EPA/600/D-90/119
THE ROLE OF MICROORGANISMS IN THE BJOREMEDIATLON
OF THE OIL SPILL IN PRINCE WILLIAM SOUND, ALASKA
by
John E. Rogers*, Rochelie Araujo*,
Parmely H. Pritchard**, and Henry H. Tabak***
^Environmental Research Laboratory
U.S. Environmental Protection Agency
Athens, Georgia 30613
**Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
***Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS. GEORGIA 30613

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THE ROLE OF MICROORGANISMS IN THE BIOREMEDIATION
OF THE OIL SPILL IN PRINCE WILLIAM SOUND, ALASKA
John E. Rogers,1 Rochelle Araujo,1
Parmely H. Pritchard,2 and Henry H. Tabak3
Hj.S. Environmental Protection Agency
College Stati6n Road
Athens, Georgia 30613-7799
2U.S. Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
3U.S. Environmental Protection Agency
RRRL, 26 W Martin Luther King Drive
Cincinnati, OH 45268
The U.S. Environmental Protection Agency's Alaska Bioremediation Project
was initiated in the aftermath of the March 24, 1989, EXXON VALDEZ oil spill.
The objective of the project was to demonstrate an alternative cleanup method
for oil-contaminated shorelines based on enhancing natural biodegradation of
the oil through the addition of nitrogen and phosphorus nutrients. This
enhancement process is a well-recognized and scientifically sound approach to
bioremediation but had never been tested on a large scale in marine
environments. The project was managed by EPA's Office of Research and
Development with financial, scientific, and logistical support from the Exxon
Company USA under the authority of the Federal Technology Transfer Act. The
report describes the microbial aspects of the EPA field study in Prince
William Sound, Alaska.
Two test sites, Snug Harbor and Passage Cove, were selected for the
field demonstration project. Snug Harbor was selected to serve as a beach
with oil contamination that approximated the degree of contamination remaining
after a heavily oiled beach had been physically washed. Physical washing was
the major cleanup available for testing early in the summer. In July, a
second site was selected that had been physically washed by the Exxon
operations. This site, Passage Cove, served as the main reference beach for
the large-scale application of fertilizers and as a means to evaluate a
sprayer system for fertilizer application.
Criteria for the selection of the test sites were based on six desired
features:
•	Typical shoreline of Prince William Sound, i.e., mixed sand and gravel
and cobblestone beaches
•	Sufficient area with fairly uniform distribution of sand, gravel, and
cobble for the test plots
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•	Protected embayment with adequate staging areas and sufficient size to
support several test and control plots
•	Uniform oil contamination
•	Minimal impact from freshwater inputs
•	Shoreline with a gradual vertical rise
SNUG HARBOR PROJECT SITE
Snug Harbor is located on the southeastern side of Knight Island. The
shoreline utilized for the demonstration is located on the western side of
this harbor. The area is surrounded by mountains, reaching an elevation of
approximately 2000 feet, with steep vertical ascents. Major sources of
freshwater runoff are from precipitation and snowmelt, which is typical of
islands in Prince William Sound. Although other shorelines in Snug Harbor
were heavily contaminated with oil, it appeared that little oil was being
released to the water, thus minimizing the prospect of reoiling on the beaches
chosen for treatment and reference plots.
Table 1 identifies the beach types, dimensions, and treatment. Each
plot was divided into 21 blocks, with 7 blocks in 3 tidal zones: high,
intermediate, and low.
Table 1. Description of Fertilizer Treatment Demonstration Plots at Snug
Harbor
Beach Name
Eagle
Otter
Otter
Beach Type
Sand, gravel
Sand, gravel
Sand, gravel
Fertilizer
Treatment
None-reference
Oleophilic
fertilizer
Water-soluble
fertilizer
Length (m) Depth (m)
21	12
21	12
35
12
Seal
Cobble
Water-soluble
fertilizer
28
12
Seal
Cobble
Oleophilic
fertilizer
28
12
Seal
Cobble
None-reference
21
2

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Oil contamination in the test area represented a continuous band along
the length of the beach. This band was approximately 15 to 20 meters wide and
corresponded roughly with the average boundaries of the high and low tides
observed in Snug Harbor, To determine the approximate distribution of oil on
the beach, samples of beach material from one of the designated mixed sand and
gravel plots were taken on May 25, 1989. The samples were extracted, and the
oil weight and chemical composition were determined. It was clear from the
oil residue weights and ratios of C17/pristane and C18/phythane at two
different depths that some weathering of the oil had occurred. Oil
concentrations varied considerably, ranging from a high of 67,000 mg/kg of
beach material to a low of 37 found in the-top 10 cm of the beach. Changes in
the ratios, relative to' fresh Prudhoe Bay crude, were also apparent in some
samples, indicating biodegradation of the oil. Changes were quite variable,
but biodegradation appeared to be occurring at the lower depths.
PASSAGE COVE PROJECT SITE
Passage Cove is located on the northwestern side of Knight Island. This
site was originally heavily contaminated with oil and was physical washed by
Exxon. Even after physical washing, considerable amounts of oil remained at
this site, mostly spread uniformly over the .surface of rocks and in the beach
material below the rocks. Pools of oil and mousse-like material were minimal
on the surface. Contamination was apparent to about 50 cm below the beach
surface. All of the beach areas tested were cobblestone set on a mixed sand
and gravel base. Table 2 lists Passage Cove beach designations and plot
dimensions and fertilizer treatments.
Table 2. Description of Fertilizer Treatment Demonstration Plots at Passage
Cove
Beach Name
Raven
Beach Type
Cobble over
mixed sand and
gravel
Nutrient
Applications
None-reference
Length (m) Depth Cm)
28	21
Tern
Cobble and
mixed sand and
gravel granules
Oleophilic and
slow-release
35
21
Kittiwake
Cobble over
mixed sand and
gravel
Nutrient solution
sprinkler system
28
21
Guillemot
Mixed sand and
gravel with
patchy cobble
Oleophilic and
granules slow-
release
21
3

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MICROBIOLOGY OF SNUG HARBOR
Test beaches at Snug Harbor were moderately contaminated. Visually, the
cobble plots, had a thin coating of dry, sticky, black oil covering rock
surfaces and gravel areas under the cobble. Oil did not penetrate more than a
few centimeters below the gravel surface. In mixed sand and gravel plots, oil
was well distributed over exposed surface areas and commonly was found at 20
to 30 cm below the surface. In many areas of the test plots, small patches of
thick oil and mousse could be found. This material was very viscous and mixed
with extensive amounts of debris.
Approximately 8 to 10 days following oleophilic fertilizer application
to the cobble beach plot, reductions in the amount of oil on rock surfaces
were visually apparent. The reduction was particularly evident from the air.
The plot was a clean rectangle on the beach surface surrounded by oiled areas.
The contrast also was impressive at ground level; there was a precise
demarkation between fertilizer-treated and untreated areas.
Close examination of this treated cobble plot showed that much of the
oil on the surface of the rocks was gone. Considerable amounts of oil still
remained under rocks and in the mixed gravel below these rocks. The remaining
oil was not dry and dull as was the case with oil in other areas of the beach,
but appeared softened and more liquid. It also was very sticky, with no
tendency to come off the rocks. At the time of these observations, no oil
slicks or oily materials were observed leaving the beach during tidal
flushing.
The mixed sand and gravel beach treated with oleophilic fertilizer also
appeared to have reduced amounts of oil in an 8 to 10 day period. However,
differences between treated and untreated plots were not as dramatic as on the
similarly treated cobble beach. Loss of subsurface oil in treated areas was
also visually apparent. Reduction of oil contamination was particularly
evident at sampling times, as noticeably less oil remained on sampling
equipment used on this beach plot.
At this time, all other plots looked as oiled as they did at the
beginning of the field study. There were essentially no visual indications of
oil removal on plots treated with slow-release fertilizer briquettes.
Over the next 2 to 3 weeks, the cleaned rectangle on the cobble beach
remained clearly visible. Oil below the rocks remained but was less and less
apparent and untreated reference plots appeared relatively unchanged. The
oleophilic-treated mixed sand and gravel plot actually showed a greater loss
of oil, appearing increasingly cleaner.
Six to eight weeks after fertilizer application, the contrast between
the treated and untreated areas on the cobble beach narrowed. This was due to
reoiling from subsurface material concurrent with the slow removal of oil on
the beach material surrounding the plot. However, it was evident that the
total amount of oil on the treated plots had decreased substantially relative
to reference plots. The corresponding mixed sand and other plots still had
observable oil contamination but generally less than that seen at the
beginning of the study.
4

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Toward the end of the summer, the area used for the bioremediatiori study
became steadily cleaner, as did most of the areas surrounding the test plots.
This was attributed to several storms and more frequent rainfall. A heavily
contaminated.area to the south that had never been treated, remained heavily
contaminated.
Determinations of numbers of oil-degrading bacteria present in beach
materials were made at each sampling of Snug Harbor, using all 21 sediment
samples taken from each test plot to evaluate sediment chemistry. Numbers of
degraders were assessed by serially diluting each sample in a minimal salts
medium containing ammonium and phosphate, adding a small quantity of oil to
each dilution, and incubating the dilutions for 21 days. The highest dilution
series showing degradation was then scored, and a calculation was made based
on dilution to extinction of the total oil degraders in the undiluted sample.
Although similar in design to a single tube MPN procedure, the dilution to
extinction procedure should not be mistaken for such.
Results from these studies are shown in Table 3. The values reported
are the log10 mean and standard deviation of 18 to 21 dilution series for each
mixed sand and gravel plot (no cobble beach material was analyzed). When a
plot was sampled on two separate days, the results represented eight to ten
dilution series per day. Table 3 has been keyed to indicate the number of
determinations within a plot in which every dilution in the series was
positive for oil degradation. The greater the number of positive dilutions,
the greater the underestimation of the relative oil-degrading population.
Results suggested that an increase in oil-degrading microorganisms
occurred within the oleophilic fertilizer-treated plots between the time zero
and 9 days after application. The results from the water-soluble treatment
showed the same trend, but the differences in both cases were not
statistically supportable. For unexplained reasons, oil degraders increased
more than 100- to 200-fold on day 31 in control and water-soluble fertilizer-
treated plots.
It was concluded from the available data that an increase in oil-
degrading microorganisms may have occurred as a result of fertilizer
application, but it could not be statistically verified. The apparent
increase in organism populations in the fertilized plots at day 9 corresponds
to the high level of nutrients seen immediately following the application of
nutrients. In these tests, the presence of high numbers of oil-degrading
bacteria in the control beaches made it difficult to detect subtle differences
in the numbers of degrading organisms between treatments.
MICROBIOLOGY OF PASSAGE COVE
Original oil contamination in Passage Cove was heavy. Following
complete physical washing, oil was well distributed over most of the surface
of all cobble and all gravel under the cobble. The oil was black, dry, and
dull with considerable stickiness. It was spread as a thin layer over the
beach material. Relatively few patches of pooled oil or mousse were present
but where they were present the oil was thick and viscous. Oil also was
found at depth in the beach', generally 30 to 40 cm below the surface. It was
well distributed within the beach material.

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Table 3. Relative Concentrations (Logl0 of the Cell Numbers/g of Beach
Material) of Oil-Degrading Microorganisms in Snug Harbor Mixed Sand and Gravel
Test Plots® •
Sampling Dateb
Before Application	Davs
6/8/89	0
Fertilizer
Control	Water Soluble	Oleophilic
6.58	5.95
±1.00'	+/-1.29
6/11/89
6.16
±0.89
5.80
±0.91
After Application
6/17/89	9
6/24/89	16
7/8/89
30
>6.24*
5.96
±0.83
6.61
±1.34
>6.62*
5.86
±1.15
>6.91v
5.96
+/-1.10
5.86
+/-0.67
7/9/89
31
>8.47*
>9.39*~-
aNo. of dilution series positive in all dilution tubes (0-25%); *(25-50);
**(50-75) .
bSamp Les on 6/8/89 and 6/11/89 are preapplication of the fertilizer.
Within approximately 2 weeks following application of oleophilic
fertilizer and slow release granular fertilizer, it became apparent that the
treated beach was considerably cleaner relative to the reference plots. In
contrast to the observations at Snug Harbor, not only did the rock surfaces
look cleaner but the oil under the rocks and on the gravel below was also
disappearing. In another 2 weeks, oil could be found only in isolated patches
and at 10 cm and below in the subsurface. At no time were oil slicks or oily
material seen leaving the beach area. During this time, no loss of oil from
the rock surfaces was apparent in the reference plot.
The beach treated with fertilizer solution from the sprinkler system
behaved in a very similar manner to the oleophilic-granule-treated plot; that
is, it became clean. The only difference was that it lagged behind the
oleophilic/granular-treated beach by about 10 to 14 days. By the end of
August, both beaches - - the oleopni.1 ic and fertilizer solution treated beaches
looked equally clean. In contract, the reference plot appeared very much as
it did in the beginning of the field study. Oil in the subsurface still

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remained in all plots. In the fertilizer-treated plots, however, oil was
apparent visually only below a depth of 20 to 30 cm.
The number of oil-degrading bacteria present on beach materials also
was determined for Passage Cove. Samples of beach material were taken from
grids 1, .3, 5, 7, 8, 10, 12, 14, 15, 17, 19, and 21. Numbers of degraders
were assessed by a modification of the dilution to extinction method used for
Snug Harbor. Five replicate dilution series were prepared from the initial
1:10 dilution. The relative numbers of bacteria in each sample was an average
of the five replicate dilution series.
Results from these studies are shown in Table 4, The values reported
are the log10 normal mean and standard deviation of 11-12 dilution series for
each mixed sand and gravel sample. Results suggested that no consistent
increase in oil-degrading microorganisms occurred as a result of fertilizer
application. This means that, even in the plot treated with nutrient
solutions from a sprinkler system where nutrient exposure to the bacteria
should be optimized, no increase in oil-degrading microorganisms occurred.
This could be the result of a relatively constant sloughing of microbial
biomass from the surfaces of the beach material, perhaps as caused by tidal
flushing action. Grazing by protozoans also could keep the microbial numbers
at a specific density. The presence of high numbers of oil-degrading bacteria
in the reference beaches made it difficult to detect subtle differences in the
numbers of degrading organisms between treatments.
Table 4. Relative Concentration (Log10 of the cell number/g of beach material)
of Oil-Degrading Microorganisms in Passage Cove.
Sampling Date
Before Application
07/22/89
After Application
08/06/89
08/19/89
Refe rence
6.44
±1.44
5.32
±1.12
6.60
+1.83
Plots Fertilizer-Treated
Water
Soluble
Oleophilic &
Water soluble
6.31
±1.36
6.44
±1.33
5.78
±1.45
5.71
±0.67
5.47
±1.34
5.66
±0.35
MICROBIOLOGY OF UNCONTAMINATED BEACHES
In early August, several' beaches that had not been affected by the oil
spill were sampled to determine relative levels of oil-degrading

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microorganisms. Samples were collected from the high, mid, and low tide areas
at each beach, The bacterial densities are shown in Table 5. The range in
concentration of oil-degrading organisms was much greater than that observed
for oil-impacted beaches. It is clear that the number of oil degraders in
uncontaminated areas were 1000-100,000 times lower than in contaminated areas.
Thus, the presence of oil causes a significant enrichment of oil degrading
microorganisms.
Table 5. Relative Concentration (Log10 of the cell numbers/g of beach material
and standard deviation) of Oil-Degrading Microorganisms in Samples from
Beaches That Were Not Impacted by Oil.
S ite
High Tide
Mid Tide
Low Tide
Tatitlek
2.41
4.31
6.11

± .58
±1.14
±2.05
Fish Bag
<1.51
<1.31
<2.71
Snug Corner Cove
2.31
. 2.51'
<1.11

± .54
± .55

Hell's Hole
<2.11
2.51
< .91


± .89

Commander Cover
4,51
1.31
3.11
±1.14	± .45
MECHANISM OF ACTION OF INIPOL-ENHANCED OIL DEGRADATION
A laboratory study was conducted to investigate the mechanism by which
the Inipol fertilizer enhanced oil degradation. Numbers of oil-degrading
microorganisms and oleic acid-degrading microorganisms were specifically
examined along with changes in oil composition. The study was performed in a
manner that would, to some extent, simulate environmental conditions, i.e., no
shaking and daily water change to simulate tidal flushing. Results are
currently available for oil-degrading and oleic acid-degrading microbial
populations,
The experimental design is shown in Figure 1. Studies were conducted in
chemically clean (I-Chem) jars, each containing approximately 200 g of oiled
rocks and either seawater, defined nutrient medium, or sodium chloride
solution (20%). The defined nutrient medium (DNM) used in these tests
contained (per liter of distilled water): NaCL (24g) MgS04-7H20 (1.0 g) KCL
(0.7 g) KH2P04 (2.0 g). Na2HP04 (3.0 g) , and NH4N03 (1.0 g) , The pH of the
medium was adjusted to 7.4 with 1.0 N NaOH following autoclaving. For sterile
systems, the oil - contaminated rocks were autoclaved in I-Chem jars. This
removed the water from the oil", but did not remove the oil from the rocks.
Inipol application consisted of dripping 3 ml of Inipol (sterile) over the
8

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rock surface and allowing the treated rocks to Incubate for 3 hours before
filling the jars with the appropriate aqueous phase (about 100 ml). Except
for the jar containing unautoclaved seawater, sterile medium (seawater,
defined nutrient medium, or NaCl solution) was used in each microcosm.
Subsamples of 1.0 ml for bacterial enumeration were collected from all jars at
24-hour intervals. Oleic acid- degrading bacteria were enumerated on oleic
acid-containing agar plates supplemented with nitrogen and phosphorous. Oil-
degrading bacteria were enumerated by the dilution to extinction technique.
After collection of bacterial enumeration samples, the aqueous phase from one
set of jars was decanted into a sterile I-Chem jar and replaced with fresh
sterile medium (fresh seawater was added to the nonsterile seawater jar). The
decanted solution was frozen for analysis of residual oil components.
Flask
1A Seawater 		
2A Artificial Seawater
3A NaCl	—		
4A Seawater 				
5A Artificial Seawater
6A NaCL		
•Sterile
¦Nonsterile
>Inipol added
IB Seawater			
2B Artificial	Seawater
3B NaCl		
4B Seawater 		
5B Artificial Seawater
6B NaCL			
•Sterile
•Nonsterile
•No Inipol added
Note: Two parallel series were either incubated, as originally planned, or the
aqueous phase was replaced each day.
Figure 1. Experimental Design for Laboratory Microcosm Study
The results from these studies indicated that the addition of Inipol led
to a substantial increase in the number of organisms capable of growth on
oleic acid-agar plates (Figure 2). High background concentrations of oleic
acid-degrading bacteria were observed in the water even before Inipol
treatment.
Because the aqueous phase at each water change was sterilized, the
number of oleic acid degraders possibly reflected those that sloughed off the
oiled rocks during a 24-hour period. However, no obvious differences were
observed for the different aqueous phases. Similar results were observed In
systems that did not have daily water changes.
9

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~ Control
S3 Inipol-Treated
Defined
Nutrient
Medium
c
3
O
O
75
O
.2
33
o
O
Seawater % ,,
I

I
1

ll
1

1


4	5
Days
1
1
I
ss
I
I
I
Days
I
1

1
1
1
1
I
Saline
Solution
i	«•
O
r '•
3	•
nj	4 '
|	4
3
I
1

I
I
I

1



2	3	4	8	«	7	IS
Days
Figure 2. Effect of Inipol on the relative numbers of oleic acid-degrading bacteria in jars containing
oiled rocks and seawater, defined nutrient medium, or saline solution. Incubated with
daily change of water.
10'

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Results from the enumeration of oil-degrading organisms (Figure 3)
indicated that, in all cases, the populations increased to a high value by day
3 and then decreased to an intermediate but variable level for the following 6
days. Similar results were seen in those jars that did not have a daily water
change. Although all the samples showed a peak after 3 days of incubation,
jars containing only seawater appeared to have the fewest microorganisms in
the 6 days following the 3-day peak. Chemical analysis of the water samples
is being performed. Information on how effectively the enriched oleic acid
degraders can degrade the oil also is forthcoming.
Inipol increased the number of oleic acid-degrading bacteria in flask
studies designed to approximate field conditions. This situation would
theoretically result in competition for available nutrients between oleic
acid-degrading and oil-degrading bacteria. This competition could explain the
decrease in oil-degrading bacteria following their initial rise after
initiation of the experiment. Tests of oleic acid-degrading bacteria
currently are being conducted to determine the percentage that are also
hydrocarbon degraders. Supplying dissolved nutrients in addition to those
nutrients in Inipol did not seem to affect the oleic acid- and oil-degrading
bacterial populations.
11

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® 9 •
tfi w "
8	•
tr
9	7 ¦
Seawater I
"2 <•
2
£ 3-
a
S 2.
JC
!?
i
I
1
i
I
i
1
I

~ Control
^ Inipol-Treated
i
I
i

10 -
Q> ft.
I
Defined f 4 =
Nutrient J
Medium 1
i i
§> ;
x
i
l

i
i
I
I
1
10
i i
i
?
Saline | * ~
Solution 2 «-
nc 9 «¦
o ^
I
I
1
1

I
i
I
i
§
i
i
i
i
i

1
i
i
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l
I
Days
Figure 3. Effect of inipol on the relative numbers of oil-degrading microorganisms in jars containing
oiled rocks and seawater, defined nutrient medium, or saline solution. Incubated with daily
change o. water.

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TECHNICAL REPORT DATA
(Please reed Instructions on the reverse before eompte'
1, REPORT NO.
EPA/600/0-90/119
2.
3.
4, TITLE AND SUBTITLE
THE ROLE OF MICROORGANISMS
IN THE BIOREMEDIATION OF THE
5 REPORT DATE
OIL SPILL IN PRINCE WILLIAM SOUND, ALASKA

6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
John. E. Rogers*, Rochelle Araujo*, Parmely H. Prit-
chard** and Henry H. Tabak***
B. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
^Environmental Research Laboratory, USEPA, Athens GA
**Environmental Research Laboratory, USEPA, Gulf
Breeze FL
***Risk Reduction Engineering Laboratory, Cincinnati OH
10. PROGRAM ELEMENT NO.
ABWD1A
11. CONTRACT/GRANT NO.
12, SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Athens, GA
13. TYPE OF REPORT AND PERIOD COVERED
Office of Research and Development
U.S. Environmental Protection Agency
Athens GA 30613-7799

14, SPONSORING AGENCY CODE
EPA/600/01
16. SUPPLEMENTARY NOTES
1#. ABSTRACT
The U.S. Environmental Protection Agency's Alaskan Bioremediation Project was
initiated in the aftermath of the March 24, 1989, EXXON VALDEZ oil spill. The ob-
jective of the project was to demonstrate an alternative cleanup method for oil-
contaminated shorelines based on enhancing natural biodegradation of the oil through
the addition of nitrogen and phosphorus nutrients. This enhancement process is a
well-recognized and scientifically sound approach to bioremediation but had never
been tested on a large scale in marine environments. The project was managed by
EPA's Office of Research and Development with financial, scientific, and logistical
support from the Exxon Company USA under the authority of the Federal Technology
Transfer Act. This paper covers the microbial aspects of the EPA field study in
Prince, William Sound, Alaska.
17,
KEY WORDS AND DOCUMENT ANALYSIS


a. DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group



18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (nils Report)
UNCLASSIFIED
21. NO. OF PAGES
13
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R.v, 4-77) previous
EDITION IS OBSOLETE
i

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