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Applied and Environmental Microbiology, January 1999, p. 95-101, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Effect of Toxic Metals on Indigenous Soil beta -Subgroup Proteobacterium Ammonia Oxidizer Community Structure and Protection against Toxicity by Inoculated Metal-Resistant Bacteria

John R. Stephen,1 Yun-Juan Chang,1 Sarah J. Macnaughton,1 George A. Kowalchuk,2 Kam T. Leung,1 Cissy A. Flemming,1 and David C. White1,3,*

Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37932-25751; Netherlands Institute of Ecology, 6666 ZG Heteren, The Netherlands2; and Biological Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 378313

Received 8 April 1998/Accepted 17 July 1998

Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide. Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation. In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the Proteobacteria (beta -subgroup ammonia oxidizers). In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria. Effects on nitrogen cycling were measured by monitoring the NH3 and NH4+ levels in soil samples. The gene encoding the alpha -subunit of ammonia monooxygenase (amoA) was selected as a functional molecular marker for the beta -subgroup ammonia oxidizing bacteria. Community structure comparisons were performed with clone libraries of PCR-amplified fragments of amoA recovered from contaminated and control microcosms for 8 weeks. Analysis was performed by restriction digestion and sequence comparison. The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR. All amoA gene fragments recovered grouped with sequences derived from cultured Nitrosospira. These comprised four novel sequence clusters and a single unique clone. Specific changes in the community structure of beta -subgroup ammonia oxidizers were associated with the addition of metals. These changes were not seen in the presence of the inoculated metal-resistant bacteria. Neither treatment significantly altered the total number of beta -subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls. Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.

* Corresponding author. Mailing address: Center for Environmental Biotechnology, University of Tennessee, 10515 Research Dr., Suite 300, Knoxville, TN 37932-2575. Phone: (423) 974 8001. Fax: (423) 974 8027. E-mail: MILIPIDS{at}AOL.COM.

Applied and Environmental Microbiology, January 1999, p. 95-101, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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