The management of marine and estuarine sediments contaminated with toxic organic compounds, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), is a significant problem with far-reaching economic and ecological consequences. Enhancement of microbial degradation of PCDD/Fs in situ is an attractive remediation alternative that could potentially detoxify sediments, avoid the problematic redistribution of contaminants that is associated with dredging, and decrease the cost of sediment management. Anaerobic dechlorination of PCDD/Fs has been reported in marine and estuarine sediments; however, rates are slow and the activity may be the result of a combination of both respiratory and cometabolic processes. For in situ bioremediation, it would be desirable to stimulate respiratory dechlorination, which is typically associated with higher dechlorination rates. 

The objectives of this project were to identify environmental conditions and amendments that enhance and accelerate dechlorination of PCDD/Fs by indigenous microbial populations and to identify the organisms responsible for the dechlorination using biomolecular methods.

Researchers characterized the PCDD/F-dechlorinating capability of indigenous dehalogenating bacteria in estuarine and marine sediments. Specific amendments and their combinations that prime or accelerate the dechlorination of PCDD/Fs by dehalorespiring or cometabolizing bacteria were identified using an existing PCDD/F-dechlorinating culture and new enrichments. The effect of different redox conditions and competitive terminal electron accepting processes (e.g., sulfate reduction, iron reduction, and methanogenesis) on the dechlorination and further transformation of PCDD/Fs was determined. Complementary molecular techniques including cellular phospholipid fatty acid profiling, terminal restriction fragment length polymorphism (TRFLP), and cloning and sequencing 16S rRNA genes associated with the TRFLP peaks along with traditional cultural approaches were used to identify and characterize specific PCDD/F-dechlorinating bacteria. Bioprocess modeling was then used to describe the effects of different amendment strategies on the dehalogenating populations.

Results from this project show that anaerobic dehalogenation of PCDD/Fs was readily promoted in estuarine, marine, and freshwater sediments from several sites. Co-amendment with more soluble halogenated aromatic compounds such as halogenated phenols, chlorinated benzenes, and chlorinated anisoles as “priming” agents was found to greatly enhance the rates of PCDD/F dechlorination. This stimulation was brought about in the presence of added electron donors lactate and propionate to ensure reduced conditions and adequate reducing equivalents to stimulate dechlorination. In highly organic sediments with adequate reducing power, it is possible that these halogenated additives may be just as effective without additional electron donors. PCDD dechlorination was, in general, more easily stimulated and proceeded to a greater extent in sediments previously contaminated with other chlorinated compounds than in less contaminated or pristine sediments. Results suggest that halogenated aromatic compounds with structural similarity to PCDD/Fs simulate bacteria with the ability to dechlorinate these contaminants. Of critical importance is that these amendments stimulate the desirable lateral dechlorination of PCDD/Fs (i.e., removal of chlorines at position 2 and 3) that ultimately results in detoxification of the compounds. A lateral dechlorination pathway dominated in most of the sediments and conditions examined. 

While the process has much potential, a key aspect of the technology – the nature and capability of the intrinsic microbial community – must also be understood at a fundamental level.  In 2003, researchers reported that Dehalococcoides strain CBDB1 could dechlorinate selected PCDDs. A result of this project was finding that Dehalococcoides ethenogenes strain 195, originally isolated on tetrachloroethene, has the potential to dechlorinate many different types of chlorinated aromatic compounds, such as PCDD/Fs, polychlorinated biphenyls (PCBs), and chlorinated naphthalenes. Strain 195 carried out a lateral, detoxification dechlorination pathway on the PCDD/F congeners examined.

Results with Strain 195 coupled with recent findings that some species of dehalogenating bacteria have multiple dehalogenases and dechlorination capabilities suggest that halogenated compounds other than PCDD/Fs could be used to enrich and isolate organisms that have activity on PCDD/Fs. Within the Chloroflexi, which includes both Dehalococcoides spp. and other yet to be described genera, there appears to be several strains with the ability to reductively dechlorinate PCBs and other halogenated compounds as electron acceptors. These findings may explain results that show the stimulation of PCDD/F dechlorination in a variety of sediments upon addition of simpler halogenated compounds. Identification of naturally occurring or less toxic halogenated co-amendments that are completely degraded in the sediment environment is desirable for field application; however, enrichment of bacteria for bioaugmentation purposes under controlled conditions could use volatile compounds such as tetrachloroethene.

In addition to determining that a well-known dehalorespirer can dechlorinate PCDD/Fs, a variety of molecular approaches was used to identify and characterize the bacteria involved in PCDD/F dechlorination. For example, polymerase chain reaction (PCR) coupled to TRFLP analysis was used to explore microbial community differences between different sites exhibiting PCDD dechlorination. PCR using primers specific for dehalogenase genes was also used to delineate the functional genes present in actively dechlorinating communities. Future studies will focus on developing these methods further for identifying and monitoring PCDD/F-dechlorinating bacteria under in situ conditions. For example, real-time PCR analysis will be used to quantify total bacteria, Dehalococcoides ethenogenes 195, and reductive dehalogenase genes from Dehalococcoides ethenogenes 195 in bioaugmented and non-bioaugmented sediments. This basic understanding of microbial processes and the ability to identify and track the activity of specific strains or genes is essential for the design of site-specific solutions for PCDD/F bioremediation and sediment restoration projects.

Data collected from enrichment cultures undergoing different amendment strategies and the accompanying microbial community characterization were used for the development of a biological process model to describe the stimulatory effect of different enhancement methods under different conditions and at different sites. Predictive modeling of San Diego Bay sediments under methanogenic and sulfidogenic conditions suggested that hydrogen concentration was a major factor controlling the activity of dechlorinating bacteria under different redox conditions.

Sediment treatment is currently limited primarily to dredging with ex situ treatment or sequestration. New methods are needed for in situ containment and degradation of contaminants to decrease the cost of long-term sediment management. Anaerobic reductive dehalogenation offers a promising approach towards eventual detoxification and complete degradation of halogenated contaminant mixtures. In situ bioremediation combined with in-place containment through capping could avoid the problematic redistribution of contaminants that is associated with dredging and, where feasible, offer a more cost-effective treatment alternative to dredging. Developing amendment technologies for enhanced microbial dehalogenation and understanding how amendment placement and mixing stimulates dehalogenation and impacts the fate and transport of organohalide mixtures is thus a high priority for the successful management of contaminated sediments. This project expanded understanding of the microorganisms that carry out dehalogenation of PCDD/Fs in sediments and produced a variety of techniques that can be used to enhance this activity. Ongoing work by the project team will enable field verification of the effectiveness of these approaches.