Molecular analysis of bacteria cultured from uranium mining waste piles

The main problem of the direct molecular approaches for studying environmental samples is the so called preferential PCR amplification which can mask the presence of some DNA templates in the samples.
For this reason the application of the classical approach of the enrichment cultures, in parallel to the molecular retrievals, for studies on bacterial diversity in the environment may be helpful. Of course, here the limitation to analyze only those strains of the community which are able to grow individually exists. It is a compromise to analyze enriched biofilm cultures or mixed cultures instead of pure cultures, because most of the bacteria in the extreme environments are living in consortia which are described as symbiotic [1, 2]. The identification of the members of such mixed cultures using the 16S rDNA retrieval may provide very important information in addition to those, derived by the direct methods, about the bacteria present in the samples studied.
Our studies on phylogenetic diversity of bacteria cultured as pure and/or mixed cultures from the uranium wastes are presented in Table 1.
From these results one can see that bacterial strains known for their ability to biotransform metals were recovered from the samples. For example, different types of Acidithiobacillus ferrooxidans, which are oxidizing iron and sulfur compounds and Leptospirillum ferrooxidans , oxidizing iron, were cultured. These two groups of bacteria play a significant role in the mobilization of many metals and radionuclides in the wastes, because as a result of their physiological activity large amounts of H2SO4 are produced and the pH in their surrounding is extremely low.
Individual cultures representing sulfate reducing bacteria of the genus Desulfovibrio which are able to reduce also uranium and other metals were cultured from the uranium mining waste samples as well.
In addition, strains belonging to the species Pseudomonas stutzeri, Pseudomonas migulae, Erwinia herbicola (g-Proteobacteria) as well as isolates related to Agrobacterium and Sphingomonas (a-Proteobacteria) were cultured.
In addition to the above described individual bacterial isolates several enrichment mixed cultures were recovered. One of them, called initially "JG1", from which the sulfate reducing Desulfovibrio isolate JG1 was purified, consisted of 11 diverse representatives of Clostridium (see the series of clones IrT-JG1 affiliated to the Bacillus/Clostridium group in Table 1) and the two clones IrT-JG1-58 and IrT-JG1-71 closely related to the isolate Desulfovibrio JG1.
Formerly we have reported that the mixed culture of "JG1" is able to reduce and precipitate amounts of about 1.5 g of U(VI) per g of dry weight bacterial biomass from a liquid medium independently on its pH value in the range between 2.8 and 6.0 [3]. Our recent results demonstrated, however, that the U(VI) reducing capacity of the pure culture of Desulfovibrio sp. JG1 does not differ significantly from those published for the other uranium reducing bacteria, Desulfovibrio vulgaris, D. desulfuricans, and Geobacter metallireducens which is about five times lower and, in addition, it is pH dependent with an optimum of pH 6.8 [4, 5]. This indicates that the higher capability of the mixed culture "JG1" to precipitate uranium at a wider range of environmental conditions (pH for example) is a result of the combined function of the different bacterial members of which it consists. We suppose that in addition to the U(VI) reduction performed by the above mentioned natural strains of Desulfovibrio sp. (JG1, IrT-JG1-58 and JrT-JG1-71) some other processes occur, as bio-precipitation or/and bio-minearalisation stimulated by the metabolic functions of the described Clostridia present in the mixed culture.
Another mixed culture "JG14" consisted of different a-Proteobacteria (Agrobacterium and Rhizobium) and a microdiverse population of environmental strains of Stenotrophomonas maltopilia (d-Proteobacteria). ...