Bio-sustainable Control of the Blue Stain Fungi Aureobasidium pullulans on Exterior Wood Coatings PhD Thesis by Jonas Stenbæk Supervisor Associated prof. Bo Jensen Section of Microbiology, Department of Biology, Faculty of Science University of Copenhagen This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen Cover page: eSEM image of Aureobasidium pullulans (image: Peter Falkman and Jonas Stenbæk). This is for my mother Anne September 22nd 1957 - July 27th 2015 Abstract Imminent requirements and demands to the composition of protective wood coatings and the use of commercial biocides lead to an increased and necessary interest by the paint industry to find and develop alternative solutions within substitutions of raw materials and new innovative anti-fungal strategies. Besides comprehensive future regulations in the requirements and laws within the use of fossil-based raw materials, companies also have to take the increasing consumer awareness of environmental responsibility into account necessitating the companies to use more environmentally friendly solutions in the production, composition and service life of the protective coatings. Today’s commercial biocides in the wood coating industry, e.g. IPBC, are proven environmental toxic and exposed to future regulations or indeed complete exclusions so new bio-sustainable alternative solutions are demanded. In this PhD study, a number of alternative and innovative solutions and strategies have been investigated and a number of possible new solutions and reviews are presented. The focus has been on the control of blue stain fungi e.g. Aureobasidium pullulans that lead to discoloration on processed wood and wood protective coatings. In this study, a number of anionic compounds like fatty acid-based emulsifiers have been screened for anti-fungal properties. The best candidate, a Sodium Caproyl Lactylate, has further been tested in wood panel tests and the mode of action on the fungal cell membrane has been investigated. The compound exhibits promising anti-fungal properties, however, more in-depth investigation on the compound and closely similar compounds are recommended before an actual and useable technology are accessible. Other compounds like silica-encapsulated enzymes and nanoclay have also been investigated and interesting preliminary results makes ground for further studies based on promising anti-fungal properties when added to a standard coating without commercial biocides and tested in environmental mould growth chambers. As a non-additive biocidal solution the structure of the topography on the surface on the coating has been studied. In this study, manipulation of the micro structure on the coating surface has been performed and revealed that small scratches-structure (between 4-20 µm) had an inhibitory effect on the attachment on fungal conidia from moulds while bigger structure scratches (>50 µm) did not seem to have a notable effect. Abstract A comprehensive study in the role of an interfacial protein, hydrophobin, in the surface growth of Aureobasidium pullulans resulted in bioinformatic identification of two hydrophobin genes, first ever described in a blue stain fungi. The full genome of Aureobasidium pullulans (De Bary) P268 was sequenced and the bioinformatic information led to molecular gene manipulation in the attempt to clarify the role of hydrophobins in surface growth of Aureobasidium pullulans. Overall, this PhD thesis presents a range of studies in alternative and bio-sustainable solutions and strategies in controlling attacks of blue stain fungi on wood protective coatings. The results provide a state-of-art platform for further research and development of alternative options in the control of moulds in coatings. Resumé Stigende krav til sammensætningen af træbeskyttende coatings og kommercielle biocider forventes at føre til en øget interesse hos coating-industrien for både at udvikle alternative løsninger inden for substitution af råmaterialer og for at finde nye, innovative strategier inden for svampebekæmpelse. Udover de stigende krav og regler inden for brugen af fossilt baserede råvarer, er virksomhederne også nødt til at tage hensyn til forbrugernes øgede bevidsthed om miljøansvarlige forbrugsvaner. Dette nødvendiggør, at virksomhederne fremover i højere grad må anvende mere miljøvenlige løsninger inden for produktion, produktindhold og produktets levetid. Nutidens kommercielt anvendte biocider i coating-industrien, eksempelvis IPBC, er giftige for miljøet og vil forventeligt på et tidspunkt blive udsat for restriktioner eller ultimative forbud. Nye bio-bæredygtige alternative løsninger efterspørges derfor allerede på nuværende tidspunkt af industrien. Dette ph.d.-studie undersøger og præsenterer en række alternative og innovative løsninger samt strategier inden for bæredygtige og miljøvenlige biocider, som er mulige fremtidige løsninger. Fokus har været på bekæmpelse af blåsplint svampe, som eksempelvis Aureobasidium pullulans, der fører til misfarvning af forarbejdet træ og træbeskyttelse, herunder udendørs træmaling. I dette studie er en række anioniske forbindelser, som fedtsyre-baserede emulgatorer, blevet screenet for svampehæmmende egenskaber. Den bedste kandidat - en Sodium Caproyl Lactylat (SCL) - er blevet testet yderligere i træpanels-tests. Derudover er virkemåden af SCL på svampens cellemembran blevet undersøgt, og samlet set udviser SCL lovende svampebekæmpende egenskaber. Dog anbefales en udvidet undersøgelse af SCL, og dens nært beslægtede stoffer, før en endelig og brugbar teknologi kan introduceres. Andre additiver, herunder silica-indkapslede enzymer og nanoclay, er også blevet undersøgt, og foreløbige interessante resultater kan danne grundlag for yderligere studier. Dette er baseret på lovende svampehæmmende egenskaber, når additiverne blandes i en standard coating uden indhold af kommercielle biocider og efterfølgende testes i skimmelvækst-kamre. Coating-topografien i mikro-skala er blevet undersøgt med henblik på at belyse eventuelle muligheder for svampehæmmende effekter uden brug af additiver. I dette studie er Resumé mikrostruktur på coating-overfladen blevet manipuleret, så den har fået en præcis ridser- struktur. Det viste sig, at en fin typografi (mellem 4-20 µm) havde en hæmmende virkning på vedhæftningen af konidier fra skimmelsvampe, mens større struktur-ridser (> 50 µm) ikke syntes at have en mærkbar hæmmende virkning. En omfattende undersøgelse af et overfladeaktivt protein, kaldet hydrofobin, havde fokus på proteinets rolle ved overfladevækst hos blåsplint-svampen Aureobasidium pullulans. Undersøgelsen resulterede i bioinformatisk identifikation af to hydrofobin-gener i Aureobasidium pullulans. Det er de første hydrofobiner, som er beskrevet i en blåsplintssvamp. Aureobasidium pullulans (De Bary) P268 er blevet sekventeret, og det fulde genom er nu tilgængeligt. Bioinformatiske oplysninger førte til molekylærbiologisk genmanipulation i forsøget på at klarlægge hydrofobinernes rolle ved overfladevækst hos Aureobasidium pullulans. Samlet set præsenterer denne ph.d.-afhandling både en række studier i alternative og bio- bæredygtige løsninger samt strategier for kontrol af svampeangreb fra blåsplints svampe på træbeskyttende coatings. Resultaterne giver en state-of-art platform for yderligere forskning og udvikling af alternative og innovative biocider. Table of Contents Table of contents 1 Preface and Acknowledgements 3 List of Manuscripts 5 Conference Proceedings 6 PhD Course Portfolio 9 Teaching and Assistant Supervision 10 Change of Scientific Environment 11 Abbreviations 12 Chapter 1: Background and Aims 15 1.1 Superior Bio Based Coating System for Exterior Wood Applications 1.2 The overall aim: Control of A. pullulans in bio-sustainable coating systems for ex. wood 1.2.1 Time line 1.2.2 Structure of this PhD thesis 1.3 References Chapter 1 Chapter 2: Introduction 21 2.1 Blue Stain and protective coatings 2.2 IPBC 2.3 Aureobasidium pullulans 2.3.1 Morphology and life cyclus. 2.3.2 Pullulan 2.4 Methods and materials 2.4.1 Strains 2.4.2 Full-genome sequences 2.4.3 Growth tests 2.4.4 Wood panel tests in Environmental Chamber 2.4.5 Protoplasts 2.5 Chapter 2 references Chapter 3: Fatty acid-based emulsifiers: possible biological agents 43 3.1 Introduction 3.2 Candidates 3.2.1 Lactylates 3.3 Mode of action 3.3.1 Supported lipid bilayers (SLB) 3.3.2 Quartz Crystal Microbalance with Dissipation (QCM-D) 3.4 Preliminary results 3.5 Conclusions and perspectives for further research 3.6 References Chapter 3 1 Table of Contents Chapter 4: Enzymes, Nano clay and other additives 55 4.1 Introduction 4.2 Enzymes 4.2.1 Pullulanase 4.2.2 Glucanex 4.2.3 Aerogel – encapsulation of enzymes. 4.3 Nanoclay 4.4 Materials and Methods 4.4.1 Enzymes 4.4.2 Nanoclay 4.5 Preliminary results and discussions 4.5.1 Enzymes 4.5.1 Nanoclay 4.6 Conclusions and perspectives for further research 4.7 References Chapter 4 Chapter 5: Topography; Surface Structure on Coatings 71 5.1 Introduction 5.2 Materials and Methods 5.3 Conclusions and perspectives for further research Chapter 6: Hydrophobins; a Unique Protein in Fungal Surface Living 73 6.1 Introduction 6.1.1 Nature of the Hydrophobins 6.1.2 Classification and protein structure 6.1.3 Properties 6.1.4 Aims of the work related to hydrophobins 6.2 Results and discussion 6.2.1 Analysis of the Aureobasidium pullulans strain P268 hydrophobins 6.2.2 Disruption of the hydrophobin encoding genes, hfbA and hfbB 6.2.2.1 Evaluation of potential double gene