Tânia Rosado

Methyl syringate: an efficient phenolic mediator for bacterial and fungal laccases.

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and (1)H NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzymes properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed.

Microbial communities analysis assessed by pyrosequencing--a new approach applied to conservation state studies of mural paintings.

The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium, but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.

Material Characterization and Biodegradation Assessment of Mural Paintings: Renaissance Frescoes from Santo Aleixo Church, Southern Portugal

The aim of this work is the material characterization of wall paintings and biodegradation assessment, including the analysis of microbial growth and the effect of microbial proliferation, in view of their conservation. The methodology was applied to the study of frescoes dated from 1531, located in the ancient parish church of Santo Aleixo, Southern Portugal. The combined use of optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDS) and μ- X-ray diffraction (XRD) showed that the painting palette is composed of red and yellow ochers, malachite, azurite, and bone black. The pigments do not show signs of chemical deterioration, except for malachite, which transformed to black copper oxide (tenorite). The microbiological study allowed the identification of several bacterial strains (e.g., Gram+ cocci, Gram+ bacilli, Actinomycetes sp.), yeast strains, and filamentous fungi of the genera Penicillium, Cladosporium and Aspergillus, among others of the microflora present in the paintings. Their metabolic activity is primarily responsible for the physical disruption of paint layers and underlying mortars. The combined approach using SEM analysis and enzymatic dehydrogenase measurement allowed the evaluation of microflora proliferation and diagnosis of the biodeterioration of the mural paintings. Additionally, the effect of some commercial biocides was evaluated for the predominant strains in order to select the most efficient biocide.

Characterizing Microbial Diversity and Damage in Mural Paintings

Mural paintings are some of the oldest and most important cultural expressions of mankind and play an important role for the understanding of societies and civilizations. These cultural assets have high economic and cultural value and therefore their degradation has social and economic impact. The present work presents a novel microanalytical approach to understand the damages caused by microbial communities in mural paintings. This comprises the characterization and identification of microbial diversity and evaluation of damage promoted by their biological activity. Culture-dependent methods and DNA-based approaches like denaturing gradient gel electrophoresis (DGGE) and pyrosequencing are important tools in the isolation and identification of the microbial communities allowing characterization of the biota involved in the biodeterioration phenomena. Raman microspectrometry, infrared spectrometry, and variable pressure scanning electron microscopy coupled with energy-dispersive X-ray spectrometry are also useful tools for evaluation of the presence of microbial contamination and detection of the alteration products resulting from metabolic activity of the microorganisms. This study shows that the degradation status of mural paintings can be correlated to the presence of metabolically active microorganisms.

Natural limestone discolouration triggered by microbial activity—a contribution

Colour is a major argument that drives the decision of an architect in a specific architecture project and one of the most important characteristics and perceptible aspects of natural building stones. “Blue” limestones are building rocks, with different geological ages, typically used in several countries, and are known for their vulnerability to alteration, which causes colour change and the occurrence of unaesthetic patterns. Owing to this vulnerability, the conservation-restoration works in monuments, or new buildings constructed with “blue” limestone is extremely costly. Considering that the main limitation of this lithological variation is the chromatic change, a multidisciplinary approach was envisaged in this study to allow a closer insight into the chemical and mineralogical alterations and the microbial communities. Results obtained suggest that the inorganic alteration in the “blue” limestone may create favourable conditions for microbial growth and could lead to an increment in deterioration process.

Green mitigation strategy for cultural heritage: bacterial potential for biocide production

Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.