Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site

Abstract

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

FIG. 1.

Stratigraphy of borehole logs. Borehole logs collected from the in situ treatment plot installed at the Old Rifle site after 40 days of acetate injection.

FIG. 2.

Layout of the in situ test plot installed at Old Rifle. (a) Schematic layout of Old Rifle UMTRA site indicating location of monitoring wells and sediment cores. (b) Schematic layout indicating sampling depth positions on multilevel samplers (MLS) installed in monitoring wells B02, M02, M03, M08, and M13 and core subsections for collected sediment cores.

FIG. 3.

Geochemical and molecular analysis of groundwater. Concentrations of SO₄, • (mM); Fe(II), ○ (μM); U(VI), ▪ (μM); acetate, ▴ (mM); and bromide, ▵ (μM) in multilevel sampler wells (B02, M03, M08, and M13) at day 38 of acetate injection in the Rifle aquifer (depths of 4.2, 4.5, 5, 5.3, 5.7, and 6.1 m from surface). For simplicity the zero acetate measure in the background well was omitted from the figure. Pie charts show percentages of total 16S rRNA genes in the corresponding wells sampled from 5 m below the ground surface. Locations and sampling detail were as shown in Fig. 2 and described in the text. The various bacterial taxa are represented based on their relative occurrence in groundwater samples. The designation “other δ-proteob.” was applied to 16S rRNA gene sequences that fell into the Deltaproteobacteria but did not group with the Geobacteraceae, the Desulfovibrionaceae, or the three families comprising the order Desulfobacterales (Desulfoarculaceae, Desulfobacteraceae, and Desulfobulbaceae). The designation unclassified indicates sequences that could not be unequivocally classified with any one group due to insufficient similarity.

FIG. 4.

Geochemical and molecular analysis of sediment. Acid volatile sulfide (AVS) (•; μmol/g) and Fe(II)/Fe(total) (○) in cores P11 to P15 (at depths of 4, 4.6, and 5 m from the surface) collected on day 40 of acetate injection in the Rifle aquifer (see Fig. 2 and text for details). The key for designations of bacterial taxa and groups within the pie charts are the same as for Fig. 3.

FIG. 5.

Phylogenetic tree showing the relationship of Geobacteraceae clones to 16S rRNA gene sequences of previously described representative sequences. For clarity, one representative sequence for each location (i.e., core/depth or monitoring well) within each cluster was selected. Branch points were supported by maximum-likelihood and neighbor-joining distance analysis. Bootstrap values were calculated for 100 replicates, and values for key branch points are shown. The scale bar represents the expected number of changes per sequence position.

FIG. 6.

Phylogenetic tree showing relationships between detected deltaproteobacterial sulfate-reducing bacterial sequences and representative sequences. Details are as for Fig. 5 and in the text.