Date of Award


Document Type


Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering and Science


Freedman, David L

Committee Member

Carraway , Elizabeth R

Committee Member

Henson , John M


A former tar plant site in the southeastern
U.S. is one of hundreds across the country that is contaminated with polycyclic
aromatic hydrocarbons (PAHs). The 2007
CERCLA Priority List of Hazardous Substances ranks PAHs as the eighth most
prominent hazardous substance at National Priority List sites. However, benzo(a)pyrene
is the only PAH that is regulated under the Safe Drinking Water Act, with a maximum
contaminant level of 0.2 µg/l. As with most sites, monitored natural
attenuation was the preferred bioremediation approach for this site, assuming
it could be shown that the PAHs were undergoing biodegradation. Field data from the site suggested that the
contaminated groundwater was stable, i.e., the PAHs were not migrating. However, the field data was not sufficiently
conclusive to warrant monitored natural attenuation as a remedy. In such cases, laboratory studies may be
conducted to fill in gaps left by the field data. The focus of this thesis was to evaluate PAH
biodegradation in samples taken from the site using microcosms. The research
objectives were as follows: 1) To evaluate the potential for aerobic and
anaerobic biodegradation of PAHs at the tar plant site using microcosms
prepared with crushed carbonate bedrock and groundwater from two locations; 2) To
determine the distribution of products formed during biodegradation of [14C]naphthalene,
including 14CO2 and 14C-labeled soluble
metabolites; and 3) To evaluate the potential of sulfate to enhance in situ
anaerobic biodegradation of naphthalene.

Since naphthalene was present at considerably higher levels than the other
PAHs (acenaphthene, fluorene and phenanthrene), its fate was evaluated in more depth
via the use of [14C]naphthalene.

Fractured rock and groundwater samples were
collected from two locations: location 1 was heavily contaminated with PAHs
(i.e., 7.5 mg/L concentrations in the groundwater); and location 2 was less
contaminated (i.e., less than approximately 1 mg/L in the groundwater). Two sets of microcosms were evaluated. Set I was designed to compare the extent of
PAH biodegradation under aerobic and anaerobic conditions, for both
locations. Set II microcosms were
designed to enhance anaerobic degradation by biostimulation with sulfate. For the Set I microcosms, four treatments
were prepared: i) live microcosms, designed to simulate in situ conditions; ii)
live microcosms with approximately 0.45 µCi of [14C]naphthalene; iii)
killed controls, to determine if activity observed in the live microcosms was a
consequence of biotic or abiotic processes; and iv) killed controls with
approximately 0.45 µCi of [14C]naphthalene. Each treatment was
prepared in triplicate, yielding 12 microcosms per redox condition per
location, or a grand total of 48 microcosms.

Biodegradation of naphthalene,
acenaphthene, fluorene and phenanthrene was observed under aerobic conditions
for both locations 1 and 2. The first order decay rate for naphthalene was
greater than or equal to 0.19 d-1 for location 1 and approximately 0.053
d-1 for location 2. The
decrease in naphthalene coincided with a decrease in the percentage of oxygen
in the headspace of the live treatments.
Nevertheless, delivering oxygen to the contaminated areas of this site
would be challenging, due in part to the high costs associated with aeration
and the low solubility of oxygen.

Analysis of the distribution of 14C
in the aerobic microcosms confirmed that mineralization was the predominant
fate process, with a higher percentage of 14CO2 detected in
the live microcosms from location 1 (81%) versus 2 (45%). The majority of the 14C remaining
in the killed controls was due to [14C]naphthalene.

Under anaerobic conditions,
naphthalene concentrations in the location 1 live microcosms decreased over the
first 23 days of incubation by approximately 50%, followed by a stall in
activity. After determining that the low
level of sulfate initially available in the groundwater had been depleted, more
was added on days 397 and 486. About
one-half of the added sulfate was consumed (presumptively by reduction to
sulfide). Nevertheless, stimulation of
sulfidogenesis did not result in any further anaerobic biodegradation of
naphthalene. With location 2, there was
no conclusive evidence in support of anaerobic biodegradation of PAHs. These microcosms had an Eh above
-110 mV that was not conducive to methanogenesis. After more than a year of incubation, there was
still no indication that methanogenic conditions (and possibly commensurate
fermentation of naphthalene) were likely to develop in the location 2 anaerobic

In the location 1 anaerobic
microcosms, approximately 21% of the [14C]naphthalene was recovered
as 14CO2 in the live treatment, while the location 2
microcosms did not show any significant 14CO2 accumulation.
The majority of the 14C remaining in the killed controls was due to
[14C]naphthalene. In the live
anaerobic microcosms for location 2 and in the killed controls, approximately
6-8% of the 14C added was identified as soluble nonstrippable
residue (sNSR). Detection of similar
levels of [14C]sNSR in the water controls suggests this material may
represent an impurity in the [14C]naphthalene stock solution. [14C]sNSR in the location 1 live
microcosms was higher (approximately 21%), suggesting that at least some of
this material was a product of [14C]naphthalene biodegradation.

The results from Set I for
location 1 suggested that the availability of sulfate may play a significant
role in the extent of anaerobic naphthalene biodegradation. To test this further, Set II was prepared. For the Set II microcosms, six anaerobic treatments
for location 1 were prepared: i) live microcosms with sulfate added, designed
to evaluate the potential for enhancing in situ biodegradation; ii) live
microcosms with sulfate and approximately 0.45 µCi of [14C]naphthalene
dissolved in methanol; iii) live microcosms with sulfate and molybdate added,
in order to inhibit sulfate reducing activity; iv) live microcosms with sulfate,
molybdate and approximately 0.45 µCi of [14C]naphthalene dissolved
in methanol; v) killed controls with sulfate added, to determine if activity
observed in the live microcosms was a consequence of biotic or abiotic
processes; and vi) killed controls with sulfate and approximately 0.45 µCi of [14C]naphthalene
dissolved in methanol. Each treatment
was prepared in triplicate, yielding a grand total of 18 microcosms.

After 122 days of incubation,
biodegradation activity was not observed for any of the PAHs, in spite of the
fact that sulfidogenic conditions did develop.

These results cast doubt on the opportunity to enhance in situ anaerobic
biodegradation of the two and three ring PAHs simply by addition of