Enumeration and characterization of iron(III)-reducing microbial communities from acidic subsurface sediments contaminated with uranium(VI)

Abstract

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml(-1) for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.

FIG. 1.

Map of sample sites at the U.S. Department of Energy-NABIR Field Research Center, Oak Ridge, Tennessee. The contaminated plot is 7 by 25 m just south of the S-3 ponds, and contamination extends to a depth of approximately 22.8 m below the surface.

FIG. 2.

MPN counts of Fe(III)-reducing bacteria (FeRB) at pH 7 (A) and pH 4 to 5 (B) from the contaminated and background sites. W, washed sediment; UNW, unwashed sediment. Background cores are labeled 302-02 and 302-05, and contaminated cores are labeled 30, 32, and 33. Only counts greater than or equal to 20 cells/ml are included in this graph.

FIG. 3.

Uranium(VI) reduction by Fe(III)-reducing enrichment cultures from contaminated Field Research Center subsurface sediments.

FIG. 4.

Phylogenetic affiliations of 16S rRNA cloned genes obtained from pH 7 enrichment cultures of background (top) and contaminated (bottom) sediments.

FIG. 5.

Phylogenetic tree of 16S rRNA genes cloned from Fe(III)-reducing enrichments from background and contaminated subsurface sediments cultivated at pH 7 and pH 4 to 5. The scale bar equals a 10% difference in nucleotide sequence. The cloned genes are named according to the pH, carbon source, and sediment source. Names containing the numbers 302 are from background sediment, while those with numbers 030, 032, and 034 are from contaminated sediment.

FIG. 6.

Electropherograms of the 5′-terminal restriction fragments of HaeIII-digested 16S rRNA genes amplified from contaminated and background Fe(III)-reducing MPN tubes (A and B) and cloned 16S rRNA genes (C).

FIG. 7.

Quantification of Anaeromyxobacter and Geobacter 16S gene sequences extracted from sediments at contaminated (FWB032, FWB034) and background (FWB302) sites by MPN-PCR.