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, 9, 1985
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Microbial Community and in situ Bioremediation of Groundwater by Nitrate Removal in the Zone of a Radioactive Waste Surface Repository

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Microbial Community and in situ Bioremediation of Groundwater by Nitrate Removal in the Zone of a Radioactive Waste Surface Repository

Alexey V Safonov et al. Front Microbiol.

Abstract

The goal of the present work was to investigate the physicochemical and radiochemical conditions and the composition of the microbial community in the groundwater of a suspended surface repository for radioactive waste (Russia) and to determine the possibility of in situ groundwater bioremediation by removal of nitrate ions. Groundwater in the repository area (10-m depth) had elevated concentrations of strontium, tritium, nitrate, sulfate, and bicarbonate ions. High-throughput sequencing of the V3-V4/V4 region of the 16S rRNA gene revealed the presence of members of the phyla Proteobacteria (genera Acidovorax, Simplicispira, Thermomonas, Thiobacillus, Pseudomonas, Brevundimonas, and uncultured Oxalobacteraceae), Firmicutes (genera Bacillus and Paenibacillus), and Actinobacteria (Candidatus Planktophila, Gaiella). Canonical correspondence analysis suggested that major contaminant - nitrate, uranium, and sulfate shaped the composition of groundwater microbial community. Groundwater samples contained culturable aerobic organotrophic, as well as anaerobic fermenting, iron-reducing, and denitrifying bacteria. Pure cultures of 33 bacterial strains belonging to 15 genera were isolated. Members of the genera Pseudomonas, Rhizobium, Cupriavidus, Shewanella, Ensifer, and Thermomonas reduced nitrate to nitrite and/or dinitrogen. Application of specific primers revealed the nirS and nirK genes encoding nitrite reductases in bacteria of the genera Pseudomonas, Rhizobium, and Ensifer. Nitrate reduction by pure bacterial cultures resulted in decreased ambient Eh. Among the organic substrates tested, sodium acetate and milk whey were the best for stimulation of denitrification by the microcosms with groundwater microorganisms. Injection of these substrates into the subterranean horizon (single-well push-pull test) resulted in temporary removal of nitrate ions in the area of the suspended radioactive waste repository and confirmed the possibility for in situ application of this method for bioremediation.

Keywords: 16S rRNA gene; denitrifying bacteria; high-throughput sequencing; in situ groundwater bioremediation; microbial ecology; nirS and nirK genes; nitrate removal; surface radioactive waste repository.

Figures

FIGURE 1
FIGURE 1
Schematic representation of the suspended surface repository for liquid radioactive waste (A) and location of the observation wells (B) outside the zone of waste spreading (well A) and in the contamination zone (wells B, C, D, and E). The direction of groundwater movement is indicated by an arrow. (1) sandy protective layer; (2) plant layer; (3) upper clay screen; (4) crushed stone; (5) sandy soil; (6) pulp; (7) gravel-sand rammed layer; (8) clay screen; (9) waterproof layer; (10) zone of waste spread.
FIGURE 2
FIGURE 2
Taxonomic classification of bacterial 16S rRNA gene fragments in the libraries from groundwater samples at the phylum level (at the class level for Proteobacteria) using the RDP classifier.
FIGURE 3
FIGURE 3
Heatmap analysis of the dominant genera distribution in seven groundwater samples. The double hierarchical dendrogram shows microbial distribution in these samples. The relative values for microbial genera are marked by colors from green to red designating the least abundant to most abundant. Abundance is expressed as the number of targeted sequences to the total number of sequences from each groundwater sample.
FIGURE 4
FIGURE 4
Phylogenetic tree of the 16S rRNA genes of pure cultures isolated from groundwater (shown in boldface). The libraries containing the OTUs (phylotypes) closely related to the 16S rRNA genes of the isolates (over 98% similarity) are shown in parentheses. The scale shows the evolutionary distance corresponding to five nucleotide replacements per 100 nucleotides. The numerals indicate the branching order determined by bootstrap analysis of 100 alternative trees (the values above 80 were accepted as significant). Accession numbers of 16S rRNA genes of isolated strains are given in Supplementary Table S3. α-PB, Alphaproteobacteria; β-PB, Betaproteobacteria, and γ-PB, Gammaproteobacteria.
FIGURE 5
FIGURE 5
Concentrations of HCO3, NO3, and NO2 in groundwater from wells D (A) and B (B) before and after injection of organic substrates into the subsurface horizon.
FIGURE 6
FIGURE 6
Canonical correspondence analysis (CCA) showing the correlation between bacterial diversity at phylum or class level in the 16S rRNA gene libraries of the microorganisms from groundwater and geochemical parameters of the groundwater samples.
FIGURE 7
FIGURE 7
Canonical correspondence analysis showing the correlation between bacterial diversity at the genus level and geochemical parameters of the groundwater samples.

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