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Polyphasic Characterisation of the Bacterial Community Associated with Biodeterioration of Mural Paintings

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UNIVERSITEIT GENT Faculteit Wetenschappen Vakgroep Biochemie, Fysiologie & Microbiologie Polyphasic characterisation of the bacterial community associated with biodeterioration of mural paintings Polyfasische karakterisering van de bacteriële gemeenschap geassocieerd met biodeterioratie van muurschilderingen Jeroen HEYRMAN Academiejaar 2002-2003 Promotor: Prof. Dr. ir. J. Swings Proefschrift voorgelegd tot het behalen Co-promotor: Prof. Dr. P. De Vos van de graad van Doctor in de Wetenschappen (Biologie) DANKWOORD Toegegeven, er is ook wat genetica mee gemoeid, maar het feit blijft dat het leven van een mens voor een groot deel wordt bepaald door de plaats waar zijn wieg staat. Persoonlijk is mijn leven op dat vlak bijzonder gunstig begonnen. Allereerst wil ik dan ook mijn ouders bedanken. Het lijstje met redenen is zo groot dat ik graag dit proefschrift aan hen wil opdragen. Na vele rimpelloze jaren onder moeders vleugels samen met drie beminnelijke zussen, was ik volgens de middelbare school klaar voor verdere studie. Een moeilijke keuze want mijn interesses lagen nogal breed. Het werd biologie in Gent en dat heb ik mij nooit beklaagd want naast een licentiaatsdiploma leverde het mij ook de vrouw van mijn leven op. Brita en ik, vanaf 1e Kan. een team, deden samen onze scriptie op het 9e van de Ledeganckstraat. Johan en Lynda wil ik hierbij nogmaals bedanken voor de fijne tijd en de positieve reacties op ons werk waardoor we aangespoord werden om aan de universiteit te blijven. Via Johan kwam ik in contact met Jean en het Laboratorium voor Microbiologie, nu al weer meer dan vier jaar mijn werkplek. Jean en Paul wil ik beiden heel hartelijk bedanken voor de geboden mogelijkheden en hun vertrouwen in mij door de jaren heen. Het was/is altijd heel aangenaam werken, vaak heb ik mogen meegenieten van hun jovialiteit en meer dan gulle lach. Jean wil ik meer specifiek nog bedanken voor de grote zelfstandigheid die ik van hem kreeg en de goedkeuring van mijn kleurencombinaties (sommige werden “flashy” genoemd). Paul wil ik extra bedanken voor de flexibele werksfeer en de snelle verbeteringen. Alle collega’s van het Laboratorium voor Microbiologie wil ik bedanken voor de fijne werksfeer waar ik zelf heel veel belang aan hecht. Vier en een half jaar op het 4e en 8e gevuld met veel leuke momenten en verrijkende gesprekken o.a. in ‘In den Zeven Billekes’ (oftewel het koffielokaal). In I Dankwoord verband met mijn proefschrift wil ik Margo extra bedanken voor het aanleren van verschillende technieken, Joris voor de inwijding in EU-project- rapportering en An(ke) voor de hulp bij het verder beschrijven van de nieuwe soorten. I would like to thank the colleagues of the MICROCORE-project for the fruitfull collaboration, their hospitality and the pleasant time we shared. Some typical food and meals will also be remembered, such as the delicious salmorejo in Sevilla, the gingerbread men on the day of Saint-Nicolas in Oldenburg and the spaghetti on the china service of the countess of Herberstein. Mijn (schoon)familie en vrienden wil ik bedanken voor hun interesse, maar ook voor de verstrooiing. Het leven is meer dan doctoreren alleen. Naast de thuis waar je geboren wordt, is je verdere levenspartner natuurlijk eveneens van grote invloed op je leven. Ook hier kan ik mijzelf gelukkig prijzen, ‘één uit de duizend’ mag gerust aangehaald worden. Heel veel dankje’s zijn dan ook gericht aan Brita. In verband met dit proefschrift wil ik haar extra bedanken voor het nauwkeurig nalezen op fouten, het geven van het goede voorbeeld en het bewaren van het juiste evenwicht tussen besprekingen over ‘het werk’ en het leven van alledag. Tenslotte wil ik ons eerste kindje bedanken voor het goede gevoel dat hij/zij mij nu al geeft. Duizendmaal dank – Thanks a million Jeroen Heyrman Gent, 13 juni 2002 II CONTENTS Dankwoord I-II Contents III List of non-standard abbreviations V Chapter 1 General introduction 1-22 Chapter 2 The use of fatty acid methyl ester analysis (FAME) 25-36 for the identification of heterotrophic bacteria present on three mural paintings showing severe damage by microorganisms Chapter 3 16S rDNA sequence analysis of bacterial isolates 39-49 from biodeteriorated mural paintings in the Servilia tomb (necropolis of Carmona, Seville, Spain) Chapter 4 Comparative analyses of the bacterial diversity on 51-69 two different biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis Chapter 5 Analysis of the bacterial community associated with 71-79 severe biological damage on the mural paintings of Saint-Martin church (Greene-Kreiensen, Germany) Chapter 6 Brachybacterium fresconis sp. nov., and Brachy- 81-92 bacterium sacelli sp. nov., isolated from deteriorated parts of a medieval wall painting of the chapel of castle Herberstein (Austria) Chapter 7 Halomonas muralis sp. nov., a new species isolated 95-106 from microbial biofilms colonising the walls and murals of the Saint-Catherine chapel (castle Herberstein, Austria) Chapter 8 Virgibacillus carmonensis sp. nov., Virgibacillus 109-132 necropolis sp. nov. and Virgibacillus picturae sp. nov., three new species isolated from deteriorated mural paintings, transfer of the species of the genus Salibacillus to Virgibacillus, as Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov., and emended description of the genus Virgibacillus Chapter 9 Bacillus decolorationis sp. nov., a new species 135-144 isolated from biodeteriorated parts of the mural paintings at the Servilia tomb (Roman necropolis of Carmona, Spain) and the Saint-Catherine chapel (castle Herberstein, Austria) Chapter 10 Concluding remarks 147-158 Summary 161-165 Samenvatting 167-171 References 173-189 Annex 1 – list of strains 191-194 Annex 2 – project descriptions 195-196 Curriculum vitae 197-200 III IV LIST OF NON-STANDARD ABBREVIATIONS Media BRII Bunt and Rovira, medium 2 MA marine agar PY-BHI peptone-yeast extract medium supplemented with brain heart infusion PYGV medium containing peptone, yeast extract, glucose and vitamin solution RCA reinforced clostridial agar TSA trypticase soy agar Materials and methods cfu colony forming units CTAB hexadecyltrimethyl ammonium bromide DGGE denaturing gradient gel electrophoresis EDTA ethylene-diamine-tetra-acetic acid EMBL European molecular biology laboratory FAME fatty acid methyl ester HPLC high-performance liquid chromatography MK Menaquinone UPGMA unweighted pair group method with arithmetic averages RDP ribosomal database project rep repetitive extragenic palindrome SD standard deviation SDS sodium dodecyl sulfate Tm melting temperature TLC thin-layer chromatography Culture collections ATCC American type culture collection CCM Czech collection of microorganisms CECT colección Española de cultivos tipo CIP collection de l’institut Pasteur DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen BBCM/LMG Belgian co-ordinated collections of microorganisms / laboratorium voor microbiologie Gent V CHAPTER ONE General introduction 1.1. Mural paintings (Mora et al., 1984) Below, a brief description of the most commonly used painting techniques, some general trends in the history of mural paintings and some conservation problems of conservation are given. Fresco techniques: fresco refers to any painting executed on fresh plaster whilst still moist in such a way that the pigments are fixed by the carbonisation of the lime (calcium hydroxide) contained in the plaster ground. The pigment mixed with water (fresco puro) or with lime water, or even milk of lime (lime fresco painting), is brushed onto the surface of the rendering or onto whitewash with a lime base. When the latter begins to dry, the saturated calcium hydroxide solution migrates towards the surface where it reacts with the carbon dioxide in the air to form calcium carbonate, as the water evaporates [Ca(OH)2 + CO2 = CaCO3 + H2O]. During the course of this reaction, the pigments become bound by crystallisation of the superficial carbonate. The term fresco can also be used for paintings executed with the addition of tempera medium (see further), such as casein, as long as it is applied on the fresh plaster and the pigments are fixed by carbonisation of the lime. Techniques a secco: This term groups all forms of painting executed on dry plaster or whitewash, where the pigments are fixed by a medium with which they are mixed before their application. The typical technique of mural painting a secco is painting with lime, which consists of applying pigments mixed with milk of lime onto a dry ground previously dampened in order to 1 Chapter one promote adherence. The lime mixed with the pigments acts as a binder. In addition to painting with milk of lime, there are two other main types of technique a secco: tempera and oil. The term tempera applies to techniques where the pigments are mixed with a medium, either aqueous or in emulsion, that fixes the pigments as the medium dries. The main tempera binders used in mural paintings are egg, casein, animal glue and certain vegetable gums. For oil mural paintings, linseed oil and poppy-seed oil have been employed. The oldest mural paintings date from about 30,000 years B.C. They are positive or negative imprints of hands applied to the walls of caves. Later in the Magdalenian period (about 12,000 years ago) the Palaeolithic rock art reached its full development, as demonstrated by the masterpieces at Altamira (ES) and Lascaux (F). In the Neolithic period (about 8000 – 3000 years B.C.), painting began to be associated with architecture. The natural irregular surface of rock was replaced by the plane of the wall, constructed and squared by man, and usually covered with clay rendering that served as a ground for the painting. The Neolithic technique was perfected in the great agrarian civilisations, e.g. Egypt and Mesopotamia. The origin of the first a fresco painting is disputed. However, one can say that in Greece the fresco technique was really introduced and refined and that the technique was further perfected in Rome by the general use of a final polishing.
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  • Ep 2434019 A1

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    (19) & (11) EP 2 434 019 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 28.03.2012 Bulletin 2012/13 C12N 15/82 (2006.01) C07K 14/395 (2006.01) C12N 5/10 (2006.01) G01N 33/50 (2006.01) (2006.01) (2006.01) (21) Application number: 11160902.0 C07K 16/14 A01H 5/00 C07K 14/39 (2006.01) (22) Date of filing: 21.07.2004 (84) Designated Contracting States: • Kamlage, Beate AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 12161, Berlin (DE) HU IE IT LI LU MC NL PL PT RO SE SI SK TR • Taman-Chardonnens, Agnes A. 1611, DS Bovenkarspel (NL) (30) Priority: 01.08.2003 EP 03016672 • Shirley, Amber 15.04.2004 PCT/US2004/011887 Durham, NC 27703 (US) • Wang, Xi-Qing (62) Document number(s) of the earlier application(s) in Chapel Hill, NC 27516 (US) accordance with Art. 76 EPC: • Sarria-Millan, Rodrigo 04741185.5 / 1 654 368 West Lafayette, IN 47906 (US) • McKersie, Bryan D (27) Previously filed application: Cary, NC 27519 (US) 21.07.2004 PCT/EP2004/008136 • Chen, Ruoying Duluth, GA 30096 (US) (71) Applicant: BASF Plant Science GmbH 67056 Ludwigshafen (DE) (74) Representative: Heistracher, Elisabeth BASF SE (72) Inventors: Global Intellectual Property • Plesch, Gunnar GVX - C 6 14482, Potsdam (DE) Carl-Bosch-Strasse 38 • Puzio, Piotr 67056 Ludwigshafen (DE) 9030, Mariakerke (Gent) (BE) • Blau, Astrid Remarks: 14532, Stahnsdorf (DE) This application was filed on 01-04-2011 as a • Looser, Ralf divisional application to the application mentioned 13158, Berlin (DE) under INID code 62.