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Table 2 (continued). Phenotypic features of presumably novel isolates detected in this study.

Table 2 (continued). Phenotypic features of presumably novel isolates detected in this study.

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Fig. 1. Distribution of median viable counts on plates inoculated with cave silver samples upon five weeks of incubation at 30°C with respect to growth medium. The box-plots show minimum, maximum, the 25 and 75 percentile and median. The growth medium with significant effect on viable counts (Fisher’s least significant difference P < 0.05) is indicated by an asterisk. We did a phylogenetic analysis on the representative sequences of OTUs for 80 isolates obtained in this study and relevant environmental sequences present in the GenBank database (Figs. 2, 3). The levels of diversity identified using cultivation dependent and independent approaches were notably different. Actinobacteria made up the dominant group among cultivated strains (47%) and among the environmental sequences recovered from Pajsarjeva jama cave silver samples (16%, Pasi¢ et al., 2010). Unfortunately, we could not cultivate Actinobacterial phylotypes that were dominant in environmental 16S rRNA gene libraries (Fig. 2). Ins tead, the OTUs represented by 16S rRNA sequences of isolates C3 (7.5% of all isolates obtained), F2 (2.5%), C10 (2. 5%), E4 (3.8%) and D11 (2.5%) were 299% similar to 16S rRNA sequences of Rhodococcus wratislaviensis, Rhodococcus erythropolis, Kocuria kristinae, Microbacterium paraoxidans, and Agrococcus citreus, respectively. OTUs represented by isolates All (16.3%), D8 (2 The 5%),
Fig. 1. Distribution of median viable counts on plates inoculated with cave silver samples upon five weeks of incubation at 30°C with respect to growth medium. The box-plots show minimum, maximum, the 25 and 75 percentile and median. The growth medium with significant effect on viable counts (Fisher’s least significant difference P < 0.05) is indicated by an asterisk. We did a phylogenetic analysis on the representative sequences of OTUs for 80 isolates obtained in this study and relevant environmental sequences present in the GenBank database (Figs. 2, 3). The levels of diversity identified using cultivation dependent and independent approaches were notably different. Actinobacteria made up the dominant group among cultivated strains (47%) and among the environmental sequences recovered from Pajsarjeva jama cave silver samples (16%, Pasi¢ et al., 2010). Unfortunately, we could not cultivate Actinobacterial phylotypes that were dominant in environmental 16S rRNA gene libraries (Fig. 2). Ins tead, the OTUs represented by 16S rRNA sequences of isolates C3 (7.5% of all isolates obtained), F2 (2.5%), C10 (2. 5%), E4 (3.8%) and D11 (2.5%) were 299% similar to 16S rRNA sequences of Rhodococcus wratislaviensis, Rhodococcus erythropolis, Kocuria kristinae, Microbacterium paraoxidans, and Agrococcus citreus, respectively. OTUs represented by isolates All (16.3%), D8 (2 The 5%),
ND — no colonies were detected upon eight-week incubation at 30°C. Table 1. Total viable counts (CFU/g) obtained from pretreated cave silver samples.
ND — no colonies were detected upon eight-week incubation at 30°C. Table 1. Total viable counts (CFU/g) obtained from pretreated cave silver samples.
Fig. 2. ML tree showing the phylogenetic position of OTU representatives (prefix ‘cave bacterium PJ') belonging to Actinobacteria. Numbers at nodes (>75%) indicate percentages of 1000 bootstrap resamplings. Sequences with prefix ‘uncultured actinobacterium clone PJM' originate from previous study (Pa§&ié et al., 2010).
Fig. 2. ML tree showing the phylogenetic position of OTU representatives (prefix ‘cave bacterium PJ') belonging to Actinobacteria. Numbers at nodes (>75%) indicate percentages of 1000 bootstrap resamplings. Sequences with prefix ‘uncultured actinobacterium clone PJM' originate from previous study (Pa§&ié et al., 2010).
with respect to sample colour (Table 3). Twelve isolates originated from the yellow sample, 29 from grey, 15 from white and 24 from a pink coloured sample. Common to all samples were the abundant groups belonging to the genera Agrobacterium and Streptomyces, while isolates belonging to Rhodococcus, Pseudomonas, Paenibacillus, Micrococcus and Arthrobacter were present in at least medium and isolates related to Agrococcus only on SEA. The pre-treatments did not increase the diversity of isolates obtained in pure culture. In fact, only a single isolate was obtained exclusively from a pre-treated sample (microwave irradiation, a non-Streptomyces actinomycete). Some differences in diversity of pure cultures were observed if the isolates were grouped
with respect to sample colour (Table 3). Twelve isolates originated from the yellow sample, 29 from grey, 15 from white and 24 from a pink coloured sample. Common to all samples were the abundant groups belonging to the genera Agrobacterium and Streptomyces, while isolates belonging to Rhodococcus, Pseudomonas, Paenibacillus, Micrococcus and Arthrobacter were present in at least medium and isolates related to Agrococcus only on SEA. The pre-treatments did not increase the diversity of isolates obtained in pure culture. In fact, only a single isolate was obtained exclusively from a pre-treated sample (microwave irradiation, a non-Streptomyces actinomycete). Some differences in diversity of pure cultures were observed if the isolates were grouped
Fig. 3. ML tree showing the phylogenetic position of OTU representatives (prefix 'cave bacterium PJ') belonging to Proteobacteria and Firmicutes Numbers at nodes (>75%) indicate percentages of 1000 bootstrap resamplings. Sequences with prefixes ‘uncultured gamma proteobacterium clone PJM' and ‘uncultured alpha proteobacterium clone PJM' originate from a previous study (Pasic¢ et al., 2010).
Fig. 3. ML tree showing the phylogenetic position of OTU representatives (prefix 'cave bacterium PJ') belonging to Proteobacteria and Firmicutes Numbers at nodes (>75%) indicate percentages of 1000 bootstrap resamplings. Sequences with prefixes ‘uncultured gamma proteobacterium clone PJM' and ‘uncultured alpha proteobacterium clone PJM' originate from a previous study (Pasic¢ et al., 2010).
Table 3. The similarities of 16S rRNA sequences of OTU representatives detected in this study to 16S rRNA sequences present in GenBank. Presumably novel taxa (S97% sequence identity to cultivated species) are indicated in bold. “Abbreviations Y, W, G and P correspond to yellow, white, grey and pink cave microbial colony sample.
Table 3. The similarities of 16S rRNA sequences of OTU representatives detected in this study to 16S rRNA sequences present in GenBank. Presumably novel taxa (S97% sequence identity to cultivated species) are indicated in bold. “Abbreviations Y, W, G and P correspond to yellow, white, grey and pink cave microbial colony sample.
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