EPA/600/D-90/208
Protocol for Testing Bioremediation Products
Against Weathered Alaskan Crude Oil
by
Albert D. Venosa^, John R. Haines^, Wipawan Nisamaneepong^,
Rakesh Govind^, Sali1 Pradhan^, and Belal Siddique^
1Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH
^Engi neering and Economics Research Institute
Reston, VA 22091
^University of Cincinnati
Cincinnati, OH
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EPA Form 2220-1 (Rev. 4-77) (Reverie)
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Bacterial degradation of petroleum hydrocarbons has been known and recog-
nized for decades. The subject has been reviewed comprehensively in the lit-
erature (Atlas, 1981; Cooney, 1984; Floodgate, 1984), the most recent one
appearing this year by Leahy and Colwell (1990). Vestal et al_^ (1984)
reported that, although oil degraders comprise approximately 1% of the total
heterotrophic population in unpolluted waters, the oil degrader population
increases to as high as 10% in response to a spill. In 1989, research con-
ducted by the U.S. Environmental Protection Agency in Prince William Sound
demonstrated that microbial communities on the contaminated beaches were
highly competent in their ability to degrade the Prudoe Bay crude that was
spilled from the Exxon Valdez (Pritchard et aU., 1990). The purpose of the
latter study was to determine if application of water soluble and oleophilic
nutrients could enhance the natural biodegradation rate.
After the EPA study showed that bioremediation of oil-polluted beaches
was enhanced by the addition of fertilizer, the question then arose whether
further enhancement was possible with the addition of microbial inocula pre-
pared from oil degrading populations not indigenous to Alaska. Seeding experi-
ments have been done in previous studies with mixed results (Leahy and
Colwell, 1990). In a recent study, Dott et al^ (1989) compared nine
commercial mixed bacterial cultures to activated sludge microorganisms for
their ability to degrade fuel oil in laboratory microcosms. They found that
\
fuel oil degradation by the naturally occurring bacteria in activated sludge
did not depend on nor was it enhanced by the application of highly adapted
commercially available cultures. Most success has been achieved when chemos-
tats or fermenters are used to control conditions or reduce competition from
indigenous microflora (Wong and Goldsmith, 1988).
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2
In February, 1990, the U.S. Environmental Protection Agency issued a pub-
lic solicitation for proposals to the bioremediation industry on testing the
efficacy of commercial microbial products for enhancing degradation of
weathered Alaskan crude oil. The Agency commissioned the National Environmen-
tal Technology Applications Corporation (NETAC), a non-profit corporation ded-
icated to the commercialization of environmental technologies, to convene a
panel of experts to review the proposals and choose those that offered the
most promise for success in the field. Forty proposals were submitted, and 11
were selected for the first phase of a two-tiered testing protocol (only 10
were tested because one company did not participate). The laboratory testing
consisted of electrolytic respirometers set up to measure oxygen uptake over
time and shake flask microcosms to measure oil degradation and microbial
growth. If one or more products were found effective, the second tier would
take place, consisting of small field plots on an actual contaminated beach in
Prince William Sound in the summer of 1990. This paper discusses the first
phase of testing, the laboratory microcosms and respirometric evaluations.
The objective of the laboratory protocol was to determine if commercial
bioremediation products can enhance the biodegradation of weathered crude oil
to a degree significantly better than that achievable by simple fertilizer
application. Testing was conducted in a controlled and closed environment
designed to give quick results under ideal conditions. It was not meant to
1
simulate the open environment of the oiled beaches of Prince William Sound,
where conditions are in a constant state of flux with respect to tidal cycles
and washout, temperature variation, climatic changes, freshwater/saltwater
interactions, etc. The organisms inside the respirometer vessels were in
continuous contact with the oil, seawater, and nutrients added initially, and
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3
the seawater was not replenished every 12 hours as is the case in nature. The
test was merely a screening procedure that was designed to determine if there
was sufficient enhancement due to the commercial additives that would justify
proceeding to the next tier of testing. To proceed to the field phase, three
lines of evidence were used for decision-making: rapid onset and high rate of
oxygen uptake, substantial growth of oil degraders, and significant degrada-
tion of the aliphatic and aromatic fractions of the weathered Prudoe Bay crude
oil.
The 10 companies participating 'in the laboratory testing phase were (in
alphabetical order): Alpha Environmental, Bioversal, Elf Aquitaine, ERI-
Microbe Masters, Imbach, Microlife Technics, Polybac, Sybron, Waste Microbes,
and Woodward Clyde.
MATERIALS AND METHODS
Electrolytic Respirometry. The studies were conducted using four automa-
ted continuous oxygen-uptake measuring Voith Sapromats (Model B-12). The
instrument consists of a temperature-controlled water bath containing
measuring units; a recorder for digital indication and direct plotting of the
oxygen uptake velocity curves; and a cooling unit for the conditioning and
continuous recirculation of water bath volume. The recorder displays a digi-
tal readout of oxygen uptake and constructs a graph of the data for each
measuring unit. The cooling unit constantly recirculates water to maintain a
)
uniform temperature in the water bath. The measuring units are comprised of
12 reaction vessels each with a carbon dioxide absorber mounted inside, 12
oxygen generators each connected to its own reaction vessel by tubing, and 12
pressure indicators connected electronically to the reaction vessels. The
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4
measuring units are interconnected by tubing, forming an air-sealed system, so
that the atmospheric pressure fluctuations do not adversely affect the
results.
Depletion of oxygen by microbiological activity creates a vacuum, which
is sensed by the pressure indicator. The oxygen generator is triggered to
produce just enough oxygen to counterbalance the negative pressure. The cur-
rent used to generate the oxygen is measured by the digital recorder, and the
data are converted directly into mg/L oxygen uptake. The CO2 produced by
microbial activity is absorbed by soda lime. The nitrogen/oxygen ratio in the
gas phase above the sample is maintained throughout the experiment, and there
is no depletion of oxygen. The oxygen generators of the individual measuring
units are electrolytic cells that supply the required amount of oxygen by
electrolytic decomposition of copper sulfate/sulfuric acid solution.
A recorder/plotter constructs an oxygen uptake graph as a function of
time and displays it on the computer screen while digitally saving the data on
disc. For frequent recording and storage of oxygen uptake data, the Sapromat
B-12 recorders are interfaced to an IBM-AT personal computer via the Metrobyte
interface system. A software package allows the collection of data at 15
minute intervals.
Experimental Design. All commercial products were tested in duplicate at
the concentration recommended by the manufacturer. Each experimental respi-
1
rometer flask was charged with the following materials in the order listed:
weathered crude oil, 250 mg; 250 mL seawater from Prince William Sound; and
commercial product at the concentration specified by the manufacturer. Seawa-
ter was prepared as follows: 25 g of oiled rocks from a contaminated beach in
Prince William Sound was placed in a 4-L flask to which was added 2 L of
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5
seawater. The mixture was shaken for approximately 30 min. to wash off a
microbial inoculum from the rocks. The flask contents were allowed to settle,
and the supernatant was mixed with more seawater for use in the respirometer
vessels. The following table presents the summarized experimental design
showing all control and experimental flasks.
TABLE 1. Experimental Design for Respirometric Studies.
Reaction Vessel
Weathered
Commercial
Seawater
TOTAL
Oil
Product
TEST FLASKS:
TPn
+
+
+
20
pl,2
+
-
+
2
CONTROL FLASKS:
cPn
-
+
+
20
CF1,2
-
-
+
2
Cj-inoculum
-
-
+
2
C2-no nutrients
+
-
+
2
TOTAL
48
Tpn = duplicate commercial product flasks (n = 10)
Fi 2 = fertilizer flasks (mineral N and P nutrients)
i
Cpn> Cpi^2 = no °il controls for products and fertilizer
Cj, C2 = inoculum and no-nutrient controls
Flasks Fj and F2 represented simple inorganic fertilizer application and
contained the following ingredients (mg/L final concentration): KH2PO4, 6.33;
K2HPO4, 16.19; Na2HP04, 24.86; NH4C1, 38.5; MgS04.7H20, 45; CaCl2, 55;
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6
FeCl3.6H2O, 2.5. The following additional trace elements were included in the
formulation (ng/L final concentration): MnS04-H20, 60.4; H3BO3, 114.4;
ZnS04.7H20, 85.6; and (NH4)6M07024, 69.4. This formulation was a modified
version of the standard OECD medium used widely in respirometric studies. The
modifications were in the final levels of N and P, which were reduced to 10
mg/L each, and the yeast extract was eliminated from the formulation.
All respirometer flasks were incubated at 15°C in the dark and continu-
ously stirred by magnetic stirrers. The first set of control flasks (Cpn,
Cpi,2) represented background oxygen uptake of the product and seawater
without oil. Results from these flasks were subtracted from the appropriate
test flasks to obtain the oxygen uptake on the weathered oil. The inoculum
control represented the endogenous oxygen uptake of the organisms from the
washed beach material and the seawater alone. The no-nutrient control repre-
sented the oxygen uptake of the organisms from the washed beach material and
seawater on weathered oil without any external source of nutrient addition
(i.e., background nutrient levels from Prince William Sound).
Flask Experiments. Shaker flask microcosms duplicating the respirometer
flasks were set up to assess the quantitative changes in oil composition by
chromatographic separation of the individual components. Although it is pos-
sible to remove samples from the respirometer flasks, it was deemed more pru-
dent not to disturb the respirometric"runs but instead have the flask
microcosms with proportionately higher levels of oil, commercial products,
etc., to facilitate sampling for and precision/accuracy of the analytical
chemistry. Table 2 summarizes the flask microcosm design.
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7
TABLE 2. Experimental Design for the Flask Microcosm Studies.
Reaction Vessel
Weathered
Commercial
Seawater
TOTAL
Oil
Product
TEST FLASKS:
TPn
+
+
+
20a
Spn
+
sterile
+
ga
Tpn^b
sterile
+
sterile
ga
F1 > 2
+
-
+
2a
CONTROL FLASKS:
cPn
-
+
+
10b
CF1
-
-
+
lb
Ci-inoculum
-
-
+
lb
C2*no nutrients
+
-
+
2a
TOTAL
54
Tpn = duplicate commercial products (n = 10), non-sterile system
Spn = sterile products in non-sterile seawater/oil, non-duplicated
TpnSb = non-sterile products in sterile seawater/oil, non-duplicated
Fi,2 = fertilizer (mineral N and P nutrients) in non-sterile system
Cpn> ^F1 = no controls for products and fertilizer
\
C}, C2 = inoculum and no-nutrient controls
a = microbiological and chemical analysis
b = microbiological analysis only
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8
The test flasks corresponded exactly to the 22 test flasks listed in
Table 1 (plus more; see below) but with the following modifications: flask
size, 2.0 L; seawater, 1.0 L; weathered oil, 10,000 mg/L (rather than
1,000); and commercial products or inorganic nutrients, 10 times the level
used in the respirometer flasks. The higher concentration of weathered oil
was used to improve the final sensitivity of the chemical analyses.
In addition to the 22 test flasks mentioned above, 18 supplemental micro-
cosms were set up. These microcosms represented 9 sterile product controls,
which determined whether the enhancement was due to the microorganisms or to
the nutrients or metabolites in the product, and 9 sterile background controls
(i.e., sterile oil and seawater, but non-sterile product) to evaluate the
effect of competition from naturally occurring organisms. Sterilization of
materials was accomplished by autoclaving at 121°C for 15 min. Samples for
analytical chemistry and microbiology were taken from all test and the no-
nutrient control flasks at each scheduled sampling event. The other control
flasks listed in Table 2 (i.e., those with no oil) were sampled and measured
for microbial density changes.
There were three sampling events for analytical chemistry and microbiol-
ogy: day 0, day 11, and day 20. These events were determined by the shape of
the oxygen uptake curves from the respirometry experiments. Soon after
start-up, it became clear that use of-2 L shaker flasks did not allow for
1
representative sampling, because the oil formed tar balls in many of the
flasks. Consequently, the large flasks were abandoned in favor of 250 mL
capacity flasks containing 100 mL of oil/seawater/products with proportion-
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9
ately reduced levels of each material. Each flask was sacrificed at the
indicated sampling time by mixing the contents with methylene chloride and
performing the extraction on the entire mixture.
Analytical Chemistry. The oil constituents were analyzed by measuring
the aliphatic and aromatic fractions of oil extracts. The methylene chloride
extracts were passed through a silica gel column and eluted with hexane to
analyze the alkane fraction and a 1:1 mixture of hexane and benzene to analyze
the aromatic fraction. Aliphatic fractions were measured by gas chromatogra-
phy using a flame ionization detector. The aromatic fractions were character-
ized by gas chromatography/mass spectrometry (GS/MS).
Nutrients were also analyzed. The nitrogen species NH3-N, N02~-N, and
N03"-N were determined by U.S. EPA methods (1979). The NH3-N method was No.
350.1, and the N02"-N/N03~-N method was No. 353.1. Microbiological testing
included growth of oil degraders on oil agar (Bushnell-Haas medium supplem-
ented with Prudoe Bay crude oil).
RESULTS
Nutrient Levels in Each Product. The same mineral nutrients were added
to those product flasks that required them, as specified by the product man-
ufacturers. The ammonia-nitrogen concentrations measured in each product
flask at day 0 are summarized in Table 3.
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10
TABLE 3. NH3-N Levels in Each Product Flask at the Start of the Experiment.
PRODUCT NH3-N NUTRIENTS
mg/L ADDED
8.0 YES
2.1 NO
1080.0 NO
11.8 YES
11.3 YES
10.0 YES
24.9 YES
426.0 NO
0.5 NO
1.5 NO
6.9 YES
*FR = mineral fertilizer
Respirometry. Space does not permit presentation of the oxygen uptake
curves for all 10 products. Only the two products selected from the results
of this study and used in the subsequent field test are shown. The other data
will be published in a separate publication.
I
Figure 1 summarizes the net oxygen uptake curve for Product E compared to
that for the mineral nutrients and the no-nutrient control. Net uptake means
total uptake minus uptake in the no oil control. Figure 2 presents the
results for Product G. The lag period observed for the mineral nutrients was
about 5 days, while the lag periods for Products E and G were approximately 2
A
B
C
D
E
F
G
H
I
J
FR*
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11
and 4 days, respectively. The rate of increase in oxygen uptake appeared
greater for Product E than either Product G or mineral nutrients. The final
plateau in total oxygen uptake was slightly less than 500 mg/L for both Prod-
ucts E and G, while the mineral nutrients gave a total cumulative oxygen
uptake at about 340 mg/L.
An elemental analysis of the weathered crude oil was conducted late in
the study by a contract laboratory. From that analysis, the molecular weight
and theoretical oxygen demand (ThOD) of the oil were computed. The molecular
weight was 925, and the resulting empirical formula was calculated to be
^26^50^2^0.l5•29H20. Using this formula, the ThOD was computed to be approxi-
mately 1300 mg/L. Thus, only about 38% of the ThOD was accounted for by the
flasks containing Products E and G, and even less (approximately 26%) by the
mineral nutrients alone. These results are presently being analyzed, and
interpretations will be forthcoming in later publications.
Microbiological Results. Growth of oil degraders for the inorganic
nutrients compared to Products E and G are summarized in Figures 3 and 4,
respectively. Oil degrader populations increased approximately 2.5 orders of
magnitude within 11 days in the presence of mineral nutrients, about the same
for Product E (Figure 3), and about 3 orders of magnitude for Product G (Fig-
ure 4). Growth in all flasks levelled off after 11 days. The same increases
in oil degrader counts were observed for both products whether or not the
product or the background was first sterilized. This indicates that the
indigenous Alaskan populations were not inhibited by the product microorgan-
isms, nor were the product organisms inhibited by the indigenous populations.
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12
This was not true for some of the other products (data not shown). For
example, in some cases, good growth occurred in the flasks containing sterile
product or sterile background but not when both were present together.
Total Alkane Reduction. A summary of the total alkane degradation data
at day 11 and 20 is presented in Figure 5. With respect to the results from
day 11 (top half of Figure 5), better degradation was observed in every case
when the commercial products were first sterilized, suggesting that the indig-
enous Alaskan populations are doing most if not all of the bioremediation. In
contrast, worse degradation occurred in every case when the background was
first sterilized, which allowed only the product organisms to degrade the
alkane constituents. Enhancement was observed in several cases compared to
mineral nutrients (discussed in more detail below), suggesting that the prod-
ucts exhibiting the enhancement were providing metabolites or some other form
of nutritional benefit that was lacking in the mineral nutrient flask. By day
20 (bottom half of Figure 5), all products except Products F and I caught up,
giving greater than 85% reduction in the total alkane levels in the flasks.
However, most of the flasks containing oil and seawater that were first ster-
ilized still significantly lagged behind the non-sterile systems.
Total Aromatics Reduction. A summary of the total aromatics reduction
data at day 11 and 20 is presented in Figure 6. Differences are less clear
among the products, although Products-C, F, H, and I gave total reductions
l
considerably less than mineral nutrients. By day 20 (bottom half of Figure
6), aromatic reduction by Product C was somewhat closer to the others, while
Products F, H, and I substantially lagged. Excellent removal of aromatics was
observed in all other flasks.
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13
Statistical Analysis. Figure 7 summarizes the statistical differences
between each product compared to mineral nutrients. Comparisons were made
using Tukey's Studentized Range Test (Tukey, 1953) for detecting differences
in mean percent removal of alkanes in 11 days. Only Products E and G gave
statistically significantly better removals than inorganic fertilizer after 11
days. Six of the other products gave results no different from mineral nutri-
ents, while two actually gave worse removals. The latter results suggest that
the products may have been toxic to the biomass at the levels used in the
closed microcosms.
DISCUSSION
Results from all three lines of evidence, i.e., respirometry, microbiol-
ogy, and chemistry, supported the decision to field test only Products E and
G. It appears from all the available evidence that the indigenous Alaskan
microorganisms were primarily responsible for the biodegradation in the closed
microcosms and respirometer vessels, and that any enhancement provided by
products E and G might have been due simply to metabolites,. nutrients, or
co-substrates present fortuitously in the products. This study was designed
to enable decisions to be made regarding which commercial products may be
useful in stimulating bioremediation in the field. In that regard, the objec-
tive was accomplished. Many questions remain unanswered, however, and further
research is being planned to increaseour knowledge base regarding oil spill
I
bioremediation enhancement using commercial inocula.
LITERATURE CITED
1. Atlas, R. M. 1981. "Microbial degradation of petroleum hydrocarbons:
an environmental perspective." Microbiol. Rev. 45: 180-209.
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14
2. Cooney, J. J. "The fate of petroleum pollutants in freshwater ecosys-
tems." In R. M. Atlas (ed.), Petroleum Microbiology, Macmillan Publish-
ing Co., New York.
3. Dott, W., D. Feidieker, P. Kampfer, H. Schleibinger, and S. Strechel.
1989. "Comparison of autochthonous bacteria and commercially available
cultures with respect to their effectiveness in fuel oil degradation."
J. Ind. Microbiol. 4: 365-374.
4. Floodgate, G. D. 1984. "The fate of petroleum in marine ecosystems."
In R. M. Atlas (ed.), Petroleum Microbiology, Macmillan Publishing Co.,
New York.
5. Leahy, J. G. and R. R. Colwell. 1990. "Microbial degradation of hydro-
carbons in the envrironment." Microbiol. Rev. 54: 305-315.
6. Organization for European Cooperation and Development, "Method 301C,
ready biodegradability: modified miti test (i) adopted may 12, 1981 and
method 302c, inherent biodegradability: modified miti test (ii), adopted
may 12, 1981." In "Guidelines for Testing of Chemicals, Section 3, Deg-
radation and Accumulation." Director of Information, OECD, Paris,
France.
7. Pritchard, P. H., R. Araujo, J. R. Clark, L. D. Claxton, R. B. Coffin, C.
F. Costa, J. A. Glaser, J. R. Haines, D. T. Heggem, F. V. Kremer, S. C.
McCutcheon, J. E. Rogers, and A. -D. Venosa. 1990. "Interim report: oil
l
spill bioremediation project." U.S. Environmental Protection Agency,
Office of Research and Development, Washington, D.C.
8. Tukey, J. W. 1953. The problem of multiple comparisons. 396 pp.
Princeton University, Princeton, NJ.
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15
9. U.S. Environmental Protection Agency. 1979. "Methods for chemical
analysis of water and wastes." EPA 600/4-79-020, Washington, D.C.
10. Vestal, J. R., J. J. Cooney, S. Crow, and J. Berger. 1984. In R. M.
Atlas (ed.), Petroleum microbiology, Macmillan Publishing Co., New York.
11. Wong, A. D. and C. D. Goldsmith. 1988. "The impact of a chemostat
discharge containing oil degrading bacteria on the biological kinetics of
a refinery activated sludge process." Water Sci. Technol. 20: 131-136.
-------
Figure 1. Oxygen uptake curves for Product E and mineral nutrients.
Figure 2. Oxygen uptake curves for Product G and mineral nutrients.
Figure 3. Increase in oil degraders in mineral nutrients and Product E micro-
cosms .
Figure 4. Increase in oil degraders in mineral nutrients and Product G micro-
cosms.
Figure 5. Total alkane reduction in the product flasks.
Figure 6. Total aromatic reduction in the product flasks.
Figure 7. Statistical analysis of day 11 alkane removals.
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INORGANIC — NO NUTRIENT PRODUCT E
700
CD
600
500
400
300
200
100
30
60
20
50
0
10
40
Figure 1.
TIME, DAYS
Oxygen uptake curves for Product E and mineral nutrients.
-------
INORGANIC NO NUTRIENT PRODUCT G
CD
E
111
*
<
h-
CL
D
H
<
-J
D
2
D
O
700
600
500
400
300
200
100
0
-
-
-
-
-
i i i i
//
/r
10
20
30
40
TIME, DAYS
Figure 2. Oxygen uptake curves for Product G and mineral nutrients,
50
60
-------
GROWTH OF OIL DEGRADERS
INORGANIC
I MOROANIC
INOftOAMC
NO Oft.
-I
£
N
CO
££
LU
O
<
DC
(3
LU
O
_J
o
Ll
O
1 o9
108
107
10#
105
104
10'
10*
:
E
I
l
A
1 /
1 1 1 1
1 1 t l
10
20
30
40
50
PRODUCT E
NOW- I I ¦ I STSVJE ¦ ¦ ¦ I BTETOLE
STBULE PftOOUCT BACKORND
!!¦ NO OIL
10'
10'
107
10*
10'
104
10*
10'
I
6
/
I-,
| ~
* ¦ *•••
TjJF *
V /
~
~
~
~
i
! *
*
I
I
K
1 I t 1 .
tilt
¦ i t i
ttii
.. i .. i i ;
10
20
30
40
50
Figure 3.
TIME, DAYS
Increase in oil degraders in mineral nutrients and Product E micro-
cosms .
-------
GROWTH OF OIL DEGRADERS
INORGANIC
I NOROANIC
INOMANC
NO CHL
CO
oc
LU
Q
<
DC
CD
LU
a
Ll
O
109
108
107
1 o6
108
104
103
10*
10
20
30
40
50
PRODUCT G
HOW—
8TBRL£
I ST&aUE
proouct
I &TERUE
BACKORND
!!¦ no oil
10®
10"
10'
10*
10*
104
10'
10*
s
i
9
tf it
* 1»*#
M
J* <
s* ~
~
i
! /v
\
}
Yi
!/ ^
itii
1 1 1 1
..J L , 1 J
i i t t
l i . i
10 20 30 40
TIME, DAYS
50
Figure 4. Increase in oil degraders in mineral nutrients and Product G micro-
cosms .
-------
TOTAL ALKANE REDUCTION — DAY 1 1
NON-STERILE
STERILE
PRODUCT
STERILE
BACKGROUND
100
CO
HI
z
<
*
-I
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A B C D
E F G H I J FR 00
PRODUCT
TOTAL ALKANE REDUCTION -- DAY 20
NON-STERILE STERILE V////A RTFRli F
PRODUCT BACKGROUND
ABCDEFGH I J FR 00
PRODUCT
Figure 5. Total alkane reduction in the product flasks.
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TOTAL AROMATICS REDUCTION -- DAY 11
100
ABCDEFGH I JFR
PRODUCT
TOTAL AROMATICS REDUCTION -- DAY 20
PRODUCT
Figure 6. Total aromatic reduction in the product flasks.
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TUKEY'S STUDENTIZED RANGE TEST FOR DETECTING
DIFFERENCES IN MEAN PERCENT REMOVAL OF ALKANES BY
PRODUCTS IN 11 DAYS
PRODUCT % REMOVAL SIGNIFICANTLY DIFFERENT
FROM INORGANIC NUTRIENTS*
E
94.5
YES
G
93.6
YES
B
87.9
NO
A
75.9
NO
D
74.2
NO
FR
68.4
NO
C
67.8
NO
J
59.9
NO
H
49.5
NO
F
33.3
YES
1
27.9
YES
'MINIMUM DETECTION DIFFERENCE = 21.3% AT 5% SIGNIFICANCE LEVEL
Note that products E and G have mean % removals significantly greater
than inorganic nutrients (FR), while products F and I have mean %
removals significantly less than inorganic.
Figure 7. Statistical analysis of day 11 alkane removals.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before compter'
1. REPORT NO. 2.
EPA/600/D-90/208
3- PB91-137018
4. TITLE AND SUBTITLE
Protocol for Testing Bioremediation Products Against
Weathered Alaskan Crude Oil
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOB(S) 1-1 2 3
AD Venosa^, JR Haines , W^Nisamaneepong , R Govind ,
S Pradhan , & B Siddiaque
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
1.RREL, USEPA, Cincinnati, OH 45268
2.Eng & Econ Res Inst, Reston, VA 22091
3.University of Cinti, Cincinnati, OH 45221
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Risk Reduction Engineering Laboratory--Cincinnati, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Complete
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Albert D. Venosa, FTS: 684-7668; Com:513/569-7668
Protocol for Testing Bioremediation Products Against Weathered Alaskan Crude Oil
16. ABSTRACT
In the summer of 1989, EPA and Exxon Corp. conducted a joint field study to determine
if natural biodegradation of the Prudoe Bay crude oil spilled from the Exxon Valdez
could be accelerated by application of oleophilic and water soluble fertilizers.
Numerous private firms have since submitted proposals to have their microbial pro-
ducts tested for bioremediation enhancement. EPA commissioned the National Environ-
mental Technology Applications Corporation (NETAC) to coordinate an effort to select
and test commercial products for efficacy against Alaskan crude oil. A panel of
experts was assembled to review the proposals, and nine products were selected for
the first tier of testing.
The experiments were conducted at the Risk Reduction Engineering Laboratory in
Cincinnati. Three lines of evidence were used to select the final products for
further testing: cumalative oxygen uptake via electrolytic respirometry, microbial
growth, and compositional analysis of treated oil by GC and GC/MS. The commercial
products were compared against oleophilic and inorganic fertilizers in a comprehen-
sive protocol incorporating sterile and non-sterile controls. Respirometric vessels
and shaker flask microcosms were set up for the comparative testing using weathered
oil and natural seawater from Prince William Sound. This paper presents the protocol
the test results, and conclusions derived from the study.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTI FI E RS/OPEN ENDED TERMS
c. COSATI Field/Group
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R«v. 4-77) previous edition is obsolete
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