Bacteria from Uranium waste piles and their interactions with uranium(VI) and other metals

Bacterial diversity was studied in drain waters and soil samples drawn from different sites and depths of three uranium mill-tailings - Schlema/Alberoda, Gittersee/ Coschütz (Germany), and Shiprock, New Mexico (USA) as well as in samples drawn from two uranium mining waste depository sites, "Haberlandhalde" near the town of Johanngeorgenstadt and Deponie B1, Germany. For this study both culture-dependent and culture-independent i.e. direct molecular approaches were applied.
The direct molecular analyses involved PCR based rep-APD, 16S rDNA and RISA retrievals [1, 2, 3].
Three parallel rDNA clone libraries (16S rDNA I, 16S rDNA II, and RISA) were constructed for each of the samples studied using three different sets of PCR primer pairs (see Fig. 1).Fig. 1. Clone libraries constructed for the soil and water samples from the uranium wastes analysed

The clones of each of the above mentioned three libraries constructed from the same sample usually indicated the presence of different, not overlapping bacterial groups. This demonstrates the necessity of using more than one PCR primer pair in the construction of the environmental rDNA libraries, because as mentioned by others [4] in such complex mixtures of DNA templates the regions flanking the PCR amplification products are strongly influencing the effectivity and the preferences of the PCR reaction. As a result of these so called biases of the preferential PCR on the end of each amplification an enrichment with particular fragments preferred by the process occurs.
For example, in some samples of the uranium wastes the RISA retrieval demonstrated the predominance of the aerobic ammonia oxidisers Nitrosomonas (b-Proteobacteria) and of the anaerobic chemolithotrophic ammonia oxidizing bacteria named Anammox (Planctomycetales) which were "overseen" by the 16S rDNA retrievals.
The direct molecular approach has demonstrated that, in general, the bacterial populations in the environmental samples studied possess different grade of variability. The highest variability was found in the soil samples drawn from depths between 2.5 m and 5 m below the surface and also in the water sediment samples. Despite the fact that the predominant bacterial species in the studied environments are in general site-specific, many samples share common bacterial groups. These groups belong mainly to the g subgroup of Pseudomonas; to the sulfate and metal reducing genera such as Desulfovibrio, and Geobacter (d-Proteobacteria); to the aerobic chemolithoautotrophs oxidizing iron and sulfur compounds (Acidithiobacillus); or to those oxidizing nitrite (Nitrospina) and ammonium (Nitrosomonas), d- and b-Proteobacteria, respectively. A large number of diverse 16S rDNA sequences was related to various not yet cultured representatives of Holophaga/Acidomicrobium and Cytophaga/ Flexibacter clusters. In addition, many 16S rDNA sequences were affiliated with novel so-called "candidate" bacterial divisions.
Both the RISA and the 16S rDNA retrievals revealed many cases of bacterial microdiversity, i.e. presence of closely related strains belonging to the same species predominating in the natural community.

Analyzsing different individual bacterial isolates as well as mixed enrichment bacterial cultures we have demonstrated the presence of strains belonging to the genera Acidithiobacillus, Agrobacterium, Bacillus, Clostridium, Desulfovibrio, Leptospirillum, Pseudomonas, Rhizobium, Stenotrophomonas, etc. in the samples of the uranium wastes. The exact phylogenetic affiliation of the individual strains studied was performed by the use of the 16S rDNA RFLP and sequence analyses. Many of the strains cultured represent novel bacterial species within the above mentioned genera. In addition, several pure cultures of isolates which are closely related to some previously uncultured bacterial species were cultured.
Interactions of several of the natural uranium waste isolates with uranium (VI), in particular, sor...