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, 7 (7), 1286-98

Enrichment of Specific Protozoan Populations During in Situ Bioremediation of Uranium-Contaminated Groundwater

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Enrichment of Specific Protozoan Populations During in Situ Bioremediation of Uranium-Contaminated Groundwater

Dawn E Holmes et al. ISME J.

Abstract

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

Figures

Figure 1
Figure 1
Microbial community analyses of groundwater samples collected during the Fe(III)-reducing phase of the 2010 field experiment. (a) Estimates of bacterial and protozoan cell numbers calculated from 16S rRNA and protozoan β-tubulin (b2t) gene copy numbers estimated by qPCR (Pearson's correlation, r=0.90, P=0.015). (b) The number of Breviata β-tubulin and Geobacter citrate synthase (gltA) gene copies per μg total DNA (r=0.51, P=0.02). (c) Percentage breakdown of bacterial 16S rRNA gene sequences detected in clone libraries constructed from genomic DNA extracted from groundwater. (d) Percentage breakdown of protozoan 18S rRNA gene sequences detected in clone libraries constructed from genomic DNA extracted from groundwater.
Figure 2
Figure 2
Phylogenetic tree comparing 18S rRNA gene sequences detected in the groundwater to sequences from previously characterized protozoa.
Figure 3
Figure 3
Analysis of bacterial and protozoan communities in the groundwater during the Fe(III)-reducing phase of the 2011 field experiment. (a) Estimates of bacterial and protozoan cell numbers calculated from 16S rRNA and protozoan β-tubulin (b2t) gene copy numbers estimated by qPCR (Pearson's correlation, r=0.59, P=0.02 (b) The number of Breviata β-tubulin (b2t) and Geobacter citrate synthase (gltA) gene copies per μg total DNA (r=0.63, P=0.01). (c) Percentage of Breviata 18S and Geobacter 16S rRNA gene sequences in clone libraries compared to Fe(II) concentrations (r=0.68, P=0.01). (d) The number of Geobacter gltA and Breviata β-tubulin mRNA transcripts normalized against the number of Geobacter gltA and Breviata β-tubulin gene copies (r=0.75, P=0.1).
Figure 4
Figure 4
Analysis of the bacterial and protozoan communities found in the groundwater during the sulfate-reducing phase of the 2011 field experiment. (a) H2S concentration (μℳ) and the number of dsrA gene copies per μg total DNA (r=0.67, P=0.002). (b) Percentage breakdown of bacterial 16S rRNA gene sequences detected in clone libraries. (c) Proportion of Peptococcaceae 16S rRNA gene sequences compared with the number of dsrA gene copies per μg total DNA (r=0.79, P=0.002).
Figure 5
Figure 5
Phylogenetic tree comparing 16S rRNA gene sequences from sulfate-reducing Peptococcaceae species detected in the groundwater to sequences from previously described sulfate-reducing prokaryotes.
Figure 6
Figure 6
Analysis of the protozoan community associated with the sulfate-reducing phase of the 2011 field experiment. (a) Percentage breakdown of protozoan 18S rRNA gene sequences detected in clone libraries. (b) Percentage of Hexamitidae 18S and Peptococcaceae 16S rRNA gene sequences in clone libraries compared with sulfide concentrations. Peptococcaceae vs Hexamitidae rRNA sequences (r=0.64, P=0.02); Hexamitidae vs H2S concentrations (r=0.67, P=0.02). (c) Comparison of Hexamita β-tubulin (b2t) and Desulfosporosinus dsrA gene copies per μg total DNA (P=0.57, P=0.03). (d) The number of Desulfosporosinus dsrA and Hexamita β-tubulin mRNA transcripts normalized against the number of dsrA and β-tubulin gene copies (r=0.53, P=0.05).

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