Molecular biology tools for identification and quantification of perchlorate-reduction genes in biotreatment applicatins

Perchlorate contamination of drinking water sources in the United States is widespread and represents a public health concern. Biological treatment is an attractive option because perchlorate-reducing bacteria (PRB) are ubiquitous in the environment and can reduce perchlorate completely to chloride. Treatment of perchlorate-contaminated water in fixed-bed bioreactors has been demonstrated at the laboratory- and pilot-scale. However, full-scale development of reliable biological drinking water treatment processes requires a better understanding of the microbial ecology and activity of perchlorate-reducing communities in bioreactors. The objective of this research was to develop molecular biology tools (MBTs) to quantify PRB and expression of genes required for complete perchlorate reduction (pcrA and cld). The development of MBTs targeting specific genes requires that the sequence of the genes be known. In this work, an MBT called prokaryotic Suppression Subtractive Hybridization (SSH) PCR complementary DNA (cDNA) Subtraction was developed to rapidly isolate target genes for sequencing. This new tool was developed and validated using the model bacterium Pseudomonas putida mt-2 and the model pollutant toluene. For this system, over 90% of the isolated gene fragments encoded toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were identified. Based on these results, prokaryotic SSH PCR cDNA Subtraction shows promise as a targeted method for gene identification; however, application to a PRB did not yield new pcrA and cld sequences. Therefore, to support the development of biological perchlorate treatment processes, quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) assays targeting pcrA and cld were developed using existing sequences. The qPCR and RT-qPCR assays were applied to a laboratory-scale bioreactor and two pilot-scale bioreactors treating perchlorate-contaminated water. Higher quantities of perchlorate reduction genes and transcripts generally were observed when bioreactor performance was superior. Although no quantitative correlations were established, these assays detected differences in the quantity of PRB and changes in gene expression levels during the course of bioreactor operation and between bioreactors with different performance levels. Furthermore, these assays provided an additional line of evidence that microbial perchlorate reduction was occurring. This marks the first application of qPCR assays to quantify perchlorate reduction genes and transcripts in bioreactors.