A limited microbial consortium is responsible for extended bioreduction of uranium in a contaminated aquifer

Abstract

Subsurface amendments of slow-release substrates (e.g., emulsified vegetable oil [EVO]) are thought to be a pragmatic alternative to using short-lived, labile substrates for sustained uranium bioimmobilization within contaminated groundwater systems. Spatial and temporal dynamics of subsurface microbial communities during EVO amendment are unknown and likely differ significantly from those of populations stimulated by soluble substrates, such as ethanol and acetate. In this study, a one-time EVO injection resulted in decreased groundwater U concentrations that remained below initial levels for approximately 4 months. Pyrosequencing and quantitative PCR of 16S rRNA from monitoring well samples revealed a rapid decline in groundwater bacterial community richness and diversity after EVO injection, concurrent with increased 16S rRNA copy levels, indicating the selection of a narrow group of taxa rather than a broad community stimulation. Members of the Firmicutes family Veillonellaceae dominated after injection and most likely catalyzed the initial oil decomposition. Sulfate-reducing bacteria from the genus Desulforegula, known for long-chain fatty acid oxidation to acetate, also dominated after EVO amendment. Acetate and H(2) production during EVO degradation appeared to stimulate NO(3)(-), Fe(III), U(VI), and SO(4)(2-) reduction by members of the Comamonadaceae, Geobacteriaceae, and Desulfobacterales. Methanogenic archaea flourished late to comprise over 25% of the total microbial community. Bacterial diversity rebounded after 9 months, although community compositions remained distinct from the preamendment conditions. These results demonstrated that a one-time EVO amendment served as an effective electron donor source for in situ U(VI) bioreduction and that subsurface EVO degradation and metal reduction were likely mediated by successive identifiable guilds of organisms.

Fig. 1.

Time series of bacterial (filled circles) and archaeal (open squares) Margalef richness indices and Shannon diversity indices averaged for all analyzed libraries at each time point. Error bars represent 1 standard deviation.

Fig. 2.

Comparisons of groundwater bacterial community structures and composition during the EVO experiment. Sample names indicate the monitoring well identifier (ID) and the sampling time point relative to EVO amendment. (A) Samples clustered according to abundance-based Sorenson's indices of 16S rRNA gene libraries normalized to 2,500 reads each. d, days. (B) Pie charts indicate the mean relative abundance of 16S rRNA gene pyrosequencing reads, classified at the division or class level, for samples corresponding to the indicated clades. (C) The heat map indicates the sum of the relative abundance of those 16S rRNA gene reads classified as members of the indicated taxonomic groups. Relative abundances for the 15 most abundant families or candidate phyla are shown for each sample. (D) The heat map indicates the concentrations of various dissolved constituents in groundwater samples collected at the same time as the microbiological samples.

Fig. 3.

Temporal and spatial distributions of the 15 most abundant bacterial OTUs detected in groundwater. Relative abundances of sequences belonging to each OTU, detected in each of the 8 monitoring wells at 7 different time points, are represented according to circle diameter. Circle colors indicate the duration (days) relative to EVO injection.

Fig. 4.

Comparisons of groundwater archaeal community structures and composition during the EVO experiment. (A) Samples clustered according to abundance-based Sorenson's indices of 16S rRNA gene libraries normalized to 650 reads each. Sample names are the monitoring well ID and the sampling time point relative to SRS amendment. (B) Pie charts indicate the mean relative abundances of 16S rRNA gene pyrosequencing reads, classified at the division level, for samples corresponding to the indicated clades. (C) The heat map indicates the sum of the relative abundance of those 16S rRNA gene reads classified as members of the indicated taxonomic groups. Relative abundances for the 10 most abundant families or environmental clone groups are shown for each sample.

Fig. 5.

Characterization of sulfate-reducing and methanogenic populations using dsrB and 16S rRNA gene analyses. (a) Number of bacterial 16S rRNA copies detected in groundwater using quantitative PCR on RNA extracts. (b) Number of dsrB mRNA transcript copies detected in groundwater using quantitative PCR on RNA extracts. (c) Sum of the relative abundances of 16S rRNA genes detected by pyrosequencing classified as members of the Desulfobacteraceae, Desulfovibrionaceae, and Desulfobulbaceae. (d) Fraction of archaeal 16S rRNA gene copies, relative to the sum of archaeal and bacterial gene copies, detected in groundwater using quantitative PCR on DNA extracts. (e) Sum of the relative abundances of 16S rRNA genes detected by pyrosequencing classified as members of known methanogen groups (Methanobacteraceae, Methanoregulaceae, Methanospirillaceae, and Methanosarcinaceae). n.a., not analyzed; pyro., pyrosequencing.

Fig. 6.

Ordination diagrams from canonical correspondence analyses of bacterial abundances (response variables indicated by black crosses) and geochemistry data (illustrated by vectors). (a) Relative abundance of dominant OTUs. (b) Relative abundances of dominant families. Samples are represented by colored symbols coded according to clustering results shown in Fig. 2. OTU abbreviations: 1, OTU 3 (Pelosinus); 2, OTU 90 (Desulforegula); 3, OTU 18 (Pelosinus); 4, OTU 2 (Ruminococcaceae); 5, OTU 2732 (OD1); 6, OTU 1814 (Comamonadaceae); 7, OTU 1843 (Vogesella); 8, OTU 6 (Bacteroidetes); 9, OTU 25 (Geobacter); 10, OTU 43 (Geobacter); 11, OTU 273 (Proteobacteria); 12, OTU 68 (Geobacter); 13, OTU 276 (Brevundimonas); 14, OTU 294 (Proteobacteria); 15, OTU 100 (Ruminococcaceae); 16, OTU 2990 (Neisseriaceae); 17, OTU 1852 (Veillonellaceae); 18, OTU 2762 (unclassified); 19, OTU 1857 (Desulfobacteraceae); 20, OTU 1898 (Geobacter); 21, OTU 17077 (Rhodospirillaceae). Family abbreviations: Anaer, Anaerolineaceae; Campy, Campylobacteraceae; Caulo, Caulobacteraceae; Comam, Comamonadaceae; Dbact, Desulfobacteraceae; Dbulb, Desulfobulbaceae; Dvibr, Desulfovibrionaceae; Geoba, Geobacteraceae; Neiss, Neisseriaceae; OD1, candidate phylum OD1; Pseud, Pseudomonadaceae; Rhodo, Rhodocyclaceae; Rumin, Ruminococcaceae; SR1, candidate phylum SR1; Veill, Veillonellaceae.

Fig. 7.

Conceptual model of EVO degradation and metal reduction in subsurface environments based on the relative abundance of representative abundant OTUs and known physiologies of closely allied species or genera.