Identification and control of fungal contamination in ancient heritage documents Nuno Miguel da Costa Pinheiro Meneses Mesquita Setembro 2013 Dissertação apresentada à Universidade de Coimbra para a obtenção do grau de Doutor em Biologia, especialidade em Biologia Molecular, realizada sob a orientação científica do Professor Doutor António Manuel Santos Carriço Portugal e co-orientação da Professora Doutora Helena Maria de Oliveira Freitas do Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra. This research was funded by Fundação para a Ciência e a Tecnologia (FCT) and the European Social Fund through the Programa Operacional Potencial Humano (POPH) of the National Strategic Reference Network, with the reference SFRH/BD/41287/2007. This work was also funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional Fatores de Competitividade (POFC-COMPETE) and national funds, through FCT under the project with the reference PTDC/HAH/65262/2006. Acknowledgements To Fundação para a Ciência e a Tecnologia for sponsoring this work with a PhD grant (SFRH/BD/41287/2007). To my supervisor, Professor António Portugal, for his trust in having me as a PhD student; for his friendship, advice and utmost support in so many different matters. To Professor Helena Freitas, my co-supervisor, for the open-minded ideas and advice. To the Centre for Functional Ecology of the University of Coimbra, for allowing me to perform and develop my thesis. To Professor Teresa Gonçalves, for all the interesting conversations and support. To João Loureiro for his support and advice during the flow cytometry experiences. To my lab partners, in alphabetical order (to prevent malicious sample contaminations in the future): Diana, Elisa, Gabriela, Helena, Hugo, John, Sandra, Susana and Trovão. To the Archive of the University of Coimbra, for providing the sample documents and site for most of this work. A special thank you for the precious support and unceasing interest and readiness of Dr. Ana Maria Bandeira, and to Dr. Maria José Azevedo Santos, for her interest and cooperation. To the LETAL team from the ITN, a very friendly and supporting group who have helped me so much in the course of this thesis. A special thank you to Sandra Cabo-Verde, Inês Nunes, Luísa Alves, Telma Silva and to, of course, their remarkable leader, idea-generating and co- supervisor of my thesis Dr. Maria Luísa Botelho. To Professor Katja Sterflinger and Dr. Guadalupe Piñar from the BOKU in Vienna (Austria) for their support, opinions and advice. To my family, for the unceasing support in all the matters that really matter. Special love to my mom, dad and sis, and to Aurora and Miguel for making my days brighter. Finally, to all other friends that keep have always supported me, that won´t be mad for not being individually referenced here! Table of Contents ABSTRACT .................................................................................................................................. 1 RESUMO .................................................................................................................................... 5 THESIS OUTLINE .......................................................................................................................... 9 GENERAL INTRODUCTION ............................................................................................................ 15 Fungi ................................................................................................................................. 17 Mycotoxins ....................................................................................................................... 19 Damage and damage assessment .................................................................................... 21 Foxing ............................................................................................................................... 24 Sampling and analysis ...................................................................................................... 25 Support materials ............................................................................................................. 27 Biodeterioration Control ................................................................................................... 30 Contamination spreading and vectors .............................................................................. 41 Objectives ......................................................................................................................... 45 CHAPTER 1 – FUNGAL DIVERSITY IN ANCIENT DOCUMENTS: A CASE-STUDY ON THE ARCHIVE OF THE UNIVERSITY OF COIMBRA. ................................................................................................................................ 49 1.1 Introduction ................................................................................................................ 51 1.2 Materials and methods ............................................................................................... 52 1.3 Results ......................................................................................................................... 54 1.4 Discussion ................................................................................................................... 58 1.5 Acknowledgments ....................................................................................................... 59 CHAPTER 2 - ASSESSING THE FUNGAL DIVERSITY IN AN ANCIENT PARCHMENT: THE PATHOGENIC POTENTIAL OF EXISTING MYCOFLORA AS A THREAT TO THE HEALTH OF USERS. ........................................................... 63 2.1 Introduction ................................................................................................................ 65 2.2 Materials and methods ............................................................................................... 67 2.3 Results ......................................................................................................................... 69 2.4 Discussion ................................................................................................................... 73 2.5 Acknowledgments ....................................................................................................... 76 CHAPTER 3 - ASSESSING GAMMA RADIATION EFFECTS IN A CLADOSPORIUM CLADOSPORIOIDES STRAIN ISOLATED FROM AN ANCIENT DOCUMENT: CFU-COUNT VS. GROWTH PARAMETERS. .......................................... 79 3.1 Introduction ............................................................................................................... 81 3.2 Materials and methods .............................................................................................. 82 3.3 Results ........................................................................................................................ 85 3.4 Conclusion .................................................................................................................. 89 CHAPTER 4 – FLOW CYTOMETRY AS A TOOL TO ASSESS THE EFFECTS OF GAMMA RADIATION ON THE VIABILITY, GROWTH AND METABOLIC ACTIVITY OF FUNGAL SPORES. ................................................................. 95 4.1 Introduction ............................................................................................................... 97 4.2 Materials and Methods .............................................................................................. 99 4.3 Results and Discussion ............................................................................................. 102 4.4 Conclusions .............................................................................................................. 110 CHAPTER 5 - CAN ARTHROPODS ACT AS VECTORS OF FUNGAL DISPERSION IN HERITAGE COLLECTIONS? A CASE STUDY ON THE ARCHIVE OF THE UNIVERSITY OF COIMBRA. ............................................................. 115 5.1 Introduction ............................................................................................................. 117 5.2 Materials and methods ............................................................................................ 119 5.3 Results ...................................................................................................................... 125 5.4 Discussion ................................................................................................................ 127 5.5 Acknowledgements .................................................................................................. 133 GENERAL DISCUSSION ............................................................................................................. 137 CONCLUSIONS ........................................................................................................................ 147 Future prospects ............................................................................................................ 148 REFERENCES .......................................................................................................................... 153 Abstract Archives, libraries and museums worldwide protect and conserve different historical collections. These buildings frequently present suitable conditions for the growth of fungi and bacteria, which are able to deteriorate documents by exploring small niches made available in items from these collections. This multidisciplinary work