Sunlight-exposed biofilm microbial communities are naturally resistant to chernobyl ionizing-radiation levels

Abstract

Background: The Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well.

Methodology/principal findings: To test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates.

Conclusions/significance: Our results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation.

Figure 1. Sampling sites.

The red stars indicate the two major sampling areas in Northern Ireland and Chernobyl (Ukraine). The map on the right shows the location of the sampling sites in the Chernobyl area according to the background level of radiation (µSv/h) (adapted from [47]). The photographs at the bottom show some examples of biofilms growing on concrete walls or pillars that were sampled.

Figure 2. Relative distribution of major bacterial and eukaryotic taxa in SSU rRNA gene libraries from concrete-associated biofilm microbial communities.

Figure 3. Phylogenetic tree of SSU rDNA sequences from Chernobyl concrete biofilms belonging to the Proteobacteria.

The tree was reconstructed by maximum likelihood using 767 non-ambiguously aligned positions. Bootstrap values higher than 50% are given at nodes. Colored circles show the presence of the OTU in the different samples, and the internal number corresponds to the number of occurrences of sequences in the corresponding gene libraries. Unc., uncultured.

Figure 4. Phylogenetic tree of bacterial SSU rDNA sequences to the exclusion of Proteobacteria retrieved from sunlight-exposed biofilms in Chernobyl.

The tree was reconstructed by maximum likelihood using 634 non-ambiguously aligned positions. Bootstrap values higher than 50% are given at nodes. Colored circles show the presence of the OTU in the different samples, and the internal number corresponds to the number of occurrences of sequences in the corresponding gene libraries. Unc., uncultured.

Figure 5. Phylogenetic tree of eukaryote SSU rDNA sequences from Chernobyl sunlight-exposed biofilms.

The tree was reconstructed by maximum likelihood using 1,199 non-ambiguously aligned positions. Bootstrap values higher than 50% are given at nodes. Colored circles show the presence of the OTU in the different samples, and the internal number corresponds to the number of occurrences of sequences in the corresponding gene libraries. Unc., uncultured.

Figure 6. Cluster analysis of DGGE fingerprints for bacteria (A) and eukaryotes (B) in Chernobyl concrete biofilms.

The histogram shows the relative levels of radioactivity measured in situ in Chernobyl samples (µSv/h).

Figure 7. Percentage of sequence identity at SSU rRNA genes and adjacent ITS regions in the same Rubrobacter OTU from different populations.

Note that the scale at the ordinates begins at 92% to maximize potential differences between samples and markers. The bars correspond to the standard deviation.

Figure 8. Relationship between SSU rRNA and the corresponding ITS variability (number of differing nucleotides) by comparison with Northern Ireland control sequences.

Each graph corresponds to a sampled location in the Chernobyl area. Each color represents a particular reference sequence: red (Ref-127), green (Ref-128), blue (Ref-2).

Figure 9. Adjusted means by population of the number of differing nucleotides as compared to the control Northern Ireland sequences (Ref-127, Ref-128, Ref-2 from left to right).

The Y-axis represents the number of expected differences when assuming a similar level of SSU rRNA variation set to the general mean (i.e. ca. 13 differing nucleotides).