| dc.description.abstract |
<p>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. </p><p>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. </p><p>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. </p><p> 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. </p><p> 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. </p><p> 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.</p>
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