Bacterial and archaeal communities' composition of uranium-contaminated soils in Bulgaria

The uranium mining waste piles, mill tailing, and disposal sites in several regions of Bulgaria are highly contaminated with toxic metals as a result of the U mining and milling performed in the past. In order to suggest appropriate bioremediation strategies for such environments, the phylogenetic composition and structure of the microbial communities in two uranium-contaminated sites were characterized by using molecular approaches. Three soil samples were studied: two of them were collected from different depths of a U mill tailings (Metallurg Buhovo, located in western Bulgaria), and the third - from a former U mining site (mine Sliven, South-Eastern Bulgaria). 16S rRNA gene libraries were generated from total community DNA, using universal bacterial and archaeal oligonucleotide primer sets. Four hundred fifty bacterial clones were analyzed and grouped in sixty eight operational taxonomic units (OTUs). Three hundred ninety two archaeal clones of the clone libraries from the two U mill tailings soil samples were organized in fourteen archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while the archaeal sequences were less diverse. Most of the bacterial sequences were affiliated with Proteobacteria from Alpha- Beta-, and Gamma- subdivisions. 16S rRNA gene sequences representing Bacteroidetes, Acidobacteria, Verrucomicrobia, Actinobacteria, and Gemmatimonadete were found in various numbers as well. Our results showed that the composition of the microbial communities in the two different studied ecosystems was diverse and environment-specific. Acidobacteria and Bacteroidetes were predominant in the soil samples collected from the U mill tailings, whereas Gammaproteobacteria and Actinobacteria were characteristic for the soil from the U mining waste. All archaeal 16S rRNA gene sequences retrieved from the U mill tailings soil samples were affiliated with the group 1.1b of the mesophilic soil Crenarchaeota which still does not contain any cultured representatives. Closely related 16S rRNA gene sequences were repeatedly found earlier in soil samples from different U mining waste piles and mill tailings in the east part of Germany.
Taken into account the significance of Crenarchaeota in the global nitrogen cycle, the studied soil samples were assayed also for the presence of ammonia monooxygenase subunit A (amoA) gene. Using PCR primers targeting archaeal amoA genes, two clone libraries were constructed and analyzed.