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Applied and Environmental Microbiology, March 2005, p. 1433-1444, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1433-1444.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

Chang-Ping Yu,1 Rajiv Ahuja,1 Gary Sayler,2 and Kung-Hui Chu1,2*

Department of Civil & Environmental Engineering,1 Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee2

Received 8 March 2004/ Accepted 7 October 2004

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17{alpha}-estradiol, 17ß-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.

* Corresponding author. Mailing address: Department of Civil & Environmental Engineering, 110B Perkins Hall, University of Tennessee, Knoxville, TN 37996. Phone: (865) 974-7708. Fax: (865) 974-2669. E-mail: khchu{at}utk.edu.

Applied and Environmental Microbiology, March 2005, p. 1433-1444, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1433-1444.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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