International Biodeterioration & Biodegradation 46 (2000) 229–239 www.elsevier.com/locate/ibiod Comparative analyses of the bacterial diversity on two dierent biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis Claudia Gurtner a ; ∗ , Jeroen Heyrman b , Guadalupe Pi˜ nar a , Werner Lubitz a , Jean Swings b; c , Sabine R olleke a a Institute of Microbiology and Genetics, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria b Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium c BCCM=LMG Culture Collection, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 13 April 2000; received in revised form 19 May 2000; accepted 19 June 2000 Abstract The bacterial diversity associated with two dierent biodeteriorated wall paintings in Herberstein (Austria) and Greene (Germany) was investigated and compared using a molecular approach combining ngerprinting by DGGE (denaturing gradient gel electrophoresis) with the screening of 16S rDNA clone libraries by DGGE and sequencing. In total, 70 16S rDNA sequences were obtained. Twenty-three sequences were phylogenetically aliated with genera of the Actinobacteria, namely Arthrobacter, Actinobispora, Amycolata, Asiospo- rangium, Frankia, Geodermatophilus, Nocardioides, Promicromonospora, Pseudonocardia, Rubrobacter, Streptomonospora, Saccha- ropolyspora, Sphaerobacter and Thermocrismum. Twenty-seven sequences were aliated with genera of the Proteobacteria, namely Aquaspirillum, Chromohalobacter, Deleya, Erythrobacter, Halomonas, Porphyrobacter, Pseudomonas, Rhizobium, Salmonella and unidentied -Proteobacteria. Nineteen sequences were aliated with unidentied Cytophagales. One sequence was aliated with the Chloroexaceae group. Most genera were present in more than one sample. The bacterial communities present on the two dierent wall paintings showed only similarities in members of unidentied Cytophagales and of the genera Frankia, Geodermatophilus and Arthrobacter. Cultivation experiments for one sample were carried out in parallel to the molecular approach. Isolates were clustered by FAME (fatty acid methyl ester analysis) and representative members of each cluster were additionally analyzed by DGGE. No similar organisms could be detected by the cultivation approach and the molecular approach. Isolates were phylogenetically aliated to the genera Bacillus, Paenibacillus, Micrococcus, Staphylococcus, Methylobacterium and Halomonas. The sequence of the isolated Halomonas diered from the Halomonas sequences, which were obtained by the molecular approach. The combined approach of molecular and culturing techniques gives a truer picture of all bacterial organisms on= in a surface than either alone. c  2000 Elsevier Science Ltd. All rights reserved. Keywords: DGGE; Wall paintings; Biodeterioration; 16S rDNA 1. Introduction It is a well-established knowledge that wall paintings can be attacked and destroyed by microorganisms. Wall paintings provide a variety of ecological niches, allowing primary colonization by photoautotrophic and chemolitho- autotrophic bacteria as well as secondary growth by het- erotrophic bacteria. Nutrients for heterotrophic bacteria are ∗ Corresponding author. Tel.: 0043-1-4277-54675; fax: 0043-1-4277- 54674. E-mail address: gurtifox@gem.univie.ac.at (C. Gurtner). available from metabolites of autotrophic bacteria, from airborne organic contamination and dripping water, from animal faeces and, last but not least, from organic com- pounds in the paint layers themselves. Concerning wall paintings, pigments are often suspended in water or oil, mostly together with organic binders such as casein, egg yolk and milk before application on the damp lime plas- ter. Organic substances may also be applied later, during restoration campaigns. Microbial-induced deterioration processes cause struc- tural as well as aesthetic damage to wall paintings. The formation of pigmented biolms, biomineralization, the 0964-8305/00/$ - see front matter c  2000 Elsevier Science Ltd. All rights reserved. PII: S0964-8305(00)00079-2