Engineering a Biofilm for the Biodegradation of Polycyclic Aromatic Hydrocarbons in Estuarine Sediment

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that accumulate in soils and sediment due to their physicochemical properties. In these environmental matrices, PAHs are predominantly transformed and degraded by the native fungal and bacterial communities. However, microbial degradation of PAHs is a slow process that requires engineered approaches to improve degradation rates to meet remediation criteria.

Engineered bioremediation approaches consist of altering the microbial community by either increasing cell concentrations of specific, targeted organisms or by introducing catabolic genes that confer for a phenotype that can degrade the target contaminant. This approach is called bioaugmentation and is generally applied using the former strategy. Biostimulation is another method, which includes the addition of nutrients that may be limited to microorganisms and can help grow the indigenous microbial community and accelerate contaminant degradation. However, biostimulation is not a targeted approach and may stimulate the entire microbial community, not just organisms capable of degrading the target contaminant.

Bioaugmentation of sediments is challenging due to constraints surrounding the longevity, stability, and delivery of microorganisms. To address the limitations of this remediation approach, the work within this dissertation outlines methods for developing a consortium of PAH-degrading bacteria coordinated within a stable community, as well as a technology for delivering this consortium to creosote contaminated sediments.

The first objective was to identify and isolate PAH-degrading bacteria from creosote contaminated sediment. Sediment was collected from sites along the Elizabeth River, VA and a 16S rRNA amplicon library of sequences was analyzed to generally evaluate the influence of chemical contamination on the bacterial community structure. To detect PAH-degrading organisms within sediment communities, DNA-SIP using uniformly labeled stable isotopes of phenanthrene and fluoranthene were prepared in incubations with Republic Creosoting site sediment. Clones derived from this experiment revealed one prominent degrader of phenanthrene and two prominent fluoranthene degrading bacteria. In an attempt to isolate these and other PAH-degrading organisms for laboratory evaluation, culture-based methods were employed and resulted in the successful isolation of 6 unique bacteria, including one strain which was detected in the DNA-SIP experiments. Overall, it was determined that PAH-degrading bacteria exist in Republic Creosoting site sediments, although not in significant relative abundance compared to other bacteria. This finding suggests that these contaminated sediments could be a good candidate for a bioaugmentation approach.

Most of the research on bioremediation has focused on organisms in isolation and existing in a free-floating, or planktonic, cellular state. The second objective of this dissertation was to confirm the PAH-degrading capabilities of isolated bacteria and to coordinate these organisms into a biofilm structure, which provides protection and additional community benefits to participating microorganisms. To this end, we employed a high-throughput, reproducible assay to confirm whether or not isolated bacteria are capable of coordinating within a biofilm. We also used culture-based methods and performed incubations with multiple types of PAHs to determine if the isolated organisms can interact with PAHs of various size and ring number. Finally, we used a metabolic assay for the novel application of assessing the respiration capacity of the isolated PAH-degrading bacteria in the biofilm conformation, to determine if these organisms are metabolically active when they are situated within a biofilm. We found that all of the organisms isolated were capable of forming a biofilm that was metabolically active. Many of these organisms demonstrated the ability to degrade phenanthrene and fluoranthene, but only a few showed the potential for degrading pyrene. These results confirmed that the isolated organisms from Republic site sediment can degrade PAHs and form a biofilm structure, which will be beneficial for their application to sediments in a bioaugmentation strategy.

The final aim of this work was to evaluate the use of an activated-carbon amendment based technology for the delivery of a bacterial consortium to PAH-contaminated sediment. While validated for use as a remediation technology and delivery strategy for organisms capable of degrading polychlorinated biphenyls (PCBs), this approach has not yet been tested for use with sediments contaminated with PAHs.