Galina Radeva

Bacterial diversity in soil samples from two uranium waste piles as determined by rep-APD, RISA and 16S rDNA retrieval

The bacterial diversity in two uranium waste piles was studied. Total DNA was recovered from a large number of soil samples collected from different sites and depths in the piles using two procedures for direct lysis. Significant differences in the bacterial composition of the samples were revealed by the use of rep-APD, RISA and 16S ARDREA. The 16S rDNA analyses showed that the uranium wastes were dominated by Acidithiobacillus ferrooxidans and by several Pseudomonas species classified in the γ-subdivision of the Proteobacteria. The three kinds of A. ferrooxidans 16S and IGS rDNA specific fragments that were found corresponded to the three phylogenetic groups recognised in this species. This microdiversity probably reflects the genetic adaptation of the uranium waste strains to different concentrations of heavy metals.

Community level physiological profiles of bacterial communities inhabiting uranium mining impacted sites.

Bacterial activity and physiological diversity were characterized in mining and milling impacted soils collected from three abandoned uranium mine sites, Senokos, Buhovo and Sliven, using bacterial dehydrogenase activity and Biolog (EcoPlate) tests. The elemental composition of soils revealed high levels of uranium and heavy metals (sum of technogenic coefficients of contamination; TCC(sum) pollution as follows: Sliven (uranium - 374 mg/kg; TCC(sum) - 23.40) >Buhovo (uranium - 139.20mg/kg; TCC(sum) - 3.93) >Senokos (uranium - 23.01 mg/kg; TCC(sum) - 0.86). The physiological profiles of the bacterial community level were site specific, and indicated intensive utilization of polyols, carbohydrates and carboxylic acids in low and medium polluted environments, and i-erithrytol and 2-hydroxy-benzoic acid in the highly polluted environment of Sliven waste pile. Enzymes which take part in the biodegradation of recalcitrant substances were more resistant to pollution than these from the pathways of the easily degradable carbon sources. The Shannon index indicated that the physiological diversity of bacteria was site specific but not in line with the levels of pollution. A general tendency of increasing the importance of the number of utilizable substrates to bacterial physiological diversity was observed at less polluted sites, whereas in highly polluted sites the evenness of substrate utilization rate was more significant. Dehydrogenase activity was highest in Senokos upper soil layer and positively correlated (p<0.01) with the soil organic matter content. The bacterial activity (EcoPlate) and physiological diversity (Shannon index) correlated significantly and negatively with As, Cu, Zn, Pb and U, and Co, Cr, Ni and Mn, respectively. We concluded that the observed site specific shifts in bacterial communities were complex due to both the environmental peculiarities and the bacterial tolerance to the relevant level of pollution, rather than a strong indication of uranium and heavy metals toxicity.

Effects of long-term radionuclide and heavy metal contamination on the activity of microbial communities, inhabiting uranium mining impacted soils

Ore mining and processing have greatly altered ecosystems, often limiting their capacity to provide ecosystem services critical to our survival. The soil environments of two abandoned uranium mines were chosen to analyze the effects of long-term uranium and heavy metal contamination on soil microbial communities using dehydrogenase and phosphatase activities as indicators of metal stress. The levels of soil contamination were low, ranging from ‘precaution’ to ‘moderate’, calculated as Nemerow index. Multivariate analyses of enzyme activities revealed the following: (i) spatial pattern of microbial endpoints where the more contaminated soils had higher dehydrogenase and phosphatase activities, (ii) biological grouping of soils depended on both the level of soil contamination and management practice, (iii) significant correlations between both dehydrogenase and alkaline phosphatase activities and soil organic matter and metals (Cd, Co, Cr, and Zn, but not U), and (iv) multiple relationships between the alkaline than the acid phosphatase and the environmental factors. The results showed an evidence of microbial tolerance and adaptation to the soil contamination established during the long-term metal exposure and the key role of soil organic matter in maintaining high microbial enzyme activities and mitigating the metal toxicity. Additionally, the results suggested that the soil microbial communities are able to reduce the metal stress by intensive phosphatase synthesis, benefiting a passive environmental remediation and provision of vital ecosystem services.

Localization of nif genes on large plasmids in Rhizobium galegae

Plasmid DNA of six strains of Rhizobium galegae was blotted onto nitrocellulose and hybridized with the 4.8 kb PstI fragment of pRme4lb, a megaplasmid carrying the nifH and the nifD genes of Rhizobium meliloti. DNA sequences homologous to the nif genes were localized on the megaplasmid or on the large plasmid bands of the R. galegae strains tested. In three of the strains analysed the nif genes were located on the megaplasmids. In the other three strains investigated, which also possessed megaplasmids, the nif genes were located on the smaller plasmids.

Molecular Characterization of the Archaeal Diversity in Vlasa Hot Spring, Bulgaria, by using 16S rRNA and Glycoside Hydrolase Family 4 Genes

ABSTRACT Biodiversity in the archaeal community from Vlasa hot spring, Velingrad, Bulgaria was investigated by sequence analysis of PCR amplified fragments of 16S rDNA and a metabolic gene of glycoside hydrolase 4 family (GH4). The 16S rRNA gene analysis demonstrated that the spring was inhabited predominantly by Crenarchaeota affiliated to two orders, Desulfurococcales (families Pyrodictiaceae and Desulfurococcaceae) and Thermoproteales. Almost half of the 16S rDNA clones were affiliated with hyperthermophilic anaerobic sulfate reducer Thermosphaera aggregans. Five 16S rDNA sequences were under cut off value of 97% homology to those of Genbank database and suggested the existence of novel phylogenetic units in the community. The archaeal diversity of the studied hot spring was further analyzed through sequence analysis of metagenomicaly cloned GH4 gene fragments. The comparative 16SrDNA and GH4 phylogenetic analyses demonstrated good correlation of the phylogenetic tree topology from both approaches, corresponding to the affiliation of the identified 16S rDNA sequences predominantly to organotrophic metabolizing taxons. The opportunity for simultaneous application of two molecular approaches, 16S rDNA and metabolite genes analyses for in dept characterization of environmental samples and directed metagenomic identification and cloning of metabolite genes of industrial interest is discussed.

Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria

Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

Soil Bacterial Abundance and Diversity of Uranium Impacted Area in North Western Pirin Mountain

ABSTRACT The former uranium mine Senocos (Blagoevgrad district, Bulgaria) has been exploited until 1991 when it was closed and later (1994–1997) reclaimed. Recently, the reclamation is compromised due to the erosion of protective layers and the mine wastes continue to affect the area resulting in increased radiation and uranium concentration in mine area. Soil bacterial abundance in more radioactive environment of mine remains unchanged (on average ((8.52±5.1) x 108 cell g−1) compared to the control ((8.76±3.5) x 108 cell g−1) in contrast to their dehydrogenase activity which decreases more than two times. Physiological and species diversity of bacterial mine communities are also affected by the pollution as it is expressed by lower values of Shannon indices compared to the control communities. All carbon sources in BIOLOG assay except 2-hydroxy benzoic acid are available for control bacterial communities and the number of unavailable carbon sources increases linearly with increasing the concentration of uranium in soil. The high levels of radionuclide pollution decrease the biodiversity to 3–4 species (66% of total) in the most polluted point of the mine territory (SPS(5)).

Microbial Diversity in Zostera Sp. Beds in South-Western Black Sea Region Analyzed by Amplified Ribosomal DNA Restriction Enzyme Analysis (ARDRA)

ABSTRACT Recent declines in sea grass distribution in south-western Black sea coast arise the need of understanding microbial community relationships in sea grass beds that might affect the viability of these plants. Several factors and their impact on bacterial community were investigated, including the presence or absence of vegetation, depth into sediment, and a gradient of eutrophication formed from pollution point-source. Amplified Ribosomal DNA Restriction enzyme Analysis (ARDRA) showed similar community profiles, thus demonstrating no difference in dominant bacterial members in connection with vegetation. Although highly diverse, sediment community showed also stable annual tendency. This suggested no effect of anthropogenic impact on the main characteristics of bottom sediments in the Zostera endangered meadows.