Perchlorate is a stable, soluble contaminant that occurs in the environment due to anthropogenic and natural activities. Some microorganisms can reduce perchlorate to innocuous chloride, and that potential can be harnessed for the biological treatment of perchlorate-contaminated waters. The discovery and application of biomarkers has the potential to improve the design, performance, and monitoring of such biological treatment processes for the removal of perchlorate and other compounds relevant to the Department of Defense (DoD).

The overall objective of this project was to develop molecular biological tools that are useful in assessing biodegradation potential at perchlorate-contaminated field sites and for monitoring the transcriptional activity of perchlorate-reducing bacteria in biological treatment systems. Specific objectives were (1) to refine quantitative assays for genes associated with microbial perchlorate reduction and to apply quantitative assays on bench-scale and pilot-scale perchlorate-reducing bioreactors; and (2) to improve the protocol for Prokaryotic Suppression Subtractive Hybridization Polymerase Chain Reaction Complementary Deoxyribonucleic Acid Subtraction (hereafter termed Prokaryotic cDNA Subtraction), a focused gene discovery technique that was used to expand the database of perchlorate-related gene sequences for biomarker development.

This project was conducted in two phases with a separate Final Report for each phase. Phase I was focused on demonstrating that the combination of prokaryotic cDNA subtraction and reverse transcription real-time PCR (rtRT-PCR) can be used to rapidly identify functional gene biomarkers from unsequenced environmental microorganisms. Phase II was divided into seven tasks focused on quantifying cld and pcrA genes or transcripts in pure cultures and in bench- and full-scale perchlorate-reducing bioreactors and on improving the prokaryotic cDNA subtraction protocol and deploying it on a perchlorate-reducing isolate to identify perchlorate-related gene sequences.

The transcription of cld and pcrA was examined in a bench-scale perchlorate-reducing bioreactor. The concentration of cld transcripts was 2- to 8-fold lower under near-failure conditions (<25% perchlorate removal) than during normal operation (>50% perchlorate removal) in the bioreactor, which suggests that the near-failure in the bioreactor might have been due in part to a loss of perchlorate-reducing bacteria from the reactor.

The presence of cld and pcrA was examined in two pilot-scale perchlorate-reducing bioreactors. The concentrations of cld were 3- to 4-fold higher in the bioreactor that removed 90% of the influent perchlorate as compared to the bioreactor that removed 63% of the influent perchlorate, which suggests that a larger perchlorate-reducing bacterial community was present in the reactor that demonstrated superior perchlorate removal. Based on these results, it was concluded that the concentrations of cld genes and transcripts are likely to be a good indicator of bioreactor health.

Since the data from these studies demonstrated that the concentration of pcrA genes and transcripts were at least one order of magnitude less than the concentration of cld genes and transcripts in each bioreactor, further studies investigated the potential for bias in the existing pcrA quantitative real-time PCR (qPCR) assay. The results showed that mismatches between the template and primer near the 5’ end of the primer caused pcrA quantities to be substantially underestimated, often by one or more orders of magnitude. Thus, new pcrA primer sets were designed targeting several perchlorate-reducing strains: Dechloromonas sp. PC1, Dechloromonas aromatica RBC, Dechloromonas agitata CKB, and Azospira sp. KJ. Each primer set amplified pcrA in its target strain well, but did not amplify pcrA from the other tested strains. Therefore, these primer sets might be deployed in parallel to more accurately quantify pcrA in mixed microbial communities.

Since relatively few perchlorate-related gene sequences are known, primers and probes for interrogating the potential for perchlorate reduction at a contaminated site or the transcriptional activity of perchlorate-reducing bacteria in a treatment system have been narrowly designed. Prokaryotic cDNA subtraction is a focused gene discovery technique that can be used to identify genes that are up-regulated under a condition of interest (e.g., perchlorate-reducing conditions). Thus, this technique could be used to expand the database of gene sequences related to perchlorate reduction to facilitate improved biomarker design. Key to the successful application of the technique is avoiding the identification of ribosomal ribonucleic acid (rRNA) sequences rather than the transcripts of interest in the messenger RNA (mRNA). Two modifications in the prokaryotic cDNA subtraction protocol were demonstrated to decrease the identification of rRNA sequences in the test organism (perchlorate-reducing strain JDS4 in the Rhodocyclaceae family). The first modification (sequential mRNA purification with two kits using different capture oligonucleotides for rRNA) was deployed in a prokaryotic cDNA subtraction on JDS4. The modified protocol was successful, and three perchlorate-related genes were identified: perchlorate reductase alpha subunit precursor, perchlorate reductase A, and perchlorate reductase B. In particular, the identified pcrA sequence had seven mismatches with the existing pcrA qPCR primer set, which means that we have taken an important first step in developing an expanded functional gene sequence database related to perchlorate reduction.

The quantitative assays for cld and pcrA that were refined in this project can now be directly applied to more accurately and sensitively monitor the potential for biological perchlorate reduction at a contaminated site or the transcriptional activity of perchlorate-reducing bacteria in a treatment system; thus, these assays will be beneficial at DoD sites and drinking-water treatment plants practicing biological perchlorate reduction. The successful application of prokaryotic cDNA subtraction to identify perchlorate-related genes means that this protocol can now be applied to other prokaryotes that transform contaminants of interest to DoD, including 1,4-dioxane, TNT, DNT, and RDX, thereby leading to new or improved biomarkers for biodegradation of these contaminants.