Sílvia Oliveira Sequeira Licenciada em Conservação e Restauro [Nome completo do autor] [Habilitações Académicas] Fungal biodeterioration of paper: Development of safer and [Nome completo do autor]accessible conservation treatments [Habilitações Académicas] Dissertação para obtenção do Grau de Doutor em Conservação e Restauro do Património, [Nome[Título completoda Tese] do autor] Especialidade em Ciências da Conservação [Habilitações Académicas] Dissertação para obtenção do Grau de Mestre em Orientador: Doutora Maria Filomena Macedo Dinis, Professora Auxiliar, FCT-UNL [Engenharia[Nome completo Informática] do autor] [HabilitaçõesCo-orientadores: Académicas] Doutor Eurico José Cabrita, Professor Auxiliar, FCT-UNL Doutor Allan J. L. Phillips, Investigador Principal, FCT-UNL [Nome completo do autor] Júri: [Habilitações Académicas] Presidente: Doutor Fernando Jorge da Silva Pina, Professor Catedrático, FCT-UNL Doutor António Manuel Santos Carriço Portugal, Professor Auxiliar, FCT-UC [Nome completoArguentes: do autor] Doutor Rogério Manuel dos Santos Simões, Professor Associado, UBI [Habilitações Académicas] Doutora Maria Filomena Macedo Dinis, Professora Auxiliar, FCT-UNL Doutora Maria da Conceição Lopes Casanova, Investigadora Auxiliar dos Museus da Universidade de Lisboa Vogais: Doutora Ana Isabel S. C. Delgado Martins, Investigadora Auxiliar dos Museus da [Nome completo do autor] Universidade de Lisboa Doutor César António T. Laia, Investigador Auxiliar do LAQV-REQUIMTE, FCT-UNL [Habilitações Académicas] Abril, 2016 Fungal biodeterioration of paper: development of safer and accessible conservation treatments Copyright © Sílvia Oliveira Sequeira, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa. A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. iii Dedicated to my family v Acknowledgments This PhD was developed at the Conservation and Restoration Department, FCT, Universidade NOVA de Lisboa, (DCR, FCT-UNL); at the Tropical Research Institute (IICT); at the Chemistry Department, FCT-UNL; and at VICARTE Research Unit, UNL. This multidisciplinary work was supported by Fundação para a Ciência e Tecnologia - Ministério da Educação e Ciência (FCT-MEC), with a doctoral grant (SFRH/BD/72216/2010) and the project UID/EAT/00729/2013; and by Unidade de Ciências Biomoleculares Aplicadas - UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). A PhD thesis is always the result of a multitude of collaborations and interdisciplinary work. I would like to express my sincere gratitude to everyone that contributed to the achievements presented in this thesis. To my supervisor Prof. Filomena Macedo, for all the guidance, helpful suggestions, companionship and constant encouragement. To my co-supervisor Prof. Eurico Cabrita, for all the support, valuable advice and friendship. To my co-supervisor Dr. Alan Phillips, for all the important inputs and wisdom. To my thesis advisory committee (Comissão de Acompanhamento de Tese), Prof. António Portugal and Prof. César Laia, for the discussion and valuable advice on the PhD Thesis Plan Evaluation. To all the kind people who made available the respective techniques and instruments and for all the support in analysing the results: Prof. António Candeias and Dr. Gabriela Carvalho (DGPC), for the artificial ageing chamber; Prof. Cesar Laia (DQ, FCT-UNL), for the DLS analyses; Prof. Fernando Pina and Prof. João Carlos Lima (DQ, FCT-UNL), for the photochemistry analyses and discussion; Prof. João Lopes (Faculty of Pharmacy, Univ. of Lisbon), for the analysis of FTIR results; Dr. José Carlos Rodrigues (IICT), for the ATR-FTIR analyses; Prof. José Paulo Sampaio (DCV, FCT-UNL), for the autoclave; Prof. Márcia Vilarigues (DCR, FCT-UNL), for the EDXRF analyses; Prof. Maria João Melo (DCR, FCT-UNL), for the FTIR analyses and discussion; Dr. Miguel Silva (DCR, FCT-UNL), for the ATR-FTIR analyses; and Prof. Rogério Simões (DQ, UBI), for the support with the Folding Endurance analyses and hospitality in UBI. I also thank all the other people who helped me during the experimental work, as well as their valuable comments. To all the participants of the questionnaire, Prof. Luís Ramos for the helpful discussions with the statistical analyses of the obtained results and Conservation DistList manager for the questionnaire distribution and information regarding the number of registered users. To Dr. Ana Cannas and Prof. Conceição Casanova, for all the support in the work developed in IICT and encouragement to start this endeavour. To Professor Telmo Verdelho for the images of the biodeteriorated song book. vii To all my colleagues from labs 302/304 in DQ, the people from DCR and the people from VICARTE, for the companionship and support. A special thank you to Aline Aquino, for her friendship and for helping me in those days where two hands were not enough. To all my PhD colleagues, especially to Ana Margarida Silva, Ângela Ferraz, Élia Roldão, Filipa Pereira, Marta Félix, Mathilda Larson, Susana Belchior and Susana Sá, for all the companionship and shared concerns. A special thank you to Catarina Pinheiro for the text revisions and precious advice during these PhD years. To Arminda Fortes, Catarina Gonçalves, Catarina Mateus, Laura Moura, Laura Domingues, Manuela Carvalho, Marta Costa, Rogério Abreu, Tiago Rodrigues and all my colleagues and friends in IICT. It has always been a pleasure working with you. To my dear professor and friend Luis Elias Casanovas, for being an incredible teacher and human being. To my family and friends for all the distractions from the PhD. My special appreciation and heartiest thanks goes to my parents, for being an extraordinary example and support in what really matters, and to my husband for all the patience and encouragement in this everlasting journey. viii “(…) libraries should be towards the east, for their purposes require the morning light: in libraries the books are in this aspect preserved from decay; those that are towards the south and west are injured by the worm and by the damp, which the moist winds generate and nourish, and spreading the damp, make the books mouldy.” Marcus Vitruvius Pollio (1st century BC) (Pollio, 1826) ix Part of the content of this PhD Dissertation has already been published in peer-reviewed international journals included in the Science Citation Index (SCI): Sequeira, S. O., Coutinho, M. L., Lima, J. C., Phillips, A. J. L., Macedo, M.F., Cabrita, E. J., ‘4-MUF- NAG for fungal biomass determination: scope and limitations in the context of biodeterioration studies’. Jounral of Cultural Heritage (in press). DOI: 10.1016/j.culher.2016.06.002. Sequeira, S. O., Phillips, A. J. L., Cabrita, E. J., Macedo, M.F. (2016) ‘Ethanol as an antifungal treatment for paper: Short-term and long-term effects’. Studies in Conservation (in press). DOI: 10.1080/00393630.2015.1137428. Sequeira, S. O., Cabrita, E. J. and Macedo, M. F. (2014) ‘Fungal Biodeterioration of Paper: How are Paper and Book Conservators Dealing with it ? An International Survey.’ Restaurator, 35(2) pp. 181–199. DOI: 10.1515/rest-2014-0005. Sequeira, S., Cabrita, E. J. and Macedo, M. F. (2012) ‘Antifungals on paper conservation: An overview.’ International Biodeterioration & Biodegradation, 74, pp. 67–86. DOI: 10.1016/j.ibiod.2012.07.011. xi Abstract Great part of the History of mankind is registered in the form of documents or works of art on paper support. Paper can be deteriorated due to physical, chemical and biological agents. Within microorganisms, fungi are the major paper biodeteriogens. Throughout history, several toxic methods have been used to prevent and stop fungal deterioration on paper based materials. More recently, a growing concern about environmental and health issues has led to the research on new antifungal alternatives, with lower toxicity. However, the existent antifungal methods and compounds still have drawbacks in terms of efficacy, health hazards, damaging effects on paper, or lack of thorough testing. In this context, the present thesis focused on testing and developing accessible antifungal treatments with low toxicity, which could prevent the long term paper deterioration. The selection of antifungal compounds was made taking into account the results from a literature review on antifungals used on paper conservation, a survey to paper conservators, and a review of antifungals used in cosmetics and pharmaceutical industries, having as a basic premise their low toxicity. Aspergillus niger, Chaetomium globosum, Cladosporium cladosporioides, Penicillium chrysogenum and Penicillium corylophilum were selected as test fungal species. Fungal growth on paper was evaluated by measuring colonization areas and biomass dry weight determination. A formulation containing parabens and calcium propionate (PBs+CP) presented the best antifungal activity on paper samples, followed by a formulation containing clotrimazole and calcium hydroxide nanoparticles (CLT+NPs), and ultimately 70% ethanol (70%EtOH). Before