BIOTECHNOLOGIES FOR RESTORATION OF CULTURAL HERITAGE

Abstract In recent years, operators in the restoration sector are adding to their historical-artistic competences also scientific knowledge in order to find solutions more and more effective and respectful toward the cultural heritage, the operator and the environment. Among the different scientific branches, biotechnologies allow for an innovative and precise approach to the complexity of the problems that the restorer has to face in his own daily work. Biotechnology research in the field of cultural heritage develops in two directions: on the one hand focuses on the development of accurate diagnostic techniques, useful for the correct identification and characterization of alterations and biodeteriogens; on the other hand focuses on the development of innovative restoration methods, based on the employment of new products. The employment of biotechnologies in restoration of cultural heritage is the main topic of the present PhD doctoral thesis, which deals with both of the aforementioned sides. In Chapter 1 a review on the employment of biotechnologies in the field of cultural heritage is presented, considering both diagnostic techniques for characterization of biodeteriogens, and the use of microorganisms and enzymes for restoration. The first part of the thesis focuses on a microbial product, based on sulfate-reducing bacteria (SRB) belonging to D. vulgaris species, applied for the removal of sulfate crusts from artwork surfaces. In studies carried out in the last decades, this product has turned out to be very promising and favorable, compared to traditional restoration techniques, thanks to its capability of combining effectiveness to selectivity and safety for the restorer and the environment. Such a technology, original, innovative and sustainable, has been successfully experimented on important artworks. In Chapter 2 a review on the current knowledge of Desulfovibrio genus is presented, in particular concerning its physiology, biochemistry and biotechnological applications. Nevertheless, the D. vulgaris-based product presented four limitations: i) low production yields and inability of long-term conservation of D. vulgaris biomass; ii) lack of an appropriate method for monitoring of abundance and activity of the biomass; iii) time-consuming application technique; iv) application limited to stone surfaces. The overcoming of the above mentioned limitations has been the aim of the first part of the present work. A research work, structured in different phases, has been conducted for the optimization of the production process and the development of a method for the long-term conservation of the bacterial biomass (Chapter 3). Initially the laboratory protocol has been set up on small volumes at liter scale, in order to define the growth curve of the bacterium and evaluate its metabolic response to different substrates and growth conditions. subsequently the fermentative process has been transferred from the flask to 5 lt fermentor, optimizing the control of pH and H2S concentration. H2S is the main metabolic product in the fermentative process of SRB, its accumulation is toxic for bacteria, leading to unfavorable growth conditions. These improvements allowed a significant increase in biomass production, from a concentration of 1*108 cell/ml in 120h of fermentation in flask, to the concentration of 3*109 cell/ml in 72h of fermentation in bioreactor. For the long-term conservation of D. vulgaris biomass, freeze-drying has been carried out, testing the effectiveness of different cryoprotective agents. Among them, the best in terms of cell viability post-rehydration resulted to be lactose, which ensured the stability of the product for a minimum of 6 months. Chapter 4 deals with the development of new molecular approaches for monitoring of D. vulgaris biomass concentration and viability since traditionally employed methods, such as Most Probable Number (MPN) and microscope counting, resulted unsuitable. The research focused on the set up of a method applicable not only to liquid cultures, but also to cells embedded in the delivery system used for the applications of D. vulgaris cells on the surfaces during the biorestoration treatment. Among all the tested methods, the most effective and suitable resulted to be the spectrophotometric measurement of the fluorescence specifically emitted by the prosthetic group of bisulfite reductase, a key enzyme in dissimilatory sulfate reduction. The results showed that fluorescence emission is proportional to viable cells present in liquid culture as well as when embedded in the delivery system. Real-time PCR quantification of the SRB-specific dsr gene allowed to significantly quantify D. vulgaris cells in liquid culture, but when applied on cells embedded in the delivery system the detection limit (107 cell/ml) was too high to make this method efficient. The development of new methodologies for the application of D.vulgaris-based product, aimed at the reduction of time and number of required applications for the removal of sulfations, has been conducted on the funeral monument realized in memory of ‘Neera’, the poetess Anna Zuccari, located in the Cimitero Monumentale in Milan (Chapter 5). Besides biological treatment, two other methods have been tested: chemical treatment, based on the non-ionic detergent Tween 20 and a combined treatment, consisting in a chemical pre-treatment followed by the biological treatment. The combined method resulted to be effective in the removal of the black crust, without altering the underlying stone, obtaining a 70% reduction in cleaning time. Moreover, the combined method preserved all the advantages of the biocleaning approach: selectivity toward the alteration and respectfulness toward the original material. For the purpose of extending this biocleaning approach to substrates other than stone artworks, such as mural paintings, an experimentation has been carried out on two scenes belonging to the pictorial cycle decorating ‘Queen Teodolinda Chapel’ in Monza Cathedral (Chapter 6). The applicability test on surfaces characterised by fragility, such as pigmented surfaces, is of primary importance for the further development of this technology. The obtained results can be regarded very promising, in terms of sulfations removal and respectfulness towards such a delicate surface. However, this study has to be considered merely preliminary and incomplete, and further research must be conducted in order to verify the compatibility of the treatment with different kinds of materials, such as pigments. The last part of this thesis focuses on diagnostic methodologies for the identification and characterisation of biodeteriogens from two artworks. In the past, microorganisms responsible for deterioration of cultural assets were identified through conventional methods based on the cultivation of potential biodeteriogen microorganisms and their identification and phenotypic characterisation. Here molecular biology technologies independent from bacteria cultivation were employed, which complete and expand the information provided by the cultivation-dependent approach. These methodologies have been employed for the analysis of an acrylic monochrome painting on canvas realised by the artist E. Castellani (Chapter 7) and of a paper print realized in the 17th century, conserved in the Monastery of “S. Maria al Carrobiolo” in Monza (Chapter 8). The acrylic monochrome painting on canvas presented alterations characterised by yellow-earth/red point areas of different extensions, spread on the whole posterior surface. Molecular analyses on the total microbial community have been carried out through Denaturing Gradient Gel Electrophoresis (DGGE) method. Among the identified bacteria, the most abundant were Bacillus subtilis, Bacillus licheniformis and Deinococcus gobiensis; whereas among fungi, Leptosphaerulina and Penicillium genera. Culture-dependent techniques confirmed the dominance of bacteria belonging to Bacillus genus, whilst no fungal species has been isolated. However, presence of fungi was confirmed by microscope analysis, which allowed the visualization of fungal hyphae and spores in all the samples. Considering morphology and dimensions, these structures visualized by microscope were ascribable to mycelia and spores of fungi belonging to Penicillium genus, confirmed also by comparison with literature images. Afterwards the characterization of microbial community, the activity of four different biocides on the potential biodeteriogens was evaluated. Biotin N, Biotin R, New Des 50 and Amuchina (in single and mixed) were tested toward the single microbial isolates and the whole microbial community. According to the antibiogram test, the combination of Biotin R 4% in ethyl acetate + Biotin N 4% in white spirit resulted to be the most effective in terms of inhibiting activity, both on single strains and on the whole bacterial community. The paper print realized in the 17th century, conserved in the Monastery of “S. Maria al Carrobiolo” in Monza, presented whitenings and small dark spots, on the obverse and on the reverse side, respectively. In this case, a microbial investigation was executed through culture-dependent techniques for the isolation of bacteria and fungi, in order to characterize the possible deteriogens and determine their phylogenetic affiliation. The results demonstrated a negligible presence of bacteria. The most frequently cultured strains belonged to the Staphylococcus genus, which is associated to human skin, and to the Sphingomonas genus, which is an environmental bacterium, which have never been associated to biodeteriogen activity. As concerns fungi, the results showed a dominant presence of Neurospora pannonica, both on the obverse and on the reverse side of the print. In summary, the research emphasized the importance of biotechnologies in the field of cultural heritage. The optimization of D. vulgaris-based product, described in the first part of the work, has been successful, therefore this result underlines the importance of research for the improvements of biotechnological methodologies employed in restoration. The overall results suggest that further research is required for additional enhancement of this sulfates removal methodology and for the development of novel approaches, more and more effective and convenient, to be used in the field of cultural heritage.