Essential oils to contrast biodeterioration of the external marble of Florence Cathedral.

The search for more sustainable strategies to contrast biodeterioration of stone cultural heritage has been developing in recent years to find alternatives to synthetic biocides, since their toxicity and potential impact on the environment and health. In this study, the application of the oregano and thyme essential oils (EOs) was tested to control microbial growth on the external marble of Florence Cathedral affected by extended darkening. Before in situ application, preliminary tests were carried out to evaluate the interference of the EOs with marble (colorimetric and water absorption assays on marble specimens) and their efficacy in inhibiting marble microbiota (sensitivity test on nutrient media). EOs inhibited the whole cultivable microbiota sampled from the Cathedral marble at a very low concentration, while they did not interfere with colour and water absorption capability of uncolonised marble samples when applied as a 2 % solution. Then the two EOs and the commercial biocide Biotin T were used in in situ trials on marble in two outdoor study sites of Florence Cathedral. The effectiveness of the treatments was assessed through short- and mid-term evaluation by multidisciplinary in situ non-invasive (colorimetric and ATP assays, microscopy) and ex situ (microbial viable titer) tests. Concerning results, we found a good correspondence between parameters for evaluation of viability (bacterial and fungi viable titer) and activity (ATP determination) and some correspondence among these and microscopy and colorimetry. Considering the whole data, treatments with oregano and thyme EOs were effective against microbial community, in more cases comparably to the commercial biocide. Some differences found, particularly by viable titer, in the two study sites or in bacterial and fungal components of the microbiota can be partly attributed to differences in structure and colonization pattern of the microbial community due to the peculiar climatic conditions of the differently exposed study areas.

Taxonomic resolution of the genus Cyanothece (Chroococcales, Cyanobacteria), with a treatment on Gloeothece and three new genera, Crocosphaera, Rippkaea, and Zehria.

The systematics of single-celled cyanobacteria represents a major challenge due to morphological convergence and application of various taxonomic concepts. The genus Cyanothece is one of the most problematic cases, as the name has been applied to oval-shaped coccoid cyanobacteria lacking sheaths with little regard to their phylogenetic position and details of morphology and ultrastructure. Hereby we analyze an extensive set of complementary genetic and phenotypic evidence to disentangle the relationships among these cyanobacteria. We provide diagnostic characters to separate the known genera Cyanothece, Gloeothece, and Aphanothece, and provide a valid description for Crocosphaera gen. nov. We describe two new genera, Rippkaea and Zehria, to characterize two distinct phylogenetic lineages outside the previously known genera. We further describe 13 new species in total including Cyanothece svehlovae, Gloeothece aequatorialis, G. aurea, G. bryophila, G. citriformis, G. reniformis, Gloeothece tonkinensis, G. verrucosa, Crocosphaera watsonii, C. subtropica, C. chwakensis, Rippkaea orientalis, and Zehria floridana to recognize the intrageneric diversity as rendered by polyphasic analysis. We discuss the close relationship of free-living cyanobacteria from the Crocosphaera lineage to nitrogen-fixing endosymbionts of marine algae. The current study includes several experimental strains (Crocosphaera and "Cyanothece") important for the study of diazotrophy and the global oceanic nitrogen cycle, and provides evidence suggesting ancestral N2 -fixing capability in the chroococcalean lineage.

A multi-analytical approach for the study of red stains on heritage marble.

Red stains are a common discolouration on stone cultural heritage all over the world. These are very difficult to remove and little is known about the reddish pigmentation. Numerous red stains were mapped on the Baptistery of San Giovanni in Florence, one of the most important monuments in Italy. This paper is focused on red stains on marble stone and the results of a detailed multidisciplinary approach are presented. Several analytical and investigation techniques (such as optical microscopy, X-ray fluorescence mapping, X-ray micro-tomography, micro-Raman spectroscopy, scanning electron microscopy with energy dispersive spectroscopy, microbial isolation, colorimetric measurements and isotopic analyses) were used to better understand the origin and processes involved in this kind of alteration. Analyses of the red stains led us to believe the presence of minium (lead tetroxide) and Pb are usually concentrated in the spaces between calcite grains. Red stains of Pb isotopic composition also overlap with data from Sardinian mines. These preliminary data seem to reinforce the suggestion of a source of lead from some metallic items (during restoration campaigns between 1938 and 1944, damaged parts were removed and replaced, and the new marble cladding was fixed with iron brackets treated with minium).

Sporosarcina pasteurii use in extreme alkaline conditions for recycling solid industrial wastes.

The ureolytic bacteria are one of the most efficient organisms able to produce high amounts of carbonate that easily react with the free calcium ions from the environment. Sporosarcina pasteurii, a robust microbe in alkaline environments, was tested in this work for its potential use in an eco-cementation process that involves the biomediated calcite precipitation (BCP). Bacterial behavior in extreme alkaline environment (pH values of 9-13) was tested in controlled laboratory conditions and in the presence of solid industry wastes, such as Cement Kiln Dust (CKD) and Lime Kiln Dust (LKD), by evaluating the enzymatic activity and the calcite precipitation capacity. Grain consolidation potential of S. pasteurii was tested for one type of CKD mixed with ground granulated blast-furnace slag (GGBS), with possible bioclogging and biocementation applications. The results revealed the formation of stable biocalcite in the presence of CKD, with a performance depending on the pH-value and free calcium ion content. The BCP induced by S. pasteurii and the recycling of solid wastes, such as CKD with high lime content, is a promising way for different bioclogging and biocementation applications, with benefits in construction costs and reduction of environmental pollution.

Alternative nutrient sources for biotechnological use of Sporosarcina pasteurii.

The potential use of Sporosarcina pasteurii in possible biotechnological applications on a large scale (ground improvement, consolidation of building structures and ornamental stone, or in developing bio-materials for the building industry), is based on its ability to produce high amounts of carbonate in a short period of time via urea hydrolysis. Industrial biomass production would have a low environmental impact and would be most economical if the standard growth media could be replaced with alternative nutrient sources, such as byproducts or wastes from other industries, or other low cost ingredients. The use of cost effective ingredients must guarantee ureolytic activities and growth conditions that are comparable to those resulting from the standard nutrient medium. In this work, three types of alternative media were tested for growing the ureolytic active bacteria S. pasteurii: (1) alternative nutrient sources such as industrial wastes resulting from the dairy and brewery industries, (2) fertilizer urea as an alternative urea substitute, and (3) different types of poultry manure based fertilizers as nutrient and urea substitutes. The comparison between the standard media, the nutrient alternatives and urea substitutes was possible by taking the protein concentration and nitrogen content into account. Bacterial activity was evaluated in terms of biomass changes over time (CFU, optical density, ATP measurements) and indirect estimation of the enzyme production (Nessler assay, conductivity measurement). The results revealed that some of the dairy wastes tested, such as whey and buttermilk, are potential alternative nutrients for bacterial development, while the urea fertilizer is perfectly suitable as an economical substitute for pure laboratory grade urea.

Biodiversity of phototrophic biofilms dwelling on monumental fountains.

Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical-chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of micro-environmental conditions for each fountain.