Gene Expression Correlates with Process Rates Quantified for Sulfate- and Fe(III)-Reducing Bacteria in U(VI)-Contaminated Sediments

Abstract

Though iron- and sulfate-reducing bacteria are well known for mediating uranium(VI) reduction in contaminated subsurface environments, quantifying the in situ activity of the microbial groups responsible remains a challenge. The objective of this study was to demonstrate the use of quantitative molecular tools that target mRNA transcripts of key genes related to Fe(III) and sulfate reduction pathways in order to monitor these processes during in situ U(VI) remediation in the subsurface. Expression of the Geobacteraceae-specific citrate synthase gene (gltA) and the dissimilatory (bi)sulfite reductase gene (dsrA), were correlated with the activity of iron- or sulfate-reducing microorganisms, respectively, under stimulated bioremediation conditions in microcosms of sediments sampled from the U.S. Department of Energy's Oak Ridge Integrated Field Research Challenge (OR-IFRC) site at Oak Ridge, TN, USA. In addition, Geobacteraceae-specific gltA and dsrA transcript levels were determined in parallel with the predominant electron acceptors present in moderately and highly contaminated subsurface sediments from the OR-IFRC. Phylogenetic analysis of the cDNA generated from dsrA mRNA, sulfate-reducing bacteria-specific 16S rRNA, and gltA mRNA identified activity of specific microbial groups. Active sulfate reducers were members of the Desulfovibrio, Desulfobacterium, and Desulfotomaculum genera. Members of the subsurface Geobacter clade, closely related to uranium-reducing Geobacter uraniireducens and Geobacter daltonii, were the metabolically active iron-reducers in biostimulated microcosms and in situ core samples. Direct correlation of transcripts and process rates demonstrated evidence of competition between the functional guilds in subsurface sediments. We further showed that active populations of Fe(III)-reducing bacteria and sulfate-reducing bacteria are present in OR-IFRC sediments and are good potential targets for in situ bioremediation.

Keywords: active metal-reducing bacteria; active sulfate-reducing bacteria; transcript level; uranium reduction.

Figure 1

Expression of dsrA (A) and gltA (B) genes related to electron acceptor reduction in ethanol-biostimulated microcosms. The lower panels show treatments amended with molybdate as an inhibitor of sulfate reduction. Data are means ± standard deviations of triplicate determinations on a pooled sample from triplicate microcosms.

Figure 2

Expression of dsrA (A) and gltA (B) genes related to electron acceptor reduction in acetate-biostimulated microcosms. The lower panels show treatments amended with molybdate as an inhibitor of sulfate reduction. Data are means ± standard deviations of triplicate determinations on a pooled sample from triplicate microcosms.

Figure 3

Electron donor utilization biostimulated microcosms. (A) Ethanol only treatment, (B) ethanol with molybdate, (C) acetate-only, and (D) acetate with molybdate; molybdate was added as a sulfate reduction inhibitor. Data are means ± standard deviations of triplicate microcosms.

Figure 4

Geochemistry (A,B) and levels of dsrA and gltA transcripts (C) in the depth interval from 1.23 to 15.08 m of Area 2 borehole FB107. Values for transcript levels are means ± standard deviations of triplicate determinations on a pooled sample from triplicate borehole sediment samples. Nitrate, sulfate, pH, and iron data were reported previously as supplemental material in Green et al. (2010). Uranium was not analyzed.

Figure 5

Geochemistry (A,B) and levels of dsrA and gltA transcripts (C) in the depth interval from 5 to 7 m of Area 3 borehole FB124. Data for transcript levels are means ± standard deviations of triplicate determinations on a pooled sample from triplicate borehole sediment samples. Nitrate, sulfate, pH, iron, and uranium data were reported previously as supplemental material in Green et al. (2010).

Figure 6

(A) Phylogenetic tree indicating the relationship of selected citrate synthase gene (gltA) mRNA sequences retrieved from OR-IFRC Area 2 sediment ethanol-amended microcosms (OR-IFRC), Area 2 in situ borehole sediments (OR-IFRC2), and Area 3 in situ borehole sediments (OR-IFRC3) and (B) Phylogenetic tree indicating the relationship of selected sulfate-reducing bacteria-specific 16S rRNA sequences retrieved from OR-IFRC Area 2 in situ borehole sediments (OR-IFRC16S). The scale bar indicates the estimated number of base changes per nucleotide sequence position. The accession numbers of the sequences are indicated.