Environ. Sci. Technol.
2010, 44, 7613–7621
Biodegradability of Lingering Crude
Oil 19 Years after the Exxon Valdez
Oil Spill
A L B E R T D . V E N O S A , * ,† P A B L O C A M P O , ‡
AND MAKRAM T. SUIDAN‡
U.S. Environmental Protection Agency, Cincinnati,
Ohio 45268, and Department of Civil and Environmental
Engineering, University of Cincinnati, Cincinnati, Ohio 45221
Received April 1, 2010. Revised manuscript received July
19, 2010. Accepted August 18, 2010.
In 2001 and 2003, geospatial surveys of lingering oil were
conducted in Prince William Sound (PWS) resulting in a prediction
of significant acreage being contaminated with substantial
subsurface oil from the 1989 Exxon Valdez oil spill (EVOS). In
2007, other researchers developed a mass weathering index (MWI)
based on the degree of weathering of PAHs normalized to
conserved biomarkers: if the degree of weathering of oil is 70%
or more, further attempts at bioremediation would be
unjustified. The objective of our study was to measure the
biodegradability of the 19-year lingering oil in laboratory
microcosms. Samples of beach substrate were collected from
representative sites in PWS contaminated with oil residues
of varying weathering states according to the MWI model. Enough
sacrificial microcosms were set up to accommodate two
treatments for each site (natural attenuation and biostimulation).
Results indicated that lingering oil is biodegradable. Nutrient
additionstimulatedbiodegradationcomparedtonaturalattenuation
in all treatments regardless of the degree of weathering. The
most weathered oil according to the MWI was the most
biodegradable. Substantial biodegradation occurred in the
natural attenuation microcosms due to the high sediment Total
Kjeldahl Nitrogen (TKN), which served as a nitrogen source
for biodegradation. Most of the observed biodegradation was
due to the presence of dissolved oxygen. Nitrogen was a
limiting factor but oxygen was the predominant one.
Introduction
In 2001, scientists from the National Oceanic and Atmo-
spheric Administration (NOAA) conducted a geospatial
Shoreline Cleanup Assessment Team (SCAT) survey of
lingering oil in Prince William Sound (PWS) and estimated
that about 11.3 ha of surface and subsurface oil remained on
the shoreline from the 1989 Exxon Valdez oil spill (1). Of that,
7.8 ha were estimated to be contaminated with a subsurface
oil mass of approximately 56 000 kg. Much of the lingering
oil was present in subsurface sediments in the middle to low
intertidal zone of the oiled beaches. Although substantial
weathering had occurred, high concentrations of potentially
toxic and mutagenic contaminants remained (i.e., chrysenes
and other high molecular weight polycyclic aromatic hy-
drocarbons or PAHs), suggesting that this lingering oil
* Corresponding author phone: 513-569-7668; fax: 513-569-7620;
e-mail: venosa.albert@epa.gov.
†
U.S. Environmental Protection Agency.
‡
University of Cincinnati.
10.1021/es101042h  2010 American Chemical Society
Published on Web 08/31/2010
continues to pose a threat to organisms that encounter it.
Note: weathering is defined as all physical, chemical, and
biological processes that alter oil composition, properties,
and behavior in the environment. These processes include
spreading, evaporation, dissolution, photooxidation, disper-
sion, and emulsification. The residues were classified using
the standard descriptive terms adopted in PWS: LOR, MOR,
and HOR, representing light, moderate, and heavy oil residue,
respectively (1).
Based on the 2001 survey, two follow-on surveys were
conducted (2, 3). Oil was found on 14 of 32 beaches surveyed.
The volume amounted to an estimated 0.43 ha covered by
surface oil and 1.52 ha containing subsurface oil. Thus, about
half the oil was present in the biologically rich lower intertidal
zone where otters and ducks could be exposed to it during
foraging.
For biodegradation of oil constituents to occur in the field,
several conditions must be met. First, a competent population
of oil degrading bacteria must be present at the oil-water
interface. In addition, an electron acceptor and nutrients
must be available to the degrading bacteria. Limited avail-
ability of any of these factors can lead to rate limitations.
Limitations on biodegradation rates due to low nutrient
concentrations could have played a part in the oil lingering
for such a long time in the subsurface. Previous research has
shown that nutrient concentrations between about 2-10 mg
N/L are required to support near-maximum oil biodegrada-
tion rates (4-8), and a 1994 field study in Delaware Bay (9)
showed that nitrogen concentrations of 3-6 mg N/L in the
interstitial pore water stimulated hydrocarbon biodegrada-
tion by 2- to 3-fold over a moderately high natural attenuation
rate where the average interstitial nutrient concentrations
averaged 0.8 mg N/L. These reported minimum nitrogen
concentrations are far higher than the average concentrations
of 0.2 mg/L observed in PWS pore water samples (10, 11).
Exxon-Mobil scientists also reported an increase in the
biodegradation rate of the Exxon Valdez oil in PWS beaches
when the pore water nitrogen increased from about 0.2 mg
N/L to 1 mg N/L due to nutrient addition (10).
Another likely factor contributing to the persistence of
subsurface oil is armoring (12), which stabilizes beaches by
protecting the underlying sediments from wave energy
reworking. Many of the PWS shorelines are unusual partly
due to the uplift caused by the 1964 Alaska earthquake, which
moved formerly subtidal sediments into the intertidal zone.
The resulting broad sediment grain size distribution was
poorly sorted, and wave action tended to transport smaller
particles either to landward gravel-cobble berms or seaward
where wave energy is low. This leads to “armoring” of the
beach wherein larger cobbles and boulders on the surface
protect smaller-grained sediments beneath them where the
oil is sequestered (12).
Biodegradation of crude oil requires about 0.04 g N/g oil
(4) and about 3 g O2/g oil (5). Based on unpublished data
from J. Short (personal communication, 2008), we calculated
average oil concentrations in HOR beaches of approximately
15 ( 18 g oil/kg sediment. Despite the patchiness of the oil,
approximately 0.6-1.3 g N/kg sediment and 45-99 g O2/kg
sediment would be required to support complete biodeg-
radation of oil. PWS seawater contains only about 0.2 mg
N/L (10, 13, 14) and about 8-9 mg O2/L. Therefore, at least
3 000-11 000 L of seawater must come into contact with
each kilogram of contaminated sediment to provide sufficient
nutrients and oxygen to support complete biodegradation
of the initial oil. Since only about 0.2-0.3 L seawater/kg
sediment is available in the pore water, assuming a porosity
VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7613
of ∼20-30%, biodegradation requires continual exchange
of pore water with the surrounding seawater. Incomplete
exchange of beach pore water with the surrounding seawater
during each tidal cycle would limit the extent of biodegradation.
It has also been observed during the biodegradation of
poorly soluble hydrocarbons that the microorganisms act at
the oil-water interface (15-17). Thus, one needs to account
for the bioavailability of oil to the degrading microbial
communities. That is, the biodegradation rate could be
limited by the mass-transfer rate of the biodegradable
components of oil, such as the PAHs, to the oil-water
interface (18, 19) where uptake by microorganisms occurs.
The bioavailability, and subsequently the biodegradation rate,
can also be limited by the presence of an interfacial barrier,
such as a dense mineral coating on the oil surface or a high-
viscosity layer at the oil-water interface (20).
In 2007, researchers proposed an index designed to
estimate the degree to which oil that has undergone
weathering is sufficient to determine whether or not to pursue
further treatment of lingering oil (21). This index is based on
the biomarker-normalized total polycyclic aromatic hydro-
carbon (TPAH) concentration at time t divided by the
biomarker-normalized TPAH concentration at time 0 and is
termed the mass weathering index (MWI). In this context,
the authors meant weathering by all means as defined earlier.
However, since the lingering PAHs after 19 years are
composed almost entirely of semivolatile organics that are
also poorly soluble in water, the context of weathering in
this paper is limited almost exclusively to biodegradation.
The authors argued that, if the MWI is 70% or more, then
further attempts at bioremediation would be futile since the
weathering is so severe that additional biostimulation effort
would not be justified.
The objective of this research was to determine the
biodegradability of the lingering oil in PWS 19 years following
the original spill under conditions where nutrients and oxygen
are not limiting factors. Three different sites in PWS were
selected based on their calculated MWI values. This laboratory
microcosm study was designed to provide evidence to
support a recommendation for or against bioremediation of
the remaining crude oil through addition of nutrients and/
or oxygen at selected sites in PWS.
Experimental Section
Site Selection and Sampling. Three sites were selected based
on their computed MWI, namely, KN114A (60° 29.09′, -147°
43.41′) from Knight Island (MWI ) 76%), SM006B (60° 31.67′,
-147° 23.10′) from Smith Island (MWI of 60%), and EL107
(60° 32.95′, -147° 33.45′) from Eleanor Island (MWI ) 30%).
These MWI calculations were made by Short (personal
communication, 2008) from his first SCAT survey, using TPAH
normalized to the sum of C2-3-chrysenes from 11-day old
tanker oil (see eq 1 in the Results and Discussion section).
Samples of oil-contaminated subsurface sediment were
collected in volumes of 50 L from each site. These 50 L samples
were collected by digging five separate pits at each site to a
depth that encompassed the oiled layer (typically around
10-40 cm below the surface), sieving them to remove
sediment particles greater than 1 cm, and placing them in
60 L metal drums previously washed with Alconox and clean
seawater. The drums were placed into overpack drums, iced
with glacial ice, and shipped overnight to the University of
Cincinnati (UC) for the microcosm study. Once the oiled
layer was reached, the oiled interval was collected. This
process was repeated five times at each site or until the
required volume of oiled sediment had been collected. At
EL107, an additional 20 L sample of uncontaminated
sediment was collected and processed in the same manner.
This sample served as a positive control (described below).
Seawater (50 L, 70 L at EL107) for use in the microcosms was
7614 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 19, 2010
collected at each site, packed in individual drums, iced with
glacial ice, and sent to UC. The glacial ice was still present
upon arrival.
Setup of Microcosms. A total of 117 microcosms and
accompanying reservoirs were fabricated and deployed,
comprising triplicate sacrificial units per sampling event per
treatment. The seven treatments included a natural attenu-
ation (NA) control treatment and a nutrient-amended
treatment for each of the three sites plus the nutrient-
amended positive control. The NA treatment would provide
an estimate of natural biodegradability in the presence of
nutrients naturally present in the seawater and/or sediment.
Since the oil had been lingering in these sediments for 19+
years, we assumed that little or no biodegradation would
occur in these microcosms.
A schematic diagram of the 316-stainless steel microcosms
is shown in Supporting Information (SI) Figure S1. Two
microcosms were arranged per wooden platform. Each
microcosm had a separate reservoir situated above it and
open to the atmosphere. A two-head peristaltic pump
withdrew seawater at a flow rate of 2 mL/min from the bottom
of each microcosm to the overhead reservoir, which operated
via a siphon mechanism. As the seawater approached the
level of the exit pipe in the overhead reservoir, the siphon
broke, and the water flowed rapidly into the microcosm
below. This occurred six times per day for each microcosm.
The purpose was to simulate intertidal zone submergence
of the oiled layer but not semidiurnal tidal inundations. It
was also to provide gentle mixing and reaeration of the
seawater to preclude bulk anoxic or anaerobic conditions.
Each microcosm contained 100 mL of 2 mm and 100 mL of
3 mm diameter glass beads at the bottom to prevent sediment
fines from exiting the unit and clogging the lines. Each
microcosm contained an 800 mL volume of sediment
(weighing approximately 1.5 kg) from one of the four field
sites placed atop the glass beads.
SI Table S1 summarizes the completely randomized
experimental design. The six sampling events were 0, 14, 28,
56, 112, and 168 days. All microcosms were housed in a
constant temperature room set at 15 °C, which approximates
the average daily temperature in PWS during the summer.
The 117 microcosms were deployed and labeled randomly
throughout the room. After day 0, three microcosms per
treatment were sacrificed (total of 21 altogether) for oil
analysis at each of the remaining five sampling events.
Positive Control. The triplicate positive control micro-
cosms consisted of the same 800-mL volume of clean,
uncontaminated sediment from the EL107 site. To each
positive control sediment, we added weathered crude oil
known from prior experience to be biodegradable (Alaska
North Slope Crude previously distilled at 512 °F or 272 °C)
at a concentration of 5 g/kg. This crude oil mimics fresh ANS
crude that has undergone approximately 10-14 days of
weathering on the open sea as all the light-end hydrocarbons
had been removed by the heat of distillation. The purpose
of the positive control was to determine whether some other
characteristic of the seawater, oil, or sediments would
preclude the growth of the bacteria, assuming a biodegrad-
able hydrocarbon substrate was present. Examples might
include lack of availability of oil or oil that is too weathered
to support effective biodegradation.
Extraction and Analysis of Hydrocarbons. On a desig-
nated sampling day, specified microcosms were sacrificed
for later chemical analysis. Approximately 600 of the 800 mL
of sediment from each microcosm was placed in a separate
aluminum canister and frozen until extraction. On the day
of extraction, the canisters were thawed to room temperature,
and four subsamples of 40 g each were removed for dry weight
analysis. The sediment subsamples were dried in an oven
overnight at 105 °C, then cooled in a desiccator and finally
weighed on an analytical balance. The cycle of heating,
cooling, desiccating, and weighing was repeated until a
constant weight was obtained (22). Four more subsamples
of 80 g each were first mixed with anhydrous Na2SO4 to
remove excess moisture and extracted for 18 h with 250 mL
dichloromethane (DCM) on a Soxhlet apparatus, concen-
trated to 10 mL by nitrogen blowdown, and then analyzed
by gas chromatography/mass spectrometry (GC/MS) (23).
The GC/MS analysis was performed using a 6890 gas
chromatograph coupled with a 5973 mass spectrometer from
Agilent (Palo Alto, CA). Chromatographic separation was
achieved on a J&W DB-5 MS column (30 m × 0.25 mm, 0.25
µm) (Palo Alto, CA). Analytes were detected in the selected
ion monitoring (SIM) mode. Total extractable hydrocarbons
(TEH) was quantified using a Hewlett-Packard 5890 GC Series
II equipped with a flame ionization detector (Wilmington,
DE, USA) (24).
The seawater of each microcosm (700-800 mL) was
transferred to a separatory funnel and the corresponding
reservoir and tubing extracted with 40 and 10 mL of DCM,
respectively. These solvent rinses were added to the funnel,
which was sealed and shaken vigorously for 1 min. The
seawater was serially extracted two more times with 50 mL
of fresh DCM. The three extracts were combined, concen-
trated to 10 mL, and analyzed by GC/MS and GC/FID.
Nutrients. The phosphorus source was sodium tripoly-
phosphate (Na5P3O10). The P-source was added only once at
a concentration of 25 mg/L. The source of nitrogen for the
biodegradation experiments was potassium nitrate (KNO3).
During the course of the experiment, nitrate-N was measured
in two randomly selected microcosms in each nutrient-
amended treatment by ion exchange chromatography (25).
If the concentration of nitrate-N declined to <5 mg/L, KNO3
was added back to 10 mg/L in all nutrient-amended
microcosms to replenish the used up nitrogen. Shortly after
startup, we discovered that the microcosms required the
addition of 50 mg/L nitrate-N to meet the unexpectedly high
nitrogen demand. This demand slowly diminished during
the course of the study. To replenish evaporated seawater,
distilled water was added to prevent increases in salinity
that could possibly inhibit further biodegradation. Total
Kjeldahl Nitrogen (TKN) in the sediment was measured in
each microcosm at days 0, 14, 28, 56, 112, and 168 by the
Hach method (26).
Sulfide and Volatile Solids (VS). Sulfide in the sediments
was determined by EPA Method 9030B (27). Sediment VS
was measured as the difference between the weight of the
dry solids at 105 °C and the weight of those solids after ignition
at 550 °C (28).
Statistical Analysis. Nonlinear regression using a Wald-
based test statistic was used to fit the exponential model’s
slope parameters from each site and treatment group. For
between-treatment comparisons at a given site, a common
intercept was fit along with different slope terms. For
between-site comparisons within a treatment (NA or nu-
trients), separate slope and intercept parameters were fit to
the data. All analyses were performed using the subroutine
SAS PROC NLIN, generated with SAS/STAT software (29).
Results and Discussion
Mass Weathering Index. The equation for the MWI (21, 30)
is shown below:
% loss of x ) [1 - (Cx/Cbiomarker)s/(Cx/Cbiomarker)evos] × 100
(1)
where x ) TPAH, Cx ) mass concentration of x (mg/kg
sediment), Cbiomarker ) mass concentration of conserved
species (in this case, hopane), s ) sample, and evos ) EVOS
cargo oil.
TABLE 1. Atlas-Bragg’s Mass Weathering Index (MWI) Computed
by NOAA Compared to MWI Computed in This Study
MWI computed MWI computed
source
by NOAAa in this studyb
KN114A 76 67.9
SM006B 60 60.0
EL107 30 46.9
ANS521 46.3
a
Based on normalizing to C2-C4-chrysenes on 11-day
old tanker oil. b Based on normalizing to hopane on orig-
inal fresh tanker oil.
Table 1 summarizes the MWI computed by NOAA for
11-day old tanker oil compared to our computation for fresh
tanker oil (kindly supplied by S. Miles, LSU). The calculation
of MWI by NOAA differed from ours since NOAA used 11-
day old EVOS cargo oil TPAH normalized to the sum of C2-4-
chrysenes for the denominator in eq 1, while we used hopane-
normalized fresh EVOS cargo oil. Despite this computational
difference, the two estimations are in approximate agreement.
EL107 and ANS521 were almost identical in terms of
weathering as estimated by the MWI, suggesting that, in terms
of TPAH, EL107 was weathered to the same degree as oil that
has been distilled at 272 °C to mimic oil that has weathered
for only 10-14 days on the open sea.
Figure 1 is a comparison of GC/MS results (y-axis) for the
three sites and ANS521 to the fresh cargo oil (x-axis) used in
the positive control (all data hopane-normalized). Were there
exact agreement between these groups, the data would fall
on the dashed 45° line with a slope of 1, indicating an MWI
of 0. The KN114A TPAH data were more weathered than the
ANS 521 oil (linear regression slope of 0.67 with nine PAHs
below the 45° line), the SM006B TPAH were less so (linear
regression slope of 0.81 with 7 PAHs below the 45° line),
while the EL107 TPAH were close to ANS521 (linear regression
slope of 0.99, differing in Phe and C1- and C2-Phe). The
symbols falling below the 45° lines are labeled in each panel,
identifying the specific PAH corresponding to those symbols.
The five naphthalenes were not included in any of the panels
in Figure 1 as their volatility is greater than that of other
PAHs, and thus did not conform to the 45° line.
Figure 2 summarizes the biodegradation results of all
treatments. The best fit first-order equations are shown
adjacent to the corresponding legend symbols (data were fit
with a common intercept). The y-intercepts for each of the
three sites show the differences in initial oil concentrations,
with KN114A having the least amount of PAHs, EL107 the
most, and SM006B an intermediate level. These results agree
with the MWI for each site (Figure 1). The graphs indicate
that the oil was biodegradable regardless of the degree of
weathering as measured by the MWI. In fact, the biodeg-
radation rate coefficients were of the same magnitude as
those measured for the NA treatments in the 1994 Delaware
field study (10), where the average temperature was ap-
proximately twice that of this study. Unexpectedly, in all
cases the oil was biodegraded significantly even in the NA
treatments where no nutrients were added to the micro-
cosms. As noted by the p-values in SI Table S2, the
biodegradation rate coefficient for the nutrient treatment
was significantly higher than the NA treatment in all cases
(p ) 0.0081 for KN114A, 0.0006 for SM006B, and 0.0001 for
EL107). Thus, nutrients significantly stimulated biodegrada-
tion rates compared to NA rates.
The positive control, depicted in panel d of Figure 2,
behaved differently from the other treatments. The biodeg-
radation rates for TPAHs were low, similar to the NA
treatments for the three sites. These results imply that the
microbial population was initially different from those found
VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7615
FIGURE 1. Comparison of the hopane-normalized TPAH profile of the weathered oil samples from the three PWS sites and ANS 521
oil vs the fresh cargo oil (dashed lines signify theoretically perfect fit, while solid lines signify actual fit). Naphthalenes were not
included due to their high volatility. All data were hopane-normalized. Labels on the graph adjacent to symbols show the individual
PAHs in the oil that deviate from the concentrations of PAHs in the cargo oil. Unlabeled symbols are the concentrations of the
remaining PAHs that agree with their concentrations in the cargo oil.

FIGURE 2. Hopane-normalized TPAH biodegradation as a function of time in all treatments. In panels a-c, the symbol for the natural
attenuation treatment (NA) is an open circle, whereas the symbol for the nutrient treatment is a closed circle. In the positive control
(panel d), the total alkanes are shown as open circles while the TPAHs are shown as closed circles.

at oiled sites and that the oil-degrading population was
possibly smaller (microbiological enumerations were not
performed). Upon acute exposure to 5 g of ANS 521/kg
sediment, the putative low numbers of these microbial
communities may have been further lowered by the sudden
exposure to oil. Nonetheless, the results from the positive
control samples confirmed that the experimental system
successfully demonstrated active biodegradation taking
place.
Differential Biodegradation Rates of Individual PAHs.
To determine if the PAHs in this microcosm study were
biodegraded rather than breaking down or washing out as
a result of another factor, we calculated the rate coefficients
for each PAH constituent in all treatments. These rates are
presented in SI Table S3. Not only did first-order rate
coefficients decrease for most as the degree of alkylation
increased within the homologous PAH series, but the PAHs
concerned also followed the order of biodegradation men-
tioned above. Rates were generally higher (p < 0.05) for
nutrient treatments compared to the corresponding NA
treatment within a given site, further confirming that nutrient
addition enhanced biodegradation. These observations

7616 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 19, 2010
clearly indicate that the observed disappearance of PAHs
was due to biodegradation rather than unknown factors.
Paired comparisons of TPAH rate coefficients among the
three sites for each treatment (NA vs nutrients) in SI Table
S4 shows that the statistical ranking of the rate coefficients
was KN114A > EL107 > SM006B (p , 0.01 in all instances)
for both the NA and nutrient treatments. KN114A, the most
weathered site, had the highest TPAH biodegradation rate.
Another confirmation that biodegradation was the mecha-
nism responsible for disappearance of the PAH is that the
concentration of the biomarker hopane remained constant
throughout the study (data not shown).
Biodegradation of Hopane-Normalized TEH. Biodeg-
radation of hopane-normalized TEH is presented in SI Figure
S2 and paired rate coefficients compared in SI Table S2. Rates
were lower for TEH than for TPAH in all instances as TEH
includes unresolved compounds influencing the analytical
results. For all three cases, SI Table S2 shows that the TEH
rate coefficients for the nutrient treatment were higher than
the corresponding natural attenuation treatment, but in only
one case (KN114A) was the difference not significant (p )
FIGURE 3. Total Kjeldahl nitrogen (TKN) concentrations in the
0.174). SI Table S4 summarizes the paired comparisons of
natural attenuation and nutrient-amended treatments for the
TEH biodegradation rates between the various sites for NA duration of the study (no NA treatment was run for the EL107
and for nutrients. The only significant difference observed clean sediment). Error bars represent (1 standard deviation
was between SM006B and EL107 for the NA treatment (p ) unit based on the three independent microcosm measurements
0.018). This is in marked contrast to the TPAH comparisons at each sampling event.
in which all paired comparisons were statistically significant
(p < 0.05). to nonpolar hydrocarbons, the rate of biodegradation of
Nitrogen Demand and TKN Measurements. That bio-
remaining oil in contact with nutrients is unlikely to be
degradation in the NA treatments was significant raised
nutrient limited. Our data suggest that addition of nitrate
concern for finding a possible mechanism of action. Within
and tripolyphosphate to our microcosms did indeed stimulate
2 days following startup, the nitrate-N demand in all
higher biodegradation rates than the already high natural
microcosms was high (nitrate-N was undetectable in the
attenuation rates, so in the field nutrients were limiting at
seawater despite our adding it to all nutrient-amended
least to some degree.
microcosms). These results required increasing the nitrate-N
The conditions in our microcosms were highly conducive
to 50 mg/L every 2-3 days during the first weeks of the
to bioremediation. We ensured that oxygen, which consis-
investigation. This nitrogen demand eventually plateaued
tently exceeded 4 mg/L in the recycled aqueous phase, was
during the course of the study. SI Figure S3 depicts the
not limiting in any of the treatments. Similarly, nitrogen was
cumulative consumption of nitrate-N over time for the three
nutrient-amended microcosms. The average cumulative not limiting in the nutrient-amended microcosms. The latter
nitrate-N consumed in the nutrient-amended KN114A, was of specific concern in determining the potential bio-
SM006B, and EL107 microcosms was 115, 516, and 324 mg/ degradability of the oil irrespective of any weathering that
kg sediment, respectively. it may have undergone. The sediment used in the microcosms
We measured the TKN in the sediments to determine if had been disrupted during excavation, sieving, and mixing,
this could explain these results. Figure 3 shows the TKN levels thus exposing the oil and increasing its bioavailability to the
in all microcosms at each of the sampling events. The initial degrading community. We agree with the authors’ conclu-
TKNs ranged from 350 to 540 mg/kg sediment. These sions (30) that the oil has been sequestered in a low
concentrations were unexpectedly high, indicating the pres- permeability layer in the subsurface below a higher perme-
ence of natural organic matter (NOM) in the sediments of ability layer, as found and confirmed more recently (31).
the beaches selected for this study. Consequently, since the However, since we found the oil to be highly biodegradable,
only source of nitrogen in those microcosms was the biogenic our data suggest the sequestered oil may be amenable to
NOM in the sediments, this would be the most likely bioremediation with disruption of the low permeability zone.
explanation for the observed PAH biodegradation. Another The rates of PAH biodegradation under conditions of
significant observation in Figure 3 is that the TKN declined excess nitrogen were 0.028 d-1 for KN114A, 0.0099 d-1 for
but only in the nutrient-amended microcosms (shown in SM006B, and 0.017 d-1 for EL107 (Figure 2 and SI Table S2).
the lower curve in each panel of Figure 3). This compares favorably with rates measured in a previous
In a recent article (30), the authors conducted detailed field study in Delaware (10) where we measured TPAH
surveys of subsurface oil at numerous locations in PWS and biodegradation at 0.029 d-1 in the nutrient-amended plots.
concluded that the MWI they measured was too high (>70% The temperature in our microcosm study was 15 °C, whereas
relative to the original cargo oil) at the sites to support 30 °C was the average temperature during the Delaware field
biostimulation (nutrient addition) to remove lingering oil, a study. While others have reported decreases in the overall
conclusion that is clearly refuted by the data presented here. rates of biodegradation over time (30, 32, 33), such observa-
The oil at the three sites was highly biodegradable regardless tions are consistent with first-order reactions. First order
of the degree of weathering as estimated by the MWI. In fact, connotes rates of biodegradation that are dependent on the
the biodegradation rate coefficient was highest for KN114A, concentration of the substrate (“substrate” in this sense is
which had the highest MWI (most weathered) of all three the sum total of all hydrocarbons in the oil). Consequently,
sites. This suggests that the MWI is an oversimplification if the substrate concentration decreases, the biodegradation
that is not predictive of potential in situ bioremediation rate does the same. When the data showed that higher
success of treating the lingering oil. The authors further molecular weight compounds degrade more slowly than
concluded that, based on the relatively high ratio of nitrate-N lower molecular weight compounds (SI Table S3), this

VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7617

FIGURE 4. Biodegradation of hopane-normalized total chrysenes
for each site (dashed lines signify natural attenuation microcosms,
solid lines signify nutrient-treated microcosms, and error bars
represent (1 standard deviation unit based on the three
independent microcosm measurements at each sampling event).
indicated that such compounds are more difficult for
microorganisms to degrade.
The results of an ecologically relevant study that used
semipermeable membrane devices (SPMD) to evaluate the
P450 1A (CYP1A) induction potential of various potential
PWS hydrocarbon sources in juvenile rainbow trout were
recently published (34, 35). The investigators reported that
increased CYP1A activity was strongly associated with PAHs
from EVOS oil, especially the 4-ring planar chrysene ho-
mologues. One of the sites monitored was KN114A, which,
according to the MWI, was the site with the most weathered
oil in our study. Figure 4 summarizes the biodegradability
of the hopane-normalized total chrysenes plotted as a
function of time. The figure shows that the chrysene
homologues were most heavily biodegraded in the nutrient-
treated KN114A site (73.4%) compared to the same treatment
for the other sites (35.1% for SM006B and 41.1% for EL107)
where the oil was less weathered according to the index.
This is further evidence that the weathering of EVOS oil
predicted by the MWI not only is an inadequate indicator of
biodegradability but also does not account for potential
CYP1A induction activity (and thus ecotoxicity exposure) 19
years after the spill.
As presented earlier, the sediments from the three study
sites in PWS had a high TKN content (540 mg/kg sediment
for KN114A, 350 mg/kg for SM006B, and 480 mg/kg for
PSW3A4). On average, this amount of TKN provides
sufficient nitrogen for PAH biodegradation to occur,
assuming oxygen is sufficient to support aerobic PAH
degradation. However, as little to moderate TPAH bio-
degradation has occurred in these sediments since the
input of oil, it is likely that oxygen failed to reach the oiled
subsurface layer except at the interface between the higher
permeability zone and the low permeability oiled zone.
This was not a concern in our aerobic microcosms, which
contained disturbed sediment that had been excavated,
sieved, and mixed. The TKN naturally present in the
sediments provided the nitrogen necessary for the partial
degradation of PAHs in the NA treatments (84.6% depletion
of hopane-normalized TPAH for KN114A, 48.6% for
SM006B, and 71.0% for EL107 in the 168 day duration of
the study). In addition, a further depletion of TPAH
occurred in all microcosms when excess nitrate-N was
supplied (total of 95.3% for KN114A, 81.1% for SM006B,
and 91.8% for EL107).
7618 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 19, 2010
The biomass of the PWS sediments as measured by volatile
solids (VS) was 37.4 g/kg sediment for KN114A, 24.8 g/kg for
SM006B, and 33.7 g/kg for EL107. Biomass will decay even
in an anaerobic or anoxic environment with the release of
CO2 and ammonia. In the undisturbed sediments of PWS,
significant biodegradation of PAHs would be unlikely as PAHs
do not biodegrade rapidly under anaerobic or anoxic
conditions, especially if nitrate is the electron acceptor
(36-39). However, in our microcosms not only did we observe
substantial PAH biodegradation in the absence of exogenous
nitrogen addition (NA treatments, Figure 2), but we also
observed significant consumption of TKN when excess nitrate
was added (Figure 3, solid lines). The VS measured in our
microcosm sediments and its associated TKN would normally
provide an additional demand for the available oxygen in
the seawater. However, the addition of nitrate to the nutrient-
amended microcosms supplied sufficient additional oxygen
from the nitrate to satisfy the extra competition for an electron
acceptor. We propose that the disappearance of TKN in the
presence of exogenously added nitrate-N may be explained
by denitrification mechanisms occurring within anoxic
microenvironments in the microcosms.
To provide further evidence for this hypothesis, we plotted
the oxygen content of the nitrate consumed in each treatment
as a function of the difference between the total PAH degraded
in the nutrient and NA treatments (Figure 5). The slope of
the linear regression line for the three points, which refer to
the three PWS study sites (labeled on the graph), is 58.2 mg
nitrate-oxygen consumed per mg PAH biodegraded beyond
what was degraded in the NA microcosms. The theoretical
oxygen demand (ThOD) of PAH is 3 mg oxygen/mg PAH (5),
so the 58.2 mg oxygen measured as the slope of the regression
line in Figure 5 is substantially in excess of this theoretical
demand. This oxygen can only be used for the breakdown
of the organic matter in the sediments, since PAHs do not
degrade substantially under denitrifying conditions. Thus,
the conclusion from this is that nitrate in our microcosms
was acting not only as a nitrogen source for biodegradation
of PAHS but also as an electron acceptor for the degradation
of the natural organic matter in the sediments during
denitrification. Even though denitrification only occurs under
anoxic conditions, it is plausible that pockets of anoxic
microenvironments existed within the sediments of the
microcosms. It is also plausible that the absence of oxygen
within the layers of organic matter and oil covering the
sediments facilitated the utilization of nitrate by facultative
microorganisms and led to the consumption of organic
matter, as evidenced by the decline in TKN.
Lending further credence to the denitrification argument
was the sulfide content measured in the sediments. Sulfide
was present at 10.4 ( 1.1 mg/kg sediment in KN114A, 11.1
( 0.2 mg/kg in SM006B, 13.1 ( 8.1 mg/kg in EL107, and 11.2
( 2.7 mg/kg in the clean EL107 sediment. The presence of
sulfide in all four sediments, from the anaerobic reduction
of sulfate to sulfide by sulfate reducing bacteria (40), suggests
that the sediments used in the microcosm study were in a
reduced state (anaerobic or anoxic) when collected and sent
to the laboratory.
One additional issue merits discussion. The ThOD of the
organic matter in the three sediments is assumed to be ∼1.4
mg oxygen/mg VSoxidized (41). Based on the total TKN removed
in each treatment and assuming that VS and TKN degrade
proportionately, the ThOD of the biodegraded VS in the
sediments was calculated to be 16.1 g oxygen/kg sediment
for KN114A; 15.7 g oxygen/kg sediment for SM006B; and
12.9 g oxygen/kg sediment for EL107, all of which are much
higher than the ThODs for the TPAH in those same sedi-
ments (49 mg oxygen/kg sediment for KN114A, 193 mg
oxygen/kg sediment for SM006B, and 299 mg oxygen/kg
sediment for EL107). This suggests that nitrate supplied
FIGURE 5. Nitrate-oxygen consumed as a function of the difference in TPAH biodegraded between natural attenuation and nutrient
treated sediments (error bars represent (1 standard deviation unit based on the three independent microcosm measurements at
each sampling event).
FIGURE 6. Cumulative Total Kjeldahl nitrogen (TKN) consumed as a function of cumulative nitrate-N consumed.
oxygen for the oxidation of the NOM in the sediments. This
is supported by Figure 6, which shows total TKN consumed
as a function of total nitrate-N consumed for each of the
three sites. Although the relationships are not perfect, in all
instances, consumption of TKN increased in conjunction
with nitrate-N consumption. Since ammonia-N is the
preferred nitrogen source for microbial growth (42), and as
ammonia-N is released when TKN is consumed, the most
likely explanation for both nitrate and TKN consumption
increasing concurrently is that nitrate is being used both as
a nitrogen source for the oil degraders and as an electron
acceptor for the heterotrophic microorganisms growing on
the organic matter in the sediments.
Even though the above hypothesis is plausible, it was
not considered by Atlas and Bragg (21, 30) in their
discussion of the implications of nutrient limitations in
Prince William Sound. Yet substantial surface and sub-
surface plant biomass exists throughout PWS, and the
decay of this biomass adds additional organic matter to
the extant sediment annually. Thus the addition of excess
nitrate to the sediments in PWS could accelerate the
breakdown of the organic matter under denitrifying
conditions, resulting in greater porosity (which is equiva-
lent to mild disruption of the low permeability oiled zone)
and thus increased contact of the sequestered oil with
oxygen and nutrients. Research is needed to test this
hypothesis. If research shows that addition of nitrate alone
does not enhance biodegradation, then perhaps some
physical disruption of the low permeability zone may be
appropriate to achieve the accelerated treatment. Research
is needed to evaluate the effectiveness of pumping seawater
below the oiled layer at sufficient velocity to perturb this
zone and allow channels to form in the oiled layer. Delivery
of electron acceptor in the pumped seawater in the form
of hydrogen peroxide and nitrate should also be evaluated.
This may be possible with minimal ecological impact of
the affected areas. The nitrate would serve the dual purpose
of providing nitrogen for oil degradation and electron
acceptor for the breakdown of the sediment organic carbon,
as our study showed. To our knowledge, this approach has
not been put forth in the literature.
Acknowledgments
Funding for this project was provided by the Exxon Valdez
Oil Spill Trustee Council. We thank Mr. Joseph Holmes of
Research Planning, Inc. (RPI), who led the field crew in the
collection of the samples from the three sites in Prince
William Sound and shipped them to the laboratory in
Cincinnati. Dr. Jacqueline Michel, President of RPI, helped
develop the sampling plan. Mr. David Janka was the captain
of charter vessel Auklet that was used by the field crew.
We are grateful to Mr. Dan Radigan and Dr. Seungloon
VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7619
Chung (University of Cincinnati), who fabricated the
microcosms and assisted in sampling and conducting the
environmental analyses (TKNs and nutrients). We ac-
knowledge Mr. Larry Wetzel of U.S. EPA-Cincinnati, who
fabricated the infrastructure housing the microcosms and
Ms. Edith Holder, who helped with the initial loading of
the microcosms and the subsequent sacrificial sampling.
We thank Dr. Dennis W. King of Statking Consulting, Inc.,
who conducted the statistical analyses.
Supporting Information Available
Four tables and Three figures. The tables summarize the
experimental design and all statistical rate comparisons; The
figures show the schematic of a microcosm, the summary of
the TEH data, and the cumulative nitrate consumption data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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