To determine the efficacy of a Polymerase Chain Reaction (PCR) assay in determining the genetic potential for in situ bioremediation of polychlorinated biphenyl (PCB)-contaminated sediments in the Great Lakes.
To investigate the capacity of sediments having different molecular genetic profiles in their ability to degrade complex PCB mixtures.

PCBs are a family of xenobiotic compounds that are ubiquitous and persistent pollutants. Thus, PCBs are a common target of sediment remediation efforts. Microbial degradation of PCBs offers an environmentally sound and economically favorable alternative to conventional means of remedaition such as dredging. This project describes the development of a PCR-based assay to determine the potential for PCB bioremediation by the resident microbial consortium in contaminated sediments. By PCR and RT-PCR of nucleic acids extracted from Lake Erie sediments, we can amplify the bphA1 gene encoding the large subunit of biphenyl diooxygenase. Since work in other labs have shown that the BphA1 protein dictates (in part) PCB congener specificity, our assay may prove to be a useful screen for endemic catabolic activities for PCB mixtures in contaminated sediments.

PCBs are notable among environmental contaminants in terms of both their widespread dispersal and potential toxicity. Towards the objective of remediating PCB-contaminated sediments, emphasis has been on dredging and subsequent disposal of the materials to secure landfills. However, not only is this process extremely costly, it may also be ineffective; recent data indicate that upwards of 75% of the PCB content of dredged sediments may be lost to volatilization as the sediments dry. Indeed, there is current debate over whether to proceed with a dredging operation to remediate PCB present in the Hudson River that were derived from manufacturing activities at GE during a 30 year period prior to 1977. An assay such as that described in this proposal would help address this controversy.

In sum, the capacity of bacteria to degrade PCB, both in situ and ex situ, has long been recognized yet seldom exploited in remediation efforts. In assessing the genetic potential for remediation of PCB congener mixtures in contaminated sediments, we expect to provide a rapid screening mechanism with which to gauge the feasibility of bioremediation as a viable cleanup option. Overall, we anticipate that the results of this study could one day assist appropriate agencies in determining the most fiscally-prudent and efficient approach to adopt for remediating PCB-contaminated sediments.

Prior work has shown that we can obtain total DNA and RNA from aquatic sediments, and that the nucleic acids are suitable for PCR and Reverse Transcriptase-PCR. We have also developed a series of degenerate PCR primers that recognize specific conserved regions of the bphA1 gene. Such regions flank variable sequences that encode domains of the biphenyl dioxygenase Fe-S large subunit protein. This protein exists in variable forms in nature that confer substrate specificity to different PCB congeners, thus heterogeneity in bphA1 in situ should be regarded a positive attribute, as it will increase the likelihood that multiple PCB congeners are targeted for degradation in a particular sediment zone. Selected bphA1 sequences will be amplified, cloned and sequenced. Genetic variability in bphA1 will be further investigated using microcosms of sediments obtained from sites having different bphA1 profiles. Adding PCB congeners (Arochlor 1248) to these sediments and monitoring total PCB catabolism will enhance both our understanding of the bphA1 alleles best adapted to degrade particular PCB, and the environmental conditions (eg. amendment with biphenyl) in which this can occur most rapidly.

Field sampling of Lake Erie and the Maumee River in 2002 has yielded a specific signature of bphA1 sequences by DGGE. The latter site was chosen due to a persisting problem of PCB cotamination from landfill sites. Interestingly, comparison of genetic variability of bphA1 sequences from the Maumee AOC appears to be distinct from the genetic profiling of sequenced amplified from PCB-contaminated sediments from Lake Hartwell, SC. We are currently preparing to publish this study.

We have completed sediment sampling from Lake Erie during Summer 2002, and we are analyzing bphA1 genetic profiles of the endemic bacteria by PCR. As a comparison, we also have acquired PCB-contaminated sediment from Lake Hartwell, SC. Analysis of bphA1 genetic profiles from two geographically distinct sites may yield additional information regarding the required genetic diversity to degrade the widest assortment of PCB congeners. We have currently a series of RT-PCR products from both sites that are being compared side-by-side by denaturing gradient gel electrophoresis (DGGE), which will provide a direct measure for genetic variability. Those sediments exhibiting the highest degree of variability will be selected for microcosm experiments in which the rate of PCB congener degradation can be assessed. Indeed, we are currently examining PCB-spiked sediment microcosms from both Maumee AOC and Lake Hartwell sites to see whether a specific genetic profile is linked to degradation of a specific PCB congener.