecological dynamics. Within this framework, we present the most relevant microbial pigments at the mineral-air interface and discuss some of the evolutionary landscapes that necessitate pigments as adaptive strategies for resource allocation and survivability. We report several pigment features that reflect SAB communities\' structure and function, as well as pigment ecology in the context of microbial life-history strategies and coexistence theory. Finally, we conclude the study of pigment-based ecology by presenting its potential application and some of the key challenges in the research.

Stone architectural heritage exposed outdoor represents a challenging habitat for biological growths; nevertheless, biocolonization on heritage structure is ubiquitous and represents a major mechanism of alteration. However, the identification of specific microorganisms with known reactivity towards the stone substrate does not necessarily imply that a biodeterioration process is in progress and, in specific conditions, bioprotection effects have been highlighted as a result of colonization. The main objective of the present research is to evaluate the biofilm formation on different lithotypes exposed to similar environmental polluted conditions, and to investigate whether the presence of subaerial biofilms can be associated to an increased magnitude of deterioration of the colonized surfaces with respect to the not colonized ones. In particular, the research examines the extensive biological colonization of the stone surfaces of the façade of the Cathedral of Monza (Italy). Four metamorphic stones widely used in the façade and showing rather different compositional, mineralogical and microstructural features were studied. The state of conservation of the stones was characterized under the mineralogical and compositional point of view by X-ray diffraction and Fourier Transformed infrared analysis. The microstructure of colonized substrates and of reference not colonized ones was studied by means of optical and electron microscopy, to comparatively evaluate the damage extent and weathering patterns in both conservative conditions. The structure and the architecture of biofilms growing on different lithic surfaces were investigated by CLSM in both fluorescence and reflection modes. Captured images were analyzed for 3D reconstructions of biofilm samples. The biovolumes were also calculated to estimate the total biomass. The results indicate that the four lithotypes showed different colonization extents. However, even in presence of extensive biological growth, chemical-physical deterioration mechanisms caused by environmental exposure were largely responsible for deterioration. A relationship between compositional and surface morphological features and biocolonization was also observed.

Bioreceptivity is a fundamental concept in the ornamental stone industry and in the fields of cultural heritage and civil engineering to understand the susceptibility of stone constructions to biological colonisation and subsequent biodeterioration. However, a bioreceptivity index (BI) has not yet been established for any construction material. The aim of the present study is developing a simple, robust and well-founded BI for granitic rocks. For this purpose, a standardised laboratory protocol was used to grow phototrophic biofilms on several varieties of granite. The colonisation was then assessed by chlorophyll fluorescence and colour measurements. Based on the results thus obtained, a BI including two components (BIgrowth and BIcolour) is proposed. BIgrowth quantifies the extent of the biological growth and BIcolour quantifies the colour change undergone by the stone due to the colonisation, which can be considered the bioreceptivity perceptible to the human eye. The values of BI, BIgrowth and BIcolour were fitted to a scale of 0-10, thus enabling qualitative classification of the lithotypes according to their primary bioreceptivity. Eleven varieties of granite commonly used as construction material and with a honed surface finish (one variety with three additional surface finishes: polished, sawn and sanded) were thus assigned the corresponding BI, which represents a new quality factor for the stone industry. The index can therefore be used by end-users as a decision-making tool in the selection of appropriate lithotypes for building and/or ornamental purposes.

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.

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.

Outdoor stoneworks sustain biofilm formation and are constantly at risk of deterioration by micro-organisms. In this study, the biofilm microflora of historic limestone tombstones located in a highly polluted urban environment (Cambridge, MA) and in a less polluted location (Lexington, MA) were compared using comprehensive RNA-based molecular analyses of 16S rRNA gene sequences as well as sequences of genes for different pathways of sulphur metabolism (soxB, apsA, dsrA). The metabolically active micro-organisms detected by denaturing gradient gel electrophoresis analysis of 16S rRNA fragments were predominantly represented by cyanobacteria (belonging to the family Nostocaceae and to the genus Chroococcidiopsis) in both polluted and unpolluted environments. The investigation of soxB, apsA, dsrA transcripts reflected the abundance and the diversity of sulphur-oxidizing and sulphate-reducing bacteria in the Cambridge samples in comparison with the Lexington samples. The investigation revealed that in addition to phototrophic sulphur bacteria belonging to the genera Thiocapsa, Halochromatium, Allochromatium, Thiococcus and Thermochromatium, other sulphate-oxidizing prokaryotes (e.g. the genus Thiobacillus) as well as sequences of Deltaproteobacteria from the genus Desulfovibrio occurred at the polluted urban site. The interactions between the main functional groups retrieved from the limestone tombstones were discussed.
CONCLUSIONS: The biofilm microflora inhabiting historic limestones are a multi-component open ecosystem sensitively reacting to all environmental factors including air pollutants. Little is known about specific target groups that are active in the biofilm and their physiological functions. For the first time, transcripts involved in important energy-yielding processes were investigated to reveal the metabolic capabilities of the microflora in response to atmospheric sulphur pollution. This work provides novel and important information about the ecology of limestone tombstone microbiota and its complex interaction with the external environment.