The use of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present on three mural paintings showing severe damage by microorganisms

Abstract

Mural paintings in Carmona (Spain), Herberstein (Austria) and Greene (Germany), showing visible deterioration by microorganisms, were sampled to investigate the biodiversity of the heterotrophic bacteria present. Four hundred twenty-eight bacterial strains were isolated from which 385 were characterized by fatty acid methyl ester analysis (FAME). The isolates were grouped into 41 clusters on the basis of their FAME profiles, 20 isolates remained ungrouped. The majority (94%) of the isolates comprised the gram-positive bacteria and the main clusters were identified as Bacillus sp., Paenibacillus sp., Micrococcus sp., Arthrobacter sp. and Staphylococcus sp. Other clusters contain nocardioform actinomycetes and gram-negative bacteria, respectively. A cluster of the latter contained extreme halotolerant bacteria isolated in Herberstein. The FAME profiles of this cluster showed a high similarity with Halomonas.

Brachybacterium fresconis sp. nov. and Brachybacterium sacelli sp. nov., isolated from deteriorated parts of a medieval wall painting of the chapel of Castle Herberstein (Austria).

“…Analysis of the fatty acid content of the cell wall was performed as described previously (Heyrman et al, 1999), starting from cells grown on TSA for 48 h. Purified cell wall preparations were obtained by the method of Schleifer & Kandler (1972). The amino acids and peptides in peptidoglycan hydrolysates were analysed as described by Groth et al (1999a).…”

“…Four of the wall-painting strains (LMG 20333 and 20335-20337) originated from a paint layer sample of black discoloration located in the chancel vault of the chapel ; nine strains (LMG 20338-20346) originated from black stripes in the upper area of the nave's north wall. The strains were isolated from trypticase soy agar (TSA ; BBL) and R2A agar (Difco) with or without added salt ; the isolation procedure used was described previously (Heyrman et al, 1999). Isolated strains were further grown on marine agar (MA ; Difco) at 28 mC.…”

“…The solvent was 0n02 M NH % H # PO % with 1n5 % acetonitrile (pH 4n0). Non-methylated λ phage DNA was used as the calibration reference.DNA-DNA hybridization was performed using a modification of the microplate method (Ezaki et al, 1989), as described by Willems et al (2001).Analysis of the fatty acid content of the cell wall was performed as described previously (Heyrman et al, 1999), starting from cells grown on TSA for 48 h. Purified cell wall preparations were obtained by the method of Schleifer & Kandler (1972). The amino acids and peptides in peptidoglycan hydrolysates were analysed as described by Groth et al (1999a).…”

“…Four of the wall-painting strains (LMG 20333 and 20335-20337) originated from a paint layer sample of black discoloration located in the chancel vault of the chapel ; nine strains (LMG 20338-20346) originated from black stripes in the upper area of the nave's north wall. The strains were isolated from trypticase soy agar (TSA ; BBL) and R2A agar (Difco) with or without added salt ; the isolation procedure used was described previously (Heyrman et al, 1999 Total genomic DNA was purified for 16S rDNA sequencing and REP-PCR using a slight modification of the method of Pitcher et al (1989), as described by Heyndrickx et al (1996). For determination of the GjC content and DNA-DNA hybridization, approximately 1 g biomass was harvested from agar plates and prepared further using a combination of the protocols of Marmur (1961) and Pitcher et al (1989), as described by Logan et al (2000).…”

Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov., three novel 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

“…To obtain a better insight into the microbial community involved in biodeterioration of mural paintings, three sites (including the necropolis at Carmona, Spain, and the SaintCatherine chapel of the castle at Herberstein, Austria) were sampled and investigated as part of a European research project. In a previous study (Heyrman et al, 1999), 385 strains, isolated from the different mural painting sites and samples, were analysed by fatty acid methyl ester (FAME) GC. This resulted in a grouping of the strains based on their fatty acid profiles.…”