Project Abstract

Detection of Microorganisms Capable of Anaerobic Degradation of Hazardous Substancesin Natural Environments

Principal Investigator

Sara E. Silverstone
California State University at Bakersfield

Goal

The goals of this project are to develop a quantitative method for in situ monitoring of anaerobic toluene, PCE, and ethylbenzene- degrading microbes using fluorescence-labelled oligonucleotide probes and to apply this method to field and laboratory studies of contaminated soils.

Rationale

The Stanford laboratories participating in the WRHSRC have isolated a number of novel microorganisms capable of degrading a variety of hazardous organic substances. The use of specific microorganisms in bioremediation requires knowledge about the competitive behavior of the strains and ways to stimulate their growth. In order to identify and study these organisms in soil microcosms and assess the feasibility of using them in bioaugmentation experiments, it is necessary to have a rapid and efficient method of detecting and enumerating the bacteria. To the extent that such organisms can be utilized in bioremediation, this research will be an important contribution to the design of hazardous waste cleanup strategies.

Approach

The approach is to construct fluorescent and digoxigenin-labelled, strain-specific 16S rRNA-directed oligonucleotide probes. Probe target sites are selected based on database searches for unique sequences. Hybridization conditions for each probe are optimized and strain-specificity of the probes is assessed. Protocols are being developed for the use of these probes in whole-cell hybridization in soils. Probes will be used to monitor the effects of environmental and nutritional parameters upon the relative population densities, metabolic state and spatial distribution of target organisms in soil microcosms and at contaminated field sites.

Status

Two 16S rRNA probe target sites have been selected for the anaerobic toluene-degrader PRTOL-1. Optimization of hybridization conditions and assessment of the probes stain-specificity is in progress. These experiments are being conducted using both dot-blots of total nucleic acid and whole cell hybridization.