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Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

Anne M. Spain,¹ Aaron D. Peacock,² Jonathan D. Istok,³ Mostafa S. Elshahed,^1, Fares Z. Najar,⁴ Bruce A. Roe,⁴ David C. White,² and Lee R. Krumholz^1*

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma,¹ Center for Biomarker Analysis, University of Tennessee, Knoxville, Tennessee,² Department of Civil Engineering, Oregon State University, Corvallis, Oregon,³ Advanced Center for Genome Technology, University of Oklahoma, Norman, Oklahoma⁴

Received 9 February 2007/ Accepted 30 May 2007

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.

Published ahead of print on 8 June 2007.

Present address: Oklahoma State University, Stillwater, OK.

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