Diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site

Abstract

Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from delta-Proteobacteria, gram-positive organisms, and the Nitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum and Desulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within delta-Proteobacteria were mainly recovered from low-uranium (< or =302 ppb) samples. One Desulfotomaculum-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organism's possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.

FIG. 1

Map of the UMTRA Shiprock mill tailing site.

FIG. 2

PLFA profile of UMTRA Shiprock groundwater samples. (A) Community abundance as measured by total bacterial biomass level. ▪, SRB-metal reducer biomass measured as picomoles per milliliter of mid-chain branched saturates (MBSats) and branched monounsaturates (BrMonos). □, the remaining bacterial biomass measured as total PLFA, with normal saturates over 18 carbons in length, polynoics (eukaryote PLFA), SRB-metal reducer PLFA, and trace quantities of PLFA of unknown structure excluded. (B) Community composition as categorized via SRB-metal reducer PLFA structural group.

FIG. 3

Restriction digestion analysis of DSR clones with MspI. Cloned DSR gene fragments were amplified from the cloning vector by using primers directed at the T7 and M13 reverse RNA polymerase binding sites, producing a fragment with approximately 70 bp of vector sequence on each end. The vector sequence contained no MspI recognition sites. Products were digested with a twofold excess of MspI for 1 h at 37°C, analyzed by electrophoresis on a 2% agarose gel with TAE buffer, and visualized by ethidium bromide fluorescence.

FIG. 4

Neighbor-joining analysis of DSR α-subunit fragments selected from clone libraries as frequently occurring MspI digestion patterns. Sequences prefixed “UMTRAdsr” were generated during this study. The prefix is followed by the sample well number, which is followed by the clone number. Clones were selected from libraries on the basis of MspI patterns to provide a preliminary survey of the most commonly recovered DSR gene sequences from each sample. Nucleotide sequence accession numbers are given in parentheses for the following organisms: Desulfobulbus rhabdoformis (AJ250473), Desulfovibrio desulfuricans (AJ289157), Desulfococcus multivorans (AJ277107), Desulfotomaculum thermocisternum (F074396), Archaeoglobus profundus (AF071499), Thermodesulfovibrio yellowstonii (U58122), Desulfobotulus sapovorans (U58120), Desulfovibrio sp. strain P1B2 (U58116), Desulfotomaculum ruminis (U58118), Desulfococcus multivorans (U58126), Desulfonema limicola (U58128), Desulfobacter latus (U58124), Desulfovibrio vulgaris (U16723), Archaeoglobus fulgidus (M95624), and Desulfovibrio simplex (U78738). Gene sequences from Desulfotomaculum luciae, D. acetoxidans, D. putei, D. aeronauticum, D. nigrificans, and Desulfovibrio desulfuricans were generated in this study under accession no. AY015493 to AY015499. The numbers on the tree refer to bootstrap values on 100 replicates; only values above 30 are given. Scale bar represents 10% estimated change.

FIG. 5

(A) Neighbor-joining analysis of some DSR β-subunit sequences from UMTRA Shiprock site clones and reference pure culture organisms. Sequences prefixed “UMTRAdsr” were generated during this study. Nucleotide sequence accession numbers are given in parentheses for the following organisms: Desulfobulbus rhabdoformis (AJ250473), Desulfococcus multivorans (U58127), Desulfotomaculum thermocisternum (AF074396), Archaeoglobus profundus (AF071499), Thermodesulfovibrio yellowstonii (U58123), Desulfobotulus sapovorans (U58121), Desulfovibrio sp. strain PT-2. (U58115), Desulfotomaculum ruminis (U58119), Desulfonema limicola (U58129), Desulfobacter latus (U58125), Desulfovibrio vulgaris (U16723), Archaeoglobus fulgidus (M95624), Desulfobacter vibrioformis (AJ250472), Desulfobacterium autotrophicum (Y15478), DSR clone 50 (AF179329), and DSR clone 8037 (AF179336). Gene sequences from Desulfotomaculum luciae, D. acetoxidans, D. putei, D. aeronauticum, and D. nigrificans were generated in this study under accession no. AY015577 to AY015581. (B) Neighbor-joining analysis of near full-length 16S rDNA from pure cultures. Nucleotide sequence accession numbers are given in parentheses for the following organisms: Desulfotomaculum ruminis (Y11572), D. putei strain SMCCW 459 (AF053929), D. aeronauticum (X98407), D. acetoxidans (Y11566), D. thermocisternum (U33455), D. nigrificans (X62176), D. luciae (AF069293), Desulfococcus multivorans ATCC 33890 (M34405), Desulfonema limicola (U45990), Desulfobacter latus (M34414), Desulfobotulus sapovorans (M34402), Desulfovibrio vulgaris (M34399), D. desulfuricans (AF098671), Archaeoglobus profundus. (AF272841), Desulfovibrio sp. strain zt10e (AF109469), Archaeoglobus fulgidus (Y00275), and D. desulfuricans sp. strain Norway 4 (M37312). The numbers on the trees refer to bootstrap values on 100 replicates; only values above 30 are given. The scale bar represents 10% estimated change.

FIG. 6

(A) Sequence distribution for 70 DSR clone sequences (α-subunit) obtained from high-uranium [samples 610, 608, 615, and 826; U(VI) from 2,847.5 to 1,687 ppb], medium-uranium (samples 624, 617, and 828; U(VI) from 1,205 to 342.7 ppb), and low-uranium samples (857, 853, 626, and 648; U(VI) from 301 to 0.001 ppb). (B) SRB population structure in different sample wells as measured by DSR sequences recovered and their affiliation with different cluster of SRB.