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International Biodeterioration & Biodegradation (1995) 311-321

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Bioremediation of Petroleum Pollutants
Ronald M. Atlas
Department of Biology, University of Louisville, Louisville, KY 40292, USA
ABSTRACT
Hydrocarbon-degrading microorganisms are ubiquitously distributed in soil
and aquatic environments. Populations of hydrocarbon-degraders normally
constitute less than 1% of the total microbial communities, but when oil
pollutants are present these hydrocarbon-degrading populations increase,
typically to 10% of the community. With regard to rates of natural degra-
dation, these typically have been found to be low and limited by environ-
mental factors. Rates reported for pristine marine waters typically are less
than 0=03g/m3/day. I n adapted communities rates of hydrocarbon degra-
dation of 0.5-50g/m3jday have been reported. Bioremediation tries to raise
the rates of degradation found naturally to significantly higher rates. The
two general approaches that have been tested for the bioremediation of
marine oil spills are the application of fertilizer to enhance the abilities of
the indigenous hydrocarbon-utilizing bacteria and the addition of naturally
occurring adapted microbial hydrocarbon-degraders by seeding. Bior-
emediation, accomplished by the application of fertilizer to enhance the
abilities of the indigenous hydrocarbon-utilizing bacteria, was successfully
applied for the treatment of the 1989 Alaskan oil spill in Prince William
Sound, Alaska. Seeding with adapted nonindigenous microbial hydrocarbon
degraders was tested on smaller spills in Texas-such as the Mega Borg
spill- but bioremediation to remove petroleum pollutants by seeding has
yet to be demonstrated as efficacious infield trials. The spill of more than
200,000 barrels of crude oil from the oil tanker Exxon Valdez in Prince
William Sound, Alaska, as well as smaller spills in Texas-such as the
Mega Borg spill-have been treated by bioremediation to remove petro-
leum pollutants. The Exxon Valdez spill formed the basis for a major study
on bioremediation through fertilizer application and the largest application
of this emerging technology. Three types of nutrient supplementation were
tested: water-soluble (23:2 N:P garden fertilizer formulation): slow-
317
318 R. M. Atlas
release (Customblen); and oleophilic (Inipol EAP 22). Each fertilizer was
tested in laboratory simulations and in field demonstration plots to deter-
mine the efficacy of nutrient supplementation. The use of Inipol EAP22
(oleophilic microemulsion with urea as a nitrogen source, laureth phosphate
as a phosphate source, and oleic acid as a carbon source) and Customblen
(slow-release calcium phosphate, ammonium phosphate, and ammonium
nitrate within a polymerized vegetable oil coating) was approved for
shoreline treatment and was used as a major part of the cleanup effort.
Multiple regression models showed that nitrogen applications were effective
in stimulating the rates of biodegradation.
INTRODUCTION
Bioremediation is an acceleration of the natural fate of oil pollutants and
hence a natural or green solution to the problem of oil pollutants that
causes minimal (if any) ecological effects. There are two approaches used
for bioremediation. The first relies upon the metabolic capacities of the
indigenous microbial populations. Bioremediation is accomplished by
environmental modification, for example through aeration or fertilizer
application, to overcome factors that limit the rates of hydrocarbon
biodegradation by the indigenous microbial populations. In the second
approach, exogenous microbial populations are added. The seed cultures
are selected for their hydrocarbon-degradation activities. Bioremediation
has not yet been shown to be effective for the treatment of open water oil
following a spill, but is a cost-effective approach to oil spill cleanup of
oiled shoreline environments.
To demonstrate that a bioremediation technology is potentially useful,
it is important that the ability to enhance the rates of hydrocarbon
biodegradation be demonstrated under controlled conditions. This gener-
ally requires both laboratory and in situ experiments to demonstrate the
efficacy of a bioremediation treatment. The evaluation of hydrocarbon
biodegradation in situ is far more difficult than in laboratory studies. The
analysis of residual hydrocarbons is especially complicated since the
distribution of oil in the environment is typically patchy, and therefore, a
high number of replicate samples must be obtained in order to obtain
statistically valid results.
Because of the problems with quantitation of hydrocarbon recovery
from field sites, ratios of hydrocarbons within the complex hydrocarbon
mixture have been used to assess the degree of biodegradation. In parti-
cular, the fact that hydrocarbon-degrading microorganisms usually
degrade pristane and phytane at much lower rates than n-alkanes has
permitted the use of pristane or phytane as internal recovery standards
Bioremediation of petroleum pollutants 319
(Atlas, 1981). These measurements assume that pristane and phytane
remain undegraded and, therefore, by determining the ratio of straight-
chain alkanes to these highly branched alkanes, it is possible to estimate
the extent to which microorganisms have attacked the hydrocarbons in the
petroleum mixture. However, in situations where pristane or phytane is
degraded at similar rates to straight-chain alkanes, this assumption is
invalid and alternative internal standards, such as hopanes, are required.
METHODOLOGIES FOR BIOREMEDIATION
Seeding involves the introduction of microorganisms into the natural
environment for the purpose of increasing the rate or extent, or both, of
biodegradation of pollutants. The rationale for this approach is that indi-
genous microbial populations may not be capable of degrading the wide
range of potential substrates present in such complex mixtures as petro-
leum. By adding a large biomass of hydrocarbon-degraders, the rates of
hydrocarbon biodegradation can be increased if the added cultures are
able to survive and express their hydrocarbon-degradation activities in the
environments to which they are added. This assumes that they can
outcompete the indigenous microbial populations and that they have
superior capacities. Seeding with specific cultures of oil-degrading bacteria
will fail to enhance the hydrocarbon degradation capability of natural
environments if they do not survive or if they are displaced by indigenous
hydrocarbon-degrading microorganisms. The National Environmental
Technology Assessment Corporation (NETAC) has been established to
establish protocols to test the safety and efficacy of such seed cultures.
To be effective, seeding would have to be applied early if the purpose
was to reduce the lag period before indigenous populations began to
attack the oil. This would require actively growing cultures and a huge
biomass. Seeding could also be used to extend the range of compounds
that would be attacked. Indigenous populations generally fail to attack
asphaltics and polynuclear aromatics with four or more rings. Seed
cultures could be found to attack such compounds. However, this may not
be required since asphaltics have minimal solubilities and effects on biota
and most crude oils have very limited concentrations of higher molecular
weight polynuclear aromatics.
Since microorganisms require nitrogen, phosphorus and other mineral
nutrients for incorporation into biomass, the availability of these nutrients
within the area of hydrocarbon degradation is critical. Concentrations of
available nitrogen and phosphorus in seawater generally are severely
limiting to microbial hydrocarbon degradation (Atlas, 1981; Leahy &
320 R. M. Atlas
Colwell, 1990). Laboratory experiments with an olephilic fertilizer have
demonstrated significant enhancement of oil biodegradation (Bragg et al.,
1993a; Chianelli et al., 1991); in some experiments 60% of added oil was
biodegraded in fertilized flasks compared to 38% in unfertilized ones
within 60 days (LaDousse & Tramier, 1991).
CASE STUDIES
Exxon Valdez seeding trials
In the initial effort to identify cultures that might be applied to the
cleanup effort in Prince William Sound following the Exxon VaZdez acci-
dental oil spillage, products from 10 companies were selected for labora-
tory phase testing by the USEPA (Venosa, 1991; Venosa et al., 1991a,b).
Some products delayed biodegradation. Most degradation, when it
occurred, started after a 3-5 day lag period and reached significant levels
after 20-30 days.
Of the products tested, two were selected for further field testing in
Prince William Sound on shorelines impacted by the Exxon Valdez spill.
In the field trials, four small plots were used to assess the effectiveness of
seeding (Venosa et al., 1991c, in press). These field trials failed to demon-
strate enhanced oil biodegradation by these products. There were no
significant differences between the four plots during a 27-day trial period.
It must be noted, however, that the oil was already highly degraded by the
time these field trials were conducted and that environmental variability
makes it difficult to observe statistically significant differences between
experimental and reference sites when relatively few samples are collected
and analyzed.
Mega Borg spill seeding trials
Biotreatment of the Mega Borg spill off the Texas coast consisted of
applying a seed culture produced by Alpha Corporation. The Texas
General Land Office has reported that the use of the Alpha culture on the
Mega Borg spill and also on a spillage from the Apex barge that impacted
the Marrow marsh was effective at removing significant amounts of oil
(Mauro, 1990a,b; Mauro & Wynne, 1990). Independent observations
indicated that treated oil changed in physical appearance and may have
been emulsified as a result of addition of the Alpha product; chemical
analyses on samples from impacted and reference sites failed to demon-
strate that treatment with the Alpha product enhanced rates of petroleum
Bioremediation of petroleum pollutants 321
biodegradation; no significant differences in Cl8/phytane ratios that
would indicate biodegradation enhancement were detected between Alpha
treated and untreated sites (Means, 199 1). Thus, scientifically valid
conclusions can not be reached substantiating the effectiveness of seeding
of open water spills. Equally true, no harmful effects occurred. Clearly,
well designed and extensive experiments, with appropriate controls, will be
needed if the efficacy of seeding open water oil spills is ever to be resolved.
Exxon V&fez nutrient enrichment trials
The Exxon Valdez spill formed the basis for a major study on bior-
emediation through fertilizer application and the largest application of
this emerging technology (Pritchard, 1990). Three types of nutrient
supplementation were considered: water-soluble (23:2 N:P garden fertili-
zer formulation): slow-release (isobutylenediurea and Customblen); and
oleophilic (Inipol EAP 22). Each fertilizer was tested in laboratory simu-
lations and in field demonstration plots. Consideration was also given to
potential adverse ecological effects, particularly eutrophication due to
algal blooms and toxicity to fish and invertebrates.
The application of the oleophilic fertilizer to field test plots produced
very dramatic results, stimulating biodegradation so that the surfaces of
the oil-blackened rocks on the shoreline turned white and were essentially
oil-free within 10 days after treatment (Fig. 1). The striking results
strongly supported the idea that oil degradation in Prince William Sound
was nutrient limited and that fertilizer application was a useful bior-
emediation strategy.
Exxon V..k.kz nutrient shoreline treatment
The use of Inipol EAP22 (oleophilic micro-emulsion with urea as a nitro-
gen source, laureth phosphate as a phosphate source, and oleic acid as a
carbon source) and Customblen (slow-release calcium phosphate, ammo-
nium phosphate, and ammonium nitrate within a polymerized vegetable
oil coating) was approved for shoreline treatment and was used as a major
part of the cleanup effort. In approximately 2-3 weeks, oil on the surfaces
of cobble shorelines treated with Inipol EAP22 and Customblen was
degraded so that these shorelines were visibly cleaner than non-bior-
emediated shorelines (Pritchard & Costa, 1991).Tests demonstrated that
fertilizer application sustained higher numbers of oil-degrading micro-
organisms in oiled shorelines and that rates of biodegradation were
enhanced as evidenced by the chemical changes detected in recovered oil
from treated and untreated reference sites (Chianelli et al., 1991).
322 R. M. Atlas
Fig. 1. Photograph showing results of Inipol EAP22 application to a shoreline in Prince
William Sound. The white window square in the photograph was treated with oleophilic
fertilizer 10 days earlier. While the surrounding untreated area remained black and oil-
covered, the treated area was relatively oil free.
Exxon Valdez efficacy monitoring
Monitoring by a joint Exxon, USEPA, and State of Alaska Department of
Conservation team of the oil-degrading microbial populations and
measuring the rates of oil degradation activities showed that a fivefold
increase in rates of oil biodegradation. typically followed fertilizer appli-
cation (Prince et al., 1990). Despite sampling and interpretation compli-
cations resulting from the high variability in oil distribution on the
beaches, it was possible to show statistically that oil biodegradation (as
measured by changes in residue weights and oil chemistry) was signifi-
cantly greater on the beach treated with the fertilizer solution than it was
on the control beach. After 45 days, approximately three to four times
more oil remained on the control test beach than on the fertilizer solution-
treated beach. This corresponds to an enhanced biodegradation rate of
about two- to threefold.
Laboratory experiments indicated that hopane was not biodegraded by
the indigenous Prince William Sound microorganisms. Hence, hopane
could be used as a conserved internal standard within the oil recovered
Bioremediation of petroleum pollutants 323
from the test shorelines and changes in the ratios of a hydrocarbon
component to hopane over time could be used as the basis for determining
the rate of biodegradation of that component. Using hopane as a
conserved internal marker, the monitoring program also demonstrated
that all the resolvable chemical components of the oil, even four-ring
polynuclear aromatic hydrocarbons, including substituted chrysenes, were
being degraded in the beach sediment, albeit at differing rates (Prince et
al., 1990). When subsurface oil was treated by oleophilic fertilizer in
combination with a slow-release fertilizer (6-12 in. deep in the mixed sand
and gravel), the oil was degraded to a greater extent compared to
untreated reference beaches. Both polynuclear aromatic and aliphatic
hydrocarbons were biodegraded more rapidly in the fertilized than in the
control shoreline sediments (Fig. 2). This was an important finding, since
there was concern that biodegradation might remove only some of the
components of the spilled oil.
Multiple regression models showing good overall correlations to tit the
data were obtained using only three variables with their regression para-
2:0:
20 40 60 80 100 120
lime (Days)
Fig. 2. Changes in total resolvable hydrocarbon (A) and polynuclear aromatic hydro-
carbon (B) concentrations relative to hepane in subsurface control and bioremediated KN-
135 shorehnes. Fertilizer was applied to the control at 70 days. The R-square in these
analyses was > 0.8, and the probability > F= 0.0000, both showing that the tit was highly
significant.
324 R. M. Atlas
meters: ratio of average nitrogen concentration per unit of oil load,
ln(1 - polar fraction), and time (Bragg et al., 1993a,b, 1994). The linear
regression equation for the model shown in was ln(tota1 hydrocarbons)/
hopane) = a + b( 1 - polar fraction of the residual oil) + c(nitrogen
concentration/total amount of residual oil load) + d(time). For the surface
oil the amount of previous biodegradation was critical for determining the
success of bioremediation. The most important parameter that affected
the rates and extent of degradation in subsurface oil, as shown by the
multiple regression analyses, was the ratio of nitrogenous nutrients in the
interstitial water to oil load. Based on the measurements reported in the
EPA in separate tests at Disk Island in Prince William Sound, it seems
likely that nitrogen delivery to the interstitial water can sometimes be
quite low, even with high treatment delivery to the beach surface.
As a result of the EPA-Exxon and joint monitoring projects, bior-
emediation of oil-contaminated beaches was shown to be a safe cleanup
technology. This was based on monitoring the quality of nearshore waters
for toxicity to sensitive marine species, such as Mysid shrimp, in the vici-
nity of shorelines treated with fertilizers; evaluating potential stimulation
of algal growth by measuring chlorophyll concentrations in those near-
shore waters; and testing for oil release into nearshore waters from shor-
elines treated with fertilizers. The addition of fertilizers caused no
eutrophication, no acute toxicity to sensitive marine test species, and did
not cause the release of undegraded oil residues from the beaches.
DISCUSSION
Studies on seeding with nonindigenous organisms have not provided
convincing evidence that this is an efficacious approach to the bior-
emediation of oil pollutants in marine environments. To be effective, seed
cultures must compete with indigenous hydrocarbon-degrading micro-
organisms. They would have to be applied quickly if the aim was to
shorten he lag period before the indigenous populations adapted to the
presence of oil. In general, the rate-limiting factors for polluting oil
following marine spills are environmental and not due to the enzymatic
capacities of the indigenous hydrocarbon-degrading microorganisms.
Nutrient addition has been demonstrated to overcome a critical rate
limiting factor in aerobic oil-contaminated marine environments. Rates of
stimulation have been typically about three to five times natural rates of
oil biodegradation. Greater stimulation might be achieved by higher levels
of nutrient addition, but this could risk ecological side-effects such as
toxicity to marine life and eutrophication with associated algal blooms.
Bioremediation of petroleum pollutants 325
Because of its effectiveness, bioremediation became the major treatment
method for removing oil pollutants from the impacted shorelines of Prince
William Sound. The success of the field demonstration program has now
set the stage for the consideration of bioremediation as a key component
(but not the sole component) in any cleanup strategy developed for future
oil spills.
No national coordinated response plan for oil spill bioremediation
currently exists that would create and test a scheme for using bior-
emediation in dealing with oil spilled anywhere in the US, including open
ocean areas, harbors, shorelines, estuaries, rivers, or on land.
At the present time, there are no definitive scientific data that unequi-
vocally demonstrate that addition of large quantities of oil-degrading
bacteria to oil in the open ocean leads to removal of the oil by biode-
gradation. Addition of microorganisms to oil in controlled laboratory
studies has been shown to enhance biodegradation, but conclusive data
from field experiments are not available and will be difficult to obtain.
Generally, microbial degradation of oil, even under the most favourable
of laboratory conditions, takes weeks to months. The purpose and docu-
mented result of the risk of bioremediation is to enhance degradation over
extended time periods, rather than to achieve short-term or immediate
results.
Using bioremediation to remove pollutants is inexpensive as compared
to physical methods for decontaminating the environment that are extra-
ordinarily expensive. Over $1 million a day was spent on physical washing
of shorelines in an attempt that was only partially successful to clean up
the oiled rocks of Prince William Sound, Alaska using water washing and
other physical means after the Exxon tanker ran aground there. Actual
costs for bioremediation of hundreds of miles of shoreline was probably
less than $1 million, excluding the efficacy and safety testing costs that
were about $10 million.
Bioremediation, though, is not the solution for all environmental
pollution problems. Like other technologies, bioremediation has limita-
tions as to the materials that can be treated, conditions at the treatment
site, and the time that is available for the treatment.
CONCLUSIONS
Laboratory data provide reliable predictions of bioremediation perfor-
mance with rates of biodegradation in the field being comparable to those
measured in laboratory tests. Field analyses require comparison of
compositional changes to a relatively nondegradable component of the oil
326 R. M. Atlas
and regression analyses to assess the efficacy of bioremediation given the
patchy distribution of oil in the environment following an oil spill, such as
multiple regression analyses of changes in oil composition relative to
hopane as a function of nitrogen concentration in the sediment pore water
and of oil polars content. The monitoring of bioremediation following the
Exxon Vuldez spill provides statistically sign&ant measures of bior-
emediation effectiveness with high levels of confidence. These analyses
show that differences in biodegradation rates resulted mainly from differ-
ent levels of nutrients in sediment pore waters. Higher rates could have
been achieved by adding greater concentrations of nutrients. Results
suggest that increasing the frequency of application and the fertilizer
dosage rate, consistent with maintaining levels safe to biota, could further
increase the rate of oil biodegradation. The strong correlation between
nitrogen concentration in sediment pore water and the rate of oil degra-
dation suggests improved monitoring techniques for optimizing perfor-
mance in future bioremediation applications in intertidal environments. In
other shoreline environments, concern may need to be given to oxygen
concentrations since rapid hydrocarbon degradation is an obligately
aerobic process.
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