THE TWO SIDES OF MICROORGANISMS IN CULTURAL HERITAGE CONSERVATION

Biodeterioration is any irreversible change in the properties of a material caused by the activity of organisms belonging to different systematic groups. Microorganisms (bacteria, fungi, yeasts, algae and lichens) are considered one of the most important biodeteriogens, thanks to their ability to colonise the surface of both organic and inorganic artworks. Cultural heritage biodeterioration commonly results from the complex interaction established by different kind of microorganisms co-existing simultaneously and the substrate. But then, microorganisms can also be considered biological agents rich in virtues; in fact, they can be used for the conservation of stone works of art. The potential of microorganisms for effective bioremediation of deteriorated cultural heritage materials is gradually being unveiled, and promising results of this methodology have been obtained in field sites. As a consequence, it should not come as a surprise that conservators are forever asking microbiologists how advances in biotechnologies could contribute to the conservation of heritage. The aim of this work was to investigate the two sides of microorganisms in the cultural heritage conservation, starting from some case-studies. In particular the objectives were to:  characterise the microbial communities on parchment manuscripts together with the study of the microbial airborne communities and the environmental physical conditions in close proximity to where the manuscripts are stored, suggesting some general guidelines for the correct management of historical documents;  reduce the biological treatment time and assess the effects of a chemical and sulfate-reducing bacteria (SRB) co-treatment for the bioremoval of black crusts and grey deposits on a marble statue;  broaden the fields of application of biocleaning technologies on stone conservation, proposing a valid alternative to the traditional cleaning methodologies for the bioremoval of synthetic polymers from stone monuments. The first step to assess the microbiological risk of a cultural heritage object is to identify the entire microbial community colonising art objects, using non-invasive sampling, or sampling that needs only small amounts of material. Most of the literature dealing with microbial spoilage on documents use invasive sampling methods and culture approaches for the identification of the species causing spoilage. However, traditional culture methods can be time-consuming, do not always succeed in isolating microbial agents, and do not necessarily provide exhaustive information on the real microbial community load because only a small fraction of the microorganisms can be cultivated. In addition, aerobiological investigations of conservation environments are also helpful in choosing interventions aimed at establishing the sources of contamination and preventing the microbiological deterioration of artworks. During the last decade, in fact, the environmental conditions of the museum exhibition facilities and storage areas have been shown to be the most crucial factor, concerning the preservation of collections and artifacts. Until now few microbiological studies have been conducted on parchment. Chapter 3 reports the evaluation of the microbial risk for the conservation of seven 16th century manuscripts written on finely illuminated parchment. The aims of the work were: (a) to clarify any relationship between the presence of an active microbial community and discolouration, (b) to study microbial air quality and environmental conditions in the repository, and (c) to investigate the relationship between airborne and surface-associated microbial communities. For the first time, the microbial community on historical parchment has been investigated by both non-invasive sampling and fully culture-independent approaches, coupling an aerobiological monitoring of the repository by an exclusively biomolecular approach. On discolouration putatively caused by microbial colonisation, two non-invasive sampling techniques were chosen: nitrocellulose membrane and fungi-tape. The nitrocellulose membranes were gently pressed onto the surface of the manuscripts to collect the cells; the DNA was directly extracted from the membrane and culture-independent molecular approach based on PCR-DGGE was adopted to fully characterise the surface-associated communities. Fungi-tape was chosen for investigating the spatial distribution of microorganisms, staining the tape with two fluorescent dyes (SYTO9 and Fluorescent Brightener 28, to label bacteria and chitin in the walls of fungi, respectively). Epifluorescence microscopy observations of the tape samples, coupled with ATP assay on the surfaces of the manuscript, showed a low contamination level and that the discolouration was not related to currently active microbial colonisation. The airborne microflora was sampled with a MAS-100 portable bioaerosol sampler, for monitoring the microbial loads, and the AGI-30 impinger, for fully characterising airborne communities with culture-independent molecular methods. Potential biodeteriogens, as the fungus Aspergillus fumigatus, and microorganisms ecologically related to humans, as Candida sp., were found, suggesting the need to control the conservation environment and improve handling procedures. The proteololytic activity of A. fumigatus, often isolated from libraries and museums, represents a potential risk for library materials; besides all these potential deterioration effects, A. fumigatus is an opportunistic human pathogen. Air microbial loads and thermo-hygrometric measurements showed that the repository was not suitable to prevent parchment microbial deterioration, suggesting the environmental remediation of the repository and the routine monitoring of air. In addition to microorganisms, other important causes of stone deterioration are physical and chemical agents; a chief factor in the weathering of stone monuments is the atmospheric pollution. Sulfur dioxide is one of the major gaseous components of polluted atmospheres in urban areas; when dissolved in water, sulfur dioxide forms sulfurous acid, which is oxidised to sulfuric acid, that attacks stone resulting in the formation of gypsum. During the crystallization of gypsum, airborne organic pollutants, carbonaceous particles and dust are accumulated at the surface of buildings and trapped in the mineral matrix, resulting in the formation of the black crusts. The black crust formation causes a heavy decay phenomenon, due to solubilization and recrystallization of calcium sulfate. Mechanical and chemical treatments are traditionally adopted for the black crust removal, but they are not always selective; moreover, when there are different weathering forms on a stone surface, it is difficult to use a single cleaning procedure. In this respect, an enormous contribution in the preservation of stonework can come from the microbial world. In Chapter 4 the removal of sulfate-based crusts from stone artworks using an alternative cleaning technology employing the sulfate-reducing bacteria (SRB) is discussed. SRB reduce sulfate to gaseous hydrogen sulfide and, in the last decade, they have been effectively used for the removal of gypsum and black crust. This biological methodology has been proven to be an effective cleaning procedure if compared with the traditional cleaning techniques. However, some concerns still remain: it is a time-consuming process when the crust is thick, and it is not clear how the biotechnological approach can fit within a complex conservation treatment. To address these challenges, the effects of an SRB strain (Desulfovibrio vulgaris subsp. vulgaris ATCC 29579) coupled with the non-ionic detergent Tween 20 pretreatment was studied on a stone column affected by black crusts. Stereomicroscope observations, X-ray diffraction, Fourier Transformed Infrared Spectroscopy and Environmental Scanning Electron Microscopy analyses were performed. The coupling of the two cleaning treatments removed the black crust without affecting the original sound marble, with 38% reduction in cleaning time. The combined method was later applied to the one-century-old artistic marble statue, a funeral monument realized by Lina Arpesani in 1921 in memory of ‘Neera’, the poetess Anna Zuccari. The chemical, mineralogical and morphological analyses confirmed that the statue was weathered by sulfate-based crusts and grey deposits. A barium sulfate layer was found in the black crust stratigraphy, most probably deriving from an undocumented conservation treatment employing the barium hydroxide technique. The detergent Tween 20 used alone effectively removed the grey deposit, but not the black crust. However the co-treatment synergy resulted in the complete removal of the black crust layers, with the added advantage, compared to the biocleaning alone, of fewer biological applications and a 70% reduction in total cleaning time, but still retaining all the advantages of the biocleaning approach. Bacteria were also able to reduce the barium sulfate being completely removed by the microbiological method. Over the past decades, synthetic resins have been applied on stone monuments, both as consolidants, protectives and adhesives, to enhance their long-term preservation. Although it is generally thought that synthetic resins are less prone to chemical, physical and biological deterioration than other organic products, there are some reports in the conservation of cultural heritage literature claiming the contrary. The chemical degradation of synthetic polymers include changes in chemical structure, reduction or increase in molecular weight due to chain scission and crosslinking respectively. Microorganisms are also able of degrading synthetic resins, producing enzymes that can attack the resins, causing changes in the viscosity, pH and colour of polymer emulsions. At present, the traditional way for removing a degraded synthetic polymer is the use of mixtures of solvents, that pose some health risks. In Chapter 5 a method to select bacteria able to remove synthetic polymers from cultural heritage surfaces is proposed; synthetic polymers, in fact, can act as a growth substrate for microorganisms. The ability of five bacteria to attack Paraloid B72, the most commonly used polymer in conservation treatments, was evaluated by optical and scanning electron microscopy observations, weight loss measurements, Fourier Transform Infrared Spectroscopy and Differential Scanning Calorimetric analysis. As all these techniques have proved to be very useful techniques in both polymer science and microbiological investigations, they were applied to evaluate naturally aged Paraloid B72 susceptibility to bacteria. Pseudomonas rne (PA01), P. stutzeri (ATCC 23856), P. putida (DeFENS collection, isolated from wastewater treatment plant), Escherichia coli (ATCC 25404) and Bacillus licheniformis (DeFENS collection, isolate from a biodeteriorated acrylic painting on canvas by a contemporary artist) were the test bacteria used as inocula for biodegradation tests. Although none of the bacteria were able to attack Paraloid B72, the methodology developed can be applied to select other bacteria with this ability. Therefore the results offer insightful guidance to a better design of bioremoval experiments of synthetic resins used in conservation. Biotechnology has been proved promising for a selective and environmental/health safe approach in the cultural heritage field, for the removal of altered layers on stone monuments. Consequently, each study that relates with the use of viable microorganisms for bioremoval is an advance in the cultural heritage conservation field. In conclusion, this project shows that: • non-invasive sampling coupled with selective molecular techniques have proved effective for an exhaustive investigation of both stained surfaces of historical documents and air quality of the repository where the documents are stored, and very useful for their correct management; • bioremediation techniques, using SRB, can fit within a complex conservation treatment using a soft detergent Tween 20, resulting in homogeneous, satisfactory and rapid removal of black crusts; • bioremediation techniques, promising in the cultural heritage preservation, can be implemented, opening new horizons of applications, e.g. the removal of synthetic coatings from stone monuments. Biotechnology for the preservation and restoration of culturally relevant artworks not only deals with the identification of the living organisms by molecular-based techniques but also with the development of bio-based methodologies that contribute to the biocleaning of weathered artworks. Presenting three exemplary case-studies, this thesis makes a contribution to cultural heritage preservation and restoration.